Among recent advances in cancer treatment, it’s hard to think of something with the sheer reach and impact of immunotherapy, a new but rapidly developing field which seeks to leverage the immune system—arguably, humanity’s oldest weapon—in the fight against tumors. Dr. Stanley Riddell, a professor in the Translational Science and Therapeutics Division at Fred Hutch, and his team stand at the forefront of cancer immunotherapy research; a recent publication from the lab in Blood Advances led by former postdoc Dr. Isabel Leung tackles an important technical hurdle in the field.
In their study, Leung and colleagues focused on a particular method called CAR (Chimeric Antigen Receptor)- T cell therapy. In essence, CAR-T cell therapy works by equipping a patient’s T cells to recognize and eliminate tumor cells. Researchers accomplish this by extracting T cells and genetically engineering them to express a synthetic chimeric antigen receptor (CAR)—a membrane-bound protein which recognizes a specific antigen present on the tumor cells and initiates downstream signaling to spur the T cell into action. Once expanded and reintroduced into the patient, CAR-T cells are able to recognize the tumor cells as targets and go to work as a ‘living drug.’ This therapy has proved transformative in for many previously difficult to treat cancers, but it leaves researchers and doctors with an important decision: which tumor antigens should they engineer the T cells to target? Traditionally, CAR-T cells are engineered to target a single antigen; however, patient tumors are often heterogenous in their antigen expression, and cancers often overcome so-called monospecific CAR-T cells and relapse. Thus, a new generation of CAR-T cell therapies features multispecific CAR-T cells—T cells engineered to recognize multiple tumor antigens. But do these multispecific CAR-T cell therapies actually work better than monospecific ones? And how should they be designed for best effect?
The research team tackled this question by considering the treatment of B cell malignancies with bispecific CAR-T cells targeting two antigens: CD19, a surface protein expressed on normal and cancerous B cells and considered a mainstay CAR-T cell target, and a different protein complex called CD79ab, which Leung and colleagues confirm is expressed in a variety of B cell cancer cell lines and patient tumor samples. Designing monospecific CARs against three individual targets (CD19, CD79a, and CD79b), the team confirmed that they had anti-tumor activity in mouse xenograft models. They also saw, however, that tumors were able to escape detection by these CARs by losing expression of the respective antigen over time.
As a next step, the authors sought to examine whether bispecific CARs targeting CD19 and CD79a or CD79b would be more effective than their monospecific counterparts. They designed bispecific CARs using three fundamentally different approaches: tandem CARs, where the T cells express a single receptor containing a CD19 recognition domain fused to a CD79a/b recognition domain, bicistronic CARs, which express two different receptors on their surface (one for CD19 and one for CD79a/b), and pooled CARs, in which monospecific CARs against CD19 are simply mixed together with monospecific CARs against CD79a/b. To test the effectiveness of these CARs at preventing tumor therapy escape, they contrived a system which involved ‘doping’ mouse xenograft cells with a small proportion (1%) of CD19 knockout cancer cells. As expected, treating these tumors with monospecific CARs against CD19 led to eventual expansion of the CD19-knockout cells and relapse of the tumor; however, mice receiving the tandem and bicistronic CARs showed less tumor growth and lived longer.
During the course of their study, Leung and colleagues noticed a strange trend: the tandem and bicistronic CARs, when stimulated by purified CD19 and CD79 proteins in vitro, produced lower amounts of inflammatory cytokine molecules relative to their monospecific counterparts. Indeed, by creating mouse xenograft models with varying proportions of wild-type, CD19-null, and CD79-null cancer cells, the authors confirmed in vivo that tandem and bicistronic CARs actually perform worse against tumors expressing only a single target antigen (CD19 or CD79ab). What could explain this counterintuitive result? Some more digging led the team to an interesting conclusion: the CD19 and CD79ab recognition domains in tandem CARs—which are attached to the same receptor in close proximity—may actually hinder each other’s antigen binding, while the CD19 and CD79ab receptors on bicistronic CARs—which are present on the same cell but otherwise separate—may compete for the same pool of downstream signaling molecules, damping the overall T cell response to either antigen alone!
In all, the study sheds light on an important trade-off when considering multispecific CAR-T cell therapies: benefits with regards to antigen specificity come at the cost of reduced antigen sensitivity. While bispecific CAR-T cells show promise as a more effective treatment strategy against tumors expressing multiple antigens, tumor antigen composition should weigh into the choice of CAR-T cell therapy; the simple assumption that ‘more receptors is better’ won’t cut it. With its careful investigation of different CAR configurations and rigorous tumor model work, the study also sets the stage for further technical development of CAR-T cell therapy to expand their reach in cancer treatment.
The spotlighted research was funded by the National Institutes of Health and the Leukemia and Lymphoma Society.
Fred Hutch/University of Washington/Seattle Children’s Cancer Consortium members Drs. Shivani Srivastava, Cecilia Yeung, and Stanley Riddell contributed to this study.
Leung, I., Templeton, M. L., Lo, Y., Rajan, A., Stull, S. M., Garrison, S. M., Salter, A. I., Smythe, K. S., Correnti, C. E., Srivastava, S., Yeung, C. C. S., & Riddell, S. R. (2023). Compromised antigen binding and signaling interfere with bispecific CD19 and CD79a chimeric antigen receptor function. Blood Advances, 7(12), 2718–2730. https://doi.org/10.1182/bloodadvances.2022008559