Mark Leick, MD

Findings from a germline genetics study of CAR T cell therapy could help guide how researchers select donor T cells for allogeneic, "off the shelf" CAR T products. The results also point to a new way to identify genes that could improve future CAR T cell designs.

Mark B. Leick, MD, a medical oncologist and physician scientist at Massachusetts General Hospital who led the study, spoke with HCPLive about the clinical and translational implications of the findings, which linked germline variants in STXBP2 to CAR T cell toxicity and variants in PTPN22 to greater CAR T cell expansion.

Selecting Donor T Cells for CAR T Therapy, With Mark B. Leick, MD

"I think the places where it can become relevant [is that] there's a shift now to do, rather than sort of autologous CAR T cells, where we generate CAR T cells from each patient for themselves, allogeneic CAR T cells, where we take T cells from one healthy person," Leick told HCPLive.

Leick pointed to several advantages of allogeneic, or donor derived, CAR T cell products over the autologous approach, in which a patient's own T cells are used to manufacture their therapy. Chemotherapy can leave a cancer patient's own T cells less fit and less able to kill target cells, and the cancer itself can further suppress T cell function. Manufacturing autologous CAR T cells from a sick patient's own cells can also take weeks to months, whereas healthy donor cells could theoretically be manufactured in advance and kept frozen and ready to use.

"Up until now, there's been relatively little thought put into how do you choose those donor T cells," Leick said. He noted that although some functional assays are used, a single donor's T cells can be used to manufacture CAR T products for dozens or hundreds of patients, raising the stakes of that selection process. Leick said the study's findings suggest researchers developing allogeneic CAR T products should factor genetic variants like those identified in the study into how they screen and choose donor T cells.

Natural Human Variation as an Alternative to CRISPR Screening

Beyond donor selection, Leick said the findings offer a new strategy for identifying genes that could be engineered into future CAR T cell products to improve their efficacy. Historically, the field has leaned on synthetic screening approaches, generally using CRISPR-Cas9, in which a gene is knocked out in a pool of CAR T cells before the cells are tested in a mouse model or in vitro to see whether the edit improves CAR T cell function.

"This has yielded some interesting results, but the problem is we don't actually know what happens when you put that in a human," Leick said.

Leick said the advantage of his team's approach is that it draws on natural variation that already exists within the human population. Because all of the patients in the ZUMA-1 and ZUMA-7 trials reached adulthood, the variants identified in the study are known to be compatible with survival, unlike CRISPR edited genes with unknown effects in humans. That allowed investigators to use real clinical outcomes, rather than a mouse model or in vitro system, to evaluate the effects of specific genetic variants on CAR T cell activity.

Leick's team is now pursuing the PTPN22 variants identified in the study as a target for engineering into other CAR T cell products. Two patients in ZUMA-1 who carried the same PTPN22 mutation had the highest CAR T cell expansion of the roughly 100 patients in the trial, which Leick said suggests the variant confers a gain of function advantage in the context of CAR T cell therapy.

Limitations Tied to Ancestry and a Single CAR T Product

Leick said the study carries 2 notable limitations. Germline genetics analyses require comparing genetic variation among patients of comparable ancestry, and because clinical trial accrual in the United States skews toward patients of European ancestry, the study's population was limited mostly to that group. Too few non-European patients were enrolled to support a meaningful analysis, and Leick said his team would like to extend the work to more diverse populations.

The study also focused on a single CAR T cell product, axicabtagene ciloleucel, in patients with lymphoma, so the findings may not generalize to other CAR T cell products or disease types. Leick said his lab is now working to determine whether the findings extend to other diseases and CAR T cell contexts.

Editor’s Note: Leick reports relevant disclosures with BioNTech, Cabaletta Bio, and Adaptimmune.

Leick MB, Sun B, Birocchi F, et al. Genomic correlates of clinical CAR T cell activity. Sci Immunol. 2026;11(121). doi:10.1126/sciimmunol.aef4134

Neelapu SS, Locke FL, Bartlett NL, et al. Axicabtagene ciloleucel CAR T-cell therapy in refractory large B-cell lymphoma. N Engl J Med. 2017;377(26):2531-2544.

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Genetic variants tied to CAR T toxicity and expansion could guide how researchers screen healthy donors for allogeneic CAR T products.

Germline genetic variants may help explain why CAR T cell therapy produces such variable toxicity across patients with aggressive lymphoma, according to findings published in Science Immunology.1 Investigators identified a specific gene, STXBP2, whose variants were enriched among patients who developed severe toxicity after treatment with axicabtagene ciloleucel.

"CAR T cells are different than every other form of therapy that came before," said Mark B. Leick, MD, medical oncologist and physician-scientist at Massachusetts General Hospital and the study's corresponding author. Unlike a standardized drug, Leick noted, CAR T cells are manufactured from a patient's own T cells, carrying all of that patient's inherited genetic variation into the final product.

A Genetic Signature May Predict Who Gets Sick From CAR T Cell Therapy

A Rare Disease Pointed Investigators Toward STXBP2

Investigators performed whole genome sequencing on patients with aggressive lymphoma treated with axicabtagene ciloleucel in the ZUMA-1 (n = 86 evaluable) and ZUMA-7 (n = 150 evaluable) clinical trials, in partnership with Kite Pharma.1 The hypothesis was rooted in hemophagocytic lymphohistiocytosis (HLH), a rare inherited hyperinflammatory syndrome that shares key biochemical features with cytokine release syndrome (CRS), the most common toxicity of CAR T cell therapy.1 Leick and colleagues reasoned that milder, adult-onset forms of the same genetic defects that cause HLH might also be driving toxicity in some CAR T cell recipients.

Among the ZUMA-1 toxicity cohort (n = 39), 6 patients (15.4%; 95% CI, 5.9%-30.1%) carried putative deleterious variants in STXBP2, a gene involved in lymphocyte degranulation, compared with none in the 39-patient control cohort (OR, >1.44; nominal P = .025).1 The finding was not replicated in ZUMA-7, which investigators attributed to that cohort's lower baseline inflammation and fewer prior treatments relative to the more heavily pretreated ZUMA-1 population.

CRISPR Experiments Confirmed a Mechanistic Role

To confirm a mechanistic role for the gene, investigators used CRISPR-Cas9 to knock out STXBP2 in CAR T cells manufactured from healthy donors.

The STXBP2-deficient cells showed impaired degranulation and cytotoxicity, along with increased inflammatory cytokine production when cocultured with tumor targets, mirroring the cytokine profile seen in variant carriers in the clinical cohort.

Investigators noted that the ZUMA-1 and ZUMA-7 cohorts were predominantly of European ancestry, limiting conclusions in non-European populations, and that the number of STXBP2 variant carriers identified was small, warranting confirmation in larger, more diverse cohorts.

Germline STXBP2 variants may predict severe CAR T cell toxicity, Mark B. Leick, MD, and colleagues find

 suggest germline genetic variants may help explain why CAR T cell therapy outcomes vary so widely between patients.

Investigators performed whole genome sequencing on patients with aggressive lymphoma treated with axicabtagene ciloleucel in the ZUMA-1 and ZUMA-7 clinical trials, in partnership with Kite Pharma.

Analysis focused initially on genes associated with hemophagocytic lymphohistiocytosis (HLH), a rare hyperinflammatory syndrome with biochemical similarities to cytokine release syndrome (CRS), the most common toxicity of CAR T cell therapy.

Investigators found that variants in STXBP2 were enriched among patients with high-grade toxicity in the ZUMA-1 cohort, a finding supported by in vitro experiments showing that STXBP2-deficient CAR T cells had impaired degranulation and increased inflammatory cytokine production.

Extending the analysis genome-wide, variants in ADAMTSL3 were linked to protection from toxicity, while variants in PTPN22 were strongly associated with greater CAR T cell expansion, across both ZUMA-1 and ZUMA-7.

HCPLive spoke with Mark B. Leick, MD, a medical oncologist and physician scientist at Massachusetts General Hospital who led the study, about its design and what the findings could mean for CAR T cell manufacturing and patient management going forward.

Q&A: Germline Variants May Predict CAR T Cell Toxicity, With Mark Leick, MD

HCPLive: What motivated you and your colleagues to investigate whether inherited germline genetics could influence outcomes after CAR T cell therapy?

Leick: CAR T cells are different from every other form of therapy that came before. It's not like a Bayer aspirin, where every dose is chemically identical. CAR T cells are manufactured from a patient's own T cells, so they carry all of that patient's ancestral genetic polymorphisms and mutations. Because of this, how CAR T cells behave varies from patient to patient in ways we can't totally understand.

Going into this study, we had some idea of which patients were at high risk of toxicity. Patients with a large tumor burden, or higher baseline levels of inflammation, tend to be more likely to have serious side effects. But beyond that, we didn't have a good understanding of why some patients go through catastrophic toxicities and others don't. It's a little like COVID-19, where some patients end up on a ventilator with severe immune inflammation and others sail through with minimal side effects.

Our hypothesis was founded on a rare disease called hemophagocytic lymphohistiocytosis (HLH), which classically affects pediatric patients, usually kids under age 5. In that disease, patients are born with defects in how their lymphocytes, T cells, and NK cells kill virally infected cells. When those lymphocytes can't kill effectively, they become continually activated and trigger an inflammatory cascade that can become life-threatening.

In the CAR T cell field, we've long recognized that the biochemical features of HLH and CRS after CAR T cell therapy are very similar, and the toxicity mechanism is thought to be similar as well: in HLH it's an inactivated T cell trying to kill a virally infected target, and in CAR T cells it's a CAR T cell trying to kill a tumor target, but the end result is the same, cytokines are produced that activate the myeloid system and drive high fevers, low blood pressure, and other symptoms.

Since HLH has a genetic basis, and we now know less severe forms of these mutations can present later in life, we hypothesized that some of those less severe variants might be present in patients who develop severe toxicity after CAR T cell therapy.

HCPLive: Can you walk our audience through what you eventually found?

Leick: We partnered with Kite Pharma, the largest producer of CAR T cell products, which gave us access to DNA samples, CAR T cell level measurements, clinical data, and serum biochemical data from patients across the ZUMA-1 and ZUMA-7 clinical trials of axicabtagene ciloleucel in patients with aggressive lymphoma.

We performed whole genome sequencing, but because these early CAR T cell trials only included around 100 patients, we needed a narrow hypothesis rather than an unrestricted genome-wide search. So we focused on fewer than 20 genes associated with HLH.

We defined a combined toxicity endpoint that captured patients with high-grade CRS, high-grade neurologic toxicity, or those who received tocilizumab, the antidote for CRS, since those patients likely would have had higher-grade toxicity without it. Among that pool, we found 6 patients with variants in the gene STXBP2, which is involved in lymphocyte degranulation, compared with none in the control cohort without high-grade toxicity.

We followed up with wet lab studies, comparing CAR T cells from patients with the variant to CAR T cells from controls, and found that cells with STXBP2 variants became more inflamed when they encountered target cells. We also introduced the CAR T cell gene and the specific variants into healthy donor T cells and saw the same result in vitro.

We then sought to replicate this finding in the ZUMA-7 cohort, an independent group of patients treated with the same CAR T cell product, but earlier in their line of therapy and with less inflammation overall. We didn't see the same STXBP2 finding there, which we think reflects that these patients had less severe disease and fewer prior treatments.

With both cohorts available, we could also look beyond toxicity. We found that variants in PTPN22 were strongly correlated with CAR T cell proliferation across both ZUMA-1 and ZUMA-7. PTPN22 helps regulate normal T cell biology and serves as a brake on T cell receptor activation, so there's a plausible mechanism: lose the brake, and T cells become more activated and proliferate more.

In fact, 2 patients had the exact same mutation, and that led them to have the highest CAR T cell expansion of all the 100 patients in ZUMA-1, suggesting that there is some sort of gain of function advantage to having that mutation in the context of CAR T.

HCPLive: Is there anything else you'd like to highlight, including any limitations?

Leick: I'd add a couple of limitations. Germline genetics analysis requires comparing genetic variation within populations of comparable ancestry, and because clinical trial accrual in the United States skews toward patients of European ancestry, our study was limited mostly to that population. We only had a couple of non-European patients, not enough to support any meaningful analysis. We'd love to study this in more diverse populations.

Our study was also focused on one product, axicabtagene ciloleucel, so these findings may not apply to other CAR T cell products or disease types beyond lymphoma. We're working in the lab now to understand whether this applies more broadly.

As for clinical implications, this doesn't mean patients with these variants got severely ill. No one died in either trial, and even knowing a patient had these variants, we likely wouldn't manage them differently since we're already fairly good at treating these toxicities. Where I think this becomes more relevant is in choosing donor T cells for allogeneic CAR T cell products, since a single donor's cells can be used to manufacture products for dozens or hundreds of patients, and in using natural human genetic variation as an alternative to CRISPR-based screens to identify genes, like PTPN22, that could be engineered into future CAR T cell products to improve efficacy.

Leick MB, Sun B, Birocchi F, et al. Genomic correlates of clinical CAR T cell activity. 

. 2026;11(121). doi:10.1126/sciimmunol.aef4134

Articles

Whole-genome sequencing in aggressive lymphoma treated with axicabtagene ciloleucel implicates germline variation as a contributor to interpatient heterogeneity in CAR T efficacy and toxicity. An HLH-informed candidate approach associated STXBP2 variants with severe CRS/neurologic toxicity in ZUMA-1, supported by in vitro evidence of impaired degranulation and heightened cytokine release. Genome-wide analyses identified ADAMTSL3 variants linked to reduced toxicity and PTPN22 variants correlated with greater CAR T expansion across ZUMA-1 and ZUMA-7. Generalizability is constrained by ancestry and product specificity.

New research links germline genetic variants to CAR T cell toxicity risk and expansion in lymphoma patients treated with axi-cel.