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. Author manuscript; available in PMC: 2024 Dec 5.
Published in final edited form as: Semin Hematol. 2023 Dec 5;60(5):329–337. doi: 10.1053/j.seminhematol.2023.11.007

CD19 CAR-T cell therapy for Relapsed or Refractory Diffuse Large B Cell Lymphoma; Why does it Fail?

Hannah Kinoshita 1,2, Catherine M Bollard 1,2, Keri Toner 1,2
PMCID: PMC10964476  NIHMSID: NIHMS1949494  PMID: 38336529

Abstract

Chimeric antigen receptor T (CAR-T) cell therapy is an effective treatment for relapsed or refractory diffuse large B cell lymphoma (DLBCL) with three CD19 targeting products now FDA-approved for this indication. However, up to 60% of patients ultimately progress or relapse following CAR-T cell therapy. Mechanisms of resistance to CAR-T cell therapy in patients with DLBCL are likely multifactorial and have yet to be fully elucidated. Determining patient, tumor and therapy-related factors that may predict an individual’s response to CAR-T cell therapy requires on-going analysis of data from clinical trials and real-world experience in this population. In this review we will discuss the factors identified to-date that may contribute to failure of CAR-T cell therapy in achieving durable remissions in patients with DLBCL.

Diffuse Large B-Cell Lymphoma (DLBCL) is responsive to chemoimmunotherapy with long-term remissions in about 60% of patients.[1] For patients with refractory or relapsed disease, chimeric antigen receptor (CAR) T cell therapy (CAR-T) has proven an efficacious treatment. Studies have demonstrated objective responses in about 70% of patients, however, up to 60% of patients ultimately progress or relapse.[2–6] Determining patient-specific and therapy-related factors that predict individual response to CAR-T cells in patients with DLBCL is still required to identify those patients most likely to benefit. Three CAR-T products targeting CD19 have been FDA-approved for the treatment of DLBCL, and the respective phase II and phase III trials have informed many of these factors. Multiple real-world studies have further evaluated outcomes with CAR-T in relapsed and refractory (r/r) DLBCL, contributing to the quickly growing knowledge of how to best utilize adoptive cellular therapy. In this review, we will discuss factors that have been implicated in the failures of CAR-T cell treatment in patients with r/r DLBCL (Figure 1).

Figure 1: Factors associated with outcomes following CAR-T cell therapy for DLBCL.

Figure 1:

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Factors related to the CAR-T cell product, tumor size, type and microenvironment as well as to the host themselves are implicated in predicting overall disease response and survival following CAR-T cell therapy. Generated in BioRender.

Factors related to the CAR-T cell product

CAR-T structure

Various factors in CAR-T structure and function have been implicated in responses as well as toxicities. The first generation of CAR-T was comprised of an extracellular single chain variable fragment (scFv), which recognizes the antigen in an MHC-unrestricted manner. The scFv is connected to a transmembrane region and an intracellular signaling domain, which is derived from the CD3ζ protein of an endogenous T cell receptor.[7–9] Second generation CAR-T has incorporated a costimulatory domain (4–1BB and CD28 being the two most common) between the transmembrane and intracellular signaling domains, allowing the second signal to increase T cell activation.[9, 10] Third generation CAR-T has included multiple costimulatory domains (Figure 2). The hinge links the antigen-recognizing scFv to the transmembrane protein and are derived from either CD8 or CD28.[11] Each component has the ability to affect the function and persistence of the CAR-T construct.[12]

Figure 2: CAR-T cell structure.

Figure 2:

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Current FDA-approved CAR-T for DLBCL include second generation CAR-T targeting CD19 surface antigen. Changes in antigen binding, hinge, transmembrane and costimulatory domains all may contribute to effectiveness of CAR-T product. Generated in BioRender.

FDA-approved CAR-T constructs for DLBCL

There are currently three FDA-approved CAR-T products for the treatment of DLBCL targeting surface antigen CD19; axicabtagene ciloleucel (axi-cel), tisagenlecleucel (tisa-cel), and lisocabtagene maraleucel (liso-cel). These products first demonstrated efficacy when used as salvage therapy for relapsed/refractory (r/r) DLBCL [2, 13–15] and have since been evaluated in randomized clinical trials as second-line therapy as compared to autologous stem cell transplant (ASCT).[6, 13, 15, 16] The phase II trial, ZUMA-1, evaluating axi-cel which has a CD28 transmembrane and costimulatory domain, demonstrated an objective response rate (ORR) of 82% (complete remission (CR) of 58%) with a 5 year overall survival (OS) of 42.6%, while complete responders had an OS of 64.4%.[17, 18] The phase II trial JULIET evaluating tisa-cel, generated with a CD8 transmembrane and 4–1BB costimulatory domain, demonstrated an ORR of 52%, but comparably, 60% of the responding patients maintained a clinical response at 40 months.[5] The TRANSCEND phase II trial evaluating liso-cel, which also utilizes the 4–1BB costimulatory domain with a CD28 transmembrane domain, reported an ORR of 73%. Liso-cel is uniquely manufactured whereby CD4+ and CD8+ T cell populations are selected, transduced separately, and then combined to create the final product.[6] Phase III studies compared each product to the second-line standard of care (ASCT) with ZUMA-7 (axi-cel) and TRANSFORM (liso-cel) demonstrating higher complete response rates (CRR) and superior event-free survival (EFS) while BELINDA (tisa-cel) showed no differences in CRR or EFS.[13, 15, 19, 20]

While all three FDA-approved constructs and various other CD19 directed CAR-T products have demonstrated efficacy, direct head-to-head comparison has been difficult due to lack of prospective comparative clinical trials and differing methods of evaluation in prior phase II and III trials. By identifying specific construct characteristics and external factors which may improve CAR-T function in vivo, we can prospectively develop more efficacious constructs.

CAR-T kinetics

CAR-T cells in vivo undergo periods of expansion, contraction and proliferation. These periods have significant variability between patients and CAR-T products.[21] The CD28 costimulatory domain, present in axi-cel, demonstrates a faster signal with greater peak amplitude, while the 4–1BB costimulation (tisa-cel and liso-cel) tends toward longer persistence.[10] It has been historically prescribed that increased expansion leads to toxicities, including both cytokine release syndrome (CRS) and immune effector cell-associated neurotoxicity syndrome (ICANS), and that durable remissions are associated with prolonged proliferation and persistence, though these generalities hold nuances between products and have not been consistently demonstrated. Currently, there are not standardized methods to evaluate CAR-T persistence and kinetics, and the lack of reporting leads to inability to accurately compare kinetics across various CAR-T products across clinical trials.[22] The two main methods of detection of CAR-T post infusion are flow cytometry and quantitative PCR,[22] including droplet digital PCR (ddPCR).[23]

CAR-T cell expansion

CAR-T cell expansion has been associated with initial response in multiple clinical trials. Axi-cel demonstrates rapid expansion within the first 7–14 days of infusion[16] and has been shown to correlate with objective and durable responses.[24] In the phase III clinical trial of tisa-cel, the geometric mean in vivo peak expansion was twice as high in patients who had a response compared to non-responders.[15] Though not consistent across studies, a longer EFS was also observed in patients who had a higher than median peak expansion.[15] Liso-cel also demonstrated increased peak expansion and area under the curve (AUC) in responders as opposed to non-responders in the TRANSCEND trial.[6]

Several studies have demonstrated that increased expression of markers of exhaustion in the T cell product correlate with decreased efficacy in vivo.[25–27] Deng et al reported that exhaustion signatures (PD-1 and IFN-γ) were more common in CD8+ T cell populations in patients with PR/PD, while immune memory signatures were higher in patients with CR.[26] However, in at least one study, increased levels of PD1+LAG3+ in CD4+ CAR-T cells in peripheral blood (as opposed to the product) post-infusion correlated with increased EFS, [28] possibly related to increased T cell activation during peak expansion in vivo. Ideally, CAR-T cells would not exhibit functional exhaustion prior to infusion but effect maximal peak activation in vivo to achieve an optimal clinical response.

CAR-T cell persistence

Liso-cel demonstrated long-term persistence at 1 year in 35 (52%) of 67 patients in the phase II trial.[6] Persistence of tisa-cel was also observed with a detectable CAR transgene present in 98% of patients at the 4 month assessment.[5] Of the patients who relapsed following tisa-cel as second-line therapy, 18 of the 34 evaluable patients still had quantifiable CAR transgene levels in the peripheral blood around the time of relapse.[15]

A single institution study evaluating 92 patients sought to compare CAR-T persistence utilizing ddPCR with outcomes in patients with r/r B cell lymphomas.[21] Only 2 of the 55 patients who had received tisa-cel had a negative CAR-T ddPCR within the first 6 months. This was opposed to axi-cel, in which 73% of patients (n =11) had undetectable CAR-T constructs 6 months after infusion.[21] Patients with CAR-T persistence had fewer relapses (29% vs 60% p=0.0336), and CAR-T persistence at 6 months was associated with longer progression free survival (PFS).[21] While CAR-T cell persistence in the peripheral blood was associated with longer PFS, there was no association with OS. However, it is acknowledged that the sample sizes in each group are relatively small.[21] A larger study (n=305) found that patients treated with tisa-cel were at more risk for an event as compared to patients who received axi-cel,[2] but confirmed CAR-T cell persistence in the peripheral blood was associated with longer PFS.

B-cell recovery, or loss of B cell aplasia, has been used in clinical trials and in the post licensure “real-world” setting as a surrogate for persistence of CD19-directed CAR-T therapy. The TRANSCEND trial evaluating liso-cel demonstrated that 75% of patients with ongoing clinical responses had evidence of B cell recovery at 24 months. However, some patients with response had B-cell recovery by 9 months. Thus, there are additional factors to consider since B cell recovery may not be the only indicator of CAR-T persistence in vivo, and/or persistence may not be as necessary to maintain the anti-tumor response past a certain time point in patients with lymphoma.[6] As additional constructs are introduced into clinical trials, it will be even more imperative to have a standard by which to compare CAR-T kinetics, including persistence, and correlation with disease response.[22]

Pre-manufacturing processes and the final immunophenotype of the CAR-T product likely also play a role in the potency of the CAR-T product in vivo. Gauthier and colleagues from the Fred Hutchinson Cancer Center report a single center retrospective analysis evaluating 129 patients with r/r mature B non-Hodgkin Lymphoma (NHL) comparing axi-cel, tisa-cel and an investigational product.[29] They identified lower efficacy with tisa-cel as compared to axi-cel. Interestingly, it was noted that the efficacy of the CAR-T product was differentially impacted by the pre-leukapheresis absolute lymphocyte count (ALC), with a stronger association between ALC and in vivo efficacy of tisa-cel as opposed to axi-cel products in vivo.[29] Abiding by recommended washout guidelines prior to apheresis and (if clinically able) the timing of the apheresis procedure to maximize ALC prior to collection is optimal. Additional research into leukapheresis materials and how they correlate to final product potency is ongoing to enhance manufacturing processes for a variety of CAR-T products.[26, 30]

External factors to improve CAR-T function

In phase III clinical trials, tisa-cel had the longest manufacturing time as compared to axi-cel and liso-cel, which could potentially lead to worse outcomes.[13] Decreased manufacturing time allows for more rapid access to treatment while also minimizing additional toxicities from bridging therapies. In a multi-institution consortium study evaluating the real-world results of axi-cel in a cohort of 275 patients, receiving bridging therapy was associated with poorer OS.[31] A single institution evaluation of 124 patients who received axi-cel, demonstrated no effect of bridging therapy on the ORR [32] however, those who received radiation therapy (RT) as bridging therapy did have an improved ORR as compared to those who received systemic chemotherapy. Further, radiation is known to increase tumor antigen expression which may aid in homing of antigen-specific T cell therapy, as well as affect effector function and proliferation of T cells.[32] Additional studies are needed to further elucidate the effect of radiation on CAR-T function and proliferation, but this could serve as an additional mechanism to improve efficacy in patients who require bridging therapies.

Host Factors

Age

Increased age is a poor prognostic factor in outcomes of DLBCL[33]. In patients with r/r DLBCL, approximately 50% are over the age of 70 years[34] and have limited available treatment options as many centers would not consider this group fit to undergo the second-line standard of care (SOC) of high dose (HD) chemotherapy followed by ASCT. The 2-year OS in patients >70 years is only 19%[34], indicating a need to better evaluate the role for and long-term outcomes of alternative non-transplant therapies including CAR-T cell therapy in this population. However, due to strict eligibility criteria, only 23.6–24% of patients in the phase II clinical trials of axi-cel and tisa-cel were over 65 years old[17, 25]. In the more inclusive trial of liso-cel[6], in which 42% of patients were over 65 years of age, subgroup analysis showed that CRR was comparable in patients over 65 years [60.2% (95%CI50.3–69.5)] versus those under 65 years [48% (95%CI39.7–56.3)][6]. In real-world retrospective analyses of patients receiving commercially available axi-cel and other CAR-T therapies, the distribution of age better reflected the true patient population with 51.7–62% of patients reported as ≥60 years of age [31, 35]. Paradoxically, multivariate analysis identified that age <60 years was associated with poorer response rate and PFS than in patients ≥60 years following axi-cel infusion[31]. Further, in a phase III, multi-site randomized controlled trial of primary r/r DBCL in ASCT-eligible patients comparing second-line axi-cel to the SOC, a longer EFS was observed in patients ≥65 years in the axi-cel group with the proportion of patients ≥65 years accounting for 28% of patients in the axi-cel group and 32% in the SOC group[19]. Of note, only 64 (35.7%) of the 179 patients assigned to the SOC group ultimately received HD chemotherapy due to various reasons including progression of disease and insufficient response to salvage chemotherapy, whereas 94% of patients assigned to the axi-cel arm received the infusion[19] highlighting the challenges with standard therapy. Representation of patients over 65 years of age was similar in the tisa-cel and liso-cel phase III trials with 33.3% and 39% of patients reported as over 65 years in the CAR-T groups and 28.8% and 27% in the SOC groups, however age was not identified as a relevant covariate in the exploratory post-hoc analysis of tisa-cel and was not evaluated in the liso-cel trial[15, 20]. These data suggest that increased age alone (over 65 years) is not associated with poorer outcomes following CAR-T cell therapy and that it may be better tolerated, with higher treatment success compared to attempting salvage therapy followed by HD chemotherapy and ASCT in this population. However, further studies are needed to evaluate outcomes in patients ≥65 years more comprehensively, particularly in patients with primary r/r DLBCL.

Although much less common than in adults, 4–10% of pediatric patients with mature B cell lymphomas will have refractory disease or relapse[36–39] and have a similarly dismal prognosis with a 5-year survival rate of 10–30%[36–39]. Although the distribution, aggressiveness of disease and disease subtypes differ from the adult population, these patients are also in need of novel therapies to improve outcomes. Pediatric patients were excluded from the adult CAR-T clinical trials but given the significant successes of CD19 directed CAR-T cell therapy for other B cell malignancies in pediatric patients and the benefits seen in r/r DLBCL in adults, there are currently several open trials evaluating CD19 CAR-T cell therapy for mature B cell and Burkitt lymphoma in the pediatric population[40]. Preliminary results from phase I/II trials suggest that there may be efficacy for pediatric patients with mature B cell NHL with a tolerable safety profile, but we will need to await the results of the trials before making definitive conclusions.

International Prognostic Index

To standardize the evaluation of the negative prognostic factors identified in patients with newly diagnosed NHL, a prognostic model focusing on factors related to tumor growth, stage and invasive potential as well as patient-specific factors, including age, serum LDH level and performance status was developed[33]. The international prognostic index (IPI) has been routinely used in newly diagnosed patients diagnosed with DLBCL to risk-stratify this heterogeneous population and was used to classify patients in the early phase trials of CD19 CAR-T for r/r DLBCL, with higher IPI scores historically conferring a poorer prognosis[15, 17, 25]. However, there was no association between IPI score ≤2 versus >2 and response rates in the phase II clinical trials for axi-cel and tisa-cel with the proportion of patients with an IPI score at enrollment of 0–2 reported as 52%[17]and <2 in 26.8%[25], respectively. In the phase III trial of tisa-cel, an IPI score of ≥2 was associated with receiving bridging therapy but there did not appear to be an association with modified EFS or best overall response in multivariate analysis[15]. In a real-world analysis of axi-cel, in which 45.6% of patients had an IPI ≤2, univariate analyses demonstrated that IPI 0–2 was associated with a better 12-month PFS (58%) and OS (80%) compared to higher scoring patients (PFS 37%; OS 57%)[31]. Further studies that are designed to detect differences in outcomes based on IPI will need to be conducted before making inferences on the utility of IPI in predicting prognosis following CAR-T cell therapy. However, certain factors that have previously been identified as poor prognosticators included in the IPI, such as age >65 years may not hold true in the era of immunotherapy for r/r DLBCL. These data suggest that categorizing patients based on the IPI alone may be insufficient to identify patients likely to benefit from CAR-T cell therapy.

Obesity

Obesity is associated with poorer outcomes in cancer therapy, particularly with conventional chemotherapy. However, obese patients are often excluded from or under-represented in early phase clinical trials. To address these concerns with respect to CAR-T cell therapy, a group at the Mayo clinic conducted a retrospective study evaluating baseline characteristics, patterns in prescribing lymphodepleting chemotherapy, CAR-T associated toxicities and clinical response as well as OS in obese and non-obese adult patients with r/r DLBCL who received axi-cel therapy[41]. In this cohort of 78 patients, only 28% of patients were defined as overweight (BMI 25–29.93kg/m2) and 24% of patients were obese (BMI≥30kg/m2). When obese versus non-obese patients were compared, the only differences identified were with respect to measures of renal function and a reduction in the dose of lymphodepleting chemotherapy administered. Fludarabine dose <80% of the standard dose was the only factor associated with survival outcome further highlighting the importance of LD chemotherapy in CAR-T cell therapy and the need for standardization in obese patients. Specifically, there was no difference in 12-month EFS, OS, best clinical response, cumulative rate of relapse or toxicity associated with CAR-T between the obese versus non-obese groups[41]. These findings are consistent with other studies that found no effect of obesity on outcomes following CAR-T cell therapy used in pediatric and adult patients for the treatment of hematologic malignancies[42]. However, another retrospective study evaluating onset and rate of CRS, following CAR-T cell therapy (axi-cel, tisa-cel or KTE-X19) found an increased odds in developing CRS grade ≥2 in patients with a BMI ≥27.05 kg/m2, (P=0.007), waist to height ratio ≥0.594 cm/m2 (P=0.01), waist circumference ≥99.23 cm (P=0.04) and visceral fat deposits ≥144.3 cm2 (P=0.02)[43]. Together, this data suggests that obese patients should not be excluded from clinical trials of CAR-T cell therapy based on BMI alone but that reporting of obesity as a potential factor in outcomes and toxicity remains important to identify specific risks and supportive care measures that may be taken to ensure the best outcomes for all patients.

Race and Ethnicity

The effect of race and ethnicity on outcomes following immunotherapy is not well-understood. In a retrospective evaluation of pediatric and adult CAR-T cell trials for B cell malignancies, race and ethnicity were not associated with CRR, OS and neurotoxicity but Hispanic patients were found to have a higher risk of severe CRS compared to white non-Hispanic patients[42]. When reported, the majority of patients (79.5–83%) on clinical trials of CAR-T cell therapy for r/r DLBCL were white[15, 19], which is consistent with other clinical trials, making it challenging to determine if these results can be generalized to the population as a whole. Expansion of access to novel agents to under-represented populations and evaluation of disparities which may result in poorer outcomes therefore is an important area of investigation.

Host Inflammatory State

Pro-inflammatory states in the host may impact the effective expansion of CAR-T cells in vivo. Circulating markers of inflammation and a hypoxic tumor microenvironment; ferritin, interleukin-6 (IL-6) and LDH were found to be associated with pre-treatment tumor burden[24]. Although there was no direct association of these markers of inflammation with absolute CAR-T cell expansion in vivo, ferritin and LDH may have had a slight association with lower CAR-T cell expansion when normalized to pretreatment tumor burden. Further, elevated LDH and IL-6 were associated with lower durable response rates and elevated baseline IL-6 is associated with a decreased objective response[24] indicating that a pro-inflammatory and high tumor burden state prior to CAR-T cell infusion portends a poorer response. Further evaluation is required to determine if this relationship is directly driven by reduced CAR-T cell expansion and survival in vivo and whether dampening the inflammatory response in the host pre- and post-CAR-T cell infusion would mitigate these poorer response rates. However, it does not appear that post-infusion use of glucocorticoids and tocilizumab in the setting of CRS influence PFS or response rates[17, 31].

Pretreatment Burden

Nastoupil and colleagues from 17 US institutions reported a median of 3 lines of pretreatment in their cohort of adults with r/r DLBCL with 74.5% of patients having received 3 or more lines of treatment prior to CAR-T cell therapy[31]. In this population, fewer than 3 lines of therapy was not associated with a difference in the 12-month best clinical response or PFS[31]. In another heavily pretreated cohort, regardless of the number of prior lines of therapy, patients who received CAR-T cell therapy had significantly better response rates and median OS and PFS than patients receiving alternate therapies[35]. The ORR with either CAR-T or alternate therapy was lower in patients who had received 4 or more prior lines of therapy, with a median number of lines of therapy prior to this analysis of 5 for CAR-T patients and 4 for those receiving alternate therapies[35] however, these findings are limited by the heterogeneity of alternate therapies employed. Clinical response, 2-year PFS and 2-year OS following CAR-T as second-line therapy for refractory or relapsed disease within 1 year of primary therapy were similar to the more heavily pretreated cohort receiving axi-cel in ZUMA-1[19] and were superior to the SOC for both axi-cel and liso-cel[19, 20]. However, most patients (73–76%) in the phase III second-line trials with axi-cel and liso-cel had primary refractory disease indicating a higher likelihood of being refractory to second-line chemotherapy[19, 20]. Slightly fewer patients (66%) had primary refractory disease in the tisa-cel trial that failed to show superiority over the SOC[15]. Patients who failed SOC, were allowed to cross-over to receive axi-cel or liso-cel in these studies, but had a lower EFS following CAR-T cell infusion as a third-line intervention than patients who received CAR-T as second-line treatment[19, 20] suggesting that those with primary chemotherapy-refractory disease may benefit from early transition to CAR-T.

Intestinal Microbiome

Effects of the intestinal microbiome on T cell immunity[44] as well as on the anti-tumor response to standard chemotherapy[45, 46] and immunomodulating therapies[47] (including cellular therapies)[48] have been investigated. A multi-center retrospective study evaluating adult patients with B cell malignancies receiving CD-19 CAR-T cell therapies identified differences in the intestinal microbiome. Specifically, they identified Clostridia species and exposure to broad-spectrum antibiotics as factors associated with clinical response and toxicity following CAR-T[48]. Larger scale studies may provide additional insight into the effects of the intestinal microbiome (as well as antibiotic use and nutrition) on responses to immunotherapy and potential mitigation strategies.

Tumor-specific Factors

Tumor Microenvironment

Efficacy of CAR-T cell therapy in patients with solid tumors is challenged by the hostile tumor microenvironment (TME), with physical and immunosuppressive barriers that may limit tumor infiltration and homing of CAR-T cells to the target site. In a small cohort of patients receiving a CD19-directed murine CAR-T for B-NHL, gene expression profiling of patient tumor samples before and after CAR-T cell administration demonstrated a decrease in chemokines (CCL2, CXCL8, CXCL12, CCL3, CCL4 and CCL5) that reduce recruitment of immunosuppressive pro-tumorigenic cells such as tumor-associated macrophages (TAMs), T regulatory cells (Tregs) and myeloid-derived suppressor cells (MDSCs) in patients achieving CR after CAR-T cell therapy[49]. Patients with a PR following CAR-T displayed higher levels of immunosuppressive cytokines (IL-10 and TFGF-B1) and increased expression of surface markers found on immunosuppressive MDSCs and TAMs[49], especially the M2 phenotype, that have been shown to inhibit T cell function[50].

Another study of pre- and post-CAR-T tumor biopsy samples from patients enrolled on the ZUMA-1 trial demonstrated higher T helper cell density in patients pre- and post-CAR-T who achieved CR[51]. Further, analysis of tumor immune contexture (characterized by three measures; immunoscore TL, TCE+ and Immunosign 21) demonstrated that changes in the TME gene expression profile changes rapidly (i.e. within 2 weeks of CAR-T cell infusion) and corresponds with clinical response in patients achieving CR. Specifically, these patients displayed a rapid upregulation of cytotoxic T cell-related genes[51]. Further, evaluation of germline mutations associated with T cell dysfunction were evaluated in a small cohort of patients (n=48) receiving CAR-T cell therapy using whole exome sequencing[52]. Germline mutations related to T cell function were discovered to be more common in patients with lower expansion and shorter persistence of CAR-T cells following infusion. The clinical response rates were also lower in this group[52] suggesting that there may be identifiable mutations associated with T cell dysfunction that confer primary resistance to CAR-T cell therapy. The interaction of the immediate tumor microenvironment and the host immune system are clearly important in determining patient response to immunotherapy and further investigation into this complex relationship may provide key information to identify patients at higher risk of failure following CAR-T cell therapy. Further, methods to extrinsically manipulate the TME with CAR-T constructs engineered to secrete cytokines that favor an immunologically active TME are under investigation.

CD19 Antigen Escape

As with other CAR-T cell therapies, one of the mechanisms potentially leading to resistance and/or relapse is antigen loss. CD19 surface marker expression by immunohistochemistry (IHC) and immunofluorescence staining of available tissue samples at baseline (n=96) and at the time of relapse (n=21) after treatment with axi-cel from patients on the ZUMA-1 trial[17] demonstrated that CD19 expression varied in the baseline samples but was mostly positive. In the post-relapse samples, loss of CD19 expression was identified in 25% of paired pre- and post-treatment samples and absence of CD19 expression was identified in 33% of relapsed patient samples. These findings were supported by another group that demonstrated 30% (n=18/61) of evaluable post-relapse samples were CD19 negative by IHC or flow cytometry[53]. The majority of samples in the first cohort showed preservation of other B cell markers including CD20, CD22 and CD79a as well as transcription factor PAX5[54], demonstrated isolated loss of CD19 expression as a means of antigen escape in DLBCL patients treated with CD19 directed CAR-T cell therapy. However, interestingly, CD19 negativity at the time of relapse or progressive disease after axi-cel therapy was not associated with inferior OS[53].

In B cell malignancies, multiple mechanisms of resistance have been identified that may also occur in B cell lymphomas following antigen-specific immunotherapy including selection of a pre-existing target antigen-negative clone, acquired mutations and post-transcriptional alternative splicing leading to complete loss of the antigen or the epitope recognized by CAR-T cells[55–57]. Downregulation or partial antigen loss likely also affect response to CAR-T cell therapy with studies showing that CAR-T cell cytokine production and cytotoxicity are reduced in the presence of low antigen expression on leukemic cell lines in vitro.[57] However, these mechanisms are less well-understood and need to be further evaluated in DLBCL particularly as expression at baseline is heterogeneous[58]. CAR-mediated trogocytosis is another mechanism purported to reduce cell surface antigen following exposure to CAR T cells whereby CD-19 targeting CAR-T cells extract the surface target (CD19) and demonstrate CD19+ expression after co-culture with leukemia cell lines concordant with reduced expression on the leukemia cells in vitro and may lead to fratricide[55]. The degree of trogocytosis that occurs following CAR-T has been demonstrated to depend on initial tumor cell antigen density, CAR design[55] and target affinity in vitro, with lower affinity CARs demonstrating reduced trogocytosis in leukemia and lymphoma cells[59]. Strategies to overcome antigen loss as a mechanism of resistance in these patients are therefore now being investigated for B cell leukemia including use of multi-antigen specific products and combinatorial approaches aimed at inducing or enhancing epitope spreading following CAR-T cell infusion (Table 1).

Table 1.

Proposed mechanisms of resistance to CAR-T cell therapy and potential strategies to mitigate treatment failures.

Mechanisms of Resistance Potential Strategies to Mitigate CAR-T Failure
Inadequate CAR-T cell product potency Use of allogeneic donors
Optimization of CAR construct design
Identification and selection of optimal T cell
phenotypic combination to generate CAR-T
Post-infusion activation of CAR-T
Combination with immune checkpoint inhibitors
High Tumor Burden Standardization of bridging therapy
Use of bridging radiation therapy
Enhance peak CAR-T cell expansion in vivo
Antigen Escape Multi-antigen targeting CAR-T constructs
Immunosuppressive TME Armored CAR-T
Host Inflammatory State Standardization/optimal timing of steroids and tocilizumab for treatment of CRS
Early transition to CAR-T treatment
Impaired Host Immunity Analysis of T cell function prior to leukapheresis Early collection of T cells in patients at high-risk of relapse
Optimized techniques for T cell expansion in CAR-T manufacture
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TME (tumor microenvironment); CAR-T (chimeric antigen receptor T cell)

Tumor type and cell of origin

DLBCL is a heterogeneous group of NHL of B cell origin including primary mediastinal B cell lymphoma (PMBCL), central nervous system DLBCL, cutaneous DLBLC, DLBCL not otherwise specified and DLBCL transformed from follicular lymphoma (tFL). In the initial phase II clinical trials for axi-cel and tisa-cel, there was no association with outcomes based on tumor histologic subtype[17, 25] and this was confirmed in a real-world study of axi-cel in a more heterogenous population[31]. However, in the phase II liso-cel trial, duration of response and PFS were longer in patients with PMBCL and tFL[6]. As second-line treatment, EFS following axi-cel therapy was prolonged compared to SOC for both activated B cell and germinal center molecular subtypes[19] but these studies were not designed to detect differences in outcomes based on cell of origin.

Molecular subtype

Molecular subtypes of DLBCL known to have poorer prognoses including high-grade, double or triple hit lymphomas with dual rearrangement of MYC and BCL2 +/−BCL6 and double expressor lymphomas with overexpression of MYC and BCL2 proteins were detected in 13–17%[6, 25] of patients and were not predictive of resistance in patients treated on clinical trial with tisa-cel[25], liso-cel[6] or with commercially available axi-cel[31]. In the phase III trials, more patients were identified as having double or triple hit lymphoma (24–31%)[19, 20] and were shown to have superior outcomes with axi-cel compared to SOC in subgroup analysis[19] indicating that double or triple hit lymphomas may not be more resistant to CAR-T cell therapy than other NHL subtypes.

TP53 mutations

Mutations in the TP53 gene are found in approximately 20–30% of patients with DLBCL[60, 61]. It has been identified as a negative prognostic factor and may be associated with resistance to conventional cancer drugs[60, 61]. The mechanism of action of CAR-T cell therapy differs significantly from conventional therapies and may be less impacted by these mutations. A small study of TP53 mutations in patients receiving CAR-T (n=29) and conventional therapies (n=141) for DLBCL and high-grade B cell lymphoma showed that while TP53 mutation was an independent factor predicting poorer OS in the conventional treatment group, there was no difference based on TP53 mutation in the group treated with CAR-T cell therapy[62]. However, in a larger cohort of patients with r/r DLBCL treated with CAR-T cell therapy, TP53 alterations were associated with inferior response rate and OS, with a 1-year OS in TP53-altered LBCL of 44% (95%CI 29–67) compared to 76% (95%CI 65–89) in wild-type controls[63]. Interestingly, this finding was more pronounced in the patients treated with a CAR-T product with 4–1BB compared to CD28 co-stimulation. Further, patients with TP53 alterations demonstrated significant downregulation of interferons γ and α and apoptosis pathways that mediate CAR-T cell cytotoxicity [63] suggesting a biologic mechanism for resistance. Therefore, future studies examining TP53 alterations as a mechanism of resistance to CAR-T cell therapy in this and other disease processes are needed.

Tumor Burden

In the landmark phase II clinical trials, there was a wide range of disease burden in enrolled patients. Patients receiving axi-cel on the ZUMA-1 trial were not allowed to receive bridging chemotherapy other than glucocorticoids thereby excluding patients with rapidly progressive and bulky disease who were unable to wait for CAR-T manufacture. Although the initial analysis of bulky disease did not reveal an effect on outcome, only 16% of patients had bulky disease defined as tumor size >10cm[17]. When the ZUMA-1 data was re-examined on a continuous scale, lower tumor burden prior to infusion was a strong predictor of durable response[24]. In both early trials of tisa-cel and liso-cel trials, a larger proportion of patients had higher tumor burden owing to the allowance for bridging therapy prior to CAR-T[6, 25] but yielded similar findings; patients with smaller tumor volume (measured categorically as less than or greater than 100ml or 50cm3) tended to have better overall response rates though these differences were not significant[6, 25]. These findings were supported by real-world experience of a small number of patients treated with axi-cel in which patients with a high metabolic tumor volume (MTV) had significantly inferior PFS (HR=3.296, 95%CI 1.42–7.64) and OS (HR=6.68, 95% CI 2.56–17.32) and that a lower MTV (median 35.1mL) was predictive of a better ORR and CR[64]. A small single center review (n=34) categorized tumor burden using three criteria; response evaluation criteria in lymphoma (RECIL), Lugano criteria and MTV and found an association between higher MTV (above 330mL) and poorer PFS[65]. There was no impact of MTV on OS and no significant relationship between tumor burden measured by the other criteria and PFS and OS[65].

There are conflicting data with respect to toxicities associated with higher tumor burden. In one analysis, tumor burden was associated with a higher risk of neurotoxicity[31] which is consistent with data known from CD19 CAR-T trials in leukemia. However, in a smaller cohort of patients (n= 48) receiving axi-cel for LBCL there was no association of tumor burden with neurotoxicity or CRS. As is proposed in other CAR-T cell therapies, presence of the target antigen is likely necessary to drive the early expansion of CAR-T cells following infusion. A higher tumor burden did not directly correlate with increased CAR-T expansion in this analysis[24], however patients with durable responses had a higher peak CAR-T-to-tumor burden ratio when compared to non-responders or those who relapsed within year of infusion[24] suggesting that the relationship between CAR-T expansion and tumor burden may be important to monitor rather than CAR-T expansion and persistence alone. The heterogeneous methods used to quantify tumor burden across studies makes it difficult to discern generalizable relationships with toxicities or clinical response outcomes. However, based on the available data, lower tumor burden is likely associated with better clinical outcomes but in patients with a high tumor burden, a high peak CAR-T level may overcome resistance and increase the likelihood of a durable response.

Lactate Dehydrogenase (LDH)

Lactate dehydrogenase (LDH) is used as a surrogate marker of tumor burden and aggressiveness of disease and is a factor in the IPI and AAIPI scoring tools. In the phase II clinical trial of liso-cel that reported LDH independently, elevated levels were associated with reduced objective and complete responses[6]. This finding was supported by a number of studies of commercial CAR-T cell therapy as a third or later-line intervention demonstrating that elevated pretreatment LDH was independently associated with shorter PFS and OS and reduced response rates[31, 35, 66, 67] and that PFS and OS following CAR-T cell therapy in patients with elevated LDH were not superior to alternate therapies[35]. Elevated LDH has also been described as an indicator of an immunosuppressive environment with impaired T cell function in patients receiving cancer therapy and may predict a poor intratumoral immune response[68, 69] and may reduce CAR-T cell expansion in vivo[24]. The interaction between tumor and pre-treatment burden with the host immune and inflammatory state and the effect this has on CAR-T expansion and function requires further study.

Circulating tumor DNA (ctDNA)

Markers of clonality in B cell malignancies are detectable as unique VDJ recombinations in the immunoglobulin gene. These tumor-specific VDJ clonotypes can be detected as cell-free DNA in the plasma of patients with LBCL and are being evaluated as a potential biomarker for disease to assess treatment response and predict outcomes. Baseline ctDNA obtained prior to LD-chemotherapy correlated with other markers of disease severity including disease stage ≥3, elevated LDH[70], tumor MTV[71] and IPI[70, 71]. Durable responders were also found to have lower baseline ctDNA levels prior to LD-chemotherapy compared to patients with progressive disease[70, 71] indicating that pretreatment ctDNA levels may be useful in predicting response to CAR-T cell therapy and patient selection.

Summary

Multiple host, tumor and product-specific factors determine efficacy of CAR-T therapy for r/r DLBCL and research is ongoing into potential strategies to mitigate therapy failures (Table 1). Comparing each factor independently has been difficult due to varying enrollment criteria and confounding factors in phase II and phase III trials. Real-world studies have attempted to elucidate reasons for CAR-T failure, but larger prospective trials are still needed. The data available to-date indicate that factors such as older age that contribute to poorer prognosis after relapse with conventional therapies may prove to be less important with better tolerability and clinical outcomes following early transition to CAR-T cell therapy. Rapid expansion followed by persistence of CAR-T until at least 6 months contributes to the most durable responses and identification of the optimal CAR-T construct to achieve this potency with limited toxicity is paramount to improving outcomes. The immune suppressive TME, especially in the setting of a large tumor burden, contributes to poor T cell persistence in vivo and treatment resistance and may be another area for manipulation to improve longer-term outcomes. More in-depth analyses of patient immune and inflammatory states as well as biomarkers such as ctDNA will likely become more useful as these evaluations are more widely available in the clinic setting. Further research is required to elucidate specific features that predict response to treatment and establish methods to mitigate mechanisms of resistance.

Acknowledgements

This work is supported in part from a Cancer Grand Challenge Award OT2CA280965-01 from NCI and Cancer Research UK (CMB), the Leukemia Lymphoma Society (CMB), a Mark Foundation Momentum Fellow Award (HK), Hyundai Hope on Wheels Awards (HK, KT) and a Ruth L. Kirschstein National Research Service Award Institutional Research Training Grant awarded to the Children’s Research Institute Hematology Training Program by the National Heart, Lung, and Blood Institute of the National Institutes of Health (5T32HL110841-08) (HK).

Footnotes

Conflict-of-interest disclosure: C.M.B. has stock or ownership in Cabaletta Bio, Catamaran Bio, and Neximmune and had an equity interest in Mana Therapeutics. She also serves on the DSMB of SOBI.

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