Tom van Meerten

BACKGROUND: The prognosis of patients with early relapsed or refractory large B-cell lymphoma after the receipt of first-line chemoimmunotherapy is poor. METHODS: In this international, phase 3 trial, we randomly assigned, in a 1:1 ratio, patients with large B-cell lymphoma that was refractory to or had relapsed no more than 12 months after first-line chemoimmunotherapy to receive axicabtagene ciloleucel (axi-cel, an autologous anti-CD19 chimeric antigen receptor T-cell therapy) or standard care (two or three cycles of investigator-selected, protocol-defined chemoimmunotherapy, followed by high-dose chemotherapy with autologous stem-cell transplantation in patients with a response to the chemoimmunotherapy). The primary end point was event-free survival according to blinded central review. Key secondary end points were response and overall survival. Safety was also assessed. RESULTS: A total of 180 patients were randomly assigned to receive axi-cel and 179 to receive standard care. The primary end-point analysis of event-free survival showed that axi-cel therapy was superior to standard care. At a median follow-up of 24.9 months, the median event-free survival was 8.3 months in the axi-cel group and 2.0 months in the standard-care group, and the 24-month event-free survival was 41% and 16%, respectively (hazard ratio for event or death, 0.40; 95% confidence interval, 0.31 to 0.51; P<0.001). A response occurred in 83% of the patients in the axi-cel group and in 50% of those in the standard-care group (with a complete response in 65% and 32%, respectively). In an interim analysis, the estimated overall survival at 2 years was 61% in the axi-cel group and 52% in the standard-care group. Adverse events of grade 3 or higher occurred in 91% of the patients who received axi-cel and in 83% of those who received standard care. Among patients who received axi-cel, grade 3 or higher cytokine release syndrome occurred in 6% and grade 3 or higher neurologic events in 21%. No deaths related to cytokine release syndrome or neurologic events occurred. CONCLUSIONS: Axi-cel therapy led to significant improvements, as compared with standard care, in event-free survival and response, with the expected level of high-grade toxic effects. (Funded by Kite; ZUMA-7 ClinicalTrials.gov number, NCT03391466.).

We have previously defined a panel of fully human CD20 mAb. Most of these were unexpectedly efficient in their ability to recruit C1q to the surface of CD20-positive cells and mediate tumor lysis via activation of the classical pathway of complement. This complement-dependent cytotoxicity (CDC) potency appeared to relate to the unusually slow off-rate of these human Abs. However, we now present epitope-mapping data, which indicates that all human mAb bind a novel region of CD20 that may influence CDC potency. Epitope mapping, using both mutagenesis studies and overlapping 15-mer peptides of the extracellular loops of CD20, defined the amino acids required for binding by an extensive panel of mouse and human mAb. Binding by rituximab and mouse CD20 mAb, had an absolute requirement for alanine and proline at positions 170 and 172, respectively, within the large extracellular loop of CD20. Surprisingly, however, all of the human CD20 mAb recognize a completely novel epitope located N-terminally of this motif, also including the small extracellular loop of CD20. Thus, although off-rate may influence biological activity of mAb, another critical factor for determining CDC potency by CD20 mAb appears to be the region of the target molecule they recognize. We conclude that recognition of the novel epitope cooperates with slow off-rate in determining the activity of CD20 Ab in activation of complement and induction of tumor cell lysis.

BACKGROUND: In an analysis of the primary outcome of this phase 3 trial, patients with early relapsed or refractory large B-cell lymphoma who received axicabtagene ciloleucel (axi-cel), an autologous anti-CD19 chimeric antigen receptor T-cell therapy, as second-line treatment had significantly longer event-free survival than those who received standard care. Data were needed on longer-term outcomes. METHODS: In this trial, we randomly assigned patients with early relapsed or refractory large B-cell lymphoma in a 1:1 ratio to receive either axi-cel or standard care (two to three cycles of chemoimmunotherapy followed by high-dose chemotherapy with autologous stem-cell transplantation in patients who had a response). The primary outcome was event-free survival, and key secondary outcomes were response and overall survival. Here, we report the results of the prespecified overall survival analysis at 5 years after the first patient underwent randomization. RESULTS: A total of 359 patients underwent randomization to receive axi-cel (180 patients) or standard care (179 patients). At a median follow-up of 47.2 months, death had been reported in 82 patients in the axi-cel group and in 95 patients in the standard-care group. The median overall survival was not reached in the axi-cel group and was 31.1 months in the standard-care group; the estimated 4-year overall survival was 54.6% and 46.0%, respectively (hazard ratio for death, 0.73; 95% confidence interval [CI], 0.54 to 0.98; P = 0.03 by stratified two-sided log-rank test). This increased survival with axi-cel was observed in the intention-to-treat population, which included 74% of patients with primary refractory disease and other high-risk features. The median investigator-assessed progression-free survival was 14.7 months in the axi-cel group and 3.7 months in the standard-care group, with estimated 4-year percentages of 41.8% and 24.4%, respectively (hazard ratio, 0.51; 95% CI, 0.38 to 0.67). No new treatment-related deaths had occurred since the primary analysis of event-free survival. CONCLUSIONS: At a median follow-up of 47.2 months, axi-cel as second-line treatment for patients with early relapsed or refractory large B-cell lymphoma resulted in significantly longer overall survival than standard care. (Funded by Kite; ZUMA-7 ClinicalTrials.gov number, NCT03391466.).

The effectiveness of chimeric Ag receptor (CAR)-transduced T (CAR-T) cells has been attributed to supraphysiological signaling through CARs. Second- and later-generation CARs simultaneously transmit costimulatory signals with CD3ζ signals upon ligation, but may lead to severe adverse effects owing to the recognition of minimal Ag expression outside the target tumor. Currently, the threshold target Ag density for CAR-T cell lysis and further activation, including cytokine production, has not yet been investigated in detail. Therefore, we determined the threshold target Ag density required to induce CAR-T cell responses using novel anti-CD20 CAR-T cells with a CD28 intracellular domain and a CD20-transduced CEM cell model. The newly developed CD20CAR-T cells demonstrated Ag-specific lysis and cytokine secretion, which was a reasonable level as a second-generation CAR. For lytic activity, the threshold Ag density was determined to be ∼200 molecules per target cell, whereas the Ag density required for cytokine production of CAR-T cells was ∼10-fold higher, at a few thousand per target cell. CD20CAR-T cells responded efficiently to CD20-downregulated lymphoma and leukemia targets, including rituximab- or ofatumumab-refractory primary chronic lymphocytic leukemia cells. Despite the potential influence of the structure, localization, and binding affinity of the CAR/Ag, the threshold determined may be used for target Ag selection. An Ag density below the threshold may not result in adverse effects, whereas that above the threshold may be sufficient for practical effectiveness. CD20CAR-T cells also demonstrated significant lytic activity against CD20-downregulated tumor cells and may exhibit effectiveness for CD20-positive lymphoid malignancies.

PURPOSE: The use of the CD20-specific antibody rituximab has greatly improved the response to treatment of CD20+ follicular lymphoma. Despite the success of rituximab, resistance has been reported and prognostic markers to predict individual response are lacking. The level of CD20 expression on tumors has been related to response, but results of several studies are contradictory and no clear relationship could be established. Complement-dependent cytotoxicity (CDC) and antibody-dependent cellular cytotoxicity (ADCC) are thought to be important effector mechanisms, but the exact mechanism of rituximab-mediated cell kill is still unknown. Importantly, no data have been reported on the combined contribution of CDC and ADCC. EXPERIMENTAL DESIGN: We have developed a system of clonally related CEM-CD20 cells by retroviral transfer of the human CD20 cDNA (n = 90). This set of cells, with the CD20 molecule as the only variable, was used to study the importance of CD20 expression level on rituximab-mediated CDC, ADCC, and the combination. RESULTS: We show a sigmoidal correlation of CD20 expression level and rituximab-mediated killing via CDC but not ADCC. On both high and low CD20-expressing cells, all CD20 molecules were translocated into lipid rafts after rituximab binding. Furthermore, CDC and ADCC act simultaneously and CDC-resistant cells are sensitive to ADCC and vice versa. CONCLUSIONS: These findings suggest that CDC depends on CD20 expression level and that both CDC and ADCC act complementary. These data give new insights into novel strategies to improve the efficacy of CD20-specific antibodies for the treatment of CD20+ tumors.