Larry W. Kwak

Chimeric antigen receptor (CAR) T-cell therapy has revolutionized the therapeutic landscape for relapsed or refractory lymphoid malignancies, achieving remarkable rates of durable remission. Despite this success, significant variability among patients in clinical responses and treatment-related toxicities remains a critical challenge, highlighting an urgent need for robust predictive biomarkers. Key intrinsic CAR T-cell attributes predictive of therapeutic efficacy and safety include the composition of memory T-cell subsets, particularly central memory and stem cell memory T-cell populations, CAR density and transduction efficiency, cytokine production profiles with emphasis on polyfunctionality, and metabolic fitness. Additionally, the systemic immune contexture significantly modulates outcomes, including baseline systemic inflammatory cytokines, presence of regulatory immune cell populations, and the pretreatment immunosuppressive tumor microenvironment. Recent advances in single-cell transcriptomics, comprehensive proteomic profiling, and cytokine polyfunctionality assays have provided greater resolution for identifying predictive biomarkers and optimizing therapeutic strategies. High-dimensional immunophenotyping combined with advanced machine learning methods enables automated CAR T-cell manufacturing quality control and precise immunological synapse quantification. Furthermore, tumor antigen (epitope) spreading after CAR T-cell therapy has risen as a provisional biomarker indicating broadened antitumor immunity and potentially sustained remission. Integrating these emerging biomarkers and advanced multiomic approaches into clinical practice can refine patient stratification, enhance CAR T-cell manufacturing processes, and improve therapeutic outcomes in patients with lymphoid malignancies.

Neutralizing antibodies are established correlates of protection against SARS-CoV-2, yet T-cell responses are also thought to contribute substantially to limiting disease severity and enhancing durability of protection. To examine whether cellular immunity alone can confer protection, we engineered DNA vaccines encoding modified Spike proteins, including C-terminal truncation (SARS-CoV-2-ΔC, ΔC) and cleavage-site–deleted, linker-inserted (SARS-CoV-2-Linker-ΔT, Linker-ΔT) variants, with or without genetic fusion to MIP3α, which has been shown to enhance targeting of antigen-presenting cells (APC) and preferentially induce T-cell responses. In BALB/c mice, ΔC constructs induced non-neutralizing Spike- and RBD-binding antibodies across variants, as well as robust CD4 + and CD8 + T cell responses, whereas Linker-ΔT elicited strong Th1-skewed cellular immunity in the absence of humoral responses. In K18-hACE2 mice antibody neutralizing activity was not detected by any of the vaccines, and none conferred protection following lethal virus challenge, despite robust specific T-cell cytokine responses. These results support vaccine designs incorporating chemokine fusion to enhance T-cell priming, but cellular responses alone are insufficient for SARS-CoV-2 protection. Integrating such APC-targeting strategies with structural modifications that preserve pre-fusion neutralizing epitopes may be worthwhile.

Lymphoplasmacytic lymphoma (LPL) is an incurable low-grade lymphoma with no standard therapy. Nine asymptomatic patients treated with a first-in-human, neoantigen DNA vaccine experienced no dose limiting toxicities (primary endpoint, NCT01209871). All patients achieve stable disease or better, with one minor response, and median time to progression of 72+ months. Post-vaccine single-cell transcriptomics reveal dichotomous antitumor responses, with reduced tumor B-cells (tracked by unique B cell receptor) and their survival pathways, but no change in clonal plasma cells. Downregulation of human leukocyte antigen (HLA) class II molecules and paradoxical upregulation of insulin-like growth factor (IGF) by the latter suggest resistance mechanisms. Vaccine therapy activates and expands bone marrow T-cell clonotypes, and functional neoantigen-specific responses (secondary endpoint), but not co-inhibitory pathways or Treg, and reduces protumoral signaling by myeloid cells, suggesting favorable perturbation of the tumor immune microenvironment. Future strategies may require combinations of vaccines with agents targeting plasma cell subpopulations, or blockade of IGF-1 signaling or myeloid cell checkpoints.

Chimeric antigen receptor (CAR) T-cell therapy has emerged as a promising immunotherapeutic strategy for eradicating human cancers. Their therapeutic success and durability of clinical responses hinges, in large part, on their functional capacity, including the ability of these engineered cells to simultaneously expand and persist after infusion into patients. CD19 CAR T-cell polyfunctionality, assessing the simultaneous functions of cytokine production, proliferation, and cytotoxicity has been reported to correlate with clinical outcomes. Assay optimization is potentially limited by the heterogeneous nature of CAR T-cell infusion products and target specificity. We optimized a single-cell platform for polyfunctionality using CAR T-cell products manufactured from healthy donors, engineered against a novel target, B-cell–activating factor receptor (BAFF-R) and validated the protocol using CD19 CAR T cells. We observed distinct qualitative differences between BAFF-R and CD19 CAR T cells relative to the proportions of stimulatory vs effector cytokines, based on target antigen density, and, generally, CD19 CAR T cells exhibited lower indices of polyfunctionality. Finally, we applied our assay to the autologous BAFF-R CAR T-cell product generated from the first patient with non-Hodgkin lymphoma treated in an ongoing clinical trial who had progressed after prior CD19 CAR T-cell therapy. We observed robust indicators of polyfunctionality, which correlated with successful CAR T-cell expansion after infusion and achievement of durable complete remission ongoing after 18 months. The precise identification of factors determining the role of BAFF-R CAR T-cell fitness in toxicity and clinical outcome will require the application of this robust assay in the analysis of additional treated patients. This trial was registered at www.ClinicalTrials.gov as #NCT05370430.

Lymphoplasmacytic lymphoma (LPL) is an incurable low-grade B-cell lymphoma of the bone marrow. Despite a cumulative risk of progression, there is no approved therapy for patients in the asymptomatic phase. We conducted a first-in-human clinical trial of a novel therapeutic DNA idiotype neoantigen vaccine in nine patients with asymptomatic LPL. Treatment was well tolerated with no dose limiting toxicities. One patient achieved a minor response, and all remaining patients experienced stable disease, with median time to disease progression of 61+ months. Direct interrogation of the tumor microenvironment by single-cell transcriptome analysis revealed an unexpected dichotomous antitumor response, with significantly reduced numbers of clonal tumor mature B-cells, tracked by their unique BCR, and downregulation of genes involved in signaling pathways critical for B-cell survival post-vaccine, but no change in clonal plasma cell subpopulations. Downregulation of HLA class II molecule expression suggested intrinsic resistance by tumor plasma cell subpopulations and cell-cell interaction analyses predicted paradoxical upregulation of IGF signaling post vaccine by plasma cell, but not mature B-cell subpopulations, suggesting a potential mechanism of acquired resistance. Vaccine therapy induced dynamic changes in bone marrow T-cells, including upregulation of signaling pathways involved in T-cell activation, expansion of T-cell clonotypes, increased T-cell clonal diversity, and functional tumor antigen-specific cytokine production, with little change in co-inhibitory pathways or Treg. Vaccine therapy also globally altered cell-cell communication networks across various bone marrow cell types and was associated with reduction of protumoral signaling by myeloid cells, principally non-classical monocytes. These results suggest that this prototype neoantigen vaccine favorably perturbed the tumor immune microenvironment, resulting in reduction of clonal tumor mature B-cell, but not plasma cell subpopulations. Future strategies to improve clinical efficacy may require combinations of neoantigen vaccines with agents which specifically target LPL plasma cell subpopulations, or enable blockade of IGF-1 signaling or myeloid cell checkpoints.

Summary: A dendritic cell/myeloma fusion vaccine, given with lenalidomide and GM-CSF, did not result in a statistically significant increase in CR rates at 1 year posttransplant but was associated with a significant increase in circulating multiple myeloma–reactive lymphocytes indicative of tumor-specific immunity. See related article by Chung et al., p. 4784

Eight MCL Cell lines were cultured for 48 hours in the presence of different concentrations of CFZ. Cell growth was assessed by 3H-thymidine incorporation assay. The IC50 were determined by constructing a dose-response curve. Averages from 3 independent experiments are shown.

Eight MCL Cell lines were cultured for 48 hours in the presence of different concentrations of CFZ. Cell growth was assessed by 3H-thymidine incorporation assay. The IC50 were determined by constructing a dose-response curve. Averages from 3 independent experiments are shown.

Related Article from Cancer Vaccines: Moving toward Prevention?

PDF - 1267KB, The established MCL cell lines, JVM-2, JVM-13, MAVER, NCEB-1, and Z-138 were treated with 20 nM CFZ for 0, 6, 12, or 24 hours and apoptosis was detected by Annexin V-binding assays. Representative histograms (left panel) and percentages of apoptotic cells (right panel) are shown.