CAR density influences antitumoral efficacy of BCMA CAR T cells and correlates with clinical outcome

Identification of new markers associated with long-term efficacy in patients treated with CAR T cells is a current medical need, particularly in diseases such as multiple myeloma. In this study we address the impact of CAR density on the functionality of BCMA-CAR T cells. Functional and transcriptional studies demonstrate that CAR T cells with high expression of the CAR construct show an increased tonic signaling with upregulation of exhaustion markers, increased in vitro cytotoxicity but a decrease in in vivo BM infiltration. Characterization of Gene Regulatory Networks using scRNA-seq identified regulons associated to activation and exhaustion upregulated in CARHigh T cells, providing mechanistic insights behind differential functionality of these cells. Finally, we demonstrate that patients treated with CAR T cell products enriched in CARHigh T cells show a significantly worse clinical response in several hematological malignancies. In summary, our work demonstrates that CAR density plays an important role in CAR T activity with significant impact on clinical response.

[1]  W. Wang,et al.  Anti-BCMA CAR T-cell therapy CT103A in relapsed or refractory AQP4-IgG seropositive neuromyelitis optica spectrum disorders: phase 1 trial interim results , 2023, Journal of the Neurological Sciences.

[2]  S. Usmani,et al.  Determinants of Response and Mechanisms of Resistance of CAR T-cell Therapy in Multiple Myeloma. , 2021, Cancer discovery.

[3]  Gregory M. Chen,et al.  Integrative bulk and single-cell profiling of pre-manufacture T-cell populations reveals factors mediating long-term persistence of CAR T-cell therapy. , 2021, Cancer discovery.

[4]  Jin-Yuan Ho,et al.  Promoter usage regulating the surface density of CAR molecules may modulate the kinetics of CAR-T cells in vivo , 2021, Molecular therapy. Methods & clinical development.

[5]  S. Shen-Orr,et al.  Shaping Functional Avidity of CAR T Cells: Affinity, Avidity, and Antigen Density That Regulate Response , 2021, Molecular Cancer Therapeutics.

[6]  A. Rosenwald,et al.  Homozygous BCMA gene deletion in response to anti-BCMA CAR T cells in a patient with multiple myeloma , 2021, Nature Medicine.

[7]  D. Miklos,et al.  Tumor burden, inflammation, and product attributes determine outcomes of axicabtagene ciloleucel in large B-cell lymphoma. , 2020, Blood advances.

[8]  Michael R. Green,et al.  Characteristics of anti-CD19 CAR T cell infusion products associated with efficacy and toxicity in patients with large B cell lymphomas , 2020, Nature Medicine.

[9]  J. Bluestone,et al.  The CD28-Transmembrane Domain Mediates Chimeric Antigen Receptor Heterodimerization With CD28 , 2020, bioRxiv.

[10]  Omer Dushek,et al.  Engineering AvidCARs for combinatorial antigen recognition and reversible control of CAR function , 2020, Nature Communications.

[11]  Weian Zhao,et al.  CAR-T design: Elements and their synergistic function , 2020, EBioMedicine.

[12]  A. Regev,et al.  A Distinct Transcriptional Program in Human CAR T Cells Bearing the 4-1BB Signaling Domain Revealed by scRNA-Seq. , 2020, Molecular therapy : the journal of the American Society of Gene Therapy.

[13]  V. Ponomarev,et al.  Defining an Optimal Dual-Targeted CAR T-cell Therapy Approach Simultaneously Targeting BCMA and GPRC5D to Prevent BCMA Escape-Driven Relapse in Multiple Myeloma. , 2020, Blood cancer discovery.

[14]  S. Jagannath,et al.  Update of CARTITUDE-1: A phase Ib/II study of JNJ-4528, a B-cell maturation antigen (BCMA)-directed CAR-T-cell therapy, in relapsed/refractory multiple myeloma. , 2020 .

[15]  A. McLellan,et al.  Promoter choice: Who should drive the CAR in T cells? , 2020, bioRxiv.

[16]  Jianhao Peng,et al.  A single-cell gene regulatory network inference method for identifying complex regulatory dynamics across cell phenotypes , 2020, bioRxiv.

[17]  J. Myklebust,et al.  Tuning the Antigen Density Requirement for CAR T Cell Activity. , 2020, Cancer discovery.

[18]  R. Flavell,et al.  mRNA destabilization by BTG1 and BTG2 maintains T cell quiescence , 2020, Science.

[19]  Aviv Madar,et al.  Single residue in CD28-costimulated CAR T cells limits long-term persistence and antitumor durability. , 2020, The Journal of clinical investigation.

[20]  Wei-Ting Hwang,et al.  T-cell phenotypes associated with effective CAR T-cell therapy in postinduction vs relapsed multiple myeloma. , 2019, Blood advances.

[21]  E. Wherry,et al.  Defining ‘T cell exhaustion’ , 2019, Nature Reviews Immunology.

[22]  Hui Hu,et al.  Transcriptome and Regulatory Network Analyses of CD19-CAR-T Immunotherapy for B-ALL , 2019, Genom. Proteom. Bioinform..

[23]  X. Bian,et al.  Genome-wide analysis identifies NR4A1 as a key mediator of T cell dysfunction , 2019, Nature.

[24]  A. Yoshimura,et al.  Nr4a transcription factors limit CAR T cell function in solid tumors , 2019, Nature.

[25]  M. Sadelain,et al.  Calibration of CAR activation potential directs alternative T cell fates and therapeutic potency , 2018, Nature Medicine.

[26]  J. Maher,et al.  Strategies to Address Chimeric Antigen Receptor Tonic Signaling , 2018, Molecular Cancer Therapeutics.

[27]  J. Heath,et al.  Preinfusion polyfunctional anti-CD19 chimeric antigen receptor T cells are associated with clinical outcomes in NHL. , 2018, Blood.

[28]  C. Mackall,et al.  Tumor Antigen Escape from CAR T-cell Therapy. , 2018, Cancer discovery.

[29]  M. Sadelain,et al.  Chimeric Antigen Receptor Therapy. , 2018, The New England journal of medicine.

[30]  W. Fisher,et al.  Enhancing the Potency and Specificity of Engineered T Cells for Cancer Treatment. , 2018, Cancer discovery.

[31]  G. Gaud,et al.  Regulatory mechanisms in T cell receptor signalling , 2018, Nature Reviews Immunology.

[32]  Hans Bitter,et al.  Determinants of response and resistance to CD19 chimeric antigen receptor (CAR) T cell therapy of chronic lymphocytic leukemia , 2018, Nature Medicine.

[33]  A. Goldrath,et al.  The Transcription Factor Runx3 Establishes Chromatin Accessibility of cis-Regulatory Landscapes that Drive Memory Cytotoxic T Lymphocyte Formation. , 2018, Immunity.

[34]  R. Hagedoorn,et al.  A Jurkat 76 based triple parameter reporter system to evaluate TCR functions and adoptive T cell strategies , 2018, Oncotarget.

[35]  Z. Izsvák,et al.  Efficient Non-viral Gene Delivery into Human Hematopoietic Stem Cells by Minicircle Sleeping Beauty Transposon Vectors , 2018, Molecular therapy : the journal of the American Society of Gene Therapy.

[36]  K. Davis,et al.  Tisagenlecleucel in Children and Young Adults with B‐Cell Lymphoblastic Leukemia , 2018, The New England journal of medicine.

[37]  J. Melenhorst,et al.  Enhancing CAR T cell persistence through ICOS and 4-1BB costimulation. , 2018, JCI insight.

[38]  Stuart A. Sievers,et al.  Function of Novel Anti-CD19 Chimeric Antigen Receptors with Human Variable Regions Is Affected by Hinge and Transmembrane Domains. , 2017, Molecular therapy : the journal of the American Society of Gene Therapy.

[39]  J. Orange,et al.  Tonic 4-1BB Costimulation in Chimeric Antigen Receptors Impedes T Cell Survival and Is Vector-Dependent. , 2017, Cell reports.

[40]  R. Orentas,et al.  Tumor Antigen and Receptor Densities Regulate Efficacy of a Chimeric Antigen Receptor Targeting Anaplastic Lymphoma Kinase. , 2017, Molecular therapy : the journal of the American Society of Gene Therapy.

[41]  Benjamin D. Greenbaum,et al.  Innate Immune Landscape in Early Lung Adenocarcinoma by Paired Single-Cell Analyses , 2017, Cell.

[42]  Mithat Gönen,et al.  Targeting a CAR to the TRAC locus with CRISPR/Cas9 enhances tumour rejection , 2017, Nature.

[43]  Rugang Zhang,et al.  SATB1 Expression Governs Epigenetic Repression of PD‐1 in Tumor‐Reactive T Cells , 2017, Immunity.

[44]  Kyle Bittinger,et al.  INSPIIRED: Quantification and Visualization Tools for Analyzing Integration Site Distributions , 2016, Molecular therapy. Methods & clinical development.

[45]  Kyle Bittinger,et al.  INSPIIRED: A Pipeline for Quantitative Analysis of Sites of New DNA Integration in Cellular Genomes , 2016, Molecular therapy. Methods & clinical development.

[46]  S. Heimfeld,et al.  Immunotherapy of non-Hodgkin’s lymphoma with a defined ratio of CD8+ and CD4+ CD19-specific chimeric antigen receptor–modified T cells , 2016, Science Translational Medicine.

[47]  Howard Y. Chang,et al.  Lineage-specific and single cell chromatin accessibility charts human hematopoiesis and leukemia evolution , 2016, Nature Genetics.

[48]  Standley,et al.  Arid5a regulates naive CD4+ T cell fate through selective stabilization of Stat3 mRNA , 2016, The Journal of experimental medicine.

[49]  Brian Keith,et al.  Distinct Signaling of Coreceptors Regulates Specific Metabolism Pathways and Impacts Memory Development in CAR T Cells. , 2016, Immunity.

[50]  D. Maloney,et al.  Chimeric antigen receptor-modified T cells derived from defined CD8+ and CD4+ subsets confer superior antitumor reactivity in vivo , 2015, Leukemia.

[51]  R. Kaplan,et al.  4-1BB Costimulation Ameliorates T Cell Exhaustion Induced by Tonic Signaling of Chimeric Antigen Receptors , 2015, Nature Medicine.

[52]  Matthew J. Frigault,et al.  Identification of Chimeric Antigen Receptors That Mediate Constitutive or Inducible Proliferation of T Cells , 2015, Cancer Immunology Research.

[53]  I. Amit,et al.  Massively Parallel Single-Cell RNA-Seq for Marker-Free Decomposition of Tissues into Cell Types , 2014, Science.

[54]  S. Holland,et al.  A critical role for STAT3 transcription factor signaling in the development and maintenance of human T cell memory. , 2011, Immunity.

[55]  S. Pai,et al.  GATA3 and the T-cell lineage: essential functions before and after T-helper-2-cell differentiation , 2009, Nature Reviews Immunology.

[56]  B. Barlogie,et al.  International uniform response criteria for multiple myeloma , 2006, Leukemia.

[57]  J. Dausset,et al.  In vivo, RFX5 binds differently to the human leucocyte antigen‐E, ‐F, and ‐G gene promoters and participates in HLA class I protein expression in a cell type‐dependent manner , 2004, Immunology.

[58]  W. Reith,et al.  A Functionally Essential Domain of RFX5 Mediates Activation of Major Histocompatibility Complex Class II Promoters by Promoting Cooperative Binding between RFX and NF-Y , 2000, Molecular and Cellular Biology.

[59]  Reviewers' comments: Reviewer #1, expertise in TCR repertoire in immunotherapy (Remarks to the Author): Using single-cell RNA sequencing and TCRB gene sequencing, Alyssa Sheih and colleagues have presented here single-cell transcriptional profiling of adoptively transferred CD19-specific CD8+ CAR-T , 2019 .

[60]  H. Einsele,et al.  Enhanced CAR T-cell engineering using non-viral Sleeping Beauty transposition from minicircle vectors , 2017, Leukemia.

[61]  D. Speiser,et al.  Molecular profiling of CD8 T cells in autochthonous melanoma identifies Maf as driver of exhaustion , 2015, The EMBO journal.

[62]  K. Anstey,et al.  Cc-by-nc-nd 4.0 International License , 2022 .