[en] Multiple myeloma (MM) is an incurable hematologic malignancy arising from clonal plasma cells, with poor long-term outcomes due to inevitable relapse after conventional therapies. Chimeric antigen receptor (CAR) T-cell immunotherapy targeting B-cell maturation antigen (BCMA) has shown remarkable efficacy in relapsed patients. Conventional CARs employ single-chain variable fragments (scFvs), whereas single-domain antibodies (sdAb or VHHs) offer advantages such as small size, high stability, and potentially reduced immunogenicity. We designed and evaluated a novel anti-BCMA nanoCAR-T based on the VHH Nb17, compared with the conventional scFv-based CAR-T CT103a. Nb17 demonstrated strong BCMA binding and was incorporated into a CAR construct. Both nanoCAR-T and CT103a were generated via lentiviral transduction of primary T cells. Their cytotoxicity, cytokine secretion, degranulation, memory phenotype, and gene expression were assessed in vitro, along with antitumor activity in vivo. Nb17-nanoCAR-T demonstrated specific cytotoxicity, cytokine release (IL-2, TFNa, IFNg), and CD107a degranulation comparable to CT103a. Transcriptomic analysis revealed overlapping pathways between both CARs. Upon rechallenge, both CARs showed enhanced proliferation compared with untransduced T cells. In vivo, Nb17-nanoCAR-T and CT103a eradicated tumors in NSG mice. These findings demonstrate Nb17-nanoCAR-T exhibits potent anti-myeloma efficacy comparable to scFv-based CAR-T, supporting its potential as a promising therapeutic alternative.
Di Valentin, Emmanuel ; Université de Liège - ULiège > GIGA > GIGA Platforms - Viral Vectors
Baron, Frédéric ; Université de Liège - ULiège > Département des sciences cliniques
GREGOIRE, Céline ; Centre Hospitalier Universitaire de Liège - CHU > > Service d'hématologie clinique
Ehx, Grégory ; Université de Liège - ULiège > Département des sciences cliniques ; Walloon Excellence in Life Sciences and Biotechnology (WELBIO) Department, WEL Research Institute, 1300 Wavre, Belgium
Nguyen, Tham Thi; Laboratory of Hematology, GIGA Institute, University of Liège, 4000 Liège, Belgium
Caers, Jo ; Université de Liège - ULiège > Département des sciences cliniques > Hématologie
Belgian Foundation Against Cancer F.R.S.-FNRS - Fonds de la Recherche Scientifique Télévie
Funding text :
This research was funded by the Foundation Against Cancer, the ERA-NET TRANSCAN-3 (EC co-funded call 2021, SmartCAR-T), the Fonds National de la Recherche Scientifique, (FNRS, Belgium), Télévie-FNRS, and the Fonds Spéciaux de la Recherche (University of Liège).
Laubach J. Richardson P. Anderson K. Multiple Myeloma Annu. Rev. Med. 2011 62 249 264 10.1146/annurev-med-070209-175325
Went M. Duran-Lozano L. Halldorsson G.H. Gunnell A. Ugidos-Damboriena N. Law P. Ekdahl L. Sud A. Thorleifsson G. Thodberg M. et al. Deciphering the genetics and mechanisms of predisposition to multiple myeloma Nat. Commun. 2024 15 1 15 10.1038/s41467-024-50932-7 39103364
Graf K.C. Davis J.A. Cendagorta A. Granger K. Gaffney K.J. Green K. Hess B.T. Hashmi H. “Fast but not so furious”: A condensed step-up dosing schedule of teclistamab for relapsed/refractory multiple myeloma eJHaem 2024 5 793 797 10.1002/jha2.906 39157607
Pan D. Richter J. Management of Toxicities Associated with BCMA, GPRC5D, and FcRH5-Targeting Bispecific Antibodies in Multiple Myeloma Curr. Hematol. Malign-Rep. 2024 19 237 245 10.1007/s11899-024-00740-z 39145912
Guedan S. Ruella M. June C.H. Emerging Cellular Therapies for Cancer Annu. Rev. Immunol. 2019 37 145 171 10.1146/annurev-immunol-042718-041407
Utkarsh K. Srivastava N. Kumar S. Khan A. Dagar G. Kumar M. Singh M. Haque S. CAR-T cell therapy: A game-changer in cancer treatment and beyond Clin. Transl. Oncol. 2024 26 1300 1318 10.1007/s12094-023-03368-2
American Association for Cancer Research First-Ever CAR T-cell Therapy Approved in U.S Cancer Discov. 2017 7 10 10.1158/2159-8290.CD-NB2017-126
Jagannath S. Lin Y. Goldschmidt H. Reece D. Nooka A. Senin A. Rodriguez-Otero P. Powles R. Matsue K. Shah N. et al. KarMMa-RW: Comparison of idecabtagene vicleucel with real-world outcomes in relapsed and refractory multiple myeloma Blood Cancer J. 2021 11 116 10.1038/s41408-021-00507-2
Mu W. Long X. Cai H. Chen C. Hu G. Lou Y. Xing S. Wang D. Wang J. Xiao M. et al. A Model Perspective Explanation of the Long-Term Sustainability of a Fully Human BCMA-Targeting CAR (CT103A) T-Cell Immunotherapy Front. Pharmacol. 2022 13 803693 10.3389/fphar.2022.803693
Li D. Que Y. Ding S. Hu G. Wang W. Mao X. Wang Y. Li C. Huang L. Zhou J. et al. Anti-BCMA CAR-T cells therapy for a patient with extremely high membrane BCMA expression: A case report J. Immunother. Cancer 2022 10 e005403 10.1136/jitc-2022-005403
Xu J. Melenhorst J.J. CT103A, a forward step in multiple myeloma immunotherapies Blood Sci. 2021 3 59 61 10.1097/BS9.0000000000000068 35402835
Wang Q. Wei R. Guo S. Min C. Zhong X. Huang H. Cheng Z. An alternative fully human anti-BCMA CAR-T shows response for relapsed or refractory multiple myeloma with anti-BCMA CAR-T exposures previously Cancer Gene Ther. 2023 31 420 426 10.1038/s41417-023-00712-0 38102463
Li C. Wang D. Song Y. Huang H. Li J. Chen B. Liu J. Dong Y. Hu K. Liu P. et al. CT103A, a novel fully human BCMA-targeting CAR-T cells, in patients with relapsed/refractory multiple myeloma: Updated results of phase 1b/2 study (FUMANBA-1) J. Clin. Oncol. 2023 41 8025 10.1200/JCO.2023.41.16_suppl.8025
Pampusch M.S. Haran K.P. Hart G.T. Rakasz E.G. Rendahl A.K. Berger E.A. Connick E. Skinner P.J. Rapid Transduction and Expansion of Transduced T Cells with Maintenance of Central Memory Populations Mol. Ther.-Methods Clin. Dev. 2020 16 1 10 10.1016/j.omtm.2019.09.007
Song H.W. Prochazkova M. Shao L. Traynor R. Underwood S. Black M. Fellowes V. Shi R. Pouzolles M. Chou H.-C. et al. CAR-T cell expansion platforms yield distinct T cell differentiation states Cytotherapy 2024 26 757 768 10.1016/j.jcyt.2024.03.003
Abdo L.D.M. Barros L.R.C. Viegas M.S. Marques L.V.C. Ferreira P.D.S. Chicaybam L. Bonamino M.H. Development of CAR-T cell therapy for B-ALL using a point-of-care approach OncoImmunology 2020 9 1752592 10.1080/2162402X.2020.1752592
Brilhante-Da-Silva N. Sousa R.M.D.O. Arruda A. dos Santos E.L. Marinho A.C.M. Stabeli R.G. Fernandes C.F.C. Pereira S.D.S. Camelid Single-Domain Antibodies for the Development of Potent Diagnosis Platforms Mol. Diagn. Ther. 2021 25 439 456 10.1007/s40291-021-00533-7
Duray E. Lejeune M. Baron F. Beguin Y. Devoogdt N. Krasniqi A. Lauwers Y. Zhao Y.J. D’hUyvetter M. Dumoulin M. et al. A non-internalised CD38-binding radiolabelled single-domain antibody fragment to monitor and treat multiple myeloma J. Hematol. Oncol. 2021 14 183 10.1186/s13045-021-01171-6
Abebe E.C. Shiferaw M.Y. Admasu F.T. Dejenie T.A. Ciltacabtagene autoleucel: The second anti-BCMA CAR T-cell therapeutic armamentarium of relapsed or refractory multiple myeloma Front. Immunol. 2022 13 991092 10.3389/fimmu.2022.991092
Smith R. Bringing cell therapy to tumors: Considerations for optimal CAR binder design Antib. Ther. 2023 6 225 239 10.1093/abt/tbad019 37846297
Hanssens H. Meeus F. Gesquiere E.L. Puttemans J. De Vlaeminck Y. De Veirman K. Breckpot K. Devoogdt N. Anti-Idiotypic VHHs and VHH-CAR-T Cells to Tackle Multiple Myeloma: Different Applications Call for Different Antigen-Binding Moieties Int. J. Mol. Sci. 2024 25 5634 10.3390/ijms25115634 38891821
Zhou J. Liu J. Hu G. Yang Y. Meng G. Gao W. Wang Y. Niu P. Chimeric Antigen Receptor (CAR) Binding to BCMA, and Uses Thereof US20200376030A1 3 December 2020
Fan X. Zhuang Q. Zhao Y. Yang L. Fang X. Xu C. Single Domain Antibodies and Chimeric Antigen Receptors Targeting BCMA and Methods of Use Thereof WO2021121228A1 24 June 2021
Wang Y. Yuan W. Guo S. Li Q. Chen X. Li C. Liu Q. Sun L. Chen Z. Yuan Z. et al. A 33-residue peptide tag increases solubility and stability of Escherichia coli produced single-chain antibody fragments Nat. Commun. 2022 13 4614 10.1038/s41467-022-32423-9
Zeghal M. Matte K. Venes A. Patel S. Laroche G. Sarvan S. Joshi M. Rain J.-C. Couture J.-F. Giguère P.M. Development of a V5-tag–directed nanobody and its implementation as an intracellular biosensor of GPCR signaling J. Biol. Chem. 2023 299 105107 10.1016/j.jbc.2023.105107 37517699
Beghein E. Gettemans J. Nanobody Technology: A Versatile Toolkit for Microscopic Imaging, Protein–Protein Interaction Analysis, and Protein Function Exploration Front. Immunol. 2017 8 771 10.3389/fimmu.2017.00771
Lee D.H. Cervantes-Contreras F. Lee S.Y. Green D.J. Till B.G. Improved Expansion and Function of CAR T Cell Products from Cultures Initiated at Defined CD4:CD8 Ratios Blood 2018 132 3334 10.1182/blood-2018-99-111576
Nasiri F. Kozani P.S. Rahbarizadeh F. T-cells engineered with a novel VHH-based chimeric antigen receptor against CD19 exhibit comparable tumoricidal efficacy to their FMC63-based counterparts Front. Immunol. 2023 14 1063838 10.3389/fimmu.2023.1063838
Cheng J. Ge T. Zhu X. Wang J. Zeng Y. Mu W. Cai H. Dai Z. Jin J. Yang Y. et al. Preclinical development and evaluation of nanobody-based CD70-specific CAR T cells for the treatment of acute myeloid leukemia Cancer Immunol. Immunother. 2023 72 2331 2346 10.1007/s00262-023-03422-6
Xiong Q. Wang H. Shen Q. Wang Y. Yuan X. Lin G. Jiang P. The development of chimeric antigen receptor T-cells against CD70 for renal cell carcinoma treatment J. Transl. Med. 2024 22 368 10.1186/s12967-024-05101-1
Hanssens H. Meeus F. De Vlaeminck Y. Lecocq Q. Puttemans J. Debie P. De Groof T.W.M. Goyvaerts C. De Veirman K. Breckpot K. et al. Scrutiny of chimeric antigen receptor activation by the extracellular domain: Experience with single domain antibodies targeting multiple myeloma cells highlights the need for case-by-case optimization Front. Immunol. 2024 15 1389018 10.3389/fimmu.2024.1389018
Temple W.C. Nix M.A. Naik A. Izgutdina A. Huang B.J. Wicaksono G. Phojanakong P. Serrano J.A.C. Young E.P. Ramos E. et al. Framework humanization optimizes potency of anti-CD72 nanobody CAR-T cells for B-cell malignancies J. Immunother. Cancer 2023 11 e006985 10.1136/jitc-2023-006985
De Munter S. Ingels J. Goetgeluk G. Bonte S. Pille M. Weening K. Kerre T. Abken H. Vandekerckhove B. Nanobody Based Dual Specific CARs Int. J. Mol. Sci. 2018 19 403 10.3390/ijms19020403
Maude S.L. Frey N. Shaw P.A. Aplenc R. Barrett D.M. Bunin N.J. Chew A. Gonzalez V.E. Zheng Z. Lacey S.F. et al. Chimeric antigen receptor T cells for sustained remissions in leukemia N. Engl. J. Med. 2014 371 1507 1517 Erratum in N. Engl. J. Med. 2016, 374, 998. https://doi.org/10.1056/NEJMx160005 10.1056/NEJMoa1407222 25317870
Gardner R.A. Finney O. Annesley C. Brakke H. Summers C. Leger K. Bleakley M. Brown C. Mgebroff S. Kelly-Spratt K.S. et al. Intent-to-treat leukemia remission by CD19 CAR T cells of defined formulation and dose in children and young adults Blood 2017 129 3322 3331 10.1182/blood-2017-02-769208 28408462
Fischer J.W. Bhattarai N. CAR-T Cell Therapy: Mechanism, Management, and Mitigation of Inflammatory Toxicities Front. Immunol. 2021 12 693016 10.3389/fimmu.2021.693016 34220853
King M.A. Covassin L. Brehm M.A. Racki W. Pearson T. Leif J. Laning J. Fodor W. Foreman O. Burzenski L. et al. Human peripheral blood leucocyte non-obese diabetic-severe combined immunodeficiency interleukin-2 receptor gamma chain gene mouse model of xenogeneic graft-versus-host-like disease and the role of host major histocompatibility complex Clin. Exp. Immunol. 2009 157 104 118 10.1111/j.1365-2249.2009.03933.x
Covassin L. Laning J. Abdi R. Langevin D.L. Phillips N.E. Shultz L.D. Brehm M.A. Human peripheral blood CD4 T cell-engrafted non-obese diabetic-scid IL2rγ null H2-Ab1 tm1Gru Tg (human leucocyte antigen D-related 4) mice: A mouse model of human allogeneic graft-versus-host disease Clin. Exp. Immunol. 2011 166 269 280 10.1111/j.1365-2249.2011.04462.x
Wu S. Luo Q. Li F. Zhang S. Zhang C. Liu J. Shao B. Hong Y. Tan T. Dong X. et al. Development of novel humanized CD19/BAFFR bicistronic chimeric antigen receptor T cells with potent antitumor activity against B-cell lineage neoplasms Br. J. Haematol. 2024 205 1361 1373 10.1111/bjh.19631
Xia B. Lin K. Wang X. Chen F. Zhou M. Li Y. Lin Y. Qiao Y. Li R. Zhang W. et al. Nanobody-derived bispecific CAR-T cell therapy enhances the anti-tumor efficacy of T cell lymphoma treatment Mol. Ther.-Oncolytics 2023 30 86 102 10.1016/j.omto.2023.07.007
Kueberuwa G. Gornall H. Alcantar-Orozco E.M. Bouvier D. Kapacee Z.A. Hawkins R.E. Gilham D.E. CCR7+ selected gene-modified T cells maintain a central memory phenotype and display enhanced persistence in peripheral blood in vivo J. Immunother. Cancer 2017 5 14 10.1186/s40425-017-0216-7
