Design of a novel NanoCAR construct that compares well with a conventional ScFv-based CAR targeting BCMA on multiple myeloma

Design of a novel NanoCAR construct that compares well with a conventional ScFv-based CAR targeting BCMA on multiple myeloma | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Design of a novel NanoCAR construct that compares well with a conventional ScFv-based CAR targeting BCMA on multiple myeloma Mégane Jassin, Chloé Onkelinx, Valentina Bocuzzi, Bianca E Silva, and 12 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7693534/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract 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. 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 typically employ single-chain variable fragments (scFvs), whereas single-domain antibodies (sdAb or V H Hs) offer advantages such as small size, high stability, and potentially reduced immunogenicity. The aim of this research is to design and evaluate the activity of a novel anti-BCMA nanoCAR-T based on the V H H Nb17, and that compares well with a standard one with the conventional CAR-T CT103a. Nb17 was validated for strong BCMA binding on MM cell lines and incorporating into a CAR construct. Both nanoCAR-T and CT103a CAR-T were generated via lentiviral transduction of primary T cells. Their functional properties, including cytotoxicity, cytokine secretion, degranulation, memory phenotype, and gene expression, were assessed in vitro, and their antitumor efficacy was tested in vivo in NSG mice model. Nb17-nanoCAR-T demonstrated specific killing of MM cells, robust cytokine release (IL-2, TFNa, IFNg), and CT107a degranulation comparable to CT103a. Transcriptomic analysis revealed overlapping pathways between the two CAR-T products. Upon rechallenge with MM cells, both showed enhanced proliferation and reduced exhaustion compared with untransduced T cells. In vivo, Nb17-nanoCAR-T and CT103a eradicated MM1.S tumors, although both yet inducing graft-versus-host disease (GVHD)-like toxicity in a subset of mice. MM BCMA Nanobody CAR T-cells Immunotherapy Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Introduction Multiple myeloma (MM) is a hematologic malignancy characterized by the proliferation of clonal plasma cells in the bone marrow (BM). Although still incurable, survival has improved with proteasome inhibitors, immunomodulatory drugs, and, more recently, immunotherapeutic approaches [ 1 ]. B-cell maturation antigen (BCMA), highly expressed on the surface of MM cells [ 2 ], is a major therapeutic target for bispecific T-cell engagers and chimeric antigen receptor (CAR) T-cells [ 3 – 4 ]. CAR-T cells are genetically modified T cells that express chimeric receptors capable of recognizing tumor-associated antigens independently of MHC presentation [ 5 – 6 ]. In 2017, the US Food and Drug Administration (FDA) approved the first CAR-T therapy for B-cell malignancies [ 5 – 7 ]. The first anti-BCMA CAR-T, idecabtagene vicleucel, was approved in March 2021 [ 8 ], followed in 2022 by CT103a, a fully human CAR containing a single-chain variable fragment (ScFv) from a phage display library for MM treatment [ 9 ]–[ 12 ]. The phase 1/2b clinical studies showed CT103a demonstrated a favorable safety profile with no new risk observed with longer follow-up in patients [ 13 ]. CAR-T cells can be generated through retroviral or lentiviral transduction, CRISPR-Cas9 gene editing, lipid nanoparticles, or non-viral transposon-mediated methods. Standard CAR-T cell manufacturing involves T-cell activation, CAR transduction, and in vitro expansion prior to infusion, typically using anti-CD3/CD28 stimulation and cytokines such as IL-2 or IL-7 and IL-15 [ 14 ]–[ 16 ]. Camelid-derived variable heavy chain domains (V H H or nanobodies) are antibody fragment consisting of a monomeric variable domain (sdAb) offering high specificity, small size, robust structure, and enhanced stability in comparison to conventional antibodies. Their unique properties support therapeutic and diagnostic applications [ 17 ]–[ 19 ]. In 2022, the FDA approved nanoCAR-T therapy, Ciltacabtagene autoleucel (Cilta-cel), whose antigen-binding domain consists of two V H Hs capable of binding two distinct BCMA epitopes [ 20 ]–[ 22 ]. This study aims to design and evaluate the activity of a novel anti-BCMA-nanoCAR-T that compares well with a standard one CT103a. This study seeks to provide insight that may inform the optimization of next-generation CAR-T therapies for MM. Materials and Methods Cell lines and primary T cells MM (LP-1, RPMI-8226, MM1.S, OPM-2, MOLP-2, KMS-12-BM) and leukemia (K562) cells were cultured in Roswell Park Memorial Institute (RPMI 1640) (Lonza, Verviers, Belgium) supplemented with 10% fetal bovine serum (FBS; Sigma-Aldrich, St-Louis, MO, USA), 2 mM L-glutamine (Lonza, Verviers, Belgium) and 100 U/mL penicillin-streptomycin (P/S; Lonza, Verviers, Belgium). Primary T cells were cultured in CTS™ OpTmizer™ T Cell expansion SFM with supplement (Thermo Fisher Scientific, Waltham, MA, USA), 2 mM L-glutamine (Lonza, Verviers, Belgium) and 100 U/mL penicillin-streptomycin (P/S; Lonza, Verviers, Belgium) and 5 ng/mL of IL-2 (Peprotech, Neuilly-sur-Seine, France). All cells were cultured at 37°C in 5% CO 2 . PBMCs and T Cells Isolation Human peripheral blood mononuclear cells (PBMCs) were obtained from buffy coats (Croix-Rouge de Belgique, Liège, Belgium) from healthy adult volunteers with informed consent. PBMCs were isolated by Ficoll-Paque density centrifugation (GE Healthcare, Freiburg, Germany) and T cells were purified using the EasySep Human T Cell Isolation Kit (Stemcell Technologies, Vancouver, Canada) according to the manufacturer’s protocol. Lentivral Vector Production and Cell transduction DNA sequences for ScFv and V H H antigen-binders were obtained from publicly available sources [ 23 ]. Lenti-X 293T cells were co-transfected with lentiviral gene transfer plasmids (ScFv-CT103a-hPGK-puro or V H H-nanoCARNb17-hPGK-puro) designed with VectorBuilder (China) and packaging plasmids (psPAX2 and a VSV-G-encoding plasmid). Lentiviral supernatants were collected 48h-96h post transfection, filtered, concentrated, titrated with qPCR Lentivirus Titration (Titer) Kit (ABM®, LV900, Richmond, BC, Canada), and used to transduce T cells to generate CAR-T cells using a multiplicity of infection (MOI) of 10. Transduction efficiency was analyzed by flow cytometry using a V5-AlexaFluor647 antibody (Thermo Fisher Scientific, Waltham, MA, USA). Cancer cell lines were similarly transduced with GFP or GFP/luciferase vectors (CMV-GFP-hPGK-blasti or CMV-GFP-luciferase-hPGK-blasti), and GFP + cells were sorted using the Sony MA900 (Sony Biotechnology, San Jose, California, USA). Sequences of CT103a and Nb17 are listed in Table S1 . Flow Cytometry staining and analysis Cells were incubated for 60 min at 4°C with titrated antibody concentrations before analysis on a FACS Canto TM II (BD Biosciences, San Jose, CA, USA), a CytoFLEX (Beckman Coulter, Brea, California, USA), or an LSRFortessa™ (BD Biosciences, San Jose, CA, USA). The following antibodies were used: anti-human CD3-BV605 (UCHT1, BD Biosciences, CA, USA), anti-human CD45-BV711 (HI30, BD Biosciences, CA, USA), anti-mouse CD45-Pe-Cy5.5 (30-F11, BD Biosciences, CA, USA), anti-human CD4-PerCP-Cy5.5 (RPA-R4, Sony Biotechnology, San Jose, CA, USA), anti-human CD8-PE-Cy7 (HIT8a, Biolegend, San Diego, CA, USA), anti-human CD45RA-BV510 (HI100, BD Biosciences, CA, USA), anti-human CD62L-AlexaFluor700 (DREG-56, Biolegend, San Diego, CA, USA), anti-human CD27-PE (M-T271, BD Biosciences, San Jose, CA, USA), anti-human CD107a (H4A3, Biolegend, San Diego, CA, USA). To discriminate live from dead cells, samples were then incubated 10 min at 4°C with a Fixable Viability Dye eFluor™ 780 (1:10,000 dilution; Invitrogen, Thermo Fisher Scientific). For the detection of V H H Nb17 binding, cells were incubated with an anti-V H H antibody (GenScript Biotech, Piscataway, New Jersey, USA) followed by an anti-rabbit-AlexaFluor647 secondary antibody (Biolegend, San Diego, CA, USA). All analysis were performed using FlowJo™ software V10.1 (BD Biosciences, Ashland, OR, USA). Quantification of BCMA or CAR cell surface expressions and binding capacity of VH Nb17 MM cell lines were labelled with a PE-conjugated anti-BCMA antibody, while primary T and CAR-T cells were labelled with PE-conjugated BCMA soluble protein (ACROBiosystems, Basel, Switzerland). BCMA or CAR expression levels were quantified with the BD Quantibrite™ Beads (BD Biosciences, San Jose, CA, USA) according to the manufacturer’s instructions. This kit contains beads conjugated at four levels of PE intensity, each corresponding to a defined number of PE molecules per bead. Based on the generated standard curve, the number of PE molecules per cell was calcutated after labelling with the PE-labelled antibody or soluble protein. Cellular Cytotoxicity Assays and CAR-T phenotyping and characterization: Flow Cytometry CAR-T-dependent tumor cell cytotoxicity was quantified by flow cytometry. GFP transduced target cells were mixed in co-culture with effector CAR-T cells at different effector-to-target ratios (1:3, 1:1, 3:1) in 96-well plates in RPMI 1640 supplemented with 10% FBS, 2 mM L-glutamine and 100 U/mL penicillin-streptomycin without IL-2 at 37°C in a humidified atmosphere with 5% CO 2 . After 24h to 48h, target cell viability was assessed by counting live cells using Fixable Viability Dye eFluor™ 780. CAR-T were phenotyped and characterized by antibody labeling as described above. Cellular Cytotoxicity Assays and cytokine productions: ELISA CAR-T-dependent tumor cell cytokine production was quantified by ELISA. GFP transduced target cells were mixed in co-culture with effector CAR-T cells at a ratio effector-to-target 1:1 in 96-well plates in RPMI 1640 supplemented with 10% FBS, 2 mM L-glutamine and 100 U/mL penicillin-streptomycin without IL-2 at 37°C in a humidified atmosphere with 5% CO 2 . After 24h, cells were centrifuged and supernatents were collected to analyse the IL-2, IFNγ, TNFα productions by ELISA Max™ Deluxe Set from Biolegend (San Diego, CA, USA) according to the manufacturer’s protocol. Absorbances were read using the Plate Reader Tristar 2 S LB942 number 2076 from Berthold Technologies (Bad Wildbad, Germany). Bulk-RNA sequencing RNA-seq samples were collected at three key co-culture timepoints: 0, 4, and 16h of co-culture of CAR-T or untransduced T cells with MM1.S. After these timepoints, CD3 + CAR + (CAR-T) and CD3 + CAR − (T cells) were sorted by fluorescence-activated cell sorting (Sony MA900 flow cell sorter) using CD3-BV605 (UCHT1, BD Biosciences, CA, USA) and/or V5-AlexaFluor647 (Thermo Fisher Scientific, Waltham, MA, USA) antibodies. Bulk-RNA samples were prepared from sorted cells using the kit RNeasy® Mini Kit (Qiagen, Hilden, Germany) according to the manufacturer’s protocol. Samples were purified after treatment with DNAse I (Qiagen, Hilden, Germany) and further purified using Zymo columns (Zymo Research, California, USA). Libraries were sequenced on the Illumina NovaSeg 6000. Raw sequencing reads were quality-controlled with FastQC (v0.12.1) to remove adapters and low-quality bases. Cleaned reads were pseudo-aligned to the human reference genome (GRCh38.99) using kallisto (v0.51.1). Gene-level counts were generated with R package tximport (v1.34.0 based on Ensembl gene annotations). Downstream analyses were performed in RStudio (R v4.4.2). Normalization and differential expression analyses were conducted using the DESeq2 package (v1.46.0). Genes with adjusted p-value (Benjamini–Hochberg FDR) 1 were considered significantly differentially expressed. Gene set enrichment analysis (GSEA) was performed with fgsea package (v1.32.0) using Hallmark gene sets. Visualization of results was performed with ggplot2 (v3.5.1). In vivo NSG mouse model Ethical approval for the in vivo part of the study was obtained from the institutional animal ethical board (Commission d’Ethique Animale Universitaire de Liège). NSG mice received 2-G -irradiation on day − 1, followed by tail vein injection of 1x10 6 MM1.S-GFP/luc on day 0. Tumor growth was followed using bioluminescence imaging with the Xenogen IVIS 200 with 3 mg/mice luciferin injection every 7 to 10 days. Sixteen to 19 days after tumor injection, when tumor signal was detectable, 2x10 6 CAR-T were injected intravenously in the tails. Mice were monitored for tumor growth and survival for up to 90 days. Additionally, blood samples were collected via tail vein every 20 days after CAR-T injections to analyze CAR-T phenotype and trafficking using flow cytometry. Two independent cohorts were conducted. In cohort 1 (n = 15), mice were distributed into four groups, MM only (n = 3), MM + Mock T cells (n = 4), MM + CT103a (n = 4), and MM + nanoCAR-T cells (n = 4). In cohort 2 (n = 12 mice), mice were distributed into three groups, MM only (n = 2), MM + CT103a (n = 5), and MM + nanoCAR-T cells (n = 5). In each cohort, all T-cell products were derived from a single healthy donor. Statistical analysis GraphPad Prism 8.0.1. (GraphPad Software, Inc., USA) was used to perform statistical analysis and graph plotting. Comparisons between two datasets were conducting using paired or unpaired Student’s t-tests, with parametric or non-parametric versions applied based on the distribution of the data thank to the Shapiro-Wilk normality test (e.g., Gaussian, non-Gaussian). Statistical significance was defined as p < 0.05 (*) and defined as follows: p < 0.01 (**), p < 0.001 (***), p < 0.0001 (****). p-values above 0.05 were considered non-significant (ns). Results Construction of a nanoCAR sequence containing V H H Nb17 To evaluate the performance of a V H H-based CAR relative to the scFv-based CT103a, we designed a nanoCAR with an analogous backbone. Both constructs were second-generation CARs containing a CD8 hinge, a transmembrane domain, and a 4-1BB co-stimulatory region. A V5 tag was added for flow cytometry detection. The nanoCAR sequence was composed of SFFVprom-V H H-V5tag-CD8α-(4-1BB)-CD3ζ, while CT103a sequence was SFFVprom-V5tag-VH-linker-VL-CD8α-(4-1BB)-CD3ζ (Fig. 1 A). Both CAR-T cells were generated through lentiviral transduction at the same MOI (Fig. 1 B). Although nanoCAR transduction efficiency was slightly lower than CT103a, resulting transduced T cells exhibited equal stable CAR surface expression (Fig. 1 C). To incorporate the V H H Nb17 in our nanoCAR sequence, we assessed its specific binding capacity to cell-surface BCMA. BCMA expression was quantified by flow cytometry across various human MM cell lines before incubating them with Nb17. BCMA expression was highest in MOLP-2 (2772 BCMA/cell), followed by RPMI-8226 (2034 BCMA/cell), MM1.S (1942 BCMA/cell), KMS-12-BM (1834 BCMA/cell) and LP-1 (1375 BCMA/cell). OPM-2 showed the lowest expression (Fig. 1 D). Despite variability in BCMA expression, Nb17 demonstrated specific binding to BCMA (Fig. 1 E). Thus, we successfully designed a persistent and stable nanoCAR capable of specifically targetting cell-surface BCMA. In vitro efficacy of CT103a and nanoCAR in killing BCMA + MM cell lines We compared the cytotoxic activity of nanoCAR, CT103a and untransduced (Mock) T cells against BCMA + MM cells. Each effector population was co-cultured with MM1.S-GFP + for 24–48 hours, and tumor killing was assessed by quantifying the GFP signal using flow cytometry at various effector-to-target (E:T) ratios of 1:3, 1:1 and 3:1. Both CAR-T cells significantly reduced the number of GFP + tumor cells compared with Mock T cells, with enhanced cytotoxicity observed at 48 hours (Figs. 2 A, 2 B, Supplementary Data Fig I). To assess antigen specificity, CAR-T and Mock T cells were co-cultured at a 1:1 ratio with GFP + -BCMA + MM cell lines (MM1.S, RPMI-8226 and LP-1) or with a GFP + -BCMA − K562 24–48 hours. Both CT103a and nanoCAR mediated robust cytotoxicity against BCMA + targets, particularly after 48 hours (Figs. 2 C-E). K562 viability slightly decreased in co-culture, likely reflecting basal IFNγ production by T/CAR-T cells (Fig. 6 ). Together, these results confirm the therapeutic potential of CT103a and nanoCAR in selectively targeting BCMA-expressing malignancies. Persistence of CAR-T killing ability following repeated antigen challenges To assess functional persistence, we evaluated the long-term activity of CT103a and nanoCAR through repeated tumor challenges. Mock or CAR-T cells were co-cultured with GFP + tumor cell lines (MM1.S, RPMI-8226, LP-1 and K562) and rechallenged thrice with intervals of 48 hours. Tumor killing was quantified by flow cytometry, while CAR-T proliferation was determined by live T-cell counts. Both CAR-Ts eradicated BCMA + cell lines after each rechallenge, whereas Mock T cells failed to eliminate tumors, and untreated cancer cells proliferated (Figs. 3 A, 3 B). In contrast, BCMA − K562 showed no significant cytotoxicity when co-cultured with either Mock or CAR-T cells. Regarding expansion, both CT103a and nanoCAR proliferated robustly when repeatedly exposed to BCMA + targets, but not with K562 cells or in tumor-free conditions. Mock T cells exhibited minimal proliferation, under all conditions (Fig. 3 C). We conclude that both CT103a and nanoCAR T cells exhibit long-term efficacy, suggesting their potential for long-term tumor control. CAR-T cell differentiation into CD4-CD8 and central memory subsets with enhanced CD107a surface expression After 24 hours of co-culture with MM1.S, RPMI-8226, LP-1 and K562, CD4 and CD8 levels were assessed. Both subsets remained stable across groups, with CD4 representing ~ 60% and CD8 ~ 20–30% of the T-cell population, consistent with reported distributions [ 24 ]. To assess differentiation, CT103a and nanoCAR-T cells were co-cultured with MM1.S for 24–48 hours, and expression of CD27, CD45RA and CD62L was analyzed using flow cytometry. Compared to non-activated PBMC-derived T cells, Mock, CT103a and nanoCAR presented increased frequency of CD27 + -CD45RA − cells, indicating a memory T cell phenotype. Within this population, CD62L distinguished central memory (TCM, CD62L + ) from effector memory (TEM, CD62L − ) subsets (Fig. 5 A). Upon antigen exposure, CAR-T cells displayed dynamic shifts: at 24 hours, TEM increased, reflecting active cytotoxicity, while at 48 hours, subsets rebalanced between TCM and TEM, indicating restoration of memory phenotype post-target elimination. Mock T cells without antigen largely retained a TCM phenotype, underscoring the role of antigen engagement in effector differentiation (Fig. 5 B). Degranulation was assessed via CD107a expression 6 hours after co-culture with MM1.S, RPMI-8226, LP-1, and K562 (Fig. 5 C) by flow cytometry. Both CT103a and nanoCAR showed higher CD107a levels than Mock T cells, including with K562, likely reflecting basal activation. Overall, CD107a expression confirms the robust potential of both CAR constructs and their functional readiness to engage and kill target cells, regardless of BCMA specificity or antigen-independent activation. CT103a and nanoCAR produce cytokines when co-cultured with MM cell lines Cytokine secretion is a key aspect of CAR-T functionality. After 24 hours of co-culture with BCMA + MM1.S, RPMI-8226, or LP-1, both CT103a and nanoCAR-T cells produced high levels of IL-2, IFNγ and TNFα (Fig. 6 A, Supplementary Data, Fig II). In contrast, co-culture with BCMA − K562 cells induced minimal IFNγ and no IL-2 or TNFα from either CAR or Mock T cells (Fig. 6 A). These results demonstrate antigen-specific activation and confirm the functional responsiveness of both CAR constructs to BCMA + targets. Gene expression studies of CT103a and nanoCAR co-cultured with MM cells To gain deeper insights into CAR-T activity, we performed bulk RNA sequencing and compared the transcriptional profiles across the different conditions (CT103a, nanoCAR, Mock) after incubation with MM1.S (Supplementary Data, Fig III). Given that critical biological events occur rapidly after the encounter between CAR-T cells and target cells, we selected early time points (0, 4, and 16h post-incubation) for this analysis. We used Mock T cells as negative control and CT103a as positive control. To explore the transcriptional responses of CT103a and nanoCAR, we first visualized the top 50 overexpressed genes of both CAR-Ts compared to Mock at 16h using heatmaps (Fig. 7 A). Both CAR-T constructs demonstrated upregulation of key genes associated with T cell activation and proliferation (e.g. IL17REL , TNFRS9 ( 4-1BB )) or effector functions (e.g. IL5 , IL2 , IFNG ). Complementing this analysis, volcano plots revealed minimal differences between CT103a and nanoCAR across all time points (0h, 4h, 16h). In contrast, both CAR-T cell types exhibited a significant increase in the number of upregulated and downregulated genes at 4h and 16h compared to Mock T cells, consistent with CAR-specific activation (Fig. 7 B). Venn diagrams further illustrated that most of upregulated and downregulated genes at 4h and 16h were shared between CT103a and nanoCAR, underscoring their comparable transcriptional programs (Fig. 7 C). Gene set enrichment analysis (GSEA) provided further insights into CAR-T cell activation. Hallmark (Supplementary Data, Fig IV A)) and KEGG (Supplementary Data, Fig IV B)) pathway analyses revealed significant enrichment of pathways related to cytokine production, cytokine-cytokine interactions, and proliferation in CT103a and nanoCAR compared to Mock at 4h. This enrichment reflected CAR-specific functionality. However, these pathways were no longer differentially expressed by 16h, likely due to basal cytokine production by pre-activated Mock T cells masking CAR-specific effects. These findings suggested that cytokine-related signaling at early time points predominantly arises from CAR-specific mechanisms, while the distinction diminished over time due to baseline activation in Mock cells. Overall, while CT103a and nanoCAR demonstrated similar transcriptional profiles, their distinct gene signatures relative to Mock revealed shared mechanisms of antigen-specific activation. CT103a and nanoCAR can eliminate tumor cells in vivo We examined the anti-tumor efficacy of both CT103a and nanoCAR-T cells in a NSG mouse model using the Xenogen IVIS 200 imaging system and flow cytometry (Supplementary Data Fig IV). Mice were intravenously injected with MM1.S-GFP-Luc and subsequently treated with Mock T cells, CT103a or nanoCAR T cells via intravenous injection 16 to 19 days after tumor inoculation once tumor signals became detectable by bioluminescence. In cohort 1, all mice injected with MM1.S-GFP-Luc cells and left untreated or treated with Mock T cells succumbed to disease within 30–40 days, confirming the aggressiveness of the model (Figs. 9 A and 9 B). CT103a-treated mice died by day 36 to 46. In contrast, all four mice treated with nanoCAR-T cells survived beyond day 90, with three out of four mice being completely tumor-free, and one mouse showing residual disease. In cohort 2, both CT103a and nanoCAR-T treatments induced complete tumor regression within 10 days of CAR-T cell infusion (Figs. 9 A and 9 B). Mice were subsequently monitored for long-term survival. Four out of five nanoCAR-T-treated mice died before day 90 due to ascites, which may have resulted from hepatic and/or cardiac insufficiencies, maybe also due to graft-versus-host disease-like based on visual symptoms and the high amounts of hCD45 + cells in the peripheral blood and/or in the BM even if low amounts of V5 + (CAR-T + ) cells analyzed by flow cytometry the day of their sacrifice were detected (Fig C). Among CT103a-treated mice, one also developed ascites and died prematurely, while another survived until the end of the study but developed signs of hepatic insufficiency such as jaundice. Both mice had high levels of hCD45 + cells and low amounts of V5 + (Fig C). Consequently, we split the Kaplan-Meier survival curves in two, one showing the MM-specific mortality and the other one showing the global mortality (Figs. 9 D). We next evaluated CD4 and CD8 T-cell subsets, as well as the proportion of CD62L + cells among CD27 + CD45RA − memory population (Fig. 9 E). We found no significant differences in phenotype between CT103a and nanoCAR-T cells. These findings suggest that while both CT103a and nanoCAR-T cells display potent anti-MM activity, nanoCAR-T cells may additionally induce xenoreactivity against murine tissues. Discussion Our study provided a detailed characterization of two anti-BCMA CAR-T therapies, CT103a (ScFv-based) and nanoCAR (V H H-based), for MM treatment. Despite their structural difference, both CAR constructs demonstrated comparable cytotoxic activity, persistence and transcriptional responses, underscoring shared tumor-specific activation mechanisms. Previous studies compared ScFv-based CAR T and nanoCAR T cells. These showed the smaller and more stable structure of V H H can reduce steric hindrance, which is particularly advantageous for bispecific CAR T constructs. These studies also suggested nanoCAR-T cells demonstrated comparable tumor killing efficacy, particularly in the context of bispecific targeting where their compact structure allowed for better antigen engagement and improved therapeutic efficacy [ 25 ]–[ 30 ]. While not eradicating BCMA − cells, such as the K562 leukemia cell line, both CT103a and nanoCAR specifically targeted BCMA + MM cell lines (MM1.S, RPMI-8226 and LP-1). This specific anti-tumor activity was maintained over prolonged periods and following repeated antigen exposures, which supports potential for long-term therapeutic efficacy. Moreover, key cytokines, including IL-2, TFNα and IFNγ, were produced in response to BCMA-positive targets, highlighting the specificity of both CAR-T constructs [ 31 ] [ 32 ]. T cell subsets are defined by specific surface markers reflecting their differentiation state and functional capacity. Compared to non-activated PBMC-derived T cells, all CAR-T and Mock T cells exhibited a memory phenotype (CD27⁺CD45RA⁻). Moreover, CAR-T cells showed dynamic phenotypic shifts between TCM and TEM. At 24 hours, an increase in TEM frequency was observed, consistent with active cytotoxic engagement. By 48 hours, the distribution shifted back toward TCM, suggesting recovery without target cells maintained a TCM-dominant phenotype over time. These results demonstrate that CT103a and nanoCAR-T cells possess phenotypic plasticity, enabling a transition between effector and memory states in response to antigen, a property associated with long-term persistence and functional resilience. Moreover, their functional readiness was further reinforced by the expression of degranulation markers such as CD107a. These characteristics were essential for effective CAR-T treatments, providing insights into how CAR-T cells maintain robust and extended antitumor activity over time [ 33 ]. Transcriptional analyses revealed minimal differences between CT103a and nanoCAR during early activation, with both exhibiting upregulated genes linked to T cell activation, proliferation and effector functions. Common pathways, such as cytokine production and cytokine-cytokine interactions, were enriched at 4h but diminished by 16h, possibly reflecting baseline cytokine production in pre-activated T cells. In vivo results demonstrate that both CT103a and nanoCAR-T cells exhibit significant anti-MM activity. The long-term survival and minimal residual disease observed in nanoCAR-T treated mice highlight the therapeutic potential of V H H-based CAR-T designs, particularly in aggressive MM models. Notably, NanoCAR-T cells displayed prolonged survival in both the BM and peripheral blood. In contrast, the limited peripheral circulation in CT103a-treated mice suggest distinct capacities in persistence and in vivo expansion between the two CAR constructs. While cohort 2 further supports the potent anti-tumor activity of both CAR-T therapies, the occurrence of ascites in several nanoCAR-T-treated mice may suggests possible xenoreactivity against murine tissues, pointing to the importance of carefully assessing potential safety concerns. Importantly, this alloreactivity is likely a consequence of the xenogeneic setting, and nanoCAR-T cells would be expected to be safe in an autologous clinical context. These in vivo results, combined with phenotypic and functional profiling, underscore the potential of VHH-based CAR-T therapies as a promising strategy for the treatment of BCMA + MM. In conclusion, our results showed that CT103a and nanoCAR are promising CAR-T treatments for BCMA-positive MM. Both constructs demonstrated significant cytotoxic activity, persistence and favorable transcriptional profiles, indicating their potential as effective therapeutic approaches. Notably, compared to traditional scFvs, the integration of nanobodies, such as the V H H Nb17, in CAR designs could provide several advantages including smaller size, higher stability and the ability to target distinct epitopes or antigens with reduced steric hindrance. These benefits could improve CAR-T cell targeting accuracy, increasing their overall efficacy, especially in full tumor microenvironments. Future research could focus on optimizing bispecific CAR-T cells, which would target multiple antigens to reduce the possibility of antigen escape and increase treatment durability [ 34 ] [ 35 ]. Moreover, exploring the integration of nanobodies in combination with immune checkpoint inhibitors or other immune-modulating treatments could enhance CAR-T functionality, overcoming limitations like immune resistance. Finally, the potential for engineering CAR-T cells with a more defined "memory-like" phenotype could offer strategies to improve long-term persistence, reducing the need for repeated treatments [ 36 ]. These next steps would move beyond initial proof-of-concept studies to more clinical and mechanistic understanding, facilitating the translation of these promising CAR-T therapies into the clinic. Abbreviations MM Multiple myeloma BCMA B-cell maturation antigen BM Bone marrow CAR Chimeric antigen receptor Cilta-cel Ciltacabtagene autoleucel FDA Food and Drug Administration GSEA Gene set enrichment analysis IL-2 Interleukin-2 MOI Multiplicity of infection ScFv Single-chain variable fragment SdAb Single domain antibody SCFV Single-chain variable fragment SD Standard deviation TCM Central memory T cell TEM Effector memory T cell V H H Heavy chain variable domains Declarations Funding Funding: The laboratory of Haematology is supported by the Foundation Against Cancer, the Intergroup Francophone du Myélome, the Fonds National de la Recherche Scientifique, (FNRS, Belgium), Télévie-FNRS and the Fonds Spéciaux de la Recherche (University of Liège). Competing interests This research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. Ethical approval Ethical approval for the in vivo part of the study was obtained from the institutional animal ethical board (Commission d’Ethique Animale Universitaire de Liège). Author Contribution MJ wrote the manuscript and SO, EDV, FB, CG, GE, TN, JC, provided additional input for the manuscript. All authors read and approved the manuscript. References J. Laubach, P. Richardson, and K. Anderson, “Multiple myeloma,” Annu. Rev. Med. , vol. 62, pp. 249–264, 2011, doi: 10.1146/annurev-med-070209-175325. M. Went et al. , “Deciphering the genetics and mechanisms of predisposition to multiple myeloma,” Nat. Commun. , vol. 15, no. 1, 2024, doi: 10.1038/s41467-024-50932-7. K. C. Graf et al. , “ ‘Fast but not so furious’ : A condensed step‐up dosing schedule of teclistamab for relapsed/refractory multiple myeloma ,” eJHaem , vol. 5, no. 4, pp. 793–797, 2024, doi: 10.1002/jha2.906. D. Pan and J. Richter, “Management of Toxicities Associated with BCMA, GPRC5D, and FcRH5-Targeting Bispecific Antibodies in Multiple Myeloma,” Curr. Hematol. Malig. Rep. , 2024, doi: 10.1007/s11899-024-00740-z. S. Guedan, M. Ruella, and C. H. June, “Emerging Cellular Therapies for Cancer,” Annu. Rev. Immunol. , vol. 37, pp. 145–171, 2019, doi: 10.1146/annurev-immunol-042718-041407. K. Utkarsh et al. , “CAR-T cell therapy: a game-changer in cancer treatment and beyond,” Clin. Transl. Oncol. , vol. 26, no. 6, pp. 1300–1318, 2024, doi: 10.1007/s12094-023-03368-2. “First-Ever CAR T-cell Therapy Approved in U.S.,” Cancer Discov. , vol. 7, p. 10, 2017, doi: https://doi.org/10.1158/2159-8290.CD-NB2017-126. S. Jagannath et al. , “KarMMa-RW: comparison of idecabtagene vicleucel with real-world outcomes in relapsed and refractory multiple myeloma,” Blood Cancer J. , vol. 11, no. 6, pp. 1–9, 2021, doi: 10.1038/s41408-021-00507-2. W. Mu et al. , “A Model Perspective Explanation of the Long-Term Sustainability of a Fully Human BCMA-Targeting CAR (CT103A) T-Cell Immunotherapy,” Front. Pharmacol. , vol. 13, no. February, pp. 1–9, 2022, doi: 10.3389/fphar.2022.803693. D. Li et al. , “Anti-BCMA CAR-T cells therapy for a patient with extremely high membrane BCMA expression: a case report,” J. Immunother. Cancer , vol. 10, no. 9, pp. 1–5, 2022, doi: 10.1136/jitc-2022-005403. J. Xu and J. J. Melenhorst, “CT103A, a forward step in multiple myeloma immunotherapies,” Blood Sci. , vol. 3, no. 2, pp. 59–61, 2021, doi: 10.1097/BS9.0000000000000068. Q. Wang et al. , “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. , vol. 31, no. 3, pp. 420–426, 2024, doi: 10.1038/s41417-023-00712-0. C. Li 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. , vol. 41, no. 16_suppl, pp. 8025–8025, 2023, doi: 10.1200/jco.2023.41.16_suppl.8025. M. S. Pampusch et al. , “Rapid Transduction and Expansion of Transduced T Cells with Maintenance of Central Memory Populations,” Mol. Ther. Methods Clin. Dev. , vol. 16, no. March, pp. 1–10, 2020, doi: 10.1016/j.omtm.2019.09.007. H. W. Song et al. , “CAR-T cell expansion platforms yield distinct T cell differentiation states,” Cytotherapy , vol. 26, no. 7, pp. 757–768, 2024, doi: 10.1016/j.jcyt.2024.03.003. L. de Macedo Abdo et al. , “Development of CAR-T cell therapy for B-ALL using a point-of-care approach,” Oncoimmunology , vol. 9, no. 1, 2020, doi: 10.1080/2162402X.2020.1752592. S. Muyldermans, “Nanobodies: Natural single-domain antibodies,” Annu. Rev. Biochem. , vol. 82, pp. 775–797, 2013, doi: 10.1146/annurev-biochem-063011-092449. A. C. M. M. Nairo Brilhante-da-Silva, Rosa Maria de Oliveira Sousa, Andrelisse Arruda, Eliza Lima Dos Santos, “Camelid Single-Domain Antibodies for the Development of Potent Diagnosis Platforms,” Mol. Diagn. Ther. , vol. 25, no. 4, pp. 439–456, 2021, doi: 10.1007/s40291-021-00533-7. E. Duray et al. , “A non-internalised CD38-binding radiolabelled single-domain antibody fragment to monitor and treat multiple myeloma,” J. Hematol. Oncol. , vol. 14, no. 1, pp. 1–13, 2021, doi: 10.1186/s13045-021-01171-6. E. Chekol Abebe, M. Yibeltal Shiferaw, F. Tadele Admasu, and T. Asmamaw Dejenie, “Ciltacabtagene autoleucel: The second anti-BCMA CAR T-cell therapeutic armamentarium of relapsed or refractory multiple myeloma,” Front. Immunol. , vol. 13, no. September, pp. 1–15, 2022, doi: 10.3389/fimmu.2022.991092. R. Smith, “Bringing cell therapy to tumors: Considerations for optimal CAR binder design,” Antib. Ther. , vol. 6, no. 4, pp. 225–239, 2023, doi: 10.1093/abt/tbad019. H. Hanssens et al. , “Anti-Idiotypic VHHs and VHH-CAR-T Cells to Tackle Multiple Myeloma: Different Applications Call for Different Antigen-Binding Moieties,” Int. J. Mol. Sci. , vol. 25, no. 11, 2024, doi: 10.3390/ijms25115634. W. H. Zhao et al. , “A phase 1, open-label study of LCAR-B38M, a chimeric antigen receptor T cell therapy directed against B cell maturation antigen, in patients with relapsed or refractory multiple myeloma,” J. Hematol. Oncol. , vol. 11, no. 1, pp. 1–8, 2018, doi: 10.1186/s13045-018-0681-6. M. Dong Hoon Lee, B.S., Francisco Cervantes-Contreras, Sang Yun Lee, PhD, Damian J. Green, MD, Brian G. Till, “Improved Expansion and Function of CAR T Cell Products from Cultures Initiated at Defined CD4:CD8 Ratios,” Blood , vol. 132, no. Supplement 1, 2018, doi: https://doi.org/10.1182/blood-2018-99-111576. F. Nasiri, P. Safarzadeh Kozani, and F. Rahbarizadeh, “T-cells engineered with a novel VHH-based chimeric antigen receptor against CD19 exhibit comparable tumoricidal efficacy to their FMC63-based counterparts,” Front. Immunol. , vol. 14, no. February, pp. 1–13, 2023, doi: 10.3389/fimmu.2023.1063838. J. Cheng et al. , “Preclinical development and evaluation of nanobody-based CD70-specific CAR T cells for the treatment of acute myeloid leukemia,” Cancer Immunol. Immunother. , vol. 72, no. 7, pp. 2331–2346, 2023, doi: 10.1007/s00262-023-03422-6. Q. Xiong et al. , “The development of chimeric antigen receptor T-cells against CD70 for renal cell carcinoma treatment,” J. Transl. Med. , vol. 22, no. 1, pp. 1–13, 2024, doi: 10.1186/s12967-024-05101-1. H. Hanssens 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. , vol. 15, no. April, pp. 1–18, 2024, doi: 10.3389/fimmu.2024.1389018. W. C. Temple et al. , “Framework humanization optimizes potency of anti-CD72 nanobody CAR-T cells for B-cell malignancies,” J. Immunother. Cancer , vol. 11, no. 11, pp. 1–16, 2023, doi: 10.1136/jitc-2023-006985. S. De Munter et al. , “Nanobody based dual specific CARs,” Int. J. Mol. Sci. , vol. 19, no. 2, pp. 1–11, 2018, doi: 10.3390/ijms19020403. S. L. Maude et al. , “Chimeric Antigen Receptor T Cells for Sustained Remissions in Leukemia,” N. Engl. J. Med. , vol. 371, no. 16, pp. 1507–1517, 2014, doi: 10.1056/nejmoa1407222. M. C. J. Rebecca A. Gardner, Olivia Finney, Colleen Annesley, Hannah Brakke, Corinne Summers, Kasey Leger, Marie Bleakley, Christopher Brown, Stephanie Mgebroff, Karen S. Kelly-Spratt, Virginia Hoglund, Catherine Lindgren, Assaf P. Oron, Daniel Li, Stanley R. Ridd, “Intent-to-treat leukemia remission by CD19 CAR T cells of defined formulation and dose in children and young adults,” Blood , vol. 129, no. 25, pp. 3322–3331, 2017, doi: https://doi.org/10.1182/blood-2017-02-769208. J. W. Fischer, “CAR-T Cell Therapy: Mechanism, Management, and Mitigation of Inflammatory Toxicities,” Front. Immunol. , vol. 12, no. June, pp. 1–9, 2021, doi: 10.3389/fimmu.2021.693016. S. Wu 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. , no. April, pp. 1361–1373, 2024, doi: 10.1111/bjh.19631. B. Xia et al. , “Nanobody-derived bispecific CAR-T cell therapy enhances the anti-tumor efficacy of T cell lymphoma treatment,” Mol. Ther. Oncolytics , vol. 30, no. September, pp. 86–102, 2023, doi: 10.1016/j.omto.2023.07.007. G. Kueberuwa et al. , “CCR7+ selected gene-modified T cells maintain a central memory phenotype and display enhanced persistence in peripheral blood in vivo,” J. Immunother. Cancer , vol. 5, no. 1, pp. 1–14, 2017, doi: 10.1186/s40425-017-0216-7. Additional Declarations No competing interests reported. Supplementary Files ResearchMeganeJassinSupplementaryData.docx Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-7693534","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":526016859,"identity":"0ceb65d7-af6a-495b-b394-faa7f6a8caac","order_by":0,"name":"Mégane Jassin","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABCUlEQVRIiWNgGAWjYBADZhBxgKGCQYYNxGJsIFrLGQYeorVAAGMbAw8DIS267e0XPxdUMLDz9x9/eLhw3mEePvb2iw8Yd9jg1GJ25kyx9IwzDMwSN3IMDs/cdpiHjedMsQHjmTTcWm7kJEjztgH9coOH4TAvSItETpoEY9th3Fruv0n+zfuPgVn+/PEHh3nnALXIv0n/wdj2H48t7MekeRsYmA0OJBgc5m0A2cJ+DBgOB/D4JYfNmueYBLMhyC88x9KBfslhlkg8k4xby/Hjj2/z1Ngky50//vgzT421nHz78YcfPu6ww6mFgYHHAEhIJKOKJODRwMDA/gBE2mGIjIJRMApGwSiAAQArlFO6ad1VgQAAAABJRU5ErkJggg==","orcid":"","institution":"University of Liège","correspondingAuthor":true,"prefix":"","firstName":"Mégane","middleName":"","lastName":"Jassin","suffix":""},{"id":526016860,"identity":"53064179-0751-494c-ac8f-59f2b1dc94aa","order_by":1,"name":"Chloé Onkelinx","email":"","orcid":"","institution":"University of Liège","correspondingAuthor":false,"prefix":"","firstName":"Chloé","middleName":"","lastName":"Onkelinx","suffix":""},{"id":526016861,"identity":"bc6480b8-4bd5-4ccc-9ac5-301a7278d157","order_by":2,"name":"Valentina Bocuzzi","email":"","orcid":"","institution":"University of Liège","correspondingAuthor":false,"prefix":"","firstName":"Valentina","middleName":"","lastName":"Bocuzzi","suffix":""},{"id":526016862,"identity":"a9bf8c33-fef4-4656-8d6a-4ac21b162da4","order_by":3,"name":"Bianca E Silva","email":"","orcid":"","institution":"University of Liège","correspondingAuthor":false,"prefix":"","firstName":"Bianca","middleName":"E","lastName":"Silva","suffix":""},{"id":526016864,"identity":"bb0b78dd-f4e5-4dcd-835f-5eab8e406c91","order_by":4,"name":"Oswin Kwan","email":"","orcid":"","institution":"University of Liège","correspondingAuthor":false,"prefix":"","firstName":"Oswin","middleName":"","lastName":"Kwan","suffix":""},{"id":526016866,"identity":"205dc18c-a4e2-4972-98dd-133076c5c25c","order_by":5,"name":"Alix Block","email":"","orcid":"","institution":"University of Liège","correspondingAuthor":false,"prefix":"","firstName":"Alix","middleName":"","lastName":"Block","suffix":""},{"id":526016868,"identity":"2d4f55e0-dec9-48ca-be81-2d081e72dd69","order_by":6,"name":"Sophie Dubois","email":"","orcid":"","institution":"University of Liège","correspondingAuthor":false,"prefix":"","firstName":"Sophie","middleName":"","lastName":"Dubois","suffix":""},{"id":526016869,"identity":"ea30041e-98f8-407d-a708-688fee432f1b","order_by":7,"name":"Coline Daulne","email":"","orcid":"","institution":"University of Liège","correspondingAuthor":false,"prefix":"","firstName":"Coline","middleName":"","lastName":"Daulne","suffix":""},{"id":526016870,"identity":"4df1dd74-2dd0-4753-9a7f-f20fca7dc4a8","order_by":8,"name":"Guillaume Marcion","email":"","orcid":"","institution":"University of Liège","correspondingAuthor":false,"prefix":"","firstName":"Guillaume","middleName":"","lastName":"Marcion","suffix":""},{"id":526016872,"identity":"f0f55b18-a757-4ae9-be55-310343f3a394","order_by":9,"name":"Sandra Ormenese","email":"","orcid":"","institution":"University of Liège","correspondingAuthor":false,"prefix":"","firstName":"Sandra","middleName":"","lastName":"Ormenese","suffix":""},{"id":526016874,"identity":"fe68ef0d-8a79-42e8-9574-77f055d38816","order_by":10,"name":"Emmanuel Valentin","email":"","orcid":"","institution":"University of Liège","correspondingAuthor":false,"prefix":"","firstName":"Emmanuel","middleName":"","lastName":"Valentin","suffix":""},{"id":526016876,"identity":"1a278251-a254-416d-82fa-6cd1070882f4","order_by":11,"name":"Frédéric Baron","email":"","orcid":"","institution":"University of Liège","correspondingAuthor":false,"prefix":"","firstName":"Frédéric","middleName":"","lastName":"Baron","suffix":""},{"id":526016878,"identity":"e3e05aab-978e-41a6-94f4-d525c41be109","order_by":12,"name":"Céline Grégoire","email":"","orcid":"","institution":"University of Liège","correspondingAuthor":false,"prefix":"","firstName":"Céline","middleName":"","lastName":"Grégoire","suffix":""},{"id":526016880,"identity":"4be4ba84-8837-412f-92f0-a4f1f8a8cc0e","order_by":13,"name":"Grégory Ehx","email":"","orcid":"","institution":"University of Liège","correspondingAuthor":false,"prefix":"","firstName":"Grégory","middleName":"","lastName":"Ehx","suffix":""},{"id":526016881,"identity":"06e9be57-b728-4588-ac09-827cbafb4bf2","order_by":14,"name":"Thi Tham Nguyen","email":"","orcid":"","institution":"University of Liège","correspondingAuthor":false,"prefix":"","firstName":"Thi","middleName":"Tham","lastName":"Nguyen","suffix":""},{"id":526016882,"identity":"cf50c51b-c7de-4b66-a4bd-f871bc611fdf","order_by":15,"name":"Jo Caers","email":"","orcid":"","institution":"University of Liège","correspondingAuthor":false,"prefix":"","firstName":"Jo","middleName":"","lastName":"Caers","suffix":""}],"badges":[],"createdAt":"2025-09-23 11:08:19","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7693534/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7693534/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":93246605,"identity":"0fce8fc4-b92e-423a-8c7a-5f0b42879140","added_by":"auto","created_at":"2025-10-10 15:15:28","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":2657540,"visible":true,"origin":"","legend":"","description":"","filename":"ResearchMeganeJassinCancerImmunologyImmunotherapy.docx","url":"https://assets-eu.researchsquare.com/files/rs-7693534/v1/7347fe79acb6f58aefb2ec27.docx"},{"id":93247262,"identity":"64156237-f1e2-4edc-9d9b-d632f45e9af4","added_by":"auto","created_at":"2025-10-10 15:23:28","extension":"json","order_by":1,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":14866,"visible":true,"origin":"","legend":"","description":"","filename":"89fc472e3a184f858d930ed6013a27d0.json","url":"https://assets-eu.researchsquare.com/files/rs-7693534/v1/453a0ff6d4351830b69b47a9.json"},{"id":93246599,"identity":"15a0bdde-cc63-469f-96f2-79ed4cbdce15","added_by":"auto","created_at":"2025-10-10 15:15:28","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":1237899,"visible":true,"origin":"","legend":"","description":"","filename":"ResearchMeganeJassinSupplementaryData.docx","url":"https://assets-eu.researchsquare.com/files/rs-7693534/v1/b0b1285cbb52b393a92d69d0.docx"},{"id":93244523,"identity":"9b6242b3-f38c-4251-a388-26b716c56eff","added_by":"auto","created_at":"2025-10-10 15:07:28","extension":"xml","order_by":3,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":109033,"visible":true,"origin":"","legend":"","description":"","filename":"89fc472e3a184f858d930ed6013a27d01enriched.xml","url":"https://assets-eu.researchsquare.com/files/rs-7693534/v1/dabb06858d6212c82c82f42e.xml"},{"id":93244533,"identity":"5abb2eec-7035-4422-82bb-3a64c2eed9ae","added_by":"auto","created_at":"2025-10-10 15:07:28","extension":"png","order_by":4,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":131396,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-7693534/v1/48348149ac5733eeede9cede.png"},{"id":93247261,"identity":"1cb2c126-df86-44c3-b122-72c736f546f8","added_by":"auto","created_at":"2025-10-10 15:23:28","extension":"png","order_by":5,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":308105,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-7693534/v1/34ac377cfb52176820a24e24.png"},{"id":93246603,"identity":"301f56fe-7c82-4af0-9d3f-2139ec14debf","added_by":"auto","created_at":"2025-10-10 15:15:28","extension":"png","order_by":6,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":223629,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-7693534/v1/108e83e2791172be9e4bc30d.png"},{"id":93244535,"identity":"3ae7a658-3660-497c-9991-9e71f63552dc","added_by":"auto","created_at":"2025-10-10 15:07:28","extension":"png","order_by":7,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":55850,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-7693534/v1/96574d203ff8fac1c44805b9.png"},{"id":93244544,"identity":"59b86b31-3abd-491e-888c-aeb5c9ad3983","added_by":"auto","created_at":"2025-10-10 15:07:28","extension":"png","order_by":8,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":357298,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-7693534/v1/e097c2895b99222820943588.png"},{"id":93246607,"identity":"382e9239-0fad-41f4-8999-86e330b033b9","added_by":"auto","created_at":"2025-10-10 15:15:28","extension":"png","order_by":9,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":38853,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage6.png","url":"https://assets-eu.researchsquare.com/files/rs-7693534/v1/5ff74c1f0809f8c2702cf498.png"},{"id":93244542,"identity":"b67b1886-2e68-4eae-9fe2-686223a29cc6","added_by":"auto","created_at":"2025-10-10 15:07:28","extension":"png","order_by":10,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":395867,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage7.png","url":"https://assets-eu.researchsquare.com/files/rs-7693534/v1/f2b579f94e309a5fb35c568d.png"},{"id":93246610,"identity":"770a32af-b17f-4940-ab25-bf1112895f5b","added_by":"auto","created_at":"2025-10-10 15:15:28","extension":"png","order_by":11,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":1058415,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage8.png","url":"https://assets-eu.researchsquare.com/files/rs-7693534/v1/a7723dcbf94807909b6153c5.png"},{"id":93244539,"identity":"26b0cc94-e9ca-4845-8b80-8cf9554e4969","added_by":"auto","created_at":"2025-10-10 15:07:28","extension":"png","order_by":12,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":33720,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-7693534/v1/57d63a27b8952695f0c21be8.png"},{"id":93244543,"identity":"bf902820-0fee-48d7-b62d-9f762eccd776","added_by":"auto","created_at":"2025-10-10 15:07:28","extension":"png","order_by":13,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":53304,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-7693534/v1/168bf6e719bb53bdf88f5222.png"},{"id":93244536,"identity":"443d31d7-820c-421d-9a8c-0497a5b2d849","added_by":"auto","created_at":"2025-10-10 15:07:28","extension":"png","order_by":14,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":57653,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-7693534/v1/77553be53aefd85a6668c82b.png"},{"id":93244548,"identity":"b8715129-1340-44ac-ac37-d9ed985a4ac6","added_by":"auto","created_at":"2025-10-10 15:07:28","extension":"png","order_by":15,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":12130,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-7693534/v1/8cb6765e462b8dfa0783c0f6.png"},{"id":93244537,"identity":"d9e3876d-49b2-4dd2-aeb9-98df9efbc3e4","added_by":"auto","created_at":"2025-10-10 15:07:28","extension":"png","order_by":16,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":81964,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-7693534/v1/2308887504a7de23354e456d.png"},{"id":93246609,"identity":"9f2472f5-b7e8-459e-9f6c-99f8110e0c5a","added_by":"auto","created_at":"2025-10-10 15:15:28","extension":"png","order_by":17,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":13461,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage6.png","url":"https://assets-eu.researchsquare.com/files/rs-7693534/v1/87f27fc6c68fd0040e29643c.png"},{"id":93246611,"identity":"660ace49-44b4-431f-9a74-91ef07db6289","added_by":"auto","created_at":"2025-10-10 15:15:28","extension":"png","order_by":18,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":82006,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage7.png","url":"https://assets-eu.researchsquare.com/files/rs-7693534/v1/f994f991af524fb423c6394c.png"},{"id":93244550,"identity":"e71d471c-7e20-4733-b03e-11f7f0161886","added_by":"auto","created_at":"2025-10-10 15:07:28","extension":"png","order_by":19,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":159613,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage8.png","url":"https://assets-eu.researchsquare.com/files/rs-7693534/v1/251cc63b8ae8791a1ef8184a.png"},{"id":93244545,"identity":"e98a3958-dddd-4e85-9ff1-6f453039489f","added_by":"auto","created_at":"2025-10-10 15:07:28","extension":"xml","order_by":20,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":107596,"visible":true,"origin":"","legend":"","description":"","filename":"89fc472e3a184f858d930ed6013a27d01structuring.xml","url":"https://assets-eu.researchsquare.com/files/rs-7693534/v1/2e9732384064648e6882c288.xml"},{"id":93244546,"identity":"b9f1e40d-5482-45bd-aea1-445f602ff867","added_by":"auto","created_at":"2025-10-10 15:07:28","extension":"html","order_by":21,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":117897,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-7693534/v1/e169ab559ebd2517e44c3444.html"},{"id":93244521,"identity":"16f3aea0-4ad2-4046-9a37-982e441fc88e","added_by":"auto","created_at":"2025-10-10 15:07:28","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":384158,"visible":true,"origin":"","legend":"\u003cp\u003eConstruction of a nanoCAR sequence containing V\u003csub\u003eH\u003c/sub\u003eH Nb17. \u003cstrong\u003eA.\u003c/strong\u003e Schematic diagram of the second-generation CAR, the FDA-approved scFv-based CAR-T CT103a, and our nanoCAR construct containing V\u003csub\u003eH\u003c/sub\u003eH Nb17. \u003cstrong\u003eB. \u003c/strong\u003eRate of lentiviral transduction of T cells to generate CT103a and nanoCAR evaluated using flow cytometry. \u003cstrong\u003eC\u003c/strong\u003e. Analysis of CAR expression stability on the T cell surface at 3 and 8 days post-transduction. \u003cstrong\u003eD\u003c/strong\u003e. Quantification of the number of cell surface BCMA molecules on MM cell lines: MM1.S, RPMI-8226, LP-1, OPM-2, MOLP-2 and KMS-12-BM. \u003cstrong\u003eE\u003c/strong\u003e. Validation of V\u003csub\u003eH\u003c/sub\u003eH Nb17 binding to cell-surface BCMA across various MM cell lines. Results are presented as the mean value and standard deviation (SD) from several independent experiments (n = 19 for B., n = 3 for C., n = 14 for D., n = 4 for E.). **: p \u0026lt; 0.01. ***: p \u0026lt; 0.001. ****: p \u0026lt; 0.0001, determined by Mann-Whitney test.\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-7693534/v1/d8a713ee56e543931e12578c.png"},{"id":93244519,"identity":"2ae04c45-7af1-492c-8816-948a84b0e053","added_by":"auto","created_at":"2025-10-10 15:07:28","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1078333,"visible":true,"origin":"","legend":"\u003cp\u003eIn vitro efficacy of CT103a and nanoCAR in killing BCMA\u003csup\u003e+\u003c/sup\u003e MM cell lines. \u003cstrong\u003eA.\u003c/strong\u003e In vitro cytotoxicity assays after 24 or 48 hours of MM1.S-GFP at different effector-to-target (E:T) ratios (1:3, 1:1, 3:1). The mean value and SD from nine biological replicates were shown (n = 9). **: p \u0026lt; 0.01. ***: p \u0026lt; 0.001. ****: p \u0026lt; 0.0001, determined by t-tests. \u003cstrong\u003eB.\u003c/strong\u003e Representative histograms from flow cytometry of in vitro cytotoxicity assays after 48 hours of MM1.S-GFP at (E/T) ratio 1/1. \u003cstrong\u003eC\u003c/strong\u003e., \u003cstrong\u003eD.\u003c/strong\u003e, \u003cstrong\u003eE.\u003c/strong\u003e In vitro cytotoxicity assays (GFP from total cells C., total viability of cells D., GFP signal remaining from alive cells E.) after 24h or 48h of various GFP\u003csup\u003e+\u003c/sup\u003e-BCMA\u003csup\u003e+\u003c/sup\u003e MM cell lines (MM1.S, RPMI-8226 and LP-1) or a GFP leukemia cell line (K562) with Mock T cells, CT103a or nanoCAR in a 1:1 ratio. Results were presented as the mean value and SD of three replicates for three different donors (n = 3 for each donor).\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-7693534/v1/773483a0f29d4b9c38ad703b.png"},{"id":93244522,"identity":"e6d4af05-159f-46fc-8294-50a02844f7c4","added_by":"auto","created_at":"2025-10-10 15:07:28","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":620124,"visible":true,"origin":"","legend":"\u003cp\u003ePersistence of CAR-T killing ability following repeated antigen rechallenges \u003cstrong\u003eA.\u003c/strong\u003e Representative histograms of GFP signal from flow cytometry of the killing capacity of CT103a and nanoCAR after four consecutive rechallenges every 2 days with MM1.S. \u003cstrong\u003eB. \u003c/strong\u003eEvaluation by flow cytometry of killing capacity (GFP\u003csup\u003e+\u003c/sup\u003e signal) of CT103a and nanoCAR by repeatedly rechallenging them four times, every 2 days, with various BCMA\u003csup\u003e+\u003c/sup\u003e MM cell lines (MM1.S, RPMI-8226 and LP-1) or a BCMA\u003csup\u003e- \u003c/sup\u003eleucemia cell line (K562). Results were shown as the mean value and SD of two replicates for 2 different donors. \u003cstrong\u003eC.\u003c/strong\u003e Evaluation by flow cytometry (CD3\u003csup\u003e+\u003c/sup\u003e signal) of proliferation of CT103a and nanoCAR following four repeated challenges every 2 days with BCMA\u003csup\u003e+\u003c/sup\u003e MM cell lines (MM1.S, RPMI-8226 and LP-1) or K562. Results were shown as the mean value and SD of two replicates for 2 different donors.\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-7693534/v1/136b7873162d4a46dfefdd85.png"},{"id":93247265,"identity":"cdb592cb-43af-4fcb-a220-207ae5a945e6","added_by":"auto","created_at":"2025-10-10 15:23:28","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":190175,"visible":true,"origin":"","legend":"\u003cp\u003eTypical CD4/CD8 distribution in CAR-T cell preparations. \u003cstrong\u003eA\u003c/strong\u003e. Evaluation of CD4 and CD8 levels after 24 hours of co-culture with MM1.S, RPMI-8226, LP-1 and K562. The mean value and SD from 3 biological replicates of 3 different donors were shown (n = 3 for each donor).\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-7693534/v1/264e66d3f87b452c35f13734.png"},{"id":93247263,"identity":"bb6b3c35-67d3-4208-813f-1faae589a238","added_by":"auto","created_at":"2025-10-10 15:23:28","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":695631,"visible":true,"origin":"","legend":"\u003cp\u003eCAR-T cell differentiation into central or effector memory subsets with enhanced CD107a surface expression in vitro \u003cstrong\u003eA.\u003c/strong\u003e Flow cytometry gating strategy to evaluate the frequency of central and effector memory CAR-T cells during co-culture with MM1.S compared to non-activated PBMC-derived T cells. \u003cstrong\u003eB.\u003c/strong\u003e Evaluation by flow cytometry of memory phenotype (CD27\u003csup\u003e+\u003c/sup\u003e CD45RA\u003csup\u003e-\u003c/sup\u003e) after 24h to 48h of co-culture with MM1.S. (left graph), followed by the analysis of central (CD62L\u003csup\u003e+\u003c/sup\u003e) or effector (CD62L\u003csup\u003e-\u003c/sup\u003e) rates after 24h (dark or light blue) to 48h (dark or ligth red) of co-culture of non-activated T cells, Mock T cells, CT103a, and nanoCAR.\u0026nbsp; \u003cstrong\u003eC\u003c/strong\u003e. Evaluation by flow cytometry of the degranulation marker CD107a after 6 hours of co-culture with MM1S, RPMI-8226, LP-1 and K562.\u003c/p\u003e","description":"","filename":"floatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-7693534/v1/33b5ecdd17ae5104682208d5.png"},{"id":93246608,"identity":"eb535c4f-1490-4819-9b01-fad27cf0a069","added_by":"auto","created_at":"2025-10-10 15:15:28","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":227535,"visible":true,"origin":"","legend":"\u003cp\u003eCT103a and nanoCAR produce cytokines when co-cultured with MM cell lines \u003cstrong\u003eA.\u003c/strong\u003e Measurements of cytokine production (IL-2, IFNγ, TNFα) after 24 hours of co-culture with MM1.S or K562 cells. Results expressed as mean value and SD from five independent experiments, utilizing a total of seven different donors in triplicate, (n = 3 for each of the seven donors). **: p \u0026lt; 0.01. ***: p \u0026lt; 0.001. ****: p \u0026lt; 0.0001, determined by Mann-Whitney test.\u003c/p\u003e","description":"","filename":"floatimage6.png","url":"https://assets-eu.researchsquare.com/files/rs-7693534/v1/93e41c285650532aab842cce.png"},{"id":93244526,"identity":"5a5d9cd0-f390-4d91-9b96-7bbdeacf9304","added_by":"auto","created_at":"2025-10-10 15:07:28","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":653866,"visible":true,"origin":"","legend":"\u003cp\u003eCT103a and nanoCAR express similar set of genes and pathways (bulk RNA seq) when co-cultured with MM cells \u003cstrong\u003eA.\u003c/strong\u003e List the top 50 overexpressed genes, based on adjusted p-value, of CT103a and nanoCAR compared to activated Mock T cells for three different donors using heatmaps. \u003cstrong\u003eB.\u003c/strong\u003eList of the significant upregulated and downregulated genes of 0h, 4h and 16h post-co-culture with MM1.S using Volcano plots. These plots compared CT103a with Mock used as a negative control, nanoCAR with Mock used as a negative control, and nanoCAR with CT103a used as a reference. \u003cstrong\u003eC.\u003c/strong\u003e Evolution of the number of upregulated or downregulated genes between CT103a and nanoCAR after 0h, 4h, 16h of co-ulture using Venn diagramms.\u003c/p\u003e","description":"","filename":"floatimage7.png","url":"https://assets-eu.researchsquare.com/files/rs-7693534/v1/67c5cf9bad8173e5c9066a6b.png"},{"id":93244528,"identity":"fed73fde-fc80-4ba9-9fb9-187679e14a29","added_by":"auto","created_at":"2025-10-10 15:07:28","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":1130708,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFig 9\u003c/strong\u003e CT103a and nanoCAR-T cells display anti-MM activity in vivo in a murine model. \u003cstrong\u003eA.\u003c/strong\u003e Representative regions of interest (ROI) from bioluminescence imaging (Xenogen IVIS 200) illustrating tumor progression over time. \u003cstrong\u003eB.\u003c/strong\u003e Representative bioluminescent images of mice from each treatment group (MM, Mock, CT103a, and nanoCAR) at different timepoints.\u003cstrong\u003e C. \u003c/strong\u003eFlow cytometry analysis of immune reconstitution in peripheral blood and BM of mice from the second cohort by analysing frequency of human CD45\u003csup\u003e+\u003c/sup\u003e (hCD45) cells and V5\u003csup\u003e+\u003c/sup\u003e (CAR-T\u003csup\u003e+\u003c/sup\u003e) cells among hCD45\u003csup\u003e+\u003c/sup\u003epopulations.\u003cstrong\u003e D. \u003c/strong\u003eKaplan-Meier survival curves of treated mice showing cause-specific mortality: MM-related mortality (left) and non-MM related mortality (right). \u003cstrong\u003eE.\u003c/strong\u003e Flow cytometry analysis of immune reconstitution in peripheral blood and BM of CD4-CD8 subsets, and TCM or ECM phenotypes. \u0026nbsp;Left: distribution of CD4\u003csup\u003e+\u003c/sup\u003e and CD8\u003csup\u003e+\u003c/sup\u003e T cells. Right: frequency of CD62L\u003csup\u003e+\u003c/sup\u003e cells among CD27\u003csup\u003e+\u003c/sup\u003eCD45\u003csup\u003e-\u003c/sup\u003e memory T cells. Each dot represents one mouse, bars indicate median values.\u003c/p\u003e","description":"","filename":"floatimage8.png","url":"https://assets-eu.researchsquare.com/files/rs-7693534/v1/7beb9a2fd087e68a522efb22.png"},{"id":93440112,"identity":"0bb396d9-c33e-4ae9-8009-96daf5976960","added_by":"auto","created_at":"2025-10-13 22:32:24","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":5999901,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7693534/v1/6f1e552a-dd8d-49c8-9202-cd91738a4850.pdf"},{"id":93246598,"identity":"8b794d15-7dc8-4e5e-a30c-0ccd8a4101b4","added_by":"auto","created_at":"2025-10-10 15:15:28","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":1237899,"visible":true,"origin":"","legend":"","description":"","filename":"ResearchMeganeJassinSupplementaryData.docx","url":"https://assets-eu.researchsquare.com/files/rs-7693534/v1/a0e19fe5d5d6a09c771208d2.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Design of a novel NanoCAR construct that compares well with a conventional ScFv-based CAR targeting BCMA on multiple myeloma","fulltext":[{"header":"Introduction","content":"\u003cp\u003eMultiple myeloma (MM) is a hematologic malignancy characterized by the proliferation of clonal plasma cells in the bone marrow (BM). Although still incurable, survival has improved with proteasome inhibitors, immunomodulatory drugs, and, more recently, immunotherapeutic approaches [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. B-cell maturation antigen (BCMA), highly expressed on the surface of MM cells [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e], is a major therapeutic target for bispecific T-cell engagers and chimeric antigen receptor (CAR) T-cells [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. CAR-T cells are genetically modified T cells that express chimeric receptors capable of recognizing tumor-associated antigens independently of MHC presentation [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. In 2017, the US Food and Drug Administration (FDA) approved the first CAR-T therapy for B-cell malignancies [\u003cspan additionalcitationids=\"CR6\" citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. The first anti-BCMA CAR-T, idecabtagene vicleucel, was approved in March 2021 [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e], followed in 2022 by CT103a, a fully human CAR containing a single-chain variable fragment (ScFv) from a phage display library for MM treatment [\u003cspan additionalcitationids=\"CR10 CR11\" citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]\u0026ndash;[\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. The phase 1/2b clinical studies showed CT103a demonstrated a favorable safety profile with no new risk observed with longer follow-up in patients [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eCAR-T cells can be generated through retroviral or lentiviral transduction, CRISPR-Cas9 gene editing, lipid nanoparticles, or non-viral transposon-mediated methods. Standard CAR-T cell manufacturing involves T-cell activation, CAR transduction, and in vitro expansion prior to infusion, typically using anti-CD3/CD28 stimulation and cytokines such as IL-2 or IL-7 and IL-15 [\u003cspan additionalcitationids=\"CR15\" citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]\u0026ndash;[\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eCamelid-derived variable heavy chain domains (V\u003csub\u003eH\u003c/sub\u003eH or nanobodies) are antibody fragment consisting of a monomeric variable domain (sdAb) offering high specificity, small size, robust structure, and enhanced stability in comparison to conventional antibodies. Their unique properties support therapeutic and diagnostic applications [\u003cspan additionalcitationids=\"CR18\" citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]\u0026ndash;[\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. In 2022, the FDA approved nanoCAR-T therapy, Ciltacabtagene autoleucel (Cilta-cel), whose antigen-binding domain consists of two V\u003csub\u003eH\u003c/sub\u003eHs capable of binding two distinct BCMA epitopes [\u003cspan additionalcitationids=\"CR21\" citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]\u0026ndash;[\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eThis study aims to design and evaluate the activity of a novel anti-BCMA-nanoCAR-T that compares well with a standard one CT103a. This study seeks to provide insight that may inform the optimization of next-generation CAR-T therapies for MM.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003eCell lines and primary T cells\u003c/h2\u003e\u003cp\u003eMM (LP-1, RPMI-8226, MM1.S, OPM-2, MOLP-2, KMS-12-BM) and leukemia (K562) cells were cultured in Roswell Park Memorial Institute (RPMI 1640) (Lonza, Verviers, Belgium) supplemented with 10% fetal bovine serum (FBS; Sigma-Aldrich, St-Louis, MO, USA), 2 mM L-glutamine (Lonza, Verviers, Belgium) and 100 U/mL penicillin-streptomycin (P/S; Lonza, Verviers, Belgium). Primary T cells were cultured in CTS\u0026trade; OpTmizer\u0026trade; T Cell expansion SFM with supplement (Thermo Fisher Scientific, Waltham, MA, USA), 2 mM L-glutamine (Lonza, Verviers, Belgium) and 100 U/mL penicillin-streptomycin (P/S; Lonza, Verviers, Belgium) and 5 ng/mL of IL-2 (Peprotech, Neuilly-sur-Seine, France). All cells were cultured at 37\u0026deg;C in 5% CO\u003csub\u003e2\u003c/sub\u003e.\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003ePBMCs and T Cells Isolation\u003c/h3\u003e\n\u003cp\u003eHuman peripheral blood mononuclear cells (PBMCs) were obtained from buffy coats (Croix-Rouge de Belgique, Li\u0026egrave;ge, Belgium) from healthy adult volunteers with informed consent. PBMCs were isolated by Ficoll-Paque density centrifugation (GE Healthcare, Freiburg, Germany) and T cells were purified using the EasySep Human T Cell Isolation Kit (Stemcell Technologies, Vancouver, Canada) according to the manufacturer\u0026rsquo;s protocol.\u003c/p\u003e\n\u003ch3\u003eLentivral Vector Production and Cell transduction\u003c/h3\u003e\n\u003cp\u003eDNA sequences for ScFv and V\u003csub\u003eH\u003c/sub\u003eH antigen-binders were obtained from publicly available sources [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. Lenti-X 293T cells were co-transfected with lentiviral gene transfer plasmids (ScFv-CT103a-hPGK-puro or V\u003csub\u003eH\u003c/sub\u003eH-nanoCARNb17-hPGK-puro) designed with VectorBuilder (China) and packaging plasmids (psPAX2 and a VSV-G-encoding plasmid). Lentiviral supernatants were collected 48h-96h post transfection, filtered, concentrated, titrated with qPCR Lentivirus Titration (Titer) Kit (ABM\u0026reg;, LV900, Richmond, BC, Canada), and used to transduce T cells to generate CAR-T cells using a multiplicity of infection (MOI) of 10. Transduction efficiency was analyzed by flow cytometry using a V5-AlexaFluor647 antibody (Thermo Fisher Scientific, Waltham, MA, USA). Cancer cell lines were similarly transduced with GFP or GFP/luciferase vectors (CMV-GFP-hPGK-blasti or CMV-GFP-luciferase-hPGK-blasti), and GFP\u003csup\u003e+\u003c/sup\u003e cells were sorted using the Sony MA900 (Sony Biotechnology, San Jose, California, USA). Sequences of CT103a and Nb17 are listed in Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e.\u003c/p\u003e\n\u003ch3\u003eFlow Cytometry staining and analysis\u003c/h3\u003e\n\u003cp\u003eCells were incubated for 60 min at 4\u0026deg;C with titrated antibody concentrations before analysis on a FACS Canto\u003csup\u003eTM\u003c/sup\u003eII (BD Biosciences, San Jose, CA, USA), a CytoFLEX (Beckman Coulter, Brea, California, USA), or an LSRFortessa\u0026trade; (BD Biosciences, San Jose, CA, USA). The following antibodies were used: anti-human CD3-BV605 (UCHT1, BD Biosciences, CA, USA), anti-human CD45-BV711 (HI30, BD Biosciences, CA, USA), anti-mouse CD45-Pe-Cy5.5 (30-F11, BD Biosciences, CA, USA), anti-human CD4-PerCP-Cy5.5 (RPA-R4, Sony Biotechnology, San Jose, CA, USA), anti-human CD8-PE-Cy7 (HIT8a, Biolegend, San Diego, CA, USA), anti-human CD45RA-BV510 (HI100, BD Biosciences, CA, USA), anti-human CD62L-AlexaFluor700 (DREG-56, Biolegend, San Diego, CA, USA), anti-human CD27-PE (M-T271, BD Biosciences, San Jose, CA, USA), anti-human CD107a (H4A3, Biolegend, San Diego, CA, USA). To discriminate live from dead cells, samples were then incubated 10 min at 4\u0026deg;C with a Fixable Viability Dye eFluor\u0026trade; 780 (1:10,000 dilution; Invitrogen, Thermo Fisher Scientific). For the detection of V\u003csub\u003eH\u003c/sub\u003eH Nb17 binding, cells were incubated with an anti-V\u003csub\u003eH\u003c/sub\u003eH antibody (GenScript Biotech, Piscataway, New Jersey, USA) followed by an anti-rabbit-AlexaFluor647 secondary antibody (Biolegend, San Diego, CA, USA).\u003c/p\u003e\u003cp\u003eAll analysis were performed using FlowJo\u0026trade; software V10.1 (BD Biosciences, Ashland, OR, USA).\u003c/p\u003e\n\u003ch3\u003eQuantification of BCMA or CAR cell surface expressions and binding capacity of VH Nb17\u003c/h3\u003e\n\u003cp\u003eMM cell lines were labelled with a PE-conjugated anti-BCMA antibody, while primary T and CAR-T cells were labelled with PE-conjugated BCMA soluble protein (ACROBiosystems, Basel, Switzerland). BCMA or CAR expression levels were quantified with the BD Quantibrite\u0026trade; Beads (BD Biosciences, San Jose, CA, USA) according to the manufacturer\u0026rsquo;s instructions. This kit contains beads conjugated at four levels of PE intensity, each corresponding to a defined number of PE molecules per bead. Based on the generated standard curve, the number of PE molecules per cell was calcutated after labelling with the PE-labelled antibody or soluble protein.\u003c/p\u003e\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003eCellular Cytotoxicity Assays and CAR-T phenotyping and characterization: Flow Cytometry\u003c/h2\u003e\u003cp\u003eCAR-T-dependent tumor cell cytotoxicity was quantified by flow cytometry. GFP transduced target cells were mixed in co-culture with effector CAR-T cells at different effector-to-target ratios (1:3, 1:1, 3:1) in 96-well plates in RPMI 1640 supplemented with 10% FBS, 2 mM L-glutamine and 100 U/mL penicillin-streptomycin without IL-2 at 37\u0026deg;C in a humidified atmosphere with 5% CO\u003csub\u003e2\u003c/sub\u003e. After 24h to 48h, target cell viability was assessed by counting live cells using Fixable Viability Dye eFluor\u0026trade; 780. CAR-T were phenotyped and characterized by antibody labeling as described above.\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eCellular Cytotoxicity Assays and cytokine productions: ELISA\u003c/h3\u003e\n\u003cp\u003eCAR-T-dependent tumor cell cytokine production was quantified by ELISA. GFP transduced target cells were mixed in co-culture with effector CAR-T cells at a ratio effector-to-target 1:1 in 96-well plates in RPMI 1640 supplemented with 10% FBS, 2 mM L-glutamine and 100 U/mL penicillin-streptomycin without IL-2 at 37\u0026deg;C in a humidified atmosphere with 5% CO\u003csub\u003e2\u003c/sub\u003e. After 24h, cells were centrifuged and supernatents were collected to analyse the IL-2, IFNγ, TNFα productions by ELISA Max\u0026trade; Deluxe Set from Biolegend (San Diego, CA, USA) according to the manufacturer\u0026rsquo;s protocol. Absorbances were read using the Plate Reader Tristar\u003csup\u003e2\u003c/sup\u003e S LB942 number 2076 from Berthold Technologies (Bad Wildbad, Germany).\u003c/p\u003e\n\u003ch3\u003eBulk-RNA sequencing\u003c/h3\u003e\n\u003cp\u003eRNA-seq samples were collected at three key co-culture timepoints: 0, 4, and 16h of co-culture of CAR-T or untransduced T cells with MM1.S. After these timepoints, CD3\u003csup\u003e+\u003c/sup\u003eCAR\u003csup\u003e+\u003c/sup\u003e (CAR-T) and CD3\u003csup\u003e+\u003c/sup\u003eCAR\u003csup\u003e\u0026minus;\u003c/sup\u003e (T cells) were sorted by fluorescence-activated cell sorting (Sony MA900 flow cell sorter) using CD3-BV605 (UCHT1, BD Biosciences, CA, USA) and/or V5-AlexaFluor647 (Thermo Fisher Scientific, Waltham, MA, USA) antibodies. Bulk-RNA samples were prepared from sorted cells using the kit RNeasy\u0026reg; Mini Kit (Qiagen, Hilden, Germany) according to the manufacturer\u0026rsquo;s protocol. Samples were purified after treatment with DNAse I (Qiagen, Hilden, Germany) and further purified using Zymo columns (Zymo Research, California, USA). Libraries were sequenced on the Illumina NovaSeg 6000. Raw sequencing reads were quality-controlled with \u003cem\u003eFastQC\u003c/em\u003e (v0.12.1) to remove adapters and low-quality bases. Cleaned reads were pseudo-aligned to the human reference genome (GRCh38.99) using \u003cem\u003ekallisto\u003c/em\u003e (v0.51.1). Gene-level counts were generated with R package \u003cem\u003etximport\u003c/em\u003e (v1.34.0 based on Ensembl gene annotations). Downstream analyses were performed in RStudio (R v4.4.2). Normalization and differential expression analyses were conducted using the \u003cem\u003eDESeq2\u003c/em\u003e package (v1.46.0). Genes with adjusted p-value (Benjamini\u0026ndash;Hochberg FDR)\u0026thinsp;\u0026lt;\u0026thinsp;0.05 and the absolute value of the fold-change\u0026thinsp;\u0026gt;\u0026thinsp;1 were considered significantly differentially expressed. Gene set enrichment analysis (GSEA) was performed with \u003cem\u003efgsea\u003c/em\u003e package (v1.32.0) using Hallmark gene sets. Visualization of results was performed with \u003cem\u003eggplot2\u003c/em\u003e (v3.5.1).\u003c/p\u003e\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u003ch2\u003eIn vivo NSG mouse model\u003c/h2\u003e\u003cp\u003eEthical approval for the in vivo part of the study was obtained from the institutional animal ethical board (Commission d\u0026rsquo;Ethique Animale Universitaire de Li\u0026egrave;ge). NSG mice received 2-G -irradiation on day \u0026minus;\u0026thinsp;1, followed by tail vein injection of 1x10\u003csup\u003e6\u003c/sup\u003e MM1.S-GFP/luc on day 0. Tumor growth was followed using bioluminescence imaging with the Xenogen IVIS 200 with 3 mg/mice luciferin injection every 7 to 10 days. Sixteen to 19 days after tumor injection, when tumor signal was detectable, 2x10\u003csup\u003e6\u003c/sup\u003e CAR-T were injected intravenously in the tails. Mice were monitored for tumor growth and survival for up to 90 days. Additionally, blood samples were collected via tail vein every 20 days after CAR-T injections to analyze CAR-T phenotype and trafficking using flow cytometry. Two independent cohorts were conducted. In cohort 1 (n\u0026thinsp;=\u0026thinsp;15), mice were distributed into four groups, MM only (n\u0026thinsp;=\u0026thinsp;3), MM\u0026thinsp;+\u0026thinsp;Mock T cells (n\u0026thinsp;=\u0026thinsp;4), MM\u0026thinsp;+\u0026thinsp;CT103a (n\u0026thinsp;=\u0026thinsp;4), and MM\u0026thinsp;+\u0026thinsp;nanoCAR-T cells (n\u0026thinsp;=\u0026thinsp;4). In cohort 2 (n\u0026thinsp;=\u0026thinsp;12 mice), mice were distributed into three groups, MM only (n\u0026thinsp;=\u0026thinsp;2), MM\u0026thinsp;+\u0026thinsp;CT103a (n\u0026thinsp;=\u0026thinsp;5), and MM\u0026thinsp;+\u0026thinsp;nanoCAR-T cells (n\u0026thinsp;=\u0026thinsp;5). In each cohort, all T-cell products were derived from a single healthy donor.\u003c/p\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\u003ch2\u003eStatistical analysis\u003c/h2\u003e\u003cp\u003eGraphPad Prism 8.0.1. (GraphPad Software, Inc., USA) was used to perform statistical analysis and graph plotting. Comparisons between two datasets were conducting using paired or unpaired Student\u0026rsquo;s t-tests, with parametric or non-parametric versions applied based on the distribution of the data thank to the Shapiro-Wilk normality test (e.g., Gaussian, non-Gaussian). Statistical significance was defined as p\u0026thinsp;\u0026lt;\u0026thinsp;0.05 (*) and defined as follows: p\u0026thinsp;\u0026lt;\u0026thinsp;0.01 (**), p\u0026thinsp;\u0026lt;\u0026thinsp;0.001 (***), p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001 (****). p-values above 0.05 were considered non-significant (ns).\u003c/p\u003e\u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\u003ch2\u003eConstruction of a nanoCAR sequence containing V\u003csub\u003eH\u003c/sub\u003eH Nb17\u003c/h2\u003e\u003cp\u003eTo evaluate the performance of a V\u003csub\u003eH\u003c/sub\u003eH-based CAR relative to the scFv-based CT103a, we designed a nanoCAR with an analogous backbone. Both constructs were second-generation CARs containing a CD8 hinge, a transmembrane domain, and a 4-1BB co-stimulatory region. A V5 tag was added for flow cytometry detection. The nanoCAR sequence was composed of SFFVprom-V\u003csub\u003eH\u003c/sub\u003eH-V5tag-CD8α-(4-1BB)-CD3ζ, while CT103a sequence was SFFVprom-V5tag-VH-linker-VL-CD8α-(4-1BB)-CD3ζ (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). Both CAR-T cells were generated through lentiviral transduction at the same MOI (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). Although nanoCAR transduction efficiency was slightly lower than CT103a, resulting transduced T cells exhibited equal stable CAR surface expression (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC). To incorporate the V\u003csub\u003eH\u003c/sub\u003eH Nb17 in our nanoCAR sequence, we assessed its specific binding capacity to cell-surface BCMA. BCMA expression was quantified by flow cytometry across various human MM cell lines before incubating them with Nb17. BCMA expression was highest in MOLP-2 (2772 BCMA/cell), followed by RPMI-8226 (2034 BCMA/cell), MM1.S (1942 BCMA/cell), KMS-12-BM (1834 BCMA/cell) and LP-1 (1375 BCMA/cell). OPM-2 showed the lowest expression (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD). Despite variability in BCMA expression, Nb17 demonstrated specific binding to BCMA (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE). Thus, we successfully designed a persistent and stable nanoCAR capable of specifically targetting cell-surface BCMA.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e\u003ch2\u003eIn vitro efficacy of CT103a and nanoCAR in killing BCMA\u003csup\u003e+\u003c/sup\u003e MM cell lines\u003c/h2\u003e\u003cp\u003eWe compared the cytotoxic activity of nanoCAR, CT103a and untransduced (Mock) T cells against BCMA\u003csup\u003e+\u003c/sup\u003e MM cells. Each effector population was co-cultured with MM1.S-GFP\u003csup\u003e+\u003c/sup\u003e for 24\u0026ndash;48 hours, and tumor killing was assessed by quantifying the GFP signal using flow cytometry at various effector-to-target (E:T) ratios of 1:3, 1:1 and 3:1. Both CAR-T cells significantly reduced the number of GFP\u003csup\u003e+\u003c/sup\u003e tumor cells compared with Mock T cells, with enhanced cytotoxicity observed at 48 hours (Figs.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA, \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB, Supplementary Data Fig I). To assess antigen specificity, CAR-T and Mock T cells were co-cultured at a 1:1 ratio with GFP\u003csup\u003e+\u003c/sup\u003e-BCMA\u003csup\u003e+\u003c/sup\u003e MM cell lines (MM1.S, RPMI-8226 and LP-1) or with a GFP\u003csup\u003e+\u003c/sup\u003e-BCMA\u003csup\u003e\u0026minus;\u003c/sup\u003e K562 24\u0026ndash;48 hours. Both CT103a and nanoCAR mediated robust cytotoxicity against BCMA\u003csup\u003e+\u003c/sup\u003e targets, particularly after 48 hours (Figs.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC-E). K562 viability slightly decreased in co-culture, likely reflecting basal IFNγ production by T/CAR-T cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e). Together, these results confirm the therapeutic potential of CT103a and nanoCAR in selectively targeting BCMA-expressing malignancies.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e\u003ch2\u003ePersistence of CAR-T killing ability following repeated antigen challenges\u003c/h2\u003e\u003cp\u003eTo assess functional persistence, we evaluated the long-term activity of CT103a and nanoCAR through repeated tumor challenges. Mock or CAR-T cells were co-cultured with GFP\u003csup\u003e+\u003c/sup\u003e tumor cell lines (MM1.S, RPMI-8226, LP-1 and K562) and rechallenged thrice with intervals of 48 hours. Tumor killing was quantified by flow cytometry, while CAR-T proliferation was determined by live T-cell counts. Both CAR-Ts eradicated BCMA\u003csup\u003e+\u003c/sup\u003e cell lines after each rechallenge, whereas Mock T cells failed to eliminate tumors, and untreated cancer cells proliferated (Figs.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA, \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). In contrast, BCMA\u003csup\u003e\u0026minus;\u003c/sup\u003e K562 showed no significant cytotoxicity when co-cultured with either Mock or CAR-T cells.\u003c/p\u003e\u003cp\u003eRegarding expansion, both CT103a and nanoCAR proliferated robustly when repeatedly exposed to BCMA\u003csup\u003e+\u003c/sup\u003e targets, but not with K562 cells or in tumor-free conditions. Mock T cells exhibited minimal proliferation, under all conditions (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC). We conclude that both CT103a and nanoCAR T cells exhibit long-term efficacy, suggesting their potential for long-term tumor control.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec17\" class=\"Section2\"\u003e\u003ch2\u003eCAR-T cell differentiation into CD4-CD8 and central memory subsets with enhanced CD107a surface expression\u003c/h2\u003e\u003cp\u003eAfter 24 hours of co-culture with MM1.S, RPMI-8226, LP-1 and K562, CD4 and CD8 levels were assessed. Both subsets remained stable across groups, with CD4 representing\u0026thinsp;~\u0026thinsp;60% and CD8\u0026thinsp;~\u0026thinsp;20\u0026ndash;30% of the T-cell population, consistent with reported distributions [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e].\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eTo assess differentiation, CT103a and nanoCAR-T cells were co-cultured with MM1.S for 24\u0026ndash;48 hours, and expression of CD27, CD45RA and CD62L was analyzed using flow cytometry. Compared to non-activated PBMC-derived T cells, Mock, CT103a and nanoCAR presented increased frequency of CD27\u003csup\u003e+\u003c/sup\u003e-CD45RA\u003csup\u003e\u0026minus;\u003c/sup\u003e cells, indicating a memory T cell phenotype. Within this population, CD62L distinguished central memory (TCM, CD62L\u003csup\u003e+\u003c/sup\u003e) from effector memory (TEM, CD62L\u003csup\u003e\u0026minus;\u003c/sup\u003e) subsets (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). Upon antigen exposure, CAR-T cells displayed dynamic shifts: at 24 hours, TEM increased, reflecting active cytotoxicity, while at 48 hours, subsets rebalanced between TCM and TEM, indicating restoration of memory phenotype post-target elimination. Mock T cells without antigen largely retained a TCM phenotype, underscoring the role of antigen engagement in effector differentiation (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB). Degranulation was assessed via CD107a expression 6 hours after co-culture with MM1.S, RPMI-8226, LP-1, and K562 (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC) by flow cytometry. Both CT103a and nanoCAR showed higher CD107a levels than Mock T cells, including with K562, likely reflecting basal activation. Overall, CD107a expression confirms the robust potential of both CAR constructs and their functional readiness to engage and kill target cells, regardless of BCMA specificity or antigen-independent activation.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec18\" class=\"Section2\"\u003e\u003ch2\u003eCT103a and nanoCAR produce cytokines when co-cultured with MM cell lines\u003c/h2\u003e\u003cp\u003eCytokine secretion is a key aspect of CAR-T functionality. After 24 hours of co-culture with BCMA\u003csup\u003e+\u003c/sup\u003e MM1.S, RPMI-8226, or LP-1, both CT103a and nanoCAR-T cells produced high levels of IL-2, IFNγ and TNFα (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA, Supplementary Data, Fig II). In contrast, co-culture with BCMA\u003csup\u003e\u0026minus;\u003c/sup\u003e K562 cells induced minimal IFNγ and no IL-2 or TNFα from either CAR or Mock T cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA). These results demonstrate antigen-specific activation and confirm the functional responsiveness of both CAR constructs to BCMA\u003csup\u003e+\u003c/sup\u003e targets.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec19\" class=\"Section2\"\u003e\u003ch2\u003eGene expression studies of CT103a and nanoCAR co-cultured with MM cells\u003c/h2\u003e\u003cp\u003eTo gain deeper insights into CAR-T activity, we performed bulk RNA sequencing and compared the transcriptional profiles across the different conditions (CT103a, nanoCAR, Mock) after incubation with MM1.S (Supplementary Data, Fig III). Given that critical biological events occur rapidly after the encounter between CAR-T cells and target cells, we selected early time points (0, 4, and 16h post-incubation) for this analysis. We used Mock T cells as negative control and CT103a as positive control.\u003c/p\u003e\u003cp\u003eTo explore the transcriptional responses of CT103a and nanoCAR, we first visualized the top 50 overexpressed genes of both CAR-Ts compared to Mock at 16h using heatmaps (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eA). Both CAR-T constructs demonstrated upregulation of key genes associated with T cell activation and proliferation (e.g. \u003cb\u003eIL17REL\u003c/b\u003e, \u003cb\u003eTNFRS9\u003c/b\u003e (\u003cb\u003e4-1BB\u003c/b\u003e)) or effector functions (e.g. \u003cb\u003eIL5\u003c/b\u003e, \u003cb\u003eIL2\u003c/b\u003e, \u003cb\u003eIFNG\u003c/b\u003e). Complementing this analysis, volcano plots revealed minimal differences between CT103a and nanoCAR across all time points (0h, 4h, 16h). In contrast, both CAR-T cell types exhibited a significant increase in the number of upregulated and downregulated genes at 4h and 16h compared to Mock T cells, consistent with CAR-specific activation (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eB). Venn diagrams further illustrated that most of upregulated and downregulated genes at 4h and 16h were shared between CT103a and nanoCAR, underscoring their comparable transcriptional programs (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eC).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eGene set enrichment analysis (GSEA) provided further insights into CAR-T cell activation. Hallmark (Supplementary Data, Fig IV A)) and KEGG (Supplementary Data, Fig IV B)) pathway analyses revealed significant enrichment of pathways related to cytokine production, cytokine-cytokine interactions, and proliferation in CT103a and nanoCAR compared to Mock at 4h. This enrichment reflected CAR-specific functionality. However, these pathways were no longer differentially expressed by 16h, likely due to basal cytokine production by pre-activated Mock T cells masking CAR-specific effects. These findings suggested that cytokine-related signaling at early time points predominantly arises from CAR-specific mechanisms, while the distinction diminished over time due to baseline activation in Mock cells. Overall, while CT103a and nanoCAR demonstrated similar transcriptional profiles, their distinct gene signatures relative to Mock revealed shared mechanisms of antigen-specific activation.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec20\" class=\"Section2\"\u003e\u003ch2\u003eCT103a and nanoCAR can eliminate tumor cells in vivo\u003c/h2\u003e\u003cp\u003eWe examined the anti-tumor efficacy of both CT103a and nanoCAR-T cells in a NSG mouse model using the Xenogen IVIS 200 imaging system and flow cytometry (Supplementary Data Fig IV). Mice were intravenously injected with MM1.S-GFP-Luc and subsequently treated with Mock T cells, CT103a or nanoCAR T cells via intravenous injection 16 to 19 days after tumor inoculation once tumor signals became detectable by bioluminescence. In cohort 1, all mice injected with MM1.S-GFP-Luc cells and left untreated or treated with Mock T cells succumbed to disease within 30\u0026ndash;40 days, confirming the aggressiveness of the model (Figs.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e9\u003c/span\u003eA and \u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e9\u003c/span\u003eB). CT103a-treated mice died by day 36 to 46. In contrast, all four mice treated with nanoCAR-T cells survived beyond day 90, with three out of four mice being completely tumor-free, and one mouse showing residual disease. In cohort 2, both CT103a and nanoCAR-T treatments induced complete tumor regression within 10 days of CAR-T cell infusion (Figs.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e9\u003c/span\u003eA and \u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e9\u003c/span\u003eB). Mice were subsequently monitored for long-term survival. Four out of five nanoCAR-T-treated mice died before day 90 due to ascites, which may have resulted from hepatic and/or cardiac insufficiencies, maybe also due to graft-versus-host disease-like based on visual symptoms and the high amounts of hCD45\u003csup\u003e+\u003c/sup\u003e cells in the peripheral blood and/or in the BM even if low amounts of V5\u003csup\u003e+\u003c/sup\u003e (CAR-T\u003csup\u003e+\u003c/sup\u003e) cells analyzed by flow cytometry the day of their sacrifice were detected (Fig C). Among CT103a-treated mice, one also developed ascites and died prematurely, while another survived until the end of the study but developed signs of hepatic insufficiency such as jaundice. Both mice had high levels of hCD45\u003csup\u003e+\u003c/sup\u003e cells and low amounts of V5\u003csup\u003e+\u003c/sup\u003e (Fig C). Consequently, we split the Kaplan-Meier survival curves in two, one showing the MM-specific mortality and the other one showing the global mortality (Figs.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e9\u003c/span\u003eD). We next evaluated CD4 and CD8 T-cell subsets, as well as the proportion of CD62L\u003csup\u003e+\u003c/sup\u003e cells among CD27\u003csup\u003e+\u003c/sup\u003eCD45RA\u003csup\u003e\u0026minus;\u003c/sup\u003e memory population (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e9\u003c/span\u003eE). We found no significant differences in phenotype between CT103a and nanoCAR-T cells. These findings suggest that while both CT103a and nanoCAR-T cells display potent anti-MM activity, nanoCAR-T cells may additionally induce xenoreactivity against murine tissues.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eOur study provided a detailed characterization of two anti-BCMA CAR-T therapies, CT103a (ScFv-based) and nanoCAR (V\u003csub\u003eH\u003c/sub\u003eH-based), for MM treatment. Despite their structural difference, both CAR constructs demonstrated comparable cytotoxic activity, persistence and transcriptional responses, underscoring shared tumor-specific activation mechanisms. Previous studies compared ScFv-based CAR T and nanoCAR T cells. These showed the smaller and more stable structure of V\u003csub\u003eH\u003c/sub\u003eH can reduce steric hindrance, which is particularly advantageous for bispecific CAR T constructs. These studies also suggested nanoCAR-T cells demonstrated comparable tumor killing efficacy, particularly in the context of bispecific targeting where their compact structure allowed for better antigen engagement and improved therapeutic efficacy [\u003cspan additionalcitationids=\"CR26 CR27 CR28 CR29\" citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]\u0026ndash;[\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eWhile not eradicating BCMA\u003csup\u003e\u0026minus;\u003c/sup\u003e cells, such as the K562 leukemia cell line, both CT103a and nanoCAR specifically targeted BCMA\u003csup\u003e+\u003c/sup\u003e MM cell lines (MM1.S, RPMI-8226 and LP-1). This specific anti-tumor activity was maintained over prolonged periods and following repeated antigen exposures, which supports potential for long-term therapeutic efficacy. Moreover, key cytokines, including IL-2, TFNα and IFNγ, were produced in response to BCMA-positive targets, highlighting the specificity of both CAR-T constructs [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e] [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eT cell subsets are defined by specific surface markers reflecting their differentiation state and functional capacity. Compared to non-activated PBMC-derived T cells, all CAR-T and Mock T cells exhibited a memory phenotype (CD27⁺CD45RA⁻). Moreover, CAR-T cells showed dynamic phenotypic shifts between TCM and TEM. At 24 hours, an increase in TEM frequency was observed, consistent with active cytotoxic engagement. By 48 hours, the distribution shifted back toward TCM, suggesting recovery without target cells maintained a TCM-dominant phenotype over time. These results demonstrate that CT103a and nanoCAR-T cells possess phenotypic plasticity, enabling a transition between effector and memory states in response to antigen, a property associated with long-term persistence and functional resilience. Moreover, their functional readiness was further reinforced by the expression of degranulation markers such as CD107a. These characteristics were essential for effective CAR-T treatments, providing insights into how CAR-T cells maintain robust and extended antitumor activity over time [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eTranscriptional analyses revealed minimal differences between CT103a and nanoCAR during early activation, with both exhibiting upregulated genes linked to T cell activation, proliferation and effector functions. Common pathways, such as cytokine production and cytokine-cytokine interactions, were enriched at 4h but diminished by 16h, possibly reflecting baseline cytokine production in pre-activated T cells.\u003c/p\u003e\u003cp\u003eIn vivo results demonstrate that both CT103a and nanoCAR-T cells exhibit significant anti-MM activity. The long-term survival and minimal residual disease observed in nanoCAR-T treated mice highlight the therapeutic potential of V\u003csub\u003eH\u003c/sub\u003eH-based CAR-T designs, particularly in aggressive MM models. Notably, NanoCAR-T cells displayed prolonged survival in both the BM and peripheral blood. In contrast, the limited peripheral circulation in CT103a-treated mice suggest distinct capacities in persistence and in vivo expansion between the two CAR constructs. While cohort 2 further supports the potent anti-tumor activity of both CAR-T therapies, the occurrence of ascites in several nanoCAR-T-treated mice may suggests possible xenoreactivity against murine tissues, pointing to the importance of carefully assessing potential safety concerns. Importantly, this alloreactivity is likely a consequence of the xenogeneic setting, and nanoCAR-T cells would be expected to be safe in an autologous clinical context. These in vivo results, combined with phenotypic and functional profiling, underscore the potential of VHH-based CAR-T therapies as a promising strategy for the treatment of BCMA\u003csup\u003e+\u003c/sup\u003e MM.\u003c/p\u003e\u003cp\u003eIn conclusion, our results showed that CT103a and nanoCAR are promising CAR-T treatments for BCMA-positive MM. Both constructs demonstrated significant cytotoxic activity, persistence and favorable transcriptional profiles, indicating their potential as effective therapeutic approaches. Notably, compared to traditional scFvs, the integration of nanobodies, such as the V\u003csub\u003eH\u003c/sub\u003eH Nb17, in CAR designs could provide several advantages including smaller size, higher stability and the ability to target distinct epitopes or antigens with reduced steric hindrance. These benefits could improve CAR-T cell targeting accuracy, increasing their overall efficacy, especially in full tumor microenvironments. Future research could focus on optimizing bispecific CAR-T cells, which would target multiple antigens to reduce the possibility of antigen escape and increase treatment durability [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e] [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. Moreover, exploring the integration of nanobodies in combination with immune checkpoint inhibitors or other immune-modulating treatments could enhance CAR-T functionality, overcoming limitations like immune resistance. Finally, the potential for engineering CAR-T cells with a more defined \"memory-like\" phenotype could offer strategies to improve long-term persistence, reducing the need for repeated treatments [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. These next steps would move beyond initial proof-of-concept studies to more clinical and mechanistic understanding, facilitating the translation of these promising CAR-T therapies into the clinic.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eMM\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Multiple myeloma\u003c/p\u003e\n\u003cp\u003eBCMA\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;B-cell maturation antigen\u003c/p\u003e\n\u003cp\u003eBM\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Bone marrow\u003c/p\u003e\n\u003cp\u003eCAR\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Chimeric antigen receptor\u003c/p\u003e\n\u003cp\u003eCilta-cel\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;Ciltacabtagene autoleucel\u003c/p\u003e\n\u003cp\u003eFDA\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;Food and Drug Administration\u003c/p\u003e\n\u003cp\u003eGSEA\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Gene set enrichment analysis\u003c/p\u003e\n\u003cp\u003eIL-2\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Interleukin-2\u003c/p\u003e\n\u003cp\u003eMOI\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;Multiplicity of infection\u003c/p\u003e\n\u003cp\u003eScFv\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Single-chain variable fragment\u003c/p\u003e\n\u003cp\u003eSdAb\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;Single domain antibody\u003c/p\u003e\n\u003cp\u003eSCFV\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Single-chain variable fragment\u003c/p\u003e\n\u003cp\u003eSD\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;Standard deviation\u003c/p\u003e\n\u003cp\u003eTCM\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Central memory T cell\u003c/p\u003e\n\u003cp\u003eTEM\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Effector memory T cell\u003c/p\u003e\n\u003cp\u003eV\u003csub\u003eH\u003c/sub\u003eH \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Heavy chain variable domains\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003cp\u003eFunding: The laboratory of Haematology is supported by the Foundation Against Cancer, the Intergroup Francophone du My\u0026eacute;lome, the Fonds National de la Recherche Scientifique, (FNRS, Belgium), T\u0026eacute;l\u0026eacute;vie-FNRS and the Fonds Sp\u0026eacute;ciaux de la Recherche (University of Li\u0026egrave;ge).\u003c/p\u003e\u003c/p\u003e\u003cp\u003e\u003ch2\u003eCompeting interests\u003c/h2\u003e\u003cp\u003eThis research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.\u003c/p\u003e\u003c/p\u003e\u003cp\u003e\u003ch2\u003eEthical approval\u003c/h2\u003e\u003cp\u003e Ethical approval for the in vivo part of the study was obtained from the institutional animal ethical board (Commission d\u0026rsquo;Ethique Animale Universitaire de Li\u0026egrave;ge).\u003c/p\u003e\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eMJ wrote the manuscript and SO, EDV, FB, CG, GE, TN, JC, provided additional input for the manuscript. All authors read and approved the manuscript.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eJ. Laubach, P. Richardson, and K. Anderson, \u0026ldquo;Multiple myeloma,\u0026rdquo; \u003cem\u003eAnnu. Rev. Med.\u003c/em\u003e, vol. 62, pp. 249\u0026ndash;264, 2011, doi: 10.1146/annurev-med-070209-175325.\u003c/li\u003e\n\u003cli\u003eM. Went \u003cem\u003eet al.\u003c/em\u003e, \u0026ldquo;Deciphering the genetics and mechanisms of predisposition to multiple myeloma,\u0026rdquo; \u003cem\u003eNat. Commun.\u003c/em\u003e, vol. 15, no. 1, 2024, doi: 10.1038/s41467-024-50932-7.\u003c/li\u003e\n\u003cli\u003eK. C. Graf \u003cem\u003eet al.\u003c/em\u003e, \u0026ldquo; \u0026lsquo;Fast but not so furious\u0026rsquo; : A condensed step‐up dosing schedule of teclistamab for relapsed/refractory multiple myeloma ,\u0026rdquo; \u003cem\u003eeJHaem\u003c/em\u003e, vol. 5, no. 4, pp. 793\u0026ndash;797, 2024, doi: 10.1002/jha2.906.\u003c/li\u003e\n\u003cli\u003eD. Pan and J. Richter, \u0026ldquo;Management of Toxicities Associated with BCMA, GPRC5D, and FcRH5-Targeting Bispecific Antibodies in Multiple Myeloma,\u0026rdquo; \u003cem\u003eCurr. Hematol. Malig. Rep.\u003c/em\u003e, 2024, doi: 10.1007/s11899-024-00740-z.\u003c/li\u003e\n\u003cli\u003eS. Guedan, M. Ruella, and C. H. June, \u0026ldquo;Emerging Cellular Therapies for Cancer,\u0026rdquo; \u003cem\u003eAnnu. Rev. Immunol.\u003c/em\u003e, vol. 37, pp. 145\u0026ndash;171, 2019, doi: 10.1146/annurev-immunol-042718-041407.\u003c/li\u003e\n\u003cli\u003eK. Utkarsh \u003cem\u003eet al.\u003c/em\u003e, \u0026ldquo;CAR-T cell therapy: a game-changer in cancer treatment and beyond,\u0026rdquo; \u003cem\u003eClin. Transl. Oncol.\u003c/em\u003e, vol. 26, no. 6, pp. 1300\u0026ndash;1318, 2024, doi: 10.1007/s12094-023-03368-2.\u003c/li\u003e\n\u003cli\u003e\u0026ldquo;First-Ever CAR T-cell Therapy Approved in U.S.,\u0026rdquo; \u003cem\u003eCancer Discov.\u003c/em\u003e, vol. 7, p. 10, 2017, doi: https://doi.org/10.1158/2159-8290.CD-NB2017-126.\u003c/li\u003e\n\u003cli\u003eS. Jagannath \u003cem\u003eet al.\u003c/em\u003e, \u0026ldquo;KarMMa-RW: comparison of idecabtagene vicleucel with real-world outcomes in relapsed and refractory multiple myeloma,\u0026rdquo; \u003cem\u003eBlood Cancer J.\u003c/em\u003e, vol. 11, no. 6, pp. 1\u0026ndash;9, 2021, doi: 10.1038/s41408-021-00507-2.\u003c/li\u003e\n\u003cli\u003eW. Mu \u003cem\u003eet al.\u003c/em\u003e, \u0026ldquo;A Model Perspective Explanation of the Long-Term Sustainability of a Fully Human BCMA-Targeting CAR (CT103A) T-Cell Immunotherapy,\u0026rdquo; \u003cem\u003eFront. Pharmacol.\u003c/em\u003e, vol. 13, no. February, pp. 1\u0026ndash;9, 2022, doi: 10.3389/fphar.2022.803693.\u003c/li\u003e\n\u003cli\u003eD. Li \u003cem\u003eet al.\u003c/em\u003e, \u0026ldquo;Anti-BCMA CAR-T cells therapy for a patient with extremely high membrane BCMA expression: a case report,\u0026rdquo; \u003cem\u003eJ. Immunother. Cancer\u003c/em\u003e, vol. 10, no. 9, pp. 1\u0026ndash;5, 2022, doi: 10.1136/jitc-2022-005403.\u003c/li\u003e\n\u003cli\u003eJ. Xu and J. J. Melenhorst, \u0026ldquo;CT103A, a forward step in multiple myeloma immunotherapies,\u0026rdquo; \u003cem\u003eBlood Sci.\u003c/em\u003e, vol. 3, no. 2, pp. 59\u0026ndash;61, 2021, doi: 10.1097/BS9.0000000000000068.\u003c/li\u003e\n\u003cli\u003eQ. Wang \u003cem\u003eet al.\u003c/em\u003e, \u0026ldquo;An alternative fully human anti-BCMA CAR-T shows response for relapsed or refractory multiple myeloma with anti-BCMA CAR-T exposures previously,\u0026rdquo; \u003cem\u003eCancer Gene Ther.\u003c/em\u003e, vol. 31, no. 3, pp. 420\u0026ndash;426, 2024, doi: 10.1038/s41417-023-00712-0.\u003c/li\u003e\n\u003cli\u003eC. Li \u003cem\u003eet al.\u003c/em\u003e, \u0026ldquo;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).,\u0026rdquo; \u003cem\u003eJ. Clin. Oncol.\u003c/em\u003e, vol. 41, no. 16_suppl, pp. 8025\u0026ndash;8025, 2023, doi: 10.1200/jco.2023.41.16_suppl.8025.\u003c/li\u003e\n\u003cli\u003eM. S. Pampusch \u003cem\u003eet al.\u003c/em\u003e, \u0026ldquo;Rapid Transduction and Expansion of Transduced T Cells with Maintenance of Central Memory Populations,\u0026rdquo; \u003cem\u003eMol. Ther. Methods Clin. Dev.\u003c/em\u003e, vol. 16, no. March, pp. 1\u0026ndash;10, 2020, doi: 10.1016/j.omtm.2019.09.007.\u003c/li\u003e\n\u003cli\u003eH. W. Song \u003cem\u003eet al.\u003c/em\u003e, \u0026ldquo;CAR-T cell expansion platforms yield distinct T cell differentiation states,\u0026rdquo; \u003cem\u003eCytotherapy\u003c/em\u003e, vol. 26, no. 7, pp. 757\u0026ndash;768, 2024, doi: 10.1016/j.jcyt.2024.03.003.\u003c/li\u003e\n\u003cli\u003eL. de Macedo Abdo \u003cem\u003eet al.\u003c/em\u003e, \u0026ldquo;Development of CAR-T cell therapy for B-ALL using a point-of-care approach,\u0026rdquo; \u003cem\u003eOncoimmunology\u003c/em\u003e, vol. 9, no. 1, 2020, doi: 10.1080/2162402X.2020.1752592.\u003c/li\u003e\n\u003cli\u003eS. Muyldermans, \u0026ldquo;Nanobodies: Natural single-domain antibodies,\u0026rdquo; \u003cem\u003eAnnu. Rev. Biochem.\u003c/em\u003e, vol. 82, pp. 775\u0026ndash;797, 2013, doi: 10.1146/annurev-biochem-063011-092449.\u003c/li\u003e\n\u003cli\u003eA. C. M. M. Nairo Brilhante-da-Silva, Rosa Maria de Oliveira Sousa, Andrelisse Arruda, Eliza Lima Dos Santos, \u0026ldquo;Camelid Single-Domain Antibodies for the Development of Potent Diagnosis Platforms,\u0026rdquo; \u003cem\u003eMol. Diagn. Ther.\u003c/em\u003e, vol. 25, no. 4, pp. 439\u0026ndash;456, 2021, doi: 10.1007/s40291-021-00533-7.\u003c/li\u003e\n\u003cli\u003eE. Duray \u003cem\u003eet al.\u003c/em\u003e, \u0026ldquo;A non-internalised CD38-binding radiolabelled single-domain antibody fragment to monitor and treat multiple myeloma,\u0026rdquo; \u003cem\u003eJ. Hematol. Oncol.\u003c/em\u003e, vol. 14, no. 1, pp. 1\u0026ndash;13, 2021, doi: 10.1186/s13045-021-01171-6.\u003c/li\u003e\n\u003cli\u003eE. Chekol Abebe, M. Yibeltal Shiferaw, F. Tadele Admasu, and T. Asmamaw Dejenie, \u0026ldquo;Ciltacabtagene autoleucel: The second anti-BCMA CAR T-cell therapeutic armamentarium of relapsed or refractory multiple myeloma,\u0026rdquo; \u003cem\u003eFront. Immunol.\u003c/em\u003e, vol. 13, no. September, pp. 1\u0026ndash;15, 2022, doi: 10.3389/fimmu.2022.991092.\u003c/li\u003e\n\u003cli\u003eR. Smith, \u0026ldquo;Bringing cell therapy to tumors: Considerations for optimal CAR binder design,\u0026rdquo; \u003cem\u003eAntib. Ther.\u003c/em\u003e, vol. 6, no. 4, pp. 225\u0026ndash;239, 2023, doi: 10.1093/abt/tbad019.\u003c/li\u003e\n\u003cli\u003eH. Hanssens \u003cem\u003eet al.\u003c/em\u003e, \u0026ldquo;Anti-Idiotypic VHHs and VHH-CAR-T Cells to Tackle Multiple Myeloma: Different Applications Call for Different Antigen-Binding Moieties,\u0026rdquo; \u003cem\u003eInt. J. Mol. Sci.\u003c/em\u003e, vol. 25, no. 11, 2024, doi: 10.3390/ijms25115634.\u003c/li\u003e\n\u003cli\u003eW. H. Zhao \u003cem\u003eet al.\u003c/em\u003e, \u0026ldquo;A phase 1, open-label study of LCAR-B38M, a chimeric antigen receptor T cell therapy directed against B cell maturation antigen, in patients with relapsed or refractory multiple myeloma,\u0026rdquo; \u003cem\u003eJ. Hematol. Oncol.\u003c/em\u003e, vol. 11, no. 1, pp. 1\u0026ndash;8, 2018, doi: 10.1186/s13045-018-0681-6.\u003c/li\u003e\n\u003cli\u003eM. Dong Hoon Lee, B.S., Francisco Cervantes-Contreras, Sang Yun Lee, PhD, Damian J. Green, MD, Brian G. Till, \u0026ldquo;Improved Expansion and Function of CAR T Cell Products from Cultures Initiated at Defined CD4:CD8 Ratios,\u0026rdquo; \u003cem\u003eBlood\u003c/em\u003e, vol. 132, no. Supplement 1, 2018, doi: https://doi.org/10.1182/blood-2018-99-111576.\u003c/li\u003e\n\u003cli\u003eF. Nasiri, P. Safarzadeh Kozani, and F. Rahbarizadeh, \u0026ldquo;T-cells engineered with a novel VHH-based chimeric antigen receptor against CD19 exhibit comparable tumoricidal efficacy to their FMC63-based counterparts,\u0026rdquo; \u003cem\u003eFront. Immunol.\u003c/em\u003e, vol. 14, no. February, pp. 1\u0026ndash;13, 2023, doi: 10.3389/fimmu.2023.1063838.\u003c/li\u003e\n\u003cli\u003eJ. Cheng \u003cem\u003eet al.\u003c/em\u003e, \u0026ldquo;Preclinical development and evaluation of nanobody-based CD70-specific CAR T cells for the treatment of acute myeloid leukemia,\u0026rdquo; \u003cem\u003eCancer Immunol. Immunother.\u003c/em\u003e, vol. 72, no. 7, pp. 2331\u0026ndash;2346, 2023, doi: 10.1007/s00262-023-03422-6.\u003c/li\u003e\n\u003cli\u003eQ. Xiong \u003cem\u003eet al.\u003c/em\u003e, \u0026ldquo;The development of chimeric antigen receptor T-cells against CD70 for renal cell carcinoma treatment,\u0026rdquo; \u003cem\u003eJ. Transl. Med.\u003c/em\u003e, vol. 22, no. 1, pp. 1\u0026ndash;13, 2024, doi: 10.1186/s12967-024-05101-1.\u003c/li\u003e\n\u003cli\u003eH. Hanssens \u003cem\u003eet al.\u003c/em\u003e, \u0026ldquo;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,\u0026rdquo; \u003cem\u003eFront. Immunol.\u003c/em\u003e, vol. 15, no. April, pp. 1\u0026ndash;18, 2024, doi: 10.3389/fimmu.2024.1389018.\u003c/li\u003e\n\u003cli\u003eW. C. Temple \u003cem\u003eet al.\u003c/em\u003e, \u0026ldquo;Framework humanization optimizes potency of anti-CD72 nanobody CAR-T cells for B-cell malignancies,\u0026rdquo; \u003cem\u003eJ. Immunother. Cancer\u003c/em\u003e, vol. 11, no. 11, pp. 1\u0026ndash;16, 2023, doi: 10.1136/jitc-2023-006985.\u003c/li\u003e\n\u003cli\u003eS. De Munter \u003cem\u003eet al.\u003c/em\u003e, \u0026ldquo;Nanobody based dual specific CARs,\u0026rdquo; \u003cem\u003eInt. J. Mol. Sci.\u003c/em\u003e, vol. 19, no. 2, pp. 1\u0026ndash;11, 2018, doi: 10.3390/ijms19020403.\u003c/li\u003e\n\u003cli\u003eS. L. Maude \u003cem\u003eet al.\u003c/em\u003e, \u0026ldquo;Chimeric Antigen Receptor T Cells for Sustained Remissions in Leukemia,\u0026rdquo; \u003cem\u003eN. Engl. J. Med.\u003c/em\u003e, vol. 371, no. 16, pp. 1507\u0026ndash;1517, 2014, doi: 10.1056/nejmoa1407222.\u003c/li\u003e\n\u003cli\u003eM. C. J. Rebecca A. Gardner, Olivia Finney, Colleen Annesley, Hannah Brakke, Corinne Summers, Kasey Leger, Marie Bleakley, Christopher Brown, Stephanie Mgebroff, Karen S. Kelly-Spratt, Virginia Hoglund, Catherine Lindgren, Assaf P. Oron, Daniel Li, Stanley R. Ridd, \u0026ldquo;Intent-to-treat leukemia remission by CD19 CAR T cells of defined formulation and dose in children and young adults,\u0026rdquo; \u003cem\u003eBlood\u003c/em\u003e, vol. 129, no. 25, pp. 3322\u0026ndash;3331, 2017, doi: https://doi.org/10.1182/blood-2017-02-769208.\u003c/li\u003e\n\u003cli\u003eJ. W. Fischer, \u0026ldquo;CAR-T Cell Therapy: Mechanism, Management, and Mitigation of Inflammatory Toxicities,\u0026rdquo; \u003cem\u003eFront. Immunol.\u003c/em\u003e, vol. 12, no. June, pp. 1\u0026ndash;9, 2021, doi: 10.3389/fimmu.2021.693016.\u003c/li\u003e\n\u003cli\u003eS. Wu \u003cem\u003eet al.\u003c/em\u003e, \u0026ldquo;Development of novel humanized CD19/BAFFR bicistronic chimeric antigen receptor T cells with potent antitumor activity against B-cell lineage neoplasms,\u0026rdquo; \u003cem\u003eBr. J. Haematol.\u003c/em\u003e, no. April, pp. 1361\u0026ndash;1373, 2024, doi: 10.1111/bjh.19631.\u003c/li\u003e\n\u003cli\u003eB. Xia \u003cem\u003eet al.\u003c/em\u003e, \u0026ldquo;Nanobody-derived bispecific CAR-T cell therapy enhances the anti-tumor efficacy of T cell lymphoma treatment,\u0026rdquo; \u003cem\u003eMol. Ther. Oncolytics\u003c/em\u003e, vol. 30, no. September, pp. 86\u0026ndash;102, 2023, doi: 10.1016/j.omto.2023.07.007.\u003c/li\u003e\n\u003cli\u003eG. Kueberuwa \u003cem\u003eet al.\u003c/em\u003e, \u0026ldquo;CCR7+ selected gene-modified T cells maintain a central memory phenotype and display enhanced persistence in peripheral blood in vivo,\u0026rdquo; \u003cem\u003eJ. Immunother. Cancer\u003c/em\u003e, vol. 5, no. 1, pp. 1\u0026ndash;14, 2017, doi: 10.1186/s40425-017-0216-7.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"MM, BCMA, Nanobody, CAR T-cells, Immunotherapy","lastPublishedDoi":"10.21203/rs.3.rs-7693534/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7693534/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThese findings demonstrate Nb17-nanoCAR-T exhibits potent anti-myeloma efficacy comparable to scFv-based CAR-T, supporting its potential as a promising therapeutic alternative.\u003c/p\u003e\n\u003cp\u003eMultiple 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 typically employ single-chain variable fragments (scFvs), whereas single-domain antibodies (sdAb or V\u003csub\u003eH\u003c/sub\u003eHs) offer advantages such as small size, high stability, and potentially reduced immunogenicity. The aim of this research is to design and evaluate the activity of a novel anti-BCMA nanoCAR-T based on the V\u003csub\u003eH\u003c/sub\u003eH Nb17, and that compares well with a standard one with the conventional CAR-T CT103a.\u003c/p\u003e\n\u003cp\u003eNb17 was validated for strong BCMA binding on MM cell lines and incorporating into a CAR construct. Both nanoCAR-T and CT103a CAR-T were generated via lentiviral transduction of primary T cells. Their functional properties, including cytotoxicity, cytokine secretion, degranulation, memory phenotype, and gene expression, were assessed in vitro, and their antitumor efficacy was tested in vivo in NSG mice model.\u003c/p\u003e\n\u003cp\u003eNb17-nanoCAR-T demonstrated specific killing of MM cells, robust cytokine release (IL-2, TFNa, IFNg), and CT107a degranulation comparable to CT103a. Transcriptomic analysis revealed overlapping pathways between the two CAR-T products. Upon rechallenge with MM cells, both showed enhanced proliferation and reduced exhaustion compared with untransduced T cells. In vivo, Nb17-nanoCAR-T and CT103a eradicated MM1.S tumors, although both yet inducing graft-versus-host disease (GVHD)-like toxicity in a subset of mice.\u003c/p\u003e","manuscriptTitle":"Design of a novel NanoCAR construct that compares well with a conventional ScFv-based CAR targeting BCMA on multiple myeloma","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-10-10 15:07:23","doi":"10.21203/rs.3.rs-7693534/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"c42a816f-7a9a-4d1f-805f-9d38e4778d49","owner":[],"postedDate":"October 10th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-10-13T22:24:13+00:00","versionOfRecord":[],"versionCreatedAt":"2025-10-10 15:07:23","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7693534","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7693534","identity":"rs-7693534","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}