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. 2026 Jul 2;26:1125. doi: 10.1186/s12885-026-16352-0

Human single domain antibody-based CAR-T cells targeting BAFF-R demonstrate promising preclinical activity in B-cell malignancies

Cuiling Zhang 1,#, Qian Luo 2,#, Sungui Wu 3, Le Xu 2, Qiaoe Wei 2, Yanying Zhang 2, Chaoyang Guan 1, Xiaoqing Dong 1, Yang Hong 1,✉, Taochao Tan 2,✉, Bing Chen 1,✉
PMCID: PMC13595693  PMID: 42387444

Abstract

Background

Single-chain antibody-based CAR-T cells targeting BAFF-R have demonstrated antitumor effects against human B-cell malignancies and can overcome CD19 antigen loss. However, CARs built on antigen-specific single-chain antibody variable fragments may have limitations, e.g., large CAR binding domain size. To improve the function of BAFF-R CAR-T cells, we designed CARs with only a fully human heavy-chain variable domain.

Methods

A fully human antibody phage display library was used to select anti- BAFF- R clones using protein/cell alternate panning. The screened clones were grafted into a second-generation CAR to generate CAR-T cells. CD107a degranulation, luciferase-based cytolysis, repeat antigen stimulation, activation markers, and exhaustion markers were used to detect the function of CAR-T cells in vitro. Xenografts were established in NPG mice following intravenous injection of Jeko-1 cells. Tumor burden and survival of mice were recorded weekly.

Results

Three single-domain heavy chain antibodies, namely Clone #5, Clone #77, and Clone #80, were selected for further evaluation. CAR-T cells constructed with these three candidate clones demonstrated specific target cell activation and effective cytotoxicity in vitro. Clone #5 and Clone #77 CAR-T cells exhibited superior cytotoxic capacity and expansion ability. No significant difference was observed in the expression levels of exhaustion markers on the surface of the three CAR-T cell types. In vivo studies revealed that Clone #5 CAR-T cells displayed enhanced antitumor efficacy compared to other groups, resulting in prolonged survival.

Conclusion

We developed a novel BAFF-R CAR-T cell product structured on a fully human single-domain antibody. This product demonstrated promising preclinical activity and may provide a potential alternative treatment for patients with B-cell malignancies.

Supplementary Information

The online version contains supplementary material available at https://doi.org/10.1186/s12885-026-16352-0.

Keywords: B-cell malignancies, Fully human, Single-domain antibody, BAFF-R, CAR- T

Background

B-cell malignancies comprise a group of heterogeneous tumors that occur at various stages of B-cell differentiation, primarily including B-cell lymphoma and B-cell leukemia. The prognosis of these patients has significantly improved over the years due to the adoption of novel treatments; nonetheless, some patients still suffer recurrence or are refractory, facing dismal survival [1–3]. Chimeric antigen receptor T (CAR-T) cell therapies, specifically targeting CD19, have demonstrated remarkable efficacy in treating relapsed and refractory B-cell malignancies, with an overall response rate of 53% to 83% [4–8]. Despite this, there are still a subset of patients who do not respond to CD19 CAR-T therapies or relapse after receiving them [9]. The prognosis for these patients remains bleak, compounded by the absence of consensus on optimal management strategies [10]. The failure of CD19 CAR- T therapies can be attributed to a variety of factors, one of which is the loss of the CD19 antigen [11, 12]. To overcome this limitation, it’s critical to look for novel therapeutic targets. These targets offer the potential to counter CD19 antigen escape and enable combination with CD19 in a multi-target CAR approach for improved treatment outcomes.

Currently, several targets for B-cell malignancies, such as CD20, CD22, and ROR1, are being actively explored [13]. However, few antigens fully meet the criteria for an ideal target due to persistent challenges in limiting off-target effects for safety and in overcoming antigen escape to prevent early relapse [13]. B-cell activating factor-receptor (BAFF-R), encoded by the TNFRSF13C gene, is one of the key membrane protein receptors involved in B-cell differentiation and proliferation [14]. It is expressed almost exclusively on B cells [15] and is commonly overexpressed in various B-cell malignancies [16–18], which establishes it as an attractive therapeutic target with a low risk of severe off-tumor toxicity. Critically, as BAFF-R signaling is essential for B-cell survival, its loss is less tolerable for malignant cells, thereby reducing antigen escape risk compared to targets like CD19 [19]. According to the research by Qin et al. [20], antigen-specific single-chain antibody variable fragments (scFv)-based CAR-T cells targeting BAFF-R (PMB-101) exhibited antitumor benefits against human B-cell malignancies, even when the tumor cells had lost their CD19 antigen. Similarly, the novel BAFF-R CAR-T (MC10029 CAR-T) based on scFv showed identical outcomes [21]. However, CARs constructed using scFv may lead to certain adverse effects, such as potential anti-CAR immunogenicity and protein aggregation [22]. In contrast, single-domain heavy chain exhibits lower immunogenicity in clinical applications due to its high sequence similarity to the human variable heavy chain, and its hydrophilic modifications within the framework regions circumvent aggregation issues [23]. Moreover, compared to single-domain heavy chain, the larger size of scFv makes it less spatially advantageous to contact specific small or partially concealed cell surface antigens [24]. Additionally, evidence showed that single-domain heavy chain-expressing CAR-T cells achieve functional equivalence to conventional scFv-based CAR-T cells [24]. Thus, we designed BAFF-R CARs that contained only a fully human heavy-chain variable domain (FHVH) and evaluated the performance of BAFF-R CAR-T cells. Our results showed that BAFF-R CAR-T cells based on FHVH exhibited satisfactory anti-tumor efficacy and represented a promising treatment for B-cell malignancies.

Methods

Cell lines

Human hematological tumor cell lines including Jeko-1 (mantle cell lymphoma), Nalm6 (pre-B acute lymphoblastic leukemia cell), RS4 (B cell acute lymphoblastic leukemia), Molt4 (chronic B cell leukemia), CCRF-CEM (T cell acute lymphoblastic leukemia), THP1 (acute myeloid leukemia) and K562 (chronic myelogenous leukemia) were cultured in RPMI-1640 medium (Gibco) containing 10% fetal bovine serum (FBS; Gibco). Mec-1 (chronic B cell leukemia) was cultured in IMDM medium (Gibco) containing 10% FBS. K562-BAFF-R cells were engineered by lentiviral transduction of the BAFF-R gene into parental K562 cells, followed by antibiotic selection and single-cell cloning to establish a monoclonal line with stable, high-level BAFF-R expression as a positive control. The expression levels of BAFF-R on different hematological tumor cell lines were assessed using PE-conjugated mouse anti-human BAFF-R antibody (clone: 11C1, BioLegend) and isotype antibody (clone: MOPC-21, Biolegend). A MACSQuant Analyzer 10 flow cytometer (Miltenyi Biotec) was used for data acquisition, and FlowJo V10 (Tree Star) was used for data analyses and graphs. BAFF-R positive and negative cell lines were stably transfected with firefly luciferase by lentivirus.

OVCAR3 (ovarian adenocarcinoma), KATO III (gastric carcinoma), HCT-116 (colorectal carcinoma), HEPG2 (hepatocellular carcinoma), MDA-MB-468 (breast adenocarcinoma), PANC-1 (pancreatic epithelioid carcinoma), A375 (melanoma cancer), 293T (embryonic kidney), and NCI-H460 (large cell lung cancer) was cultured in DMEM medium (Gibco) containing 10% FBS. All cell lines (Table S1) were purchased from the American Tissue Culture Collection (ATCC; Manassas, VA, USA) and verified before use.

Plasmid construction

A fully human antibody phage display library (IMARS, IASO Biotherapeutics) was used to select anti-BAFF-R clones using optimal protein/cell alternate panning. IMARS platform contains not only regular antibody formats like scFv but also proprietary human single-domain antibodies (in separate sub-libraries). Four rounds of bead panning were carried out using recombinant biotinylated human BAFF-R protein (BAFF-R his) (Kactus) as the target antigen and K562-BAFF-R cells as the target cells. The phage clones were first screened for their ability to bind to BAFF-R antigen via enzyme-linked immunosorbent assay (ELISA) using plates coated with BAFF-R his and streptavidin (Pierce). After incubation with anti-M13 phage mouse antibody (abcam) and anti-mouse horseradish peroxidase antibody (clone: Poly4053, Biolegend), plates were read at 450 nm by using a microplate reader (BioTek). Flow cytometry was used to evaluate their binding affinities to K562-BAFF-R and K562 cell lines after they were stained with anti-M13 phage mouse antibody and FITC anti-mouse IgG antibody (clone: Poly4060, Biolegend). The screened clones were sent for sequencing. Then, the specificity of these clones was further tested by ELISA using plates coated with BAFF-R his, human BAFF-R protein (BAFF-R Fc) (ACRO Biosystem), recombinant biotinylated human CD19 Protein (CD19 Fc) (Kactus), biotinylated human CD70 protein (CD70 his) (ACRO Biosystem) and streptavidin. Their binding affinities to BAFF-R+ and BAFF-R− cell lines were further confirmed using flow cytometry. The final screened clones were integrated into a second-generation CAR with CD8α hinge/transmembrane region, 4‐1BB costimulatory domain, and CD3ζ intracellular signaling domain. The CAR fragment was downstream attached to the truncated epidermal growth factor receptor (EGFRt) [25] through a T2A sequence, facilitating the detection of CAR-T cells.

Antibody affinity measurement

The Octet96e system (ForteBio) was used to evaluate the binding affinity of anti-BAFF-R antibodies. In brief, the anti-BAFF-R antibodies were diluted to 20 µg/ml and loaded onto the biosensors at 0.8 nM. The binding kinetics of the BAFF-R antigen were observed at various antigen concentrations ranging from 200 to 3.13 nM, following a 180-second equilibration period. Each concentration was measured for 60 s of association and 60 s of disassociation. The 1:1 binding site model (Fortebio data analysis version 11.1) was used to analyze the binding kinetics.

Generation of BAFF-R CAR-T cells

Lentivirus was packaged by the tri-plasmid system containing a transfer plasmid, GAG-POL plasmid psPAX2, and VSV-G plasmid (pMD2.G). The lentiviral supernatants were harvested and concentrated at 24 h post transfection. 293T cells and PE-labelled human BAFF-R protein (ACRO Biosystems) was used to detect viral titer via flow cytometry. Human peripheral blood mononuclear cells (hPBMCs) were obtained from Milestone Biotechnologies. CD3 + T cells were isolated from hPBMCs using CD3 microbeads (Miltenyi Biotec) and activated with Dynabeads Human T-Activator CD3/CD28 (Thermo Fisher). The activated T cells were cultured in OpTmizer™ CTS™ T-cell expansion basal medium (Gibco) containing 10% FBS, 200 IU/ml IL-2 (SL Pharm), and 1×GluMax (Gibco). After 24 h, T cells were transduced with a lentiviral vector encoding the BAFF-R CAR at a multiplicity of infection of 2. The duration of T-cell expansion post-transduction was about 1 week, then FITC-conjugated CD4 (clone: SK3, Biolegend), BV421-conjugated CD8 (clone: SK1, Biolegend), APC-conjugated mouse anti-human EGFR antibody (clone: AY13, Biolegend) and PE-labelled human BAFF- R protein were used to measure transduction efficiencies via flow cytometry.

CD107a degranulation assay

BAFF-R CAR-T cells or non-transduced (mock) T cells were co-cultured with different target cells at a 1:1 effector to target (E: T) ratio in a 96-well U-bottom plate. To assess degranulation, a staining solution containing PE-conjugated mouse anti-human CD107a antibody (clone H4A3, Biolegend) and monensin (Biolegend) was prepared by mixing 1.5 µL of the antibody, 0.2 µL of monensin, and 50 µL of RPMI-1640 medium supplemented with 10% FBS. This solution was added to each well, which already contained 100 µL of target cells and 100 µL of effector cells. After 4 h of incubation, CAR-T cells were stained with BV421-conjugated mouse anti-human CD8 antibody and APC-conjugated mouse anti-human EGFR antibody. The degranulation assay was evaluated by flow cytometry.

Luciferase-based cytolysis assay

In white opaque 96-well plates, BAFF-R CAR-T cells or mock T cells were co-cultured with different target cells transfected with firefly luciferase at specified E: T ratios in triplicate. After 24 h, luciferase assay substrate was added to each well, and the fluorescence values were measured using a microplate reader.

Repeat antigen stimulation assay

BAFF-R CAR-T cells or mock T cells were co-cultured with different target cells treated with mitomycin C one day earlier at a 4:1 E: T ratio. On day 5, T cells were counted to obtain data for the first round of repeated stimulation. Fold expansion was calculated as (the number of viable CAR-T cells or mock T cells on Day 5) divided by (the number of viable CAR-T cells or mock T cells on Day 1). Repeat the above steps using the stimulated CAR-T cells and new mitomycin C-treated target cells until the number of CAR-T cells was insufficient to support the next round of experiments. Cumulative fold expansion was determined by multiplying the fold expansion of each round ([fold expansionn] × [fold expansionn+1] …).

Detection of activation and exhaustion markers

To detect activation markers, BAFF-R CAR-T cells or mock T cells were co-cultured with Jeko-1 cells at a 1:1 E: T ratio. After 24 h of incubation, cells were stained with BV510-conjugated CD4 (clone: SK3, Biolegend), BV421-conjugated CD8, APC-conjugated EGFR antibody, FITC-conjugated CD69 (clone: FN50, Biolegend) and PerCP/Cyanine5.5-conjugated CD25 (clone: BC96, Biolegend). The assay was evaluated by flow cytometry.

To detect exhaustion markers, BAFF-R CAR-T cells or mock T cells were co-cultured with mitomycin-C-treated Jeko-1 cells at a 4:1 E: T ratio. After three rounds of stimulation, cells were stained with PE-conjugated CD8 (clone: SK1, Biolegend), PE/Cyanine7-conjugated EGFR antibody (clone: AY13, Biolegend), APC-conjugated LAG3 (clone: 7H2C65, Biolegend), BV510-conjugated PD1 (clone: NAT105, Biolegend) and BV421-conjugated TIM3 (clone: F38-2E2, Biolegend). The assay was evaluated by flow cytometry.

Mouse xenograft models

NPG mice were acquired from Charles River (Beijing, China) and housed in the experimental animal center of ClinBridge Biotech (Nanjing, China) under specific pathogen-free conditions. NPG mice are highly immunodeficient mice established by backcrossing Il2rg knockout mice with NOD-scid mice. The Institutional Animal Care and Use Protocol (AP-C210507.45) approved the animal experiments.

A total of 45 female NPG mice aged 5–6 weeks were fed a vacuum-sealed, Co⁶⁰-γ-irradiated pelleted diet for 1 week. Each mouse was injected with 1 × 106 Jeko-1 cells transfected with firefly luciferase through the tail vein. Four days later, the tumor load was detected by bioluminescence imaging (PerkinElmer). To ensure a balanced average tumor burden across cohorts, 40 mice were randomly distributed into 8 groups (n = 5 per group) based on their baseline bioluminescence signals. Group assignment was performed by generating random numbers using the RAND function in Excel. Evaluators were blinded to the allocation sequence during group assignment. After one day, each group received a tail vein injection of 200 µL of either PBS, 4 × 10⁶ mock T cells, or 4 × 10⁶ CAR‑T cells, including 1499 (PMB101), 1500 (VAY736), 1533 (BAFF‑R#1), 1537 (BAFF‑R#5), 2050 (BAFF‑R#77), and 2053 (BAFF‑R#80). The PBS and MockT groups served as negative controls, the 1499 and 1500 groups as positive controls, and the remaining four groups (1533, 1537, 2050, 2053) as experimental groups.

The mice were weighed twice a week and imaged once weekly. Their blood was collected per week from the submandibular vein, and the order was randomized. CAR expression was detected using APC-conjugated mouse anti-human EGFR antibody. The living state was observed and recorded daily. Clinical observations included body weight, food intake, activity level, fur condition, and the presence of any signs of distress (e.g., lethargy, hunched posture, orbital tightening).

Animals were immediately euthanized if they exhibited signs of severe distress, which were defined as: body weight loss exceeding 20% of baseline weight, severe lethargy, or an inability to access food or water. At the end of the experiment, the mice were euthanized by carbon dioxide (CO2) inhalation. Specifically, euthanasia was performed in a dedicated chamber with a gradual CO2 displacement rate of 20% of the chamber volume per minute to minimize animal distress. Euthanasia was confirmed by the cessation of respiration for at least 2 min, followed by cervical dislocation as a secondary physical method to ensure death.

Graphs and statistical analysis

Data analyses and graphs were performed using the GraphPad Prism software version 7. All data were presented as mean ± SD, except for mice body weight data and mice peripheral blood data, which were shown as mean ± SEM. Continuous data were first tested for normality using the Shapiro–Wilk test. For comparisons between two independent groups, the unpaired t‑test was applied when data met normality and variance homogeneity assumptions; otherwise, the Mann–Whitney U test was used. For comparisons among three or more groups, one‑way analysis of variance (ANOVA) was performed. When two categorical factors were involved, two‑way ANOVA was used. Survival data were compared using the log‑rank test. A two‑sided P < 0.05 was considered statistically significant. P-values are represented as follows: not significant (ns), *P < 0.05, **P < 0.01, ***P < 0.001, or ****P < 0.0001.

Results

Screening of fully human BAFF-R-specific clones and construction of BAFF-R CARs

A diagram of BAFF-R specific clone screening process was shown in Fig. 1A. The fully human phage antibody library was used for affinity panning to enrich the clones bound to the BAFF-R antigen (Table S2). A total of 182 monoclonal clones were selected for primary screening by ELISA (Figure S1A) and fluorescence-activated cell sorting (FACS) analysis (Figure S1B), of which 93 clones that were specific and double-positive were sequenced, yielding 29 distinct monoclonal sequences. To verify whether the 29 clones had good binding specificity to a variety of BAFF-R+/- antigens and cell lines, ELISA and FACS were utilized again and 18 unique binding clones were obtained. The representative clones were shown in Fig. 1B and C and Figure S2. The 18 clones were then constructed into CAR-T cells and functionally assessed via CD107a degranulation and luciferase-based cytolysis assays. Based on these functional assays, four clones (#1, #5, #77, and #80) were ultimately identified (Figure S3). A schematic of their CAR structures was presented in Fig. 2A, with three of the clones (#5, #77, #80) being single-domain heavy chain antibodies and the remaining one (#1) being a fully human scFv. The PMB-101 CAR molecule—an scFv antibody from City of Hope’s autologous anti-BAFF-R CAR-T therapy—was used as a reference. The CAR was constructed using a second-generation design, which represents the most prevalent architecture in current clinical applications.

Fig. 1.

Fig. 1

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Screening of fully human BAFF-R-specific clones. A Schematic of BAFF- R‐specific clones discovery process. B Binding activities of representative phage clones via ELISA. VAY736 phage was used as the positive control, and KO7 was used as the negative control. The optical density at 450 nm was measured to assess different antigens binding by staining with anti-M13 phage mouse antibody and anti-mouse horseradish peroxidase antibody. The data are presented as mean ± SD of triplicates. ****P < 0.0001 (two-way ANOVA). C Binding activities of representative phage clones via flow cytometry (data from one experiment). VAY736 phage was used as the positive control, and KO7 was used as the negative control. The phage clones were stained with anti-M13 phage mouse antibody and FITC anti-mouse IgG antibody after being incubated with K562-BAFF-R cell lines and K562 cell lines

Fig. 2.

Fig. 2

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Construction of BAFF-R CARs and transduction efficiencies of CAR-T cells. A Schematic overview of the BAFF-R-CARs construct. Schematic structures containing the anti‐BAFF-R clones, a CD8α hinge/transmembrane region, a 4‐1BB costimulatory domain, and a CD3ζ T cell activation domain linked to the truncated EGFR by T2A. 1499 (PMB-101 CAR) and 1500 (VAY736 CAR) were used as the positive control. PMB-101 is a proven BAFF-R CAR-T cell therapy, and VAY 736 is a human monoclonal antibody to BAFF-R. B Comparison of viral titer in each group. The data are presented as mean ± SD of triplicates. ns: not significant (one-way ANOVA). C Proportion of CD4 + and CD8 + cells in each group 7 days after viral transduction. The data are presented as mean ± SD of triplicates. ns: not significant (two-way ANOVA). D Transduction efficiencies of CAR-T cells stained with APC-conjugated mouse anti-human EGFR antibody and PE-labelled human BAFF-R protein. The data are presented as mean ± SD of triplicates. ns: not significant (two-way ANOVA). E Proportion of CAR-T cells in CD4 + and CD8 + cells. The data are presented as mean ± SD (n = 6) of triplicates. ns: not significant, **P < 0.01, ***P < 0.001, ****P < 0.0001 (two-way ANOVA)

To further measure the three heavy chain antibodies’ affinities to the BAFF-R antigen, the Octet96e system was used. The result showed that clone #5 (KD = 1.49 × 10− 8M), clone #77 (KD = 6.54 × 10− 8M), and clone #80 (KD = 3.28 × 10− 9M) showed slightly lower but comparable binding affinity to BAFF-R protein when compared to PMB-101 (KD = 3.64 × 10− 9M) (Figure S4).

Clones with good specificity to both BAFF-R antigens and cell lines were used to construct CAR-T cells. Comparison of viral titer in each group was shown in Fig. 2B. Proportion of CD4 + and CD8 + cells was detected 7 days after viral transduction, and no significant difference was observed among the groups, indicating a balanced coexistence of these two subsets (Fig. 2C).The transduction efficiencies of CAR-T cells ranged from 20% to 50%, and there was no significant difference between the results measured by the EGFR antibody and BAFF-R protein (Fig. 2D). On day 7 after viral transduction, the proportion of CAR-T cells in CD4 + cells was significantly higher than that in CD8 + cells (Fig. 2E).

Degranulation and cytotoxicity of BAFF-R CAR-T cells in vitro

Using K562-BAFF-R as a positive control, cell-surface BAFF-R expression was detected on various hematological malignancy cell lines, among which Jeko-1, RS4, Nalm6, and Mec-1 cells were BAFF-R positive, while THP1, CCRF-CEM, and Molt4 cells were BAFF-R negative (Fig. 3A).

Fig. 3.

Fig. 3

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Degranulation and cytotoxicity of BAFF-R CAR-T cells in vitro. A Validation of BAFF-R expression on cell lines via flow cytometry. Blue histograms represent the expression of BAFF-R on the cell surface stained with PE-conjugated mouse anti-human BAFF-R antibody. Grey histograms represent the negative control stained with isotype antibody. B The degranulation assay of BAFF-R CAR-T cells stimulated with the target cells at a 1:1 effector to target (E: T) ratio for 4 h. Plots are gated on CD8+ EGFR+ cells. The data represents mean ± SD for three donors. *P < 0.05, **P < 0.01, ***P < 0.001 (two-way ANOVA). C Luciferase-based cytotoxicity of BAFF-R CAR-T cells incubated with target cells at different E: T ratios for 24 h. The data are presented as mean ± SD for three donors. *P < 0.05 (two-way ANOVA)

CD107a expression on the cell surface is a marker of cytotoxic CD8 + T cell degranulation, which can be used to evaluate the immune cell antitumor activity. By co-incubating BAFF-R CAR-T cells with BAFF-R +/- expressing cell lines, we found that the expression levels of CD107a in all BAFF-R CAR-T cells were significantly higher under the stimulation of BAFF-R positive target cells, and no BAFF-R CAR-T cells were found to be nonspecifically activated in response to the stimulation of BAFF- R negative cells (Fig. 3B). Representative flow cytometry plots were shown in Figure S5. In addition, to assess the safety of BAFF-R CAR- T cells, various cancer cells from different human tissues were co-cultured with BAFF-R CAR-T cells, and no obvious CD107a released in all BAFF-R CAR-T cells under the stimulation of these cancer cells, except for Nalm-6 cells (Figure S6).

To evaluate the tumor cell killing ability, BAFF-R CAR-T cells were co-cultured with BAFF-R +/- expressing target cells at different E: T ratios. The results showed that all BAFF-R CAR-T cells could selectively kill tumor cells expressing BAFF-R antigen, whereas they were not cytotoxic against the BAFF-R negative cell lines (Fig. 3C). In contrast to mock T cells, all BAFF-R CAR-T cells exhibited considerable cytotoxicity when co-cultured with Jeko-1 and Nalm6 cells, even at a very low E: T ratio (P < 0.05) (Fig. 3C). Among the four candidate CAR-T cell constructs, 1533 (BAFF-R#1) showed the strongest in vitro cytotoxicity. For the three constructs based on a single-domain heavy-chain antibody, 2053 (BAFF-R#88) exhibited lower killing capacity, while no significant difference was observed between 1537 (BAFF-R#5) and 2050 (BAFF-R#77) (Fig. 3C).

Activation, expansion, and exhaustion of BAFF-R CAR-T cells in vitro

CD69 and CD25 are early and late activation markers of T cells. By co-incubating BAFF-R CAR-T cells with Jeko-1 cells, we found that the expression levels of both CD25 and CD69 were significantly elevated on the surface of CD4⁺ and CD8⁺ CAR-T cells in all groups (Fig. 4A, Figure S7), indicating effective activation of these CAR-T cells.

Fig. 4.

Fig. 4

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Activation, expansion, and exhaustion of BAFF-R CAR-T cells in vitro. A Expression of the T cell activation markers CD69 and CD25 on BAFF-R CAR-T cells after a 24-hour co-incubation with Jeko-1 cells at 1:1 E: T ratio. Cells were stained with BV510-conjugated CD4, BV421-conjugated CD8, APC-conjugated EGFR antibody, FITC-conjugated CD69 and PerCP/Cyanine5.5-conjugated CD25. The data are presented as mean ± SD for three donors. ns: not significant, *P < 0.05, **P < 0.01, ****P < 0.0001 (t test or Mann-Whitney test). B Repeated antigen stimulation expansion assay of BAFF-R CAR-T cells stimulated by mitomycin-C-treated Jeko-1 cells at 4:1 E: T ratio. The data are presented as mean ± SD of triplicates. ***P < 0.001 (two-way ANOVA). C Expression of the T cell exhaustion markers LAG3, PD1, and TIM3 on BAFF-R CAR-T cells after two rounds of stimulation with mitomycin-C-treated Jeko-1 cells at 4:1 E: T ratio. Cells were stained with PE-conjugated CD8, PE/Cyanine7-conjugated EGFR antibody, APC-conjugated LAG3, BV510-conjugated PD1 and BV421-conjugated TIM3. The data are presented as mean ± SD of triplicates. ns: not significant (two-way ANOVA)

To evaluate the proliferation ability of BAFF-R CAR-T cells in vitro, mitomycin C-treated Jeko-1 cells were used to repeatedly stimulate CAR-T cells. The results revealed that 2050 (BAFF-R #77) and 1537 (BAFF-R #5) CAR-T cells demonstrated a notable amplification capability as the number of stimulation rounds increased, especially at the 5th round of stimulation (Fig. 4B). Clone #77 showed the strongest expansion, with average fold increases of 4.19, 23.66, 82.12, 175.49, and 563.82 from rounds 1 to 5, respectively. Its fold expansion became significantly greater than that of clone #5 as early as the third stimulation round (P < 0.05). Clone #5 itself displayed robust proliferation, with average fold increases of 4.15, 21.93, 53.05, 73.38, and 297.7 across the five rounds, and also showed a significantly higher fold expansion than the 1500 (VAY736) CAR-T cells at the third round (P < 0.05). In contrast, clone #1 exhibited insufficient proliferative capacity, with cell numbers declining to an extent that precluded continuation of the experiment beyond two rounds of stimulation. After two rounds of stimulation, exhaustion markers (LAG3, PD1, and TIM3) of T cells were detected, and we found that there was no significant difference in the expression levels of LAG3, PD1, and TIM3 on the surface of the four candidate BAFF-R CAR-T cells (Fig. 4C, Figure S8).

Antitumor activity of BAFF-R CAR-T cells in vivo

The experimental setup for the mouse xenograft models is shown in Fig. 5A. A tumor-bearing NPG mouse model was established by injecting Jeko-1 cells via the tail vein. Five days later, BAFF-R CAR- T cells were infused into each mouse. Tumor burden was monitored longitudinally using in vivo bioluminescence imaging, as shown in Fig. 5B. After 6 weeks of observation, we found that 1537 (BAFF-R #5) CAR-T cells exhibited obvious in vivo anti-tumor effects (Fig. 5C). Compared to the 2050 (BAFF-R #77) CAR-T cell treatment group, in which all mice survived till day 42, the tumor burden of mice in the 1537 (BAFF-R #5) CAR-T cell treatment group was significantly lower (P < 0.05) (Fig. 5C). On day 49, the mice in the 1537 CAR-T cell treatment group had a considerably longer survival period than those in the PBS group (P < 0.05) (Fig. 5D).

Fig. 5.

Fig. 5

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Antitumor activity of BAFF-R CAR-T cells in vivo. A The schematic diagram of the experimental setup for mouse xenograft models. BLI: Bioluminescence imaging. B Bioluminescence images and survival of mice in each group at the indicated time points. C The average radiance quantification of each group measured at the indicated time points. The data are displayed as mean ± SD (n = 5 mice per group). ****P < 0.0001(two-way ANOVA). D Overall Kaplan-Meier survival curve. E Relative frequency of BAFF-R CAR-T cells (EGFR+) in peripheral blood of mice in each group at the indicated time points. The data are presented as mean ± SEM (n = 5 mice per group). **P < 0.01(two-way ANOVA). F Body weight curve. The data are presented as mean ± SEM (n = 5 mice per group)

Blood was collected weekly from mice to track changes in CAR-T cell proportions in vivo. The results showed that the proportion of CAR-T cells in peripheral blood of mice in the 1533 (BAFF-R #1) and the 1537 (BAFF-R #5) CAR-T cell treatment groups increased steadily over time (Fig. 5E, Figure S9). Meanwhile, compared to the 2050 (BAFF-R #77) CAR-T cell treatment group, the 1537 (BAFF-R #5) CAR-T cell treatment group had a higher proportion of CAR+ cells on day 41(Fig. 5E). The body weight curve revealed that the body weights of mice in all BAFF-R CAR-T treatment groups were basically stable (Fig. 5F).

Discussion

For the CAR-T treatment of B-cell malignancies, CD19 CAR-T cells are currently the most extensively studied. However, the persistent challenge of tumor recurrence drives the ongoing pursuit of novel therapeutic targets [13]. BAFF-R has emerged as a promising target [26–28]. As a member of the tumor necrosis factor family, BAFF-R plays a pivotal role in B cell survival, maturation, and differentiation through its interaction with the B-cell activating factor ligand [29]. BAFF-R signaling predominantly activates the NF-κB pathway and concurrently relieves the inhibition of spleen tyrosine kinase, thereby enhancing the activation of the B cell receptor signaling pathway [30]. Instead of CD19 CAR-T cells, targeting BAFF-R may result in less severe B cell aplasia since early-stage B cells do not express BAFF-R [28]. Currently, the BAFF-R CAR-T cells based on the scFv have demonstrated promising clinical efficacy and safety, showing ability to overcome CD19 antigen loss [31].

In recent years, nanoantibodies based on single-domain heavy chain variable antigen-binding domains have attracted great interest [32, 33]. They can overcome some limitations of monoclonal antibodies by virtue of their small size, simple structure, strong antigen-binding affinity, and extraordinary durability under severe conditions [34]. Furthermore, they are easier to modify, manufacture, and purify, making them more suitable for clinical application [35]. Currently, CAR-T cells with FHVH have shown promising antitumor activities [36, 37] and powerful clinical efficacy [38]. Therefore, we developed a novel BAFF-R CAR-T cell product that contained only a FHVH.

Based on the fully human phage antibody library, we screened three fully human single-domain heavy chain antibodies (Clone #5, Clone #77, and Clone #80) targeting BAFF- R. They all displayed the capacity to specifically bind to different BAFF-R antigens and BAFF-R positive cell lines. We then incorporated them into CAR-T cells. All three clones were efficiently expressed on the surface of T cells. In vitro experiments revealed that three FHVH-based CAR-T cells demonstrated adequate target cell-specific activation, while 1537 (BAFF-R #5) and 2050 (BAFF-R #77) CAR-T cells displayed stronger cytotoxic capacity and expansion ability. In animal studies, 1537 (BAFF-R #5) CAR-T cells exhibited enhanced anti-tumor activity and more effectively delayed tumor progression. Furthermore, these CAR-T cells maintained a stable, increasing proportion in the peripheral blood of mice. Thus, the Clone #5 was selected for further study. We subsequently developed CD19/BAFF-R dual-targeted CAR-T cells. The dual-targeted CAR-T cells were functionally compared to CD19 and BAFF-R single-targeted CAR-T cells in B-cell malignancies with loss of CD19 or BAFF-R antigens, and the result showed that 1537 (BAFF-R #5) CAR-T cells could overcome CD19 antigen escape [39]. Furthermore, we noted that the bicistronic BAFF/CD19 CAR outperformed the tandem CAR design [39]. This advantage may be attributed to the physical separation and independent expression of the two binding domains, which likely helps maintain their optimal conformations. This observation warrants further investigation.

In addition to the three FHVH-based CAR-T cells, we identified one scFv-based CAR- T cell, 1533 (BAFF-R #1), which also exhibited potent efficacy. 1499 (PMB-101) and 1500 (VAY736) CAR-T cells were initially used as positive controls. However, in this study, the 1499 (PMB-101) CAR-T cells did not achieve the expected optimal efficacy. This outcome may be attributed to factors related to the manufacturing process; for instance, we did not classify CAR-T cell subtypes as Qin et al. [16] did. Furthermore, 1500 (VAY736) CAR-T cells showed poor anti-tumor performance in vivo, suggesting that VAY736 may not be suitable for CAR-T cell construction. Compared to FHVH-based CAR-T cells, scFv-based CAR-T cells seemed to perform weaker efficacy, but the difference was not significant.

There are still some limitations in this study. While our study successfully identified BAFF-R-specific CAR-T clones through functional screening, more assays to rigorously dissect antigen-specific versus background cytotoxicity are needed to define the safety profile. We only confirmed the efficacy of 1537 (BAFF-R #5) CAR-T cells in mice modeled with Jeko-1 cells for several weeks. For other types of B-cell malignancies and longer efficacy, more experiments are needed for further confirmation. Furthermore, although no significant side effects had been observed in animal experiments, the safety of 1537(BAFF-R #5) CAR-T cells has yet to be verified due to the use of immunodeficient mice and the absence of cytokine analyses. Future studies should be conducted against more complex animal models or humanized mouse models to assess the therapeutic potential of BAFF-R CAR-T cells in a more clinically relevant context. Lastly, this study has not fully established the advantages of FHVH-based CAR-T cells over scFv-based CAR-T cells. Further experiments are required to comprehensively compare the functional properties of these two types of CAR-T cells.

Conclusions

Taken together, we developed a novel BAFF-R CAR-T cell product based on FHVH. This product demonstrates promising efficacy both in vitro and in mouse models. BAFF-R CAR-T cells based on FHVH may offer a potentially effective treatment option for patients with B-cell malignancies.

Supplementary Information

Supplementary Material 1. (7.4MB, docx)

Acknowledgements

The authors would like to thank all of the volunteers for donating PBMCs, Xuefeng Wu for preparing the plasmids, Bang Shao for guiding the animal experiments, and Dr. Jianwei Liu and Meng Xie for their assistance with the in vitro experiments.

Abbreviations

CAR-T

Chimeric antigen receptor T

BAFF-R

B-cell activating factor-receptor

scFv

Antigen-specific single-chain antibody variable fragments

FHVH

Fully human heavy-chain variable domain

ELISA

Enzyme-linked immunosorbent assay

FACS

Fluorescence-activated cell sorting

EGFRt

Truncated epidermal growth factor receptor

hPBMCs

Human peripheral blood mononuclear cells

E: T

Effector to target

Authors’ contributions

Bing Chen, Taochao Tan, Cuiling Zhang, and Qian Luo designed this study. Cuiling Zhang, Qian Luo, Sungui Wu, Le Xu, Qieoe Wei, Yanying Zhang and Yang Hong performed the experiments, collected, and analyzed the data. Cuiling Zhang wrote the manuscript. Bing Chen, Taochao Tan, Yang Hong, Chaoyang Guan, and Xiaoqing Dong reviewed and revised this manuscript. All the authors have read and approved the final manuscript.

Funding

This work was supported by the Social Development Project of Jiangsu Science and Technology Plan (Nos. BE2023656 and BE2017602), the Key Research Project of Jiangsu Provincial Health Commission (ZDB2020021), and the Basic Research Program of Jiangsu Province (BK20250226).

Data availability

The data supporting the findings of this study are available from the corresponding authors upon reasonable request.

Declarations

Ethics approval and consent to participate

Milestone Biotechnologies (China) provided the PBMCs from healthy donors, which are commercial products. According to Article 32 of the “Ethical Review Measures for Life Sciences and Medical Research Involving Humans” issued by the National Science and Technology Ethics Committee, this study is exempt from ethical review. The animal experiments were approved by the Institutional Animal Care and Use Protocol (AP-C210507.45).

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.

Footnotes

Publisher’s note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Cuiling Zhang and Qian Luo contributed equally to this work.

Contributor Information

Yang Hong, Email: hughhong@live.com.

Taochao Tan, Email: taochao.tan@iasobio.com.

Bing Chen, Email: chenbing1@nju.edu.cn.

References

  • 1.Barraclough A, Hawkes E, Sehn LH, Smith SM. Diffuse large B-cell lymphoma. Hematol Oncol. 2023. 10.1002/hon.3202. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 2.Eyre TA, Cheah CY, Wang ML. Therapeutic options for relapsed/refractory mantle cell lymphoma. Blood. 2022;139(5):666–77. 10.1182/blood.2021013326. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 3.Aldoss I, Shah BD, Park JH, Muffly L, Logan AC, Brown P, et al. Sequencing antigen-targeting antibodies and cellular therapies in adults with relapsed/refractory B-cell acute lymphoblastic leukemia. Am J Hematol. 2023;98(4):666–80. 10.1002/ajh.26853. [DOI] [PubMed] [Google Scholar]
  • 4.Al-Juhaishi T, Ahmed S. Selecting the Optimal CAR-T for the Treatment of B-Cell Malignancies. Curr Hematol Malig Rep. 2021;16(1):32–9. 10.1007/s11899-021-00615-7. [DOI] [PubMed] [Google Scholar]
  • 5.Shah BD, Ghobadi A, Oluwole OO, Worel N, McGuirk JP, Holte H, et al. Two-year follow-up of KTE-X19 in patients with relapsed or refractory adult B-cell acute lymphoblastic leukemia in ZUMA-3 and its contextualization with SCHOLAR-3, an external historical control study. J Hematol Oncol. 2022;15(1):170. 10.1186/s13045-022-01379-0. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 6.Locke FL, Ghobadi A, Jacobson CA, Miklos DB, Lekakis LJ, Oluwole OO, et al. Long-term safety and activity of axicabtagene ciloleucel in refractory large B-cell lymphoma (ZUMA-1): a single-arm, multicentre, phase 1–2 trial. Lancet Oncol. 2019;20(1):31–42. 10.1016/S1470-2045(18)30864-7. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 7.Schuster SJ, Tam CS, Borchmann P, Worel N, McGuirk JP, Holte H, et al. Long-term clinical outcomes of tisagenlecleucel in patients with relapsed or refractory aggressive B-cell lymphomas (JULIET): a multicentre, open-label, single-arm, phase 2 study. Lancet Oncol. 2021;22(10):1403–15. 10.1016/S1470-2045(21)00375-2. [DOI] [PubMed] [Google Scholar]
  • 8.Abramson JS, Palomba ML, Gordon LI, Lunning MA, Wang M, Arnason J, et al. Lisocabtagene maraleucel for patients with relapsed or refractory large B-cell lymphomas (TRANSCEND NHL 001): a multicentre seamless design study. Lancet. 2020;396(10254):839–52. 10.1016/S0140-6736(20)31366-0. [DOI] [PubMed] [Google Scholar]
  • 9.Vercellino L, Di Blasi R, Kanoun S, Tessoulin B, Rossi C, D’Aveni-Piney M, et al. Predictive factors of early progression after CAR T-cell therapy in relapsed/refractory diffuse large B-cell lymphoma. Blood Adv. 2020;4(22):5607–15. 10.1182/bloodadvances.2020003001. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 10.Martin Y, Franz JK, Agha ME, Lazarus HM. Failure of CAR-T cell therapy in relapsed and refractory large cell lymphoma and multiple myeloma: An urgent unmet need. Blood Rev. 2023;60:101095. 10.1016/j.blre.2023.101095. [DOI] [PubMed] [Google Scholar]
  • 11.Strati P, Neelapu SS. CAR-T failure: beyond antigen loss and T cells. Blood. 2021;137(19):2567–8. 10.1182/blood.2020010462. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 12.Kinoshita H, Bollard CM, Toner K. CD19 CAR-T cell therapy for relapsed or refractory diffuse large B cell lymphoma: Why does it fail? Semin Hematol. 2023;60(5):329–37. 10.1053/j.seminhematol.2023.11.007. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 13.Vanegas YM, Mohty R, Gadd ME, Luo Y, Aljurf M, Qin H, et al. CAR-T cell Therapies for B-cell Lymphoid Malignancies: Identifying Targets Beyond CD19. Hematol Oncol Stem Cell Ther. 2022;15(3):81–93. 10.56875/2589-0646.1026. [DOI] [PubMed] [Google Scholar]
  • 14.Smulski CR, Eibel H. BAFF and BAFF-Receptor in B Cell Selection and Survival. Front Immunol. 2018;9:2285. 10.3389/fimmu.2018.02285. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 15.Dong Z, Song JY, Thieme E, Anderson A, Oh E, Cheng WA, Kuang BZ, Lee V, Zhang T, Wang Z, et al. Generation of a humanized afucosylated BAFF-R antibody with broad activity against human B-cell malignancies. Blood Adv. 2023;7(6):918–32. 10.1182/bloodadvances.2022008560. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 16.Rodig SJ, Shahsafaei A, Li B, Mackay CR, Dorfman DM. BAFF-R, the major B cell-activating factor receptor, is expressed on most mature B cells and B-cell lymphoproliferative disorders. Hum Pathol. 2005;36(10):1113–9. 10.1016/j.humpath.2005.08.005. [DOI] [PubMed] [Google Scholar]
  • 17.Novak AJ, Grote DM, Stenson M, Ziesmer SC, Witzig TE, Habermann TM, et al. Expression of BLyS and its receptors in B-cell non-Hodgkin lymphoma: correlation with disease activity and patient outcome. Blood. 2004;104(8):2247–53. 10.1182/blood-2004-02-0762. [DOI] [PubMed] [Google Scholar]
  • 18.Nakamura N, Hase H, Sakurai D, Yoshida S, Abe M, Tsukada N, et al. Expression of BAFF-R (BR 3) in normal and neoplastic lymphoid tissues characterized with a newly developed monoclonal antibody. Virchows Arch. 2005;447(1):53–60. 10.1007/s00428-005-1275-6. [DOI] [PubMed] [Google Scholar]
  • 19.Budde E, Del Real M, Baird J, Chen L, Song J, Wang X, Thiruvengadam S, Hu M, Macias A, Marcucci E, et al. BAFFR-CAR T cells (PMB-CT01) demonstrate durable responses and manageable toxicities in relapsed/refractory B-cell lymphomas with prior CD19-directed therapy failure or CD19-negative disease. Blood. 2025;146(Supplement 1):268–268. 10.1182/blood-2025-268.40674121 [DOI] [Google Scholar]
  • 20.Qin H, Dong Z, Wang X, Cheng WA, Wen F, Xue W, et al. CAR T cells targeting BAFF-R can overcome CD19 antigen loss in B cell malignancies. Sci Transl Med. 2019;11(511). 10.1126/scitranslmed.aaw9414. [DOI] [PMC free article] [PubMed]
  • 21.Luo Y, Qie Y, Gadd ME, Manna A, Rivera-Valentin R, To T, et al. Translational development of a novel BAFF-R CAR-T therapy targeting B-cell lymphoid malignancies. Cancer Immunol Immunother. 2023;72(12):4031–47. 10.1007/s00262-023-03537-w. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 22.Rahbarizadeh F, Ahmadvand D, Moghimi SM. CAR T-cell bioengineering: Single variable domain of heavy chain antibody targeted CARs. Adv Drug Deliv Rev. 2019;141:41–6. 10.1016/j.addr.2019.04.006. [DOI] [PubMed] [Google Scholar]
  • 23.Asaadi Y, Jouneghani FF, Janani S, Rahbarizadeh F. A comprehensive comparison between camelid nanobodies and single chain variable fragments. Biomark Res. 2021;9(1):87. 10.1186/s40364-021-00332-6. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 24.Lam N, Trinklein ND, Buelow B, Patterson GH, Ojha N, Kochenderfer JN. Anti-BCMA chimeric antigen receptors with fully human heavy-chain-only antigen recognition domains. Nat Commun. 2020;11(1):283. 10.1038/s41467-019-14119-9. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 25.Paszkiewicz PJ, Frassle SP, Srivastava S, Sommermeyer D, Hudecek M, Drexler I, et al. Targeted antibody-mediated depletion of murine CD19 CAR T cells permanently reverses B cell aplasia. J Clin Invest. 2016;126(11):4262–72. 10.1172/JCI84813. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 26.Turazzi N, Fazio G, Rossi V, Rolink A, Cazzaniga G, Biondi A, et al. Engineered T cells towards TNFRSF13C (BAFFR): a novel strategy to efficiently target B-cell acute lymphoblastic leukaemia. Br J Haematol. 2018;182(6):939–43. 10.1111/bjh.14899. [DOI] [PubMed] [Google Scholar]
  • 27.Li G, Zhang Q, Liu Z, Shen H, Zhu Y, Zhou Z, et al. TriBAFF-CAR-T cells eliminate B-cell malignancies with BAFFR-expression and CD19 antigen loss. Cancer Cell Int. 2021;21(1):223. 10.1186/s12935-021-01923-x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 28.Wong DP, Roy NK, Zhang K, Anukanth A, Asthana A, Shirkey-Son NJ, et al. A BAFF ligand-based CAR-T cell targeting three receptors and multiple B cell cancers. Nat Commun. 2022;13(1):217. 10.1038/s41467-021-27853-w. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 29.McAllister E, Jellusova J. BAFF signaling in B cell metabolism. Curr Opin Immunol. 2021;71:69–74. 10.1016/j.coi.2021.05.011. [DOI] [PubMed] [Google Scholar]
  • 30.Schweighoffer E, Tybulewicz VL. BAFF signaling in health and disease. Curr Opin Immunol. 2021;71:124–31. 10.1016/j.coi.2021.06.014. [DOI] [PubMed] [Google Scholar]
  • 31.Aldoss I, Wang X, Chen L, Song J, Sandhu K, Macias A, Sanani N, Barva B, Guan M, Espinosa R, et al. BAFFR-CAR T cells (PMB-CT01) show promising safety and anti-leukemia efficacy in relapsed/refractory B-cell ALL patients after CD19-targeted therapy failure, including CD19-negative disease. Blood. 2025;146(Supplement 1):813–813. 10.1182/blood-2025-813. [DOI] [Google Scholar]
  • 32.Jovcevska I, Muyldermans S. The Therapeutic Potential of Nanobodies. BioDrugs. 2020;34(1):11–26. 10.1007/s40259-019-00392-z. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 33.Sun S, Ding Z, Yang X, Zhao X, Zhao M, Gao L, et al. Nanobody: A Small Antibody with Big Implications for Tumor Therapeutic Strategy. Int J Nanomed. 2021;16:2337–56. 10.2147/IJN.S297631. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 34.Jin BK, Odongo S, Radwanska M, Magez S, Nanobodies. A Review of Generation, Diagnostics and Therapeutics. Int J Mol Sci. 2023;24(6). 10.3390/ijms24065994. [DOI] [PMC free article] [PubMed]
  • 35.Verhaar ER, Woodham AW, Ploegh HL. Nanobodies in cancer. Semin Immunol. 2021;52:101425. 10.1016/j.smim.2020.101425. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 36.Dai Z, Mu W, Zhao Y, Cheng J, Lin H, Ouyang K, et al. T cells expressing CD5/CD7 bispecific chimeric antigen receptors with fully human heavy-chain-only domains mitigate tumor antigen escape. Signal Transduct Target Ther. 2022;7(1):85. 10.1038/s41392-022-00898-z. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 37.Dai Z, Mu W, Zhao Y, Zhao Y, Jia X, Liu J, et al. The rational development of CD5-targeting biepitopic CARs with fully human heavy-chain-only antigen recognition domains. Mol Ther. 2021;29(9):2707–22. 10.1016/j.ymthe.2021.07.001. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 38.Mikkilineni L, Natrakul DA, Lam N, Manasanch EE, Mann J, Weissler KA, et al. Rapid anti-myeloma activity by T cells expressing an anti-BCMA CAR with a human heavy-chain-only antigen-binding domain. Mol Ther. 2024;32(2):503–26. 10.1016/j.ymthe.2023.12.018. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 39.Wu S, Luo Q, Li F, Zhang S, Zhang C, Liu J, et al. Development of novel humanized CD19/BAFFR bicistronic chimeric antigen receptor T cells with potent antitumor activity against B-cell lineage neoplasms. Br J Haematol. 2024. 10.1111/bjh.19631. [DOI] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Supplementary Material 1. (7.4MB, docx)

Data Availability Statement

The data supporting the findings of this study are available from the corresponding authors upon reasonable request.

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