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Construction of tetravalent bispecific Tandab (CD3/BCMA)-secreting human umbilical cord mesenchymal stem cells and its efficiency in the treatment of multiple myeloma

Stem Cell Research & Therapy volume 16, Article number: 69 (2025) Cite this article

Abstract

Background

Highly efficient targeted therapy is urgently needed for multiple myeloma (MM). Mesenchymal stem cells (MSCs) are an attractive candidate of cell-based, targeted therapy due to their inherent tumor tropism. However, there is still no MSCs-based tandem diabody for treating MM.

Methods

Here, we designed a dual-target therapeutic system in which human umbilical cord MSCs (UCMSCs) were engineered to produce and deliver Tandab (CD3/BCMA), a tetravalent bispecific tandem diabody with two binding sites for CD3 and two for B-cell maturation antigen (BCMA). Western blot and flow cytometry were used to confirm the lentivirus-mediated construction of UCMSCs for diabody expression. The tropism of MSCs towards H929 cells in vitro was determined by migration assays, and the in vivo homing capacity of MSCs was analyzed by immunofluorescence staining. The activation and antitumor efficacy of human T cells mediated by MSCs secreting Tandab (CD3/BCMA) were evaluated in vitro. Finally, an MM xenograft NOD/SCID mouse model was established to investigate the therapeutic effect in vivo.

Results

We successfully constructed MSCs that can continuously secrete bioactive Tandab (CD3/BCMA), whereby lentiviral transduction did not affect the morphology, proliferation, and lineage differentiation potential of the MSCs. The tropism of MSC-Tandab for MM was verified both in vitro and in vivo. Furthermore, MSC-Tandab promoted the expansion and activation of primary human T cells and induced healthy donor T cells to selectively eliminate BCMA-positive cell lines and primary blasts from patients but not BCMA-negative cells. A similar ability was also observed in the patient-derived T cells. Finally, MSC-Tandab significantly alleviated the MM xenograft tumor burden in NOD/SCID mice without toxic side effects in vivo, whereby the cytokine levels (IFN-γ) in the peripheral blood (PB) were higher in the MSC-Tandab group, and the tumor infiltration of T cells was significantly enhanced.

Conclusions

These results suggest that UCMSCs releasing Tandab (CD3/BCMA) are a promising new tool for the treatment of MM, opening a new avenue for the development of cell-based therapy.

Background

Multiple myeloma (MM) is a malignant plasma-cell disorder that accounts for 10–15% of all hematological cancers [1, 2]. Treatment options for MM have substantially improved and include the use of proteasome inhibitors, immunomodulatory drugs, monoclonal antibodies, and stem-cell transplantation [3]. Despite these therapeutic achievements, MM remains practically incurable, and new therapies are urgently needed to overcome the inevitable resistance to current agents, in particular therapies that address novel targets and/or those with new mechanisms of action

Recent advances in T-cell-redirecting bispecific antibodies (T-BsAbs) suggest that redirecting T cells to tumor-specific surface antigens may be an effective means to harness the immune system to induce cancer cell death, thus creating meaningful and long-lasting clinical responses [4, 5]. However, bispecific antibodies present several challenges in both their clinical use and production. For example, certain Fc-free bispecific antibody constructs exhibit a short half-life, necessitating daily continuous intravenous infusion, which results in inconvenience for patients and increases the risk of infusion-related infections [6]. Additionally, off-target toxicities (mainly cytokine release syndrome and neurotoxicity), due to the expression of the targeted antigen on non-tumor cells, is a major concern for patients undergoing systemic treatment with T-BsAbs [7, 8]. Furthermore, the manufacturing process of recombinant antibodies is associated with issues such as poor stability and the tendency to aggregate over time [9]. Conversely, building a biological system to effectively deliver bispecific antibodies in vivo, thereby leading to their accumulation in tumor sites over a longer period, may be a perfect solution for these problems.

Due to their inherent tumor tropism, mesenchymal stem cells (MSCs) isolated from bone marrow (BM), umbilical cord, Wharton’s jelly, adipose tissue, placenta, or skeletal tissues have been intensively investigated as cellular vectors for tumor biotherapy [10,11,12]. Among these sources, human umbilical cord-derived MSCs (UCMSCs) are comparatively facile to isolate and expand, and the harvesting procedure is more consistent and yields a greater number of relevant cells than other adult or fetal tissues [13]. These characteristics indicate that UCMSCs may be a promising targeted delivery system for a variety of anticancer agents. In our previous study, a tetravalent bispecific tandem diabody (Tandab) with two binding sites for CD3 and two for BCMA, Tandab (CD3/BCMA), was established [14]. Although Tandab (CD3/BCMA) exhibited good specificity and efficacy both in vitro and in vivo, and its homodimeric design prolonged the drug's half-life, systemic distribution of antibody-based drugs can still lead to certain side-effects. An effective targeted delivery system for these drugs can enhance their local concentration within tumors, thereby improving the efficacy of immunotherapy while reducing the incidence of adverse reactions. Based on these encouraging developments, here we investigated if MSCs are suitable for engineering to express our recombinant antibody, with the aim to take advantage of their potential as cell-based carriers to deliver Tandab (CD3/BCMA) to myeloma lesions.

In this study, we exploited the feasibility and efficacy of using genetically modified UCMSCs that constitutively secreted Tandab (CD3/BCMA) (MSC-Tandab) for the treatment of multiple myeloma. Our results showed that MSC-Tandab could induce specific lysis of BCMA-positive MM cells and primary blasts from patients in the presence of T cells in vitro, while also reducing xenograft tumor growth without toxic effects on normal tissues in NOD/SCID mice in vivo.

Materials and methods

Cell lines and primary patient samples

HEK293T, H929, MM.1S, Jurkat, and Kasumi-1 cell lines were purchased from American Type Culture Collection. All cells were cultured in a humidified chamber at 37℃ with an atmosphere comprising 5% CO2.

Bone marrow mononuclear cells (BMMNCs) were obtained from patients with newly diagnosed MM (NDMM, n = 3), and relapsed/refractory MM (RRMM, n = 3) who visited the Department of Hematology, Jiangxi Provincial People's Hospital (Jiangxi, China). The characteristics of the patients with MM involved in this study are shown in Tables 2 and 3. Primary MM tumor cells were isolated from BMMNCs using anti-CD138 microbeads (Miltenyi Biotec, Germany) and cultured in Iscove's Modified Dulbecco's Medium (IMDM) supplemented with 15% FBS, 100 ng/mL rhFLT3-L, 100 ng/mL rhSCF and 50 ng/mL rhTPO (MCE, China).

PBMC isolation

With informed consent, human peripheral blood mononuclear cells (PBMCs) were isolated from peripheral blood of healthy volunteers by Ficoll-Hypaque density-gradient centrifugation. Cells from the interphase were washed twice with phosphate-buffered saline solution (PBS) followed by incubation in erythrocyte lysis buffer for 10 min at 4℃. Then, the cells were washed twice and cultured in lymphocyte medium KBM581 (Corning, USA) supplemented with 10% FBS and 50 IU/mL human interleukin-2 (IL-2) (R&D systems, USA) for 72 h. Peripheral blood B cells were isolated from PBMCs using anti-CD19 microbeads (Miltenyi Biotec, Germany) and cultured in RPMI 1640 (Gibco, USA) supplemented with 10% FBS.

Isolation of MSCs

MSCs were isolated from human umbilical cord Wharton’s jelly as previously described [15]. MSCs were cultured using the MesenCult™ Proliferation Kit (Stem cell, USA). When cells reached 80–90% confluence, they were detached using a 0.125% trypsin–EDTA solution (Gibco, USA) and re-seeded using the same growth medium for subsequent passages. For all experiments, early passage MSCs (3P to 5P) were used.

Lentivirus production

We previously designed the lentiviral vector pCDH-Tandab (CD3/BCMA) to express the Tandab (CD3/BCMA) fusion fragment with a hexa-histidine (6 × His) tag and green fluorescent protein (GFP) [14]. The same vector with only GFP but no antibody coding sequences was used as negative control. Infectious lentiviral particles were prepared by transfected HEK293T cells with psPAX2 and pMD2G using Attractene Transfection Reagent (QIAGEN, Germany), harvested after 72 h, and concentrated through ultracentrifugation (20,000 × g, 2.5 h, 4℃).

Transduction and viability of transduced MSCs

MSCs were plated at a density of 8000 cells/cm2 in a T-75 plastic culture flask and incubated overnight at 37℃. On the next day, culture medium was removed and 8 mL of fresh medium containing lentivirus at a multiplicity of infection (MOI) of 8 with 8 μg/mL of polybrene (Sigma-Aldrich, USA) was added. The virion-containing medium was removed after 8 h, and fresh culture medium was added. MSCs were incubated for another 72 h and observed under a fluorescence microscope, after which the efficiency of infection was determined by flow cytometry. Western blot analysis was applied to detect the expression of Tandab protein. The concentrations of MSC-secreted Tandab in the supernatants were measures using a His-tag ELISA detection kit (GenScript, USA).

To assess the influence of lentiviral transduction on MSCs, the viability of transduced MSCs was assessed. MSCs infected with Lenti-empty vector or Lenti-Tandab (CD3/BCMA) were named MSC-EV and MSC-Tandab, respectively. And wild-type MSCs were included as control. These three kinds of MSCs were plated into 96-well culture plates at a density of 3000 cells per well. After 72 h of incubation, 10 μL of CCK-8 solution (Dojindo, Japan) was added to each well. The 96-well plates were incubated at 37℃ in an incubator with 5% CO2 for an additional 2–4 h, after which the absorbance was measured at 450 nm using a microplate reader (Thermo, USA).

MSC differentiation assay

To verify whether lentiviral transduction affected the lineage differentiation ability of UCMSCs, untreated MSCs and transduced MSCs were cultured in adipogenic and osteogenic differentiation medium (Procell, China). For adipogenic differentiation, the MSCs were maintained in human UCMSC lipid-induced differentiation medium. Three weeks later, the cells were fixed and stained with oil red O. For osteogenic differentiation, the MSCs were cultured in an osteogenic induction differentiation medium for 3 weeks. At the end of incubation, the cells were assayed for calcium deposition by alizarin red S staining. In addition, RNA was isolated from cells after the incubation, and the expression levels of differentiation-related genes were determined by real-time PCR on an ABI Prism 7500 detection system (Applied Biosystems, USA). The primers used for real-time PCR were summarized in Table 1.

Table 1 Primer sequence for genes in real-time PCR
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Western blot

To detect the expression of Tandab (CD3/BCMA) fusion protein, MSC-Tandab, MSC-EV and MSCs were cultured in 6-well plates for 72 h, after which the supernatants and cell lysates were harvested. Protein samples were loaded onto 10% SDS-PAGE gels, electrophoresed and then transferred to PVDF membranes. The blocked membranes were incubated with a mouse anti-His primary antibody (MBL, Japan) overnight, followed by a rabbit anti-mouse horseradish peroxidase (HRP)-conjugated secondary antibody (Cell Signaling Technology, USA) for 1 h. Finally, the bands were visualized using ECL western blotting detection substrate (PerkinElmer, USA) and imaged on a chemiluminescence imaging system.

Cell binding assay

MSCs were infected with lentivirus for 72 h, after which the culture supernatants were collected to detect the binding activity of Tandab (CD3/BCMA) by flow cytometry. The BCMA-positive cell lines H929 and MM.1S, as well as the CD3-positive cell lines Jurkat were employed for analysis of binding activity of Tandab (CD3/BCMA). The BCMA- and CD3- negative Kasumi-1 cells were included as a negative control. For the direct-binding assay, 1 × 106 cells were incubated with the collected supernatant in a volume of 200 μL for 30 min at room temperature. After washing three times with PBS, the cells were incubated with 200 μL Alexa Fluor 488 labeled anti-His tag antibody (MBL, Japan) at 0.5 μg/mL for an additional 30 min. Then, the stained cells were washed and analyzed by flow cytometry. Cells without any supernatant were used as negative control. For the competition assay, 1 × 106 cells were suspended in 200 μL of the collected supernatant and mixed with 5 μL of APC-conjugated 19F2 or FITC-conjugated HIT 3a (BioLegend, USA) for 30 min at room temperature. After washing three times and resuspending in PBS, the cells were analyzed by flow cytometry.

In vitro migration assay

The migration ability of MSCs in vitro was determined using 8 μm pore-size membrane inserts with 6.5 mm diameter (Corning, USA). Following 24 h after transduction, 4 × 104 MSCs were seeded into the top chamber in 400 μL of serum-free medium. The previous day, H929 cells were plated at a density of 6 × 105 cells per well in the lower chamber in RPMI-1640 medium containing 10% FBS. Culture medium (CM) served as a negative control. After 24 h of incubation at 37℃, cells that had not migrated from the upper side of the membrane were scraped off with a cotton swab, and cells in the lower side of the membrane were stained with 0.1% crystal violet at 37℃ for 45 min. The number of cells that had migrated to the lower side of the membrane was quantified in five randomly selected microscopic fields. The experiments were performed in triplicate.

MSC in vivo tumor tropism test

6-week-old female NOD/SCID mice were obtained from SPF (Jiangsu) GemPharmatech and housed in air-conditioned rooms maintained at a constant temperature of 22 ± 2 °C and 55 ± 5% humidity, with a 12-h light/dark cycle. Mice were implanted subcutaneously with 5 × 106 H929 cells under the right flank. The longest (L) and widest (W) diameters of the tumors were recorded using a vernier caliper by an investigator blinded to the group allocation. Tumor volumes were measured twice a week with a modified ellipsoidal formula: L × W2 × 0.5. Two weeks later, when tumors reached 150–200 mm3, the mice were randomly allocated into two groups (three mice per group) using a random number generator to ensure unbiased group assignment as follows: (a) MSC-EV; (b) MSC-Tandab. 6 female NOD/SCID mice were used in this study.1 × 106 genetically modified MSCs were injected into the mice via the tail vein in a volume 0.2 ml of PBS. A total of three injections were administered once every week. Eighteen days after the first injection, the mice were euthanized by cervical dislocation following isoflurane anesthesia, after which the localization of recombinant protein (anti-His tag antibody, MBL, Japan) and MSC-Tandab (anti-CD90 antibody, Abcam, UK) was examined by immunofluorescence staining. Tumor tissues and the main organs were inspected by confocal microscopy, with all procedures performed in the dark. No animals were excluded from this experiment. The work has been reported in line with the ARRIVE guidelines 2.0.

Cytotoxicity, activation, and proliferation of T cells

The culture supernatants of MSC, MSC-EV and MSC-Tandab were used in the following experiments. MM.1S cells (target cells) were seeded into 96-well plates (1 × 104 cells/well). The next day, PBMCs (effector cells) pretreated with IL-2 for 72 h were added at different effector: target (E:T) ratios ranging from 10:1 to 1:1, followed by the addition of 100 μL of the supernatant. The specific cytotoxicity toward MM.1S cells was measured via the lactate dehydrogenase assay 8 h later using the CytoTox 96 nonradioactive cytotoxicity kit (Promega, USA) following the manufacturer’s protocol. Then, the cells and culture supernatants from each well were separated. The cells were stained with FITC-human CD3 antibody, PE-human CD25 antibody, APC-human CD69 antibody, and PE/CY7-human CD107a antibody (BioLegend, USA) for 30 min at room temperature and analyzed by flow cytometry to detect activated T cells. The levels of interleukin-2 (IL-2), interferon-γ (IFN-γ) and tumor necrosis factor-α (TNF-α) in the supernatants were measured using corresponding ELISA kits (Neobioscience, China). For analysis of T-cell proliferation, the T cells were labeled with CFSE-Far Red (Thermo, USA) and analyzed by flow cytometry.

In vitro co-culture killing experiments

To mimic the therapeutic process of MSC-Tandab in vivo, a co-culture system using transwell plates with a 0.4 μm pore-size membrane (cells cannot passage) was established, in which Tandab (CD3/BCMA) secreted from MSC-Tandab could migrate to the upper insert to trigger cytotoxicity of T cells against BCMA-positive cells. MSC-Tandab, MSC-EV and MSCs were seeded into 24-well culture plates (lower chamber) at a density of 2 × 104 cells per well and incubated for 72 h. Then, target tumor cells labeled with CFSE (Abcam, UK) and pre-activated PBMCs were added to the equilibrated inserts (upper chamber). After co-culture for 24 h, the cell pellets in the inserts were stained with 7-AAD (Abcam, UK) followed by flow cytometry. The expression of activation surface markers CD25 and CD69 as well as the degranulation markers CD107a in T cells was detected by flow cytometry under the same conditions as the co-culture system with unlabeled tumor cells. The supernatants in the inserts were collected to assay the cytokines produced in the co-culture system (IL-2, IFN-γ and TNF-α) using ELISA kits (Neobioscience, China). For analysis of T-cell proliferation, the T cells were labeled with CFSE-Far Red (Thermo, USA) and analyzed by flow cytometry.

Growth inhibition of multiple myeloma xenografts in vivo

6-week-old female NOD/SCID mice were obtained from SPF (Jiangsu) GemPharmatech and housed in air-conditioned rooms maintained at a constant temperature of 22 ± 2 °C and 55 ± 5% humidity, with a 12-h light/dark cycle. A total of 5 × 106 H929 cells were implanted subcutaneously into the right flank of mice. Following 13 days after xenografting, when tumor size reached 100–200 mm3, the mice were randomly allocated into five groups (five mice per group) using a random number generator to ensure unbiased group assignment as follows: (a) PBS; (b) PBMC; (c) MSC + PBMC; (d) MSC-EV + PBMC; (e) MSC-Tandab + PBMC. 25 female NOD/SCID mice were used in this study. MSCs were injected intravenously into the tumor-bearing mice (day 13 and 20; 1 × 106 per mouse) followed by administration of IL-2 pre-activated PBMC 3 days later (day 16 and 23; 1 × 107 per mouse). The body weight was recorded twice a week after the MSC injection. The longest (L) and widest (W) diameters of the tumors were recorded using a vernier caliper by an investigator blinded to the group allocation. Tumor volumes were measured twice a week with a modified ellipsoidal formula: L × W2 × 0.5. Once the tumor volume reached 2000 mm3, all mice were euthanized by cervical dislocation following isoflurane anesthesia. Subsequently, their organs and peripheral blood will be collected for further experiments. No animals were excluded from this experiment. Cytokine levels (IFN-γ) in the peripheral blood were analyzing by ELISA. Tissue samples of the tumor, lungs, and liver were removed to detect apoptosis via TUNEL staining. Lymphocyte infiltration was detected by CD3 staining. For the histopathological inspection, organs were stained with hematoxylin and eosin. The work has been reported in line with the ARRIVE guidelines 2.0.

Statistical analysis

Data are represented as means ± SD. Statistical analyses were performed using GraphPad Prism (GraphPad Software Inc., USA). Significance was assayed using the independent-samples t-test. Statistical significance was indicated with asterisks: *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001.

Results

The construction of MSC-Tandab (CD3/BCMA)

We previously constructed a tetravalent tandem diabody, Tandab (CD3/BCMA), which has two binding sites for CD3 and two for BCMA [14]. The full length of the coding sequence (1533 base pairs) was inserted into the lentiviral expression vectors pCDH-CMV-EF1-copGFP. The Tandab gene expression cassette was composed in the order: CD3VL-GGGGS-BCMAVH-4 × GGGGS-BCMAVL-GGGGS-CD3VH-6 × His. MSCs were isolated from human umbilical cord and transduced with lentivirus encoding Tandab (CD3/BCMA) with copGFP (MSC-Tandab) or alone copGFP (MSC-EV). Under the microscope, MSC-Tandab and MSC-EV cells were spindle-shaped and growing adherently to culture plates. Both of the modified cells showed spontaneous green fluorescence under an inverted fluorescence microscope, suggesting that they were successfully transduced (Fig. 1A). The transduction efficiency was also assessed by flow cytometry analysis, which indicated that more than 62% of the MSCs were successfully transduced (Fig. 1B). Importantly, no significant alterations in cell survival were observed among MSC-Tandab, MSC-EV, and wild-type MSCs, suggesting that the lentiviral construct had no influences on the modified MSCs (Fig. 1C). Next, western blot was used to detect the expression of Tandab. Both in the cell lysate and in the culture supernatant of MSC-Tandab, Tandab protein with a molecular weight of 106 kDa was detected on non-reducing status, while a 53-kDa protein was also detected under reducing status, which demonstrated that Tandab (CD3/BCMA) was highly expressed in MSC-Tandab but not in MSC-EV (Fig. 1D, Fig. S1), as expected. Additionally, the Tandab (CD3/BCMA) released into the culture supernatants of MSC-Tandab was detected by ELISA. The level of secreted Tandab (CD3/BCMA) reached a peak at day 9 and was detectable even at day 15 after transduction (Fig. 1E). In addition, the results of adipogenic and osteogenic differentiation assays showed that the introduction of Tandab did not change the differentiation potential of the MSCs (Fig. S2A and B).

Fig. 1
figure 1

Expression of recombinant fusion proteins in engineered MSCs. (A) The morphology and fluorescence microscopy imaging of UCMSCs transfected with Tandab-expressing or empty lentiviral vector. Scale bar = 50 μm. (B) Flow cytometry analysis of copGFP-positive UCMSCs. (C) Cell survival of UCMSCs with or without lentivirus transduction was assessed using the CCK8 assay. (D) Western blot analysis was employed to determine the protein expression of Tandab (CD3/BCMA) in UCMSCs using anti-His tag antibodies following 3 days after transduction. Target bands are indicated by blue arrows. BME, β-mercaptoethanol. Lane 1, MSCs transfected with empty vector (negative control); lane 2, MSCs transfected with vector expressing Tandab (CD3/BCMA). Full-length blots/gels are presented in Supplementary Figure S1. (E) Transduced UCMSCs consistently secreted Tandab (CD3/BCMA). MSC-Tandab and MSC-EV were cultured in a 24-well plate (4 × 104 cells/well), and the level of Tandab (CD3/BCMA) released into the culture supernatant was measured by ELISA at the indicated time points. Data are shown as the means ± SD of the three repeated experiments

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Functional validation of Tandab (CD3/BCMA) secreted from infected MSCs

The supernatants of MSCs infected with lentivirus for 72 h were collected for the following experiments. The direct binding assays verified that the Tandab (CD3/BCMA) in the supernatants of MSC-Tandab could bind to BCMA-positive H929 and MM.1S cells as well as CD3-positive Jurkat cells (Fig. 2A). No binding was detectable on human acute myeloid leukemia cell line Kasumi-1, which expresses neither BCMA nor CD3 (Fig. S3A, B). Furthermore, a competitive binding assay was conducted and the Tandab (CD3/BCMA) in the supernatants of MSC-Tandab was shown to significantly prevent anti-BCMA antibody and anti-CD3 antibody from binding to H929 and Jurkat cells (Fig. 2B).

Fig. 2
figure 2

Identification of Tandab (CD3/BCMA) in the culture supernatants. The culture supernatants of UCMSCs infected with different lentiviruses for 72 h were collected for the direct-binding assay and competitive-binding assay (A-B). (A) Direct binding activities of the culture supernatants for human CD3 on Jurkat cells, as well as human BCMA on MM.1S and H929 cells was detected using flow cytometry. The FITC-His tag antibody was used as the secondary antibody. (B) Competitive binding activities of culture supernatants with commercial APC-human BCMA antibody and FITC-human CD3 antibody were detected on H929 and Jurkat cells using flow cytometry. (C) PBMCs and MM.1S cells were co-cultured in the collected supernatants of the infected UCMSCs at a ratio of 10:1 for 24 h, after which the cell compartment of the co-culture system was collected and stained with FITC-human CD3 antibody, PE-human CD25 antibody, APC-human CD69 antibody, and PE/CY7-human CD107a antibody to detect activated T cells using flow cytometry. (D) Supernatants of the co-culture system were analyzed for IL-2, IFN-γ, and TNF-α using ELISA. (E) Representative flow cytometry analysis of the proliferation of PBMCs (labeled with CellTrace™ Far Red) after co-cultur with MM.1S at an effector to target (E:T) ratio of 10:1 in the collected supernatants of the infected UCMSCs for 72 h (left) and statistical analysis of the data (right). (F) PBMCs and MM.1S cells were co-cultured in the collected supernatants of the infected UCMSCs at different E:T ratios ranging from 10:1 to 2:1 for 8 h, and the specific cytotoxicity toward MM.1 s cells was measured via the lactate dehydrogenase assay using the CytoTox 96 nonradioactive cytotoxicity kit. **P < 0.01, ***P < 0.001, ****P < 0.0001 compared with the corresponding MSC-EV group. The results in (C–F) represent the means ± SD from three separate experiments

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After MM.1S (target cells) and PBMCs (effector cells) were co-cultured in the medium containing the indicated supernatants separately for 24 h, Tandab (CD3/BCMA) in the supernatants induced T cell activation, which was reflected by the upregulation of activation markers CD25 and CD69 as well as the degranulation marker CD107a in T cells (Fig. 2C). As further hallmarks of T cell activation upon tumor lysis, the levels of IL-2, IFN-γ and TNF-α in the co-culture medium containing Tandab (CD3/BCMA) were also remarkably increased (Fig. 2D). Moreover, T cell proliferation was substantially higher in the MSC-Tandab group compared to the corresponding control (Fig. 2E). To investigate tumor lysis mediated by the indicated supernatants, a nonradioactive cytotoxicity assay was performed. The results showed that target cells (T) were efficiently eliminated by effector cells (E) in the medium containing Tandab (CD3/BCMA), and lysis of target cells proceed after increasing the E:T ratio, which resulted in enhanced cytotoxicity (Fig. 2F).

Migration capacity of UCMSCs toward multiple myeloma in vitro

To investigate the migration capacity of transduced MSCs toward multiple myeloma, migration assays were performed in vitro using transwell plates. Culture medium (CM) without H929 cells served as a negative control for chemotaxis, and unmodified MSCs were used as a positive control of migrating cells. We confirmed that the transduced MSCs migrated towards H929 in a similar pattern to that of unmodified MSCs (Fig. 3A and B). These results indicated that H929 cells were able to stimulate the migration of MSCs and the migration capacity of MSCs was not affected by lentiviral transduction.

Fig. 3
figure 3

Homing ability of genetically modified UCMSCs were verified in vitro and in vivo. (A) The migration ability of all three UCMSCs lines was assessed using the transwell migration assay. Representative images of migrated UCMSCs stained with crystal violet after transduction with different lentiviral vectors for 24 h. H929 cells were used to trigger the migration of stem cells (MSC-Tandab, MSC-EV and MSC), and the CM group served as a negative control. Scale bar = 50 μm. (B) Statistical analysis of the migrated UCMSCs represented by the means ± SD from three separate experiments. (C) Schematic representation of the process of H929 xenograft establishment and MSC-Tandab treatment by tail vein injection. (D) Mice were sacrificed and the distribution of fusion proteins and MSCs in tumor tissues was determined by immunofluorescence staining with an anti-His antibody (green) and anti-CD90 antibody (red). Blue (DAPI) indicates nuclei. White arrows indicate the co-localization of Tandab (CD3/BCMA) and MSCs. Scale bars, a: 200 μm, b: 20 μm. n = 3 tumors

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In order to verify the homing ability of MSCs in vivo, MSC-Tandab or MSC-EV were injected into the NOD/SCID mice bearing H929-xenograft by tail vein once every week for a total of three times (Fig. 3C). Eighteen days after the first injection, the tissues were removed and observed under a confocal microscope. The results of immunofluorescence staining showed that the transduced MSCs migrated and effectively accumulated at tumor the site, while only a small amount of fusion protein could be detected in the lungs and the liver (Fig. S4). In the MSC-Tandab group, recombinant Tandab (CD3/BCMA) fusion protein accumulated in tumor tissues. Furthermore, co-staining for the His-tag and the MSC marker CD90 showed that the two signals substantially co-located in tumor tissues, indicating that MSC-Tandab could infiltrate the tumors and produce His-tagged Tandab (CD3/BCMA) (Fig. 3D, Fig. S5).

MSC-Tandab exhibited selective cytotoxicity in BCMA-positive MM cells in vitro

Next, we validated the anti-MM effects of MSC-Tandab in vitro by co-culturing BCMA-positive (MM.1S) or BCMA-negative cells (Kasumi-1) with engineered MSCs or CM in the presence of PBMCs. CFSE was utilized to identify the target cell population. Tumor lysis was quantified based on residual CFSE+ 7-AAD+ tumor cells detected by flow cytometry. After 24 h of incubation, significant lysis of MM.1S cells was detected by flow cytometry (Fig. 4A). The upregulation of CD69 and CD25 activation markers, as well as the CD107a degranulation marker, was observed on CD3-positive cells (Fig. 4B). The concentrations of IL-2, IFN-γ and TNF-α in the supernatant were 1.24 ± 0.89, 103.58 ± 21.57 and 2.650 ± 1.19 ng/mL, respectively, all of which were significantly higher than in the MSC-EV group (Fig. 4C). Similarly, the PBMC proliferation ratio was substantially higher in the MSC-Tandab group compared to the corresponding control (Fig. 4D). However, there was no obvious change in Kasumi-1 cells and peripheral B cells after similar treatments (Fig. S6, S7).

Fig. 4
figure 4

Cytotoxicity of T cells towards MM.1S cells mediated by Tandab (CD3/BCMA) secreted from UCMSCs. In order to functionally validate the MSC-secreted Tandab (CD3/BCMA), a co-culture system using transwell plates with a 0.4-μm pore-size membrane was established. MSCs were plated into 24-well plates at a density of 2 × 104 cells per well after transduction with the lentiviral vector. Seventy-two hours later, MM.1S cells were labeled with CFSE. Then, PBMCs and labeled MM.1S cells (effector to target (E:T) ratio of 10:1) were added to the equilibrated inserts. After co-culture for 24 h, cells in the inserts were labeled with 7-AAD and detected by flow cytometry. (A) Specific lysis of MM.1S cells. (B) Activation surface markers CD25, CD69, and CD107a of T cells. (C) Cytokines including IL-2, IFN-γ, and TNF-α in the supernatant were measured using ELISA kits. (D) Representative flow cytometry analysis of the proliferation of PBMCs (labeled with CellTrace™ Far Red) after co-culture with MM.1S at an E:T ratio of 10:1 in the collected supernatants of the infected UCMSCs for 72 h (left) and statistical analysis of the data (right). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 compared with the corresponding MSC-EV group. The data represent the means ± SD from three repeated experiments

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MSC-Tandab mediated the lysis of primary MM cells by healthy donor T cells

The efficacy of MSC-Tandab was further tested on BMMNCs from 3 MM patients (Table 2). The expressions of BCMA was detected by flow cytometry (Fig. S8). After 24 h of co-culture, MSC-Tandab successfully mediated the killing of tumor cells by healthy donor T cells at an E:T ratio of 2:1. CFSE was utilized to identify the target cell population. Tumor lysis was calculated based on residual CFSE+ 7-AAD+ tumor cells detected by flow cytometry (Fig. 5A). In addition, we observed the upregulation of the CD25 and CD69 activation markers, as well as the CD107a degranulation marker (Fig. 5B). Co-culture experiments also revealed that MSC-Tandab induced T cells to secrete higher levels of cytokines than was observed with MSC-EV and MSCs (Fig. 5C).

Table 2 Patient information
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Fig. 5
figure 5

MSC-Tandab mediated lysis of primary MM cells by healthy donor T cells. (A) MM BMMNCs (labeled with CFSE) were incubated with healthy donor T cells (effector to target (E:T) ratio of 2:1) for 24 h in the presence of each of the three UCMSCs lines in a co-culture system using transwell plates with 0.4-μm pore-size membrane. Representative flow cytometry analysis of the specific lysis of CD138 + primary MM cells (left) and statistical analysis of the data (right), (n = 3). (B) Representative flow cytometry analysis of CD25+, CD69+, and CD107a+ subpopulations of healthy donor T cells after co-culture with MM BMMNCs in the presence of each of the three UCMSCs lines in a co-culture system using transwell plates with a 0.4-μm pore-size membrane (left) and statistical analysis of the data (right). (C) Quantification of cytokines (IL-2, TNF-α, and IFN-γ) released into the culture supernatants by healthy donor T cells as a result of activation and killing of tumor cells mediated by the three UCMSCs lines. *P < 0.05, **P < 0.01, ***P < 0.001 compared with the corresponding MSC-EV group. The data represent the means ± SD from three repeated experiments

Full size image

MSC-Tandab induced the activation of T cells derived from MM patients and mediated the killing of autologous MM cells

To test whether MSC-Tandab could activate T cells from MM patients and mediate the lysis of MM cells, T cells and MM tumor cells were sorted from BMMNCs samples of 3 MM patients (Table 3) and co-cultured with MSC-Tandab at an E:T ratio of 2:1 for 24 h. The expression of BCMA was detected by flow cytometry (Fig. S8). Remarkably, MM-derived T cells could also efficiently lyse their own MM tumor cells in the presence of MSC-Tandab (Fig. 6A). Moreover, MSC-Tandab could induce the activation of T cells derived from MM patients, as evidenced by upregulation of the activation markers CD25 and CD69, the degranulation marker CD107a, as well as IL-2, TNF-α and IFN-γ (Fig. 6B and C). Taken together, MSC-Tandab induced the functional activation of MM-derived T cells, resulting in potent lysis of MM cells, offering hope for future clinical applications.

Table 3 Patient information
Full size table
Fig. 6
figure 6

MSC-Tandab induced the activation of T cells derived from MM patients and mediated the killing of their own MM cells. (A) MM BMMNCs (labeled with CFSE) were incubated with MM-derived T cells (effector to target (E:T) ratio of 2:1) for 24 h in the presence of each of the three UCMSCs lines in a co-culture system using transwell plates with a 0.4 μm pore-size membrane. Representative flow cytometry analysis of the specific lysis of CD138+ primary MM cells (left) and statistical analysis of the data (right), (n = 3). (B) Representative flow cytometry analysis of CD25+, CD69+, and CD107a+ subpopulations among T cells derived from MM patients after co-culture with their own MM BMMNCs in the presence of each of the three UCMSC lines in a co-culture system using transwell plates with a 0.4 μm pore-size membrane (left) and statistical analysis of the data (right). (C) Quantification of cytokines (IL-2, TNF-α, and IFN-γ) released into the culture supernatants by patient-derived T cells as a result of activation and killing of their own MM cells mediated by each of the three UCMSC lines. *P < 0.05, **P < 0.01 compared with the corresponding MSC-EV group. The data represent the means ± SD from three repeated experiments

Full size image

Antitumor potential of MSC-Tandab against H929 xenograft tumors

To evaluate the antitumor potential of MSC-Tandab in vivo, a H929 xenograft model in NOD/SCID mice was established, in which MSCs and PBMCs were injected intravenously, as shown in Fig. 7A. The animals were sacrificed on day 27, after which tumor tissues were dissected and weighed for analysis. Compared with the mice in the control group (MSC-EV + PBMC), tumor weight and tumor volume were decreased in mice treated with MSC-Tandab (Fig. 7B and C). Moreover, no significant differences were detected in tumor size between the T and MSC-EV treated groups, indicating that wild-type MSCs had no direct effects on MM. In addition, the serum cytokine levels of MM xenograft NOD/SCID mice on day 27 were also analyzed. The results showed that the IFN-γ levels were higher in the MSC-Tandab group than in the MSC-EV group (51.54 vs. 35.29 pg/ml, p = 0.045;) (Fig. 7D). In addition, no obvious changes in the body weight of the mice were observed during the treatment (Fig. 7E). Confocal microscopy was performed to verify the presence of apoptotic cells and the infiltration of T cells in tumor tissues. Extensive apoptosis was detected in the tumor tissues of mice treated with MSC-Tandab, while the apoptotic rate was much lower in other groups. Lymphocytes labeled with CD3 were observed in tumor tissues of the MSC-Tandab group (Fig. 7F, Fig. S9). These data suggest that genetically modified MSCs selectively migrated to the tumor sites and released Tandab (CD3/BCMA), which could trigger T cells to kill tumor cells. What’s more, no significant apoptosis was detected in the lung and the liver tissues of all treatment groups (Fig. S10). Importantly, pathohistological analysis indicated that this treatment did not damage the main organs, such as the liver, spleen, and kidneys (Fig. 7G).

Fig. 7
figure 7

Tumor-suppressing effects of MSC-Tandab against MM xenografts in NOD/SCID mice. (A) Schematic diagram of in vivo evaluation of MSC-Tandab. NOD/SCID mice were injected subcutaneously with H929 cells (5 × 106 per mouse) into the right flank. Following 13 days after xenografting (day 13), MSCs (1 × 106 per mouse) were injected via the tail vein. The Mice received an injection of PBMCs (1 × 107 per mouse) on day 16. The second treatment was administered on day 20. All mice were sacrificed on day 27. (B) The tumor weights of different groups were measured at the end of treatment. n = 5 tumors. (C) The tumor volume was calculated using the formula (width2 × length × 0.5). n = 5 tumors. (D) Serum cytokine levels on day 27. n = 5 tumors. (E) Changes in the body weight of H929 xenograft models during the treatment. n = 5 tumors. (F) Representative confocal microscopy images for the tumor tissues in each treatment group on the day 27. Green (anti-CD3) indicates T cells, red (TUNEL) indicates apoptosis in tumors, and blue (DAPI) indicates nuclei. The dashed boxes indicate areas of apoptosis, while the white arrows denote T cell infiltration. Scale bar = 50 μm. n = 5 tumors. (G) Histological analysis of organs from the xenograft model mice. On day 27, organs including the liver, spleen, and kidneys were dissected and fixed prior to hematoxylin and eosin (HE) staining. Scale bar = 100 μm. n = 5 tumors. ns, not significant, *P < 0.05, ***P < 0.001 compared with the corresponding MSC-EV + T group

Full size image

Discussion

With the emergence of monoclonal antibodies and development of genetic engineering techniques, T-BsAbs therapy has entered a phase of rapid development, during which a large number of recombinant therapeutic molecules were constructed [16]. However, several limitations hinder the effectiveness of these molecules in clinical practice, including low antibody concentrations at the tumor sites and high production costs. Thus, introducing an efficient and targeted delivery system for therapeutic antibodies may enhance the efficacy of treatment. In this study, we engineered UCMSCs to secrete Tandab (CD3/BCMA) via lentiviral transduction and investigated the therapeutic efficacy of MSC-Tandab in multiple myeloma xenografts in NOD/SCID mice. The results demonstrated that intravenously injected engineered MSCs were able to specifically migrate to the tumor site and secrete Tandab (CD3/BCMA), thereby recruiting T cells to elicit a potent antitumor immune response.

Cell-based therapy has emerged as a novel approach to treat malignancies. MSCs hold great promise for clinical applications in the treatment of various diseases owing to their multilineage differentiation potential and immunosuppressive properties. Furthermore, the tumor tropism of MSCs has led to their utilization as attractive delivery vehicles for a wide spectrum of antitumor agents [17, 18]. BCMA has been widely recognized as a molecular marker of multiple myeloma, as it is specifically expressed on tumor cells. A tetravalent bispecific tandem diabody with two binding sites for CD3 and two for BCMA, Tandab (CD3/BCMA), with high specificity and affinity has been created in our previous work [14]. In the current study, we successfully constructed a Tandab lentivirus and transduced UCMSCs to produce MSC-Tandab (CD3/BCMA).

MSCs were selected as the vehicle for gene therapy due to their inherent tumor tropism. According to previous studies, stem cells are usually enriched in blood-rich organs such as the heart, lungs, and liver following intravenous injection, and the number of stem cells that infiltrate tumor tissues is limited [19]. However, there is increasing evidence that MSCs are able to migrate to tumor sites or areas of inflammation [20, 21]. In the current study, the tumor tropism of MSC-Tandab was demonstrated in vitro and in vivo, showing that the MM cells effectively attract MSC-Tandab, which can migrate to tumor sites effectively. Owing to blood circulation, recombinant protein molecules cannot maintain a high concentration for long to exert their anticancer effects [22]. Using MSCs as a vehicle to directly target tumors and constantly release recombinant molecules can effectively solve these problems.

MSCs are also an attractive choice for cellular drug delivery vehicles as they have stable genetic traits. It has been shown that introduction of adenovirus, lentivirus, retrovirus, and nano-magnetic particles into MSCs does not affect their tumor tropism and multi-directional differentiation potential [23]. Indeed, we demonstrated that transduction of UCMSCs with the Tandab-expressing lentiviral vector did not affect their morphology, differentiation potential and proliferation ability. Furthermore, to mimic the therapeutic mechanism of MSC-Tandab in vivo, we designed a co-culture system using transwell plates, in which Tandab (CD3/BCMA) secreted by MSC-Tandab could diffuse to the upper insert to trigger cytotoxicity of T cells against BCMA-positive cells. Analysis of the lysis of tumor cells and released cytokines confirmed our initial hypothesis. In addition to their tumor tropism to tumors and stable genetic background, their immunogenicity of MSC is low, as they do not express major histocompatibility complex I (MHC I), MHC II and co-stimulatory molecules. Therefore, these cells can effectively evade attacks from the immune system and sustainably express the fusion protein at tumor sites. More importantly, MSCs have an intrinsic ability to effectively secrete recombinant molecules. Indeed, we demonstrated in this study that the recombinant secretion level of MSC-Tandab (CD3/BCMA) continuously increased over several days of culture, reaching the peak titer of 8031 ± 487 pg/ml at day 9, and was detectable even at day 15, suggesting that the secretion of the Tandab protein was continuous and stable. This result was in accordance with other studies that reported the highest secretion level at 6–9 days, ranging from 1500 to 8247 pg/ml, with constant increases for 15 to 30 days after transduction [24, 25]. These features, together with their easy acquisition and amplification, make MSCs a preferred choice of cellular delivery vehicles for clinical use.

In spite of several advantages, the effect of MSCs on tumor growth remains the subject of debate, either promoting or inhibiting tumor development, which might be due to the factors related to the different sources of MSCs, the ratio of each cell population used in animal models, or alternative administration routes and similar differences [26,27,28]. In our in vitro co-culture experiments, we observed that the MSC group induced significant cytotoxicity against the MM cell line H929 compared to the blank group (32% vs. 67%). Given that we used transwell chambers with a pore size of 0.4 μm, which prevents direct contact of MSCs with effector and target cells, it stands to reason that the observed tumor-killing effect on H929 cells may be mediated by the paracrine secretion of certain factors by the MSCs. This result was consistent with previous finding that UCMSCs can significantly inhibit both MM proliferation and tumor growth in vitro and in vivo through the secretion of soluble factors [29]. These results further illustrate the potential of MSCs as effective delivery vehicles in the treatment of MM. Importantly, genetically modified MSCs demonstrated good in vivo safety. Although some MSCs were able to home to the liver and lungs to release small amounts of Tandab (CD3/BCMA), no tissue damage was observed. This may be related to the specific cytotoxicity of Tandab. Although concerns about MSC’s role in tumor may continue for some time, their application as a cellular carrier for anticancer agents is being actively investigated [30, 31].

Compared to the original bispecific antibody Tandab (CD3/BCMA), MSC-Tandab offers several advantages. MSC-Tandab can home to tumor sites and continuously secrete Tandab, circumventing storage and stability issues commonly associated with antibody drug production. Furthermore, while some MSCs may become trapped in tissues such as the lungs and liver, their ability to preferentially migrate to tumors helps minimize systemic distribution of the antibody drug, thereby reducing the incidence of adverse reactions. However, the MSC drug delivery system also faces challenges. Although most preclinical studies suggest that stem cell therapy is effective, many clinical trials, particularly those targeting hematological malignancies, remain in the early stages, indicating that there is still much work to be done in utilizing MSCs as drug delivery vehicles.

Conclusions

In summary, we successfully established a novel dual-target system for the treatment of multiple myeloma. Its potent inhibitory effect on multiple myeloma has been demonstrated both in vitro and in vivo. This dual-target system is an important complement to conventional antibody-based targeted molecular therapeutic strategies, laying an important theoretical and experimental basis for the clinical application of modified MSCs.

Availability of data and materials

The datasets used during the current study are available from the corresponding author on reasonable request.

Abbreviations

MM:

Multiple myeloma

MSCs:

Mesenchymal stem cells

UCMSCs:

Umbilical cord-derived mesenchymal stem cells

BCMA:

B-cell maturation antigen

PB:

Peripheral blood

T-BsAbs:

T-cell-redirecting bispecific antibodies

BM:

Bone marrow

Tandab:

Tandem diabody

BMMNCs:

Bone marrow mononuclear cells

NDMM:

Newly diagnosed multiple myeloma

RRMM:

Relapsed/refractory multiple myeloma

PBMCs:

Peripheral blood mononuclear cells

PBS:

Phosphate-buffered saline

GFP:

Green fluorescent protein

6 × His:

Hexa-histidine

MOI:

Multiplicity of infection

CM:

Culture medium

IL-2:

Interleukin-2

IFN-γ:

Interferon-γ

TNF-α:

Tumor necrosis factor-α

GGGGS:

Gly-Gly-Gly-Gly-Ser residues

BME:

β-Mercaptoethanol

References

  1. Siegel RL, Miller KD, Wagle NS, Jemal A. Cancer statistics, 2023. CA Cancer J Clin. 2023;73(1):17–48.

    Article  PubMed  Google Scholar 

  2. Silberstein J, Tuchman S, Grant SJ. What Is Multiple Myeloma? JAMA. 2022;327(5):497.

    Article  PubMed  Google Scholar 

  3. Röllig C, Knop S, Bornhäuser M. Multiple myeloma. Lancet Lond Engl. 2015;385(9983):2197–208.

    Article  Google Scholar 

  4. Swan D, Murphy P, Glavey S, Quinn J. Bispecific antibodies in multiple myeloma: opportunities to enhance efficacy and improve safety. Cancers Basel. 2023;15(6):1819.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Tian Z, Liu M, Zhang Y, Wang X. Bispecific T cell engagers: an emerging therapy for management of hematologic malignancies. J Hematol OncolJ Hematol Oncol. 2021May 3;14(1):75.

    Article  CAS  Google Scholar 

  6. Przepiorka D, Ko CW, Deisseroth A, Yancey CL, Candau-Chacon R, Chiu HJ, et al. FDA approval: blinatumomab. Clin Cancer Res Off J Am Assoc Cancer Res. 2015;21(18):4035–9.

    Article  CAS  Google Scholar 

  7. Cosenza M, Sacchi S, Pozzi S. Cytokine release syndrome associated with T-cell-based therapies for hematological malignancies: pathophysiology, clinical presentation, and treatment. Int J Mol Sci. 2021;22(14):7652.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Jain T, Litzow MR. No free rides: management of toxicities of novel immunotherapies in ALL, including financial. Blood Adv. 2018;2(22):3393–403.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Abstract 55: Generation of half-life extended anti-CD33 BiTE® antibody constructs compatible with once-weekly dosing | Cancer Research | American Association for Cancer Research [Internet]. [cited 2024 Sep 25]. Available from: https://aacrjournals.publicaciones.saludcastillayleon.es/cancerres/article/77/13_Supplement/55/621265/Abstract-55-Generation-of-half-life-extended-anti

  10. Sanz L, Compte M, Guijarro-Muñoz I, Álvarez-Vallina L. Non-hematopoietic stem cells as factories for in vivo therapeutic protein production. Gene Ther. 2012;19(1):1–7.

    Article  CAS  PubMed  Google Scholar 

  11. Aliperta R, Cartellieri M, Feldmann A, Arndt C, Koristka S, Michalk I, et al. Bispecific antibody releasing-mesenchymal stromal cell machinery for retargeting T cells towards acute myeloid leukemia blasts. Blood Cancer J. 2015;5(9): e348.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Heo JR, Hwang KA, Kim SU, Choi KC. A potential therapy using engineered stem cells prevented malignant melanoma in cellular and xenograft mouse models. Cancer Res Treat. 2019;51(2):797–811.

    Article  CAS  PubMed  Google Scholar 

  13. Kim DW, Staples M, Shinozuka K, Pantcheva P, Kang SD, Borlongan CV. Wharton’s jelly-derived mesenchymal stem cells: phenotypic characterization and optimizing their therapeutic potential for clinical applications. Int J Mol Sci. 2013;14(6):11692–712.

    Article  PubMed  PubMed Central  Google Scholar 

  14. Xiong M, Liu R, Lei X, Fan D, Lin F, Hao W, et al. A novel CD3/BCMA bispecific T-cell redirecting antibody for the treatment of multiple myeloma. J Immunother. 2022;45(2):78–88.

    Article  CAS  PubMed  Google Scholar 

  15. Ma L, Yong FX, Lin CB, Law F, Wu JX, Yang LY, et al. Human umbilical cord Wharton’s Jelly-derived mesenchymal stem cells differentiation into nerve-like cells. Chin Med J (Engl). 2005;118(23):1987–93.

    CAS  PubMed  Google Scholar 

  16. Zinn S, Vazquez-Lombardi R, Zimmermann C, Sapra P, Jermutus L, Christ D. Advances in antibody-based therapy in oncology. Nat Cancer. 2023;4(2):165–80.

    Article  PubMed  Google Scholar 

  17. Stoff-Khalili MA, Rivera AA, Mathis JM, Banerjee NS, Moon AS, Hess A, et al. Mesenchymal stem cells as a vehicle for targeted delivery of CRAds to lung metastases of breast carcinoma. Breast Cancer Res Treat. 2007;105(2):157–67.

    Article  PubMed  Google Scholar 

  18. Sm K, Jy L, Si P, Ch J, Jh O, et al. Gene therapy using TRAIL-secreting human umbilical cord blood-derived mesenchymal stem cells against intracranial glioma. Cancer Res [Internet]. 2008;68(23):9614.

    Article  Google Scholar 

  19. Nystedt J, Anderson H, Tikkanen J, Pietila M, Hirvonen T, Takalo R, et al. Cell surface structures influence lung clearance rate of systemically infused mesenchymal stromal cells. Stem cells. 2013;31(2):317–26.

    Article  CAS  PubMed  Google Scholar 

  20. Greco SJ, Rameshwar P. Mesenchymal stem cells in drug/gene delivery: implications for cell therapy. Ther Deliv. 2012;3(8):997–1004.

    Article  CAS  PubMed  Google Scholar 

  21. Kidd S, Spaeth E, Dembinski JL, Dietrich M, Watson K, Klopp A, et al. Direct evidence of mesenchymal stem cell tropism for tumor and wounding microenvironments using in vivo bioluminescent imaging. Stem Cells. 2009;27(10):2614–23.

    Article  CAS  PubMed  Google Scholar 

  22. Blanco B, Ramírez-Fernández Á, Alvarez-Vallina L. Engineering immune cells for in vivo secretion of tumor-specific t cell-redirecting bispecific antibodies. Front Immunol. 2020;11:1792.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Myers TJ, Granero-Molto F, Longobardi L, Li T, Yan Y, Spagnoli A. Mesenchymal stem cells at the intersection of cell and gene therapy. Expert Opin Biol Ther. 2010;10(12):1663–79.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Yan F, Li X, Li N, Zhang R, Wang Q, Ru Y, et al. Immunoproapoptotic molecule scFv-Fdt-tBid modified mesenchymal stem cells for prostate cancer dual-targeted therapy. Cancer Lett. 2017;402:32–42.

    Article  CAS  PubMed  Google Scholar 

  25. Zhang X, Yang Y, Zhang L, Lu Y, Zhang Q, Fan D, et al. Mesenchymal stromal cells as vehicles of tetravalent bispecific Tandab (CD3/CD19) for the treatment of B cell lymphoma combined with IDO pathway inhibitor d-1-methyl-tryptophan. J Hematol OncolJ Hematol Oncol. 2017;10(1):56.

    Article  Google Scholar 

  26. Rhee KJ, Lee J, Eom Y. Mesenchymal stem cell-mediated effects of tumor support or suppression. Int J Mol Sci. 2015;16(12):30015–33.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. Ribeiro A, Laranjeira P, Mendes S, Velada I, Leite C, Andrade P, et al. Mesenchymal stem cells from umbilical cord matrix, adipose tissue and bone marrow exhibit different capability to suppress peripheral blood B, natural killer and T cells. Stem Cell Res. 2013. https://doiorg.publicaciones.saludcastillayleon.es/10.1186/scrt336.

    Article  Google Scholar 

  28. Zhong W, Zhu Z, Xu X, Zhang H, Xiong H, Li Q, et al. Human bone marrow-derived mesenchymal stem cells promote the growth and drug-resistance of diffuse large B-cell lymphoma by secreting IL-6 and elevating IL-17A levels. J Exp Clin Cancer Res CR. 2019;38(1):73.

    Article  PubMed  Google Scholar 

  29. Ciavarella S, Caselli A, Tamma AV, Savonarola A, Loverro G, Paganelli R, et al. A peculiar molecular profile of umbilical cord-mesenchymal stromal cells drives their inhibitory effects on multiple myeloma cell growth and tumor progression. Stem Cells Dev. 2015;24(12):1457–70.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. Shi Y, Du L, Lin L, Wang Y. Tumour-associated mesenchymal stem/stromal cells: emerging therapeutic targets. Nat Rev Drug Discov. 2017;16(1):35–52.

    Article  CAS  PubMed  Google Scholar 

  31. Mesenchymal stem cell-mediated cancer therapy: A dual-targeted strategy of personalized medicine - PubMed [Internet]. [cited 2024 Nov 28]. Available from: https://pubmed.ncbi.nlm.nih.gov/22180830/

Download references

Acknowledgements

The authors declare that they have not use AI-generated work in this manuscript.

Funding

This work was supported by the National Natural Science Foundation of China (NO. 82400238), the Jiangxi Provincial Natural Science Foundation (No. 20224BAB216062), the Jiangxi Provincial Health Technology Project (No. 202410140), and the CAMS Innovation Fund for Medical Sciences (CIFMS: 2021-I2M-1–041).

Author information

Author notes

  1. Mengshang Xiong, Chunfang Kong and Yang Lu have contributed equally to this work.

Authors and Affiliations

  1. Department of Hematology, Jiangxi Provincial People’s Hospital, The First Affiliated Hospital of Nanchang Medical College, Nanchang, 330006, China

    Mengshang Xiong, Chunfang Kong, Weirong Ding, Tingting Zhang, Wei Zuo, Lixia Cao, Qiling Lu, Anna Li, Bo Ke & Caishui Wan

  2. Jiangxi Province Key Laboratory of Hematologic Diseases, Nanchang, 330006, China

    Mengshang Xiong, Chunfang Kong, Weirong Ding, Tingting Zhang, Wei Zuo, Lixia Cao, Qiling Lu, Anna Li, Bo Ke & Caishui Wan

  3. State Key Laboratory of Experimental Hematology, National Clinical Research Center for Blood Diseases, Haihe Laboratory of Cell Ecosystem, Institute of Hematology & Blood Diseases Hospital, Chinese Academy of Medical Sciences & Peking Union Medical College, Tianjin, 300020, China

    Yang Lu

  4. Tianjin Institutes of Health Science, Tianjin, 301600, China

    Yang Lu

  5. Obstetrics and Gynecology Department, Shanggao County People’s Hospital, Yichun, 331100, Jiangxi, China

    Jiaojun Liu

  6. Jiangxi Medical College, Nanchang University, Nanchang, 330006, China

    Chaoyu Li

  7. Institute of Clinical Medicine, Jiangxi Provincial People’s Hospital, The First Affiliated Hospital of Nanchang Medical College, Nanchang, 330006, China

    Chaoyu Li

  8. Jiangxi Province Key Laboratory of Immunology and Inflammation, Jiangxi Provincial People’s Hospital, Nanchang, 330001, China

    Liting Ding & Yutao Yan

  9. Medical College of Nanchang University, Nanchang, 330006, Jiangxi, China

    Bo Ke & Caishui Wan

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Contributions

CW, BK conceived, designed, and supervised the study, reviewed, and approved the manuscript. MX, CK and YL performed all the experiments, analyzed the data, and wrote the manuscript. JL, WD, TZ, and WZ provided experimental design and guidance. LC, QL, AL, and CL helped with the mice experiments. LD and YY helped in blood samples preparation. All authors read and approved the final manuscript.

Corresponding authors

Correspondence to Bo Ke or Caishui Wan.

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Ethics approval and consent to participate

All animal experiments received approval from the ethical committee of Nanchang Medical College (Project title: Human mesenchymal stem cells as vehicles of tetravalent bispecific Tandab for the treatment of multiple myeloma combined with gamma-secretase inhibitors and its mechanism study; Approval number: NYLLSC20240701; Date of approval: 01/07/2024). Human MSCs, PBMC, and CD138+ BMMNCs were respectively isolated from umbilical cord, peripheral blood, and bone marrow with informed patient consent in accordance with the ethical committee of Jiangxi Provincial People’s Hospital (Project title: Human mesenchymal stem cells as vehicles of tetravalent bispecific Tandab for the treatment of multiple myeloma combined with gamma-secretase inhibitors and its mechanism study; Approval number: 2024 (81); Date of approval: 11/11/2024).

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Supplementary Information

Supplementary Material 1: Fig. S1 Uncropped blot Tandab (CD3/BCMA) corresponding to Fig. 1F.

13287_2025_4212_MOESM2_ESM.tif

Supplementary Material 2: Fig. S2 Identification of MSCs and transduced MSCs. (A) Representative images showing the in vitro differentiation of MSCs and transduced MSCs into adipogenic and osteogenic lineages. Scale bar = 50 μm. (B) Relative quantification of gene expression after lineage differentiation in MSCs and transduced MSCs. The mRNA levels were normalized to GAPDH and compared with the MSCs group. ns, not significant, *P < 0.05. The data represent the means ± SD from three repeated experiments.

13287_2025_4212_MOESM3_ESM.pdf

Supplementary Material 3: Fig. S3 Binding specificity of Tandab (CD3/BCMA) in the culture supernatants. (A) Direct binding activities of commercial APC-human BCMA antibody and FITC-human CD3 antibody toward Kasumi-1 cells. (B) The culture supernatants of UCMSCs transduced with different lentiviral vectors for 72 h were collected for the direct-binding assay. Direct binding activities of the culture supernatants toward Kasumi-1 cells were assessed using flow cytometry. The FITC-His tag antibody was used as the secondary antibody.

13287_2025_4212_MOESM4_ESM.pdf

Supplementary Material 4: Fig. S4 The distribution of fusion proteins and MSCs in lung and liver tissues were assessed by immunofluorescence staining. Red (anti-CD90) indicates MSCs, green (anti-His) indicates Tandab (CD3/BCMA), and blue (DAPI) indicates nuclei. White arrows indicate the co-localization of Tandab (CD3/BCMA) and MSCs. Scale bar, a: 200 μm, b: 20 μm.

13287_2025_4212_MOESM5_ESM.pdf

Supplementary Material 5: Fig. S5 Each fluorescence channel in Fig. 3D. DAPI (blue), anti-CD90 (red), anti-His (green). Scale bar = 200 μm.

13287_2025_4212_MOESM6_ESM.pdf

Supplementary Material 6: Fig. S6 Cytotoxicity of T cells toward Kasumi-1 cells mediated by Tandab (CD3/BCMA) secreted from UCMSCs. A co-culture system using transwell plates with a 0.4 μm pore-size membrane was established. MSCs were plated into 24-well plates at a density of 2 × 104 cells per well after lentiviral transduction. Seventy-two hours later, Kasumi-1 cells were labeled with CFSE. Then, PBMCs and labeled Kasumi-1 cells (effector to target (E:T) ratio of 10:1) were added to the equilibrated inserts. After co-culture for 24 h, cells in the inserts were labeled with 7-AAD and detected by flow cytometry. (A) Specific lysis of Kasumi-1 cells. (B) Activation surface markers CD25, CD69, and CD107a of T cells. (C) Cytokines including IL-2, IFN-γ, and TNF-α in the supernatant were measured using ELISA kits. (D) Representative flow cytometry analysis on the proliferation of PBMCs (labeled with CellTrace™ Far Red) after co-cultured with Kasumi-1 cells at an E:T ratio of 10:1 in the collected supernatants of the transduced UCMSCs for 72 h (left) and statistical analysis of the data (right). The data represent the means ± SD from three repeated experiments.

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Supplementary Material 7: Fig. S7 Cytotoxicity of T cells toward peripheral blood B cells mediated by MSC-Tandab. A co-culture system using transwell plates with a 0.4 μm pore-size membrane was established. MSCs were plated into 24-well plates at a density of 2 × 104 cells per well after lentiviral transduction. Seventy-two hours later, peripheral blood B cells from healthy donors were labeled with CFSE. Then, PBMCs and labeled B cells (effector to target (E:T) ratio of 10:1) were added to the equilibrated inserts. After co-culture for 24 h, cells in the inserts were labeled with 7-AAD and detected by flow cytometry. The data represent the means ± SD from three repeated experiments.

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Supplementary Material 8: Fig. S8 Representative flow cytometry analysis of the expression of BCMA on BMMNCs from six MM patients. Black, negative control; red, anti-BCMA antibody.

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Supplementary Material 9: Fig. S9 Each fluorescence channel in Fig. 7F. DAPI (blue), TUNEL (red), anti-CD3 (green). The dashed boxes indicate areas of apoptosis, while the white arrows denote T cell infiltration. Scale bar = 50 μm.

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Supplementary Material 10: Fig. S10 Apoptosis in lung (A) and liver tissues (B) in each treatment group was analyzed by TUNEL staining. Red (TUNEL) indicates apoptosis in tissues and blue (DAPI) indicates nuclei. The dashed boxes indicate areas of apoptosis. Scale bar = 200 μm.

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Xiong, M., Kong, C., Lu, Y. et al. Construction of tetravalent bispecific Tandab (CD3/BCMA)-secreting human umbilical cord mesenchymal stem cells and its efficiency in the treatment of multiple myeloma. Stem Cell Res Ther 16, 69 (2025). https://doiorg.publicaciones.saludcastillayleon.es/10.1186/s13287-025-04212-w

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  • DOI: https://doiorg.publicaciones.saludcastillayleon.es/10.1186/s13287-025-04212-w

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Stem Cell Research & Therapy

ISSN: 1757-6512