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, 12 (1), 46

Bispecific CD3-HAC Carried by E1A-engineered Mesenchymal Stromal Cells Against Metastatic Breast Cancer by Blocking PD-L1 and Activating T Cells

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Bispecific CD3-HAC Carried by E1A-engineered Mesenchymal Stromal Cells Against Metastatic Breast Cancer by Blocking PD-L1 and Activating T Cells

Yuanyuan Yang et al. J Hematol Oncol.

Abstract

Background: PD-1/PD-L1 blockade can confer durable benefits in the treatment of metastatic cancers, but the response rate remains modest and potential adverse effects occur sometimes. Concentrating immunotherapeutic agents at the site of disease was believed to break local immune tolerance and reduce systemic toxicity. E1A-engineered mesenchymal stromal cell (MSC.E1A) was an attractive transfer system that preferentially homing and treating cancer metastasis, through which the tumor cells were modified by locally replicated adenoviruses to release CD3-HAC, a bifunctional fusion protein that anti-CD3 scfv linked with high-affinity consensus (HAC) PD-1. Subsequently, CD3-HAC, wbich was bound on PD-L1-positive breast cancer cells, recruited T cells to exhibit a potent antitumor immunity incombination with immune checkpoint blockade.

Methods: We constructed the CD3-HAC gene driven by human telomerase reverse transcriptase (hTERT) promoter into an adenoviral vector and the E1A gene into the lentiviral vector. The homing property of MSCs in vivo was analyzed with firefly luciferase-labeled MSCs (MSC.Luc) by bioluminescent imaging (BLI). The cytotoxicity of T cells induced by CD3-HAC towards PD-L1-positive cells was detected in vitro and in vivo in combination with 5-FU.

Results: Our data suggest that CD3-HAC could specifically bind to PD-L1-positive tumor cells and induce lymphocyte-mediated lysis effectively both in vitro and in vivo. The intervention with HAC diminished the effects of PD-1/PD-L1 axis on T cells exposed to MDA-MB-231 cells and increased lymphocytes activation. MSCs infected by AdCD3-HAC followed by LentiR.E1A could specially migrate to metastasis of breast cancer and produce adenoviruses in the tumor sites. Furthermore, treatment with MSC.CD3-HAC.E1A in combination with 5-FU significantly inhibited the tumor growth in mice.

Conclusions: This adenovirus-loaded MSC.E1A system provides a promising strategy for the identification and elimination of metastasis with locally released immuno-modulator.

Keywords: CD3-HAC; Immunotherapy; MSC; Metastatic; PD-L1.

Conflict of interest statement

Ethics approval and consent to participate

All animal studies were performed in accordance with guidelines under the Animal Ethics Committee of the Institute of Hematology & Hospital of Blood Diseases, Chinese Academy of Medical Sciences & Peking Union Medical College.

Consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.

Publisher’s Note

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

Figures

Fig. 1
Fig. 1
Specific binding capacities of CD3-HAC to PD-L1-positive cells and CD3-positive cells. a Schematic representation of adenoviral expression vector for CD3-HAC. hTERT, promoter of human telomerase reverse transcriptase; SP, signal peptide, a murine kappa light-chain leader peptide; His6, hexa-histidine tag; G4S, Gly-Gly-Gly-Gly-Ser residues. b, c PD-L1 expression on breast cancer cells (MDA-MB-231 and MCF-7) detected by flow cytometry and immunofluorescence. (a) Negative control. (b) PE-labeled anti-PD-L1 antibody. Blue (nuclei); red (anti-PD-L1). Scale bar, 10 μm. d Competitive binding activity of purified CD3-HAC with anti-PD-L1 antibody on MDA-MB-231 cells. (a) Negative control. (b) CD3-HAC+anti-PD-L1 antibody. (c) Anti-PD-L1 antibody alone. e Binding specificity of CD3-HAC to Jurkat cells. (a) Negative control. (b) CD3-HAC. f, g Flow cytometry and immunofluorescence analyses performed on breast tumor cell lines infected by AdCD3-HAC to detect the binding specificities of CD3-HAC with anti-His antibody. Blue (nuclei). Green (GFP) indicates the cells infected by adenovirus. Red (anti-His for CD3-HAC). Scale bar, 35 μ m
Fig. 2
Fig. 2
The specific interaction between PBMCs and PD-L1-positive cell lines induced by CD3-HAC. a LDH release assays performed on MDA-MB-231 (PD-L1hi) cells or MCF-7 (PD-L1low) cells infected by various adenovirus in the presence of PBMCs in different effector to target (E:T) ratios for 10 h. b Cytokines including IL-2, IFN-γ, and TNF-α in the co-culture supernatants at E:T ratio (10:1) were detected by ELISA kits. c Flow cytometry analysis on CD69 and CD25 expression on T cells after co-cultured with adenovirus loaded MDA-MB-231 cells for 24 h. d Flow cytometry analysis on the proliferation of PBMCs (labeled with CellTrace™ Far Red) after co-cultured with adenovirus-loaded MDA-MB-231 at E:T radio (10:1) at indicated time. e Representative pictures of PBMC proliferation assay. f Dynamic observation of cell interaction by living cells workstation. Green-colored cells were AdCD3-HAC-transfected MDA-MB-231, and others were PBMCs. Scale bar, 50 μm. SD shown, n ≥ 3. One-way ANOVA with Tukey’s post-test in all cases. ** P < 0.01; ***P < 0.001; ****P < 0.0001; compared with Adtrack group
Fig. 3
Fig. 3
Lymphocytes cytotoxicity mediated by AdCD3-HAC was enhanced by 5-FU through upregulation of CAR and αvβ3. a ELISA analyses for IFN-γ on supernatants from 231.CD3scfv cells incubated with PBMCs for 3 days followed by that floating cells were harvested for second-round co-incubation with or without HAC for 5 days. b Jurkat T cells were incubated either alone or in co-culture with various virus-loaded MDA-MB-231 cells at the ratio of 1:10 for 10 h. Apoptosis of Jurkat cells was determined by flow cytometry using FITC-Annexin V. c Representative images show the percentages of apoptotic Jurkat cells. d Cytotoxicity of PBMCs to MDA-MB-231 cells infected with different low MOIs of AdCD3-HAC (E:T = 10:1) with or without 5-FU. e Cytokines including IL-2, IFN-γ, and TNF-α in the corresponding co-culture supernatants from d were detected by ELISA. f Flow cytometry analysis performed on the infection efficiencies of adenovirus to MDA-MB-231 cells, which were pretreated with 5-FU (0, 0.25, and 0.5 μg/mL) for 48 h, at different MOIs. gi Flow cytometry analysis on the expression level of CAR, αvβ3, and PD-L1 on the surface of MDA-MB-231 cells and MCF-7 cells treated with low doses of 5-FU for 48 h. j Mean fluorescence intensity of PD-L1 on MDA-MB-231 cells treated with 5-FU. SD shown, n ≥ 3. Two-tailed unpaired t test for a and j and one-way ANOVA with Tukey’s post-test for b, e, f, g, h, and i. *P < 0.05; **P < 0.01; ***P < 0.001;****P < 0.0001
Fig. 4
Fig. 4
Release of adenovirus by co-infected MSCs and the re-infection ability for tumor cells. a Droplet Digital PCR analysis on adenoviral DNA in the intracellular and supernatant from MSC.Adtrack.E1A at indicated time. The infection of LentiR.E1A was set as 0 h. b Total DNA numbers of adenovirus (intracellular plus supernatant) in MSC.Adtrack.E1A. c Electron micrographs showing viral particles in MSCs 48 h after co-infection. Adenovirus particles were indicated by white arrow. MSC.Adtrack.LentiR. maintained integral cell morphology (left panel); MSC.Adtrack.E1A lysed to release the new packaged adenoviral particles (middle panel); high magnification of MSC.Adtrack.E1A (right panel). d Flow cytometry analysis on the re-infection efficiency of new released adenoviruses to MDA-MD-231 cells after co-cultured with different amount of MSC.Adtrack.E1A for 72 h with or without 5-FU. SD shown, n ≥ 3. One-way ANOVA with Tukey’s post-test for d. ** P < 0.01; ***P < 0.001; compared with group in the absence of 5-FU
Fig. 5
Fig. 5
MSCs migrate to metastatic breast cancer in vivo. a Frozen sections of the lungs from Luc-231 tumor-bearing and tumor-free mice sacrificed 24 h after MCS.AdLuc.LentiR infusion were stained with anti-PD-L1 (red) for lung metastasis, anti-Luc (green) for luciferase expressed by MSCs, and DAPI (blue). White arrows indicate the co-localization of lung metastatic sites and MSCs. Scale bar, 50 μm. b Representative images show the homing capability of different virus-loaded MSCs to tumor sites. MSC.AdLuc.E1A or MSC.AdLuc.LentiR. were intravenously injected into mice hosting MDA-MB-231 in the lung or tumor-free control. Luciferase signal was monitored by bioluminescence imaging using Xenogen imaging system at indicated time. c Quantification of luciferase activity of MSCs in the lungs of MDA-MB-231 tumor-bearing or tumor-free mice at different time after MSCs infusion. Relative Luc activity (RLA) = Log2 [(luciferase ROI of the mice infused with MSC.AdLuc.E1A or MSC.AdLuc.LentiR.)/(luciferase ROI of control mice infused with PBS)], (n = 3)
Fig. 6
Fig. 6
Dual viruses-loaded MSCs delivering CD3-HAC to metastatic niche. a Frozen sections of the lungs from 231-Luc tumor-bearing mice sacrificed at indicted time points after MSC.Adtrack.LentiR. or MSC.Adtrack.E1A infusion showed the change of gene expression of engineered MSC in vivo. Blue (nuclei). Green (GFP) represented the cells infected by adenovirus. Red (anti-Luc) represented MDA-MB-231 cells. White (DsRed) represented the expression of E1A. Pictures of each channel were displayed in Additional file 1: Fig. S9. b MSC.CD3-HAC.E1A or MSC.Adtrack.E1A were injected intravenously into 231-Luc hosting mice, followed by PBMCs infusion after 2 days. IFN-γ+ cells were detected in tumor sites. White arrows indicated the interaction between the IFN-γ+ cells and adenovirus-infected tumor cells. Green (GFP) represented the cells infected by adenovirus. Red (Cell Trace™ Far Red) represented PBMCs. White (anti-IFN-γ). Scar bar, 50 μm
Fig. 7
Fig. 7
Tumor suppression of MSC.CD3-HAC.E1A in combination with 5-FU against MDA-MB-231 lung metastasis. a Design and timeline for tumor therapy. BALB/c nude mice were injected with MDA-MB-231-Luc cells (Luc-231, 1 × 106 per mouse) via tail vein. Seven days later (day 1), MSCs (1 × 106 per mouse) were administrated intravenously into the Luc-231 tumor-bearing mice. Then, mice received intravenous injection of PBMCs (5 × 106 per mouse) on day 4. Besides, 5-FU was given (i.p., 20 mg/kg) every other day along the treatment. The same treatment was performed another two times as indicated. i.p., intraperitoneal. b IVIS imaging of MDA-MB-231-Luc cells were shown for all groups. IVIS imaging was taken before (day 0) and after MSCs treatment (day 10 and day 20). Quantification of luciferase signals in the lung on day 10 (c) and day 20 (d). e Mouse survival after MSC.CD3-HAC.E1A treatment (n = 7 per group). In c, relative growth index = luciferase read on day 10 /luciferase read on day 0. In d, lung metastasis index = log10 [(luciferase read of the tested mice)/(luciferase read of average for tumor-free mice)]. *P <0.05 and **P <0.01 compared with the PBS group. In e, P = 0.0108, MSC.CD3-HAC.E1A+PBMC versus PBS; P = 0.0161, MSC.CD3-HAC.E1A+PBMC+5-FU versus PBS. Median survival (days): PBS, 60; 5-FU, 61; MSC+PBMC, 56; MSC.CD3-HAC.E1A+PBMC, 69; MSC.CD3-HAC.E1A+PBMC+ 5-FU, 89

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