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. 2023 Mar 8;15(6):1647.
doi: 10.3390/cancers15061647.

The First-In-Class Anti-AXL×CD3ε Pronectin™-Based Bispecific T-Cell Engager Is Active in Preclinical Models of Human Soft Tissue and Bone Sarcomas

Nicoletta Polerà  1 , Antonia Mancuso  1 , Caterina Riillo  1 , Daniele Caracciolo  1 , Stefania Signorelli  1 , Katia Grillone  1 , Serena Ascrizzi  1 , Craig A Hokanson  2 , Francesco Conforti  3 , Nicoletta Staropoli  1 , Luigia Gervasi  1 , Maria Teresa Di Martino  1 , Mariamena Arbitrio  4 , Giuseppe Nisticò  5 , Roberto Crea  2   5 , Pierosandro Tagliaferri  1 , Giada Juli  1 , Pierfrancesco Tassone  1   6
Affiliations

The First-In-Class Anti-AXL×CD3ε Pronectin™-Based Bispecific T-Cell Engager Is Active in Preclinical Models of Human Soft Tissue and Bone Sarcomas

Nicoletta Polerà et al. Cancers (Basel). .

Abstract

Sarcomas are heterogeneous malignancies with limited therapeutic options and a poor prognosis. We developed an innovative immunotherapeutic agent, a first-in-class Pronectin™-based Bispecific T-Cell Engager (pAXL×CD3ε), for the targeting of AXL, a TAM family tyrosine kinase receptor highly expressed in sarcomas. AXL expression was first analyzed by flow cytometry, qRT-PCR, and Western blot on a panel of sarcoma cell lines. The T-cell-mediated pAXL×CD3ε cytotoxicity against sarcoma cells was investigated by flow cytometry, luminescence assay, and fluorescent microscopy imaging. The activation and degranulation of T cells induced by pAXL×CD3ε were evaluated by flow cytometry. The antitumor activity induced by pAXL×CD3ε in combination with trabectedin was also investigated. In vivo activity studies of pAXL×CD3ε were performed in immunocompromised mice (NSG), engrafted with human sarcoma cells and reconstituted with human peripheral blood mononuclear cells from healthy donors. Most sarcoma cells showed high expression of AXL. pAXL×CD3ε triggered T-lymphocyte activation and induced dose-dependent T-cell-mediated cytotoxicity. The combination of pAXL×CD3ε with trabectedin increased cytotoxicity. pAXL×CD3ε inhibited the in vivo growth of human sarcoma xenografts, increasing the survival of treated mice. Our data demonstrate the antitumor efficacy of pAXL×CD3ε against sarcoma cells, providing a translational framework for the clinical development of pAXL×CD3ε in the treatment of human sarcomas, aggressive and still-incurable malignancies.

Keywords: AXL; BTCE; bispecific T-cell engager; cancer; immunotherapy; pronectins™; sarcomas.

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Conflict of interest statement

R.C. and C.A.H. are associated with Protelica, Inc. that is the owner of Pronectins™ patent. The other authors declare no conflict of interest.

Figures

Figure 1
Figure 1
AXL expression. (A) Schematic representation of AXL receptor tyrosine kinase structure. It is composed of two immunoglobulin (Ig)-like domains that characterize extracellular domains, two fibronectin type III (FNIII) domains, a transmembrane domain and a kinase domain that is intracellular. (B) Flow cytometry analysis of AXL expression of sarcoma cells. (C) Representative FACS overlays between unstained (empty) and stained (full red) sample of each cell line. (D) Quantification of antibody-binding capacity (ABC) assay. (E) AXL-relative mRNA level determined by qRT-PCR and normalized on GAPDH housekeeping. (F) Western blot of AXL total form reported in a collection panel of sarcoma cells. The uncropped blots are shown in Figure S3.
Figure 2
Figure 2
Redirected T-lymphocyte cytotoxicity by pAXL×CD3ε in sarcoma cell lines. (A) FACS analysis of 7-AAD(7-amino-actinomycin D)-positive cells. (B) HT-1080 and Rh-30 treated with increasing concentrations of pAXL×CD3ε (0.1 µg/mL, 1 µg/mL and 2.5 µg/mL), and as a negative control 2.5 µg/mL of pBCMA×CD3ε after 72 h. Each group has been compared to the 0.1 µg/mL group for statistical analysis. Results are normalized to the recorded vehicle values. (C) Cell Titer-Glo luminescent cell viability (%) assay performed on sarcoma cell lines without effector cells, following pAXL×CD3ε 72 h treatment. (C) Positive cells (relative percentage %) of sarcoma cell lines co-cultured with peripheral blood mononuclear cells (PBMCs) and treated with different concentrations of pAXL×CD3ε or vehicle for 72 h. Each group has been compared to the 0.1 µg/mL group for statistical analysis. Results are normalized to the recorded vehicle values. (D) Imaging of CAL-72 stably expressing green fluorescent protein (GFP) in untreated cells (vehicle) and 2.5 µg/mL of pAXL×CD3ε-treated cells after 72 h. Nuclei were stained with DAPI and microscopies were performed at 10-fold magnification. (E) Percentage of stably expressing CAL-72 GFP viable cells and median fluorescence intensity (MFI) of GFP analyzed by flow cytometry. PBMCs were obtained from 3 healthy donors and results are expressed as the mean value of triplicate experiments from each donor. * p < 0.0332; ** p < 0.0021; *** p < 0.0002; **** p < 0.0001.
Figure 3
Figure 3
Functional experiments on CD4–CD8 gated T cells. Surface early and late activation markers (CD69 and CD25), cytokine release (IFN-γ) and cytolytic enzyme (Granzyme B) on CD4 and CD8-positive T lymphocytes from at least 3 donors co-cultured with Rh-30, HT-1080 and CAL-72 sarcoma cell lines at 10:1 E:T ratio, in the presence of different concentrations of pAXL×CD3ε. Each result is expressed as the mean value of triplicate experiments obtained from each donor. * p < 0.0332; ** p < 0.0021; *** p < 0.0002.
Figure 4
Figure 4
Degranulation assay on activated T lymphocytes. (A) Histogram quantification of CD107a-positive T cells from at least 3 donors, co-cultured with Rh-30, HT-1080 and CAL-72 sarcoma cell lines and treated with 0.1 µg/mL and 1 µg/mL of pAXL×CD3ε for 72 h. (B) Representative dot plots of CD107a-positive T lymphocytes analyzed by flow cytometry. Each result is expressed as the mean value of triplicate experiments obtained from each donor. * p < 0.0332.
Figure 5
Figure 5
pAXL×CD3ε sensitizes chemotherapeutic drug activity in vitro. (A) Redirected cytotoxicity T-cell of pAXL×CD3ε in SAOS-2, analyzed in co-culture experiments using 1 µg/mL of pAXL×CD3ε, 0.2 nM of trabectedin and their combination (Combo) for 72 h. (B) Representative dot plots of SAOS-2 viability reduction (%) after treatment with pAXL×CD3ε and trabectedin alone or in combination. PBMCs were obtained from 3 healthy donors and results are expressed as the mean value of triplicate experiments from each donor. * p < 0.0332.
Figure 6
Figure 6
pAXL×CD3ε in vivo activity. (A) Experimental timeline of in vivo study on a sarcoma xenograft model. (B) Tumor volume curve of mice treated with pAXL×CD3ε 0.1 mg/kg or vehicle alone. Results obtained from each group have been compared at the same timepoint for statistical purposes. (C) Survival curves (Kaplan–Meier) of mice treated with pAXL×CD3ε or vehicle. (D) Representative FACS dot plots of T-cell engraftment evaluated on the day of sacrifice on intracardiac blood samples collected from mice. CD3-positive cells were evaluated on gated CD45-positive lymphocytes. (E) WB analysis of cleaved caspase-3 and cleaved PARP in whole-cell protein extracts from representative retrieved xenografts. GAPDH was used as a loading control. The uncropped blots are shown in Figure S3. (F) IHC staining of CD3 lymphocytes performed on tumors explanted from mice treated with vehicle or pAXL×CD3ε 0.1 mg/kg at 20-fold magnification. * p < 0.0332; ** p < 0.0021.
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References

    1. Agnoletto C., Caruso C., Garofalo C. Heterogeneous circulating tumor cells in sarcoma: Implication for clinical practice. Cancers. 2021;13:2189. doi: 10.3390/cancers13092189. - DOI - PMC - PubMed
    1. Siegel R.L., Miller K.D., Wagle N.S., Jemal A. Cancer statistics, 2023. CA A Cancer J. Clin. 2023;73:17–48. doi: 10.3322/caac.21763. - DOI - PubMed
    1. Damerell V., Pepper M.S., Prince S. Molecular mechanisms underpinning sarcomas and implications for current and future therapy. Signal Transduct. Target. Ther. 2021;6:246. doi: 10.1038/s41392-021-00647-8. - DOI - PMC - PubMed
    1. Petrella A., Storey L., Hulbert-Williams N.J., Fern L.A., Lawal M., Gerrand C., Windsor R., Woodford J., Bradley J., O’Sullivan H. Fear of Cancer Recurrence in Patients with Sarcoma in the United Kingdom. Cancers. 2023;15:956. doi: 10.3390/cancers15030956. - DOI - PMC - PubMed
    1. Pushpam D., Garg V., Ganguly S., Biswas B. Management of refractory pediatric sarcoma: Current challenges and future prospects. OncoTargets Ther. 2020;13:5093. doi: 10.2147/OTT.S193363. - DOI - PMC - PubMed

Grants and funding

This manuscript has been supported by Institutional funds at Department of Experimental and Clinical Medicine (T27), University Magna Graecia of Catanzaro.
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