The Collagen Receptor uPARAP as a Therapeutic Target for Mesothelioma

Abstract

This work focuses on a preclinical study of novel targeted therapy for mesothelioma.
Mesothelioma is a very aggressive cancer disease with very low overall survival rates. Asbestos plays a critical role in the cascade of molecular events leading to mesothelioma carcinogenesis, and its exposure is causative in more than 80% of the cases. However, despite the use of many asbestos minerals being prohibited in industrialized nations, the mesothelioma incidence rates are increasing in the world. Furthermore, with no targeted therapies approved for mesothelioma, a novel potential target leading to an effective treatment modality would fulfill a crucial need in the clinic.
The initiation of this project was based on indications of elevated urokinase plasminogen activator receptor-associated protein (uPARAP) expression in mesothelioma. uPARAP is a collagen receptor that contributes to several mechanisms (presented in this thesis), notably including extracellular
matrix remodeling. Many properties of uPARAP provide an advantage for it to be a successful candidate as an antibody-drug conjugate (ADC) target, such as favorable internalizing kinetics, rapid recycling, robust intracellular trafficking, and, importantly, suitable expression patterns (high expression in certain cancers with only limited expression in healthy tissue). Therefore, the thesis work was focused on the expression of uPARAP in mesothelioma and the potential of utilizing this receptor as a therapeutic target in this disease.
The first study (Article I) presents results that substantiate the high uPARAP expression in mesothelioma. This information was available at the mRNA level from publicly available databases from the start but was shown in this work at the protein level and analyzed in detail employing immunohistochemical assessment of whole tissue sections and tissue microarrays. uPARAP
expression was demonstrated in all main mesothelioma subtypes (epithelioid, biphasic, and sarcomatoid), where it was found to be upregulated compared to benign reactive mesothelial proliferation (RMP). Specimens from sarcomatoid and biphasic tumors seemed to show a higher uPARAP expression than tumors of the epithelial subtype on average, but there was a large variation within each subtype. Furthermore, neither the patient´s history with respect to chemotherapy nor the history of asbestos exposure nor the tumor genotype in terms of the deletion of the tumor suppressor protein BAP1 were found to be essential elements in uPARAP expression. Altogether, it was concluded that the mesothelioma subtype was not sufficient for the stratification of patients
for a putative uPARAP-directed treatment, which should rather be based on an individual assessment of the expression of the target. In the in vitro part of this study, we first demonstrated the uPARAP expression in several mesothelioma cell lines and their capacity for internalization of anti-uPARAP antibody, utilizing flow cytometry and confocal microscopy techniques. After this, we investigated whether it would be possible to target these cells in vitro, specifically with a uPARAPdirected ADC. To do this, we prepared ADCs, conjugating anti-uPARAP antibody as well as a nontargeting control antibody to a highly potent cytotoxin (PNU). The resulting ADCs were assayed on cultured mesothelioma cells. This assay revealed efficient eradication of mesothelioma cells with low concentrations of the uPARAP-targeted ADC. Furthermore, the pronounced specificity of the
uPARAP-targeting reagent was documented by comparison with the non-targeting ADC. Altogether, this study provided an important part of a proof-of-concept for this type of treatment in vitro.

In the second study (Manuscript I), building up on our initial results, we advanced to the preclinical assessment of uPARAP-targeted ADCs in subcutaneous mesothelioma models in mice. We established biphasic H-meso-1 cell line-derived (CDX) and NCI-Meso79 patient-derived (PDX) mouse
models. Then, we demonstrated the anti-tumor activity of a uPARAP-targeting ADC. For this study, we employed an anti-uPARAP antibody conjugated to the cytotoxin monomethyl auristatin E (MMAE), which has been clinically approved in the context of ADCs against other protein targets.
This uPARAP-targeting ADC was tested in two doses, 6 mg/kg and 10 mg/kg. The ADC treatment resulted in a pronounced delay in the CDX tumor growth (low and high dose treatments) and a tumor volume reduction, showing an even more pronounced effect in the PDX model (only high dose treatment tested). In all of these cases, the effect on tumor growth was significant compared to all control groups (non-targeted ADC, unconjugated mAb, and saline). The results obtained in this study were promising in terms of uPARAP-ADC efficiency, and at the same time, the setup made it possible to obtain some impression of potential target-specific side effects since the antibody obtained is cross-reactive between human and mouse uPARAP. We found no initial side effects of treatment in these preclinical models but did observe a weight drop in the mice during the longterm
follow-up period when using high-dose uPARAP-ADC. However, additional studies with nontumor-bearing mice with an immunocompetent background suggested that this effect was mouse strain dependent and that a further complicating factor was induced by the invasive growth of the tumor itself in the described long-term studies.
The last study (presented as a separate project in this thesis) had the aim to perform a preliminary investigation of uPARAP as a target for a novel chimeric antigen receptor (CAR) T cell therapy.
Initially, we produced a recombinant single chain variant fragment (scFv), derived from a monoclonal antibody against uPARAP and showed its functional binding. Later, the CAR was constructed using two orientations of the antigen-binding regions from the same antibody. The vector plasmids containing those constructs were used to produce CAR T cells from peripheral blood mononuclear cells (PBMCs) from different healthy donors. The initial functional results of CAR T cells
(effector function and cytokine production) appeared promising. However, further productions of CAR T cells derived from different donor PBMCs, performed to increase the proliferation of those cells, did not provide similar functional outcomes. This study is, for now, inconclusive to determine uPARAP’s potential in such kind of therapy for mesothelioma.
This thesis has delivered promising findings across a diverse range of contexts with relevance for mesothelioma research: immunohistochemical evaluation of patient materials with respect to the current potential target, in vitro assays, and, eventually, animal studies. Altogether, this study may contribute to paving the way for the development of a successful treatment modality targeting uPARAP as a potential remedy for mesothelioma.
Original languageDanish
Number of pages134
Publication statusPublished - 2024

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