Preclinical development of a uPAR-specific Antibody-Drug Conjugate for pancreatic cancer therapy

Antibody-drug conjugates (ADCs) have marked a breakthrough in the era of targeted therapies, with 14 FDA-marketed agents over a decade, and more than hundreds under advanced pre- and clinical investigation. Though most of the approvals have been originally granted for hematological malignancies, the treatment of solid tumors has posed considerable challenges in terms of target
antigen (Ag) selection and intratumoral drug delivery. While the human epidermal growth factor receptor 2 (HER2) remains the main validated ADC target in a few solid tumors, particularly breast cancer, research efforts currently aim at exploring alternative targets to expand the oncological indications that might benefit from this new generation of drugs.
In this perspective, a promising reemerging target is the urokinase plasminogen activator receptor or uPAR. Known as a key regulator of the extracellular proteolysis driving metastatic progression in most aggressive cancers, the tumor-restricted overexpression of this receptor and its endocytic activity
have only been recently appreciated as an attractive gateway for delivering tumor-selective cytotoxic insults. Indeed, the initially explored strategies involving traditional functional antagonism were largely unsuccessful due to a combination of factors, including the species-specificity of uPAR interactions and lack of cross-reactive agents, the evolving landscape of alleged uPAR interactors, and cancer compensatory mechanisms driving therapeutic resistance.
uPAR ubiquitous expression offers versatile opportunities for targeted cytotoxic treatment in virtually all human cancers, particularly those with aggressive phenotypes and paucity of druggable targets, like pancreatic ductal adenocarcinoma (PDAC). This potential is further supported by the lack of
overt phenotypes concerning uPAR deficiency. In the review article, presented as the first work in this thesis, I outlined the paradigm shift in the therapeutic landscape of uPAR-targeting modalities, focusing on currently explored cytotoxic-based approaches. An introductory section on salient biological and structural features of uPAR, along with a thorough illustration of the receptor
expression profile in human physiology and cancer, provides a concise overview of the key properties of uPAR from a therapeutic standpoint.
Study II constitutes the core of my Ph.D. research work. Here, we explored uPAR as a potential ADC target for PDAC therapy, where no approved targeted therapies currently exist. The unique expression pattern of uPAR in this cancer type, namely its superior differential expression (e.g., tumor-vs-normal
tissue) over all other cancer types and combined stromal expression, renders it particularly ideal for an ADC-based modality in PDAC. The study starts with selecting the optimal monoclonal antibody (mAb) candidate for such an approach. As part of this initial screening process, we conducted cellular
uptake assays to determine the internalization potential of a panel of high-affinity uPAR-mAb binders.
From this group, we selected Kor-1 due to its superior uptake and ability to cross-react with human and mouse uPAR. We then thoroughly analyzed Kor-1's binding and uptake profile in a target panel of human and murine PDAC cells through flow cytometry and confocal microscopy-based studies.
Subsequently, we developed a site-specific ADC using Kor-1 and a highly potent cytotoxic payload.
The conjugate's relative structural properties and in vitro cytotoxicity were analyzed through cell viability assays and mechanistic studies. The results showed that this conjugate displayed potent and uPAR-specific cytotoxic activity across the target cell panel, which correlated with both uptake and target-expression levels.
Expanding on the promising in vitro data, we next assessed the ADC in vivo efficacy and safety in xenograft and syngeneic allograft mouse models of PDAC. Encouragingly, we observed potent antitumor responses in both models, with the xenografted mice showing complete and sustained tumor regression and long-term survival benefits. While some animals experienced relapse towards
the end of the study, tumor growth stabilized in all but one case. Notably, the allograft model, which accurately mimics the notorious stromal barrier to tumor penetration and antitumor immunity of human PDAC, also demonstrated significant tumor regression, albeit without complete remission.
To gain insight into the ADC's impact on the tumor microenvironment (TME) in the immunocompetent allograft-bearing mice, we combined immunohistochemistry (IHC) and multicolor flow cytometry. This preliminary investigation revealed immunomodulatory potential by our ADC that engaged both adaptive and innate immune players. By alleviating the suppressive TME
constraints on antitumor immunity, our ADC holds promise as a candidate therapeutic option for PDAC. Our findings open the possibility of exploring combinatorial approaches with other immunotherapies, including immune checkpoint inhibitors (ICI), which have been unsuccessful in PDAC thus far.
Furthermore, our IHC analysis also demonstrated that none of the tumor models lost uPAR expression after treatment, ruling out the development of drug resistance via decreased or lost antigen expression as the reason for the observed tumor relapse or partial responses.
Importantly, the ADC displayed a tolerable safety profile with dose-limiting toxicities involving the typical payload-associated myelosuppression, which we addressed by optimizing the ADC dosage via pilot dose-scouting experiments.
Encouraged by the translational potential of the hybridoma-derived lead candidate mAb, Kor-1, revealed in study II, we proceeded, in study III, with the generation and characterization of a recombinant version of the mAb (rec-wt). We established a transient recombinant expression platform, based on CHO (Chinese Hamster Ovary) cells, to facilitate mAb production and allow for
genetic engineering. We also examined the potential Fab (Fragment antigen binding) N-glycosylation profile by standard computational analysis and identified two potential additional N-glycan sites in the Fragment variable (Fv) region of the HC. Considering the negative impact that glycosylation
heterogeneity may have on the therapeutic value and commercialization of mAb-based therapeutics, we engineered an Fv N-glycan deficient recombinant mAb version (rec-mut) to obtain a more homogeneous product. Thus, we successfully produced and purified large batches of both rec-mAb versions and validated their uPAR-binding properties and thermostability by Western Blot (WB) and
nano-differential scanning fluorimetry (nano-DSF) analyses. These preliminary results indicate that a glycan-devoid mAb variant will likely preserve its functional attributes following a future humanization process. Furthermore, we demonstrated that rec-wt-Kor-1, when incorporated in an ADC construct, shows similar attributes and in vitro activity as the original hybdridoma-Kor-1-based
ADC, further supporting its use in our future research activities.
The presented research work is the first to validate uPAR as a putative ADC target in PDAC. It also demonstrates the translational potential of our developed drug conjugate as a novel therapeutic candidate for PDAC and similarly aggressive cancers. In this perspective, the established recombinant production and optimization strategies may assist the future development and engineering of Kor-1 to create customizable mAb derivatives as uPAR-specific therapeutics or tools with various applications.
We recently filed a patent on Kor-1 mAb and derived drug-conjugate, along with two other valuable anti-uPAR antibody candidates described in Study II. Therefore, sensitive information, namely the specific mAb sequences, will not be disclosed.