BACKGROUND Pembrolizumab is a potent humanized immunoglobulin G4 (IgG4) monoclonal antibody (mAb) with high specificity of binding to the programmed cell death 1 (PD 1) receptor, thus inhibiting its interaction with programmed cell death ligand 1 (PD-L1) and programmed cell death ligand 2 (PD-L2). Based on preclinical in vitro data, pembrolizumab has high affinity and potent receptor blocking activity for PD 1. Pembrolizumab has an acceptable preclinical safety profile and is in clinical development as an intravenous (IV) immunotherapy for advanced malignancies. Keytruda® (pembrolizumab) is indicated for the treatment of patients across a number of indications because of its mechanism of action to bind the PD-1 receptor on the T cell. For more details on specific indications refer to the Investigator brochure (IB).
Enfortumab Vedotin (EV) is an ADC comprised of a fully human anti-Nectin-4 IgG1 kappa mAb conjugated to the small molecule microtubule disrupting agent, MMAE, via a protease cleavable maleimidocaproyl vc linker. Conjugation takes place on cysteine residues that comprise the interchain disulfide bonds of the antibody to yield a product with a drug to antibody ratio of approximately 3:8.
EV binds to the V domain of Nectin-4 protein. In the presumed mechanism of action, the drug binds to the Nectin-4 protein on the cell surface and is internalized, causing proteolytic cleavage of the vc linker and intracellular release of MMAE. Free MMAE subsequently disrupts tubulin polymerization and leads to mitotic arrest.
PHARMACEUTICAL AND THERAPEUTIC BACKGROUND
Pembrolizumab. The importance of intact immune surveillance function in controlling outgrowth of neoplastic transformations has been known for decades. Accumulating evidence shows a correlation between tumor-infiltrating lymphocytes in cancer tissue and favorable prognosis in various malignancies. In particular, the presence of CD8+ T-cells and the ratio of CD8+ effector T cells/FoxP3+ regulatory T-cells (T-regs) correlates with improved prognosis and long-term survival in solid malignancies, such as ovarian, colorectal, and pancreatic cancer; hepatocellular carcinoma; malignant melanoma; and renal cell carcinoma. Tumor-infiltrating lymphocytes can be expanded ex vivo and reinfused, inducing durable objective tumor responses in cancers such as melanoma.
The PD-1 receptor-ligand interaction is a major pathway hijacked by tumors to suppress immune control. The normal function of PD-1, expressed on the cell surface of activated T cells under healthy conditions, is to down-modulate unwanted or excessive immune responses, including autoimmune reactions. PD-1 (encoded by the gene Pdcd1) is an immunoglobulin (Ig) superfamily member related to cluster of differentiation 28 (CD28) and cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) that has been shown to negatively regulate antigen receptor signaling upon engagement of its ligands (PD L1 and/or PD-L2).
The structure of murine PD-1 has been resolved. PD-1 and its family members are type I transmembrane glycoproteins containing an Ig-variable-type (IgV type) domain responsible for ligand binding and a cytoplasmic tail responsible for the binding of signaling molecules. The cytoplasmic tail of PD-1 contains 2 tyrosine-based signaling motifs, an immunoreceptor tyrosine-based inhibition motif, and an immunoreceptor tyrosine-based switch motif. Following T-cell stimulation, PD-1 recruits the tyrosine phosphatases, SHP-1 and SHP-2, to the immunoreceptor tyrosine-based switch motif within its cytoplasmic tail, leading to the dephosphorylation of effector molecules such as CD3 zeta (CD3ζ), protein kinase C-theta (PKCθ), and zeta-chain-associated protein kinase (ZAP70), which are involved in the CD3 T-cell signaling cascade. The mechanism by which PD-1 down-modulates T cell responses is similar to, but distinct from, that of CTLA-4, because both molecules regulate an overlapping set of signaling proteins. As a consequence, the PD 1/PD-L1 pathway is an attractive target for therapeutic intervention in Collecting Duct Carcinoma and Renal Medullary Carcinoma.
Enfortumab Vedotin Nectin-4 is a 66 kDa type I transmembrane protein that belongs to the Nectin family of adhesion molecules. It is composed of an ECD containing 3 Ig-like subdomains, a transmembrane helix, and an intracellular region. Nectins are thought to mediate Ca2+-independent cell-cell adhesion via both homophilic and heterophilic trans- interactions at adherens junctions where they can recruit cadherins and modulate cytoskeletal rearrangements. Sequence identity of Nectin-4 to other Nectin family members is low and ranges from 25% to 30% in the ECD.
The 3 Ig-like subdomains in the ECD of Nectin-4 are designated V, C1, and C2. The C1 domain is responsible for cisplatin-interaction (homodimerization), while V domains of most Nectin molecules contribute to trans-interaction and cell-cell adhesion.
Nectin-4 was originally identified by bioinformatics and cloned from human trachea. In humans, Nectin-4 is normally expressed in keratinocytes of the skin, sweat glands, hair follicles, transitional epithelium of the bladder, salivary gland ducts, esophagus, breast, and stomach. Nectin-4 was identified as markedly upregulated in urothelial carcinoma using suppression subtractive hybridization on a pool of urothelial carcinoma specimens. Immunohistochemical characterization of expression in multiple tumor specimens demonstrated high levels of Nectin-4 in bladder, breast, pancreatic, lung, ovarian and other cancers.
Enfortumab Vedotin in Combination with Pembrolizumab. Combining PD-1/PD-L1 inhibitors with a novel therapy, such as EV, may improve patient outcomes. Data from preclinical studies of brentuximab vedotin (a CD30-directed ADC comprising the same linker and MMAE payload as EV), shows potential to induce ICD, antigen presentation, and tumor immune infiltration. These results suggest that the effects are due to MMAE. Treatment with brentuximab vedotin in vitro and in preclinical models has been shown to induce hallmarks of ICD. ICD is characterized by induction of the endoplasmic reticulum stress response and subsequent surface presentation of DAMPs immune stimulatory molecules. These DAMPs induce innate immune migration and activation into the tumor microenvironment.
Based on the potential enhancement of immune response, it is hypothesized that combining EV with pembrolizumab will result in improved response leading to prolonged PFS and OS in patients with Collecting Duct Carcinoma and Renal Medullary Carcinoma with minimal overlapping toxicities between the 2 agents.
RATIONALE FOR THE TRIAL AND SELECTED POPULATION Non-clear cell renal cell carcinomas (nccRCC) comprise several rare and poorly described diseases. Among them, Renal medullary carcinoma (RMNC) represents less than 0,5% and Collecting Duct Carcinoma (CDC) represents 1% of all renal cell carcinomas. Both are characterized by an aggressive clinical behaviour and a particular poor prognosis. Both tumors are under-represented in prospective randomized trials predominantly including clear-cell histotype. Thus, a standard of treatment for these rare and aggressive histologies has not been defined yet. Based on the data of a small phase II study with responses of 23%, the more used first line therapy in both cases is a platinum-based cytotoxic chemotherapy that has the ability to control the disease but only for a limited time. More recently, the prospective BONSAI trial met its primary endpoint showing encouraging efficacy of cabozantinib in first line with an objective response (ORR) of 35% in 25 patients with metastatic CDC (mCDC). Immune-checkpoint inhibitors (ICI) such as Pembrolizumab in combination with Tyrosine Kinase Inhibitors are now recommended as standard-of-care options for clear-cell renal cell carcinoma due to the relevant results in term of antitumor activity and Overall Survival (OS). Cases of excellent response to ICI are reported in literature also in previously treated mCDC patients. Enfortumab Vedotin showed encouraging activity in different tumour types and when combined with pembrolizumab showed relevant results in term of ORR and PFS. More recently, the combination of EV and pembrolizumab in the EV-103/KEYNOTE-869 (NCT03288545) study has demonstrated dramatic improvement in ORR in cisplatin- ineligible patients with locally advanced and metastatic urothelial carcinoma, with a preliminary ORR of 73% (95% CI: 58, 85) regardless of PD-L1 expression level.
Due to the mechanism of action of Enfortumab Vedotin, the expression of NECTIN-4 in CDC is under analysis in the CICERONE trial (NCT05372302). Unpublished results from this trial, state that NECTIN-4 is expressed in 30% of CDC tissue. Furthermore, biology of CDC and of Urothelial Carcinoma is similar, and this support the expression of NECTIN-4 in this orphan disease and the activity of EV.
Based on the encouraging data regarding the use of ICI in clear cell renal cell carcinoma, the investigators hypothesize that in these histotypes it may be useful to evaluate in more depth the action of drugs that act on the immune system in combination with Antibody-Drug Conjugate (ADC).
PLANNED EXPLORATORY BIOMARKER RESEARCH Analysis on biomarkers is exploratory by nature and will be performed retrospectively after the main study analysis is completed. Tissue assessments will include the identification of somatic mutations in CDC and the assessment of the mutational load by focused exome analysis, as well as the identification of transcript fusions by RNA sequencing.
Plasma and viable peripheral blood mononuclear cell (PBMC) will be isolated and stored frozen for subsequent analysis. PBMC will be studied for immune cell profile by multicolor cytofluorimetry (including frequency and activation state of antitumor and immunosuppressive cell subsets of both innate and adaptive immunity), associated with gene-expression profiling and Cibersort analysis for assessing the activation state of the immune cell subsets.
