Profound Functional Suppression of Tumor-Infiltrating T-Cells in Ovarian Cancer Patients Can Be Reversed Using PD-1-Blocking Antibodies or DARPin® Proteins

Abstract

PD-1/PD-L1 blockade has revolutionized the field of immunooncology. Despite the relative success, the response rate to anti-PD-1 therapy requires further improvements. Our aim was to explore the enhancement of T-cell function by using novel PD-1-blocking proteins and compare with clinically approved monoclonal antibodies (mAbs). We isolated T-cells from the ascites and tumor of 17 patients with advanced epithelial ovarian cancer (EOC) and analyzed the effects using the mAbs nivolumab and pembrolizumab and two novel engineered ankyrin repeat proteins (DARPin® proteins). PD-1 blockade with either mAb or DARPin® molecule significantly increased the release of IFN-γ, granzyme B, IL-2, and TNF-α, demonstrating successful reinvigoration. The monovalent DARPin® protein was less effective compared to its bivalent equivalent, demonstrating that bivalency brings an additional benefit to PD-1 blockade. Overall, we found a higher fold increase of lymphokine secretion in response to the PD-1 blockade by tumor-derived T-cells; however, the absolute amounts were significantly lower compared to the release from ascites-derived T-cells. Our results demonstrate that PD-1 blockade can only partially reinvigorate functionally suppressed T-cells from EOC patients. This warrants further investigation preferably in combination with other therapeutics. The study provides an early pilot proof-of-concept for the potential use of DARPin® proteins as eligible alternative scaffold proteins to block PD-1.

Revolutionization in Cancer Therapeutics via Targeting Major Immune Checkpoints PD-1, PD-L1 and CTLA-4

“…Currently, approximately more than 2000 clinical trials encompassing PD-1 inhibition are being sustained by NCI. Pembrolizumab and nivolumab are IgG4 antibodies that have high PD-1 binding efficacy and low affinity for Fc receptors and complements [ 28 ]. Both antibodies have a similar structure except for the difference in the Ag binding component of the antibody variable domain.…”

Directed Evolution of PD-L1-Targeted Affibodies by mRNA Display

“…PD-L1 has been targeted with multiple molecular scaffolds including adnectins (fibronectin domain scaffolds), nanobodies, conjugated Fab fragments, ankyrin repeat proteins (against PD-1), and nonblocking single-domain antibodies . While these mid- to high-molecular-weight scaffolds often show superior affinity, their slow systemic clearance renders them susceptible to the same disadvantages as full-length antibodies in molecular imaging applications.…”

“…Low-molecular-weight polypeptide scaffolds offer a compromise between small peptides and large protein scaffolds. These scaffolds can be utilized for ligand design and capture many of the affinity-based benefits of monoclonal antibodies , and nonblocking single-domain antibodies. The bundled triple α-helical affibody scaffold, derived from the immunoglobulin (Ig) binding Z-domain of protein A, provides a favorable combination of evolvability, size, and ease of synthesis/expression .…”

Thermostable designed ankyrin repeat proteins (DARPins) as building blocks for innovative drugs

“…Importantly, this potential extends beyond areas of applications that have classically been "occupied" by recombinant immunoglobulins. The DARPin scaffold was shown to serve as an alternative 5 , as a complementation 6 and as an expansion of what is possible with binders derived from immunoglobulins 7,8 . Translation of academic research in DARPin technology towards pharmaceutical benefits has been predominantly steered by Molecular Partners, who provided the fundamental clinical validation of the scaffold 9 .…”