Multiple Myeloma (MM) is a devastating malignancy that evades immune destruction using multiple mechanisms. The NKp44 receptor interacts with PCNA (Proliferating Cell Nuclear Antigen) and may inhibit NK cells’ functions. Here we studied in vitro the expression and function of PCNA on MM cells. First, we show that PCNA is present on the cell membrane of five out of six MM cell lines, using novel anti-PCNA mAb developed to recognize membrane-associated PCNA. Next, we stained primary bone marrow (BM) mononuclear cells from MM patients and showed significant staining of membrane-associated PCNA in the fraction of CD38⁺CD138⁺ BM cells that contain the MM cells. Importantly, blocking of the membrane PCNA on MM cells enhanced the activity of NK cells, including IFN-γ-secretion and degranulation. Our results highlight the possible blocking of the NKp44-PCNA immune checkpoint by the mAb 14-25-9 antibody to enhance NK cell responses against MM, providing a novel treatment option.

Harnessing immune effector cells to benefit cancer patients is becoming more and more prevalent in recent years. However, the increasing number of different therapeutic approaches, such as chimeric antigen receptors and armored chimeric antigen receptors, requires constant adjustments of the transgene expression levels. We have previously demonstrated it is possible to achieve spatial and temporal control of transgene expression as well as tailoring the inducing agents using the Chimeric Antigen Receptor Tumor Induced Vector (CARTIV) platform. Here we describe the next level of customization in our promoter platform. We have tested the functionality of three different minimal promoters, representing three different promoters’ strengths, leading to varying levels of CAR expression and primary T cell function. This strategy shows yet another level of CARTIV gene regulation that can be easily integrated into existing CAR T systems.

Receptor tyrosine kinases (RTKs) are major players in signal transduction, regulating cellular activities in both normal regeneration and malignancy. Thus, many RTKs, c-Kit among them, play key roles in the function of both normal and neoplastic cells, and as such constitute attractive targets for therapeutic intervention. We thus sought to manipulate the self-association of stem cell factor (SCF), the cognate ligand of c-Kit, and hence its suboptimal affinity and activation potency for c-Kit. To this end, we used directed evolution to engineer SCF variants having different c-Kit activation potencies. Our yeast-displayed SCF mutant (SCF_M) library screens identified altered dimerization potential and increased affinity for c-Kit by specific SCF-variants. We demonstrated the delicate balance between SCF homo-dimerization, c-Kit binding, and agonistic potencies by structural studies, in vitro binding assays and a functional angiogenesis assay. Importantly, our findings showed that a monomeric SCF variant exhibited superior agonistic potency vs. the wild-type SCF protein and vs. other high-affinity dimeric SCF variants. Our data showed that action of the monomeric ligands in binding to the RTK monomers and inducing receptor dimerization and hence activation was superior to that of the wild-type dimeric ligand, which has a higher affinity to RTK dimers but a lower activation potential. The findings of this study on the binding and c-Kit activation of engineered SCF variants thus provides insights into the structure–function dynamics of ligands and RTKs.