A single-chain-Fv-based immunofluorometric assay specific for the CEA variant NCA-2

The disagreement between various immunoassays for the tumour marker carcinoembryonic antigen (CEA) is well known, and the discrepancies are explained by the co-measurement of the structurally related non-specific cross-reacting antigen 2 (NCA-2). Until now, this hypothesis has only been proven indirectly, as no NCA-2 specific assay has been available. This thesis describes the production of a high affinity single-chain antibody fragment (scFv) raised by using phage display technology. The scFv was used to construct an immunometric assay that specifically recognises NCA-2. A phagemid library was constructed from splenic mRNA obtained from a mouse immunised with NCA-2 purified from human meconium. Following phage rescue and three rounds of selection on solid phase coated with NCA-2, several clones, which displayed scFvs with high specificity and affinity for NCA-2, were isolated. These were sub-cloned into an expression vector for high-level expression of soluble scFv. Based on BIAcore analysis, a scFv with low koff and a KD of 10-10 mol/l was selected for europium labelling and employed as the tracer molecule for the NCA-2 - specific immunofluorometric assay. This novel assay was applied for the quantification of NCA-2 in sera from 35 patients with elevated CEA levels. The measured NCA-2 correlated fairly well (R2=0.82) with the values obtained by indirect methods based on the differences between serum values measured by NCA-2 cross-reactive and CEA specific immunoassays

CAR T Cell Therapy: A Game Changer in Cancer Treatment

The development of novel targeted therapies with acceptable safety profiles is critical to successful cancer outcomes with better survival rates. Immunotherapy offers promising opportunities with the potential to induce sustained remissions in patients with refractory disease. Recent dramatic clinical responses in trials with gene modified T cells expressing chimeric antigen receptors (CARs) in B-cell malignancies have generated great enthusiasm. This therapy might pave the way for a potential paradigm shift in the way we treat refractory or relapsed cancers. CARs are genetically engineered receptors that combine the specific binding domains from a tumor targeting antibody with T cell signaling domains to allow specifically targeted antibody redirected T cell activation. Despite current successes in hematological cancers, we are only in the beginning of exploring the powerful potential of CAR redirected T cells in the control and elimination of resistant, metastatic, or recurrent nonhematological cancers. This review discusses the application of the CAR T cell therapy, its challenges, and strategies for successful clinical and commercial translation

Intrinsic functional potential of NK-Cell subsets constrains retargeting driven by chimeric antigen receptors

Natural killer (NK) cells hold potential as a source of allogeneic cytotoxic effector cells for chimeric antigen receptor (CAR)-mediated therapies. Here, we explored the feasibility of transfecting CAR-encoding mRNA into primary NK cells and investigated how the intrinsic potential of discrete NK-cell subsets affects retargeting efficiency. After screening five second- and third-generation anti-CD19 CAR constructs with different signaling domains and spacer regions, a third-generation CAR with the CH2-domain removed was selected based on its expression and functional profiles. Kinetics experiments revealed that CAR expression was optimal after 3 days of IL15 stimulation prior to transfection, consistently achieving over 80% expression. CAR-engineered NK cells acquired increased degranulation toward CD19+ targets, and maintained their intrinsic degranulation response toward CD19− K562 cells. The response of redirected NK-cell subsets against CD19+ targets was dependent on their intrinsic thresholds for activation determined through both differentiation and education by killer cell immunoglobulin-like receptors (KIR) and/or CD94/NKG2A binding to self HLA class I and HLA-E, respectively. Redirected primary NK cells were insensitive to inhibition through NKG2A/HLA-E interactions but remained sensitive to inhibition through KIR depending on the amount of HLA class I expressed on target cells. Adaptive NK cells, expressing NKG2C, CD57, and self-HLA–specific KIR(s), displayed superior ability to kill CD19+, HLA low, or mismatched tumor cells. These findings support the feasibility of primary allogeneic NK cells for CAR engineering and highlight a need to consider NK-cell diversity when optimizing efficacy of cancer immunotherapies based on CAR-expressing NK cells