Affinity maturation of T-cell receptor-like antibodies for Wilms tumor 1 peptide greatly enhances therapeutic potential.

WT1126 (RMFPNAPYL) is a human leukocyte antigen-A2 (HLA-A2)-restricted peptide derived from Wilms tumor protein 1 (WT1), which is widely expressed in a broad spectrum of leukemias, lymphomas and solid tumors. A novel T-cell-receptor (TCR)-like single-chain variable fragment (scFv) antibody specific for the T-cell epitope consisting of the WT1/HLA-A2 complex was isolated from a human scFv phage library. This scFv was affinity-matured by mutagenesis combined with yeast display and structurally analyzed using a homology model. This monovalent scFv showed a 100-fold affinity improvement (dissociation constant (KD)=3 nm) and exquisite specificity towards its targeted epitope or HLA-A2(+)/WT1(+) tumor cells. Bivalent scFv-huIgG1-Fc fusion protein demonstrated an even higher avidity (KD=2 pm) binding to the T-cell epitope and to tumor targets and was capable of mediating antibody-dependent cell-mediated cytotoxicity or tumor lysis by chimeric antigen receptor-expressing human T- or NK-92-MI-transfected cells. This antibody demonstrated specific and potent cytotoxicity in vivo towards WT1-positive leukemia xenograft that was HLA-A2 restricted. In summary, T-cell epitopes can provide novel targets for antibody-based therapeutics. By combining phage and yeast displays and scFv-Fc fusion platforms, a strategy for developing high-affinity TCR-like antibodies could be rapidly explored for potential clinical development.

Retargeting NK92 cells using an HLA-A2-restricted, EBNA3C-specific chimeric antigen receptor.

Advances in adoptive cell immunotherapy have led to several promising options for cancer patients. Single-chain variable fragments (scFvs) were isolated from a human phage display library by panning on recombinant human leukocyte antigen (HLA)-A2-peptide complexes. A scFv (EBNA Clone 315) specific for HLA-A2 carrying a 10 amino acid peptide (LLDFVRFMGV) derived from the Epstein-Barr virus latent protein EBNA3C was fully characterized. EBNA Clone 315 displayed exquisite specificity toward its targeted T-cell epitope (TCE) and did not cross-react with the free peptide, HLA-A2 complexes, which carried irrelevant peptides, or HLA-A2(-) cells. Furthermore, after engineering into a scFv-Fc fusion protein, we were able to determine its affinity, detection sensitivity, and ability to induce antibody-dependent cellular cytotoxicity (ADCC). As a proof-of-principle, a chimeric antigen receptor (CAR) version of EBNA Clone 315 was used to reprogram NK92MI cells. CAR-expressing NK92MI cells showed highly specific and potent cytotoxicity toward the targeted TCE, with detection sensitivity of approximately 25 molecules and cytolytic capacity threefold greater than scFv-Fc-mediated ADCC. For the first time, we show the successful reprogramming of non-T cells toward a specific TCE using a CAR.