The T1-84C supernatant was tested only against Flu and p53RMP peptides (right panel). single letters underneath the corresponding codons, with * representing the stop codon.(TIFF) pone.0176642.s001.tiff (2.8M) GUID:?6AFEDC53-3DB2-4AF8-A452-72063E303A81 S1 Table: ELISA reactivity used to assign domain specificity. (DOCX) pone.0176642.s002.docx (52K) GUID:?C7551B14-1A28-48F0-AE1E-EF2B389B0BC7 S2 Table: Epitope mapping results from individual fusions. (DOCX) pone.0176642.s003.docx (80K) GUID:?E01AB111-082D-44FF-A362-F0DCB12CE9BF Data Availability StatementAll relevant data are within the paper. Abstract Therapeutic monoclonal antibodies targeting cell surface or secreted antigens are Rabbit Polyclonal to SLC25A12 among the most effective classes of novel immunotherapies. However, the majority of human proteins and established cancer biomarkers are intracellular. Peptides derived from these intracellular proteins are presented on the cell surface by major histocompatibility complex class I (MHC-I) and can be targeted by a novel class of T-cell receptor mimic (TCRm) antibodies that recognise similar epitopes to T-cell receptors. Humoural immune responses to MHC-I tetramers rarely generate TCRm antibodies and many antibodies recognise the 3 domain of MHC-I and 2 microglobulin (2m) that are not directly involved in presenting the target peptide. Here we describe the production of functional chimeric human-murine HLA-A2-H2Dd tetramers and modifications that increase their bacterial expression and refolding efficiency. These chimeric tetramers were successfully used to generate TCRm antibodies against two epitopes derived from wild type tumour suppressor p53 (RMPEAAPPV and GLAPPQHLIRV) that have been used in vaccination studies. Immunisation with chimeric tetramers yielded no antibodies recognising the human 3 domain and 2m and generated TCRm antibodies capable of specifically recognising the target peptide/MHC-I complex in fully human tetramers and on the cell surface of peptide pulsed T2 cells. Chimeric tetramers represent novel immunogens for TCRm antibody production and may also improve the yield of tetramers for groups using these reagents to monitor CD8 T-cell immune responses in HLA-A2 transgenic mouse models of immunotherapy. Introduction Manipulating the host immune system to enable and/or to enhance anti-tumour resistance with the goal of eradicating cancer cells via immunotherapy combinations is already making an indispensible contribution to cancer treatment regimens. Cancer immunotherapy Tartaric acid has been further revitalised by Tartaric acid the introduction of immune checkpoint blockers (e.g. monoclonal antibodies to CTLA-4, PD-1 and PDL-1) that activate T cells, and by recent developments in chimeric antigen receptor (CAR) T-cell therapy that specifically direct effector T cells to the tumour.[1, 2] T-cell mediated cellular immunity plays a pivotal role in tumour rejection. Through their surface T-cell receptor (TCR), T cells recognise short 9-10mer peptide epitopes presented by major histocompatibility complex (MHC) class I proteins on the surface of cells. Enhancing the numbers and activity of tumour-infiltrating lymphocytes (TILs), which are mostly composed of T lymphocytes, were among the early approaches immunologists and oncologists tested to treat cancer.[3] Monoclonal T cells Tartaric acid and soluble T-cell receptors (TCRs) have been intensively studied for anti-tumour immunotherapy, as exemplified by Immunocore, Adaptimmune and Altor Biosciences soluble and membrane-associated TCR therapeutics.[4C6] The idea that targeting single epitopes derived from cancer-specific or cancer-related antigens is sufficient to treat cancer has fuelled research efforts to identify suitable epitopes and has opened up many new routes for developing novel anti-cancer immunotherapy agents.[7] An innovative class of antibodies with binding specificities similar to that of TCRs, so-called TCR mimic, or TCR-like antibodies, has been developed in recent years to target cancer T-cell epitopes.[8, 9] These antibodies recognise cancer derived T-cell epitopes in the context of MHC-I restriction in a manner similar to that of T cells. However, instead of activating T cells to release cytokines and to mediate cell-cell contact-dependent cell killing, TCRm antibodies bind cancer cells Tartaric acid and engage the innate immune system, including complement, natural killer cells and macrophages, to kill cancer cells. TCRm phage antibodies without the ability to Tartaric acid engage the innate immune system have also been explored as candidates for delivery of antibody drug conjugates (ADC) because MHC-I complexes are internalised.[10] Compared to adoptive T-cell based immunotherapy, therapeutic monoclonal antibodies have a more mature scalable manufacturing platform. They do not need to be produced for individual use, and therefore can be provided as off-the-shelf drugs for patients at much lower cost and with wider availability. Antibody therapy also allows for control of treatment dosage and the adjustment of treatment regimens depending on the patients responses. Multiple antibody drugs such as rituximab, bevacizumab, trastuzumab have proven utility for cancer therapy.[11] However, the production of TCRm antibodies is a challenging process.[8, 9] A contributing factor is that the target peptide-specific epitopes only represent a small area within the whole MHC-I/peptide complex. Furthermore TCRm antibody epitopes are comprised of contributions from both the MHC-I molecule and the peptide. Much like TCRs, binding to an adequate number of proteins within the prospective.