Circulating T-cells that have passed thymic selection generally bear T-cell receptors (TCRs) with sub-optimal affinity for cancer-associated antigens, resulting in a limited ability to detect and eliminate tumor cells. Engineering TCRs to increase their affinity for cancer targets is a promising strategy for generating T-cells with enhanced potency for adoptive immunotherapy in cancer patients. However, this manipulation also risks generating cross-reactivity to antigens expressed by normal tissue, with potentially serious consequences. Testing in animal models might not detect such cross-reactivity due to species differences in the antigenic repertoire. To mitigate the risk of off-target toxicities in future clinical trials, we therefore developed an extensive in vitro testing strategy. This approach involved systematic substitution at each position of the antigenic peptide sequence using all natural amino acids to generate a profile of peptide specificity (“X-scan”). The likelihood of off-target reactivity was investigated by searching the human proteome for sequences matching this profile, and testing against a panel of primary cell lines. Starting from a diverse panel of parental TCRs, we engineered several affinity-enhanced TCRs specific for the cancer-testis antigen MAGE-A10. Two of these TCRs had affinities and specificities which appeared to be equally optimal when tested in conventional biochemical and cellular assays. The X-scan method, however, permitted us to select the most specific and potent candidate for further pre-clinical and clinical testing.

The use of the PCR method for routine testing has increased dramatically during recent years. Most assays involve co-amplification either of an internal control, of several alleles at a given locus or of a variety of bands produced by low-stringency primer annealing. In such multiplex reactions, certain products will often amplify preferentially. Amplimers that are more sensitive can be outcompeted under suboptimal PCR conditions, leading to assignment of false negatives. Optimization of PCR parameters such as temperature steps, relative concentrations of primers and their annealing temperature do not alone ensure against false negatives when caused by stable double-stranded DNA (dsDNA) regions in the amplified sequence. A two-step strategy to solve this problem is presented in this paper: (i) titration of the PCR with NaCl as a model inhibitor to establish the critical range within which false negatives occur; (ii) titration of the PCR with a dsDNA-destabilizing additive under false-negative-inducing conditions until the relative amplification efficiencies of co-amplified fragments are adjusted. Betaine is introduced as a novel and efficient cosolute. These measures to achieve reliable PCR typing of a difficult target should be useful for many qualitative and quantitative multiplex PCR applications.

Patients with hepatocellular carcinoma (HCC) have a poor prognosis and limited therapeutic options. Alpha‐fetoprotein (AFP) is often expressed at high levels in HCC and is an established clinical biomarker of the disease. Expression of AFP in nonmalignant liver can occur, particularly in a subset of progenitor cells and during chronic inflammation, at levels typically lower than in HCC. This cancer‐specific overexpression indicates that AFP may be a promising target for immunotherapy. We verified expression of AFP in normal and diseased tissue and generated an affinity‐optimized T‐cell receptor (TCR) with specificity to AFP/HLA‐A*02+ tumors. Expression of AFP was investigated using database searches, by qPCR, and by immunohistochemistry (IHC) analysis of a panel of human tissue samples, including normal, diseased, and malignant liver. Using in vitro mutagenesis and screening, we generated a TCR that recognizes the HLA‐A*02‐restricted AFP158‐166 peptide, FMNKFIYEI, with an optimum balance of potency and specificity. These properties were confirmed by an extension of the alanine scan (X‐scan) and testing TCR‐transduced T cells against normal and tumor cells covering a variety of tissues, cell types, and human leukocyte antigen (HLA) alleles. Conclusion: We have used a combination of physicochemical, in silico, and cell biology methods for optimizing a TCR for improved affinity and function, with properties that are expected to allow TCR‐transduced T cells to differentiate between antigen levels on nonmalignant and cancer cells. T cells transduced with this TCR constitute the basis for a trial of HCC adoptive T‐cell immunotherapy.

The strong association of HLA B27 with spondyloarthropathies contrasts strikingly with most autoimmune diseases, which are HLA class II associated and thought to be mediated by CD4+ T lymphocytes. By introducing a human-derived HLA B27-restricted TCR into HLA B27 transgenic mice, we have obtained a functional TCR transgenic model, GRb, dependent on HLA B27 for response. Surprisingly, HLA B27 supported CD4+ as well as CD8+ T cell responses in vivo and in vitro. Further, HLA B27-restriced CD4+ T cells were capable of differentiation into a range of Th1 and Th2 T cell subsets with normal patterns of cytokine expression. The transgenic T cells were also able to enhance clearance of recombinant vaccinia virus containing influenza nucleoprotein in vivo. This is the first description of a human HLA class I-restricted TCR transgenic line. The existence of CD4+ MHC class I-restricted T cells has significant implications for immune regulation in autoimmunity and, in particular, in HLA B27-associated arthritis. We believe that this model provides a novel system for the study of unusual T cell behavior in vivo.