No abstract

T-cell interaction with a target cell is a key event in the adaptive immune response and primarily driven by T-cell receptor (TCR) recognition of peptide-MHC (pMHC) complexes. TCR avidity for a given pMHC is determined by number of MHC molecules, availability of coreceptors, and TCR affinity for MHC or peptide, respectively, with peptide recognition being the most important factor to confer target specificity. Here we present high-resolution crystal structures of 2 Fab antibodies in complex with the immunodominant NY-ESO-1 157–165 peptide analogue (SLLMWITQV) presented by HLA-A*0201 and compare them with a TCR recognizing the same pMHC. Binding to the central methionine-tryptophan peptide motif and orientation of binding were almost identical for Fabs and TCR. As the MW “peg” dominates the contacts between Fab and peptide, we estimated the contributions of individual amino acids between the Fab and peptide to provide the rational basis for a peptide-focused second-generation, high-affinity antibody library. The final Fab candidate achieved better peptide binding by 2 light-chain mutations, giving a 20-fold affinity improvement to 2–4 nM, exceeding the affinity of the TCR by 1,000-fold. The high-affinity Fab when grafted as recombinant TCR on T cells conferred specific killing of HLA-A*0201/NY-ESO-1 157–165 target cells. In summary, we prove that affinity maturation of antibodies mimicking a TCR is possible and provide a strategy for engineering high-affinity antibodies that can be used in targeting specific pMHC complexes for diagnostic and therapeutic purposes.

To determine the expression of cancer testis (CT) genes and antibody responses in a nonselected population of patients with primary breast cancer, we investigated the composite expression of 11 CT genes by RT-PCR in fresh biopsies of 100 consecutive cases of primary breast carcinoma and by immunohistology in selected RT-PCR-positive cases. Antibody responses against 7 CT antigens were analyzed using recombinant antigen expression on yeast surface. In 98 evaluable cases, SCP-1 and SSX-4 were expressed most frequently (both 65%), followed by HOM-TES-85/CT-8 (47%), GAGE (26%), SSX-1 (20%), NY-ESO-1 (13%), MAGE-3 (11%), SSX-2 (8%), CT-10 (7%), MAGE-4 (4%) and CT-7 (1%). One CT gene was expressed by 90% of the cases; 79% expressed 2, 48% 3, 29% 4, 12% 5, 6% 6, 3% 7, 2% 8 and one case coexpressed 9 antigens. Of 100 serum samples screened for CT antigen-specific antibodies, antibodies against NY-ESO-1 were detected in 4 patients, against SCP-1 in 6 patients and against SSX-2 in 1 patient, while no antibodies were detected against MAGE-3, CT-7 and CT-10. Expression of CT genes or antibody responses was not correlated with clinical parameters (menopausal status, tumor size, nodal involvement, grading, histology and estrogen receptor status) or the demonstration of CT gene expression at the protein level, by immunohistology. Our results show that breast carcinomas are among the tumors with the most frequent expression of CT antigens, rendering many patients potential candidates for vaccine trials. ' 2005 Wiley-Liss, Inc.

Mutations in the adenomatous polyposis coli (APC) gene are linked to the dysplastic transformation of colorectal polyps and represent an early step in the development of colorectal tumors. Ninety-four percent of all mutations result in the expression of a truncated APC protein lacking the C-terminal region. The C-terminal region of the APC protein may have a tumor suppressor function as its absence appears to be linked to the development of dysplastic lesions. Recently, we discovered and characterized a protein called RP1 which binds specifically to the C-terminal region of the APC protein. We show now that RP1 and the other known members of the EB/RP family (EB1 and RP3) also bind directly to tubulin, both in vitro and in vivo. Immunohistochemical analyses reveal a distinct staining pattern during interphase as well as an association of RP1/EB1 with mitotic microtubule structures. The previously described puncta of the APC protein at the leading edge of membrane protrusions contact microtubule fibers that contain RP1 or EB1. Int.

Fibroblast activation protein (FAP), as described so far, is a type II cell surface serine protease expressed by fibroblastic cells in areas of active tissue remodelling such as tumour stroma or healing wounds. We investigated the expression of FAP by fibroblast-like synoviocytes (FLSs) and compared the synovial expression pattern in rheumatoid arthritis (RA) and osteoarthritis (OA) patients. Synovial tissue from diseased joints of 20 patients, 10 patients with refractory RA and 10 patients with end-stage OA, was collected during routine surgery. As a result, FLSs from intensively inflamed synovial tissues of refractory RA expressed FAP at high density. Moreover, FAP expression was co-localised with matrix metalloproteinases (MMP-1 and MMP-13) and CD44 splice variants v3 and v7/8 known to play a major role in the concert of extracellular matrix degradation. The pattern of signals appeared to constitute a characteristic feature of FLSs involved in rheumatoid arthritic joint-destructive processes. These FAP-expressing FLSs with a phenotype of smooth muscle actin-positive myofibroblasts were located in the lining layer of the synovium and differ distinctly from Thy-1-expressing and non-proliferating fibroblasts of the articular matrix. The intensity of FAP-specific staining in synovial tissue from patients with RA was found to be different when compared with end-stage OA. Because expression of FAP by RA FLSs has not been described before, the findings of this study highlight a novel element in cartilage and bone destruction of arthritic joints. Moreover, the specific expression pattern qualifies FAP as a therapeutic target for inhibiting the destructive potential of fibroblast-like synovial cells.