Andreas Wadle

Fibroblast activation protein is expressed by rheumatoid myofibroblast-like synoviocytes

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.

EB/RP gene family encodes tubulin binding proteins

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. J. Cancer 81:275–284, 1999.

Prospective study on the expression of cancer testis genes and antibody responses in 100 consecutive patients with primary breast cancer.

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.

Rational development of high-affinity T-cell receptor-like antibodies

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-1157–165 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-1157–165 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.

Inhibiting HLA–B27 homodimer–driven immune cell inflammation in spondylarthritis

OBJECTIVE Spondylarthritides (SpA), including ankylosing spondylitis (AS), are common inflammatory rheumatic diseases that are strongly associated with positivity for the HLA class I allotype B27. HLA-B27 normally forms complexes with β(2) -microglobulin (β(2) m) and peptide to form heterotrimers. However, an unusual characteristic of HLA-B27 is its ability to form β(2) m-free heavy chain homodimers (HLA-B27(2) ), which, unlike classic HLA-B27, bind to killer cell immunoglobulin-like receptor 3DL2 (KIR-3DL2). Binding of HLA-B27(2) to KIR-3DL2-positive CD4+ T and natural killer (NK) cells stimulates cell survival and modulates cytokine production. This study was undertaken to produce an antibody to HLA-B27(2) in order to confirm its expression in SpA and to inhibit its proinflammatory properties. METHODS We generated monoclonal antibodies by screening a human phage display library positively against B27(2) and negatively against B27 heterotrimers. Specificity was tested by enzyme-linked immunosorbent assay (ELISA), surface plasmon resonance (SPR) assay, and fluorescence-activated cell sorting (FACS) analysis of B27(2) -expressing cell lines and peripheral blood mononuclear cells (PBMCs) and synovial fluid mononuclear cells (SFMCs) from patients with SpA. Functional inhibition of KIR-3DL2-B27(2) interactions was tested using cell lines and PBMCs from patients with SpA. RESULTS Monoclonal antibody HD6 specifically recognized recombinant HLA-B27(2) by ELISA and by SPR assay. HD6 bound to cell lines expressing B27(2) . FACS revealed binding of HD6 to PBMCs and SFMCs from patients with AS but not from controls. HD6 inhibited both the binding of HLA-B27(2) to KIR-3DL2 and the survival and proliferation of KIR-3DL2-positive NK cells. Finally, HD6 inhibited production of the proinflammatory disease-associated cytokine interleukin-17 by PBMCs from patients with AS. CONCLUSION These results demonstrate that antibody HD6 has potential for use in both the investigation and the treatment of AS and other B27-associated spondylarthritides.

Serological identification of breast cancer‐related antigens from a Saccharomyces cerevisiae surface display library

A yeast cell surface display technology was used for the isolation and characterization of tumor antigens recognised by autologous or allogeneic breast cancer serum. More than 100 clones recognized by patient serum were isolated using high‐through‐put fluorescence activated cell sorting. Combined serological and sequence analysis confirmed that a number of proteins known to be overexpressed in breast cancer tissue could be detected. A recently identified small breast epithelial mucin almost exclusively expressed in mammary gland tissue was isolated as a mutated protein variant. Subsequent serological analysis using the yeast expression system for the wild‐type and mutant form showed a strong recognition by patient sera, whereas no significant recognition was observed for the respective prokaryotically expressed proteins. The small breast epithelial mucin is present to a large extent in a membrane bound format and might be used for tumor targeting strategies.

Radioimmunotherapy of Fibroblast Activation Protein Positive Tumors by Rapidly Internalizing Antibodies

Purpose: Fibroblast activation protein (FAP) is a serine protease that has emerged as a promising target for cancer therapy, either by direct abrogation of its proinvasive activity or by specific targeting of FAP-expressing cells with cytotoxic immunoconjugates. We aimed to select novel human–mouse cross-reactive antibodies and to test suitability for tumor therapy as radioimmunoconjugates in a preclinical model. Experimental Design: Human Fab fragments that bind to human and murine FAP were selected from an antibody phage library. Two candidates (ESC11 and ESC14) were engineered into fully human IgG1 antibodies and further characterized. We investigated the intracellular trafficking of ESC11 and ESC14 in live cells by confocal microscopy and analyzed the biodistribution and therapeutic effects of anti-FAP antibodies labeled with the β-emitting radionuclide 177Lu in a melanoma xenograft nude mouse model. Results were compared with vF19, a humanized variant of an anti-FAP antibody that has been previously used in clinical trials. Results: The two antibodies bound selectively to both human and mouse FAP, with affinities in the low nanomolar range. Binding to FAP-expressing melanoma cells resulted in rapid internalization of FAP-antibody complexes. 177Lu-labeled ESC11 specifically accumulated in melanoma xenografts in vivo, with a higher tumor uptake than ESC14 and vF19. Radioimmunotherapy with 8 MBq 177Lu-labeled anti-FAP antibodies delayed growth of established tumors, whereas 177Lu-ESC11 extended mouse survival more pronounced than 177Lu-ESC14 and 177Lu-vF19. Conclusion: Our results show the potential of ESC11 and ESC14 as potent radioimmunoconjugates or antibody–drug conjugates for diagnostic and therapeutic use in patients with FAP-expressing tumors. Clin Cancer Res; 18(22); 6208–18. ©2012 AACR.

Serological immune response to cancer testis antigens in patients with pancreatic cancer

Serological screening approaches have allowed for the identification of a large number of potentially relevant tumor antigens in cancer patients. Within this group, cancer testis antigens represent promising targets for cancer immunotherapy, since they are widely expressed in a variety of human cancer entities. In pancreatic cancer, however, there are only few data available about the expression pattern and serological response to cancer testis antigens and other serological‐defined tumor antigens. Therefore, we investigated the IgG antibody response against 11 cancer testis antigens (SCP‐1, GAGE, LAGE‐1a,‐1b, CT‐7, NY‐ESO‐1, SSX‐1‐5) recombinantly expressed on yeast surface (RAYS) in patients with pancreatic cancer (n = 96), chronic pancreatitis (n = 18) and healthy donors (n = 48). We found in 14% of all patients antibody responses to SCP‐1, but not to other cancer testis antigens (GAGE, LAGE‐1a,‐1b, CT‐7, NY‐ESO‐1, SSX‐1‐5). Antibody response correlated with the expression of SCP‐1 in the primary tumor of the respective patient as shown by RT‐PCR, immunohistochemistry and Western blot. In contrast, no serological response to cancer testis antigens was observed in healthy donors. The humoral immune response against SCP‐1 was associated with the size of tumor, but not with other clinico‐pathological parameters such as histology, stage, presence of lymph node metastases, grading, age, gender or gemcitabine treatment. In conclusion, antibody response to cancer testis antigen SCP‐1 is found in a proportion of pancreatic carcinoma patients. These results indicate that identification of additional tumor antigens by serological screening of tumor cDNA expression libraries by RAYS is a promising goal in pancreatic cancer.

Structure-Activity Profiles of Ab-Derived TNF Fusion Proteins

TNF application in humans is limited by severe side effects, including life-threatening symptoms of shock. Therefore, TNF can be successfully applied as a tumor therapeutic reagent only under conditions that prevent its systemic action. To overcome this limitation, genetic fusion of TNF to tumor-selective Abs is a favored strategy to increase site-specific cytokine targeting. Because wild-type TNF displays its bioactivity as noncovalently linked homotrimer, the challenge is to define structural requirements for a TNF-based immunokine format with optimized structure-activity profile. We compared toxicity and efficacy of a dimerized CH2/CH3 truncated IgG1-TNF fusion protein and a single-chain variable fragment-coupled TNF monomer recognizing fibroblast-activating protein. The former construct preserves its dimeric structure stabilized by the natural disulfide bond IgG1 hinge region, while the latter trimerizes under native conditions. Analysis of complex formation of wild-type TNF and of both fusion proteins with TNFR type 1 (TNF-R1) using surface plasmon resonance correlated well with in vitro and in vivo toxicity data. There is strong evidence that TNF subunits in a trimeric state display similar toxicity profiles despite genetic fusion to single-chain variable fragment domains. However, LD50 of either immunodeficient BALB/c nu/nu or immunocompetent BALB/c mice was significantly decreased following administration of TNF in the formation of IgG1-derived dimeric fusion protein. Reduction of unspecific peripheral complexation of TNF-R1 resulted in higher anticancer potency by immunotargeting of fibroblast-activating protein-expressing xenografts. The broader therapeutic window of the IgG1-derived TNF fusion protein favors the dimeric TNF-immunokine format for systemic TNF-based tumor immunotherapy.

MHC-peptide-specific antibodies reveal inefficient presentation of an HLA-A*0201-restricted, Melan-A-derived peptide after active intracellular processing

MHC‐peptide‐specific Fab antibodies binding to HLA‐A*0201 complexes presenting the wild‐type EAAGIGILTV (EAA) or analogue Melan‐A 10‐mer ELAGIGILTV (ELA) peptide were generated to study efficacy of peptide processing and presentation. None of the selected Fab antibodies detected the naturally processed EAA/HLA‐A*0201 complex on melanoma tumor cells, confirming the known low peptide number on the cell surface. To study the effect of peptide presentation and processing in more detail, genes coding for the A27L‐mutated Melan‐A protein or the processed ELA peptide were introduced into HLA‐A*0201+ B cells by infection with the respective recombinant vaccinia virus construct producing equimolar amounts of GFP‐ubiquitin directly linked to the fragment of interest. Correlating GFP expression to actual numbers of peptide presented, 110–260 ELA peptides had to be synthesized to be presented by a single MHC class I antigen‐peptide complex. This number increased 10‐ to 20‐fold when ELA peptide presentation from the A27L‐mutated full length Melan‐A protein was studied, since 1600–5200 GFP molecules needed to be synthesized for the detection of one ELA peptide. Our results indicate that peptide processing rather than presentation is the rate‐limiting step in our experimental setting and is much more ineffective for Melan‐A than has been previously shown for other MHC class I‐restricted epitopes.