Gerhard Moldenhauer

Of mice and men: hybridoma and recombinant antibodies

Thousands of mouse monoclonal antibodies have been produced from hybridomas over the past 25 years. The same technique can now be used to clone human antibodies from transgenic mice. Full-length antibodies and recombinant fragments engineered for various diagnostic and therapeutic applications can be obtained in reasonably large amounts after expression in mammalian cells, milk and plants.

Editing of the HLA-DR-peptide repertoire by HLA-DM

Antigenic peptide loading of classical major histocompatibility complex (MHC) class II molecules requires the exchange of the endogenous invariant chain fragment CLIP (class II associated Ii peptide) for peptides derived from antigenic proteins. This process is facilitated by the non‐classical MHC class II molecule HLA‐DM (DM) which catalyzes the removal of CLIP. Up to now it has been unclear whether DM releases self‐peptides other than CLIP and thereby modifies the peptide repertoire presented to T cells. Here we report that DM can release a variety of peptides from HLA‐DR molecules. DR molecules isolated from lymphoblastoid cell lines were found to carry a sizeable fraction of self‐peptides that are sensitive to the action of DM. The structural basis for this DM sensitivity was elucidated by high‐performance size exclusion chromatography and a novel mass spectrometry binding assay. The results demonstrate that the overall kinetic stability of a peptide bound to DR determines its sensitivity to removal by DM. We show that DM removes preferentially those peptides that contain at least one suboptimal side chain at one of their anchor positions or those that are shorter than 11 residues. These findings provide a rationale for the previously described ligand motifs and the minimal length requirements of naturally processed DR‐associated self‐peptides. Thus, in endosomal compartments, where peptide loading takes place, DM can function as a versatile peptide editor that selects for high‐stability MHC class II‐peptide complexes by kinetic proofreading before these complexes are presented to T cells.

VEGF expression by mesenchymal stem cells contributes to angiogenesis in pancreatic carcinoma.

Little is known about the factors that enable the mobilisation of human mesenchymal stem cells (MSC) from the bone marrow into the blood stream and their recruitment to and retention in the tumour. We found specific migration of MSC towards growth factors present in pancreatic tumours, such as PDGF, EGF, VEGF and specific inhibitors Glivec, Erbitux and Avastin interfered with migration. Within a few hours, MSC migrated into spheroids consisting of pancreatic cancer cells, fibroblasts and endothelial cells as measured by time-lapse microscopy. Supernatant from subconfluent MSC increased sprouting of HUVEC due to VEGF production by MSC itself as demonstrated by RT-PCR and ELISA. Only few MSCs were differentiated into endothelial cells in vitro, whereas in vivo differentiation was not observed. Lentiviral GFP-marked MSCs, injected in nude mice xenografted with orthotopic pancreatic tumours, preferentially migrated into the tumours as observed by FACS analysis of green fluorescent cells. By immunofluorescence and intravital microscopic studies, we found the interaction of MSC with the endothelium of blood vessels. Mesenchymal stem cells supported tumour angiogenesis in vivo, that is CD31+ vessel density was increased after the transfer of MSC compared with siVEGF-MSC. Our data demonstrate the migration of MSC toward tumour vessels and suggest a supportive role in angiogenesis.

HLA-DM Acts as a Molecular Chaperone and Rescues Empty HLA-DR Molecules at Lysosomal pH

HLA-DM (DM) is a nonclassical MHC class II molecule that interacts with classical MHC II molecules in acidic compartments. During this association DM is supposed to catalyze the release of invariant chain (Ii)-derived CLIP peptides, as well as other peptides bound with low kinetic stability. Here we provide evidence that in lysosomal compartments of B cells a considerable fraction of DM is stably associated with empty DR alphabeta dimers, thereby preventing their functional inactivation and aggregation. Upon encounter with cognate peptide, the DM-associated DR molecules can be rapidly loaded and no longer bind to DM. Thus, DM seems to act as a dedicated class II-specific chaperone. In view of the suggested shortage of DM-resistant self-peptides in the loading compartment, empty class II molecules that are chaperoned by DM may enable the antigen-processing system to respond promptly to the challenge by newly entering antigens.

High level production of soluble single chain antibodies in small-scale Escherichia coli cultures

We have investigated the effect of growth and induction conditions on the production of soluble single-chain Fv antibody fragments in Escherichia coli under the control of wt lac promoter. The scFv was directed into the periplasmic space by a pelB leader sequence. Addition of sucrose to the medium gave a 15-25-fold increase in the yield of soluble scFv-phOx (3.0 mg/l) for bacterial shake-tube cultures and an increase of 80-150-fold (16.5 mg/l) for shake-flask cultures. Using flask culture in the presence of 0.4 M sucrose, a significant amount of scFv was released into the medium. We found that the scFv could be made to accumulate in the periplasm or be secreted into the medium by simply changing the incubation conditions and the concentration of the inducer. The ratio between soluble antibody fragments and insoluble scFv aggregates proved to be dependent on the strength of the promoter. Lowering the incubation temperature below 20 degrees C had no effect on the yield of soluble antibody fragments in the periplasm, but they were no longer secreted into the medium. An example of high level production in shake-flask cultures and one-step purification by immobilized metal affinity chromatography (IMAC) is described for a soluble scFv specific for the T cell surface antigen CD3. The biological activity of the purified anti-CD3 scFv was demonstrated by flow cytometry. This method should be especially useful for the functional screening of a large number of clones in small-scale cultures.

A Complex of EpCAM, Claudin-7, CD44 Variant Isoforms, and Tetraspanins Promotes Colorectal Cancer Progression

High expression of EpCAM and the tetraspanin CO-029 has been associated with colorectal cancer progression. However, opposing results have been reported on CD44 variant isoform v6 (CD44v6) expression. We recently noted in rat gastrointestinal tumors that EpCAM, claudin-7, CO-029, and CD44v6 were frequently coexpressed and could form a complex. This finding suggested the possibly that the complex, rather than the individual molecules, could support tumor progression. The expression of EpCAM, claudin-7, CO-029, and CD44v6 expression was evaluated in colorectal cancer (n = 104), liver metastasis (n = 66), and tumor-free colon and liver tissue. Coexpression and complex formation of the molecules was correlated with clinical variables and apoptosis resistance. EpCAM, claudin-7, CO-029, and CD44v6 expression was up-regulated in colon cancer and liver metastasis. Expression of the four molecules did not correlate with tumor staging and grading. However, coexpression inversely correlated with disease-free survival. Coexpression was accompanied by complex formation and recruitment into tetraspanin-enriched membrane microdomains (TEM). Claudin-7 contributes to complex formation inasmuch as in the absence of claudin-7, EpCAM hardly associates with CO-029 and CD44v6 and is not recruited into TEMs. Notably, colorectal cancer lines that expressed the EpCAM/claudin-7/CO-029/CD44v6 complex displayed a higher degree of apoptosis resistance than lines devoid of any one of the four molecules. Expression of EpCAM, claudin-7, CO-029, and CD44v6 by themselves cannot be considered as prognostic markers in colorectal cancer. However, claudin-7–associated EpCAM is recruited into TEM and forms a complex with CO-029 and CD44v6 that facilitates metastasis formation. (Mol Cancer Res 2007;5(6):553–67)

Sulforaphane targets pancreatic tumour-initiating cells by NF-κB-induced antiapoptotic signalling

Background and aims: Emerging evidence suggests that highly treatment-resistant tumour-initiating cells (TICs) play a central role in the pathogenesis of pancreatic cancer. Tumour necrosis factor-related apoptosis-inducing ligand (TRAIL) is considered to be a novel anticancer agent; however, recent studies have shown that many pancreatic cancer cells are resistant to apoptosis induction by TRAIL due to TRAIL-activated nuclear factor-κB (NF-κB) signalling. Several chemopreventive agents are able to inhibit NF-κB, and favourable results have been obtained—for example, for the broccoli compound sulforaphane—in preventing metastasis in clinical studies. The aim of the study was to identify TICs in pancreatic carcinoma for analysis of resistance mechanisms and for definition of sensitising agents. Methods: TICs were defined by expression patterns of a CD44+/CD24−, CD44+/CD24+ or CD44+/CD133+ phenotype and correlation to growth in immunodeficient mice, differentiation grade, clonogenic growth, sphere formation, aldehyde dehydrogenase (ALDH) activity and therapy resistance. Results: Mechanistically, specific binding of transcriptionally active cRel-containing NF-κB complexes in TICs was observed. Sulforaphane prevented NF-κB binding, downregulated apoptosis inhibitors and induced apoptosis, together with prevention of clonogenicity. Gemcitabine, the chemopreventive agents resveratrol and wogonin, and the death ligand TRAIL were less effective. In a xenograft model, sulforaphane strongly blocked tumour growth and angiogenesis, while combination with TRAIL had an additive effect without obvious cytotoxicity in normal cells. Freshly isolated patient tumour cells expressing markers for TICs could be sensitised by sulforaphane for TRAIL-induced cytotoxity. Conclusion: The data provide new insights into resistance mechanisms of TICs and suggest the combination of sulforaphane with TRAIL as a promising strategy for targeting of pancreatic TICs.

Bispecific CD3×CD19 diabody for T cell-mediated lysis of malignant human B cells

For the treatment of minimal residual disease in patients with leukemias and malignant lymphomas, we constructed a heterodimeric diabody specific for human CD19 on B cells and CD3ϵ chain of the T cell receptor complex. The bispecific diabody was expressed in Escherichia coli using a vector containing a dicistronic operon for co‐secretion of VH3‐VL19 and VH19‐VL3 single‐chain Fv fragments (scFv). It was purified in one step by immobilized metal affinity chromatography (IMAC) from the periplasmic extract and culture medium. Flow cytometry experiments revealed specific interactions of the diabody with both CD3 and CD19 positive cells, to which it bound with affinities close to those of the parental scFvs. It was less stable than anti‐CD3 scFv but more stable than anti‐CD19 scFv when incubated in human serum at 37°C. In cytotoxicity tests, the diabody proved to be a potent agent for retargeting peripheral blood lymphocytes to lyse tumor cells expressing the CD19 antigen. The efficiency of cell lysis compared favorably with that obtained with a bispecific antibody (BsAb) of the same dual specificity that was prepared by the quadroma technique. Int. J. Cancer 77:763–772, 1998.

Efficient Inhibition of Intra-Peritoneal Tumor Growth and Dissemination of Human Ovarian Carcinoma Cells in Nude Mice by Anti-L1-Cell Adhesion Molecule Monoclonal Antibody Treatment

The L1 cell adhesion molecule is implicated in the control of proliferation, migration, and invasion of several tumor cell types in vitro. Recently, L1 overexpression was found to correlate with tumor progression of ovarian carcinoma, one of the most common causes of cancer-related deaths in gynecologic malignant diseases. To evaluate L1 as a potential target for ovarian cancer therapy, we investigated the effects of anti-L1 monoclonal antibodies (chCE7 and L1-11A) on proliferation and migration of L1-positive human SKOV3ip ovarian carcinoma cells in vitro and the therapeutic efficacy of L1-11A against i.p. SKOV3ip tumor growth in nude mice. In vitro, both anti-L1 antibodies efficiently inhibited the proliferation of SKOV3ip cells as well as other L1-expressing tumor cell lines (renal carcinoma, neuroblastoma, and colon carcinoma). On two cell lines, hyper-cross-linking of L1-11A with a secondary antibody was necessary for significant inhibition of proliferation, indicating that cross-linking of L1 is required for the antiproliferative effect. L1-negative prostate carcinoma cells were not influenced by antibody treatment. Biweekly treatment of ovarian carcinoma-bearing mice with L1-11A led to a dose-dependent and significant reduction of tumor burden (up to -63.5%) and ascites formation (up to -75%). This effect was associated with reduced proliferation within the tumors. L1-directed antibody-based inhibition of peritoneal growth and dissemination of human ovarian carcinoma cells represents important proof-of-principle for the development of a new therapy against one of the leading gynecologic malignant diseases.

Di-, tri- and tetrameric single chain Fv antibody fragments against human CD19: effect of valency on cell binding.

Single chain variable fragments (scFv) of the murine monoclonal antibody HD37 specific to human B‐cell antigen CD19 were constructed by joining the VH and VL domains with linkers of 18, 10, 1 and 0 residues. ScFv‐18 formed monomers, dimers and small amounts of tetramers; scFv‐10 formed dimers and small amounts of tetramers; scFv‐1 formed exclusively tetramers; scFv‐0 formed exclusively trimers. The affinities of the scFv‐10 (diabody) and scFv‐1 (tetrabody) were approximately 1.5‐ and 2.5‐fold higher, respectively, than that of the scFv‐0 (triabody). The tetrabody displayed a significantly prolonged association with cell‐bound antigen (t 1/2 cell surface retention at 37°C of 26.6 min) compared to both the diabody (13.3 min) and triabody (6.7 min). This increase in avidity of the tetrabody combined with its larger size could prove to be particularly advantageous for imaging and the immunotherapy of B‐cell malignancies.