Holger Bartsch

Retargeting of T cells to prostate stem cell antigen expressing tumor cells: comparison of different antibody formats.

Prostate cancer (PCa) is the most common malignant disease in men. Novel treatment options are needed for patients after development of metastatic, hormone‐refractory disease or for those who have failed a local treatment. The prostate stem cell antigen (PSCA) is expressed in >80% of primary PCa samples and bone metastases. Its expression is increased both in androgen‐dependent and independent prostate tumors, particularly in carcinomas of high stages and Gleason scores. Therefore, PSCA is an attractive target for immunotherapy of PCa by retargeting of T cells to tumor cells.

Simultaneous engagement of the activatory receptors NKG2D and CD3 for retargeting of effector cells to CD33-positive malignant cells

Simultaneous engagement of the activatory receptors NKG2D and CD3 for retargeting of effector cells to CD33-positive malignant cells

Unexpected recombinations in single chain bispecific anti-CD3-anti-CD33 antibodies can be avoided by a novel linker module.

CD33 is an attractive immunotarget on the surface of tumor cells from patients with acute myeloid leukemia (AML). In a first attempt for immunotargeting of AML blasts we constructed two bispecific antibodies in the single chain bispecific diabody (scBsDb) format by fusing the variable domains of monoclonal antibodies directed against CD3 and CD33. Unfortunately, protein expression of both scBsDbs resulted in varying mixtures of fragmented and full length proteins. As the non-functional fragments competed with the functional full length antibodies we tried to understand the reason for the fragmentation. We found that the anti-CD3 and anti-CD33 antibody genes show striking sequence homologies: during B cell development the same V(h) J558 heavy and V(l) kk4 light chain genes were selected. Moreover, the closely related D genes DSP2 (9 and 11) were combined with the same JH4 gene. And finally, during VJ recombination of the light chain the same JK5 element was selected. These homologies between the two monoclonal antibodies were the reason for recombinations in the cell lines generated for expression of the scBsDbs. Finally, we solved this problem by (i) rearranging the order of the heavy and light chains of the anti-CD3 and anti-CD33 domains, and (ii) a replacement of one of the commonly used glycine serine linkers with a novel linker domain. The resulting bispecific antibody in a single chain bispecific tandem format (scBsTaFv) was stable and capable of redirecting T cells to CD33-positive tumor cells including AML blasts of patients.

A Novel Modular Antigen Delivery System for Immuno Targeting of Human 6-sulfo LacNAc-Positive Blood Dendritic Cells (SlanDCs)

Background Previously, we identified a major myeloid-derived proinflammatory subpopulation of human blood dendritic cells which we termed slanDCs (e.g. Schäkel et al. (2006) Immunity 24, 767–777). The slan epitope is an O-linked sugar modification (6-sulfo LacNAc, slan) of P-selectin glycoprotein ligand-1 (PSGL-1). As slanDCs can induce neoantigen-specific CD4+ T cells and tumor-reactive CD8+ cytotoxic T cells, they appear as promising targets for an in vivo delivery of antigens for vaccination. However, tools for delivery of antigens to slanDCs were not available until now. Moreover, it is unknown whether or not antigens delivered via the slan epitope can be taken up, properly processed and presented by slanDCs to T cells. Methodology/Principal Findings Single chain fragment variables were prepared from presently available decavalent monoclonal anti-slan IgM antibodies but failed to bind to slanDCs. Therefore, a novel multivalent anti-slanDC scaffold was developed which consists of two components: (i) a single chain bispecific recombinant diabody (scBsDb) that is directed on the one hand to the slan epitope and on the other hand to a novel peptide epitope tag, and (ii) modular (antigen-containing) linker peptides that are flanked at both their termini with at least one peptide epitope tag. Delivery of a Tetanus Toxin-derived antigen to slanDCs via such a scBsDb/antigen scaffold allowed us to recall autologous Tetanus-specific memory T cells. Conclusions/Significance In summary our data show that (i) the slan epitope can be used for delivery of antigens to this class of human-specific DCs, and (ii) antigens bound to the slan epitope can be taken up by slanDCs, processed and presented to T cells. Consequently, our novel modular scaffold system may be useful for the development of human vaccines.

Generation of single-chain bispecific green fluorescent protein fusion antibodies for imaging of antibody-induced T cell synapses.

There is growing interest in the development of novel single-chain bispecific antibodies for retargeting of immune effector T cells to tumor cells. Until today, functional fusion constructs consisting of a single-chain bispecific antibody and a fluorescent protein were not reported. Such molecules could be useful for an in vivo visualization of this retargeting process. Recently, we established two novel single-chain bispecific antibodies. One is capable of retargeting T cells to CD33, and the other is capable of retargeting T cells to the prostate stem cell antigen (PSCA). CD33 is an attractive immunotarget on the surface of tumor cells from patients with acute myeloid leukemia (AML). The PSCA is a potential target on prostate cancer cells. Flanking the reading frame encoding the green fluorescent protein (GFP) with a recently described novel helical linker element allowed us to establish novel single-chain bispecific fusion antibodies. These fluorescent fusion antibodies were useful to efficiently retarget T cells to the respective tumor cells and visualize the formation of immune synapses between effector and target cells.

Isolation of rat cDNA clones coding for the autoantigen SS-B/La: detection of species-specific variations

Clones of cDNA coding for the autoantigen La (or SS-B) were isolated from a library made from rat liver. A comparison of the rat La cDNA (encoding from nt 38 to 1281 for rat La protein) with the sequences known for human and bovine La protein resulted in the identification of species-specific inserts. The inserts seem to be the result of multiplication of flanking sequences during evolution. In addition to these variations, we observed that rat La cDNAs exhibit non-canonical polyadenylation sites. Finally, a databank search resulted in the identification of a DNA sequence originally termed as TAG or TSG20X (GenBank accession No. X61893) which represents the C terminus of mouse La/SS-B protein.

Autoimmunity as a Result of Escape from RNA Surveillance

In previous studies, we detected a frame shift mutation in the gene encoding the autoantigen La of a patient with systemic lupus erythematosus. The mutant La mRNA contains a premature termination codon. mRNAs that prematurely terminate translation should be eliminated by RNA quality control mechanisms. As we find Abs specific for the mutant La form in ∼30% of sera from anti-La-positive patients, we expected that mutant La mRNAs circumvent RNA control and the expression of mutant La protein could become harmful. Indeed, real-time PCR, immunostaining, and immunoblotting data of mice transgenic for the mutant La form show that mutant La mRNAs are not repressed in these animals and are translated to mutant La protein. In addition to the mutant La protein, we detected a minor portion of native human La in the mutant La-transgenic mice. Therefore, ribosomal frame shifting may allow the mutant La mRNA to escape from RNA control. Interestingly, expression of the mutant La mRNA results in a lupus-like disease in the experimental mice. Consequently, escape of mutant La mRNA from RNA control can have two effects: it 1) results in the expression of an immunogenic (neo)epitope, and 2) predisposes to autoimmunity.

Detection of Autoantibodies to Tumour-Associated Antigens in Sera of Patients with Systemic Autoimmunity Using a Novel Protein Microblot Array

It is well known that sera of patients with systemic autoimmunity contain autoantibodies to nuclear antigens. It is also known that patients with systemic autoimmunity have an increased risk for the development of tumours. Interestingly, tumour patients frequently develop autoantibodies and there is a growing list of potential tumour‐associated antigens. It is, however, not known whether or not patients with systemic autoimmunity also develop antibodies to tumour‐associated antigens. Here we describe the development of a novel multiprotein array allowing us to screen for autoantibodies to 30 different tumour‐associated antigens in parallel. Using this novel assay, we found that the frequency of autoantibodies to the selected tumour‐associated antigens is increased between 2‐ and 14‐fold in patients with systemic autoimmunity compared with an age‐matched control group.

Coomassie-Brilliant Blue Staining of Polyacrylamide Gels

Over the past, a series of staining procedures for proteins were published. The most commonly used staining dye for proteins is still Coomassie-Brilliant Blue. The major reason is Coomassie-Brilliant Blue staining is simple, fast, and sensitive. As Coomassie-Brilliant Blue is almost insoluble in water, a series of procedures including colloidal aqueous procedures were described.

Cell type-specific expression of endogenous cardiac Troponin I antisense RNA in the neonatal rat heart

Since the number of detected natural antisense RNA is growing, investigations upon the expression pattern of the antisense RNA become more important. As we focused our work on natural occurring antisense transcripts in human and rat heart tissues, we were interested in the question, whether the expression pattern of antisense and sense RNA can vary in different cell types of the same tissue. In our previous analysis of total neonatal rat heart tissue, we demonstrated the co-expression of both cTnI RNA species in this tissue. Now we investigated the expression of antisense and sense RNA quantitatively in neonatal cardiomyocytes (NCMs) and neonatal cardiac fibroblasts (NCFs). Performing northern blot as well as RT-PCR, we could detect natural antisense and sense RNA transcripts of cTnI in NCM and NCF implying that these transcripts are co-expressed in both cell types. The absolute amounts of the RNA transcripts were higher in NCM. Both RNA species showed identical sizes in the northern blot. Quantification by real-time PCR revealed a higher relative level of natural antisense RNA in NCF compared to NCM which points out to a cell type-specific expression of sense and antisense RNA. Our observations suggest that antisense RNA transcription may contribute to a cell type-specific regulation of the cTnI gene.