Construction of miniantibodies for the in vivo study of human autoimmune diseases in animal models

<p>Abstract</p> <p>Background</p> <p>Phage display antibody libraries have been made from the lymphocytes of patients suffering from autoimmune diseases in which the antibodies are known to play a role in the pathogenesis or are important for the diagnosis of the disease. In the case of Celiac Disease, the immune response is directed against the autoantigen tissue transglutaminase. However, despite numerous studies, the role of these antibodies in the pathogenesis of this disease has not been elucidated.</p> <p>Results</p> <p>We were able to engineer specific anti-transglutaminase antibody fragments in the form called "miniantibody". These are produced by genetic fusion of anti-tTG scFv to Human, Mouse or Rat Fc domains, making them suitable for in vivo expression. The results obtained here indicate that the miniantibody molecule is efficiently secreted, and that the reactivity to the antigen is retained even after fusion to heterologous Fc domains. Further analysis demonstrate that the molecule is secreted as homodimeric, mimicking original antibody structure. Finally, the in vivo expression in mice leads to detectable serum levels with no apparent gross immune response by the host.</p> <p>Conclusion</p> <p>In this work we demonstrated the usefulness of a method for the in vivo expression of miniantibodies specific to transglutaminase, corresponding to the autoimmune specificity of Celiac Disease. This can be proposed as a general method to study the pathogenic role of autoimmune antibodies in autoimmune diseases.</p

Autologous anti-GD2 CAR T cells efficiently target primary human glioblastoma

Glioblastoma (GBM) remains a deadly tumor. Treatment with chemo-radiotherapy and corticosteroids is known to impair the functionality of lymphocytes, potentially compromising the development of autologous CAR T cell therapies. We here generated pre-clinical investigations of autologous anti-GD2 CAR T cells tested against 2D and 3D models of GBM primary cells. We detected a robust antitumor effect, highlighting the feasibility of developing an autologous anti-GD2 CAR T cell-based therapy for GBM patients

Targeting GD2-positive glioblastoma by chimeric antigen receptor empowered mesenchymal progenitors

Tumor targeting by genetically modified mesenchymal stromal/stem cells (MSCs) carrying anti-cancer molecules represents a promising cell-based strategy. We previously showed that the pro-apoptotic agent tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) can be successfully delivered by MSCs to cancer sites. While the interaction between TRAIL and its receptors is clear, more obscure is the way in which MSCs can selectively target tumors and their antigens. Several neuroectoderm-derived neoplasms, including glioblastoma (GBM), sarcomas, and neuroblastoma, express high levels of the tumor-associated antigen GD2. We have already challenged this cell surface disialoganglioside by a chimeric antigen receptor (CAR)-T cell approach against neuroblastoma. With the intent to maximize the therapeutic profile of MSCs delivering TRAIL, we here originally developed a bi-functional strategy where TRAIL is delivered by MSCs that are also gene modified with the truncated form of the anti-GD2 CAR (GD2 tCAR) to mediate an immunoselective recognition of GD2-positive tumors. These bi-functional MSCs expressed high levels of TRAIL and GD2 tCAR associated with a robust anti-tumor activity against GD2-positive GBM cells. Most importantly, the anti-cancer action was reinforced by the enhanced targeting potential of such bi-functional cells. Collectively, our results suggest that a truncated anti-GD2 CAR might be a powerful new tool to redirect MSCs carrying TRAIL against GD2-expressing tumors. This affinity-based dual targeting holds the promise to combine site-specific and prolonged retention of MSCs in GD2-expressing tumors, thereby providing a more effective delivery of TRAIL for still incurable cancers

New tags for recombinant protein detection and O-glycosylation reporters.

Monoclonal antibodies (mAbs), because of their unique specificity, are irreplaceable tools for scientific research. Precise mapping of the antigenic determinants allows the development of epitope tagging approaches to be used with recombinant proteins for several purposes. Here we describe a new family of tags derived from the epitope recognized by a single highly specific mAb (anti-roTag mAb), which was obtained from a pool of mAbs reacting with the rotavirus nonstructural protein 5 (NSP5). The variable regions of the anti-roTag mAb were identified and their binding capacity verified upon expression as a single-chain/miniAb. The minimal epitope, termed roTag, was identified as a 10 amino acid sequence (SISSSIFKNE). The affinity of the anti-roTag/roTag interaction was found to be comparable to that of the anti-SV5/SV5 tag interaction. roTag was successfully used for detection of several recombinant cytosolic, secretory and membrane proteins. Two additional variants of roTag of 10 and 13 amino acids containing O-glycosylation susceptible sites (termed OG-tag and roTagO) were constructed and characterised. These tags were useful to detect proteins passing through the Golgi apparatus, the site of O-glycosylation

Efficient Detection of Proteins Retro-Translocated from the ER to the Cytosol by In Vivo Biotinylation

Retro-translocation from the ER to the cytosol of proteins within the secretory pathway takes place on misfolded molecules that are targeted for degradation by the cytosolically located 26S proteasome complex. Retro-translocation occurs also for other proteins (such as calreticulin) that, despite being synthesized and transported to the ER, are in part dislocated to the cytosol. We have taken advantage of the E. coli derived biotin-ligase (BirA) expressed in the cytosol of mammalian cells to specifically biotin-label in vivo proteins within the secretory pathway that undergo retro-translocation. We validated the method using four different proteins that are known to undergo retro-translocation upon different conditions: the human trans-membrane protein MHC class-I a chain (MHC-Ia), the Null Hong Kong mutant of the secretory a1 anti-trypsin (NHKa1AT), the immunoglobulin heavy chain (HC) and the ER chaperone calreticulin (Crt). We observed specific monobiotinylation of cytosolically dislocated molecules, resulting in a novel, reliable way of determining the extent of retrotranslocation

The HPV-18 E7 CKII phospho acceptor site is required for maintaining the transformed phenotype of cervical tumour-derived cells.

The Human Papillomavirus E7 oncoprotein plays an essential role in the development and maintenance of malignancy, which it achieves through targeting a number of critical cell control pathways. An important element in the ability of E7 to contribute towards cell transformation is the presence of a Casein Kinase II phospho-acceptor site within the CR2 domain of the protein. Phosphorylation is believed to enhance E7 interaction with a number of different cellular target proteins, and thereby increase the ability of E7 to enhance cell proliferation and induce malignancy. However, there is little information on how important this site in E7 is, once the tumour cells have become fully transformed. In this study, we have performed genome editing of the HPV-18 E7 CKII recognition site in C4-1 cervical tumour-derived cells. We first show that mutation of HPV18 E7 S32/S34 to A32/A34 abolishes CKII phosphorylation of E7, and subsequently we have isolated C4-1 clones containing these mutations in E7. The cells continue to proliferate, but are somewhat more slow-growing than wild type cells, reach lower saturation densities, and are also more susceptible to low nutrient conditions. These cells are severely defective in matrigel invasion assays, partly due to downregulation of matrix metalloproteases (MMPs). Mechanistically, we find that phosphorylation of E7 plays a direct role in the ability of E7 to activate AKT signaling, which in turn is required for optimal levels of MMP secretion. These results demonstrate that the E7 CKII phospho-acceptor site thus continues to play an important role for E7's activity in cells derived from cervical cancers, and suggests that blocking this activity of E7 could be expected to have therapeutic potential

O-glycosylated tags.

<p>(A) WB of supernatants of 293T cells transfected with the reporter protein tagged with OG-tag, P-roTag or roTagO (previously indicated as 9–18, 11–21 and 9–21, respectively) treated (T) or not (Ctrl) with a glycosidase mix containing Neuraminidase, β1-3 Galactosidase and β-NAc-hexosaminidase. (B) WB of cellular extracts (E) and supernatants (S) of HEK 293T cells transfected with the reporter protein tagged with OG-tag and roTagO with or without the ER retention signal KDEL. In all panels blots were developed, as indicated, with anti-SV5 or anti-roTag/1F2.</p

Mapping of mAb 1F2 epitope and related tags.

<p>(A) Scheme of the reporter protein used (scFv) tagged with the peptides shown in the right panel. The amino acid sequence present in NSP5 is highlighted in blue. (B) – (E) WB of cellular extracts (indicated as E) and supernatants (indicated as S) of HEK 293T cells transfected with the reporter protein tagged with: (B) an irrelevant amino acid sequence (Ctrl) or peptide 9–24, (C) peptides 9–24, S22G or 9–21, (D) peptide 12–24, (E) peptides 10–21, 11–21 or 12–21, (F) peptides 11–21 or 12–21 (here also indicated as P-roTag and roTag, respectively), (G) peptides 9–20, 9–19 and 9–18. In all panels blots were developed, as indicated, with anti-SV5 or 1F2. (F) Equal dilutions of cell extracts used in (E) analyzed by WB and densitometric analysis. The plot shows values for the non-saturating linear range part.</p

mAb 1F2 detection of NSP5 N-terminal deletion mutants.

<p>(A) NSP5 N-terminal sequence; deleted amino acids are indicated in red. (B) and (C) WB of cellular extracts of HEK 293T cells transfected with NSP5 N-terminal deletion mutants or empty vector (Ctrl), developed with polyclonal anti-NSP5 serum (B) or 1F2 (C).</p

Sequences and properties of roTag epitopes family.

<p>Sequences and properties of roTag epitopes family.</p