In Silico and In Vitro Evaluation of Bevacizumab Biosimilar MB02 as an Antitumor Agent in Canine Mammary Carcinoma.

Canine mammary carcinomas (CMC) are associated with major aggressive clinical behavior and high mortality. The current standard of care is based on surgical resection, without an established effective treatment scheme, highlighting the urgent need to develop novel effective therapies. Vascular endothelial growth factor (VEGF) is a key regulator of tumor angiogenesis and progression in the majority of solid cancers, including human and canine mammary carcinomas. The first therapy developed to target VEGF was bevacizumab, a recombinant humanized monoclonal antibody, which has already been approved as an anticancer agent in several human cancers. The goal of this work was to establish the therapeutic value of MB02 bevacizumab biosimilar in CMC. First, through different in silico approaches using the MUSCLE multiple-sequence alignment tool and the FoldX protein design algorithm, we were able to predict that canine VEGF is recognized by bevacizumab, after showing an extremely high sequence similarity between canine and human VEGF. Further, by using an ELISA-based in vitro binding assay, we confirmed that MB02 biosimilar was able to recognize canine VEGF. Additionally, canine VEGF-induced microvascular endothelial cell proliferation was inhibited in a concentration-dependent manner by MB02 biosimilar. These encouraging results show a high potential for MB02 as a promising therapeutic agent for the management of CMC.

Coronavirus nucleocapsid protein is an RNA chaperone.

RNA chaperones are nonspecific nucleic acid binding proteins with long disordered regions that help RNA molecules to adopt its functional conformation. Coronavirus nucleoproteins (N) are nonspecific RNA-binding proteins with long disordered regions. Therefore, we investigated whether transmissible gastroenteritis coronavirus (TGEV) N protein was an RNA chaperone. Purified N protein enhanced hammerhead ribozyme self-cleavage and nucleic acids annealing, which are properties that define RNA chaperones. In contrast, another RNA-binding protein, PTB, did not show these activities. N protein chaperone activity was blocked by specific monoclonal antibodies. Therefore, it was concluded that TGEV N protein is an RNA chaperone. In addition, we have shown that purified severe acute respiratory syndrome (SARS)-CoV N protein also has RNA chaperone activity. In silico predictions of disordered domains showed a similar pattern for all coronavirus N proteins evaluated. Altogether, these data led us to suggest that all coronavirus N proteins might be RNA chaperones.

Recombinant dimeric small immunoproteins neutralize transmissible gastroenteritis virus infectivity efficiently in vitro and confer passive immunity in vivo.

Small immunoproteins (SIPs) are single-chain molecules comprising the variable regions of an antibody assembled in a single polypeptide (scFv) and joined to the immunoglobulin heavy-chain dimerizing domain. To investigate the potential of these molecules to provide protection against enteric infections when supplied orally, SIPs were generated against Transmissible gastroenteritis virus (TGEV), a highly pathogenic porcine virus. Different variants of TGEV-specific SIPs were created, of epsilon and alpha isotypes, by exploiting the dimerizing domains epsilonCH4 and alphaCH3 of human and swine origin. Transfected cells secreted these recombinant mini-antibodies efficiently, mainly as dimers stabilized covalently by inter-chain disulphide bridges. The specificity and functionality of the recombinant TGEV-specific SIPs were determined by in vitro binding, neutralization and infection-interference assays. The neutralization indices of the TGEV-specific SIPs were all very similar to that of the original TGEV-specific mAb, thus confirming that the immunological properties have been preserved in the recombinant SIPs. In vivo protection experiments on newborn piglets have, in addition, demonstrated a strong reduction of virus titre in infected tissues of animals treated orally with TGEV-specific SIPs. It has therefore been demonstrated that it is possible to confer passive immunization to newborn pigs by feeding them with recombinant SIPs.

Use of virus vectors for the expression in plants of active full-length and single chain anti-coronavirus antibodies.

To extend the potential of antibodies and their derivatives to provide passive protection against enteric infections when supplied orally in crude plant extracts, we have expressed both a small immune protein (SIP) and a full-length antibody in plants using two different plant virus vectors based on potato virus X (PVX) and cowpea mosaic virus (CPMV). The alphaSIP molecule consisted of a single chain antibody (scFv) specific for the porcine coronavirus, transmissible gastroenteritis virus (TGEV) linked to the alpha-CH3 domain from human IgA. To express the full-length IgA, the individual light and heavy chains from the TGEV-specific mAb 6A.C3 were inserted into separate PVX constructs and plants were co-infected with both constructs. Western blot analysis revealed the efficient expression of both the SIP and IgA molecules. Analysis of crude plant extracts revealed that both the plant-expressed alphaSIP and IgA molecules could bind to and neutralize TGEV in tissue culture, indicating that active molecules were produced. Oral administration of crude extracts from antibody-expressing plant tissue to 2-day-old piglets showed that both the alphaSIP and full-length IgA molecules can provide in vivo protection against TGEV.

An antibody derivative expressed from viral vectors passively immunizes pigs against transmissible gastroenteritis virus infection when supplied orally in crude plant extracts.

To investigate the potential of antibody derivatives to provide passive protection against enteric infections when supplied orally in crude plant extracts, we have expressed a small immune protein (SIP) in plants using two different plant virus vectors based on potato virus X (PVX) and cowpea mosaic virus (CPMV). The epsilonSIP molecule consisted of a single-chain antibody (scFv) specific for the porcine coronavirus transmissible gastroenteritis virus (TGEV) linked to the epsilon-CH4 domain from human immunoglobulin E (IgE). In some constructs, the sequence encoding the epsilonSIP molecule was flanked by the leader peptide from the original murine antibody at its N-terminus and an endoplasmic reticulum retention signal (HDEL) at its C-terminus to allow the expressed protein to be directed to, and retained within, the endoplasmic reticulum. Western blot analysis of samples from Nicotiana clevelandii or cowpea tissue infected with constructs revealed the presence of SIP molecules which retained their ability to dimerize. The analysis of crude plant extracts revealed that the plant-expressed epsilonSIP molecules could bind to and neutralize TGEV in tissue culture, the levels of binding and neutralization reflecting the level of expression. Oral administration of crude extracts from SIP-expressing plant tissue to 2-day-old piglets demonstrated that the extracts which showed the highest levels of in vitro neutralization could also provide in vivo protection against challenge with TGEV.