CHO-GMT recombinant protein expression

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The present invention provides modified cells for producing proteins with modified glycosylation patterns. Proteins produced in such cells, and the use of such proteins in medicine, and particularly in the treatment of cancer, is also provided.

The present invention relates to the field of biotechnology and molecular biology. In particular, the present invention relates to mammalian cell lines and their use in protein expression in. The present invention also relates to such proteins for use in medicine.

The antigen mannosylation is an antigen effective method for enhancing immunogenicity, since it enhances the antigen presenting cells (APC), especially dendritic cells (DC) are antigen uptake and presentation. Apostolopoulos and his colleagues focused on the target DC MUCl mannose-binding receptors. Has MUCl peptide conjugated to yeast mannan DC to enhance uptake. They have demonstrated under oxidizing conditions by antigen conjugated to a carbohydrate polymer mannan induced able to induce a high cytotoxic T- lymphocytes (CTL) in the Tl-type response. Some clinical trials research traitor with conjugated to oxidized mannan MUCl GST fusion peptide. However, due to the presence of immunodominant epitopes irreversible modification may therefore mannose glycosylated antigen for traditional chemical methods are not always reliable. Expression of soluble recombinant protein mannosylation need to introduce glycosylation sites on the protein in yeast, also require complex characterization or optimization step. In order to produce reliable mannosylation antigen needs to address these issues.

Tn- glycosylated glycopeptide MUCl

The main problem above strategy is to tumor-associated glycans and MUCl derived peptides separated. When using a not-glycosylated MUCl derived peptide as a vaccine, the immune response against not only glycosylated MUCl and not for abnormal sugar in the cancer cells express the glycosylated MUC1. By using these non-glycosylated peptide immunization to produce MUCl strong humoral immunity against attempts usually failed, partly due to tolerance. To overcome this problem, a set of recombinant human glycosyltransferase synthesized having cancer-associated antigens MUClTR glycopeptide by chemical enzymatic method (Serensen et al., 2006). Conjugated to KLH having different densities of Tn and STn sugar shaped MUCl glycopeptide aroused the most intense and breast cancer cell lines expressed MUCl reactive antibody response. Humoral immune response induced cancer cell lines showed strong specificity, which indicates that there is hope glycopeptides designed as a cancer vaccine. Caused by the immune response against a sugar binding peptide epitopes, and peptide sequence and carbohydrate structures are very important for the antigen.

wild-type CHO-Kl cells to produce recombinant MUCl

have been generated and reported 16 TR containing recombinant MUCl. Because it is generated in the MUC1,0- glycan wild type CHO-Kl cells is different from the tumor-associated antigen ST and diST. Thus, the recombinant MUCl CHO cells from wild-type different from MUCl expressed on the surface of cancer cells chemically.

The above-mentioned study based on defects

The above-mentioned study based on each item has its flaws. STn-KLH contains only 0- glycan not containing MUCl peptide. Instead, BLP25 liposome vaccine (Stimuvax) contains only peptide not containing glycans. Conjugated to mannan peptides seem MUCl enhanced DC antigen uptake. DC on in vivo targeting of C- type lectin receptor is an effective strategy to increase vaccine efficacy. However, this method has the same problem because of its lack of connection with a tumor associated peptide MUCl o- glycans. As has been demonstrated not glycosylated peptide MUCl cause humoral response, the antibody does not recognize glycosylated MUCl. The series has proved MUCl 20 amino acid repeat motif of GSTA with only immature short glycans- when starting presenting a highly immunodominant epitope (Tarp et al ·, 2007). Vlad et al. (2002) have demonstrated in the course of a tumor antigen MUCl DC glycopeptides for processing will not remove 0- glycans. DC swallow the case within glycopeptides, they are processed into smaller peptides and carbohydrates without removing these smaller peptides presented on MHCII molecules. Obtained in MHCII groove binding (binding grove) in glycopeptides can trigger abnormal glycosylation MUCl Acknowledge glycopeptide specific CD4T cells. In addition, 0 glycosylation also affected the DC on MUCl intracellular processing. MUCl intracellular cleavage site located between a tandem repeat of 20 amino acids Thr-Ser peptide bond. However, if any one of the amino acid residues are glycosylated, then cut in on the site namely stop. All these data suggest that MUCl glycosylated peptides and different antigens MUCl not glycosylated peptides represent, therefore, MUCl peptides used in vaccines should be properly glycosylated. Chemically synthesized enzyme Tn / STnMUCl glycopeptide an important step forward, because they can cause cancer-specific antibody response against -MUCI. But it is difficult to ensure that each batch to get the same glycosylation pattern. The need for new ways to be more cost-effective way to continuous production in the peptide sequence and glycosylation form the same vaccine precision analog MUCl structure.

Dendritic cells (DC) and C- type lectins bind mannose macrophages can significantly enhance antigen uptake and processing

DC is the most effective antigen presenting cells (APC) and having a central role in guiding the adaptive immune response. DC at the outer surface and the inner surface of the body to specifically bind to an antigen presented to them in the lymphoid organs of lymphocytes. DC uptake receptors on can significantly increase the efficiency of antigen capture and processing. Many of these uptake receptors are mannose-specific C- type lectins, such as Langerhans cell-specific protein (Iangerin) (CD207), DC-SIGN (CD209) and mannose receptor (CD206). Another type of APC-- macrophages also use mannose-specific uptake as C- type lectin receptors (Gazi et al., 2009). Therefore, the vaccine mannosylation has become an antigen specifically target DC and macrophages to enhance the effectiveness of the antigen effective method. As mentioned above, the yeast mannan and MUCl peptide conjugate to enhance the immunogenicity. In addition to mannan, it has proved mannose N- glycans capped recombinant protein can also be on the mannose-specific C- type lectin identified and macrophages swallow. A successful examples from the use of decomposition GlcCer in macrophages recombinant glucocerebrosidase for patients with Gaucher disease treatment. Recombinant glucocerebrosidase produced by the wild-type CHO cells only when they are continuously exoglycosidases process until exposed N- glycans (Man 3GlcNAc2) mannose residues can be effectively captured by macrophages. Having dysfunction of GnT I gene CHO glycosylation mutants of recombinant glucocerebrosidase contain mannose-terminated N- glycans (Man5GlcNAc2) . The product can also be C- type lectin on the surface of macrophages specific uptake (pick up). The present invention's goal is to produce MUCl in GnT I mutant CHO cell lines, the GnT I mutant CHO cell lines will produce MUCl contain mannose-terminated N- glycans. This feature can greatly enhance its effectiveness as an anticancer vaccine.

In some clinical trials have MUCl as a cancer immunotherapy target. Biomira has developed Theratope, which is used to treat breast cancer. Theratope is a synthetic vaccine, which STn disaccharide is chemically linked to a protein carrier - keyhole limpet hemocyanin (KLH) on. Since the specificity of the peptide backbone containing TR separate STn epitope strong enough to cause abnormal sugar for cancer on the base of MUCl immune response, thus Theratop failed in a Phase III trial. BLP25 liposomal vaccine (Stimuvax) containing derivatized non-glycosylated peptides from TR a. Currently, III trial of Merck's ongoing Stimuvax. Stimuvax potential problem is not glycosylated peptide MUCl Unlike glycosylated MUC1 expressed on cancer cells chemically. Binding of TR unglycosylated high affinity antibody can not recognize cancer-related MUCl. Has MUCl mannose glycosylated peptides as vaccine research traitor.

TR already containing the five peptides conjugated to mannan yeast to increase dendritic cells (DC) uptake. DC and mannose-binding lectin C- type macrophages can significantly enhance antigen uptake and processing. However, this peptide also lacks MUCl o- tumor-associated glycans. Has passed the MUClcDNA delivered to the patient carried a gene therapy attempt. Because not express the transgene in the patient's cancer cells, thus resulting 0- glycan MUCl on normal cells and 0-glycan same. Therefore, this vaccine is likely MUCl tolerated by the immune system. In short, all of these problems are of MUCl policies derived peptide glycosylation can not be a tumor-associated short 0- glycans. Chemically synthesized enzyme multimeric Tn / STnMUCl glycopeptide has caused -MUCl specific anti-cancer antibody response and no tolerance, thereby indicating the importance of 0- glycans. However, the chemical synthesis of Tn / STn MUCl very expensive and difficult to produce a large number of the same product.