The present disclosure relates to a method of obtaining a cell where fucosylation pathways are modified, leading to production of partially fucosylated and non-fucosylated protein products, specifically antibodies from the cell. The present disclosure employs the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) technology. The method of the present disclosure targets the Fut8 gene and GMD gene in a cell. Such products are used in developing therapeutics and biomarkers, and in diagnosis and prognosis of diseases.

The present invention, in the field of bioengineering and biotechnology, relates to a method for preparing a recombinant antibody with a unique glycan profile produced by a genome-edited CHO host cell. Specifically, according to a method of the present invention, the TALEN technology is used to edit the FUT8 gene in CHO cells that have been adapted for serum-free suspension growth. The edited CHO host cells can produce recombinant antibodies with a unique glycan profile. The unique glycan profile can be characterized by non-fucosylated N-linked oligosaccharide chains of the antibodies, extremely low N-glycosylation heterogeneity and uniform carbohydrate chains. The antibody prepared by the method of the invention exhibit significantly increased ADCC and greater stability.

A population of antibodies, wherein less than 80% of the oligosaccharides covalently attached to the population of the antibodies via N297 residues thereof comprise a core fucose residue; and wherein the population of the antibodies comprises an antibody which Fc region comprises K338A and T437R mutations, or K248E and T437R mutations.

The present invention, in the field of bioengineering and biotechnology, relates to a method for preparing a recombinant antibody with a unique glycan profile produced by a genome-edited CHO host cell. Specifically, according to a method of the present invention, the TALEN technology is used to edit the FUT8 gene in CHO cells that have been adapted for serum-free suspension growth. The edited CHO host cells can produce recombinant antibodies with a unique glycan profile. The unique glycan profile can be characterized by non-fucosylated N-linked oligosaccharide chains of the antibodies, extremely low N-glycosylation heterogeneity and uniform carbohydrate chains. The antibody prepared by the method of the invention exhibit significantly increased ADCC and greater stability.

The invention discloses a recombinant protein (P-selectin glycoprotein ligand-1 and Neural Retina-specific Leucine Zipper) PSGL-1-NRL chimeric protein comprising a Selectin Binding domain and a non-covalent dimerization domain, which is a leucine zipper and is more preferably the leucine zipper domain of the human or mouse Neural Retina-specific Leucine Zipper. The chimeric protein further comprises a covalent dimerization domain with at least one cysteine suitable to form a disulfide bridge with another chimeric protein to form a homodimer. In the chimeric protein, the PSGL-1 domain corresponds to the extracellular region of Human PSGL-1 and is more preferably the selectin binding region of the mature protein. The chimeric protein is correctly post-translationally modified and is efficiently expressed in a mammalian system. It is sulfated, O-linked glycosylated and sialylated and binds P, E and L selectin, allowing in vivo and in vitro targeting for diagnostic or therapeutic purposes.

The disclosure provides an expression vector (e.g., AAV vector) comprising a nucleic acid sequence encoding (1) the heavy and/or light chain of an antibody and (2) one or more shRNA sequences targeting fucosyltransferase-8 (FUT8).

The present invention provides methods and compositions for stable genetic modification of cultured mammalian cells. The genetic modifications can be used to produce cultured mammalian cells for therapeutic or diagnostic purposes.

The invention discloses a recombinant protein (P-selectin glycoprotein ligand-1 and Neural Retina-specific Leucine Zipper) PSGL-1-NRL chimeric protein comprising a Selectin Binding domain and a non-covalent dimerization domain, which is a leucine zipper and is more preferably the leucine zipper domain of the human or mouse Neural Retina-specific Leucine Zipper. The chimeric protein further comprises a covalent dimerization domain with at least one cysteine suitable to form a disulfide bridge with another chimeric protein to form a homodimer.

In the chimeric protein, the PSGL-1 domain corresponds to the extracellular region of Human PSGL-1 and is more preferably the selectin binding region of the mature protein.

The chimeric protein is correctly post-translationally modified and is efficiently expressed in a mammalian system. It is sulfated, O-linked glycosylated and sialylated and binds P, E and L selectin, allowing in vivo and in vitro targeting for diagnostic or therapeutic purposes.

The present disclosure relates to methods of obtaining cell with disrupted fucosylation and obtaining afucosylated protein. The present disclosure employs the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) technology in a protein producing cell line to produce afucosylated protein. The resulting protein, specifically the resulting monoclonal antibody is completely afucosylated and reveals higher degree of antibody dependent cellular cytotoxicity.

Provided methods for producing an afucosylated antibody, the afucosylated antibodies and composition thereof, and cells for producing antibodies. The method comprises introducing a nucleic acid encoding at least one modified enzyme of the fucosylation pathway to a host cell to produce the afucosylated antibody in the host cell. The afucosylated antibodies produced by the disclosed methods have increased ADCC activity and would not suppress their CDC and safety.