CLIAMS:None ,TagSPECI:Title
Modification of therapeutic protein expressing CHO cell line to over-express improved versions of biologics.
Technical field
The present invention pertains to using gene disruption technologies to knock out essential gene(s) for fucose biosynthesis in CHO cell lines expressing therapeutic proteins. CHO cell lines thus modified will produce defucosylated monoclonal antibody and/or other proteins. Specific glycan residues of therapeutic proteins directs efficiency of drug molecules in various disease indications towards better treatment outcome. In case of therapeutic monoclonal antibody drugs fucosylation at the Fc region determines specific immune functions responsible for drug molecule efficiency. Absence of fucose moiety in antibody glycan chain directly affects antibody dependent cellular cytotoxicity through natural killer cells and hence higher efficacious biologic product. The defucosylated molecules could be used for development of protein reagents that target biomarkers for the diagnosis and prognosis of various diseases.
Background information and prior art
Glycosylation in eukaryotes has been studied intensively for decades as the most common covalent protein modification mechanism (Varki et al 2009). About 1-2% of the human transcriptome (about 250-500 glycogenes) has been predicted to translate proteins which are responsible for glycosylation (Campbell and Yarema 2005). Glycosylation plays many key biological functions such as protein folding, stability, intracellular and inter-cellular trafficking, cell-cell and cell matrix Interaction.
There are four distinct groups of Glycoproteins : N-linked, O-linked, glycosaminoglycans, and glycosylphosphatidylinositol-anchored proteins. N-linked glycosylation occurs through side chain amide nitrogen of asparagine residue, while O-linked glycosylation uses the oxygen atom in the side chain of serine or threonine residues. N-linked glycosylation takes place in the amino acid sequence of Asn-X-Ser/Thr, where X can be any amino acid except proline and aspartic acid (Helenius and Aebi 2004).

Fucose (6-deoxy-L-galactose) is a monosaccharide that is present in many glycoproteins and glycolipids present in vertebrates, invertebrates, plants, and bacteria. Fucosylation is the process of transferring fucose residue to various proteins and oligosaccharides. Fucosylation is regulated by several molecules, including fucosyltransferases, guanosine diphosphate (GDP)-fucose synthetic enzymes, and GDP-fucose transporter(s). Large number of fucosylated glycoproteins are secretary proteins or membrane proteins on the cell surface. A potent example of fucosylated glycoprotein is fucosylated alpha-fetoprotein (AFP), an important cancer biomarker (Simm, 1979).

Human IgG1 antibody is a highly fucosylated glycoprotein. Two N- linked biantennary oligosaccharides consisted of core hepta saccharide with variable addition of fucose, galactose, bisecting N-acetylglucosamine and sialic acid are present at Asn-297 of IgG1. Antibody glycosylation leads to unique biological functions known as “effector functions” - antibody dependent cellular cytotoxicity (ADCC) and complement dependent cytotoxicity (CDC). ADCC is a cell mediated immune system where immune cells (like natural killer cells) will lyse the target cells identified through antibodies against cell surface antigens. The effector function of IgG molecule is defined by the interaction of antibody Fc region with leukocyte receptors, known as Fc?Rs or interactions with complement components. The composition of the oligosaccharide structure is critically important for effector function through Fc?R binding (Shields et al. 2002; Shinkawa et al. 2003; Niwa et al. 2004; Niwa, Shoji-Hosaka, et al. 2004; Yamane-Ohnuki et al. 2004;). Crystal structure analysis of human IgG1 revealed intricate interaction of the oligosaccharide chains with the CH2 domain (Harris et al. 1998; Radaev et al. 2001).

Efficiency of ADCC mechanism is considerably dependent on the level of antibody fucosylation; lower the fucosylation higher the rate of ADCC. Therefore, loss of fucosylation has significant biological consequences. The loss could be due to non-functional fucosyltransferase enzymes, resulting in nonfucosylation of cellular proteins. The absence of fucose from the primary N-acetylglucosamine results in the IgG1 antibody having increased binding affinity for the Fc?RIIIa receptor, with consequent increase of 50 – 100 times higher efficacy of ADCC (Shinkawa et al. 2003) . Improvement of ADCC with nonfucosylated IgG is directly proportional to the increased affinity for Fc?RIIIa — this allows the nonfucosylated IgG Fc to overcome the competition from high concentrations of fucosylated IgG in normal serum. Plausible rationale for the increased affinity of non-fucosylated IgG Fc for Fc?RIIIa may be the reduction or absence of steric inhibition at the receptor–ligand interface (Harris, 1998; Radaev, 2001)

In mammalian expression systems, GDP-fucose, an essential substrate of fucosylation, is synthesized in the cytoplasm through de novo and salvage pathways. In the de novo pathway of fucosylation, GDP-fucose is synthesized through conversion of GDP-mannose to GDP-4-keto-6-deoxy-mannose, catalyzed by the enzyme GDP-mannose 4,6-dehydratase (GMD). This GDP-Fucose is then transported inside the golgi and used as a substrate for protein fucosylation by the enzyme ?1-6 fucosyltransferase. The enzyme transfers the fucose moiety from GDP-fucose to N-acetylglucosamine of the N-glycan chain (Miyoshi, 1999).These critical enzymes, GDP-mannose 4,6-dehydratase and ?1-6 fucosyltransferase are encoded by GMD and Fut8 genes, respectively. Disruption of gene function through various means will lead to production of nonfucosylated proteins and/or antibodies (Naoko Yamane-Ohnuki, 2004).

A very new approach for targeted genome editing to introduce deletions, insertions and specific sequence changes in a wide range of organisms and cell types is the use of clustered, regularly interspaced, short palindromic repeat (CRISPR) technology (Pennisi, 2013). This is based on a class of RNA-guided endonucleases known as Cas9 from the microbial adaptive immune system found in Streptococcus pyogenes. Cas9 nuclease is directed to specific sites on the genome by guide RNAs (gRNAs). The Cas9/gRNA complex binds to a 20bp target sequence that is followed by a 3bp protospacer activation motif (PAM) NGG or NAG on the specific gene that needs to be edited (Jinek, 2012; Mali, 2013). Thus, the binding of this whole complex creates double stranded breaks (DSBs). A crucial step in targeted genome editing at genomic loci that need to be modified, is the introduction of these DSBs. Once, DSBs are introduced, they are repaired either by non homologous end joining (NHEJ) or homology directed repair (HDR). NHEJ is known for the efficient introduction of insertion/deletion mutations (indels) that in turn cause disruption of the translational reading frame of the target coding sequence or at binding sites of trans-acting factors in promoters or enhancers. On the other hand, HDR mediated repair can insert specific point mutations or sequences at the target locus. Thus, co-transfection of cell types with vectors that express the Cas9 nulcease and the gRNAs targeted to a specific gene locus can efficiently knock down the expression of target genes. The expected frequency of mutations at these specific sites ranges from >1% to 50% (Sander 2014). Selection of mutants can be performed by simple screening using sequencing without the use of drug resistance marker selection. In order to increase the specificity of gene disruption, we use a mutant Cas9 (D10A) that is guided by two guide RNAs for a single gene locus and that introduces two single stranded breaks or nicks. This also reduces the chances of non-specific binding at other random sites (Ran 2013). We will use a vector encoding Cas9-D10A and the 2 gRNAs to cause efficient gene knock-out.
Recently a family of proteins known as Transcription activator like effectors (TALE) were identified from plant pathogen Xanthomonas which binds effector specific DNA sequences and activate transcription (Boch, 2009; Moscou, 2009). Customised TALEs binding to specific DNA sequences provide an unique platform to introduce targeted genome manipulation through transcription activator/repressor and through endonucleases. The central region of the TALE contains 34 amino acid repeats which provide DNA sequence specificity; the 12th and 13th amino acid positions are critical for recognizing specific DNA sequences and are known as repeat variable diresidue (RVD). RVD is specific to a simple cipher like, NI = A, HD = C, NG = T, NN = G or A (Boch, 2009; Moscou, 2009). The repeats could be assembled in a TALE expression vector and coexpressed with a nuclease FokI endonuclease catalytic domain to create TALE nuclease (TALEN). Such TALENs once expressed in cells will bind sequence specifically and will create double strand break; the DNA break will be repaired by Non Homologous End Joining (NHEJ). During such cellular processes mutations either deletions and/or insertion within the gene sequence will render non functional protein products. TALEN technology is highly efficient in creating gene knock out in multiple system.
We will target the GMD and/or the Fut8 genomic loci in monoclonal antibody and/or other protein expressing cell lines. The specific gene(s) will be targeted using CRISPR/CAS9 and /or TALEN technologies. The expected outcome would deliver cell lines expressing therapeutic proteins that are defucosylated. The ranges of therapeutic proteins include monoclonal antibodies and/or other protein products. Defucosylated forms of therapeutic proteins thus achieved in mammalian cell lines where fucose biosynthesis is impaired or abolished may have clinical advantages over the fucosylated forms due to the enhanced efficiency of ADCC.
This application targets to disrupt either or both genes upstream and downstream of the key biochemical steps involving GDP-Fucose.

Brief Description
Therapeutic IgG1 monoclonal antibodies contain fucose residues at the N-acetylglucosamine through ?-1,6 linkage. De novo synthesis of GDP-fucose, in the fucose biosynthetic pathway involves conversion of GDP-Mannose to GDP-4-keto-6-deoxy-mannose by the enzyme GDP-mannose 4,6-dehydratase (GMD). The final fucosylation step for the transfer of fucose from GDP-fucose to N-acetylglucosamine residue is catalyzed by ?-1,6 fucosyltransferase. The above crucial enzymes are coded by the GMD and Fut8 genes, respectively. The invention aims to introduce mutations (insertions/deletions) at specific positions of either or both GMD and FUT8 codon sequences through CRISPR/Cas9 and TALEN technologies in mammalian cell line expressing therapeutic proteins. We have obtained both the GMD and FUT8 coding sequences from the CHO genomic database and demarcated position of each exon. gRNAs that introduce 2 single stranded breaks, recognized by a mutant form of Cas9 (D11A) at GMD and Fut8 sequences were designed. We hypothesize that insertions/deletions caused by CRISPR/Cas9 genome editing would knock down the functionality of GMD and Fut 8 genes. Gene targeting using CRISPR technology would be a novel approach to alter CHO cell line expressing therapeutic protein of interest. CRISPR/Cas9 vector transfected cells will be screened through functional assays. Selected clones will be confirmed through sequencing of genomic loci for mutations.
Therapeutic IgG1 monoclonal antibodies contain fucose residues at the N-acetylglucosamine through a-1,6 linkage. The invention aims to introduce mutations at critical amino acid positions of the FUT8 codon sequence through TALENs. It has been reported that Arg365 and Arg366 in human Fut8 gene plays important role in catalytic function of a-1,6 fucosyltranfease (Takahashi 2000). Few other critical amino acids were also reported to be conserved in Fut8 gene across species. We have compared FUT8 amino acid sequence from CHO genomic database and confirmed that these critical amino acids are conserved in CHO cell line as well. Sequence specific TALENs were designed targeting these amino acid motifs to introduce genomic modifications.
We hypothesize that mutation of these critical amino acids will provide complete disruption of Fut8 gene functionality. Gene targeting using TALEN technology would be a novel approach to create Fucose knock out CHO cell line expressing therapeutic protein of interest. TALEN transfected cells will be screened through Fut8 gene functionality assays. Selected clones will be confirmed through sequencing of genomic Fut8 loci for mutations. Gene targeting using TALEN technology would be a novel approach to modify CHO cell line expressing therapeutic protein of interest.
Detailed description of the invention
Complete GMD and Fut8 gene loci from the CHO cell genome will be analyzed from publicly available CHO genome database. The accession numbers NM_001246696.1 and XM_003501735.2 correspond to GMD and Fut8 genes, respectively. The target sequences encompass the complete coding sequence required for expression of both the GMD and Fut8 gene products, which are GDP-mannose 4,6-dehydratase and ?-1,6 fucosyltransferase, respectively. Success of the CRISPR/Cas9 technology sometimes depends on targeting sites on the first few exons of the coding sequence. This is done to avoid partial fucosylation that can be caused by truncated or partially functional protein. The GMD and Fut8 genes will be targeted with CRISPR/Cas9 knock down constructs (Seq ID 1 to Seq ID 4). Single plasmid vectors pD1401 that express both the Cas9 (D10A) nuclease under the control of a strong mammalian CMV promoter and respective gRNAs under a RNA polymerase promoter will be constructed. The CRISPR/Cas9 constructs will be transiently transfected in CHO cells; and plated in 96 well plates for single colony generation. Each clone will then be screened for defucosylation using lens culinaris agglutinin (LCA) based selection assay. LCA binds fucosylated cell membrane proteins leading to endocytosis and cell death. This enables selection for fucosylation associated gene disruptions, as LCA can no longer bind to cells devoid of fucosylation. Hence, these cells survive (Malphettes, 2010). Fluorescein labeled LCA will be used to confirm fucose knock out clones through flow cytometry assays. Finally, the genomic sequence at both the GMD and/or Fut8 loci will be analyzed for any mutations caused by CRISPR/Cas9 based gene editing. These mutations could involve deletions or insertions and thereby introduce frame shift mutations in the GMD and FUT8 codon sequence.
Fut8 gene has been extensively studied for the catalytic activity and substrate specificity. Multiple studies confirmed FUT8 enzyme functionalities through site directed mutagenesis studies of critically important amino acid residues in the catalytic domain. Two Arginine residues at positions 365 and 366 along with Asp-368, Lys- 369 and Glu-373 revealed most dramatic reduction of FUT8 catalytic activity as measured through fluorescence based assays (Takahashi, 2000 and Ihara, 2007). Sequence ID 5 indicates the corresponding genomic DNA sequence for TALEN target. The nucleotide sequence corresponding to Arg 365, Arg 366, Asp 368, Lys 369, Glu 373 are indicated in bold and underline. TALENs specifically targeting the amino acid codon sequences in genomic locations will be designed, synthesized and cloned in expression vectors like pCDNA3.1. The TALEN constructs will be transiently transfected in CHO cells; the cells will be plated in 96 well plates for single colony generation. Each clone will then be screened for Fut8 gene expression using fluorescence based Lens Culinaris assay as well as LCA based selection assay. Finally, the genomic sequence at the Fut8 loci will be analyzed for any mutation carried out through TALENs. These mutations could involve deletions or insertions and thereby introducing frame shift mutation of the FUT 8 codon sequence.
The fucose knock out CHO cell line (s) expressing therapeutic proteins will thus be generated for producing the protein of interest. Defucosylated therapeutic proteins produced from the abovementioned cell line(s) include monoclonal antibodies, peptides, fusion proteins of therapeutic purposes, biomarker development, diagnostic and prognosis uses.
One example of intended use
Trastuzumab is a monoclonal antibody that is used as a therapeutic drug in breast cancer patients. The drug binds cell surface receptors known as Human epidermal growth factor receptor 2 (Her2) on tumor cells and blocks its downstream signaling pathways. The CRISPR/CAS and/or TALEN technologies will be used to develop fucose knock out CHO cell expressing Trastuzumab monoclonal antibody. Defucosylated Trastuzumab monoclonal antibody thus produced using the gene disruption technologies will be functionally different compared to the innovator product with respect to antibody glycosylation pattern. The product will be purified according to established protocols and guidelines. Defucosylated Trastuzumab biobetter will result in higher level of ADCC and thereby expected to have better therapeutic benefits for breast cancer patients. Thus, a biobetter Trastuzumab product for therapeutic use would be developed.

Sequence listing if relevant
Sequence ID 1
Sequence ID 2
Sequence ID 3
Sequence ID 4

Sequence ID 5

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