Patent Grant
11267899
This application is a National Stage application under 35 U.S.C. § 371 of International Application No. PCT/IB2016/052774 having an International Filing Date of May 13, 2016, which claims the benefit of priority from Indian Application Serial No. 194/CHE/2015, having a filing date of May 13, 2015.
The disclosures of the prior applications are considered part of (and are incorporated by reference in) the disclosure of this application.
This application contains a Sequence Listing that has been submitted electronically as an ASCII text file named SEQ.txt. The ASCII text file, created on Feb. 10, 2020, is 45,066 bytes in size. The material in the ASCII text file is hereby incorporated by reference in its entirety.
The present disclosure pertains to the field of biotechnology, genetic engineering and immunology. Particularly, the present disclosure relates to developing cell lines where specific biological pathways are modified. Such modifications are in the enzymes of the cell, particularly in enzymes involved in glycosylation of proteins. The present disclosure develops protein expression systems wherein specific modification of glycan chain of the protein is achieved. The specific modification of the glycan chain produces afucosylated proteins, including antibodies. The present disclosure employs the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) technology in a protein producing cell line, specifically an antibody producing cell line. The resulting monoclonal antibody is completely afucosylated and reveals higher degree of antibody dependent cellular cytotoxicity.
Glycosylation in eukaryotes has been studied intensively for decades as the most common covalent post translational protein modification mechanism. About 1-2% of the human transcriptome (about 250-500 glycogenes) is predicted to translate proteins which are responsible for glycosylation (Campbell and Yarema 2005). Glycosylation of cellular proteins plays many key biological functions such as protein folding, stability, intracellular and inter-cellular trafficking, cell-cell interaction 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 the side chain amide nitrogen of asparagine residues, and 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). O-linked glycosylation uses the oxygen atom in the side chain of serine or threonine residues.
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 a 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). A large number of fucosylated glycoproteins are secretory proteins or membrane proteins on the cell surface.
The most prominent change in oncology drug development in the last 20 years has been the shift from classic cytotoxics to drugs that affect signaling pathways implicated in cancer, known as “Monoclonal Antibodies” or mAbs. Improvement of monoclonal antibody therapeutics through technologies described here will pave the way for better clinical outcome for patients.
Human IgG1 antibody is a highly fucosylated glycoprotein. Two N-linked biantennary oligosaccharides consisting 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).
The effector function of IgG molecule is defined by the interaction of antibody Fc region with leukocyte receptors, known as FcγRs, or interactions with complement components. The composition of the oligosaccharide structure is critically important for effector function through FcγR 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 has revealed intricate interaction of the oligosaccharide chains with the CH2 domain (Harris et al. 1998; Radaev et al. 2001).
The efficiency of the ADCC mechanism is considerably dependent on the level of antibody fucosylation. The lower the fucosylation, the higher is the rate of ADCC. Therefore, loss of fucosylation has significant biological consequences. The loss could be due to non-functional fucosyltransferase enzymes, resulting in non-fucosylation of cellular proteins. The absence of fucose from the primary N-acetylglucosamine results in the IgG1 antibody having increased binding affinity for the FcγRIIIα receptor, with consequent increase of 50-100 times higher efficacy of ADCC. Improvement of ADCC with non-fucosylated IgG is directly proportional to the increased affinity for FcγRIIIα, which allows the non-fucosylated 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 FcγRIIIα 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 α1-6 fucosyltransferase (encoded by FUT8 gene).
Non-fucosylated forms of therapeutic antibodies developed in mammalian platforms, where fucose biosynthesis is impaired, may have clinical advantage over the fucosylated forms due to the enhanced efficiency of ADCC towards target tumor cells.
Historically, gene knock out systems completely depended on Homologous recombination (HR) mediated targeted mutation, deletion and/or insertion. The HR system, although very specific, is highly inefficient, as thousands of clones need to be screened to find one mutated clone. Moreover, deleting allelic variations would take even further time and much larger screening.
Zinc Finger Nuclease (ZFN) is one of the most frequently used techniques for gene disruption. It requires three bases at the DNA level for each zinc finger tandem array. Moreover, target site overlap and cross-talk between individual fingers in a zinc-finger array considerably complicate the production of sequence-specific ZFNs. Additionally, major drawback of ZFNs includes elaborate and time-consuming experimental selection process to identify the ZFN motifs for specific DNA sequence recognition.
There are methods in the prior art for disruption of Fut8 genomic locus. However, none of the methods target the specific location on the FUT8 genomic locus by the CRISPR technology to produce afucosylated proteins from cells that encode for such proteins.
The present disclosure overcomes the disadvantages or limitations associated with methods of the prior art by using the CRISPR technology to target a specific location on the FUT8 genomic locus, which results in complete disruption of the gene and related function, providing a cell that produces non-fucosylated proteins.
Accordingly, the present disclosure relates to a cell comprising a gene encoding antibody involved in Antibody-dependent cell-mediated cytotoxicity (ADCC) and vector comprising a CRISPR DNA binding domain selected from the group consisting of SEQ ID No. 11 and SEQ ID No. 13 or combination thereof; a method of obtaining a fucose knockout cell, said method comprising a step of transfecting a cell with a CRISPR nuclease construct to obtain a fucose knockout cell; a method of obtaining afucosylated protein, said method comprising step of obtaining a protein expressed by the fucose knockout cell obtained by the method mentioned above; an afucosylated protein; a composition comprising the protein as mentioned above, optionally along with pharmaceutically acceptable excipient; afucosylated protein as mentioned above for use in managing disorder selected from the group consisting of cancer, autoimmune disorder, viral infectious disease, bacterial infectious disease, inflammation and tumour or combinations thereof; and a method of managing disorder selected from the group consisting of cancer, autoimmune disorder, viral infectious disease, bacterial infectious disease, inflammation and tumour or combinations thereof, said method comprising step of administering the afucosylated protein as mentioned above, to a subject in need thereof
The features of the present disclosure will become fully apparent from the following description taken in conjunction with the accompanying drawings. With the understanding that the drawings depict only several embodiments in accordance with the disclosure and are therefore, not to be considered limiting of its scope, the disclosure will be described with additional specificity and detail through use of the accompanying drawings:
The present disclosure relates to a cell comprising a gene encoding antibody involved in Antibody-dependent cell-mediated cytotoxicity (ADCC) and vector comprising a CRISPR DNA binding domain selected from the group consisting of SEQ ID No. 11 and SEQ ID No. 13 or combination thereof.
In an embodiment of the present disclosure, the vector further comprises a nuclease.
In another embodiment of the present disclosure, the cell is an antibody producing cell; and the antibody is selected from the group consisting of anti-CD20 antibody, anti-EGFR antibody, anti-Her2 antibody, anti-CD19 antibody, anti-LAG3 antibody, anti-CD40 antibody, anti-EpHA3 antibody, anti-HIV neutralizing antibody, anti-HCV neutralizing antibody and anti-dengue neutralizing antibody.
In yet another embodiment of the present disclosure, the cell is selected from the group consisting of COS, CHO-S, CHO-K1, CHO-K1 GS (−/−), CHO-DG44, CHO-DUXB11, CHO-DUKX, CHOK1SV, VERO, MDCK, W138, V79, B14AF28-G3, BHK, HaK, NS0, SP2/0-Ag14, HeLa, HEK293-F, HEK293-H, HEK293-T, YB23HL.P2.G11.16Ag.20, perC6, antibody producing Hybridoma cell, embryonic stem cell, Namalwa cell, insect cell line from Spodoptera fugiperda (Sf), Pichia, Saccharomyces and Schizosaccharomyces.
In still another embodiment of the present disclosure, the cell is a CHO cell; and wherein the antibody is anti-Her2 antibody.
The present disclosure also relates to a method of obtaining a fucose knockout cell, said method comprising a step of transfecting a cell with a CRISPR nuclease construct to obtain a fucose knockout cell.
In an embodiment of the present disclosure, the cell comprises a gene encoding antibody involved in Antibody-dependent cell-mediated cytotoxicity (ADCC).
In another embodiment of the present disclosure, the antibody is selected from the group consisting of anti-CD20 antibody, anti-EGFR antibody, anti-Her2 antibody, anti-CD19 antibody, anti-LAG3 antibody, anti-CD40 antibody, anti-EpHA3 antibody, anti-HIV neutralizing antibody, anti-HCV neutralizing antibody and anti-dengue neutralizing antibody.
In yet another embodiment of the present disclosure, the CRISPR nuclease construct comprises CRISPR DNA binding domain selected from the group consisting of SEQ ID No. 11 and SEQ ID No. 13 or combination thereof.
In still another embodiment of the present disclosure, the CRISPR-nuclease construct disrupts Fut8 gene of the cell; and wherein the Fut8 gene sequence is disrupted by cleavage at Exon 7.
In still another embodiment of the present disclosure, the cell is selected from the group consisting of COS, CHO-S, CHO-K1, CHO-K1 GS (−/−), CHO-DG44, CHO-DUXB11, CHO-DUKX, CHOK1SV, VERO, MDCK, W138, V79, B14AF28-G3, BHK, HaK, NS0, SP2/0-Ag14, HeLa, HEK293-F, HEK293-H, HEK293-T, YB23HL.P2.G11.16Ag.20, perC6, antibody producing Hybridoma cell, embryonic stem cell, Namalwa cell, insect cell line from Spodoptera fugiperda (Sf), Pichia, Saccharomyces and Schizosaccharomyces.
The present disclosure also relates to a method of obtaining afucosylated protein, said method comprising step of obtaining a protein expressed by the fucose knockout cell obtained by the method mentioned above.
In an embodiment of the present disclosure, the protein is an antibody involved in Antibody-dependent cell-mediated cytotoxicity (ADCC).
In another embodiment of the present disclosure, the antibody is selected from the group consisting of anti-CD20 antibody, anti-EGFR antibody, anti-Her2 antibody, anti-CD19 antibody, anti-LAG3 antibody, anti-CD40 antibody, anti-EpHA3 antibody, anti-HIV neutralizing antibody, anti-HCV neutralizing antibody and anti-dengue neutralizing antibody.
In yet another embodiment of the present disclosure, the antibody is an anti-Her2 antibody.
In still another embodiment of the present disclosure, the cell is selected from the group consisting of COS, CHO-S, CHO-K1, CHO-K1 GS (−/−), CHO-DG44, CHO-DUXB11, CHO-DUKX, CHOK1SV, VERO, MDCK, W138, V79, B14AF28-G3, BHK, HaK, NS0, SP2/0-Ag14, HeLa, HEK293-F, HEK293-H, HEK293-T, YB23HL.P2.G11.16Ag.20, perC6, antibody producing Hybridoma cell, embryonic stem cell, Namalwa cell, insect cell line from Spodoptera fugiperda (Sf), Pichia, Saccharomyces and Schizosaccharomyces.
The present disclosure also relates to an afucosylated protein.
In an embodiment of the present disclosure, the protein is an antibody involved in Antibody-dependent cell-mediated cytotoxicity (ADCC).
In another embodiment of the present disclosure, the protein is an antibody selected from the group consisting of anti-CD20 antibody, anti-EGFR antibody, anti-Her2 antibody, anti-CD19 antibody, anti-LAG3 antibody, anti-CD40 antibody, anti-EpHA3 antibody, anti-HIV neutralizing antibody, anti-HCV neutralizing antibody and anti-dengue neutralizing antibody.
In yet another embodiment of the present disclosure, the antibody is an anti-Her2 antibody.
In still another embodiment of the present disclosure, the protein is obtained by the method as mentioned above.
The present disclosure also relates to a composition comprising the protein as mentioned above, optionally along with pharmaceutically acceptable excipient.
The present disclosure also relates to Afucosylated protein as mentioned above, for use in managing disorder selected from the group consisting of cancer, autoimmune disorder, viral infectious disease, bacterial infectious disease, inflammation and tumour or combinations thereof.
The present disclosure also relates to a method of managing disorder selected from the group consisting of cancer, autoimmune disorder, viral infectious disease, bacterial infectious disease, inflammation and tumour or combinations thereof, said method comprising step of administering the afucosylated protein as mentioned above to a subject in need thereof.
In an embodiment of the present disclosure, the method of managing disorder and use in managing disorder is in a suitable subject, including man.
In the present disclosure, a cell without fucosylation activity is also referred to as “Fucose Knockout” of “FKO” cell.
The terms “non-fucosylated antibody”, “afucosylated antibody” 0% fucosylated antibody”, “afucosylated monoclonal antibody”, afucosylated Mab” and “100% non-fucosylated antibody” have the same scope and meaning, and are used interchangeably throughout the disclosure.
The present disclosure relates to production of non-fucosylated or afucosylated proteins, from cell encoding said protein.
In an embodiment, the cell produces non-fucosylated monoclonal antibody.
In a non-limiting embodiment, the non-fucosylated antibody is IgG1 monoclonal antibody.
In a non-limiting embodiment, the non-fucosylated antibody is anti-Her2 monoclonal antibody.
The terms “Innovator”, “Herclon”, “Trastuzumab Innovator”, “Innovator Herclon”, have the same meaning and scope and are used interchangeably. All the terms relate to an anti-Her2 antibody, which is the first available and commercialized antibody for use in Breast Cancer.
The present disclosure employs the CRISPR technology to disrupt Fut8 gene and produce non-fucosylated proteins.
The CRISPR (Clustered, Regularly Interspaced, Short Palindromic Repeat) system is an adaptable, naturally occurring immune mechanism, used by many bacteria to protect themselves from foreign nucleic acids, such as viruses or plasmids. CRISPRs are segments of prokaryotic DNA containing short repetitions of base sequences, followed by short segments of “spacer DNA”. This Spacer DNA is foreign DNA obtained from previous exposures to a bacterial virus or plasmid. A set of enzymes called Cas (CRISPR-associated proteins) enzymes are found in association with these CRISPR sequences, and Cas are nucleases which can precisely snip DNA.
The bacterium copies the genetic material in each spacer DNA into an RNA molecule. Cas enzymes then take up one of the RNA molecules, which are referred to as the guide RNAs (gRNA). Together they form the CRISPR-Cas system. When the system encounters DNA from a virus that matches the CRISPR RNA, the RNA hybridises to the DNA sequence and the Cas enzyme then cleaves the DNA in two, preventing the virus from replicating.
There are various Cas enzymes that work in conjunction with CRISPR, but the most well-known and frequently employed in genetic engineering is Cas9 nuclease, which is derived from Streptococcus pyogenes. Together, they form the CRISPR/Cas9 system, called the type II CRISPR system.
Cas9 has been shown to be a key player in certain CRISPR mechanisms, specifically type II CRISPR systems where only one Cas protein is required. In this system, the endonuclease Cas9 participates in the cleaving of the target DNA. The Cas9 function is dependent on presence of two nuclease domains, a RuvC-like nuclease domain located at the amino terminus and a HNH-like nuclease domain that resides in the mid-region of the protein.
For site specific DNA recognition and cleavage, the nuclease Cas9 must complex with two RNA sequences, a crRNA (CRISPR RNA) and a separate trans-activating crRNA (tracrRNA or trRNA), that is partially complementary to the crRNA. The tracrRNA is required for crRNA maturation from a primary transcript encoding multiple pre-crRNAs. This occurs in the presence of RNase III and Cas9. During the cleavage of target DNA, the HNH and RuvC-like nuclease domains of the Cas9 nuclease cut both DNA strands, generating double-stranded breaks (DSBs). The recognition sites are defined by 20-nucleotide target sequence within an associated crRNA transcript. The HNH domain cleaves the complementary strand, while the RuvC domain cleaves the non-complementary strand. The double-stranded endonuclease activity of Cas9 also requires that a short conserved sequence, (2-5 nts) known as Protospacer-Associated Motif (PAM), follows immediately 3′—of the crRNA complementary sequence in the target DNA. The requirement of PAM sequence is obligatory for CRISPR/Cas function.
In general, a two vector system is used for CRISPR mediated gene editing, 1) a Cas9 endonuclease and 2) a complex of crRNA (CRISPR RNA) and tracrRNA (trans-activating crRNA). When these two constructs are co-expressed in mammalian cells, they form a complex and are recruited to target DNA sequence. The crRNA and tracrRNA are combined to form a chimeric guide RNA (gRNA) with the same function—to guide Cas9 to target gene sequences.
Homologous recombination mediated gene editing technologies are the first of its kind to be used for gene editing. However, frequency of successful events is very rare using HR, 1 in every 3×104 cells.
In recent days, Zinc finger nuclease is becoming popular as it allows higher specificity of targeting with higher frequency of successful mutant events. It uses DNA binding proteins with nuclease activity, that binds to DNA and creates site-specific DSBs. While effective, these methods require extensive protein engineering tools to be successful and thereby limit flexibility in targeting complex genome sequences. The adaptation of CRISPR for mammalian cells has revolutionized genome editing with higher accuracy and ease of designing. Unlike ZFN, CRISPR/Cas does not require protein engineering for every gene being targeted.
The CRISPR system only requires a few simple DNA constructs to encode the gRNA and Cas9.
Although it is rare for a 20 bp gRNA sequence to have 100% homology at multiple sites throughout the genome, sgRNA-Cas9 complexes are tolerant of several mismatches in their targets. Cas9 has been reported to bind multiple locations in genome nonspecifically. However, it creates DNA double strand break only at a handful of those sites. Experimental data also suggests that certain levels of mismatch at the DNA target site allows DNA double strand break. Therefore, strategies for increasing CRISPR/Cas specificity are pursued.
One such observation is a point mutation of Aspartate to Alanine (D10A) mutation at the RuvC catalytic domain resulting in single strand breaks (nicks) instead of double strand breaks. The mutant Cas9 is known as Cas9n. Using Cas9n at two neighbouring DNA target site allows DNA nicks at close proximity, and if the target sites are appropriately spaced, it creates a double strand break.
Therefore, the specificity of DSB creation is higher, which is eventually repaired by NHEJ mechanism. Nonspecifically bound Cas9n creates only nicks which is generally repaired through HR mediated repair and rarely causes mutation or off target effects. In this disclosure, Cas9n and CRISPR are used to knockout Fut8 gene.
In an embodiment of the present disclosure, the nuclease used in the CRISPR complex is a Cas nuclease.
In an embodiment of the present disclosure, the nuclease used in the CRISPR complex is a Cas9 nuclease.
In an embodiment of the present disclosure, the nuclease used in the CRISPR complex is a Cas9n nuclease.
In an embodiment of the present disclosure, the CRISPR-nuclease construct is a CRISPR Cas construct.
In the present disclosure, CRISPR-Cas construct upon expression in a cell provides CRISPR-Cas complex.
In the present disclosure, the terms CRISPR-Cas complex and CRISPR-Cas system have the same meaning and scope and are used interchangeably.
The present disclosure relates to a method for obtaining non-fucosylated protein, by disruption or inactivation of the fucosylating machinery in a cell.
In an embodiment, the protein is an antibody.
In a preferred but non-limiting embodiment, the antibody is a monoclonal antibody.
In the most preferred embodiment, the antibody is an anti-Her2 monoclonal antibody.
The present disclosure particularly relates to disruption or inactivation of the FUT8 gene in a cell. The FUT8 gene encodes the enzyme α-1,6 fucosyltransferase.
In an embodiment of the present disclosure, the cell is a cell that already expresses a protein.
In an embodiment of the present disclosure, the cell is a cell that already expresses an antibody.
In an embodiment of the present disclosure, the cell is a cell that is known to expresses a protein.
In an embodiment of the present disclosure, the cell is a cell that is known to express an antibody.
In an embodiment of the present disclosure, the cell is a cell that expresses an antibody, and a gene encoding an antibody is introduced into the cell.
In an embodiment, the antibody produced by the method of the present disclosure is a therapeutic antibody.
In an embodiment, the cell is a eukaryotic cell.
In an embodiment, the cell is mammalian cell.
In embodiments of the present disclosure, the cell line is selected from the group consisting of COS, CHO-S, CHO-K1, CHO-DG44, CHO-DUXB11, CHO-DUKX, CHOK1SV, VERO, MDCK, W138, V79, B14AF28-G3, BHK, HaK, NS0, SP2/0-Ag14, HeLa, HEK293-F, HEK293-H, HEK293-T, YB23HL.P2.G11.16Ag.20, perC6, Hybridoma cell which produces antibody, embryonic stem cell, Namalwa cell, insect cell line from Spodoptera fugiperda (Sf), Pichia, Saccharomyces and Schizosaccharomyces.
In a non-limiting embodiment, the cell is Chinese Hamster Ovary cell.
In a non-limiting embodiment, the cell is Chinese Hamster Ovary S (CHO-S) cell.
In a non-limiting embodiment of the present disclosure, the cell is a cell with Glutamine synthetase knockout (GS−/−), preferably a CHO S cell with Glutamine synthetase knockout (GS−/−).
In an embodiment, the CHO S cell is an antibody producing cell.
In an embodiment, the CHO S cell produces Anti Her2 monoclonal antibody.
GPEx® technology, used in an embodiment of the present disclosure, is a well known technique and is based on a highly efficient retroviral vector that transfects 100% of target cells. The advantage of GPEx® is:
A person skilled in the art would know how to use GPEx® technology in conjunction with the methods of the present disclosure.
In an embodiment, the method of the present disclosure relates to using parental CHO-S cell line overexpressing Anti-Her2 antibody to obtain Fucose KO cell line and subsequently afucosylated Anti-her2 antibody product development, the method comprising the following steps:
The methods of the present disclosure are useful for developing any monoclonal antibody developed against targets which require ADCC for functionality. If any of these targets are produced in a CHO S (or other cell type), a similar strategy of Fucose knock out, as seen in the methods of the present disclosure, could be adopted.
Antibody-dependent cell-mediated cytotoxicity (ADCC) is a cell mediated immune system where immune cells (like natural killer cells) lyse the target cells identified through antibodies against cell surface antigens. A person skilled in the art would know which antibody would be involved in the ADCC mechanism.
Examples of specific antibodies that may be produced using the methods of the present disclosure are in from the field of oncology, viz monoclonal antibodies against targets like CD20, EGFR, Her2, CD19, LAG3, CD40, EpHA3 etc.
A monoclonal antibody may also be produced towards the treatment of autoimmune disorder Rheumatoid Arthritis.
Also, neutralizing antibody against antigens of HIV, HCV, Dengue and other diseases where the infectious organisms are removed through ADCC mechanism, may also be produced by the methods of the present disclosure.
In the present disclosure, the term “managing” or “management” or “manage” of a disorder, includes therapeutic and prophylactic activities. It includes the treatment and healing of a disease or disorder, or ill effects or side effects of the disease or the disorder. It also includes prevention of further progress of the disease or disorder, or ill effects or side effects of the disease or the disorder. It further includes maintenance of the optimum state of an individual.
In an embodiment of the present disclosure, the afucosylated monoclonal antibody of the present disclosure is used in—
In an embodiment, the cell is referred to as a “Fucose Knockout” cell or “FKO” cell or “Fucose Knockout” platform or “FKO” platform.
In an embodiment, the cell is referred to as a Recombinant cell.
In an embodiment, CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)—Cas complex is used to disrupt or inactivate the Fucosylation pathway of a cell.
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 α-(1-6) fucosyltransferase. The enzyme transfers the fucose moiety from GDP-fucose to N-acetyl glucosamine of the N-glycan chain.
In an embodiment, CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)—Cas complex) is used to disrupt the Fut8 gene encoding the α-1,6 fucosyltransferase enzyme.
In an embodiment of the present disclosure, the N-terminal catalytic region of fucosyl transferase enzyme is targeted by CRISPR/Cas complex.
In a particular embodiment, Exon 7 of the gene sequence of Fut8 is targeted by CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)—Cas complex.
In an embodiment of the present disclosure, the Fu.cosyltransferase enzyme is mutated at an amino acid position selected from the group consisting of amino acid sequence at the beta 2 strand and amino acid sequence at 3H2 helix region, both coded by exon 7 coding sequence.
The resulting clones may result in premature translation stop, therefore absence of downstream amino acids such as Arg-365, Arg-366, Asp-368, Lys-369, Glu-373, Tyr-382, Asp-409, Asp-410, Asp-453, Ser-469 and combinations thereof provides a non-functional Fut8 gene.
The CRISPR/Cas constructs are designed as a two vector system, in general. One construct codes for the Cas9 endonuclease expression and the second vector expresses the gRNA—which is made up of the crRNA and tracrRNA. The crRNA is usually designed as a 20 nucleotide long fragment that recognizes the target sequence, depending on proper positioning of tracrRNA, PAM sequence and the functional complex of crRNA-Cas9-tracrRNA. In certain cases, one single vector expresses both gRNA and the Cas9 protein for higher activity and ease of use. Target recognition specificity comes from the crRNA design.
In embodiments of the present disclosure, the DNA binding domain is also referred to as the DNA recognition domain.
In an embodiment of the present disclosure, polynucleotides encoding said CRISPR/Cas complex are also provided, as are cells comprising said polynucleotides.
In a particular embodiment, nucleotides encoding for the DNA binding domain of CRISPR Cas9 complex are provided. In another embodiment, nucleotides encoding for the nuclease domain of CRISPR Cas9 complex are provided.
In an embodiment, the nuclease is Cas9.
In another embodiment, the nuclease is Cas9n (nickase) D10A mutant.
In an embodiment of the present disclosure, the CRISPR/Cas complex recognizes target site in FUT8 gene. In an embodiment of the present disclosure, the nuclease is a homing endonuclease. In another embodiment, the nuclease is a meganuclease. It is also known that the specificity of homing endonucleases and meganucleases can be engineered to bind non-natural target sites. Further, in exemplary embodiments, homing endonucleases include I-Scel, I-CeuI, PI-PspI, PI-Sce, I-ScelY, I-CsmI, I-PanI, I-SceII, I-PpoI, I-SceIII, I-CreI, I-TevI, I-TevII and I-TevIII. Their recognition sequences are known.
In an embodiment, a combination of one or more of the above-mentioned nucleases is used with the DNA binding domain of the CRISPR-Cas protein complex.
In an embodiment, transfection is used to introduce a CRISPR/Cas complex into a cell. Though a lipofection protocol is provided as an exemplary embodiment, any method of transfection known to one skilled in the art is equally applicable to the methods of the present disclosure.
In another embodiment, the present disclosure provides methodologies for producing recombinant proteins in any host cell where the host cell has endogenous FUT8 gene expression which is targeted through CRISPR/Cas technology to disrupt endogenous FUT8 gene as described herein. The resulting cell line is null for FUT8 gene expression and is further used for expression of gene of interest.
In the present disclosure, seventeen FUT8 knock out clonal cell lines are created from a screen of less than 120 clonal cell lines generated after transfection with pD1401 (gRNA 514-553) CRISPR/Cas complex. In comparison, only three FUT8 −/− cell lines could be selected from approximately 120,000 clonal cell lines as reported in the art.
The specificity, safety and simplicity of the protocol are some of the advantages offered by CRISPR/Cas complex and the method of the present disclosure over the prior art methods. CRISPR mediated gene disruption provides a unique advantage of specificity of target locus that allows customized CRISPR/Cas complex to recognize user defined target sequence of any complexity. CRISPR/Cas complex are more effective than ZFN in terms of genome editing efficiency and significantly less toxic, thereby allowing higher efficiency in generating mutant clones against a particular locus. In the present disclosure, FUT8 genomic loci are targeted for sequence specific modification through CRISPR gRNAs.
The methodology described herein has achieved an efficiency of more than 3% success rate of generating FUT8 knock out CHO-S cell line expressing anti Her2 antibody. is This unanticipated achievement following the methodology and the specific CRISPR constructs of the present disclosure has vastly improved the FUT8 knock out cell line development.
Also, in an embodiment, the present disclosure has used only one set of CRISPR constructs targeting a very specific genomic location in the FUT8 DNA sequence of the anti Her2 antibody expressing cell line. Surprisingly, the CRISPR/Cas complex results in not only disrupting the targeted amino acids but also produced long deletions which introduced frame shift mutations and premature stop codon. Thereby, the present disclosure has achieved FUT8 knock out cell lines from the anti Her2 antibody expressing cell line with very minimal DNA modifications at the target locus as well as large genome level modifications at the targeted FUT8. Generation of such a large number of FUT8 knock out cell lines is unexpected, considering the small number of clonal populations screened for fucose knock out phenotype. This surprising achievement provides a unique method where any monoclonal antibody expressing cell line could be used for generating fucose knock out lines and thereby an afucosylated monoclonal antibody product could be generated. The method allows screening of only few cell lines to achieve multiple FUT8 knock out cell lines to establish best performing clonal lines for over expression of afucosylated monoclonal antibody.
In embodiments, inactivating a FUT8 gene in a cell line, results in a cell line which produces proteins at higher levels.
In certain embodiments, inactivating a FUT8 gene provides a cell line in which one or more activities (functions) of a protein is increased, as compared to proteins produced in cells where the FUT8 gene is not inactivated.
In an embodiment, the non-fucosylated antibody exhibits greater effector function than a corresponding fucosylated antibody.
In an embodiment, the non-fucosylated antibody exhibits more efficacious therapeutic properties than a corresponding fucosylated antibody.
In an embodiment, the non-fucosylated antibody exhibits higher Antibody dependent Cellular Toxicity (ADCC) than a corresponding fucosylated antibody.
In an embodiment, the non-fucosylated antibody exhibits up to about 7 to 20 folds higher Antibody dependent Cellular Toxicity (ADCC) than a corresponding fucosylated antibody.
In a non-limiting embodiment, the non-fucosylated antibody exhibits about 8.95 folds higher Antibody dependent Cellular Toxicity (ADCC) than a corresponding fucosylated antibody.
In the present disclosure, the methods, preparation and use of the proteins disclosed employ, unless otherwise indicated, conventional techniques in molecular biology, biochemistry, computational chemistry, cell culture, recombinant DNA technology, Polymerase Chain Reaction (PCR) and related fields. These techniques, their principles, and requirements are explained in the literature and known to a person skilled in the art. The techniques for determining nucleic acid and amino acid sequence identity are known to one skilled in the art.
In the present disclosure, the cell with the disrupted fucosylation machinery is a cell that naturally produces antibodies A “functional fragment” of a protein, polypeptide or nucleic acid is a protein, polypeptide or nucleic acid whose sequence is not identical to the full-length protein, polypeptide or nucleic acid, yet retains the same function as the full-length protein, polypeptide or nucleic acid.
The term “antibody” used here includes both polyclonal and monoclonal antibody preparations and also includes the following: Chimeric antibody molecules, F(ab′)2 and F(ab) fragments, Fv molecules, single chain Fv molecules (ScFv), dimeric and trimeric antibody fragments, minibodies, humanized monoclonal antibody molecules, human antibodies, fusion proteins comprising Fc region of antibody and any functional fragments arising out of these molecules, where derivative molecules retain immunological functionality of the parent antibody molecule.
The term “monoclonal antibody” in the present disclosure, refers to an antibody composition having a homogeneous antibody population. The antibody is not limited to the species or source of the antibody or by the manner in which it is made. The term encompasses whole immunoglobulins as well as fragments such as Fab, F(ab′)2, Fv, and other fragments, as well as chimeric and humanized homogeneous antibody populations that exhibit immunological binding properties of the parent monoclonal antibody molecule.
It is to be noted that clones/cells of the present disclosure are referred to by terms such as CR2TMCHO 1A, CR2TMCHO 1B etc., which are internal denominations and do not represent any particular feature of the cell. These cell lines are developed using pD1401 (gRNA 514-553) CRISPR/Cas complex.
In an embodiment, a composition comprising the non-fucosylated antibody, optionally along with a pharmaceutically acceptable carrier or additive or excipient is provided. Pharmaceutically acceptable carrier or additive or excipient is determined by the composition being administered, as well as by the particular method used to administer the composition and is known to a person skilled in the art.
All sequences provided in the present disclosure are read in the 5′ to 3′ direction, unless stated otherwise.
Excipients are important for achieving protein stabilization and improving other qualities of biologics. A variety of excipients are added to compositions to stabilize proteins, act as antimicrobials, aid in the manufacture of the dosage form, control or target drug delivery, and minimize pain upon injection.
Excipients can be broadly divided into five categories based on their modes of action:
The biological material used in the present disclosure is obtained from outside India.
Rationale for Targeting Specific Genomic Sequence in FUT 8 Locus
FUT8 is comprised of three domains, an N-terminal coiled-coil domain, a catalytic domain, and a C-terminal SH3 domain.
Fut8 protein structure is studied extensively to understand the functional domain of the enzyme amino acid sequence. Three dimensional crystal structure of FUT8 enzyme revealed 15 strands and 16 helices. There are at least three regions, N terminus (residues 68-107), C-terminus (573-575) and residues 368-372 which are disordered.
The putative catalytic domain of the FUT8 enzyme is consisted of two domains, an open sheet alpha/beta domain and the Rossmann fold widely known for nucleotide binding region. The alpha/beta domain consisted of five helices and three beta strands, which are alpha 4, 3H1, 3H2, 3H3, beta 1, beta 2 and beta3 strands. The domain is located in the N terminus of the protein sequence. There is no clear evidence how the N terminus catalytic domain is responsible for enzyme functionality.
The Rossmann fold is located downstream at residue 359-492 and contains several alpha helix and beta strands. A series of residues Arg 365, Arg 366, Asp-368, Lys-369, Glu-373, Tyr-382, Asp-409, Asp-410, Asp-453, and Ser-469 play an important role in catalytic domain of FUT8 enzyme.
Ten amino acid residues, Arg 365, Arg 366, Asp-368, Lys-369, Glu-373, Tyr-382, Asp-409, Asp-410, Asp-453, and Ser-469 of human FLITS enzyme protein are conserved among various species, including vertebrates, insectnematode and ascidian.
Rationale for Targeting FUT8 Genes in CHO S Cell Line
Fucose knock out platform is useful to achieve non fucosylated monoclonal antibody molecule. In many instances, developing completely non-fucosylated antibody is a preferred outcome and therefore strategies are made in this disclosure to create complete knock out of Fucose biosynthetic pathway genes.
Fut8 enzyme functions downstream of GDP-Fucose biosynthesis step and is the last enzymatic step for fucosylation of cellular proteins in golgi. Fucosylation precursors from both de novo and salvage pathway use FUT8 enzyme for final fucose moiety transfer. Therefore, knocking out Fut8 gene essentially stops both de novo and salvage pathway of cellular protein fucosylation. This approach results in 100% defucosylation of proteins, including monoclonal antibodies produced in the Fut8 knock out cell line.
The CRISPR/Cas target sequences are targeted in this region. Fut 8 exon7 genomic locus, respective amino acid sequence and position of important structural motifs and CRISPR target locations are depicted in
This targeting is not a random selection, but has been arrived at, in the present disclosure, by experimentation to determine the highly specific location on the gene or enzyme, the disruption of which ensures that partial fucosylation that is caused by truncated or partially functional enzyme is avoided.
The Rossmann fold on the other hand, is located downstream at residue 359-492 and contains several alpha helix and beta strands. A series of residues Arg 365, Arg 366, Asp-368, Lys-369, Glu-373, Tyr-382, Asp-409, Asp-410, Asp-453, and Ser-469 play an important role in catalytic domain of FUT8 enzyme.
Thus, targeting the region equivalent to the active site of the enzyme ensures complete disruption of the Fut8 gene and provides efficacious results in comparison to either a technique that is unable to target a precise location on the Fut8 gene or a technique that targets another location on the Fut8 gene, which might result in partial disruption of Fut8 gene and enzyme activity. A cell with partially functional fucosylated machinery produces partially fucosylated proteins, which exhibits lower therapeutic functions as compared to non-fucosylated proteins.
The cells produced by the method of the present disclosure produce completely or 100% non-fucosylated proteins, including 100% non-fucosylated antibodies.
The present disclosure introduces mutations at critical amino acid positions at the catalytic site of the FUT8 codon sequence through CRISPR/Cas complex. The CRISPR design is aimed to primarily target the N-terminal catalytic domain, specifically the beta 2 strand and the 3H2 helix region by incorporating single stranded breaks. The cellular DNA repair system introduces nucleotide changes while carrying out the single stand break repair and creates non-functional FUT8 enzyme.
The CRISPR system is well known for deletion and insertion in a localized manner and therefore creates frame-shift mutation at the targeted exon7 and inserts stop codons. Introduction of stop codons ensures premature translation termination and the downstream Rossmann fold is excluded from enzyme structure, resulting in non-functional FUT8 enzyme.
In an embodiment of the present disclosure, the subsequent genomic DNA analysis of the modified CHO-S cell lines reveals deletion, insertion, stop codon as well as frame shift mutations. Thus, the present disclosure envisages disruption of Fut8 gene and Fucosyltransferase enzyme by targeting amino acid positions in the beta 2 strand and the 3H2 helix through deletions, insertions and/or frame shift mutations.
The resulting clones may result in premature translation stop, therefore causing extensive changes in critical downstream sequences such as Arg-365, Arg-366, Asp-368, Lys-369, Glu-373, Tyr-382, Asp-409, Asp-410, Asp-453, Ser-469 and combinations thereof.
In the present disclosure, the FUT8 amino acid sequence from CHO-S genomic database is analyzed and it is confirmed that these critical amino acids are conserved in the FUT8 gene derived from CHO-S cell line as well. Sequence specific CRISPR/Cas complex is designed, targeting gene sequences upstream of these amino acid motifs to introduce genomic modifications. It is analysed how altering amino acid sequences upstream of the critical FUT8 enzyme catalytic domain disrupts the enzyme function.
It is stated that mutation of these critical amino acids provides complete disruption of FUT8 gene functionality. Gene targeting using CRISPR/Cas technology is a novel approach to create a Fucose knock out cell line platform. CRISPR/Cas transfected cells are screened through FUT8 gene functionality assays. Selected clones are confirmed through sequencing of genomic FUT8 loci for mutations. The mutant fucose knock out CHO-S cell line is then used for expressing non-fucosylated therapeutic proteins, including non-fucosylated therapeutic monoclonal antibodies or part of antibody.
CRISPR/Cas constructs specifically targeting the amino acid codon sequences in genomic locations are designed, and cloned in expression vectors, for e.g. pD1401 or pD1301 depending on the type of Cas9 gene. The CRISPR/Cas complex is transiently transfected in CHO-S cells; the cells are plated in 96 well plates for single colony generation. Each clone is then screened for fucosylation of cellular proteins using fluorescence based Lens Culinaris Agglutinin assay (LCA). Clones positive for FUT8 gene disruption are further tested through enzymatic assays and kinetic analysis of mutant alleles of FUT8 gene. Finally, the genomic sequence at the FUT8 loci is analyzed for any mutation carried out through CRISPR/Cas. These mutations involve deletions or insertions, thereby introducing frame shift mutations of the FUT8 codon sequence, and rendering the sequence disrupted and the enzymes non-functional.
The fucose knock out Anti Her2 antibody expressing cell line obtained from the method of the present disclosure is used as a cell line for expressing afucosylated anti Her2 antibody product for therapeutic purposes, and any other uses. The method has been used to develop afucosylated Antiher2 antibody product.
Any CHO cell line which produces Anti Her2 Antibody at a higher titer is selected as the starting cell line. The said cell line is transfected with the CRISPR-Cas complex against FUT8 gene and is selected for afucosylation through fluorescence based Lens Culinaris Agglutinin assay. Clonal cell lines positive for fluorescence based Lens Culinaris Agglutinin assay are developed and used for expression of afucosylated AntiHer2 antibody product.
The present disclosure describes the characterization of completely Afucosylated Anti Her2 antibody product and its biological significance. The present disclosure describes significant improvement of the Afucosylated AntiHer2 antibody interaction with FcγRIII receptor compared to innovator Trastuzumab molecule. The corresponding Antibody Dependent Cellular Cytotoxicity (ADCC) assay clearly established the improved efficacy of the afucosylated AntiHer2 Antibody. This improvement has translated to more than 9 fold cytotoxicity of Her2 overexpressing breast cancer cell lines compared to Trastuzumab. The results are significant improvement as this Afucosylated AntiHer2 antibody product is more efficacious in breast cancer treatment in patients.
The Afucosylated AntiHer2 antibody therefore represents a more efficious molecule for improved treatment options for breast cancer patients in terms of—
The present disclosure is further described with reference to the following examples, which are only illustrative in nature and should not be construed to limit the scope of the present disclosure in any manner.
Reagent Preparation
Advanced DMEM Complete Growth Medium—500 ml
Reagent Preparation
Materials & Equipment
The objective of this example is to design CRISPR/Cas complex for specific inactivation of FUT8 allele.
1.1—CRISPR Constructs
CRISPR 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 20 bp target sequence that is followed by a 3 bp 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 nuclease 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 is performed by simple screening using sequencing, without the use of drug resistance marker selection. In order to increase the specificity of gene disruption, the present disclosure uses 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. A vector encoding Cas9-D10A and the 2 gRNAs are used to cause efficient gene knock-out.
The Fut8 genomic loci is targeted for sequence specific deletions through CRISPR/CAS9 technology and generate defucosylated mammalian expression systems.
Cricetulus griseus or Chinese Hamster fucosyltransferase 8 (Fut8) mRNA (3126 bp) is derived from NCBI Reference Sequence: XM_003501735.1, also represented by SEQ ID No. 1 of the present disclosure.
CAGGTTGCTGCTCTGGCTTAGGCCATCTATGACCCTGGTGGTGTTTTCA
TTCACTATAAGTCCTTCCCATCTTTATTAACTGAGCAAGTTCAG
ctagt
aattttagagaccgaggttcaagcaataacacctatctctgcaataccg
tgtggctttcttcaatgtcttacatcctaaggaaaggaag
CATGTAGAG
CCCAGGAAGCACAGGACAAGAAAGCTGCCTCCTTGTATCACCAGGAAGA
TCTTTTTGTAAGAGTCATCACAGTATACCAGAGAGACTAATTTTGTCTG
AAGCATCATGTGTTGAAACAACAGAAACTTATTTTCCTGTGTGGCTAAC
TAGAACCAGAGTACAATGTTTCCAATTCTTTGAGCTCCGAGAAGACAGA
AGGGAGTTGAAACTCTGAAAATGCGGGCATGGACTGGTTCCTGGCGTTG
GATTATGCTCATTCTTTTTGCCTGGGGGACCTTATTGTTTTATATAGGT
GGTCATTTGGTTCGAGATAATGACCACCCTGACCATTCTAGCAGAGAAC
TCTCCAAGATTCTTGCAAAGCTGGAGCGCTTAAAACAACAAAATGAAGA
CTTGAGGAGAATGGCTGAGTCTCTCCG
aataccagaaggccctattgat
caggggacagctacaggaagagtccgtgttttagaagaacagcttgtta
aggccaaagaacagattgaaaattacaagaaacaagctaggaatg
ATCT
GGGAAAGGATCATGAAATCTTAAGGAGGAGGATTGAAAATGGAGCTAAA
GAGCTCTGGTTTTTTCTACAAAGTGAATTGAAGAAATTAAAGAAATTAG
AAGGAAACGAACTCCAAAGACATGCAGATGAAATTCTTTTGGATTTAGG
ACATCATGAAAG
gtctatcatgacagatctatactacctcagtcaaaca
gatggagcaggtgagtggcgggaaaaagaagccaaagatctgacagagc
tggtccagcggagaataacatatctgcag
AATCCCAAGGACTGCAGCAA
AGCCAGAAAGCTGGTATGTAATATCAACAAAGGCTGTGGCTATGGATGT
CAACTCCATCATGTGGTTTACTGCTTCATGATTGCTTATGGCACCCAGC
GAACACTCATCTTGGAATCTCAGAATTGGCGCTATGCTACTGGAGGATG
GGAGACTGTGTTTAGACCTGTAAGTGAGACATGCACAGACAGGTCTGGC
CTCTCCACTGGACACTGGTCAG
gtgaagtgaaggacaaaaatgttcaag
tggtcgagctccccattgtagacagcctccatcctcgtcctccttactt
acccttggctgtaccagaagaccttgcagatcgactcctgagagtccat
ggtgatcctgcagtgtggtgggtatcccagtttgtcaaatacttgatcc
gtccacaaccttggctggaaagggaaatagaagaaaccaccaagaagct
tggcttcaaacatccagttattgg
AGTCCATGTCAGACGCACTGACAAA
GTGGGAACAGAAGCAGCCTTCCATCCCATTGAGGAATACATGGTACACG
TTGAAGAACATTTTCAGCTTCTCGAACGCAGAATGAAAGTGGATAAAAA
AAGAGTGTATCTGGCCACTGATGACCCTTCTTTGTTAAAGGAGGCAAAG
ACAAA
gtactccaattatgaatttattagtgataactctatttcttggt
cagctggactacacaaccgatacacagaaaattcacttcggggcgtgat
cctggatatacactttctctcccaggctgacttccttgtgtgtactttt
tcatcccag
GTCTGTAGGGTTGCTTATGAAATCATGCAAACACTGCATC
CTGATGCCTCTGCAAACTTCCATTCTTTAGATGACATCTACTATTTTGG
AGGCCAAAATGCCCACAACCAGATTGCAGTTTATCCTCACCAACCTCGA
ACTAAAGAGGAAATCCCCATGGAACCTGGAGATATCATTGGTGTGGCTG
GAAACCATTGGAATGGTTACTCTAAAGGTGTCAACAGAAAACTAGGAAA
AACAGGCCTGTACCCTTCCTACAAAGTCCGAGAGAAGATAGAAACAGTC
AAATACCCTACATATCCTGAAGCTGAAAAATAGAGATGGAGTGTAAGAG
ATTAACAACAGAATTTAGTTCAGACCATCTCAGCCAAGCAGAAGACCCA
GACTAACATATGGTTCATTGACAGACATGCTCCGCACCAAGAGCAAGTG
GGAACCCTCAGATGCTGCACTGGTGGAACGCCTCTTTGTGAAGGGCTGC
TGTGCCCTCAAGCCCATGCACAGTAAAATAATGTACTCACACATAACAT
ACAAATGGATTATTTTCTACTTTGCCCTTTAAATATTCTGTCCCCATGA
AACAAACACTGCCACATTATGTAATTTAAGTGACACAGACGTTTTGTGT
GAGACTTCAAACATGGTGCCTATATCTGAGAGACCTCTGTGATTTACTG
AGAAGATGAGAACAGCTCCCTTCTGTGGGGAAGTTGGTTCTTAGTCAGT
GGTGGACTGGCCACTGAATTCACTGCAATCAACAGATTCAGAATGAGAA
TGGATGTTTTTCCTTTATATGGTTGTCTGGATTTTTTTTAAAGTAATTT
CATCAGTTCAGTTCATCCACCTCATTAATAAATGAAGGAATATACCAAT
AAAATCAAATGAAATATTCACTGTCCATTAGGAAGTTTTATAAAACAAT
GCCATGAACAAAAAATTCTTTAGTACTCAATGTTTCTGGACATTCTCTT
TGATAACAAAAATAAATTTTAAAAAGGAATTTTGTAAAGTTTCTGGGAT
TCTGTATCACTGGATGATGTAGTTATAAGCTTTGTAGTAGAAATATGGG
AAGTGGGTTTATAGCTTTTAAGATTTTTTTCTACTTTTGTCCTACTTTT
TCTATTTCTGATAGAATAATCATATTTCAAGAGAAGCATTGGTCCCCTC
TAATACTAGTAACTGCCTTTAGTCATGCATATTATATGAAGTTGCTAAG
AACACGCTTTGGGGGAGGTGTTCACTCTCTTAGTTTGATATTGTTGACT
TGATATAATTGAATGAAATAGTCATTCTCTTGCTTCCAG
Alternative exons are represented in upper and lower case letters
Fut8 protein structure is studied extensively to understand the functional domain of the enzyme. Three dimensional crystal structure of FUT8 enzyme revealed 15 strands and 16 helices.
Amino acid sequence of FUT8 gene is provided in
The CRISPR/Cas binding regions are designed in such a way that the specificity of site recognition is high and at the same time the CRISPR/Cas complex carries out the intended DNA single strand break.
In an embodiment, Cas9n (D10A mutant of Cas9 endonuclease) is used for the CRISPR/Cas complex. The Cas9n endonuclease causes single strand DNA break. The two CRISPR recognition sites (5′ recognition site and 3′ recognition site are spaced at 5 base pair distance, allowing two single stand breaks at close proximity. The resulting breaks allow the NHEJ process of DNA break repair and that introduces mutations in this region.
The CRISPR construct has two unique 20 basepair CRISPR recognition sequences flanked by gRNA scaffolds in tandem with U6 promoter elements for efficient expression of the gRNA sequences. The unique design allows one single vector to express two separate gRNA scaffolds and two unique CRISPR recognition sequences on the genomic DNA.
The nucleotide sequence of wild type Cas9 gene is provided in Seq ID No. 2.
The nucleotide sequence of the Cas9n endonuclease is provided in Seq ID No. 3.
The CRISPR/Cas design is uniquely positioned to target beta 2 strand and the 3H2 helix region by incorporating single stranded breaks. The design is compatible with two single strand breaks at close proximity, thereby imparting higher specificity of target recognition as NHEJ repair mechanism occurs only at these targeted genomic locations. Nonspecific single stand breaks, if created are usually repaired by homologous recombination which is accurate and rarely creates any mutation.
The primary target of the present disclosure is to create mutations at the N-terminal catalytic domain, the beta 2 strand and 3H2 helix. Insertion and deletions through CRISPR/Cas at this location makes the FUT8 enzyme non-functional. In addition, frame shift mutations also cause premature translation stop codons, the Rossmann fold which is downstream of this region does not express then. Amino acid residues at Rossmann fold such as Arg 365, Arg 366, Asp-368, Lys-369, Glu-373, Tyr-382, Asp-409, Asp-410, Asp-453, and Ser-469 are very important for FUT8 functionality. The truncated enzyme is non-functional and leads to Fucose Knock out cell line.
The CHO whole cell genome shotgun sequencing data with accession number NW_003613860 for the Fut8 gene corresponds to a total of 161634 bp. The Pubmed accession number for the coding region or mRNA of the Fut8 gene is XM 003501735.1. The mRNA sequence, as shown in
The Spidey alignment tool (http://www.ncbi.nlm.nih.gov/spidey/spideyweb.cgi) is used to identify the exons in the genomic DNA by aligning the mRNA sequence with the genomic DNA sequence. A total of 11 exons with the boundaries as shown in Table 3 are identified.
FUT8 enzyme functionalities through site directed mutagenesis studies of critically important amino acid residues in the catalytic domain has been confirmed.
A 100% identity between the genomic DNA and mRNA sequence is observed. Organization of the Fut8 gene showing all the 11 exons and position of the gRNAs targeting exon7 is shown in
In the present disclosure, CRISPR/Cas9 technology target sites are localized to the first few exons of the Fut8 gene. This is done to avoid partial fucosylation that can be caused by truncated or partially functional enzyme.
Exon-7 (CDS-5) nucleotide sequence of Fut8 is represented by SEQ ID No. 4 of the present disclosure.
Exon-7 (CDS-5) amino acid sequence of Fut8 of CHO cell is represented by SEQ ID No. 5 of the present disclosure. The targeted amino acid positions in the protein/peptide sequence are underlined.
RYATGGWETVFRPVSETCTDRSGLSTGHWS
1.2—Sequence of Interest in Fut8 Gene to be Targeted Using CRISPR
In Exon 7 of the Fut8 gene, the sequences provided below are used to bind to CRISPR-Cas complex.
The target sequence in Fut8 gene that is used in an embodiment of the present method is shown in
This method of the present disclosure uses Cas9n (nickase mutant) in targeting Fut8 genomic sequence, exon 7 with CRISPR/Cas system. The Cas9n endonuclease makes single stand break (SSB) in opposite strand of DNA. The CRISPR/Cas recognition sequences in the upper and lower strands are underlined. Corresponding single strand break sites are indicated as black arrow heads. The three nucleotide PAM sequences are indicated in bold letters.
The CRISPR/Cas vector construct for this design is termed as pD1401 gRNA (514-553).
The 5′ and 3′ CRISPR recognition sequence is indicated in small and italicized, two separate sites complementary to this recognition sequence are recognized at the FUT8 genomic sequence. The sequence represented with bold letters indicate gRNA scaffold sequence for CRISPR/Cas complex to get engaged.
attccaagatgagtgttcgc
GTTTTAGAGCTAGAAATAGCAAGTTAA
AATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGT
GCTTTTTTGCTCCGCGGCACGAGAACTCAAAGCCCCGGGGCCTGGGT
GTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATC
AACTTGAAAAAGTGGCACCGAGTCGGTGC
GTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATC
AACTTGAAAAAGTGGCACCGAGTCGGTGC
The construct map is provided in
1.3—CRISPR/Cas Complex Synthesis
Two components must be introduced and/or expressed in cells or an organism to perform CRISPR based genome editing: the Cas9 nuclease; and a ‘guide RNA’ (gRNA).
A twenty nucleotides long recognition sequence at the 5′ end of the gRNA directs Cas9 to a specific target DNA site using standard RNA-DNA complementarity base pairing rules. These target sites must lie immediately 5′ of a PAM sequence that matches the canonical form 5-NGG.
A mutant Cas9 nuclease (D10A), known as Cas9n is used to target Fut8 Exon 7 locus, the constructs create single strand breaks instead of a double strand DNA break. This design is aimed to improve specificity of CRISPR/Cas constructs.
In case of single strand breaks, two DNA target sites are targeted at close proximity where single stand break or nicks happen in opposite DNA stands. Thereby, it recruits DNA repair machinery (NHEJ) to repair the DNA damage. Recruiting two gRNA/Cas9n complex at a specific interval to initiate DNA repair improves the specificity to the targeted site. Nonspecific binding of only one of the gRNA/Cas9n complex to unrelated sites causes nicks which are usually repaired through homologous recombination based repair with very low rate of mutation. Therefore, this approach increases the specificity of targeting the Fut8 gene.
Unique regions of FUT 8 gene are targeted based on the enzyme structure information in a way to abolish enzyme catalytic function or by disrupting higher order structure.
The important features of the vectors are:
1.4—the Complete Process of Obtaining CRISPR Construct is Composed of the Following Steps:
The monoclonal antibody product could be produced in any Chinese Hamster ovary cell line using any technology. Examples for such technologies include but not are restricted to GPEx™, UCOE™, SURE™ technology, Light Path™ technology and many others. In this particular example, the anti Her2 antibody gene is expressed in Chinese Hamster Ovary (CHO-S) cell line utilizing the GPEx technology. GPEx® is based on a highly efficient retroviral vector that transfects 100% of target cells. The advantage of GPEx® is
Anti Her2 antibody heavy and light chain protein sequences are obtained from open-access public sources. The corresponding heavy and light chain gene sequences are codon-optimized using proprietary codon-enhancement system. The codon-optimized sequences for antibody heavy and light chain genes are synthesized by commercial vendor and cloned in pUC57 vector. The heavy and light chain sequences are represented by SEQ ID Nos. 16 and 17.
The heavy chain amino acid sequence of Anti-her2 antibody—SEQ ID No. 16
The light chain amino acid sequence of Antiher2 antibody—SEQ ID No. 17
Both genes coding DNA sequences (CDS) are synthesized with flanking Hind III and Xho I cloning sites for facilitated cloning. The pUC57 heavy and light chain encoding plasmids are digested with Hind III and Xho I restriction enzymes to isolate the two CDS. Anti Her2 antibody heavy and light chain DNA fragments are cloned into GPEx® plasmid pCS-newMCS-WPRE and digested with the same two enzymes for retroviral vector based expression. The new plasmid encoding full-length AntiHer2 antibody light chain CDS is named pCS-HighwayLC-WPRE and the validity of the CDS of both constructs is confirmed by DNA sequencing.
These two retroviral expression vectors are used for development of Anti Her2 antibody expressing producer cell lines. Production cell lines are made by performing multiple rounds of transduction of the Chinese Hamster Ovary (CHO-S) parental cell line with retroviral vector containing Anti Her2 antibody sequences coding for light chain (LC) and heavy chain (HC). Six transductions are performed in all. The pooled populations are seeded into 96-well cell culture plates to establish clonal cell lines that originated from single cells. The clonal cell lines are screened by ELISA for protein titer. The top thirty clones are expanded, tested in triplicate T175 flasks for growth and productivity and are cryopreserved. The top clone is tested for productivity by fed batch analyses in shake flasks.
The AntiHer2 antibody producer cell lines are used for generating afucosylated AntiHer2 antibody using the CRISPR/Cas construct targeting FUT8 gene, specifically Exon 7 of the Fut8 gene, of the parental CHO-S cell line. In this example, one of the high producing clonal cell lines is used for obtaining afucosylated AntiHer2.
This example contains procedure for transfection of CHO S cell line over expressing AntiHer2 Antibody. The CHO S cell line is transfected with the CRISPR/Cas construct targeting the FUT8 gene described in previous section. It also describes the method of selection and confirmation of single cell stable cell lines for developing FUT8 knock out CHO-S cell line over expressing AntiHer2 Antibody using CRISPR/Cas technology, and selection of positive clones by flow-cytometry based functional assay.
3.1—Transfection of CHO Cell Line with CRISPR Construct
This example contains transfection procedure for CHO-S cell line over expressing Anti Her2 antibody with CRISPR constructs. DNA concentration ranging from 0.5 μg to 5 μg are tested for various incubation times for e.g., 4 hrs, 24 hrs and 48 hrs. Liposome and modified liposome mediated transfection reagents are tested for e.g., Lipofectamine 2000, Lipofectamine 3000, Lipofectamine LTX with Plus' reagent, MIRUS TransIT X2, MIRUS TransIT 2020, MIRUS TransIT 293, MIRUS TransIT CHO transfection kit. Multiple DNA to transfection reagent ratios (μg: μl) are also tested. The optimum transfection efficiency is achieved using 1:5 DNA to transfection reagent ratio, 24 hrs incubation and Lipofectamine LTX with Plus™ reagent. Optimization experiments are performed with RFP expressing plasmid DNA.
Transfection efficiency is calculated by the following formula:
Transfection efficiency=(Number of RFP expressing cells/Total number of cells)*100
Optimized transient transfection efficiency is about 50-70% in the Anti Her2 Antibody expressing CHO-S cell line.
Transfection Protocol:
CHO-S cells are seeded at more than 90% viability and at a density of 2×106 cells/well in a 6 well tissue culture plate and allowed to adhere for 24 hrs. pD1401 (gRNA 514-553) CRISPR Cas construct is transfected using Lipofectamine LTX with Plus™ reagent. 10 μg of construct is used with 1:5 DNA to transfection reagent ratio. The cells are incubated for 20-24 hrs after transfection. Prior to transfection DNA quantity and quality is estimated by UV spectrophotometry. A 260/280 value DNA represents quality and protein contamination. The ratio of absorbance at 260 nm and 280 nm is used to assess the purity of DNA. A 260/280>1.8 is generally accepted as “pure” or good quality DNA. 3-4 μl of DNA sample is placed on the micro cuvette and DNA concentration is estimated using Eppendorf Biophotometer D30 against suitable controls.
CRISPR DNA and Lipofectamine LTX solution are diluted, mixed gently and incubated for 5-10 minutes at 20-25° C. DNA and transfection reagent dilutions (3 ml) are mixed and incubated for 20-30 minutes at Room Temperature for complex formation. The media is aspirated from the wells. 3 ml of DNA and transfection reagent complex is added drop wise to the plated cells.
The cells are incubated for 24 hours at 37° C. in a 5% CO2 Incubator. The cells are centrifuged at 1400 rpm for 4 min and the spent media is removed. The pellet is re-suspended in 5-10 ml complete media/125 ml Erlenmeyer shake flask and incubated at 37° C. in a 5% CO2 Incubator. Cell count is taken every 2-3 days and viability is monitored. Fresh media change is given every 2-3 days. RCB (Research Cell Banks) is prepared when cells attain a viability of 95%.
3.2—Single Cell Cloning
Serial Dilution and Plating:
Cells are splitted at a density of 0.25×106 cells/ml one day prior to cloning. Cell count is taken by Vicell XR automated cell counter and ensured viability more than 90% on the day of plating. Cell suspension is serially diluted to obtain a cell density of 1 cell/well. Dilutions are made at 1:10 ratio in each step. Cells are plated in Power CHO 2CD media with 10% FBS, 4 mM Glutamine, 1× PenStrep and 1× Clona Cell CHO ACF (animal component free) supplement in a non-treated 96 well round bottom plate in 100 μl/well. Plates are scanned under inverted phase contrast microscope to identify single cells. Wells containing single cell are visually confirmed and marked for further monitoring. Wells containing 2 cells and 6 cells are also marked and kept for amplification. The plates are incubated at 37° C. in a 5% CO2 Incubator with 75% relative humidity for 10-15 days.
3.3—Amplification of Clones and Mini-Pools in 24 Well Plate:
After 10-15 days the clones are amplified from one well of 96 well plate to one well of 24 well plate in one ml culture volume. Media used for amplification is Power CHO2 CD with 10% FBS 4 mM Glutamine and 1× PenStrep. The 24 well plates are incubated at 37° C. in a 5% CO2 Incubator with 75% relative humidity for 2-3 days.
3.4—Amplification of Clones and Mini-Pools in 6 Well Plates:
The clones and mini-pools are then amplified from one well of 24 well plate to one well of 6 well plate in 3 ml culture volume. The 6 well plates are incubated at 37° C. in a 5% CO2 Incubator with 75% relative humidity for 4-5 days. Media used for amplification is Power CHO2 CD with 4 mM Glutamine and 1× PenStrep. From this step onwards, FBS free media is used for amplification.
3.5—Screening by Flow Cytometry
LCA-FITC binding profile of respective clones is determined and two 6 cell mini-pools (CR2TMCHO 6A, CR2TMCHO 6B) and two single cell clones (CR2TMCHO 1A, CR2TMCHO 1B) are selected for further studies. The details of LCA-FITC binding assay are described in the below section.
3.6—Amplification of Clones and Mini-Pools in 125 ml Erlenmeyer Shake Flasks
The clones and mini-pools are further amplified into 10 ml culture volume in a flat bottom 125 ml Erlenmeyer shake flask. The flasks are incubated at 37° C. in a 5% CO2 Incubator shaker at 120 rpm with 75% relative humidity for 4-5 days. After that, the clones are amplified to 30 ml culture volume in 125 ml Erlenmeyer shake flasks and incubated at 37° C. in a 5% CO2 Incubator shaker at 120 rpm with 75% relative humidity for 4-5 days. Power CHO2 CD media is used for amplification with 4 mM Glutamine and 1× PenStrep solution. Finally cell count, viability and LCA-FITC binding profile are determined. Cell supernatants are harvested by centrifugation at 1400 rpm for 4 minutes and used for protein A purification and further characterization. Research Cell Banks are prepared and cryopreserved following established protocols.
Fluorescein isothio cyanate (FITC) is a flurochrome conjugated to LCA. Therefore, presence of fucosylated proteins on cell membrane of control CHO S cells is recognized by fluorescein conjugated LCA. These cells fluoresce brighter in specific flow cytometer channel. The fluorescence observed is represented as fluorescence unit. The cells where fucose pathway is disrupted, the knockout lines are not able to produce fucosylated cellular proteins and hence the cell membrane proteins are non fucosylated. Testing these cells with Fluorescein-LCA conjugate results in fluorescence comparable to background. Therefore, the Fucose knock out cells fluoresce at a much lower level compared to control CHO S cell line.
Fluorescein Lens Culinary Agglutinin (LCA-FITC) stock 5 mg/ml is diluted to get 2 μg/ml final concentration in assay buffer (DPBS containing 2% BSA). Cells are spun at 1400 rpm for 4 minutes using Eppendorf minispin centrifuge. The media is aspirated and the pellet is re-suspended in 0.25-1 ml of assay buffer containing 2 μg/ml LCA-FITC. CHO S control cells are re-suspended in 0.25-1 ml of assay buffer alone (unstained control) and 0.25-1 ml of assay buffer containing 2 μg/ml LCA-FITC (stained control). All the samples are diluted to get 0.1-0.2×106 cells/ml in final assay buffer. The samples are then incubated in dark on ice for 30 minutes. Then each sample is analyzed by BD Accuri C6 benchtop flow cytometer for data acquisition. Data analysis is done using Accuri C6 software.
Fluorescein-streptavidin (Strep-FITC) negative staining is also performed. Fluorescein streptavidin (Strep-FITC) stock 1 mg/ml is diluted to get 2 μg/ml final concentration in assay buffer (DPBS containing 2% BSA). Cells are spun at 1400 rpm for 4 minutes using Eppendorf minispin centrifuge. Media is aspirated and the pellet is re-suspended in assay buffer containing 2 μg/ml Strep-FITC. CHO S control cells are re-suspended in assay buffer alone (unstained control), assay buffer containing 2 μg/ml LCA-FITC (stained control) and assay buffer containing 2 μg/ml Strep-FITC. All the samples are diluted to get 0.1-0.2×106 cell/ml in 0.25-1 ml assay buffer. The samples are then incubated in dark on ice for 30 minutes. Then each sample is analyzed by BD Accuri C6 benchtop flow cytometer for data acquisition. Data analysis is done using Accuri C6 software. The fluorescence data is presented in below Table 6.
LCA-FITC Binding Profile of Single Cell Clones and Minipool Populations:
LCA-FITC Binding Profile of Transfected Pool Over Multiple Passages:
The CHO S cell line expressing Anti Her2 Antibody is transfected with CRISPR/Cas construct and is analyzed for Fucose knock out phenotype. The transfected cell line pools are analyzed for multiple passages to understand the stability of Fucose knock out phenotype.
The data suggests the transfected pools maintains Fucose knock out phenotype over multiple generations (
The results indicate that the uniqueness of the CRISPR/Cas system designed and used in this disclosure has the ability to transform large number of cells (more than 82% of cells) to Fucose knock out cells. The uniqueness of the constructs and the unique methods are useful because the methodology and the CRISPR/Cas system are usable in any antibody over-expressing cell line to create a novel Afucosylated antibody product. This is a novel approach to create Afucosylated monoclonal antibody which is much simpler, very rapid and highly robust. This surprising achievement allows this methodology and CRISPR/Cas system to develop novel Afucosylated antibody products against any target antigen.
In comparison, for developing Afucosylated monoclonal antibodies, a special Fucose knock out platform needs to be created and be completely characterized before expressing monoclonal antibody genes in that platform. Processes of developing such Fucose knock out platforms are very complex and time consuming and success rate is quite minimal.
The simplification of this process enables development of Afucosylated monoclonal antibodies in a very short period of time from any CHO S cell line expressing any monoclonal antibody.
Therefore the methodologies described here and the CRISPR/Cas systems designed and developed in this disclosure are considered to be efficacious.
0.5×10{circumflex over ( )}6 cells/ml are seeded in 125 ml Erlenmeyer shake flasks. Viable cell count is performed using Vi-cell XR cell viability analyser on Day 1, 2, 3, and 4. Respective growth curves are generated and depicted in
CRISPR/Cas transfected CHO S cell lines selected through functional assay, namely LCA-FITC flow cytometry assay are used for genomic sequence analysis. The FUT 8 genomic locus of Chinese Hamster is well reported in literature (NW_003613860) and is used as wild type sequence to understand type of gene modification in each cell line clone. The objective of this example is to analyse genomic DNA sequencing results obtained from CHO S cell lines transfected with CRISPR/Cas system and expressing Afucosylated AntiHer2 antibody product. All cell lines reported here are clonal cell lines and are selected from LCA-FITC flow cytometry assay.
Briefly, the selected clonal cell lines are grown in appropriate growth conditions for genomic DNA isolation, purified genomic DNA is used for PCR amplification using primers flanking the FUT8 target locus, the PCR amplified product is then purified and cloned in a suitable vector using E. coli competent cells, resulting ampicillin resistant E. coli colonies are selected and cultured, plasmid DNA are isolated from each bacterial clone, approximately 5-10 individual bacterial colonies are tested per clonal cell lines through automated sequencing to understand the type of modification at the FUT8 target genomic locus.
Following reagents and solutions are used to carry out genome sequencing of the selected clones
The entire genome sequencing protocol is divided in following four processes
Genomic DNA Isolation from Selected Clones
Clonal CHO S cell lines are grown in Power CHO2 CD media with 4 mM glutamine, 100 units/ml Penicillin and 100 μg/ml Streptomycin in shake flasks with 120 rpm at 37° C. in presence of 5% CO2 and 75% relative humidity in controlled condition incubators. The cell growth is observed every day and viability is monitored. Cells are harvested at greater than 90% viability. On the day of isolation, culture media is removed by centrifuging the cell population. Cells are then mixed with 10 ml of DPBS and centrifuged at 1500 rpm for 5 min. The spent media is removed and cell pellet is resuspended in 10 ml DPBS. Cells are washed again using centrifugation at 1500 rpm for 5 min. DPBS is removed completely by aspiration. The final cell pellet is used for genomic DNA isolation.
Genomic DNA is isolated from CHO S parental cells and CHO S cell lines transfected with CRISPR/Cas system expressing Afucosylated AntiHer2 Antibody and selected through LCA flow cytometry assay. Commercially available QIAGEN gDNA extraction kit is used for isolating genomic DNA following manufacturers protocol.
PCR Strategy Design
Genomic DNA sequence of Chinese Hamster is analysed from publicly available database sequence NW_003613860. FUT8 Exon 7 DNA sequences and partial intron sequence is used for designing PCR strategy to amplify the FUT8 target locus.
Primers are designed based on primer length, PCR product length, GC content, melting temperature and potential homoduplex and heteroduplex formation. Primers are designed flanking the FUT8 target locus as provided below. The amplified PCR product is intended for mutation analysis due to CRISPR mediated SSB and subsequent DNA repair. Following nucleotide sequence represents the region of interest with primer sequences in bold letters.
Fut8 Exon 7 and associated intron sequences used for PCR primer design: SEQ ID NO. 20
aagaaataagctgaatcagctctgacttattgtgtgattttcaatac
TTGGAATCTCAGAATTGGCGCTATGCTACTGGAGGATGGGAGACTGT
gttaggtcagattagcac
Introns are represented from base 21 to base 106 and from base 345 to base 371 in lower case letters. Exon 7 is represented from base 107 to base 344 in upper case letters. The primer binding sites of Left and Right are underlined.
Primer Design for Identifying the INDEL by PCR
Genomic PCR is performed using QIAGEN gDNA extraction kit using the following primers mentioned in table 7.
The following section provides experimental details for PCR product generation from CHO S genomic DNA from control cell lines and CHO S clonal cell lines expressing afucosylated AntiHer2 antibody, cloning of PCR products in E coli competent cells and sequencing of cloned PCR products.
Optimization of PCR Condition—
The experiments are designed to standardize PCR conditions. The parameters tested include, genomic DNA concentration (from 100 ng to 1000 ng), primer concentrations (2 nmole to 20 nmole), PCR annealing temperature (from 55.8° C. to 62.9° C.) and time (20 secs to 50 secs), PCR product extention time (30 secs to 60 secs) and PCR cycle number is set at 30 cycles. Arrived optimized condition is described in following section.
PCR reactions are carried out using proof reading polymerase Phusion polymerase to ensure that PCR mediated mutations are limited. Following PCR amplification cycles, Taq polymerase enzyme is added in the mix for tailing. The tailing step is important as the extra base added to the PCR products allows direct cloning in sequencing vector described in the next section. In order to add dATP overhangs to PCR product for cloning in TA cloning vector, the Phusion polymerase amplified product is incubated with Taq DNA polymerase for 20 minutes at 72° C.
Cross Checking the Genomic DNA Sample by PCR—
Genomic DNA PCR products are analysed in agarose gel electrophoresis and the product length is confirmed using a molecular weight standard. PCR samples with clear amplification profile are used in the next processing step.
PCR Product Gel Elution Using QIAGEN Kit—
The amplified PCR products are loaded in freshly prepared 1% agarose gel and electrophoresed at 100V for one hour to separate amplified PCR products from unused primers and any other dimers produced during the amplification process. The amplified products are excised from gel and eluted using commercially available Qiagen gel elution kit. DNA is eluted with highly pure molecular biology grade water.
Cloning of PCR Products in Sequencing Vectors—
Agarose gel purified PCR amplified products are then used for cloning in commercially available pTZ57R/T vector through DNA ligation process. Conditions for DNA ligation have been standardized previously.
Transformation of Ligated Sample pTZ57R/T+CRISPR(PCR) in DH5alpha E. coli Competent Cells—
Ligated DNA is transformed in E. coli DH5alpha competent cells, available commercially. Transformation protocol as described by manufacturer is followed to achieve high level transformation efficiency. After transformation, the E. coli cells are grown in presence of Ampicillin antibiotic for growth of transformed colonies.
Inoculation of Transformed Cells (pTZ57R/T+CRISPR(PCR)) into LB Media with Ampicillin—
Each separate colony is inoculated in LB+Ampicillin broth in 5 ml culture volume and grown overnight for plasmid DNA isolation.
Isolation of Plasmid DNA (pTZ57R/T+CRISPR(PCR) from DH5alpha Transformed Cells—
4.5 ml of overnight grown cultures are used for plasmid DNA isolation using commercially available QIAGEN plasmid DNA isolation kit following manufacturers protocol. The plasmid DNA is eluted with highly pure molecular biology grade water.
Cross Checking of Plasmids for the Presence of Insert—
Each plasmid preparation is tested for presence of insert using suitable restriction enzyme digestion followed by agarose gel electrophoresis. The size of insert is compared with suitable molecular weight standards.
PCR Reaction
First, the double-stranded DNA template is denatured at a high temperature at 94° C. Sequence-specific primers mentioned in the Table 7 are then annealed (60.4° C.) to sites flanking the target sequence. A thermostable DNA polymerase (Phusion polymerase) extends (72° C.) the annealed primers, thereby doubling the amount of the original DNA sequence. This newly synthesized product then becomes an additional template for subsequent cycles of amplification. These three steps are repeated for 30 cycles, resulting in a 109 fold increase in target DNA concentration. In order to add dATP overhangs to PCR product for cloning in TA cloning vector, the PCR Phusion polymerase amplified product is incubated with Taq polymerase for 20 minutes at 72° C.
The PCR product is further modified with Taq DNA polymerase for tailing. Final PCR product is then electrophoresed in agarose gel for elution of amplified fragment. The amplified fragments are then gel eluted using QIAEX II Gel extraction kit.
Ligation
PCR amplified and gel eluted products are ligated in commercially available pTZ57R/T vector. Ligation protocol is described as follows
The above ligation mix is incubated at 4° C. overnight and 50% of ligated mix is transformed into DH5alpha E. coli competent cells by heat shock method.
Transformation of Ligated Sample into Bacterial Cell by Heat Shock Method
The purpose to transform bacterial cells is to clone and propagate the plasmid DNA. 20 μL aliquot of competent E. coli cells (DH5alpha) are taken from −80° C. freezer and thawed on ice for 5 minutes. 50% of ligated sample (pTZ57R/T+CRISPR(PCR) is added to the competent cells and gently mixed and incubated on ice for 20 minutes. The mix containing tube is placed on water bath/dry bath at 42° C. for 50 seconds. The tube is placed back on ice for 2 minutes. 0.950 ml of 37° C. warmed LB broth (without ampicillin antibiotic), is incubated at 37° C., 220 rpm for 1 hour, in shaker. 100 μL of the resulting culture is spread on warmed LB+ ampicillin culture plates. The plates are incubated overnight at 37° C. incubator.
Plasmid DNA Isolation from Bacterial Cells Using QIAPrep Spin Miniprep
The purpose of this procedure is to grow/culture bacteria that contain a specific DNA plasmid, which is used in following experiments. 5 mL of LB+ ampicillin broth is added into autoclaved tubes, isolated bacterial colonies are inoculated from the culture plates to the LB broth+Ampicillin culture tubes. Tubes are incubated at 220 rpm, at 37° C. overnight (approximately 16-18 hours depending on the growth of the bacterium). Overnight culture of 4.5 mL is centrifuged at 13 rpm for 1 minute. Plasmid DNA is isolated using commercially available QIAGEN plasmid isolation kit. Plasmid DNA is eluted with highly pure molecular biology grade water and stored at −20° C. freezer until further use.
Positive Clones Selected Using Restriction Digestion with EcoR I-HF and Hind III-HF Enzymes
Plasmid DNA thus isolated is tested for presence of insert, in this case the PCR amplified fragment. The pTZ57R/T vector contains multiple restriction enzyme sites flanking the cloned PCR product. The restriction sites EcoRI and HindIII are selected for restriction digestion as described in below table. The reaction is carried out at 37° C. for 2 hours for complete digestion of the plasmid DNA. Following restriction digestion, the mixture is electrophoresed in 1% agarose gel for 1 hour. The PCR product insert, if present, separates from pTZ57R/T vector backbone and the confirmed bacterial clones are used for DNA sequencing.
The present disclosure depicts the representative restriction enzyme digestion of PCR amplified product in pTZ57R/T vector to confirm presence of inserts from different CHO S cell lines. Plasmid DNA preparations from independent bacterial clones are digested with EcoRI and HindIII restriction enzymes flanking the PCR fragment cloned in the vector and the mixture is electrophoresed in 1% agarose gel. The size of resulting DNA fragments are estimated from the DNA molecular weight standards. The bacterial clones are used for automated sequencing to know the status of FUT8 locus in different CHO S cell lines.
Sequencing—
The confirmed plasmids are then sequenced with specific sequencing primers present in the pTZ57R/T vector backbone. Sequence data is generated in automated DNA sequencing instruments following appropriate protocols. Sequencing is carried out with both forward and reverse sequencing primers to ensure proper sequence information.
DNA Sequence Analysis—
DNA sequencing data from all plasmids are analyzed. DNA sequence from plasmid DNA derived from CHO S parental cell line and various CRISPR/Cas mediated FUT8 knock out CHO S clonal cell lines are compared and differences in DNA sequences are identified. From each CHO S cell line clone, PCR products are generated and cloned in E. coli. Multiple E. coli clones are sequenced to confirm nucleotide sequence modification at the target genomic locus.
CHO S parental cell line—sequence of Exon-7 of FUT8 gene (SEQ ID No. 20) is in upper case. Intron sequence is in lower case and underlined.
aagaaataagctgaatcagctctgacttattgtgtgattttcaatac
gttaggtcagattagcac
CHO-S FUT8 knockout clonal cell line sequences are provided below. It is observed that the Exon 7 sequence is mutated in the cell lines.
Sequence data is collected from analysis of 5-15 independent bacterial clones sequenced with both forward and reverse sequencing primers. The sequencing data suggests deletions of variable lengths in multiple clones compared to CHO S parental cell line. Deletion of bases is observed in clone CR2TMCHO 1A and CR2TMCHO 1B allelic versions. All deletions are located at the CRISPR/Cas target site.
The data suggests various INDELs present at the FUT8 genomic locus in CHO S cell lines transfected with CRISPR/Cas system targeting FUT8 gene and overexpressing Afucosylated AntiHer2 Antibody. In many cases, it is observed that there are modifications at the targeted bases. Such diversity of genomic modification through CRISPR/Cas complex is possible due to endogenous DNA single strand breaks at close proximity and repair through non homologous end joining. All of these cell lines are selected through functional screening assay, namely LCA-FITC flow cytometry assay. The results also imply high efficiency of the functional assays to isolate and identify CHO S FUT8 knock out cell lines over expressing AntiHer2 antibody.
It is also revealed that the design of the CRISPR/Cas complex depicted in this disclosure is highly efficacious as this one pair of CRISPR/Cas complex with the Cas9n endonuclease provides a highly sequence specific gene alteration at the targeted FUT8 locus in CHO S cell lines.
Composite analysis of the sequence data is used to identify potential FUT8 knock out CHO S cell lines where FUT8 genomic target locus is modified through deletion and/or insertions (INDELs). The DNA sequences are then aligned to show distinct differences.
Amino Acid Sequence Analysis of the CHO S Cell Lines Over Expressing AntiHer2 Antibody Transfected with CRISPR/Cas System Targeting FUT8 Gene
Genomic DNA sequence at the FUT8 locus of CHO S parental cell line and CHO S cell lines over expressing Anti Her2 antibody and transfected with CRISPR/Cas system targeting FUT8 gene are further analyzed to understand the impact of DNA sequence INDEL on FUT8 protein status. DNA sequences at the targeted FUT8 locus is translated into amino acid sequences using vertebrate codon bias. The amino acid sequence of exon7 region is studied closely and the results are summarized in below table 12. When compared to CHO S parental cell line, the FUT8 knock out cell lines revealed modifications involving frame shift mutations and introduction of stop codons.
In many instances, frame shift mutations are observed, which alter the C-terminal region of the FUT8 protein to make it non-functional enzyme. In addition, stop codon is introduced as an effect of frame shift mutation and thereby the FUT8 protein is truncated and non-functional in these clones.
Furthermore, it is observed that the selection of target amino acids in the FUT8 protein sequence is highly effective. Targeting conserved amino acids at positions of wild type FUT8 protein with only one pair of CRISPR/Cas complex has created mutations at the targeted locus in multiple knock out cell lines (
All Fucose Knock out clonal CHO S cell lines over expressing Afucosylated AntiHer2 antibody and confirmed through the genome sequencing protocols described above are stored in liquid nitrogen storage as Research Cell Banks and Working Cell Banks. Multiple vials of Research Cell Banks and Working Cell Banks are stored in liquid nitrogen storage for future use.
A vial of working cell bank containing Fucose Knock out clonal CHO S cell line over expressing Afucosylated AntiHer2 antibody is used to start the 2 liter Bioreactor culture protocol. The cells are thawed and seed train cultures are started in 150 ml baffled shake flask. The culture is further expanded to 250 ml baffled shake flask over a period of 11 days for inoculation of 2 L bioreactor. For each inoculum step a starting cell density of about 0.3 to about 0.5×106 viable cells per milliliter is used.
The production bioreactor is run in fed-batch mode. The bioreactor is a stirred tank reactor with a working volume of about 1.3 L to about 2 L. The media used is a commercially available chemically defined media. Aeration is performed by sparging. Process parameters such as pH, temperature, dissolved oxygen and agitation rate are controlled automatically. Some medium components are fed as separate sterile solutions to the bioreactor. These comprise glucose, amino acids, vitamins, and trace elements. After several days of fermentation with continuous monitoring of many in-process control parameters such as cell density, viability, pH, dissolved oxygen and metabolite levels such as Glucose, Glutamine, Lactate, Ammonia, Glutamate, IgG and LDH, the batch is harvested on day 10 with around 80% viability. The intact cells and cell debris are removed from the culture broth by centrifugation. The profile of in-process parameters are depicted below.
As shown in
It is well known that cultured mammalian cells such as CHO S cell line culture frequently consume nutrients like glucose and glutamine beyond their stoichiometric needs and secrete the waste products lactate and ammonia in recombinant CHO S cell lines. Carbohydrates are the main source of energy in the medium. The most commonly used carbohydrate in mammalian cell culture is glucose. It provides the cells with energy and acts as the major source of carbon for synthesis of proteins. Amino acids, which form the building blocks required by the cell to produce proteins, form an important constituent. Concentration of amino acids in the media greatly influences the maximum cell density that a culture can achieve. Essential amino acids like L-Glutamine act as a nitrogen source for nucleotides and also as an alternate source of metabolism. The profile for glucose and glutamine consumption during fermentation run has been shown in
Ammonium ion has a stronger impact on cell culture than lactate because high concentration of ammonium ion can inhibit growth and impair productivity. The precise mechanism of ammonium ion toxicity in cells is not clear, even though this effect has been shown to depend on pH. In the fermentation run, ammonium concentration is obtained as around 8 mmol/L as shown in
The present disclosure describes the optimal growth conditions required for over expression of Afucosylated AntiHer2 antibody product at 2 liter fed batch bioreactor. The results revealed that these unique cell lines developed through transfection of CRISPR/Cas system maintain major biochemical parameters monitored during 2 liter fed batch bioreactor production. This observation again confirms that using the unique CRISPR/Cas system, the methodology has successfully developed a novel Afucosylated monoclonal antibody product from a CHO S cell line over expressing AntiHer2 antibody.
The CRISPR/Cas system and the methodology described herein has the potential to develop Afucosylated monoclonal antibody from any CHO S cell line over expressing any monoclonal antibody.
The CHO S cell line over expressing AntiHer2 antibody is genetically modified to disrupt the Fucose biosynthetic pathway using CRISPR/Cas system targeting the FUT8 genomic locus. Cell culture material from 2 liter fed batch bioreactor production is harvested after 10 days of culture with more than 80% viability. The culture is processed for purification of Afucosylated AntiHer2 Antibody, the procedure is described below.
Quality attributes of monoclonal antibody or bio therapeutic proteins are highly affected by both process and product related impurities. Modern downstream purification processes are increasingly driven by economic factors. It's mandate to shorten the time to deliver the product to market means that the process development must be fast and inexpensive without compromising quality. Keeping this in view, a good product development workflow is very essential and will ensure a robust process where critical parameters have been identified and are under control. The concept of Quality by Design (QbD) with the support of Process analytical technology (PAT) in both upstream and downstream early stage process development has been employed in recent years by many biopharmaceutical companies.
The downstream process development of monoclonal antibodies (typically at the laboratory scale) involves optimization and integration of several unit operations that provide the desired purity, product quality, throughput and yield. CHO (Chinese Hamster Ovary) cells are widely used in monoclonal antibodies production, where proteins expressed using chemically defined media in fed batch mode. Other cell lines used for the production of therapeutic monoclonal antibodies include NS0, SP2/0 and PER.C6.
CHO cells are most commonly used for manufacturing of number of mAb products. In recent years, efforts made to increase the productivity by controlling fermentation process parameters and efficient feeding strategy. Once the CHO cell culture is harvested, the harvest is subjected to clarification to remove cells, cell debris, media components and impurities. This is accomplished by any of the methods namely centrifugation, depth filtration or tangential flow filtration (microfiltration). Each clarification method has its own advantages as well as drawbacks. Centrifugation step was used in earlier days for clarification. The clarification efficiency of the centrifugation process depends upon many factors which includes centrifuge feed rate, G-force, bowl geometry, operating pressures, discharge frequency and ancillary equipment used in the transfer of cell culture to the centrifuge. The cell culture characteristics such as total cell density and culture viability during the culture process and at harvest will also affect separation performance. The next method of choice is Tangential flow filtration, where the cell culture flows tangential to the microporous membrane under set pressure limit that leads to the separation of cells, cell debris and insoluble particles. A series of concentration and diafiltration steps is followed in cell separation. Control of pressure is very critical, since the pressure force can lyse the cell that leads to increased release of intracellular proteins, host cell DNA, proteases etc. Most commonly used pore size is 0.22 micron and 0.11 micron also used in recent years.
Depth filtration is the most commonly used clarification tool. Due to multiple mechanisms such as electrokinetic adsorption, size exclusion and hydrophobic interaction, this type of clarification step has been used extensively to separate or remove cells, cell debris, process contaminants and soluble as well as insoluble impurities. Depth filtration also helps in removal of host cell DNA. In recent days, precipitation/flocculation-based pretreatment of cell culture fluid had been employed to reduce the load onto the clarification stage. Initially in laboratory scale, optimization of filter type, pore size, surface area and flux will be done and then scaled up to pilot scale.
After clarification, the clarified sample is subjected to filtration using a filter of 0.22 μm pore size. The filtered sample is loaded onto Protein A affinity chromatography column, where the bound molecules are eluted by low pH buffer. Protein A affinity chromatography is a well-established capture step, which gives purity above 95% with highest recovery. Optimum acidic pH for monoclonal antibodies elution should be studied experimentally, since mAbs have a tendency to aggregate at low pH. The eluted protein is subjected to virus inactivation (low pH) and neutralization to remove viruses. There are number of Protein A resins commercially available and few are alkali tolerant with high protein binding capacity. Commercial Protein A resins are available from various suppliers namely GE Healthcare, Repligen, Merck Millipore, Tosoh Bioscience etc.
One or two chromatography polishing steps are generally employed to remove both product related impurities such as product isoforms, clipped species, charge variants and process related impurities such as host cell proteins (HCP), host cell DNA (HCD), endotoxins, adventitious viruses, protein A leachates. Generally, either anion or cation exchange chromatography is employed as a second chromatography step. Although, hydrophobic interaction chromatography, mixed mode chromatography and hydroxyapatite chromatography are used in few cases. The purification process flow includes two virus removal steps, one is low pH inactivation post capture Pro A chromatography stage and second one is nanofiltration (virus filtration) step after final polishing chromatography. In recent years, efforts have been made to use either ion exchange chromatography or mixed mode chromatography as a capture step instead of Pro A Chromatography. Anionic membrane absorbers are also used in downstream processing of mAbs as negative binding mode, where protein elute in flow through and impurities bind to the membrane. Lastly, the purified product after final polishing chromatography step is concentrated and diafiltered into the final formulation buffer using tangential flow filtration.
The present disclosure describes the methodologies to purify Afucosylated AntiHer2 antibody produced from CHO S cells transformed with CRISPR/Cas mediated FUT8 knock out cell line using Protein A Capture Chromatography and other purification technologies.
Clarification:
About 1410 ml of KO CHO cell culture harvest with viability of 79.7% is subjected to centrifugation to remove cells and cell debris. The supernatant with the turbidity of 22.5 NTU is collected carefully and passed though ZetaPlus BC25 depth filter (25 cm2 Area) to remove host cell proteins and suspended solid particles. The secondary clarified sample is filtered using 0.22 μm filter (47 mm Area) and the filtrate collected in a sterile bottle to load on Protein A affinity column.
Affinity Chromatography:
12 miligram of Carboxypeptidase B is added to around 1400 milliliter of clarified sample (2400 miligram) and incubated at 37° C. for 1 hour to remove C-terminal lysine variants. After incubation, the clarified sample is loaded onto pre-equilibrated MabSelectSure ˜70 ml XK 26/20 column in equilibration buffer (30 mM Sodium Phosphate buffer, 150 mM NaCl pH 7.20±0.20) at 8.8 ml/min flow rate. Around 1550 ml of flow through is collected. Next Passage is through equilibration buffer to remove loosely bound impurities and host cell proteins. Next Passage is through 1 column volume of high salt ish buffer (30 mM Sodium Phosphate buffer, 1 M NaCl pH 7.20±0.20) followed by 3 column volume of low pH Ish buffer (30 mM Sodium Phosphate buffer, 50 mM NaCl pH 6.0±0.20) to remove impurities. Bound proteins are eluted by elution buffer (30 mM Phosphate buffer, 50 mM NaCl pH 3.0±0.20).
Virus Inactivation:
The pH of Protein A elution is adjusted to 3.0±0.2 for virus inactivation and the sample is incubated for 30 minutes at 24° C.±2° C. The sample is then neutralized to pH 6.0±0.2 using 1M Tris to purify further.
Anion Exchange Chromatography:
After virus inactivation, sample at pH 6.0 is diluted using purified water to reduce the conductivity and again adjusted the pH to 6.±0.2. Diluted sample (˜1000 milliliter) is loaded on pre-equilibrated XK16/40 Q Sepharose 6 FF column, collected flowthrough, where the product eluted in flow through since this chromatography step is set at negative binding mode. This step is followed by elution of bound impurities, host cell DNA and truncated species by elution buffer (30 mM Sodium Phosphate buffer, 1 M NaCl pH 7.20±0.20) in single step (100% of Concentration B). The elution is collected as single fraction.
Cation Exchange Chromatography:
Flow Through from AEX chromatography is loaded on pre-equilibrated XK26/40 Capto S Column in Equilibration buffer (50 mM Histidine buffer pH 6.0, ˜2.23 milli siemens). Flow through sample is collected. Next passage is through equilibration buffer of ˜2 column volume to remove traces of impurities. Elution is started from 0 to 20% Concentration B (Elution buffer: 50 mM Histidine buffer pH 6.0, ˜14.50 milli siemens) in single step (˜2.5 mS to ˜7.30 milli siemens) followed by 20 to 22.5% in single step (˜7.30 milli siemens to ˜7.50 milli siemens). Gradient elution is started from 22.5% to 80% in gradient (20 column volume gradient length). The fractions are collected of about 6 ml size. 100% Concentration B is then passed followed by high salt elution buffer (30 mM Sodium Phosphate buffer pH 7.20 with 1 M NaCl).
Data Analysis
The secondary clarified sample reveals turbidity less than 5 nephlometric turbidometry unit (NTU) after clarification by depth filtration. The Protein A Affinity Chromatography profile (
AEX Chromatography (
In CEX Chromatography step (
Multiple analytical methods are used to characterize the Afucosylated AntiHer2 Antibody product produced from the CHO S cell line transformed with CRISPR/Cas system targeting FUT8 gene knock out.
Following methodologies are used for characterization of Afucosylated Anti Her2 Antibody product.
Isoelectric focusing methods are widely applied for the separation of proteins based on their charge difference. Isoelectric focusing (IEF) is an electrophoretic technique for separation of proteins based on their isoelectric point (pI). The principle of isoelectric focusing is that in a pH gradient, the sample components migrate towards the anode or the cathode to the pH values, where their net charges are become zero (their pI).
Method Protocol
The samples used in the study for identification of charge variants are Trastuzumab (obtained from Roche), and afucosylated Anti-HER2 antibody developed from CHO S cell line transfected with CRISPR/Cas system targeting FUT8 gene. All samples are diluted and kept at a concentration of 1 mg/ml using ultra-pure milli Q water as diluent and 20 μg protein samples are loaded on to IEF sample applicator. Standard IEF conditions are used. 1M sodium hydroxide and 1M ortho-phosphoric acid are used as the cathode buffer and anode buffer respectively. Gel mixture used in this study is 5%. After the run, the gels are fixed with 2% trichloroacetic acid for 30 minutes. Coomassie brilliant blue solution is used for staining solution.
2. SDS Page Analysis
Antibody samples are analyzed by SDS-PAGE under reducing and non-reducing conditions using 10% (v/v) resolving gels. Tris Glycine SDS PAGE is carried out with the supplier's protocol (Biorad). Polyacrylamide gels are prepared from acrylamide and bis-acrylamide stock solution (29:1), Stacking buffer (1M Tris-HCl pH 6.8), Resolving buffer (2M Tris-HCl pH 8.8), 10% SDS, APS and TEMED as the catalyst. Stacking gel is used at 4% concentration.
5 μg of the antibody samples are mixed with 5× sample buffer (1M Tris-HCl pH 6.8, 10% SDS, β-mercaptoethanol (in case of reducing condition), 50% glycerol, 2% (w/v) bromophenol blue). The mixture is then heated for 5 min and loaded on to the gel. The SDS-PAGE of antibody samples is carried out at a voltage of 150 volts for 60 minutes. Protein Marker Standard (Biorad) with molecular weight ranged between 10-250 kDa is used. Protein staining is performed with Coomasie brilliant blue 0.2% (w/v). The stained gels are then washed with a mixture of methanol:acetic acid solution (40%: 10%) and then photographed by Canon digital camera to visualize the protein bands.
3. Immunoblotting
Antibody samples that are resolved in SDS-polyacrylamide gels are transferred to a PVDF membrane. After blocking with 1% (w/v) BSA in 0.05% Tween/PBS (PBST) for 30 minutes, the membrane is ished 1 time with PBST for 10 min and incubated further with a 1:5000 dilution of Goat anti-mouse IgG secondary antibody HRP conjugated antibody for 30 minutes at RT and detected using DAB substrate.
4. Quantification of Afucosylated Anti-Her2 Antibody by Her2 Binding ELISA
Anti-HER2 IgG1 Trastuzumab, a humanized anti-human epidermal growth factor receptor-2 (HER2) monoclonal antibody, is used for treatment of metastatic breast cancer patients overexpressing HER2 on tumor cells. Quantification of anti-HER2 monoclonal antibody can be carried out using different methods, for example, an ELISA based method. Human Her2/ErbB2 Protein (Human Her2, His Tag) Thr 23-Thr 652 (Accession #AAA75493) produced in human 293 cells (HEK293) at ACROBiosystems is used to coat on the 96 well plates. The receptor is Human Her2; His Tag is fused with a poly-histidine tag at the C-terminus, and has a calculated MW of 70.2 kDa. The predicted N-terminus is Thr 23. DTT-reduced Protein migrates as 110-115 kDa in SDS-PAGE due to glycosylation
Method Protocol
100 μl HER 2 protein (Acro bio systems, US) is coated onto the 96 well plates (cell culture delta surface treated plates) at a concentration of 1 μg/ml (diluted with 100 mM bicarbonate buffer, pH 9.6). Plate is kept for overnight incubation at 2-4° C. followed by wash with wash buffer (PBS+0.05% Tween 20) 2×200 μl. Nonspecific binding is blocked with blocking buffer (wash buffer+0.1% BSA) for 2 hours (200 μl) and washed with wash buffer twice. 100 μl of standard/samples are added and kept for incubation for 1.5 hours. Samples and standards are serially diluted in two folds with the range of 1.953-250 ng/ml. Secondary antibody is added to the plates and kept for incubation for 1 hour. TMB is used as a substrate. And the plates are read at 450 nm with 620 nm as background. All the measurements are done in duplicates. The sample/standard concentrations measured are determined using the four parameter nonlinear regression curve fitting program to generate the binding curves (Gen 5).
5. Cell Based Binding ELISA
Cell based ELISA is one of the method to estimate the binding potency of specific target molecule with specific receptor expressing cells. SKBR3 and BT474 cell lines (obtained from ATCC) over express HER2 receptors on cell surface. The cell lines are used to evaluate the binding potency of anti-HER2 antibody. Below protocol is followed in cell based ELISA assay,
Method Protocol
Two cell lines are used in this study, namely SKBR3 and BT474 cells. SKBR3 and BT474 cells are passaged and maintained in the RPMI 1640 and DMEM medium respectively, supplemented with 10% FBS at 37° C. and 5% CO2. The cells are plated at a density of 5000 cells per well (100 μL) in 96 well plate delta treated surface and allowed to adhere overnight at 37° C. and 5% CO2. The following day the cells are washed with 2×200 μL cold PBS to remove the non-adherent particles. Next the cells are fixed with 100 μL of 10% formalin and incubated for 10 minutes at room temperature. The fixing solution is removed and washed with 2×200 μL of cold PBS and the plates are blocked with 200 μL of blocking solution [PBS+Tween 20 (0.05%)+0.1% BSA] for one hour. The plates are washed with 2×200 μL of wash buffer (PBS+0.05% Tween 20) to remove the excess blocking solution. 100 μL of sample/standard added in each well and incubated for 30 minutes in plate shaker. The plates are ished with 2×200 μL of ish buffer (PBS+0.05% Tween 20) to remove the non-specific interaction. 100 μL of secondary antibody (anti human Fc IgG1) is added and incubated for 1 hour. 100 μL of TMB substrate is added and the color developed is read at 450 nm with 620 nm as background. All the samples/standard is done in duplicates. The sample/standard concentrations measured are determined using the four parameter nonlinear regression curve fitting program to generate the binding curves (Gen 5). Table 13 shows the Sample/standard concentrations used for both cell lines (BT474 and SKBR3)
6. Protein Concentration Measurements
The protein concentration is determined by Protein-A affinity chromatography using MAbPac Protein-A column (12 μm; 4×35 mm) on an Shimadzu Prominence-i HPLC. The mobile phase is Dulbecco's Phosphate Buffered Saline at a flow rate of 2.5 mL/min. The monoclonal antibody is eluted with 3% glacial acetic acid gradient. The column temperature is 25° C. and detection is at 280 nm. Chromatograms are integrated using Lab Solution Software and the concentration is estimated using standard curve plotted with Area under the peak against concentration of known standard.
7. Ion Exchange Chromatography
Charge variants are separated on a 4.0×250 mm Dionex ProPac cation exchange (IEC) column WCX-10. The mobile phase-A is 10 mM MES buffer at pH 6.8 and mobile phase-B is 10 mM IVIES buffer at pH 6.8 with 250 mM Sodium Chloride and the flow rate is 1.0 mL/min with run time of 21 min. The column temperature is 30° C. and detection is at 280 nm. Chromatograms are integrated using Lab Solution Software and relative percent peak area obtained.
8. Enzymatic N-Glycan Release and Labeling by Glycoworks RapiFluor-Ms N-Glycan Kit
The sample preparation for N-Glycan profiling is performed using GlycoWorks RapiFluor-MS N-Glycan Kit (Waters, Milford, Mass., USA). The kit has four steps.
STEP 1: Rapid Deglycosylation,
STEP 2: Rapid Labeling of Glycosylamines,
STEP 3: HILIC Clean-Up of Labeled Glycosylamines,
STEP 4: Preparing Labeled Glycan for HILIC-FLR UPLC analysis.
9. UPLC-Fluorescence HILIC N-Glycan Profiling
2-AB derivatised N-glycans are separated by ultra-performance liquid chromatography (UPLC) with fluorescence detection on a Waters Acquity™ UPLC H-Class instrument consisting of a Quaternary solvent manager, sample manager and fluorescence detector under the control of Empower 3 chromatography workstation software (Waters, Milford, Mass., USA). The HILIC separations are performed using Thermo Accucore-150-Amide-HILIC column, 100×2.1 mm i.d., 2.6 μm particles, using a linear gradient of 80-60% acetonitrile at 1 mL/min in 26 minutes. Solvent A is 50 mM Ammonium Formate which is adjusted to pH4.4 with ammonia solution and Solvent B is acetonitrile. The injection volume of sample is 10 μL. Samples are maintained at 5° C. prior to injection and the column temperature is 60° C. The fluorescence detection excitation/emission wavelengths are ex=265 nm and em=425 nm, respectively.
10. Flow Cytometry Analysis
Specific binding of the mAb samples to HER2 antigen is investigated using indirect flow cytometry method. BT-474 cell line is obtained from ATCC which expresses high levels of HER2 receptors on its cell surface. BT-474 cells are cultured in DMEM/F12 Growth medium containing 10% FBS.
For the experiment, cells are trypsinized with 0.25% Trypsin-EDTA. After centrifugation at 1000 rpm for 3 min, pellet is suspended in 2% BSA in DPBS buffer to achieve a desired concentration of 0.1 million cells/ml. Then the cells are treated with varying concentration viz., 0.05 mg/ml, 0.025 mg/ml and 0.00125 mg/ml of biotin labelled Innovator Herclon and Anti-HER2 Antibody. Biotinylation of the antibody samples are performed using EZ SulfoLink Biotinylation kit (Thermo Scientific).
Volume of the antibody samples and cells are maintained at ratio of 1:1 and incubated at RT for 1 hr. 20 μl of 0.1 mg/ml Strep-FITC is added to the reaction mixture and incubated on ice bath for 30 min. All treated samples are analyzed with BD Accuri Flow Cytometer (BD Biosciences).
11. SPR Binding Kinetics
Binding kinetics studies for different charge variants are performed by Surface Plasmon Resonance (SPR) using a Biacore 3000 instrument. Variants and main species are analyzed for HER-2 binding kinetics and Fc gamma RIII (CD16a) binding kinetics. HER2 and CD16a ligands are immobilized on CM5 chip (GE Healthcare) using standard amine coupling procedure described by the manufacturer protocol.
12. Her2 Binding Kinetics
HER2, also known as neu and c-erbB2, is a member of the epidermal growth factor (EGF) receptor or HER family of tyrosine kinase receptors that also includes HER1 (EGFR/c-erbB1), HER3 (c-erbB3) and HER4 (c-erbB4). Overexpression of HER2 is frequently observed in breast and ovarian cancers and is associated with an unfavorable prognosis.
HER2 ecto-domine receptor (Sino Biologicals) is diluted to 2 ug/mL in Acetate buffer pH5.0 and the amine coupling protocol followed as mentioned in GE amine coupling procedure. The ligand immobilization is aimed for 500 RU with flow cell 2 (FC2) and flow cell 1 (FC1) serves as background without any ligand. All anti-HER2 Mabs are diluted with the running buffer (EMS P+1× buffer from GE Healthcare) in different concentrations from 100 nM to 6.25 nM with 2 fold dilutions. These individual concentrations are injected in duplicate for 5 minutes of association kinetics and then only running buffer is passed for 15 minutes of dissociation kinetics and the surface is regenerated with 30 sec injection of glycine buffer pH 1.5, 90 sec of glycine buffer pH 2.0 and with running buffer stabilization time of 1 minute.
Therapeutic antibodies are typically molecules of the IgG class and comprise an antigen-binding fragment (Fab) that engages the tumor cell antigen and a crystalline fragment (Fc) that binds to Fc gamma receptor (FcγR) on an effector cell such as a natural killer (NK) cell, monocyte, or macrophage. Fcγ receptors are well known molecule of the Immunoglobulin superfamily familiar to be involved in antibody-dependent cellular cytotoxicity (ADCC). The contribution of IgG Fc-mediated effector functions in ADCC has motivated the efforts being made to enhance interactions with Fc gamma receptors (FcγRs) for the development and manufacturing of therapeutic antibodies.
FcγRIII (CD16a V158) valine variant is used in this study, Ectodomain of CD16a receptor is procured from Acro bio systems, USA. All Afucosylated anti-HER2 antibody is serially diluted with the running buffer (HBS EP+1× buffer from GE Healthcare) in different concentrations from 0.833 μM to 0.05 μM (2 fold dilution). Analyte kinetics is determined using the Langmuir binding kinetics (1:1 binding model).
13. Antiproliferation Assay
BT474 cell line is used in the study and the cells are propagated and maintained using DMEM medium supplemented with 10% FBS at 37° C. and 5% CO2. 2000 cells per well is used in the study. The sample/standard dilutions are performed in DMEM assay media (containing 2% FBS). 100 μL of sample/standard is added in 96 well plates along with 100 μL of cells and kept for 6 days incubation at 37° C., 5% CO2. After 6 days, 30 μL of Alamar blue is added and kept for 6 hours incubation. Then the plates are read at excitation wavelength of 530 nm and excitation wavelength of 590 nm. Four point nonlinear regression curve is obtained from PLA software. Below Table 14 shows the Sample/standard concentrations used for BT474 Antiproliferation assay
Isolation of Effector Cells
Peripheral blood mononuclear cells (PBMC) are isolated by ficoll gradient centrifugation from heparinized blood. Cells are ished in DPBS (1×) and then again in complete media and then adjusted to the cell concentrations required to obtain the optimal effector:target (E:T) ratios.
14. ADCC Assay
BT-474 target cells (10,000 cells per well) are mixed with afucosylated monoclonal antibody samples in RPMI 1640 assay medium and distributed in 96 well plate. After 30 min of incubation, effector cells are added at a ratio of 1:20 (BT474 cells to effector cells) to cell and drug reaction mixture. Assay plate is incubated in CO2 incubator at 370 C for 7 hours. The dead cell protease levels (indicator of cell death) released by the cells are measured using Cytotoxglo assay kit (Promega). After incubation, plates are kept on shaker for 30 minutes for uniform mixing. 20 μl of lysis buffer added to the control well for determining maximum cytotoxicity level. 20 μl of the Cytotoxglo reagent is added to each sample and control wells and incubated for 15 minutes at room temperature. Luminescence is measured using Biotek ELISA reader. Percentage Cytotoxicity values are calculated as follows: [(Test Sample−Low Control)/(Maximum Control−Low Control)]×100. Low control represents spontaneous cell death and Maximum control represents complete cell death.
Results and Discussion
The prospect of the afucosylated anti Her2 antibody are tested and characterized with the series of analytical experiments. Afucosylated AntiHer2 antibody is compared with Trastuzumab to compare biological activity.
IEF
IEF is performed to identify the charge variants (acidic and basic species) present. Four bands are clearly visible in innovator. The main band (
SDS Page
Under non-reducing conditions, a single major band corresponding to a molecular weight (MW) of 150 kDa is observed indicating the intact antibody (
Western Blotting
Western Blotting analysis is performed for the specific detection of the antibody samples. Trastuzumab is used for the evaluation of and comparison of protein patterns. The non-reduced antibody samples show a single distinct band when probed with secondary antibody. However, in the case of reduced samples, only a single band of heavy chain (around 50 kDa) is detected and not the light chain of the monoclonal antibody. This is because the secondary antibody that is used is specific only against Fc region of the antibody and not the light chain region. In
Anti Her2 Binding ELISA
Serially diluted different concentrations (60 ng/mL to 0.48 ng/mL) of Trastuzumab and Afucosylated anti-HER2 antibodies are analyzed for binding efficiency in HER2 coated ELISA plates. Observed result (
Cell Based ELISA
Cell based ELISA (
Protein Concentration Measurements
The prototype Protein A column is used to measure the monoclonal antibody titer from harvested cell culture (HCC) till Drug Product stage. In the chromatogram (
Ion Exchange Chromatography
The peaks of the ion exchange profiles are typically denoted into three distinct components. Early and late-eluting peaks are called acidic and basic variants, respectively. The most abundant peak is designated as the main peak. The ProPac WCX-10 column provides excellent peak efficiencies and high resolution for acidic and basic variant analysis of monoclonal antibodies as shown in
HILIC N-Glycan Profiling
One of the important quality attributes for therapeutic monoclonal antibodies is Glycosylation profile. To check the glycan distributions in Trastuzumab and Afucosylated Anti-Her2 Antibody products are analyzed by HILIC N-glycan profiling method. The major glycan such as G0, G1, G1′, G2, G0F, G1F, G1F′ and G2F are listed in the following Table 17 for Trastuzumab and Afucosylated Anti-Her2 Antibody product. Overall percentage of afucosylated glycan for Trastuzumab and Afucosylated Anti-Her2 Antibody samples are 8.95% and 100%, respectively. Glycan profile for Trastuzumab and Afucosylated Anti-Her2 Antibody is shown in
Flow Cytometry
This method provides a means of measuring the ligand binding affinity of Her2 receptors expressed on BT-474, a breast cancer cell line through a flow cytometry-based assay. The level of receptor binding by the biotinylated antibody samples are assessed by probing with FITC labeled Streptavidin and measuring the level of fluorescence using a flow cytometer. The Afucosylated Anti-Her2 Antibody product shows a shift in their peak towards right side in comparison with ‘unstained cells only’ control indicating the specificity of these samples towards Her2 receptors on breast cancer cells. The overlay data (
SPR Data
SPR data (
Antiproliferation Assay
The antiproliferation assay (
Antibody Dependent Cellular Cytotoxicity (ADCC)Assay with PBMC Samples
Large numbers of tumor antigenic targets are currently being studied for possible monoclonal antibody mediated treatment. Traditional monoclonal antibody therapeutics is developed in CHO S cell expression systems, which has an intrinsic fucosylation activity through fucose biosynthetic pathway.
The methodologies and CRISPR/Cas system described in this disclosure therefore bring novel product development approaches where any monoclonal antibody product which has poor biological efficiency due to fucosylation is significantly improved by developing an afucosylated version of the monoclonal antibody. The novel approaches described here, open up new product development routes to develop afucosylated monoclonal antibodies from existing high expression eukaryotic protein expression platforms.
| Number | Date | Country | Kind |
|---|---|---|---|
| 194/CHE/2015 | May 2015 | IN | national |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/IB2016/052774 | 5/13/2016 | WO | 00 |
| Publishing Document | Publishing Date | Country | Kind |
|---|---|---|---|
| WO2016/181357 | 11/17/2016 | WO | A |
| Number | Name | Date | Kind |
|---|---|---|---|
| 20190112358 | Prasad | Apr 2019 | A1 |
| Number | Date | Country |
|---|---|---|
| 1705251 | Sep 2006 | EP |
| WO2015010114 | Jan 2015 | WO |
| WO2016075662 | May 2016 | WO |
| WO2016181357 | Nov 2016 | WO |
| Entry |
|---|
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| Carlotta Ronda et al., “Accelerating genome editing in CHO cells using CRISPR Cas9 and CRISPy, a web-based target finding tool”, Biotechnology and Bioengineering, vol. 111, No. 8, Aug. 22, 2014, pp. 1604-1616. |
| International Search Report and Written Opinion dated Sep. 28, 2016 for Int'l. Appln. No. PCT/IB2016/052774 (20 pgs.). |
| International Preliminary Report on Patentability dated Sep. 5, 2017 for Int'l. Appln. No. PCT/IB2016/052774 (28 pgs.). |
| Written Opinion dated Jun. 16, 2017 for Int'l. Appln. No. PCT/IB2016/052774 (14 pgs.). |
| Number | Date | Country | |
|---|---|---|---|
| 20180171028 A1 | Jun 2018 | US |
