Patent Application
20240010752
This application claims the benefit of priority of Singapore patent application No. 10202009841Y, filed 2 Oct. 2020, the contents of it being hereby incorporated by reference in its entirety for all purposes.
The present invention generally relates to the field of biotechnology in particular to a nucleotide based expression system. In particular, the present invention relates to an expression system for antigen binding molecules for dual screening purposes.
Antigen binding molecules such as antibodies are one of the fastest growing class of biotherapeutic molecules. One such example of antibodies is immunoglobulin G (IgG), which consists of two heavy chain (HC) and two light chain (LC) polypeptides.
Developing therapeutic antibodies remains a technically challenging, extremely time-consuming and costly process. The process of developing the correct antibodies is often unsuccessful as the antibodies might be ineffective or simply comprise the incorrect sequences, therefore resulting in a high attrition rate and a high risk of failure. The process of developing therapeutic antibodies starts with discovery of antibodies with specific binding affinity and desirable functionalities, followed by production of antibodies in mammalian cells to provide enough high-quality materials for preclinical and clinical studies before commercialization. Antibody discovery has relied on either in vivo immunization of animals or in vitro display-based technologies, such as phage display, bacterial display and mammalian cell display. Chinese hamster ovary cells (CHO) are the dominant mammalian cells for antibody production due to their capability to perform proper assembly of complexes and human-like glycosylation. The transition from the antibody development to the production, which involves re-cloning of antibody genes and changes in antibody format and production host cells, not only results in increased time and cost, but also failure of many antibody candidates.
In view of the above, there is a need to provide an expression system that permits screening of proteins, to enable the production of antigen binding molecules such as antibodies, and to keep the production of incorrectly processed antibody species to the minimum.
In one aspect, the present disclosure refers to an expression system for an antigen binding molecule, wherein the antigen binding molecule is either secretable or membrane-bound, comprising:
In another aspect, the present disclosure refers to an expression system for an antigen binding molecule, wherein the antigen binding molecule is either secretable or membrane-bound, comprising:
In another aspect, the present disclosure refers to a vector comprising the expression system as disclosed herein.
In another aspect, the present disclosure refers to a host cell comprising the expression system or the vector as disclosed herein.
In another aspect, the present disclosure refers to a kit comprising the expression system, the vector, or the host cell as disclosed herein.
In yet another aspect, the present disclosure refers to a method for detecting the presence of one or more secreted antibodies and/or one or more surface-bound antibodies in a sample, the method comprising:
In yet another aspect, the present disclosure refers to a method for detecting the presence of one or more secreted antibodies and/or one or more surface-bound antibodies in a sample, the method comprising:
In yet another aspect, the present disclosure refers to the expression system, the vector, the host cell or the kit as disclosed herein for use in screening antibody libraries or antibody production
The invention will be better understood with reference to the detailed description when considered in conjunction with the non-limiting examples and the accompanying drawings, in which:
Secreted recombinant IgGs are practical for production purposes, while cell-surface displayed antibodies are useful for screening/ sorting via FACS. The expression systems in use today show either high levels of membrane antibodies and little to no secretable antibodies, or vice versa, low levels of membrane antibodies and high levels secretable antibodies. Such unbalanced levels of expression of membrane and secretable antibodies cannot satisfy the need for a smooth transition from antibody discovery to production, and ultimately leads to an inefficient system for antibody screening and/or production.
In view of the above problems, there is a need to provide a more effective expression system that permits simultaneous cell surface display and secretion of the same protein at optimal ratios through the use of engineered peptides with different cleavage sites with different cleavage efficiencies. Such an expression system can be used for dual screening purpose and for more efficient antibody production. The inventors of the present disclosure have found expression systems for an antigen binding molecule, wherein the antigen binding molecule is either secretable or membrane-bound. The term “expression system” used herein refers to DNA construct that are designed to produce a protein, or an RNA (ribonucleic acid), either inside or outside a cell. The expression system can exist on its own or be incorporated into a vector. Exemplary expression systems include, but are not limited to, mammalian expression system, insect expression system, yeast expression system, bacteria expression system, algae expression system or a cell-free expression system. The expression system can comprise the following components including, but not limited to, a promoter, one or more genes of interest, one or more identification tags. In one example, expression system can be used for dual screening purpose to screen for the expression levels of the secretable and/or membrane-bound antigen binding molecule.
The term “antigen binding molecule” used herein refers to an antibody, an antibody fragment or other protein construct, such as a domain.
In one example, the present disclosure provides an expression system for an antigen binding molecule, wherein the antigen binding molecule is either secretable or membrane-bound, comprising:
In another example, the present disclosure provides an expression system for an antigen binding molecule, wherein the antigen binding molecule is either secretable or membrane-bound, comprising:
The term “polynucleotide” used herein refers to a nucleotide sequence that encodes for the product of interest, or a fragment, derivative, mutein, or variant thereof. A polynucleotide includes DNA molecules (e.g., cDNA or genomic DNA), RNA molecules (e.g., mRNA), analogs of the DNA or RNA generated using nucleotide analogs (e.g., peptide nucleic acids and non-naturally occurring nucleotide analogs), and hybrids thereof. The nucleic acid molecule can be single-stranded or double-stranded. The nomenclature of the polynucleotide is dependent on the product of interest that the polynucleotide it encodes. For example, a first or second antigen binding polynucleotide is the nucleotide sequence that encodes the first or second part of the antigen binding molecule as disclosed herein; a first or second cleavage polynucleotide is the nucleotide sequence that encodes the first or second cleavage site as disclosed herein; an anchor polynucleotide is the nucleotide sequence that encodes a membrane anchor polypeptide as disclosed herein. The same nomenclature would apply to any other polynucleotides as disclosed herein.
The first antigen binding polynucleotide can be at least 15 nucleotides in length. In another example, the first antigen binding polynucleotide can be, but is not limited to, a length of about 15 to 1500 nucleotides, or about 50, about 100, about 200, about 300, about 400, about 500, about 600, about 700, about 800, about 900, about 1000, about 1100, about 1200, about 1300, about 1400, or about 1500 nucleotides in length.
The first antigen binding polynucleotide encodes for a first part of the antigen binding molecule. In one example, the first part of the antigen binding molecule is an antibody or a fragment thereof. In another example, the first part of the antigen binding molecule is an antibody heavy chain. In another example, the first part of the antigen binding molecule is an antibody light chain.
The first antigen binding molecule can be at least 5 amino acids in length. In another example, the first antigen binding molecule can be, but is not limited to, a length of about 5 to 500 amino acids, or about 50, about 100, about 150, about 200, about 250, about 300, about 350, about 400, about 450, or about 500 amino acid residues in length.
The expression system of the present disclosure can further comprise a second antigen binding polynucleotide encoding a second part of the antigen binding molecule.
The second antigen binding polynucleotide can be at least 15 nucleotides in length. In another example, the second binding polynucleotide can be, but is not limited to, a length of about 15 to 700 nucleotides, or about 50, about 100, about 200, about 300, about 400, about 500, about 600, or about 700 nucleotides in length.
The second antigen binding polynucleotide encodes for a second part of the antigen binding molecule. In one example, the second part of the antigen binding molecule is an antibody or a fragment thereof. In another example, the second part of the antigen binding molecule is an antibody light chain. In another example, the second part of the antigen binding molecule is an antibody heavy chain.
The second antigen binding molecule can be at least 5 amino acids in length. In another example, the second antigen binding molecule can be, but is not limited to, a length of about 5 to 250 amino acids, or about 50, about 100, about 150, about 200 or about 250 amino acid residues in length.
Where the expression system encodes more than one cleavage site, the first cleavage polynucleotide of the expression system can be at least 9 nucleotides in length. In another example, the first cleavage polynucleotide can be, but is not limited to, a length of about 9 to 30 nucleotides, or about 10, about 15, about 20, about 25 or about 30 nucleotides in length. In a preferred example, the first cleavage polynucleotide is 12 nucleotides in length.
The first cleavage polynucleotide encodes for a first cleavage site. The first cleavage site can be at least 3 amino acids in length. In another example, the first cleavage site can be, but is not limited to, a length of about 3 to 10 amino acids, or about 3, 4, 5, 6, 7, 8, 9 or 10 amino acid residues in length. In a preferred example, the first cleavage site is 4 amino acids in length.
In one example, the first cleavage site is a minimal Furin cleavage consensus sequence. The term “Furin” used herein refers to a ubiquitous subtilisin-like proprotein convertase which cleaves proteins containing its recognition site. The terms not limiting to “Furin consensus sequence”, “minimal cleavage site”, “minimal Furin cleavage consensus sequence” or “Furin recognition site” as used herein refer to an amino acid sequence of RXKR (SEQ ID NO: 1) or RXRR (SEQ ID NO: 2), wherein X can be any amino acids. Furin recognises a protein that comprises a Furin consensus sequence, which leads to the cleavage of the protein that comprises the Furin consensus sequence. The cleavage can take place in, for example, the Golgi.
In one example, the Furin consensus sequence is selected from a group consisting of RRKR (SEQ ID NO: 3), RRRR (SEQ ID NO: 4), RSKR (SEQ ID NO: 5), RSRR (SEQ ID NO: 6), RKKR (SEQ ID NO: 7), RKRR (SEQ ID NO: 8), RQKR (SEQ ID NO: 9), RQRR (SEQ ID NO: 10), RTKR (SEQ ID NO: 11), RTRR (SEQ ID NO: 12), REKR (SEQ ID NO: 13), RERR (SEQ ID NO: 14), RDKR (SEQ ID NO: 15), RDRR (SEQ ID NO: 16), RHKR (SEQ ID NO: 17), RHRR (SEQ ID NO: 18), RFKR (SEQ ID NO: 19), RFRR (SEQ ID NO: 20), RAKR (SEQ ID NO: 21), RARR (SEQ ID NO: 22), RNKR (SEQ ID NO: 23), RNRR (SEQ ID NO: 24), RCKR (SEQ ID NO: RCRR (SEQ ID NO: 26), RGKR (SEQ ID NO: 27), RGRR (SEQ ID NO: 28), RIKR (SEQ ID NO: 29), RIRR (SEQ ID NO: 30), RLKR (SEQ ID NO: 31), RLRR (SEQ ID NO: 32), RMKR (SEQ ID NO: 33), RMRR (SEQ ID NO: 34), RPKR (SEQ ID NO: 35), RPRR (SEQ ID NO: 36), RWKR (SEQ ID NO: 37), RWRR (SEQ ID NO: 38), RYKR (SEQ ID NO: 39), RYRR (SEQ ID NO: 40), RVKR (SEQ ID NO: 41) and RVRR (SEQ ID NO: 42). In a preferred example, the Furin consensus sequence is RRKR (SEQ ID NO: 3).
The second cleavage polynucleotide of the expression system can be at least 10 nucleotides in length. In another example, the first cleavage polynucleotide can be, but is not limited to, a length of about 10 to 200 nucleotides, or about 10, about 20, about 30, about 40, about 50, about 60, about 70, about 80, about 90, about 100, about 110, about 120, about 130, about 140, about 150, about 160, about 170, about 180, about 190 or about 200 nucleotides in length. In a preferred example, the second cleavage polynucleotide is 15 nucleotides in length. In another preferred example, the second cleavage polynucleotide is 57 nucleotides in length.
The second cleavage polynucleotide encodes for a second cleavage site. The second cleavage site is a self-processing cleavage site. The term “self-processing cleavage site” as used herein refers to a peptide sequence that has self-cleaving or self-processing ability, wherein the peptide does not require an external molecule or enzyme such as a protease to cleave the peptide sequence. The second cleavage site can be at least 4 amino acids in length. In another example, the first cleavage site can be, but is not limited to, a length of about 4 to 50 amino acids, about 10 to 20 amino acids, about 20 to 30 amino acids, about 30 to 40 amino acids, about 40 to 50 amino acids, or about 4, 5, 6, 7, 8, 9, 10, 11, 12. 13, 14, 15, 16, 17, 18, 19, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 46, 47, 48, 49 or 50 amino acid residues in length. In one example, the first cleavage site is amino acids in length. In another example, the first cleavage site is 19 amino acids in length.
In one example, the self-processing second cleavage site is a 2A polypeptide or a fragment thereof. The terms not limiting to “2A polypeptide”, “2A peptide”, “2A protein” used herein refer to a peptide that mediates “self-cleavage” or “self-processing” of proteins during translation in eukaryotic cells. The 2A polypeptide is, for example, usually 18-25 amino-acid (aa) in length, and originates from viruses. 2A polypeptide is capable of self-cleaving, which occurs co-translationally between, for example, between the last two amino acids, glycine and proline. In one example, the 2A polypeptide or a fragment thereof is selected from a group consisting of P2A, F2A, E2A and T2A, or a fragment thereof. F2A is a 2A peptide derived from foot-and-mouth disease virus; E2A is a 2A peptide derived from equine rhinitis A virus; T2A is a 2A peptide derived from Thosea asigna virus; P2A is a 2A peptide derived from porcine teschovirus-1. In another example, the 2A polypeptide or a fragment thereof is a P2A polypeptide or a fragment thereof. In one example, the P2A polypeptide is ATNFSLLKQAGDVEENPGP (SEQ ID NO: 43). In another example, the F2A polypeptide is APVKQTLNFDLLKLAGDVESNPGP (SEQ ID NO: 44). In another example, the E2A polypeptide is QCTNYALLKLAGDVESNPGP (SEQ ID NO: 45). In another example, the T2A polypeptide is EGRGSLLTCGDVEENPGP (SEQ ID NO: 46). The corresponding nucleotide sequences are shown in Table 1 below.
Where the expression system encodes one cleavage site, the expression system comprises a cleavage polynucleotide encoding a cleavage site comprising a Furin consensus sequence as disclosed herein, and a 2A polypeptide fragment thereof. It would also be understood that the 2A polypeptide fragment thereof refers to a section of the 2A polypeptide as disclosed herein. In one example, the 2A polypeptide fragment thereof comprises a section of at least 3 amino acids from the 2A polypeptide. In another example, the 2A polypeptide fragment thereof comprises a section of, but is not limited to, about 3-10 amino acids, or about 3, 4, 5, 6, 7, 8, 9, or 10 amino acids from the 2A polypeptide. In another example, the 2A polypeptide fragment thereof is selected from a group consisting of P2A, F2A, E2A and T2A fragment thereof. In another example, the 2A polypeptide fragment thereof is a P2A polypeptide fragment thereof.
In one example, the 2A polypeptide fragment thereof can be the first 3-10 amino acids of the 2A polypeptide as disclosed herein, or the last 3-10 amino acids of the 2A polypeptide as disclosed herein. In another example, the 2A polypeptide fragment thereof is the first 5 amino acids of a P2A, F2A, E2A or T2A polypeptide. In another example, the P2A polypeptide fragment thereof is the first 5 amino acids of a P2A polypeptide. In another example, the 2A polypeptide fragment thereof is ATNFS (SEQ ID NO: 51).
The 2A polypeptide or the 2A polypeptide fragment thereof encoded by the expression system comprises one or more mutations. An amino acid mutation to, for example, proline or glycine in a 2A polypeptide or the 2A polypeptide fragment thereof can influence the secondary structure of the 2A polypeptide or the 2A polypeptide fragment thereof, by constraining or providing high flexibility to peptide chains, respectively.
In expression system that encodes more than one cleavage site, such mutations can control the cleavage efficiencies of the first cleavage site and the second cleavage site to modulate ratio of production of the secretable antigen binding molecule versus the membrane-bound antigen binding molecule. In one example, the 2A polypeptide or a fragment thereof comprises one or more mutations in amino acid residue 1, 2, 3, 4 or 5. It would be understood that the amino acid residue 1, 2, 3, 4 or 5 refers to the amino acid residue 1, 2, 3, 4 or 5 of the 2A polypeptide or fragment thereof. In one example, amino acid residue 1, 2, 3, 4 or 5 of the 2A polypeptide or the fragment thereof is mutated to any one selected from the group consisting of glycine, proline, alanine, arginine, histidine, lysine, aspartic acid, glutamic acid, serine, threonine, asparagine, glutamine, cysteine, selenocysteine, valine, isoleucine, leucine, methionine, phenylalanine, tyrosine and tryptophan. In another example, amino acid residue 1, 2, 3, 4 or 5 of the 2A polypeptide or fragment thereof is mutated to glycine, proline or alanine.
In one example, the one or more mutations in 2A polypeptide or a fragment thereof is selected from the group consisting of A1P, A1G, T2G, T2P, N3P, F4P, S5P, N3A and F4A. If the mutations occur in the P2A polypeptide, the mutations result in the following P2A polypeptide sequences: PTNFSLLKQAGDVEENPGP (SEQ ID NO: 109), GTNFSLLKQAGDVEENPGP (SEQ ID NO: 101), AGNFSLLKQAGDVEENPGP (SEQ ID NO: 108), APNFSLLKQAGDVEENPGP (SEQ ID NO: 107), ATPFSLLKQAGDVEENPGP (SEQ ID NO: 103), ATNPSLLKQAGDVEENPGP (SEQ ID NO: 105), ATNFPLLKQAGDVEENPGP (SEQ ID NO: 106), ATAFSLLKQAGDVEENPGP (SEQ ID NO: 104) and ATNASLLKQAGDVEENPGP (SEQ ID NO: 102) respectively. In another example, the mutation in 2A polypeptide or a fragment thereof comprises A1P, A1G, T2G or T2P. In another example, the mutation in 2A polypeptide or a fragment thereof is A1P. In another example, the mutation in 2A polypeptide or a fragment thereof is A1G. In another example, the mutation in 2A polypeptide or a fragment thereof is T2G. In another example, the mutation in 2A polypeptide or a fragment thereof is T2P.
In the expression system that encodes one cleavage site, such mutations can control the cleavage efficiencies of the cleavage site to modulate ratio of production of the secretable antigen binding molecule versus the membrane-bound antigen binding molecule. In another example, the 2A polypeptide fragment thereof comprises one or more mutations in amino acid residue 1, 2, 3, 4 or 5. It would be understood that the amino acid residue 1, 2, 3, 4 or 5 refers to the amino acid residue 1, 2, 3, 4 or 5 of the 2A polypeptide fragment thereof. In one example, amino acid residue 1, 2, 3, 4 or 5 of the 2A polypeptide fragment thereof is mutated to any one selected from the group consisting of glycine, proline, alanine, arginine, histidine, lysine, aspartic acid, glutamic acid, serine, threonine, asparagine, glutamine, cysteine, selenocysteine, valine, isoleucine, leucine, methionine, phenylalanine, tyrosine and tryptophan. In another example, amino acid residue 1, 2, 3, 4 or 5 of the 2A polypeptide fragment thereof is mutated to glycine, proline or alanine. In another example, the one or more mutations in 2A polypeptide fragment thereof is selected from the group consisting of A1P, A1G, T2G, T2P, N3P, F4P, S5P, N3A and F4A. The mutations result in the following 2A polypeptide fragment sequences: PTNFS (SEQ ID NO: 60), GTNFS (SEQ ID NO: 52), AGNFS (SEQ ID NO: 59), APNFS (SEQ ID NO: 58), ATPFS (SEQ ID NO: 54), ATNPS (SEQ ID NO: 56), ATNFP (SEQ ID NO: 57), ATAFS (SEQ ID NO: 55) and ATNAS (SEQ ID NO: 53) respectively. In another example, the mutation in 2A polypeptide fragment thereof is A1P. In another example, the mutation in 2A polypeptide fragment thereof is A1G. In another example, the mutation in 2A polypeptide fragment thereof is T2G. In another example, the mutation in 2A polypeptide fragment thereof is T2P.
The expression system can comprise a first and second cleavage polynucleotide encoding a first cleavage site comprising a Furin consensus sequence and a second cleavage site comprising a 2A polypeptide or fragment thereof respectively, and can include different combinations of the Furin consensus sequence and 2A polypeptide or fragment thereof as disclosed herein. In one example, the first and second cleavage sites comprises a sequence selected from a group consisting of RXKRPTNFSLLKQAGDVEENPGP (SEQ ID NO: 139), RXKRGTNFSLLKQAGDVEENPGP (SEQ ID NO: 131), RXKRAGNFSLLKQAGDVEENPGP (SEQ ID NO: 138), RXKRAPNFSLLKQAGDVEENPGP (SEQ ID NO: 137), RXKRATPFSLLKQAGDVEENPGP (SEQ ID NO: 133), RXKRATNPSLLKQAGDVEENPGP (SEQ ID NO: 135), RXKRATNFPLLKQAGDVEENPGP (SEQ ID NO: 136), RXKRATAFSLLKQAGDVEENPGP (SEQ ID NO: 134), RXKRATNASLLKQAGDVEENPGP (SEQ ID NO: 132), RXKRATNFSLLKQAGDVEENPGP (SEQ ID NO: 130), RXRRATNFSLLKQAGDVEENPGP (SEQ ID NO: 140), RXRRGTNFSLLKQAGDVEENPGP (SEQ ID NO: 141), RXRRATNASLLKQAGDVEENPGP (SEQ ID NO: 142), RXRRATPFSLLKQAGDVEENPGP (SEQ ID NO: 143), RXRRATAFSLLKQAGDVEENPGP (SEQ ID NO: 144), RXRRATNPSLLKQAGDVEENPGP (SEQ ID NO: 145), RXRRATNFPLLKQAGDVEENPGP (SEQ ID NO: 146), RXRRAPNFSLLKQAGDVEENPGP (SEQ ID NO: 147), RXRRAGNFSLLKQAGDVEENPGP (SEQ ID NO: 148), and RXRRPTNFSLLKQAGDVEENPGP (SEQ ID NO: 149). In another example, the first and second cleavage sites comprises a sequence selected from a group consisting of RRKRPTNFSLLKQAGDVEENPGP (SEQ ID NO: 119), RRKRGTNFSLLKQAGDVEENPGP (SEQ ID NO: 111), RRKRAGNFSLLKQAGDVEENPGP (SEQ ID NO: 118), RRKRAPNFSLLKQAGDVEENPGP (SEQ ID NO: 117), RRKRATPFSLLKQAGDVEENPGP (SEQ ID NO: 113), RRKRATNPSLLKQAGDVEENPGP (SEQ ID NO: 115), RRKRATNFPLLKQAGDVEENPGP (SEQ ID NO: 116), RRKRATAFSLLKQAGDVEENPGP (SEQ ID NO: 114), RRKRATNASLLKQAGDVEENPGP (SEQ ID NO: 112), RRKRATNFSLLKQAGDVEENPGP (SEQ ID NO: 110), RRRRATNFSLLKQAGDVEENPGP (SEQ ID NO: 120), RRRRGTNFSLLKQAGDVEENPGP (SEQ ID NO: 121), RRRRATNASLLKQAGDVEENPGP (SEQ ID NO: 122), RRRRATPFSLLKQAGDVEENPGP (SEQ ID NO: 123), RRRRATAFSLLKQAGDVEENPGP (SEQ ID NO: 124), RRRRATNPSLLKQAGDVEENPGP (SEQ ID NO: 125), RRRRATNFPLLKQAGDVEENPGP (SEQ ID NO: 126), RRRRAPNFSLLKQAGDVEENPGP (SEQ ID NO: 127), RRRRAGNFSLLKQAGDVEENPGP (SEQ ID NO: 128), and RRRRPTNFSLLKQAGDVEENPGP (SEQ ID NO: 129). Examples of such cleavage sites can be found in the expression system shown in
The expression system can comprise a cleavage polynucleotide encoding a cleavage site comprising a Furin consensus sequence and a 2A polypeptide fragment thereof can include different combinations of the Furin consensus sequence and a 2A polypeptide fragment thereof as disclosed herein. In one example, the cleavage site comprises a sequence selected from a group consisting of RXKRPTNFS (SEQ ID NO: 90), RXKRGTNFS (SEQ ID NO: 82), RXKRAGNFS (SEQ ID NO: 89), RXKRAPNFS (SEQ ID NO: 88), RXKRATPFS (SEQ ID NO: 84), RXKRATNPS (SEQ ID NO: 86), RXKRATNFP (SEQ ID NO: 87), RXKRATAFS (SEQ ID NO: 85), RXKRATNAS (SEQ ID NO: 83), RXKRATNFS (SEQ ID NO: 81), RXRRATNFS (SEQ ID NO: 91), RXRRGTNFS (SEQ ID NO: 92), RXRRATNAS (SEQ ID NO: 93), RXRRATPFS (SEQ ID NO: 94), RXRRATAFS (SEQ ID NO: 95), RXRRATNPS (SEQ ID NO: 96), RXRRATNFP (SEQ ID NO: 97), RXRRAPNFS (SEQ ID NO: 98), RXRRAGNFS (SEQ ID NO: 99), and RXRRPTNFS (SEQ ID NO: 100). In another example, the cleavage site comprises a sequence selected from a group consisting of RRKRPTNFS (SEQ ID NO: 70), RRKRGTNFS (SEQ ID NO: 62), RRKRAGNFS (SEQ ID NO: 69), RRKRAPNFS (SEQ ID NO: 68), RRKRATPFS (SEQ ID NO: 64), RRKRATNPS (SEQ ID NO: 66), RRKRATNFP (SEQ ID NO: 67), RRKRATAFS (SEQ ID NO: 65), RRKRATNAS (SEQ ID NO: 63), RRKRATNFS (SEQ ID NO: 61), RRRRATNFS (SEQ ID NO: 71), RRRRGTNFS (SEQ ID NO: 72), RRRRATNAS (SEQ ID NO: 73), RRRRATPFS (SEQ ID NO: 74), RRRRATAFS (SEQ ID NO: 75), RRRRATNPS (SEQ ID NO: 76), RRRRATNFP (SEQ ID NO: 77), RRRRAPNFS (SEQ ID NO: 78), RRRRAGNFS (SEQ ID NO: 79), and RRRRPTNFS (SEQ ID NO: 80). In another example, the cleavage site sequence is RRKRPTNFS (SEQ ID NO: 70). In another example, the cleavage site sequence is RRKRGTNFS (SEQ ID NO: 62). In another example, the cleavage site sequence is RRKRAGNFS (SEQ ID NO: 69). In another example, the cleavage site sequence is RRKRAPNFS (SEQ ID NO: 68). In one example, the cleavage site sequence is RRKRATNFS (SEQ ID NO: 61). In another example, the cleavage site sequence is RRKRATNAS (SEQ ID NO: 63). In another example, the cleavage site sequence is RRKRATPFS (SEQ ID NO: 64). In another example, the cleavage site sequence is RRKRATAFS (SEQ ID NO: 65). In another example, the cleavage site sequence is RRKRATNPS (SEQ ID NO: 66). In another example, the cleavage site sequence is RRKRATNFP (SEQ ID NO: 67). Examples of such cleavage sites can be found in the expression system shown in
The anchor polynucleotide of the expression system can be at least 20 nucleotides in length. In another example, the first cleavage polynucleotide can be, but is not limited to, a length of about 20 to 130 nucleotides, or about 10, about 20, about 30, about 40, about 50, about 60, about 70, about 80, about 90, about 100, about 110, about 120 or about 130 nucleotides in length. In a preferred example, the first cleavage polynucleotide is 111 nucleotides in length.
The anchor polynucleotide encodes for a membrane anchor polypeptide. The membrane anchor polypeptide can be at least 7 amino acids in length. In another example, the membrane anchor polypeptide can be, but is not limited to, a length of about 7 to 45 amino acids, about 15 to 40 amino acids, or about 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44 or 45 amino acid residues in length. In a preferred example, the membrane anchor polypeptide is 37 amino acids in length.
In one example, the membrane anchor polypeptide comprises glycophospholipid transmembrane domain (GPI), platelet-derived growth factor receptor (PDGFR) beta chain transmembrane domain (PTM), or immunoglobulin C2-type extracellular-transmembrane-cytosolic domains of murin B7-1 antigen. In another example, the membrane anchor polypeptide is glycophospholipid transmembrane domain (GPI). The terms not limiting to “glycophospholipid transmembrane domain”, “glycophospholipid membrane anchor”, “GM” or “GPI” used herein refer to membrane proteins that are anchored to a membrane in a cell by a structure comprising phosphatidylinositol, carbohydrate, and ethanolamine. In one example, the glycophospholipid transmembrane domain (GPI) is a glycosidylphosphatidylinositol membrane anchor derived from human decay-accelerating factor. In one example, the amino acid sequence of glycophospholipid transmembrane domain (GPI) is SEQ ID NO: 175.
One purpose of the expression system of the present disclosure is to screen for the expression levels of the secretable and/or membrane-bound antigen binding molecule, otherwise known as dual screening. To modulate ratio of production of the secretable antigen binding molecule versus the membrane-bound antigen binding molecule, cleavage efficiencies of the cleavage sites, for example, the first cleavage site and the second cleavage site, can be controlled by, for example, the type of cleavage sites and/or the mutations present in the cleavage site. Depending on the efficiencies of the cleavage sites, complete cleavage or incomplete cleavage can occur to the proteins that are produced by the expression system of the present disclosure.
The terms not limiting to “incomplete cleavage” or “partial cleavage” used herein refer to antigen binding molecules in one cell that are differentially cleaved at the Furin and/or 2A cleavage sites, resulting in a mixture of: antigen binding molecule that is cleaved only at the Furin cleavage site resulting in a secretable antigen binding molecule; antigen binding molecule that is cleaved only at the 2A cleavage site resulting in an incorrect secretable antigen binding molecule; antigen binding molecule that is cleaved at both Furin and 2A cleavage sites; and antigen binding molecule that are not cleaved at both Furin and 2A cleavage sites resulting in a membrane bound antigen binding molecule, as shown in the schematic presented in
The term “complete cleavage” as used herein refers to all antigen binding molecules in one cell that are cleaved at both the Furin cleavage site and the 2A cleavage site, resulting in a secretable antigen binding molecule, as shown in the schematic presented in
The term “membrane bound antigen binding molecule” as used herein refers to an antigen binding molecule comprising a first and second part of the antigen binding molecule, a Furin consensus sequence, a 2A polypeptide or a fragment thereof and a membrane anchor polypeptide derived from the expression system as disclosed herein that is produced and released from a cell. In a one example, the membrane bound antigen binding molecule comprises a light and heavy chain of an antibody, RXKR (SEQ ID NO: 1) or RXRR (SEQ ID NO: 2) sequence, a P2A polypeptide or a fragment thereof and a membrane anchor polypeptide. In a preferred example, the membrane bound antigen binding molecule comprises a light and heavy chain of an antibody, RRKR (SEQ ID NO: 3) sequence, a P2A polypeptide or a fragment thereof and a membrane anchor polypeptide.
The levels of membrane bound antigen binding molecule is determined by the display level. The term “display level” used herein refers to the expression of a membrane-bound antigen binding molecule on a cell surface, wherein the membrane-bound antigen binding molecule comprises: a first antigen binding polynucleotide encoding a first part of the antigen binding molecule; a second antigen binding polynucleotide encoding a second part of the antigen binding molecule.
The terms “secretable antigen binding molecule”, “correct product” or “desired product” used herein refer to an antigen binding molecule comprising a first and second part of the antigen binding molecule derived from the expression system as disclosed herein that is produced and released from a cell. In a preferred example, the secretable antigen binding molecule comprises a light and heavy chain of an antibody.
The terms not limiting to “incorrect secretable antigen binding molecule”, “incorrect product” or “undesired product” used herein refer to an antigen binding molecule comprising a first and second part of the antigen binding molecule, a Furin consensus sequence and a 2A polypeptide or a fragment thereof derived from the expression system as disclosed herein that is produced and released from a cell. In a preferred example, the incorrect secretable antigen binding molecule comprises a light and heavy chain of an antibody, RRKR (SEQ ID NO: 3) sequence and a P2A polypeptide or a fragment thereof.
The expression system of the present disclosure can further comprise one or more internal ribosome entry site (IRES) polynucleotide. In one example, the one or more IRES polynucleotide is before the first antigen binding polynucleotide. In another example, the one or more IRES polynucleotide is after the anchor polynucleotide. In one example, the nucleotide sequence of the one or more IRES polynucleotide is SEQ ID NO: 181. In one example, the one or more IRES polynucleotide encodes for a wild-type encephalomyocarditis virus (EMCV) internal ribosome entry site (IRES).
The expression system of the present disclosure can also further comprise one or more operably linked promoter sequences. The term “operably linked” refers to a functional linkage between a transcription regulating nucleotide sequence (for example, a promoter sequence) and other nucleotide sequences. Thus, the transcription regulating nucleotide sequence may regulate transcription and/or translation of other nucleotide sequences. In one example, the one or more operably linked promoter sequence is selected from a group consisting of ChiP, human CMV, murine CMV, SV40, human EF promoters. In a preferred example, the promoter sequence is ChiP. ChiP is a chimeric promoter consisting of murine cytomegalovirus (CMV) enhancer, human CMV core promoter and human CMV intron A. In one example, the nucleotide sequence of the one or more operably linked promoter sequences is SEQ ID NO: 182.
Different combinations of expressions systems can be generated. In one example, the expression system comprises: a second antigen binding polynucleotide encoding a light chain antibody; a first antigen binding polynucleotide encoding a heavy chain antibody; a first cleavage polynucleotide encoding a Furin consensus sequence RXKR (SEQ ID NO: 1) or RXRR (SEQ ID NO: 2); a second cleavage polynucleotide encoding a 2A polypeptide or a fragment thereof; an anchor polynucleotide encoding a membrane anchor polypeptide; wherein the 2A polypeptide or a fragment thereof comprises one or more mutations. In another example, the expression system comprises a polynucleotide encoding an operably linked promoter sequence; a second antigen binding polynucleotide encoding a light chain antibody; a first IRES polynucleotide; a first antigen binding polynucleotide encoding a heavy chain antibody; a first cleavage polynucleotide encoding a Furin consensus sequence RXKR (SEQ ID NO: 1) or RXRR (SEQ ID NO: 2); a second cleavage polynucleotide encoding a 2A polypeptide or a fragment thereof; an anchor polynucleotide encoding a membrane anchor polypeptide; a second IRES polynucleotide; wherein the 2A polypeptide or a fragment thereof comprises one or more mutations.
In another example, the expression system comprises: a second antigen binding polynucleotide encoding a light chain antibody; a first antigen binding polynucleotide encoding a heavy chain antibody; a cleavage polynucleotide encoding a Furin consensus sequence RXKR (SEQ ID NO: 1) or RXRR (SEQ ID NO: 2) and a 2A polypeptide fragment thereof; an anchor polynucleotide encoding a membrane anchor polypeptide; wherein the 2A polypeptide fragment thereof comprises one or more mutations. In another example, the expression system comprises: a second antigen binding polynucleotide encoding a light chain antibody; a first antigen binding polynucleotide encoding a heavy chain antibody; a cleavage polynucleotide encoding RRKRGTNFS (SEQ ID NO: 62); an anchor polynucleotide encoding a membrane anchor polypeptide. In another example, the expression system comprises: a second antigen binding polynucleotide encoding a light chain antibody; a first antigen binding polynucleotide encoding a heavy chain antibody; a cleavage polynucleotide encoding RRKRAPNFS (SEQ ID NO: 68); an anchor polynucleotide encoding a membrane anchor polypeptide. In another example, the expression system comprises: a second antigen binding polynucleotide encoding a light chain antibody; a first antigen binding polynucleotide encoding a heavy chain antibody; a cleavage polynucleotide encoding RRKRATNAS (SEQ ID NO: 63); an anchor polynucleotide encoding a membrane anchor polypeptide. In another example, the expression system comprises: a second antigen binding polynucleotide encoding a light chain antibody; a first antigen binding polynucleotide encoding a heavy chain antibody; a cleavage polynucleotide encoding RRKRATPFS (SEQ ID NO: 64); an anchor polynucleotide encoding a membrane anchor polypeptide. In another example, the expression system comprises: a second antigen binding polynucleotide encoding a light chain antibody; a first antigen binding polynucleotide encoding a heavy chain antibody; a cleavage polynucleotide encoding RRKRATAFS (SEQ ID NO: 65); an anchor polynucleotide encoding a membrane anchor polypeptide. In another example, the expression system comprises: a second antigen binding polynucleotide encoding a light chain antibody; a first antigen binding polynucleotide encoding a heavy chain antibody; a cleavage polynucleotide encoding RRKRATNPS (SEQ ID NO: 66); an anchor polynucleotide encoding a membrane anchor polypeptide. In another example, the expression system comprises: a second antigen binding polynucleotide encoding a light chain antibody; a first antigen binding polynucleotide encoding a heavy chain antibody; a cleavage polynucleotide encoding RRKRATNFP (SEQ ID NO: 67); an anchor polynucleotide encoding a membrane anchor polypeptide. In another example, the expression system comprises: a second antigen binding polynucleotide encoding a light chain antibody; a first antigen binding polynucleotide encoding a heavy chain antibody; a cleavage polynucleotide encoding RRKRAGNFS (SEQ ID NO: 69); an anchor polynucleotide encoding a membrane anchor polypeptide. In another example, the expression system comprises: a second antigen binding polynucleotide encoding a light chain antibody; a first antigen binding polynucleotide encoding a heavy chain antibody; a cleavage polynucleotide encoding RRKRPTNFS (SEQ ID NO: 70); an anchor polynucleotide encoding a membrane anchor polypeptide.
In yet another example, the expression system comprises: a polynucleotide encoding an operably linked promoter sequence; a second antigen binding polynucleotide encoding a light chain antibody; a first IRES polynucleotide; a first antigen binding polynucleotide encoding a heavy chain antibody; a cleavage polynucleotide encoding a Furin consensus sequence RXKR (SEQ ID NO: 1) or RXRR (SEQ ID NO: 2) and a 2A polypeptide fragment thereof; an anchor polynucleotide encoding a membrane anchor polypeptide; a second IRES polynucleotide; wherein the 2A polypeptide fragment thereof comprises one or more mutations. In one example, the expression system comprises: a polynucleotide encoding an operably linked promoter sequence; a second antigen binding polynucleotide encoding a light chain antibody; a first IRES polynucleotide; a first antigen binding polynucleotide encoding a heavy chain antibody; a cleavage polynucleotide encoding RRKRGTNFS (SEQ ID NO: 62); an anchor polynucleotide encoding a membrane anchor polypeptide; a second IRES polynucleotide. In one example, the expression system comprises: a polynucleotide encoding an operably linked promoter sequence; a second antigen binding polynucleotide encoding a light chain antibody; a first IRES polynucleotide; a first antigen binding polynucleotide encoding a heavy chain antibody; a cleavage polynucleotide encoding RRKRAPNFS (SEQ ID NO: 68); an anchor polynucleotide encoding a membrane anchor polypeptide; a second IRES polynucleotide. In one example, the expression system comprises: a polynucleotide encoding an operably linked promoter sequence; a second antigen binding polynucleotide encoding a light chain antibody; a first IRES polynucleotide; a first antigen binding polynucleotide encoding a heavy chain antibody; a cleavage polynucleotide encoding RRKRATNAS (SEQ ID NO: 63); an anchor polynucleotide encoding a membrane anchor polypeptide; a second IRES polynucleotide. In one example, the expression system comprises: a polynucleotide encoding an operably linked promoter sequence; a second antigen binding polynucleotide encoding a light chain antibody; a first IRES polynucleotide; a first antigen binding polynucleotide encoding a heavy chain antibody; a cleavage polynucleotide encoding RRKRATPFS (SEQ ID NO: 64); an anchor polynucleotide encoding a membrane anchor polypeptide; a second IRES polynucleotide. In one example, the expression system comprises: a polynucleotide encoding an operably linked promoter sequence; a second antigen binding polynucleotide encoding a light chain antibody; a first IRES polynucleotide; a first antigen binding polynucleotide encoding a heavy chain antibody; a cleavage polynucleotide encoding RRKRATAFS (SEQ ID NO: 65); an anchor polynucleotide encoding a membrane anchor polypeptide; a second IRES polynucleotide. In one example, the expression system comprises: a polynucleotide encoding an operably linked promoter sequence; a second antigen binding polynucleotide encoding a light chain antibody; a first IRES polynucleotide; a first antigen binding polynucleotide encoding a heavy chain antibody; a cleavage polynucleotide encoding RRKRATNPS (SEQ ID NO: 66); an anchor polynucleotide encoding a membrane anchor polypeptide; a second IRES polynucleotide. In one example, the expression system comprises: a polynucleotide encoding an operably linked promoter sequence; a second antigen binding polynucleotide encoding a light chain antibody; a first IRES polynucleotide; a first antigen binding polynucleotide encoding a heavy chain antibody; a cleavage polynucleotide encoding RRKRATNFP (SEQ ID NO: 67); an anchor polynucleotide encoding a membrane anchor polypeptide; a second IRES polynucleotide. In one example, the expression system comprises: a polynucleotide encoding an operably linked promoter sequence; a second antigen binding polynucleotide encoding a light chain antibody; a first IRES polynucleotide; a first antigen binding polynucleotide encoding a heavy chain antibody; a cleavage polynucleotide encoding RRKRAGNFS (SEQ ID NO: 69); an anchor polynucleotide encoding a membrane anchor polypeptide; a second IRES polynucleotide. In one example, the expression system comprises: a polynucleotide encoding an operably linked promoter sequence; a second antigen binding polynucleotide encoding a light chain antibody; a first IRES polynucleotide; a first antigen binding polynucleotide encoding a heavy chain antibody; a cleavage polynucleotide encoding RRKRPTNFS (SEQ ID NO: 70); an anchor polynucleotide encoding a membrane anchor polypeptide; a second IRES polynucleotide.
In one example, the expression system as disclosed herein comprises any one of the sequences selected from the group consisting of SEQ ID NOs: 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187. 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205 and 206. In one example, the expression system comprises SEQ ID NO: 170. In another example, the expression system comprises SEQ ID NO: 154. In another example, the expression system comprises SEQ ID NO: 168. In another example, the expression system comprises SEQ ID NO: 166. In another example, the expression system comprises SEQ ID NO: 169. In another example, the expression system comprises SEQ ID NO: 153. In another example, the expression system comprises SEQ ID NO: 167. In another example, the expression system comprises SEQ ID NO: 165.
There is provided a vector comprising the expression system as disclosed herein. The term “vector” as used herein refers to a means, typically a nucleic acid, of transporting and expressing a target gene in a host cell. For example, the vector may include a plasmid vector, a cosmid vector, or a virus vector, such as a bacteriophage vector, an adenovirus vector, a retrovirus vector, and an adeno-associated virus vector. The recombinant vector may be prepared by manipulating a plasmid, a phage, or a virus known in the art.
There is also provided a host cell comprising the expression system or vector as disclosed herein. The host cell may be prokaryotic or eukaryotic host cells. The host cell, which is capable of stably and continuously cloning or expressing the expression system or vector, may be any host cell known in the art.
There is also provided a kit comprising the expression system, vector or host cell as disclosed herein. The kit can further comprise, but is not limited to, buffer or cell culture media known in the art.
The expression system as disclosed herein can be used in a method for detecting the presence of secreted antibodies and/or surface-bound antibodies in a sample. In one example, the method for detecting the presence of one or more secreted antibodies and/or one or more surface-bound antibodies in a sample comprises: providing an expression system as disclosed herein; delivering said expression system to one or more target cells, wherein said target cell transcribes said expression system, wherein once transcribed, said first cleavage site is cleaved in a first plurality of first part of the antigen binding molecule, so that said first plurality of first part of the antigen binding molecule do not comprise said membrane anchor polypeptide, and are thereby secreted by said target cell, and wherein said first and second cleavage sites are not cleaved in a second plurality of first part of the antigen binding molecule, so that said second plurality of first part of the antigen binding molecule comprise said membrane anchor polypeptide, and are thereby bound to the surface of said target cell; and detecting the presence or absence of said first plurality of first part of the antigen binding molecule secreted by said target cell and/or detecting a quantity of said second plurality of first part of the antigen binding molecule on the surface of said target cell.
In another example, the method for detecting the presence of one or more secreted antibodies and/or one or more surface-bound antibodies in a sample comprises: providing an expression system as disclosed herein; delivering said expression system to one or more target cells, wherein said target cell transcribes said expression system, wherein once transcribed, said cleavage site is cleaved in a first plurality of first part of the antigen binding molecule, so that said first plurality of first part of the antigen binding molecule do not comprise said membrane anchor polypeptide, and are thereby secreted by said target cell, and wherein said cleavage sites are not cleaved in a second plurality of first part of the antigen binding molecule, so that said second plurality of first part of the antigen binding molecule comprise said membrane anchor polypeptide, and are thereby bound to the surface of said target cell; and detecting the presence or absence of said first plurality of first part of the antigen binding molecule secreted by said target cell and/or detecting a quantity of said second plurality of first part of the antigen binding molecule on the surface of said target cell.
The expression system, vector, host cell or kit as disclosed herein thus has different applications. In one example, the expression system, vector, host cell or kit as disclosed herein is for use in antibody discovery. In another example, the expression system, vector, host cell or kit as disclosed herein is for use in screening antibody libraries. In another example, the expression system, vector, host cell or kit as disclosed herein is for use in antibody humanization. In another example, the expression system, vector, host cell or kit as disclosed herein is for use in affinity maturation. In another example, the expression system. vector, host cell or kit as disclosed herein is for use in antibody production, including but not limited to monoclonal antibodies or polyclonal antibodies.
As used in this application, the singular form “a,” “an,” and “the” include plural references unless the context clearly dictates otherwise. For example, the term “a genetic marker” includes a plurality of genetic markers, including mixtures and combinations thereof.
As used herein, the terms “increase” and “decrease” refer to the relative alteration of a chosen trait or characteristic in a subset of a population in comparison to the same trait or characteristic as present in the whole population. An increase thus indicates a change on a positive scale, whereas a decrease indicates a change on a negative scale. The term “change”, as used herein, also refers to the difference between a chosen trait or characteristic of an isolated population subset in comparison to the same trait or characteristic in the population as a whole. However, this term is without valuation of the difference seen.
As used herein, the term “about” in the context of concentration of a substance, size of a substance, length of time, or other stated values means +/−5% of the stated value, or +/−4% of the stated value, or +/−3% of the stated value, or +/−2% of the stated value, or +/−1% of the stated value, or +/−0.5% of the stated value.
Throughout this disclosure, certain embodiments may be disclosed in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the disclosed ranges. Accordingly, the description of a range should be considered to have specifically disclosed all the possible sub-ranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed sub-ranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 3, 4, 5, and 6. This applies regardless of the breadth of the range.
The invention illustratively described herein may suitably be practiced in the absence of any element or elements, limitation or limitations, not specifically disclosed herein. Thus, for example, the terms “comprising”, “including”, “containing”, etc. shall be read expansively and without limitation. Additionally, the terms and expressions employed herein have been used as terms of description and not of limitation, and there is no intention in the use of such terms and expressions of excluding any equivalents of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the invention claimed. Thus, it should be understood that although the present invention has been specifically disclosed by preferred embodiments and optional features, modification and variation of the inventions embodied therein herein disclosed may be resorted to by those skilled in the art, and that such modifications and variations are considered to be within the scope of this invention.
The invention has been described broadly and generically herein. Each of the narrower species and subgeneric groupings falling within the generic disclosure also form part of the invention. This includes the generic description of the invention with a proviso or negative limitation removing any subject matter from the genus, regardless of whether or not the excised material is specifically recited herein.
Other embodiments are within the following claims and non-limiting examples. In addition, where features or aspects of the invention are described in terms of Markush groups, those skilled in the art will recognize that the invention is also thereby described in terms of any individual member or subgroup of members of the Markush group.
A master clone for display and secretion of antibodies needs to have a single integration site which is able to provide stable and high antibody expression and allows efficient targeted integration of antibody genes. The process of generating such a master clone is summarized in
To develop CHO master clone, the suspension CHO K1 cells were transfected with a pTag vector, wherein the pTag vector comprised an EGFP reporter gene and a zeocin resistant gene linked by an EMCV IRES variant (IRESv18). The EGFP-IRES-Zeo cassette is flanked by FLP recombinase recognition sites, a mutant FRT variant (F3) and the wild type FRT (Fwt). An ATG-lacking puromycin resistant gene was placed downstream of Fwt for future selection of correct cassette exchange. The transfected cells were selected in a medium containing zeocin to generate a stably transfected pool. The stably transfected pool was enriched for high EGFP producing cells by fluorescence-activated cell sorting (FACS). Clones were isolated using limiting dilution and screened for high EGFP expression by fluorescence-activated cell sorting (FACS), and integration of one copy of pTag vector was analyzed by southern blotting. A primary master clone Z2A4 was confirmed to have one copy of pTag. This primary master clone was co-transfected with a pExchange vector containing the hygromycin resistant gene and a vector expressing Flpe. The transfected cells were selected in medium containing hygromycin.
The cells in which GFP gene was replaced with hygromycin resistant gene survived the selection. Limiting dilution was carried out again to identify a master clone Z2A4-18 containing one copy of hygromycin resistant gene. The single gene integration was confirmed by southern blot and targeted locus amplification analysis.
The CHO K1 master clone was grown in a protein-free medium (maintenance media) consisting of 50% HyQ PF (GE Healthcare Life Sciences) and 50% CD CHO (Thermo Fisher Scientific) supplemented with 1 g/L sodium carbonate (Sigma), 6 mM glutamine (Sigma) and 0.1% Pluronic F-68 (Thermo Fisher Scientific) in a humidified Kuhner shaker (Adolf Kühner AG) with 8% CO2 at 37° C. Routine subculture was conducted every 3 to 4 days by seeding cells at density of 3×105 cells/mL in 15 mL of fresh medium in 125 mL shake flasks (Corning). Cell density and viability were determined by trypan blue exclusion method on Vi-Cell XR viability analysers (Beckman Coulter).
The CHO K1 master clone was co-transfected with one or two appropriate targeting vectors and a vector expressing FLPe using Amaxa SG Cell Line 4D-Nucleofector X Kit and program FF-137 (Lonza). In each transfection, 1×107 cells were transfected with 5 μg of targeting plasmid vector and 5 μg of FLPe plasmid vector in circular format. The transfected cells were then re-suspended in 2 mL of maintenance media preloaded in 6-well suspension culture plates (NUNC) and incubated in the static IncuSafe incubators (Sanyo). At 24 hours post-transfection, they were collected by centrifugation (100×g, 5 min) and re-suspended in 15 mL of protein-free maintenance medium in 125 mL shake flasks in the humidified Kuhner shaker (Adolf Kühner AG) with 8% CO2 at 37° C. Four days later, the transfected cells were subjected to selection in the maintenance media containing Puromycin (InvivoGen) at 20 μg/mL. Selection was continued for two weeks by passaging in the selection medium every 3 to 4 days. Stably transfected cell pools were deemed established when cell viabilities recovered over 95%.
Stably transfected cell pools expressing antibodies were subjected to 14-day fed-batch production by seeding 30 mL of cultures at viable cell density of 3×105 cells/mL in 50 mL tube spin (TPP) in the humidified Kuhner shaker (Adolf Kühner AG) with 8% CO2 at 37° C. 3mL of Ex-Cell Advanced CHO Feed 1 (with glucose) (Sigma) were added at day 3, 5, 7, 9 and 11. 400 μL 45% (w/v) D-glucose (Sigma) were added when glucose level was dropped below 2 g/L during fed-batch cultures. Cell density, viability and antibody titer were monitored at day 3, 5, 7, 9, 11 and 14 using the Vi-Cell XR viability analyzer (Beckman Coulter) and an IMMAGE 800 immunochemistry system (Beckman Coulter), respectively. The IMMAGE 800 immunochemistry system utilized anti-human Fc region antibodies for IgG quantification. The specific mAb productivity (qP) in the exponential phase of cultures was calculated as the mAb concentration at day 7 divided by the integrated viable cell density (IVCD) which was determined based on the trapezoidal method.
Purification of the harvested culture supernatant was performed using a Tricorn column packed with MabSelect SuRe on a ÄKTA AVANT equipped with a UV detector at 280 nm and 254 nm. Prior to loading sample, the column was equilibrated using Dulbecco PBS at pH 7.4. The cultures were filtered (0.22 μm) without pH adjustment prior to loading into the column. Elution was done using 100 mM sodium acetate (acetic acid and sodium hydroxide) at pH 3.6. The purified samples were neutralized to pH 6.0 with 1 M Tris base buffer. Flow rate was set at 5.0 mL/min. Ultrafiltration concentrators, Vivaspin 20, 3000 MWCO PES were used to concentrate the purified sample to a concentration greater than 15 mg/mL. Centrifugation was performed using swing bucket at 2200 g and at 4° C.
Prior to SDS-PAGE separation of the purified mAbs, 4 μg of each purified mAb sample was denatured by boiling in the presence of 25 mM reducing agent DTT (Bio-Rad Laborotories, 161-0611) for reduced gel and absence of reducing agent for non-reducing gel at 95° C. for 10 minutes in the 1XLaemmli buffer [62.5 mM Tris-HCl, pH 6.8, 10.5% glycerol (BDH, 101186), 2% SDS (Bio Rad, 161-0148), 0.01% Bromophenol Blue(PlusOne, 17-1329-01). The reduced and non-reduced denatured mAb protein samples were separated by Bio-Rad Mini-PROTEAN® TGX™ polyacrylamide precast gels (4-15%) for 30 minutes at 200 Volt, and stained with 0.1% Coomassie blue R-250 (Pierce, 20278) in 50% methanol, 10% acetic acid, 40% H2O (V/V). The gel was destained with 10% methanol and 5% acetic acid and 30% ethanol, and then scanned on Imagescanner III (GE Healthcare).
To determine the antibody display levels on cell surface, culture supernatant containing 1×107 cells were collected from the stably transfected pools generated using different dual display and secretion targeting vectors at exponential growth phase. The culture supernatant was then centrifuged at 400 g for 5 minutes to remove the supernatant completely. The cell pellet was re-suspended using 500 μL of cold PBS by pipetting up and down for several times and centrifuged again to remove PBS solution. Subsequently the cells were re-suspended in 500 uL of Anti Human IgG (gamma chain specific)—FITC antibody produced in Goat (Sigma, F0132) diluted 100 times in PBS containing 3% BSA (Bovine Serum Albumin) and then incubated at 4° C. for 30 minutes (on ice) in the dark. Next, the cell solution was centrifuged to remove the solution and washed a few times using PBS. Finally, the cells were re-suspended in 500 μL cold PBS and proceed to flow cytometry analysis on a BD FACSCalibur.
To validate that the generated master clone Z2A4-18 allowed efficient integration of one copy of gene per cell, it was co-transfected with the pTarget-DsRed vector, pTarget-EGFP vector and the vector expressing Flpe (
To evaluate the master clone for expressing monoclonal antibodies and exchange efficiency, the master clone was co-transfected with a targeting vector pTarget-DsRedHER2 containing DsRed, LC and HC (
Next, one set of targeting vectors was designed, wherein the display and secretion of full-length IgG antibodies in CHO cells were tested (
The control targeting vector without the membrane anchor had high level secretion of antibodies into the culture medium but very little display of antibodies on the cell surface (
To increase the ratios of display to secretion of antibodies from the RRKR-P2A-GPI vector, point mutations of P2A were created, wherein the point mutations include the individual residue to glycine, proline or alanine (
The 9 identified variants with point mutations which increased the display level from the F-P2A-GM vectors located at the first to fifth amino acid of P2A. It was indicated that the 5 flanking amino acids downstream of R-X-K-R (SEQ ID NO: 1) or R-X-R-R (SEQ ID NO: 2) were conserved and may play roles in determining the furin cleavage efficiency. To understand how the 9 variants with point mutations affect the furin cleavage efficiency, one set of targeting vectors with the heavy chain (HC) linked with GPI through furin cleavage sequence variants was constructed. These furin cleavage sequence variant-GPI vectors, or variant expression system, comprises the first 5 amino acids from the N-terminus of P2A variants downstream of RRKR (SEQ ID NO: 3). The variant expression system are: RRKR-(ATNFS)-GPI, RRKR-(GTNFS)-GPI, RRKR-(ATNAS)-GPI, RRKR-(ATPFS)-GPI, RRKR-(ATAFS)-GPI, RRKR-(ATNPS)-GPI, RRKR-(ATNFP)-GPI, RRKR-(APNFS)-GPI, RRKR-(AGNFS)-GPI, RRKR-(PTNFS)-GPI. The RRKR-P2A-GPI vectors, or main expression systems containing same mutations were included for comparison. The RRKR-P2A-GPI vectors are RRKR-(ATNFS)P2A-GPI, RRKR-(GTNFS)P2A-GPI, RRKR-(ATNAS)P2A-GPI, RRKR-(ATPFS)P2A-GPI, RRKR-(ATAFS)P2A-GPI, RRKR-(ATNPS)P2A-GPI, RRKR-(ATNFP)P2A-GPI, RRKR-(APNFS)P2A-GPI, RRKR-(AGNFS)P2A-GPI, RRKR-(PTNFS)P2A-GPI (
The expression system as disclosed herein has different applications. For example, the expression system can be used in antibody discovery, and can replace hybridoma and single B cell cloning to raise antibodies from immunized mice. This allows the rapid identification and production of antibodies from human blood for treatment of infectious disease, such as COVID-19.
The expression systems as disclosed herein can also be used for antibody humanization and affinity maturation, as well as antibody production. The expression system allows for rapid cell line development for producing monoclonal antibodies. This can allow for cell line development for homogenous production of polyclonal antibodies, such as recombinant IVIG for treatment of immunodeficiency disease.
A mammalian expression system is developed to permit simultaneous cell surface display and secretion of the same protein at different ratios (
Cleavage at both the minimal furin recognition sequence and 2A peptide results in the heavy chain (HC) polypeptides without attachment of 2A residues and membrane anchor, which assembles with light chain (LC) polypeptides to form secreted antibodies. Cleavage at 2A but not at RRKR results in secreted incorrect antibodies in which HC polypeptides are attached with 2A residues. Incomplete cleavage at both 2A and the minimal furin recognition sequence results in heavy chain (HC) polypeptides fused with membrane anchor, which assembles with light chain (LC) polypeptides to form membrane-bounded antibodies. Obtaining varied ratios of display to secretion of antibodies requires controlled cleavage efficiency of furin and 2A at different levels.
In addition, the cleavage at the minimal furin recognition sequence needs to be controlled at higher efficiency than at 2A peptide to ensure secreted antibodies without attachment of 2A residues to the C-terminus of HC polypeptide. Many types of 2A peptides have been identified from virus. Different 2A peptides have different cleavage efficiency. The cleavage efficiency of 2A and the minimal furin recognition sequence are also affected by their flanking amino acid sequence. Different combinations of the minimal furin recognition sequence and the different types of 2A peptides were screened for simultaneous display and secretion of antibodies. The results indicated that E2A has the highest cleavage efficiency followed by T2A, F2A and P2A. In one example, the targeting vector containing the RRKR-P2A combination secretes correct antibodies without 2A residues attached to the HC polypeptides. However, the display level from the vector with the wild-type P2A peptide is too low to be used for screening antibody binding affinities. To increase the relative amount of cell surface to the secreted antibody, one set of P2A variants was generated with different cleavage efficiencies by point mutations. It was observed that the first five amino acids of P2A are critical for the cleavage efficiency of both furin and P2A. Nine specific point mutations, A1G, T2G, A1P, T2P, N3P, F4P, S5P, N3A and F4A, which inhibit cleavage of furin and P2A at different efficiencies, increased the display level ranging from 9% to 80% of the control vector which linked HC directly with the membrane anchor. The secreted products from the two targeting vectors containing Al P and T2G respectively, which gave the highest display level of 80% and 50% of the control vector, contain incorrect product with 2A residues attached to the HC polypeptide due to lower cleavage at RRKR than at P2A. All the targeting vectors containing the other point mutations secreted correct antibody products. Point mutations of the first five amino acids to other amino acids or mutation at other sites have no effect on cleavage at both RRKR (SEQ ID NO: 3) and P2A cleavage or only inhibit cleavage of P2A. The targeting vectors containing these mutations did not change the display level as the membrane anchor can still be removed by cleavage at RRKR. To cover a wider range of ratios of display to secretion and ensuring secretion of correct product, a high through screening of a library of the main and variant expression systems in which the first five amino acids of P2A are randomized to identify variants with controlled cleavage efficiencies at RRKR and P2A (
The expression system using RRKR-P2A enables secretion of product without attachment of 2A residues to the heavy chain (HC) polypeptides. This expression system also has the advantage of obtaining higher secretion at comparable or even higher display levels than those obtained by using the minimal furin recognition sequence. This is because furin cleavage occurs at Golgi. Attachment of the membrane anchor to the HC polypeptides could result in improper folding at ER which leads to unfolded protein response (UPR) and thus earlier cell death in fed-batch cultures. In contrast, 2A “self-cleavage” occurs co-transnationally. Removing the membrane anchor on some HC polypeptides before entering ER could reduce ER stress and thus enhance the viability and secreted antibody titer in fed-batch cultures.
In contrast to the other expression systems, the expression system of the present disclosure allows simultaneous display and secretion of antibody. Some point mutations of P2A in the expression system of the present disclosure also show increased display levels because these mutations inhibited cleavage of at both RRKR and P2A. By linking the heavy chain (HC) and the membrane anchor through furin cleavage sequence variants (designed by inclusion of the first 5 amino acids of P2A variants), it allows secretion and display at different ratios. Compared to the other expression systems, it has the advantage of avoiding secretion of the incorrect product with 2A residues attached to the heavy chain (HC) polypeptides.
In summary, the inventors have developed a platform technology which provides antibody discovery and production capabilities in one system. Such a platform consists of three key components: 1) a CHO master clone containing a predetermined genomic site that provides stable and high-level gene expression, 2) a targeting vector which allows simultaneous display and secretion of antibodies, and 3) a library consisting of diverse antibodies (
| Number | Date | Country | Kind |
|---|---|---|---|
| 10202009841Y | Oct 2020 | SG | national |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/SG2021/050592 | 10/1/2021 | WO |
