Engineered furin cleavage sequences for co-expression of polygenes in mammalian cells

Abstract

The present disclosure relates to expression systems of antigen binding molecules. In particular, the present disclosure relates to expression systems comprising plasmid vectors for high level and homogeneous co-expression of multiple genes, such as, but not limited to, light and heavy chains of antibodies, in cells.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to Singapore Provisional Application No. 10202300591X, filed on March 6, 2023, the contents of which are incorporated herein by reference in their entirety for all purposes. Technical Field

[0003] The present disclosure relates to, but is not limited to, an expression system for antigen binding molecules. Specifically, the present disclosure relates to an expression system comprising a plasmid vector for co-expressing multiple genes (such as, but not limited to, light and heavy chains of antibodies) at high levels and homogeneously in cells.

[0004] background

[0005] Displaying full-length monoclonal antibodies (mAbs) on the surface of mammalian cells requires tightly coupled co-expression of light and heavy chain genes in a single vector and is an important aspect of various biotechnology applications such as antibody production and development. Traditionally, achieving surface display of mAbs on mammalian cells involves the use of a variety of molecular tools with multiple promoters (MPs) and internal ribosome entry sites (IRES) strategies.

[0006] The use of MP and IRES to co-express the light and heavy chains of mAbs results in low or heterogeneous expression of antibodies in transfected cells. Therefore, it is necessary to develop expression systems that address and alleviate these limitations to enhance the secretion of antibodies of the correct size without compromising the titer level of secreted antibodies and the homogeneity of antibody display. It is necessary to provide improved expression systems comprising plasmid vectors for high-level and homogeneous co-expression of multiple genes (such as mAb light and heavy chains) in cells.

[0007] summary

[0008] In one aspect, the present disclosure relates to an expression system for an antigen binding molecule, comprising:

[0009] - a first polynucleotide encoding a first portion of an antigen binding molecule;

[0010] - a second polynucleotide encoding a first cleavage site comprising the amino acid sequence ZR-X1-X2-R-X3-X4-X5-X6 (SEQ ID NO: 256), wherein the second polynucleotide is located downstream of the first polynucleotide;

[0011] - a third polynucleotide encoding a second cleavage site comprising the 2A polypeptide or a fragment thereof, wherein the third polynucleotide is located downstream of the second polynucleotide; and

[0012] - a fourth polynucleotide encoding the second portion of the antigen binding molecule, wherein the fourth polynucleotide is located downstream of the third polynucleotide,

[0013] wherein when the first and second cleavage sites are cleaved, the antigen binding molecule comprising the first and second parts of the antigen binding molecule is released,

[0014] wherein Z is R, P, G, A, K, N, Q, H, I, L, M, F, S, T, W, Y, or V, and

[0015] wherein X1, X2, X3, X4, X5, and X6 are R, P, G, A, D, C, E, K, N, Q, H, I, L, M, F, S, T, W, Y, or V.

[0016] In one aspect, the present disclosure relates to an expression system for an antigen binding molecule, wherein the antigen binding molecule is secreted or membrane-bound, the expression system comprising:

[0017] - a first polynucleotide encoding a first portion of an antigen binding molecule;

[0018] - a second polynucleotide encoding a first cleavage site comprising the amino acid sequence ZR-X1-X2-R-X3-X4-X5-X6 (SEQ ID NO: 256), wherein the second polynucleotide is located downstream of the first polynucleotide;

[0019] - a third polynucleotide encoding a second cleavage site comprising the 2A polypeptide or a fragment thereof, wherein the third polynucleotide is located downstream of the second polynucleotide;

[0020] - a fourth polynucleotide encoding the second portion of the antigen binding molecule, wherein the fourth polynucleotide is located downstream of the third polynucleotide;

[0021] - a fifth polynucleotide encoding a third cleavage site comprising a furin cleavage sequence variant, the variant comprising:

[0022] (i) the furin consensus sequence RXKR (SEQ ID NO: 18); or

[0023] (ii) the furin consensus sequence RXRR (SEQ ID NO: 19),

[0024] wherein the fifth polynucleotide is located downstream of the fourth polynucleotide; and

[0025] - a sixth polynucleotide encoding a membrane-anchored polypeptide, wherein the sixth polynucleotide is located downstream of the fifth polynucleotide;

[0026] wherein when the first, second and third cleavage sites are cleaved, a secreted antigen-binding molecule comprising the first and second portions of the antigen-binding molecule is released;

[0027] wherein when the first and second cleavage sites are cleaved and the third cleavage site is not cleaved, the membrane-bound antigen-binding molecule comprising the first and second parts of the antigen-binding molecule, the third cleavage site and the membrane-anchored polypeptide is released,

[0028] where Z is R, P, G, A, K, N, Q, H, I, L, M, F, S, T, W, Y or V,

[0029] wherein X1, X2, X3, X4, X5, and X6 are R, P, G, A, D, C, E, K, N, Q, H, I, L, M, F, S, T, W, Y, or V, and

[0030] wherein X is any amino acid.

[0031] In one aspect, the present disclosure relates to a vector comprising the expression system disclosed herein.

[0032] In one aspect, the present disclosure relates to a host cell comprising an expression system disclosed herein or a vector disclosed herein.

[0033] In one aspect, the present disclosure relates to a kit comprising an expression system disclosed herein, a vector disclosed herein, or a host cell disclosed herein.

[0034] In one aspect, the present disclosure relates to an expression system disclosed herein, a vector disclosed herein, a host cell disclosed herein, or a kit disclosed herein for screening antibody libraries or antibody production.

[0035] In one aspect, the present disclosure relates to a method of producing one or more secreted antigen binding molecules and / or one or more membrane-bound antigen binding molecules, comprising culturing a host cell disclosed herein under culture conditions suitable for producing one or more secretable antigen binding molecules and / or one or more membrane-bound antigen binding molecules.

[0036] In one aspect, the present disclosure relates to a method for detecting the presence of one or more secreted antigen binding molecules and / or one or more membrane-bound antigen binding molecules, the method comprising:

[0037] - providing an expression system disclosed herein;

[0038] - delivering the expression system to one or more target cells;

[0039] wherein the target cell transcribes the expression system into one or more amino acid sequences,

[0040] wherein when all cleavage sites in the one or more amino acid sequences are cleaved, the target cell secretes one or more secreted antigen binding molecules comprising the first and second parts of the antigen binding molecule,

[0041] - detecting the presence or absence of one or more secreted antigen binding molecules and / or one or more membrane-bound antigen binding molecules.

[0042] BRIEF DESCRIPTION OF THE DRAWINGS

[0043] The present disclosure will be better understood when considered with reference to the detailed description taken in conjunction with the non-limiting examples and accompanying drawings, in which:

[0044] Figure 1 (including Figure 1A and Figure 1B ) is an overview of the recombinase-mediated cassette exchange (RMCE) strategy and the design of targeting vectors for co-expression of mAb light and heavy chains. Figure 1A Schematic diagram of the CHO targeted integration platform for high-throughput screening of antibody libraries. Figure 1B Schematic diagram of the targeting vector for co-expression of mAb light and heavy chains via IRES, MP, and 2A peptide in the context of the CHO targeted integration platform. hCMV, human cytomegalovirus (CMV) major immediate early gene enhancer and promoter; mCMV, murine CMV enhancer and promoter; ChiP, a chimeric promoter consisting of the mCMV enhancer, the human CMV major immediate early gene promoter, and intron A; Flpe, enhanced flippase; HYGR, a cDNA encoding the hygromycin resistance gene; (ATG-)Pur, a puromycin resistance gene with the ATG start codon removed; F and F3, wild-type and mutant flippase recognition targets; pA, SV40 polyadenylation signal; IRES, wild-type encephalomyocarditis virus (EMCV) internal ribosome entry site (IRES); LC, light chain cDNA; HC, heavy chain cDNA; DRS, DNA regulatory element, such as IRES, promoter or FCS-2A peptide; FCS, engineered furin cleavage sequence for simultaneous display and secretion of antibodies; FCSm, minimal furin cleavage sequence, RRKR; FCS1, engineered FCS variant RRKRSVDTS; GPI, DNA encoding a glycosylphosphatidylinositol membrane anchor molecule derived from human decay-accelerating factor; T2A, 2A peptide from T. lucidum virus.

[0045] Figure 2 (including Figure 2A 、 Figure 2B 、 Figure 2C and Figure 2D ) shows various comparative data for cells transfected with IRES, MP, Fm-2A, F1-2A, F2-2A, and F3-2A targeting vectors. Figure 2A are line graphs showing the viability of cells transfected with IRES, MP, Fm-2A, F1-2A, F2-2A, and F3-2A targeting vectors. Figure 2BFigure 2 is flow cytometry analysis data showing the homogeneity level of antibody display in stable cell pools transfected with IRES, MP, Fm-2A, F1-2A, F2-2A, and F3-2A targeting vectors. Figure 2C is a bar graph showing secreted antibody titers in fed-batch cultures of cells transfected with IRES, MP, Fm-2A, F1-2A, F2-2A, and F3-2A targeting vectors. Figure 2D is an SDS-PAGE gel showing the mass of secreted antibodies from cells transfected with IRES, MP, Fm-2A, F1-2A, F2-2A, and F3-2A targeting vectors as determined by SDS-PAGE under reducing conditions.

[0046] Figure 3 (including Figure 3A 、 Figure 3B 、 Figure 3C 、 Figure 3D 、 Figure 3E 、 Figure 3F and Figure 3G ) shows the complete mass spectrometry (MS) analysis data of light and heavy chain polypeptides expressed by different targeting vectors. Figure 3A Shown are intact MS analysis data for light and heavy chain polypeptides expressed from a control (IRES)-mediated targeting vector. Figure 3B Shown are the complete MS analysis data of light and heavy chain polypeptides expressed by the Fm-2A-mediated targeting vector. Figure 3C Shown are the complete MS analysis data of light and heavy chain polypeptides expressed by the F1-2A-mediated targeting vector. Figure 3D Shown are the complete MS analysis data of light and heavy chain polypeptides expressed by the F2-2A-mediated targeting vector. Figure 3E Shown are the complete MS analysis data of light and heavy chain polypeptides expressed by the F3-2A-mediated targeting vector. Figure 3F Shown is a magnified image of the complete MS analysis data of the light chain polypeptide expressed by the F3-2A-mediated targeting vector. Figure 3G The percentage cleavage efficiency of the Fm, F1, F2 and F3 furin cleavage sequences is shown. The cleavage efficiency of the furin cleavage sequence is determined as the intensity of the cleaved light chain species divided by the intensity of the total light chain species, which includes the cleaved light chain and the uncleaved light chain to which the 2A peptide is attached.

[0047] Figure 4 (including Figure 4A and Figure 4B ) shows the evaluation of the cleavage efficiency of 16 FCS variants. Figure 4A The amino acid sequences of 16 different FCS variants are shown. Figure 4B Shown are SDS-PAGE analyses of secreted antibodies expressed from targeting vectors containing combinations of different FCS variants and 2A under reducing conditions.

[0048] Figure 5 (including Figure 5A 、 Figure 5B 、 Figure 5C 、 Figure 5D 、 Figure 5E and Figure 5F ) shows the evaluation of the cleavage efficiency of 95 FCS variants. Figure 5A The amino acid sequences of 95 different FCS variants are shown. Figure 5B Shown is an SDS-PAGE analysis of secreted antibody expressed from a targeting vector containing a unique mutation at position P5 of the furin cleavage variant F19-F37. Figure 5C Shown is an SDS-PAGE analysis of secreted antibody expressed from a targeting vector containing a unique mutation at position P1' of the furin cleavage variant F38-F56. Figure 5D Shown is an SDS-PAGE analysis of secreted antibodies expressed from targeting vectors containing unique mutations at position P2' of the furin cleavage variants F57-F75. Figure 5E Shown is an SDS-PAGE analysis of secreted antibody expressed from a targeting vector containing a unique mutation at position P3' of the furin cleavage variant F76-F94. Figure 5F Shown is an SDS-PAGE analysis of secreted antibody expressed from a targeting vector containing a unique mutation at position P4' of the furin cleavage variant F95-F113.

[0049] Details

[0050] The present disclosure describes an expression system comprising a plasmid vector containing an engineered furin cleavage sequence for high-level and homogeneous co-expression of multiple genes, such as the light and heavy chains of mAbs, within cells.

[0051] In one aspect, the present disclosure relates to an expression system for an antigen binding molecule, comprising:

[0052] - a first polynucleotide encoding a first portion of an antigen binding molecule;

[0053] - a second polynucleotide encoding a first cleavage site comprising the amino acid sequence ZR-X1-X2-R-X3-X4-X5-X6 (SEQ ID NO: 256), wherein the second polynucleotide is located downstream of the first polynucleotide;

[0054] - a third polynucleotide encoding a second cleavage site comprising the 2A polypeptide or a fragment thereof, wherein the third polynucleotide is located downstream of the second polynucleotide; and

[0055] a fourth polynucleotide encoding the second part of the antigen binding molecule, wherein the fourth polynucleotide is located downstream of the third polynucleotide, wherein when the first and second cleavage sites are cleaved, the antigen binding molecule comprising the first and second parts of the antigen binding molecule is released,

[0056] wherein Z is R, P, G, A, K, N, Q, H, I, L, M, F, S, T, W, Y, or V, and

[0057] wherein X1, X2, X3, X4, X5, and X6 are R, P, G, A, D, C, E, K, N, Q, H, I, L, M, F, S, T, W, Y, or V.

[0058] In one example of the aforementioned aspects, the present disclosure relates to an expression system for an antigen binding molecule, comprising:

[0059] - a first polynucleotide encoding a first portion of an antigen binding molecule;

[0060] - a second polynucleotide encoding a first cleavage site comprising the amino acid sequence RR-X1-X2-R-X3-X4-X5-X6 (SEQ ID NO: 1), wherein the second polynucleotide is located downstream of the first polynucleotide;

[0061] - a third polynucleotide encoding a second cleavage site comprising the 2A polypeptide or a fragment thereof, wherein the third polynucleotide is located downstream of the second polynucleotide; and

[0062] - a fourth polynucleotide encoding the second part of the antigen binding molecule, wherein the fourth polynucleotide is located downstream of the third polynucleotide, wherein when the first and second cleavage sites are cleaved, the antigen binding molecule comprising the first and second parts of the antigen binding molecule is released.

[0063] In one example, the term "expression system" as used herein refers to a DNA construct designed to produce protein or RNA (ribonucleic acid) intracellularly or extracellularly. The expression system can exist independently or be integrated into a vector. Exemplary expression systems include but are not limited to mammalian expression systems, insect expression systems, yeast expression systems, bacterial expression systems, algae expression systems or cell-free expression systems. The expression system can comprise the following components, including but not limited to a promoter, one or more target genes and one or more identification tags. In one example, the expression system can be used for dual screening purposes, to screen the expression level of secretory and / or membrane-bound antigen binding molecules.

[0064] In one example, the term "antigen binding molecule" as used herein refers to an antibody or other protein construct, such as a domain. In one example, the antigen binding molecule described herein is an antibody. In one example, the first part of the antigen binding molecule is the light chain or other protein construct of the antibody. In one example, the antigen binding molecule described herein is the light chain or heavy chain variable region of the antibody. In one example, the antigen binding molecule described herein is the heavy chain variable region or the light chain variable region. In a specific example, the antigen binding molecule described herein is the light chain variable region of the antibody.

[0065] In one example, the expression system of antigen binding molecules as described herein comprises the first polynucleotide of the first part encoding antigen binding molecules.In one example, the first polynucleotide encodes the light chain of antibody.Term " polynucleotide " used herein refers to the nucleotide sequence encoding target product or its fragment, derivative, mutant protein or variant.Polynucleotide includes DNA molecule (for example, cDNA or genomic DNA), RNA molecule (for example, mRNA), analog (for example, peptide nucleic acid and non-natural nucleotide analog) and hybrid thereof of the DNA or RNA produced using nucleotide analogs.Nucleic acid molecule can be single-stranded or double-stranded.In one example, polynucleotide can encode the first part or second part, the first or second or third cleavage site or membrane anchored polypeptide of antigen binding molecules.

[0066] In one example, the expression system of antigen binding molecules as herein described comprises the second polynucleotide encoding the first cleavage site. In one example, the first cleavage site comprises a furin cleavage sequence (FCS). In one example, the first cleavage site comprises a minimum furin cleavage sequence, and its flank is one or more additional amino acids, wherein the additional amino acids can be any amino acids. Amino acid can be natural amino acids or non-natural amino acids. In one example, the first cleavage site comprises the amino acid sequence ZR-X1-X2-R-X3-X4-X5-X6 (SEQ ID NO:256), wherein X1-X6 can be any amino acids. In one example, X1-X6 is natural amino acids or non-natural amino acids or a combination thereof. In one example, X1-X6 is natural amino acids. In one example, natural amino acid is arginine (R), proline (P), glycine (G), alanine (A), aspartic acid (D), cysteine ​​(C), glutamic acid (E), lysine (K), asparagine (N), glutamine (Q), histidine (H), isoleucine (I), leucine (L), methionine (M), phenylalanine (F), serine (S), threonine (T), tryptophan (W), tyrosine (Y) or valine (V). The term " furin " as used herein refers to a general subtilisin-like proprotein convertase that cuts proteins containing its recognition site. The term used herein is not limited to " furin consensus sequence ", " minimal cleavage site ", " minimal furin cleavage consensus sequence " or " furin recognition site ", refers to the amino acid sequence of RXKR (SEQ ID NO: 18) or RXRR (SEQ ID NO: 19), wherein X can be any amino acid. In one example, X is naturally occurring amino acid. In one example, X is arginine (R). Furin recognizes proteins that comprise a furin consensus sequence, which results in the cutting of proteins that comprise a furin consensus sequence. For example, cutting can occur in the Golgi apparatus. In one example, the amino acid sequence of the first cleavage site comprises the amino acid sequence RR-X1-X2-R-X3-X4-X5-X6 (SEQ ID NO:1). In one example, the second polynucleotide encoding comprises the first cleavage site of the amino acid sequence RRRKRSVDT (SEQ ID NO:2). In one example, the cleavage site comprising the amino acid sequence RRRKRSVDT (SEQ ID NO:2) corresponds to FCS variant F2. In one example, the second polynucleotide encoding comprises the first cleavage site of the amino acid sequence RRRKRAVDT (SEQ ID NO:3).In one example, the cleavage site comprising the amino acid sequence RRRKRAVDT (SEQ ID NO: 3) corresponds to FCS variant F4. In one example, the second polynucleotide encodes a first cleavage site comprising the amino acid sequence RRRKRSLDT (SEQ ID NO: 4). In one example, the cleavage site comprising the amino acid sequence RRRKRSLDT (SEQ ID NO: 4) corresponds to FCS variant F5. In one example, the second polynucleotide encodes a first cleavage site comprising the amino acid sequence RRRKRALDT (SEQ ID NO: 5). In one example, the cleavage site comprising the amino acid sequence RRRKRALDT (SEQ ID NO: 5) corresponds to FCS variant F6. In one example, the second polynucleotide encodes a first cleavage site comprising the amino acid sequence RRRKRSVST (SEQ ID NO: 6). In one example, the cleavage site comprising the amino acid sequence RRRKRSVST (SEQ ID NO: 6) corresponds to FCS variant F7. In one example, the second polynucleotide encodes a first cleavage site comprising the amino acid sequence RRRKRAVST (SEQ ID NO: 7). In one example, the cleavage site comprising the amino acid sequence RRRKRAVST (SEQ ID NO: 7) corresponds to FCS variant F8. In one example, the second polynucleotide encoding comprises the first cleavage site of the amino acid sequence RRRKRALST (SEQ ID NO: 8). In one example, the cleavage site comprising the amino acid sequence RRRKRALST (SEQ ID NO: 8) corresponds to FCS variant F9. In one example, the second polynucleotide encoding comprises the first cleavage site of the amino acid sequence RRRKRSLST (SEQ ID NO: 9). In one example, the cleavage site comprising the amino acid sequence RRRKRSLST (SEQ ID NO: 9) corresponds to FCS variant F10. In one example, the second polynucleotide encoding comprises the first cleavage site of the amino acid sequence RRRKRSVDL (SEQ ID NO: 10). In one example, the cleavage site comprising the amino acid sequence RRRKRSVDL (SEQ ID NO: 10) corresponds to FCS variant F11. In one example, the second polynucleotide encoding comprises the first cleavage site of the amino acid sequence RRRKRAVDL (SEQ ID NO: 11).In one example, the cleavage site comprising the amino acid sequence RRRKRAVDL (SEQ ID NO: 11) corresponds to FCS variant F12. In one example, the second polynucleotide encodes the first cleavage site comprising the amino acid sequence RRRKRSLDL (SEQ ID NO: 12). In one example, the cleavage site comprising the amino acid sequence RRRKRSLDL (SEQ ID NO: 12) corresponds to FCS variant F13. In one example, the second polynucleotide encodes the first cleavage site comprising the amino acid sequence RRRKRALDL (SEQ ID NO: 13). In one example, the cleavage site comprising the amino acid sequence RRRKRALDL (SEQ ID NO: 13) corresponds to FCS variant F14. In one example, the second polynucleotide encodes the first cleavage site comprising the amino acid sequence RRRKRSVSL (SEQ ID NO: 14). In one example, the cleavage site comprising the amino acid sequence RRRKRSVSL (SEQ ID NO: 14) corresponds to FCS variant F15. In one example, the second polynucleotide encodes the first cleavage site comprising the amino acid sequence RRRKRAVSL (SEQ ID NO: 15). In one example, the cleavage site comprising the amino acid sequence RRRKRAVSL (SEQ ID NO: 15) corresponds to FCS variant F16. In one example, the second polynucleotide encodes a first cleavage site comprising the amino acid sequence RRRKRALSL (SEQ ID NO: 16). In one example, the cleavage site comprising the amino acid sequence RRRKRALSL (SEQ ID NO: 16) corresponds to FCS variant F17. In one example, the second polynucleotide encodes a first cleavage site comprising the amino acid sequence RRRKRSLSL (SEQ ID NO: 17). In one example, the cleavage site comprising the amino acid sequence RRRKRSLSL (SEQ ID NO: 17) corresponds to FCS variant F18. In one example, the second polynucleotide is located downstream of the first polynucleotide. In one example, the second polynucleotide encodes a first cleavage site comprising the amino acid sequence PRRKRSVDT (SEQ ID NO: 66). In one example, the cleavage site comprising the amino acid sequence PRRKRSVDT (SEQ ID NO: 66) corresponds to FCS variant F19.In one example, the second polynucleotide encodes a first cleavage site comprising the amino acid sequence GRRKRSVDT (SEQ ID NO: 67). In one example, the cleavage site comprising the amino acid sequence GRRKRSVDT (SEQ ID NO: 67) corresponds to FCS variant F20. In one example, the second polynucleotide encodes a first cleavage site comprising the amino acid sequence ARRKRSVDT (SEQ ID NO: 68). In one example, the cleavage site comprising the amino acid sequence ARRKRSVDT (SEQ ID NO: 68) corresponds to FCS variant F21. In one example, the second polynucleotide encodes a first cleavage site comprising the amino acid sequence KRRKRSVDT (SEQ ID NO: 69). In one example, the cleavage site comprising the amino acid sequence KRRKRSVDT (SEQ ID NO: 69) corresponds to FCS variant F22. In one example, the second polynucleotide encodes a first cleavage site comprising the amino acid sequence NRRKRSVDT (SEQ ID NO: 70). In one example, the cleavage site comprising the amino acid sequence NRRKRSVDT (SEQ ID NO: 70) corresponds to FCS variant F23. In one example, the second polynucleotide encodes a first cleavage site comprising the amino acid sequence DRRKRSVDT (SEQ ID NO: 71). In one example, the cleavage site comprising the amino acid sequence DRRKRSVDT (SEQ ID NO: 71) corresponds to FCS variant F24. In one example, the second polynucleotide encodes a first cleavage site comprising the amino acid sequence CRRKRSVDT (SEQ ID NO: 72). In one example, the cleavage site comprising the amino acid sequence CRRKRSVDT (SEQ ID NO: 72) corresponds to FCS variant F25. In one example, the second polynucleotide encodes a first cleavage site comprising the amino acid sequence ERRKRSVDT (SEQ ID NO: 73). In one example, the cleavage site comprising the amino acid sequence ERRKRSVDT (SEQ ID NO: 73) corresponds to FCS variant F26. In one example, the second polynucleotide encodes a first cleavage site comprising the amino acid sequence QRRKRSVDT (SEQ ID NO: 74). In one example, the cleavage site comprising the amino acid sequence QRRKRSVDT (SEQ ID NO: 74) corresponds to FCS variant F27.In one example, the second polynucleotide encodes a first cleavage site comprising the amino acid sequence HRRKRSVDT (SEQ ID NO: 75). In one example, the cleavage site comprising the amino acid sequence HRRKRSVDT (SEQ ID NO: 75) corresponds to FCS variant F28. In one example, the second polynucleotide encodes a first cleavage site comprising the amino acid sequence IRRKRSVDT (SEQ ID NO: 76). In one example, the cleavage site comprising the amino acid sequence IRRKRSVDT (SEQ ID NO: 76) corresponds to FCS variant F29. In one example, the second polynucleotide encodes a first cleavage site comprising the amino acid sequence LRRKRSVDT (SEQ ID NO: 77). In one example, the cleavage site comprising the amino acid sequence LRRKRSVDT (SEQ ID NO: 77) corresponds to FCS variant F30. In one example, the second polynucleotide encodes a first cleavage site comprising the amino acid sequence MRRKRSVDT (SEQ ID NO: 78). In one example, the cleavage site comprising the amino acid sequence MRRKRSVDT (SEQ ID NO: 78) corresponds to FCS variant F31. In one example, the second polynucleotide encodes a first cleavage site comprising the amino acid sequence FRRKRSVDT (SEQ ID NO: 79). In one example, the cleavage site comprising the amino acid sequence FRRKRSVDT (SEQ ID NO: 79) corresponds to FCS variant F32. In one example, the second polynucleotide encodes a first cleavage site comprising the amino acid sequence SRRKRSVDT (SEQ ID NO: 80). In one example, the cleavage site comprising the amino acid sequence SRRKRSVDT (SEQ ID NO: 80) corresponds to FCS variant F33. In one example, the second polynucleotide encodes a first cleavage site comprising the amino acid sequence TRRKRSVDT (SEQ ID NO: 81). In one example, the cleavage site comprising the amino acid sequence TRRKRSVDT (SEQ ID NO: 81) corresponds to FCS variant F34. In one example, the second polynucleotide encodes a first cleavage site comprising the amino acid sequence WRRKRSVDT (SEQ ID NO: 82). In one example, the cleavage site comprising the amino acid sequence WRRKRSVDT (SEQ ID NO: 82) corresponds to FCS variant F35.In one example, the second polynucleotide encodes a first cleavage site comprising the amino acid sequence YRRKRSVDT (SEQ ID NO: 83). In one example, the cleavage site comprising the amino acid sequence YRRKRSVDT (SEQ ID NO: 83) corresponds to FCS variant F36. In one example, the second polynucleotide encodes a first cleavage site comprising the amino acid sequence VRRKRSVDT (SEQ ID NO: 84). In one example, the cleavage site comprising the amino acid sequence VRRKRSVDT (SEQ ID NO: 84) corresponds to FCS variant F37. In one example, the second polynucleotide encodes a first cleavage site comprising the amino acid sequence RRRKRPVDT (SEQ ID NO: 85). In one example, the cleavage site comprising the amino acid sequence RRRKRPVDT (SEQ ID NO: 85) corresponds to FCS variant F38. In one example, the second polynucleotide encodes a first cleavage site comprising the amino acid sequence RRRKRGVDT (SEQ ID NO: 86). In one example, the cleavage site comprising the amino acid sequence RRRKRGVDT (SEQ ID NO: 86) corresponds to FCS variant F39. In one example, the second polynucleotide encodes a first cleavage site comprising the amino acid sequence RRRKRAVDT (SEQ ID NO: 87). In one example, the cleavage site comprising the amino acid sequence RRRKRAVDT (SEQ ID NO: 87) corresponds to FCS variant F40. In one example, the second polynucleotide encodes a first cleavage site comprising the amino acid sequence RRRKRRVDT (SEQ ID NO: 88). In one example, the cleavage site comprising the amino acid sequence RRRKRRVDT (SEQ ID NO: 88) corresponds to FCS variant F41. In one example, the second polynucleotide encodes a first cleavage site comprising the amino acid sequence RRRKRKVDT (SEQ ID NO: 89). In one example, the cleavage site comprising the amino acid sequence RRRKRKVDT (SEQ ID NO: 89) corresponds to FCS variant F42. In one example, the second polynucleotide encodes a first cleavage site comprising the amino acid sequence RRRKRNVDT (SEQ ID NO: 90). In one example, the cleavage site comprising the amino acid sequence RRRKRNVDT (SEQ ID NO: 90) corresponds to FCS variant F43.In one example, the second polynucleotide encodes a first cleavage site comprising the amino acid sequence RRRKRDVDT (SEQ ID NO: 91). In one example, the cleavage site comprising the amino acid sequence RRRKRDVDT (SEQ ID NO: 91) corresponds to FCS variant F44. In one example, the second polynucleotide encodes a first cleavage site comprising the amino acid sequence RRRKRCVDT (SEQ ID NO: 92). In one example, the cleavage site comprising the amino acid sequence RRRKRCVDT (SEQ ID NO: 92) corresponds to FCS variant F45. In one example, the second polynucleotide encodes a first cleavage site comprising the amino acid sequence RRRKREVDT (SEQ ID NO: 93). In one example, the cleavage site comprising the amino acid sequence RRRKREVDT (SEQ ID NO: 93) corresponds to FCS variant F46. In one example, the second polynucleotide encodes a first cleavage site comprising the amino acid sequence RRRKRQVDT (SEQ ID NO: 94). In one example, the cleavage site comprising the amino acid sequence RRRKRQVDT (SEQ ID NO: 94) corresponds to FCS variant F47. In one example, the second polynucleotide encodes a first cleavage site comprising the amino acid sequence RRRKRHVDT (SEQ ID NO: 95). In one example, the cleavage site comprising the amino acid sequence RRRKRHVDT (SEQ ID NO: 95) corresponds to FCS variant F48. In one example, the second polynucleotide encodes a first cleavage site comprising the amino acid sequence RRRKRIVDT (SEQ ID NO: 96). In one example, the cleavage site comprising the amino acid sequence RRRKRIVDT (SEQ ID NO: 96) corresponds to FCS variant F49. In one example, the second polynucleotide encodes a first cleavage site comprising the amino acid sequence RRRKRLVDT (SEQ ID NO: 97). In one example, the cleavage site comprising the amino acid sequence RRRKRLVDT (SEQ ID NO: 97) corresponds to FCS variant F50. In one example, the second polynucleotide encodes a first cleavage site comprising the amino acid sequence RRRKRMVDT (SEQ ID NO: 98). In one example, the cleavage site comprising the amino acid sequence RRRKRMVDT (SEQ ID NO: 98) corresponds to FCS variant F51.In one example, the second polynucleotide encodes a first cleavage site comprising the amino acid sequence RRRKRFVDT (SEQ ID NO: 99). In one example, the cleavage site comprising the amino acid sequence RRRKRFVDT (SEQ ID NO: 99) corresponds to FCS variant F52. In one example, the second polynucleotide encodes a first cleavage site comprising the amino acid sequence RRRKRTVDT (SEQ ID NO: 100). In one example, the cleavage site comprising the amino acid sequence RRRKRTVDT (SEQ ID NO: 100) corresponds to FCS variant F53. In one example, the second polynucleotide encodes a first cleavage site comprising the amino acid sequence RRRKRWVDT (SEQ ID NO: 101). In one example, the cleavage site comprising the amino acid sequence RRRKRWVDT (SEQ ID NO: 101) corresponds to FCS variant F54. In one example, the second polynucleotide encodes a first cleavage site comprising the amino acid sequence RRRKRYVDT (SEQ ID NO: 102). In one example, the cleavage site comprising the amino acid sequence RRRKRYVDT (SEQ ID NO: 102) corresponds to FCS variant F55. In one example, the second polynucleotide encodes a first cleavage site comprising the amino acid sequence RRRKRVVDT (SEQ ID NO: 103). In one example, the cleavage site comprising the amino acid sequence RRRKRVVDT (SEQ ID NO: 103) corresponds to FCS variant F56. In one example, the second polynucleotide encodes a first cleavage site comprising the amino acid sequence RRRKRSPDT (SEQ ID NO: 104). In one example, the cleavage site comprising the amino acid sequence RRRKRSPDT (SEQ ID NO: 104) corresponds to FCS variant F57. In one example, the second polynucleotide encodes a first cleavage site comprising the amino acid sequence RRRKRSGDT (SEQ ID NO: 105). In one example, the cleavage site comprising the amino acid sequence RRRKRSGDT (SEQ ID NO: 105) corresponds to FCS variant F58. In one example, the second polynucleotide encodes a first cleavage site comprising the amino acid sequence RRRKRSADT (SEQ ID NO: 106). In one example, the cleavage site comprising the amino acid sequence RRRKRSADT (SEQ ID NO: 106) corresponds to FCS variant F59.In one example, the second polynucleotide encodes a first cleavage site comprising the amino acid sequence RRRKRSRDT (SEQ ID NO: 107). In one example, the cleavage site comprising the amino acid sequence RRRKRSRDT (SEQ ID NO: 107) corresponds to FCS variant F60. In one example, the second polynucleotide encodes a first cleavage site comprising the amino acid sequence RRRKRSKDT (SEQ ID NO: 108). In one example, the cleavage site comprising the amino acid sequence RRRKRSKDT (SEQ ID NO: 108) corresponds to FCS variant F61. In one example, the second polynucleotide encodes a first cleavage site comprising the amino acid sequence RRRKRSNDT (SEQ ID NO: 109). In one example, the cleavage site comprising the amino acid sequence RRRKRSNDT (SEQ ID NO: 109) corresponds to FCS variant F62. In one example, the second polynucleotide encodes a first cleavage site comprising the amino acid sequence RRRKRSDDT (SEQ ID NO: 110). In one example, the cleavage site comprising the amino acid sequence RRRKRSDDT (SEQ ID NO: 110) corresponds to FCS variant F63. In one example, the second polynucleotide encodes a first cleavage site comprising the amino acid sequence RRRKRSCDT (SEQ ID NO: 111). In one example, the cleavage site comprising the amino acid sequence RRRKRSCDT (SEQ ID NO: 111) corresponds to FCS variant F64. In one example, the second polynucleotide encodes a first cleavage site comprising the amino acid sequence RRRKRSEDT (SEQ ID NO: 112). In one example, the cleavage site comprising the amino acid sequence RRRKRSEDT (SEQ ID NO: 112) corresponds to FCS variant F65. In one example, the second polynucleotide encodes a first cleavage site comprising the amino acid sequence RRRKRSQDT (SEQ ID NO: 113). In one example, the cleavage site comprising the amino acid sequence RRRKRSQDT (SEQ ID NO: 113) corresponds to FCS variant F66. In one example, the second polynucleotide encodes a first cleavage site comprising the amino acid sequence RRRKRSHDT (SEQ ID NO: 114). In one example, the cleavage site comprising the amino acid sequence RRRKRSHDT (SEQ ID NO: 114) corresponds to FCS variant F67.In one example, the second polynucleotide encodes a first cleavage site comprising the amino acid sequence RRRKRSIDT (SEQ ID NO: 115). In one example, the cleavage site comprising the amino acid sequence RRRKRSIDT (SEQ ID NO: 115) corresponds to FCS variant F68. In one example, the second polynucleotide encodes a first cleavage site comprising the amino acid sequence RRRKRSLDT (SEQ ID NO: 116). In one example, the cleavage site comprising the amino acid sequence RRRKRSLDT (SEQ ID NO: 116) corresponds to FCS variant F69. In one example, the second polynucleotide encodes a first cleavage site comprising the amino acid sequence RRRKRSMDT (SEQ ID NO: 117). In one example, the cleavage site comprising the amino acid sequence RRRKRSMDT (SEQ ID NO: 117) corresponds to FCS variant F70. In one example, the second polynucleotide encodes a first cleavage site comprising the amino acid sequence RRRKRSFDT (SEQ ID NO: 118). In one example, the cleavage site comprising the amino acid sequence RRRKRSFDT (SEQ ID NO: 118) corresponds to FCS variant F71. In one example, the second polynucleotide encoding comprises the first cleavage site of the amino acid sequence RRRKRSSDT (SEQ ID NO: 119). In one example, the cleavage site comprising the amino acid sequence RRRKRSSDT (SEQ ID NO: 119) corresponds to FCS variant F72. In one example, the second polynucleotide encoding comprises the first cleavage site of the amino acid sequence RRRKRSTDT (SEQ ID NO: 120). In one example, the cleavage site comprising the amino acid sequence RRRKRSTDT (SEQ ID NO: 120) corresponds to FCS variant F73. In one example, the second polynucleotide encoding comprises the first cleavage site of the amino acid sequence RRRKRSWDT (SEQ ID NO: 121). In one example, the cleavage site comprising the amino acid sequence RRRKRSWDT (SEQ ID NO: 121) corresponds to FCS variant F74. In one example, the second polynucleotide encodes a first cleavage site comprising the amino acid sequence RRRKRSYDT (SEQ ID NO: 122). In one example, the cleavage site comprising the amino acid sequence RRRKRSYDT (SEQ ID NO: 122) corresponds to FCS variant F75.In one example, the second polynucleotide encodes a first cleavage site comprising the amino acid sequence RRRKRSVPT (SEQ ID NO: 123). In one example, the cleavage site comprising the amino acid sequence RRRKRSVPT (SEQ ID NO: 123) corresponds to FCS variant F76. In one example, the second polynucleotide encodes a first cleavage site comprising the amino acid sequence RRRKRSVGT (SEQ ID NO: 124). In one example, the cleavage site comprising the amino acid sequence RRRKRSVGT (SEQ ID NO: 124) corresponds to FCS variant F77. In one example, the second polynucleotide encodes a first cleavage site comprising the amino acid sequence RRRKRSVAT (SEQ ID NO: 125). In one example, the cleavage site comprising the amino acid sequence RRRKRSVAT (SEQ ID NO: 125) corresponds to FCS variant F78. In one example, the second polynucleotide encodes a first cleavage site comprising the amino acid sequence RRRKRSVRT (SEQ ID NO: 126). In one example, the cleavage site comprising the amino acid sequence RRRKRSVRT (SEQ ID NO: 126) corresponds to FCS variant F79. In one example, the second polynucleotide encoding comprises the first cleavage site of the amino acid sequence RRRKRSVKT (SEQ ID NO: 127). In one example, the cleavage site comprising the amino acid sequence RRRKRSVKT (SEQ ID NO: 127) corresponds to FCS variant F80. In one example, the second polynucleotide encoding comprises the first cleavage site of the amino acid sequence RRRKRSVNT (SEQ ID NO: 128). In one example, the cleavage site comprising the amino acid sequence RRRKRSVNT (SEQ ID NO: 128) corresponds to FCS variant F81. In one example, the second polynucleotide encoding comprises the first cleavage site of the amino acid sequence RRRKRSVCT (SEQ ID NO: 129). In one example, the cleavage site comprising the amino acid sequence RRRKRSVCT (SEQ ID NO: 129) corresponds to FCS variant F82. In one example, the second polynucleotide encodes a first cleavage site comprising the amino acid sequence RRRKRSVET (SEQ ID NO: 130). In one example, the cleavage site comprising the amino acid sequence RRRKRSVET (SEQ ID NO: 130) corresponds to FCS variant F83.In one example, the second polynucleotide encodes a first cleavage site comprising the amino acid sequence RRRKRSVQT (SEQ ID NO: 131). In one example, the cleavage site comprising the amino acid sequence RRRKRSVQT (SEQ ID NO: 131) corresponds to FCS variant F84. In one example, the second polynucleotide encodes a first cleavage site comprising the amino acid sequence RRRKRSVHT (SEQ ID NO: 132). In one example, the cleavage site comprising the amino acid sequence RRRKRSVHT (SEQ ID NO: 132) corresponds to FCS variant F85. In one example, the second polynucleotide encodes a first cleavage site comprising the amino acid sequence RRRKRSVIT (SEQ ID NO: 133). In one example, the cleavage site comprising the amino acid sequence RRRKRSVIT (SEQ ID NO: 133) corresponds to FCS variant F86. In one example, the second polynucleotide encodes a first cleavage site comprising the amino acid sequence RRRKRSVLT (SEQ ID NO: 134). In one example, the cleavage site comprising the amino acid sequence RRRKRSVLT (SEQ ID NO: 134) corresponds to FCS variant F87. In one example, the second polynucleotide encoding comprises the first cleavage site of the amino acid sequence RRRKRSVMT (SEQ ID NO: 135). In one example, the cleavage site comprising the amino acid sequence RRRKRSVMT (SEQ ID NO: 135) corresponds to FCS variant F88. In one example, the second polynucleotide encoding comprises the first cleavage site of the amino acid sequence RRRKRSVFT (SEQ ID NO: 136). In one example, the cleavage site comprising the amino acid sequence RRRKRSVFT (SEQ ID NO: 136) corresponds to FCS variant F89. In one example, the second polynucleotide encoding comprises the first cleavage site of the amino acid sequence RRRKRSVST (SEQ ID NO: 137). In one example, the cleavage site comprising the amino acid sequence RRRKRSVST (SEQ ID NO: 137) corresponds to FCS variant F90. In one example, the second polynucleotide encodes a first cleavage site comprising the amino acid sequence RRRKRSVTT (SEQ ID NO: 138). In one example, the cleavage site comprising the amino acid sequence RRRKRSVTT (SEQ ID NO: 138) corresponds to FCS variant F91.In one example, the second polynucleotide encodes a first cleavage site comprising the amino acid sequence RRRKRSVWT (SEQ ID NO: 139). In one example, the cleavage site comprising the amino acid sequence RRRKRSVWT (SEQ ID NO: 139) corresponds to FCS variant F92. In one example, the second polynucleotide encodes a first cleavage site comprising the amino acid sequence RRRKRSVYT (SEQ ID NO: 140). In one example, the cleavage site comprising the amino acid sequence RRRKRSVYT (SEQ ID NO: 140) corresponds to FCS variant F93. In one example, the second polynucleotide encodes a first cleavage site comprising the amino acid sequence RRRKRSVVT (SEQ ID NO: 141). In one example, the cleavage site comprising the amino acid sequence RRRKRSVVT (SEQ ID NO: 141) corresponds to FCS variant F94. In one example, the second polynucleotide encodes a first cleavage site comprising the amino acid sequence RRRKRSVDP (SEQ ID NO: 142). In one example, the cleavage site comprising the amino acid sequence RRRKRSVDP (SEQ ID NO: 142) corresponds to FCS variant F95. In one example, the second polynucleotide encoding comprises the first cleavage site of the amino acid sequence RRRKRSVDG (SEQ ID NO: 143). In one example, the cleavage site comprising the amino acid sequence RRRKRSVDG (SEQ ID NO: 143) corresponds to FCS variant F96. In one example, the second polynucleotide encoding comprises the first cleavage site of the amino acid sequence RRRKRSVDA (SEQ ID NO: 144). In one example, the cleavage site comprising the amino acid sequence RRRKRSVDA (SEQ ID NO: 144) corresponds to FCS variant F97. In one example, the second polynucleotide encoding comprises the first cleavage site of the amino acid sequence RRRKRSVDR (SEQ ID NO: 145). In one example, the cleavage site comprising the amino acid sequence RRRKRSVDR (SEQ ID NO: 145) corresponds to FCS variant F98. In one example, the second polynucleotide encodes a first cleavage site comprising the amino acid sequence RRRKRSVDK (SEQ ID NO: 146). In one example, the cleavage site comprising the amino acid sequence RRRKRSVDK (SEQ ID NO: 146) corresponds to FCS variant F99.In one example, the second polynucleotide encoding comprises the first cleavage site of the amino acid sequence RRRKRSVDN (SEQ ID NO: 147). In one example, the cleavage site comprising the amino acid sequence RRRKRSVDN (SEQ ID NO: 147) corresponds to FCS variant F100. In one example, the second polynucleotide encoding comprises the first cleavage site of the amino acid sequence RRRKRSVDD (SEQ ID NO: 148). In one example, the cleavage site comprising the amino acid sequence RRRKRSVDD (SEQ ID NO: 148) corresponds to FCS variant F101. In one example, the second polynucleotide encoding comprises the first cleavage site of the amino acid sequence RRRKRSVDC (SEQ ID NO: 149). In one example, the cleavage site comprising the amino acid sequence RRRKRSVDC (SEQ ID NO: 149) corresponds to FCS variant F102. In one example, the second polynucleotide encoding comprises the first cleavage site of the amino acid sequence RRRKRSVDE (SEQ ID NO: 150). In one example, the cleavage site comprising the amino acid sequence RRRKRSVDE (SEQ ID NO: 150) corresponds to FCS variant F103. In one example, the second polynucleotide encoding comprises the first cleavage site of the amino acid sequence RRRKRSVDQ (SEQ ID NO: 151). In one example, the cleavage site comprising the amino acid sequence RRRKRSVDQ (SEQ ID NO: 151) corresponds to FCS variant F104. In one example, the second polynucleotide encoding comprises the first cleavage site of the amino acid sequence RRRKRSVDH (SEQ ID NO: 152). In one example, the cleavage site comprising the amino acid sequence RRRKRSVDH (SEQ ID NO: 152) corresponds to FCS variant F105. In one example, the second polynucleotide encoding comprises the first cleavage site of the amino acid sequence RRRKRSVDI (SEQ ID NO: 153). In one example, the cleavage site comprising the amino acid sequence RRRKRSVDI (SEQ ID NO: 153) corresponds to FCS variant F106. In one example, the second polynucleotide encodes a first cleavage site comprising the amino acid sequence RRRKRSVDL (SEQ ID NO: 154).In one example, the cleavage site comprising the amino acid sequence RRRKRSVDL (SEQ ID NO: 154) corresponds to FCS variant F107. In one example, the second polynucleotide encoding comprises the first cleavage site of the amino acid sequence RRRKRSVDM (SEQ ID NO: 155). In one example, the cleavage site comprising the amino acid sequence RRRKRSVDM (SEQ ID NO: 155) corresponds to FCS variant F108. In one example, the second polynucleotide encoding comprises the first cleavage site of the amino acid sequence RRRKRSVDF (SEQ ID NO: 156). In one example, the cleavage site comprising the amino acid sequence RRRKRSVDF (SEQ ID NO: 156) corresponds to FCS variant F109. In one example, the second polynucleotide encoding comprises the first cleavage site of the amino acid sequence RRRKRSVDS (SEQ ID NO: 157). In one example, the cleavage site comprising the amino acid sequence RRRKRSVDS (SEQ ID NO: 157) corresponds to FCS variant F110. In one example, the second polynucleotide encodes a first cleavage site comprising the amino acid sequence RRRKRSVDW (SEQ ID NO: 158). In one example, the cleavage site comprising the amino acid sequence RRRKRSVDW (SEQ ID NO: 158) corresponds to FCS variant F111. In one example, the second polynucleotide encodes a first cleavage site comprising the amino acid sequence RRRKRSVDY (SEQ ID NO: 159). In one example, the cleavage site comprising the amino acid sequence RRRKRSVDY (SEQ ID NO: 159) corresponds to FCS variant F112. In one example, the second polynucleotide encodes a first cleavage site comprising the amino acid sequence RRRKRSVDV (SEQ ID NO: 160). In one example, the cleavage site comprising the amino acid sequence RRRKRSVDV (SEQ ID NO: 160) corresponds to FCS variant F113.

[0067] In one example, the expression system of the antigen-binding molecules described herein comprises a third polynucleotide. In one example, the third polynucleotide encodes a second cleavage site comprising a 2A polypeptide or a fragment thereof. In one example, the second cleavage site is a 2A polypeptide or a fragment thereof. As used herein, the terms "2A polypeptide," "2A peptide," and "2A protein" are not limited to peptides that mediate protein "self-cleavage" or "self-processing" during translation in eukaryotic cells. For example, 2A polypeptides are typically 18-25 amino acids long and are derived from viruses. 2A polypeptides are capable of self-cleavage, which occurs during co-translation, for example, between the last two amino acids, glycine and proline. In one example, the 2A polypeptide or fragment thereof is selected from the group consisting of P2A, F2A, E2A, and T2A, or fragments thereof. F2A is a 2A peptide derived from foot-and-mouth disease virus; E2A is a 2A peptide derived from equine rhinitis virus A; T2A is a 2A peptide derived from scutellaria avium virus; and P2A is a 2A peptide derived from porcine teschovirus type 1. In another example, the 2A polypeptide or fragment thereof is a P2A polypeptide or fragment thereof. In one example, the second polypeptide encoding the first cleavage site needs to be linked to the 2A peptide to ensure efficient co-expression of the antibody light and heavy chain genes. In another example, the 2A polypeptide or fragment thereof is a T2A polypeptide or fragment thereof. In one example, the P2A polypeptide is ATNFSLLKQAGDVEENPGP (SEQ ID NO: 20). In another example, the F2A polypeptide is APVKQTLNFDLLKLAGDVESNPGP (SEQ ID NO: 21). In another example, the E2A polypeptide is QCTNYALLKLAGDVESNPGP (SEQ ID NO: 22). In another example, the T2A polypeptide is EGRGSLLTCGDVEENPGP (SEQ ID NO: 23). In one example, cleavage of a 2A polypeptide such as T2A (EGRGSLLTCGDVEENPGP; SEQ ID NO: 23) occurs at the last two amino acids, G and P. In one example, if there is no furin cleavage sequence or variant thereof in front of the 2A polypeptide, the amino acid sequence EGRGSLLTCGDVEENPG (SEQ ID NO: 24) is linked to the first portion of the antigen binding molecule (e.g., the light chain of an antibody). In one example, the second polynucleotide is located downstream of the first polynucleotide.

[0068] In one example, the expression system of antigen binding molecules as herein described comprises the 4th polynucleotide of the second part of encoding antigen binding molecules.In one example, the second part of antigen binding molecules is the heavy chain of antibody or other protein constructs.In another example, the second part of antigen binding molecules is the heavy chain of antibody or other protein constructs.In one example, the 4th polynucleotide is located downstream of the 3rd polynucleotide.In one example, when the first and second cleavage sites are cut, the antigen binding molecules comprising the first and second parts of antigen binding molecules are released.

[0069] In one aspect, the present disclosure relates to an expression system for an antigen binding molecule, wherein the antigen binding molecule is secreted or membrane-bound, comprising:

[0070] - a first polynucleotide encoding a first portion of an antigen binding molecule;

[0071] - a second polynucleotide encoding a first cleavage site comprising the amino acid sequence ZR-X1-X2-R-X3-X4-X5-X6 (SEQ ID NO: 256), wherein the second polynucleotide is located downstream of the first polynucleotide;

[0072] - a third polynucleotide encoding a second cleavage site comprising the 2A polypeptide or a fragment thereof, wherein the third polynucleotide is located downstream of the second polynucleotide;

[0073] - a fourth polynucleotide encoding the second portion of the antigen binding molecule, wherein the fourth polynucleotide is located downstream of the third polynucleotide;

[0074] - a fifth polynucleotide encoding a third cleavage site comprising a furin cleavage sequence variant, the variant comprising:

[0075] (i) the furin consensus sequence RXKR (SEQ ID NO: 18) and at least the first five amino acids from the 2A polypeptide; or

[0076] (ii) the furin consensus sequence RXRR (SEQ ID NO: 19) and at least the first five amino acids from the 2A polypeptide,

[0077] wherein the fifth polynucleotide is located downstream of the fourth polynucleotide; and

[0078] - a sixth polynucleotide encoding a membrane-anchored polypeptide, wherein the sixth polynucleotide is located downstream of the fifth polynucleotide;

[0079] wherein when the first, second and third cleavage sites are cleaved, a secreted antigen-binding molecule comprising the first and second portions of the antigen-binding molecule is released;

[0080] wherein when the first and second cleavage sites are cleaved and the third cleavage site is not cleaved, the membrane-bound antigen-binding molecule comprising the first and second parts of the antigen-binding molecule, the third cleavage site and the membrane-anchored polypeptide is released,

[0081] where Z is R, P, G, A, K, N, Q, H, I, L, M, F, S, T, W, Y or V,

[0082] wherein X1, X2, X3, X4, X5, and X6 are R, P, G, A, D, C, E, K, N, Q, H, I, L, M, F, S, T, W, Y, or V, and

[0083] wherein X is any amino acid.

[0084] In one example of the aforementioned aspects, the present disclosure relates to an expression system for an antigen binding molecule, wherein the antigen binding molecule is secreted or membrane-bound, comprising:

[0085] - a first polynucleotide encoding a first portion of an antigen binding molecule;

[0086] - a second polynucleotide encoding a first cleavage site comprising the amino acid sequence RR-X1-X2-R-X3-X4-X5-X6 (SEQ ID NO: 1), wherein the second polynucleotide is located downstream of the first polynucleotide;

[0087] - a third polynucleotide encoding a second cleavage site comprising the 2A polypeptide or a fragment thereof, wherein the third polynucleotide is located downstream of the second polynucleotide;

[0088] - a fourth polynucleotide encoding the second portion of the antigen binding molecule, wherein the fourth polynucleotide is located downstream of the third polynucleotide;

[0089] - a fifth polynucleotide encoding a third cleavage site comprising a furin cleavage sequence variant, the variant comprising:

[0090] (i) the furin consensus sequence RXKR (SEQ ID NO: 18) and at least the first five amino acids from the 2A polypeptide; or

[0091] (ii) the furin consensus sequence RXRR (SEQ ID NO: 19) and at least the first five amino acids from the 2A polypeptide,

[0092] wherein the fifth polynucleotide is located downstream of the fourth polynucleotide; and

[0093] - a sixth polynucleotide encoding a membrane-anchored polypeptide, wherein the sixth polynucleotide is located downstream of the fifth polynucleotide;

[0094] wherein when the first, second and third cleavage sites are cleaved, a secreted antigen-binding molecule comprising the first and second portions of the antigen-binding molecule is released;

[0095] Wherein when the first and second cleavage sites are cleaved and the third cleavage site is not cleaved, the membrane-bound antigen-binding molecule comprising the first and second parts of the antigen-binding molecule, the third cleavage site and the membrane-anchored polypeptide is released.

[0096] In one example, an antigen-binding molecule produced by or derived from an expression system disclosed herein can be secreted from a cell and can be referred to as a "secreted antigen-binding molecule." In one example, the term "secreted antigen-binding molecule" as used herein refers to an antigen-binding molecule that can be secreted from a cell, wherein the antigen-binding molecule comprises the first and second parts of an antigen-binding molecule derived from an expression system disclosed herein.

[0097] In one example, the antigen binding molecules produced by or derived from the expression system disclosed herein can be anchored or bound to the cell membrane and can be referred to as "membrane-bound antigen binding molecules". In one example, the term "membrane-bound antigen binding molecules" used herein refers to antigen binding molecules that can be anchored or bound to the cell membrane, wherein the antigen binding molecules comprise the first and second parts of the antigen binding molecules, the third cleavage site and a membrane-anchored polypeptide derived from the expression system disclosed herein. The level of membrane-bound antigen binding molecules is determined by the display level. The term "display level" used herein refers to the expression of membrane-bound antigen binding molecules on the cell surface, wherein the membrane-bound antigen binding molecules comprise the first and second parts of the antigen binding molecules.

[0098] In one example, the antigen binding molecules produced by expression system disclosed herein or derived from this expression system can be the antigen binding molecules retained in the cell, wherein it is not secreted from the cell or is captured on the cell surface. In one example, if the signal peptide on the antigen binding molecules is removed, the antigen binding molecules can remain in the cell. In one example, if the furin consensus sequence or its variant between the second portion (such as the heavy chain of antibody) and the membrane anchored polypeptide (such as GPI) of the antigen binding molecules is removed, the antigen binding molecules can remain in the cell. In one example, if the furin consensus sequence or its variant between the second portion (such as the heavy chain of antibody) and the membrane anchored polypeptide (such as GPI) of the antigen binding molecules is eliminated or the cleavage function of its variant is eliminated, the antigen binding molecules can remain in the cell. In one example, the furin consensus sequence or its variant and GPI affect the amount of the antigen binding molecules captured by the cell surface.

[0099] In one example, the expression system of the antigen binding molecule includes a fifth polynucleotide encoding a third cleavage site comprising a furin cleavage sequence variant, wherein the antigen binding molecule is secreted or membrane-bound. In one example, the third cleavage site comprises a furin cleavage sequence variant, the variant comprising a furin consensus sequence RXKR (SEQ ID NO: 18) or RXRR (SEQ ID NO: 19) and at least the first amino acid derived from a 2A polypeptide, at least the first two amino acids derived from a 2A polypeptide, at least the first three amino acids derived from a 2A polypeptide, at least the first four amino acids derived from a 2A polypeptide, or at least the first five amino acids derived from a 2A polypeptide. In one example, the third cleavage site comprises a furin cleavage sequence variant, the variant comprising a furin consensus sequence RXKR (SEQ ID NO: 18) or RXRR (SEQ ID NO: 19) and at least the first five amino acids derived from a 2A polypeptide.

[0100] In one example, the expression system of the antigen binding molecule comprises a sixth polynucleotide encoding a membrane-anchored polypeptide, wherein the antigen binding molecule is secreted or membrane-bound. In one example, the sixth polynucleotide is located downstream of the fifth polynucleotide. In one example, when the first, second, and third cleavage sites are cut, the secretory antigen binding molecules comprising the first and second parts of the antigen binding molecule are released. In one example, when the first and second cleavage sites are cut and the third cleavage site is not cut, the membrane-bound antigen binding molecules comprising the first and second parts of the antigen binding molecule, the third cleavage site, and the membrane-anchored polypeptide are released. In one example, the fifth polynucleotide does not need to be connected to the 2A peptide to simultaneously display and secrete the antibody.

[0101] In one example, the expression system of the antigen binding molecules described herein comprises a second polynucleotide encoding a first cleavage site comprising the amino acid sequence ZR-X1-X2-R-X3-X4-X5-X6 (SEQ ID NO: 256). In one example, Z is arginine (R), proline (P), glycine (G), alanine (A), lysine (K), asparagine (N), glutamine (Q), histidine (H), isoleucine (I), leucine (L), methionine (M), phenylalanine (F), serine (S), threonine (T), tryptophan (W), tyrosine (Y), or valine (V); X3 is any one of serine (S), alanine (A), aspartic acid (D), or glutamic acid (E); X4 is any one of valine (V), alanine (A), isoleucine (I), leucine (L), or threonine (T); X5 is any one of arginine (R), proline (P), glycine (G), alanine (A), aspartic acid (D), cysteine ​​(C), glutamic acid (E), or threonine (T); In one embodiment, Z is an amino acid residue of the present invention, wherein the amino acid residue of the present invention ... In one example, the expression system of the antigen binding molecules described herein includes a second polynucleotide encoding a first cleavage site, wherein the first cleavage site includes an amino acid sequence RR-X1-X2-R-X3-X4-X5-X6 (SEQ ID NO: 1). In one example, X1-X6 can be any amino acid. In one example, X1 is arginine (R), X2 is lysine (K) or arginine (R), and X3, X4, X5 and X6 are any of proline (P), glycine (G), alanine (A), aspartic acid (D), cysteine ​​(C), glutamic acid (E), lysine (K), asparagine (N), glutamine (Q), histidine (H), isoleucine (I), leucine (L), methionine (M), phenylalanine (F), serine (S), threonine (T), tryptophan (W), tyrosine (Y) or valine (V).In one example, X1 is arginine (R), X2 is lysine (K) or arginine (R), X3 is serine (S) or alanine (A), X4 is valine (V) or leucine (L), X5 is aspartic acid (D) or serine (S), and X6 is threonine (T) or leucine (L). In one example, X3 is any one of proline (P), glycine (G), alanine (A), aspartic acid (D), glutamic acid (E), histidine (H), isoleucine (I), leucine (L), methionine (M), phenylalanine (F), or serine (S); X4 is any one of proline (P), alanine (A), isoleucine (I), leucine (L), threonine (T), or valine (V); X5 is any one of arginine (R), proline (P), glycine (G), alanine (A), aspartic acid (D), cysteine ​​(C), glutamic acid (E), lysine (K), asparagine (N), glutamine (Q), histidine (H), or threonine (T). ), isoleucine (I), leucine (L), methionine (M), phenylalanine (F), serine (S), threonine (T), tryptophan (W), tyrosine (Y) or valine (V); and X6 is any one of arginine (R), proline (P), glycine (G), alanine (A), aspartic acid (D), cysteine ​​(C), glutamic acid (E), lysine (K), asparagine (N), glutamine (Q), histidine (H), isoleucine (I), leucine (L), methionine (M), phenylalanine (F), serine (S), threonine (T), tryptophan (W), tyrosine (Y) or valine (V).In one example, Z is any one of arginine (R), proline (P), glycine (G), alanine (A), lysine (K), asparagine (N), glutamine (Q), histidine (H), isoleucine (I), leucine (L), methionine (M), phenylalanine (F), serine (S), threonine (T), tryptophan (W), tyrosine (Y), or valine (V); X3 is any one of serine (S), alanine (A), aspartic acid (D), or glutamic acid (E); X4 is any one of valine (V), alanine (A), isoleucine (I), leucine (L), or threonine (T); X5 is any one of arginine (R), proline (P), glycine (G), alanine (A), aspartic acid (D), cysteine ​​(C), or threonine (T); , glutamic acid (E), lysine (K), asparagine (N), glutamine (Q), histidine (H), isoleucine (I), leucine (L), methionine (M), phenylalanine (F), serine (S), threonine (T), tryptophan (W), tyrosine (Y) or valine (V); and X6 is any one of arginine (R), proline (P), glycine (G), alanine (A), aspartic acid (D), cysteine ​​(C), glutamic acid (E), lysine (K), asparagine (N), glutamine (Q), histidine (H), isoleucine (I), leucine (L), methionine (M), phenylalanine (F), serine (S), threonine (T), tryptophan (W), tyrosine (Y) or valine (V).

[0102] In one example, the first cleavage site encoded by the second polynucleotide comprises the amino acid sequence set forth in SEQ ID NOs: 2-17 and SEQ ID NOs: 66-160. In one example, the first cleavage site encoded by the second polynucleotide comprises the amino acid sequence set forth in SEQ ID NO: 2. In one example, the first cleavage site encoded by the second polynucleotide comprises the amino acid sequence set forth in SEQ ID NO: 3. In one example, the first cleavage site encoded by the second polynucleotide comprises the amino acid sequence set forth in SEQ ID NO: 4. In one example, the first cleavage site encoded by the second polynucleotide comprises the amino acid sequence set forth in SEQ ID NO: 5. In one example, the first cleavage site encoded by the second polynucleotide comprises the amino acid sequence set forth in SEQ ID NO: 6. In one example, the first cleavage site encoded by the second polynucleotide comprises the amino acid sequence set forth in SEQ ID NO: 7. In one example, the first cleavage site encoded by the second polynucleotide comprises the amino acid sequence set forth in SEQ ID NO: 8. In one example, the first cleavage site encoded by the second polynucleotide comprises the amino acid sequence set forth in SEQ ID NO: 9. In one example, the first cleavage site encoded by the second polynucleotide comprises the amino acid sequence set forth in SEQ ID NO: 10. In one example, the first cleavage site encoded by the second polynucleotide comprises the amino acid sequence set forth in SEQ ID NO: 11. In one example, the first cleavage site encoded by the second polynucleotide comprises the amino acid sequence set forth in SEQ ID NO: 12. In one example, the first cleavage site encoded by the second polynucleotide comprises the amino acid sequence set forth in SEQ ID NO: 13. In one example, the first cleavage site encoded by the second polynucleotide comprises the amino acid sequence set forth in SEQ ID NO: 14. In one example, the first cleavage site encoded by the second polynucleotide comprises the amino acid sequence set forth in SEQ ID NO: 15. In one example, the first cleavage site encoded by the second polynucleotide comprises the amino acid sequence set forth in SEQ ID NO: 16. In one example, the first cleavage site encoded by the second polynucleotide comprises the amino acid sequence set forth in SEQ ID NO: 17. In one example, the first cleavage site encoded by the second polynucleotide comprises the amino acid sequence set forth in SEQ ID NO: 66. In one example, the first cleavage site encoded by the second polynucleotide comprises the amino acid sequence set forth in SEQ ID NO: 67. In one example, the first cleavage site encoded by the second polynucleotide comprises the amino acid sequence set forth in SEQ ID NO: 68. In one example, the first cleavage site encoded by the second polynucleotide comprises the amino acid sequence shown in SEQ ID NO:69.In one example, the first cleavage site encoded by the second polynucleotide comprises the amino acid sequence set forth in SEQ ID NO: 70. In one example, the first cleavage site encoded by the second polynucleotide comprises the amino acid sequence set forth in SEQ ID NO: 71. In one example, the first cleavage site encoded by the second polynucleotide comprises the amino acid sequence set forth in SEQ ID NO: 72. In one example, the first cleavage site encoded by the second polynucleotide comprises the amino acid sequence set forth in SEQ ID NO: 73. In one example, the first cleavage site encoded by the second polynucleotide comprises the amino acid sequence set forth in SEQ ID NO: 74. In one example, the first cleavage site encoded by the second polynucleotide comprises the amino acid sequence set forth in SEQ ID NO: 75. In one example, the first cleavage site encoded by the second polynucleotide comprises the amino acid sequence set forth in SEQ ID NO: 76. In one example, the first cleavage site encoded by the second polynucleotide comprises the amino acid sequence set forth in SEQ ID NO: 77. In one example, the first cleavage site encoded by the second polynucleotide comprises the amino acid sequence set forth in SEQ ID NO: 78. In one example, the first cleavage site encoded by the second polynucleotide comprises the amino acid sequence set forth in SEQ ID NO: 79. In one example, the first cleavage site encoded by the second polynucleotide comprises the amino acid sequence set forth in SEQ ID NO: 80. In one example, the first cleavage site encoded by the second polynucleotide comprises the amino acid sequence set forth in SEQ ID NO: 81. In one example, the first cleavage site encoded by the second polynucleotide comprises the amino acid sequence set forth in SEQ ID NO: 82. In one example, the first cleavage site encoded by the second polynucleotide comprises the amino acid sequence set forth in SEQ ID NO: 83. In one example, the first cleavage site encoded by the second polynucleotide comprises the amino acid sequence set forth in SEQ ID NO: 84. In one example, the first cleavage site encoded by the second polynucleotide comprises the amino acid sequence set forth in SEQ ID NO: 85. In one example, the first cleavage site encoded by the second polynucleotide comprises the amino acid sequence set forth in SEQ ID NO: 86. In one example, the first cleavage site encoded by the second polynucleotide comprises the amino acid sequence set forth in SEQ ID NO: 87. In one example, the first cleavage site encoded by the second polynucleotide comprises the amino acid sequence set forth in SEQ ID NO: 88. In one example, the first cleavage site encoded by the second polynucleotide comprises the amino acid sequence set forth in SEQ ID NO: 89. In one example, the first cleavage site encoded by the second polynucleotide comprises the amino acid sequence shown in SEQ ID NO:90.In one example, the first cleavage site encoded by the second polynucleotide comprises the amino acid sequence set forth in SEQ ID NO: 91. In one example, the first cleavage site encoded by the second polynucleotide comprises the amino acid sequence set forth in SEQ ID NO: 92. In one example, the first cleavage site encoded by the second polynucleotide comprises the amino acid sequence set forth in SEQ ID NO: 93. In one example, the first cleavage site encoded by the second polynucleotide comprises the amino acid sequence set forth in SEQ ID NO: 94. In one example, the first cleavage site encoded by the second polynucleotide comprises the amino acid sequence set forth in SEQ ID NO: 95. In one example, the first cleavage site encoded by the second polynucleotide comprises the amino acid sequence set forth in SEQ ID NO: 96. In one example, the first cleavage site encoded by the second polynucleotide comprises the amino acid sequence set forth in SEQ ID NO: 97. In one example, the first cleavage site encoded by the second polynucleotide comprises the amino acid sequence set forth in SEQ ID NO: 98. In one example, the first cleavage site encoded by the second polynucleotide comprises the amino acid sequence set forth in SEQ ID NO: 99. In one example, the first cleavage site encoded by the second polynucleotide comprises the amino acid sequence set forth in SEQ ID NO: 100. In one example, the first cleavage site encoded by the second polynucleotide comprises the amino acid sequence set forth in SEQ ID NO: 101. In one example, the first cleavage site encoded by the second polynucleotide comprises the amino acid sequence set forth in SEQ ID NO: 102. In one example, the first cleavage site encoded by the second polynucleotide comprises the amino acid sequence set forth in SEQ ID NO: 103. In one example, the first cleavage site encoded by the second polynucleotide comprises the amino acid sequence set forth in SEQ ID NO: 104. In one example, the first cleavage site encoded by the second polynucleotide comprises the amino acid sequence set forth in SEQ ID NO: 105. In one example, the first cleavage site encoded by the second polynucleotide comprises the amino acid sequence set forth in SEQ ID NO: 106. In one example, the first cleavage site encoded by the second polynucleotide comprises the amino acid sequence set forth in SEQ ID NO: 107. In one example, the first cleavage site encoded by the second polynucleotide comprises the amino acid sequence set forth in SEQ ID NO: 108. In one example, the first cleavage site encoded by the second polynucleotide comprises the amino acid sequence set forth in SEQ ID NO: 109. In one example, the first cleavage site encoded by the second polynucleotide comprises the amino acid sequence set forth in SEQ ID NO: 110. In one example, the first cleavage site encoded by the second polynucleotide comprises the amino acid sequence shown in SEQ ID NO:111.In one example, the first cleavage site encoded by the second polynucleotide comprises the amino acid sequence set forth in SEQ ID NO: 112. In one example, the first cleavage site encoded by the second polynucleotide comprises the amino acid sequence set forth in SEQ ID NO: 113. In one example, the first cleavage site encoded by the second polynucleotide comprises the amino acid sequence set forth in SEQ ID NO: 114. In one example, the first cleavage site encoded by the second polynucleotide comprises the amino acid sequence set forth in SEQ ID NO: 115. In one example, the first cleavage site encoded by the second polynucleotide comprises the amino acid sequence set forth in SEQ ID NO: 116. In one example, the first cleavage site encoded by the second polynucleotide comprises the amino acid sequence set forth in SEQ ID NO: 117. In one example, the first cleavage site encoded by the second polynucleotide comprises the amino acid sequence set forth in SEQ ID NO: 118. In one example, the first cleavage site encoded by the second polynucleotide comprises the amino acid sequence set forth in SEQ ID NO: 119. In one example, the first cleavage site encoded by the second polynucleotide comprises the amino acid sequence set forth in SEQ ID NO: 120. In one example, the first cleavage site encoded by the second polynucleotide comprises the amino acid sequence set forth in SEQ ID NO: 121. In one example, the first cleavage site encoded by the second polynucleotide comprises the amino acid sequence set forth in SEQ ID NO: 122. In one example, the first cleavage site encoded by the second polynucleotide comprises the amino acid sequence set forth in SEQ ID NO: 123. In one example, the first cleavage site encoded by the second polynucleotide comprises the amino acid sequence set forth in SEQ ID NO: 124. In one example, the first cleavage site encoded by the second polynucleotide comprises the amino acid sequence set forth in SEQ ID NO: 125. In one example, the first cleavage site encoded by the second polynucleotide comprises the amino acid sequence set forth in SEQ ID NO: 126. In one example, the first cleavage site encoded by the second polynucleotide comprises the amino acid sequence set forth in SEQ ID NO: 127. In one example, the first cleavage site encoded by the second polynucleotide comprises the amino acid sequence set forth in SEQ ID NO: 128. In one example, the first cleavage site encoded by the second polynucleotide comprises the amino acid sequence set forth in SEQ ID NO: 129. In one example, the first cleavage site encoded by the second polynucleotide comprises the amino acid sequence set forth in SEQ ID NO: 130. In one example, the first cleavage site encoded by the second polynucleotide comprises the amino acid sequence set forth in SEQ ID NO: 131. In one example, the first cleavage site encoded by the second polynucleotide comprises the amino acid sequence shown in SEQ ID NO:132.In one example, the first cleavage site encoded by the second polynucleotide comprises the amino acid sequence set forth in SEQ ID NO: 133. In one example, the first cleavage site encoded by the second polynucleotide comprises the amino acid sequence set forth in SEQ ID NO: 134. In one example, the first cleavage site encoded by the second polynucleotide comprises the amino acid sequence set forth in SEQ ID NO: 135. In one example, the first cleavage site encoded by the second polynucleotide comprises the amino acid sequence set forth in SEQ ID NO: 136. In one example, the first cleavage site encoded by the second polynucleotide comprises the amino acid sequence set forth in SEQ ID NO: 137. In one example, the first cleavage site encoded by the second polynucleotide comprises the amino acid sequence set forth in SEQ ID NO: 138. In one example, the first cleavage site encoded by the second polynucleotide comprises the amino acid sequence set forth in SEQ ID NO: 139. In one example, the first cleavage site encoded by the second polynucleotide comprises the amino acid sequence set forth in SEQ ID NO: 140. In one example, the first cleavage site encoded by the second polynucleotide comprises the amino acid sequence set forth in SEQ ID NO: 141. In one example, the first cleavage site encoded by the second polynucleotide comprises the amino acid sequence set forth in SEQ ID NO: 142. In one example, the first cleavage site encoded by the second polynucleotide comprises the amino acid sequence set forth in SEQ ID NO: 143. In one example, the first cleavage site encoded by the second polynucleotide comprises the amino acid sequence set forth in SEQ ID NO: 144. In one example, the first cleavage site encoded by the second polynucleotide comprises the amino acid sequence set forth in SEQ ID NO: 145. In one example, the first cleavage site encoded by the second polynucleotide comprises the amino acid sequence set forth in SEQ ID NO: 146. In one example, the first cleavage site encoded by the second polynucleotide comprises the amino acid sequence set forth in SEQ ID NO: 147. In one example, the first cleavage site encoded by the second polynucleotide comprises the amino acid sequence set forth in SEQ ID NO: 148. In one example, the first cleavage site encoded by the second polynucleotide comprises the amino acid sequence set forth in SEQ ID NO: 149. In one example, the first cleavage site encoded by the second polynucleotide comprises the amino acid sequence set forth in SEQ ID NO: 150. In one example, the first cleavage site encoded by the second polynucleotide comprises the amino acid sequence set forth in SEQ ID NO: 151. In one example, the first cleavage site encoded by the second polynucleotide comprises the amino acid sequence set forth in SEQ ID NO: 152. In one example, the first cleavage site encoded by the second polynucleotide comprises the amino acid sequence shown in SEQ ID NO:153.In one example, the first cleavage site encoded by the second polynucleotide comprises the amino acid sequence set forth in SEQ ID NO: 154. In one example, the first cleavage site encoded by the second polynucleotide comprises the amino acid sequence set forth in SEQ ID NO: 155. In one example, the first cleavage site encoded by the second polynucleotide comprises the amino acid sequence set forth in SEQ ID NO: 156. In one example, the first cleavage site encoded by the second polynucleotide comprises the amino acid sequence set forth in SEQ ID NO: 157. In one example, the first cleavage site encoded by the second polynucleotide comprises the amino acid sequence set forth in SEQ ID NO: 158. In one example, the first cleavage site encoded by the second polynucleotide comprises the amino acid sequence set forth in SEQ ID NO: 159. In one example, the first cleavage site encoded by the second polynucleotide comprises the amino acid sequence set forth in SEQ ID NO: 160. In one example, the first cleavage site encoded by the second polynucleotide comprises the amino acid sequence set forth in SEQ ID NOs: 2-17, 66-70, 74-84, 86, 87, 91, 93, 95-99, 104, 106, 115, 116, 120, 123-129, and 130- 160. In one example, the first cleavage site encoded by the second polynucleotide comprises the amino acid sequence set forth in SEQ ID NOs: 2, 87, 91, 93, 106, 115, 116, and 120.

[0103] In one example, the expression system of the antigen binding molecules described herein further comprises a polynucleotide encoding the linker Serine-Glycine-Serine-Glycine (SGSG) between the second and third polynucleotides.

[0104] In one example, the third cleavage site of the fifth polynucleotide of the expression system described herein comprises a furin cleavage sequence variant comprising the furin consensus sequence RXKR (SEQ ID NO: 18). In one example, the third cleavage site of the fifth polynucleotide of the expression system described herein comprises a furin cleavage sequence variant comprising the furin consensus sequence RXRR (SEQ ID NO: 19). In one example, the third cleavage site of the fifth polynucleotide of the expression system described herein comprises a furin cleavage sequence variant comprising the furin consensus sequence RXKR (SEQ ID NO: 18) and at least the first five amino acids derived from a 2A polypeptide. In one example, the third cleavage site of the fifth polynucleotide of the expression system described herein comprises a furin cleavage sequence variant comprising the furin consensus sequence RXRR (SEQ ID NO: 19) and at least the first five amino acids derived from a 2A polypeptide. In one example, the third cleavage site of the fifth polynucleotide of the expression system described herein comprises a furin cleavage sequence variant comprising the furin consensus sequence RXKR (SEQ ID NO: 18) and the first five amino acids derived from a 2A polypeptide. In one example, the third cleavage site of the fifth polynucleotide of the expression system described herein comprises a furin cleavage sequence variant comprising the furin consensus sequence RXRR (SEQ ID NO: 19) and the first five amino acids derived from a 2A polypeptide. In one example, the first five amino acids derived from a 2A polypeptide comprise one or more point mutations. In one example, the third cleavage site comprises a furin cleavage sequence variant comprising the furin consensus sequence RXKR (SEQ ID NO: 18) or RXRR (SEQ ID NO: 19) and the first five amino acids derived from a 2A polypeptide, wherein the first five amino acids derived from a 2A polypeptide comprise one or more point mutations. In one example, the first five amino acids derived from a 2A polypeptide comprise one point mutation. In one example, the first five amino acids derived from a 2A polypeptide comprise two point mutations. In one example, the first five amino acids derived from a 2A polypeptide comprise three point mutations. In one example, the first five amino acids derived from a 2A polypeptide comprise four point mutations. In one example, the first five amino acids derived from a 2A polypeptide comprise five point mutations. In one example, the first five amino acids are derived from a 2A polypeptide selected from the group consisting of P2A, F2A, E2A, and T2A. In one example, the first five amino acids are derived from P2A. In one example, the first five amino acids are derived from T2A.In one example, the first five amino acids are from P2A, and one or more point mutations are selected from the group consisting of A1P, A1G, T2G, T2P, N3P, N3A, F4P, F4A, and S5P, as shown in Table 1 below. In one example, the FCS variants listed in Table 1 are positioned between the antibody heavy chain and the membrane anchor molecule for simultaneous display and secretion of the antibody. In one example, the fifth polynucleotide is positioned downstream of the fourth polynucleotide. It should also be understood that a 2A polypeptide fragment refers to a segment of a 2A polypeptide disclosed herein. In one example, a 2A polypeptide fragment comprises a segment of at least 3 amino acids from a 2A polypeptide. In another example, a 2A polypeptide fragment includes, but is not limited to, a segment of about 3 to about 10 amino acids, or about 3, 4, 5, 6, 7, 8, 9, or 10 amino acids from a 2A polypeptide. In another example, a 2A polypeptide fragment is selected from P2A, F2A, E2A, and T2A fragments thereof. In another example, the 2A polypeptide fragment is a P2A polypeptide fragment. In another example, the 2A polypeptide fragment is a T2A polypeptide fragment. In one example, the 2A polypeptide fragment can be the first 3-10 amino acids of a 2A polypeptide disclosed herein, or the last 3-10 amino acids of a 2A polypeptide disclosed herein. In another example, the 2A polypeptide fragment is the first five amino acids of a P2A, F2A, E2A, or T2A polypeptide. In another example, the P2A polypeptide fragment is the first five amino acids of a P2A polypeptide. In another example, the T2A polypeptide fragment is the first five amino acids of a T2A polypeptide. In one example, the term "point mutation" refers to a mutation that replaces, inserts, or deletes a single amino acid from an amino acid sequence.

[0105] Table 1

[0106]

[0107] In one example, the membrane anchor polypeptide encoded by the sixth polynucleotide comprises a glycophospholipid transmembrane domain (GPI), a platelet-derived growth factor receptor (PDGFR) β chain transmembrane domain (PTM) or an immunoglobulin C2 type extracellular-transmembrane-cytoplasmic domain of a mouse B7-1 antigen. In another example, the membrane anchor polypeptide is a glycophospholipid transmembrane domain (GPI). The terms used herein are not limited to "glycophospholipid transmembrane domain", "glycophospholipid membrane anchor molecule", "GM" or "GPI" and refer to membrane proteins that are anchored to the cell membrane by a structure comprising phosphatidylinositol, a carbohydrate and ethanolamine. In one example, the glycophospholipid transmembrane domain (GPI) is a glycosylphosphatidylinositol membrane anchor molecule derived from human decay accelerating factor.

[0108] In one example, the second polynucleotide encoding of the expression system disclosed herein has a first cleavage site with high or improved cleavage efficiency. In one example, the second polynucleotide encoding first cleavage site has a higher cleavage efficiency of at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% than an expression system comprising a second polynucleotide encoding a first cleavage site comprising a furin consensus sequence RXKR (SEQ ID NO: 18) or RXRR (SEQ ID NO: 19).

[0109] In one example, the expression system of the antigen binding molecules described herein can improve the accuracy of producing or releasing antigen binding molecules, and the antigen binding molecules include the first part of the antigen binding molecules with the correct polypeptide sequence. In one example, the expression system disclosed herein can release antigen binding molecules, and the antigen binding molecules include the first part of the antigen binding molecules with the correct polypeptide sequence, wherein the first part of the antigen binding molecules does not contain one or more residual amino acids from the second cleavage site after cleavage at the first and second cleavage sites.

[0110] In one example, the expression system of the antigen binding molecules described herein can improve the homogeneity of the generated antigen binding molecules. In one example, the term "homogeneity" refers to the fact that cleavage at the first cleavage site results in a major species of the first part of the antigen binding molecule (e.g., Figure 3D and Figure 3G Instead of resulting in multiple species of the first portion of the antigen binding molecule (e.g., as shown in FIG. 1 ), one major species of antibody light chain without the linked 2A peptide is present. Figure 3E 、 Figure 3F and Figure 3G In one example, the expression system disclosed herein is capable of increasing the homogeneity of the produced antigen-binding molecules, wherein homogeneity is achieved when a plurality of such antigen-binding molecules are produced, wherein each of the plurality of antigen-binding molecules comprises a first portion having the same amino acid sequence and / or molecular weight.

[0111] In one example, the expression system of the antigen binding molecules described herein is delivered to the target cell before transcription. In one example, the target cell is an animal cell, a yeast cell, a plant cell, an insect cell or a fungal cell. In one example, the target cell is an animal cell. In one example, the animal cell is a mammalian cell.

[0112] In one example, the expression system of the antigen binding molecules described herein includes a second polynucleotide having a nucleotide sequence shown in SEQ ID NO:35-50 and SEQ ID NO:161-255. In one example, the expression system of the antigen binding molecules described herein includes a second polynucleotide having a nucleotide sequence shown in SEQ ID NO:35. In one example, the expression system of the antigen binding molecules described herein includes a second polynucleotide having a nucleotide sequence shown in SEQ ID NO:36. In one example, the expression system of the antigen binding molecules described herein includes a second polynucleotide having a nucleotide sequence shown in SEQ ID NO:37. In one example, the expression system of the antigen binding molecules described herein includes a second polynucleotide having a nucleotide sequence shown in SEQ ID NO:38. In one example, the expression system of the antigen binding molecules described herein includes a second polynucleotide having a nucleotide sequence shown in SEQ ID NO:39. In one example, the expression system of the antigen binding molecules described herein includes a second polynucleotide having a nucleotide sequence shown in SEQ ID NO:40. In one example, the expression system of the antigen binding molecules described herein includes a second polynucleotide having a nucleotide sequence shown in SEQ ID NO:41. In one example, the expression system of the antigen binding molecules described herein comprises a second polynucleotide having a nucleotide sequence shown in SEQ ID NO: 42. In one example, the expression system of the antigen binding molecules described herein comprises a second polynucleotide having a nucleotide sequence shown in SEQ ID NO: 43. In one example, the expression system of the antigen binding molecules described herein comprises a second polynucleotide having a nucleotide sequence shown in SEQ ID NO: 44. In one example, the expression system of the antigen binding molecules described herein comprises a second polynucleotide having a nucleotide sequence shown in SEQ ID NO: 45. In one example, the expression system of the antigen binding molecules described herein comprises a second polynucleotide having a nucleotide sequence shown in SEQ ID NO: 46. In one example, the expression system of the antigen binding molecules described herein comprises a second polynucleotide having a nucleotide sequence shown in SEQ ID NO: 47. In one example, the expression system of the antigen binding molecules described herein comprises a second polynucleotide having a nucleotide sequence shown in SEQ ID NO: 48. In one example, the expression system of the antigen binding molecules described herein comprises a second polynucleotide having a nucleotide sequence shown in SEQ ID NO: 49. In one example, the expression system of the antigen binding molecules described herein comprises a second polynucleotide having a nucleotide sequence shown in SEQ ID NO: 50. In one example, the expression system of the antigen-binding molecule described herein comprises a second polynucleotide having a nucleotide sequence shown in SEQ ID NO: 161.In one example, the expression system of the antigen binding molecules described herein includes a second polynucleotide having a nucleotide sequence shown in SEQ ID NO: 162. In one example, the expression system of the antigen binding molecules described herein includes a second polynucleotide having a nucleotide sequence shown in SEQ ID NO: 163. In one example, the expression system of the antigen binding molecules described herein includes a second polynucleotide having a nucleotide sequence shown in SEQ ID NO: 164. In one example, the expression system of the antigen binding molecules described herein includes a second polynucleotide having a nucleotide sequence shown in SEQ ID NO: 165. In one example, the expression system of the antigen binding molecules described herein includes a second polynucleotide having a nucleotide sequence shown in SEQ ID NO: 166. In one example, the expression system of the antigen binding molecules described herein includes a second polynucleotide having a nucleotide sequence shown in SEQ ID NO: 167. In one example, the expression system of the antigen binding molecules described herein includes a second polynucleotide having a nucleotide sequence shown in SEQ ID NO: 168. In one example, the expression system of the antigen binding molecules described herein includes a second polynucleotide having a nucleotide sequence shown in SEQ ID NO: 169. In one example, the expression system of the antigen binding molecules described herein includes a second polynucleotide having a nucleotide sequence shown in SEQ ID NO: 170. In one example, the expression system of the antigen binding molecules described herein includes a second polynucleotide having a nucleotide sequence shown in SEQ ID NO: 171. In one example, the expression system of the antigen binding molecules described herein includes a second polynucleotide having a nucleotide sequence shown in SEQ ID NO: 172. In one example, the expression system of the antigen binding molecules described herein includes a second polynucleotide having a nucleotide sequence shown in SEQ ID NO: 173. In one example, the expression system of the antigen binding molecules described herein includes a second polynucleotide having a nucleotide sequence shown in SEQ ID NO: 174. In one example, the expression system of the antigen binding molecules described herein includes a second polynucleotide having a nucleotide sequence shown in SEQ ID NO: 175. In one example, the expression system of the antigen binding molecules described herein includes a second polynucleotide having a nucleotide sequence shown in SEQ ID NO: 176. In one example, the expression system of the antigen binding molecules described herein includes a second polynucleotide having a nucleotide sequence shown in SEQ ID NO: 177. In one example, the expression system of the antigen-binding molecule described herein comprises a second polynucleotide having a nucleotide sequence as shown in SEQ ID NO: 178. In one example, the expression system of the antigen-binding molecule described herein comprises a second polynucleotide having a nucleotide sequence as shown in SEQ ID NO: 179.In one example, the expression system of the antigen binding molecules described herein includes a second polynucleotide having a nucleotide sequence shown in SEQ ID NO: 180. In one example, the expression system of the antigen binding molecules described herein includes a second polynucleotide having a nucleotide sequence shown in SEQ ID NO: 181. In one example, the expression system of the antigen binding molecules described herein includes a second polynucleotide having a nucleotide sequence shown in SEQ ID NO: 182. In one example, the expression system of the antigen binding molecules described herein includes a second polynucleotide having a nucleotide sequence shown in SEQ ID NO: 183. In one example, the expression system of the antigen binding molecules described herein includes a second polynucleotide having a nucleotide sequence shown in SEQ ID NO: 184. In one example, the expression system of the antigen binding molecules described herein includes a second polynucleotide having a nucleotide sequence shown in SEQ ID NO: 185. In one example, the expression system of the antigen binding molecules described herein includes a second polynucleotide having a nucleotide sequence shown in SEQ ID NO: 186. In one example, the expression system of the antigen binding molecules described herein includes a second polynucleotide having a nucleotide sequence shown in SEQ ID NO: 187. In one example, the expression system of the antigen binding molecules described herein includes a second polynucleotide having a nucleotide sequence shown in SEQ ID NO: 188. In one example, the expression system of the antigen binding molecules described herein includes a second polynucleotide having a nucleotide sequence shown in SEQ ID NO: 189. In one example, the expression system of the antigen binding molecules described herein includes a second polynucleotide having a nucleotide sequence shown in SEQ ID NO: 190. In one example, the expression system of the antigen binding molecules described herein includes a second polynucleotide having a nucleotide sequence shown in SEQ ID NO: 191. In one example, the expression system of the antigen binding molecules described herein includes a second polynucleotide having a nucleotide sequence shown in SEQ ID NO: 192. In one example, the expression system of the antigen binding molecules described herein includes a second polynucleotide having a nucleotide sequence shown in SEQ ID NO: 193. In one example, the expression system of the antigen binding molecules described herein includes a second polynucleotide having a nucleotide sequence shown in SEQ ID NO: 194. In one example, the expression system of the antigen binding molecules described herein includes a second polynucleotide having a nucleotide sequence shown in SEQ ID NO: 195. In one example, the expression system of the antigen binding molecule described herein comprises a second polynucleotide having a nucleotide sequence shown in SEQ ID NO: 196. In one example, the expression system of the antigen binding molecule described herein comprises a second polynucleotide having a nucleotide sequence shown in SEQ ID NO: 197.In one example, the expression system of the antigen binding molecules described herein includes a second polynucleotide with a nucleotide sequence shown in SEQ ID NO: 198. In one example, the expression system of the antigen binding molecules described herein includes a second polynucleotide with a nucleotide sequence shown in SEQ ID NO: 199. In one example, the expression system of the antigen binding molecules described herein includes a second polynucleotide with a nucleotide sequence shown in SEQ ID NO: 200. In one example, the expression system of the antigen binding molecules described herein includes a second polynucleotide with a nucleotide sequence shown in SEQ ID NO: 201. In one example, the expression system of the antigen binding molecules described herein includes a second polynucleotide with a nucleotide sequence shown in SEQ ID NO: 202. In one example, the expression system of the antigen binding molecules described herein includes a second polynucleotide with a nucleotide sequence shown in SEQ ID NO: 203. In one example, the expression system of the antigen binding molecules described herein includes a second polynucleotide with a nucleotide sequence shown in SEQ ID NO: 204. In one example, the expression system of the antigen binding molecules described herein includes a second polynucleotide with a nucleotide sequence shown in SEQ ID NO: 205. In one example, the expression system of the antigen binding molecules described herein includes a second polynucleotide with a nucleotide sequence shown in SEQ ID NO: 206. In one example, the expression system of the antigen binding molecules described herein includes a second polynucleotide with a nucleotide sequence shown in SEQ ID NO: 207. In one example, the expression system of the antigen binding molecules described herein includes a second polynucleotide with a nucleotide sequence shown in SEQ ID NO: 208. In one example, the expression system of the antigen binding molecules described herein includes a second polynucleotide with a nucleotide sequence shown in SEQ ID NO: 209. In one example, the expression system of the antigen binding molecules described herein includes a second polynucleotide with a nucleotide sequence shown in SEQ ID NO: 210. In one example, the expression system of the antigen binding molecules described herein includes a second polynucleotide with a nucleotide sequence shown in SEQ ID NO: 211. In one example, the expression system of the antigen binding molecules described herein includes a second polynucleotide with a nucleotide sequence shown in SEQ ID NO: 212. In one example, the expression system of the antigen binding molecules described herein includes a second polynucleotide with a nucleotide sequence shown in SEQ ID NO: 213. In one example, the expression system of the antigen-binding molecule described herein comprises a second polynucleotide having a nucleotide sequence as shown in SEQ ID NO: 214. In one example, the expression system of the antigen-binding molecule described herein comprises a second polynucleotide having a nucleotide sequence as shown in SEQ ID NO: 215.In one example, the expression system of the antigen binding molecules described herein includes a second polynucleotide having a nucleotide sequence shown in SEQ ID NO: 216. In one example, the expression system of the antigen binding molecules described herein includes a second polynucleotide having a nucleotide sequence shown in SEQ ID NO: 217. In one example, the expression system of the antigen binding molecules described herein includes a second polynucleotide having a nucleotide sequence shown in SEQ ID NO: 218. In one example, the expression system of the antigen binding molecules described herein includes a second polynucleotide having a nucleotide sequence shown in SEQ ID NO: 219. In one example, the expression system of the antigen binding molecules described herein includes a second polynucleotide having a nucleotide sequence shown in SEQ ID NO: 220. In one example, the expression system of the antigen binding molecules described herein includes a second polynucleotide having a nucleotide sequence shown in SEQ ID NO: 221. In one example, the expression system of the antigen binding molecules described herein includes a second polynucleotide having a nucleotide sequence shown in SEQ ID NO: 222. In one example, the expression system of the antigen binding molecules described herein includes a second polynucleotide having a nucleotide sequence shown in SEQ ID NO: 223. In one example, the expression system of the antigen binding molecules described herein includes a second polynucleotide having a nucleotide sequence shown in SEQ ID NO: 224. In one example, the expression system of the antigen binding molecules described herein includes a second polynucleotide having a nucleotide sequence shown in SEQ ID NO: 225. In one example, the expression system of the antigen binding molecules described herein includes a second polynucleotide having a nucleotide sequence shown in SEQ ID NO: 226. In one example, the expression system of the antigen binding molecules described herein includes a second polynucleotide having a nucleotide sequence shown in SEQ ID NO: 227. In one example, the expression system of the antigen binding molecules described herein includes a second polynucleotide having a nucleotide sequence shown in SEQ ID NO: 228. In one example, the expression system of the antigen binding molecules described herein includes a second polynucleotide having a nucleotide sequence shown in SEQ ID NO: 229. In one example, the expression system of the antigen binding molecules described herein includes a second polynucleotide having a nucleotide sequence shown in SEQ ID NO: 230. In one example, the expression system of the antigen binding molecules described herein includes a second polynucleotide having a nucleotide sequence shown in SEQ ID NO: 231. In one example, the expression system of the antigen binding molecules described herein comprises a second polynucleotide having a nucleotide sequence shown in SEQ ID NO: 232. In one example, the expression system of the antigen binding molecules described herein comprises a second polynucleotide having a nucleotide sequence shown in SEQ ID NO: 233.In one example, the expression system of the antigen binding molecules described herein includes a second polynucleotide having a nucleotide sequence shown in SEQ ID NO: 234. In one example, the expression system of the antigen binding molecules described herein includes a second polynucleotide having a nucleotide sequence shown in SEQ ID NO: 235. In one example, the expression system of the antigen binding molecules described herein includes a second polynucleotide having a nucleotide sequence shown in SEQ ID NO: 236. In one example, the expression system of the antigen binding molecules described herein includes a second polynucleotide having a nucleotide sequence shown in SEQ ID NO: 237. In one example, the expression system of the antigen binding molecules described herein includes a second polynucleotide having a nucleotide sequence shown in SEQ ID NO: 238. In one example, the expression system of the antigen binding molecules described herein includes a second polynucleotide having a nucleotide sequence shown in SEQ ID NO: 239. In one example, the expression system of the antigen binding molecules described herein includes a second polynucleotide having a nucleotide sequence shown in SEQ ID NO: 240. In one example, the expression system of the antigen binding molecules described herein includes a second polynucleotide having a nucleotide sequence shown in SEQ ID NO: 241. In one example, the expression system of the antigen binding molecules described herein includes a second polynucleotide with a nucleotide sequence shown in SEQ ID NO: 242. In one example, the expression system of the antigen binding molecules described herein includes a second polynucleotide with a nucleotide sequence shown in SEQ ID NO: 243. In one example, the expression system of the antigen binding molecules described herein includes a second polynucleotide with a nucleotide sequence shown in SEQ ID NO: 244. In one example, the expression system of the antigen binding molecules described herein includes a second polynucleotide with a nucleotide sequence shown in SEQ ID NO: 245. In one example, the expression system of the antigen binding molecules described herein includes a second polynucleotide with a nucleotide sequence shown in SEQ ID NO: 246. In one example, the expression system of the antigen binding molecules described herein includes a second polynucleotide with a nucleotide sequence shown in SEQ ID NO: 247. In one example, the expression system of the antigen binding molecules described herein includes a second polynucleotide with a nucleotide sequence shown in SEQ ID NO: 248. In one example, the expression system of the antigen binding molecules described herein includes a second polynucleotide with a nucleotide sequence shown in SEQ ID NO: 249. In one example, the expression system of the antigen-binding molecule described herein comprises a second polynucleotide having a nucleotide sequence as shown in SEQ ID NO: 250. In one example, the expression system of the antigen-binding molecule described herein comprises a second polynucleotide having a nucleotide sequence as shown in SEQ ID NO: 251.In one example, the expression system of the antigen binding molecules described herein comprises a second polynucleotide having a nucleotide sequence shown in SEQ ID NO: 252. In one example, the expression system of the antigen binding molecules described herein comprises a second polynucleotide having a nucleotide sequence shown in SEQ ID NO: 253. In one example, the expression system of the antigen binding molecules described herein comprises a second polynucleotide having a nucleotide sequence shown in SEQ ID NO: 254. In one example, the expression system of the antigen binding molecules described herein comprises a second polynucleotide having a nucleotide sequence shown in SEQ ID NO: 255.

[0113] In one aspect, the present disclosure relates to vectors, including expression systems disclosed herein. As used herein, the term "vector" refers to a means, typically a nucleic acid, for transporting and expressing a target gene in a host cell. For example, vectors can include plasmid vectors, cosmid vectors, or viral vectors, such as phage vectors, adenoviral vectors, retroviral vectors, and adeno-associated viral vectors. Recombinant vectors can be prepared by manipulating plasmids, phages, or viruses known in the art.

[0114] In one aspect, the present disclosure relates to a host cell comprising an expression system disclosed herein or a vector disclosed herein. In one example, a host cell comprising an expression system or vector as disclosed herein is provided. The host cell can be a prokaryotic or eukaryotic host cell. The host cell capable of stably and continuously cloning or expressing the expression system or vector can be any host cell known in the art.

[0115] In one aspect, the present disclosure relates to a kit comprising an expression system disclosed herein, a vector disclosed herein, a host cell disclosed herein, additional buffers and / or reagents required for the use of the kit, and / or instructions for use of the kit and any other relevant information. Those skilled in the art of molecular biology know or can determine the content of buffers and / or reagents commonly used in the art. In one example, the present disclosure relates to a kit comprising an expression system disclosed herein. In one example, the present disclosure relates to a kit comprising a vector disclosed herein. In one example, the present disclosure relates to a kit comprising a host cell disclosed herein. In one example, the expression system, vector or host cell provided in the kit described herein can be provided in a separate container comprising components independently distributed in one or more containers.

[0116] In one aspect, the present disclosure relates to the use of expression systems disclosed herein, vectors disclosed herein, host cells disclosed herein, or kits disclosed herein for screening antibody libraries or antibody production. In one example, the present disclosure relates to the use of expression systems disclosed herein for screening antibody libraries or antibody production. In one example, the present disclosure relates to the use of vectors disclosed herein for screening antibody libraries or producing antibodies. In one example, the present disclosure relates to the use of host cells disclosed herein for screening antibody libraries or producing antibodies. In one example, the present disclosure relates to the use of kits disclosed herein for screening antibody libraries or producing antibodies. In one example, the present disclosure relates to the use of expression systems disclosed herein, vectors disclosed herein, host cells disclosed herein, or kits disclosed herein for screening antibody libraries. In one example, screening antibody libraries includes, but is not limited to, identifying and selecting antibodies with desired properties (e.g., binding specificity or affinity). Those skilled in the art know how to use methods known in the art (e.g., but not limited to SDS-PAGE, ELISA, Western blotting, flow cytometry, and immunohistochemistry) to screen antibody libraries using expression systems, vectors, host cells, or kits disclosed herein. In one example, the present disclosure relates to expression systems disclosed herein, vectors disclosed herein, host cells disclosed herein, or kits disclosed herein for use in producing antibodies. In one example, antibody production using an expression system, vector, host cell, or kit generally includes, but is not limited to, the following steps, such as cloning the antibody gene into an expression vector, and culturing the host cell for protein expression. Those skilled in the art know how to use methods known in the art (such as, but not limited to, antibody cloning, host cell transformation, antibody purification, and quantification) to produce antibodies using expression systems, vectors, host cells, or kits disclosed herein.

[0117] On the one hand, the present disclosure relates to a method for producing one or more secretory antigen binding molecules and / or one or more membrane-bound antigen binding molecules, which is included in culturing host cells disclosed herein under culture conditions suitable for producing one or more secretory antigen binding molecules and / or one or more membrane-bound antigen binding molecules. In one example, the present disclosure relates to a method for producing one or more secretory antigen binding molecules and one or more membrane-bound antigen binding molecules, which is included in culturing host cells disclosed herein under suitable culture conditions, so that one or more secretable antigen binding molecules and / or one or more membrane-bound antigen binding molecules are produced. In one example, the present disclosure relates to a method for producing one or more secretory antigen binding molecules, which is included in culturing host cells disclosed herein under culture conditions suitable for producing one or more secretable antigen binding molecules. In one example, the present disclosure relates to a method for producing one or more membrane-bound antigen binding molecules, which is included in culturing host cells disclosed herein under culture conditions suitable for producing one or more membrane-bound antigen binding molecules.

[0118] In one example, the method for producing one or more secretory antigen binding molecules and / or one or more membrane-bound antigen binding molecules disclosed herein also includes reclaiming one or more secretory antigen binding molecules. In one example, the method for producing one or more secretable antigen binding molecules disclosed herein also includes reclaiming one or more secretory antigen binding molecules. In one example, the method for producing one or more membrane-bound antigen binding molecules disclosed herein also includes reclaiming one or more secretory antigen binding molecules. In one example, reclaiming secretory antigen binding molecules generally includes but is not limited to separating and purifying molecules from culture medium or cell extracts. Those skilled in the art are familiar with conventional methods for reclaiming antigen binding molecules, such as but not limited to protein affinity chromatography, ion exchange chromatography, size exclusion chromatography, precipitation and ultrafiltration.

[0119] In one aspect, the present disclosure relates to a method for detecting the presence of one or more secreted antigen binding molecules or one or more membrane-bound antigen binding molecules, or one or more secretable antigen binding molecules and one or more membrane-bound antigen binding molecules, the method comprising:

[0120] - providing an expression system disclosed herein;

[0121] - delivering the expression system to one or more target cells;

[0122] wherein the target cell transcribes the expression system into one or more amino acid sequences,

[0123] wherein when all cleavage sites in the one or more amino acid sequences are cleaved, the target cell secretes one or more secreted antigen binding molecules comprising the first and second portions of the antigen binding molecule,

[0124] wherein when the first and second cleavage sites of the one or more amino acid sequences are cleaved and the third cleavage site of the one or more amino acid sequences is not cleaved, the one or more membrane-bound antigen-binding molecules comprising the first and second portions of the antigen-binding molecule bind to the surface of the target cell; and

[0125] - detecting the presence or absence of one or more secreted antigen binding molecules and / or one or more membrane-bound antigen binding molecules.

[0126] In one example, the present disclosure relates to a method for detecting the presence of one or more secreted antigen binding molecules and / or one or more membrane-bound antigen binding molecules disclosed herein, wherein:

[0127] (i) detecting the presence or absence of one or more secreted antigen binding molecules using an enzyme-linked immunosorbent assay (ELISA) or a surrogate binding assay; and / or

[0128] (ii) using flow cytometry to detect the presence or absence of one or more membrane-bound antigen binding molecules, wherein the first portion of the antigen binding molecule, the second portion of the antigen binding molecule and / or the antigen specific to the antigen binding molecule are stained prior to the flow cytometry analysis. In one example, one or more secreted antigen binding molecules and / or one or more membrane-bound antigen binding molecules can be detected using any conventional method known in the art. Conventional methods for detecting secreted antigen binding molecules are well known to those skilled in the art, such as, but not limited to, SDS-PAGE, ELISA, Western blotting, immunofluorescence, protein microarrays, and surface plasmon resonance. Conventional methods for detecting membrane-bound antigen binding molecules are also well known to those skilled in the art, such as, but not limited to, flow cytometry, immunohistochemistry, immunoprecipitation, surface plasmon resonance, SDS-PAGE, Western blotting, and fluorescence activated cell sorting (FACS). It will be readily understood by those skilled in the art that flow cytometry analysis comprises passing cells labeled with a fluorescent marker through a laser beam, and using a sensor to detect emitted fluorescence and scattered light. Standard laboratory protocols and commercially available flow cytometers are well known to those skilled in the art and are used to perform flow cytometric analysis for a variety of applications, including immunophenotyping, cell sorting, and the study of various cellular functions.

[0129] As used in this application, the singular forms "a", "an" and "the" include plural references unless the context clearly dictates otherwise. For example, the term "primer" includes a plurality of primers, including mixtures and combinations thereof.

[0130] As used herein, the terms "increase" and "decrease" refer to the relative change in the selected trait or characteristic in a subset of a population compared to the same trait or characteristic present in the entire population. Thus, an increase indicates a change on a positive scale, while a decrease indicates a change on a negative scale. As used herein, the term "change" also refers to the difference between the same trait or characteristic in a separate subset of a population compared to the selected trait or characteristic in the population as a whole. However, this term does not assess the difference observed.

[0131] As used herein, in the context of a concentration of a substance, a size of a substance, a length of time, or other specified value, the term "about" means ±5% of the specified value, or ±4% of the specified value, or ±3% of the specified value, or ±2% of the specified value, or ±1% of the specified value, or ±0.5% of the specified value.

[0132] Throughout this disclosure, certain embodiments can be disclosed in range format. It should be understood that the description of range format is merely for convenience and brevity and should not be construed as a rigid limitation on the disclosed range. Therefore, it should be considered that the description of a range has specifically disclosed all possible subranges and each numerical value within the range. For example, it should be considered that a description of a range such as from 1 to 6 has specifically disclosed subranges 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, and individual digits within the range, such as 1, 2, 3, 4, 5, and 6. This applies regardless of how broad the range is.

[0133] The disclosure described illustratively herein can be suitably implemented in the absence of any one element or elements, a limitation or limitations not specifically disclosed herein. Therefore, for example, the terms "comprise", "include", "contain" etc. should be interpreted expansively without limitation. In addition, the terms and expressions used herein have been used as descriptive and non-restrictive terms, and any equivalents or parts thereof of the features shown and described are not intended to be excluded in the use of these terms and expressions, but it should be recognized that various modifications are possible within the scope of the disclosure required. Therefore, it should be understood that although the disclosure has been specifically disclosed by preferred embodiments and optional features, those skilled in the art can modify and change the disclosure contained in the disclosed herein, and these modifications and changes are considered to be within the scope of the disclosure.

[0134] The present disclosure has been described broadly and generally herein. Each narrower species and subgeneric grouping of the disclosure belonging to a genus also forms part of the present disclosure. This includes description of a genus of the present disclosure accompanied by a proviso or negative limitation removing any subject matter from that genus, regardless of whether the removed material is specifically recited herein.

[0135] Other embodiments are within the following claims and non-limiting examples.In addition, where features or aspects of the disclosure are described in terms of Markush groups, those skilled in the art will recognize that the disclosure is also thereby described in terms of any individual member or subgroup of members of the Markush group. Example

[0136] method

[0137] Cell culture and culture media for maintaining the CHO K1 master cell line (MCL)

[0138] CHO K1 master cell line (MCL) was generated by nucleofection of landing pad vector into CHO K1 cells (ATCC) and screening of single copy integrated clones by Southern blotting. The landing pad vector expresses the hygromycin resistance gene (HYG) using a chimeric promoter (ChiP) consisting of the murine CMV enhancer (M11788), the hCMV core promoter, and the hCMV intron A (M60321). The HYG expression cassette is flanked by FRT3 and FRT. An impaired puromycin resistance gene lacking a start codon ((ATG-)Puro) followed by a simian virus 40 (SV40) polyadenylation signal (pA) was placed downstream of the FRT to select for correct cassette exchange via RMCE ( Figure 1A ). MCLs were confirmed to contain only one copy of the landing pad vector at a single integration site by Southern blotting and targeted locus amplification (TLA) analysis (Cergentis). MCLs were grown in a protein-free medium (maintenance medium) consisting of 50% HyQ PF (GE Healthcare Life Sciences) and 50% CD CHO (ThermoFisher) supplemented with 1 g / L sodium carbonate (Sigma), 6 mM glutamine (Sigma), and 0.1% Pluronic F-68 (Thermo Fisher) at 37°C in a humidified Kuhner shaker (Adolf Kühner Ag) with 8% CO2. Routine subculture was performed every three to four days, with cells plated at 3 × 10 5Cells were seeded at a density of 10 cells / mL in 15 mL of fresh culture medium in a 125 mL shake flask (Corning). Cell density and viability were determined by trypan blue exclusion on a Vi-Cell XR viability analyzer (Beckman Coulter).

[0139] Generation of stable mAb-producing cell lines by recombinase-mediated cassette exchange (RMCE) and random integration

[0140] Using Amaxa SG cell line MCL were co-transfected with the appropriate targeting vector and a vector expressing FLPe using the X kit and procedure FF-137 (Lonza). In each transfection, 1 × 10 7 cells. The transfected cells were then resuspended in 2 mL of maintenance medium preloaded in a 6-well suspension culture plate (NUNC™) and incubated in a static incubator (IncuSafe, Sanyo). 24 hours after transfection, the cells were collected by centrifugation (100 × g, 5 min) and resuspended in 15 mL of protein-free maintenance medium in a 125 mL shake flask at 37 ° C with 8% CO2 in a humidified Kuhner shaker (Adolf Kühner AG). After 4 days, the transfected cells were screened in maintenance medium containing 20 μg / mL puromycin (InvivoGen). Selection was continued for two weeks by passage in selective medium every three to four days. When cell viability recovered to more than 95%, a stably transfected cell pool was considered to have been established.

[0141] The three polycistronic vectors expressing B4GALT1, ST6GAL1, and the combination of these two genes were further transfected into the target pool already expressing the combination of B4GALT1 and ST6GAL1 or the combination of B4GALT1 and MGAT5. The protocol was the same as that described for RMCE, but with slight modifications. In each transfection, 5 μg of linearized plasmid was transfected into 1 × 10 7 After overnight incubation in 2 mL of protein-free medium in a 6-well suspension culture plate (NUNC™), the transfected cells were harvested by centrifugation at 100 × g for 5 minutes and resuspended in 15 mL of protein-free medium supplemented with 20 μg / mL blasticidin (Thermofisher Scientific). Subsequent passages were performed in selection medium every three to four days until cell viability recovered to over 95%.

[0142] Characterization of growth and productivity of stabilization pools

[0143] The cells were shaken at 3 × 10 5 30mL culture was inoculated in a 50mL centrifuge tube (TPP) with a viable cell density of 10 cells / mL, and a 7-day batch feed production was performed on the stable cell pool. 3mL Ex-Cell Advanced CHO Feed 1 (containing glucose) (SAFC, Sigma) and 400μL 45% (w / v) D-glucose (Sigma) were added on the fifth day. Cell density, viability and antibody titer were monitored using a Vi-CellXR viability analyzer (Beckman Coulter) and an IMMAGE 800 immunochemistry system (Beckman Coulter) on the third, fifth and seventh days, respectively. The IMMAGE 800 immunochemistry system utilizes anti-human Fc region antibodies for IgG quantification. The specific mAb productivity (qP) of the culture exponential phase was calculated as the difference in mAb concentration between the fifth and seventh days divided by the total viable cell density (IVCD) determined based on the trapezoidal method. 10 million cells of two groups were collected from each culture on the 5th day for analysis of mRNA and protein levels, respectively. Flow cytometry was performed on a BD FACSCalibur on day 5 to confirm homogeneous expression of DsRed protein in the stable pool. Flow cytometric data were analyzed using FlowJo software. Culture supernatants were harvested on day 7, centrifuged at 5000 × g for 10 min to remove cells, and used for N-glycan analysis.

[0144] SDS-PAGE

[0145] Purified samples were subjected to SDS-PAGE followed by Coomassie blue staining to assess the presence of different species in the samples. NuPAGE in MOPS buffer under reducing and non-reducing conditions was performed according to the manufacturer's protocol. TM 2 μg-3 μg of each sample was resolved on a 4-12% Bis-Tris protein gel (Thermo Fisher Scientific). The gel was then incubated in a fixing buffer (50% methanol and 10% acetic acid) for 10 min and then stained with 0.1% Coomassie blue stain for 10 minutes on an orbital shaker. Destaining was performed using 30% ethanol until the background was clear. Gel images were taken using a ChemiDoc imaging system (Biorad).

[0146] Complete MS

[0147] The molecular masses of all samples were measured using a quadrupole time-of-flight (QTOF) mass analyzer coupled to a reversed-phase LC system. 4 μL (125 ng / μL) of each sample was directly injected onto a C4 column coupled to the QTOF mass spectrometer for analysis, and mass spectrometry acquisition was set to a mass range of 1000 to 4000 m / z. The column temperature was maintained at 60°C. The bound mAb was eluted at a flow rate of 50 μL / min for 12 minutes using the LC gradient shown in Table 3. The first 3 minutes of the 12-minute LC gradient were for on-column desalting, followed by a 9-minute linear gradient for mAb separation and column re-equilibration. The MS method consisted of two phases: during on-column desalting, the ion spray voltage (ISVF) was set to 0 V for 3 minutes to avoid spraying salts into the MS, and then the ISVF was set to 5500 V for 9 minutes for sample analysis. Protein Metrics Intact Mass Software V3.1-19 was used for MS data processing, including spectral deconvolution, mass reconstruction, and analysis of glycoforms and other PTMs. Peaks representing major glycoforms or subunits and their corresponding molecular weights were derived from deconvoluted mass spectra of biosimilar and reference batches. The relative ratios of the major glycoform and subunit peaks for each batch (i.e., the relative intensity percentage of each peak) were defined as the intensity relative to the highest intensity peak in each individual sample batch.

[0148] Flow cytometry

[0149] Targeted cells were stained with anti-human IgG (γ-chain specific) FITC conjugate to quantify bound antibodies on the cell surface using flow cytometry.

[0150] result

[0151] Incorporation of IRES-mediated, MP-mediated, and 2A peptide-mediated targeting vectors into landing pads in CHO master clones

[0152] A CHO targeted integration platform for high-throughput screening of antibody libraries was developed as a basis for integrating IRES-mediated targeting vectors, MP-mediated targeting vectors, and 2A peptide-mediated targeting vectors ( Figure 1A). The CHO targeted integration platform consists of two key components: 1) a CHO master clone containing a single copy landing pad at a genomic site that provides stable and high-level gene expression; and 2) a targeting vector that allows for simultaneous display and secretion of antibodies. The targeting vector uses IRES to express antibody light chains and antibody heavy chains in one transcript. The glycosylphosphatidylinositol membrane anchor molecule (GimmunoPI) is connected to the C-terminus of the heavy chain through an engineered FCS with incomplete cleavage efficiency, enabling simultaneous display and secretion of antibodies. The integration of single-copy targeting vectors carrying different antibody genes into the landing pad of each CHO master cell is achieved by FLP / FRT-based RMCE or CRISPR knock-in. It has been demonstrated that the CHO targeted integration platform is capable of screening an antibody library consisting of more than millions of molecules and producing secreted antibodies with a titer of approximately 200 mg / L in fed-batch cultures. The CHO targeted integration platform provides a powerful tool for improving the efficiency of antibody discovery and engineering. However, the low antibody titers provided by the CHO targeted integration platform itself limit its wider application in developability and functional studies.

[0153] When the IRES-mediated targeting vector is integrated into the landing pad of the CHO master clone, the light chain gene is under the control of the built-in promoter and translated by the classic cap-dependent mechanism, while the heavy chain gene is driven by IRES and translated by a cap-independent mechanism. The efficiency of IRES-driven cap-independent translation is lower than that of cap-dependent translation, resulting in low expression of the heavy chain gene and, thereby, low antibody titer. In order to further improve the secreted antibody titer, the inventors of the present disclosure compared the co-expression of light and heavy chains of IRES, MP and 2A peptide in the context of the CHO targeted integration platform ( Figure 1B ). When MP is used to express multiple genes in a vector, each gene is driven by its own promoter and transcribed separately. A possible disadvantage of using MP-mediated targeting vectors is transcriptional interference, in which one active transcription unit inhibits the expression of another unit, which may result in lower expression of one gene than another. In contrast, the light chain and heavy chain connected by the 2A peptide are expressed in a single open reading frame, and co-translational "self-cleavage" occurs between the last two amino acids GP at the C-terminus of the 2A polypeptide, producing equal amounts of light chain and heavy chain polypeptides, which is beneficial to improving antibody expression levels. It is well known in the art that furin is a ubiquitous subtilisin-like proprotein convertase with a minimal cleavage site for RXR / KR, and protein cleavage occurs in the Golgi apparatus. In order to remove the 2A residues (otherwise these residues would be attached to the light chain), a minimal FCS (Fm), RRKR, is inserted upstream of the 2A peptide. In addition, an SGSG linker is inserted between Fm and the 2A peptide to enhance the cleavage efficiency of the 2A peptide.

[0154] Comparative data for cells transfected with IRES, MP, Fm-2A, F1-2A, F2-2A, and F3-2A targeting vectors

[0155] Each of the three targeting vectors IRES, MP and Fm-2A was co-transfected with a vector expressing Flpe into a CHO master clone. The transfected cells were incubated in an incubator for 5 days to allow RMCE to occur. The transfected cells were subsequently passaged in a culture medium containing puromycin to select transfected cells in which the targeting vector was correctly integrated into the landing pad. When the transfected cell pool was recovered, it was stained with an anti-human IgG (γ chain specific) FITC conjugate to quantify the cell surface binding antibody by flow cytometry. The productivity of the stable transfection pool of each targeting vector in 14-day fed-batch culture was also characterized. The culture supernatant was harvested at the end of the fed-batch phase and purified by protein A. The purified antibodies of each targeting vector were analyzed using SDS PAGE under reducing conditions. When the transfected cells were passaged in the selective culture medium, the viability of the transfected cells gradually decreased and then slowly recovered. The cell pools transfected with the IRES and Fm-2A targeting vectors recovered more than 95% viability within 18 days, whereas the cell pools generated with the MP targeting vector required an additional 3 days to recover ( Figure 2A Flow cytometric analysis of cell surface displayed antibodies showed that the IRES and Fm-2A vectors gave homogeneous expression, whereas the cell pool generated using the MP vector had heterogeneous expression, indicated by two peaks in the histogram ( Figure 2B Compared with IRES-mediated targeting vectors, MP vectors produced slightly lower titers in fed-batch culture, whereas Fm-2A increased the secreted antibody titer by approximately one-fold ( Figure 2C However, the light chain polypeptide expressed by Fm-2A was larger than that expressed by IRES and MP vectors. MS analysis confirmed that the 2A residue was attached to the light chain, indicating that the cleavage at Fm was unsuccessful ( Figure 3B ).

[0156] The amino acids flanking the minimal cleavage site RXR / KR affect furin cleavage efficiency. The inventors of the present disclosure designed three FCS variants F1, F2, and F3 to include conservative amino acids around RRKR in the 2A targeting vector to improve cleavage efficiency ( Figure 1B Compared with the IRES targeting vector, the three newly designed targeting vectors F1-2A, F2-2A, and F3-2A required similar time to generate stable pools ( Figure 2A ). Antibodies displayed on the cell surface from these three 2A vectors were homogeneous ( Figure 2BIn fed-batch cultures, the antibody titers of F1-2A, F2-2A, and F3-2A were similar to those of the Fm-2A vector. SDS-PAGE analysis showed that the light chain polypeptides produced by F1-2A, F2-2A, and F3-2A were smaller than those from Fm-2A and were similar in size to those expressed from IRES and MP targeting vectors ( Figure 2D Further analysis of molecular weight revealed that the product expressed from F1-2A contained 60% of the light chain polypeptide with 2A removed, while the products expressed from F2-2A and F3-2A contained 90% of the light chain polypeptide with 2A removed. However, the light chain polypeptide from F3-2A was heterogeneous due to the different number of amino acid residues attached to the furin cleavage sequence. Overall, F2-2A was the best for co-expression of antibody light and heavy chains in terms of secreted antibody titer and quality.

[0157] Evaluation of cleavage efficiency of FCS variants

[0158] The inventors further designed 15 other FCS variants, F4 to F18, with RRKR flanked by R at P5, S / A at P1', V / L at P2', D / S at P3', and T / L at P4' ( Figure 4A Using the same procedure described above, the combinations of these 15 variants and 2A were tested for co-expression of light and heavy chains in dual display and secretion targeting vectors. SDS-PAGE analysis showed that the light and heavy chain polypeptides expressed from these FCS variants-2A were of the same size as those expressed from the control IRES vector ( Figure 4B ).

[0159] To investigate the effects of different amino acid residues at various positions within the furin cleavage sequence, the inventors designed 95 variants by changing one amino acid at a time in the RRRKRSVDT (SEQ ID NO: 2) sequence while keeping the other amino acids unchanged ( Figure 5A ). These variants were tested for co-expression of the antibody light chain and the antibody heavy chain in dual display and secretion targeting vectors following the procedures described in the methods section above. Notably, replacing the amino acid at position P5 from arginine to aspartic acid (R to D) [FCS variant F24], arginine to cysteine ​​(R to C) [FCS variant F25], or arginine to glutamic acid (R to E) [FCS variant F26] resulted in low cleavage efficiency, while changing it to other amino acid types did not affect cleavage efficiency ( Figure 5B). At the P1' position, replacing serine (S) with alanine (A) [FCS variant F40], aspartic acid (D) [FCS variant F44] or glutamic acid (E) [FCS variant F46] maintains high cleavage efficiency, while replacing serine (S) with other amino acids leads to reduced cleavage efficiency. Similarly, at the P2' position, replacing valine (V) with leucine (L) [FCS variant F69], threonine (T) [FCS variant F73], isoleucine (I) [FCS variant F68] or alanine (A) [FCS variant F59] maintains high cleavage efficiency, while replacing it with other amino acids leads to reduced cleavage efficiency. Mutations in P3' and P4' do not affect cleavage efficiency. Figures 4 and 5 show that the 20 amino acids most conducive to improving cleavage efficiency are serine (S), alanine (A), aspartic acid (D), and glutamic acid (E) at the P1' position, valine (V), leucine (L), threonine (T), isoleucine (I), and alanine (A) at the P2' position, and excluding aspartic acid (D), cysteine ​​(C), and glutamic acid (E) at the P5 position. Any combination of these amino acids at the P5, P1', and P2' positions in the FCS variant with RX-(K / R)-R at the P4, P3, P2, and P1 positions results in an FCS variant with high cleavage efficiency. It was found that when a combination of amino acids providing high cleavage efficiency at the P5, P1', and P2' positions in the FCS variant and RX-(K / R)-R at the P4, P3, P2, and P1 positions is used, any amino acid at the P3' and P4' positions still produces an FCS variant with high cleavage efficiency.

[0160] discuss

[0161] Displaying full-length mAbs on the surface of mammalian cells requires that light and heavy chain genes be co-expressed in a single vector in a tightly coupled manner. This can be achieved by using a combination of MP, IRES, and a furin cleavage sequence and a 2A peptide (F-2A). Compared to IRES and MP, F-2A linked genes are expressed in a single open reading frame, producing equal amounts of different genes. The inventors of the present disclosure compared the co-expression of antibody light and heavy chains of IRES, MP, and F-2A peptides to simultaneously display and secrete antibodies in a CHO targeted integration platform. In each targeting vector, the antibody light chain is arranged as the first gene, followed by IRES, MP, or F-2A to drive the expression of the heavy chain. The antibody heavy chain is further connected to a membrane anchor molecule via an engineered furin cleavage sequence with reduced cleavage efficiency. In order to remove 2A residues (otherwise these residues would attach to the light chain expressed by the 2A targeting vector), a minimal furin cleavage sequence (Fm) RRKR is inserted upstream of the 2A peptide. In addition, an SGSG linker is inserted between Fm and 2A to enhance the cleavage efficiency of the 2A peptide. Compared to the use of IRES and MP, Fm-2A provides enhanced antibody secretion levels and homogeneity of cell surface antibody display. However, most secreted antibodies have 2A residues connected to the C-terminus of the light chain polypeptide, indicating that cleavage at Fm is ineffective. To overcome this problem, the inventors designed an engineered furin cleavage sequence variant with RRKR flanked by highly conserved amino acids. The combination of these newly designed furin cleavage sequence variants and 2A peptides enhances the secretion of antibodies with the correct size without compromising the secreted antibody titer level and the homogeneity of antibody display.

[0162] The amino acid of the minimum furin cleavage sequence RXR / KR flank affects furin cleavage efficiency. In order to improve the cleavage efficiency of furin, a large amount of tests need to be carried out to identify amino acid whose type and specific combination. The inventors of the present disclosure have found that in naturally occurring furin cleavage sequence, adding amino acid around RXR / KR can enhance cleavage efficiency. Therefore, the design of furin cleavage sequence based on this principle demonstrates that furin cleavage efficiency improves.

[0163] Notable features of the methods disclosed herein include:

[0164] 1. The expression vector described herein comprises at least the following domains:

[0165] -An antibody light chain variable region;

[0166] -ZR-X1-X2-R-X3-X4-X5-X6;

[0167] -2A; and

[0168] -The heavy chain variable region of an antibody.

[0169] 2. Plasmid expression vectors containing an engineered furin cleavage sequence for high-level and homogeneous co-expression of multiple genes, such as the light and heavy chains of mAbs, in cells.

[0170] The expression system disclosed herein has the following advantages:

[0171] 1. Compared with conventional methods known in the art, the expression system described herein can produce higher levels of cell surface displayed antibodies (membrane bound) and secreted antibodies.

[0172] 2. Compared with conventional methods known in the art, the expression system described herein is capable of producing secreted antibodies with high homogeneity.

[0173] 3. The improved expression system capable of co-expressing antibody light and heavy chains with high cleavage efficiency is used for antibody library screening to provide a more efficient method for the development of therapeutic antibodies compared with expression systems known in the art.

[0174] 4. An improved expression system capable of co-expressing antibody light and heavy chains with high cleavage efficiency can more efficiently produce therapeutically specific and functional antibody proteins for the development of mRNA-based therapies.

[0175] Sequence Listing

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Claims

1. An expression system for an antigen-binding molecule, comprising: - a first polynucleotide encoding the first part of the antigen binding molecule; - a second polynucleotide encoding a first cleavage site comprising the amino acid sequence ZR-X1-X2-R-X3-X4-X5-X6 (SEQ ID NO: 256), wherein the second polynucleotide is located downstream of the first polynucleotide; - a third polynucleotide encoding a second cleavage site comprising the 2A polypeptide or a fragment thereof, wherein the third polynucleotide is located downstream of the second polynucleotide; and - a fourth polynucleotide encoding the second portion of the antigen binding molecule, wherein the fourth polynucleotide is located downstream of the third polynucleotide, wherein when the first cleavage site and the second cleavage site are cleaved, the antigen-binding molecule comprising the first part and the second part of the antigen-binding molecule is released, wherein Z is R, P, G, A, K, N, Q, H, I, L, M, F, S, T, W, Y, or V, and wherein X1, X2, X3, X4, X5, and X6 are R, P, G, A, D, C, E, K, N, Q, H, I, L, M, F, S, T, W, Y, or V.

2. An expression system for an antigen-binding molecule, wherein the antigen-binding molecule is secreted or membrane-bound, comprising: - a first polynucleotide encoding the first part of the antigen binding molecule; - a second polynucleotide encoding a first cleavage site comprising the amino acid sequence ZR-X1-X2-R-X3-X4-X5-X6 (SEQ ID NO: 256), wherein the second polynucleotide is located downstream of the first polynucleotide; - a third polynucleotide encoding a second cleavage site comprising the 2A polypeptide or a fragment thereof, wherein the third polynucleotide is located downstream of the second polynucleotide; - a fourth polynucleotide encoding the second portion of the antigen binding molecule, wherein the fourth polynucleotide is located downstream of the third polynucleotide; - a fifth polynucleotide encoding a third cleavage site comprising a furin cleavage sequence variant comprising: (i) the furin consensus sequence RXKR (SEQ ID NO: 18); or (ii) the furin consensus sequence RXRR (SEQ ID NO: 19), wherein the fifth polynucleotide is located downstream of the fourth polynucleotide; and - a sixth polynucleotide encoding a membrane-anchored polypeptide, wherein the sixth polynucleotide is located downstream of the fifth polynucleotide; wherein when the first cleavage site, the second cleavage site, and the third cleavage site are cleaved, the secretory antigen-binding molecule comprising the first and second parts of the antigen-binding molecule is released; wherein when the first cleavage site and the second cleavage site are cleaved and the third cleavage site is not cleaved, the membrane-bound antigen-binding molecule comprising the first and second parts of the antigen-binding molecule, the third cleavage site and the membrane-anchored polypeptide is released, where Z is R, P, G, A, K, N, Q, H, I, L, M, F, S, T, W, Y or V, wherein X1, X2, X3, X4, X5, and X6 are R, P, G, A, D, C, E, K, N, Q, H, I, L, M, F, S, T, W, Y, or V, and wherein X is any amino acid.

3. The expression system of claim 1 or 2, wherein Z is R, and the amino acid sequence of the first cleavage site comprises the amino acid sequence RR-X1-X2-R-X3-X4-X5-X6 (SEQ ID NO: 1).

4. The expression system of any one of claims 1 to 3, wherein X1 is R, X2 is K or R, and X3, X4, X5, and X6 are any of P, G, A, D, C, E, K, N, Q, H, I, L, M, F, S, T, W, Y, or V.

5. The expression system according to any one of claims 1 to 4, wherein the first cleavage site comprises the amino acid sequence shown in SEQ ID NOs: 2 to 17 and SEQ ID NOs: 66 to 160.

6. The expression system of any one of claims 1 to 5, wherein X3 is any one of P, G, A, D, E, H, I, L, M, F, or S; X4 is any one of P, A, I, L, T, or V; X5 is any one of R, P, G, A, D, C, E, K, N, Q, H, I, L, M, F, S, T, W, Y, or V; and X6 is any one of R, P, G, A, D, C, E, K, N, Q, H, I, L, M, F, S, T, W, Y, or V.

7. The expression system of any one of claims 1-6, wherein the first cleavage site comprises the amino acid sequence shown in SEQ ID NO: 2-17, 66-70, 74-84, 86, 87, 91, 93, 95-99, 104, 106, 115, 116, 120, 123-129 and 130-160.

8. The expression system of any one of claims 1-7, wherein Z is R, P, G, A, K, N, Q, H, I, L, M, F, S, T, W, Y, or V; X is any of S, A, D, or E; X is any of V, A, I, L, or T; X is any of R, P, G, A, D, C, E, K, N, Q, H, I, L, M, F, S, T, W, Y, or V; and X is any of R, P, G, A, D, C, E, K, N, Q, H, I, L, M, F, S, T, W, Y, or V.

9. The expression system according to any one of claims 1 to 8, wherein the first cleavage site comprises the amino acid sequence shown in SEQ ID NO: 2, 87, 91, 93, 106, 115, 116 and 120. 10 . The expression system according to claim 1 , further comprising a polynucleotide encoding a linker SGSG between the second polynucleotide and the third polynucleotide.

11. The expression system of claim 2, wherein the fifth polynucleotide encodes a third cleavage site comprising a furin cleavage sequence variant comprising: (i) the furin consensus sequence RXKR (SEQ ID NO: 18) and at least the first five amino acids from the 2A polypeptide; or (ii) the furin consensus sequence RXRR (SEQ ID NO: 19) and at least the first five amino acids from the 2A polypeptide.

12. The expression system of claim 11, wherein the fifth polynucleotide encodes a third cleavage site comprising a furin cleavage sequence variant comprising: (i) the furin consensus sequence RXKR (SEQ ID NO: 18) and the first five amino acids derived from a 2A polypeptide, wherein the first five amino acids derived from the 2A polypeptide comprise one or more point mutations; or (ii) the furin consensus sequence RXRR (SEQ ID NO: 19) and the first five amino acids derived from a 2A polypeptide, wherein the first five amino acids derived from the 2A polypeptide comprise one or more point mutations.

13. The expression system of claim 12, wherein the one or more point mutations are selected from the group consisting of A1P, A1G, T2G, T2P, N3P, N3A, F4P, F4A, and S5P.

14. The expression system of any one of claims 1-13, wherein the 2A polypeptide is selected from the group consisting of P2A, F2A, E2A and T2A, wherein optionally the 2A polypeptide is T2A.

15. The expression system according to any one of claims 1 to 14, wherein the membrane-anchored polypeptide is a glycophospholipid transmembrane domain (GPI), a platelet-derived growth factor receptor (PDGFR) β chain transmembrane domain (PTM), or an immunoglobulin C2 type extracellular-transmembrane-cytoplasmic domain or a murine B7-1 antigen, wherein optionally the membrane-anchored polypeptide is GPI.

16. The expression system of any one of claims 1-15, wherein the second polynucleotide encodes a first cleavage site that has at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% higher cleavage efficiency compared to an expression system comprising a second polynucleotide encoding a first cleavage site comprising the furin consensus sequence RXKR (SEQ ID NO: 18) or RXRR (SEQ ID NO: 19).

17. The expression system of any one of claims 1 to 16, wherein the expression system is capable of releasing an antigen binding molecule comprising a first portion of the antigen binding molecule having a correct polypeptide sequence, wherein the first portion of the antigen binding molecule does not contain one or more residual amino acids from the second cleavage site after cleavage at the first and second cleavage sites.

18. The expression system of any one of claims 1 to 17, wherein the expression system is capable of increasing the homogeneity of the produced antigen binding molecules, wherein homogeneity is achieved when a plurality of antigen binding molecules are produced, wherein each of the plurality of antigen binding molecules comprises a first portion having the same amino acid sequence and / or the same molecular weight.

19. The expression system of any one of claims 1-18, wherein the expression system is delivered to a target cell prior to transcription, wherein optionally the target cell is an animal cell, and wherein optionally the animal cell is a mammalian cell.

20. The expression system according to any one of claims 1-19, wherein the second polynucleotide comprises the nucleotide sequence shown in SEQ ID NOs: 35-50 and SEQ ID NOs: 161-255. A vector comprising the expression system according to any one of claims 1 to 20.

22. A host cell comprising the expression system according to any one of claims 1 to 20 or the vector according to claim 21.

23. A kit comprising the expression system according to any one of claims 1 to 20, the vector according to claim 21 or the host cell according to claim 22.

24. The expression system according to any one of claims 1 to 20, the vector according to claim 21, the host cell according to claim 22, or the kit according to claim 23, for use in screening an antibody library or antibody production.

25. A method for producing one or more secretory antigen binding molecules and / or one or more membrane-bound antigen binding molecules, comprising culturing the host cell of claim 22 under culture conditions suitable for producing the one or more secretory antigen binding molecules and / or one or more membrane-bound antigen binding molecules.

26. The method of claim 25, further comprising recovering the one or more secreted antigen binding molecules.

27. A method for detecting the presence of one or more secreted antigen binding molecules and / or one or more membrane-bound antigen binding molecules, the method comprising: - Providing an expression system according to any one of claims 1 to 20; - delivering the expression system to one or more target cells; wherein the target cell transcribes the expression system into one or more amino acid sequences, wherein when all cleavage sites in the one or more amino acid sequences are cleaved, the target cell secretes one or more secretory antigen-binding molecules comprising the first and second parts of the antigen-binding molecule, wherein when the first and second cleavage sites in the one or more amino acid sequences are cleaved and the third cleavage site in the one or more amino acid sequences is not cleaved, one or more membrane-bound antigen-binding molecules bind to the surface of the target cell, the membrane-bound antigen-binding molecules comprising the first portion and the second portion of the antigen-binding molecule; and - detecting the presence or absence of said one or more secreted antigen binding molecules and / or one or more membrane-bound antigen binding molecules.

28. The method of claim 27, wherein: (i) detecting the presence or absence of the one or more secreted antigen binding molecules using an enzyme-linked immunosorbent assay (ELISA) or a surrogate binding assay; and / or (ii) detecting the presence or absence of the one or more membrane-bound antigen-binding molecules using flow cytometry analysis, wherein the first portion of the antigen-binding molecule, the second portion of the antigen-binding molecule and / or the antigen for which the antigen-binding molecule is specific are stained prior to the flow cytometry analysis.

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