Methods for increasing expression of recombinant proteins

Abstract

Herein is reported a nucleic acid comprising in operably linked form a nucleic acid encoding a selection marker, a nucleic acid encoding a self-cleaving peptide sequence, and a nucleic acid encoding a protein protease inhibitor. Further reported is a method for the recombinant production of heterologous polypeptides using the nucleic acid according to the invention as well as the cells comprising the nucleic acid. Further reported is the use of the nucleic acid according to the invention for increasing the amount of a heterologous polypeptide recombinantly produced by reducing protease cleavage.

Description

[0001] The present invention relates to the field of recombinant protein production. More particularly, the present invention relates to the co-expression of a protein protease inhibitor (such as BPTI or aprotinin) in mammalian cells for recombinant heterologous polypeptides to increase the amount of intact (i.e., not protease-cleaved) recombinant heterologous polypeptides, which can be obtained from the culture of said mammalian cells. Background Art

[0002] For the commercial production of therapeutic biologics (such as antibodies), an ideal characteristic is to maximize the expression yield of the therapeutic agent. The expression yield directly depends on the production titer and indirectly on the amount of therapeutically relevant by-products. These by-products can be, for example, incorrectly assembled therapeutic polypeptides, incorrectly folded therapeutic polypeptides, or degraded therapeutic polypeptides.

[0003] For the recombinant production of therapeutic biologics, mammalian cells such as CHO and HEK cells are commonly used. Due to their origin, these cells also produce proteases that can cleave the therapeutic agent. In cases where the therapeutic agent is susceptible to cleavage by the endogenous proteases of the production cells, the cleaved (i.e., degraded) therapeutic agent is obtained as a therapeutically relevant by-product.

[0004] This problem has been addressed in different ways in the art.

[0005] WO 99 / 10503 reports a method for the recombinant production of a serine protease using a recombinant nucleic acid encoding a zymogen precursor of the serine protease, wherein a non-autocatalytic cleavage site naturally present in the zymogen precursor is replaced with an autocatalytic cleavage site that is recognized by the active form of the protease, and thereby the zymogen precursor is cleaved into the active form.

[0006] WO 02 / 61064 reports a method for the recombinant production of trypsin using a nucleic acid encoding a trypsinogen having an enterokinase recognition site in the propeptide sequence, the trypsinogen being in a form that can be secreted by a host cell, and the method comprising culturing cells comprising said nucleic acid under conditions capable of secreting the expression product into the culture medium, thereby selecting conditions such that at least substantially prevents autocatalytic cleavage of the propeptide sequence.

[0007] CN111607615 reported a method for preparing a bivalent subunit vaccine against porcine epidemic diarrhea (PEDV) and transmissible gastroenteritis virus of swine (TGEV) diseases. This method uses the PEDV-T2A-TGEV-S sequence and includes culturing insect cells containing the said sequence in a medium, which includes culturing a mixture of serine protease inhibitor aprotinin, aspartic protease inhibitor pepstatin a, aminopeptidase inhibitor bestatin, cysteine protease inhibitor E-64, and serine / cysteine protease inhibitor leupeptin for 24 hours to obtain a culture containing protease inhibitors, and continuously culturing the culture containing protease inhibitors for 48 hours to obtain a culture containing the expressed protein.

[0008] WO 2016 / 118775 reported a method for producing retroviral producer cells for stably producing cocal envelope pseudotyped retroviral vectors, which includes transfecting cells with a plasmid encoding cocal envelope, self-cleaving peptide, and selection marker.

[0009] WO 2021 / 183946 reported an expression vector, which includes a nucleic acid and a selectable marker gene. The nucleic acid includes an open reading frame encoding a protein of interest, followed by a self-cleaving peptide that can induce ribosomal skipping during translation.

[0010] CN110835632 reported an expression cassette, which includes a promoter, an sgRNA framework sequence of mutant Cas9 derived from Saccharomyces cerevisiae, human uracil glycosylase inhibitor, T2A self-splicing polypeptide, green fluorescent protein, and BGH polyA sequence.

[0011] CN 108441516 reported a lentiviral CMV-CBh dual promoter-modified vector pLenti-CMV-3FLAG-EGFP-PGK-mCherry-T2A-Puro.

[0012] CN 112626121 reported a triple selectable marker antibody expression vector, which includes expression cassette I and expression cassette II, where expression cassette II is adjacent to expression cassette I in a forward arrangement. In expression cassette I, the puromycin resistance gene is coupled downstream of the antibody light chain gene through an IRES sequence, and then mouse glutamine synthetase is coupled downstream of the puromycin resistance gene through an E2A polypeptide. In expression cassette II, the dihydrofolate reductase gene DHFR is coupled downstream of the antibody heavy chain gene through an IRES sequence.

[0013] WO 2019 / 157099 reported a nasal spray pharmaceutical preparation, which includes adrenaline or its salt, and includes one or more absorption enhancers, such as aprotinin.

[0014] An oral pharmaceutical composition is reported in WO 2019 / 239405, which comprises a therapeutic peptide or therapeutic protein up to 100 kilodaltons, a divalent cation chelator, and an isolated recombinantly expressed Bowman-Birk inhibitor (BBI), wherein the recombinantly expressed BBI is expressed in a Pichia pastoris expression system.

[0015] WO 2008 / 005847 reports that FVIII can be expressed in a medium containing a protease inhibitor such as aprotinin, for example, in an amount of about 0.01 to about 5%, or about 0.5 to about 1.0% (vol / vol) (aprotinin, 15 - 30 trypsin inhibitor units (TIU) / ml, Sigma) or the activity units of other protease inhibitors in corresponding amounts.

[0016] WO 2008 / 135501 reports a method for producing factor VIII polypeptide by culturing mammalian cells expressing factor VIII polypeptide, wherein the cell culture medium comprises soybean trypsin inhibitor.

[0017] WO 2018 / 22032 reports a method for recombinantly producing debrilase, which comprises culturing host cells containing one or more vectors, wherein the one or more vectors encode debrilase and a protein inhibitor of debrilase in an expressible form under conditions where debrilase and the protein inhibitor of debrilase are expressed, and the protein inhibitor of debrilase is selected from the group consisting of: aprotinin, plasmin streptocin (I16.001), protease inhibitor type 2K (PIN2K, I20.001), colicin (I11.001), trypsin inhibitor MCTI - 1 (I07.001), ascidian trypsin inhibitor (I05.001), peptidase inhibitor 5 (κPI - actitoxin - Avd3a, I02.026), and tissue factor pathway inhibitor 2 inhibitor unit 1 (TFPI2, I02.013).

[0018] WO 2021 / 170839 reports the production of a recombinant protein, polypeptide, or peptide in algae using aprotinin as a carrier, wherein aprotinin and the recombinant protein, polypeptide, or peptide are fused together to form a fusion protein.

[0019] Therefore, there is a need for improved methods for recombinantly producing therapeutic polypeptides. Summary of the Invention

[0020] This article reports a method for producing heterologous polypeptides by recombinant mammalian cells, wherein the heterologous polypeptide is susceptible to cleavage by endogenous proteases produced by the recombinant mammalian cells, and thus the cleavage of the heterologous polypeptide by the endogenous proteases is reduced by co-expression of a protein protease inhibitor.

[0021] Accordingly, this article reports a nucleic acid comprising a first part encoding a selectable marker and a second part encoding a protein protease inhibitor, wherein the two parts are linked by an internal ribosome entry site (IRES) or an intein or a self-cleaving peptide sequence. This article also reports the use of the nucleic acid according to the invention in the recombinant production of heterologous polypeptides and a cell comprising the nucleic acid according to the invention. This article further reports the use of the nucleic acid according to the invention for increasing the yield of a recombinantly produced heterologous polypeptide and its use for reducing protease cleavage of a recombinant heterologous polypeptide during production in mammalian cells.

[0022] The present invention is at least partly based on the finding that the combination of a selectable marker and a protein protease inhibitor in a single, i.e., the same, cistron, such as using an IRES or an intein or a self-cleaving peptide sequence, is advantageous and provides improvements. Among them, these improvements are at least a reduction in protease cleavage during the recombinant production (expression) of heterologous polypeptides in mammalian cells, and thus an increase in production (yield). At the same time, the expression of the recombinant heterologous polypeptide is not reduced compared to cells that do not comprise the nucleic acid according to the invention.

[0023] The present invention is at least partly based on the finding that co-expression of a protein protease inhibitor and a recombinant heterologous polypeptide reduces the amount (yield) of the heterologous polypeptide compared to cells that do not express the protein protease inhibitor.

[0024] The present invention is at least partly based on the finding that knockout of a single protease or all variants of a single protease in cells expressing a heterologous polypeptide does not result in the same increase in the amount (yield) of the recombinant heterologous polypeptide as the use of the nucleic acid according to the invention. Without being bound by this theory, it is assumed that in cases where cleavage is observed during the recombinant production of a heterologous polypeptide, it is usually caused by more than one protease.

[0025] The present invention is further at least partly based on the finding that the promoter used to drive the expression of the nucleic acid according to the invention should not be a strong promoter, i.e., it should not be, for example, the CMV promoter, the human EF1a promoter or the CAG promoter. It has been found that using a promoter with medium strength (such as, for example, the SV40 promoter, the PGK1 promoter or the Ubc promoter) is advantageous, and the SV40 promoter is a preferred promoter.

[0026] It should be noted that in the following aspects and embodiments, the term "self-cleaving peptide sequence" can be replaced by the term "IRES" or the term "intein" without departing from the present invention. These are also specific aspects and embodiments of the present invention.

[0027] When an IRES element is placed between two genes, it mediates cap-independent translation of the second gene.

[0028] Inteins are typically flanked by two genes and are co-translated into a single polypeptide before splicing occurs, removing the intein to join the flanking proteins with new bonds. Mutations in the intein sequence inhibit splicing while retaining cleavage.

[0029] One aspect of the present invention is an (isolated) nucleic acid that comprises the following elements in an operably linked form in the 5' to 3' or 3' to 5' direction:

[0030] a) a nucleic acid encoding a selectable marker,

[0031] b) a nucleic acid encoding a self-cleaving peptide sequence, and

[0032] c) a nucleic acid encoding a protein protease inhibitor.

[0033] In certain embodiments of all aspects and embodiments, the nucleic acid further comprises the following elements in an operably linked form:

[0034] d) a promoter upstream (5') of the nucleic acid of a) (in the 5' to 3' direction) or c) (in the 3' to 5' direction),

[0035] e) a polyadenylation signal sequence downstream (3') of the nucleic acid of c) (in the 5' to 3' direction) or a) (in the 3' to 5' direction), and

[0036] f) an optional terminator sequence downstream (3') of the nucleic acid of e).

[0037] In certain embodiments of all aspects and embodiments, the nucleic acid encoding a selectable marker is selected from the group consisting of a nucleic acid encoding puromycin acetyltransferase, histidinol dehydrogenase, thymidine kinase, hygromycin B phosphotransferase, dihydrofolate reductase, blastocidin deaminase, glutamine synthetase, G418 resistance gene, bleomycin resistance gene, and aminoglycoside 3'-phosphotransferase.

[0038] In certain embodiments of all aspects and embodiments, the nucleic acid encoding a selectable marker encodes puromycin acetyltransferase or a functional variant thereof that is capable of inactivating / modifying puromycin.

[0039] In certain embodiments of all aspects and embodiments, the nucleic acid encoding the selection marker encodes the amino acid sequence of SEQ ID NO:01 (SMAA; Uniprot P13249 (PUAC_STRAD), excluding the N-terminal methionine residue) that can inactivate puromycin / modify puromycin or a functional variant thereof.

[0040] In certain embodiments of all aspects and embodiments,

[0041] - the nucleic acid encoding the selection marker has the nucleotide sequence of SEQ ID NO:02 (SMNN), or

[0042] - the nucleic acid encoding the selection marker is a variant of the nucleotide sequence of SEQ ID NO:02 that encodes a selection marker having the amino acid sequence of SEQ ID NO:01, or

[0043] - the nucleic acid encoding the selection marker encodes a functional variant of SEQ ID NO:01 that can inactivate puromycin / modify puromycin.

[0044] In certain embodiments of all aspects and embodiments, the self-cleaving peptide sequence is a viral 2-A self-cleaving peptide sequence that can cause ribosomal skipping or a functional variant thereof.

[0045] In certain embodiments of all aspects and embodiments, the self-cleaving peptide sequence is selected from the group consisting of: P2A, T2A, F2A, E2A, A2A, D2A, I2A, self-cleaving peptides of SEQ ID NO:64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84 or functional variants thereof.

[0046] In certain embodiments of all aspects and embodiments, the self-cleaving peptide sequence is T2A that can cause ribosomal skipping or a functional variant thereof.

[0047] In certain embodiments of all aspects and embodiments, the nucleic acid encoding the self-cleaving peptide sequence encodes the amino acid sequence of SEQ ID NO:14 (SCAA) that can cause ribosomal skipping or a functional variant thereof.

[0048] In certain embodiments of all aspects and embodiments,

[0049] - the nucleic acid encoding the self-cleaving peptide sequence has the nucleotide sequence of SEQ ID NO:15 (SCNN), or

[0050] - the nucleic acid encoding the self-cleaving peptide sequence is a variant of the nucleotide sequence of SEQ ID NO:15 that encodes a self-cleaving peptide sequence having the amino acid sequence of SEQ ID NO:14 (SCAA), or

[0051] - The nucleic acid encoding the self-cleaving peptide sequence encodes a functional variant of SEQ ID NO:14 (SCAA) that can cause ribosomal frameshifting.

[0052] In certain embodiments of all aspects and embodiments, the protein protease inhibitor inhibits one or more proteases selected from the group consisting of: ADAMS, ADAMTS, such as ADAM8; ADAM9; ADAM10; ADAM12; ADAM15; ADAM17 / TACE; ADAMDEC1; ADAMTS1; ADAMTS4; ADAMTS5; aspartic proteases, such as BACE or renin; aspartic cathepsins, such as cathepsin D or cathepsin E; caspases, such as caspase 1, caspase 2, caspase 3, caspase 4, caspase 5, caspase 6, caspase 7, caspase 8, caspase 9, caspase 10 or caspase 14; cysteine cathepsins, such as cathepsin B, cathepsin C, cathepsin K, cathepsin L, cathepsin S, cathepsin V / L2, cathepsin X / Z / P; cysteine proteases, such as Cruzipain; legumain; Otubain-2; KLKs, such as KLK4, KLK5, KLK6, KLK7, KLK8, KLK10, KLK11, KLK13 or KLK14; metalloproteases, such as Meprin; neprilysin; PSMA; BMP-1; MMPs, such as MMP1, MMP2, MMP3, MMP7, MMP8, MMP9, MMP10, MMP11, MMP12, MMP13, MMP14, MMP15, MMP16, MMP17, MMP19, MMP20, MMP23, MMP24, MMP26 or MMP27, serine proteases, such as activated protein C, cathepsin A, cathepsin G, chymotrypsin, coagulation factor proteases (such as FVIIa, FIXa, FXa, FXIa, FXIIa), elastase, granzyme B, glycylbenzoylarginine amide hydrolase, HtrA1, human neutrophil elastase, lactoferrin, Marapsin, NS3 / 4A, PACE4, plasmin, PSA, tPA, thrombin, neutrophil elastase, uPA; type II transmembrane serine proteases (TTSPs), such as DESC1, DPP-4, FAP, Hepsin, proteinase 2, proteinase, TMPRSS2, TMPRSS3 and TMPRSS4.

[0053] In certain embodiments of all aspects and embodiments, the nucleic acid encoding a protein protease inhibitor is selected from the group consisting of: BPTI, aprotinin, leupeptin (LLR), pepstatin (iVVVStaASta), SPINT1, SPINK1, HAI-1, HAI-2, or a functional variant thereof.

[0054] In certain embodiments of all aspects and embodiments, the nucleic acid encoding a protein protease inhibitor encodes BPTI or a functional variant thereof that is capable of inhibiting one or more serine proteases.

[0055] In certain embodiments of all aspects and embodiments, the nucleic acid encoding a protein protease inhibitor encodes the amino acid sequence of SEQ ID NO:86 (AprotAA) or a functional variant thereof that is capable of inhibiting one or more serine proteases.

[0056] In certain embodiments of all aspects and embodiments,

[0057] - the nucleic acid encoding a protein protease inhibitor has the nucleotide sequence of SEQ ID NO:87 (AprotNN), or

[0058] - the nucleic acid encoding a protein protease inhibitor is a variant of the nucleotide sequence of SEQ ID NO:87 (AprotNN) that encodes a protein protease inhibitor having the amino acid sequence of SEQ ID NO:86 (AprotAA), or

[0059] - the nucleic acid encoding a protein protease inhibitor encodes a functional variant of SEQID NO:86 (AprotAA) that is capable of inhibiting one or more serine proteases.

[0060] In certain embodiments of all aspects and embodiments, the promoter is the SV40 (simian virus 40) promoter or a functional variant thereof having the same (equivalent) or lower promoter strength.

[0061] In certain embodiments of all aspects and embodiments, the promoter has the nucleotide sequence of SEQ ID NO:102 (PromNN) or a functional variant thereof having the same or lower promoter strength.

[0062] In certain embodiments of all aspects and embodiments, the polyadenylation signal sequence is the bGH (bovine growth hormone) polyadenylation signal sequence.

[0063] In certain embodiments of all aspects and embodiments, the polyadenylation signal sequence has the nucleotide sequence of SEQ ID NO:100 (PolyANN).

[0064] In certain embodiments of all aspects and embodiments, a terminator sequence is present and is the hGT (human growth hormone terminator) sequence.

[0065] In certain embodiments of all aspects and embodiments, a terminator sequence is present and has the nucleotide sequence of SEQ ID NO:101 (TermNN).

[0066] One aspect of the invention is a (recombinant) cell comprising a nucleic acid according to the invention.

[0067] In certain embodiments of all aspects and embodiments, the nucleic acid according to the invention is stably integrated into the genome of the cell / into the chromosome of the cell.

[0068] In certain embodiments of all aspects and embodiments, a single copy of the nucleic acid according to the invention is integrated into the genome of the cell / into the chromosome of the cell.

[0069] In certain embodiments of all aspects and embodiments, the cell further comprises one or more nucleic acid sequences encoding a (recombinant) heterologous polypeptide.

[0070] In certain embodiments of all aspects and embodiments, the one or more nucleic acid sequences encoding a (recombinant) heterologous polypeptide are stably integrated into the genome of the cell / into the chromosome of the cell.

[0071] In certain embodiments of all aspects and embodiments, the one or more nucleic acid sequences encoding a (recombinant) heterologous polypeptide are integrated into the genome of the cell / into a single locus in one chromosome of the cell at a single locus.

[0072] In certain embodiments of all aspects and embodiments, the nucleic acid according to the invention and the one or more nucleic acid sequences encoding a (recombinant) heterologous polypeptide are integrated into the genome of the cell / into the chromosome of the cell at the same locus.

[0073] In certain embodiments of all aspects and embodiments, the cell further comprises one or more nucleic acid sequences encoding a (recombinant) heterologous polypeptide, the heterologous polypeptide comprising one or more protease-cleavable amino acid sequences / protease recognition sequences.

[0074] In certain embodiments of all aspects and embodiments, the cell further comprises one or more nucleic acid sequences encoding a (recombinant) heterologous polypeptide, the heterologous polypeptide comprising one or more amino acid sequences cleavable by a serine protease / a serine protease recognition sequence.

[0075] In certain embodiments of all aspects and embodiments, the cell further comprises one or more nucleic acid sequences encoding a (recombinant) heterologous polypeptide, the heterologous polypeptide comprising one or more protease-cleavable amino acid sequences selected from the group consisting of: ADAMS, ADAMTS, such as ADAM8; ADAM9; ADAM10; ADAM12; ADAM15; ADAM17 / TACE; ADAMDEC1; ADAMTS1; ADAMTS4; ADAMTS5; aspartic proteases, such as BACE or renin; aspartic cathepsins, such as cathepsin D or cathepsin E; caspases, such as caspase 1, caspase 2, caspase 3, caspase 4, caspase 5, caspase 6, caspase 7, caspase 8, caspase 9, caspase 10 or caspase 14; cysteine cathepsins, such as cathepsin B, cathepsin C, cathepsin K, cathepsin L, cathepsin S, cathepsin V / L2, cathepsin X / Z / P; cysteine proteases, such as Cruzipain; legumains; Otubain-2; KLKs, such as KLK4, KLK5, KLK6, KLK7, KLK8, KLK10, KLK11, KLK13 or KLK14; metalloproteases, such as Meprin; neprilysin; PSMA; BMP-1; MMPs, such as MMP1, MMP2, MMP3, MMP7, MMP8, MMP9, MMP10, MMP11, MMP12, MMP13, MMP14, MMP15, MMP16, MMP17, MMP19, MMP20, MMP23, MMP24, MMP26 or MMP27, serine proteases, such as activated protein C, cathepsin A, cathepsin G, chymotrypsin, coagulation factor proteases (such as FVIIa, FIXa, FXa, FXIa, FXIIa), elastase, granzyme B, guanidobenzoate hydrolase, HtrA1, human neutrophil elastase, lactoferrin, Marapsin, NS3 / 4A, PACE4, plasmin, PSA, tPA, thrombin, neutrophil elastase, uPA; type II transmembrane serine proteases (TTSPs), such as DESC1, DPP-4, FAP, Hepsin, proteinase 2, proteinase, TMPRSS2, TMPRSS3 or TMPRSS4; and any combination thereof.

[0076] In certain embodiments of all aspects and embodiments, the recombinant heterologous polypeptide is an antibody comprising one or more protease-cleavable amino acid sequences / protease recognition sequences.

[0077] In certain embodiments of all aspects and embodiments, the cell is a mammalian cell.

[0078] In certain embodiments of all aspects and embodiments, the cell is a CHO cell or a HEK cell or a BHK cell.

[0079] In certain embodiments of all aspects and embodiments, the cell is a CHO-K1 cell.

[0080] One aspect of the invention is a method for producing a (recombinant) heterologous polypeptide, wherein the method comprises the following steps:

[0081] - Culturing a cell according to the invention in a culture medium to produce a (recombinant) heterologous polypeptide,

[0082] - Recovering the (recombinant) heterologous polypeptide from the cell or the culture medium, and

[0083] - Optionally, purifying the (recombinant) heterologous polypeptide by one or more chromatographic steps.

[0084] In certain embodiments of all aspects and embodiments, the culturing is carried out (for at least some time / days) in the presence of puromycin, histidinol, ganciclovir, hygromycin, methionine sulfoximine, blasticidin, methotrexate, geomycin, bleomycin or neomycin (G418).

[0085] In certain embodiments of all aspects and embodiments, the culturing is carried out (for at least some time / days) in the presence of puromycin or a functional variant thereof.

[0086] In certain embodiments of all aspects and embodiments, the amount of the uncut (recombinant) heterologous polypeptide recovered is increased compared to a method using a cell that does not comprise a nucleic acid according to the invention.

[0087] One aspect of the invention is the use of a nucleic acid according to the invention for reducing the proteolytic cleavage of a (recombinant) heterologous polypeptide during recombinant production in mammalian cells.

[0088] One aspect of the invention is the use of a nucleic acid according to the invention for increasing the amount of intact / non-proteolytically cleaved (recombinant) heterologous polypeptide recovered from the culture of mammalian cells.

[0089] The invention at least encompasses the following independent aspects and dependent embodiments:

[0090] 1. A nucleic acid, which comprises the following elements in an operably linked form:

[0091] a) A nucleic acid encoding a selectable marker,

[0092] b) A nucleic acid encoding a self-cleaving peptide sequence, and

[0093] c) Nucleic acids encoding a protein protease inhibitor.

[0094] 2. The nucleic acid according to Example 1, wherein the element has the sequence of a)-b)-c) in the 5' to 3' direction.

[0095] 3. The nucleic acid according to Example 1, wherein the element has the sequence of c)-b)-a) in the 5' to 3' direction.

[0096] 4. The nucleic acid according to any one of Examples 1 to 3, wherein the nucleic acid further comprises the following elements in an operably linked form:

[0097] d) A promoter upstream (5') of the first element,

[0098] e) A polyadenylation signal sequence downstream (3') of the last element, and

[0099] f) Optionally, a terminator sequence downstream (3') of the nucleic acid of e).

[0100] 5. The nucleic acid according to any one of Examples 1 to 4, wherein the nucleic acid encoding a selection marker is selected from the group consisting of: nucleic acids encoding puromycin acetyltransferase, histidinol dehydrogenase, thymidine kinase, hygromycin B phosphotransferase, dihydrofolate reductase, blastocidin S deaminase, glutamine synthetase, G418 resistance gene, bleomycin resistance gene, and aminoglycoside 3'-phosphotransferase.

[0101] 6. The nucleic acid according to any one of Examples 1 to 5, wherein the nucleic acid encoding a selection marker encodes puromycin acetyltransferase or a functional variant thereof capable of inactivating / modifying puromycin.

[0102] 7. The nucleic acid according to any one of Examples 1 to 6, wherein the nucleic acid encoding a selection marker encodes the amino acid sequence of SEQ ID NO:01 or a functional variant thereof capable of inactivating / modifying puromycin.

[0103] 8. The nucleic acid according to any one of Examples 1 to 7, wherein

[0104] - the nucleic acid encoding a selection marker has the nucleotide sequence of SEQ ID NO:02, or

[0105] - the nucleic acid encoding a selection marker is a variant of the nucleotide sequence of SEQ ID NO:02 encoding a selection marker having the amino acid sequence of SEQ ID NO:01, or

[0106] - The nucleic acid encoding the selection marker encodes a functional variant of SEQ ID NO:01 that can inactivate puromycin / modify puromycin.

[0107] 9. The nucleic acid according to any one of embodiments 1 to 8, wherein the nucleic acid encoding the selection marker encodes the amino acid sequence of SEQ ID NO:01.

[0108] 10. The nucleic acid according to any one of embodiments 1 to 9, wherein the self-cleaving peptide sequence is a viral 2A self-cleaving peptide sequence capable of causing ribosomal skipping or a functional variant thereof.

[0109] 11. The nucleic acid according to any one of embodiments 1 to 10, wherein the self-cleaving peptide sequence is selected from the group consisting of: P2A, T2A, F2A, E2A, A2A, D2A, I2A, the self-cleaving peptides of SEQ ID NO:64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84 or functional variants thereof.

[0110] 12. The nucleic acid according to any one of embodiments 1 to 11, wherein the self-cleaving peptide sequence is T2A capable of causing ribosomal skipping or a functional variant thereof.

[0111] 13. The nucleic acid according to any one of embodiments 1 to 12, wherein the self-cleaving peptide sequence is T2A.

[0112] 14. The nucleic acid according to any one of embodiments 1 to 13, wherein the nucleic acid encoding the self-cleaving peptide sequence encodes the amino acid sequence of SEQ ID NO:14 capable of causing ribosomal skipping or a functional variant thereof.

[0113] 15. The nucleic acid according to any one of embodiments 1 to 14, wherein

[0114] - the nucleic acid encoding the self-cleaving peptide sequence has the nucleotide sequence of SEQ ID NO:15, or

[0115] - the nucleic acid encoding the self-cleaving peptide sequence is a variant of the nucleotide sequence of SEQ ID NO:15 encoding a self-cleaving peptide sequence having the amino acid sequence of SEQ ID NO:14, or

[0116] - the nucleic acid encoding the self-cleaving peptide sequence encodes a functional variant of SEQ ID NO:14 capable of causing ribosomal skipping.

[0117] 16. The nucleic acid according to any one of embodiments 1 to 15, wherein the nucleic acid encoding the self-cleaving peptide sequence encodes the amino acid sequence of SEQ ID NO:14.

[0118] 17. The nucleic acid according to any one of embodiments 1 to 16, wherein the protein protease inhibitor inhibits one or more proteases selected from the group consisting of: ADAMS, ADAMTS, such as ADAM8; ADAM9; ADAM10; ADAM12; ADAM15; ADAM17 / TACE; ADAMDEC1; ADAMTS1; ADAMTS4; ADAMTS5; aspartic proteases, such as BACE or renin; aspartic cathepsins, such as cathepsin D or cathepsin E; caspases, such as caspase 1, caspase 2, caspase 3, caspase 4, caspase 5, caspase 6, caspase 7, caspase 8, caspase 9, caspase 10 or caspase 14; cysteine cathepsins, such as cathepsin B, cathepsin C, cathepsin K, cathepsin L, cathepsin S, cathepsin V / L2, cathepsin X / Z / P; cysteine proteases, such as Cruzipain; legumain; Otubain-2; KLK, such as KLK4, KLK5, KLK6, KLK7, KLK8, KLK10, KLK11, KLK13 or KLK14; metalloproteases, such as Meprin; enkephalinase; PSMA; BMP-1; MMP, such as MMP1, MMP2, MMP3, MMP7, MMP8, MMP9, MMP10, MMP11, MMP12, MMP13, MMP14, MMP15, MMP16, MMP17, MMP19, MMP20, MMP23, MMP24, MMP26 or MMP27, serine proteases, such as activated protein C, cathepsin A, cathepsin G, chymotrypsin, coagulation factor proteases (such as FVIIa, FIXa, FXa, FXIa, FXIIa), elastase, granzyme B, guanidino benzoic acid enzyme, HtrA1, human neutrophil elastase, lactoferrin, Marapsin, NS3 / 4A, PACE4, plasmin, PSA, tPA, thrombin, neutrophil protease, uPA; type II transmembrane serine proteases (TTSP), such as DESC1, DPP-4, FAP, Hepsin, proteinase 2, proteinase, TMPRSS2, TMPRSS3 and TMPRSS4.

[0119] 18. The nucleic acid according to any one of embodiments 1 to 17, wherein the nucleic acid encoding the protein protease inhibitor is selected from the group consisting of: BPTI, aprotinin, leupeptin (LLR), pepstatin (iVVVStaASta), SPINT1, SPINK1, HAI-1, HAI-2 or functional variants thereof.

[0120] 19. The nucleic acid according to any one of embodiments 1 to 18, wherein the nucleic acid encoding the protein protease inhibitor encodes BPTI or a functional variant thereof capable of inhibiting one or more serine proteases.

[0121] 20. The nucleic acid according to any one of embodiments 1 to 19, wherein the nucleic acid encoding the protein protease inhibitor encodes BPTI.

[0122] 21. The nucleic acid according to any one of embodiments 1 to 20, wherein the nucleic acid encoding the protein protease inhibitor encodes the amino acid sequence of SEQ ID NO: 86 or SEQ ID NO: 177 or a functional variant thereof capable of inhibiting one or more serine proteases.

[0123] 22. The nucleic acid according to any one of embodiments 1 to 21, wherein

[0124] - the nucleic acid encoding the protein protease inhibitor has the nucleotide sequence of SEQ ID NO: 87 or SEQ ID NO: 178, or

[0125] - the nucleic acid encoding the protein protease inhibitor is a variant of the nucleotide sequence of SEQ ID NO: 87 encoding the protein protease inhibitor having the amino acid sequence of SEQ ID NO: 86, or

[0126] - the nucleic acid encoding the protein protease inhibitor encodes a functional variant of SEQ ID NO: 86 capable of inhibiting one or more serine proteases.

[0127] 23. The nucleic acid according to any one of embodiments 1 to 22, wherein the nucleic acid encoding the protein protease inhibitor encodes the amino acid sequence of SEQ ID NO: 86 capable of inhibiting one or more serine proteases.

[0128] 24. The nucleic acid according to any one of embodiments 4 to 23, wherein the promoter is the SV40 promoter or a functional variant thereof having the same or lower promoter strength.

[0129] 25. The nucleic acid according to any one of embodiments 4 to 24, wherein the promoter is the SV40 promoter.

[0130] 26. The nucleic acid according to any one of embodiments 4 to 25, wherein the promoter has the nucleotide sequence of SEQ ID NO: 102.

[0131] 27. The nucleic acid according to any one of embodiments 4 to 26, wherein the polyadenylation signal sequence is the bGH polyadenylation signal sequence.

[0132] 28. The nucleic acid according to any one of embodiments 4 to 27, wherein the polyadenylation signal sequence has the nucleotide sequence of SEQ ID NO: 100.

[0133] 29. The nucleic acid according to any one of embodiments 4 to 28, wherein a terminator sequence is present and is the hGT terminator sequence.

[0134] 30. The nucleic acid according to any one of embodiments 4 to 29, wherein a terminator sequence is present and has the nucleotide sequence of SEQ ID NO: 101.

[0135] 31. A vector comprising the nucleic acid according to any one of embodiments 1 to 30 and optionally additional regulatory elements.

[0136] 32. A cell comprising the nucleic acid according to any one of embodiments 1 to 30 or the vector according to embodiment 31.

[0137] 33. The cell according to embodiment 32, wherein the nucleic acid is stably integrated into the genome of the cell / the chromosome of the cell.

[0138] 34. The cell according to any one of embodiments 32 to 33, wherein a single copy of the nucleic acid is integrated into the genome of the cell / the chromosome of the cell.

[0139] 35. The cell according to any one of embodiments 32 to 34, wherein the cell further comprises one or more nucleic acid sequences encoding a heterologous polypeptide.

[0140] 36. The cell according to embodiment 35, wherein the one or more nucleic acid sequences encoding a heterologous polypeptide are stably integrated into the genome of the cell / the chromosome of the cell.

[0141] 37. The cell according to any one of embodiments 35 to 36, wherein the one or more nucleic acid sequences encoding a heterologous polypeptide are integrated into the genome of the cell / a single site in one chromosome of the cell at a single site.

[0142] 38. The cell according to any one of embodiments 35 to 37, wherein the nucleic acid according to any one of embodiments 1 to 30 or the plasmid according to embodiment 31 and the one or more nucleic acid sequences encoding a heterologous polypeptide are integrated into the genome of the cell / into the chromosome of the cell at the same site.

[0143] 39. The cell according to any one of embodiments 35 to 38, wherein the heterologous polypeptide comprises one or more protease-cleavable amino acid sequences / protease recognition sequences.

[0144] 40. A cell according to any one of embodiments 35 to 39, wherein the heterologous polypeptide comprises one or more amino acid sequences cleavable by a serine protease / serine protease recognition sequence.

[0145] 41. A cell according to any one of embodiments 32 to 40, wherein the cell further comprises one or more nucleic acid sequences encoding a recombinant heterologous polypeptide, the heterologous polypeptide comprising one or more amino acid sequences cleavable by a protease selected from the group consisting of: ADAMS, ADAMTS, such as ADAM8; ADAM9; ADAM10; ADAM12; ADAM15; ADAM17 / TACE; ADAMDEC1; ADAMTS1; ADAMTS4; ADAMTS5; aspartic proteases, such as BACE or renin; aspartic cathepsins, such as cathepsin D or cathepsin E; caspases, such as caspase 1, caspase 2, caspase 3, caspase 4, caspase 5, caspase 6, caspase 7, caspase 8, caspase 9, caspase 10 or caspase 14; cysteine cathepsins, such as cathepsin B, cathepsin C, cathepsin K, cathepsin L, cathepsin S, cathepsin V / L2, cathepsin X / Z / P; cysteine proteases, such as Cruzipain; legumains; Otubain-2; KLKs, such as KLK4, KLK5, KLK6, KLK7, KLK8, KLK10, KLK11, KLK13 or KLK14; metalloproteases, such as Meprin; neprilysin; PSMA; BMP-1; MMPs, such as MMP1, MMP2, MMP3, MMP7, MMP8, MMP9, MMP10, MMP11, MMP12, MMP13, MMP14, MMP15, MMP16, MMP17, MMP19, MMP20, MMP23, MMP24, MMP26 or MMP27, serine proteases, such as activated protein C, cathepsin A, cathepsin G, chymotrypsin, coagulation factor proteases (such as FVIIa, FIXa, FXa, FXIa, FXIIa), elastase, granzyme B, guanidobenzoate hydrolase, HtrA1, human neutrophil elastase, lactoferrin, Marapsin, NS3 / 4A, PACE4, plasmin, PSA, tPA, thrombin, neutrophil elastase, uPA; type II transmembrane serine proteases (TTSPs), such as DESC1, DPP-4, FAP, Hepsin, proteinase 2, proteinase, TMPRSS2, TMPRSS3 or TMPRSS4; and any combination thereof.

[0146] 42. A cell according to any one of embodiments 35 to 41, wherein the heterologous polypeptide is an antibody comprising one or more protease-cleavable amino acid sequences.

[0147] 43. A cell according to any one of embodiments 32 to 42, wherein the cell is a mammalian cell.

[0148] 44. A cell according to any one of embodiments 32 to 43, wherein the cell is a CHO cell or a HEK cell or a BHK cell.

[0149] 45. A cell according to any one of embodiments 32 to 44, wherein the cell is a CHO-K1 cell.

[0150] 46. A method for producing a heterologous polypeptide in a recombinant cell, the method comprising the steps of:

[0151] - culturing a cell according to any one of embodiments 35 to 45 in a culture medium to produce a heterologous polypeptide,

[0152] - recovering the heterologous polypeptide from the cell or the culture medium, and

[0153] - optionally, purifying the heterologous polypeptide by one or more chromatography steps.

[0154] 47. The method according to embodiment 46, wherein the culturing is carried out in the presence of puromycin, histidinol, ganciclovir, hygromycin, methionine sulfoximine, blasticidin, methotrexate, geneticin, bleomycin or neomycin (G418).

[0155] 48. The method according to any one of embodiments 46 to 47, wherein the culturing is carried out in the presence of puromycin or a functional variant thereof.

[0156] 49. The method according to any one of embodiments 46 to 48, wherein the culturing is carried out in the presence of puromycin.

[0157] 50. The method according to any one of embodiments 46 to 49, wherein the amount of the uncleaved heterologous polypeptide recovered is increased as compared to a method using a cell that does not contain the nucleic acid according to any one of embodiments 1 to 30 or the vector according to embodiment 31.

[0158] 51. Use of the nucleic acid according to any one of embodiments 1 to 30 or the vector according to embodiment 31 for reducing protease cleavage of a heterologous polypeptide during recombinant production in mammalian cells.

[0159] Use of a nucleic acid according to any one of embodiments 1 to 30 or a vector according to embodiment 31 for increasing the amount of intact / non-protease-cleaved heterologous polypeptide recovered from mammalian cells in which the heterologous polypeptide is recombinantly expressed.

[0160] In addition to the various aspects and embodiments depicted and claimed herein, the subject matter disclosed herein also relates to other aspects and embodiments having other combinations of the features disclosed and claimed herein. Thus, the specific features presented herein, particularly as presented as aspects or embodiments, may be combined with each other in other ways within the scope of the subject matter disclosed herein such that the subject matter disclosed herein includes any suitable combination of the features disclosed herein. For purposes of illustration and description, a description of specific embodiments of the disclosed subject matter has been presented. It is not intended to be exhaustive or to limit the disclosed subject matter to the embodiments disclosed. BRIEF DESCRIPTION OF THE DRAWINGS

[0161] Figure 1 Composition of the product obtained from (a) a cell line expressing an exemplary heterologous protein in the absence of BPTI compared to (b) a cell line co-expressing BPTI, as measured by CE-SDS.

[0162] Figure 2 Percentage of intact main product and cleaved main product produced by different culture methods; cells without inhibitor (circles); co-expression of a protease-specific inhibitor (asterisks); knockout of a specific protease (one variant alone (light color) or in combination (dark color)) (crosses).

[0163] Figure 3 Supernatant product concentration of cell cultures without protease inhibitor and cultures co-expressing a soluble protease-specific inhibitor.

[0164] Figure 4 Percentage of total product concentration (sum of uncleaved and cleaved main products) and main product of cell cultures without protease inhibitor (triangles) and cultures co-expressing a soluble protease-specific inhibitor using a medium-strength SV40 promoter (circles).

[0165] Figure 5 Percentage of total product concentration (sum of uncleaved and cleaved main products) and uncleaved product of cell cultures without protease inhibitor and cultures co-expressing a soluble protease-specific inhibitor using a medium-strength SV40 promoter (triangles: cells without inhibitor; circles: cells with co-expression of protease inhibitor driven by a medium-strength SV40 promoter).

[0166] Figure 6The percentage of the total product concentration (= sum of cleaved and uncleaved products) of cell cultures co-expressing a soluble protease-specific inhibitor and cultures to which the protein serine protease inhibitor BPTI was added to the culture medium (triangles: cells without inhibitor; circles: cells with BPTI added) to the total main product (sum of uncleaved and cleaved main products).

[0167] Figure 7 The percentage of the uncleaved product to the total product concentration (sum of uncleaved and cleaved main products) of cell cultures co-expressing a soluble protease-specific inhibitor and cultures to which the protein serine protease inhibitor BPTI was added to the culture medium (triangles: cells without inhibitor; circles: cells with BPTI added).

[0168] Figure 8 The titers (as determined by Protein A chromatography) and relative amounts (as determined by reduced CE-SDS) of cleaved and uncleaved products under different culture conditions. Detailed Description

[0169] Useful methods and techniques for carrying out the present invention are described, for example, in Ausubel, F.M. (ed.), Current Protocols in Molecular Biology, Volumes I to III (1997); Glover, N.D. and Hames, B.D. eds., DNA Cloning: A Practical Approach, Volumes I and II (1985), Oxford University Press; Freshney, R.I. (ed.), Animal Cell Culture – a practical approach, IRL Press Limited (1986); Watson, J.D. et al., Recombinant DNA, Second Edition, CHSL Press (1992); Winnacker, E.L., From Genes to Clones; N.Y., VCH Publishers (1987); Celis, J. ed., Cell Biology, Second Edition, Academic Press (1998); Freshney, R.I., Culture of Animal Cells: A Manual of Basic Technique, Second Edition, Alan R. Liss, Inc., N.Y. (1987).

[0170] Derivatives of nucleic acids can be generated using recombinant DNA technology. Such derivatives can be modified, for example, at one or several nucleotide positions by substitution, alteration, exchange, deletion, or insertion. The modification or derivatization can be carried out, for example, by means of site-directed mutagenesis. Such modifications can be readily carried out by a person skilled in the art (see, for example, Sambrook, J. et al., Molecular Cloning: A laboratory manual (1999) Cold Spring Harbor Laboratory Press, New York, USA; Hames, B.D. and Higgins, S.G., Nucleic acid hybridization – a practical approach (1985) IRL Press, Oxford, England).

[0171] It must be noted that, as used herein and in the appended claims, the singular forms "a", "an", and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a cell" includes a plurality of such cells and their equivalents known to those skilled in the art, and so forth. Similarly, the terms "a / an", "one or more", and "at least one" can be used interchangeably herein. It should also be noted that the terms "comprising", "including", and "having" can be used interchangeably.

[0172] The term "about" means a range of + / - 20% of the value that follows. In certain embodiments, the term "about" means a range of + / - 10% of the value that follows. In certain embodiments, the term "about" means a range of + / - 5% of the value that follows.

[0173] The term "including" also includes the term "consisting of".

[0174] Nucleic acid according to the invention

[0175] The present invention is at least partially based on the discovery that the combination of a selection marker and a protein protease inhibitor in a single cistron, for example, using a self-cleaving peptide sequence or an IRES for ligation, is advantageous and provides improvements. These improvements especially reduce protease cleavage during the recombinant production (expression) of heterologous polypeptides in mammalian cells and thereby increase the production volume (yield). At the same time, the expression titer of the recombinant heterologous polypeptide is not reduced compared to cells that do not include the nucleic acid according to the invention.

[0176] It has been found that during the recombinant production of polypeptides comprising one or more amino acid sequences cleavable by a serine protease (i.e., comprising one or more serine protease recognition (amino acid) sequences), cleavage of said sequences has occurred.

[0177] The present invention is exemplified hereinafter using an N-terminal Fab domain inserted 2+1 bispecific antibody (TCB) which has additional functional groups linked by a peptide linker comprising one or more amino acid sequences cleavable by a serine protease / serine protease recognition sequence. This is presented only as an example of the method according to the invention and should not be construed as a limitation thereof. The true scope is set forth in the appended claims.

[0178] Cleavage occurs independently of the cells expressing the heterologous polypeptide. The degree of cleavage in different cells used for expression is as shown in Table 1 below and Figure 1 as indicated.

[0179] Table 1: Cleavage is dependent on the recombinant cells.

[0180] Cell Cleavage product of the total product ExpiCHO-S 3％ HEK293 9％ CHO-K1 34％

[0181] Generally, cleavage of recombinant polypeptides during production cannot be attributed to a single protease of the cells. Thus, it is expected that by co-expressing a soluble protease-specific inhibitor to reduce the activity of a single protease, the amount of cleaved recombinant polypeptide will not be significantly reduced. However, by co-expressing a protease-specific inhibitor, the fraction of intact (i.e., uncleaved) recombinant polypeptide can be increased from about 38% ( Figure 2 circles in, x-axis) to more than 50% ( Figure 2 asterisks in, x-axis), while the fraction of cleaved recombinant polypeptide can be reduced from about 11% ( Figure 2 circles in, y-axis) to about 4.5% ( Figure 2 asterisks in, y-axis). For comparison, the knockout of the corresponding protease in the corresponding cell pool (one variant alone or both in combination) is shown. The knockout results in a cell population with heterogeneous knockouts (e.g., homozygous, heterozygous, no knockout), thus resulting in intermediate cleavage levels ( Figure 2 crosses in).

[0182] However, at the same time, the total polypeptide titre decreases sharply by about 30% from 1000 mg / L to 700 mg / L, so overall no improvement in yield can be achieved (see Figure 3 ).

[0183] It has been found that the reduction in total titer can be overcome by reducing the promoter strength of the promoter operably linked (i.e., driving its expression) to the coding sequence of the protease inhibitor. More specifically, by using a medium-strength SV40 promoter instead of a high-strength CMV promoter, the reduction in total titer was reduced while maintaining the inhibition of cleavage (including an increase in the uncleaved antibody heavy chain at the protease cleavage site from 84% to 94%). This is as Figure 4 (Total titer vs. total main product) Figure 5 (Total titer vs. uncleaved heavy chain) shown (triangles: cells without inhibitor; circles: cells with co-expression of protease inhibitor driven by medium-strength SV40 promoter).

[0184] Further unexpectedly, it was found that by adding a genus-specific protease inhibitor to the culture medium, comparable results to those obtained by co-expressing a protease-specific inhibitor, i.e., the degree of cleavage reduction, could not be achieved, although a better effect was expected from the genus-specific protease inhibitor. This has been exemplified by adding the protein serine protease inhibitor BPTI (aprotinin) to the culture medium (4 μM on days 3 and 10 of a 14-day fed-batch culture; see Figure 6 and Figure 7 ; circles represent without protease inhibitor; squares with protease inhibitor). The uncleaved antibody chain with protease cleavage site only increased by 82% to 89% ( Figure 7 ), while co-expression using the SV40 promoter led to an increase in the uncleaved antibody chain with protease cleavage site from 84% to 94% ( Figure 5 ).

[0185] Now it has been unexpectedly found that even further improvements in titer and cleavage prevention can be achieved by combining the protease inhibitor with a selection marker in a monocistronic expression cassette. This is shown in Figure 8and in Table 2. An exemplary combination of the protein protease inhibitor BPTI (aprotinin) and the selectable marker puromycin acetyltransferase linked by a T2A self-cleaving peptide sequence is used. It can be seen that, on the one hand, the total titer (cleaved + uncleaved) increases (average 2578 mg / mL (2385 - 2770 mg / mL) vs. average 1862 mg / mL (1133 - 2365 mg / mL); in the supernatant determined by protein A chromatography) and the relative titer (total titer multiplied by the main product (monomer) content determined by SEC) increases (average 1501 mg / mL (1485 - 1517 mg / mL) vs. average 625 mg / mL (491 - 675 mg / mL) in the supernatant determined by sequential protein A and SEC chromatography). This increase in relative titer is achieved by increasing the total titer (as determined by protein A chromatography) and simultaneously maintaining or even improving the ratio of cleaved / uncut antibody heavy chains including the protease cleavage site (as determined by CE-SDS).

[0186] Table 2: Expression yield and relative titer.

[0187]

[0188]

[0189] Thus, in one specific implementation taught by the present invention, the monocistronic expression cassette according to the present invention comprises a polynucleotide sequence encoding two polypeptides linked by a linker, the linker comprising a sequence capable of inducing ribosome skipping (i.e., self-cleavage).

[0190] In certain embodiments of all aspects and embodiments, the nucleic acid according to the present invention comprises a first nucleic acid encoding a selectable marker and a second nucleic acid encoding a protein protease inhibitor combined by a linker sequence encoding a self-cleaving peptide sequence.

[0191] Thus, an independent aspect of the present invention is an (isolated) nucleic acid which, in the 5' to 3' direction, comprises the following elements in operable linkage:

[0192] a) a nucleic acid encoding a selectable marker,

[0193] b) a nucleic acid encoding a self-cleaving peptide sequence, and

[0194] c) a nucleic acid encoding a protein protease inhibitor.

[0195] In certain embodiments of all aspects and embodiments, the nucleic acid further comprises the following elements in operable linkage:

[0196] d) a promoter upstream (5') of the nucleic acid in a),

[0197] e) a polyadenylation signal sequence downstream (3') of the nucleic acid of c), and

[0198] f) an optional terminator sequence downstream (3') of the nucleic acid of e).

[0199] In a preferred embodiment of all aspects and embodiments, the protease inhibitor inhibits plasminogen activator and / or is BPTI (aprotinin) (SEQ ID NO: 86; AQRPDFCLEPPYTGPCKARMIRYFYNAKAGLCQPFVYGGCRAKRNNFKSSEDCMRTCGGA) or a plasminogen activator inhibitor type 1 (PAI-1) - derived peptide EEIIMD (SEQ ID NO: 88).

[0200] In a preferred embodiment of all aspects and embodiments, the self - cleaving peptide sequence is the T2A self - cleaving peptide sequence or a functional variant thereof that induces ribosomal skipping.

[0201] In certain embodiments of all aspects and embodiments, the T2A self - cleaving peptide sequence comprises the amino acid sequence EGRGSLLTCGDVEENPGP (SEQ ID NO: 14), which can be encoded by the nucleic acid sequence GAGGGCAGAGGAAGTCTGCTAACATGCGGTGACGTCGAGGAGAATCCTGGCCCA (SEQ ID NO: 15).

[0202] In certain embodiments of all aspects and embodiments, the linker sequence further comprises a spacer sequence before / upstream of the self - cleaving peptide sequence. In certain embodiments, the spacer sequence comprises the amino acid sequence SGRSGGG (SEQ ID NO: 03), which can be encoded by the nucleic acid sequence TCCGGAAGATCTGGCGGCGGA (SEQ ID NO: 90).

[0203] In certain embodiments of all aspects and embodiments, the linker further comprises an amino acid sequence corresponding to a furin cleavage site. Furin is a protease that can cleave a protein precursor before it is secreted into the trans-Golgi. Furin cleaves at the C-terminus of its recognition sequence. A furin cleavage sequence can be added to remove amino acid residues at the C-terminus of a protein upstream of the self-cleaving peptide sequence. Different furin recognition sequences (or "furin cleavage sites") have been developed. These include, but are not limited to, RXKR (SEQ ID NO:91) or RXRR (SEQ ID NO:92) and RXXR (SEQ ID NO:93), where X is any naturally occurring amino acid. In certain embodiments, the furin cleavage site has the recognition sequence RQKR (SEQ ID NO:94). In certain embodiments, the furin cleavage site has the recognition sequence X1RX2X3R (SEQ ID NO:95), where X1 is K or R, X2 is any naturally occurring amino acid, and X3 is K or R. The appropriate furin cleavage site for the present invention can be selected based on the knowledge of the present invention and in combination with the knowledge in the art.

[0204] In certain embodiments of all aspects and embodiments, the linker comprises a nucleic acid sequence encoding a combination of a furin cleavage site and a 2A self-cleaving peptide sequence. In certain embodiments, the linker comprises a nucleic acid sequence encoding a furin cleavage site and an F2A self-cleaving peptide sequence, a furin cleavage site and an E2A self-cleaving peptide sequence, a furin cleavage site and a P2A self-cleaving peptide sequence, or a furin cleavage site and a T2A self-cleaving peptide sequence. In certain embodiments, the linker comprises a nucleic acid sequence encoding a furin cleavage site and a T2A self-cleaving peptide sequence. The appropriate combination for the present invention can be selected based on the knowledge of the present invention and in combination with the knowledge in the art.

[0205] In certain embodiments of all aspects and embodiments, the linker may further comprise a spacer sequence between the furin cleavage site and the 2A self-cleaving peptide sequence. Various spacer sequences are known in the art. In certain embodiments, the spacer sequence is a glycine-serine (GS) spacer sequence, such as (GS) n , (GSGGS) n (SEQ ID NO:06) and (GGGS) n(SEQ ID NO:07), where n represents an integer of at least 1. In certain embodiments, the spacer sequences are selected from GGSG (SEQ ID NO:08), GGSGG (SEQ ID NO:09), GSGSG (SEQ ID NO:10), GSGGG (SEQ ID NO:11), GGGSG (SEQ ID NO:12), GSSSG (SEQ ID NO:13), GGGGS (SEQ ID NO:99), etc. Suitable spacer sequences for the present invention can be selected based on the knowledge of the present invention and combined with the knowledge in the art.

[0206] In certain embodiments of all aspects and embodiments, the nucleic acid according to the present invention comprises a nucleic acid encoding puromycin acetyltransferase and a nucleic acid encoding BPTI separated by a furin cleavage site-(G4S)2-T2A self-cleaving peptide sequence (F-G4S2-T2A linker). The F-G4S2-T2A linker has the amino acid sequence RAKRGGGGSGGGGSEGRGSLLTCGDVEENPGP (SEQ ID NO:106) and can be encoded by the nucleic acid sequence AGAGCCAAGCGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGAGGGCAGAGGAAGTCTGCTAACATGCGGTGACGTCGAGGAGAATCCTGGCCCA (SEQ ID NO:107).

[0207] In certain alternative embodiments above, the self-cleaving peptide sequence is an F2A self-cleaving peptide sequence. In certain embodiments, the F2A self-cleaving peptide sequence comprises the amino acid sequence VKQTLNFDLLKLAGDVESNPGP (SEQ ID NO:44), which can be encoded by the nucleic acid sequence (SEQ ID NO:45) GTGAAACAGACTTTGAATTTTGACCTTCTCAAGTTGGCGGGAGACGTGGAGTCCAACCCAGGGCCG.

[0208] A "functional fragment" of a polypeptide, such as an antibody, a self-cleaving peptide sequence, an enzyme, a selectable marker, or a nucleic acid, is a polypeptide or nucleic acid whose sequence is not identical to that of the corresponding full-length polypeptide or nucleic acid, but retains the same function as the full-length polypeptide or nucleic acid. Thus, the term "functional fragment" encompasses variants of the full-length polypeptide or nucleic acid that have the same or fewer residues than the corresponding full-length molecule, i.e., are shorter or longer, and / or contain one or more amino acid or nucleotide substituents. Methods for determining the function of nucleic acids (e.g., coding function, ability to hybridize to another nucleic acid) and polypeptides are well known in the art. See, e.g., Ausubel et al., supra; Fields et al. (1989) Nature 340:245-246; US 5,585,245 and WO 98 / 44350.

[0209] Cells and methods according to the invention

[0210] The present invention is exemplified by CHO cells and using targeted integration. This is presented only by way of illustration of the present invention and should not be construed in any way as limiting. Any other eukaryotic or mammalian cells and any other transfection / integration methods can be used. The true scope of the present invention is set forth in the claims.

[0211] For example, in targeted integration (TI), site-specific recombination is used to introduce exogenous nucleic acid into a specific locus within the genome of a mammalian TI host cell. This can be used to generate recombinant cells according to the present invention. TI is an enzyme-catalyzed process in which the sequence located at the integration site within the genome is exchanged for the exogenous nucleic acid. One system for effecting this nucleic acid exchange is the Cre-lox system. The enzyme that catalyzes the exchange is Cre recombinase. The sequences to be exchanged are defined by the positions of at least two lox(P) sites within the genome and within the exogenous nucleic acid. These lox(P) sites are recognized by Cre recombinase. No more is required, i.e., no ATP, etc.

[0212] A suitable mammalian TI host cell for the example method for generating recombinant cells according to the present invention is a CHO cell carrying a landing site integrated at a single site within a genomic locus of the cell, wherein the landing site comprises three heterospecific loxP sites for Cre recombinase-mediated DNA recombination.

[0213] More specifically, the heterospecific loxP sites are L3, LoxFas, and 2L (see, for example, Lanza et al., Biotechnol. 7 (2012) 898-908; Wong et al., Nucleic Acids Res. 33 (2005) e147), where L3 and 2L flank the landing site at the 5'-end and 3'-end, respectively, and LoxFas is located between the L3 and 2L sites. The landing site further contains a dicistronic unit that links the expression of a selection marker via an IRES to the expression of the fluorescent GFP protein, allowing for the stabilization of the landing site by positive selection and allowing for the selection of the absence of the site after transfection and Cre recombination (negative selection). The green fluorescent protein (GFP) is used to monitor the recombinase-mediated cassette exchange (RMCE) reaction.

[0214] Such a configuration of the landing site as outlined in the foregoing paragraph allows for the synchronous integration of two vectors, for example, the synchronous integration of a pre-vector carrying the L3 and LoxFas sites and a post-vector carrying the LoxFas and 2L sites. Functional elements of a selection marker that are different from those of the selection marker present in the landing site and also different from those of the selection marker in the nucleic acid according to the invention have been distributed between the two vectors: the promoter and the start codon are located on the pre-vector, while the coding region and the polyadenylation signal sequence are located on the post-vector. Only the correct recombinase-mediated integration of the nucleic acids from the two vectors induces resistance against the corresponding selection agent.

[0215] Thus, two vectors are designed: a first vector (pre-vector) and a second vector (post-vector). The two vectors comprise different expression cassettes. The number of expression cassettes is generally not limited, but typically each vector independently has between 1 and 4 expression cassettes. One of the expression cassettes, for example in the post-vector, comprises the nucleic acid according to the invention.

[0216] In the current example of expressing an N-terminal Fab domain with additional functional groups linked by a peptide linker inserted into a 2+1 bispecific antibody, a pre-vector including the following sequence is used: the L3 site, an expression cassette for the first heavy chain, a first expression cassette for the first light chain, a second expression cassette for the first light chain, and a promoter, the start codon of a selection marker different from puromycin acetyltransferase, and the LoxFas site, and a post-vector including the LoxFas site, the coding region and the polyadenylation signal sequence of the selection marker, an expression cassette encoding the second heavy chain, an expression cassette encoding the second light chain, an expression cassette comprising the nucleic acid according to the invention, and the 2L site. They are integrated into CHO-K1 TI host cells using dual RMCE, cultured, and the heterologous antibody product is isolated from the supernatant. The corresponding data are presented in the previous paragraph.

[0217] Thus, in certain embodiments of all aspects and embodiments, nucleic acids according to the invention and one or more nucleic acids encoding heterologous polypeptides have been integrated into mammalian TI host cells by recombinase-mediated cassette exchange (RMCE). Recombinant mammalian cells according to the invention, such as recombinant CHO cells, are thereby obtained, in which the expression cassette has been integrated into a single locus in the genome.

[0218] In certain embodiments of all aspects and embodiments, the integrated landing site comprises at least one selectable marker. In certain embodiments, the integrated landing site comprises first, second, and third recombination recognition sequences (RRSs) and at least one selectable marker. In certain embodiments, the selectable marker is located between the first RRS and the second RRS. In certain embodiments, two RRSs flank the at least one selectable marker, i.e., the first RRS is located 5' (upstream), and the second RRS is located 3' (downstream) of the selectable marker. In certain embodiments, the first RRS is adjacent to the 5' end of the selectable marker, and the second RRS is adjacent to the 3' end of the selectable marker. In certain embodiments, the landing site comprises a first RRS, a second RRS, and a third RRS, and at least one selectable marker located between the first RRS and the third RRS.

[0219] In certain embodiments of all aspects and embodiments, the first, second, and third RRSs are the L3 (SEQ ID NO:96), LoxFas (SEQ ID NO:98), and 2L (SEQ ID NO:97) sites.

[0220] In certain embodiments of all aspects and embodiments, the CHO cells are CHO-K1 cells.

[0221] Accordingly, one aspect of the invention is a method for preparing a recombinant cell expressing a heterologous polypeptide, the method comprising:

[0222] a) providing a targeted integration host cell comprising an exogenous nucleotide sequence integrated at a locus in the genome of a host cell, wherein the exogenous nucleotide sequence comprises first and second recombination recognition sequences flanking at least one first selectable marker and a third recombination recognition sequence positioned between the first and second recombination recognition sequences, and all recombination recognition sequences are different;

[0223] b) Introduce a first vector comprising two recombination recognition sequences that match the first and third recombination recognition sequences on the integrated exogenous nucleotide sequence into the cells provided in a), wherein the two recombination recognition sequences flank two to four expression cassettes (exogenous nucleotide sequences) and at least (a part of) one second selection marker, and a second vector comprising two recombination recognition sequences that match the second and third recombination recognition sequences on the integrated exogenous nucleotide sequence, wherein the two recombination recognition sequences flank two to four (additional) expression cassettes (exogenous nucleotide sequences), and wherein at least one expression cassette comprises a nucleic acid according to the invention;

[0224] c) Introduce simultaneously into i) the first and second vectors of b); or ii) subsequently use one or more recombinases in sequence,

[0225] wherein one or more recombinases recognize the recombination recognition sequences of the first and second vectors; (and optionally wherein one or more recombinases perform two recombinase-mediated cassette exchanges); and

[0226] d) Select recombinant host cells that express the second selection marker and secrete the bispecific antibody,

[0227] thereby preparing recombinant cells that express a heterologous polypeptide.

[0228] In certain embodiments of all aspects and embodiments, the first and / or second vector comprises an expression cassette that comprises a nucleic acid according to the invention.

[0229] In certain embodiments of all aspects and embodiments, the heterologous polypeptide is a multispecific antibody. In a preferred embodiment, the heterologous polypeptide is a bispecific antibody.

[0230] In certain embodiments of all aspects and embodiments, each of the first and second vectors comprises at least one exogenous nucleotide sequence encoding an antibody light chain and at least one exogenous nucleotide sequence encoding an antibody heavy chain.

[0231] In certain embodiments of all aspects and embodiments, the first and / or second vector comprises one exogenous nucleotide sequence encoding an antibody light chain and one exogenous nucleotide sequence encoding an antibody heavy chain.

[0232] In certain embodiments of all aspects and embodiments, the first and / or second vector comprises one exogenous nucleotide sequence encoding an antibody light chain and one exogenous nucleotide sequence encoding an antibody heavy chain, wherein the exogenous nucleotide sequence encoding the antibody heavy chain is upstream (5') of the exogenous nucleotide sequence encoding the antibody light chain.

[0233] In certain embodiments of all aspects and embodiments, the first and / or second vector comprises an exogenous nucleotide sequence encoding an antibody light chain and an exogenous nucleotide sequence encoding an antibody heavy chain, wherein the antibody light chain and the antibody heavy chain have domain crossover.

[0234] In certain embodiments of all aspects and embodiments, the first vector comprises an exogenous nucleotide sequence encoding an antibody light chain and an exogenous nucleotide sequence encoding an antibody heavy chain, wherein the antibody light chain and the antibody heavy chain have domain crossover.

[0235] In a preferred embodiment of all aspects and embodiments, the first vector comprises an exogenous nucleotide sequence encoding an antibody light chain and an exogenous nucleotide sequence encoding an antibody heavy chain, wherein the antibody light chain and the antibody heavy chain have domain crossover and the exogenous nucleotide sequence encoding the antibody light chain having domain crossover is upstream (5') of the exogenous nucleotide sequence encoding the antibody heavy chain having domain crossover.

[0236] In a preferred embodiment of all aspects and embodiments, the first vector comprises an exogenous nucleotide sequence encoding an antibody light chain and an exogenous nucleotide sequence encoding an antibody heavy chain, wherein the antibody light chain and the antibody heavy chain have domain crossover and the exogenous nucleotide sequence encoding the antibody heavy chain having domain crossover is upstream (5') of the exogenous nucleotide sequence encoding the antibody light chain having domain crossover.

[0237] In an embodiment of all aspects and embodiments, the first vector comprises a promoter sequence operably linked to the codon ATG, wherein the upstream side of the promoter sequence is flanked (two) exogenous nucleotide sequences (i.e., located downstream thereof), and the downstream side of the ATG codon is flanked by a recombination recognition sequence (i.e., located upstream thereof); and the second vector comprises a selection marker lacking an ATG transcription start codon, the upstream side of which is flanked by a recombination recognition sequence and the downstream side is flanked (two) exogenous nucleotide sequences.

[0238] A further aspect of the present invention is a method for producing a heterologous polypeptide, the method comprising:

[0239] a) providing a recombinant cell according to the present invention;

[0240] b) culturing the recombinant cell of a) and recovering the heterologous polypeptide from the cell or the culture medium;

[0241] c) optionally, purifying the heterologous polypeptide by one or more chromatographic steps;

[0242] and thereby producing the heterologous polypeptide.

[0243] Cell

[0244] Any mammalian cell can be used to generate the recombinant cells according to the invention, which can be used in the method according to the invention. In addition, any mammalian cell can be used, independent of the integration method, i.e., for random integration (RI) as well as TI.

[0245] Examples of useful mammalian cells are human amniotic fluid cells (e.g., CAP-T cells as described in Woelfel, J. et al., BMC Proc. 5 (2011) P133); monkey kidney CV1 line transformed by SV40 (COS-7); human embryonic kidney line (HEK293 or HEK293T cells, as described in Graham, F.L. et al., J. Gen Virol. 36 (1977) 59-74); baby hamster kidney cells (BHK); mouse Sertoli cells (TM4 cells, as described in Mather, J.P., Biol. Reprod. 23 (1980) 243-252); monkey kidney cells (CV1); African green monkey kidney cells (VERO-76); human cervical carcinoma cells (HELA); dog kidney cells (MDCK); buffalo rat hepatocytes (BRL 3A); human lung cells (W138); human hepatocytes (Hep G2); mouse mammary tumor (MMT060562); TRI cells (as described in Mather, J.P. et al., Annals N.Y. Acad. Sci. 383 (1982) 44-68); MRC 5 cells; and FS4 cells. Other useful mammalian cells include Chinese hamster ovary (CHO) cells, including DHFR-CHO cells (Urlaub, G. et al., Proc. Natl. Acad. Sci. USA 77 (1980) 4216-4220); and myeloma cells, such as Y0, NS0, and Sp2 / 0. For a review of certain mammalian cells suitable for antibody production, see, e.g., Yazaki, P. and Wu, A.M., Methods in Molecular Biology, Vol. 248, Lo, B.K.C. (ed.), Humana Press, Totowa, NJ (2004), pp. 255-268.

[0246] As used herein, the term "recombinant cell" refers to a mammalian cell comprising an exogenous nucleic acid. Such recombinant mammalian cells are cells into which at least a nucleic acid according to the invention has been introduced, including progeny of such cells. In certain embodiments, the recombinant cell is a mammalian cell comprising a nucleic acid according to the invention and one or more additional nucleic acids encoding a heterologous polypeptide. Thus, the term "recombinant cell comprising a nucleic acid encoding a heterologous polypeptide" refers to a recombinant mammalian cell comprising one or more exogenous nucleic acids integrated into the genome of a mammalian cell and capable of expressing a heterologous polypeptide as well as the nucleic acid according to the invention. In certain embodiments, the recombinant cell is a mammalian cell comprising one or more exogenous nucleic acids at a single locus integrated within a locus of the genome of the cell. In a preferred embodiment, the recombinant cell is a mammalian cell comprising a nucleic acid according to the invention and one or more additional exogenous nucleic acids at a single locus integrated within a locus of the genome of the cell, wherein the integrated nucleic acids comprise first, second, and third recombination recognition sequences located between a first and a second recombination recognition sequence, and all of the recombination recognition sequences are different.

[0247] "Recombinant cell" further encompasses cells that have been genetically modified, such as, for example, cells comprising a nucleic acid according to the invention and expressing a heterologous polypeptide of interest and that can be used for recombinant production of said heterologous polypeptide of interest at any scale. For example, "recombinant cell" refers to a cell into which a nucleic acid according to the invention and one or more nucleic acids encoding a heterologous polypeptide of interest have been stably introduced into the genome. For example, a "recombinant mammalian cell comprising a nucleic acid according to the invention and one or more nucleic acids encoding a heterologous polypeptide" can be a mammalian cell that has undergone recombinase-mediated cassette exchange (RMCE), wherein a nucleic acid according to the invention and a coding sequence for a polypeptide of interest have been stably introduced into the genome of the mammalian cell.

[0248] "Recombinant cell" further includes primary transformed cells and progeny derived therefrom, regardless of the number of passages. For example, the progeny may not be identical to the parental cell in nucleic acid content but may contain mutations. Mutant progeny having the same function or biological activity as the function or biological activity selected or screened for in the originally transformed cell are encompassed.

[0249] "Isolated cell" refers to a cell that has been separated from the components of its natural environment.

[0250] "Isolated nucleic acid" refers to a nucleic acid molecule that has been separated from the components of its natural environment.

[0251] In certain embodiments of all aspects and embodiments, the mammalian cells are, for example, Chinese hamster ovary (CHO) cells (such as CHO K1, CHO DG44, etc.), human embryonic kidney (HEK) cells, lymphoblastoid cells (such as, Y0, NS0, Sp2 / 0 cells) or human amniotic fluid cells (such as CAP-T, etc.). In a preferred embodiment of all aspects and embodiments, the cells are CHO cells.

[0252] Regarding TI, any known or future mammalian cells that are suitable for TI and include landing sites as described herein integrated at a single site within a genomic locus can be used in the present invention. Such cells can be designated as mammalian TI host cells. In certain embodiments, the mammalian TI host cells are hamster cells, human cells, rat cells or mouse cells that contain landing sites as described herein. In a preferred embodiment, the mammalian TI host cell is a CHO cell. In certain embodiments, the mammalian TI host cell is a Chinese hamster ovary (CHO) cell, a CHO K1 cell, a CHO K1SV cell, a CHO DG44 cell, a CHO DUKXB-11 cell, a CHO K1S cell or a CHO K1M cell that contains a landing site as described herein integrated at a single site within a genomic locus.

[0253] Antibody

[0254] General information regarding the nucleotide sequences of human immunoglobulin light and heavy chains is given in: Kabat, E.A. et al., Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, MD (1991).

[0255] The term "antibody" herein is used in its broadest sense and encompasses various antibody structures, including but not limited to full-length antibodies, monoclonal antibodies, multispecific antibodies (such as bispecific antibodies) and antibody-antibody fragment-fusions, and combinations thereof, provided that they contain one or more amino acid sequences that are endogenous to the mammalian cells used for expression during recombinant production and are subject to protease cleavage in the absence of a nucleic acid or a functional variant thereof according to the present invention.

[0256] The term "full-length antibody" refers to an antibody having a structure substantially similar to a native antibody. A full-length antibody comprises two full-length antibody light chains and two full-length antibody heavy chains, each full-length antibody light chain comprising a light chain variable region and a light chain constant domain in the N-terminal to C-terminal direction, and each full-length antibody heavy chain comprising a heavy chain variable region, a first heavy chain constant domain, a hinge region, a second heavy chain constant domain, and a third heavy chain constant domain in the N-terminal to C-terminal direction. In contrast to a native antibody, a full-length antibody may further comprise immunoglobulin domains, such as one or more additional scFvs conjugated to one or more termini of different chains of the full-length antibody, or heavy chain or light chain Fab fragments, or scFabs, provided that only a single fragment is conjugated to each terminus. These conjugates are also encompassed by the term full-length antibody.

[0257] The term "antibody binding site" refers to a pair of heavy chain variable domains and light chain variable domains. To ensure proper binding to an antigen, these variable domains are homologous variable domains, i.e., belong together. An antibody binding site comprises at least three HVRs (e.g., in the case of VHH) or three to six HVRs (e.g., in the naturally occurring case, i.e., a conventional antibody having a VH / VL pair). Generally, the amino acid residues of the antibody responsible for antigen binding form the binding site. These residues are normally contained within a pair of antibody heavy chain variable domains and the corresponding antibody light chain variable domains. The antigen binding site of an antibody comprises amino acid residues from the "hypervariable region" or "HVR". The "framework" or "FR" regions are those variable domain regions other than the hypervariable region residues as defined herein. Thus, the light chain and heavy chain variable domains of an antibody comprise, from the N-terminus to the C-terminus, the regions FR1, HVR1, FR2, HVR2, FR3, HVR3, and FR4. In particular, the HVR3 region of the heavy chain variable domain is the region that contributes most to antigen binding and defines the antibody binding specificity. A "functional binding site" is capable of binding to its target. The term "binds to" means that in certain embodiments the binding site binds to its target in an in vitro assay in a binding assay. Such a binding assay can be any assay so long as the binding event can be detected. "Binding" can be determined, for example, using an ELISA assay.

[0258] As used herein, the term "hypervariable region" or "HVR" refers to each of the following: a region of an antibody variable domain that is hypervariable in sequence ("complementary determining region" or "CDR") and / or forms a structurally defined loop ("hypervariable loop") and / or contains antigen contact residues ("antigen contact points") including amino acid residue extensions. Generally, an antibody comprises six HVRs; three in the heavy chain variable domain VH (H1, H2, H3), and three in the light chain variable domain VL (L1, L2, L3).

[0259] HVRs include

[0260] (a) Hypervariable loops present at amino acid residues 26 - 32 (L1), 50 - 52 (L2), 91 - 96 (L3), 26 - 32 (H1), 53 - 55 (H2), and 96 - 101 (H3) (Chothia, C. and Lesk, A.M., J. Mol. Biol. 196 (1987) 901 - 917);

[0261] (b) CDRs present at amino acid residues 24 - 34 (L1), 50 - 56 (L2), 89 - 97 (L3), 31 - 35b (H1), 50 - 65 (H2), and 95 - 102 (H3) (Kabat, E.A. et al., Sequences of Proteins of Immunological Interest, 5th ed. Public Health Service, National Institutes of Health, Bethesda, MD (1991), NIH Publication 91 - 3242.);

[0262] (c) Antigen - contact points present at amino acid residues 27c - 36 (L1), 46 - 55 (L2), 89 - 96 (L3), 30 - 35b (H1), 47 - 58 (H2), and 93 - 101 (H3) (MacCallum et al. J. Mol. Biol. 262:732 - 745 (1996)); and

[0263] (d) Combinations of (a), (b), and / or (c), including amino acid residues 46 - 56 (L2), 47 - 56 (L2), 48 - 56 (L2), 49 - 56 (L2), 26 - 35 (H1), 26 - 35b (H1), 49 - 65 (H2), 93 - 102 (H3), and 94 - 102 (H3).

[0264] Unless otherwise specified, HVR residues and other residues (e.g., FR residues) in the variable domains are numbered herein according to Kabat et al., supra.

[0265] The "class" of an antibody refers to the type of constant domain or constant region (preferably the Fc region) possessed by the heavy chain of the antibody. Antibodies are mainly divided into five classes: IgA, IgD, IgE, IgG, and IgM, and some of these classes can be further divided into subclasses (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The heavy - chain constant domains corresponding to the different classes of immunoglobulins are designated α, δ, ε, γ, and μ, respectively.

[0266] The term "heavy chain constant region" refers to an immunoglobulin heavy chain that contains constant domains, namely, the CH1 domain, the hinge region, the CH2 domain, and the CH3 domain. In one embodiment, the human IgG constant region extends from Ala118 to the carboxyl terminus of the heavy chain (according to the Kabat EU index numbering). However, the C-terminal lysine (Lys447) of the constant region may or may not be present (according to the Kabat EU index numbering). The term "constant region" refers to a dimer containing two heavy chain constant regions that may be covalently linked to each other via hinge region cysteine residues to form interchain disulfide bonds.

[0267] The term "heavy chain Fc region" refers to the C-terminal region of an immunoglobulin heavy chain that contains at least a portion of the hinge region (the middle and lower hinge regions), the CH2 domain, and the CH3 domain. In one embodiment, the human IgG heavy chain Fc region extends from Asp221 or Cys226 or Pro230 to the carboxyl terminus of the heavy chain (according to the Kabat EU index numbering). Thus, the Fc region is smaller than the constant region but is identical to it in the C-terminal portion. However, the C-terminal lysine (Lys447) of the heavy chain Fc region may or may not be present (according to the Kabat EU index numbering). The term "Fc region" refers to a dimer containing two heavy chain Fc regions that may be covalently linked to each other through hinge region cysteine residues to form interchain disulfide bonds.

[0268] As used in this application, the term "valence" refers to the number of binding sites present in an antibody. Thus, the terms "bivalent", "tetravalent", and "hexavalent" refer to the presence of two binding sites, four binding sites, and six binding sites, respectively, in an antibody.

[0269] "Monospecific antibody" refers to an antibody having a single binding specificity, i.e., specifically binding to one antigen. Monospecific antibodies can be prepared as full-length antibodies or antibody fragments (e.g., F(ab')2) or combinations thereof (e.g., full-length antibody plus additional scFv or Fab fragments). Monospecific antibodies do not need to be monovalent, i.e., monospecific antibodies can contain more than one binding site that specifically binds to one antigen. For example, natural antibodies are monospecific but bivalent.

[0270] "Multispecific antibody" means having binding specificities with respect to at least two different epitopes on the same antigen or two different antigens. Multispecific antibodies can be prepared as full-length antibodies or antibody fragments (e.g., Fab bispecific antibodies) or combinations thereof (antibody-antibody fragment-fusions, e.g., a full-length antibody conjugated to an additional scFv or Fab fragment). Multispecific antibodies are at least bivalent, i.e., contain two antigen-binding sites. In addition, multispecific antibodies are at least bispecific. Thus, a bivalent bispecific antibody is the simplest form of a multispecific antibody. Engineered antibodies having two, three, or more (e.g., four) functional antigen-binding sites have also been reported (see, e.g., US2002 / 0004587).

[0271] In certain embodiments of all aspects and embodiments, the cell produces the multispecific antibody as a heterologous polypeptide. In certain embodiments, one of the binding specificities of the multispecific antibody is directed against a first antigen and the other is directed against a different second antigen. In certain embodiments, the multispecific antibody binds to two different epitopes of the same antigen. In certain embodiments, the second epitope on the same antigen is a non-overlapping epitope. In certain embodiments, the antibody is a bispecific antibody. In a preferred embodiment, the bispecific antibody is a trivalent bispecific antibody or a bivalent bispecific antibody.

[0272] Techniques for preparing multispecific antibodies include, but are not limited to, recombinant co-expression of two immunoglobulin heavy chain-light chain pairs with different specificities (see Milstein, C. and Cuello, A.C., Nature 305 (1983) 537-540, WO 93 / 08829, and Traunecker, A. et al., EMBO J. 10 (1991) 3655-3659), and "knob-in-hole" engineering (see, e.g., US 5,731,168). Multispecific antibodies can also be prepared by engineering electrostatic steering effects for preparing antibody Fc-heterodimer molecules (WO 2009 / 089004); crosslinking two or more antibodies or fragments (see, e.g., US 4,676,980; and Brennan, M. et al., Science 229 (1985) 81-83); producing bispecific antibodies using leucine zippers (see, e.g., Kostelny, S.A. et al., J. Immunol. 148 (1992) 1547-1553); using common light chain technology to circumvent light chain mispairing problems (see, e.g., WO 98 / 50431); using specific techniques for preparing bispecific antibody fragments (see, e.g., Holliger, P. et al., Proc. Natl. Acad. Sci. USA 90 (1993) 6444-6448); and preparing trispecific antibodies as described, e.g., in Tutt, A. et al., J. Immunol. 147 (1991) 60-69).

[0273] Also included herein are engineered antibodies having three or more antigen-binding sites, including, for example, "Octopus antibody" or DVD-Ig (see, e.g., WO 2001 / 77342 and WO 2008 / 024715). Other examples of multispecific antibodies having three or more antigen-binding sites can be found in WO 2010 / 115589, WO 2010 / 112193, WO 2010 / 136172, WO 2010 / 145792, and WO 2013 / 026831. Bispecific antibodies or antigen-binding fragments thereof also include "dual action Fab" or "DAF" (see, e.g., US2008 / 0069820 and WO 2015 / 095539).

[0274] Multispecific antibodies can also be provided in an asymmetric form, where there is domain crossover in one or more binding arms with the same antigen specificity, i.e., by swapping VH / VL domains (see, e.g., WO 2009 / 080252 and WO 2015 / 150447), CH1 / CL domains (see, e.g., WO 2009 / 080253), or entire Fab arms (see, e.g., WO 2009 / 080251, WO 2016 / 016299, see also Schaefer et al., Proc. Natl. Acad. Sci. USA 108 (2011) 1187-1191, and Klein et al., MAbs 8 (2016) 1010-1020).

[0275] In a preferred embodiment of all aspects and embodiments, the multispecific antibody comprises Fab fragments, where the variable or constant regions of the heavy and light chains are exchanged, i.e., where in one chain, the heavy chain VH variable domain is conjugated directly or via a peptide linker to the light chain CL constant domain, and in the corresponding other chain, the light chain VL variable domain is conjugated directly or via a peptide linker to the heavy chain CH1 constant domain.

[0276] Thus, the domain-swapped Fab fragment comprises a polypeptide chain composed of the light chain variable region (VL) and the heavy chain constant region 1 (CH1), and a polypeptide chain composed of the heavy chain variable region (VH) and the light chain constant region (CL).

[0277] Asymmetric Fab arms can also be engineered by introducing charged or uncharged amino acid mutations at the domain interface to direct correct Fab pairing. See, e.g., WO 2016 / 172485.

[0278] The antibody or fragment can also be a multispecific antibody as described in WO 2009 / 080254, WO 2010 / 112193, WO 2010 / 115589, WO2010 / 136172, WO 2010 / 145792, or WO 2010 / 145793.

[0279] The antibody or its fragment can also be a multispecific antibody as described in WO 2012 / 163520.

[0280] Various additional molecular forms for multispecific antibodies are known in the art and are included herein (see, e.g., Spiess et al., Mol. Immunol. 67 (2015) 95-106).

[0281] Bispecific antibodies are generally antibody molecules that specifically bind to two different, non-overlapping epitopes on the same antigen or to two epitopes on different antigens.

[0282] In certain embodiments of all aspects and embodiments, the bispecific antibody is selected from the group of bispecific antibodies consisting of:

[0283] - Domain - swapped 1 + 1 bispecific antibody (CrossMab);

[0284] Such an antibody is a bispecific full - length IgG antibody comprising a pair of first light and first heavy chains and a pair of second light and second heavy chains, wherein the pair of first light and first heavy chains comprises a first Fab fragment and the pair of second light and second heavy chains comprises a second Fab fragment,

[0285] wherein in the first Fab fragment

[0286] a) only the CH1 and CL domains are swapped with each other (i.e., the light chain of the first Fab fragment comprises the VL and CH1 domains and the heavy chain of the first Fab fragment comprises the VH and CL domains);

[0287] b) only the VH and VL domains are swapped with each other (i.e., the light chain of the first Fab fragment comprises the VH and CL domains while the heavy chain of the first Fab fragment comprises the VL and CH1 domains); or

[0288] c) the CH1 and CL domains and the VH and VL domains are swapped with each other (i.e., the light chain of the first Fab fragment comprises the VH and CH1 domains and the heavy chain of the first Fab fragment comprises the VL and CL domains);

[0289] wherein the second Fab fragment comprises a light chain comprising the VL and CL domains and a heavy chain comprising the VH and CH1 domains;

[0290] wherein both the first heavy chain and the second heavy chain contain CH3 domains, and the two CH3 domains are engineered in a complementary manner by corresponding amino acid substitutions to support heterodimerization of the first heavy chain and the second heavy chain (in a preferred embodiment, one CH3 domain contains a knob mutation and the corresponding other CH3 domain contains a hole mutation;

[0291] - C - terminal Fab domain - fused 2 + 1 bispecific antibody (BS);

[0292] Such an antibody is a bispecific full - length IgG antibody that comprises

[0293] a) a full - length antibody comprising two pairs each having a full - length antibody light chain and a full - length antibody heavy chain, wherein the binding site formed by each pair of the full - length heavy chain and full - length light chain specifically binds to a first antigen, and

[0294] b) an additional Fab fragment, wherein the additional Fab fragment is fused to the C - terminus of one of the heavy chains of the full - length antibody, and the binding site of the additional Fab fragment specifically binds to a second antigen,

[0295] The additional Fab fragments that specifically bind to the second antigen include domain crossover such that a) the variable light chain domain (VL) and the variable heavy chain domain (VH) are replaced with each other, or b) the constant light chain domain (CL) and the constant heavy chain domain (CH1) are replaced with each other;

[0296] - Bispecific single - arm single - chain antibody (OaMab);

[0297] Such antibodies are bispecific single - arm single - chain antibodies that include a first binding site that specifically binds to a first epitope or antigen and a second binding site that specifically binds to a second epitope or antigen, whereby the individual chains are as follows

[0298] - Light chain (including variable light chain domain and constant light chain domain);

[0299] - Combined light / heavy chain (including variable light chain domain, constant light chain domain, peptide linker, variable heavy chain domain, CH1 domain, hinge region, CH2 domain, and CH3 with knob or hole mutations) in N - terminal to C - terminal order

[0300] - Heavy chain (including variable heavy chain domain, CH1 domain, hinge region, CH2 domain, and CH3 domain with hole or knob mutation) in N - terminal to C - terminal order));

[0301] - Bispecific two - arm single - chain antibody;

[0302] Such antibodies are bispecific two - arm single - chain antibodies that include a first binding site that specifically binds to a first epitope or antigen and a second binding site that specifically binds to a second epitope or antigen, whereby the individual chains are as follows

[0303] - Combined light / heavy chain 1 (including variable light chain domain 1, constant light chain domain, peptide linker, variable heavy chain domain 1, CH1 domain, hinge region, CH2 domain, CH3 domain with knob or hole mutation);

[0304] - Combined light / heavy chain 2 (including variable light chain domain 2, constant light chain domain, peptide linker, variable heavy chain domain 2, CH1 domain, hinge region, CH2 domain, CH3 domain with hole or knob mutation);

[0305] - N - terminal Fab domain - inserted 2 + 1 bispecific antibody (TCB);

[0306] Such antibodies are bispecific full - length antibodies that have additional heavy - chain N - terminal binding sites and domain exchange and include

[0307] - First and second Fab fragments, wherein each binding site of the first and second Fab fragments specifically binds to a first antigen,

[0308] - A third Fab fragment, wherein the binding site of the third Fab fragment specifically binds to a second antigen, and wherein the third Fab fragment includes a domain crossover such that the variable light chain domain (VL) and the variable heavy chain domain (VH) are replaced with each other, and

[0309] - An Fc region, which includes a first Fc region polypeptide and a second Fc region polypeptide,

[0310] wherein each of the first and second Fab fragments includes a heavy chain fragment and a full-length light chain,

[0311] wherein the C-terminus of the heavy chain fragment of the first Fab fragment is fused to the N-terminus of the first Fc region polypeptide,

[0312] wherein the C-terminus of the heavy chain fragment of the second Fab fragment is fused to the N-terminus of the variable light chain domain of the third Fab fragment, and the C-terminus of the CH1 domain of the third Fab fragment is fused to the N-terminus of the second Fc region polypeptide;

[0313] - An antibody-polymer-fusion;

[0314] Such an antibody is a fusion polypeptide that includes

[0315] (a) An antibody heavy chain and an antibody light chain, and

[0316] (b) A first fusion polypeptide that includes, in the N-terminus to C-terminus direction, a first portion of a non-antibody polymer polypeptide, an antibody heavy chain CH1 domain or an antibody light chain constant domain, an antibody hinge region, an antibody heavy chain CH2 domain, and an antibody heavy chain CH3 domain; and a second fusion polypeptide that includes, in the N-terminus to C-terminus direction, a second portion of the non-antibody polymer polypeptide and, in the case where the first polypeptide includes an antibody heavy chain CH1 domain, an antibody light chain constant domain or, in the case where the first polypeptide includes an antibody light chain constant domain, an antibody heavy chain CH1 domain,

[0317] wherein

[0318] (i) The antibody heavy chain of (a) and the first fusion polypeptide of (b), (ii) the antibody heavy chain of (a) and the antibody light chain of (a), and (iii) the first fusion polypeptide of (b) and the second fusion polypeptide of (b) are each independently covalently linked to each other by at least one disulfide bond,

[0319] wherein

[0320] the variable domains of the antibody heavy chain and the antibody light chain form a binding site that specifically binds to an antigen).

[0321] The CH3 domain in the heavy chain of an antibody can be modified by the "knob-into-hole" technology, which is disclosed in several examples such as WO 96 / 027011, Ridgway, J.B. et al., Protein Eng. 9 (1996) 617-621; and Merchant, A.M. et al., Nat. Biotechnol. 16 (1998) 677-681. In this method, the interaction surfaces of two CH3 domains are modified to increase the heterodimerization of these two CH3 domains and thereby increase the heterodimerization of the polypeptide comprising them. One of the two CH3 domains (of the two heavy chains) can be the "knob", and the corresponding other one the "hole". The introduction of a disulfide bridge further stabilizes the heterodimer (Merchant, A.M. et al., Nature Biotech. 16 (1998) 677-681; Atwell, S. et al., J. Mol. Biol. 270 (1997) 26-35) and improves the yield.

[0322] The mutation T366W in the CH3 domain (of the heavy chain of the antibody) is designated as the "knob mutation", and the mutations T366S, L368A, Y407V in the CH3 domain (of the heavy chain of the antibody) are designated as the "hole mutations" (according to the Kabat EU index numbering). Additional inter-chain disulfide bridges between CH3 domains can also be used (Merchant, A.M. et al., Nature Biotech. 16 (1998) 677-681), for example, by introducing the S354C mutation into the CH3 domain of the heavy chain with the "knob mutation" (designated as the "knob-cys-mutation"), and by introducing Y349C into the CH3 domain of the heavy chain with the "hole mutation" (designated as the "hole-cys-mutation") (according to the Kabat EU index numbering).

[0323] As used herein, the term "domain crossover" means that in a pair of antibody heavy chain VH-CH1 fragments and their corresponding homologous antibody light chains, i.e., in an antibody Fab (antigen-binding fragment), the domain sequences are derived from the sequences in a natural antibody, where at least one heavy chain domain is replaced by its corresponding light chain domain and vice versa. There are three common types of domain crossover: (i) crossover of the CH1 domain and the CL domain, which results in a VL-CH1 domain sequence due to domain crossover in the light chain and a VH-CL domain sequence (or a full-length antibody heavy chain with a VH-CL-hinge-CH2-CH3 domain sequence) due to domain crossover in the heavy chain fragment; (ii) crossover of the VH domain and the VL domain, which results in a VH-CL domain sequence due to domain crossover in the light chain and a VL-CH1 domain sequence due to domain crossover in the heavy chain fragment; and (iii) crossover of the full light chain (VL-CL) and the full VH-CH1 heavy chain fragment ("Fab crossover"), which results in a light chain with a VH-CH1 domain sequence due to domain crossover and a heavy chain fragment with a VL-CL domain sequence due to domain crossover (all of the foregoing domain sequences are represented in the N-terminal to C-terminal direction).

[0324] As used herein, the term "replace each other" with respect to a corresponding heavy chain domain and a light chain domain refers to the aforementioned domain crossover. Thus, when the CH1 domain and the CL domain "replace each other", it refers to the domain crossover described in item (i) below and the resulting heavy chain domain sequence and light chain domain sequence. Thus, when VH and VL "replace each other", it refers to the domain crossover described in item (ii) below; and when the CH1 and CL domains "replace each other" and the VH and VL domains "replace each other", it refers to the domain crossover described in item (iii) below. Bispecific antibodies including domain crossover are reported in, for example, WO 2009 / 080251, WO 2009 / 080252, WO 2009 / 080253, WO 2009 / 080254, and Schaefer, W. et al., Proc. Natl. Acad. Sci. USA 108 (2011) 11187-11192. Such antibodies are commonly referred to as CrossMab.

[0325] In certain embodiments, the multispecific antibody further comprises at least one Fab fragment that includes a domain crossover of the CH1 and CL domains as mentioned in item (i) above, or a domain crossover of the VH and VL domains as mentioned in item (ii) above, or a domain crossover of the VH-CH1 and VL-VL domains as mentioned in item (iii) above. In certain embodiments, in the case of a multispecific antibody having a domain crossover, the Fabs that specifically bind to the same antigen are constructed to have the same domain sequence. Thus, in the case of a multispecific antibody that includes multiple Fabs having a domain crossover, the Fabs specifically bind to the same antigen.

[0326] As used herein, the term "recombinant antibody" refers to all antibodies (chimeric antibodies, humanized antibodies, and human antibodies) prepared, expressed, created, or isolated by recombinant means such as recombinant cell preparation. This includes antibodies isolated from recombinant cells such as NS0, HEK, BHK, amniotic cells, or CHO cells.

[0327] As used herein, the term "antibody fragment" refers to a molecule other than an intact antibody, which includes a portion of an intact antibody that binds to the antigen to which the intact antibody binds, i.e., it is a functional fragment. Examples of antibody fragments include, but are not limited to, Fv; Fab; Fab'; Fab'-SH; F(ab')2; bispecific Fab; diabody; linear antibody; single-chain antibody molecule (e.g., scFv or scFab).

[0328] Recombinant method

[0329] Antibodies can be produced using recombinant methods and compositions, for example, as described in US 4,816,567. For such methods, one or more isolated nucleic acids encoding the antibody are provided.

[0330] In one aspect of the invention, a method for recombinantly producing an antibody is provided, the antibody comprising one or more cleavable amino acid sequences and being cleavable by endogenous proteases of the production cells during recombinant production in the absence of the nucleic acid according to the invention, wherein the method comprises culturing a recombinant cell according to the invention comprising the nucleic acid according to the invention and one or more nucleic acids encoding the antibody under conditions suitable for antibody expression, and optionally recovering the antibody from the recombinant cell (and / or cell culture medium), and further optionally purifying the antibody by one or more chromatographic steps.

[0331] For the recombinant production of antibodies, the nucleic acid encoding the antibody is generated / designed / synthesized and inserted into one or more vectors for further cloning and / or expression in cells. Such nucleic acids can be readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes capable of specifically binding to the genes encoding the heavy and light chains of the antibody), or such nucleic acids can be produced by recombinant methods or obtained by chemical synthesis.

[0332] Typically, for the recombinant large-scale production of a target polypeptide, such as a therapeutic antibody, recombinant cells that stably express and secrete the polypeptide are required. The overall process for generating such recombinant cells is referred to as "cell line development". In the first step of the cell line development process, a suitable mammalian cell, such as, for example, a CHO cell in certain embodiments, is transfected with one or more nucleic acids, the one or more nucleic acids including a nucleic acid according to the present invention and the nucleic acid suitable for expressing the target polypeptide. In the second step, recombinant cells that stably express a protein protease inhibitor and the target polypeptide are selected, for example, based on co-expression of a selection marker, which have been co-transfected with the nucleic acids.

[0333] The nucleotide sequence of a nucleic acid encoding a polypeptide, i.e., the coding sequence, is designated as the structural gene. Such structural genes are pure coding information. Therefore, additional regulatory elements are required for its expression. Thus, normally, the structural gene is integrated into a so-called expression cassette. The minimum regulatory elements required for an expression cassette to function in a mammalian cell are a promoter that functions in the mammalian cell, which is located upstream of the structural gene, i.e., 5', and a polyadenylation signal sequence that functions in the mammalian cell, which is located downstream of the structural gene, i.e., 3'. The promoter, the structural gene, and the polyadenylation signal sequence are arranged in an operably linked form.

[0334] In the case where the target polypeptide is a heteromeric polypeptide composed of different polypeptides, such as, for example, an antibody or a complex antibody form, not only a single expression cassette is required, but a large number of expression cassettes that are different in the structural genes they contain, i.e., at least one expression cassette is required for each of the different polypeptides (chains) of the heteromeric polypeptide (heteromeric antibody). For example, a full-length antibody is a heteropolymeric polypeptide comprising two copies of the light chain and two copies of the heavy chain. Thus, a full-length antibody is composed of two different polypeptides. Therefore, for the expression of a full-length antibody, two expression cassettes are required, one for the light chain and one for the heavy chain. For example, if the full-length antibody is a bispecific antibody (i.e., an antibody comprising two different binding sites that specifically bind to two different antigens / epitopes on the same antigen), then the two light chains, as well as the two heavy chains, are also different from each other. Thus, such a bispecific full-length antibody is composed of four different polypeptides and therefore requires four expression cassettes.

[0335] The expression cassette for the target polypeptide is then integrated into one or more so-called "expression vectors" for correct expression or "integration vectors" for targeted integration. A "vector" is a nucleic acid that provides all the elements required for amplifying the vector in bacterial cells and expressing the included structural genes in mammalian cells. Generally, an expression vector includes a prokaryotic plasmid proliferation unit, such as a prokaryotic plasmid proliferation unit for Escherichia coli, which includes an origin of replication and a prokaryotic selection marker, as well as a eukaryotic selection marker, and an expression cassette required for expressing the target structural gene. An "expression vector" or "integration vector" is a transport tool for introducing the expression cassette into mammalian cells to generate recombinant polypeptide-expressing cells.

[0336] As outlined in the previous paragraph, the more complex the polypeptide to be expressed, the higher the number of different expression cassettes required. As the number of expression cassettes increases, the size of the nucleic acid to be integrated into the cellular genome also increases. At the same time, the size of the expression vector also increases. However, the practical upper limit of the vector size is in the range of about 15 kbp, beyond which the processing and production efficiency significantly decreases. This problem can be solved by using two or more expression vectors. Thus, the expression cassettes can be split between different expression vectors, with each expression vector containing only some of the expression cassettes, resulting in a size reduction.

[0337] Cell line development (CLD) for generating recombinant cells for expressing heterologous polypeptides such as, for example, multispecific antibodies, employs nucleic acids by random integration (RI) or targeted integration (TI), which contain the corresponding expression cassettes required for expressing and producing the target heterologous polypeptide.

[0338] Generally, using RI, several vectors or fragments thereof are integrated into the cellular genome at the same or different loci.

[0339] Generally, using TI, a single copy of a transgene containing different expression cassettes is integrated into a predetermined "hot spot" in the cellular genome.

[0340] Suitable cells for generating recombinant cells for expressing (glycosylated) antibodies usually originate from multicellular organisms such as vertebrates.

[0341] Targeted integration

[0342] One method for generating recombinant mammalian cells according to the present invention to be used in the method according to the present invention is targeted integration to introduce the corresponding nucleic acid.

[0343] In certain embodiments of all aspects and embodiments, nucleic acids according to the invention and one or more nucleic acids encoding a heterologous polypeptide have been integrated into a mammalian TI host cell by single or double recombinase-mediated cassette exchange (RMCE). Thereby, recombinant mammalian cells, such as recombinant CHO cells, are obtained, in which the expression cassette has been integrated at a single locus of the genome.

[0344] The Cre-LoxP site-specific recombination system has been widely used in many biological experimental systems. Cre recombinase is a 38 kDa site-specific DNA recombinase that can recognize the 34 bp LoxP sequence. Cre recombinase is derived from bacteriophage P1 and belongs to the tyrosine family of site-specific recombinases. Cre recombinase can mediate intramolecular and intermolecular recombination between LoxP sequences. The LoxP sequence consists of an 8 bp non-palindromic core region and two flanking 13 bp inverted repeat sequences. Cre recombinase binds to the 13 bp repeat sequences, thereby mediating recombination within the 8 bp core region. Cre-LoxP-mediated recombination occurs with high efficiency and does not require any other host factors. If two LoxP sequences are placed in the same nucleotide sequence in the same orientation, Cre recombinase-mediated recombination will excise the DNA sequence located between the two LoxP sequences to form a covalently closed loop. If two LoxP sequences are placed in the same nucleotide sequence in inverted positions, Cre recombinase-mediated recombination will invert the orientation of the DNA sequence located between these two sequences. If two LoxP sequences are on two different DNA molecules and if one DNA molecule is a circular molecule, Cre recombinase-mediated recombination will cause the integration of the circular DNA sequence.

[0345] A "recombination recognition sequence" (RRS) is a nucleotide sequence recognized by a recombinase and is necessary and sufficient for a recombinase-mediated recombination event. The RRS can be used to define the position in a nucleotide sequence where a recombination event will occur.

[0346] The term "matching RRS" means that recombination has occurred between two RRSs. In certain embodiments, the two matching RRSs are the same.

[0347] In certain embodiments of all aspects and embodiments, the RRS can be recognized by Cre recombinase.

[0348] In certain embodiments of all aspects and embodiments, the RRS can be recognized by FLP recombinase.

[0349] In certain embodiments of all aspects and embodiments, the RRS can be recognized by Bxb1 integrase.

[0350] In certain embodiments of all aspects and embodiments, the RRS can be recognized by integrase.

[0351] In certain embodiments of all aspects and embodiments, both RRSs are wild-type LoxP sequences. In certain embodiments, both RRSs are mutant LoxP sequences.

[0352] In certain embodiments of all aspects and embodiments, both RRSs are wild-type FRT sequences.

[0353] In certain embodiments of all aspects and embodiments, both RRSs are mutant FRT sequences.

[0354] In certain embodiments of all aspects and embodiments, the two matching RRSs are different sequences, but can be recognized by the same recombinase.

[0355] In certain embodiments of all aspects and embodiments, the first matching RRS is a Bxb1 attP sequence and the second matching RRS is a Bxb1 attB sequence.

[0356] In certain embodiments of all aspects and embodiments, the first matching RRS is an attB sequence and the second matching RRS is an attB sequence.

[0357] When using a dual-vector combination, a "dual-plasmid RMCE" strategy or "dual-RMCE" is employed in the method according to the invention. For example, but not by way of limitation, the integrated landing site can comprise three RRSs, such as the following arrangement: wherein a third RRS ("RRS3") is present between a first RRS ("RRS1") and a second RRS ("RRS2"), and the first vector comprises two RRSs that match the first RRS and the third RRS on the integrated foreign nucleotide sequence, and the second vector comprises two RRSs that match the third RRS and the second RRS on the integrated foreign nucleotide sequence.

[0358] The dual plasmid RMCE strategy involves using three RRS sites to perform two independent RMCEs simultaneously. Thus, the landing site in mammalian TI host cells using the dual plasmid RMCE strategy includes a third RRS site (RRS3) that has no cross-activity with the first RRS site (RRS1) or the second RRS site (RRS2). The two plasmids to be targeted need the same flanking RRS sites for efficient targeting, where one plasmid (the forward) is flanked by RRS1 and RRS3, and the other expression plasmid (the reverse) is flanked by RRS3 and RRS2. Two selection markers are also required in this dual plasmid RMCE. One selection marker expression cassette is split into two parts. The forward plasmid will contain a promoter, followed by a start codon and the RRS3 sequence. The reverse plasmid will have the RRS3 sequence fused to the N-terminus of the selection marker coding region with the start codon (ATG) removed. Additional nucleotides may need to be inserted between the RRS3 site and the selection marker sequence to ensure in-frame translation of the fusion protein (i.e., an operable linkage). Only when both plasmids are correctly inserted will the complete expression cassette of the selection marker be assembled and thus confer resistance of the cells to the corresponding selection agent.

[0359] Dual plasmid RMCE involves double recombination crossover events between two heterospecific RRSs within the target genomic locus and the donor DNA molecule, which are catalyzed by recombinase. Dual plasmid RMCE is designed to introduce copies of DNA sequences from the combined forward and reverse vectors into a predetermined locus of the genome of mammalian TI host cells. RMCE can be implemented such that prokaryotic vector sequences are not introduced into the genome of mammalian TI host cells, thereby reducing and / or preventing unnecessary triggering of host immune or defense mechanisms. The RMCE procedure can be repeated with multiple DNA sequences.

[0360] In certain embodiments of all aspects and embodiments, targeted integration is achieved by two RMCEs, where two different DNA sequences (each including at least one expression cassette encoding a portion of a heterologous polypeptide and / or at least one selection marker or a portion thereof flanked by two heterospecific RRSs) are integrated into a predetermined locus of the genome of an RRS-matched mammalian TI host cell. In certain embodiments, targeted integration is achieved by multiple RMCEs, where DNA sequences from multiple vectors are all integrated into a predetermined locus of the genome of mammalian TI host cells, where each DNA sequence contains at least one expression cassette encoding a portion of a heterologous polypeptide and / or at least one selection marker or a portion thereof flanked by two heterospecific RRSs. In certain embodiments, the selection marker may be partially encoded on the first vector and partially encoded on the second vector such that expression of the selection marker is only permitted when both are correctly integrated by two RMCEs.

[0361] In certain embodiments of all aspects and embodiments, targeted integration via recombinase-mediated recombination results in the integration of a selection marker and / or different expression cassettes for the nucleic acids according to the invention, as well as multimeric polypeptides, into one or more predetermined integration sites of the genome of a host cell that does not contain sequences from a prokaryotic vector.

[0362] In certain embodiments of all aspects and embodiments, a mammalian TI host cell comprises an integrated landing site, wherein the landing site comprises two or more recombination recognition sequences (RRSs). The RRSs can be recognized by a recombinase (e.g., Cre recombinase, FLP recombinase, Bxb1 integrase, or an integrase). The RRSs can be independently selected from the group consisting of: LoxP sequences, LoxP L3 sequences, LoxP 2L sequences, LoxFas sequences, Lox511 sequences, Lox2272 sequences, Lox2372 sequences, Lox5171 sequences, Loxm2 sequences, Lox71 sequences, Lox66 sequences, FRT sequences, Bxb1 attP sequences, Bxb1 attB sequences, attP sequences, and attB sequences. If multiple RRSs must be present, the selection of each of these sequences depends on another sequence within the limits of selecting different RRSs.

[0363] Typically, a mammalian TI host cell is a mammalian cell comprising a landing site integrated at a single locus in the genome of the mammalian cell, wherein the landing site comprises a first recombination recognition sequence and a second recombination recognition sequence flanking at least one first selection marker, and a third recombination recognition sequence located between the first recombination recognition sequence and the second recombination recognition sequence, and all of the recombination recognition sequences are different.

[0364] The selectable marker may be selected from the group consisting of aminoglycoside phosphotransferase (APH) (e.g., hygromycin phosphotransferase (HYG), neomycin, and G418 APH), dihydrofolate reductase (DHFR), thymidine kinase (TK), glutamine synthetase (GS), asparagine synthetase, tryptophan synthase (indole), histidinol dehydrogenase (histidinol D), and genes encoding resistance to puromycin, blasticidin, bleomycin, phleomycin, chloramphenicol, Zeocin, and mycophenolic acid. The selectable marker may also be a fluorescent protein selected from the group consisting of green fluorescent protein (GFP), enhanced GFP (eGFP), synthetic GFP, yellow fluorescent protein (YFP), enhanced YFP (eYFP), cyan fluorescent protein (CFP), mPlum, mCherry, tdTomato, mStrawberry, J-red, DsRed monomer, mOrange, mKO, mCitrine, Venus, YPet, Emerald6, CyPet, mCFPm, Cerulean, and T-Sapphire.

[0365] An exogenous nucleotide sequence is a nucleotide sequence that is not derived from a specific cell but can be introduced into the cell by a DNA delivery method (such as, for example, by a transfection method, an electroporation method, or a transformation method). In certain embodiments, a mammalian TI host cell contains at least one landing site at one or more integration sites integrated into the genome of the mammalian cell. In certain embodiments, the landing site is integrated at one or more integration sites within a specific locus of the genome of the mammalian cell.

[0366] In certain embodiments of all aspects and embodiments, the integrated landing site includes at least one selectable marker. In certain embodiments, the integrated landing site includes first, second, and third RRSs and at least one selectable marker. In certain embodiments, the selectable marker is located between the first RRS and the second RRS. In certain embodiments, two RRSs flank at least one selectable marker, i.e., the first RRS is located 5' (upstream), and the second RRS is located 3' (downstream) of the selectable marker. In certain embodiments, the first RRS is adjacent to the 5' end of the selectable marker, and the second RRS is adjacent to the 3' end of the selectable marker. In certain embodiments, the landing site includes a first RRS, a second RRS, and a third RRS, and at least one selectable marker located between the first RRS and the third RRS.

[0367] In certain embodiments of all aspects and embodiments, the selection marker is located between a first RRS and a second RRS, and the two flanking RRSs are different. In certain embodiments, the first flanking RRS is the LoxP L3 sequence (SEQ ID NO:96), and the second flanking RRS is the LoxP 2L sequence (SEQ ID NO:97). In certain embodiments, the LoxP L3 sequence is located at the 5' end of the selection marker, and the LoxP 2L sequence is located at the 3' end of the selection marker. In certain embodiments, the first flanking RRS is a wild-type FRT sequence, and the second flanking RRS is a mutant FRT sequence. In certain embodiments, the first flanking RRS is the Bxb1 attP sequence, and the second flanking RRS is the Bxb1 attB sequence. In certain embodiments, the first flanking RRS is the attP sequence, and the second flanking RRS is the attB sequence. In certain embodiments, the two RRSs are oriented in the same direction. In certain embodiments, both RRSs are in the forward or reverse orientation. In certain embodiments, the two RRSs are oriented in opposite directions.

[0368] In certain embodiments of all aspects and embodiments, the integrated landing site includes a first selection marker and a second selection marker, flanked by two RRSs, wherein the first selection marker is different from the second selection marker. In certain embodiments, both selection markers are independently selected from the group consisting of: glutamine synthetase selection marker, thymidine kinase selection marker, HYG selection marker, and puromycin resistance selection marker. In certain embodiments, the integrated landing site includes a thymidine kinase selection marker and an HYG selection marker. In certain embodiments, the first selection marker is selected from the group consisting of: aminoglycoside phosphotransferase (APH) (e.g., hygromycin phosphotransferase (HYG), neomycin, and G418 APH), dihydrofolate reductase (DHFR), thymidine kinase (TK), glutamine synthetase (GS), asparagine synthetase, tryptophan synthase (indole), histidinol dehydrogenase (histidinol D), and genes encoding resistance to puromycin, blasticidin, bleomycin, phleomycin, chloramphenicol, Zeocin, and mycophenolic acid, and the second selection marker is selected from the group consisting of: GFP, eGFP, synthetic GFP, YFP, eYFP, CFP, mPlum, mCherry, tdTomato, mStrawberry, J-red, DsRed monomer, mOrange, mKO, mCitrine, Venus, YPet, Emerald, CyPet, mCFPm, Cerulean, and T-Sapphire fluorescent proteins. In certain embodiments, the first selection marker is a glutamine synthetase selection marker and the second selection marker is a GFP fluorescent protein. In certain embodiments, the two RRSs flanking the two selection markers are different.

[0369] In certain embodiments, the selection marker is operably linked to a promoter sequence. In certain embodiments, the selection marker is operably linked to the SV40 promoter. In certain embodiments, the selection marker is operably linked to the human cytomegalovirus (CMV) promoter.

[0370] As used herein, the term "operably linked" refers to the juxtaposition of two or more components, wherein the relationship of these components allows them to function in a desired manner. For example, if a promoter and / or enhancer is used to regulate transcription of a coding sequence, the promoter and / or enhancer is operably linked to the coding sequence. In certain embodiments, the DNA sequences that are "operably linked" are contiguous and adjacent on a single chromosome. In certain embodiments, for example, when two protein coding regions (such as a secretory leader and a polypeptide) must be joined, the sequences are contiguous, adjacent, and in the same reading frame. In certain embodiments, an operably linked promoter is located upstream of and adjacent to the coding sequence. In certain embodiments, for example, with respect to an enhancer sequence that regulates expression of a coding sequence, the two components are capable of being operably linked but are not adjacent. If an enhancer increases transcription of a coding sequence, the enhancer is operably linked to the coding sequence. An operably linked enhancer can be located upstream of, within, or downstream of the coding sequence and can be located at a considerable distance from the promoter of the coding sequence. Operable linkage can be accomplished by recombinant methods known in the art, such as using PCR methods and / or by ligation at convenient restriction sites. If convenient restriction sites do not exist, synthetic oligonucleotide linkers or adaptors can be used according to conventional practice. An internal ribosome entry site (IRES) is operably linked to an open reading frame (ORF) if it permits initiation of translation of the ORF in an internal position independent of the 5' end.

[0371] As used herein, the term "selectable marker" refers to a gene that permits the specific selection or exclusion of cells carrying that gene in the presence of the corresponding selective agent. For example, but not by way of limitation, a selectable marker may permit positive selection of host cells transformed with the selectable marker gene in the presence of the corresponding selective agent (selective culture conditions); untransformed host cells will not be able to grow or survive under the selective culture conditions. A selectable marker can be a positive marker, a negative marker, or a bifunctional marker. A positive selectable marker permits selection of cells carrying the marker, while a negative selectable marker permits the selective elimination of cells carrying the marker. A selectable marker can confer resistance to a drug or compensate for a metabolic or catabolic defect in the host cell. In prokaryotic cells, genes that confer resistance to ampicillin, tetracycline, kanamycin, or chloramphenicol can be used. Resistance genes that can be used as selectable markers in eukaryotic cells include, but are not limited to, genes for aminoglycoside phosphotransferase (APH) (e.g., hygromycin phosphotransferase (HYG), neomycin, and G418 APH), dihydrofolate reductase (DHFR), thymidine kinase (TK), glutamine synthetase (GS), asparagine synthetase, tryptophan synthase (indole), histidinol dehydrogenase (histidinol D), and genes encoding resistance to puromycin, blasticidin, bleomycin, phleomycin, chloramphenicol, Zeocin, and mycophenolic acid. Additional marker genes are described in WO92 / 08796 and WO 94 / 28143.

[0372] In addition to facilitating selection in the presence of the corresponding selective agent, a selectable marker can alternatively be a molecule that is not normally present in the cell, such as green fluorescent protein (GFP), enhanced GFP (eGFP), synthetic GFP, yellow fluorescent protein (YFP), enhanced YFP (eYFP), cyan fluorescent protein (CFP), mPlum, mCherry, tdTomato, mStrawberry, J-red, DsRed monomer, mOrange, mKO, mCitrine, Venus, YPet, Emerald, CyPet, mCFPm, Cerulean, and T-Sapphire. Cells expressing such a molecule can be distinguished from cells that do not contain the gene, for example, by the detection or absence of fluorescence emitted by the encoded polypeptide.

[0373] Protease

[0374] As used herein, the term "protease" and its grammatical equivalents refer to enzymes that catalyze the hydrolysis of covalent peptide (amide) bonds. Proteases can be subdivided into different classes, such as, for example, serine proteases and matrix metalloproteases.

[0375] Matrix metalloproteinases (MMPs) are a family of metal-dependent endopeptidases, e.g., Zn 2+ -dependent endopeptidases. They preferably cleave components of the extracellular matrix. MMPs include collagenases, stromelysins, membrane-type matrix metalloproteinases, and gelatinases. In vivo, MMPs occur in the form of inactive precursors (zymogens) that need to be cleaved to obtain the catalytically active form. MMPs are specifically regulated by tissue inhibitors of matrix metalloproteinases.

[0376] Serine proteases are widely present in prokaryotes and eukaryotes. They are characterized by the presence of a catalytically active serine residue in the active center of the enzyme. The peptide bonds cleaved by serine proteases involve the nucleophilic attack of the serine residue in the active center of the enzyme on the target peptide bond. In addition to the serine residue, histidine and aspartic acid residues may further be involved, specifically their side chains. Together they form the so-called catalytic triad common to most serine proteases.

[0377] Serine proteases include chymotrypsin, trypsin, elastase, NS3, factor Xa, granzyme B, thrombin, plasmin, urokinase, tissue plasminogen activator, prostate-specific antigen; matrix metalloproteinases include, for example, gelatinase B and gelatinase A.

[0378] Chymotrypsin acts on peptide bonds flanked by a large number of hydrophobic amino acid residues, especially phenylalanine, tryptophan, and tyrosine.

[0379] Trypsin cleaves peptide bonds flanked by positively charged amino acid residues.

[0380] Elastase hydrolyzes peptide bonds flanked by small neutral / aliphatic amino acid residues, especially alanine, methionine, glycine, and valine.

[0381] Self-cleaving peptide

[0382] Introducing a so-called self-cleaving peptide sequence between two coding sequences allows the production of two polypeptides flanking the self-cleaving peptide sequence in a form separated from a single nucleic acid.

[0383] Incorporating the self-cleaving peptide sequence into a nucleic acid and thus into the corresponding mRNA results in ribosome hopping during protein synthesis or prevents the synthesis of the peptide bond at the C-terminus (prevents the formation of a phosphodiester bond between peptides or amino acid residues or acts as a pseudo-stop codon sequence that induces the translation complex to move from one codon to the next without forming a peptide bond). Thus, a single mRNA molecule encodes multiple different / independent proteins that are generated by ribosome hopping during translation.

[0384] Two separate polypeptides are obtained by including a self-cleaving peptide, one corresponding to the upstream sequence of the self-cleaving peptide sequence and the other corresponding to the downstream sequence of the self-cleaving peptide sequence. Although it is called a self-cleaving peptide sequence, in fact neither of the resulting products includes the complete sequence, but only its N-terminal or C-terminal portion respectively. The use of the term "self-cleaving" is not intended to imply proteolytic activity.

[0385] Thus, nucleic acids encoding self-cleaving peptide sequences are arranged in such a way that they are located between and conform to two coding regions before and after them.

[0386] One type of self-cleaving peptide sequence is the viral 2A self-cleaving peptide sequence. Detailed methods for the design and use of 2A self-cleaving peptide sequences can be found in Szymczak-Workman et al. (Design and Construction of 2A Peptide-Linked Multi cistronic Vectors. Cold Spring Harb. Protoc. February 1, 2012; 2012(2):199-204), which is hereby incorporated by reference in its entirety.

[0387] As with other self-cleaving peptide sequences, the incorporation of the viral 2A self-cleaving peptide sequence results in ribosomal skipping of the peptide bond synthesis at the C-terminus of the 2A self-cleaving peptide sequence. More specifically, the peptide bond connecting the glycine amino acid residue and the proline amino acid residue at the C-terminus of the 2A self-cleaving peptide sequence is not formed. Thus, two polypeptides are obtained, one of which includes a part of the 2A self-cleaving peptide sequence at its C-terminus and the other of which includes a part of the 2A self-cleaving peptide sequence at its N-terminus (see, for example, Matsuzaki, J. et al., Sci. Rep. 5(2015)14896; Banu, N. et al., Sci. Rep. 4(2014)4166; Kim et al., PLoS One 6(2011)e18556; Donnelly, M.L. et al., J. Gen. Virol, 82(2001)1027-1101; Ryan, M.D. et al., J. Gen. Virol., 72(2001)2727-2732). Exemplary viral 2A self-cleaving peptide sequences are

[0388] - The 2A self-cleaving peptide sequence of the Adoxophyes orana fasciata nuclear polyhedrosis virus - called T2A,

[0389] - The 2A self-cleaving peptide sequence of the equine rhinitis A virus - called E2A,

[0390] - The 2A self-cleaving peptide sequence of the Theiler's murine encephalomyelitis virus - called P2A,

[0391] - The 2A self-cleaving peptide sequence of foot-and-mouth disease virus - called F2A,

[0392] - The 2A self-cleaving peptide sequence of acute bee paralysis virus - A2A,

[0393] - The 2A self-cleaving peptide sequence of Drosophila C virus - D2A,

[0394] - The 2A self-cleaving peptide sequence of infectious myonecrosis virus - I2A.

[0395] The following table shows the sequences of members of the viral 2A self-cleaving peptide sequence family. It is known in the art that adding a peptide linker sequence GSG (Gly-Ser-Gly; SEQ ID NO:04) at the N-terminus of the 2A self-cleaving peptide sequence (the N-terminus of the 2A self-cleaving peptide sequence) can increase the cleavage efficiency. The N-terminus of the 2A self-cleaving peptide means that the sequence encoding GSG is located upstream of the sequence encoding the 2A self-cleaving peptide. Generally, GSG will be adjacent to the N-terminus of the 2A self-cleaving peptide sequence. In certain embodiments, 1 to 10 other amino acid residues are inserted between GSG and the 2A self-cleaving peptide sequence. In certain embodiments, the polynucleotide sequence encoding GSG is GGC AGT GGA (SEQ ID NO:05). As with any polynucleotide encoding a peptide, due to the degeneracy of the genetic code, the nucleotide sequence can be altered without changing the encoded peptide sequence, as is known to those skilled in the art.

[0396]

[0397]

[0398]

[0399]

[0400]

[0401] Substitutions of amino acid residues are within the skill of those in the art. Thus, the term "2A self-cleaving peptide sequence" encompasses variants of the foregoing peptides that retain the desired skipping / self-cleaving activity but optionally have 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more substitutions relative to the wild-type 2A self-cleaving peptide sequence (see, e.g., Liu et al., Sci. Rep. 7 (2017) 2193).

[0402] In certain embodiments of all aspects and embodiments, the 2-A self-cleaving peptide sequence is a variant of the wild-type viral 2-A self-cleaving peptide sequence, namely SEQ ID NOs: 14-63. Such variants have at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or higher sequence identity with the wild-type viral 2-A self-cleaving peptide sequence and retain the ribosomal skipping function. In certain embodiments, at least one N-terminal amino acid or the corresponding codon of any one of SEQ ID NOs: 14-85 is deleted, such as 1, 2, 3, 4 or 5 amino acids or the corresponding number of codons, including the ranges between any two of the listed values being deleted. In certain embodiments, at least one C-terminal amino acid or the corresponding codon of any one of SEQ ID NOs: 14-85 is deleted, such as 1, 2, 3, 4 or 5 amino acids or the corresponding number of codons, including the ranges between any two of the listed values being deleted. In certain embodiments, at least 1, 2, 3, 4 or 5 amino acids or the corresponding number of codons of any one of SEQ ID NOs: 14-85, including the ranges between any two of the listed values, are substituted, such as as conservative amino acid substitutions.

[0403] In certain embodiments of all aspects and embodiments, the T2A self-cleaving peptide sequence comprises an amino acid sequence that comprises EGRGSLLTCGDVEENPGP (SEQ ID NO: 14) or a sequence having at least 70%, 80%, 90%, 95% or 99% sequence identity with the amino acid sequence comprising EGRGSLLTCGDVEENPGP (SEQ ID NO: 14) and has the ribosomal skipping function.

[0404] In certain embodiments of all aspects and embodiments, the GSG-T2A self-cleaving peptide sequence comprises an amino acid sequence that comprises GSGEGRGSLLTCGDVEENPGP (SEQ ID NO: 108) or a sequence having at least 70%, 80%, 90%, 95% or 99% sequence identity with the amino acid sequence comprising GSGEGRGSLLTCGDVEENPGP (SEQ ID NO: 108) and has the ribosomal skipping function. In certain embodiments, the GSG-T2A self-cleaving peptide sequence is encoded by a nucleic acid sequence comprising GGCAGTGGAGAGGGCAGAGGAAGTCTGCTAACATGCGGTGACGTCGA GGAGAATCCTGGCCCA (SEQ ID NO: 109).

[0405] In certain embodiments of all aspects and embodiments, the E2A self-cleaving peptide sequence comprises an amino acid sequence that comprises QCTNYALLKLAGDVESNPGP (SEQ ID NO:30) or a sequence having at least 70%, 80%, 90%, 95% or 99% identity to the amino acid sequence comprising QCTNYALLKLAGDVESNPGP (SEQ ID NO:30) and a ribosome skipping function. In certain embodiments, the GSG-E2A self-cleaving peptide sequence comprises an amino acid sequence that comprises GSGQCTNYALLKLAGDVESNPGP (SEQ ID NO:110) or a sequence having at least 70%, 80%, 90%, 95% or 99% sequence identity to the amino acid sequence comprising GSGQCTNYALLKLAGDVESNPGP (SEQ ID NO:110), and has a ribosome skipping function.

[0406] In certain embodiments of all aspects and embodiments, the F2A self-cleaving peptide comprises an amino acid sequence that comprises VKQTLNFDLLKLAGDVESNPGP (SEQ ID NO:44) or a sequence having at least 70%, 80%, 90%, 95% or 99% sequence identity to the amino acid sequence comprising VKQTLNFDLLKLAGDVESNPGP (SEQ ID NO:44), and has a ribosome skipping function. In certain embodiments, the GSG-F2A self-cleaving peptide comprises an amino acid sequence that comprises GSGVKQTLNFDLLKLAGDVESNPGP (SEQ ID NO:112) or a sequence having at least 70%, 80%, 90%, 95% or 99% sequence identity to the amino acid sequence comprising GSGVKQTLNFDLLKLAGDVESNPGP (SEQ ID NO:112), and has a ribosome skipping function.

[0407] In certain embodiments of all aspects and embodiments, the P2A self-cleaving peptide comprises an amino acid sequence that comprises ATNFSLLKQAGDVEENPGP (SEQ ID NO:24) or a sequence having at least 70%, 80%, 90%, 95% or 99% sequence identity to the amino acid sequence comprising ATNFSLLKQAGDVEENPGP (SEQ ID NO:24), and has ribosome skipping function. In certain embodiments, the GSG-P2A self-cleaving peptide comprises an amino acid sequence that comprises GSGATNFSLLKQAGDVEENPGP (SEQ ID NO:114) or a sequence having at least 70%, 80%, 90%, 95% or 99% sequence identity to the amino acid sequence comprising GSGATNFSLLKQAGDVEENPGP (SEQ ID NO:114), and has ribosome skipping function.

[0408] In certain embodiments of all aspects and embodiments, 1 to 5, or more than 5 Gly or Ser residues are added / inserted to the N-terminus and / or C-terminus of the 2-A self-cleaving peptide sequence. In certain embodiments, the amino acid residue GSG is added to the N-terminus and / or C-terminus of the 2-A self-cleaving peptide sequence

[0409] In certain embodiments of all aspects and embodiments, the 2-A self-cleaving peptide sequence is combined with a protease cleavage site (e.g., furin cleavage site (RKRR (SEQ ID NO:116) SEQ ID NO:92, where X = K); cgcaaacggaga SEQ ID NO:117) at its N-terminus or C-terminus.

[0410] Peptide linker

[0411] In certain embodiments of all aspects and embodiments, the recombinant polypeptide comprises a protease-cleavable site. In certain embodiments, the protease-cleavable site is included in a peptide linker (used interchangeably with a cleavable peptide linker). In certain embodiments, a cleavable linker or a protease-cleavable peptide linker is a peptide linker that comprises at least one peptide bond, and in a preferred embodiment, the peptide bond is within the recognition (amino acid) sequence (recognition site) of the protease. In certain embodiments, the cleavable peptide linker is a targeting substrate for the protease such that it is preferentially or specifically cleaved by the protease as compared to a peptide linker that does not contain the recognition sequence of the same protease.

[0412] In certain embodiments of all aspects and embodiments, the cleavable peptide linker comprises a recognition sequence or cleavage site for a specific protease. The recognition (amino acid) sequence is the sequence recognized by the active site of the protease, and wherein the peptide bond is cleaved by the protease.

[0413] For example, for serine proteases, the recognition sequence consists of the N-terminal and C-terminal amino acid residues of the cleaved peptide bond. These residues are designated as P4-P1 (N-terminal) and P1'-P4' (C-terminal) amino acid residues. Cleavage occurs after the P1 position, i.e., the peptide bond between amino acid residues P1 and P1' is cleaved. Generally, the recognition sequence of serine proteases is six to eight amino acid residues in length, but can be longer or even shorter depending on the specific protease. Generally, the cleavable peptide linker includes a P1-P1' cleavable bond within the recognition sequence recognized by the protease.

[0414] In certain embodiments of all aspects and embodiments, the cleavable peptide linker is a peptide linker whose cleavage by the protease is significantly higher than that of a non-targeted substrate of the same protease. Generally, the protease exhibits specificity (preference) for cleaving a specific polypeptide comprising the corresponding recognition sequence compared to another polypeptide that does not include the recognition sequence. Such specificity can be determined based on the cleavage rate constant of the sequence (e.g., the peptide linker sequence). The rate constant is a value that reflects the specificity of the protease for its substrate as well as its efficiency. Any method for determining the cleavage rate constant can be used. For example, a substrate comprising the corresponding recognition sequence is conjugated to a fluorescent moiety that is released upon cleavage by the protease. By determining the cleavage rate at different protease concentrations, the cleavage rate constant (kcat / Km) can be determined relative to a combination of a specific protease and a specific substrate. In certain embodiments, the cleavable peptide linker is a peptide linker that is cleaved by the protease at a rate greater than 1×10 7 M -1 S, or greater than 10 8 M -1 S, greater than 1×10 9 M -1 S or greater than 1×10 10 M -1 S.

[0415] In certain embodiments of all aspects and embodiments, at least one polypeptide of the multispecific antibody produced by the recombinant cell according to the invention comprises a recognition sequence of a protease, which protease includes, for example, matrix metalloproteinase (MMP), cysteine protease, serine protease, and plasmin activator. In certain embodiments, the polypeptide comprises a recognition sequence of a protease that is a protease produced by a tumor, activated immune effector cells (e.g., T cells or NK cells), or cells in the tumor microenvironment.

[0416] In certain embodiments of all aspects and embodiments, a recombinant antibody comprises at least one polypeptide, the at least one polypeptide comprising a recognition sequence specifically recognized by one or more of the following enzymes or proteases: ADAMS; ADAMTS; ADAM10; ADAM12; ADAM15; ADAM17 / TACE; ADAMDEC1; ADAMTS1; ADAMTS4; ADAMTS5; aspartic proteases such as BACE or renin; aspartic cathepsins such as cathepsin D or cathepsin E; caspases such as caspase 1, caspase 2, caspase 3, caspase 4, caspase 5, caspase 6, caspase 7, caspase 8, caspase 9, caspase 10 or caspase 14; cysteine cathepsins such as cathepsin B, cathepsin C, cathepsin K, cathepsin L, cathepsin S, cathepsin V / L2, cathepsin X / Z / P; cysteine proteases such as Cruzipain; legumains; Otubain-2; KLKs such as KLK4, KLK5, KLK6, KLK7, KLK8, KLK10, KLK11, KLK13 or KLK14; metalloproteases such as Meprin; neprilysin; PSMA; BMP-1; MMPs such as MMP1, MMP2, MMP3, MMP7, MMP8, MMP9, MMP10, MMP11, MMP12, MMP13, MMP14, MMP15, MMP16, MMP17, MMP19, MMP20, MMP23, MMP24, MMP26 or MMP27; serine proteases such as activated protein C, cathepsin A, cathepsin G, chymotrypsin, coagulation factor proteases (e.g., FVIIa, FIXa, FXa, FXIa, FXIIa), elastase, granzyme B, guanidobenzoate hydrolase, HtrA1, human neutrophil elastase, lactoferrin, Marapsin, NS3 / 4A, PACE4, plasmin, PSA, tPA, thrombin, neutrophil protease, uPA; type II transmembrane serine proteases (TTSPs) such as DESC1, DPP-4, FAP, Hepsin, proteinase 2, proteinase, TMPRSS2, TMPRSS3 or TMPRSS4.

[0417] In certain embodiments of all aspects and embodiments, the recombinant antibody comprises at least one polypeptide, the at least one polypeptide comprising a cleavable peptide linker, the cleavable peptide linker comprising an amino acid sequence recognized and cleaved by granzyme B. In certain embodiments, the recombinant antibody comprises at least one polypeptide, the at least one polypeptide comprising a cleavable peptide linker, the cleavable peptide linker comprising an amino acid sequence having the general formula P4 P3 P2P1↓P1' (SEQ ID NO:118), wherein P4 is the amino acid I, L, Y, M, F, V or A; P3 is the amino acid A, G, S, V, E, D, Q, N or Y; P2 is the amino acid H, P, A, V, G, S or T; P1 is the amino acid D or E; and P1' is the amino acid I, L, Y, M, F, V, T, S, G or A. In certain embodiments, the recombinant antibody comprises at least one polypeptide, the at least one polypeptide comprising a cleavable peptide linker, the cleavable peptide linker comprising an amino acid sequence having the general formula P4P3 P2 P1↓P1' (SEQ IDNO:118), wherein P4 is the amino acid I or L; P3 is the amino acid E; P2 is the amino acid P or A; P1 is the amino acid D; and P1' is the amino acid I, V, T, S or G.

[0418] In certain embodiments of all aspects and embodiments, the recombinant antibody comprises at least one polypeptide, the at least one polypeptide comprising a cleavable peptide linker, the cleavable peptide linker comprising the amino acid sequence LEAD (SEQ ID NO:119), LEPD (SEQID NO:120) or LEAE (SEQ ID NO:121). In certain embodiments, the recombinant antibody comprises at least one polypeptide, the at least one polypeptide comprising a cleavable peptide linker, the cleavable peptide linker comprising the amino acid sequence IEPDI (SEQ ID NO:122), LEADT (SEQ ID NO:123), IEPDG (SEQ ID NO:124), IEPDV (SEQ ID NO:125), IEPDS (SEQ IDNO:126), IEPDT (SEQ ID NO:127), IEPDP (SEQ ID NO:128), LEPDG (SEQ ID NO:129) or LEADG (SEQ ID NO:130).

[0419] In certain embodiments of all aspects and embodiments, the recombinant antibody comprises at least one polypeptide, the at least one polypeptide comprising a cleavable peptide linker, the cleavable peptide linker comprising an amino acid that is a substrate for a proteolytic enzyme.

[0420] In certain embodiments, the recombinant antibody comprises at least one polypeptide, the at least one polypeptide comprising a cleavable peptide linker, the cleavable peptide linker comprising the sequence P1QAR↓(A / V / K) (SEQ ID NO:131), wherein P1 is any amino acid.

[0421] In certain embodiments, the recombinant antibody comprises at least one polypeptide, the at least one polypeptide comprising a cleavable peptide linker, the cleavable peptide linker comprising the sequence RQAR(A / V / K) (SEQ ID NO:131, where P1 = R). In certain embodiments, the recombinant antibody comprises at least one polypeptide, the at least one polypeptide comprising a cleavable peptide linker, the cleavable peptide linker comprising the amino acid sequence RQAR (SEQ ID NO:132). In certain embodiments, the recombinant antibody comprises at least one polypeptide, the at least one polypeptide comprising a cleavable peptide linker, the cleavable peptide linker comprising the amino acid sequence RQARK (SEQ ID NO:133).

[0422] In certain embodiments, the recombinant antibody comprises at least one polypeptide, the at least one polypeptide comprising a cleavable peptide linker, the cleavable peptide linker comprising the sequence PQAR(A / V / K) (SEQ ID NO:131, where P1 = P). In certain embodiments, the recombinant antibody comprises at least one polypeptide, the at least one polypeptide comprising a cleavable peptide linker, the cleavable peptide linker comprising the amino acid sequence PQAR (SEQ ID NO:166). In a preferred embodiment, the recombinant antibody comprises at least one polypeptide, the at least one polypeptide comprising a cleavable peptide linker, the cleavable peptide linker comprising the amino acid sequence PQARK (SEQ ID NO:167).

[0423] In certain embodiments, the recombinant antibody comprises at least one polypeptide, the at least one polypeptide comprising a cleavable peptide linker, the cleavable peptide linker comprising the sequence HQAR(A / V / K) (SEQ ID NO:131, where P1 = H). In certain embodiments, the recombinant antibody comprises at least one polypeptide, the at least one polypeptide comprising a cleavable peptide linker, the cleavable peptide linker comprising the amino acid sequence HQAR (SEQ ID NO:168). In a preferred embodiment, the recombinant antibody comprises at least one polypeptide, the at least one polypeptide comprising a cleavable peptide linker, the cleavable peptide linker comprising the amino acid sequence HQARK (SEQ ID NO:169).

[0424] In certain embodiments, the recombinant antibody comprises at least one polypeptide that comprises a cleavable peptide linker, the cleavable peptide linker comprising the sequence P1MAK↓(A / V / K)(SEQ ID NO:170), where P1 is any amino acid. In certain embodiments, the recombinant antibody comprises at least one polypeptide that comprises a cleavable peptide linker, the cleavable peptide linker comprising the sequence PMAK(A / V / K)(SEQ ID NO:171, where P1 = P). In certain embodiments, the recombinant antibody comprises at least one polypeptide that comprises a cleavable peptide linker, the cleavable peptide linker comprising the amino acid sequence PMAK(SEQ ID NO:172). In a preferred embodiment, the recombinant antibody comprises at least one polypeptide that comprises a cleavable peptide linker, the cleavable peptide linker comprising the amino acid sequence PMAKK(SEQ ID NO:173).

[0425] In certain embodiments of all aspects and embodiments, the recombinant antibody comprises at least one polypeptide that comprises a cleavable peptide linker, the cleavable peptide linker comprising an amino acid that is a substrate for one or more matrix metalloproteinases (MMPs). In certain embodiments, the MMP is MMP-2. In certain embodiments, the recombinant antibody comprises at least one polypeptide that comprises a cleavable peptide linker, the cleavable peptide linker comprising a sequence having the general formula P3 P2 P1↓P1'(SEQ IDNO:134), where P3 is P, V, or A; P2 is Q or D; P1 is A or N; and P1' is L, I, or M. In certain embodiments, the recombinant antibody comprises at least one polypeptide that comprises a cleavable peptide linker, the cleavable peptide linker comprising the general formula P3 P2 P1↓P1'(SEQ ID NO:134), where P3 is P; P2 is Q or D; P1 is A or N; and P1' is L or I. In certain embodiments, the recombinant antibody comprises at least one polypeptide that comprises a cleavable peptide linker, the cleavable peptide linker comprising the amino acid sequence PAGL(SEQ ID NO:135).

[0426] In certain embodiments of all aspects and embodiments, the recombinant antibody comprises at least one polypeptide, the at least one polypeptide comprising a cleavable peptide linker, the cleavable peptide linker comprising the amino acid sequences TGLEADGSPAGLGRQARVG (SEQ ID NO:136); TGLEADGSRQARVGPAGLG (SEQ ID NO:137); TGSPAGLEADGSRQARVGS (SEQ ID NO:138); TGPAGLGLEADGSRQARVG (SEQ ID NO:139); TGRQARVGLEADGSPAGLG (SEQ ID NO:140); TGSRQARVGPAGLEADGS (SEQ ID NO:141); and TGPAGLGSRQARVGLEADGS (SEQ ID NO:142); GPAGLGLEPDGSRQARVG (SEQ ID NO:143); GGSGGGGIEPDIGGSGGS (SEQ ID NO:144); GGSGGGGLEADTGGSGGS (SEQ ID NO:145); GSIEPDIGS (SEQ ID NO:146); GSLEADTGS (SEQ IDNO:147); GGSGGGGIEPDGGGSGGS (SEQ ID NO:148); GGSGGGGIEPDVGGSGGS (SEQ ID NO:149); GGSGGGGIEPDSGGSGGS (SEQ ID NO:150); GGSGGGGIEPDTGGSGGS (SEQ ID NO:151); GGGSLEPDGSGS (SEQ IDNO:152); GPAGLGLEADGSRQARVG (SEQ ID NO:153), GGEGGGGSGGSGGGS (SEQ ID NO:154); GSSAGSEAGGSGQAGVGS (SEQ ID NO:155); GGSGGGGLEAEGSGGGGS (SEQ IDNO:156); GGSGGGGIEPDPGGSGGS (SEQ ID NO:157); TGGSGGGGIEPDIGGSGGS (SEQ ID NO:158).

[0427] Protease inhibitor

[0428] In certain embodiments of all aspects and embodiments, the protease inhibitor is selected from C1 - protease inhibitor, trypsin inhibitor, thrombin inhibitor, antithrombin - III (AT - III), heparin - cofactor - II, BPTI, aprotinin, pepstatin, leupeptin, and ε - aminocaproic acid. In a preferred embodiment, the protease inhibitor is BPTI.

[0429] In certain embodiments of all aspects and embodiments, the protease inhibitor inhibits elastase and comprises the sequence Ala - Ala - Pro - Val (SEQ ID NO:159); inhibits elastase and comprises the general structure AA1 - AA2 - AA3 - AA4 (SEQ ID NO:160), where AA1 is - Arg -, - Phe -, and - Ile - or is a bond; AA2 is - Ala -, - Phe -, - Cit -, and - Nle -; AA3 is - Trp -, - Val -, and - Tyr -; and AA4: - Phe - and - Gly -; inhibits elastase and comprises a constrained peptide or a β - hairpin peptide as shown in US 8,658,604; or inhibits elastase and comprises Pep4 (KRCCPDTCGIKCL; SEQ ID NO:161) or Pep4M (KRMMPDTMGIKML; SEQ ID NO:162).

[0430] In certain embodiments of all aspects and embodiments, the protease inhibitor inhibits matrix metalloproteinase and comprises the sequence of the inhibitory peptide reported in Ndinguri et al., Molecules 17(2012)14230 - 14248.

[0431] In certain embodiments of all aspects and embodiments, the protease inhibitor inhibits cathepsin and comprises the structure Z - Phe - Gly - NHO - Bz (Z = carboxybenzyl, Bz = benzyl); or inhibits cathepsin and comprises the structure Z - Phe - Phe - DK (SEQ ID NO:163) or Z - Phe - Phe - CHN2.

[0432] In certain embodiments of all aspects and embodiments, the protease inhibitor inhibits chymotrypsin and comprises the structure Z - Arg - Glu - Thr - Phep(OPh)2 (SEQ ID NO:164).

[0433] In certain embodiments of all aspects and embodiments of the invention, the protease inhibitor inhibits thrombin and / or coagulation factors IX and X and is selected from hirudin (MTYTDCTESGQNLCLCEGSNVCGQGNKCILGSDGEKNQCVTGEGTPKPQSHNDGDFEEIPEEYLQ; SEQ ID NO: 165) or a derivative thereof, such as lepirudin or desirudin.

[0434] In certain embodiments of all aspects and embodiments of the invention, the protease inhibitor inhibits plasminogen activator and comprises bovine pancreatic trypsin inhibitor (BPTI) (SEQ ID NO: 86; AQRPDFCLEPPYTGPCKARMIRYFYNAKAGLCQPFVYGGCRAKRNNFKSSEDCMRTCGGA) or a plasminogen activator inhibitor type 1 (PAI-1) - derived peptide EEIIMD (SEQ ID NO: 88).

[0435] As used herein, the term "protein protease inhibitor" refers to an inhibitor of a naturally occurring protease that can be produced recombinantly. In certain embodiments of all aspects and embodiments of the invention, the protein protease inhibitor is a serine protease inhibitor. In certain embodiments of all aspects and embodiments of the invention, the protein protease inhibitor is a trypsin inhibitor. In certain preferred embodiments of all aspects and embodiments of the invention, the protein protease inhibitor is a pancreatic trypsin inhibitor. In one preferred embodiment of all aspects and embodiments of the invention, the protein protease inhibitor is bovine pancreatic trypsin inhibitor (BPTI). The BPTI has the mature amino acid sequence of SEQ ID NO: 86, which is generated from the pro - form of SEQ ID NO: 177 by processing during expression and secretion.

[0436] The following examples, sequences, and figures are provided to assist in understanding the invention, the true scope of which is set forth in the appended claims. It should be understood that the procedures described may be modified without departing from the spirit of the invention.

[0437] Illustration with examples

[0438] Example 1

[0439] Generation of expression plasmids:

[0440] a) A common light - chain bispecific antibody in TCB format

[0441] As a recombinant polypeptide / protein, a bispecific antibody composed of two different immunoglobulin heavy chains (denoted as "K" (stalk chain) and "H" (socket chain), respectively) and a common light chain (denoted as "L") is used. For the heterodimerization of the heavy chains, the socket-stalk structure technology is used. The fully assembled bispecific antibody includes three copies of the light chain. Therefore, the stoichiometry of the K:H:L chains of the fully assembled antibody is 1:1:3.

[0442] The expression cassette configuration (including Cre recombinase sites) after the target is stably integrated into the genome of the host cell is L3-K-L-L-LoxFas-H-L-2L (for generating Figures 2 to 7 the data shown) or L3-K-K-L-L-LoxFas-K-L-H-2L (for generating Figure 8 and the data shown in Table 2). L3 (SEQ ID NO:96), LoxFas (SEQ ID NO:98), and 2L (SEQ ID NO:97) are heterospecific loxP sites for targeted integration.

[0443] Expression cassette

[0444] For the expression of immunoglobulin chains, a transcription unit including the following functional elements is used:

[0445] - The immediate early enhancer and promoter from human cytomegalovirus including intron A (CMV promoter) (SEQ ID NO:103),

[0446] - The human heavy chain immunoglobulin 5'-untranslated region (5'UTR),

[0447] - The murine immunoglobulin heavy chain signal sequence,

[0448] - The nucleic acid encoding the corresponding antibody chain,

[0449] - The bovine growth hormone polyadenylation sequence (BGH pA) (SEQ ID NO:100), and

[0450] - The human gastrin terminator (hGT) (SEQ ID NO:101).

[0451] In addition to the expression cassette of the immunoglobulin subunit, the shuttle plasmid also contains

[0452] - The origin of replication from vector pUC18, which allows the replication of the plasmid in Escherichia coli, and

[0453] - The β-lactamase gene, which confers ampicillin resistance in Escherichia coli.

[0454] Pre-vector

[0455] Chemically synthesize DNA fragments encoding K and L and introduce them into a shuttle plasmid using appropriate restriction sites, thereby generating complete expression cassettes for K and L. The expression cassettes for K and L are excised from their shuttle plasmids in a manner that provides them with appropriate sticky ends.

[0456] Generate a first pre-vector by combining, in a four-way ligation reaction, one DNA fragment carrying the K expression cassette and two fragments carrying the L expression cassette with a fourth fragment carrying the backbone elements of the pre-vector. The expression cassette configuration (including Cre recombinase sites) of this final pre-vector is L3-K-L-L-LoxFas.

[0457] Alternatively, generate a second pre-vector by combining, in a five-way ligation reaction, two DNA fragments carrying the K expression cassette and two fragments carrying the L expression cassette with a fifth fragment carrying the backbone elements of the pre-vector. The expression cassette configuration (including Cre recombinase sites) of this final pre-vector is L3-K-K-L-L-LoxFas.

[0458] In addition to the expression cassettes for K and L, the pre-vectors each contain

[0459] - an SV40 enhancer and early promoter (SEQ ID NO: 102) having a start codon upstream (or in other words, 5') of LoxFas to drive the expression of a resistance marker after stable integration,

[0460] - an origin of replication from vector pUC18, which permits the replication of the plasmid in Escherichia coli, and

[0461] - a β-lactamase gene, which confers ampicillin resistance in Escherichia coli.

[0462] Post-vector

[0463] Chemically synthesize a DNA fragment encoding H and introduce it into a shuttle plasmid using appropriate restriction sites, thereby generating a complete expression cassette for H. The expression cassettes for H as well as K and L (see the pre-vector section above) are excised from their shuttle plasmids in a manner that provides them with appropriate sticky ends.

[0464] Generate a first post-vector by combining, in a three-way ligation reaction, one DNA fragment carrying the H expression cassette and one fragment carrying the L expression cassette with a third fragment carrying the backbone elements of the post-plasmid. The expression cassette configuration (including Cre recombinase sites) of this final post-vector is LoxFas-H-L-2L.

[0465] Alternatively, a second post-vector is generated by combining in a four-way ligation a fragment carrying the K expression cassette, a fragment carrying the H expression cassette, and a fragment carrying the L expression cassette with a fourth fragment carrying the backbone of the post-vector. In this case, the expression cassette configuration (including the Cre recombinase sites) of the final post-vector is LoxFas-K-H-L-2L.

[0466] In addition to the immunoglobulin expression cassettes, the post-vectors each contain

[0467] - a sequence encoding puromycin acetyltransferase lacking a start codon directly downstream (or in other words, 3') of the LoxFas site (SEQ ID NO:02),

[0468] - an origin of replication from vector pUC18, which allows replication of the plasmid in Escherichia coli, and

[0469] - the β-lactamase gene, which confers ampicillin resistance in Escherichia coli.

[0470] b) HAI expression construct

[0471] To evaluate the effect of the protease inhibitor HAI-1 on the expression of the bispecific antibody described in a), a vector for additional expression of HAI-1 from a separate expression cassette was generated. The expression cassette configuration (including the Cre recombinase sites) after stable integration of the target into the host genome is L3-K-L-L-LoxFas-H-L-HAI-1-2L.

[0472] Expression cassette

[0473] As described in Example 1 a), the same immunoglobulin expression cassette was used.

[0474] To express HAI-1, different expression cassettes were generated. These expression cassettes include

[0475] - the immediate early enhancer and promoter from human cytomegalovirus including intron A (CMV promoter) (SEQID NO:103),

[0476] - the human heavy chain immunoglobulin 5'-untranslated region (5'UTR),

[0477] - a nucleic acid encoding soluble HAI-1 (SEQ ID NO:180),

[0478] - the bovine growth hormone polyadenylation sequence (BGH pA) (SEQ ID NO:100), and

[0479] - the human gastrin terminator (hGT) (SEQ ID NO:101);

[0480] or

[0481] - The enhancer and early promoter of simian virus 40 (SV40) (SEQ ID NO:102),

[0482] - The nucleic acid encoding soluble HAI-1 (SEQ ID NO:180),

[0483] - The bovine growth hormone polyadenylation sequence (BGH pA) (SEQ ID NO:100), and

[0484] - The human gastrin terminator (hGT) (SEQ ID NO:101).

[0485] In addition to the HAI-1 expression cassette, the vectors each contain

[0486] - The origin of replication from vector pUC18, which allows replication of the plasmid in Escherichia coli, and

[0487] - The β-lactamase gene, which confers ampicillin resistance in Escherichia coli.

[0488] Pre-vector

[0489] Use the pre-vector with the expression cassette configuration (including Cre recombinase sites) L3-K-L-L-LoxFas described in Example 1a).

[0490] Post-vector

[0491] Chemically synthesize the DNA fragment encoding HAI-1 and introduce it into the shuttle plasmid using appropriate restriction sites, thereby generating the complete expression cassette for HAI-1. The expression cassette for HAI-1 is excised from its shuttle plasmid in a manner that provides them with appropriate sticky ends.

[0492] Generate the final post-vector by combining in a four-way ligation reaction one DNA fragment carrying the H expression cassette (see Example 1a)), one fragment carrying the L expression cassette (see Example 1a)) and one fragment carrying the HAI-1 expression cassette with a fourth fragment carrying the vector backbone elements.

[0493] In this way, two different post-vectors were generated that express HAI-1 under the control of the CMV promoter or the SV40 enhancer and early promoter. The expression cassette configuration (including Cre recombinase sites) of the two final post-vectors is LoxFas-H-L-HAI-1-2L.

[0494] In addition to the expression cassettes for H, L and HAI-1, the post-vectors also contain

[0495] - The sequence encoding puromycin acetyltransferase lacking a start codon (SEQ ID NO:02),

[0496] - The origin of replication from vector pUC18, which allows replication of the plasmid in Escherichia coli, and

[0497] - The β-lactamase gene, which confers ampicillin resistance in Escherichia coli.

[0498] c) BPTI expression construct

[0499] To evaluate the effect of the protease inhibitor BPTI (aprotinin) on the expression of the bispecific antibody described in Example 1a), a vector for additional expression of BPTI (aprotinin) from a separate expression cassette was generated. The expression cassette configuration (including Cre recombinase sites) after stable integration of the target into the host genome was: L3-K-K-L-L-LoxFas-K-L-H-BPTI-2L.

[0500] Expression cassette

[0501] As described in Example 1a), the same immunoglobulin expression cassette configuration was used.

[0502] For the expression of BPTI (aprotinin), a transcription unit comprising the following functional elements was used:

[0503] - The enhancer and early promoter of simian virus 40 (SV40) (SEQ ID NO:102),

[0504] - The signal peptide (SEQ ID NO:175) coding sequence (SEQ ID NO:176),

[0505] - The nucleic acid encoding BPTI as a propeptide (SEQ ID NO:177),

[0506] - The bovine growth hormone polyadenylation sequence (BGH pA) (SEQ ID NO:100), and

[0507] - The human gastrin terminator (hGT) (SEQ ID NO:101).

[0508] In addition to the expression cassette, the vector also contains

[0509] - The origin of replication from vector pUC18, which allows replication of the plasmid in Escherichia coli, and

[0510] - The β-lactamase gene, which confers ampicillin resistance in Escherichia coli.

[0511] Pre-vector

[0512] Use a pre-vector with the expression cassette configuration (including Cre recombinase sites) L3-K-K-L-L-LoxFas as described in Example 1a).

[0513] Post-vector

[0514] Chemically synthesize a DNA fragment encoding BPTI (aprotinin) and introduce it into a shuttle plasmid using appropriate restriction sites, thereby generating a complete expression cassette for BPTI (aprotinin). The expression cassette for BPTI (aprotinin) is excised from its shuttle plasmid in a manner that provides them with appropriate sticky ends.

[0515] Generate a post-vector by combining in a four-way ligation reaction a DNA fragment carrying the H expression cassette (see Example 1a)), a fragment carrying the L expression cassette (see Example 1a)), and a fragment carrying the BPTI (aprotinin) expression cassette with a fourth fragment carrying the vector backbone elements. The expression cassette configuration (including Cre recombinase sites) of the final post-vector is LoxFas-K-L-H-BPTI-2L.

[0516] In addition to the expression cassettes for H, L, and BPTI, the post-vector also contains

[0517] - a sequence encoding puromycin acetyltransferase lacking a start codon (SEQ ID NO:02),

[0518] - an origin of replication from vector pUC18, which allows replication of the plasmid in Escherichia coli, and

[0519] - the β-lactamase gene, which confers ampicillin resistance in Escherichia coli.

[0520] d) Puromycin acetyltransferase-T2A-BPTI fusion expression construct

[0521] To evaluate the effect of the protease inhibitor BPTI (aprotinin) on the expression of the bispecific antibody described in Example 1a), a vector for additional expression of the BPTI amino acid sequence fused to the C-terminus of the puromycin-N-acetyltransferase amino acid sequence can be generated via a self-cleaving T2A peptide linker. The expression cassette configuration (including Cre recombinase sites) after stable integration of the target into the host genome is: L3-K-K-L-L-LoxFas-K-L-H-puroT2A-BPTI-2L.

[0522] Expression cassette

[0523] Use the same immunoglobulin expression cassette configuration as described in Example 1a).

[0524] Pre-vector

[0525] Use a pre-vector with an expression cassette configuration as described in Example 1a) (including Cre recombinase sites) L3-K-K-K-L-L-LoxFas.

[0526] Post-vector

[0527] Apply the same procedure as described in Example 1a), only different from the post-vector used. Here, the post-vector encodes a puromycin acetyltransferase-T2A-BPTI fusion protein instead of just puromycin acetyltransferase. Thus, the expression cassette configuration of the final post-vector (including Cre recombinase sites) is LoxFas-K-L-H-puroT2A-BPTI-2L.

[0528] In addition to the expression cassettes for H and L, the post-vector also contains

[0529] - a sequence encoding a puromycin acetyltransferase-GSG-T2A-GGGGS-BPTI fusion protein (SEQ ID NO:01-SEQ ID NO:04-SEQ ID NO:14-SEQ ID NO:99-SEQ ID NO:177), which lacks a start codon (SEQ ID NO:02-SEQ ID NO:05-SEQ ID NO:15-SEQ ID NO:174-SEQ ID NO:178),

[0530] - an origin of replication from vector pUC18, which allows replication of the plasmid in E. coli, and

[0531] - a β-lactamase gene, which confers ampicillin resistance in E. coli.

[0532] Example 2

[0533] Cultivation of host cells:

[0534] a) ExpiCHO-S

[0535] ExpiCHO-S cells (Thermo Fisher) were cultured according to the manufacturer's recommendations.

[0536] b) Expi293F

[0537] Expi293F cells (Thermo Fisher) were cultured according to the manufacturer's recommendations.

[0538] c) CHO-K1 TI host cell

[0539] The CHO-K1 TI host cells are cultured at 37 °C in a proprietary DMEM / F12-based medium containing 300 μg / ml hygromycin B and 4 μg / ml of a second selection marker in a humidified incubator with 85% humidity and 5% CO2. The cells are split every 3 or 4 days at a concentration of 0.3x10^6 cells / ml in a total volume of 30 ml. For culturing, 125 ml non-baffled conical shake flasks are used. The cells are shaken. The viable cell density is determined using a Cedex HiRes cell counter (Roche).

[0540] Example 3

[0541] Transfection:

[0542] a) Transient transfection of ExpiCHO-S and CHO-K1 TI host cells For transient transfection and production, an expression vector encoding a single immunoglobulin subunit is used at a molar ratio of 4(K):1(H):2(L). Transfection of ExpiCHO-S is carried out according to the protocol of the manufacturer of the ExpiFectamine TM CHO transfection kit prototype (A29128). According to the manufacturer's protocol, CHO-K1 TI host cells are transfected using a MaxCyte STX electroporation device (MaxCyte Inc., Gaithersburg) and an OC-400 electroporation cassette. 3x10^7 cells are transfected with a total of 30 μg of nucleic acid. After transfection, the cells are seeded in 30 ml of medium.

[0543] b) Stable transfection of CHO-K1 TI host cells by targeted integration For stable transfection, equimolar amounts of the pre-vector and the post-vector are mixed. The total DNA used for each transfection is 30 μg, with a plasmid ratio of 2.5:2.5:1 (pre-, post-, Cre vector).

[0544] Two days before transfection of CHO-K1, the TI host cells are seeded in fresh medium at a density of 4x10^5 cells / ml. According to the manufacturer's protocol, transfection is carried out using an OC-400 electroporation cassette with a MaxCyte STX electroporation device (MaxCyte Inc., Gaithersburg). 3x10^7 cells are transfected with a total of 30 μg of nucleic acid, i.e., transfected with 30 μg of plasmid or with a mixture of 5 μg of Cre mRNA and 25 μg of the pre-vector and the post-vector. After transfection, the cells are seeded in 30 ml of medium without the selection agent.

[0545] On the 5th day after inoculation, the cells were centrifuged and transferred at a concentration of 6x10^5 cells / ml into 80 mL of chemically defined medium containing an effective amount of puromycin (selection agent 1) and 1-(2'-deoxy-2'-fluoro-1-β-D-arabinofuranosyl-5-iodo)uracil (FIAU; selection agent 2) for the selection of stably recombinant cells. From this day on, the cells were incubated at 37°C, 150 rpm, 5% CO2 and 85% humidity. The cell density and viability of the culture were monitored regularly. When the viability of the culture began to increase again, the concentrations of selection agents 1 and 2 were reduced to approximately half of the previously used amounts. Therefore, 4x10^5 cells / ml were centrifuged and resuspended in 40 ml of selective medium II (chemically defined medium, 1 / 2 selection markers 1 and 2). The cells were incubated under the same conditions as before and did not divide either.

[0546] Fourteen to twenty-one days after the start of selection, the viability exceeded 90% and the selection was considered complete.

[0547] Example 4

[0548] Production of IgG-like protein

[0549] a) Transient production of IgG-like protein in ExpiCHO-S or CHO-K1 TI host cells

[0550] After 7 days, the cell supernatant of transiently transfected cells was harvested by centrifugation and subsequent filtration (0.2 μm filter), and the protein was purified from the harvested supernatant by the standard methods shown below.

[0551] b) Transient production of IgG-like protein in Expi293F cells

[0552] Cells were seeded in Expi293(TM) medium (Gibco, catalog number 1435101) at a density of 2.5 x 10^6 / ml. The expression vector and ExpiFectamine (Gibco, ExpiFectamine(TM) transfection kit, catalog number 13385544) were separately mixed in OptiMEM(TM) reduced-serum medium (Gibco, catalog number 11520386). After a few minutes, the two solutions were combined, mixed by pipetting, and further incubated at room temperature. The cells were added to the expression vector / ExpiFectamine solution and incubated in an orbital incubator at 37°C and 5% CO2 atmosphere for 24 hours. One day after transfection, supplements (Transfection Enhancer 1 and 2, ExpiFectamine(TM) transfection kit) were added. Four to five days later, the cell supernatant was harvested by centrifugation and subsequent filtration (0.2 μm filter), and the protein was purified from the harvested supernatant by the standard method shown below.

[0553] c) Stable production of IgG-like protein in CHO-K1 cells

[0554] Fed-batch culture

[0555] Fed-batch production cultures were carried out in shake flasks using a proprietary chemically defined medium. Cells were seeded at 2 x 10^6 cells / ml. The cultures received the proprietary feed medium on days 1, 3, and 6. The viable cell count (VCC) and percentage of cell viability in the cultures were measured on days 0, 3, 7, 10, 12, and 14 using Cedex HiRes (Roche Diagnostics GmbH, Mannheim, Germany). The product titer was measured on days 3, 5, 7, 10, 12, and 14 using a Cobas analyzer (Roche Diagnostics GmbH, Mannheim, Germany). The supernatant was harvested by centrifugation (10 min, 1000 rpm, followed by 10 min, 4000 rpm) 10, 12, or 14 days after the start of the fed-batch and clarified by filtration (0.22 μm). Protein A affinity chromatography and UV detection were used to determine the harvest titer. The product quality was determined by Caliper's LabChip (Caliper Life Sciences).

[0556] High cell density fed-batch culture

[0557] High cell density fed-batch production was carried out in an Amr 250 vessel (Sartorius Stedim) using a proprietary chemically defined medium. On day 0, cells were inoculated at 15x10^6 cells / ml. The culture received the proprietary feed medium on days 1, 3, and 6. The viable cell count (VCC) and percentage of cell viability in the culture were measured using a Cedex HiRes instrument (Roche Diagnostics GmbH, Mannheim, Germany) on days 0, 3, 7, 10, 12, and 14. The product titer was measured using a Cobas analyzer (Roche Diagnostics GmbH, Mannheim, Germany) on days 3, 5, 7, 10, 12, and 14. At 10 or 12 or 14 days after the start of the culture, the supernatant was harvested by centrifugation (10 min, 1000 rpm, followed by 10 min, 4000 rpm) and clarified by filtration (0.22 μm). Protein A affinity chromatography and UV detection were used to determine the harvest titer. The product quality was determined by Caliper's LabChip (Caliper Life Sciences).

[0558] Example 5

[0559] Protein purification:

[0560] The recombinant immunoglobulin-like protein was purified from the cell culture supernatant by affinity chromatography using MabSelectSure-Sepharose(TM) (GE Healthcare, Sweden) chromatography. The sterile-filtered cell culture supernatant was captured on MabSelect SuRe resin equilibrated with PBS buffer (10 mM sodium phosphate, 1 mM potassium phosphate, 137 mM sodium chloride, and 2.7 mM potassium chloride, pH 7.4), washed with the equilibration buffer, eluted with 25 mM citrate buffer (pH 3.0), and neutralized with 1 M Tris pH 9.

[0561] Example 6

[0562] Analytical method

[0563] a) Titer determination

[0564] The concentration of recombinant immunoglobulin in the cell culture supernatant was measured quantitatively by affinity HPLC chromatography. Briefly, the centrifuged and sterile-filtered cell culture supernatant was applied to 200 mM KH2PO4 (pH 7.4) in a Poros A / 20 column (Applied Biosystems) and eluted with 200 mM NaCl, 100 mM citric acid (pH 2.5) on a Dionex Ultimate HPLC system (Thermo Fisher Scientific). The eluted antibody was quantified by UV absorbance and peak area integration. Purified standard IgG1 antibody was used as a standard.

[0565] b) CE-SDS

[0566] Product purity and integrity were analyzed by CE-SDS under reducing and non-reducing conditions using microfluidic Labchip technology (PerkinElmer, USA). For this purpose, a 5 μl sample solution was prepared using the HT Protein Express Reagent kit according to the manufacturer's instructions and analyzed on a LabChip GXII system using an HT Protein Express chip. Data were analyzed using LabChip GX software.

[0567] c) SEC

[0568] Size exclusion chromatography (SEC) for the determination of the aggregation and oligomerization state of recombinant immunoglobulin was performed by HPLC chromatography. Briefly, the Protein A purified product was applied to a TSKgel QC-PAK GFC 300 column (Tosoh Bioscience) or Tosoh TSKgel UP-SW3000 (containing 250 mM KCl, 200 mM K2HPO4 / KH2PO4 buffer (pH 6.2)) on a Dionex HPLC system (Thermo Fisher Scientific). The eluted antibody was quantified by UV absorbance and peak area integration. BioRad Gel Filtration Standard #151–1901 was used as a gel filtration calibration standard.

Claims

1. A nucleic acid which comprises the following elements in an operably linked form: a) A nucleic acid encoding a selectable marker, b) A nucleic acid encoding a self-cleaving peptide sequence, and c) A nucleic acid encoding a protein protease inhibitor.

2. The nucleic acid according to claim 1, wherein the elements have the sequence of a)-b)-c) in the 5' to 3' direction.

3. The nucleic acid according to any one of claims 1 to 2, wherein the nucleic acid further comprises the following elements in an operably linked form: d) A promoter upstream (5') of the first element, e) A polyadenylation signal sequence downstream (3') of the last element, f) Optionally, a terminator sequence downstream (3') of the nucleic acid of e).

4. The nucleic acid according to any one of claims 1 to 3, wherein the nucleic acid encoding the selectable marker encodes puromycin acetyltransferase or a functional variant thereof capable of inactivating / modifying puromycin.

5. The nucleic acid according to any one of claims 1 to 4, wherein - the nucleic acid encoding the selectable marker has the nucleotide sequence of SEQ ID NO:02, or - the nucleic acid encoding the selectable marker is a variant of the nucleotide sequence of SEQ ID NO:02 encoding a selectable marker having the amino acid sequence of SEQ ID NO:01, or - the nucleic acid encoding the selectable marker encodes a functional variant of SEQ ID NO:01 capable of inactivating / modifying puromycin.

6. The nucleic acid according to any one of claims 1 to 5, wherein the self-cleaving peptide sequence is T2A or a functional variant thereof capable of causing ribosomal skipping.

7. The nucleic acid according to any one of claims 1 to 6, wherein - the nucleic acid encoding the self-cleaving peptide sequence has the nucleotide sequence of SEQ ID NO:15, or - the nucleic acid encoding the self-cleaving peptide sequence is a variant of the nucleotide sequence of SEQ ID NO:15 encoding a self-cleaving peptide sequence having the amino acid sequence of SEQ ID NO:14, or - the nucleic acid encoding the self-cleaving peptide sequence encodes a functional variant of SEQ ID NO:14 capable of causing ribosomal skipping.

8. The nucleic acid according to any one of claims 1 to 7, wherein the nucleic acid encoding the protein protease inhibitor encodes BPTI or a functional variant thereof capable of inhibiting one or more serine proteases.

9. The nucleic acid according to any one of claims 1 to 8, wherein - the nucleic acid encoding the protein protease inhibitor has the nucleotide sequence of SEQ ID NO:87 or SEQ ID NO:178, or - the nucleic acid encoding the protein protease inhibitor is a variant of the nucleotide sequence of SEQ ID NO:87 encoding a protein protease inhibitor having the amino acid sequence of SEQ ID NO:86, or - the nucleic acid encoding the protein protease inhibitor encodes a functional variant of SEQ ID NO:86 capable of inhibiting one or more serine proteases.

10. The nucleic acid according to any one of claims 3 to 9, wherein the promoter is the SV40 promoter.

11. A cell comprising the nucleic acid according to any one of claims 1 to 10.

12. The cell according to claim 11, wherein the cell further comprises one or more nucleic acid sequences encoding a heterologous polypeptide.

13. The cell according to claim 12, wherein the heterologous polypeptide is an antibody comprising one or more protease-cleavable amino acid sequences.

14. The cell according to any one of claims 11 to 13, wherein the cell is a CHO-K1 cell.

15. A method for producing a heterologous polypeptide in a recombinant cell, the method comprising the steps of: - culturing the cell according to any one of claims 12 to 14 in a medium to produce the heterologous polypeptide, - recovering the heterologous polypeptide from the cell or the medium, and - optionally, purifying the heterologous polypeptide by one or more chromatographic steps.

16. The method according to claim 15, wherein, The amount of the uncleaved heterologous polypeptide recovered is increased compared to a method using a cell that does not comprise the nucleic acid according to any one of claims 1 to 10.

17. Use of the nucleic acid according to any one of claims 1 to 10 for reducing protease cleavage of a heterologous polypeptide during recombinant production in mammalian cells.

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