Methods for increasing recombinant protein expression

Abstract

The present invention provides a nucleic acid comprising a nucleic acid encoding a selection marker, a nucleic acid encoding a self-cleaving peptide sequence, and a nucleic acid encoding a proteinaceous protease inhibitor, all operably linked together. A method for recombinantly producing a heterologous polypeptide using the nucleic acid according to the present invention and a cell containing the nucleic acid is also provided. The use of the nucleic acid according to the present invention to increase the amount of recombinantly produced heterologous polypeptide by reducing protease cleavage is also provided.

Description

[Technical Field]

[0001] The present invention is in the field of recombinant protein production. More particularly, the present invention relates to the co-expression of a proteinaceous protease inhibitor, such as BPTI or aprotinin, in mammalian cells recombinant to a heterologous polypeptide to increase the amount of intact, i.e., protease-uncleaved, recombinant heterologous polypeptide that can be obtained from the culture of said mammalian cells. [Background technology]

[0002] Background of the Invention For commercial production of therapeutic biologics such as antibodies, one desirable characteristic is maximizing the expression yield of the therapeutic agent. Expression yield depends directly on the production titer and indirectly on the amount of therapeutic-related by-products. These by-products can be, for example, incorrectly assembled, incorrectly folded, or degraded therapeutic polypeptides.

[0003] For the recombinant production of therapeutic biologics, mammalian cells such as CHO cells and HEK cells are usually used. Due to their origin, these cells also produce proteases that can cleave therapeutic agents. If the therapeutic agent is susceptible to cleavage by endogenous proteases of the producing cells, the cleaved, i.e., degraded, therapeutic agent is obtained as a therapeutic by-product.

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

[0005] WO 99 / 10503 (Patent Document 1) reports a process for the recombinant production of serine proteases using recombinant nucleic acids encoding a zymogen precursor of the serine protease, in which a naturally occurring non-autocatalytic cleavage site is replaced in the zymogen precursor by an autocatalytic cleavage site that is recognized by the active form of the protease, thus cleaving the zymogen precursor to its active form.

[0006] WO 02 / 61064 (Patent Document 2) reports a method for the recombinant production of trypsin, which comprises using a nucleic acid encoding trypsinogen having an enterokinase recognition site in the propeptide sequence in a form that can be secreted by host cells, and culturing cells containing the nucleic acid under conditions that allow secretion of the expression product into the culture medium, whereby the conditions are selected so that autocatalytic cleavage of the propeptide sequence is at least substantially prevented.

[0007] CN111607615 (Patent Document 3) reports a method for preparing bi-branched subunit vaccines for porcine epidemic diarrhea (PEDV) and transmissible gastroenteritis (TGEV) virus diseases using the PEDV-T2A-TGEV-S sequence, comprising culturing insect cells containing the sequence in a medium containing a mixture of the serine protease inhibitor aprotinin, the aspartic protease inhibitor pepstatin a, the aminopeptidase inhibitor bestatin, the cysteine ​​protease inhibitor E-64, and the serine / cysteine ​​protease inhibitor leupeptin for 24 hours to obtain a culture containing the protease inhibitors, and continuously culturing the culture containing the protease inhibitors for 48 hours to obtain a culture containing the expressed protein.

[0008] WO 2016 / 118775 (Patent Document 4) reports a method for producing retrovirus-producing cells that stably produce a cokal envelope pseudotyped retroviral vector, which comprises transfecting cells with a plasmid encoding the cokal envelope, a self-cleaving peptide, and a selection marker.

[0009] International Publication No. 2021 / 183946 (Patent Document 5) reports an expression vector containing a nucleic acid comprising an open reading frame encoding a protein of interest, followed by a self-cleaving peptide capable of inducing ribosomal skipping during translation, and a selectable marker gene.

[0010] The specification of CN110835632 (Patent Document 6) reports an expression cassette containing a promoter, a framework sequence of the sgRNA of a budding yeast-derived mutant Cas9, a human uracil glycosidase inhibitor, a T2A self-splicing polypeptide, a green fluorescent protein, and a BGH poly(A) sequence.

[0011] CN108441516 (Patent Document 7) reports a lentivirus CMV-CBh dual promoter modified vector pLenti-CMV-3 FLAG-EGFP-PGK-mCherry-T2A-Puro.

[0012] CN112626121 (Patent Document 8) reports a triple selection marker antibody expression vector comprising expression frame I and expression frame II, in which expression frame II is adjacent to expression frame I in a forward orientation mode, in which in expression frame I, a puromycin resistance gene is linked downstream of an antibody light chain gene via an IRES sequence, and then mouse glutamine synthetase is linked downstream of the puromycin resistance gene via an E2A polypeptide, and in expression frame II, a dihydrofolate reductase gene DHFR is linked downstream of an antibody heavy chain gene via an IRES sequence.

[0013] WO 2019 / 157099 (Patent Document 9) reports a nasal spray pharmaceutical formulation containing epinephrine or a salt thereof and one or more absorption enhancers such as aprotinin.

[0014] International Publication No. 2019 / 239405 (Patent Document 10) reports an oral pharmaceutical composition comprising a therapeutic peptide or protein of up to 100 kilodaltons, a divalent cation chelator, and isolated, recombinantly expressed Bowman-Birk inhibitor (BBI), wherein the recombinantly expressed BBI is expressed in a Pichia pastoris expression system.

[0015] WO 2008 / 005847 (Patent Document 11) reports that FVIII can be expressed in a medium containing, for example, about 0.01 to about 5%, or about 0.5 to about 1.0% (vol / vol) of a protease inhibitor such as aprotinin (Aprot., 15 to 30 trypsin inhibitor units (TIU) / ml, Sigma), or a corresponding amount of activity units of other protease inhibitors.

[0016] WO 2008 / 135501 (Patent Document 12) reports a method for producing a factor VIII polypeptide by culturing mammalian cells that express the factor VIII polypeptide, wherein the cell culture medium contains soybean trypsin inhibitor.

[0017] WO 2018 / 22032 (Patent Document 13) describes a method for the recombinant production of debrylase, which comprises culturing a host cell containing one or more vectors, wherein the one or more vectors encode an expressible form of debrylase and a proteinaceous inhibitor of debrylase under conditions in which the debrylase and the proteinaceous inhibitor of debrylase are expressed, and the proteinaceous inhibitor of debrylase is selected from the group consisting of aprotinin, plasminostreptin (I1 6.001), proteinase inhibitor type 2K (PIN2K, I20.001), ecotin (I11.001), trypsin inhibitor MCTI-1 (I07.001), ascidian trypsin inhibitor (I05.001), peptidase inhibitor 5 (KappaPI-Actitoxin-Avd3a, I02.026), and tissue factor pathway inhibitor-2 inhibitor unit 1 (TFPI2, I02.013), methods are reported.

[0018] WO 2021 / 170839 (Patent Document 14) reports the use of aprotinin as a carrier for producing recombinant proteins, polypeptides or peptides in algae, wherein the aprotinin and the recombinant protein, polypeptide or peptide are fused to each other to form a fusion protein.

[0019] Thus, there is a need for improved methods for the recombinant production of therapeutic polypeptides. [Prior art documents] [Patent documents]

[0020] [Patent Document 1] International Publication No. 99 / 10503 [Patent Document 2] WO 02 / 61064 [Patent Document 3] CN111607615 No. [Patent Document 4] International Publication No. 2016 / 118775 [Patent Document 5] International Publication No. 2021 / 183946 [Patent Document 6] No. CN110835632 [Patent Document 7] No. CN108441516 [Patent Document 8] No. CN112626121 [Patent Document 9] International Publication No. 2019 / 157099 [Patent Document 10] International Publication No. 2019 / 239405 [Patent Document 11] International Publication No. 2008 / 005847 [Patent Document 12] International Publication No. 2008 / 135501 [Patent Document 13] International Publication No. 2018 / 22032 [Patent Document 14] International Publication No. 2021 / 170839 Summary of the Invention

[0021] Reported herein is a method for the production of a heterologous polypeptide by a recombinant mammalian cell, wherein the heterologous polypeptide is susceptible to cleavage by an endogenous protease produced by the recombinant mammalian cell, and wherein cleavage of the heterologous polypeptide by the endogenous protease is reduced by co-expression of a proteinaceous protease inhibitor.

[0022] Thus, the present invention provides a nucleic acid comprising a first part encoding a selection marker and a second part encoding a proteinaceous protease inhibitor, both parts being linked by an internal ribosome entry site (IRES) or intein or self-cleaving peptide sequence.Similarly, the present invention provides a nucleic acid for use in the recombinant production of heterologous polypeptides, as well as a cell comprising the nucleic acid of the present invention.The present invention also provides a nucleic acid for increasing the production yield of recombinantly produced heterologous polypeptides, as well as a nucleic acid for reducing protease cleavage of recombinant heterologous polypeptides during production in mammalian cells.

[0023] The present invention is based, at least in part, on the finding that the combination of a selectable marker and a proteinaceous protease inhibitor in a single, i.e., co-cistron, using, for example, an IRES or an intein or a self-cleaving peptide sequence, is advantageous and provides an improvement, inter alia, a reduction in protease cleavage during recombinant production (expression) of a heterologous polypeptide, at least in mammalian cells, and thus an increase in production yield. At the same time, expression of the recombinant heterologous polypeptide is not reduced compared to cells not containing a nucleic acid according to the invention.

[0024] The present invention is based, at least in part, on the discovery that standard co-expression of a proteinaceous protease inhibitor with a recombinant heterologous polypeptide reduces the amount (yield) of the heterologous polypeptide compared to cells that do not express the proteinaceous protease inhibitor.

[0025] The present invention is based, at least in part, on the finding that knocking out 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 recombinant heterologous polypeptide as the use of nucleic acids according to the present invention. Without being bound by this theory, it is assumed that when cleavage is observed during recombinant production of a heterologous polypeptide, it is often caused by more than one protease.

[0026] The present invention is further based, at least in part, on the finding that the promoter for driving expression of the nucleic acid according to the present invention should not be a strong promoter, i.e., should not be, for example, a CMV promoter, a human EF1a promoter, or a CAG promoter. It has been found that it is advantageous to use a promoter with moderate strength, such as, for example, an SV40 promoter, a PGK1 promoter, or a Ubc promoter, and that the SV40 promoter is one preferred promoter.

[0027] It should be pointed out that the term "self-cleaving peptide sequence" in the following aspects and embodiments can be replaced by the term "IRES" or the term "intein" without departing from the invention, which are also particular aspects and embodiments of the invention.

[0028] When placed between two genes, an IRES element mediates cap-independent translation of the second gene.

[0029] Inteins are typically located adjacent to two genes that are translated together into a single polypeptide before splicing occurs, removing the intein and allowing the adjacent proteins to join together at a new junction. Mutations to the intein sequence inhibit splicing while maintaining cleavage.

[0030] One aspect of the invention is an (isolated) nucleic acid comprising the following elements in operably linked form, in a 5' to 3' or 3' to 5' orientation: 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 proteinaceous protease inhibitor.

[0031] In particular embodiments of all aspects and embodiments, the nucleic acid further comprises, in operably linked form, the following elements: d) a promoter upstream (5') of the nucleic acid of a) (5' to 3' direction) or c) (3' to 5' direction); e) a polyadenylation signal sequence downstream (3') of the nucleic acid of c) (5' to 3' direction) or a) (3' to 5' direction), and f) optionally, a terminator sequence downstream (3') of the nucleic acid of e).

[0032] In certain embodiments of all aspects and embodiments, the nucleic acid encoding the selectable marker is selected from the group consisting of nucleic acids encoding puromycin acetyltransferase, histidinol dehydrogenase, thymidine kinase, hygromycin B phosphotransferase, dihydrofolate reductase, blasticidin deaminase, glutamine synthase, zeocin resistance gene, bleomycin resistance gene, and aminoglycoside 3' phosphotransferase.

[0033] In certain embodiments of all aspects and embodiments, the nucleic acid encoding the selectable marker encodes puromycin acetyltransferase or a functional variant thereof that is capable of inactivating / modifying puromycin.

[0034] In certain embodiments of all aspects and embodiments, the nucleic acid encoding the selectable marker encodes the amino acid sequence of SEQ ID NO: 01 (SMAA; Uniprot P13249 (PUAC_STRAD) excluding the N-terminal methionine residue) or a functional variant thereof that is capable of inactivating / modifying puromycin.

[0035] In certain embodiments of all aspects and embodiments, - the nucleic acid encoding the selection marker has the nucleotide sequence of SEQ ID NO: 02 (SMNN), or - the nucleic acid encoding the selection marker is a variant of the nucleotide sequence of SEQ ID NO: 02 which encodes a selection marker having the amino acid sequence of SEQ ID NO: 01, or - the nucleic acid encoding the selection marker encodes a functional variant of SEQ ID NO: 01 that is capable of inactivating / modifying puromycin.

[0036] In certain embodiments of all aspects and embodiments, the self-cleaving peptide sequence is a viral 2-A self-cleaving peptide sequence or a functional variant thereof that is capable of ribosomal skipping.

[0037] 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, the self-cleaving peptides of SEQ ID NOs: 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, or functional variants thereof.

[0038] In certain embodiments of all aspects and embodiments, the self-cleaving peptide sequence is T2A or a functional variant thereof that is capable of ribosomal skipping.

[0039] 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), or a functional variant thereof that is capable of performing ribosomal skipping.

[0040] In certain embodiments of all aspects and embodiments, - the nucleic acid encoding the self-cleaving peptide sequence has the nucleotide sequence of SEQ ID NO: 15 (SCNN), or - the nucleic acid encoding the self-cleaving peptide sequence is a variant of the nucleotide sequence of SEQ ID NO: 15 (SCNN), which encodes a self-cleaving peptide sequence having the amino acid sequence of SEQ ID NO: 14 (SCAA), or The nucleic acid encoding the self-cleaving peptide sequence encodes a functional variant of SEQ ID NO: 14 (SCAA) that is capable of ribosomal skipping.

[0041] In certain embodiments of all aspects and embodiments, the proteinaceous protease inhibitor is an ADAMS, an ADAMTS, e.g., ADAM8; ADAM9; ADAM10; ADAM12; ADAM15; ADAM17 / TACE; ADAMDECl; ADAMTSl; ADAMTS4; ADAMTS5; an aspartic protease, e.g., BACE or renin; an aspartic cathepsin, e.g., cathepsin D or cathepsin E; a caspase, e.g., 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 ​​proteinases, such as crizipain; legumain; otubain-2; KLKS, such as KLK4, KLK5, KLK6, KLK7, KLK8, KLK10, KLK11, KLK13, or KLK14; metalloproteinases , such as meprin; neprilysin; 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, chymase, coagulation factor proteases (e.g., FVIIa, FIXa, FXa, FXIa, FXI), Ia), elastase, granzyme B, guanidinobenzotase, HtrA1, human neutrophil elastase, lactoferrin, marapsin, NS3 / 4A, PACE4, plasmin, PSA, tPA, thrombin, tryptase, uPA; type II transmembrane serine proteases (TTSPs), such as DESC1, DPP-4, FAP, hepsin, matriptase-2, matriptase, TMPRSS2, TMPRSS3, and TMPRSS4.

[0042] In certain embodiments of all aspects and embodiments, the nucleic acid encoding a proteinaceous 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.

[0043] In certain embodiments of all aspects and embodiments, the nucleic acid encoding a proteinaceous protease inhibitor encodes BPTI or a functional variant thereof capable of inhibiting one or more serine proteases.

[0044] In certain embodiments of all aspects and embodiments, the nucleic acid encoding the proteinaceous protease inhibitor encodes the amino acid sequence of SEQ ID NO: 86 (AprotAA) or a functional variant thereof capable of inhibiting one or more serine proteases.

[0045] In certain embodiments of all aspects and embodiments, - the nucleic acid encoding the proteinaceous protease inhibitor has the nucleotide sequence of SEQ ID NO: 87 (AprotNN), or - the nucleic acid encoding the proteinaceous protease inhibitor is a variant of the nucleotide sequence of SEQ ID NO: 87 (AprotNN), which encodes a proteinaceous protease inhibitor having the amino acid sequence of SEQ ID NO: 86 (AprotAA), or - the nucleic acid encoding the proteinaceous protease inhibitor encodes a functional variant of SEQ ID NO: 86 (AprotAA) that is capable of inhibiting one or more serine proteases.

[0046] In particular embodiments of all aspects and embodiments, the promoter is the SV40 (Simian Virus 40) promoter or a functional variant thereof with the same (equivalent) or lower promoter strength.

[0047] In particular 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.

[0048] In particular embodiments of all aspects and embodiments, the polyadenylation signal sequence is a bGH (bovine growth hormone) polyadenylation signal sequence.

[0049] In particular embodiments of all aspects and embodiments, the polyadenylation signal sequence has the nucleotide sequence of SEQ ID NO: 100 (PolyANN).

[0050] In particular embodiments of all aspects and embodiments, the terminator sequence is present and is the hGT (human growth hormone terminator) sequence.

[0051] In particular embodiments of all aspects and embodiments, the terminator sequence is present and has the nucleotide sequence of SEQ ID NO: 101 (TermNN).

[0052] One aspect of the present invention is a (recombinant) cell comprising a nucleic acid according to the invention.

[0053] In particular embodiments of all aspects and embodiments, the nucleic acid according to the invention is stably integrated into the genome / chromosome of the cell.

[0054] In particular embodiments of all aspects and embodiments, a single copy of the nucleic acid according to the invention is integrated into the genome / chromosome of the cell.

[0055] In certain embodiments of all aspects and embodiments, the cells further comprise one or more nucleic acid sequences encoding a (recombinant) heterologous polypeptide.

[0056] In particular embodiments of all aspects and embodiments, the one or more nucleic acid sequences encoding the (recombinant) heterologous polypeptide(s) are stably integrated into the genome / chromosome(s) of the cell.

[0057] In particular embodiments of all aspects and embodiments, the one or more nucleic acid sequences encoding the (recombinant) heterologous polypeptide(s) are integrated into the genome of the cell at a single site / at a single site within one chromosome of the cell.

[0058] In a particular embodiment of all aspects and embodiments, the nucleic acid according to the invention and the one or more nucleic acid sequences encoding the (recombinant) heterologous polypeptide are integrated into the genome / chromosome of the cell at the same site.

[0059] In certain embodiments of all aspects and embodiments, the cells further comprise one or more nucleic acid sequences encoding a (recombinant) heterologous polypeptide comprising one or more protease-cleavable amino acid sequences / protease recognition sequences.

[0060] In certain embodiments of all aspects and embodiments, the cell further comprises one or more nucleic acid sequences encoding a (recombinant) heterologous polypeptide comprising one or more amino acid sequences cleavable by a serine protease / serine protease recognition sequence.

[0061] In certain embodiments of all aspects and embodiments, the cells are selected from the group consisting of ADAMS, ADAMTS, e.g., ADAM8; ADAM9; ADAM10; ADAM12; ADAM15; ADAM17 / TACE; ADAMDECl; ADAMTS1; ADAMTS4; ADAMTS5; aspartic proteases, e.g., BACE or renin; aspartic cathepsins, e.g., cathepsin D or cathepsin E; caspases, e.g., caspase 1, caspase 2, caspase 3, caspase 4, caspase 5, caspase 6, caspase 7, caspase 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 ​​proteinases, such as crizipain; legumain; otubain-2; KLKS, such as KLK4, KLK5, KLK6, KLK7, KLK8, KLK10, KLK11, KLK13, or KLK14; metalloproteinases, such as meprin; neprilysin; PSMA; BMP-1; MMPs, such as MMP 1, 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, chymase, coagulation factor proteases (e.g., FVIIa, FIXa, FXa, FXIa, FXIIa), elastase, granzyme B, guanidinobenzotase, HtrA1, human neutrophil elastase, lac The recombinant polypeptide further comprises one or more nucleic acid sequences encoding one or more heterologous polypeptides comprising one or more amino acid sequences cleavable by a protease selected from the group consisting of: toferrin, marapsin, NS3 / 4A, PACE4, plasmin, PSA, tPA, thrombin, tryptase, uPA; type II transmembrane serine proteases (TTSPs), e.g., DESC1, DPP-4, FAP, hepsin, matriptase-2, matriptase, TMPRSS2, TMPRSS3, or TMPRSS4; and any combination thereof.

[0062] 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.

[0063] In certain embodiments of all aspects and embodiments, the cell is a mammalian cell.

[0064] In particular embodiments of all aspects and embodiments, the cell is a CHO cell or a HEK cell or a BHK cell.

[0065] In certain embodiments of all aspects and embodiments, the cells are CHO-K1 cells.

[0066] One aspect of the present invention is a method for producing a (recombinant) heterologous polypeptide, comprising the following steps: - culturing the cells according to the invention in a culture medium to produce the (recombinant) heterologous polypeptide, - recovering the (recombinant) heterologous polypeptide from the cells or the culture medium, and - optionally purifying the (recombinant) heterologous polypeptide by one or more chromatography steps, Includes.

[0067] In certain embodiments of all aspects and embodiments, the culture is performed (for at least several hours / days) in the presence of puromycin, histidinol, gancyclovir, hygromycin, methionine sulfoximine, blasticidin, methotrexate, zeocin, bleomycin, or neomycin (G418).

[0068] In certain embodiments of all aspects and embodiments, the culturing is performed in the presence of puromycin or a functional variant thereof (for at least several hours / days).

[0069] In certain embodiments of all aspects and embodiments, the amount of recovered uncleaved (recombinant) heterologous polypeptide is increased compared to methods using nucleic acid-free cells according to the present invention.

[0070] One aspect of the present invention is the use of a nucleic acid according to the invention for reducing protease cleavage of a (recombinant) heterologous polypeptide during its recombinant production in mammalian cells.

[0071] One aspect of the present invention is the use of a nucleic acid according to the invention to increase the amount of intact / non-protease-cleaved (recombinant) heterologous polypeptide recovered from mammalian cell culture.

[0072] The present invention encompasses at least the following independent aspects and dependent embodiments.

[0073] 1. The following elements, in 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 proteinaceous protease inhibitor A nucleic acid comprising:

[0074] 2. The nucleic acid of embodiment 1, wherein the element has the sequence of a)-b)-c) in the 5' to 3' direction.

[0075] 3. The nucleic acid of embodiment 1, wherein the element has the sequence c)-b)-a) in the 5' to 3' direction.

[0076] 4. The following elements, in operably linked form: d) a promoter upstream (5') of the first element; e) a polyadenylation signal sequence downstream (3') of the last element, and f) optionally a terminator sequence downstream (3') of the nucleic acid of e); The nucleic acid according to any one of embodiments 1 to 3, further comprising:

[0077] 5. The nucleic acid of any one of embodiments 1 to 4, wherein the nucleic acid encoding the selectable marker is selected from the group consisting of nucleic acids encoding puromycin acetyltransferase, histidinol dehydrogenase, thymidine kinase, hygromycin B phosphotransferase, dihydrofolate reductase, blasticidin deaminase, glutamine synthase, zeocin resistance gene, bleomycin resistance gene, and aminoglycoside 3' phosphotransferase.

[0078] 6. The nucleic acid of any one of embodiments 1 to 5, wherein the nucleic acid encoding the selectable marker encodes puromycin acetyltransferase or a functional variant thereof that is capable of inactivating / modifying puromycin.

[0079] 7. The nucleic acid of any one of embodiments 1 to 6, wherein the nucleic acid encoding the selectable marker encodes the amino acid sequence of SEQ ID NO: 01 or a functional variant thereof that is capable of inactivating / modifying puromycin.

[0080] 8. - 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, which encodes a selectable marker having the amino acid sequence of SEQ ID NO: 01, or - the nucleic acid encoding the selection marker encodes a functional variant of SEQ ID NO: 01 that is capable of inactivating / modifying puromycin, The nucleic acid according to any one of embodiments 1 to 7.

[0081] 9. The nucleic acid of any one of embodiments 1 to 8, wherein the nucleic acid encoding the selectable marker encodes the amino acid sequence of SEQ ID NO: 01.

[0082] 10. The nucleic acid of any one of embodiments 1 to 9, wherein the self-cleaving peptide sequence is a viral 2-A self-cleaving peptide sequence or a functional variant thereof capable of performing ribosomal skipping.

[0083] 11. The nucleic acid of 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 NOs: 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, or a functional variant thereof.

[0084] 12. The nucleic acid of any one of embodiments 1 to 11, wherein the self-cleaving peptide sequence is T2A or a functional variant thereof capable of ribosomal skipping.

[0085] 13. The nucleic acid of any one of embodiments 1 to 12, wherein the self-cleaving peptide sequence is T2A.

[0086] 14. The nucleic acid of 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, or a functional variant thereof capable of performing ribosomal skipping.

[0087] 15. - 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, which encodes 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 that is capable of ribosomal skipping; The nucleic acid according to any one of embodiments 1 to 14.

[0088] 16. The nucleic acid of 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.

[0089] 17. Proteinaceous protease inhibitors include ADAMS, ADAMTS, e.g., ADAM8; ADAM9; ADAM10; ADAM12; ADAM15; ADAM17 / TACE; ADAMDEC1; ADAMTS1; ADAMTS4; ADAMTS5; aspartic proteases, e.g., BACE or renin; aspartic cathepsins, e.g., cathepsin D or cathepsin E; caspases, e.g., caspase 1, caspase 2, caspase 3, caspase 4, caspase 5, caspase 6, and 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 ​​proteinases, such as crizipain; legumain; otubain-2; KLKS, such as KLK4, KLK5, KLK6, KLK7, KLK8, KLK10, KLK11, KLK13, or KLK14; metalloproteinases, 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, chymase, coagulation factor proteases (e.g., FVIIa, FIXa, FXa, FXIa, FXIIa), elastase, granzyme B, guanidinium phosphate, erythrocyte serine proteases (e.g., erythrocyte serine proteases), ... 17. The nucleic acid of any one of embodiments 1 to 16, which inhibits one or more proteases selected from the group of proteases consisting of dinobenzotase, HtrA1, human neutrophil elastase, lactoferrin, marapsin, NS3 / 4A, PACE4, plasmin, PSA, tPA, thrombin, tryptase, uPA; type II transmembrane serine proteases (TTSPs), such as DESC1, DPP-4, FAP, hepsin, matriptase-2, matriptase, TMPRSS2, TMPRSS3, and TMPRSS4.

[0090] 18. The nucleic acid of any one of embodiments 1 to 17, wherein the nucleic acid encoding a proteinaceous 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.

[0091] 19. The nucleic acid according to any one of embodiments 1 to 18, wherein the nucleic acid encoding a proteinaceous protease inhibitor encodes BPTI or a functional variant thereof capable of inhibiting one or more serine proteases.

[0092] 20. The nucleic acid of any one of embodiments 1 to 19, wherein the nucleic acid encoding a proteinaceous protease inhibitor encodes BPTI.

[0093] 21. The nucleic acid of any one of embodiments 1 to 20, wherein the nucleic acid encoding a proteinaceous 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.

[0094] twenty two. - the nucleic acid encoding the proteinaceous protease inhibitor has the nucleotide sequence of SEQ ID NO: 87 or SEQ ID NO: 178, or - the nucleic acid encoding the proteinaceous protease inhibitor is a variant of the nucleotide sequence of SEQ ID NO: 87, which encodes a proteinaceous protease inhibitor having the amino acid sequence of SEQ ID NO: 86, or - the nucleic acid encoding the proteinaceous protease inhibitor encodes a functional variant of SEQ ID NO: 86 that is capable of inhibiting one or more serine proteases; 22. The nucleic acid according to any one of embodiments 1 to 21.

[0095] 23. The nucleic acid of any one of embodiments 1 to 22, wherein the nucleic acid encoding the proteinaceous protease inhibitor encodes the amino acid sequence of SEQ ID NO: 86.

[0096] 24. The nucleic acid according to any one of embodiments 4 to 23, wherein the promoter is an SV40 promoter or a functional variant thereof having the same or lower promoter strength.

[0097] 25. The nucleic acid of any one of embodiments 4 to 24, wherein the promoter is an SV40 promoter.

[0098] 26. The nucleic acid of any one of embodiments 4 to 25, wherein the promoter has the nucleotide sequence of SEQ ID NO: 102.

[0099] 27. The nucleic acid of any one of embodiments 4 to 26, wherein the polyadenylation signal sequence is a bGH polyadenylation signal sequence.

[0100] 28. The nucleic acid of any one of embodiments 4 to 27, wherein the polyadenylation signal sequence has the nucleotide sequence of SEQ ID NO: 100.

[0101] 29. The nucleic acid of any one of embodiments 4 to 28, wherein the terminator sequence is present and is the hGT terminator sequence.

[0102] 30. The nucleic acid of any one of embodiments 4 to 29, wherein a terminator sequence is present and the nucleic acid has the nucleotide sequence of SEQ ID NO: 101.

[0103] 31. A vector comprising a nucleic acid according to any one of embodiments 1 to 30 and optionally further regulatory elements.

[0104] 32. A cell comprising a nucleic acid according to any one of embodiments 1 to 30 or a vector according to embodiment 31.

[0105] 33. The cell of embodiment 32, wherein the nucleic acid is stably integrated into the genome / chromosome of the cell.

[0106] 34. A cell according to any one of embodiments 32 to 33, wherein a single copy of the nucleic acid is integrated into the genome / chromosome of the cell.

[0107] 35. The cell of any one of embodiments 32 to 34, further comprising one or more nucleic acid sequences encoding a heterologous polypeptide.

[0108] 36. The cell of embodiment 35, wherein one or more nucleic acid sequences encoding heterologous polypeptides are stably integrated into the genome / chromosomes of the cell.

[0109] 37. A cell according to any one of embodiments 35 to 36, wherein one or more nucleic acid sequences encoding heterologous polypeptides are integrated into the genome of the cell at a single site / at a single site within one chromosome of the cell.

[0110] 38. A 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 one or more nucleic acid sequences encoding a heterologous polypeptide are integrated into the genome / chromosome of the cell at the same site.

[0111] 39. The cell of any one of embodiments 35 to 38, wherein the heterologous polypeptide comprises one or more protease-cleavable amino acid sequences / protease recognition sequences.

[0112] 40. The cell of 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.

[0113] 41. The cell is treated with an ADAMS, ADAMTS, e.g., ADAM8; ADAM9; ADAM10; ADAM12; ADAM15; ADAM17 / TACE; ADAMDEC1; ADAMTS1; ADAMTS4; ADAMTS5; an aspartic protease, e.g., BACE or renin; an aspartic cathepsin, e.g., cathepsin D or cathepsin E; a caspase, e.g., caspase 1, caspase 2, caspase 3, caspase 4, caspase 5, caspase 6, caspase 7, caspase 8, caspase 9, caspase 10, or caspase B. Ssase-14; cysteine ​​cathepsins, such as cathepsin B, cathepsin C, cathepsin K, cathepsin L, cathepsin S, cathepsin V / L2, cathepsin X / Z / P; cysteine ​​proteinases, such as crizipain; legumain; otubain-2; KLKS, such as KLK4, KLK5, KLK6, KLK7, KLK8, KLK10, KLK11, KLK13, or KLK14; metalloproteinases, 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, chymase, coagulation factor proteases (e.g., FVIIa, FIXa, FXa, FXIa, FXIIa), elastase, granzyme B, guanidinobenzotase, HtrA1, human neutrophil elastase, lactoferrin, marapsin, NS3 / 4A, PAC 41. The cell of any one of embodiments 32 to 40, further comprising one or more nucleic acid sequences encoding a (recombinant) heterologous polypeptide comprising one or more amino acid sequences cleavable by a protease selected from the group of proteases consisting of E4, plasmin, PSA, tPA, thrombin, tryptase, uPA; a type II transmembrane serine protease (TTSP), such as DESC1, DPP-4, FAP, hepsin, matriptase-2, matriptase, TMPRSS2, TMPRSS3 or TMPRSS4; and any combination thereof.

[0114] 42. The 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.

[0115] 43. The cell according to any one of embodiments 32 to 42, wherein the cell is a mammalian cell.

[0116] 44. The 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.

[0117] 45. The cell according to any one of embodiments 32 to 44, wherein the cell is a CHO-K1 cell.

[0118] 46. ​​A method for producing a heterologous polypeptide in a recombinant cell, comprising the steps of: - culturing a cell according to any one of embodiments 35 to 45 in a culture medium to produce a heterologous polypeptide, - recovering the heterologous polypeptide from the cells or the culture medium, and - optionally purifying the heterologous polypeptide by one or more chromatography steps A method comprising:

[0119] 47. The method of embodiment 46, wherein the culturing is carried out in the presence of puromycin, histidinol, ganciclovir, hygromycin, methionine sulfoximine, blasticidin, methotrexate, zeocin, bleomycin, or neomycin (G418).

[0120] 48. The method according to any one of embodiments 46-47, wherein the culture is carried out in the presence of puromycin or a functional variant thereof.

[0121] 49. The method according to any one of embodiments 46 to 48, wherein the culturing is carried out in the presence of puromycin.

[0122] 50. The method of any one of embodiments 46 to 49, wherein the amount of recovered uncleaved heterologous polypeptide is increased compared to a method using a cell that does not contain a nucleic acid of any one of embodiments 1 to 30 or a vector of embodiment 31.

[0123] 51. Use of a nucleic acid according to any one of embodiments 1 to 30 or a vector according to embodiment 31 for reducing protease cleavage of a heterologous polypeptide during its recombinant production in mammalian cells.

[0124] 52. Use of a nucleic acid according to any one of embodiments 1 to 30 or a vector according to embodiment 31 to increase the amount of intact / non-protease-cleaved heterologous polypeptide recovered from the culture of mammalian cells recombinant for said heterologous polypeptide.

[0125] In addition to the various aspects and embodiments depicted and claimed herein, the subject matter of the present disclosure is also directed to other aspects and embodiments having other combinations of the features disclosed and claimed herein. Thus, specific features presented herein, particularly as aspects or embodiments, can be combined with each other in other ways within the scope of the subject matter of the present disclosure, such that the subject matter of the present disclosure includes any suitable combination of the features disclosed herein. The descriptions of specific embodiments of the disclosed subject matter are presented for purposes of illustration and description and are not intended to be comprehensive or to limit the subject matter of the present disclosure to the disclosed embodiments. [Brief explanation of the drawings]

[0126] [Figure 1] (a) Product composition obtained from cell lines expressing exemplary heterologous proteins in the absence of BPTI compared with (b) cell lines co-expressing BPTI, as measured by CE-SDS. [Figure 2]Percentage of intact vs. cleaved main product produced in different cultures; inhibitor-free cells (circles); co-expression of one protease-specific inhibitor (stars); knockout of one specific protease, one variant individually (light) or in combination (dark) (crosses). [Figure 3] Supernatant product concentrations of cultures of cells in the absence of protease inhibitors and cultures with co-expression of soluble protease-specific inhibitors. [Figure 4] Total product concentration (sum of uncleaved and cleaved major products) versus percentage of major products for cultures of cells without protease inhibitors (triangles) and cultures with coexpression of soluble protease-specific inhibitors using a medium-strength SV40 promoter (circles). [Figure 5] Total product concentration (sum of uncleaved and cleaved major products) versus percentage of uncleaved products for cultures of cells without protease inhibitors and cultures with coexpression of soluble protease-specific inhibitors using a medium-strength SV40 promoter (triangles: cells without inhibitors; circles: cells with coexpression of protease inhibitors driven by a medium-strength SV40 promoter). [Figure 6] Total product concentration (= sum of cleaved and uncleaved products) for cultures of cells with co-expression of soluble protease-specific inhibitors and for cultures in which the proteinaceous serine protease inhibitor BPTI was added to the culture medium (triangles: cells without inhibitor; circles: cells with BPTI added) versus percentage of total main product (sum of uncleaved and cleaved main products). [Figure 7] Total product concentration (sum of uncleaved and major cleaved products) versus percentage of uncleaved products for cultures of cells with coexpression of soluble protease-specific inhibitors and cultures in which the proteinaceous serine protease inhibitor BPTI was added to the culture medium (triangles: cells without inhibitor; circles: cells with BPTI added). [Figure 8] Titers (determined by protein A chromatography) and relative amounts of cleaved and uncleaved products (determined by reducing CE-SDS) for different culture conditions. DETAILED DESCRIPTION OF THE INVENTION

[0127] Detailed Description of the Invention Useful methods and techniques for practicing the present invention are described, for example, in Ausubel, FM (ed.), Current Protocols in Molecular Biology, Vols. I-III (1997); Glover, ND, and Hames, BD, ed., DNA Cloning: A Practical Approach, Vols. I and II (1985), Oxford University Press; Freshney, RI (ed.), Animal Cell Culture—a practical approach, IRL Press Limited (1986); Watson, JD, et al., Recombinant DNA, Second Edition, CHSL Press (1992); Winnacker, EL, From Genes to Clones, NY, VCH Publishers (1987); Celis, J., ed., Cell Biology, Second Edition, Academic Press (1998); Freshney, RI, Culture of Animal Cells: A Manual of Basic Technique, second edition, Alan R. Liss, Inc., NY (1987).

[0128] The use of recombinant DNA technology makes it possible to produce derivatives of nucleic acids. Such derivatives can be modified at individual or several nucleotide positions, for example, by substitution, alteration, replacement, deletion, or insertion. Modification or derivatization can be performed, for example, by site-directed mutagenesis. Such modifications can be easily performed by those 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).

[0129] It should be noted that as used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, a reference to "a cell" includes a plurality of such cells and equivalents thereof known to those skilled in the art, and so forth. Similarly, the terms "a" (or "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.

[0130] The term "about" refers to a range of ±20% of the preceding numerical value. In certain embodiments, the term "about" refers to a range of ±10% of the preceding numerical value. In certain embodiments, the term "about" refers to a range of ±5% of the preceding numerical value.

[0131] The term "comprising" also encompasses the term "consisting of."

[0132] Nucleic acids according to the present invention The present invention is based, at least in part, on the discovery that the combination of a selectable marker and a proteinaceous protease inhibitor in a single cistron, for example using a self-cleaving peptide sequence or an IRES for linkage, is advantageous and offers improvements, particularly a reduction in protease cleavage during recombinant production (expression) of a heterologous polypeptide in mammalian cells, and thus an increase in production yield. At the same time, the expression titer of the recombinant heterologous polypeptide is not reduced compared to cells not containing a nucleic acid according to the invention.

[0133] It has been found that during the recombinant production of polypeptides which contain one or more amino acid sequences cleavable by serine proteases, i.e. which contain one or more serine protease recognition (amino acid) sequences, cleavage of said sequences already occurs.

[0134] The present invention is exemplified below using an N-terminal Fab domain-inserted 2+1 bispecific antibody (TCB) with additional functional groups linked by a peptide linker containing 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 present invention and should not be construed as limiting thereof. The true scope is set forth in the appended claims.

[0135] Cleavage occurs independently of the cell in which the heterologous polypeptide is expressed. The extent of cleavage for the different cells used for expression is shown in Table 1 below and in Figure 1.

[0136] [Table 1]

[0137] Generally, cleavage of recombinant polypeptides during production cannot be attributed to a single cellular protease. Therefore, it was expected that reducing the activity of a single protease by coexpressing a soluble protease-specific inhibitor would not significantly reduce the amount of cleaved recombinant polypeptide. However, by coexpressing a single protease-specific inhibitor, we were able to increase the fraction of intact, i.e., uncleaved, recombinant polypeptide from approximately 38% (circles, x-axis in Figure 2) to over 50% (stars, x-axis in Figure 2), while simultaneously reducing the fraction of cleaved recombinant polypeptide from approximately 11% (circles, y-axis in Figure 2) to approximately 4.5% (stars, y-axis in Figure 2). For comparison, knockout of each protease (either one variant individually or both in combination) in each cell pool is shown. The knockout resulted in a cell population with heterogeneous knockouts (e.g., homozygous, heterozygous, no knockout), which resulted in intermediate levels of ablation (crosses in Figure 2).

[0138] However, at the same time, the total polypeptide titer was dramatically reduced by about 30% from 1000 mg / L to 700 mg / L, and as a result, no overall improvement in yield could be achieved (see Figure 3).

[0139] It was found that the reduction in total titer could be overcome by reducing the promoter strength of the promoter operably linked to the protease inhibitor coding sequence, i.e., by driving expression of the protease inhibitor coding sequence. More specifically, by using a medium-strength SV40 promoter instead of a high-strength CMV promoter, the reduction in total titer was reduced while simultaneously maintaining inhibition of cleavage (uncleaved antibody heavy chain containing the protease cleavage site increased from 84% to 94%). This is shown in Figure 4 (total titer vs. total main product) and Figure 5 (total titer vs. uncleaved heavy chain) (triangles: cells without inhibitor; circles: cells with co-expression of a protease inhibitor driven by a medium-strength SV40 promoter).

[0140] Although genus-specific protease inhibitors would be expected to have a better effect, we unexpectedly found that adding genus-specific protease inhibitors to the culture medium did not achieve the same degree of reduction in cleavage as coexpressing a single protease-specific inhibitor. This is illustrated by adding the proteinaceous serine protease inhibitor BPTI (aprotinin) to the culture medium (4 µM on days 3 and 10 of a 14-day fed-batch culture; see Figures 6 and 7; circles indicate no protease inhibitor; squares indicate protease inhibitor). While we were only able to obtain an 82%-89% increase in uncleaved antibody chains with protease cleavage sites (Figure 7), coexpression using the SV40 promoter resulted in an 84%-94% increase in uncleaved antibody chains with protease cleavage sites (Figure 5).

[0141] It was now unexpectedly discovered that even further improvements in titer and cleavage prevention could be achieved by combining a protease inhibitor with a selectable marker in a monocistronic expression cassette. This is shown in Figure 8 and Table 2. An exemplary combination of the proteinaceous protease inhibitor BPTI (aprotinin) and the selectable marker puromycin acetyltransferase linked via a T2A self-cleaving peptide sequence was used. On the one hand, we can see that 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 as determined by Protein A chromatography), as well as the relative titer (total titer multiplied by the main product (monomer) content as determined by SEC) increases (average 1501 mg / mL (1485-1517 mg / mL) vs. average 625 mg / mL (491-675 mg / mL) in the supernatant as 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) while simultaneously maintaining or even improving the ratio of cleaved / uncleaved antibody heavy chains containing protease cleavage sites (as determined by CE-SDS).

[0142] [Table 2]

[0143] Thus, in one specific implementation of the teachings of the present invention, a monocistronic expression cassette according to the present invention comprises a polynucleotide sequence encoding two polypeptides linked by a linker comprising a sequence capable of inducing ribosomal skipping, i.e., self-cleavage.

[0144] In particular embodiments of all aspects and embodiments, the nucleic acid according to the invention comprises a first nucleic acid encoding a selectable marker and a second nucleic acid encoding a proteinaceous protease inhibitor connected by a linker sequence encoding a self-cleaving peptide sequence.

[0145] Thus, one independent aspect of the invention is an (isolated) nucleic acid comprising, in operably linked form, the following elements, in a 5' to 3' direction: 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 proteinaceous protease inhibitor.

[0146] In particular embodiments of all aspects and embodiments, the nucleic acid further comprises, in operably linked form, the following elements: d) a promoter upstream (5') of the nucleic acid of a); e) a polyadenylation signal sequence downstream (3') of the nucleic acid of c), and f) optionally, a terminator sequence downstream (3') of the nucleic acid of e).

[0147] In a preferred embodiment of all aspects and embodiments, the protease inhibitor inhibits plasminogen activators and / or is BPTI (aprotinin) (SEQ ID NO: 86; AQRPDFCLEPPYTGPCKARMIRYFYNAKAGLCQPFVYGGCRAKRNNFKSSEDCMRTCGGA) or the plasminogen activator inhibitor type 1 (PAI-1) derived peptide EEIIMD (SEQ ID NO: 88).

[0148] In a preferred embodiment of all aspects and embodiments, the self-cleaving peptide sequence is a T2A self-cleaving peptide sequence or a functional variant thereof that induces ribosomal skipping.

[0149] 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).

[0150] 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).

[0151] 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 cleaves protein precursors before their secretion in 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. Suitable furin cleavage sites for use in the present invention can be selected based on knowledge of the present invention in combination with knowledge in the art.

[0152] 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 T2A 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. Suitable combinations for use in the present invention can be selected based on knowledge of the present invention in combination with knowledge in the art.

[0153] 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. A variety of spacer sequences are known in the art. In certain embodiments, the spacer sequence is a glycine serine (GS) spacer sequence, for example, (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 sequence is 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 use in the present invention can be selected based on knowledge of the present invention in combination with knowledge in the art.

[0154] In certain embodiments of all aspects and embodiments, the nucleic acid according to the 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).

[0155] In certain alternative embodiments of the 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.

[0156] A "functional fragment" of a polypeptide, such as an antibody, a self-cleaving peptide sequence, an enzyme, a selection marker, or a nucleic acid, is a polypeptide or nucleic acid whose sequence is not identical to the respective full-length polypeptide or nucleic acid, but which retains the same function as the full-length polypeptide or nucleic acid. Thus, the term "functional fragment" encompasses variants of full-length polypeptides or nucleic acids that have more 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 substitutions. 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, for example, Ausubel et al. (supra); Fields et al. (1989) Nature 340:245-246; U.S. Patent No. 5,585,245 and WO 98 / 44350.

[0157] Cells and methods according to the present invention The present invention is exemplified using CHO cells and targeted integration. This is presented only to illustrate the invention and should not be construed as limiting in any way. Any other eukaryotic or mammalian cells and any other transfection / integration method can be used. The true scope of the invention is set forth in the following claims.

[0158] For example, targeted integration (TI) uses site-specific recombination 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 enzymatic process in which the sequence at the site of integration in the genome is exchanged with the exogenous nucleic acid. One system used to perform such nucleic acid exchange is the Cre-lox system. The enzyme that catalyzes the exchange is Cre recombinase. The exchanged sequence is defined by the location of at least two lox(P) sites within the genome and the exogenous nucleic acid. These lox(P) sites are recognized by Cre recombinase. No further inputs, such as ATP, are required.

[0159] Suitable mammalian TI host cells for use in this exemplification of the method according to the invention to generate recombinant cells according to the invention are CHO cells that have a landing site integrated at a single site within a locus in their genome, the landing site containing three heterospecific loxP sites for Cre recombinase-mediated DNA recombination.

[0160] More specifically, the heterospecific loxP sites are L3, LoxFas, and 2L (see, for example, Lanza et al., Biotechnol. J. 7 (2012) 898-908; Wong et al., Nucleic Acids Res. 33 (2005) e147), where L3 and 2L are adjacent to the 5' and 3' ends of the landing site, respectively, and LoxFas is located between the L3 and 2L sites. The landing site further contains a bicistronic unit that links the expression of a selection marker via an IRES to the expression of a fluorescent GFP protein, allowing for the stabilization of the landing site by positive selection and the selection of the absence of the site after transfection and Cre recombination (negative selection). Green fluorescent protein (GFP) is useful for monitoring the recombinase-mediated cassette exchange (RMCE) reaction.

[0161] This organization of the landing sites, as outlined in the previous paragraph, allows the simultaneous integration of two vectors, for example, a so-called front vector with an L3 site and a LoxFas site, and a back vector with an internal LoxFas site and a 2L site. The functional elements of the selection marker, which are different from those present in the landing sites and also in the nucleic acid according to the invention, are distributed between both vectors: the promoter and start codon are located on the front vector, and the coding region and polyadenylation signal sequence are located on the back vector. Only correct recombinase-mediated integration of the nucleic acid from both vectors induces resistance to the respective selection agents.

[0162] Thus, two vectors are designed: a first vector, a front vector, and a second vector, a back vector. Both vectors contain different expression cassettes. The number of expression cassettes is generally not limited, but is generally 1 to 4 expression cassettes per vector, independently. For example, one of the expression cassettes in the back vector contains a nucleic acid according to the present invention.

[0163] In this example, we used a front vector containing, in order, an 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, a promoter, a start codon for a selectable marker other than puromycin acetyltransferase, and a LoxFas site. The back vector contained, in order, a LoxFas site, a coding region for the selectable marker and a polyadenylation signal sequence, an expression cassette encoding the second heavy chain, an expression cassette encoding the second light chain, an expression cassette containing a nucleic acid according to the present invention, and a 2L site. These vectors were then transfected into CHO-K1 TI host cells using double RMCE, cultured, and the heterologous antibody product was isolated from the supernatant. Data for each vector are presented in the previous section.

[0164] Thus, in a particular embodiment of all aspects and embodiments, a nucleic acid according to the invention and one or more nucleic acids encoding a heterologous polypeptide are integrated into a mammalian TI host cell by double recombinase-mediated cassette exchange (RMCE), thereby obtaining a recombinant mammalian cell according to the invention, e.g., a recombinant CHO cell, in which the expression cassettes are integrated into the genome at a single locus.

[0165] 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, the two RRSs are adjacent to the at least one selectable marker. That is, the first RRS is located 5' (upstream) of the selectable marker 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 the first RRS, the second RRS, and the third RRS, and at least one selectable marker located between the first RRS and the third RRS.

[0166] In particular embodiments of all aspects and embodiments, the first, second, and third RRSs are L3 (SEQ ID NO: 96), LoxFas (SEQ ID NO: 98), and 2L (SEQ ID NO: 97) sites.

[0167] In particular embodiments of all aspects and embodiments, the CHO cells are CHO-K1 cells.

[0168] Accordingly, one aspect of the present invention is a method for preparing a recombinant cell expressing a heterologous polypeptide, comprising the steps of: a) providing a targeted integration host cell comprising an exogenous nucleotide sequence integrated at a site within a locus in the genome of the host cell, wherein the exogenous nucleotide sequence comprises a first recombination recognition sequence and a second recombination recognition sequence adjacent to at least one first selectable marker, and a third recombination recognition sequence located between the first and second recombination recognition sequences, wherein all of the recombination recognition sequences are different; b) introducing into the cell provided in a) a first vector comprising two recombination recognition sequences that match the first and third recombination recognition sequences on the integrated exogenous nucleotide sequence, wherein the two recombination recognition sequences flank (part of) two to four expression cassettes (exogenous nucleotide sequences) and at least 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 (further) expression cassettes (exogenous nucleotide sequences), at least one expression cassette comprising a nucleic acid according to the invention; c) i) simultaneously with the first and second vectors of b); or ii) subsequently introducing one or more recombinases, introducing one or more recombinases that recognize recombination recognition sequences of the first vector and the second vector (optionally, the one or more recombinases perform two recombinase-mediated cassette exchanges); and d) selecting recombinant host cells that express the second selectable marker and secrete the bispecific antibody; thereby preparing a recombinant cell that expresses the heterologous polypeptide.

[0169] In particular embodiments of all aspects and embodiments, the first or / and second vector comprises an expression cassette comprising a nucleic acid according to the invention.

[0170] 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.

[0171] In certain embodiments of all aspects and embodiments, the first and second vectors each comprise at least one exogenous nucleotide sequence encoding an antibody light chain and at least one exogenous nucleotide sequence encoding an antibody heavy chain.

[0172] In certain embodiments of all aspects and embodiments, the first or / and second vector comprises an exogenous nucleotide sequence encoding an antibody light chain and an exogenous nucleotide sequence encoding an antibody heavy chain.

[0173] 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, and the exogenous nucleotide sequence encoding the antibody heavy chain is located upstream (5') of the exogenous nucleotide sequence encoding the antibody light chain.

[0174] 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 antibody light chain and the antibody heavy chain have a domain crossover.

[0175] In certain embodiments of all aspects and embodiments, the first vector comprises one exogenous nucleotide sequence encoding an antibody light chain and one exogenous nucleotide sequence encoding an antibody heavy chain, wherein the antibody light chain and the antibody heavy chain have a domain crossover.

[0176] In one preferred embodiment of all aspects and embodiments, the first vector comprises one exogenous nucleotide sequence encoding an antibody light chain and one exogenous nucleotide sequence encoding an antibody heavy chain, wherein the antibody light chain and the antibody heavy chain have a domain crossover, and wherein the exogenous nucleotide sequence encoding the antibody light chain with the domain crossover is located upstream (5') of the exogenous nucleotide sequence encoding the antibody heavy chain with the domain crossover.

[0177] In one preferred embodiment of all aspects and embodiments, the first vector comprises one exogenous nucleotide sequence encoding an antibody light chain and one exogenous nucleotide sequence encoding an antibody heavy chain, wherein the antibody light chain and the antibody heavy chain have a domain crossover, and wherein the exogenous nucleotide sequence encoding the antibody heavy chain with the domain crossover is located upstream (5') of the exogenous nucleotide sequence encoding the antibody light chain with the domain crossover.

[0178] In one embodiment of all aspects and embodiments, the first vector comprises a promoter sequence operably linked to the codon ATG, whereby the promoter sequence is flanked upstream by (two) exogenous nucleotide sequences (i.e., located downstream of) the exogenous nucleotide sequences, and the ATG codon is flanked downstream by (i.e., located upstream of) the recombination recognition sequence, and the second vector comprises a selectable marker lacking the ATG transcription start codon, flanked upstream by the recombination recognition sequence and downstream by (two) exogenous nucleotide sequences.

[0179] A further aspect of the present invention is a method for producing a heterologous polypeptide comprising the steps of: a) providing a recombinant cell according to the invention; b) culturing the recombinant cells of a) and recovering the heterologous polypeptide from the cells or the culture medium; c) optionally purifying the heterologous polypeptide by one or more chromatography steps; thereby producing a heterologous polypeptide.

[0180] cell Any mammalian cell can be used to generate a recombinant cell according to the invention, which can be used in the method according to the invention, i.e., independent of the integration method, i.e., for random integration (RI) and TI, any mammalian cell can be used.

[0181] Examples of useful mammalian cells include human amniotic cells (e.g., CAP-T cells described in Woelfel, J. et al., BMC Proc. 5 (2011) p. 133); monkey kidney CV1 line transformed with SV40 (COS-7); human embryonic kidney lines (e.g., HEK293 cells or HEK293T cells described in Graham, FL et al., J. Gen Virol. 36 (1977) 59-74); baby hamster kidney cells (BHK); mouse Sertoli cells (e.g., TM4 cells described in Mather, JP, Biol. Reprod. 23 (1980) 243-252); monkey kidney cells (CV1); African green monkey kidney cells (VERO-76); human cervical carcinoma cells (HELA); canine kidney cells (MDCK); buffalo rat liver cells (BRL 3A); human lung cells (W138); human hepatocytes (HepG2); mouse mammary tumor (MMT060562); TRI cells, as described, for example, in Mather, JP et al., Annals NYAcad. Sci. 383 (1982) 44-68; MRC5 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 specific mammalian cells suitable for antibody production, see, for example, Yazaki, P. and Wu, AM, Methods in Molecular Biology, Vol. 248, Lo, BKC (ed.), Humana Press, Totowa, NJ (2004), pp. 255-268.

[0182] As used herein, the term "recombinant cell" refers to a mammalian cell containing an exogenous nucleic acid. Such a recombinant mammalian cell is a cell into which at least a nucleic acid according to the present invention has been introduced, including the progeny of such a cell. In certain embodiments, a recombinant cell is a mammalian cell containing a nucleic acid according to the present 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 containing one or more exogenous nucleic acids integrated into the genome of the mammalian cell and capable of expressing a heterologous polypeptide as well as a nucleic acid according to the present invention. In certain embodiments, a recombinant cell is a mammalian cell containing one or more exogenous nucleic acids integrated at a single site within a locus in the cell's genome. In a preferred embodiment, the recombinant cell is a mammalian cell containing a nucleic acid according to the present invention and one or more additional exogenous nucleic acids integrated at a single site within a locus in the cell's genome, wherein the integrated nucleic acids comprise first, second, and third recombination recognition sequences located between the first and second recombination recognition sequences, and all recombination recognition sequences are different.

[0183] "Recombinant cells" also encompass genetically modified cells, such as cells that contain a nucleic acid according to the present invention, express a heterologous polypeptide of interest, and can be used for recombinant production of the heterologous polypeptide of interest on any scale. For example, "recombinant cells" refer to cells in which a nucleic acid according to the present 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 present invention and one or more nucleic acids encoding a heterologous polypeptide" can be a mammalian cell that has been subjected to recombinase-mediated cassette exchange (RMCE), whereby the nucleic acid according to the present invention and the coding sequence of the polypeptide of interest have been stably introduced into the genome of the mammalian cell.

[0184] "Recombinant cells" further include the primary transformed cell and progeny derived therefrom without regard to the number of transfers. The progeny may not be completely identical to the parent cell in nucleic acid content, for example, but may contain mutations. Mutant progeny that have the same function or biological activity as screened or selected for in the originally transformed cell are included.

[0185] An "isolated cell" refers to a cell that has been separated from a component of its natural environment.

[0186] An "isolated nucleic acid" refers to a nucleic acid molecule that has been separated from a component of its natural environment.

[0187] In certain embodiments of all aspects and embodiments, the mammalian cell is, for example, a Chinese hamster ovary (CHO) cell (e.g., CHO K1, CHO DG44, etc.), a human embryonic kidney (HEK) cell, a lymphoid cell (e.g., Y0, NS0, Sp2 / 0 cell), or a human amniotic cell (e.g., CAP-T, etc.). In a preferred embodiment of all aspects and embodiments, the cell is a CHO cell.

[0188] For TI, any known or future mammalian cell suitable for TI that contains a landing site described herein integrated at a single site within a genomic locus can be used in the present invention. Such cells may be referred to as mammalian TI host cells. In certain embodiments, the mammalian TI host cell is a hamster cell, human cell, rat cell, or mouse cell that contains a landing site 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, CHO K1 cell, CHO K1SV cell, CHO DG44 cell, CHO DUKXB-11 cell, CHO K1S cell, or CHO K1M cell that contains a landing site described herein integrated at a single site within a genomic locus.

[0189] antibody General information regarding the nucleotide sequences of human immunoglobulin light and heavy chains is given in Kabat, EA, et al., Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, MD (1991).

[0190] The term "antibody" as used herein is used in the broadest sense, insofar as it comprises one or more amino acid sequences that are cleaved during recombinant production by proteases endogenous to the mammalian cells used for expression in the absence of a nucleic acid according to the present invention or a functional variant thereof, and encompasses a variety of antibody structures, including, but not limited to, full-length antibodies, monoclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody-antibody fragment fusions, as well as combinations thereof.

[0191] The term "full-length antibody" refers to an antibody having a structure substantially similar to that of a natural antibody. A full-length antibody comprises two full-length antibody light chains, each comprising, from N- to C-terminus, a light-chain variable region and a light-chain constant domain, and two full-length antibody heavy chains, each comprising, from N- to C-terminus, 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 contrast to natural antibodies, full-length antibodies may comprise additional immunoglobulin domains, such as one or more additional scFvs, or heavy- or light-chain Fab fragments, or scFabs conjugated to one or more of the ends of different chains of a full-length antibody (but only one fragment at each end). These conjugates are also encompassed by the term full-length antibody.

[0192] The term "antibody binding site" refers to a pair of heavy-chain variable domain and light-chain variable domain. To ensure proper binding to an antigen, these variable domains are cognate, i.e., belong together. An antibody binding site comprises at least three HVRs (e.g., in the case of a VHH) or three to six HVRs (e.g., in the case of a naturally occurring, i.e., conventional antibody with a VH / VL pair). Generally, the amino acid residues of an antibody involved in antigen binding form the binding site. These residues are usually contained in a pair of antibody heavy-chain variable domain and corresponding antibody light-chain variable domain. An antibody antigen-binding site comprises amino acid residues from "hypervariable regions" or "HVRs." "Framework" or "FR" regions are variable domain regions other than the hypervariable region residues as defined herein. Thus, the light and heavy chain variable domains of an antibody comprise, from N- to 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 binding specificity of the antibody. A "functional binding site" can bind to its target. The term "binding" refers to the binding of a binding site to its target in an in vitro assay, in a specific embodiment, in a binding assay. Such a binding assay can be any assay as long as a binding event can be detected. "Binding" can be determined, for example, using an ELISA assay.

[0193] The term "hypervariable region" or "HVR", as used herein, refers to each region of an antibody variable domain comprising stretches of amino acid residues that are hypervariable sequences ("complementarity determining regions" or "CDRs") and / or form structurally defined loops ("hypervariable loops") and / or contain residues that contact the antigen ("antigen contacts"). Typically, antibodies contain 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).

[0194] HVR includes: (a) Hypervariable loops occurring 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); (b) CDRs occurring 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.); (c) antigen contacts occurring 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 (d) 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); Any combination of (a), (b), and / or (c), including:

[0195] Unless otherwise indicated, HVR residues and other residues of the variable domain (e.g., FR residues) are numbered herein according to Kabat et al., supra.

[0196] The "class" of an antibody refers to the type of constant domain or constant region, preferably the Fc region, possessed by the heavy chain. There are five major classes of antibodies: IgA, IgD, IgE, IgG, and IgM, some of which can be further divided into "subclasses" (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The heavy chain constant domains corresponding to the different classes of immunoglobulins are called α, δ, ε, γ, and μ, respectively.

[0197] The term "heavy chain constant region" refers to the region of an immunoglobulin heavy chain containing the constant domains, i.e., the CH1 domain, hinge region, CH2 domain, and CH3 domain. In one embodiment, a human IgG constant region extends from Ala118 to the carboxyl terminus of the heavy chain (numbering according to the Kabat EU index). However, the C-terminal lysine (Lys447) of the constant region may or may not be present (numbering according to the Kabat EU index). The term "constant region" refers to a dimer comprising two heavy chain constant regions that can be covalently linked to each other via hinge region cysteine ​​residues that form interchain disulfide bonds.

[0198] The term "heavy chain Fc region" refers to the C-terminal region of an immunoglobulin heavy chain, containing at least a portion of the hinge region, 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 (Kabat EU index numbering). Thus, the Fc region is smaller than the constant region, but the C-terminal portion is identical to it. However, the C-terminal lysine (Lys447) of the heavy chain Fc region may or may not be present (Kabat EU index numbering). The term "Fc region" refers to a dimer comprising two heavy chain Fc regions that can be covalently bound to each other via hinge region cysteine ​​residues that form interchain disulfide bonds.

[0199] The term "valency," as used within this application, refers to the presence of a specific number of binding sites within an antibody. Thus, the terms "bivalent," "tetravalent," and "hexavalent" refer to the presence of two, four, and six binding sites, respectively, within an antibody.

[0200] A "monospecific antibody" refers to an antibody that has a single binding specificity, i.e., that specifically binds 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 antibodies with additional scFv or Fab fragments). Monospecific antibodies need not be monovalent; that is, they may contain more than one binding site that specifically binds to one antigen. For example, natural antibodies are monospecific but bivalent.

[0201] A "multispecific antibody" refers to an antibody that has binding specificities for 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., full-length antibodies conjugated to additional scFv or Fab fragments). Multispecific antibodies are at least bivalent, i.e., contain two antigen-binding sites. Furthermore, multispecific antibodies are at least bispecific. Thus, bivalent bispecific antibodies are the simplest form of multispecific antibodies. Engineered antibodies with two, three, or more (e.g., four) functional antigen-binding sites have been reported (see, e.g., U.S. Patent Application Publication No. 2002 / 0004587).

[0202] In certain embodiments of all aspects and embodiments, the cells produce a multispecific antibody as a heterologous polypeptide. In certain embodiments, one of the binding specificities of the multispecific antibody is for a first antigen and the other is for a different second antigen. In certain embodiments, the multispecific antibody binds to two different epitopes of the same antigen. In certain embodiments, the second epitopes on the same antigen are non-overlapping epitopes. 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.

[0203] Techniques for producing 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 "knobs-in-holes" engineering (see, e.g., U.S. Pat. No. 5,731,168). Multispecific antibodies can also be produced by manipulating electrostatic steering effects to create antibody Fc heterodimeric molecules (WO 2009 / 089004), cross-linking two or more antibodies or fragments (see, e.g., U.S. Pat. No. 4,676,980, and Brennan, M., et al., Science 229 (1985) 81-83), using leucine zippers to produce bispecific antibodies (see, e.g., Kostelny, SA, et al., J. Immunol. 148 (1992) 1547-1553), using general light chain techniques to circumvent light chain mispairing problems (see, e.g., WO 98 / 50431), using specific techniques to generate bispecific antibody fragments (see, e.g., Holliger, P., et al., Proc. Natl. Acad. Sci. USA 90 (1993) 6444-6448), and by using techniques such as those described, e.g., in Tutt, A., et al. al., J. Immunol. 147 (1991) 60-69, for the preparation of trispecific antibodies.

[0204] Also included herein are engineered antibodies, or DVD-Igs, having three or more antigen-binding sites, including, for example, "octopus antibodies" (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-acting Fabs" or "DAFs" (see, e.g., U.S. Patent Application Publication Nos. 2008 / 0069820 and WO 2015 / 095539).

[0205] Multispecific antibodies can also be provided in an asymmetric manner with domain crossovers in one or more binding arms of the same antigen specificity, i.e., by exchanging VH / VL domains (see, e.g., WO 2009 / 080252 and WO 2015 / 150447), CH1 / CL domains (see WO 2009 / 080253) or complete Fab arms (see 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).

[0206] In a preferred embodiment of all aspects and embodiments, the multispecific antibody comprises Fab fragments in which either the variable or constant regions of the heavy and light chains have been exchanged, i.e., in one chain the heavy chain VH variable domain is directly connected via a peptide linker to the light chain CL constant domain, and in the respective other chain the light chain VL variable domain is directly connected via a peptide linker to the heavy chain CH1 constant domain.

[0207] Thus, a domain-swapped Fab fragment comprises a polypeptide chain composed of a light chain variable region (VL) and a heavy chain constant region 1 (CH1), and a polypeptide chain composed of a heavy chain variable region (VH) and a light chain constant region (CL).

[0208] 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.

[0209] The antibody or fragment may also be a multispecific antibody as described in WO2009 / 080254, WO2010 / 112193, WO2010 / 115589, WO2010 / 136172, WO2010 / 145792 or WO2010 / 145793.

[0210] The antibody or fragment thereof may also be a multispecific antibody as disclosed in WO 2012 / 163520.

[0211] A variety of additional molecular formats of multispecific antibodies are known in the art and are included herein (see, e.g., Spiess et al., Mol. Immunol. 67 (2015) 95-106).

[0212] 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.

[0213] In particular embodiments of all aspects and embodiments, the bispecific antibody is selected from the group of bispecific antibodies consisting of: -Domain-swapped 1+1 bispecific antibodies (CrossMabs); Such antibodies are bispecific full-length IgG antibodies comprising a first light chain and a first heavy chain pair comprising a first Fab fragment, and a second light chain and a second heavy chain pair comprising a second Fab fragment, In the first Fab fragment: a) only the CH1 and CL domains are replaced by 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); b) only the VH and VL domains are replaced by one another (i.e. the light chain of the first Fab fragment comprises the VH and CL domains and the heavy chain of the first Fab fragment comprises the VL and CH1 domains); or c) the CH1 and CL domains and the VH and VL domains are replaced by 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); the second Fab fragment comprises a light chain comprising a VL and a CL domain, and a heavy chain comprising a VH and a CH1 domain; The first heavy chain and the second heavy chain both comprise a CH3 domain, and both CH3 domains are complementarily engineered by respective amino acid substitutions to support heterodimerization of the first heavy chain and the second heavy chain (in one preferred embodiment, one CH3 domain comprises a knob mutation and the other CH3 domain comprises a hole mutation; -C-terminal Fab domain fused 2+1 bispecific antibody (BS); Such antibodies are bispecific full-length IgG antibodies, a) one full-length antibody comprising two pairs of full-length antibody light chains and two pairs of full-length antibody heavy chains, wherein the binding site formed by each pair of full-length heavy chains and full-length light chains specifically binds to a first antigen; and b) one additional Fab fragment fused to the C-terminus of one heavy chain of the full-length antibody, the binding site of the additional Fab fragment specifically binding to a second antigen; Additional Fab fragments that specifically bind to a second antigen contain domain crossovers such that a) the light chain variable domain (VL) and the heavy chain variable domain (VH) are substituted for each other, or b) the light chain constant domain (CL) and the heavy chain constant domain (CH1) are substituted for each other; -Bispecific one-arm single-chain antibodies (OaMab); Such antibodies are bispecific one-arm single-chain antibodies comprising 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, wherein the individual chains are as follows: - a light chain (comprising a variable light domain and a light chain constant domain); - a light chain / heavy chain combination (comprising, from N-terminus to C-terminus, a variable light chain domain, a light chain constant domain, a peptide linker, a variable heavy chain domain, a CH1 domain, a hinge region, a CH2 domain, and a CH3 domain with a knob or hole mutation) - heavy chain (comprising, from N-terminus to C-terminus, the variable heavy domain, the CH1 domain, the hinge region, the CH2 domain, and the CH3 domain with a hole or knob mutation); -Bispecific two-arm single chain antibodies; Such antibodies are bispecific two-arm single chain antibodies comprising 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, wherein the individual chains are as follows: - light chain / heavy chain combination 1 (comprising, from N-terminus to C-terminus, variable light chain domain 1, light chain constant domain, peptide linker, variable heavy chain domain 1, CH1 domain, hinge region, CH2 domain, CH3 domain with knob or hole mutation); - light chain / heavy chain combination 2 (comprising, from N-terminus to C-terminus, variable light chain domain 2, light chain constant domain, peptide linker, variable heavy chain domain 2, CH1 domain, hinge region, CH2 domain, CH3 domain with hole or knob mutation); - 2+1 bispecific antibodies (TCBs) with inserted N-terminal Fab domains; Such antibodies are bispecific full-length antibodies with an additional heavy chain N-terminal binding site with domain swapping, a first Fab fragment and a second Fab fragment, wherein each binding site of the first Fab fragment and the second Fab fragment specifically binds to a first antigen; - 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 comprises a domain crossover such that the variable light domain (VL) and the variable heavy domain (VH) are substituted for each other; and - comprising an Fc region, the Fc region comprising a first Fc region polypeptide and a second Fc region polypeptide; the first Fab fragment and the second Fab fragment comprise a heavy chain fragment and a full-length light chain, respectively; 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; 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 a third Fab fragment, and the C-terminus of the CH1 domain of the third Fab fragment is fused to the N-terminus of a second Fc region polypeptide; -Antibody-multimer fusion; Such antibodies are fusion polypeptides comprising: (a) an antibody heavy chain and an antibody light chain; (b) a first fusion polypeptide comprising, from N-terminus to C-terminus, a first portion of a non-antibody multimeric 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 comprising, from N-terminus to C-terminus, a second portion of a non-antibody multimeric polypeptide, and an antibody light chain constant domain if the first polypeptide comprises the antibody heavy chain CH1 domain, or an antibody heavy chain CH1 domain if the first polypeptide comprises the antibody light chain constant domain; wherein (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; Here, the variable domains of the antibody heavy chain and the antibody light chain form a binding site that specifically binds to an antigen).

[0214] The CH3 domains of antibody heavy chains can be modified using the "knob-into-hole" technique. This technique is described in detail, with some examples, in WO 96 / 027011, Ridgway, JB, et al., Protein Eng. 9 (1996) 617-621, and Merchant, AM, et al., Nat. Biotechnol. 16 (1998) 677-681. This method involves modifying the interaction surfaces of two CH3 domains to increase heterodimerization of these two CH3 domains, thereby increasing heterodimerization of polypeptides containing them. Each of the two CH3 domains (of the two heavy chains) can be a "knob," and the other can be a "hole." The introduction of disulfide bridges further stabilizes the heterodimer (Merchant, AM, et al., Nature Biotech. 16 (1998) 677-681; Atwell, S., et al., J. Mol. Biol. 270 (1997) 26-35) and increases the yield.

[0215] The mutation T366W in the CH3 domain (of an antibody heavy chain) is designated as a "knob mutation," and the mutations T366S, L368A, and Y407V in the CH3 domain (of an antibody heavy chain) are designated as "hole mutations" (numbering according to the EU index of Kabat). Additional interchain disulfide bridges between CH3 domains (Merchant, AM et al., Nature Biotech. 16 (1998) 677-681) can also be used, for example, by introducing a S354C mutation in the CH3 domain of a heavy chain bearing a "knob mutation" (designated a "knob-cys mutation") and a Y349C mutation in the CH3 domain of a heavy chain bearing a "hole mutation" (designated a "hole-cys mutation") (numbering according to the EU index of Kabat).

[0216] The term "domain crossover" as used herein refers to deviations in domain sequence from that of a native antibody in that in a pair of antibody heavy chain VH-CH1 fragment and its corresponding cognate antibody light chain, i.e., antibody Fab (fragment-antigen binding), at least one heavy chain domain is replaced by the corresponding light chain domain, or vice versa. There are three general types of domain crossovers: (i) crossovers of CH1 and CL domains, where the domain crossover in the light chain results in a VL-CH1 domain sequence and the domain crossover in the heavy chain fragment results in a VH-CL domain sequence (or a full-length antibody heavy chain having a VH-CL-hinge-CH2-CH3 domain sequence); (ii) domain crossovers of VH and VL domains, where the domain crossover in the light chain results in a VH-CL domain sequence and the domain crossover in the heavy chain fragment results in a VL-CH1 domain sequence; and (iii) domain crossovers of a complete light chain (VL-CL) and a complete VH-CH1 heavy chain fragment ("Fab crossover"), where the domain crossover results in a light chain with a VH-CH1 domain sequence and the domain crossover results in a heavy chain fragment with a VL-CL domain sequence (all domain sequences listed above are in the N-terminal to C-terminal direction).

[0217] As used herein, the term "replaced with each other" in relation to corresponding heavy chain domains and light chain domains refers to the domain crossover described above. Thus, when CH1 and CL domains are "replaced with each other", this term refers to the domain crossover described under item (i) and the resulting heavy chain and light chain domain sequences. Thus, when VH and VL are "replaced with each other", this term refers to the domain crossover described under item (ii); and when CH1 and CL domains are "replaced with each other" and VH and VL domains are "replaced with each other", this term refers to the domain crossover described under item (iii). Bispecific antibodies comprising domain crossovers have been reported, for example, in International Publication Nos. 2009 / 080251, 2009 / 080252, 2009 / 080253, and 2009 / 080254, and Schaefer, W., et al., Proc. Natl. Acad. Sci. USA 108 (2011) 11187-11192. Such antibodies are generally referred to as CrossMabs.

[0218] In certain embodiments, a multispecific antibody also comprises at least one Fab fragment comprising either the domain crossover of the CH1 and CL domains described in item (i) above, or the domain crossover of the VH and VL domains described in item (ii) above, or the domain crossover of the VH-CH1 and VL-VL domains described in item (iii) above. In the case of a multispecific antibody with domain crossover, Fabs that specifically bind to the same antigen are constructed to have the same domain sequence in certain embodiments. Therefore, when more than one Fab with domain crossover is contained in a multispecific antibody, the Fabs specifically bind to the same antigen.

[0219] The term "recombinant antibody," as used herein, refers to all antibodies (chimeric, humanized, and human) that are prepared, expressed, produced, or isolated by recombinant means, such as recombinant cells. This includes antibodies isolated from recombinant cells, such as NS0, HEK, BHK, amniotic cells, CHO cells, etc.

[0220] As used herein, the term "antibody fragment" refers to a molecule other than an intact antibody that contains a portion of the 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, and single-chain antibody molecules (e.g., scFv or scFab).

[0221] Recombination Method Antibodies can be produced using recombinant methods and compositions, for example, as described in U.S. Patent No. 4,816,567. These methods provide one or more isolated nucleic acid(s) encoding the antibody.

[0222] In one aspect of the invention, there is provided a method for recombinantly producing an antibody comprising one or more amino acid sequences cleavable and cleaved by endogenous proteases of the producing cell during recombinant production in the absence of a nucleic acid according to the invention, the method comprising culturing a recombinant cell according to the invention comprising a nucleic acid according to the invention and one or more nucleic acids encoding the antibody under conditions suitable for expression of the antibody, optionally recovering the antibody from the recombinant cell (and / or cell culture medium), and further optionally purifying the antibody by one or more chromatography steps.

[0223] For recombinant production of an antibody, nucleic acids encoding the antibody are 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 genes encoding the antibody heavy and light chains), or can be produced by recombinant methods, or can be obtained by chemical synthesis.

[0224] Typically, recombinant mass production of a polypeptide of interest, such as a therapeutic antibody, requires recombinant cells that stably express and secrete the polypeptide. The overall process used to generate such recombinant cells is called "cell line development." In the first step of the cell line development process, suitable mammalian cells, such as CHO cells in certain embodiments, are transfected with one or more nucleic acids, including a nucleic acid according to the present invention and a nucleic acid suitable for expressing the polypeptide of interest. In the second step, recombinant cells that stably express the proteinaceous protease inhibitor and the polypeptide of interest are selected, for example, based on the co-expression of a selectable marker co-transfected with the nucleic acid.

[0225] The nucleotide sequence of a nucleic acid that encodes a polypeptide, i.e., a coding sequence, is referred to as a structural gene. Such a structural gene is purely coding information. Therefore, its expression requires additional regulatory elements. Therefore, a structural gene is usually incorporated into a so-called expression cassette. The minimum regulatory elements required for an expression cassette to be functional in mammalian cells are a promoter that is functional in mammalian cells, located upstream, i.e., 5', of the structural gene, and a polyadenylation signal sequence that is functional in mammalian cells, located downstream, i.e., 3', of the structural gene. The promoter sequence, structural gene sequence, and polyadenylation signal sequence are arranged in an operably linked form.

[0226] If the polypeptide of interest is a heteromultimeric polypeptide composed of different polypeptides, such as an antibody or a complex antibody format, not only a single expression cassette is required, but multiple expression cassettes, each containing a different structural gene, are required, i.e., at least one expression cassette for each of the different polypeptides (chains) of the heteromultimeric polypeptide (heteromultimeric antibody). For example, a full-length antibody is a heteromultimeric polypeptide containing two copies of a light chain and two copies of a heavy chain. Therefore, a full-length antibody is composed of two different polypeptides. Therefore, two expression cassettes are required for the expression of a full-length antibody, one for the light chain and one for the heavy chain. For example, if the full-length antibody is a bispecific antibody, i.e., if the antibody contains two different binding sites that specifically bind to two different antigens / epitopes on the same antigen, the two light chains and the two heavy chains are also different from each other. Therefore, such a bispecific full-length antibody is composed of four different polypeptides, and therefore four expression cassettes are required.

[0227] The expression cassette(s) for the polypeptide of interest are incorporated into one or more so-called "expression vectors" for direct expression or "integration vectors" for targeted integration. A "vector" is a nucleic acid that provides all the elements required to amplify the vector in bacterial cells and to express the contained structural gene in mammalian cells. Typically, an expression vector comprises a prokaryotic plasmid propagation unit, which, in the case of E. coli, for example, contains an origin of replication and a prokaryotic or even eukaryotic selection marker, as well as the expression cassette required for expression of the structural gene of interest. An "expression vector" or "integration vector" is a delivery vehicle for introducing an expression cassette into mammalian cells to generate a recombinant polypeptide-expressing cell.

[0228] As outlined in the previous paragraph, the more complex the polypeptide to be expressed, the greater the number of different expression cassettes required. Essentially, the size of the nucleic acid integrated into the genome of the cell increases with the number of expression cassettes. 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, above which the efficiency of manipulation and processing decreases significantly. This problem can be addressed by using two or more expression vectors. Thereby, the expression cassette can be divided between different expression vectors, each containing only a portion of the expression cassette, resulting in a reduction in size.

[0229] Cell line development (CLD) for generating recombinant cells expressing heterologous polypeptides, such as multispecific antibodies, uses either random integration (RI) or targeted integration (TI) of nucleic acids containing the respective expression cassettes required for the expression and production of the heterologous polypeptide of interest.

[0230] Using RI, multiple vectors or fragments thereof typically integrate into the genome of a cell at the same or different loci.

[0231] Using TI, typically a single copy of a transgene containing different expression cassettes is integrated into a defined "hot spot" in the cell's genome.

[0232] Suitable cells for the generation of recombinant cells for the expression of (glycosylated) antibodies are generally derived from multicellular organisms, such as vertebrates.

[0233] Targeted embedding One method for the generation of recombinant mammalian cells according to the invention to be used in the methods according to the invention is targeted integration for the introduction of the respective nucleic acid.

[0234] In certain embodiments of all aspects and embodiments, a nucleic acid according to the invention and one or more nucleic acids encoding a heterologous polypeptide are integrated into a mammalian TI host cell by single or double recombinase-mediated cassette exchange (RMCE), resulting in a recombinant mammalian cell, e.g., a recombinant CHO cell, in which the expression cassette is integrated into the genome at a single locus.

[0235] The Cre-LoxP site-specific recombination system is widely used in many biological experimental systems. Cre recombinase is a 38 kDa site-specific DNA recombinase that recognizes 34-bp LoxP sequences. Cre recombinase is derived from bacteriophage P1 and belongs to the tyrosine family of site-specific recombinases. Cre recombinase can mediate both intramolecular and intermolecular recombination between LoxP sequences. The LoxP sequence consists of an 8-bp nonpalindromic core region flanked by two 13-bp inverted repeats. Cre recombinase binds to the 13-bp repeats, 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. When two LoxP sequences are positioned in the same orientation on the same nucleotide sequence, Cre recombinase-mediated recombination will excise the DNA sequence located between the two LoxP sequences into a covalently closed loop. If two LoxP sequences are located in opposite orientations on the same nucleotide sequence, Cre recombinase-mediated recombination will reverse the orientation of the DNA sequence located between the two sequences. If two LoxP sequences are located on two different DNA molecules and one DNA molecule is circular, Cre recombinase-mediated recombination will result in the integration of the circular DNA sequence.

[0236] A "recombination recognition sequence" (RRS) is a nucleotide sequence that is recognized by a recombinase and is necessary and sufficient for a recombinase-mediated recombination event. RRSs can be used to define locations in a nucleotide sequence where recombination events are expected to occur.

[0237] The term "matched RRS" indicates that recombination occurs between two RRSs. In certain embodiments, the two matched RRSs are the same.

[0238] In certain embodiments of all aspects and embodiments, the RRS can be recognized by Cre recombinase.

[0239] In certain embodiments of all aspects and embodiments, the RRS is capable of being recognized by an FLP recombinase.

[0240] In certain embodiments of all aspects and embodiments, the RRS can be recognized by Bxb1 integrase.

[0241] In certain embodiments of all aspects and embodiments, the RRS can be recognized by φC31 integrase.

[0242] In certain embodiments of all aspects and embodiments, both RRSs are wild-type LoxP sequences. In certain embodiments, both RRSs are mutant LoxP sequences.

[0243] In certain embodiments of all aspects and embodiments, both RRSs are wild-type FRT sequences.

[0244] In certain embodiments of all aspects and embodiments, both RRSs are mutated FRT sequences.

[0245] In certain embodiments of all aspects and embodiments, the two matching RRSs are different sequences but can be recognized by the same recombinase.

[0246] 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.

[0247] In certain embodiments of all aspects and embodiments, the first matching RRS is a φC31 attB sequence and the second matching RRS is a φC31 attB sequence.

[0248] A "two-plasmid RMCE" strategy or "double RMCE" is used in the methods of the present invention when a combination of two vectors is used. For example, but not limited to, the integrated landing site can contain three RRSs, e.g., an arrangement in which a third RRS ("RRS3") is located between a first RRS ("RRS1") and a second RRS ("RRS2"), where the first vector contains two RRSs that match the first and third RRSs on the integrated exogenous nucleotide sequence, and the second vector contains two RRSs that match the third and second RRSs on the integrated exogenous nucleotide sequence.

[0249] The two-plasmid RMCE strategy involves simultaneously performing two independent RMCEs using three RRS sites. Therefore, the landing site in mammalian TI host cells using the two-plasmid RMCE strategy contains a third RRS site (RRS3) that has no cross-reactivity with either the first RRS site (RRS1) or the second RRS site (RRS2). The two targeted plasmids require identical flanking RRS sites for efficient targeting: one plasmid (front) is flanked by RRS1 and RRS3, and the other (back) is flanked by RRS3 and RRS2. Furthermore, two selectable markers are also required in two-plasmid RMCE. One selectable marker expression cassette is split into two parts. The front plasmid contains a promoter followed by an initiation codon and the RRS3 sequence. The back plasmid lacks an initiation codon (ATG) and has the RRS3 sequence fused to the N-terminus of the selectable marker coding region. To ensure in-frame translation of the fusion protein, i.e., operable linkage, additional nucleotides may need to be inserted between the RRS3 site and the selection marker sequence. Only when both plasmids are correctly inserted will the complete expression cassette for the selection marker be assembled, thereby conferring resistance to the respective selection agent to the cells.

[0250] Two-plasmid RMCE involves a recombinase-catalyzed double recombination crossover event between two heterospecific RRSs within a target genomic locus and a donor DNA molecule. Two-plasmid RMCE is designed to combine and introduce copies of DNA sequences from a front vector and a back vector into a predetermined locus in the genome of a mammalian TI host cell. RMCE can be performed such that sequences from the prokaryotic vector are not introduced into the mammalian TI host cell genome, thus reducing and / or preventing unwanted triggering of host immune or defense mechanisms. The RMCE procedure can be repeated with multiple DNA sequences.

[0251] In certain aspects and embodiments, targeted integration is achieved by two rounds of RMCE, in which two different DNA sequences are both integrated into a predetermined site in the genome of a matching RRS of the mammalian TI host cell, each DNA sequence comprising at least one expression cassette encoding a portion of a heteromultimeric polypeptide and / or at least one selectable marker or portion thereof flanked by two heterospecific RRSs. In certain embodiments, targeted integration is achieved by multiple rounds of RMCE, in which DNA sequences from multiple vectors are all integrated into a predetermined site in the genome of the mammalian TI host cell, each DNA sequence comprising at least one expression cassette encoding a portion of a heteromultimeric polypeptide and / or at least one selectable marker or portion thereof flanked by two heterospecific RRSs. In certain embodiments, the selectable marker may be partially encoded on a first vector and partially encoded on a second vector, such that only correct integration of both by dual RMCE allows expression of the selectable marker.

[0252] In certain embodiments of all aspects and embodiments, targeted integration by recombinase-mediated recombination results in the integration of the selectable marker and / or different expression cassettes for the nucleic acids of the invention, as well as the multimeric polypeptide, into one or more predetermined integration sites in the host cell genome that do not contain sequences from the prokaryotic vector.

[0253] In certain embodiments of all aspects and embodiments, the mammalian TI host cell comprises an integrated landing site, the landing site comprising two or more recombination recognition sequences (RRS). The RRS can be recognized by a recombinase, such as Cre recombinase, FLP recombinase, Bxb1 integrase, or φC31 integrase. The RRSs can be independently selected from the group consisting of LoxP, LoxP L3, LoxP 2L, LoxFas, Lox511, Lox2272, Lox2372, Lox5171, Loxm2, Lox71, Lox66, FRT, Bxb1 attP, Bxb1 attB, φC31 attP, and φC31 attB. If multiple RRSs are to be present, the selection of each sequence is dependent on the other, provided that non-identical RRSs are selected.

[0254] Typically, a mammalian TI host cell is a mammalian cell that contains a landing site for integration at a site within a locus in the genome of the mammalian cell, the landing site comprising a first recombination recognition sequence and a second recombination recognition sequence adjacent to at least one first selectable marker, and a third recombination recognition sequence located between the first recombination recognition sequence and the second recombination recognition sequence, and the recombination recognition sequences are all different.

[0255] The selectable marker may be selected from the group consisting of aminoglycoside phosphotransferases (APHs) (e.g., hygromycin phosphotransferase (HYG), neomycin, and G418 APH), dihydrofolate reductase (DHFR), thymidine kinase (TK), glutamine synthetase (GS), asparagine synthetase, tryptophan synthetase (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.

[0256] An exogenous nucleotide sequence is a nucleotide sequence that is not native to a particular cell but can be introduced into the cell by a DNA delivery method, such as transfection, electroporation, or transformation. In certain embodiments, the mammalian TI host cell comprises at least one landing site integrated into one or more integration sites in the genome of the mammalian cell. In certain embodiments, the landing site is integrated into one or more integration sites within a specific locus in the genome of the mammalian cell.

[0257] 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 a first RRS, a second RRS, and a third RRS, 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, the two RRSs are adjacent to the at least one selectable marker. That is, the first RRS is located 5' (upstream) of the selectable marker, 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.

[0258] In certain embodiments of all aspects and embodiments, the selectable marker is located between the first RRS and the second RRS, and these two flanking RRSs are different from each other. In certain preferred embodiments, the first flanking RRS is a LoxP L3 sequence (SEQ ID NO: 96), and the second flanking RRS is a LoxP 2L sequence (SEQ ID NO: 97). In certain embodiments, the LoxP L3 sequence is located 5' of the selectable marker, and the LoxP 2L sequence is located 3' of the selectable 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 a Bxb1 attP sequence, and the second flanking RRS is a Bxb1 attB sequence. In certain embodiments, the first flanking RRS is a φC31 attP sequence, and the second flanking RRS is a φC31 attB sequence. In certain embodiments, the two RRSs are positioned in the same orientation. In certain embodiments, the two RRSs are both oriented in the forward or reverse direction. In certain embodiments, the two RRSs are positioned in opposite directions.

[0259] In certain embodiments of all aspects and embodiments, the integrated landing site comprises a first selection marker and a second selection marker flanked by two RSSs, wherein the first selection marker is different from the second selection marker. In certain embodiments, both of the two selection markers are selected, independently of one another, from the group consisting of a glutamine synthetase selection marker, a thymidine kinase selection marker, a HYG selection marker, and a puromycin resistance selection marker. In certain embodiments, the integrated landing site comprises a thymidine kinase selection marker and a HYG selection marker. In certain embodiments, the first selection marker is an 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 protein. In certain embodiments, the first selectable marker is a glutamine synthetase selectable marker and the second selectable marker is a GFP fluorescent protein, hi certain embodiments, the two RRSs flanking both selectable markers are different.

[0260] In certain embodiments of all aspects and embodiments, the selectable marker is operably linked to a promoter sequence. In certain embodiments, the selectable marker is operably linked to an SV40 promoter. In certain embodiments, the selectable marker is operably linked to a human cytomegalovirus (CMV) promoter.

[0261] As used herein, the term "operably linked" refers to the juxtaposition of two or more components, in a relationship permitting them to function in a desired manner. For example, a promoter and / or enhancer is operably linked to a coding sequence if it functions to regulate the transcription of the coding sequence. In certain embodiments, DNA sequences that are "operably linked" are contiguous and adjacent on a single chromosome. In certain embodiments, when it is necessary to join two protein coding regions, e.g., a secretory leader and a polypeptide, these sequences are contiguous, adjacent, and in the same reading frame. In certain embodiments, an operably linked promoter can be 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, two components can be operably linked even though they are not adjacent. An enhancer is operably linked to a coding sequence if the enhancer increases the transcription of the coding sequence. An operably linked enhancer may be located upstream, inside, or downstream of a coding sequence, and may be located at a considerable distance from the promoter of the coding sequence. Operable linkage can be achieved by recombinant methods known in the art, for example, by using PCR methods and / or by ligation at a convenient restriction site. If a convenient restriction site does not exist, synthetic oligonucleotide adapters or linkers can be used according to conventional techniques. An internal ribosome entry site (IRES) is operably linked to an open reading frame (ORF) if it allows translation to begin at an internal position of the ORF independently of the 5' end.

[0262] As used herein, the term "selection marker" refers to a gene that allows cells carrying a certain gene to be specifically selected or specifically eliminated in the presence of a corresponding selection agent. For example, but not limited to, a selection marker can enable host cells transformed with the selection marker gene to be positively selected in the presence of the respective selection agent (under selective culture conditions); untransformed host cells would not be able to grow or survive under the selective culture conditions. A selection marker can be positive, negative, or bifunctional. A positive selection marker can allow for the selection of cells carrying the marker, while a negative selection marker can allow for the selective elimination of cells carrying the marker. A selection marker can confer resistance to a drug in a host cell or complement a metabolic or catabolic defect. In prokaryotic cells, genes that confer resistance to ampicillin, tetracycline, kanamycin, or chloramphenicol, among others, can be used. Resistance genes useful as selectable markers in eukaryotic cells include, but are not limited to, genes for aminoglycoside phosphotransferases (APHs) (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)), as well as genes encoding resistance to puromycin, blasticidin, bleomycin, phleomycin, chloramphenicol, zeocin, and mycophenolic acid. Additional marker genes are described in WO 92 / 08796 and WO 94 / 28143.

[0263] In addition to facilitating selection in the presence of a corresponding selection agent, a selection marker may alternatively be a molecule not normally present in cells, 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. For example, cells expressing such molecules can be distinguished from cells that do not harbor the gene based on the detection or absence, respectively, of fluorescence emitted by the encoded polypeptide.

[0264] Proteases As used herein, the term "protease" and its grammatical equivalents refer to an enzyme that catalyzes the hydrolysis of covalent peptide (amide) bonds. Proteases can be subdivided into different classes, such as serine proteases and matrix metalloproteinases.

[0265] Matrix metalloproteinases (MMPs) are metal-dependent, e.g., Zn 2+ MMPs are a family of dependent endopeptidases. They preferably cleave components of the extracellular matrix. MMPs include collagenases, stromelysins, membrane metalloproteinases, and gelatinases. In vivo, MMPs occur as inactive precursors (zymogens) that must be cleaved to obtain catalytically active forms. MMPs are specifically regulated by tissue inhibitors of matrix metalloproteinases.

[0266] Serine proteases are widespread in prokaryotes and eukaryotes. They are characterized by the presence of a catalytically active serine residue in the active center of the enzyme. Peptide bond cleavage by serine proteases involves nucleophilic attack of the target peptide bond by the serine residue in the active center of the enzyme. In addition to the serine residue, additional histidine and aspartic acid residues (respectively their side chains) may be involved. Together, they form the so-called catalytic triad common to most serine proteases.

[0267] Serine proteases include, among others, chymotrypsin, trypsin, elastase, NS3, factor Xa, granzyme B, thrombin, plasmin, urokinase, tissue plasminogen activator, and prostate-specific antigen. Matrix metalloproteinases include, for example, gelatinase B and gelatinase A.

[0268] Chymotrypsin acts on peptide bonds adjacent to bulky, hydrophobic amino acid residues, particularly phenylalanine, tryptophan, and tyrosine.

[0269] Trypsin cleaves peptide bonds adjacent to positively charged amino acid residues.

[0270] Elastase hydrolyzes peptide bonds adjacent to small neutral / aliphatic amino acid residues, particularly alanine, methionine, glycine, and valine.

[0271] Self-cleaving peptides The introduction of a so-called self-cleaving peptide sequence between the two coding sequences allows the production of two polypeptides flanked by the self-cleaving peptide sequence in separated form from a single nucleic acid.

[0272] Incorporation of a self-cleaving peptide sequence into a nucleic acid, and thereby into the corresponding mRNA, causes or prevents the ribosome from skipping or synthesizing a peptide bond at the C-terminus during protein synthesis (either by preventing the formation of a peptide or phosphodiester bond between amino acid residues, or by acting as a pseudo-stop codon sequence that directs the translation complex to move from one codon to the next without forming a peptide bond). Thus, a single mRNA molecule encodes multiple different / separate proteins that are produced by ribosomal skipping during translation.

[0273] By including the self-cleaving peptide, two separate polypeptides are obtained, one corresponding to the sequence upstream of the self-cleaving peptide sequence and one corresponding to the sequence downstream of the self-cleaving peptide sequence. Although it is called the self-cleaving peptide sequence, in fact, neither of the resulting products contains the entire 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.

[0274] Thus, the nucleic acids encoding the self-cleaving peptide sequences are positioned so that they are between and in-frame with the two coding regions before and after them.

[0275] One type of self-cleaving peptide sequence is the viral 2-A self-cleaving peptide sequence.Detailed methodology for the design and use of 2-A self-cleaving peptide sequence can be found in Szymczak-Workman et al. (Design and Construction of 2A Peptide-Linked Multicistronic Vectors.Cold Spring Harb.Protoc.2012 Feb 1;2012(2):199-204), which is expressly incorporated herein by reference.

[0276] Similar to other self-cleaving peptide sequences, the incorporation of the viral 2-A self-cleaving peptide sequence results in a ribosome skip, which skips the synthesis of the peptide bond at the C-terminus of the 2-A self-cleaving peptide sequence. More specifically, the peptide bond connecting the glycine and proline amino acid residues at the C-terminus of the 2-A self-cleaving peptide sequence is not formed. This results in two polypeptides, one containing a portion of the 2-A self-cleaving peptide sequence at its C-terminus and the other at its N-terminus (see, e.g., 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) el8556; Donnelly, ML, et al., J. Gen. Virol, 82 (2001) 1027-1101; Ryan, MD, et al., J. Gen. Virol, 72 (2001) 2727-2732). An exemplary viral 2-A self-cleaving peptide sequence is: the 2-A self-cleaving peptide sequence of the thosea asigna virus, designated -T2A; - the 2-A self-cleaving peptide sequence of equine rhinitis A virus, designated E2A; the porcine teschovirus-1 2-A self-cleaving peptide sequence, designated -P2A; the 2-A self-cleaving peptide sequence of foot-and-mouth disease virus, designated -F2A; -2-A self-cleaving peptide sequence of acute bee paralysis virus - A2A, -Drosophila C virus 2-A self-cleaving peptide sequence-D2A, -2-A self-cleaving peptide sequence of infectious myonecrosis virus - I2A.

[0277] The following table shows the sequences of members of the viral 2-A self-cleaving peptide sequence family. It is known in the art that adding the peptide linker sequence GSG (Gly-Ser-Gly; SEQ ID NO: 04) to the N-terminus of the 2-A self-cleaving peptide sequence (the N-terminus of the 2-A self-cleaving peptide sequence) can increase cleavage efficiency. The N-terminus of the 2-A self-cleaving peptide means that the sequence encoding GSG is upstream of the sequence encoding the 2-A self-cleaving peptide. Generally, GSG is immediately N-terminal to the 2-A self-cleaving peptide sequence. In certain embodiments, 1 to 10 additional amino acid residues are inserted between the GSG and the 2-A self-cleaving peptide sequence. In certain embodiments, the polynucleotide sequence encoding GSG is GGC AGT GGA (SEQ ID NO: 05). As with any peptide-encoding polynucleotide, the nucleotide sequence can be altered without changing the encoded peptide sequence due to the degeneracy of the genetic code, as known to those skilled in the art.

[0278] TIFF2025538565000003.tif204170TIFF2025538565000004.tif234170TIFF2025538565 000005.tif237170TIFF2025538565000006.tif239170TIFF2025538565000007.tif95170

[0279] Substitution of amino acid residues is within the skill of one in the art. Thus, the term "2-A self-cleaving peptide sequence" encompasses variants of the aforementioned 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 compared to the wild-type 2-A self-cleaving peptide sequence (see, e.g., Liu et al., Sci. Rep. 7 (2017) 2193).

[0280] 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, i.e., 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 more sequence identity to the wild-type viral 2-A self-cleaving peptide sequence and retain ribosomal skipping function. In certain embodiments, at least one N-terminal amino acid or respective codon of any one of SEQ ID NOs: 14-85 is deleted, e.g., 1, 2, 3, 4, or 5 amino acids or respective numbers of codons (including ranges between any two of the recited values). In certain embodiments, at least one C-terminal amino acid or respective codon of any one of SEQ ID NOs: 14-85 is deleted, e.g., 1, 2, 3, 4, or 5 amino acids or respective numbers of codons (including ranges between any two of the recited values). In certain embodiments, at least 1, 2, 3, 4, or 5 amino acids or respective numbers of codons (including ranges between any two of the listed values) of any one of SEQ ID NOs: 14-85 are substituted, e.g., as conservative amino acid substitutions.

[0281] In certain embodiments of all aspects and embodiments, the T2A self-cleaving peptide sequence comprises an amino acid sequence comprising EGRGSLLTCGDVEENPGP (SEQ ID NO: 14), or a sequence having at least 70%, 80%, 90%, 95%, or 99% sequence identity to an amino acid sequence comprising EGRGSLLTCGDVEENPGP (SEQ ID NO: 14) and having ribosomal skipping function.

[0282] In certain aspects and embodiments, the GSG-T2A self-cleaving peptide sequence comprises an amino acid sequence comprising GSGEGRGSLTCGDVEENPGP (SEQ ID NO: 108), or a sequence having at least 70%, 80%, 90%, 95%, or 99% sequence identity to an amino acid sequence comprising GSGEGRGSLTCGDVEENPGP (SEQ ID NO: 108) and having ribosomal skipping function. In certain embodiments, the GSG-T2A self-cleaving peptide sequence is encoded by a nucleic acid sequence comprising GGCAGTGGAGAGGGCAGAGGAAGTCTGCTAACATGCGGTGACGTCGAGGAGAATCCTGGCCCA (SEQ ID NO: 109).

[0283] In certain aspects and embodiments, the E2A self-cleaving peptide sequence comprises an amino acid sequence comprising QCTNYALLKLAGDVESNPGP (SEQ ID NO: 30), or a sequence having at least 70%, 80%, 90%, 95%, or 99% identity to an amino acid sequence comprising QCTNYALLKLAGDVESNPGP (SEQ ID NO: 30) and comprising ribosomal skipping function. In certain embodiments, the GSG-E2A self-cleaving peptide sequence comprises an amino acid sequence comprising GSGQCTNYALLKLAGDVESNPGP (SEQ ID NO: 110), or a sequence having at least 70%, 80%, 90%, 95%, or 99% identity to an amino acid sequence comprising GSGQCTNYALLKLAGDVESNPGP (SEQ ID NO: 110) and comprising ribosomal skipping function.

[0284] In certain aspects and embodiments, the F2A self-cleaving peptide comprises an amino acid sequence comprising VKQTLNFDLLKLAGDVESNPGP (SEQ ID NO: 44), or a sequence having at least 70%, 80%, 90%, 95%, or 99% identity to an amino acid sequence comprising VKQTLNFDLLKLAGDVESNPGP (SEQ ID NO: 44) and having ribosomal skipping function. In certain embodiments, the GSG-F2A self-cleaving peptide comprises an amino acid sequence comprising GSGVKQTLNFDLLKLAGDVESNPGP (SEQ ID NO: 112), or a sequence having at least 70%, 80%, 90%, 95%, or 99% identity to an amino acid sequence comprising GSGVKQTLNFDLLKLAGDVESNPGP (SEQ ID NO: 112) and having ribosomal skipping function.

[0285] In certain aspects and embodiments, the P2A self-cleaving peptide comprises an amino acid sequence comprising ATNFSLLKQAGDVEENPGP (SEQ ID NO: 24), or a sequence having at least 70%, 80%, 90%, 95%, or 99% identity to an amino acid sequence comprising ATNFSLLKQAGDVEENPGP (SEQ ID NO: 24) and having ribosomal skipping function. In certain embodiments, the GSG-P2A self-cleaving peptide comprises an amino acid sequence comprising GSGATNFSLLKQAGDVEENPGP (SEQ ID NO: 114), or a sequence having at least 70%, 80%, 90%, 95%, or 99% identity to an amino acid sequence comprising GSGATNFSLLKQAGDVEENPGP (SEQ ID NO: 114) and having ribosomal skipping function.

[0286] 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.

[0287] In certain embodiments of all aspects and embodiments, the 2-A self-cleaving peptide sequence is combined at its N- or C-terminus with a protease cleavage site, such as a furin cleavage site (RKRR (SEQ ID NO: 116 (SEQ ID NO: 92 where X=K); cgcaaacggaga SEQ ID NO: 117)).

[0288] Peptide Linker In certain embodiments of all aspects and embodiments, the recombinant polypeptide comprises a protease-cleavable site. In certain embodiments, the protease-cleavable site is comprised in a peptide linker (used interchangeably with cleavable peptide linker). In certain embodiments, the cleavable linker, or protease-cleavable peptide linker, is a peptide linker that, in one preferred embodiment, comprises at least one peptide bond within a recognition (amino acid) sequence (recognition site) of a protease. In certain embodiments, the cleavable peptide linker is a target substrate for a protease such that it is preferentially or specifically cleaved by the protease compared to a peptide linker that does not contain a recognition sequence for the same protease.

[0289] 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 the peptide bond is cleaved by the protease.

[0290] For example, in the case of serine proteases, the recognition sequence consists of amino acid residues N- and C-terminal to the peptide bond to be cleaved. 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 for serine proteases is 6-8 amino acid residues long, but can be longer or shorter depending on the particular protease. Typically, a cleavable peptide linker contains a P1-P1' cleavable bond within the recognition sequence recognized by the protease.

[0291] In certain embodiments of all aspects and embodiments, the cleavable peptide linker is one that is cleaved by a protease at a rate substantially higher than the cleavage of a non-target substrate by the same protease. Typically, a protease exhibits specificity (preference) for cleaving a particular polypeptide containing its respective recognition sequence compared to another polypeptide that does not contain the recognition sequence. Such specificity can be determined based on the rate constant of cleavage 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 rate constant of cleavage can be used. For example, substrates containing the respective recognition sequences are conjugated to a fluorogenic moiety that is released upon cleavage by the protease. By determining the rate of cleavage at different protease concentrations, the rate constant for cleavage (kcat / Km) can be determined for a particular protease-substrate combination. In certain embodiments, the cleavable peptide linker has a cleavage rate of 1×10 7 M -1 More than S or 10 8 M -1 Exceeds S, 1×10 9 M -1 More than S or 1×10 10 M -1 It is a peptide linker that is cleaved by proteases at a rate greater than S.

[0292] In certain aspects and embodiments, at least one polypeptide of a multispecific antibody produced in a recombinant cell according to the invention comprises a recognition sequence for a protease, including, for example, matrix metalloproteinases (MMPs), cysteine ​​proteases, serine proteases, and plasmin activators. In certain embodiments, the polypeptide comprises a recognition sequence for a protease that is a protease produced by a tumor, an activated immune effector cell (e.g., a T cell or an NK cell), or a cell in the tumor microenvironment.

[0293] In certain embodiments of all aspects and embodiments, the recombinant antibody comprises 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; ADAMDECl; ADAMTS1; ADAMTS4; ADAMTS5; an aspartic protease, such as BACE or renin; an aspartic cathepsin, such as cathepsin D or cathepsin E; a caspase. 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 ​​proteinases, such as crizipain; legumain; otubain-2; KLKSs, such as KLK4, KLK5, KLK6, KLK7, KLK8, KLK1 0, KLK11, KLK13, or KLK14; metalloproteinases, 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, chymase, coagulation factors Proteases (e.g., FVIIa, FIXa, FXa, FXIa, FXIIa), elastase, granzyme B, guanidinobenzotase, HtrA1, human neutrophil elastase, lactoferrin, marapsin, NS3 / 4A, PACE4, plasmin, PSA, tPA, thrombin, tryptase, uPA; type II transmembrane serine proteases (TTSPs), such as DESC1, DPP-4, FAP, hepsin, matriptase-2, matriptase, TMPRSS2, TMPRSS3, or TMPRSS4.

[0294] In certain aspects and embodiments, the recombinant antibody comprises at least one polypeptide comprising a 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 comprising a cleavable peptide linker comprising an amino acid sequence having the general formula P4 P3 P2 P1↓P1' (SEQ ID NO: 118), where P4 is amino acid I, L, Y, M, F, V, or A; P3 is amino acid A, G, S, V, E, D, Q, N, or Y; P2 is amino acid H, P, A, V, G, S, or T; P1 is amino acid D or E; and P1' is amino acid I, L, Y, M, F, V, T, S, G, or A. In certain embodiments, the recombinant antibody comprises at least one polypeptide comprising a cleavable peptide linker comprising an amino acid sequence having the general formula P4 P3 P2 P1↓P1' (SEQ ID NO: 118), wherein P4 is amino acid I or L; P3 is amino acid E; P2 is amino acid P or A; P1 is amino acid D; and P1' is amino acid I, V, T, S, or G.

[0295] In certain embodiments of all aspects and embodiments, the recombinant antibody comprises at least one polypeptide comprising a cleavable peptide linker comprising the amino acid sequence LEAD (SEQ ID NO: 119), LEPD (SEQ ID NO: 120), or LEAE (SEQ ID NO: 121). In certain embodiments, the recombinant antibody comprises at least one polypeptide comprising a 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 ID NO: 126), IEPDT (SEQ ID NO: 127), IEPDP (SEQ ID NO: 128), LEPDG (SEQ ID NO: 129), or LEADG (SEQ ID NO: 130).

[0296] In certain embodiments of all aspects and embodiments, the recombinant antibody comprises at least one polypeptide comprising a cleavable peptide linker comprising an amino acid that is a substrate for matriptase.

[0297] In certain embodiments, the recombinant antibody comprises at least one polypeptide comprising a cleavable peptide linker comprising the sequence P1QAR↓(A / V / K) (SEQ ID NO: 131), where P1 is any amino acid.

[0298] In certain embodiments, a recombinant antibody comprises at least one polypeptide comprising a cleavable peptide linker comprising the sequence RQAR(A / V / K) (SEQ ID NO: 131, where P1=R). In certain embodiments, a recombinant antibody comprises at least one polypeptide comprising a cleavable peptide linker comprising the amino acid sequence RQAR (SEQ ID NO: 132). In certain embodiments, a recombinant antibody comprises at least one polypeptide comprising a cleavable peptide linker comprising the amino acid sequence RQARK (SEQ ID NO: 133).

[0299] In certain embodiments, the recombinant antibody comprises at least one polypeptide comprising a 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 comprising a 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 comprising a cleavable peptide linker comprising the amino acid sequence PQARK (SEQ ID NO: 167).

[0300] In certain embodiments, the recombinant antibody comprises at least one polypeptide comprising a 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 comprising a 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 comprising a cleavable peptide linker comprising the amino acid sequence HQARK (SEQ ID NO: 169).

[0301] In certain embodiments, a recombinant antibody comprises at least one polypeptide comprising a cleavable peptide linker comprising the sequence P1MAK↓(A / V / K) (SEQ ID NO: 170), where P1 is any amino acid. In certain embodiments, a recombinant antibody comprises at least one polypeptide comprising a cleavable peptide linker comprising the sequence PMAK(A / V / K) (SEQ ID NO: 171, where P1=P). In certain embodiments, a recombinant antibody comprises at least one polypeptide comprising a cleavable peptide linker comprising the amino acid sequence PMAK (SEQ ID NO: 172). In a preferred embodiment, a recombinant antibody comprises at least one polypeptide comprising a cleavable peptide linker comprising the amino acid sequence PMAKK (SEQ ID NO: 173).

[0302] In certain aspects and embodiments, the recombinant antibody comprises at least one polypeptide comprising a cleavable peptide linker comprising an amino acid that is a substrate for one or more matrix metalloproteases (MMPs). In certain embodiments, the MMP is MMP-2. In certain embodiments, the recombinant antibody comprises at least one polypeptide comprising a cleavable peptide linker comprising a sequence having the general formula P3 P2 P1↓P1′ (SEQ ID NO: 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 comprising a 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 comprising a cleavable peptide linker comprising the amino acid sequence PAGL (SEQ ID NO: 135).

[0303] In certain embodiments of all aspects and embodiments, the recombinant antibody has the amino acid sequence 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); GSLE ADTGS (SEQ ID NO: 147); GGSGGGGGIEPDGGGSGGS (SEQ ID NO: 148); GGSGGGGGIEPDVGGSGGS (SEQ ID NO: 149); GGSGGGGGIEPDSGGSGGS (SEQ ID NO: 150); GGSGGGGGIEPDTGGSGGS (SEQ ID NO: 151); GGSLEPDGSGS (SEQ ID NO: 152); GPAGLLEADGSRQARVG (SEQ ID NO: 153), GGEGGGGSGGSGGGS (SEQ ID NO: 154); GSSAGSEAGGSGQAGVGS (SEQ ID NO: 155); GGSGGGGGLEAEGSGGGGS (SEQ ID NO: 156); GGSGGGGGIEPDPGGSGGS (SEQ ID NO: 157); TGGSGGGGIEPDIGGSGGS (SEQ ID NO: 158).

[0304] Protease inhibitors 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 epsilon-aminocaproic acid. In one preferred embodiment, the protease inhibitor is BPTI.

[0305] 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 AAA1-AA2-AA3-AA4 (SEQ ID NO: 160), where AAA1 is -Arg-, -Phe-, and -Ile-, or a bond; AA2 is -Ala-, -Phe-, -Cit-, and -Nle-; AA3 is -Trp-, -Val-, and -Tyr-; and AA4 is -Phe- and -Gly-; inhibits elastase and comprises a constrained or β-hairpin peptide as set forth in U.S. Pat. No. 8,658,604; or inhibits elastase and comprises Pep4 (KRCCPDTCGIKCL; SEQ ID NO: 161) or Pep4M (KRMMPDTMGIKML; SEQ ID NO: 162).

[0306] In certain embodiments of all aspects and embodiments, the protease inhibitor inhibits matrix metalloproteases and comprises the sequence of the inhibitory peptide reported in Ndinguri et al., Molecules 17 (2012) 14230-14248.

[0307] 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.

[0308] In certain embodiments of all aspects and embodiments, the protease inhibitor inhibits chymase and comprises the structure Z-Arg-Glu-Thr-Phep(OPh)2 (SEQ ID NO: 164).

[0309] In certain embodiments of all aspects and embodiments, 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.

[0310] In certain embodiments of all aspects and embodiments, the protease inhibitor inhibits plasminogen activators and includes bovine pancreatic trypsin inhibitor (BPTI) (SEQ ID NO: 86; AQRPDFCLEPPYTGPCKARMIRYFYNAKAGLCQPFVYGGCRAKRNNFKSSEDCMRTCGGA) or the plasminogen activator inhibitor type 1 (PAI-1) derived peptide EEIIMD (SEQ ID NO: 88).

[0311] As used herein, the term "proteinaceous protease inhibitor" refers to an inhibitor of a naturally occurring protease that can be recombinantly produced. In certain embodiments of all aspects and embodiments according to the present invention, the proteinaceous protease inhibitor is a serine protease inhibitor. In certain embodiments of all aspects and embodiments according to the present invention, the proteinaceous protease inhibitor is a trypsin inhibitor. In certain preferred embodiments of all aspects and embodiments according to the present invention, the proteinaceous protease inhibitor is pancreatic trypsin inhibitor. In one preferred embodiment of all aspects and embodiments according to the present invention, the proteinaceous protease inhibitor is bovine pancreatic trypsin inhibitor (BPTI). 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.

[0312] The following examples, sequences and figures are provided to aid the understanding of the present invention, the true scope of which is set forth in the appended claims. It is understood that modifications can be made in the procedures set forth without departing from the spirit of the invention. [Example]

[0313] Description of the Examples Example 1 Generation of expression plasmids: a) Common light chain bispecific antibody in TCB format The recombinant polypeptide / protein used was a bispecific antibody consisting of two different immunoglobulin heavy chains (designated "K" (knob chain) and "H" (hole chain) respectively) and a common light chain (designated "L"). Knob-into-hole technology was used for heterodimerization of the heavy chains. The fully assembled bispecific antibody contains three copies of the light chain. As a result, the stoichiometry of the K:H:L chains of the fully assembled antibody is 1:1:3.

[0314] The configuration of the expression cassette (including the Cre-recombinase site) after targeted stable integration into the host cell genome was either L3-KLL-LoxFas-HL-2L (used to generate the data shown in Figures 2-7) or L3-KKLL-LoxFas-KLH-2L (used to generate the data shown in Figure 8 and Table 2). L3 (SEQ ID NO: 96), LoxFas (SEQ ID NO: 98), and 2L (SEQ ID NO: 97) are heterospecific loxP sites used for targeted integration.

[0315] Expression cassette For the expression of immunoglobulin chains, transcription units containing the following functional elements were used: - the immediate early enhancer and promoter from human cytomegalovirus (CMV promoter) including intron A (SEQ ID NO: 103), -human heavy chain immunoglobulin 5' untranslated region (5'UTR), - mouse immunoglobulin heavy chain signal sequence, - nucleic acids encoding each antibody chain, - bovine growth hormone polyadenylation sequence (BGH pA) (SEQ ID NO: 100), and - human gastrin terminator (hGT) (SEQ ID NO: 101).

[0316] In addition to the expression cassettes for the immunoglobulin subunits, the shuttle plasmid contained: - the origin of replication from the vector pUC18, allowing replication of this plasmid in E. coli, and -The beta-lactamase gene that confers ampicillin resistance to E. coli.

[0317] Front Vector Complete expression cassettes for K and L were obtained by chemically synthesizing DNA fragments encoding K and L and introducing them into a shuttle plasmid using appropriate restriction sites. The expression cassettes for K and L were excised from their shuttle plasmids to provide them with appropriate cohesive ends.

[0318] The first front vector was generated by combining one DNA fragment carrying the expression cassette for K and two fragments carrying the expression cassette for L in a four-way ligation reaction with a fourth fragment carrying the front vector backbone elements. The expression cassette configuration of this final front vector (including the Cre recombinase site) was L3-KLL-LoxFas.

[0319] Alternatively, the first front vector was generated by combining two DNA fragments carrying the expression cassette for K and two fragments carrying the expression cassette for L in a five-way ligation reaction with a fifth fragment carrying the front vector backbone elements. The expression cassette configuration of this final front vector (including the Cre recombinase site) was L3-KKLL-LoxFas.

[0320] In addition to the K and L expression cassettes, the front vectors each contained: - SV40 enhancer and early promoter (SEQ ID NO: 102) with a start codon upstream (or in other words 5') of LoxFas to drive resistance marker expression after stable integration, - the origin of replication from the vector pUC18, allowing replication of this plasmid in E. coli, and -The beta-lactamase gene that confers ampicillin resistance to E. coli.

[0321] Back Vector The complete expression cassette for H was obtained by chemically synthesizing a DNA fragment encoding H and introducing it into a shuttle plasmid using appropriate restriction sites. The expression cassettes for H and K and L (see the front vector section above) were excised from their shuttle plasmids to provide them with appropriate cohesive ends.

[0322] The first back vector was generated by combining one DNA fragment carrying the expression cassette for H and one fragment carrying the expression cassette for L in a three-way ligation reaction with a third fragment carrying the back plasmid backbone elements. The expression cassette configuration of this final back vector (including the Cre recombinase site) was LoxFas-HL-2L.

[0323] Alternatively, a second back vector was generated by combining one fragment carrying an expression cassette for K, one fragment carrying an expression cassette for H, and one fragment carrying an expression cassette for L in a four-way ligation with a fourth fragment carrying the back vector backbone. In this case, the expression cassette configuration (including the Cre recombinase site) of the final back vector was LoxFas-KHL-2L.

[0324] In addition to the immunoglobulin expression cassettes, the back vectors each contained the following: - a sequence encoding puromycin acetyltransferase (SEQ ID NO: 02) lacking the initiation codon immediately downstream (or in other words, 3') of the LoxFas site, - the origin of replication from the vector pUC18, allowing replication of this plasmid in E. coli, and -The beta-lactamase gene that confers ampicillin resistance to E. coli.

[0325] b) HAI expression construct To evaluate the effect of the protease inhibitor HAI-1 on the expression of the bispecific antibody described in a), a vector was generated for the additional expression of HAI-1 from a separate expression cassette. The expression cassette configuration (including a Cre recombinase site) after targeted stable integration into the host genome was L3-KLL-LoxFas-HL-HAI-1-2L.

[0326] Expression cassette The same immunoglobulin expression cassette was used as described in Example 1 a).

[0327] For the expression of HAI-1, different expression cassettes were generated, which contained either: - the immediate early enhancer and promoter from human cytomegalovirus (CMV promoter) including intron A (SEQ ID NO: 103), -human heavy chain immunoglobulin 5' untranslated region (5'UTR), - a nucleic acid encoding soluble HAI-1 (SEQ ID NO: 180), - bovine growth hormone polyadenylation sequence (BGH pA) (SEQ ID NO: 100), and human gastrin terminator (hGT) (SEQ ID NO: 101); or - Simian virus 40 (SV40) enhancer and early promoter (SEQ ID NO: 102), - a nucleic acid encoding soluble HAI-1 (SEQ ID NO: 180), - bovine growth hormone polyadenylation sequence (BGH pA) (SEQ ID NO: 100), and - human gastrin terminator (hGT) (SEQ ID NO: 101).

[0328] In addition to the HAI-1 expression cassette, the vectors each contained the following: - the origin of replication from the vector pUC18, allowing replication of this plasmid in E. coli, and -The beta-lactamase gene that confers ampicillin resistance to E. coli.

[0329] Front Vector The front vector having the expression cassette configuration L3-KLL-LoxFas (including the Cre-recombinase site) described in Example 1 a) was used.

[0330] Back Vector The complete expression cassette for HAI-1 was obtained by chemically synthesizing a DNA fragment encoding HAI-1 and introducing it into a shuttle plasmid using appropriate restriction sites. The expression cassettes for HAI-1 were excised from their shuttle plasmids to provide them with appropriate cohesive ends.

[0331] The final back vector was generated by combining one DNA fragment carrying the expression cassette for H (see Example 1a), one fragment carrying the expression cassette for L (see Example 1a), and one fragment carrying the expression cassette for HAI-1 with a fourth fragment carrying the vector backbone elements in a four-way ligation reaction.

[0332] In this way, two different back vectors were generated that expressed HAI-1 under the control of either the CMV promoter or the SV40 enhancer and early promoter. The expression cassette configuration (including the Cre recombinase site) for both final back vectors was LoxFas-HL-HAI-1-2L.

[0333] In addition to the expression cassettes for H, L and HAI-1, the back vector contained the following: a sequence encoding puromycin acetyltransferase lacking an initiation codon (SEQ ID NO: 02), - the origin of replication from the vector pUC18, allowing replication of this plasmid in E. coli, and -The beta-lactamase gene that confers ampicillin resistance to E. coli.

[0334] c) BPTI expression construct To evaluate the effect of the protease inhibitor BPTI (aprotinin) on the expression of the bispecific antibody described in Example 1a), a vector was generated for the additional expression of BPTI (aprotinin) from a separate expression cassette. The expression cassette configuration (including Cre recombinase sites) after targeted stable integration into the host genome was: L3-KKLL-LoxFas-KLH-BPTI-2L.

[0335] Expression cassette The same immunoglobulin expression cassette configuration was used as described in Example 1 a).

[0336] For expression of BPTI (aprotinin), a transcription unit containing the following functional elements was used: - Simian virus 40 (SV40) enhancer and early promoter (SEQ ID NO: 102), - signal peptide (SEQ ID NO: 175) coding sequence (SEQ ID NO: 176), - a nucleic acid encoding BPTI as a propeptide (SEQ ID NO: 177), - bovine growth hormone polyadenylation sequence (BGH pA) (SEQ ID NO: 100), and - human gastrin terminator (hGT) (SEQ ID NO: 101).

[0337] In addition to the expression cassette, the vector contained the following: - the origin of replication from the vector pUC18, allowing replication of this plasmid in E. coli, and -The beta-lactamase gene that confers ampicillin resistance to E. coli.

[0338] Front Vector The front vector having the expression cassette configuration L3-KKLL-LoxFas (including the Cre-recombinase site) described in Example 1 a) was used.

[0339] Back Vector The complete expression cassette for BPTI (aprotinin) was obtained by chemically synthesizing a DNA fragment encoding BPTI (aprotinin) and introducing it into the shuttle plasmid using the appropriate restriction sites. The expression cassette for BPTI (aprotinin) was excised from the shuttle plasmid to provide appropriate cohesive ends.

[0340] The back vector was generated by combining one DNA fragment carrying the expression cassette for H (see Example 1a), one fragment carrying the expression cassette for L (see Example 1a), and one fragment carrying the expression cassette for BPTI (aprotinin) with a fourth fragment carrying the vector backbone elements in a four-way ligation reaction. The expression cassette configuration of the final back vector (including the Cre recombinase site) was LoxFas-KLH-BPTI-2L.

[0341] In addition to the expression cassettes for H, L and BPTI, the back vector contained the following: a sequence encoding puromycin acetyltransferase lacking an initiation codon (SEQ ID NO: 02), - the origin of replication from the vector pUC18, allowing replication of this plasmid in E. coli, and -The beta-lactamase gene that confers ampicillin resistance to E. coli.

[0342] d) Puromycin acetyltransferase-T2A-BPTI fusion expression construct To evaluate the effect of the protease inhibitor BPTI (aprotinin) on the expression of the bispecific antibody described in Example 1a), a vector was generated to further express the BPTI amino acid sequence fused to the C-terminus of the puromycin-N-acetyltransferase amino acid sequence by a self-cleavable T2A peptide linker. The expression cassette configuration (including a Cre recombinase site) after targeted stable integration into the host genome was as follows: L3-KKLL-LoxFas-KLH-puroT2A-BPTI-2L.

[0343] Expression cassette The same immunoglobulin expression cassette configuration was used as described in Example 1 a).

[0344] Front Vector The front vector having the expression cassette configuration L3-KKKLL-LoxFas (including the Cre-recombinase site) described in Example 1 a) was used.

[0345] Back Vector The same procedure as described in Example 1(a) was applied, except for the back vector used, which now encodes a puromycin acetyltransferase-T2A-BPTI fusion protein instead of puromycin acetyltransferase alone. As a result, the expression cassette configuration of the final back vector (including the Cre recombinase site) was LoxFas-KLH-puroT2A-BPTI-2L.

[0346] Besides the H and L expression cassettes, the back vector contained the following: - sequences (SEQ ID NO: 02-SEQ ID NO: 05-SEQ ID NO: 15-SEQ ID NO: 174-SEQ ID NO: 178) encoding puromycin acetyltransferase-GSG-T2A-GGGGS-BPTI fusion proteins (SEQ ID NO: 01-SEQ ID NO: 04-SEQ ID NO: 14-SEQ ID NO: 99-SEQ ID NO: 177) lacking a start codon, - the origin of replication from the vector pUC18, allowing replication of this plasmid in E. coli, and -The beta-lactamase gene that confers ampicillin resistance to E. coli.

[0347] Example 2 Host cell culture: a) ExpiCHO-S ExpiCHO-S cells (Thermo Fisher) were cultured according to the manufacturer's recommendations.

[0348] b) Expi293F Expi293F cells (Thermo Fisher) were cultured according to the manufacturer's recommendations.

[0349] c) CHO-K1 TI host cells CHO-K1 TI host cells were cultured in a proprietary DMEM / F12-based medium containing 300 μg / ml hygromycin B and 4 μg / ml of a second selection marker at 37°C in a humidified incubator with 85% humidity and 5% CO2. Cells were split every 3 or 4 days at a concentration of 0.3 x 10^6 cells / ml in a total volume of 30 ml. Cultures were performed in 125 ml non-baffled Erlenmeyer shake flasks. Cells were shaken. Viable cell density was determined using a Cedex HiRes Cell Counter (Roche).

[0350] Example 3 Transfection: a) Transient transfection of ExpiCHO-S and CHO-K1 TI host cells For transient transfection and production, expression vectors encoding single immunoglobulin subunits were used at a molar ratio of 4 (K):1 (H):2 (L). Transfection of ExpiCHO-S was performed according to the manufacturer's protocol for the ExpiFectamine™ CHO Transfection Kit, prototype (A29128). CHO-K1 TI host cells were transfected using a MaxCyte STX electroporation device (MaxCyte Inc., Gaithersburg) with an OC-400 electroporation cassette according to the manufacturer's protocol. A total of 30 μg of nucleic acid was transfected into 3 x 10^7 cells. After transfection, cells were seeded in 30 ml of medium.

[0351] b) Stable transfection of CHO-K1 TI host cells by targeted integration For stable transfection, equimolar amounts of front and back vectors were mixed. The total DNA used per transfection was 30 μg with a plasmid ratio of 2.5:2.5:1 (front-, back-, Cre-vector).

[0352] Two days prior to transfection, CHO-K1 TI host cells were seeded in fresh medium at a density of 4 x 10^5 cells / ml. Transfections were performed using a MaxCyte STX electroporation device (MaxCyte Inc., Gaithersburg) with an OC-400 electroporation cassette according to the manufacturer's protocol. 3 x 10^7 cells were transfected with a total of 30 μg of nucleic acid: either 30 μg of plasmid or 5 μg of Cre mRNA and 25 μg of a front and back vector mix. After transfection, cells were seeded in 30 ml of medium without selection agent.

[0353] Five days after seeding, the cells were centrifuged and transferred to 80 mL of chemically defined medium containing puromycin (selection agent 1) and 1-(2'-deoxy-2'-fluoro-1-beta-D-arabinofuranosyl-5-iodo)uracil (FIAU; selection agent 2) at effective concentrations at a concentration of 6 x 10^5 cells / mL for selection of stable recombinant cells. The cells were incubated at 37°C, 150 rpm, and maintained at 5% CO2 and 85% humidity without splitting from this day onward. The cell density and viability of the cultures were monitored periodically. When the viability of the cultures began to increase again, the concentrations of selection agents 1 and 2 were reduced to approximately half of the amounts previously used. Therefore, 4 x 10^5 cells / mL were centrifuged and resuspended in 40 mL of selection medium II (chemically defined medium, 1 / 2 selection markers 1 and 2). These cells were incubated under the same conditions as before, again without splitting.

[0354] 14–21 days after the start of selection, survival rates exceeded 90% and selection was considered complete.

[0355] Example 4 IgG-like protein production a) Transient production of IgG-like proteins in ExpiCHO-S or CHO-K1 TI host cells After 7 days, the cell supernatants of the transiently transfected cells were harvested by centrifugation and subsequent filtration (0.2 μm filter), and proteins were purified from the harvested supernatants by standard methods as follows.

[0356] b) Transient production of IgG-like proteins in Expi293F cells Cells were seeded at a density of 2.5 x 10^6 / ml in Expi293™ medium (Gibco, catalog number 1435101). The expression vector and ExpiFectamine (Gibco, ExpiFectamine™ Transfection Kit, catalog number 13385544) were mixed separately in OptiMEM™ Reduced Serum Medium (Gibco, catalog number 11520386). After a few minutes, both solutions were combined, mixed by pipetting, and further incubated at room temperature. Cells were added to the expression vector / ExpiFectamine solution and incubated for 24 hours at 37°C in a shaking incubator with a 5% CO2 atmosphere. One day after transfection, supplements (Transfection Enhancer 1 and 2, ExpiFectamine™ Transfection Kit) were added. After 4-5 days, the cell supernatant was collected by centrifugation and subsequent filtration (0.2 μm filter), and the protein was purified from the collected supernatant by standard methods as follows.

[0357] c) Stable production of IgG-like proteins in CHO-K1 cells Fed-batch culture Fed-batch production cultures were performed in shake flasks containing proprietary chemically defined medium. Cells were seeded at 2 × 10^6 cells / ml. Cultures received proprietary feed medium on days 1, 3, and 6. Viable cell counts (VCC) and percent viability of cells in the cultures were measured on days 0, 3, 7, 10, 12, and 14 using a Cedex HiRes (Roche Diagnostics GmbH, Mannheim, Germany). Product titers were measured on days 3, 5, 7, 10, 12, and 14 using a Cobas analyzer (Roche Diagnostics GmbH, Mannheim, Germany). On days 10, 12, or 14 after starting the fed-batch culture, supernatants were harvested by centrifugation (10 min, 1000 rpm, followed by 10 min, 4000 rpm) and clarified by filtration (0.22 μm). Harvest titers were determined using protein A affinity chromatography with UV detection. Product quality was determined by Caliper's LabChip (Caliper Life Sciences).

[0358] High cell density fed-batch culture High-cell-density fed-batch production cultures were performed in Ambr 250 vessels (Sartorius Stedim) containing proprietary chemically defined medium. Cells were seeded at 15 x 10^6 cells / ml on day 0. Cultures received proprietary feed medium on days 1, 3, and 6. Viable cell counts (VCC) and percent viability of cells in cultures were measured on days 0, 3, 7, 10, 12, and 14 using a Cedex HiRes instrument (Roche Diagnostics GmbH, Mannheim, Germany). Product titers were measured on days 3, 5, 7, 10, 12, and 14 using a Cobas Analyzer (Roche Diagnostics GmbH, Mannheim, Germany). On days 10, 12, or 14 after culture initiation, supernatants were harvested by centrifugation (10 min, 1000 rpm, followed by 10 min, 4000 rpm) and clarified by filtration (0.22 μm). Harvest titers were determined using Protein A affinity chromatography with UV detection. Product quality was determined by Caliper's LabChip (Caliper Life Sciences).

[0359] Example 5 Protein purification: Recombinant immunoglobulin-like proteins were purified from cell culture supernatants by affinity chromatography using MabSelectSure-Sepharose™ (GE Healthcare, Sweden). Sterile-filtered cell culture supernatants were 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 equilibration buffer, eluted with 25 mM citrate buffer, pH 3.0, and neutralized with 1 M Tris pH 9.

[0360] Example 6 Analysis method a) Titer determination The concentration of recombinant immunoglobulin in cell culture supernatant was quantitatively measured by affinity HPLC chromatography. Briefly, centrifuged and sterile-filtered cell culture supernatant was applied to a Poros A / 20 column (Applied Biosystems) in 200 mM KH2PO4, pH 7.4, 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.

[0361] b) CE-SDS The purity and integrity of the products were analyzed by CE-SDS under reducing and non-reducing conditions using microfluidic Labchip technology (PerkinElmer, USA). For this purpose, 5 μl of 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 the HT Protein Express Chip. Data were analyzed using LabChip GX Software.

[0362] c)SEC Size exclusion chromatography (SEC) to determine the aggregation and oligomeric state of recombinant immunoglobulins was performed by HPLC chromatography. Briefly, Protein A purified products were applied to a TSKgel QC-PAK GFC 300 column (Tosoh Bioscience) or a Tosoh TSKgel UP-SW3000 column in 250 mM KCl, 200 mM KHPO / KHPO buffer (pH 6.2) on a Dionex Ultimate® HPLC system (Thermo Fisher Scientific). Eluted antibodies were quantified by UV absorbance and peak area integration. BioRad Gel Filtration Standard #151-1901 served as the gel filtration calibration standard.

Claims

1. The following elements, in operably linked form: a) a nucleic acid encoding a selectable marker; b) a nucleic acid encoding a self-cleaving peptide sequence, and c) Nucleic acids encoding proteinaceous protease inhibitors A nucleic acid comprising:

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

3. The following elements, in 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); The nucleic acid of claim 1 or 2, further comprising:

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

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

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 ribosomal skipping.

7. - 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, which encodes the 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 that is capable of ribosomal skipping; The nucleic acid according to any one of claims 1 to 6.

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

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

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

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

12. The cell of claim 11 , further comprising one or more nucleic acid sequences encoding a heterologous polypeptide.

13. The cell of 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, which is a CHO-K1 cell.

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

16. 16. The method of claim 15, wherein the amount of recovered uncleaved heterologous polypeptide is increased compared to a method using a nucleic acid-free cell according to any one of claims 1 to 10.

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

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