SE544006C2

SE544006C2 - Novel combination of tis sequence and signal peptide sequence for expressing a recombinant protein

Info

Publication number: SE544006C2
Application number: SE2030082A
Authority: SE
Inventors: David Vikström; Joanne Stevenson; Kiavash Mirzadeh; Nathalie Chatzissavidou; Patrik Samuelson; Per Edebrink; Siavash Bashiri; Veronica Ramberg
Original assignee: Xbrane Biopharma Ab
Priority date: 2020-03-17
Filing date: 2020-03-17
Publication date: 2021-11-02
Also published as: SE2030082A1

Abstract

The present invention relates to DNA constructs comprising a novel TIS sequence. The TIS sequence transcribes into an RNA motif that functions as the protein translation initiation site in an mRNA transcript. The DNA construct may further comprise a nucleic acid sequence encoding signal peptide. Additionally, the DNA constructs may also comprise a nucleic acid sequence encoding a recombinant protein or one or more polypeptide chains thereof. The invention further relates to an expression vector, expression cassette and host cell which comprise said DNA constructs. Furthermore, the present invention relates to a recombinant protein expressed by the host cell as well as a method for expressing the recombinant protein.

Description

NOVEL COMBINATION OF TIS SEQUENCE AND SIGNAL PEPTIDE SEQUENCE FOREXPRESSING A RECOMBINANT PROTEIN TECHNICAL FIELD The present invention relates to DNA constructs comprising a novel TIS sequence. The DNAconstruct may also comprise a nucleic acid sequence encoding signal peptide. Additionally,the DNA constructs may also comprise a nucleic acid sequence encoding a recombinantprotein or one or more polypeptide chains thereof. The invention further relates to aneXpression vector, expression cassette and host cell Which comprise said DNA constructs.Furthermore, the present invention relates to a recombinant protein expressed by the host cell as Well as a method for eXpressing the recombinant protein.

BACKGROUND OF INVENTION The maj or determinants for the steady state protein concentration levels in recombinant celllines involve (l) the genomic integration site of the eXpression cassette, (2) promoter strength,(3) translation efficiency of the transcript and (4) protein folding and post translationalmodifications affecting degradation rates. For generation of recombinant Chinese hamsterovary (CHO) cell lines, often random integration sites that facilitate high eXpression levels arescreened through. The eXpression vector itself also contains regulatory elements such as apromoter sequence that yields high transcription and a translation initiation site (TIS) thatenables proper translation initiation [l]. Frustratingly, even though all regulatory elementsthat ought to facilitate high eXpression levels are selected for, protein yields can differ in acontext dependent manner. A solution that Would guide in the rational design When generating a recombinant cell line is therefore highly sought-after.

Since protein synthesis uses up a large fraction of the cell°s energy budget [2], evolution hastightly regulated the different phases of protein production. The eukaryotic translationinitiation phase requires tWelve initiation factors and is believed to be the rate-limiting stepduring protein synthesis [3]. This initiation phase has most likely become tightly regulatedduring evolution to minimize energy consumption. The major element regulating theefficiency of translation initiation is embedded in the mRNA nucleotide sequence surrounding the ATG start codon. This nucleotide bias Within the TIS Was discovered in the 1980”s and is commonly known as the Kozak sequence [4]. Ribosomal structural studies have since thenelucidated how the TIS sequence interacts with the ribosome, imposing conformationalchanges for translation initiation to occur [5]. Due to their biological impact, TIS sequenceswith different strengths have naturally evolved as indicators for true initiation sites and for fine tuning eXpression levels [6].

Usually, a strong naturally occurring TIS sequence is selected for when engineering theeXpression vector for recombinant production. Among other TIS variants, the mammalianconsensus sequence GCCACC preceding the ATG start codon [7] is routinely introducedduring ad hoc construct design. This sequence is prevalent for naturally found highlyexpressed genes, likely resulting in enhanced fitness of the organism by fine tuning translationinitiation rates for abundant transcripts. In contrast to natural evolution, fed-batchrecombinant eXpression conditions alter the proteome and impose a metabolic burden on cellsthat by no means mimic natural circumstances [8]. Consequently, recent studies have focusedon eXperimentally screening through TIS libraries in order to identify common sequence features that lead to optimal translation rates for recombinant eXperiments [9, l0].

However, prior art does not suggest one unique universal sequence that could be reliablyused. Consequently, there is a need for TIS sequences that could provide better technical effects than the GCCACCATG sequence.

Moreover, mammalian cells are commonly used as hosts for production of recombinantbiopharmaceuticals. Cell lines derived from Chinese hamster ovary (CHO) cells can routinelyallow for production yields in the g/L range. However, despite advances in the field,eXpression levels can vary in an unpredictable and context dependent manner, limiting therational design for obtaining a desired expression level during cell line development. In orderto solve this problem, the translation initiation site (TIS) has been altered in the present invention with the aim of ultimately inﬂuencing the titer and productivity of antibodies.

THE OBJ ECT OF THE INVETIONThe object of the invention is to increase translation initiation rates.

A further object of the invention is to increase translation initiation rates without effecting protein quality.

A further object of the invention is to increase translation initiation rates Whilst maintaining protein biosimilarity.

A further object of the invention is to increase translation initiation rates Whilst maintaining post translational modifications.

A further object of the invention is to increase translation initiation rates Whilst maintaining glycan-based post translational modifications.

A further object of the invention is to increase translation initiation rates Whilst maintaining acidic and basic distribution of species.

A further object of the invention is to increase expression of recombinant proteins.

A further object of the invention is to increase titer of recombinant proteins.

A further object of the invention is to increase titer of antibodies or fragments thereof.

A further object of the invention is to increase titer of monoclonal antibodies.

SUMMARY OF INVENTION The objects of the invention are attained by the subject-matter disclosed in the claims as Well as the subject-matter disclosed in the below aspects of the invention.

A first aspect of the invention relates to a DNA construct suitable for expressing arecombinant protein in mammalian cells, Wherein the DNA construct comprises a nucleic acid sequence of SEQ ID No l, Wherein the nucleic acid sequence of SEQ ID No l is a TIS SCqUCnCC .

In a preferred embodiment, the nucleic acid sequence of SEQ ID No l comprises:- 6 nucleotides upstream of an ATG start codon; and/or - 2 nucleotides doWnstream of an ATG start codon.

In a preferred embodiment, the DNA construct further comprises a nucleic acid sequence Which encodes a signal peptide.

In a preferred embodiment, the DNA construct further comprises a nucleic acid sequence Which encodes a recombinant protein or one or more polypeptide chains thereof.

A second aspect of the invention to relates to a DNA construct for eXpressing a recombinantprotein in mammalian cells, Wherein the DNA construct comprises:- a nucleic acid sequence of SEQ ID No l, Wherein the nucleic acid sequence of SEQID l is a TIS sequence; and - a nucleic acid sequence Which encodes a signal peptide.

In a preferred embodiment, the nucleic acid sequence Which encodes a signal peptide comprises a nucleic acid sequence of SEQ ID No 2.

In a preferred embodiment, the nucleic acid sequence of SEQ ID No l comprises:- the ATG start codon in the nucleic acid sequence Which encodes the first amino acidresidue of the signal peptide; and- the first two nucleotides doWnstream of the ATG start codon in the nucleic acid sequence Which encodes the second amino acid residue of the signal peptide.

In a preferred embodiment, the nucleic acid sequence of SEQ ID No l comprises:- the ATG start codon in SEQ ID No 2; and- the first two nucleotides doWnstream of the ATG start codon in the nucleic acid sequence of SEQ ID No 2.

A third aspect of the invention to relates to a DNA construct for eXpressing a recombinantprotein in mammalian cells, Wherein the DNA construct comprises:- a nucleic acid sequence of SEQ ID No l, Wherein the nucleic acid sequence of SEQID l is a TIS sequence;- a nucleic acid sequence Which encodes a signal peptide; and- a nucleic acid sequence Which encodes for a recombinant protein, preferably a monoclonal antibody, more preferably an IgG4 monoclonal antibody, In a preferred embodiment, the nucleic acid sequence of SEQ ID No l comprises:- 6 nucleotides upstream of an ATG start codon; and/or - 2 nucleotides doWnstream of an ATG start codon.

In a preferred embodiment, the nucleic acid sequence Which encodes the signal peptide is operably linked to the nucleic acid sequence Which encodes for the recombinant protein.

A fourth aspect of the invention relates to a DNA construct for eXpressing a recombinantprotein in mammalian cells, Wherein the DNA construct comprises:- a first and second nucleic acid sequences each comprising a nucleic acid sequence ofSEQ ID No l, Wherein the nucleic acid sequence of SEQ ID l is a TIS sequence;- a first nucleic acid sequence Which encodes a signal peptide;- a second nucleic acid sequence Which encodes a signal peptide;- a first nucleic acid sequence Which encodes a heavy chain of an antibody; and - a second nucleic acid sequence Which encodes a light chain of an antibody.

In a preferred embodiment, a nucleic acid sequence of SEQ ID No l comprises:- 6 nucleotides upstream of an ATG start codon; and/or - 2 nucleotides doWnstream of an ATG start codon.

In a preferred embodiment, a nucleic acid sequence Which encodes a signal peptide comprises a nucleic acid sequence of SEQ ID No 2.

In a preferred embodiment, the first and second nucleic acid sequences Which encode a signal peptide each comprise a nucleic acid sequence of SEQ ID No 2.

In a preferred embodiment, the first and second nucleic acid sequences each comprising anucleic acid sequence of SEQ ID No l comprise:- the ATG start codon in the first and second nucleic acid sequences Which encode thefirst amino acid residue of the signal peptide; and- the first two nucleotides doWnstream of the ATG start codon in the first and secondnucleic acid sequences Which encode the second amino acid residue of the signal peptide.

In a preferred embodiment, the first and second nucleic acid sequences comprising a nucleicacid sequence of SEQ ID No l comprise:- the ATG start codon in SEQ ID No 2 of the first and second nucleic acid sequencesWhich encode a signal peptide, and- the first two nucleotides doWnstream of the ATG start codon in SEQ ID No 2 of the first and second nucleic acid sequences Which encode a signal peptide.

In a preferred embodiment, the first nucleic acid sequence Which encodes a signal peptide isoperably linked to the first nucleic acid sequence Which encodes the heavy chain of an antibody.

In a preferred embodiment, the second nucleic acid sequence Which encodes a signal peptideis operably linked to the second nucleic acid sequence Which encodes the light chain of an antibody.

In a preferred embodiment, the first nucleic acid sequence Which encodes the heavy chain of an antibody encodes an amino acid sequence of SEQ ID No 5.

In a preferred embodiment, the second nucleic acid sequence Which encodes the light chain of an antibody encodes an amino acid sequence of SEQ ID No 7.

In a preferred embodiment, the first and second nucleic acid sequences Which encode for theheavy and light chains of an antibody, respectively, encode an amino acid sequence of SEQ ID No 5 and SEQ ID No 7, respectively.

In a preferred embodiment, the first nucleic acid sequence Which encodes the heavy chain of an antibody comprises a sequence of SEQ ID No 4.

In a preferred embodiment, the second nucleic acid sequence Which encodes for the light chain of an antibody comprises a sequence of SEQ ID No 6.

In a preferred embodiment, the first and second nucleic acid sequence Which encode theheavy and light chains of an antibody, respectively, comprise a sequence of SEQ ID No 4 andSEQ ID No 6, respectively.

In a preferred embodiment, the DNA construct comprises nucleic acid sequences of SEQ IDNo 8 and SEQ ID No 9. The resulting heavy and light chains of an antibody may eachcomprise a cleaved or an uncleaved signal peptide. In the case When the signal peptides arenot cleaved, each of said heavy and light chains having an uncleaved signal peptide comprises an amino acid sequence of SEQ ID No l0 and SEQ ID Noll, respectively.

In a preferred embodiment, the DNA construct comprises nucleic acid sequences of SEQ IDNo 12 and SEQ ID No l3. The resulting heavy and light chains of an antibody may eachcomprise a cleaved or an uncleaved signal peptide. In the case When the signal peptides arenot cleaved, each of said heavy and light chains having an uncleaved signal peptide comprises an amino acid sequence of SEQ ID No l0 and SEQ ID Noll, respectively.

A fifth aspect of the invention relates to a DNA construct comprising a nucleic acid sequenceencoding an an1ino acid sequence, Wherein the amino acid sequence comprises:- the amino acid sequence for Nivolumab (Opdivo, CAS number 946414-94-4);- a signal peptide of SEQ ID No 3 fused to the heavy chain amino acid sequence ofNivolumab; and- a signal peptide of SEQ ID NO 3 fused to the light chain amino acid sequence of Nivolumab.

In a preferred embodiment, the heavy chain amino acid sequence of Nivolumab comprises a sequence of SEQ ID No 5.

In a preferred embodiment, the light chain amino acid sequence of Nivolumab comprises a sequence of SEQ ID No 7.

In a siXth aspect of the invention relates to an eXpression vector Which comprises the DNA construct according to the any one of the first to fifth aspects of the invention.

In a preferred embodiment, the eXpression vector comprises one or more of the following nucleic acid elements: - promoter, - terrninator, - selection marker, - origin of replication, and/or - antibiotic resistance marker,Wherein the eXpression Vector further comprises at least one multiple cloning site cleavable bya restriction enzyme, preferably the restriction enzyme is EcoRI, Ndel, Notl, Xhol, PspXl,PaeR7l, Bbsl, Styl, Avrll, Banl, Acc65I, Kpnl, Eco53kl, Sacl, BamHl, Xbal, Sall, Accl,Pstl, Sbfl, Sphl or Hindlll.

In a preferred embodiment, the selection marker comprises the gene encoding dihydrofolate reductase, DHFR, or glutamine synthetase, GS.

A seventh aspect of the invention relates to an eXpression cassette Which comprises the DNA construct according to the any one of the first to fifth aspects of the invention.

An eighth aspect of the invention relates to a host cell Which comprises a DNA constructaccording to any one of the first to fifth aspects of the invention, Wherein said host cell is preferable and eukaryotic cell, more preferably a mammalian cell.In a preferred embodiment, the host cell is a Chinese hamster ovary, CHO, cell.

In a preferred embodiment, the host cell is a CHO-DG44 cell or a CHO GS* cell.

A ninth aspect of the invention relates to a recombinant protein expressed by a DNA constructaccording to any one of the first to fifth aspects of the invention. The recombinant protein may comprise one or more polypeptide chains.

In a preferred embodiment, the recombinant protein is an antibody, antibody fragment, enzyme and hormone.

In a preferred embodiment, the recombinant protein is monoclonal antibody, polyclonalantibody, chimeric antibody or a fragment of said monoclonal antibody, polyclonal antibody, chimeric antibody.

In a preferred embodiment, the recombinant protein is Nivolumab. Nivolumab is a humanIgG4 monoclonal antibody. It is a therapeutic antibody Which is sold under the brand nameOpdivo and is a medication used to treat a number of types of cancer. This includesmelanoma, lung cancer, renal cell carcinoma, Hodgkin lymphoma, head and neck cancer, colon cancer, and liver cancer. It is typically used by slow injection into a vein.

A tenth aspect of the invention relates to RNA expressed by the DNA construct according to any one of the first to fifth aspects of the invention.

An eleventh aspect of the invention relates to a method of eXpressing a recombinant protein,comprising the steps of: a. cloning one or more open reading frames encoding a recombinant protein, or one ormore polypeptide chains thereof, into one or more DNA constructs according to anyone of the first to third aspects of the invention; and b. transfecting the resulting nucleic acid sequences into a host cell, Wherein the host cell is preferably a eukaryotic cell, more preferably a mammalian cell.

In a preferred embodiment, the method further comprises the step of integrating the transfected nucleic acid sequence into the genome of the host cell.

A thirteenth aspect of the invention relates to a method of eXpressing a recombinant protein,comprising the steps of:a. cloning a DNA construct according to the fourth or fifth aspects of the invention; andb. transfecting the resulting nucleic acid sequences into a host cell, Wherein the host cell is preferably a eukaryotic cell, more preferably a mammalian cell.

A fourteenth aspect of the invention relates to a DNA construct for eXpressing a signalpeptide in any type of host cell (including prokaryotic cell, eukaryotic cell and/or yeast cell), Wherein the DNA construct comprises a nucleic acid sequence Which encodes a signal peptide, Wherein the nucleic acid sequence Which encodes a signal peptide comprises anucleic acid sequence of SEQ ID No 2. The nucleic acid sequence of SEQ ID No 2 encodes asignal peptide comprising an amino acid sequence of SEQ ID No 3. Further aspects relate to an eXpression vector, eXpression cassette and host cell Which comprise said DNA construct.

BRIEF DESCRIPTION OF THE DRAWINGS Figure l - Increased likelihood of identifying high antibody producing cell cultures for mini - pools containing a TISEVO containing a TISCON (black) or TISEVO (halloW). - chart represents titer values (g/L) on harvest day for mini-pools Figure 2 - Increased likelihood of identifying high antibody producing cell cultures for mini - pools containing a TISEVO day for rnini-pools containing a TISCON (black) or TISEVO (halloW). - chart represents mean productivity, mean Qp, (pg/c/d) on harvest Figure 3 - Titer and specific productivity comparison of monoclonal antibody (mAb)eXpressing CHO-DG44 cell lines - Final accumulated titer values (g/L) of cell lines harboringa TISCON (black dots) or TISEVO (holloW dots).

Figure 4 - Titer and specific productivity comparison of mAb eXpressing CHO-DG44 cell lines - Final accumulated specific productivity, Qp, (pg/c/d) of cell lines harboring a TISCON (black dots) or TISEVO (holloW dots).

Figure 5 - Glycan profile of commercially obtained Nivolumab (i.e. these samples have been produced Without the use of TISCON and TISEVO).Figure 6 - Glycan profile of Nivolumab variants derived from TISCON containing mini pools.Figure 7 - Glycan profile of Nivolumab variants derived from TISEVO containing mini pools.

Figure 8 - Charge distribution of Nivolumab variants derived from TISCON mini pools. The originator is labeled AAX24l4.

Figure 9 - Charge distribution of Nivolumab variants derived from TISEVO mini pools. The originator is labeled AAX24l4.

DETAILED DESCRIPTION In the art, a TIS sequence is sometimes referred to as the RNA sequence that functions as theprotein translation initiation site (TIS) in an mRNA transcript [9]. However, in the presentinvention, the term TIS sequence instead refers to the DNA sequence Which transcribes into said RNA sequence.

Since prior art studies do not suggest one unique universal sequence that could be reliablyused, the inventor has designed a novel nucleic acid TIS sequence (herein referred to asTISEVO) that provides better technical effect than the prior art GCCACCTGA sequence(herein labelled TISCON) in a recombinant eXpression system for producing an antibody in mammalian cells.

The novel TIS sequence herein referred to as TISEVO TCGGTCATGGC Which is also referred to as SEQ ID No l in the present invention. comprises the nucleic acid sequence of In the present invention, the term nucleic acid means at least two nucleotides covalentlylinked together. Moreover, the disclosure of a single strand also discloses the sequence of thecomplementary strand. Thus, a nucleic acid sequence also encompasses the complementary strand of a disclosed single strand.

The nucleic acid sequence of SEQ ID No l is comprised in a DNA construct Which is suitablefor eXpressing a recombinant protein in mammalian cells. The TISEVO nucleic acid sequenceof SEQ ID No l comprises: - 6 nucleotides (immediately) upstream of the ATG start codon (i.e. comprisesnucleotides from position -6 to -l in Which the A of the ATG start codon is position+l), as Well as - 2 nucleotides (immediately) doWnstream of the ATG start codon (i.e. comprisesnucleotides from position +4 to +5 in Which the G of the ATG start codon is position+3).

In the present invention, the term DNA construct means refers to an artificially constructedsegment of nucleic acid that is to be inserted into a host cell (e. g. via using an eXpression vector or eXpression cassette).

The TISEVO nucleic acid sequence transcribes into an RNA sequence of UCGGUCAUGGC (herein also referred to as SEQ ID No l4) that functions as the protein translation initiation site in an mRNA transcript. In other Words, the TISEVO nucleic sequence is a Kozak-like SCqUCnCC .

The above mentioned DNA construct may further comprise a nucleic acid sequence whichencodes a signal peptide. The nucleic acid sequence of SEQ ID No 1 may in such a DNAconstruct comprise: - the ATG start codon in the nucleic acid sequence which encodes the first amino acid residue of the signal peptide; and- the first two nucleotides downstream of the ATG start codon in the nucleic acid sequence which encodes the second amino acid residue of the signal peptide.

In the present invention, the term signal peptide refers to a leader peptide which is fused to theN-terrninus of the recombinant protein to be expressed. Signal peptides facilitate secretion ofthe recombinant protein from the host cell in which it is produced. Signal peptides are typically cleaved from the remainder of the upon secretion from the cell.

In an embodiment of the invention, the nucleic acid sequence which encodes the signalpeptide according to the present invention comprises a modification of the nucleic acidsequence which eXpresses a signal peptide having methionine (M) as the first amino acid atthe N-terrninus end of the signal peptide. Such signals peptides may be selected from as theones described in Kober et al [l1], Haryadi R. et al [12], US10066019, Ramezani A. et al[15] and Peng L. et al [16] which all relate to use of signal peptides for eXpression ofrecombinant proteins in eukaryotic host cells. The modification according to the presentinvention involves a change of the first codon downstream of the ATG start codon byeXchanging the first two nucleotides to GC. The resulting signal peptide will comprise MA as the first two an1ino acids at the N-terminus end of the signal peptide.

In an embodiment of the invention, the nucleic acid sequence which encodes the signalpeptide comprises a nucleic acid sequence of SEQ ID No 2. The nucleic acid sequence ofSEQ ID No 1 will in such a DNA construct comprise: - the ATG start codon in SEQ ID No 2; and - the first two nucleotides downstream of the ATG start codon in the nucleic acid sequence of SEQ ID No 2.

The DNA construct may further comprise a nucleic acid sequence which encodes arecombinant protein. In such as DNA construct, the nucleic acid sequence which encodes the signal peptide may be operably linked to the nucleic acid sequence which encodes the recombinant protein. The recombinant protein may be an antibody, a multimeric protein, amonomeric protein, enzyme and/or hormone. However, other recombinant proteins may also be envisaged.

The term antibody is in the present invention referred to a monoclonal antibody, polyclonalantibody, chimeric antibody or a fragment thereof. Such a fragment of an antibody (hereinalso refer to as antibody fragment) is a portion of an intact antibody comprising the antigen-binding site or Variable region. An antibody fragment may be a Fab fragment, Fab' fragment,Fab'-SH fragment, F(ab')2 fragment, Fd fragment, Fv fragment, diabody, triabody and/or single-chain FV (scFv) molecule.

In an embodiment of the invention, the DNA construct comprises: - two nucleic acid sequence of SEQ ID No l; - two nucleic acid sequences which encode signal peptides; and - a recombinant protein;wherein the nucleic acid sequences which encode the signal peptides may be the same ordifferent sequences, i.e. the signal peptides may be the same or different signal peptides. Theconstruction of vectors which express signal peptides which are same or different are knownin the art as described in Kober et al [l l], Haryadi R. et al [12] and Li F. et al [l3]. Therecombinant protein is preferably an antibody. Moreover, each of the nucleic acid sequenceswhich encode signal peptide is operably linked to two nucleic acid sequences (such as nucleic acid sequences encoding the heavy and light chains) of the recombinant proteins.

In an embodiment of the invention, the DNA construct comprises:- a first and second nucleic acid sequences each comprising a nucleic acid sequence ofSEQ ID No l, - a first nucleic acid sequence which encodes a signal peptide; - a second nucleic acid sequence which encodes a signal peptide; - a first nucleic acid sequence which encodes a heavy chain of an antibody; and - a second nucleic acid sequence which encodes a light chain of an antibody.

In such as DNA construct, the first and second nucleic acid sequences of SEQ ID No lcomprise:- the ATG start codon in the first and second nucleic acid sequences which encode the signal peptides; and - the first two nucleotides doWnstream of the ATG start codon in the first and second nucleic acid sequences Which encode the signal peptides.

The first nucleic acid sequence Which encodes a signal peptide is operably linked to the firstnucleic acid sequence Which encodes the heavy chain of an antibody. Similarly, the secondnucleic acid sequence Which encodes a signal peptide is operably linked to the second nucleic acid sequence Which encodes the light chain of an antibody.

In an embodiment of the invention, the DNA construct of the first and second nucleic acidsequences Which encode the signal peptides may each comprise a nucleic acid sequence ofSEQ ID No 2. In such a DNA construct, the first and second nucleic acid sequences of SEQID No l comprise: - the ATG start codon in SEQ ID No 2 of the first and second nucleic acid sequences Which encode the signal peptides,- the first two nucleotides doWnstream of the ATG start codon in SEQ ID No 2 of the first and second nucleic acid sequences Which encode the signal peptides.

The first nucleic acid sequence Which encodes the heavy chain of an antibody may encode anan1ino acid sequence of SEQ ID No 5. The second nucleic acid sequence Which encodes the light chain of an antibody may encode an amino acid sequence of SEQ ID No 7.

The first nucleic acid sequence Which encodes the heavy chain of an antibody may comprise anucleic acid sequence of SEQ ID No 4. The second nucleic acid sequence Which encodes for the light chain of an antibody may comprise a nucleic acid sequence of SEQ ID No 6.

The DNA construct may comprise a nucleic acid sequence of SEQ ID No 8 or SEQ ID No l2Wherein said sequence comprises:- a first nucleic acid sequence comprising a nucleic acid sequence of SEQ ID No l;- a first nucleic acid sequence Which encodes a signal peptide comprising a nucleic acidsequence of SEQ ID No 2; and- a first nucleic acid sequence Which encodes a heavy chain of an antibody comprising anucleic acid sequence of SEQ ID No 4;Wherein the first nucleic acid sequences of SEQ ID No l comprises:- the ATG start codon in SEQ ID No 2 of the first nucleic acid sequence Which encodesa signal peptide; and - the first two nucleotides doWnstream of said ATG start codon.

The DNA construct may comprise a nucleic acid sequence of SEQ ID No 9 or SEQ ID No 13wherein said sequence comprises:- a second nucleic acid sequence comprising a nucleic acid sequence of SEQ ID No 1;- a second nucleic acid sequence which encodes a signal peptide comprising a nucleicacid sequence of SEQ ID No 2; and- a second nucleic acid sequence which encodes a light chain of an antibody comprisinga nucleic acid sequence of SEQ ID No 6,wherein the second nucleic acid sequences of SEQ ID No 1 comprises:- the ATG start codon in SEQ ID No 2 of the second nucleic acid sequence whichencodes a signal peptide; and - the first two nucleotides downstream of said ATG start codon.

The DNA construct may comprise both of nucleic acid sequences of SEQ ID No 8 and SEQID No 9. Alternatively, the DNA construct may instead comprise both of the nucleic acidsequences of SEQ ID No 12 and SEQ ID No 13. Nucleic acids of SEQ ID No 12 and 13 differfrom nucleic acids of SEQ ID No 8 and 9, respectively, only in that nucleic acid of SEQ ID No 12 and 13 each further comprise restriction sites.

The above disclosed DNA construct may be integrated into an eXpression vector suitable fortransfecting into mammalian cells. Such a Vector may comprise the following nucleic acidelements: - promoter, - terrninator, - selection marker, - origin of replication, and/or - antibiotic resistance marker.

The expression vector may further comprise at least one multiple cloning site cleavable by arestriction enzyme such as EcoRI, NdeI, NotI, XhoI, PspXI, PaeR71, BbsI, StyI, AvrII, BanI,Acc65I, KpnI, Eco53kI, SacI, BamHI, XbaI, SalI, AccI, PstI, SbfI, SphI and/or HindIII.

Expression Vector for use with mammalian cells as well as nucleic acid elements which aretypically comprised in such eXpression Vectors are described in Li F. et al [13] and Noh S.H.et al [14] and the uses of these expression Vectors in the present invention are indicated in the below described embodiments of the expression Vector.

The expression vector may include one or more promoters. The promoter may be anypromoter that is capable of driving gene expression and regulating gene expression.Preferably, the promoter may be a promoter shown effective for expression of recombinantproteins in mammalian cells such as CHO cells. ln a further preferred embodiment, thepromoter is effective for expression of recombinant proteins in CHO-DG44 cells. A specificexample of a promoter which may be used in the present invention is a cytomegalovirus (CMV) promoter and/or elongation factor alpha (EFlot) promoter.

In expression vectors having a DNA construct comprising: - a first nucleic acid sequence which encodes a heavy chain of an antibody, - a second nucleic acid sequence which encodes a light chain of an antibody,the expression vector may comprise a first promoter for expressing the first nucleic acidsequence as a first transcript and a second promoter for expressing the second nucleic acidsequence as a second transcript. The first transcript will then be translated to a heavy chainpolypeptide and the second transcript will be translated into the light chain polypeptide andthe resulting antibody will be generated by said heavy and light chain polypeptides. In such anexpression vector, the first and second promoters may be same or different in embodiments ofthe invention. In such an expression vector, a preferred embodiment involves the use of twoCMV promoters, i.e. one for each of the first and second nucleic acid sequences encoding the heavy and light chains of an antibody.

In an embodiment of the expression vector, an intron sequence in the 5' untranslated region isincluded after the promoter(s) to increase export of transcribed mRNA to the cytoplasm fromthe nucleus of the host cell; moreover, one or more 3' polyadenylation signal sequences mayalso be included in the expression vector to maximize mRNA levels. Some examples ofpolyadenylation signal sequences which may be included in the expression vector are SV40late or early polyadenylation signal sequences and the bovine growth hormone polyadenylation sequence.

In the present invention, a metabolic selection marker such as the gene encodingdihydrofolate reductase (DHFR) may be used as selection marker in an expression vectorwhich is to be transfected into CHO-DG44 cells. The DNA construct can be amplified withthe use of Methotrexate (MTX), a DHFR inhibitor.

An alternative marker is the gene encoding Glutamine synthase (GS) which may be used as selection marker in an expression vector to be transfected into CHO GS* cells. GS catalyzes the conversion of ammonia and glutamate into glutamine, and MSX inhibits the activity of the GS protein.

An origin of replication which may be used in the present invention may be selected from apUC origin, a pBR 322 origin; a pACYC origin, a pSCl0l origin and a ColEl origin.However, derivatives of these origins of replication as well as other origins of replication usedin the art may also be used. An example of an eukaryotic origin of replication which may be used is an SV40 origin of replication.

The antibiotic resistance marker comprises a gene whose product confers resistance to anantibiotic such as chloramphenicol, ampicillin, zeocin, bleomycin, gentamycin, streptomycin,tetracycline, kanamycin and neomycin. Some examples of antibiotic resistance marker used inthe expression vector are therefore a chloramphenicol resistance gene, kanamycin resistancegene, ampicillin resistance gene, zeocin resistance gene, bleomycin resistance gene,gentamycin resistance gene, gentamycin resistance gene, streptomycin resistance gene,tetracycline resistance gene and neomycin resistance gene. The use of these antibioticresistance markers in expression vectors is known in the art, see e.g. Li F. et al [13] and US8l38324.

Other antibiotic selection markers which may be used are Puromycin acetyltransferase,Blasticidin deaminase, Histidinol dehydrogenase, Hygromycin phosphotransferase, Zeocinresistance gene, Bleomycin resistance gene and Aminoglycoside phosphotransferase. Thesemarkers use Puromycin, Blasticidin, Histidinol, Hygromycin, Zeocin, Bleomycin and Neomycin (G4l8), respectively, as selective reagents.

The expression vector may also include one or more transcription terrnination regions. Thetranscription terrnination region is typically downstream of the coding sequence to provide for efficient transcription terrr1ination.

In a specific embodiment of the invention for use in the expression of heavy and light chainsof an antibody, the expression vector of the present invention comprises the following nucleicacid elements which are also indicated in chapter “Cell line development” in Li F. et al [l3]: - first and second nucleic acid sequences encoding the heavy and light chains of an antibody to be expressed;- two CMV promoters, i.e. one for each of first and second nucleic acid sequences encoding the heavy and light chains of the antibody; - an intron sequence in the 5' untranslated region is included after each of the CMVpromoters; - 3' polyadenylation signal sequence is included after each of said 5' untranslatedregions; - gene encoding selection marker DHFR; - DNA construct comprising a TIS sequence (i.e. Kozak sequence) and a signal peptidein front of (i.e. upstream) each of the first and second nucleic acid sequences encodingthe heavy and light chains of an antibody; and - antibiotic resistance marker.

The above disclosed DNA constructs may also be included in an expression cassette suitablefor transfecting into mammalian cells. Such an expression cassette may comprise thefollowing nucleic acid elements: - promoter, - terrninator, and - selection marker.

The host cell lines used in the present invention may be developed as explained in Li F. et al[13] and Noh S.H. et al [l4]. The host cell Which hosts the above described embodiments ofthe expression vector and expression cassette is preferably a mammalian ho st cell derivedfrom human, hamster or murine cell lines. In preferred embodiments of the invention, CHO cells such as CHO-DG44 cell or a CHO GS* cell may be used.

As already discussed above, the various embodiments of DNA constructs, expression vectors,host cells and methods can be used for expressing a recombinant protein or one or morepolypeptide chains thereof. An example of a protein Which may be expressed is an antibody, antibody fragment, enzyme and hormone.

It is to be noted that, in the present invention, the singular forms "a," "and" and "the" include plural references unless the context clearly dictates otherwise. The present invention also contemplates other embodiments "comprising," "consisting of" and "consisting essentially of," the embodiments or elements presented herein, whether explicitly set forth or not.

The present invention has multiple aspects, illustrated in the non-limiting EXAMPLESsection. It should be noted that, in the EXAMPLES section, DNA constructs encoding the heavy and lights chains of Nivolumab (Opdivo®) have been cloned into an expression vector SCON SEVO. comprising TI and an expression vector comprising TI It should be understood that these examples relating to Nivolumab, while indicating preferredembodiments of the invention, are given by way of illustration only. From the above disclosedembodiments of the invention and the following examples, one skilled in the art can ascertainthe essential characteristics of this invention, and without departing from the spirit and scopethereof, can make various changes and modifications of the invention to adapt it to varioustypes of therapeutic antibodies (such as mAbs or fragments thereof) and immunoglobins (i.e.IgG, IgM, IgD, IgA and IgE). Thus, various modifications of the invention in addition tothose shown and described herein will be apparent to those skilled in the art from theforegoing description. Such modifications are also intended to fall within the scope of the appended claims.

EXAMPLES Examples l and 3 described below show that by introducing the TISEVO of SEQ ID No l intoan expression vector, the likelihood of identifying high mAb (Nivolumab) producing mini pools increased compared to mini pools transfected with the TISCON.

Unexpectedly, among the monoclonal cell lines producing > 4.2 g/L mAb, ten out of fourteenharboured the TISEVO. In addition, the top ten TISEVO cell lines yielded on average 0.56 g/Lmore mAb (Nivolumab) compared to the top ten cell lines with the commonly used TISCONwhilst the quality and biosimilarity were maintained. The present invention underlines the importance and the implications of the TIS sequence during CHO cell line development.

Moreover, Example 2 shows similar post translation modifications for Nivolumab expressedby TISEVO when compared to Opdivo® (i.e. originator Nivolumab) expressed in an expression vector system not comprising any one of TISEVO and TISCON. This clearly shows that that SEVO quality and biosimilarity of Nivolumab was maintained when TI was used.

For experimental details pertaining to the examples below, the reader is directed to theseparate MATERIALS AND METHODS section. All publications, patent applications,patents and other references mentioned in this document are incorporated by reference in their entirety.

The EXAMPLES and MATERIALS AND METHODS sections disclosed herein are illustrative only and not intended to be limiting.

Example l - TISEVO increases mAb yield in fed-batch cultivation of mini pools In order to compare implications of TIS sequence variants during cell line development inCHO-DG44, nucleotide changes were introduced to the expression vector by altering the TISsequence GCCACCATGGA (TISCON) to novel TIS sequence of TCGGTCATGGC (TISEVO).

In parallel experiments, cells were transfected via electroporation with either the TISEVO o rthe TISCON vectors (i.e. vectors comprising TISEVO or the TISCON sequences) and integrateswere selected for by seeding 4000-8000 viable cells/well in 96-well plates 24 hours aftertransfection. After screening for the best growing colonies, titers were measured in staticcultures and top mini pools were expanded and re-adapted to suspension. Top twelve minipools based on cell specific productivity (pg/cell/pay) and overall titer (g/L) were furtherevaluated in a fed batch study in shake ﬂasks. Notably, seven out of the top twelve mini poolswere cells transfected with the TISEVO. Moreover, when analysing data from the fed batchstudy, we saw clear indications of higher titer and cell specific productivity, in addition toincreased colony formation in 96-well plates, for mini-pools containing a TISEVO. The fedbatch results showed that the top three high producing mini pools had integrated vectors withthe TISEVO (figures l and 2). The average max viable cell density (VCD) / ml was lower formini pools with a TISEVO (l5.2*l06 cells/ml) compared to TISCON (20.l*l06 cells/ml) (datanot shown) and there was prolonged viability for TISEVO integrates (figures l and 2, x-axis).These results suggest that a mRNA with a TISEVO promote longevity, potentially throughrecruiting ribosomes more efficiently, thereby enhancing cell specific productivity and titer but slightly hampering VCD (less ribosomes available for rapid growth).

Example 2 - Comparable mAb glycan proﬁle and charge distribution for TISEVO and TISCON mini pools To assess if the increase in productivity and titer for cells transfected with TISEVO affectsprotein quality, mini pools were analysed for charge distribution of acidic and basic species(figures 8 and 9), glycan profiling (figures 5-7) and size distribution, i.e. post translationalmodifications, PTMs. The resulting data from Water RapiFluor-MS workﬂow, as illustratedin figure 5-9, showed similar PTM patterns and charge distribution for Nivolumab expressedby expression vector systems comprising TISEVO and TISCON, respectively, when compared toOpdivo® (i.e. originator mAb) expressed in an expression vector system not comprising anyone of TISEVO and TISCON. This clearly indicates that the alteration in translation initiation rates did neither affect protein quality nor biosimilarity.

The glycans which are disclosed in figures 5-8 have the following Oxford Notation names[l8]:- A2;- F(6)A2 (same as FA2 in reference 20);- A2[3]Gl;- A2[6]Gl;- F(6)A2[3]Gl (same as FA2[3]Gl in reference 20);- F(6)A2[6]Gl (same as FA2[6]Gl in reference 20);- F(6)A2G2 (same as FA2G2 in reference 20)- M5; and- F(6)Al.

Increased mAb production in monoclonal cell lines harbouring a TISEVO To generate monoclonal cell lines, top eight mini pools (based on titer and protein quality)were seeded as single cells using ﬂuorescence-activated cell sorting (FACS) and single cellimages were taken as a further assurance of monoclonality. The top 48 clones based onmonoclonality, cell growth and productivity were expanded and adapted to suspension culturebefore evaluation in an ambrl5 microbioreactor run. Cultures were harvested when viabilitywas < 70% or on culture day l4 at the latest. When analysing overall yield and cell specificproductivity of the mAb producing monoclonal cell lines, we observed a clear difference intiter and specific productivity between the TISEVO and TISCON containing cell lines (figures 3and 4). Among the 48 cultured clones, l4 clones gave rise to accumulated titer values 2 4.2 g/L of which l0 clones harboured a TISEVO. The highest producing cell line also contained a TISEVO and yielded 6.1 g/L mAb in a generic, non-optimised fed-batch process (figure 3).Moreover, when analysing the cell specific productivity 10 out of the 14 high producerscontained a TISEVO with the best TISEVO variant producing ca. 60 pg/c/d mAb (figure 4).Taken together, these results indicate that our rationally designed TISEVO is superiorcompared to a standard commonly used TISCON both in terms of the likelihood of identifyinghigh producers during mini pool generation as well as identifying a monoclonal DG44 cell lines with enhanced productivity and titer.MATERIALS AND METHODSVector engineering and transfection The eXpression vectors (i.e. the TISCON vector and TISEVO vector) for comparing the TISCONand TISEVO both comprised the following nucleic acid elements which are disclosed in thechapter “Cell line development” in Li F. et al [l3]: - first and second nucleic acid sequences encoding the heavy and light chains of theantibody to be expressed; - two CMV promoters, i.e. one for each of first and second nucleic acid sequencesencoding the heavy and light chains of the antibody; - an intron sequence in the 5' untranslated region is included after each of the CMVpromoters; - 3' polyadenylation (polyA) signal sequence is included after each of said 5'untranslated regions; - gene encoding selection marker DHFR; - a nucleic acid sequence encoding a signal peptide in front of (i.e. upstream) each ofthe first and second nucleic acid sequences encoding the heavy and light chains of theantibody to be expressed; - TIS sequences (i.e. Kozak sequences) upstream of the signal peptide nucleic acidsequences; and - antibiotic resistance marker.

The first and second nucleic acid sequences encoding the heavy and lights chains ofNivolumab (Opdivo®) were each cloned into an eXpression vector comprising, either two TISCON sequences, or two TISEVO sequences, as described in below paragraphs. The firstnucleic acid sequence encoding the heavy chains of Nivolumab comprises a sequence of SEQ ID No 4 while the second nucleic acid sequence encoding the light chains of Nivolumab comprises a sequence of SEQ ID No 6. Thus, the first nucleic acid sequence encodes heavychains comprising an amino acid sequence of SEQ ID No 5 while the second nucleic acid sequence encodes light chains comprising an amino acid sequence of SEQ ID No 7.

In the vector comprising the two TISCON sequences (i.e. the TISCON vector), the nucleic acidsequence encoding each of the signal peptides comprised a nucleic acid sequence foreXpressing a signal peptide of amino acid sequenceMDLLHKNMKHLWFFLLLVAAPRWVLS. This signal peptide has previously beendisclosed in Haryadi R. et al [12] and USl00660l9 for eXpressing a polypeptide chain of therapeutic antibodies.

For designing the TISEVO sequence, the GCCACC sequence of the TISCON sequence wasaltered to the TCGGTC sequence. In addition, the first codon downstream of the ATG startcodon, coding for the first amino acid of the signal peptide, was changed from GAT to GCTresulting in an amino acid substitution at that position. These combinatory changes yielded the TISEVO sequence comprising the TCGGTCATGGC nucleotide sequence (SEQ ID No l).

The eXpression vector comprising the two TISEVO sequence (SEQ ID No l) was engineered tocomprise two nucleic acid sequences each encoding the signal peptide comprising a nucleicacid sequence of SEQ ID No 2. The nucleic acid sequences of SEQ ID No l each comprised:- the ATG start codon in a sequence of SEQ ID No 2; and- the first two nucleotides downstream of the ATG start codon in a nucleic acid sequence of SEQ ID No 2.

The nucleic acid sequence of SEQ ID No 2 encodes a novel signal peptide of amino acidsequence MALLHKNMKHLWFFLLLVAAPRWVLS (SEQ ID No 3) which has previously not been disclosed in any prior art documents.

For testing the activity of TISEVO, DNA constructs comprising nucleic acid sequences of SEQID No 8 and SEQ ID No 9 encoding heavy chain and light chain of Nivolumab, respectively,were cloned into the eXpression vector. The DNA constructs used for this purpose comprisednucleic acid sequences of SEQ ID No l2 and SEQ ID No 13 which comprise (i) nucleic acidsequences of SEQ ID No 8 and SEQ ID No 9 encoding heavy and light chain of an antibody, respectively, and (ii) restriction sites which enabled cloning into the eXpression vector.

Consequently, the DNA constructs comprising the nucleic acid sequences of SEQ ID No 8and SEQ ID No l2, each comprised: - a first nucleic acid sequence comprising a nucleic acid sequence of SEQ ID No 1(TISEVOX - a first nucleic acid sequence which encodes a signal peptide comprising a nucleic acidsequence of SEQ ID No 2; and - a first nucleic acid sequence which encodes a heavy chain of an antibody comprising anucleic acid sequence of SEQ ID No 4; wherein said first nucleic acid sequence of SEQ ID No 1 comprised: - the ATG start codon in SEQ ID No 2 of the first nucleic acid sequence which encodes a signal peptide; and - the first two nucleotides downstream of said ATG start codon.

Similarly, the DNA constructs comprising the nucleic acid sequence of SEQ ID No 9 andSEQ ID No 13, each comprised:- a second nucleic acid sequence comprising a nucleic acid sequence of SEQ ID No 1(TISEVOX- a second nucleic acid sequence which encodes a signal peptide comprising a nucleicacid sequence of SEQ ID No 2; and- a second nucleic acid sequence which encodes a light chain of an antibody comprisinga nucleic acid sequence of SEQ ID No 6;wherein said second nucleic acid sequences of SEQ ID No 1 comprised:- the ATG start codon in SEQ ID No 2 of the second nucleic acid sequence whichencodes a signal peptide; and - the first two nucleotides downstream of said ATG start codon.

For testing the activity of TISCON, two DNA constructs comprising nucleic acid sequencesencoding heavy chain (SEQ ID No 4) and light chain (SEQ ID No 6) of Nivolumab,respectively, were cloned into the eXpression vector. In analogy to the above described vectorfor testing TISEVO, each of the nucleic acid sequences encoding heavy chain (SEQ ID No 4)and light chain (SEQ ID No 6) of Nivolumab were operably linked to the nucleic acidsequence eXpressing the signal peptide MDLLHKNMKHLWFFLLLVAAPRWVLS. Each ofthe nucleic acid sequences eXpressing said signal peptide were operably linked to the TIS sequence of GCCACC.

Consequently, the TISCON vector and the TISEVO vector differed only in that (in which differences in nucleic acid and amino acid sequences have been underlined): - the TISCON vector comprised two TISCON sequences of GCCACCATGGA While theTISEVO vector comprised two TISEVO sequences of ICGíGTCATGGQ; and - the TISCON vector comprised two nucleic acid sequences each eXpressing a signalpeptide of amino acid sequence MQLLHKNMKHLWFFLLLVAAPRWVLS whilethe TISEVO vector comprised two nucleic acid sequences each eXpressing a signal peptide of amino acid sequence MALLHKNMKHLWFFLLLVAAPRWVLS.

Fed-batch cultivation for mini pool and clone evaluation Standard fed-batch processes were run according to a generic process for mini pool and cloneevaluation. Cells were inoculated at a density of 3 X 105 cells/mL in 25 mL chemicallydefined production medium using 125 mL shake flasks (mini pools) or in an ambr15 microbioreactor (clones). Feed A, feed B and glucose were added according to a standard feedingregimen. Cultures were controlled for cell density, viability, product concentration, glucose and lactate. Cells were cultivated for up to 14 days or until viability dropped below 70 %.

Claims

1. DNA construct for eXpressing a recombinant protein in mammalian cells, Wherein theDNA construct comprises:- a nucleic acid sequence of SEQ ID No 1, Wherein the nucleic acid sequence of SEQ ID 1 is a TIS sequence; and - a nucleic acid sequence Which encodes a signal peptide.

2. DNA construct according to claim 1, Wherein the TIS sequence transcribes into anRNA motif that functions as the protein translation initiation site in an mRNA transcript.

3. DNA construct according to claim 1 or 2, Wherein the TIS sequence is a Kozaksequence Which transcribes into an RNA motif that functions as the protein translation initiation site in an mRNA transcript.

4. DNA construct according to any one of the claims 1-3, Wherein the nucleic acidsequence of SEQ ID 1 comprises:- 6 nucleotides upstream of an ATG start codon; and - 2 nucleotides doWnstream of an ATG start codon.

5. DNA construct according to any one of the claims 1-4, Wherein the nucleic acid sequence Which encodes a signal peptide comprises a nucleic acid sequence of SEQ ID No 2.

6. DNA construct according to any one of the claims 1-5, Wherein the nucleic acidsequence of SEQ ID No 1 comprises:- the ATG start codon in the nucleic acid sequence Which encodes the firstamino acid residue of the signal peptide; and- the first two nucleotides doWnstream of the ATG start codon in the nucleicacid sequence Which encodes the second amino acid residue of the signal peptide.

7. DNA construct according to any one of the claims 1-6, Wherein the nucleic acid sequence of SEQ ID No 1 comprises: - the ATG start codon in SEQ ID No 2; and- the first two nucleotides doWnstream of the ATG start codon in the nucleic acid sequence of SEQ ID No 2.

8. EXpression vector Which comprises the DNA construct according to any one of the claims 1-7.

9. EXpression cassette comprising the DNA construct according to any one of the claims1-7.

10. Host cell Which comprises a DNA construct according to any one of the claims 1-7, Wherein said host cell is preferably an eukaryotic cell.

11. Host cell according to claim 10, Wherein said host cell is a mammalian cell.

12. Recombinant protein expressed by a host cell according to any one of the claims 1-7Wherein said recombinant protein is preferably an antibody or an antibody fragment thereof.

13. RNA expressed by the DNA construct according to any one of the claims 1-7.

14. Method of eXpressing a recombinant protein, comprising the steps of: a. cloning one or more open reading frames encoding a recombinant protein, orone or more polypeptide chains thereof, into one or more DNA constructsaccording to any one of the claims 1-7; and b. transfecting the resulting nucleic acid sequences into a host cell, Wherein the host cell is preferably an eukaryotic cell, more preferably a mammalian cell.

15. Method according to claim 14, further comprising the step of integrating the transfected nucleic acid sequence into the genome of the host cell.