Patent Grant
11851666
This application is a Section 371 national phase entry of PCT application PCT/EP2018/072687, filed Aug. 22, 2018. This application also claims the benefit of the earlier filing date of European patent application 17187552.9, filed Aug. 23, 2017.
This application contains a Sequence Listing which has been submitted electronically in ASCII format and is herein incorporated by reference. The ASCII file, created on Nov. 17, 2020, is named 75162us-topto-20201130-CorrectedSequenceListing.txt, and is 88,940 bytes in size.
The present invention belongs to the field of biotechnology, specifically to the field of recombinant protein expression. The present invention focuses on two problems commonly encountered during recombinant protein expression, low quantity of protein expression and genetic instability of cell lines used for recombinant protein expression. The basic principle of the present invention is to introduce several expression cassettes into a cell which expression cassettes all code for the same mature recombinant protein of interest, but which expression cassettes have different nucleotide sequences. Expression cassette means a polynucleotide sequence which comprises at least a promoter sequence, a start codon, a polynucleotide sequence coding for a protein which is intended to be recombinant expressed (POI), a stop codon and a terminator.
The present invention belongs to the field of biotechnology, specifically to the field of recombinant protein expression. Furthermore the invention relates to cells modified to express higher yields of recombinant protein (protein of interest, POI) and to modified cells which are less prone to genetic instability due to re-arrangement of the genetic material introduced into said modified cells. In another aspect the invention relates to vectors, expression cassettes used to generate said modified cells, as well as methods how to generate said modified cells and methods how to manufacture recombinant proteins using said modified cells, said vectors and said expression cassettes.
Recombinant protein expression in general has two main objectives: Firstly obtaining a recombinant protein of high quality, meaning e.g. pure, low content of degradation products, homogeneous regarding amino acid sequence and posttranslational modifications, soluble, correct three-dimensional folding and having the same biological activity as compared to the native, wild-type protein. Secondly the aim is to obtain a recombinant protein in high quantity in short time, e.g. in order to save costs, time and resources during the production process.
The present invention focuses on two problems commonly encountered during recombinant protein expression, low quantity of protein expression and genetic instability of cell lines used for recombinant protein expression.
In order to obtain high quantities of recombinant protein it is commonly tried to introduce not only one copy of a so called expression cassette into the cell chosen for recombinant protein production, but to try to introduce several copies of the expression cassette into a cell and subsequently select those modified host cells which have the optimal high number of expression cassettes in order to express the maximal amount of the protein of interest (POI). This strategy has at least two drawbacks:
First, the more copies of the expression cassette are introduced into the cell the more likely it is that over time the sequences of these expression cassettes recombine with each other due to the similarity of their sequences, which promotes recombination. As a consequence rearrangements of nucleotide sequences within the modified host cell result in an instable genome of the modified host cell used for protein expression. This results in a lower recombinant protein expression of the modified cell over time. In the worst case these unwanted recombination processes result in altered sequences of the POI, thereby not only decreasing the recombinant protein expression rate, but also decreasing the quality, because the recombinant protein gets a mixture of different variants of the POI, for example truncated or mutated versions of the POI, or POI with duplicated domains and region, etc.
Second, it is commonly recognized that a high copy number of an expression cassette is no guarantee for a high expression rate of the POI. Likely, a too high number of the expression cassette results in some kind of overburden or overstrain of the molecular machinery needed for protein expression of the modified host cell and thereby the expression rate of the POI goes down once the copy number of the expression cassette within the modified host cell exceeds a certain threshold.
The basic principle of the present invention is to introduce several expression cassettes into a cell which expression cassettes all code for the same mature recombinant protein of interest, but which expression cassettes have different nucleotide sequences. One of the main advantages of the present invention is its universal applicability, which is not limited to a certain type of cells, but can be used for prokaryotic as well as eukaryotic cells.
For example the expression cassettes may have different promoters, different terminators, different signal sequences, etc. and the coding sequence of the POI may be the same in the expression cassettes, or may be different in the different expression cassettes, however the amino acid sequence of the POI is always the same. The expression cassettes may have different nucleotide sequences coding for the same POI with the same amino acid sequence by utilizing the degenerated genetic code. The same amino acid may be coded for by up to 6 different codons, and thereby it is possible to have the same amino acid sequence coded by quite different nucleotide sequences. Furthermore, if the same vector element such as an expression cassette, a selection marker, an origin of replication, etc. is used twice within a vector or is used in more than one vector, said vector element might be used in different orientation within the vector sequences. This further increases the differences of the vector sequences and thereby lowers the likely hood of recombination of said vector elements within a transfected host cell comprising these two or more identical vector elements. This further increases genetic stability of said host cell.
This strategy has at least two main advantages. At the one side the expression cassettes now have quite different nucleotide sequences and therefore they are less likely to recombine with each other. This can result in a more stable genome of the modified cell which in turn allows to have higher copy numbers of the expression cassettes within the modified cell. On the other side the protein synthesis machinery of a modified cell is less likely to be overburden or overstrain due to high expression rates of the POI, because the modified cell in parallel uses:
Besides these two aspects the present invention furthermore has a third advantage. The skilled person is not required to find out in a series of experiments, which combination of promoter and POI works best in a certain host cell to be modified, because always a set of different types of promoters is used in parallel and even if an individual promoter in a certain POI/host cell combination does not perform well, this does not necessarily have a big effect on the overall expression rate of the POI, as other, different promoters are used at the same time that can compensate for the non-optimal promoter. This can result in e.g. faster development times for modified host cells suitable for cost-effective, efficient recombinant expression of a POI.
The concept of several in parallel used vectors, each vector comprising a single, different expression cassette for the same POI, has the additional advantage, that it is more flexible as compared to the also possible concept of using vectors comprising several different expression cassettes within the same vector. With a set of different single expression cassette vectors, the skilled person can easy and quickly test various combinations of different expression cassettes, and even can easily vary the relative abundance of the individual expression cassettes, simply be simultaneously transfecting the different vectors in different quantities (amount of transfected DNA of each single expression cassette vector) into one host cell. This allows to adjust the copy-number of the individual expression cassettes in order to get an optimal result regarding genetic stability of the host cell, and/or regarding POI expression rate.
Similar advantages can be obtained if the expression cassettes have the same promoter sequences. For instance, the expression cassettes have the same promoter sequences, different nucleotide sequences coding to the identical mature amino acid sequence of the POI, and optionally different terminator sequences and/or different signal sequences, if present.
Also the different mRNAs as a result of different coding sequences of the POI have different nucleotide sequences and therefore can have different stabilities, half-lives and different secondary structures which may or may not interfere with efficient translation of the mRNA into a POI. This mechanism avoids that the overall expression rate will be low just because by chance one certain version of mRNA is instable or has an unfavourable three-dimensional structure, because other, better suited versions of mRNAs are present at the same time and compensate for that.
In general, the more copies of a nucleic acid coding for a POI are transfected into a host cell, the higher is the expression rate. However, it is known to a skilled person in the field of recombinant protein expression that there is a certain threshold to that, meaning that a certain copy number the expression rate is no longer increasing, but instead may indeed decrease. The optimal copy number usually is determined empirically for each cell or POI. The same effect is also likely observed using the protein expression strategy of the invention disclosed herein. It is expected that increasing the copy number of individual expression cassettes of the invention at a certain threshold copy-number no longer increases the protein expression rate. It is furthermore expected that increasing the number of different expression cassettes coding for the same POI amino acid sequence also has a certain threshold-number, and furthermore increasing the number of said different expression cassettes does not further increase the quantity of expressed POI. The skilled person in the field of recombinant protein expression knows how to empirically determine the optimal number of an expression cassette for a certain POI in a certain type of host cell, for example simply by measuring the quantity of expressed POI and comparing it with the copy number of expression cassettes detected in the same host cell.
One of the main advantages of the present invention is its universal applicability, independent of the type of cells used. The invention is useable for all types of cells, eukaryotic as well as prokaryotic cells. It can be used for example with mammalian cells, yeast cells, fungal cells, bacteria, etc.
In the prior art this concept is unknown. The only protein expression strategy which remotely goes into the direction of the invention described herein is the concept to express several different POI at the same time in the same host cell, for example the alpha- and the beta-chain of a T-cell receptor (WO 2016/073794), the light and the heavy chain of an antibody (WO 03/018771), L- and H-ferritin (J. Microbiol. Biotechnol, 2008, 18: 926-932), etc. However these concepts in the prior art are clearly different to the present invention in several aspects:
WO 2016/005931 describes a method to increase the protein expression in E. coli using a dual, independent cistron expression system wherein both cistrons are located within one vector. The main object of this application is to increase the expression of proteins, especially antibody fragments such as Fab fragments consisting of two polypeptide sequences. The use of the dual cistron expression system for expression of only one protein of interest is also disclosed. However also this concept is different from the present invention in several aspects:
The present invention provides the following aspects, subject-matters and preferred embodiments, which respectively taken alone or in combination, contribute to solving the object of the present invention:
Item (1): Host cell comprising three or more different types of expression cassettes, each expression cassette coding for the same Protein Of Interest (POI) with identical mature amino acid sequence, and each type of expression cassette at least is comprising a promoter sequence, a polynucleotide sequence of the coding sequence of the POI, and a terminator sequence, wherein said expression cassettes differ in, that they comprise
The titles given in front of paragraphs of this application are meant to guide through the text of the application, but are not meant and should not be understood to limit the scope of the invention in any way.
The term “and/or” wherever used herein includes the meaning of “and”, “or” and “all or any other combination of the elements connected by said term”. For example, A, B and/or C means A, B, C, A+B, A+C, B+C and A+B+C.
“Host cell” means the cell, which is used for expression of a recombinant protein. The host cell can be any type of cells such as bacterial cells, yeast cells, fungal cells, mammalian cells, human cells, cell lines such as cancer cells or cells which have been experimentally modified resulting in immortalized cells (=cells which divide an unlimited number of times, the same as cancer cells), etc.
“Expression cassette” means a polynucleotide sequence which comprises at least a promoter sequence, a start codon, a polynucleotide sequence coding for a protein which is intended to be recombinant expressed (POI), a stop codon and a terminator. An expression cassette may comprise additional regulatory and other sequences such as enhancers, signal sequences, enhancers, introns, IRES-sequences, etc. A host cell comprising three or more different expression cassettes may be a host cell which was transfected with three or more vectors, each vector comprising a different expression cassette. The resulting host cell might comprise said vectors present as plasmids within its cytosol, or might have integrated said expression cassettes and optionally further parts of said vectors into its genome. It might also be that some of the transfected vectors are integrated (partially or complete) into the genome of said host cells, whereas other of said transfected vectors are present as plasmids within the cytosol of said host cell. Said host alternatively also might have been transfected with at least one vector comprising at least three different expression cassettes within said one vector, or with mixtures of vectors comprising individual expression cassettes and at the same time transfected with vectors comprising two or more different expression cassettes.
“Transfection” of a GOI (meaning an expression cassette of a GOI) or of a vector (meaning a vector comprising at least one expression cassette of a GOI) might result in transfected host cells (or transformed host cells, which is the same), wherein said host cells have integrated said GOI or said vector into their chromosome (if said cell has only one chromosome), or said host cells have integrated said GOI or said vector into several or all of their chromosomes (if said host cell has more than one chromosome). Said GOI or vector might be integrated once or several times into said chromosome, preferably it is integrated several times into a chromosome. Preferably it is integrated in more than one chromosome of the same host cell. If a vector is integrated into a chromosome the complete sequence or only part of the sequence of said vector might get integrated into said chromosome, but at least the expression cassette of said GOI present in said vector is integrated into said chromosome. Alternatively said vector might not get integrated into a chromosome of said host cell, but might exist outside of a chromosome within the cytosol of said host cell, for example in the form of a circular, double-stranded desoxy-polynucleic acid. If said host cell is a eukaryote, more preferably if said host cell is a mammalian or a yeast or a fungal cell, most preferably is said host cell is a CHO cell or a Pichia pastoris cell, preferably said GOI or said vector is integrated into a chromosome of said host cell. If said host cell is a prokaryote, preferably a bacterial cell, more preferably an E. coli cell, said vector preferably is not integrated into the chromosome of said host cell but is located in the cytosol of said host cell.
If an expression cassette comprises two or more polynucleotide sequences coding for the protein which is intended to be recombinant expressed (POI) and said two or more polynucleotide sequences are expressed due to the function of a single promoter polynucleotide within said expression cassette, said expression cassette is still regarded as one expression cassette. Such an expression cassette for example could for example arise from the use of IRES sequences, or from the use of a bi-directional promoter. A bi-directional promoter is a promoter which results in expression of two coding sequences, one of which is located 5′ to the promoter and one is located 3′ to the promoter.
Further parts of a vector used according to the invention, which parts are not directly needed for the expression of the POI, such as for example the origin of replication (ori), antibiotic resistance gene, or metabolic selection marker, etc. are not regarded as part of the expression cassette. However also some or all of these parts of the vector might be different in different vectors. For example if several individual vectors are used according to the invention, each of these vectors might contain a different antibiotic resistance gene or a different metabolic selection marker or a different origin of replication (ori), etc. Alternatively the antibiotic resistance gene and/or the metabolic selection marker, etc. might be the same protein, but the nucleic acid sequence within the vector coding for said protein might be different due to the degenerated genetic code, but still code for the identical antibiotic resistance protein or metabolic selection marker protein.
“Coding”: A polynucleotide or sequence “codes” if it results in, if combined with appropriate regulatory sequences such as a promoter, a start codon, a stop codon and a terminator, etc., in the expression of a protein or polypeptide or peptide comprising at least 10, at least 20, at least 30, at least 50 or at least 100 amino acids connected via peptide bonds.
“Coding sequence” or “coding region” means those parts of a polynucleotide, which code for the amino acid sequence of the mature amino acid sequence. “Mature amino acid sequence” is explained a few paragraphs below.
“Open reading frame” means those parts of a polynucleotide, which code for amino acid sequences, regardless if these amino acid sequences are present in the final mature amino acid sequence or if these amino acid sequences are removed during the processing of the POI, for example amino acid sequences of a signal peptide, which are removed from the POI in order to obtain the “mature amino acid sequence”.
“Protein Of Interest”, also abbreviated POI, is a protein, polypeptide, or peptide comprising at least 10, at least 20, at least 30, at least 50, at least 100, at least 150, at least 200, at least 250 amino acids connected via peptide bonds, which POI is intended to be recombinantly expressed by use of a host cell. The POI is coded by a “Gene Of Interest” (GOI). The amino acid sequence of the POI is regarded as the “mature amino acid sequence”.
The POI can be a protein, polypeptide or peptide, which is present in nature, or a protein, polypeptide, or peptide, which is not present in nature, for example a fusion protein of two peptides, polypeptides, proteins, protein-domains, etc. present in nature, which fusion protein is not present in nature. For example the POI might be a protein present in nature fused to a His-tag, or fused to other peptides which are intended to label, or to purify the fusion protein, or fusion proteins comprising domains of two or more proteins present in nature, which domains normally are not present in nature within one protein, polypeptide or peptide, or a non-human sequence which has been “humanized” like for example humanized antibodies, etc. Humanized antibodies are for example murine antibodies, whose constant amino acid sequence part has been replaced by the corresponding amino acid sequence part of a human antibody. Therefore mature amino acid sequence in general means the final amino acid sequence intended to be manufactured by the person, who designed or performed the experiment to obtain the POI.
Consequently the mature amino acid sequence of a POI can be:
“Mature amino acid sequence” means for example the amino acid sequence of a protein after it has undergone the complete processing steps of the corresponding non-recombinant protein, polypeptide or peptide regarding its amino acid sequence. For example secretion signal sequences have been removed, the pre- or the pre-pro-form of for example a protein have been converted to the final protein, polypeptide or peptide sequence, or internal sequences within the amino acid sequence have been removed during processing. For example in the case of insulin this means: pre-pro-insulin: removal of signal sequence=pro-insulin; pro-insulin: removal of the internal C-peptide=insulin=the mature amino acid sequence in this case.
“Mature recombinant protein” means a recombinant protein comprising a mature amino acid sequence, as defined above. Introns in general do not code for a part of the mature protein, polypeptide, or peptide.
“Processing sequences” means amino acid sequences, which are removed from the protein, polypeptide or peptide in order to obtain a mature amino acid sequence, such as secretion signal sequences, signal sequences for intracellular protein targeting, pre-pro-sequences, pro-sequences, etc.
The sequence of the POI might comprise or might partially or completely lacking processing sequences. Said processing sequences are often present in proteins present in nature (native proteins, natural proteins) and are often needed for correct processing of the native protein, or for the correct physically location of the native protein in the correct location inside or outside the cell, or for the transport of the native protein, etc. A transmembrane sequence usually is not removed during processing of a protein, polypeptide or peptide and therefore is normally not regarded as processing sequence. A transmembrane sequence is only then regarded as processing sequence, if the POI is only transiently localized to the cell membrane by use of said transmembrane sequence and said transmembrane sequence is removed from the rest of the POI during the processing of the POI in order to obtain the POI.
Promoter or promoter sequences means a region of a polynucleotide, which initiates transcription of a gene or in the case of the current invention initiates the transcription of a nucleotide sequence coding for a POI. The promoter can be an “inducible promoter” or “constitutive promoter.” IRES sequences and sequences function like IRES sequences are not regarded as a promoter or a promoter sequence. “inducible promoter” refers to a promoter which can be induced by the presence or absence of certain induction factors, and “constitutive promoter” refers to an unregulated promoter which is active at all times, independent of the presence of certain induction factors, that allow for continuous transcription of its associated gene or genes. Optionally, a promoter my initiate the transcription of two or more genes if for example these two or more genes are separated by an IRES sequence. Optionally, a promoter my initiate the transcription of two genes, if said promoter for example is a bi-directional promoter.
“Degenerated genetic code” means that for a certain amino acid there are more than one nucleotide codons. For example the amino acid Cysteine can be coded for by the following two different codons: TGC or TGT, the amino acid Arginine can be coded for by the following 6 codons: CGG, CGA, CGC, CGT, AGG, AGA, etc. As a consequence the same amino acid sequence can be coded for by different nucleotide sequences. If only the individual codons are exchanged but not the amino acid for which these codons code. The degenerated genetic code is the same for almost all organisms with a few exceptions. For example human mitochondria have a different genetic code. Within this patent application “degenerated genetic code” is always meant regarding the genetic code of the specific cell or the specific organelle (such as a mitochondrion), which is intended to be use to express the POI.
“Terminator” means the same as “transcription terminator”. According to the invention a terminator is a section of nucleic acid sequence that marks the end of the nucleic acid sequence needed to code for a POI. Usually said terminator is localized shortly downstream of the stop-codon of the GOI. In prokaryotes termination includes Rho-independent as well as Rho-dependant transcription termination. Prokaryotic termination sequences used according to the invention preferably are Rho-independent termination sequences such as the T7 and the rrnB termination sequences. Rho-independent termination is also known as intrinsic terminations. Preferably in one expression cassette one or two termination sequences are used. Two combined termination sequences increase the termination efficiency. If IRES sequences are used preferably more than one termination sequence are placed between two coding sequences of the POI. Mammalian termination sequences are for example SB40-, hGH-, BGH- or rbGlob-termination sequences.
“signal sequence” means an amino acid sequence which usually is needed to direct a protein, polypeptide or peptide to be secreted into the extracellular region and which signal sequence usually is removed from the mature amino acid sequence by proteolysis. There are also signal sequences, which direct the protein, polypeptide or peptide to certain organelles of the cell. Bacterial cells also use signal sequences, for example signal sequences, which direct a POI into the periplasm. Signal sequences usually are located at the N-terminal end of an amino acid sequence, but can also be present at the C-terminal end or can be present internally, within the polypeptide sequence.
“IRES” sequences, also named “internal ribosome entry site” sequences are nucleotide sequences within the mRNA, which allow for the translation initiation within the mRNA sequence and do not depend on the 5′-end of the mRNA for initiation of the translation. So IRES sequences allow to express two or more POI from one mRNA. Alternatives for IRES sequences with the same principal function as IRES sequences are for example the 2A, P2A, T2A and the F2A sequences.
“Heterologous” protein, polypeptide, peptide sequence means that the amino acid sequence coded by a nucleotide sequence is not naturally present in the host cell. If an amino acid sequence, which is naturally present in the host cell is mutated (e.g. point mutations, insertions, deletions, fusions, etc.) the resulting mutated sequence is also regarded as heterologous sequence.
“Heterologous” polynucleotide or nucleotide sequence means that the polynucleotide or nucleotide sequence is not naturally present in the host cell. If a naturally in the host cell present polynucleotide or nucleotide sequence is modified by exchanging individual nucleotides in a way that said polynucleotide or nucleotide sequence still codes for the same amino acid sequence, such a modified polynucleotide or nucleotide sequence is regarded as heterologous.
The terms “sequence difference”, and terms like “differ”, “different”, “differing”, etc. if mentioned in connection with amino acid sequences or nucleic acid sequences are meant to be determined for example as follows:
In the present invention, reference is made e.g. to “different promoter sequences” or different nucleotide sequences coding for the (identical) mature amino acid sequence of the POI. Thus, in order to determine whether said sequences are “different”, the respective corresponding sequences (amino acid sequences or nucleotide sequences) are compared regarding their sequence identity. For instance, the promoter sequences are compared or the nucleotide sequences coding for the mature amino acid sequence of the POI.
If two or more sequences are compared regarding their sequence identity the comparison only considers a nucleotide or amino acid to be identical if exactly the same nucleotide or amino acid is present at a certain position. Especially for amino acid sequence comparisons it has to be clearly distinguished between sequence identity and sequence homology. In the present patent application in the context of sequence comparisons always sequence identity is meant, not sequence homology, except if the contrary is expressly mentioned. Homology means for example that the amino acid at a certain position within a sequence is not identical but is only similar regarding its chemical and/or biological and/or physical characteristics. Examples for such amino acids, which commonly are regarded as homologue are:
heterocyclic secondary alpha-amino acid: Proline
Sequence alignments or sequence differences for example can be determined with various methods, software and algorithms. Such determinations can be done for example using the web-services of the National Institute of Health (NIH), or using the web-services of the European Bioinformatics Institute (EMBL-EBI). “Sequence identity” or “% identity” refers to the percentage of residue matches between two protein, polypeptide, peptide, amino acid, or nucleotide sequences aligned using a standardized algorithm. Such an algorithm may insert, in a standardized and reproducible way, gaps in the sequences being compared in order to optimize alignment between two sequences, and therefore achieve a more meaningful comparison of the two sequences. Because of the different algorithms and software settings it is possible that an alignment or sequence comparison of the same two sequences using different software/algorithms does not give exactly the same result. Therefore, the software and the software settings have to be given, in order to clearly define how results were obtained.
For purposes of the present invention, the sequence identity between two sequences is determined using the NCBI BLAST program version 2.6.0 (Jan. 10, 2017), BLAST=Basic Local Alignment Search Tool, (Altschul et al., Nucleic Acids Res. (1997) 25:3389-3402). As reference sequence is always used the shorter one of the two to be compared promoter sequences. For example if a certain promoter Xshort of a sequence length of 100 nucleotides is aligned/compared to the same promoter Xlong, which is the same promoter but a longer version of said promoter of 200 nucleotides, a comparison of the two sequences Xshort and Xlong gives the following result: If the shorter sequence Xshort is the reference sequence, which is compared to the longer version of the sequence namely Xlong, then Xshort is 100% identical to Xlong. If however the longer sequence Xlong is the reference and compared to Xshort, then Xlong is only 50% identical to Xshort. Consequently in the present patent application a comparison of sequence Xshort and sequence Xlong would always be regarded as 100% identical not 50% identical, because as a reference sequence within this applications is always used the shorter one of the to be compared promoter sequences.
Sequence identity of two amino acid sequences for example can be determined with blastp, set at the following default algorithm parameters: “Max target sequences”=100, “Short queries”=“Automatically adjust to parameters for short input sequences”, “Expect threshold”=10, “Word size”=6, “Max matches in a query range”=0, “Matrix”=BLOSUM62, “Gap Costs”=“Existence: 11 Extension: 1”, “Compositional adjustments”=“Conditional compositional score matrix adjustment”, Filters and Masking: “Low complexity regions”, “Mask for lookup table only”, “Mask lower case letters”, all three filters deactivated.
Sequence identity of two nucleotide acid sequences for example can be determined with blastn, set at the following default algorithm parameters: “Max target sequences”=100, “Short queries”=“Automatically adjust to parameters for short input sequences”, “Expect threshold”=10, “Word size”=28, “Max matches in a query range”=0, “Match/Mismatch Scores”=1,−2, “Gap Costs”=“Linear”, Filters and Masking: “Low complexity regions”, “Mask for lookup table only”, both filters activated.
If nucleotides of nucleotide sequences are mentioned the abbreviations A, T, G, C, and U represent the different nucleotides. Whenever T or U as a nucleotide is mentioned T and U can be exchanged for each other, unless this does not make sense from an experimental or scientific point of view. If the terms nucleotide sequence, polynucleotide etc. are used within the application always DNA and/or RNA, or deoxynucleic acids and/or deoxyribonucleic acids are meant to the extent this makes sense from an experimental or scientific point of view.
The use of the degenerated genetic code allows to have several different nucleotide sequences, all of which code for an identical amino acid sequence. The amount of differences between two nucleotide sequences coding for the same mature protein depends on the amino acid sequence of said mature protein. Very simplified, all amino acids are coded by three nucleotides, and the last nucleotide of the codon of most amino acids can vary between Guanine (G), Cytosine (C), Alanine (A), and Thymidine (T). So most Amino acids have four different codons, each of which codes for the same amino acid. As a consequence a mature polypeptide of for example 100 amino acids length is coded by 300 nucleotides, and every third nucleotide can be mutated without changing the amino acid sequence. So in this simplified model 100 nucleotides of the total 300 nucleotides can be exchanged due to the degenerated code without changing the corresponding amino acid sequence. This simplified model results in a maximal theoretical nucleotide sequence difference of 33.3%. If it is desired that 50% of the maximal theoretical nucleotide sequence difference is desired, 50% of these 100 nucleotides, namely 50 nucleotides can be exchanged for other nucleotides, resulting in a nucleotide sequence difference of 16.65%.
In reality this calculation is a little bit more difficult. For example the maximal nucleotide sequence of the following peptide sequence can be calculated as follows:
As a result the nucleotide sequence of the sample peptide Pep1 has 24 out of 30 nucleotide positions, which can be exchanged by at least one different nucleotide without changing the amino acid sequence. The maximal different nucleotide sequence is 24/30=0.8, meaning 80% maximal nucleotide sequence difference.
If one calculates the same for Peptide Pep2 the result is as follows: Methionine and Tryptophan each have only one codon, meaning that no nucleotide can be exchange without changing the amino acid coded. All other amino acids have two, three or four different codons, but all codons have the first and second nucleotide fixed whereas only the third nucleotide can vary.
As a result the nucleotide sequence of Pep2 has only 7 out of 30 nucleotides, which can be exchanged without changing the amino acid sequence coded. The maximal different nucleotide sequence is therefore 7/30=0.23, meaning 23% maximal nucleotide sequence difference.
So the maximal variation of the nucleotide sequence without changing the amino acid sequence highly depends on the amino acid sequence of the POI. If for example it is intended, that the nucleotide difference should be 50% of the maximum nucleotide sequence difference possible in order to still get an identical mature amino acid sequence this 50%-value for Pep 1 would be 50% of 80%=40%, whereas the 50%-value for Pep2 would be 50% of 23%=11.5%.
Using this strategy a skilled person can easily calculate for any POI the % of variation from the mature nucleotide sequence, which is possible, if for example 50% of the maximum nucleotide sequence difference possible, in order to get an identical mature amino acid sequence of the POI is intended.
“Genetic stability” or alternatively also termed “genomic stability” according to the invention means that the nucleic acid sequence belonging to the genome of a host cell does not “significantly change” over time, e.g. over a certain number of cell generations or cell divisions of said host cell. Such changes can for example result from homologous recombination events of very similar or identical nucleotide sequences. If for example several identical copies of an expression cassette have been integrated into the genome of a host cell, the likelihood that later on these identical nucleotide sequences recombine with each can increase. Such recombination events for example can result in partial or complete deletion, duplication or multiplication of said expression cassettes. Also re-arrangement of said expression cassettes changes of their location within a chromosome or changes regarding their orientation within a chromosome can occur.
“Significantly change” regarding genetic stability means larger rearrangements of the genome of a host cell, such as deletion, duplication, multiplication, re-arrangement, re-location, partial deletion, partial duplication, partial-multiplication, partial re-arrangement, partial re-location, etc. of nucleotide sequences within the genome of the host cell. Such genetic instabilities can preferably effect nucleotide sequences of expression cassettes introduced into the host cell genome in order to express a POI by said host cell. A significant change of the host cell genome can effect a nucleotide sequence of at least 5 to 20, preferably at least 5 to 100, more preferably at least 5 to 500, most preferably at least 5 to 1500 nucleotide length.
Limited numbers of point mutations of for example expression cassettes, are only regarded as minor changes of the genome of the host cell. Such limited numbers of point mutations are common in nature, and normally can occur in any cell over time especially during cell division and cell ageing. Such limited numbers of point mutations are not regarded as impaired genetic stability and are not regarded as significantly change nucleic acid sequences.
The genome of the host cell according to the invention is regarded as the chromosomes, the chromosomes of mitochondria, and extra-chromosomal plasmids present in the host cell prior to introduction of expression cassettes coding for a POI. According to the invention nucleic acids such as mRNA, tRNA, rRNA, etc. are not regarded as belonging to the genome of said host cell.
Not all types of such nucleic acids belonging to the genome are present in all types of host cells. For example bacterial host cells usually do not contain mitochondrial chromosomes.
“Cell generations” according to the inventions means that one cell generation is the doubling of the number of a certain host cell. Depending on the type of host cell one cell generation may take only a few minutes, for example in the case of a bacterial host cells, or may take several hours or even several days, for example in the case of mammalian cells.
“Single chain protein” according to the invention includes proteins which comprise only one single amino acid chain. Proteins which are modified during posttranslational processing from a single chain precursor but which consist of several amino acid chains, eventually connected via disulphide bridges, such as for example human insulin, are still regarded as single chain proteins according to this invention. Such single chain proteins, which after posttranslational processing comprise two or more amino acid chains, can be easily identified by analysing the coding nucleotide sequence of said single chain protein for its open reading frame. An open reading frame is a continuous stretch of codons that do not contain a stop codon (usually a TAA, TAG or TGA in the case of deoxyribonucleic acids, or UAA, UAG or UGA in the case of ribonucleic acids) within a nucleotide sequence. The open reading frame may code for a single polypeptide chain which later on during processing of said polypeptide chain may be processed into a protein comprising two or more polypeptide chains. Such a protein, according to this invention, is still regarded as single chain protein.
“Vector” according to the invention preferably is a circular, double-stranded deoxy-poly-nucleotide, which may be linearized, for example by digestion with a restriction endonuclease which recognizes only on site within the nucleotide sequence of said vector. A vector may be manufactured by molecular biologic techniques, or may be chemically or enzymatically synthesized, using techniques known in the art.
“Resistance gene” or “resistance marker” according to the invention means a gene coding for a protein rendering a host cell resistant to the activity of toxic substance, preferably an antibiotic.
“Metabolic marker” according to the invention usually means a gene coding for a protein providing the host cell with the ability to synthesize a certain metabolite such as for example a certain amino acid, which metabolite is needed for growth or survival of the host cell.
“Selectable marker” according to the invention usually is a resistance gene, a metabolic marker, or an auxotrophic marker, but can also be for example a gene, which allows to recognize a host cell harbouring said gene, for example a gene coding for a coloured protein, or coding for an enzyme which generates or metabolizes a coloured substance, or an enzyme such as luciferase which emits light when metabolizing a substrate, etc.
A kit according to the invention is a set of materials suitable to for example to express a recombinant protein or a POI. A kit typically might contain materials such as host cells, protein expression vectors, PCR-primers suitable to detect parts of said protein expression vectors, culture media suitable to grow said host cells, chemicals and buffers suitable to transfect vectors into host cells, enzymes to perform PCR-reactions, enzymes to cut circular vectors into linear vectors, instruction manuals which explain how to use said kit or which explain for what purposes said kit is suitable, etc.
“Derivatives of cells” or derivatives of cell lines, or “derivatives of host cells” or “derivatives of host cell lines” are cells which originated from cells or host cells, wherein said cells or host cells have been manipulated in a way to, for example, contain or lack certain resistance genes, to contain or lack certain metabolic genes, to contain or lack certain genes which allow to distinguish said cells or host cells from their corresponding non-modified cells or host cells. Usually derivative of cells or host cells are genetically almost identically to the corresponding cell or host cell from which they originated (their mother cells), but are only different regarding one or very few genes, such as the types of genes mentioned above.
The host cells according to the invention in principal can be any type of cells, such as cell lines or primary cells or even mixtures of different types of cells or tissue samples, organs or whole multicellular organisms. Preferably the cells are prokaryotic or eukaryotic cell lines.
If prokaryotic cells are used according to the invention, the cells are preferably bacteria such as Escherichia coli, such as BL21, BL21(DE3), W3110, MG1655, RB791, RV308, or Bacillus megaterium, such as QM B1551, PV361, DSM319, or Pseudomonas, such as P. aeruginosa, P. putida, P. fluorescens, P. alcaligenes, P. aeruginosa PAO1-LAC, P. putida KT2440, or Streptomyces, such as S. coelicolor A3, S. avermitilis, S. griseus, S. scabies, S. lividans TK24, S. lividans 1326. Examples of E. coli include those derived from Escherichia coli K12 strain, specifically, HMS 174, HMS174 (DE3), NM533, XL1-Blue, C600, DH1, HB101, JM109, as well as those derived from B-strains, specifically BL-21, BL21 (DE3) and the like. In general also derivatives such as modified prokaryotic cells such as bacteria, are suitable for use in the invention. Such modification for example might be the deletion or inactivation of proteases, or deletion or inactivation of other genes.
If eukaryotic cells are use according to the invention, the cells are preferably yeast cells, filamentous fungal cells, insect cells, mammal cells or human cells.
Yeast cells preferably are methylotrophic yeasts (=yeast cells that can utilize methanol as a carbon and energy source) such as Komagataella pastoris=Pichia pastoris, P. methanolica, H. polymorpa, O. minuta, C. biodinii or non-methylotrophic yeasts such as Saccharomyces cerevisiae, Kluyveromyces lactis, P. Stipitis, Yarrowia lipolytica, Z. rouxii, Z. bailii, A. adeninivorans, Kluyveromyces marxianus, Schizosaccharomyces pombe and Arxula adeninivorans. Examples for Pichia pastoris strains useful in the present invention are X33 and its subtypes GS115, KM71, KM71H; CBS7435 (mut+) and its subtypes CBS7435 muts, CBS7435 mutsdeltaArg, CBS7435 mutsdeltaHis, CBS7435 mutsdeltaArg, deltaHis, CBS7435 muts PDI+, CBS 704 (=NRRL Y-1603=DSMZ 70382), CBS 2612 (=NRRL Y-7556), CBS 9173-9189 and DSMZ 70877, PPS-9010 (available from ATUM, formerly DNA2.0, Newark, CA, USA) and PPS-9016 (available from ATUM, formerly DNA2.0, Newark, CA, USA) as well as mutants thereof. In general also derivatives of such yeast cells, such as for example modified yeast cells, are suitable for use in the invention. Such modification for example might be the deletion or inactivation of yeast proteases, or the deletion or inactivation of other genes such as for example the ssn6-like gene (for details see WO2016139279A1) or the deletion of the so called killer plasmids from the yeast genome, especially from the P. pastoris or the S. cerevisiae genome (Sturmberger et al., J Biotechnol., 2016, 235:121-131).
Filamentous fungal cells are preferably Aspergillus such as, A. niger, A. oryzae, A. terreus, A. awamori, A. nidulans, or Trichoderma such as, T. reesei, T. reesei QM9414, T. reesei RUT-C30, T. reesei QM6a, T. atroviride, T. harzianum, T. virens, T. asperellum, T. longibrachiatum, or Penicillium such as P. purpurogenum, P. funiculosum, Penicillium (Talaromyces) emersonii, P. camemberti and P. roqueforti and their derivatives
Insect cells are preferably Sf9 or Sf21 cells (both from Spondoptera frugiperda), High-Five-cells (same as Hi5, same as High-Five BTI-TN-5B1-4) or Tn-368 cells (both from Trichoplusia ni), or Se301 cells (from Spondoptera exigua) and their derivatives.
Mammalian cells are preferably CHO (Chinese Hamster Ovary=CHO) cells, such as CHO-K1, CHO-DXB11, CHO-S, CHO-DG44 and their derivatives.
Human cells are preferably HEK293 (Human Embryonic Kidney=HEK) cells, such as HT-1080, PER.C6, HKB-11, CAP and HuH-7 and their derivatives.
Cells and cell lines can be obtained from various sources such as tissue culture collections such as the American Type Culture Collection (ATCC), 10801 University Boulevard, Manassas, VA 20110, USA, Deutsche Sammlung von Mikroorganismen and Zellkulturen GmbH (DSMZ), Inhoffenstraße 7B, 38124 Braunschweig, Germany, Centraalbureau voor Schimmelcultures (CBS), Uppsalalaan 8, 3584 CT Utrecht (Utrecht), Nederland, The Coli Genetic Stock Center (CGSC), 730 Kline Biology Tower, Dept. of Molecular, Cellular, and Developmental Biology, 266 Whitney Ave., PO box 208103, Yale University, New Haven, CT 06520-8103, USA or from commercial vendors such as Merck KGaA, Frankfurter Straße 250, 64293 Darmstadt, Germany, GE Healthcare, Chalfont St Giles, Buckinghamshire, Great Britain, Thermo Fischer Scientific, 168 Third Avenue, Waltham, MA USA 02451, etc.
Escherichia
coli *
E. coli
E. coli
E. coli
Saccharomyces
S. cerevisiae
cerevisiae
S. cerevisiae
S. cerevisiae
S. cerevisiae
S. cerevisiae
S. cerevisiae
S. cerevisiae
S. cerevisiae
S. cerevisiae
S. cerevisiae
Pichia
P. pastoris
pastoris**
P. pastoris
P. pastoris
P. pastoris
P. pastoris
P. pastoris
Homo
sapiens
Homo sapiens
P. pastoris
Oryctolagus
cuniculus
Rous
Sarcoma
Cytomegalo
virus
Homo
sapiens
Y. lipolytica
Y. lipolytica
Y. lipolytica
Y. lipolytica
Y. lipolytica
Y. lipolytica
Y. lipolytica
A. niger
A. nidulans
A. nidulans
A. awamori
A. oryzae
A. nidulans
A. oryzae
A. nidulans
A. nidulans
A. nidulans
A. oryzae
A. niger
A. nidulans
A. oryzae
Trichoderma
reesei
T. reesei
T. reesei
T. reesei
T. reesei
T. reesei
T. reesei
T. reesei
T. reesei
T. reesei
Bacillus
Bacillus
megaterium
megaterium
E. coli
Bacillus
amyloliquefaciens
B.
megaterium
B.
megaterium
E. coli
E. coli
E. coli
E. coli
E. coli
E. coli
E. coli
E. coli
S. cerevisiae
Saccharomyces
cerevisiae
Homo sapiens
B. subtilis
K. lactis
Saccharomyces
cerevisiae
P. pastoris
Pichia Pastoris
Homo sapiens
Saccharomyces
cerevisiae
Saccharomyces
cerevisiae
Pichia Pastoris
Saccharomyces
cerevisiae
Bacillus
megaterium
Leuconostoc
mesenteroides
Bacillus
amyloliquefaciens
Bacillus licheniformis
E. coli
E. coli
E. coli
E. coli
S. cerevisiae
S. cerevisiae
S. cerevisiae
S. cerevisiae
P. Pastoris
Pichia Pastoris
S. cerevisiae
S. cerevisiae
S. cerevisiae
Oryctolagus cuniculus
Molecular biologic techniques, such as cloning, transfection, determination of copy numbers of the transfected expression cassettes, design and chemical synthesis of vectors, use and choice of vector elements such as origins of replications, antibiotic resistances, selection markers, promoters, signal sequences, terminators, etc., cell culture techniques, protein expression techniques including viral techniques for example used for the Bacculovirus system, etc., quantitative and semi-quantitative determination of protein expression, etc. are all standard laboratory methods and are known to the skilled person. Protocols can be obtained from standard text books and laboratory manuals, for example from M. R. Green, J. Sambrook, 2013, Molecular cloning: a laboratory manual, Cold Spring Harbor, N.Y.; Current Protocols in Protein Science, John Wiley & Sons Inc. ISSN 1934-3655; Current Protocols in Molecular Biology, John Wiley & Sons Inc. ISSN 1934-3639; Advanced Technologies for Protein Complex Production and Characterization, Editor M. Cristina Vega, Springer, 2016, ISSN 0065-2598; Bacculovirus and Insect Cell Expression protocols, Third Edition, Editor David W. Murhammer, Humana Press, 2016, ISSN 1064-3745; Recombinant Gene Expression, Reviews and Protocol, Third Edition, Editor A. Lorence, Humana Press, ISSN 1064-3745, etc.
Measuring of Host Cell Expression of POI
In order to determine whether a host cell transfected with different expression cassettes according to the invention expresses higher quantities of said POI as compared to a host cell comprising the same number of expression cassettes with identical expression cassette sequences, there are known a number a standard testing systems, such as ELISA (enzyme-linked immunosorbent assay), ELIspot assays (Enzyme Linked Immuno Spot Assay), surface plasmon resonance assays (Biacore Life Science, now GE Healthcare), protein chip assays, quantitative reverse-transkriptase PCR (qRT-PCR), desitometric measurement of western-blots, coomassie blue or silver-stained SDS-PAGE gels, quantitative mass spectrometry, calculation of the peak-area under the corresponding POI-peak of a chromatogram of a POI sample, etc.). Suitable protocols for carrying out said methods are known to the skilled person and can be for instance found in M. R. Green, J. Sambrook, 2013, Molecular cloning: a laboratory manual, Cold Spring Harbor, N.Y., or in Current Protocols in Protein Science, John Wiley & Sons Inc. ISSN 1934-3655.
Measuring of Genetic Stability
Genetic stability for example can be measured by determining the copy number of different expression cassettes according to the invention in the host cells of the invention as compared to the copy number of identical expression known in the art in cassettes in host cells. Copy numbers of expression cassettes for example can be determined by quantitative PCR (qPCR). Primers for qPCR can be designed in a way that they amplify the complete or a part of the expression cassettes. If the copy number of the expression cassettes alters after a number of cell generations, this proofs genomic instability. Furthermore the sequence length of the qPCR products can be determined by for example agarose gel electrophoresis. If deletions or duplications of parts of the expression products occurred the sequence length of the qPCR products is altered accordingly, which also indicates genomic instability. Other methods to determine copy numbers of expression cassettes are for example are Southern blots or Fluorescence In Situ Hybridization (FISH). Suitable protocols for carrying out said method are known to the skilled person and can be for instance found in M. R. Green, J. Sambrook, 2013, Molecular cloning: a laboratory manual, Cold Spring Harbor, N.Y., or in Current Protocols in Molecular Biology, John Wiley & Sons Inc. ISSN 1934-3639.
Yeast vector Y391_1 xGOI in addition to vector back bone contains the following expression cassette for a GOI, which in this case is a single-chain antibody (scFV):
Yeast vector Y393_2 xGOI in addition to vector back bone contains the following expression cassette for a GOI, which in both cases codes for the same amino acid sequence of a single-chain antibody (scFV):
Yeast vector Y394_3 xGOI in addition to vector back bone contains the following expression cassette for a GOI, which in in all three case codes for the same amino acid sequence of a single-chain antibody (scFV):
Yeast vector Y395_4 xGOI in addition to vector back bone contains the following expression cassette for a GOI, which in in all four case codes for the same amino acid sequence of a single-chain antibody (scFV):
Sequences of the expression vectors from
A) Yeast vector Y391_1 xGOI (SEQ-ID NO.: 1)
B) Yeast vector Y393_2 xGOI (SEQ-ID NO.: 2)
C) Yeast vector Y394_3 xGOI (SEQ-ID NO.: 3)
D) Yeast vector Y395_4 xGOI (SEQ-ID NO.: 4)
Vector maps of the vectors used for transfection of mammalian cells (CHO cells), each vector comprising a single expression cassette, wherein the expression cassettes comprise as a GOI the sequence of a fusion protein consisting of a constant region of an antibody fused to the ligand-binding domain of a TNF-receptor 2. Each vector furthermore comprises the metabolic selection marker dihydrofolate reductase (DHFR), an enzyme which for example allows CHO (chinese hamster ovary) cells to grow in cell culture medium lacking thymidine, thereby allowing to select CHO (or other cells) transfected with DHFR-comprising vectors from non-transfected cells. Furthermore each vector comprises the sequence of the neomycin resistance gene (NeoR), which allows to select transformed cells by using the antibiotic neomycin. Furthermore each vector comprises another antibiotic resistance gene selected from Ampicillin Resistance (AmpR), Spectromycin Resistance (SpectR) and Chloramphenicol Resistance (CmR). Each vector comprises a different promoter, a different signal sequence and a different terminator sequence within the expression cassette for the GOI.
Sequences of the expression vectors from
A) Mammalian vector pNT-MG001 (SEQ-ID NO.: 5)
B) Mammalian vector pNT-MG002 (SEQ-ID NO.: 6)
C) Mammalian vector pNT-MG003 (SEQ-ID NO.: 7)
D) Mammalian vector pNT-MG004 (SEQ-ID NO.: 8)
Methods for Pichia Pastoris Cells
Generation of yeast vectors: The set of vectors contains one vector with one expression cassette, one vector with two different expression cassettes, one vector with three different expression cassettes and one vector with four different expressions cassettes. In the vector set each of the four different expression cassettes has a different nucleotide sequence of the GOI but the resulting POI has an identical mature amino acid sequence, and each of the four different expression cassettes comprises a different promoter nucleotide sequence, a different signal sequence, and a different terminator nucleotide sequence.
The four different nucleotide sequence of the POI are designed by use of the degenerated genetic code. The POI is a single chain antibody (scFV, ESBA1845=scFv=single chain variable fragment=artificial antibody fragment comprising a single polypeptide chain including its antigen binding domain). There are used 4 different variants of said scFv termed scFv_var1, scFv_var2, scFv_var3, and scFv_var4, which all code for an identical amino acid sequence but have different nucleotide sequences due to the use of the degenerated genetic code. The promoter sequences used are lectin-like protein promoter from Pichia Pastoris (pLLP), the GAP-promoter (pGAP), the ADH-promoter (pADH), and the TEF-promoter (pTEF). The secretion signal sequences used for the POI are the signal sequence of lectin-like protein from P. pastoris (LLPSS), the signal sequence of mating factor alpha-4 from S. cerevisiae (MFa4SS), the signal sequence of human serum albumin ((HSASS), and the signal sequence of mating factor alpha-2 of S. cerevisiae (MFa2SS). The termination sequences are the Alcohol dehydrogenase (ADHTT), the termination sequence of the lectin-like protein from Pichia Pastoris (LLPTT), the termination sequence of cytochrome c1 terminator (cyc1TT), and the termination sequence of Alcohol oxidase (AOXTT). The yeast cell selection marker used in all vectors is Zeocin-r, expressed by use of the ILV5-promoter, the EM72-signal sequence and the AOD terminator. The pUC ori is used in all yeast expression vectors.
Generation of Vectors
The four different expression vectors are designed as depicted in the vector maps of
Transfection of P. pastoris
The four different vectors are transfected individually into Pichia pastoris yeast cell SSS1. This yeast cells is described in patent application WO2016139279A1 and is genetically identical to Pichia pastoris CBS 7435 and identical to NRRL Y-11430, except that the ssn6-like gene is disrupted at position 807,480 of chromosome 1 of the P. pastoris CBS 7435 genome by insertion of the expression cassette as described in WO 2016/139270 A1. The complete sequence of CBS 7435 is disclosed in Journal of Biotechnology, published in 2011, Vol. 154, page 312-320 year 2011. The nucleotide sequences are published in GenBank under the following Accession Numbers: Chromosome 1: FR839628.1; Chromosome 2: FR839629.1; Chromosome 3: FR839630.1; Chromosome 4: FR839631.1; Mitochondrion: FR839632.1
Expression of POI in 48-Deep Well Plates, Semi Quantitative Measurement of POI
The transfections are streaked out and individual transformed clones are cultured in synthetic medium. After 70 hours cell culture supernatant is removed from the culture, yeast cells and cell debris is removed from the supernatant by centrifugation and 10 μl of supernatant is loaded and electrophoretically separated on SDS-PAGE (Novex NuPage 4-12%, Invitrogen) gels. After staining with coomassie blue or after silver staining of the SDS-PAGE gels the protein band of the scFv (ESBA1845), having a molecular weight of about 26 kDa is semi-quantitatively determined by scanning and densitometric measurement of the protein band in the gels. The signal intensity gives an estimate of the expression rate of the scFv protein.
Concentration of POI in the supernatant was determined by applying automated capillary electrophoreses (LabChip GXII-Touch, Perkin Elmer, Waltham, MA, USA) according to manufacturer's recommendations.
Expression of POI in P. pastoris in Shaker Flasks, Determination of Genetic Stability
The individual P. pastoris clones of are either cultured in shaker flasks for 4 weeks. The cell culture is diluted with medium when needed in order to ensure growth of the cells. Before and after this 4 week-culture the copy number of the expression cassettes is determined by for example quantitative PCR (qPCR). Optionally or in addition the sequence of the expression cassettes is determined by sequencing and the correct size of the PCR-amplified nucleic acids is determined by agarose gel electrophoresis, according to methods known in the art. These experiments are performed in order to determine genetic stability of the clones.
Methods for CHO Cells
Generation of Vectors
Four different CHO expression vectors are designed, each coding for the same POI. Two different nucleotide sequences coding for the same amino acid sequence of the POI were used (Etanercept var1 and Etanercept var2). The four different vectors each contain only one expression cassette coding for the same POI, one expression cassette for neomycin (antibiotic selection marker), an expression cassette for another antibiotic resistance, and one expression cassette for DHFR (metabolic selection marker needed for growth of the CHO cell line). Within each of the four different vectors different promoters and terminators are used for the GOI, the neomycin selection marker, and the DHFR, meaning that within a vector different promoters and terminators are used. The nucleotide sequence of the neomycin selection marker and the DHFR is identical in all four vectors. All vectors are chemically synthesized using the GeneArt synthesis service from (Geneart AG, Regensburg, Germany, now belonging to Life Technologies). Details on the vector elements of the different vectors can be found in Table 6, vector maps are depicted in
The CHO-vectors each time comprise only one expression cassette, which expression cassette is different in each of the four vectors. In detail each expression cassette uses a different promoter, a different signal sequence and a different terminator. The POI is always the same. Furthermore each vector comprises an expression cassettes for the metabolic selection marker DHFR (each time coded by the same nucleotide sequence), an expression cassette for the antibiotic selection marker Neomycin R (NeoR) (each time coded by the same nucleotide sequence), and expression cassette coding for another antibiotic selection maker which is either a different selection marker, namely Ampicillin Resistance (AmpR), Spectromycin Resistance (SpectR) or Chloramphenicol Resistance (CmR), or which selection marker is the same selection marker but inserted into the vector in different orientation, e.g. in this case the Ampicillin Resistance marker in two different orientation within vector pNT-MG001 and pNT-MG004. Furthermore all 4 vectors contain as a vector backbone a phage f1 sequence an origin of replication, either pBR322 or p16A, wherein also pBR322 is used in two different orientations within the vectors. An overview of the different vector elements of the mammalian vectors is given in Table 6 below.
The nucleotide sequences of the vectors pNT-MG001 to pNT-MG004 are given in
Table 7 shows all features of the used expression vectors Y391_1 xGOI, Y393_2 xGOI, Y394_3 xGOI, Y394_4 xGOI, pNT-MG001, pNT-MG002, pNT-MG003, and pNT-MG004.
Obtaining Stable Cell Lines
CHO (DHFR) cells are transfected with either an individual vector of the four vectors or with a mix of all four vectors. Stable transfections are performed using Amaxa Nucleofection kit (Lonza AG, Switzerland) following manufacturer's instructions. Briefly, 5×106 CHO cells are transfected with 3 μg of linearized vector DNA per transfection. All vectors are either transfected individually, or as a mix of all four vectors combined. After transfection, growth medium is added and cells are grown in a 10% CO2 atmosphere for 24-48 h at 37° C. with shaking at 110 rpm. Following the recovery of the cells, two selection rounds are performed. Firstly, cells are selected using medium containing G418, followed by selection using methotrexate (MTX) after 90% cell viability is reached. Cells are maintained under MTX selection until cell viability reaches more than 90% (usually 3-4 weeks post-transfection). Throughout the selection period, cells are cultured using fresh medium twice per week. Single cell cloning is performed a using standard limiting dilution cloning approach. Individual clones were selected based on vector copy number (i.e. at least two copies per clone).
From each transfection individual clones are selected and tested for expression rate (titer) of the POI, titer stability of the clone over time, leader peptide cleavage per clone, and genetic stability of the clone over time. With titer is meant concentration (mg/L) of recombinant POI, in this case Etanercept, in tissue culture medium.
Analysis of Vector Copy Numbers in Cell Lines
Integrated vector copy number are assessed using quantitative PCR (qPCR). Relative quantification is used to estimate the number of integrated expression constructs per clone. Repeating the copy number assessment after 3 months is also used to determine, whether copy number of the POI within the individual cell lines is stable over time. Separation of the PCR-products by agarose gel electrophoresis further allows to determine if the size of the PCR-amplified polynucleotide is stable over time, which is another indicator of genetic stability of the individual clones of the cell lines. High resolution melting analysis of PCR-products can be used to confirm the identity of the PCR products.
Analysis of Production of POI by Cell Lines
A 14-day generic fed-batch process is applied for productivity assessment. All fed-batch processes are performed in 100 mL serum-free medium. The medium is inoculated with 4×105 of viable cells/mL and cell culture is incubated in 10% CO2 atmosphere at 37° C. with shaking at 110 rpm (50 mm shaking diameter) and with temperature shift to 33° C. on day 7. Cell concentration and viability are measured using a Vi-Cell XR analyzer. Titers are measured on cultivation days 7, 10 and 14 using Cedex system (Roche Diagnostics Deutschland GmbH, Mannheim, Germany). The measurement is based on a turbidimetric method using antibodies directed against the human Fc region. Harvests are collected at the end of the fed-batch processes and purified using Protein A chromatography.
Analysis of Genetic Stability of the Cell Lines
Individual cell clones are seeded at the density of 3×105 cell/ml in 75 cm3 flasks in suspension culture in the absence of selective pressure. Productivity testing is done every 6 weeks over a period of 3 months. Expression of POI is measured using standard methods know the skilled person such as ELISA assays, ELISPOT, quantitative western blotting, quantitative mass spectrometry, surface plasmon resonance (e.g. Biacore, Sweden), etc.
Analysis of Signal Peptide Cleavage, by the Cell Lines
Analysis of the correct leader peptide cleavage is done by peptide sequencing using mass spectrometry or Edman degradation. Signal peptide miscleavage can be assessed using intact mass measurement. Protein is first de-glycosylated with N-glycosidase (PNGase) F and subsequently intact mass of the protein is analyzed using LC-MS on a high-resolution mass spectrometer. Masses are identified according to calculated theoretical masses of the protein and signal peptide adducts and proportion of miscleaved signal peptide is calculated from peak intensities.
All methods described or mentioned herein for Pichia pastoris yeast cells, CHO mammalian cells, as well as for other types of cells according to the invention, are standard methods know to the skilled person. Such methods are for example described in standard laboratory method manuals such as for instance in M. R. Green, J. Sambrook, 2013, “Molecular cloning: a laboratory manual”, Cold Spring Harbor, N.Y., or in “Current Protocols in Molecular Biology”, John Wiley & Sons Inc. ISSN 1934-3639 and “Current protocols in Protein Science”, John Wiley & Sons Inc. ISSN 1934-3655, or in other titles of the “Current Protocols” series of John Wiley & Sons Inc.
The invention does not include the by chance possible presence of two or more expression cassettes within an individual cell of a cell library, which expression cassettes comprise the same GOI but with a different coding sequence for that same expression cassette, wherein said cell library is intended to screen for an GOI coding sequence with a maximal expression rate in the cell line used for construction of the cell library.
| Number | Date | Country | Kind |
|---|---|---|---|
| 17187552 | Aug 2017 | EP | regional |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/EP2018/072687 | 8/22/2018 | WO |
| Publishing Document | Publishing Date | Country | Kind |
|---|---|---|---|
| WO2019/038338 | 2/28/2019 | WO | A |
| Number | Name | Date | Kind |
|---|---|---|---|
| 20100120623 | Jorgensen | May 2010 | A1 |
| Number | Date | Country |
|---|---|---|
| 2003018771 | Mar 2003 | WO |
| 2004033693 | Apr 2004 | WO |
| 2008077881 | Jul 2008 | WO |
| 2014088693 | Jun 2014 | WO |
| 2016005931 | Jan 2016 | WO |
| 2016139279 | Sep 2016 | WO |
| 2016073794 | May 2017 | WO |
| Entry |
|---|
| Kudla et al. 2009; Codon-sequence determinants of gene expression in Escherichia coli. Science 324; 255-258 plus Supporting Online Material pp. 1-21. |
| Mitra et al. 2016; Synonymous codons influencing gene expression in organisms, Research and Reports in Biochemistry. 2016:6 pp. 57-65. |
| Altschul, Stpehen F., et al., Nucleic Acids Research, 1997, vol. 25, No. 17, pp. 3389-3402. |
| Brachmann, Carrie Baker, et. al., Yeast, 1998, vol. 14, pp. 115-132. |
| Ho, Steven C.L., et al, PLOS, 2013, vol. 8, Issue 5. |
| Kudla, Grzegorz, et al, Science, 2009, vol. 324, pp. 255-258. |
| Kueberl, Andreas, et al., Journal of Biotechnology, 2011, No. 154, pp. 312-320. |
| Lee, Jung-Lim, et al., Journal of Microbiology and Technology, vol. 18, No. 5, 2008, pp. 926-932. |
| Sturmberger, Lukas, et al., Journal of Biotechnology., 2016, No. 235, pp. 121-131. |
| International Search Report and Written Opinion for PCT/EP2018/072687, 15 pages. |
| Number | Date | Country | |
|---|---|---|---|
| 20220025387 A1 | Jan 2022 | US |
