Codon optimization of antibody sequences

Abstract

Methods of optimizing an antibody coding sequence, comprising providing a nucleic acid sequence comprising a coding region encoding an antibody and producing at least one synonymous mutation wherein the mutation converts at least one codon into a codon more commonly used in a set of genes consisting of a plurality of nucleic acid sequences encoding antibodies are provided. Nucleic acid molecule systems comprising an optimized coding sequence are also provided.

Description

CODON OPTIMIZATION OF ANTIBODY SEQUENCESCROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of priority of Israeli Patent Application No. 305852, filed September 11, 2023, the contents of which are all incorporated herein by reference in their entirety.REFERENCE TO AN ELECTRONIC SEQUENCE LISTING

[0002] The contents of the electronic sequence listing (RMT-P-031-PCT.xml; Size: 15,718 bytes; and Date of Creation: September 10, 2024) is herein incorporated by reference in its entirety.FIELD OF INVENTION

[0003] The present invention is in the field of antibody optimization.BACKGROUND OF THE INVENTION

[0004] Antibodies are the adapted part of the immune system. Antibody genes harbor sophisticated and novel strategies for dealing with extremely large amounts of information. These include strategies for information storage, expression and evolution. Antibody genes are part of multi-gene families among others such as histone, ribosomal RNA, tRNAs and more. Because antibody genes’ development, expression and regulation are unique in many ways, it was thought decades ago, that the immune system has evolved unique genetic strategies to protect the integrity of the vertebrate organism. Other scientists disagreed and claimed that families of genes coding for antibody molecules display unusual evolutionary features that are shared by other multigene families, such as those mentioned above (Hood, et al., “The organization, expression, and evolution of antibody genes and other multigene families”, Annu Rev Genet., 1975; 9:305-53). However, unlike other gene families, the antibody gene repertoire is huge and regulated by unique mechanism of processing that enables the expression of a single gene combination for each B cell. A new method of antibody generation, as well as the improved antibodies, which takes into account the unique regulatory mechanism present in antibody genes is greatly needed.SUMMARY OF THE INVENTION

[0005] The present invention provides methods of optimizing an antibody coding sequence, comprising providing a nucleic acid sequence comprising a coding region encoding an antibody and producing at least one synonymous mutation wherein the mutation converts at least one codon into a codon more commonly used in a set of genes consisting of a plurality of nucleic acid sequences encoding antibodies are provided. Nucleic acid molecule systems comprising an optimized coding sequence are also provided.

[0006] According to a first aspect, there is provided a method of optimizing an antibody coding sequence for translation, the method comprising: a. providing a nucleic acid sequence comprising a coding region encoding an antibody; and b. producing at least one synonymous mutation in the provided sequence thereby producing an optimized sequence, wherein the synonymous mutation converts at least one codon into a codon more commonly used in a set of genes consisting of a plurality of nucleic acid sequences encoding antibodies, wherein the set of genes is not a full genome; thereby optimizing an antibody coding sequence.

[0007] According to some embodiments, the provided nucleic acid sequence encodes a variable region of an antibody heavy chain or an antibody light chain.

[0008] According to some embodiments, the at least one synonymous mutation is not made in the first 15 codons of the coding region.

[0009] According to some embodiments, the set of genes consists of a plurality of nucleic acid sequences encoding human antibodies.

[0010] According to some embodiments, the synonymous mutation comprises mutation of a codon to a preferred codon of Table 5.[Oi l] According to some embodiments, the synonymous mutation comprises at least one of: mutation of an alanine codon to GCC, mutation of an arginine codon to AGA, mutation of an asparagine codon to AAC, mutation of an aspartic acid codon to GAC, mutation of a cysteine codon to TGT, mutation of a glutamine codon to CAG, mutation of a glutamic acidcodon to GAG, mutation of glycine codon to GGG, mutation of a histidine codon to CAC, mutation of an isoleucine codon to ATC, mutation of a leucine codon to CTG, mutation of lysine codon to A AG, mutation of a phenylalanine codon to TTC, mutation of a proline codon to CCC, mutation of a serine codon to TCC, mutation of a threonine codon to ACC, mutation of a tyrosine codon to TAC and mutation of a valine codon to GTG.

[0012] According to some embodiments, the set of genes consists of a plurality of nucleic acid sequences encoding mouse antibodies.

[0013] According to some embodiments, the synonymous mutation comprises mutation of a codon to a preferred codon of Table 6.

[0014] According to some embodiments, the synonymous mutation comprises at least one of: mutation of an alanine codon to GCT, mutation of an arginine codon to AGG, mutation of an asparagine codon to AAC, mutation of a aspartic acid codon to GAT, mutation of a cysteine codon to TGC, mutation of a glutamine codon to CAG, mutation of a glutamic acid codon to GAG, mutation of glycine codon to GGA, mutation of a histidine codon to CAC, mutation of an isoleucine codon to ATC, mutation of a leucine codon to CTG, mutation of lysine codon to AAG, mutation of a phenylalanine codon to TTC, mutation of a proline codon to CCA, mutation of a serine codon to TCT, mutation of a threonine codon to ACA, mutation of a tyrosine codon to TAC and mutation of a valine codon to GTC.

[0015] According to some embodiments, the at least one synonymous mutation is at least 10 synonymous mutations.

[0016] According to some embodiments, the at least one synonymous mutation is at least 100 synonymous mutations.

[0017] According to some embodiments, the at least one synonymous mutation is every possible synonymous mutation which converts a codon to a more commonly used codon in the set of genes, with the exception of synonymous mutations within the first 15 codons of the coding region.

[0018] According to some embodiments, the every possible synonymous mutation is every possible synonymous mutation within the variable region of the antibody’s heavy chain, or every possible synonymous mutation within the variable region of the antibody’s light chain while not mutating the first 15 codons of the coding region.

[0019] According to some embodiments, every possible synonymous mutation is every possible synonymous mutation within the variable region of the antibody’s heavy chain andthe variable region of the antibody’s light chain while not mutating the first 15 codons of the coding region.

[0020] According to some embodiments, the at least one synonymous mutation increases translation of the optimized sequence as compared to the provided sequence in a target cell.

[0021] According to some embodiments, the optimizing for translation is optimizing translation of the antibody in a target cell and wherein the full genome is the full genome of the target cell.

[0022] According to some embodiments, the target cell is a mammalian cell.

[0023] According to some embodiments, the mammalian cell is a Chinese Hamster Ovary (CHO) cell.

[0024] According to some embodiments, the optimizing is optimizing translation of the antibody in CHO cells.

[0025] According to another aspect, there is provided a nucleic acid molecule system, comprising: a. a first nucleic acid molecule comprising a first coding region encoding an antibody heavy chain variable region; and b. a second nucleic acid molecule comprising a second coding region encoding a light chain variable region of the antibody; wherein at least one of the first and second coding regions comprises at least one synonymous mutation wherein the synonymous mutation converts at least one codon into a codon more commonly used in a set of genes consisting of a plurality of nucleic acid sequences encoding antibodies, wherein the set of genes is not a full genome.

[0026] According to some embodiments, the nucleic acid system is optimized for protein expression in CHO cells and wherein the full genome is a CHO cell full genome.

[0027] According to some embodiments, the at least one synonymous mutation is not made in the first 15 codons of the coding region.

[0028] According to some embodiments, the set of genes consists of a plurality of nucleic acid sequences encoding human antibodies.

[0029] According to some embodiments, the synonymous mutation comprises mutation of a codon to a preferred codon of Table 5.

[0030] According to some embodiments, the synonymous mutation comprises at least one of: mutation of an alanine codon to GCC, mutation of an arginine codon to AGA, mutation of an asparagine codon to AAC, mutation of a aspartic acid codon to GAC, mutation of a cysteine codon to TGT, mutation of a glutamine codon to CAG, mutation of a glutamic acid codon to GAG, mutation of glycine codon to GGG, mutation of a histidine codon to CAC, mutation of an isoleucine codon to ATC, mutation of a leucine codon to CTG, mutation of lysine codon to AAG, mutation of a phenylalanine codon to TTC, mutation of a proline codon to CCC, mutation of a serine codon to TCC, mutation of a threonine codon to ACC, mutation of a tyrosine codon to TAC and mutation of a valine codon to GTG.

[0031] According to some embodiments, the set of genes consists of a plurality of nucleic acid sequences encoding mouse antibodies.

[0032] According to some embodiments, the synonymous mutation comprises mutation of a codon to a preferred codon of Table 6.

[0033] According to some embodiments, the synonymous mutation comprises at least one of: mutation of an alanine codon to GCT, mutation of an arginine codon to AGG, mutation of an asparagine codon to AAC, mutation of a aspartic acid codon to GAT, mutation of a cysteine codon to TGC, mutation of a glutamine codon to CAG, mutation of a glutamic acid codon to GAG, mutation of glycine codon to GGA, mutation of a histidine codon to CAC, mutation of an isoleucine codon to ATC, mutation of a leucine codon to CTG, mutation of lysine codon to AAG, mutation of a phenylalanine codon to TTC, mutation of a proline codon to CCA, mutation of a serine codon to TCT, mutation of a threonine codon to ACA, mutation of a tyrosine codon to TAC and mutation of a valine codon to GTC.

[0034] According to some embodiments, the at least one synonymous mutation is at least 10 synonymous mutations.

[0035] According to some embodiments, the at least one synonymous mutation is at least 100 synonymous mutations.

[0036] According to some embodiments, the at least one synonymous mutation is every possible synonymous mutation which converts a codon to a more commonly used codon in the set of genes, with the exception of synonymous mutations within the first 15 codons of the coding region.

[0037] According to some embodiments, the every possible synonymous mutation is every possible synonymous mutation within the variable region of the antibody’s heavy chain, or every possible synonymous mutation within the variable region of the antibody’s light chain while not mutating the first 15 codons of the coding region.

[0038] According to some embodiments, every possible synonymous mutation is every possible synonymous mutation within the variable region of the antibody’s heavy chain and the variable region of the antibody’s light chain while not mutating the first 15 codons of the coding region.

[0039] According to another aspect, there is provided a CHO cell comprising a nucleic acid molecule system of the invention.

[0040] Further embodiments and the full scope of applicability of the present invention will become apparent from the detailed description given hereinafter. However, it should be understood that the detailed description and specific examples, while indicating preferred embodiments of the invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description.BRIEF DESCRIPTION OF THE DRAWINGS

[0041] Figure 1: Study workflow. The study included three phases: (1) Data Preprocessing, involving the collection and preprocessing of antibody, genomic, and protein family sequences; (2) Data Analysis, including filtering sequences, computing codon usage, and performing detailed analyses as described in the paper; and (3) Expressing Antibodies in CHO cells, where antibodies designed based on the analysis results are expressed and their activity is assessed.

[0042] Figures 2A-2E. Codon and amino acid analysis of unpaired human heavy chains, comparing approximately 1.9 billion sequences to the entire human genome’s coding sequences. (2A) Comparison of codon distributions for just antibodies or the entire human genome. (2B) Comparison of codon frequencies within corresponding amino acids for just antibodies or the entire human genome. (2C) Comparison of codon frequencies within corresponding amino acids, normalized against the maximum codon frequency for each amino acid for just antibodies or the entire human genome. (2D) Comparison of amino acid frequencies for just antibodies or the entire human genome. (2E) Codon preference profilebased on CAI weights, arranged in descending order of CAI values from highest (1) to lowest (0) for just antibodies or the entire human genome.

[0043] Figures 3A-3E. Codon and amino acid analysis of G0:0030234 (protein family of enzyme regulator activity) human sequences, compared to the entire human genome’s coding sequences. (3A) Comparison of codon distributions for G0:0030234 or the entire human genome. (3B) Comparison of codon frequencies within corresponding amino acids for G0:0030234 or the entire human genome. (3C) Comparison of codon frequencies within corresponding amino acids, normalized against the maximum codon frequency for each amino acid for G0:0030234 or the entire human genome. (3D) Comparison of amino acid frequencies for G0:0030234 or the entire human genome. (3E) Codon preference profile based on CAI weights, arranged in descending order of CAI values from highest (1) to lowest (0) for G0:0030234 or the entire human genome.

[0044] Figure 4: Comparative analysis of ENC in unpaired human heavy chain sequences. Comparison of ENC values between a subsample of 10,000 sequences and 100 dinucleotide-randomized antibody sequence datasets derived from the same subsample. The randomization preserves the distribution of dinucleotides and the order and content of amino acids. Note that this result is consistent across multiple other subsamples of the antibody sequence data.

[0045] Figure 5. Spatial analysis of unpaired human heavy chain sequences, examining CAI weights across the antibody regions, based on a sample of 10,000 sequences. The alignment was performed using ANARCI on a subsample containing only productive sequences with complete V(D)J regions and fully numbered amino acids by ANARCI. Codons are grouped by their corresponding amino acids, with the codon having the highest value (1) colored in yellow.

[0046] Figure 6: Spatial analysis of unpaired human heavy chain sequences, examining the JSD distance between CAI weights for each region pair of the antibody, based on a sample of 10,000 sequences. The alignment was performed using ANARCI on a subsample containing only productive sequences with complete V(D)J regions and fully numbered amino acids by ANARCI. The p-values were calculated using VCUB randomization.

[0047] Figures 7A-7G. Analysis of transiently transfected cells expressing CB6 antibody variants. (7A) Experimental workflow: 3 variants of the CB6 anti-COVID-19 antibody sequence were synthesized and then transfected into Chinese hamster ovary (CHO) cells. Subsequently, antibody expression and binding activity were measured. Created withBioRender.com. CB6 image from the RCSB PDB (RCSB.org) of PDB ID 7C01. (7B) Antibody activity as determined by ELISA, measured by optical density (OD) at 450nm following two time points: 24 and 48 hours. (7C) Average cycle threshold (CT) of quantitative reverse transcription PCR (qRT-PCR) to assess mRNA levels. (7D) Average CT of quantitative PCR (qPCR) for evaluating antibody plasmid and GAPDH copy numbers. (7E) Fold change of antibody vs. GAPDH mRNA levels (48 hours). (7F) Fold change of antibody genes vs. GAPDH copy numbers. (7G) Ratio of mRNA levels to gene copy numbers. Each result is an average of 3 biological and 2 technical repeats. Mean of the light and heavy chain expression of each variant is provided.

[0048] Figures 8A-8E. Codon and amino acid analysis of PTHR24356 (protein family of serine / threonine-protein kinase) human sequences, compared to the entire human genome’s coding sequences. (8A) Comparison of codon distributions for PTHR24356 or the entire human genome. (8B) Comparison of codon frequencies within corresponding amino acids for PTHR24356 or the entire human genome. (8C) Comparison of codon frequencies within corresponding amino acids, normalized against the maximum codon frequency for each amino acid for PTHR24356 or the entire human genome. (8D) Comparison of amino acid frequencies for PTHR24356 or the entire human genome. (8E) Codon preference profile based on CAI weights, arranged in descending order of CAI values from highest (1) to lowest (0) for PTHR24356 or the entire human genome.

[0049] Figures 9A-9E. Codon and amino acid analysis of IPR000719 (protein family of protein kinase domain) human sequences, compared to the entire human genome’s coding sequences. (9A) Comparison of codon distributions for IPR000719 or the entire human genome. (9B) Comparison of codon frequencies within corresponding amino acids for IPR000719 or the entire human genome. (9C) Comparison of codon frequencies within corresponding amino acids, normalized against the maximum codon frequency for each amino acid for IPR000719 or the entire human genome. (9D) Comparison of amino acid frequencies for IPR000719 or the entire human genome. (9E) Codon preference profile based on CAI weights, arranged in descending order of CAI values from highest (1) to lowest (0) for IPR000719 or the entire human genome.

[0050] Figures 10A-10E. Codon and amino acid analysis of G0:0003823 (protein family of antigen binding) human sequences, compared to the entire human genome’s coding sequences. (10A) Comparison of codon distributions for G0:0003823 or the entire human genome. (10B) Comparison of codon frequencies within corresponding amino acids for G0:0003823 or the entire human genome. (IOC) Comparison of codon frequencies withincorresponding amino acids, normalized against the maximum codon frequency for each amino acid for G0:0003823 or the entire human genome. (10D) Comparison of amino acid frequencies for G0:0003823 or the entire human genome. (10E) Codon preference profile based on CAI weights, arranged in descending order of CAI values from highest (1) to lowest (0) for GG:0003823 or the entire human genome.

[0051] Figures 11A-11E. Codon and amino acid analysis of GG:0005215 (protein family of transporter activity) human sequences, compared to the entire human genome’s coding sequences. (11A) Comparison of codon distributions for G0:0005215 or the entire human genome. (11B) Comparison of codon frequencies within corresponding amino acids for G0:0005215 or the entire human genome. (11C) Comparison of codon frequencies within corresponding amino acids, normalized against the maximum codon frequency for each amino acid for G0:0005215 or the entire human genome. (11D) Comparison of amino acid frequencies for G0:0005215 or the entire human genome. (HE) Codon preference profile based on CAI weights, arranged in descending order of CAI values from highest (1) to lowest (0) for GO: 0005215 or the entire human genome.

[0052] Figures 12A-12E. Codon and amino acid analysis of PTHR18945 (protein family of neurotransmitter gated ion channel) human sequences, compared to the entire human genome’s coding sequences. (12A) Comparison of codon distributions for PTHR18945 or the entire human genome. (12B) Comparison of codon frequencies within corresponding amino acids for PTHR 18945 or the entire human genome. (12C) Comparison of codon frequencies within corresponding amino acids, normalized against the maximum codon frequency for each amino acid for PTHR 18945 or the entire human genome. (12D) Comparison of amino acid frequencies for PTHR18945 or the entire human genome. (12E) Codon preference profile based on CAI weights, arranged in descending order of CAI values from highest (1) to lowest (0) for PTHR18945 or the entire human genome.

[0053] Figures 13A-13E. Codon and amino acid analysis of IPR007110 (protein family of immunoglobulin-like domain) human sequences, compared to the entire human genome’s coding sequences. (13A) Comparison of codon distributions for IPR007110 or the entire human genome. (13B) Comparison of codon frequencies within corresponding amino acids for IPR007110 or the entire human genome. (13C) Comparison of codon frequencies within corresponding amino acids, normalized against the maximum codon frequency for each amino acid for IPR007110 or the entire human genome. (13D) Comparison of amino acid frequencies for IPR007110 or the entire human genome. (13E) Codon preference profilebased on CAI weights, arranged in descending order of CAI values from highest (1) to lowest (0) for IPR007110 or the entire human genome.

[0054] Figures 14A-14E. Codon and amino acid analysis of IPR001650 (protein family of helicase, C-terminal) human sequences, compared to the entire human genome’s coding sequences. (14A) Comparison of codon distributions for IPR007110 or the entire human genome. (14B) Comparison of codon frequencies within corresponding amino acids for IPR007110 or the entire human genome. (14C) Comparison of codon frequencies within corresponding amino acids, normalized against the maximum codon frequency for each amino acid for IPR007110 or the entire human genome. (14D) Comparison of amino acid frequencies for IPR007110 or the entire human genome. (14E) Codon preference profile based on CAI weights, arranged in descending order of CAI values from highest (1) to lowest (0) for IPR007110 or the entire human genome.

[0055] Figures 15A-15B: Sequence alignments of the 3 synthesized CB6 antibody variants. (15A) Alignment between CB6MOUSE_ANTIBODIES (Query) and CB6HUMAN_ANTIBODIES (Sbjct) light chains. (15B) Alignment between CB6CHINESE_HAMSTER_GENOME (Query) and CB6HUMAN_ANTIBODIES (Sbjct) light chains.

[0056] Figure 16: Schematic of plasmid RCK-CMP22 construction. Plasmid RCK- CMP22 was assembled by simultaneously ligating 6 DNA fragments, serving as the platform for cloning the 3 synthesized CB6 antibody variants (the top portion of the schematic is continued at the bottom).

[0057] Figure 17: A summary of nucleotide changes in the variant light and heavy chain sequences.DETAILED DESCRIPTION OF THE INVENTION

[0058] The present invention, in some embodiments, provides methods of optimizing an antibody coding sequence. Nucleic acid molecule systems comprising a coding region encoding an antibody heavy chain variable region and a coding region encoding an antibody light chain variable region are also provided.

[0059] The invention is based, at least in part, on the surprising finding that antibody genes have evolved separately from other genes from the same organism to have uniquely selected for codons. It is generally considered that a sequence can be codon optimized for expression in a target cell by matching the codon bias present in that target cell. That is by producingsynonymous mutations in the coding sequence that increase the use of codons that are favored in the target cell. However, contrary to this common dogma, it has been determined that for antibodies it is more advantageous to codon optimize for the codons favored in antibodies than for the codons favored in the target cells genome. In particular, it was found that for producing antibodies in Chinese Hamster Ovary (CHO) cells, it was more advantageous to codon optimize for mouse antibody genes or even human antibody genes that it was to codon optimize for the Chinese Hamster’s genome.

[0060] By a first aspect, there is provided a method of producing an antibody coding sequence, the method comprising: a. providing a nucleic acid sequence comprising a coding region encoding an antibody; and b. producing at least one synonymous mutation in the provided sequence; thereby producing an antibody coding sequence.

[0061] In some embodiments, the method is an in vitro method. In some embodiments, the method is an ex vivo method. In some embodiments, the method is a computerized method. In some embodiments, the method is a culture method. In some embodiments, the method is a method of optimizing an antibody coding sequence. In some embodiments, optimizing is optimizing expression. In some embodiments, optimizing is optimizing translation. In some embodiments, optimizing is optimizing for target cells. In some embodiments, target cells are mammalian cells. In some embodiments, target cells are Chinese Hamster Ovary (CHO) cells. In some embodiments, optimizing is optimizing for mammalian cells. In some embodiments, optimizing is optimizing for CHO cells. In some embodiments, optimizing is optimizing protein yield. In some embodiments, the protein is an antibody.

[0062] As used herein, the term "antibody" refers to a polypeptide or group of polypeptides that include at least one binding domain that is formed from the folding of polypeptide chains having three-dimensional binding spaces with internal surface shapes and charge distributions complementary to the features of an antigenic determinant of an antigen. An antibody typically has a tetrameric form, comprising two identical pairs of polypeptide chains, each pair having one "light" and one "heavy" chain. The variable regions of each light / heavy chain pair form an antibody binding site. An antibody may be oligoclonal, polyclonal, monoclonal, chimeric, camelised, CDR-grafted, multi- specific, bi-specific, catalytic, humanized, fully human, anti- idiotypic and antibodies that can be labeled in soluble or bound form as well as fragments, including epitope-binding fragments, variantsor derivatives thereof, either alone or in combination with other amino acid sequences. An antibody may be from any species. The term antibody also includes binding fragments, including, but not limited to Fv, Fab, Fab', F(ab')2 single stranded antibody (svFC), dimeric variable region (Diabody) and disulphide-linked variable region (dsFv). In particular, antibodies include immunoglobulin molecules and immunologically active fragments of immunoglobulin molecules, i.e., molecules that contain an antigen binding site. Antibody fragments may or may not be fused to another immunoglobulin domain including but not limited to, an Fc region or fragment thereof. The skilled artisan will further appreciate that other fusion products may be generated including but not limited to, scFv- Fc fusions, variable region (e.g., VL and VH)~ Fc fusions and scFv-scFv-Fc fusions.

[0063] Immunoglobulin molecules can be of any type (e.g., IgG, IgE, IgM, IgD, IgA and IgY), class (e.g., IgGl, IgG2, IgG3, IgG4, IgAl and IgA2) or subclass.

[0064] In some embodiments, an antibody comprises a heavy chain and a light chain. In some embodiments, an antibody comprises a heavy chain variable region and a light chain variable region. In some embodiments, the coding region is an open reading frame. In some embodiments, the coding region comprises a first codon being an ATG codon. In some embodiments, the coding region encodes an antibody heavy chain. In some embodiments, the coding region encodes an antibody light chain. In some embodiments, the coding region encodes an antibody heavy chain variable region. In some embodiments, the coding region encodes an antibody light chain variable region.

[0065] In some embodiments, the coding region is 1 coding region. In some embodiments, the coding region is a plurality of coding regions. In some embodiments, the coding region is two coding regions. In some embodiments, the coding region is at least 1 coding region. In some embodiments, the coding region is at least 2 coding regions. In some embodiments, the coding region comprises a first coding region and second coding region. In some embodiments, the first coding region encodes a variable region of a heavy chain of the antibody. In some embodiments, the second coding region encodes a variable region of a light chain of the antibody. In some embodiments, the first coding region encodes the heavy chain of the antibody. In some embodiments, the second coding region encodes the light chain of the antibody.

[0066] In some embodiments, the coding region further comprises a heavy chain constant region. In some embodiments, the nucleic acid sequence further comprises a heavy chain constant region. In some embodiments, the coding region further comprises a light chainconstant region. In some embodiments, the nucleic acid sequence further comprises a light chain constant region. It will be understood by a skilled artisan that the antibody can be any type or class of antibody. This includes but is not limited to IgGl, IgG2, IgG3, and IgG4. Further, the light chain can be either kappa or lambda. Various mutations within the constant region are known. These can increase or decrease ADCC and / or CDC, improve homodimerization or heterodimerization or alter affinity for various antibody binding receptors. These mutations are all on the protein / amino acid level and thus are irrelevant to the invention, as synonymous mutations can be made for antibodies bearing any or all of these mutations.

[0067] In some embodiments, the coding region further encodes a signal peptide. In some embodiments, the signal peptide is an N-terminal signal peptide. In some embodiments, the signal peptide is an antibody heavy chain signal peptide. In some embodiments, the signal peptide is an antibody light chain signal peptide. In some embodiments, the signal peptide is a heterologous signal peptide. In some embodiments, the synonymous mutation is not made in the signal peptide. In some embodiments, the synonymous mutation is not made in the first 5, 10, 15, 20, 25, or 30 codons of the coding region. Each possibility represents a separate embodiment of the invention. In some embodiments, the synonymous mutation is not made in the first 15 codons of the coding region.

[0068] In some embodiments, the synonymous mutation converts at least one codon into another codon. In some embodiments, the synonymous mutation does not alter protein function. In some embodiments, the synonymous mutation does not alter protein sequence. Synonymous codons are provided in Table 4.

[0069] Table 4: synonymous codons

[0070] In some embodiments, the synonymous mutation converts at least one codon into a codon more commonly used in a set of genes. In some embodiments, more commonly is more frequently. In some embodiments, a more commonly used codon is a codon with a higher codon bias in the set of gene. In some embodiments, the set of genes all encode antibodies. In some embodiments, the set of genes is not a full genome. In some embodiments, a full genome is a whole genome. The use of a full genome for optimizing codons is well known in the art. For example, a non-human antibody might be codon optimized based on the full human genome or an antibody sequence that will be expressed in CHO cells might be codon optimized based on the full CHO genome. However, the instant application does not codon optimize based on a full genome but rather based on a set of genes that is less than a full genome; specifically, a set of genes consisting of sequences of antibodies. In some embodiments, the set of genes all encode antibody from the same organism. In some embodiments, the organism is humans. In some embodiments, the organism is mice. In some embodiments, the set of genes comprises a plurality of antibody genes. In some embodiments, the set of genes consists of a plurality of antibody genes. In some embodiments, the set of genes comprises a plurality of nucleic acid sequences encoding antibodies. In some embodiments, the set of genes consists of a plurality of nucleic acid sequences encoding antibodies. In some embodiments, the nucleic acid sequences encode antibody variable regions. In some embodiments, the nucleic acid sequences encode antibody heavy chain variable regions. In some embodiments, the nucleic acid sequences encode antibody light chain variable regions. In some embodiments, the nucleic acid sequences encode antibody heavy chain variable regions and light chain variable regions.

[0071] In some embodiments, the full genome is a human genome. In some embodiments, the full genome is a mouse genome. In some embodiments, the full genome is a hamster genome. In some embodiments, the full genome is a CHO genome. In some embodiments, the method produces an optimized sequence. In some embodiments, producing the synonymous mutation produces an optimized sequence. In some embodiments, optimized for translation in a target cell. In some embodiments, the full genome is the genome of the target cell. The use of CHO cells is merely a proof of concept. The instant application demonstrates that using antibodies sequences is superior to using the full genome of the target cell. Both human antibodies and mouse antibodies produced superior expression to optimization with the cell’s own genome, thus it is clear that regardless of the target cell the use of antibody sequences for optimization will be superior.

[0072] In some embodiments, the set of genes consists of a plurality of nucleic acid sequences encoding human antibody. In some embodiments, the synonymous mutation comprises mutation of a codon to a preferred codon indicated in Table 5. In some embodiments, the synonymous mutation comprises mutation of an alanine codon to GCC. In some embodiments, the synonymous mutation comprises mutation of an arginine codon to AGA. In some embodiments, the synonymous mutation comprises mutation of an asparagine codon to AAC. In some embodiments, the synonymous mutation comprises mutation of an aspartic acid codon to GAC. In some embodiments, the synonymous mutation comprises mutation of a cysteine codon to TGT. In some embodiments, the synonymous mutation comprises mutation of a glutamine codon to CAG. In some embodiments, the synonymous mutation comprises mutation of a glutamic acid codon to GAG. In some embodiments, the synonymous mutation comprises mutation of glycine codon to GGG. In some embodiments, the synonymous mutation comprises mutation of a histidine codon to CAC. In some embodiments, the synonymous mutation comprises mutation of an isoleucine codon to ATC. In some embodiments, the synonymous mutation comprises mutation of a leucine codon to CTG. In some embodiments, the synonymous mutation comprises mutation of lysine codon to AAG. In some embodiments, the synonymous mutation comprises mutation of a phenylalanine codon to TTC. In some embodiments, the synonymous mutation comprises mutation of a proline codon to CCC. In some embodiments, the synonymous mutation comprises mutation of a serine codon to TCC. In some embodiments, the synonymous mutation comprises mutation of a threonine codon to ACC. In some embodiments, the synonymous mutation comprises mutation of a tyrosine codon to TAC. Insome embodiments, the synonymous mutation comprises mutation of a valine codon to GTG.

[0073] In some embodiments, the set of genes consists of a plurality of nucleic acid sequences encoding mouse antibody. In some embodiments, the synonymous mutation comprises mutation of a codon to a preferred codon indicated in Table 6. In some embodiments, the synonymous mutation comprises mutation of an alanine codon to GCT. In some embodiments, the synonymous mutation comprises mutation of an arginine codon to AGG. In some embodiments, the synonymous mutation comprises mutation of an asparagine codon to AAC. In some embodiments, the synonymous mutation comprises mutation of an aspartic acid codon to GAT. In some embodiments, the synonymous mutation comprises mutation of a cysteine codon to TGC. In some embodiments, the synonymous mutation comprises mutation of a glutamine codon to CAG. In some embodiments, the synonymous mutation comprises mutation of a glutamic acid codon to GAG. In some embodiments, the synonymous mutation comprises mutation of glycine codon to GGA. In some embodiments, the synonymous mutation comprises mutation of a histidine codon to CAC. In some embodiments, the synonymous mutation comprises mutation of an isoleucine codon to ATC. In some embodiments, the synonymous mutation comprises mutation of a leucine codon to CTG. In some embodiments, the synonymous mutation comprises mutation of lysine codon to AAG. In some embodiments, the synonymous mutation comprises mutation of a phenylalanine codon to TTC. In some embodiments, the synonymous mutation comprises mutation of a proline codon to CCA. In some embodiments, the synonymous mutation comprises mutation of a serine codon to TCT. In some embodiments, the synonymous mutation comprises mutation of a threonine codon to ACA. In some embodiments, the synonymous mutation comprises mutation of a tyrosine codon to TAC. In some embodiments, the synonymous mutation comprises mutation of a valine codon to GTC.

[0074] In some embodiments, the coding region comprises at least 1, 2, 3, 4, 5, 7 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475 or 500 mutations. Each possibility represents a separate embodiment of the invention. In some embodiments, the coding region comprises at least 10 mutations. In some embodiments, the coding region comprises at least 100 mutations. In some embodiments, at least one mutation is at least 1, 2, 3, 4, 5, 7 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475 or 500 mutations. Each possibility represents a separate embodiment of the invention. In some embodiments, at leastone mutation is at least 10 mutations. In some embodiments, at least one mutation is at least 60 mutations. In some embodiments, at least one mutation is at least 100 mutations. In some embodiments, at least one mutation is at least 175 mutations. According to some embodiments, the coding region comprises mutation of at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45% 50%, 75% or 100% of positions of the coding region. Each possibility represents a separate embodiment of the invention.

[0075] In some embodiments, the coding region comprises mutation of at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45% 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 92%, 95%, 97% or 100% of codons that can be mutated to a more commonly used codon. Each possibility represents a separate embodiment of the invention. In some embodiments, the coding region comprises mutation of at least 10% of codons that can be mutated to a more commonly used codon. In some embodiments, the coding region comprises mutation of at least 25% of codons that can be mutated to a more commonly used codon. In some embodiments, the coding region comprises mutation of at least 50% of codons that can be mutated to a more commonly used codon. In some embodiments, the coding region comprises mutation of at least 90% of codons that can be mutated to a more commonly used codon. In some embodiments, the coding region comprises mutation of at least 100% of codons that can be mutated to a more commonly used codon. In some embodiments, more commonly used is more commonly used in the set of genes.

[0076] In some embodiments, the at least one synonymous mutation is every possible synonymous mutation. In some embodiments, every possible synonymous mutation is every possible synonymous mutation that converts a codon to a more commonly used codon in the set of genes. In some embodiments, every possible does not include mutations within the first 15 codons of the coding region. In some embodiments, every possible is every possible except mutating the first 15 codons of the coding region. In some embodiments, every possible does not include mutations within the signal peptide. In some embodiments, every possible is every possible except mutating the signal peptide.

[0077] In some embodiments, every possible synonymous mutation is every possible synonymous mutation within the variable region of the antibody. In some embodiments, every possible synonymous mutation is every possible synonymous mutation within the antibody. In some embodiments, every possible synonymous mutation is every possible synonymous mutation within the variable region of the antibody’s heavy chain. In some embodiments, every possible synonymous mutation is every possible synonymous mutationwithin the variable region of the antibody ’ s light chain. In some embodiments, every possible synonymous mutation is every possible synonymous mutation within the variable region of the antibody’s heavy chain and the variable region of the antibody’s light chain. In some embodiments, every possible synonymous mutation is every possible synonymous mutation within the antibody’s heavy chain. In some embodiments, every possible synonymous mutation is every possible synonymous mutation within the antibody’s light chain.

[0078] In some embodiments, the method produces an optimized coding region. In some embodiments, the mutation produces an optimized coding region. In some embodiments, the method produces a nucleic acid molecule comprising the optimized coding region. In some embodiments, the nucleic acid molecule is a plurality of nucleic acid molecules. In some embodiments, the nucleic acid molecule is a nucleic acid molecule system. In some embodiments, the method further comprises expressing protein from the optimized coding region. In some embodiments, the method further comprises expressing protein from the produced nucleic acid molecule. In some embodiments, expressing is producing. In some embodiments, the method further comprises contacting a cell with the optimized coding region. In some embodiments, the method further comprises contacting a cell with the produced nucleic acid molecule. In some embodiments, contacting comprises expressing the molecule in the cell.

[0079] In some embodiments, the method further comprises producing the optimized sequence. In some embodiments, the method further comprises producing a nucleic acid molecule comprises the optimized sequence. In some embodiments, the method further comprises producing an antibody from the optimized sequence. In some embodiments, the method further comprises expressing the optimized sequence in a cell. In some embodiments, the method further comprises contacting a cell with the optimized sequence. In some embodiments, the cell is a target cell. In some embodiments, the cell is a mammalian cell. In some embodiments, the cell is a CHO cell. In some embodiments, the method is a method of producing an optimized sequence. In some embodiments, the method is a method of producing an antibody. In some embodiments, producing an antibody is producing an increased yield of an antibody. In some embodiments, increased is as compared to the yield produced with the provided sequence. In some embodiments, increased is by at least 20, 30, 40, 50, 60, 70, 80, 90 or 100%. Each possibility represents a separate embodiment of the invention.

[0080] In some embodiments, expressing is transfecting a cell. In some embodiments, expressing is transducing a cell. In some embodiments, transfecting is transientlytransfecting. In some embodiments, transfecting is stably transfecting. In some embodiments, expressing is virally infecting a cell. Methods of expression protein and specifically antibodies in a cell are well known in the art and any such method can be used.

[0081] By another aspect, there is provided an improved nucleic acid sequence produced by a method of the invention.

[0082] By another aspect, there is provided a nucleic acid molecule produced by a method of the invention.

[0083] In some embodiments, the produced nucleic acid molecule is part of a vector. In some embodiments, the produced nucleic acid molecule is a vector. In some embodiments, the vector is an expression vector. In some embodiments, the expression vector is a prokaryotic expression vector. In some embodiments, the prokaryotic expression vector comprises any sequences necessary for expression of the protein encoded by the nucleic acid molecule of the invention in a prokaryotic cell. In some embodiments, the expression vector is a eukaryotic expression vector.

[0084] The term "expression" as used herein refers to the biosynthesis of a gene product, including the transcription and / or translation of said gene product. Thus, expression of a nucleic acid molecule may refer to transcription of the nucleic acid fragment (e.g., transcription resulting in mRNA or other functional RNA) and / or translation of RNA into a precursor or mature protein (polypeptide). In some embodiments, expression is protein expression.

[0085] Expressing a gene within a cell is well known to one skilled in the art. It can be carried out by, among many methods, transfection, viral infection, or direct alteration of the cell’s genome. In some embodiments, the gene is in an expression vector such as plasmid or viral vector.

[0086] A vector nucleic acid sequence generally contains at least an origin of replication for propagation in a cell and optionally additional elements, such as a heterologous polynucleotide sequence, expression control element (e.g., a promoter, enhancer), selectable marker (e.g., antibiotic resistance), poly-Adenine sequence.

[0087] The vector may be a DNA plasmid delivered via non-viral methods or via viral methods. The viral vector may be a retroviral vector, a herpesviral vector, an adenoviral vector, an adeno-associated viral vector or a poxviral vector. The promoters may be active in mammalian cells. The promoters may be a viral promoter.

[0088] In some embodiments, the coding region is operably linked to a promoter. The term “operably linked” is intended to mean that the nucleotide sequence of interest is linked to the regulatory element or elements in a manner that allows for expression of the nucleotide sequence (e.g., in an in vitro transcription / translation system or in a host cell when the vector is introduced into the host cell). In some embodiments, the promoter is a heterologous promoter. In some embodiments, the promoter is active in CHO cells. In some embodiments, the host cell is a CHO cell.

[0089] In some embodiments, the vector is introduced into the cell by standard methods including electroporation (e.g., as described in From et al., Proc. Natl. Acad. Sci. USA 82, 5824 (1985)), Heat shock, infection by viral vectors, high velocity ballistic penetration by small particles with the nucleic acid either within the matrix of small beads or particles, or on the surface (Klein et al., Nature 327. 70-73 (1987)), and / or the like.

[0090] The term "promoter" as used herein refers to a group of transcriptional control modules that are clustered around the initiation site for an RNA polymerase i.e., RNA polymerase II. Promoters are composed of discrete functional modules, each consisting of approximately 7-20 bp of DNA, and containing one or more recognition sites for transcriptional activator or repressor proteins. In some embodiments, the promoter is a constitutive promoter. In some embodiments, the promoter is an inducible promoter. In some embodiments, the promoter is a tissue specific promoter. In some embodiments, the promoter is a cell or cell type specific promoter. In some embodiments, the cell is the target cell. In some embodiments, the promoter is a heterologous promoter. In some embodiments, the promoter is active in CHO cells. In some embodiments, the host cell is a CHO cell. In some embodiments, the target cell is a CHO cell.

[0091] In some embodiments, nucleic acid sequences are transcribed by RNA polymerase II (RNAP II and Pol II). RNAP II is an enzyme found in eukaryotic cells. It catalyzes the transcription of DNA to synthesize precursors of mRNA and most snRNA and microRNA.

[0092] In some embodiments, mammalian expression vectors include, but are not limited to, pcDNA3, pcDNA3.1 (±), pGL3, pZeoSV2(±), pSecTag2, pDisplay, pEF / myc / cyto, pCMV / myc / cyto, pCR3.1, pSinRep5, DH26S, DHBB, pNMTl, pNMT41, pNMT81, which are available from Invitrogen, pCI which is available from Promega, pMbac, pPbac, pBK- RSV and pBK-CMV which are available from Strategene, pTRES which is available from Clontech, and their derivatives.

[0093] In some embodiments, expression vectors containing regulatory elements from eukaryotic viruses such as retroviruses are used by the present invention. SV40 vectors include pSVT7 and pMT2. In some embodiments, vectors derived from bovine papilloma virus include pBV-lMTHA, and vectors derived from Epstein Bar virus include pHEBO, and p2O5. Other exemplary vectors include pMSG, pAV009 / A+, pMTO10 / A+, pMAMneo- 5, baculovirus pDSVE, and any other vector allowing expression of proteins under the direction of the SV-40 early promoter, SV-40 later promoter, metallo thionein promoter, murine mammary tumor virus promoter, Rous sarcoma virus promoter, polyhedrin promoter, or other promoters shown effective for expression in eukaryotic cells.

[0094] In some embodiments, recombinant viral vectors, which offer advantages such as lateral infection and targeting specificity, are used for in vivo expression. In one embodiment, lateral infection is inherent in the life cycle of, for example, retrovirus and is the process by which a single infected cell produces many progeny virions that bud off and infect neighboring cells. In one embodiment, the result is that a large area becomes rapidly infected, most of which was not initially infected by the original viral particles. In one embodiment, viral vectors are produced that are unable to spread laterally. In one embodiment, this characteristic can be useful if the desired purpose is to introduce a specified gene into only a localized number of targeted cells.

[0095] Various methods can be used to introduce the expression vector of the present invention into cells. Such methods are generally described in Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Springs Harbor Laboratory, New York (1989, 1992), in Ausubel et al., Current Protocols in Molecular Biology, John Wiley and Sons, Baltimore, Md. (1989), Chang et al., Somatic Gene Therapy, CRC Press, Ann Arbor, Mich. (1995), Vega et al., Gene Targeting, CRC Press, Ann Arbor Mich. (1995), Vectors: A Survey of Molecular Cloning Vectors and Their Uses, Butterworths, Boston Mass. (1988) and Gilboa et at. [Biotechniques 4 (6): 504-512, 1986] and include, for example, stable or transient transfection, lipofection, electroporation and infection with recombinant viral vectors. In addition, see U.S. Pat. Nos. 5,464,764 and 5,487,992 for positive-negative selection methods.

[0096] It will be appreciated that other than containing the necessary elements for the transcription and translation of the inserted coding sequence (encoding the polypeptide), the expression construct of the present invention can also include sequences engineered to optimize stability, production, purification, yield or activity of the expressed polypeptide.

[0097] A person with skill in the art will appreciate that the antibody can also be expressed from a nucleic acid construct administered to the individual employing any suitable mode of administration, described hereinabove (i.e., in vivo gene therapy). In one embodiment, the nucleic acid construct is introduced into a suitable cell via an appropriate gene delivery vehicle / method (transfection, transduction, homologous recombination, etc.) and an expression system as needed and then the modified cells are expanded in culture and returned to the individual (i.e., ex vivo gene therapy).

[0098] By another aspect, there is provided a nucleic acid molecule system, comprising: a. a first nucleic acid molecule comprising a first coding region encoding an antibody heavy chain variable region; and b. a second nucleic acid molecule comprising a second coding region encoding a light chain variable region of said antibody.

[0099] In some embodiments, the first and second nucleic acid molecules are the same molecule. In some embodiments, the first and second nucleic acid molecules are different molecules. In some embodiments, the first molecule is a vector. In some embodiments, the second molecule is a vector. In some embodiments, the vector is an expression vector. In some embodiments, the first coding region further encodes an antibody heavy chain constant region. In some embodiments, the second coding region further encodes an antibody light chain constant region. In some embodiments, the constant region is C-terminal to the variable region. In some embodiments, the first coding region, second coding region or both further encode a signal peptide. In some embodiments, the signal peptide is an N-terminal signal peptide.

[0100] In some embodiments, the system is a kit. In some embodiments, the nucleic acid system is optimized for expression in a target cell. In some embodiments, the nucleic acid system is optimized for expression in mammalian cells. In some embodiments, the nucleic acid system is optimized for expression in CHO cells. In some embodiments, expression is protein expression. In some embodiments, expression is translation. In some embodiments, the kit further comprises CHO cells.

[0101] In some embodiments, at least one of the first and second coding regions comprises at least one synonymous mutation. In some embodiments, the first coding region comprises at least one synonymous mutation. In some embodiments, the second coding region comprises at least one synonymous mutation. In some embodiments, both the first codingregion and the second coding region each comprise at least one synonymous mutation. In some embodiments, the synonymous mutation converts at least one codon into a codon more commonly used in a set of genes.

[0102] In some embodiments, the first nucleic acid molecule is a nucleic acid molecule produced by a method of the invention. In some embodiments, the second nucleic acid molecule is a nucleic acid molecule produced by a method of the invention. In some embodiments, the first coding region comprises a coding sequence produced by a method of the invention. In some embodiments, the second nucleic acid molecule comprises a coding sequence produced by a method of the invention. In some embodiments, the first coding region consists of a coding sequence produced by a method of the invention. In some embodiments, the second nucleic acid molecule consists of a coding sequence produced by a method of the invention.

[0103] By another aspect, there is provided a cell comprising a nucleic acid molecule system of the invention.

[0104] In some embodiments, the cell is a target cell. In some embodiments, the cell is a mammalian cell. In some embodiments, the cell is a CHO cell. In some embodiments, the cell is a plurality of cells. In some embodiments, the cell is a cell in culture. In some embodiments, the cell is a tissue culture of cells. In some embodiments, the cell is a host cell. In some embodiments, the cell is a packaging cell.

[0105] By another aspect, there is provided a composition comprising a cell of the invention.

[0106] In some embodiments, the composition comprises culture media. In some embodiments, the media is adherent cell media. In some embodiments, the media is CHO media. In some embodiments, the composition is for use in producing an antibody.

[0107] By another aspect, there is provided a method of producing an antibody, the method comprising expressing a nucleic acid molecule system of the invention in a cell, thereby producing an antibody.

[0108] In some embodiments, the cell is a target cell. In some embodiments, the cell is a host cell. In some embodiments, the cell is a mammalian cell. In some embodiments, the cell is not a human cell. In some embodiments, the cell is not a mouse cell. In some embodiments, the cell is a hamster cell. In some embodiments, the hamster is a Chinese hamster. In some embodiments, the cell is a CHO cell. In some embodiments, the method further comprises producing a nucleic acid molecule system by a method of the invention.

[0109] As used herein, the term "about" when combined with a value refers to plus and minus 10% of the reference value. For example, a length of about 1000 nanometers (nm) refers to a length of 1000 nm+- 100 nm.

[0110] It is noted that as used herein and in the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a polynucleotide" includes a plurality of such polynucleotides and reference to "the polypeptide" includes reference to one or more polypeptides and equivalents thereof known to those skilled in the art, and so forth. It is further noted that the claims may be drafted to exclude any optional element. As such, this statement is intended to serve as antecedent basis for use of such exclusive terminology as "solely," "only" and the like in connection with the recitation of claim elements, or use of a "negative" limitation.

[0111] In those instances where a convention analogous to "at least one of A, B, and C, etc." is used, in general such a construction is intended in the sense one having skill in the art would understand the convention (e.g., "a system having at least one of A, B, and C" would include but not be limited to systems that have A alone, B alone, C alone, A and B together, A and C together, B and C together, and / or A, B, and C together, etc.). It will be further understood by those within the art that virtually any disjunctive word and / or phrase presenting two or more alternative terms, whether in the description, claims, or drawings, should be understood to contemplate the possibilities of including one of the terms, either of the terms, or both terms. For example, the phrase "A or B" will be understood to include the possibilities of "A" or "B" or "A and B."

[0112] It is appreciated that certain features of the invention, which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the invention, which are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable sub-combination. All combinations of the embodiments pertaining to the invention are specifically embraced by the present invention and are disclosed herein just as if each and every combination was individually and explicitly disclosed. In addition, all subcombinations of the various embodiments and elements thereof are also specifically embraced by the present invention and are disclosed herein just as if each and every such sub-combination was individually and explicitly disclosed herein.

[0113] As used in this specification and the appended claims, the singular forms “a,” “an,” and “the” include plural referents, unless the context clearly dictates otherwise. The terms“a” (or “an”) as well as the terms “one or more” and “at least one” can be used interchangeably.

[0114] Furthermore, “and / or” is to be taken as specific disclosure of each of the two specified features or components with or without the other. Thus, the term “and / or” as used in a phrase such as “A and / or B” is intended to include A and B, A or B, A (alone), and B (alone). Likewise, the term “and / or” as used in a phrase such as “A, B, and / or C” is intended to include A, B, and C; A, B, or C; A or B; A or C; B or C; A and B; A and C; B and C; A (alone); B (alone); and C (alone).

[0115] Wherever embodiments are described with the language “comprising,” otherwise analogous embodiments described in terms of “consisting of’ and / or “consisting essentially of’ are included.

[0116] Additional objects, advantages, and novel features of the present invention will become apparent to one ordinarily skilled in the art upon examination of the following examples, which are not intended to be limiting. Additionally, each of the various embodiments and aspects of the present invention as delineated hereinabove and as claimed in the claims section below finds experimental support in the following examples.

[0117] Various embodiments and aspects of the present invention as delineated hereinabove and as claimed in the claims section below find experimental support in the following examples.EXAMPLES

[0118] Generally, the nomenclature used herein and the laboratory procedures utilized in the present invention include molecular, biochemical, microbiological and recombinant DNA techniques. Such techniques are thoroughly explained in the literature. See, for example, "Molecular Cloning: A laboratory Manual" Sambrook et al., (1989); "Current Protocols in Molecular Biology" Volumes I-III Ausubel, R. M., ed. (1994); Ausubel et al., "Current Protocols in Molecular Biology", John Wiley and Sons, Baltimore, Maryland (1989); Perbal, "A Practical Guide to Molecular Cloning", John Wiley & Sons, New York (1988); Watson et al., "Recombinant DNA", Scientific American Books, New York; Birren et al. (eds) "Genome Analysis: A Laboratory Manual Series", Vols. 1-4, Cold Spring Harbor Laboratory Press, New York (1998); methodologies as set forth in U.S. Pat. Nos. 4,666,828; 4,683,202; 4,801,531; 5,192,659 and 5,272,057; "Cell Biology: A Laboratory Handbook", Volumes I-Ill Cellis, J. E., ed. (1994); "Culture of Animal Cells - A Manual of Basic Technique" by Freshney, Wiley-Liss, N. Y. (1994), Third Edition; "Current Protocols in Immunology" Volumes I-III Coligan J. E., ed. (1994); Stites et al. (eds), "Basic and Clinical Immunology" (8th Edition), Appleton & Lange, Norwalk, CT (1994); Mishell and Shiigi (eds), "Strategies for Protein Purification and Characterization - A Laboratory Course Manual" CSHL Press (1996); all of which are incorporated by reference. Other general references are provided throughout this document.Materials and Methods

[0119] Antibody data collection: The Observed Antibody Space (OAS) database was accessed, which houses millions of mammalian antibody sequences. This resource contains approximately 4.1 million unique paired antibody sequences with identified heavy and light chain connections, alongside approximately 2.4 billion unique unpaired antibody sequences, aggregating data from around 100 immune repertoire sequencing studies. Study datasets in the OAS database are organized into distinct files, referred to as data units, grouping sequences by shared metadata such as disease type. This analysis included all sequences and data units from the OAS, focusing on a subset of sequences for experimental design. Table 1 provides detailed breakdowns of the datasets and data units.

[0121] Genomic Data Collection: Genomic data was retrieved from the National Center for Biotechnology Information (NCBI) for human, mouse, rat, rabbit, rhesus, camel, and Chinese hamster, using the following accession numbers and genome release versions: Human: GCF_000001405.40 (GRCh38.pl4), Mouse: GCF_000001635.27 (GRCm39), Rat:GCF_015227675.2 (mRatBN7.2), Rabbit: GCF_009806435.1 (UM_NZW_1.0), Rhesus: GCF_003339765.1 (Mmul_10), Camel: GCF_000767855.1 (Ca_bactrianus_MBC_1.0), Chinese Hamster: GCF_000223135.1 (CriGri_1.0). All species were used in the data analysis, with the Chinese hamster genome utilized in the experimental design. Data were retrieved in October 2023.

[0122] Protein Families Data Collection: Sequences of various protein families were downloaded from Ensembl BioMart, without limited to antibodies or immune -related groups, for all relevant species: human, mouse, rat, rabbit, rhesus, camel, and Chinese hamster. The specific Ensembl datasets used are: hsapiens_gene_ensembl, mmusculus_gene_ensembl, morvegicus_gene_ensembl, ocuniculus_gene_ensembl, mmulatta_gene_ensembl, cdromedarius_gene_ensembl, and cgchoklgshd_gene_ensembl, respectively. Our selection criteria included gene ontology (GO) terms: G0:0003823 (antigen binding), G0:0030234 (enzyme regulator activity), and G0:0005215 (transporter activity), PTHR24356 (serine / threonine -protein kinase), and PTHR18945 (neurotransmitter gated ion channel), and InterPro IDs: IPR007110 (immunoglobulin-like domain), IPR001650 (helicase, C-terminal), and IPR000719 (protein kinase domain). Data were retrieved in March 2024.

[0123] Sequences Preprocessing

[0124] Antibody sequences preprocessing: Files obtained from the OAS database were processed, focusing on extracting the variable V(D)J regions of both heavy and light chains in antibody sequences. This included their amino acid translation and germline sequences. All sequences were saved in their correct reading frames. Additionally, metadata for each data unit, detailing aspects like data type, disease, and vaccine information, was preserved for subsequent analysis.

[0125] Genomic sequences preprocessing: Genomic sequences were filtered to include only those annotated as coding sequences (CDS), excluding entries with the qualifiers 'exception' and 'transl_excepf (translational exception). Sequences adhering to the standard translation table ('transl_table' = 1) were used.

[0126] Protein family sequences preprocessing: To avoid redundancy, duplicate entries were removed based on Ensembl gene IDs.

[0127] Data Analysis

[0128] Antibody Data Processing: To manage the extensive data volume from the OAS, high-performance computing (HPC) resources were employed and Dask was utilized for efficient data processing. The analysis involved species-specific filtering of relevant data units. Data units annotated as immature B-cells were excluded to focus on mature antibody sequences. Each data unit / sample of sequences was analyzed for codon usage, counts and frequencies, serving as key inputs for various analyses. For certain analyses, aggregation of counts across specific criteria (e.g., disease type) preceded computations.

[0129] Codon Frequencies Calculation: For each data unit or sequence batch analyzed, codon counts and frequencies were computed in three distinct ways:(1) Overall codon frequency: Calculating the frequency of each codon relative to the total number of codons.(2) Amino acid-specific frequency: Determining the frequency of each codon within its corresponding amino acid group.(3) Normalized amino acid-specific frequency: As per (2) but normalized against the maximum codon frequency for each amino acid.

[0130] In this process, as with all analyses conducted herein, stop codons and codons with ambiguous nucleotides were excluded. When amino acids were absent, the codon values in (2) and (3) were adjusted to a uniform frequency according to the corresponding amino acid degeneracy group, implying that adjustments in (3) resulted in a value of 1.

[0131] Codon Bias Metrics: To assess codon usage bias (CUB), multiple metrics were applied. The normalized frequencies (as outlined in the previous section, part (3)) were utilized in a manner akin to the weights used in the codon adaptation index (CAI). This method evaluates how frequently a codon is used relative to the most frequently used codon for the same amino acid. This approach provides insights into the preference of certain codons over others in the coding sequences.

[0132] Additionally, to encapsulate the codon usage diversity within a singular metric, the effective number of codons (ENC) was employed. ENC is a scalar value that quantifies the degree of codon usage bias within a coding sequence. A lower ENC value indicates a higher bias, meaning a smaller subset of codons is preferred, while a higher ENC value suggests a more uniform use of synonymous codons.

[0133] Randomization Procedures: To assess the hypothesis of selective codon usage in antibodies, a dinucleotide randomization technique was used. This method swaps codons ina given sequence while preserving the original amino acid sequence and maintaining the dinucleotide distribution. The randomized sequence is generated through multiple iterations, with each iteration randomly selecting and swapping two codons, provided these constraints are met. By keeping the dinucleotide distribution constant, the analysis focused on identifying whether codon selection is an active process influencing the antibody sequences, rather than a passive result of the existing dinucleotide composition.

[0134] To test whether changes in codon preferences across different regions of the antibody sequences are significant, vertical codon usage bias (VCUB) randomization was used. This method swaps codons within a given aligned set of sequences while preserving the original amino acid sequences. The randomized sequences are generated through multiple iterations, with each iteration randomly selecting two columns of the aligned sequences and, for each sequence, swapping two codons, provided this constraint is met.

[0135] P-value Calculation: To determine the statistical significance of differences in codon frequency distributions between two sources, an empirical p-value calculation method was employed, as follows:1. Initial sampling: Randomly sample 1,000 sequences (or genes) from each of the two sources. This sampling may include repeated selection of the same sequence.2. Distribution calculation: Calculate the codon distribution for each sample.3. Initial comparison: For practical analysis, instead of using these samples, the complete, true distributions from each source was used. Then compute the distance / correlation between these distributions using Jensen-Shannon divergence (JSD) and Spearman correlation. Additionally, for verifying the robustness of the results, use Manhattan distance (LI norm or absolute sum distance), Euclidean distance, and Kullback- Leibler (KL) divergence.4. Randomized sampling: Combine the samples to form a pool of 2,000 sequences and randomly split them into two new groups of 1,000 sequences each, mixing sequences from both sources.5. Randomized distribution calculation: Calculate the codon distribution for each group in the randomized sampling.6. Randomized comparison: Compute the distance / correlation between the two distributions from the randomized groups.7. Iteration and empirical p-value calculation: Repeat steps 4 to 6 for 1,000 iterations. The empirical p-value is the proportion of instances where the distance / correlation in the true distributions (step 3) was greater / smaller than in the random division (step 6).

[0136] For analyses involving dinucleotide randomization and ENC values, the p-value was calculated by determining the proportion at which the ENC value of randomized sequences was smaller (more biased) than that of the real sequences.

[0137] Spatial Analysis: A spatial analysis was conducted to explore positional variations in codon usage within antibodies. Initially, a sample of 10,000 sequences was randomly selected, to ensure each was productive and contained a complete V(D)J sequence, by filtering for the ‘productive’ and ‘complete vdj’ fields, respectively. However, due to the lack of complete information in many sequences, the sampled sequences were also retained without applying these filters. For the alignment of these two subsets of sequences, Clustal Omega was utilized, a tool for multiple sequence alignment (MSA). Codon usage was compared across the entire aligned sequences by applying sliding window of 13-codon length, computing the ENC value for each window.

[0138] Additionally, to analyze and compare structural regions in the antibodies, the Antigen receptor Numbering And Receptor Classification (ANARCI) tool was employed, whose output is included in the OAS, and is based on amino acid sequence positions as standardized by The International Immunogenetics Information System (IMGT). For this phase of analysis, the filtering was updated to include only those sequences where all amino acids were numbered in ANARCI. The robustness of the results was confirmed through 3 repeats.

[0139] Expressing Antibodies in CHO

[0140] Variant Design: The engineered variants are based on codons that demonstrate the highest codon usage bias (CUB), representing the most frequently used codons. This prevalence is evident in antibodies expressed in mice and within the Chinese hamster’s genome. This design approach parallels the traditional CAVRSCU optimization method, albeit referencing a distinct dataset. Specifically, all feasible codons within the open reading frames (ORFs) of the heavy and light chains were substituted with the most frequently utilized ones observed in either mouse antibodies or the Chinese hamster’s genome. Given that these newly introduced codons remain synonymous with the original ones, these alterations maintain both the protein's length and the amino acid sequence. The initial 15 codons within the sequence were deliberately preserved and left untouched. Table 2 describes the variants; Table 3 shows the exact codon positions that were substituted; Figures 15A-15B show sequence alignments of the 3 synthesized variants.

[0141] Table 2. Descriptive summary of the 3 synthesized CB6 antibody variants.

[0142] Table 3 Positions of substituted codons within the CB6 antibody variants.

[0143] DNA Cloning: Plasmid RCK-CMP22 (Fig. 16) was generated by ligating 6 DNA fragments simultaneously. The DNA fragments for CBbuuman, Antibodies. CB6Mouse_Antibodies and CHOchinese_Hamster_genome antibodie were synthesized by SenScript. These fragments were excised from their respective plasmids (pUC57) using unique restriction enzymes and were then integrated in the designated positions of pRCK-CMP22, using T4 DNA ligase (NEB). Plasmid DNA was prepared using a mini prep kit (Qiagen).

[0144] DNA transfection and clone selection: CHO-K1 cells were grown in MEM-alpha, high glucose with Glutamine supplemented with 10% FBS and 0.5% Pen-Strep (Biological Industries). Cells were split 24 hrs. before transfection, the next day they were trypsinized, washed with PBS and counted. For each transfection, 10A5 cells were seeded in 0.5 ml of growth media in 12 wells plates (Nunc). In each biological repeat, 2 ug of plasmid DNA were transfected, using FuGene (Promega) as the DNA carrier. DNA was mixed with 10 ul FuGene and 100 ul Bio-MPM-1 medium (Biological Industries) and the mixture was added to the cells. After vortexing for a few seconds, the cells were plated in 12 or 24 well plates (in 0.5 or 1 ml respectively). One hour after plating the transfected cells, 0.5ml medium were supplemented to the 12 well plates. The cells were grown in a 37°C incubator with 5% CO2 flow.

[0145] Anti-RBD bioassay: The antibodies’ expression and activity was assessed through EEISA. For the CB6 anti-Covid 19 RBD (receptor binding domain), a synthetic RBD (SinoBio) was used as the antigen. Antigen was suspended in TBST (0.15M NaCl, 30mM KC1, 250mM Tris, pH7.6, 0.05% Tween) to the desired concentration. Subsequently, 50ul (Ing / ul) of antigen were dispensed onto 96 well plates (Nunc) and incubated overnight at 4°C. Afterwards the antigen was then washed once with PBST, and blocking was carried out using 3% sterile BSA (MP) for 1 hour at room temperature (RT), followed by one wash with TBST. Supernatants from cells transfected with CB6Human_antibodies, CB6Mouse_antibodies or CHOchineese hamster genome variants, as well as cells transfected with no DNA (negative control) were added to the plate wells. Commercial anti-RBD antibodies (Sino Bio) served as a positive control. Supernatants of transfected cells in culture, or antibodies were incubated for 1 hour at RT, after which the wells were washed 3 times with PBST. Anti-human antibody conjugated to HRP was used as secondary antibody at a l:10K dilution, for 1 hour at RT, followed by 3 washes with TBST. For colorimetric development, the TMB solution was used, for 1 minute at RT, and the colorimetric reaction was terminated using TMB stop buffer (both from Southern Biotech). Color intensity was measured at 450 nm using anInfinit M nano reader (Tecan). The obtained results were then normalized to the control cells grown without DNA and to the cell count in each respective well.

[0146] RNA extraction, cDNA synthesis and qPCR: Growth media was aspirated, and cells were lysed on-plate using Trisol (acid phenol) (Ambion) per manufacturer’s instructions. The water phase which contains RNA was collected and RNA was precipitated using 3M sodium acetate and 2.5 volumes of ethanol. RNA was suspended in nucleases free water, treated with RNase free DNAse (Thermo), cleaned with Trisol and re-precipitated; its concentration was read by nanodrop. One microgram total RNA of each sample was used for cDNA synthesis using Gene Script reverse transcriptase II kit with random primers (Thermo). 1 ul of the cDNA was taken for quantitative real time PCR. qPCR was carried out with primers specific for light and heavy chains of each antibody, using Fast SYBR green master mix (Applied Biosystems) and a StepOne machine (Applied Biosystems). As a control for DNA contamination after DNAse treatment, RNA samples at the equivalent concentrations taken for cDNA synthesis were also examined by qPCR, to ensure low DNA contamination.

[0147] To assess the copy number of antibody DNA in both stable and transiently transfected cells, DNA was extracted from the organic phase of the Trisol used for cell lysis, according to the manufacturer’s instructions. DNA was cleaned, precipitated, quantified and employed in qPCR using the same primers as those used for quantitative real-time reverse transcription PCR (qRT PCR). For normalization of both mRNA and DNA by qPCR the Chinese hamster GAPDH gene was used.

[0148] Variant design based on antibody codon-usage bias: Codon usage bias (CUB) is the preferential or non-random use of synonymous codons in various organisms; it is widespread across species and serves as a code within the genetic code or the second genetic code. As such, CUB reflects the origin, mutation patterns and evolution of the species or genes, and can be used to increase gene expression levels through codon optimization.

[0149] The variants designed here are based on codons that present the highest CUB (i.e., they are the most frequent ones) in the antibodies expressed in mice or in the CHO genome - in a similar way to the “classical” Codon Adaptation Index / Relative Synonymous Codon Usage (CAI / RSCU) optimization (but with a different reference set). Specifically, all possible codons in the light and heavy ORF (if possible) were selected and replaced with the most frequent one in mouse antibodies or in the CHO genome. Since the original codonswere replaced with new synonymous ones, these changes preserve the proteins length and amino acid sequence. The first 15 codons of the sequence were preserved and not replaced.

[0150] Variant Description:

[0151] The original CB6 antibody is described in Shi et al. The nucleotide sequence encoding the heavy chain variable region mRNA can be found in GenBank accession number MT470197. The nucleotide sequence encoding the light chain variable region mRNA can be found in GenBank accession number MT470196. This sequence does not include a sequence encoding the light chain signal peptide, or indeed any signal peptide. The heavy chain variable region of the original antibody is encoded by the nucleotide sequence GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTCCAGCCTGGGGGGTCCCT GAGACTCTCCTGTGCAGCCTCTGGATTCACCGTCAGTAGCAACTACATGAGCT GGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCTCAGTTATTTATAGC GGTGGTAGCACATTCTACGCAGACTCCGTGAAGGGCAGATTCACCATCTCCAG AGACAATTCCATGAACACGCTGTTTCTTCAAATGAACAGCCTGAGAGCCGAGG ACACGGCTGTGTATTACTGTGCGAGAGTGCTACCTATGTACGGTGACTACCTTG ACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCAGCCAGCACCAAAGGC CCGAGCGTGTTTCCGCTGGCGCCGAGCAGCAAAAGCACCAGCGGCGGCACCGC GGCGCTGGGCTGCCTGGTGAAAGATTATTTTCCGGAACCGGTGACCGTGAGCT GGAACAGCGGCGCGCTGACCAGCGGCGTGCATACCTTTCCGGCGGTGCTGCAG AGCAGCGGCCTGTATAGCCTGAGCAGCGTGGTGACCGTGCCGAGCAGCAGCCT GGGCACCCAGACCTATATTTGCAACGTGAACCATAAACCGAGCAACACCAAAG TGGATAAACGCGTGGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCG TGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAA CCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGT GGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGC GTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCA CGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGC AAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAA AACTATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGC CCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTC AAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCC GGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCT TCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGT CTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAATGA (SEQ ID NO: 1). The light chain variable region of the original antibody is encoded by the nucleotide sequence GATATTGTGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGA GTCACCATCACTTGCCGGGCAAGTCAGAGCATTAGCAGGTATTTAAATTGGTA TCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATGCTGCATCCAGTT TGCAAAGTGGGGTCCCATCAAGGTTCAGTGGCAGTGGATCTGGGACAGATTTC ACTCTCACCATCAGCAGTCTGCAACCTGAAGATTTTGCAACTTACTACTGTCAA CAGAGTTACAGTACCCCTCCGGAGTACACTTTTGGCCAGGGGACCAAGCTGGA GATCAAACGAACTGTGGCTGCACCAAGCGTGTTTATCTTCCCTCCCAGCGACG AGCAGCTGAAGAGCGGCACCGCCAGCGTGGTCTGTCTCCTGAACAACTTCTAT CCCAGGGAGGCCAAGGTCCAGTGGAAAGTGGACAACGCCCTGCAAAGCGGCA ATAGCCAGGAGTCCGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCT GTCCAGCACCCTGACCCTCAGCAAGGCCGACTACGAGAAGCACAAGGTGTACG CTTGCGAGGTGACCCATCAGGGCCTGTCCAGCCCCGTGACCAAGTCCTTCAAC AGGGGCGAATGCAGCTAA (SEQ ID NO: 2).

[0152] The heavy chain variable region of the mouse optimized antibody is encoded by the nucleotide sequenceGAGGTCCAGCTGGTCGAGTCTGGAGGAGGACTGGTCCAGCCAGGAGGATCTCT GAGGCTGTCTTGCGCTGCTTCTGGATTCACAGTCTCTTCTAACTACATGTCTTG GGTCAGGCAGGCTCCAGGAAAGGGACTGGAGTGGGTCTCTGTCATCTACTCTG GAGGATCTACATTCTACGCTGATTCTGTCAAGGGAAGGTTCACAATCTCTAGG GATAACTCTATGAACACACTGTTCCTGCAGATGAACTCTCTGAGGGCTGAGGA TACAGCTGTCTACTACTGCGCTAGGGTCCTGCCAATGTACGGAGATTACCTGG ATTACTGGGGACAGGGAACACTGGTCACAGTCTCTTCTGCTTCTACAAAGGGA CCATCTGTCTTCCCACTGGCTCCATCTTCTAAGTCTACATCTGGAGGAACAGCT GCTCTGGGATGCCTGGTCAAGGATTACTTCCCAGAGCCAGTCACAGTCTCTTGG AACTCTGGAGCTCTGACATCTGGAGTCCACACATTCCCAGCTGTCCTGCAGTCT TCTGGACTGTACTCTCTGTCTTCTGTCGTCACAGTCCCATCTTCTTCTCTGGGAA CACAGACATACATCTGCAACGTCAACCACAAGCCATCTAACACAAAGGTCGAT AAGAGGGTCGAGCCAAAGTCTTGCGATAAGACACACACATGCCCACCATGCCC AGCTCCAGAGCTGCTGGGAGGACCATCTGTCTTCCTGTTCCCACCAAAGCCAA AGGATACACTGATGATCTCTAGGACACCAGAGGTCACATGCGTCGTCGTCGAT GTCTCTCACGAGGACCCAGAGGTCAAGTTCAACTGGTACGTCGATGGAGTCGA GGTCCACAACGCTAAGACAAAGCCAAGGGAGGAGCAGTACAACTCTACATACAGGGTCGTGTCTGTCCTGACAGTCCTGCACCAGGATTGGCTGAACGGAAAGGA GTACAAGTGCAAGGTCTCTAACAAGGCTCTGCCAGCTCCAATCGAGAAGACAA TCTCTAAGGCTAAGGGACAGCCAAGGGAGCCACAGGTCTACACACTGCCACCA TCTAGGGATGAGCTGACAAAGAACCAGGTCTCTCTGACATGCCTGGTCAAGGG ATTCTACCCATCTGATATCGCTGTCGAGTGGGAGTCTAACGGACAGCCAGAGA ACAACTACAAGACAACACCACCAGTCCTGGATTCTGATGGATCTTTCTTCCTGT ACTCTAAGCTGACAGTCGATAAGTCTAGGTGGCAGCAGGGAAACGTCTTCTCT TGCTCTGTCATGCACGAGGCTCTGCACAACCACTACACACAGAAGTCTCTGTCT CTGTCTCCAGGAAAGTGA (SEQ ID NO: 3). Heavy Chain: CB6_Heavy_Chain_CUBOPT_MouseAb_251_364: Optimized sequence to the most frequent codons in mouse antibodies; total number of changes: 251 codons, 364 nucleotides; Synthetic Codons: [21 24 26 27 28 29 31 33 34 35 36 38 40 42 43 44 45 47 48 49 50 54 56 57 59 60 61 63 67 68 69 72 73 74 75 76 79 80 81 82 84 85 87 89 90 91 93 96 103 105 106 108 109 110 111 115 116 117 119 122 123 126 129 131 132 134 135 136 137 138 139 140 141 143 144 145 146 148 150 151 152 153 155 156 157 158 159 160 161 162 164 167 169 172 174 175 176 177 178 181 182 183 185 186 187 188 190 192 193 194 197 198 199 202 204 205 206 207 208 209 210 211 212 213 215 216 218 223 227 228 230232 233 235 236 238 240 242 244 246 248 249 251 252 253 257 258 259 260261 265 266 268 270 271 275 276 277 278 280281 283 284 285 286 287 288 291 292 294 300 301 302 303 305 308 310 312 313 319 320 322 323 325 326 328 329 333 337 344 345 348 350 351 352 356 358 360 362 364 365 367 369 371 373 374 375 376 377 380 384 385 387 390 392 395 396 397 399 400 404 406 408 414 415 416 417 418 420 421 422 423 424 429 432 433 434 436 437 441 443 445 447 448 452 458 461 463 465 466 467],

[0153] The light chain variable region of the mouse optimized antibody is encoded by the nucleotide sequenceGATATCGTCATGACACAGTCTCCATCTTCTCTGTCTGCTTCTGTCGGAGATAGG GTCACAATCACATGCAGGGCTTCTCAGTCTATCTCTAGGTACCTGAACTGGTAC CAGCAGAAGCCAGGAAAGGCTCCAAAGCTGCTGATCTACGCTGCTTCTTCTCT GCAGTCTGGAGTCCCATCTAGGTTCTCTGGATCTGGATCTGGAACAGATTTCAC ACTGACAATCTCTTCTCTGCAGCCAGAGGATTTCGCTACATACTACTGCCAGCA GTCTTACTCTACACCACCAGAGTACACATTCGGACAGGGAACAAAGCTGGAGA TCAAGAGGACAGTCGCTGCTCCATCTGTCTTCATCTTCCCACCATCTGATGAGC AGCTGAAGTCTGGAACAGCTTCTGTCGTCTGCCTGCTGAACAACTTCTACCCAA GGGAGGCTAAGGTCCAGTGGAAGGTCGATAACGCTCTGCAGTCTGGAAACTCTCAGGAGTCTGTCACAGAGCAGGATTCTAAGGATTCTACATACTCTCTGTCTTCT ACACTGACACTGTCTAAGGCTGATTACGAGAAGCACAAGGTCTACGCTTGCGA GGTCACACACCAGGGACTGTCTTCTCCAGTCACAAAGTCTTTCAACAGGGGAG AGTGCTCTTGA (SEQ ID NO: 4). A BLAST comparison of the mouse optimized sequence and the human optimized sequence is provided in Figure 15A.

[0154] The heavy chain variable region of the CHO genome optimized antibody is encoded by the nucleotide sequenceGAGGTGCAGCTGGTGGAGAGCGGCGGCGGCCTGGTGCAGCCTGGCGGCAGCC TGAGACTGAGCTGTGCCGCCAGCGGCTTCACCGTGAGCAGCAACTACATGAGC TGGGTGAGACAGGCCCCTGGCAAGGGCCTGGAGTGGGTGAGCGTGATCTACA GCGGCGGCAGCACCTTCTACGCCGACAGCGTGAAGGGCAGATTCACCATCAGC AGAGACAACAGCATGAACACCCTGTTCCTGCAGATGAACAGCCTGAGAGCCG AGGACACCGCCGTGTACTACTGTGCCAGAGTGCTGCCTATGTACGGCGACTAC CTGGACTACTGGGGCCAGGGCACCCTGGTGACCGTGAGCAGCGCCAGCACCAA GGGCCCTAGCGTGTTCCCTCTGGCCCCTAGCAGCAAGAGCACCAGCGGCGGCA CCGCCGCCCTGGGCTGTCTGGTGAAGGACTACTTCCCTGAGCCTGTGACCGTG AGCTGGAACAGCGGCGCCCTGACCAGCGGCGTGCACACCTTCCCTGCCGTGCT GCAGAGCAGCGGCCTGTACAGCCTGAGCAGCGTGGTGACCGTGCCTAGCAGCA GCCTGGGCACCCAGACCTACATCTGTAACGTGAACCACAAGCCTAGCAACACC AAGGTGGACAAGAGAGTGGAGCCTAAGAGCTGTGACAAGACCCACACCTGTC CTCCTTGTCCTGCCCCTGAGCTGCTGGGCGGCCCTAGCGTGTTCCTGTTCCCTC CTAAGCCTAAGGACACCCTGATGATCAGCAGAACCCCTGAGGTGACCTGTGTG GTGGTGGACGTGAGCCACGAGGACCCTGAGGTGAAGTTCAACTGGTACGTGGA CGGCGTGGAGGTGCACAACGCCAAGACCAAGCCTAGAGAGGAGCAGTACAAC AGCACCTACAGAGTGGTGAGCGTGCTGACCGTGCTGCACCAGGACTGGCTGAA CGGCAAGGAGTACAAGTGTAAGGTGAGCAACAAGGCCCTGCCTGCCCCTATCG AGAAGACCATCAGCAAGGCCAAGGGCCAGCCTAGAGAGCCTCAGGTGTACAC CCTGCCTCCTAGCAGAGACGAGCTGACCAAGAACCAGGTGAGCCTGACCTGTC TGGTGAAGGGCTTCTACCCTAGCGACATCGCCGTGGAGTGGGAGAGCAACGGC CAGCCTGAGAACAACTACAAGACCACCCCTCCTGTGCTGGACAGCGACGGCAG CTTCTTCCTGTACAGCAAGCTGACCGTGGACAAGAGCAGATGGCAGCAGGGCA ACGTGTTCAGCTGTAGCGTGATGCACGAGGCCCTGCACAACCACTACACCCAG AAGAGCCTGAGCCTGAGCCCTGGCAAGTGA (SEQ ID NO: 5).CB6_Heavy_Chain_CUBOPT_CHOGnme_113_181: Optimized sequence to the mostfrequent codons in the CHO genome; total number of changes: 113 codons, 181 nucleotides; Synthetic Codons: [26 33 36 38 40 41 44 49 50 54 57 60 68 72 75 81 85 89 90 93 103 105 114 116 119 137 138 140 144 145 148 151 152 153 155 157 165 172 174 178 181 186 192 197 198 202 204 205 210 211 212 213 221 227 228 235 238 240 241 247 248 249 250 251 253 259 260 265 266 268 275 276 278 282 288 292 312 313 319 322 325 342 345 350 352 358 364 365 367 373 374 375 376 385 388 395 396 404 408 416 417 421 424 429 436 437 445 446 447 461 463 465 466],

[0155] The light chain variable region of the CHO genome optimized antibody is encoded by the nucleotide sequenceGACATCGTGATGACCCAGAGCCCTAGCAGCCTGAGCGCCAGCGTGGGCGACA GAGTGACCATCACCTGTAGAGCCAGCCAGAGCATCAGCAGATACCTGAACTGG TACCAGCAGAAGCCTGGCAAGGCCCCTAAGCTGCTGATCTACGCCGCCAGCAG CCTGCAGAGCGGCGTGCCTAGCAGATTCAGCGGCAGCGGCAGCGGCACCGACT TCACCCTGACCATCAGCAGCCTGCAGCCTGAGGACTTCGCCACCTACTACTGTC AGCAGAGCTACAGCACCCCTCCTGAGTACACCTTCGGCCAGGGCACCAAGCTG GAGATCAAGAGAACCGTGGCCGCCCCTAGCGTGTTCATCTTCCCTCCTAGCGA CGAGCAGCTGAAGAGCGGCACCGCCAGCGTGGTGTGTCTGCTGAACAACTTCT ACCCTAGAGAGGCCAAGGTGCAGTGGAAGGTGGACAACGCCCTGCAGAGCGG CAACAGCCAGGAGAGCGTGACCGAGCAGGACAGCAAGGACAGCACCTACAGC CTGAGCAGCACCCTGACCCTGAGCAAGGCCGACTACGAGAAGCACAAGGTGT ACGCCTGTGAGGTGACCCACCAGGGCCTGAGCAGCCCTGTGACCAAGAGCTTC AACAGAGGCGAGTGTAGCTGA (SEQ ID NO: 6). A BLAST comparison of the CHO genome optimized sequence and the human optimized sequence is provided in Figure 15B.

[0156] The heavy chain variable region of the human optimized antibody is encoded by the nucleotide sequenceGAGGTGCAGCTGGTGGAGTCCGGCGGCGGCCTGGTGCAGCCCGGCGGCTCCCT GCGCCTGTCCTGCGCCGCCTCCGGCTTCACCGTGTCCTCCAACTACATGTCCTG GGTGCGCCAGGCCCCCGGCAAGGGCCTGGAGTGGGTGTCCGTGATCTACTCCG GCGGCTCCACCTTCTACGCCGACTCCGTGAAGGGCCGCTTCACCATCTCCCGCG ACAACTCCATGAACACCCTGTTCCTGCAGATGAACTCCCTGCGCGCCGAGGAC ACCGCCGTGTACTACTGCGCCCGCGTGCTGCCCATGTACGGCGACTACCTGGA CTACTGGGGCCAGGGCACCCTGGTGACCGTGTCCTCCGCCTCCACCAAGGGCC CCTCCGTGTTCCCCCTGGCCCCCTCCTCCAAGTCCACCTCCGGCGGCACCGCCG CCCTGGGCTGCCTGGTGAAGGACTACTTCCCCGAGCCCGTGACCGTGTCCTGGAACTCCGGCGCCCTGACCTCCGGCGTGCACACCTTCCCCGCCGTGCTGCAGTCC TCCGGCCTGTACTCCCTGTCCTCCGTGGTGACCGTGCCCTCCTCCTCCCTGGGC ACCCAGACCTACATCTGCAACGTGAACCACAAGCCCTCCAACACCAAGGTGGA CAAGCGCGTGGAGCCCAAGTCCTGCGACAAGACCCACACCTGCCCCCCCTGCC CCGCCCCCGAGCTGCTGGGCGGCCCCTCCGTGTTCCTGTTCCCCCCCAAGCCCA AGGACACCCTGATGATCTCCCGCACCCCCGAGGTGACCTGCGTGGTGGTGGAC GTGTCCCACGAGGACCCCGAGGTGAAGTTCAACTGGTACGTGGACGGCGTGGA GGTGCACAACGCCAAGACCAAGCCCCGCGAGGAGCAGTACAACTCCACCTAC CGCGTGGTGTCCGTGCTGACCGTGCTGCACCAGGACTGGCTGAACGGCAAGGA GTACAAGTGCAAGGTGTCCAACAAGGCCCTGCCCGCCCCCATCGAGAAGACCA TCTCCAAGGCCAAGGGCCAGCCCCGCGAGCCCCAGGTGTACACCCTGCCCCCC TCCCGCGACGAGCTGACCAAGAACCAGGTGTCCCTGACCTGCCTGGTGAAGGG CTTCTACCCCTCCGACATCGCCGTGGAGTGGGAGTCCAACGGCCAGCCCGAGA ACAACTACAAGACCACCCCCCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTGT ACTCCAAGCTGACCGTGGACAAGTCCCGCTGGCAGCAGGGCAACGTGTTCTCC TGCTCCGTGATGCACGAGGCCCTGCACAACCACTACACCCAGAAGTCCCTGTC CCTGTCCCCCGGCAAGTGA (SEQ ID NO: 7). A summary of the mutated nucleotides is provided in Figure 17.

[0157] The light chain variable region of the human optimized antibody is encoded by the nucleotide sequenceGACATCGTGATGACCCAGTCCCCCTCCTCCCTGTCCGCCTCCGTGGGCGACCGC GTGACCATCACCTGCCGCGCCTCCCAGTCCATCTCCCGCTACCTGAACTGGTAC CAGCAGAAGCCCGGCAAGGCCCCCAAGCTGCTGATCTACGCCGCCTCCTCCCT GCAGTCCGGCGTGCCCTCCCGCTTCTCCGGCTCCGGCTCCGGCACCGACTTCAC CCTGACCATCTCCTCCCTGCAGCCCGAGGACTTCGCCACCTACTACTGCCAGCA GTCCTACTCCACCCCCCCCGAGTACACCTTCGGCCAGGGCACCAAGCTGGAGA TCAAGCGCACCGTGGCCGCCCCCTCCGTGTTCATCTTCCCCCCCTCCGACGAGC AGCTGAAGTCCGGCACCGCCTCCGTGGTGTGCCTGCTGAACAACTTCTACCCCC GCGAGGCCAAGGTGCAGTGGAAGGTGGACAACGCCCTGCAGTCCGGCAACTC CCAGGAGTCCGTGACCGAGCAGGACTCCAAGGACTCCACCTACTCCCTGTCCT CCACCCTGACCCTGTCCAAGGCCGACTACGAGAAGCACAAGGTGTACGCCTGC GAGGTGACCCACCAGGGCCTGTCCTCCCCCGTGACCAAGTCCTTCAACCGCGG CGAGTGCTCCTGA (SEQ ID NO: 8). A summary of the mutated nucleotides is providedin Figure 17. BLAST comparisons of the light chain variable region to the mouse optimized and CHO genome optimized antibodies are provided in Figures 15A-15B, respectively.

[0158] Demonstrating Evidence of Selection for Codon Usage in Antibodies: To computationally demonstrate that antibodies undergo selection at the level of codons, not only for a specific amino acid sequence, a database containing millions of mammalian antibody sequences (Olsen et al., “Observed Antibody Space: A diverse database of cleaned, annotated, and translated unpaired and paired antibody sequences”, Protein Science, 2022, Jan;31(l):141-146, the contents of which are herein incorporated by reference in their entirety) was analyzed. The results suggest that antibodies exhibit a preference for specific codons. Figures 2A-2E illustrate that the codon usage of antibodies is more biased than the entire genome’s coding sequences. Furthermore, this bias cannot be explained only by mutation patterns, suggesting the presence of a direct selection process driving codon usage in antibodies (Fig. 4).

[0159] The study was structured into 3 phases: data preprocessing, data analysis, and experimental validation, involving the expression of antibodies in CHO cells based on the insights from the data analysis (Fig. 1). Findings are presented from the data analysis based on the OAS database (Table 1), which indicate a selection process for codon usage in antibodies. The subsequent results subsections discuss distinct codon usage patterns of antibodies, providing further evidence supporting the discovery of a selection process. To start antibody codon frequencies are compared with genomic codon frequencies, followed by an analysis of codon usage selection in antibodies using randomization model. Variations in codon usage among antibodies from patients with different diseases are also examined, as are from patients responding to different vaccines. Additionally, complex patterns of codon usage are explored through longitudinal and spatial analyses. The final subsection presents experimental validation, demonstrating enhanced activity of antibodies designed based on these specific codon frequencies, informed by the data analysis, when expressed in CHO cells.Example 1: Antibody Codon Frequencies Are Different from Genomic Codon Frequencies

[0160] It was desired to check whether genes encoding antibodies share a codon preference, which is different than the general codon preference of the organism producing the antibodies. It was hypothesized that antibodies would have their own unique codon bias which could be exploited to codon optimize antibody sequences.

[0161] Analysis of the antibody sequence data suggests that the codon usage of antibodies is biased compared with the codon usage of the entire genome’s coding sequences (Fig. 2A- 2E) — for a given amino acid, the codon distribution or the most prevalent codon (considered as preferred as indicated by CAI (see Sharp & Li, 1987, “The codon adaptation index-a measure of directional synonymous codon usage bias, and its potential applications.” Nucleic Acids Research, 15(3), 1281-1295, the contents of which are hereby incorporated by reference in their entirety)) can vary considerably.

[0162] Although significant differences in distributions alone do not confirm the hypothesis of a selection process, this analysis took a two-pronged approach. First, the genome was compared not only to antibody protein families but also to non-antibody protein families with related functions, noting that in general, distributions from antibodies tend to show greater differences from the genome compared to those from non-antibody protein families. In particular, the following protein families were compared to the whole genome: enzyme regulator proteins (Fig. 3A-3E), serine / threonine -protein kinases (Fig. 8A-8E), protein kinase domain proteins (Fig. 9A-9E), antigen binding proteins (Fig. 10A-10E), transporter proteins (Fig. 11A-11E), neurotransmitter gated ion channels (Fig. 12A-12E), immunoglobulin-like domain containing proteins (Fig. 13A-13E), and C-terminal helicase proteins (Fig. 14A-14E). The difference in distribution for antibodies was far greater than for any other class of proteins. Notably, the protein family of antigen binding proteins (G0:0003823) did show a difference in distribution (Fig. 10E, though not as great as was observed for antibodies alone), though this was not surprising as this large category of molecules includes antibodies.

[0163] Second, a randomization model was employed to obtain statistical insights. The biased codon usage of antibodies cannot be explained only by mutation patterns — as Figure 4 shows that the antibody data displays a significant bias, evident from the reduced ENC (Wright, 1990, “The effective number of codons’ used in a gene.” Gene, 87(1), 23-29), when compared with randomized versions of the data — suggesting the presence of a direct selection process driving codon usage in antibodies.Example 2: Spatial analysis

[0164] The codon preferences across the antibody regions and positions are not constant. Figure 5 displays the CAI weights across the IMGT regions, based on a sample of aligned sequences. The preferred codons, indicated by yellow (highest CAI weight), show more variation across the regions for some amino acids and less variation for others. Noticing thisvariation, it was undertaken to determine whether these changes across positions are significant. Figure 6 presents the JSD distance between CAI weights for each region pair of the antibody. Using the VCUB randomization technique, it was found that these distances are significant, suggesting that there might be a selection process at the region level.Example 3: Improved antibody activity expressed in cho cells for antibodies designed based on antibody codon frequencies in comparison to antibodies designed based on human codon frequencies

[0165] To validate the hypothesis that the selection of codon usage leads to higher expression, 3 variants of a known antibody sequence (CB6 anti-COVID antibody, Shi R, et al., “Human neutralizing antibody targets the receptor-binding site of SARS-CoV-2”, Nature, 2020, Aug;584(7819:120-124, the contents of which are herein incorporated by reference in their entirety) were generated. This human monoclonal antibody was isolated from a COVID- 19 patient and was demonstrated to bind SARS-COV-2 receptor-binding domain (RBD) and neutralizes it. Table 2 describes the details of the variants, which include synonymous changes within the codons of both the heavy and light chains; changes that do not alter the protein sequence. Following synthesis, these variants were transfected into Chinese hamster ovary (CHO) cells and subsequently antibody expression and binding activity was measured (Fig. 7A).

[0166] Three variants of the original CB6 antibody all contain synonymous changes in both the light and heavy chains that do not alter the protein’s sequence. The compiled variant sequences were synthesized and inserted into plasmid RCK-CMP22 that was generated for this purpose (Fig. 16). The first variant contained changes in the original CB6 sequence, to match the codon usage bias for human antibodies and was named CB6a or CB6HUMAN_ANTIBODIES. The second variant contained changes to match the codon usage bias for mouse antibody genes (the closest genus to hamster that has antibodies data available) and was named CB6MOUSE_ANTIBODIES, and the third variant contained changes to match the whole genome Chinese hamster codon usage and was named CHO or CB6CHINESE_HAMSTER_GENOME. (See Fig. 17, as well as the Materials and Methods). The preferred codon used for each amino acid in each variant is provided in Tables 5-7. The plasmids harboring the three variants were transfect into Chinese hamster ovary (CHO) cells in culture.

[0167] Table 5: Preferred codons based on Human antibodies, (most preferred codon is bolded)

[0168] Table 6: Preferred codons based on Mouse antibodies. (Most preferred codon is bolded)| Vai (V) | GTT, GTC, GTA, GTG |

[0169] Table 7: Preferred codons based on Chinese hamster genome. (Most preferred codon is bolded)

[0170] A carefully controlled transient expression experiment was conducted. Following 24 and 48 hours, the binding activity of the antibodies was assessed (Fig. 7B). As expected, antibody activity was higher 48 hours post-transfection in all experiments. However, at the 48-hour time point, antibodies produced by cells transfected with the CB6MOUSE_ANTIBODIES variant exhibited significantly higher activity on average. This is an indication of a better expression, compared with cells transfected with the CB6HUMAN_ANTIBODIES or CB6CHINESE_HAMSTER_GENOME variants.

[0171] Figures 7C-7G show both mRNA and plasmid DNA levels as cycle threshold (CT) values, and as fold change relative to GAPDH gene expression. This analysis shows that while the cells transfected with the CB6MOUSE_ANTIBODIES variant (4 biological repeats) contained a low plasmid DNA copy number 48 hours post-transfection, their mRNA levels were the highest amongst the 3 variants, as opposed to cells transfected with the CB6CHINESE_HAMSTER_GENOME variant. This led to the expression of thevariant being very low, significantly lower than the human andmouse antibody variant, as shown in the mRNA to copy number ratio in Figure 7G. These results align with the observed antibody activity (Fig. 7B) and show that the CB6MOUSE_ANTIBODIES variant is most suitable for expression in CHO cells, with the CB6HUMAN_ANTIBODIES variant ranking second, while the CB6CHINESE_HAMSTER_GENOME variant, although displaying high plasmid copy number, was not expressed well. The mouse and human variants are also superior to the parental antibody sequence (CB6). These results are surprising and indicate that antibody codon bias is paramount, even over the bias of the host cell. It was expected that genes with CHO preferred codons would be most suitable for expression in CHO cells, however these results show the opposite.

[0172] This study shows that the codon usage of antibodies is consistent and different than the general codon usage of an organism. Furthermore, applying this distinct codon usage during antibody engineering demonstrates an enhancement in their expression, ultimately resulting in improved production efficiency.

[0173] Although the invention has been described in conjunction with specific embodiments thereof, it is evident that many alternatives, modifications and variations will be apparent to those skilled in the art. Accordingly, it is intended to embrace all such alternatives, modifications and variations that fall within the spirit and broad scope of the appended claims.

Claims

CLAIMS:

1. A method of optimizing an antibody coding sequence for translation, the method comprising: a. providing a nucleic acid sequence comprising a coding region encoding an antibody; and b. producing at least one synonymous mutation in said provided sequence thereby producing an optimized sequence, wherein said synonymous mutation converts at least one codon into a codon more commonly used in a set of genes consisting of a plurality of nucleic acid sequences encoding antibodies, wherein said set of genes is not a full genome; thereby optimizing an antibody coding sequence.

2. The method of claim 1, wherein said provided nucleic acid sequence encodes a variable region of an antibody heavy chain or an antibody light chain.

3. The method of claim 1 or 2, wherein said at least one synonymous mutation is not made in the first 15 codons of said coding region.

4. The method of any one of claims 1 to 3, wherein said set of genes consists of a plurality of nucleic acid sequences encoding human antibodies.

5. The method of claim 4, wherein said synonymous mutation comprises mutation of a codon to a preferred codon of Table 5.

6. The method of claim 5, wherein said synonymous mutation comprises at least one of: mutation of an alanine codon to GCC, mutation of an arginine codon to AGA, mutation of an asparagine codon to AAC, mutation of an aspartic acid codon to GAC, mutation of a cysteine codon to TGT, mutation of a glutamine codon to CAG, mutation of a glutamic acid codon to GAG, mutation of glycine codon to GGG, mutation of a histidine codon to CAC, mutation of an isoleucine codon to ATC, mutation of a leucine codon to CTG, mutation of lysine codon to AAG, mutation of a phenylalanine codon to TTC, mutation of a proline codon to CCC, mutation of a serine codon to TCC, mutation of a threonine codon to ACC, mutation of a tyrosine codon to TAC and mutation of a valine codon to GTG.

7. The method of any one of claims 1 to 3, wherein said set of genes consists of a plurality of nucleic acid sequences encoding mouse antibodies.

8. The method of claim 7, wherein said synonymous mutation comprises mutation of a codon to a preferred codon of Table 6.

9. The method of claim 5, wherein said synonymous mutation comprises at least one of: mutation of an alanine codon to GCT, mutation of an arginine codon to AGG, mutation of an asparagine codon to AAC, mutation of a aspartic acid codon to GAT, mutation of a cysteine codon to TGC, mutation of a glutamine codon to CAG, mutation of a glutamic acid codon to GAG, mutation of glycine codon to GGA, mutation of a histidine codon to CAC, mutation of an isoleucine codon to ATC, mutation of a leucine codon to CTG, mutation of lysine codon to AAG, mutation of a phenylalanine codon to TTC, mutation of a proline codon to CCA, mutation of a serine codon to TCT, mutation of a threonine codon to ACA, mutation of a tyrosine codon to TAC and mutation of a valine codon to GTC.

10. The method of any one of claims 1 to 9, wherein said at least one synonymous mutation is at least 10 synonymous mutations.

11. The method of any one of claims 1 to 10, wherein said at least one synonymous mutation is at least 100 synonymous mutations.

12. The method of any one of claims 1 to 11, wherein said at least one synonymous mutation is every possible synonymous mutation which converts a codon to a more commonly used codon in said set of genes, with the exception of synonymous mutations within the first 15 codons of the coding region.

13. The method of claim 12, wherein said every possible synonymous mutation is every possible synonymous mutation within the variable region of said antibody’s heavy chain, or every possible synonymous mutation within the variable region of said antibody’s light chain while not mutating the first 15 codons of said coding region.

14. The method of claim 12 or 13, wherein every possible synonymous mutation is every possible synonymous mutation within the variable region of said antibody’s heavy chain and the variable region of said antibody’s light chain while not mutating the first 15 codons of said coding region.

15. The method of any one of claims 1 to 14, wherein said at least one synonymous mutation increases translation of said optimized sequence as compared to said provided sequence in a target cell.

16. The method of any one of claims 1 to 15, wherein said optimizing for translation is optimizing translation of said antibody in a target cell and wherein said full genome is the full genome of said target cell.

17. The method of claim 16, wherein said target cell is a mammalian cell.

18. The method of claim 17, wherein said mammalian cell is a Chinese Hamster Ovary (CHO) cell.

19. A nucleic acid molecule system, comprising: a. a first nucleic acid molecule comprising a first coding region encoding an antibody heavy chain variable region; and b. a second nucleic acid molecule comprising a second coding region encoding a light chain variable region of said antibody; wherein at least one of said first and second coding regions comprises at least one synonymous mutation wherein said synonymous mutation converts at least one codon into a codon more commonly used in a set of genes consisting of a plurality of nucleic acid sequences encoding antibodies, wherein said set of genes is not a full genome.

20. The nucleic acid molecule system of claim 19, wherein said nucleic acid system is optimized for protein expression in CHO cells and wherein said full genome is a CHO cell full genome.

21. The nucleic acid molecule system of claim 19 or 20, wherein said at least one synonymous mutation is not made in the first 15 codons of said coding region.

22. The nucleic acid molecule system of any one of claims 19 to 21, wherein said set of genes consists of a plurality of nucleic acid sequences encoding human antibodies.

23. The nucleic acid molecule system of claim 22, wherein said synonymous mutation comprises mutation of a codon to a preferred codon of Table 5.

24. The nucleic acid molecule system of claim 23, wherein said synonymous mutation comprises at least one of: mutation of an alanine codon to GCC, mutation of an arginine codon to AGA, mutation of an asparagine codon to AAC, mutation of a aspartic acid codon to GAC, mutation of a cysteine codon to TGT, mutation of a glutamine codon to CAG, mutation of a glutamic acid codon to GAG, mutation of glycine codon to GGG, mutation of a histidine codon to CAC, mutation of an isoleucine codon to ATC, mutation of a leucine codon to CTG, mutation of lysine codon to AAG, mutation of a phenylalanine codon to TTC, mutation of a proline codon to CCC, mutation of a serine codon to TCC, mutation of a threonine codon to ACC, mutation of a tyrosine codon to TAC and mutation of a valine codon to GTG.

25. The nucleic acid molecule system of any one of claims 19 to 21, wherein said set of genes consists of a plurality of nucleic acid sequences encoding mouse antibodies.

26. The nucleic acid molecule system of claim 25, wherein said synonymous mutation comprises mutation of a codon to a preferred codon of Table 6.

27. The nucleic acid molecule system of claim 26, wherein said synonymous mutation comprises at least one of: mutation of an alanine codon to GCT, mutation of an arginine codon to AGG, mutation of an asparagine codon to AAC, mutation of a aspartic acid codon to GAT, mutation of a cysteine codon to TGC, mutation of a glutamine codon to CAG, mutation of a glutamic acid codon to GAG, mutation of glycine codon to GGA, mutation of a histidine codon to CAC, mutation of an isoleucine codon to ATC, mutation of a leucine codon to CTG, mutation of lysine codon to AAG, mutation of a phenylalanine codon to TTC, mutation of a proline codon to CCA, mutation of a serine codon to TCT, mutation of a threonine codon to ACA, mutation of a tyrosine codon to TAC and mutation of a valine codon to GTC.

28. The nucleic acid molecule system of any one of claims 19 to 27, wherein said at least one synonymous mutation is at least 10 synonymous mutations.

29. The nucleic acid molecule system of any one of claims 19 to 28, wherein said at least one synonymous mutation is at least 100 synonymous mutations.

30. The nucleic acid molecule system of any one of claims 19 to 29, wherein said at least one synonymous mutation is every possible synonymous mutation which converts acodon to a more commonly used codon in said set of genes, with the exception of synonymous mutations within the first 15 codons of the coding region.

31. The nucleic acid molecule system of claim 30, wherein said every possible synonymous mutation is every possible synonymous mutation within the variable region of said antibody’s heavy chain, or every possible synonymous mutation within the variable region of said antibody’s light chain while not mutating the first 15 codons of said coding region.

32. The nucleic acid molecule system of claim 30 or 31, wherein every possible synonymous mutation is every possible synonymous mutation within the variable region of said antibody’s heavy chain and the variable region of said antibody’s light chain while not mutating the first 15 codons of said coding region.

33. A CHO cell comprising a nucleic acid molecule system of any one of claims 19 to 32.

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