WO2012011955A2 - Compositions and methods for production and screening of monoclonal antibodies - Google Patents

Compositions and methods for production and screening of monoclonal antibodies Download PDF

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Publication number
WO2012011955A2
WO2012011955A2 PCT/US2011/001292 US2011001292W WO2012011955A2 WO 2012011955 A2 WO2012011955 A2 WO 2012011955A2 US 2011001292 W US2011001292 W US 2011001292W WO 2012011955 A2 WO2012011955 A2 WO 2012011955A2
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expression
gene expression
production
genes
antibody
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PCT/US2011/001292
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WO2012011955A3 (en
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Hua-Xin Liao
Barton F. Haynes
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Duke University
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4702—Regulators; Modulating activity
    • C07K14/4703—Inhibitors; Suppressors
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/08—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses
    • C07K16/10—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses from RNA viruses
    • C07K16/1018—Orthomyxoviridae, e.g. influenza virus
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/08—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses
    • C07K16/10—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses from RNA viruses
    • C07K16/1036—Retroviridae, e.g. leukemia viruses
    • C07K16/1045—Lentiviridae, e.g. HIV, FIV, SIV
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/78—Hydrolases (3) acting on carbon to nitrogen bonds other than peptide bonds (3.5)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/02—Fusion polypeptide containing a localisation/targetting motif containing a signal sequence
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/20—Fusion polypeptide containing a tag with affinity for a non-protein ligand
    • C07K2319/22—Fusion polypeptide containing a tag with affinity for a non-protein ligand containing a Strep-tag

Definitions

  • the present invention relates, in general, to compositions and methods for the production and screening of monoclonal antibodies and, in particular, to a dual gene expression system for expression of Ig genes and the production of stably transfected cell lines for high-throughput screening and analysis without cloning.
  • Immunoglobulin is comprised of two identical heavy- and two identical light-chains.
  • Ig heavy- and light-chain genes are produced by rearrangement of germline variable (V) and joining (J) gene segments at the light- chain locus, and by rearrangement of V, diversity (D) and J gene segments at the heavy-chain locus, respectively (Tonegawa, 1983; Diaz and Casali, 2002; Di Noia and Neuberger, 2007).
  • Ig diversity is enhanced by somatic hypermutation of the rearranged genes (Kim et al., 1981; Di Noia and Neuberger, 2007).
  • Antibody diversity allows the immune system to recognize a wide array of antigens (Honjo and Habu, 1985; Market and Papavasiliou, 2003).
  • Antibodies represent the correlates of protective immunity to infectious agents (Barreto et al., 2006). Monoclonal antibodies (mAbs) are important tools for studying pathogenesis, the protein structure of infectious agents and the correlates of protective immunity, and are essential to the development of passive antibodies
  • V H and V L Ig heavy- and light-chain variable regions
  • the expression of rearranged Ig genes as antibodies requires cloning of the amplified Ig VH and VL into eukaryotic cell expression plasmids containing a transcription regulation control element such as the CMV promoter (Boshart et al., 1985), sequences encoding the Ig leader, heavy and light-chain Ig constant regions and a poly(A) signal sequence (McLean et al., 2000; Connelly and Manley, 1988; Norderhaug et al., 1997).
  • a transcription regulation control element such as the CMV promoter (Boshart et al., 1985)
  • sequences encoding the Ig leader, heavy and light-chain Ig constant regions and a poly(A) signal sequence McLean et al., 2000; Connelly and Manley, 1988; Norderhaug et al., 1997.
  • immunization or an infection is the ability to amplify large numbers of Ig genes using a strategy that circumvents the Ig cloning step and yields sufficient quantities of transiently expressed Ig to allow functional characterization of expressed Igs.
  • Linear expression constructs generated by one-step PCR have been used for expression of vaccinia DNA topoisomerase I (Xiao et al., 2007) and HIV-1 envelope proteins (Kirchherr et al., 2007).
  • Linear Ig heavy- and light-chain gene expression cassettes have also been demonstrated to be a highly efficient strategy to rapidly express V H and VL genes isolated from sorted single B cells for high-throughput screening and analyzing of human mAbs without cloning (Liao et al. 2009).
  • production of purified mAbs in mg quantities by cloning Ig genes in plasmids, plasmids production, and generation of stably transfected cell lines are the major bottlenecks in human mAb production.
  • the present study results, at least in part, from the development of a dual gene expression system for expression of Ig genes and the production of stably transfected cell lines for high-throughput screening and analysis without cloning.
  • a strategy was designed that uses PCR and novel linear Ig heavy- and light-chain gene expression cassettes for rapid expression of Ig VH andV L genes as recombinant antibodies without cloning procedures.
  • the present invention relates to compositions and methods for the production and screening of monoclonal antibodies. More specifically, the invention relates to a dual gene expression system for expression of Ig genes and the production of stably transfected cell lines for high-throughput screening and analysis without cloning.
  • antibody and “immunoglobulin” are used interchangeably herein. These terms are well understood by those in the field, and refer to a protein consisting of one or more polypeptides that specifically binds an antigen.
  • One form of antibody constitutes the basic structural unit of an antibody. This form is a tetramer and consists of two identical pairs of antibody chains, each pair having one light and one heavy chain. In each pair, the light and heavy chain variable regions are together responsible for binding to an antigen, and the constant regions are responsible for the antibody effector functions.
  • immunoglobulin polypeptides include the kappa and lambda light chains and the alpha, gamma (IgGi, IgG 2 , IgG 3 , IgG 4 ), delta, epsilon and mu heavy chains or equivalents in other species.
  • Full-length immunoglobulin "light chains" (of about 25 kDa or about 214 amino acids) comprise a variable region of about 1 10 amino acids at the NH 2 - terminus and a kappa or lambda constant region at the COOH-terminus.
  • Full-length immunoglobulin "heavy chains” (of about 50 kDa or about 446 amino acids), similarly comprise a variable region (of about 1 16 amino acids) and one of the aforementioned heavy chain constant regions, e.g., gamma (of about 330 amino acids).
  • antibodies and immunoglobulin include antibodies or immunoglobulins of any isotype, fragments of antibodies which retain specific binding to antigen, including, but not limited to, Fab, Fv, scFv, and Fd fragments, chimeric antibodies, humanized antibodies, single-chain antibodies, and fusion proteins comprising an antigen- binding portion of an antibody and a non- antibody protein.
  • the antibodies may be detectably labeled, e.g., with a radioisotope, an enzyme which generates a detectable product, a fluorescent protein, and the like.
  • the antibodies may be further conjugated to other moieties, such as members of specific binding pairs, e.g., biotin (member of biotin- avidin specific binding pair), and the like.
  • the antibodies may also be bound to a solid support, including, but not limited to, polystyrene plates or beads, and the like. Also encompassed by the terms are Fab', Fv, F(ab') 2 , and or other antibody fragments that retain specific binding to antigen.
  • determining As used herein, the terms “determining,” “measuring,” “assessing,” and “assaying” are used interchangeably and include both quantitative and qualitative determinations.
  • polypeptide and protein are used interchangeably herein to refer to a polymeric form of amino acids of any length, which can include coded and non-coded amino acids, chemically or biochemically modified or derivatized amino acids, and polypeptides having modified peptide backbones.
  • the term includes fusion proteins, including, but not limited to, fusion proteins with a heterologous amino acid sequence, fusions with heterologous and homologous leader sequences, with or without N-terminal methionine residues;
  • fusion proteins with detectable fusion partners e.g., fusion proteins including as a fusion partner a fluorescent protein, p- galactosidase, luciferase, etc.; and the like.
  • isolated when used in the context of an isolated antibody, refers to an antibody of interest that is at least 60% free, at least 75% free, at least 90% free, at least 95% free, at least 98% free, and even at least 99% free from other components with which the antibody is associated with prior to purification.
  • a “coding sequence” or a sequence that "encodes” a selected polypeptide is a nucleic acid molecule which is transcribed (in the case of DNA) and translated (in the case of mRNA) into a polypeptide, for example, in vivo when placed under the control of appropriate regulatory sequences (or"control elements").
  • the boundaries of the coding sequence are typically determined by a start codon at the 5' (amino) terminus and a translation stop codon at the 3' (carboxy) terminus.
  • a coding sequence can include, but is not limited to, cDNA from viral, procaryotic or eucaryotic mRNA, genomic DNA sequences from viral or procaryotic DNA, and synthetic DNA sequences.
  • a transcription termination sequence may be located 3' to the coding sequence.
  • Other "control elements" may also be associated with a coding sequence.
  • a DNA sequence encoding a polypeptide can be optimized for expression in a selected cell by using the codons preferred by the selected cell to represent the DNA copy of the desired
  • Encoded by refers to a nucleic acid sequence which codes for a polypeptide sequence, wherein the polypeptide sequence or a portion thereof contains an amino acid sequence of at least 3 to 5 amino acids, more preferably at least 8 to 10 amino acids, and even more preferably at least 15 to 20 amino acids from a polypeptide encoded by the nucleic acid sequence. Also encompassed are polypeptide sequences that are immunologically identifiable with a polypeptide encoded by the sequence.
  • operably linked refers to an arrangement of elements wherein the components so described are configured so as to perform their usual function.
  • a given signal peptide that is operably linked to a polypeptide directs the secretion of the polypeptide from a cell.
  • a promoter that is operably linked to a coding sequence will direct the expression of a coding sequence.
  • the promoter or other control elements need not be contiguous with the coding sequence, so long as they function to direct the expression thereof. For example, intervening untranslated yet transcribed sequences can be present between the promoter sequence and the coding sequence and the promoter sequence can still be considered “operably linked" to the coding sequence.
  • nucleic acid construct it is meant a nucleic acid sequence that has been constructed to comprise one or more functional units not found together in nature. Examples include circular, linear, double-stranded, extrachromosomal DNA molecules (plasmids), cosmids (plasmids containing COS sequences from lambda phage), viral genomes comprising non-native nucleic acid sequences, and the like.
  • plasmids extrachromosomal DNA molecules
  • cosmids plasmids containing COS sequences from lambda phage
  • viral genomes comprising non-native nucleic acid sequences, and the like.
  • a "vector" is capable of transferring gene sequences to target cells.
  • vector construct means any nucleic acid construct capable of directing the expression of a gene of interest and which can transfer gene sequences to target cells, which can be accomplished by genomic integration of all or a portion of the vector, or transient or inheritable maintenance of the vector as an extrachromosomal element.
  • vector construct means any nucleic acid construct capable of directing the expression of a gene of interest and which can transfer gene sequences to target cells, which can be accomplished by genomic integration of all or a portion of the vector, or transient or inheritable maintenance of the vector as an extrachromosomal element.
  • expression vector means any nucleic acid construct capable of directing the expression of a gene of interest and which can transfer gene sequences to target cells, which can be accomplished by genomic integration of all or a portion of the vector, or transient or inheritable maintenance of the vector as an extrachromosomal element.
  • the term includes cloning, and expression vehicles, as well as integrating vectors.
  • An "expression cassette” comprises any nucleic acid construct capable of directing the expression of a gene/coding sequence of interest, which is operably linked to a promoter of the expression cassette.
  • Linear Ig heavy- and light-chain gene expression cassettes have been demonstrated to be a highly efficient strategy to rapidly express V H and VL genes isolated from sorted single B cells for high-throughput screening and analyzing of human mAbs without cloning (Liao et al. 2009).
  • production of purified mAbs in mg quantities by cloning Ig genes in plasmids, plasmids production, and generation of stably transfected cell lines are the major bottlenecks in human mAb production.
  • the present invention thus relates to a dual gene expression system for expression of Ig genes and the production of stably transfected cell lines for high- throughput screening and analysis without cloning.
  • the invention provides methods for producing a monoclonal antibody in a host cell. The methods involve introducing linear expression cassettes containing coding sequences for immunoglobulin heavy and light chains into a host cell and expressing a monoclonal antibody.
  • the invention further provides methods of screening a plurality of monoclonal antibodies to identify a monoclonal antibody of interest and its encoding nucleic acid.
  • the invention further provides kits for carrying out the subject methods.
  • the Example that follows describes that novel linear Ig heavy-chain and light-chain stable gene expression cassettes were constructed by PCR without cloning.
  • the gene cassettes contained dual gene expression systems for expression of Ig genes under control of C V promoter and expression of Blasticidin or Zeocin drug-resistant (R) genes under control of simian (S) CMV promoter or internal ribosomal entry site (IRES).
  • R Blasticidin or Zeocin drug-resistant
  • S simian
  • IVS internal ribosomal entry site
  • Combination of the light-chain dual gene expression cassette with a heavy-chain dual gene expression cassette containing second drug selection marker (Zeocin R ) improved the efficiency for production of purified antibodies and selecting stably transfected antibody- expression cell lines.
  • Novel linear Ig heavy-chain and light-chain stable gene expression cassettes were constructed by PCR without cloning.
  • the gene cassettes contained dual gene expression systems for expression of Ig genes under control of CMV promoter and expression of Blasticidin or Zeocin drug-resistant (R) genes under control of simian (S) CMV promoter or internal ribosomal entry site (IRES).
  • SCMV promoter and IRES were first tested for driving the Blasticidin gene in the linear light-chain gene expression cassettes for use as single drug selection, and were also effective in driving expression of the Blasitcidin R gene and development of drug-resistant 293T cell clones cultured in the presence of blasticidin (lOug/ml) with no negative effect on recombinant mAb expression.
  • the utility of the Ig light-chain stable expression cassette was established using 3 model antibodies including of HIV- 1 MPER antibody (CAP2311) and 2 influenza HA-specific antibodies (DF 1823 and DF1835). Combination of the light-chain dual gene expression cassette with a heavy-chain dual gene expression cassette containing second drug selection marker (Zeocin R ) further improved the efficiency for production of purified antibodies and selecting stably transfected antibody-expression cell lines.
  • CAP2311 HIV- 1 MPER antibody
  • 2 influenza HA-specific antibodies DF 1823 and DF1835
  • Zeocin R second drug selection marker
  • Ig gene stable expression cassettes constitute a highly efficient strategy to express rapidly Ig genes and produce stably transfected cell lines for high- throughput screening and analysis without cloning, thus allowing for extensive repertoire analysis of HIV-] -induced B cells.

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Abstract

The present invention relates, in general, to compositions and methods for the production and screening of monoclonal antibodies and, in particular, to a dual gene expression system for expression of Ig genes and the production of stably transfected cell lines for high-throughput screening and analysis without cloning.

Description

COMPOSITIONS AND METHODS FOR PRODUCTION AND SCREENING OF MONOCLONAL ANTIBODIES
This application claims the priority of US Provisional Application No. 61/366,295, filed July 21 , 2010, the entirety of which is incorporated herein by reference.
This invention was made with government support under Grant No.
AI0678501 awarded by the National Institutes of Health. The government has certain rights in the invention.
TECHNICAL FIELD
The present invention relates, in general, to compositions and methods for the production and screening of monoclonal antibodies and, in particular, to a dual gene expression system for expression of Ig genes and the production of stably transfected cell lines for high-throughput screening and analysis without cloning.
BACKGROUND
Immunoglobulin (Ig) is comprised of two identical heavy- and two identical light-chains. Ig heavy- and light-chain genes are produced by rearrangement of germline variable (V) and joining (J) gene segments at the light- chain locus, and by rearrangement of V, diversity (D) and J gene segments at the heavy-chain locus, respectively (Tonegawa, 1983; Diaz and Casali, 2002; Di Noia and Neuberger, 2007). Ig diversity is enhanced by somatic hypermutation of the rearranged genes (Kim et al., 1981; Di Noia and Neuberger, 2007). Antibody diversity allows the immune system to recognize a wide array of antigens (Honjo and Habu, 1985; Market and Papavasiliou, 2003). Antibodies represent the correlates of protective immunity to infectious agents (Barreto et al., 2006). Monoclonal antibodies (mAbs) are important tools for studying pathogenesis, the protein structure of infectious agents and the correlates of protective immunity, and are essential to the development of passive
immunotherapy and diagnostics against infectious agents. Defining the molecular aspects of human B cell repertoires to viral pathogens is critical for designing vaccines to induce broadly protective antibody responses to infections such as HIV-1 and influenza. The traditional methods used for generating human mAbs include screening Epstein-Barr virus (EBV)-transformed human B cell clones or antibody phage display libraries. These methods are often time-consuming and can have low yields of pathogen-specific mAbs. Although electroporation (Yu et al., 2008) and use of B cell activation by oCPGs (Traggiai et al., 2004) have improved the efficiency for development of EBV transformed antibody-secretion B cell lines, techniques for the isolation, sequencing and cloning of rearranged heavy- and light chain genes directly from human B cells are of interest because they provide a means to produce higher numbers of specific human mAbs.
It has been shown that rearranged Ig heavy- and light-chain variable regions (VH and VL) can be amplified from single B cells using RT-PCR (Tiller et al., 2008; Volkheimer et al., 2007;Wrammert et al., 2008), thus making it possible to produce mAbs recombinantly (Wardemann et al., 2003; oelsch et al., 2007; Tiller et al., 2008; Wrammert et al., 2008). Generally, the expression of rearranged Ig genes as antibodies requires cloning of the amplified Ig VH and VL into eukaryotic cell expression plasmids containing a transcription regulation control element such as the CMV promoter (Boshart et al., 1985), sequences encoding the Ig leader, heavy and light-chain Ig constant regions and a poly(A) signal sequence (McLean et al., 2000; Connelly and Manley, 1988; Norderhaug et al., 1997). Thus, what is needed to profile the Ig repertoire following
immunization or an infection is the ability to amplify large numbers of Ig genes using a strategy that circumvents the Ig cloning step and yields sufficient quantities of transiently expressed Ig to allow functional characterization of expressed Igs. Linear expression constructs generated by one-step PCR have been used for expression of vaccinia DNA topoisomerase I (Xiao et al., 2007) and HIV-1 envelope proteins (Kirchherr et al., 2007).
Linear Ig heavy- and light-chain gene expression cassettes have also been demonstrated to be a highly efficient strategy to rapidly express VH and VL genes isolated from sorted single B cells for high-throughput screening and analyzing of human mAbs without cloning (Liao et al. 2009). However, production of purified mAbs in mg quantities by cloning Ig genes in plasmids, plasmids production, and generation of stably transfected cell lines are the major bottlenecks in human mAb production.
The present study results, at least in part, from the development of a dual gene expression system for expression of Ig genes and the production of stably transfected cell lines for high-throughput screening and analysis without cloning. To facilitate high-throughput testing of amplified Ig VH and VL genes for antibody expression and specificity analysis, a strategy was designed that uses PCR and novel linear Ig heavy- and light-chain gene expression cassettes for rapid expression of Ig VH andVL genes as recombinant antibodies without cloning procedures.
SUMMARY OF THE INVENTION
In general, the present invention relates to compositions and methods for the production and screening of monoclonal antibodies. More specifically, the invention relates to a dual gene expression system for expression of Ig genes and the production of stably transfected cell lines for high-throughput screening and analysis without cloning.
Objects and advantages of the present invention will be clear from the description that follows. DETAILED DESCRIPTION OF THE INVENTION
Definitions
The terms "antibody" and "immunoglobulin" are used interchangeably herein. These terms are well understood by those in the field, and refer to a protein consisting of one or more polypeptides that specifically binds an antigen. One form of antibody constitutes the basic structural unit of an antibody. This form is a tetramer and consists of two identical pairs of antibody chains, each pair having one light and one heavy chain. In each pair, the light and heavy chain variable regions are together responsible for binding to an antigen, and the constant regions are responsible for the antibody effector functions.
Recognized immunoglobulin polypeptides include the kappa and lambda light chains and the alpha, gamma (IgGi, IgG2, IgG3, IgG4), delta, epsilon and mu heavy chains or equivalents in other species. Full-length immunoglobulin "light chains" (of about 25 kDa or about 214 amino acids) comprise a variable region of about 1 10 amino acids at the NH2- terminus and a kappa or lambda constant region at the COOH-terminus. Full-length immunoglobulin "heavy chains" (of about 50 kDa or about 446 amino acids), similarly comprise a variable region (of about 1 16 amino acids) and one of the aforementioned heavy chain constant regions, e.g., gamma (of about 330 amino acids).
The terms "antibodies" and "immunoglobulin" include antibodies or immunoglobulins of any isotype, fragments of antibodies which retain specific binding to antigen, including, but not limited to, Fab, Fv, scFv, and Fd fragments, chimeric antibodies, humanized antibodies, single-chain antibodies, and fusion proteins comprising an antigen- binding portion of an antibody and a non- antibody protein. The antibodies may be detectably labeled, e.g., with a radioisotope, an enzyme which generates a detectable product, a fluorescent protein, and the like. The antibodies may be further conjugated to other moieties, such as members of specific binding pairs, e.g., biotin (member of biotin- avidin specific binding pair), and the like. The antibodies may also be bound to a solid support, including, but not limited to, polystyrene plates or beads, and the like. Also encompassed by the terms are Fab', Fv, F(ab')2, and or other antibody fragments that retain specific binding to antigen.
As used herein, the terms "determining," "measuring," "assessing," and "assaying" are used interchangeably and include both quantitative and qualitative determinations.
The terms "polypeptide" and "protein" are used interchangeably herein to refer to a polymeric form of amino acids of any length, which can include coded and non-coded amino acids, chemically or biochemically modified or derivatized amino acids, and polypeptides having modified peptide backbones. The term includes fusion proteins, including, but not limited to, fusion proteins with a heterologous amino acid sequence, fusions with heterologous and homologous leader sequences, with or without N-terminal methionine residues;
immunologically tagged proteins; fusion proteins with detectable fusion partners, e.g., fusion proteins including as a fusion partner a fluorescent protein, p- galactosidase, luciferase, etc.; and the like.
As used herein the term "isolated" when used in the context of an isolated antibody, refers to an antibody of interest that is at least 60% free, at least 75% free, at least 90% free, at least 95% free, at least 98% free, and even at least 99% free from other components with which the antibody is associated with prior to purification.
A "coding sequence" or a sequence that "encodes" a selected polypeptide, is a nucleic acid molecule which is transcribed (in the case of DNA) and translated (in the case of mRNA) into a polypeptide, for example, in vivo when placed under the control of appropriate regulatory sequences (or"control elements"). The boundaries of the coding sequence are typically determined by a start codon at the 5' (amino) terminus and a translation stop codon at the 3' (carboxy) terminus. A coding sequence can include, but is not limited to, cDNA from viral, procaryotic or eucaryotic mRNA, genomic DNA sequences from viral or procaryotic DNA, and synthetic DNA sequences. A transcription termination sequence may be located 3' to the coding sequence. Other "control elements" may also be associated with a coding sequence. A DNA sequence encoding a polypeptide can be optimized for expression in a selected cell by using the codons preferred by the selected cell to represent the DNA copy of the desired
polypeptide coding sequence.
"Encoded by" refers to a nucleic acid sequence which codes for a polypeptide sequence, wherein the polypeptide sequence or a portion thereof contains an amino acid sequence of at least 3 to 5 amino acids, more preferably at least 8 to 10 amino acids, and even more preferably at least 15 to 20 amino acids from a polypeptide encoded by the nucleic acid sequence. Also encompassed are polypeptide sequences that are immunologically identifiable with a polypeptide encoded by the sequence.
"Operably linked" refers to an arrangement of elements wherein the components so described are configured so as to perform their usual function. Thus, a given signal peptide that is operably linked to a polypeptide directs the secretion of the polypeptide from a cell. In the case of a promoter, a promoter that is operably linked to a coding sequence will direct the expression of a coding sequence. The promoter or other control elements need not be contiguous with the coding sequence, so long as they function to direct the expression thereof. For example, intervening untranslated yet transcribed sequences can be present between the promoter sequence and the coding sequence and the promoter sequence can still be considered "operably linked" to the coding sequence.
By "nucleic acid construct" it is meant a nucleic acid sequence that has been constructed to comprise one or more functional units not found together in nature. Examples include circular, linear, double-stranded, extrachromosomal DNA molecules (plasmids), cosmids (plasmids containing COS sequences from lambda phage), viral genomes comprising non-native nucleic acid sequences, and the like.
A "vector" is capable of transferring gene sequences to target cells.
Typically, "vector construct," "expression vector," and "gene transfer vector," mean any nucleic acid construct capable of directing the expression of a gene of interest and which can transfer gene sequences to target cells, which can be accomplished by genomic integration of all or a portion of the vector, or transient or inheritable maintenance of the vector as an extrachromosomal element. Thus, the term includes cloning, and expression vehicles, as well as integrating vectors.
An "expression cassette" comprises any nucleic acid construct capable of directing the expression of a gene/coding sequence of interest, which is operably linked to a promoter of the expression cassette.
Compositions and Methods
Linear Ig heavy- and light-chain gene expression cassettes have been demonstrated to be a highly efficient strategy to rapidly express VH and VL genes isolated from sorted single B cells for high-throughput screening and analyzing of human mAbs without cloning (Liao et al. 2009). However, production of purified mAbs in mg quantities by cloning Ig genes in plasmids, plasmids production, and generation of stably transfected cell lines are the major bottlenecks in human mAb production.
The present invention thus relates to a dual gene expression system for expression of Ig genes and the production of stably transfected cell lines for high- throughput screening and analysis without cloning. The invention provides methods for producing a monoclonal antibody in a host cell. The methods involve introducing linear expression cassettes containing coding sequences for immunoglobulin heavy and light chains into a host cell and expressing a monoclonal antibody. The invention further provides methods of screening a plurality of monoclonal antibodies to identify a monoclonal antibody of interest and its encoding nucleic acid. Also provided by the invention are host cells containing monoclonal antibody-encoding sequences, and libraries of monoclonal antibodies for use in screening methods. The invention further provides kits for carrying out the subject methods.
The Example that follows describes that novel linear Ig heavy-chain and light-chain stable gene expression cassettes were constructed by PCR without cloning. The gene cassettes contained dual gene expression systems for expression of Ig genes under control of C V promoter and expression of Blasticidin or Zeocin drug-resistant (R) genes under control of simian (S) CMV promoter or internal ribosomal entry site (IRES). Combination of the light-chain dual gene expression cassette with a heavy-chain dual gene expression cassette containing second drug selection marker (ZeocinR) improved the efficiency for production of purified antibodies and selecting stably transfected antibody- expression cell lines.
Certain aspects of the invention can be described in greater detail in the non-limiting Example that follows. (Liao et al. 2009 is incorporated herein by reference.)
EXAMPLE
Novel linear Ig heavy-chain and light-chain stable gene expression cassettes were constructed by PCR without cloning. The gene cassettes contained dual gene expression systems for expression of Ig genes under control of CMV promoter and expression of Blasticidin or Zeocin drug-resistant (R) genes under control of simian (S) CMV promoter or internal ribosomal entry site (IRES). SCMV promoter and IRES were first tested for driving the Blasticidin gene in the linear light-chain gene expression cassettes for use as single drug selection, and were also effective in driving expression of the BlasitcidinR gene and development of drug-resistant 293T cell clones cultured in the presence of blasticidin (lOug/ml) with no negative effect on recombinant mAb expression. The utility of the Ig light-chain stable expression cassette was established using 3 model antibodies including of HIV- 1 MPER antibody (CAP2311) and 2 influenza HA-specific antibodies (DF 1823 and DF1835). Combination of the light-chain dual gene expression cassette with a heavy-chain dual gene expression cassette containing second drug selection marker (ZeocinR) further improved the efficiency for production of purified antibodies and selecting stably transfected antibody-expression cell lines.
Ig gene stable expression cassettes constitute a highly efficient strategy to express rapidly Ig genes and produce stably transfected cell lines for high- throughput screening and analysis without cloning, thus allowing for extensive repertoire analysis of HIV-] -induced B cells.
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* * *
All documents and other information sources cited herein are hereby incorporated in their entirety by reference.

Claims

WHAT IS CLAIMED IS:
1. A linear gene expression cassette comprising a dual gene expression system for expression of an Ig gene under control of CMV promoter and expression of Blasticidin or Zeocin drug-resistant (R) genes under control of simian (S) CMV promoter or internal ribosomal entry site (IRES).
2. A combination of an Ig light-chain dual gene expression cassette with an Ig heavy-chain dual gene expression cassette according to claim 1 comprising a second drug selection marker.
3. The combination according to claim 2 wherein said second drug selection marker is ZeocinR.
4. A method of producing a monoclonal antibody in a host cell comprising introducing into a host cell a linear expression cassette according to claim 1 comprising coding sequences for immunoglobulin heavy and light chains into a host cell under conditions such that said monoclonal antibody is expressed.
5. A host cell comprising the linear gene expression cassette according to claim 1.
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