Classifications

• • 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
  • C07K16/12—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria
  • C07K16/1267—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria from Gram-positive bacteria
  • C07K16/1275—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria from Gram-positive bacteria from Streptococcus (G)
• • 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
  • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
  • C12N15/09—Recombinant DNA-technology
  • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
  • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
  • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
  • C12N15/8201—Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation
  • C12N15/8214—Plastid transformation
• • 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
  • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
  • C12N15/09—Recombinant DNA-technology
  • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
  • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
  • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
  • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
  • C12N15/8242—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits
  • C12N15/8257—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits for the production of primary gene products, e.g. pharmaceutical products, interferon
  • C12N15/8258—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits for the production of primary gene products, e.g. pharmaceutical products, interferon for the production of oral vaccines (antigens) or immunoglobulins
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/10—Immunoglobulins specific features characterized by their source of isolation or production
  • C07K2317/13—Immunoglobulins specific features characterized by their source of isolation or production isolated from plants
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
  • C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2319/00—Fusion polypeptide
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2319/00—Fusion polypeptide
  • C07K2319/30—Non-immunoglobulin-derived peptide or protein having an immunoglobulin constant or Fc region, or a fragment thereof, attached thereto

Definitions

• TECHNICAL FIELD This invention relates to compositions and methods for production of multimeric proteins, including antibodies, in plants containing transformed plastids.
• transgenic plants to produce industrial or therapeutic biomolecules is one of the fastest developing areas in biotechnology.
• Recombinant proteins like monoclonal antibodies, vaccines, ho ⁇ nones, growth factors, neuropeptides, cytotoxins, serum proteins and enzymes have been expressed in nuclear transgenic plants (May et al, 1996).
• Plants provide several advantages for the production of therapeutic proteins, including lack of contamination with animal pathogens, relative ease of genetic manipulation, eukaryotic protein modification machinery and economical production. Plant genetic material is indefinitely stored in seeds, which require little or no maintenance. In particular, transgenic plants offer a number of advantages for production of recombinant/monoclonal antibodies. Plants have no immune system, therefore only one antibody species is expressed, and the absence of mammalian viruses and other pathogens provides maximum safety for humans and animals. Some types of monoclonal antibodies, such as secretory IgA (SlgA) can be produced in large quantities only in plants (Ma et al, 1995).
• secretory IgA secretory IgA
• Plants were also genetically engineered via the plastid genome to confer herbicide resistance; introduced foreign genes were maternally inherited, overcoming the problem of out-cross with weeds or other crops (Daniell et al. 1998). Plastid genetic engineering has been used to produce pharmaceutical proteins (Guda et al, 1999). Plastid genetic engineering is now extended to other useful crops (Sidorov et al, 1999; Daniell, 1999E). Nevertheless there has, until now, not been a demonstration of expression and assembly of an antibody in transgenic plastids.
• Plastids do not glycosylate their proteins. Although glycosylation is required for complement binding and effector function for some antibodies in serum, the effectiveness of antibodies at mucosal surfaces does not appear to involve glycosylation. Many single chain Ab fragments (scFv) and Fab's entirely lacking the constant regions of Ab molecule where glycosylation occurs bind to their appropriate antigen with the same affinity as the native Ab (Owen et al, 1992; Skerra et /., 1991; Skerra and Pluckthun, 1988). Non-glycosylated lull- length antibodies bind to their appropriate antigen with the same affinity as the native Ab (Boss et al, 1984). Antibodies made in plastids may have advantages for parenteral (injectable) uses, since they will not carry the potentially immunogenic plant N-linked glycans found on nuclear- encoded plantibodies.
• he plastid genome is thus an attractive target for introduction and expression of antibody genes.
• the reasons include: 1) capacity for extraordinarily high levels of foreign protein expression, 2) ability to fold, process and assemble eukaryotic proteins, 3) simpler purification, 4) containment of foreign genes through material inheritance and 5) no glycosylation.
• the signal peptide is cleaved off in the ER and stress proteins such as BiP/GRP78 and GRP94, which function as chaperonins, bind to unassembled light and heavy chains and direct their folding and assembly (Gething and Sambrook., 1992; Melnick et al, 1992).
• Disulfide bond formation is catalyzed by protein disulfide isomerase and N-linked glycans are attached in the ER and further processed in the Golgi, before the antibody is secreted from the cell.
• the present invention provides compositions and methods for the transformation of plastids of plant cells with multiple genes, and proper association or assembly of multimeric proteins that are heterologous to be plastids of plant cells.
• a plasmid construct encoding all of the individual polypeptide components of the multimeric protein is used.
• the plasmid used in the invention is made as an "expression cassette" which includes regulatory sequences.
• an expression cassette might include, operationally joined, DNA sequences coding for immunoglobulin heavy and light chains separated by a small linker containing an intervening stop codon and ribosome binding site, and control sequences positioned upstream from the 5' and downstream from the 3' ends of the coding sequences to provide expression of the coding sequences in the plastid genome. Flanking each side of this expression cassette would be DNA sequences that are homologous to a sequence of the target plastid genome. Stable integration of the heterologous coding sequences into the plastid genome of the target plant is accomplished through homologous recombination.
• the present invention achieves assembly of immunoglobulin heavy and light chains, with covalent bonding between the chains, into immunologically active immunoglobulins in the plastid.
• the expression cassette may include, operationally joined, DNA sequences coding for J chain and Secretory Components separated by a small linker containing an intervening stop codon and ribosome binding site, and control sequences positioned upstream from the 5' and downstream from the 3' ends of the coding sequences to provide expression of these coding sequences in the plastid genome.
• Homologous flanking sequences that may be the same as or different than the ones provided for the expression cassette containing the immunoglobulin heavy and light chains are similarly provided for this cassette.
• Secretory Component and J chain are also assembled with the immunoglobulin, when the heavy chain is an ⁇ (alpha) chain thereby producing secretory immunoglobulin A (SlgA).
• the antibodies produced by the present invention are antibodies which are useful for mammals, including animals and human, where it is generally accepted in the art to use antibodies in therapy.
• FIG. 1 Construction of the pLD-TP-Guy's 13 vector and PCR analysis of spectinomycin-resistant tobacco clones transformed with pLD-TP-Guy's 13.
• Figure 2A Construction of the pZS-TP-Guy's 13 vector and PCR analysis of spectinomycin resistant clones transformed with pZS-TP-Guy's 13.
• A PCR analysis of spectinomycin-resistant tobacco clones using 8P and 8M primer pair.
• B PCR analysis of spectinomycin-resistant tobacco clones using 7P and 8M primer pair.
• C The plastid pZS-TR Guy's 13 and primer annealing sites.
• Lane 1 kb ladder Lane 2, negative control without template; Lane 3, negative control untransformed plant; Lane 4, positive control previously characterized pZS-transformed plant; Lane 5, mutant clone; Lanes 6-10, transformed clones; Lane 11, the plasmid pZS-TP-Guy's 13.
• FIG. 3 Western blot analysis of antibody light chain expression in E. coli by the tobacco and universal vectors: Lane 1, molecular weight markers; Lane 2, negative control (insert in the wrong orientation); Lane 3A, XLl-Blue cells transformed with the pZS-TP-Guy's 13 vector; Lane 4A, negative control (untransformed XLl-Blue cells); Lane 3B, positive control Human IgA; Lane 4B, XLl-Blue cells transformed with the pLD-TP-Guy's 13 vector. Blots were probed with AP-conjugated goat anti-human kappa antibody.
• FIG. 4 Western blot analysis of antibody heavy chain expression in E. coli by the tobacco vector. Lane 1, molecular weight markers; Lane 2, negative control (insert in the wrong orientation); Lane 3, negative control (untransformed XLl-Blue cells); Lane 4, XLl-Blue cells transformed with the pZS-TP-Guy's 13 vector. Samples in blot A were sonicated, and those in blot B were boiled. Blots were probed with AP-conjugated goat anti-human IgA antibody. Figure 5. Steps in plastid transformation and regeneration of plastid transgenic plants.
• FIG. 1 Western blot analysis of antibody expression in Tobacco plastids.
• A Lane 1, molecular weight markers; Lanes 2-4, extracts from different transgenic plants; Lanes 5 and 7, blank, Lane 6, negative control extract from an untransformed plant; Lane 8, positive control human IgA.
• the gels were run under non-reducing conditions.
• Blot A was developed with AP- conjugated goat anti-human kappa antibodies.
• Blot B was developed using AP-conjugated goat anti-human IgA antibodies.
• FIG. 1 Western blot analysis of transgenic lines showing the assembled antibody.
• Lanes 1 and 2 extracts from transgenic plants; Lane 3, negative control extract from an untransformed plant; Lane 4 positive control human IgA.
• the gel was run under non-reducing conditions, and the blot was developed with AP-conjugated goat anti-human kappa antibody.
• FIG. 1 Southern blot analysis of the clones transformed with the pZS-TP-Guy's 13 vector. Lane C, control untransformed Petit Havana; Lanes 1-6, transgenic lines.
• Figure 9 Southern blot analysis of the clones transformed with the pLD-Guy's 13 vector. Lane C, control untransformed Petit Havana; Lanes 1-6, transgenic lines.
• FIG. 10 Northern Blot analysis of light chain transcripts in the transgenic lines transformed with the pZS-TP-Guy's 13 and the pLD-TP Guy's 13 vectors
• FIG. 11 Northern Blot analysis of heavy chain transcripts in the transgenic lines transformed with the pZS-TP-Guy's 13 and pLD-TP Guy's 13 vectors.
• A RNA gel before transfer.
• B RNA blot probed with radiolabelled heavy chain DNA probe. Lane 1, RNA ladder; Lane 2, control untransformed Petit Havana; Lanes 3-5, transgenic lines transformed with pZS- TP-Guy's 13; Lanes 6 and 7, transgenic lines transformed with pLD-TP-Guy's 13; Lane 8, post- transcriptionally silenced nuclear transformant CAR8841; Lane 9, expressing nuclear transformant CAR517; Lane 10, expressing nuclear transformant CAR532.
• a cell includes a plurality of cells, including mixtures thereof.
• variable region of an antibody refers to the variable region of the antibody's light chain or the variable region of the heavy chain either alone or in combination.
• a "polynucleotide” is a polymeric form of nucleotides of any length which contain deoxyribonucleotides, ribonucleotides, and/or their analogs.
• the term “polynucleotide” includes double-, single-stranded, and triple-helical molecules.
• any embodiment of the invention described herein that is a polynucleotide encompasses both the double-stranded form and each of two complementary single-stranded forms known or predicted to make up the double stranded form.
• polypeptide is used in its broadest sense to refer to a compound of two or more subunit amino acids. The subunits may be linked by peptide bonds.
• amino acid refers to natural and/or unnatural or synthetic amino acids, including glycine and both the D and L optical isomers.
• a peptide of three or more amino acids is commonly called an oligopeptide if the peptide chain is short. If the peptide chain is long, the peptide is commonly called a polypeptide or a protein.
• a "multimeric protein” as used herein refers to a globular protein containing more than one separate polypeptide or protein chain associated with each other to form a single globular protein in vitro or in vivo.
• the multimeric protein may consist of more than one polypeptide of the same kind to form a homodimeric or homotrimeric protein; the multimeric protein may also be composed of more than one polypeptide having distinct sequences to form, e.g., a heterodimer or a heterotrimer.
• Non-limiting examples of multimeric proteins include immunoglobulin molecules, receptor dimer complexes, trimeric G-proteins, and any enzyme complexes.
• immunoglobulin molecule or “antibody” is a polypeptide or multimeric protein containing the immunologically active portions of an immunoglobulin heavy chain and immunoglobulin light chain covalently coupled together and capable of specifically combining with antigen.
• the immunoglobulins or antibody molecules are a large family of molecules that include several types of molecules such as IgD, IgG, IgA, secretory IgA (SlgA), IgM, and IgE.
• the term "immunoglobulin molecule” includes for example hybrid antibodies or altered antibodies and fragments thereof, including but not limited to Fab fragment(s) and single-chain variable fragments (ScFv).
• Fab fragment of an immunoglobulin molecule is a multimeric protein consisting of the portion of an immunoglobulin molecule containing the immunologically active portions of an immunoglobulin heavy chain and an immunoglobulin light chain covalently coupled together and capable of specifically combining with an antigen.
• Fab fragments can be prepared by proteolytic digestion of substantially intact immunoglobulin molecules with papain using methods that are well known in the art. However, a Fab fragment may also be prepared by expressing in a suitable host cell the desired portions of immunoglobulin heavy chain and immunoglobulin light chain using methods disclosed herein or any other methods known in the art.
• ScFv fragment of an immunoglobulin molecule is a protein consisting of the immunologically active portions of an immunoglobulin heavy chain variable region and an immunoglobulin light chain variable region covalently coupled together and capable of specifically combining with an antigen. ScFv fragments are typically prepared by expressing a suitable host cell the desired portions of immunoglobulin heavy chain variable region and immunoglobulin light chain variable region using methods described herein and/or other methods known to artisans in the field.
• “Secretory component” is a fragment of an immunoglobulin molecule comprising secretory IgA as defined in US Patent No. 5,202,422 and US Patent No. 5,959,177, incorporated here by reference.
• J chain is a polypeptide that is involved in the polymerization of immunoglobulins and transport of polymerized immunoglobulins through epithelial cells. J chain is found in pentameric IgM and dimeric IgA and typically attached via disulfide bonds.
• a "protection protein” is a fragment of an immunoglobulin molecule comprising secretory IgA as defined in US Patent No. 6,046,037, incorporated herein by reference.
• Heterologous means derived from a genotypically distinct entity from that of the rest of the entity to which it is compared.
• a polynucleotide introduced by genetic engineering techniques into a different cell is a heterologous polynucleotide (and, when expressed, can encode a heterologous polypeptide).
• heterologous as applied to a multimeric protein means that the multimer is expressed in a host cell that is genotypically distinct from the host cell in which the multimer is normally expressed.
• the exemplified human IgA multimeric protein is heterologous to a plant cell.
• immunologically active refers to an immunoglobulin molecule having structural, regulatory, or biochemical functions of a naturally occurring molecule expressed in its native host cell.
• an immunologically active immunoglobulin produced in a plant cell by the methods of this invention has the structural characteristics of the naturally occurring molecule, and/or exhibits antigen binding specificity of the naturally occurring antibody that is present in the host cell in which the molecule is normally expressed.
• a “gene” refers to a polynucleotide containing at least one open reading frame that is capable of encoding a particular protein after being transcribed and translated.
• expression refers to the process by which polynucleotides are transcribed into mRNA and/or the process by which the transcribed mRNA is subsequently translated into polypeptides or proteins.
• construct refers to an artificially assembled DNA segment to be transferred into a target plant tissue or cell.
• the construct will include the gene or genes of a particular interest, a marker gene and appropriate control sequences.
• plasmid refers to an autonomous, self-replicating extrachromosomal DNA molecule.
• the plasmid constructs of the present invention contain sequences coding for heavy and light chains of an antibody. Plasmid constructs containing suitable regulatory elements are also referred to as "expression cassettes.”
• a plasmid construct can also contain a screening or selectable marker, for example an antibiotic resistance gene.
• selectable marker is used to refer to a gene that encodes a product that allows the growth of transgenic tissue on a selective medium.
• selectable markers include genes encoding for antibiotic resistance, e.g., ampicillin, kanamycin, or the like. Other selectable markers will be known to those of skill in the art.
• a "glycosylation signal sequence” is a three-amino acid sequence within a polypeptide, of the sequence N-X-S/T, where N is asparagine, X is any amino acid (except proline), S is serine, and T is threonine.
• N asparagine
• X any amino acid (except proline)
• S serine
• T threonine
• a “primer” is a short polynucleotide, generally with a free 3' OH group, that binds to a target or “template” potentially present in a sample of interest by hybridizing with the target, and thereafter promoting polymerization of a polynucleotide complementary to the target.
• a “polymerase chain reaction” (“PCR”) is a reaction in which replicate copies are made of a target polynucleotide using a "pair of primers” or a “set of primers” consisting of an "upstream” and a “downstream” primer, and a catalyst of polymerization, typically a thermally-stable DNA polymerase enzyme.
• Hybridization refers to a reaction in which one or more polynucleotides react to form a complex that is stabilized via hydrogen bonding between the bases of the nucleotide residues.
• the hydrogen bonding may occur by Watson-Crick base pairing, Hoogstein binding, or in any other sequence-specific manner.
• the complex may comprise two strands forming a duplex structure three or more strands forming a multi-stranded complex, a single self-hybridizing strand, or any combination of these.
• a hybridization reaction may constitute a step in a more extensive process, such as the initiation of a PCR reaction or the enzymatic cleavage of a polynucleotide by a ribozyme.
• a double-stranded polynucleotide can be “complementary” or “homologous” to another polynucleotide if hybridization can occur between one of the strands of the first polynucleotide and the second.
• homologous recombination refers to a process whereby two homologous double-stranded polynucleotides recombine to form a novel polynucleotide.
• transgenic plant refers to a genetically engineered plant or progeny of genetically engineered plants.
• the transgenic plant usually contains material from at least one unrelated organism, such as a virus, another plant or animal.
• a “control” is an alternative subject or sample used in an experiment for comparison purpose.
• a control can be "positive” or “negative.”
• the purpose of the experiment is to determine the presence of an exogenously introduced plasmid or the expression of a polypeptide encoded by such plasmid in a plant transformant or its progenies
• a positive control a plant or a sample from a plan, carrying such plasmid and/or expressing the encoded protein
• a negative control a plant or a sample from a plant lacking the plasmid of interest and/or expression of the polypeptide encoded by the plasmid.
• "Guy's 13" is a monoclonal antibody against the surface antigen I/II of Streptococcus mutans and is described in US Patent No. 5,518,721 and PCT/US95/16889 incorporated herein by reference.
• Humanized refers to a construct in which coding sequences for heavy and light chain variable regions from a species other than human have been fused, via genetic engineering to the coding sequences of the respective constant regions of human heavy and light chains. It also refers to the resulting antibodies.
• Codon optimization is the process of customizing a transgene so that it matches the bias of highly expressed genes in the genome in which it is to be expressed. For most amino acids there are two or more (up to six) different codons that can be used in mRNA. Every genome has a “bias” in the codons it uses, especially for highly expressed proteins. Changing the codon usage of a heterologous gene has been shown in many systems to increase the expression of that gene.
• an "operative ligand” is a polypeptide sequence that functionally interacts with or binds to another protein, polypeptide, carbohydrates or nucleic acid for a preferred function.
• an operative ligand would be ICAM-1, which binds to human rhinovirus, or an ScFv that binds to a particular epitope.
• passive immunotherapy Treatment of disease with antibodies is known as passive immunotherapy. This is distinguished from active immunotherapy, where vaccination stimulates the body's own antibody response.
• the efficacy of passive immunotherapy has been demonstrated in treatment of a number of infectious diseases, in both animals and humans.
• a major impediment to the commercialization of many types of passive immunotherapy is the need for repetitive delivery of large amounts of antibody to the site of the disease to overcome rapid clearing of the antibodies from the body.
• the production of antibodies by traditional methods is much too expensive to be practical for many types of passive immunotherapy. This is why production in plastids is such an attractive alternative.
• secretory IgA (SlgA) is the preferred antibody isotype.
• SlgA is the most abundant immunoglobulin found in the body and the most important form found in mucosal secretions, such as saliva, tears, breast milk and mucus of the bronchial, genitourinary, and digestive tracts (Kerr, 1990). It is composed of 10 polypeptides: 4 light chains, four IgA heavy chains, a J chain and a secretory component (SC), resulting in a total molecular weight of ⁇ 400 kDa. Binding of SlgA to bacterial and viral surface antigens prevents attachment of pathogens to the mucosal cells, and, once attachment is blocked, viral infection and bacterial colonization is inhibited.
• SlgA has demonstrated superiority over other antibodies for use in passive mucosal immunotherapy. It is more protease resistant than IgG or IgA, thus making it more stable in the gastrointestinal tract (Brown et al, 1970; Crottet and Corthesy, 1998, Renegar et al, 1998) and buccal mucosa (Ma et al, 1998). Recent work at Planet demonstrated that in the presence of pepsin at pH 2.5, antigen binding of an IgG antibody lasted 5 minutes versus 5 hours for the same antibody prepared as an SlgA plantibody. Such stability will be an important feature of antibodies used for the treatment of gastrointestinal tract infections, such as rotavirus and
• Clostridium difficile SlgA has twice as many binding sites than IgG, thus giving it an additional advantage where avidity is important.
• the superiority of SlgA over IgG or IgA has been demonstrated in a number of studies: 1) SlgA protected mice against group A Streptococci, but serum did not, even though the IgG had a higher titer by ELISA and opsonized cells more effectively in a mouse model (Bessen and Fischetti., 1988); 2) Mice were protected against influenza virus by intravenous injection of polymeric IgA (which was transported into nasal secretions as SlgA) while IgGl and monomeric IgA were ineffectual (Renegar and Parker, 1991); and 3) Anti gpl60 SlgA blocked transcytosis of HIV in human cells better than IgG, despite having lower specific activity (Hocini et al, 1997).
• Antibody expression in transgenic tobacco was accomplished using two plastid expression vectors pLD and pZS, as shown in Figures 1C and 2C.
• Both plastid vectors contain the 16S rRNA promoter (Prrri) driving the selectable marker gene aadA (aminoglycoside adenylyl transferase, conferring resistance to spectinomycin) followed by t epsbA 3' region (the terminator from a gene coding for photosystem II reaction center components) from the tobacco plastid genome.
• aadA aminoglycoside adenylyl transferase, conferring resistance to spectinomycin
• t epsbA 3' region the terminator from a gene coding for photosystem II reaction center components
• the tobacco vector integrates the aadA gene into the spacer region between rbcL (the gene for the large subunit of RuBisCo) and orf512 (the ccD gene) of the tobacco plastid genome.
• This vector is useful for integrating foreign genes specifically into the tobacco plastid genome; this gene order is not conserved among other plant plastid genomes.
• the universal plastid expression/integration vector uses tr «A and tral genes (plastid transfer RNAs coding for alanine and isoleucine), from the inverted repeat region of the tobacco plastid genome, as flanking sequences for homologous recombination.
• This vector can be used to transform plastid genomes of several other plant species (Daniell et al. 1998) because the flanking sequences are highly conserved among higher plants. Because the universal vector integrates foreign genes within the Inverted Repeat region of the plastid genome, it should double the copy number of antibody genes (from 5,000 to 10,000 copies per cell in tobacco). Furthermore, it has been demonstrated that homoplasmy is achieved even in the first round of selection in tobacco probably because of the presence of a plastid origin of replication within the flanking sequence in the universal vector (thereby providing more templates for integration). Because of these and several other reasons, foreign gene expression was shown to be much higher when the universal vector was used instead of the tobacco vector (Guda et al 2000).
• Example #1 An IgA Antibody against a Bacterial Surface Protein Expressed in Plastids A. Preparation of Antibody Heavy and Light Chain Expression Cassette
• the preferred heavy chain construct consists of the Guy's 13 heavy chain variable region fused to the human IgA2m(2) constant region. This heavy chain sub-isotype is resistant to the bacterial proteases that specifically target IgAl (Kerr, 1990).
• the light chain construct is a fusion of the Guy's 13 kappa chain variable region and the human kappa constant region. Expression of these two immunoglobulin chains, along with human J chain and human SC have resulted in the assembly in transgenic tobacco of a humanized Guy's 13 SlgA plantibody, which we call CaroRx.
• coding sequences were amplified, using PCR, from expression cassettes designed for nuclear expression.
• primers were engineered to incorporate a ribosome binding site utilized by the plastid protein translation machinery, and a methionine codon (in place of the signal peptides found in the nuclear expression constructs).
• H and L chain PCR products were individually cloned into the vector pCR-Script (Stratagene) and and their sequences verified.
• Both clones were cut with BamH I, creating cohesive ends at the 3 ' end of the H chain and at the 3' and 5' ends of the L chain, resulting in excision of the L chain.
• the L chain fragment was ligated adjacent to the 3' end of the H chain (with an intervening stop codon and ribosome binding site) yielding a vector, pCR-ScriptGuy's 13, that contained both, H and L chain fragments.
• Nucleotides 1-16 comprise linker sequences and a ribosome binding site.
• Nucleotides 17-1381 comprise a sequence encoding a mouse heavy chain variable/human IgA2m(2) constant hybrid with linker sequences. The native mouse signal peptide has been replaced with methionine (nt 17-19).
• the heavy chain variable region (nt 20-358) is from the murine monoclonal Guy's 13 (Smith and Lehner, 1989; US Patent No. 5,518,721 and 5,352,446, herein incorporated by reference).
• Nucleotides 1382-1408 comprise stop codon, linker sequences and a ribosome binding site.
• Nucleotides 1409-2050 comprise a sequence encoding a mouse light chain variable/human kappa constant hybrid with linker sequences. The native mouse signal peptide has been replaced with methionine (nt 1409- 1411).
• the light chain variable region (nt 1412-1731) is from the murine monoclonal Guy's 13 (Smith and Lehner 1989; US Patent No. 5,518,721 and 5,352,446).
• the sequence of the human kappa constant region (nt 1732-2050) has been previously published (Hieter et al. 1980).
• the pCR-ScriptGuy's 13 vector was digested with Xba I to excise the H/L chain insert, and the insert was ligated with Xba I-digested and dephosphorylated pLD vector (Universal vector).
• the resulting plasmid was designated as pLD-TP-Guy's 13 ( Figure 1).
• the sequences encoded are chimeric, consisting of mature variable regions from Guy's 13 heavy and light chains fused to the constant regions of human IgA2m(2) heavy chain and kappa light chain.
• Guy's 13 and pZS-TP-Guy's 13 vectors were selected on LB medium with ampicillin (100 ⁇ g/mL). Transformed colonies were tested for the presence of the correct coding sequence insert by plasmid isolation and restriction digestion.
• E. coli cells were lysed in TBS buffer (20 mM Tris-HCl, pH 8,
• Pellets were re-suspended in equal volumes of TBS buffer containing 2mM PMSF and 2X sample buffer, boiled for 5 min and electrophoresed on 12% polyacrylamide gels according to the standard procedure. The gels were blotted onto nitrocellulose membranes. The unoccupied binding sites on the blots were blocked by incubating them in blocking buffer [10 mM Tris-HCl, 0.5 M NaCl, 0.05% Twin 20 (v/v), and 5% non-fat dry milk (w/v)] at room temperature for lh. After blocking, blots were incubated with an appropriate antibody labeled with alkaline phosphatase at room temperature for 2 h.
• blocking buffer 10 mM Tris-HCl, 0.5 M NaCl, 0.05% Twin 20 (v/v), and 5% non-fat dry milk (w/v)
• Petit Havana plants were grown aseptically by germination of seeds on MSO medium containing MS salts (4.3 g/liter), B5 vitamin mixture (myo-inositol, 100 mg/liter; thiamine-HCl, 10 mg/liter; nicotinic acid, 1 mg/liter; pyridoxine-HCl, 1 mg/liter), sucrose (30g/liter) and phytagar (6 g/liter) at pH 5.8 (Ye et al, 1990).. Fully expanded, dark green leaves of about two month old plants grown under sterile conditions were used for bombardment.
• Leaves were placed abaxial side up on a Whatman No. 1 filter paper laying on RMOP medium (Daniell, 1993) in standard petri plates (100 x 15 mm) for bombardment.
• Tungsten (1 ⁇ m) or Gold (0.6 ⁇ m) microprojectiles were coated with plasmid DNA (plastid vectors) and bombardments were performed with the biolistic device PDSIOOO/He (Bio-Rad) as described by Daniell (1997). Following bombardment, petri plates were sealed with Parafilm and incubated at 24°C in the dark.
• leaves Two days after bombardment, leaves were cut into small pieces of ⁇ 5 mm 2 in size and placed on selection medium (RMOP containing 500 ⁇ g/mL of spectinomycin dihydrochloride) with the abaxial side touching the medium in deep (100 x 25 mm) petri plates ( ⁇ 6 pieces per plate).
• the regenerated spectinomycin-resistant shoots were cut into small pieces ( ⁇ 2mm 2 ) and subcloned into fresh deep petri plates ( ⁇ 5 pieces per plate) containing the same selection medium.
• Resistant shoots resulting from this second round of selection were then tested for the presence of the Guy's 13 construct (integration) using PCR (see below) and only transgenic shoots were transferred to rooting medium (MSO medium supplemented with IB A, 1 mg/L and spectinomycin dihycrochloride, 500 mg/L). These plants are designated TO plants. Rooted plants were transferred to soil and grown at 26°C under continuous lighting conditions for further analysis (Figure 5). Seed collected from TO plants were germinated on specinomycin, and then transferred to soil. These plants are designated Tl plants. Spectinomycin/streptomycin resistant clones were observed within 3-6 weeks after bombardment.
• Total DNA from unbombarded and transgenic plants was isolated using DNeasy Plant Mini Kit (Qiagen, Valencia, CA). PCR was performed in order to distinguish: a) true transformants from spontaneous mutants and b) plastid transformants from nuclear transformants. DNA was amplified using Taq PCT core kit (Qiagen, Valencia, CA), using standard protocols (Sambrook et al, 1989). Samples were amplified in the Perkin ElmerTM 92s GeneAmp PCR system 2400. PCR products were analyzed by electrophoresis on 0.8% agarose gels.
• primers 5P and 2M were used. One primer anneals to the aadA coding sequence and the other anneals to the trnA region to confirm integration of the entire gene cassette ( Figure 1C). The presence of the expected size PCR product (3.6 kb, Figure IB) confirmed that the entire gene cassette was integrated and that there were no internal deletions or loop outs during integration via homologous recombination.
• One primer (8P) anneals to the rbcL 5' gene while another anneals to the aadA gene (8M). Because the rbcL 5' primer anneals only with the plastid genome, no PCR product was obtained with nuclear transgenic plants and mutant plants using this set of primers. The presence of the expected size PCR product (2.1 kb) confirmed plastid integration of both foreign genes ( Figure 2A). Plastid transgenic plants containing the antibody H and L chain genes were subjected to a second round of selection in order to achieve homoplasmy.
• a 0.81 kb Bgl ll/BamH I fragment containing flanking sequences of the pLD vector was used as a probe for the lines transformed with the pLD-TP-Guy's 13 vector ( Figure 9).
• the probes were labeled with 32 P-dCTP using the Ready To Go kit (Pharmacia Biotech, NJ).
• the blots were prehybridized using Quickhyb prehybridization solution (Stratagene, CA). The blots were hybridized and washed according to the manufacturer's instructions.
• the native size fragment present in the non-transformed control should be absent in the transgenics.
• the presence of a large fragment (due to insertion of foreign genes within the flanking sequences) and absence of the native small fragment establishes the homoplasmic nature of our transformants (Daniell et al, 1998; Kota et al, 1999; Guda et al, 2000).
• TO lines transformed with the pLD-TP-Guy's 13 vector 4.47 kb and 7.87 kb bands were observed ( Figure 9, lanes 4-6).
• Petit Havana only the 4.47 kb band was observed ( Figure 9, lane C).
• the blots were prehybridized using Qiuckhyb prehybridization solution (Stratagene, CA). The blots were hybridized and washed according to the instructional manual (Stratagene, CA). The transcript levels were quantified using the Storm 840 phosphoimager system (Molecular Dymanics).
• RNA from plastid transformants Abundant transcripts that hybridized to both light chain and heavy chain probes were detected in RNA from plastid transformants ( Figures 9 and 10). These transcripts were larger in size than transcripts detected in nuclear transgenic plants, consistent with the presence of polycistronic transcripts in the transgenic plastids.
• the transcription levels between the nuclear transformants and plastids transformants were compared.
• the transcription levels between the plastid transfonnant lines transformed with the pZS-TP-Guy's 13 vector and the lines transformed with the pLD-TP-Guy's 13 vector were also compared.
• the plastid transformants transformed with the pLD-TP-Guy's 13 vector expressed 13/24 fold more transcripts.
• the plastid transformants transformed with the pLD-TP-Guy's 13 vector expressed two fold more transcripts than the plastid transformants transformed with the pZS-TP-Guy's 13.
• the plant leaves (100 mg) were directly ground in 2X SDS sample buffer, boiled for 4 min, briefly spun and loaded on polyacrylamide gels. Samples treated with reductant were electorphoresed on 12% acrylamide gels. Non-reduced samples were electrophoresed on 7% acrylamide gels. The gels were electro-blotted onto nitrocellulose membranes in a Trans-Blot Electrophoreic transfer cell (BioRad, CA) following the manufacturer's instructions.
• the unoccupied binding sites on the blots were blocked by incubating them in blocking buffer [10 mM Tris-HCl, 0.5 M NaCl, 0.05% Tween 20 (v/v), and 5% non-fat dry milk (w/v)] at room temperature for lh. After blocking, blots were incubated for 2 hours at room temperature with alkaline phosphatase-conjugated goat anti-human IgA or goat anti-human kappa antibody, diluted 1 :2000 in blocking buffer. Blots were washed three times at room temperature in TBS. After washing, blots were developed using the Alkaline Phosphatase Conjugate Substrate Kit (Bio-Rad, Hercules, CA) according to the manufacturer's instructions.
• Determination of antibody concentration and detection of antibody binding function is performed by ELISA. Assays are done on crude extracts of leaves made by homogenizing small samples in two volumes of extraction buffer (25 mM Tris pH 7.5, 150 mM NaCl, 10 mM EDTA, 1% sodium citrate, 1% PVPP, 0.2% sodium thiosulfate). Homogenates are centrifuged in microfuge tubes for 10 minutes to pellet plastids and assays performed in the lysed supernatant.
• the concentration of assembled antibody is determined using a double antibody sandwich ELISA.
• an antibody against kappa chain bound to the plate captures any plantibody in the extract, which is detected by antibody against IgA heavy chain (to detect assembled IgA or SlgA), or by antibody against secretory component (to detect assembled SlgA).
• Microtiter wells are coated overnight at 4 °C with goat anti-human light chain-specific antibodies (50 ⁇ l/well at 4 ⁇ g/mL in PBS). Plates are washed, then blocked with PBS + 5% non-fat dry milk 1 hour at room temperature.
• HRP substrate [0.1 M sodium citrate, pH 4.4 containing 0.0125%) hydrogen peroxide and 0.40 mg/mL 2.2'-azino-bis (3-Ethylbenzthiazoline-6-sulfonic acid)]
• HRP substrate 0.40 mg/mL 2.2'-azino-bis (3-Ethylbenzthiazoline-6-sulfonic acid)
• Color development is determined using a Benchmark Microplate Reader (Bio-Rad).
• Antibody concentrations in ⁇ g/mL) are determined by comparison with standard curve of human SlgA (Sigma), using a four-parameter logistic fit (SigmaPlot 3.0).
• SA I/II Streptococcal antigen I/II
• ELISA Streptococcal antigen I/II
• SA I/II is purified from culture supematants of Steptococcus mutans strain IB 162 by the method of Russell et al. (1980). Microtiter plates are coated with purified SA I/II (50 ⁇ L/well at 2 ⁇ g/mL) overnight at 4 °C. Plates are washed, blocked with PBS + 5% nonfat dry milk, and probed 1 hr at 37 °C with a dilution series of plant extract.
• Bound antibodies are detected using'the appropriate HRP-conjugated goat anti-human second antibody, and the plates processed exactly as described above for the double-antibody sandwich ELISA.
• a reference standard lot of Guy's 13 SlgA (produced by nuclear transgenic plants) is always tested along with test samples to control for assay to assay variation.
• Binding titer is calculated as the dilution of test antibody (normalized to 1 mg/mL as determined by the double antibody sandwich ELISA) necessary to generate an ELISA signal that is 50% of the maximum signal.
• Plastids are first isolated from a crude homogenate of leaves by a simple centrifugation step at 1500 X g. This eliminates most of the cellular organelles and proteins (Daniell et al, 1983, 1986). Then plastids are burst open by re-suspending them in a hypotonic buffer
• EDTA 1% sodium citrate, 1% PVPP, 0.2% sodium thiosulfate
• the mixture is centrifuged at 17,000 g for 60 min, and the supernatant filtered through a 0.2 ⁇ M nominal cut-off filter. Filtrate is concentrated by diafiltration using a 300-kD MWCO tangential flow cassette (Millipore Corporation). Immunoglobulins are precipitated with 40% ammonium sulfate, collected by centrifugation at 17,000 g for 15 min, and then re-suspended in phosphate buffered saline (PBS).
• PBS phosphate buffered saline
• J. Inheritance of Introduced Foreign Genes Some of the initial tobacco transformants are allowed to self-pollinate, whereas others are used in reciprocal crosses with control tobacco plants (transgenics as female acceptors and pollen donors; testing for maternal inheritance).
• Harvested seeds (Tl) are germinated on media containing spectinomycin or other appropriate selective agents. Achievement of homoplasmy and mode of inheritance can be classified by observing germination results. Homoplasmy is indicated by totally green seedlings (Daniell et al, 1998) while heteroplasmy is displayed by variegated leaves (lack of pigmentation, Svab and Maliga, 1993). Lack of variation in chlorophyll pigmentation among progeny underscore the absence of position effect, an artifact of nuclear transformation.
• Maternal inheritance is demonstrated by sole transmission of introduced genes via seed generated on transgenic plants, regardless of pollen source (green seedlings on selective media). When transgenic pollen is used for pollination of control plants, resultant progeny do not contain resistance to • chemical in selective media (appear bleached; Svab and Maliga, 1993).
• Molecular analyses PCR, Southern, and Northern confirm transmission and expression of introduced genes, and T2 seed is generated from those confirmed plants.
• Codon optimization has been used previously to successfully increase the level of transgenic protein in plants (McBride et al., 1995; Rouwendal et al., 1997 Horvath et al., 2000). In the case of a ⁇ -(l,3-l,4)-glucanase expressed in barley, codon optimization resulted in at least a 50-fold increase in expression (Horvath et al., 2000). Two factors contribute to codon bias in all organisms. One is the overall composition of the genome, which contributes to a bias in degenerate positions of codons (Bernardi et al., 1986). In tobacco plastid non-coding regions, the AT content is 69.6%).
• An AT-rich crylA gene (encoding a Bacillus thuringiensis toxin) accumulated to much higher levels in plastids than the same gene having nuclear codon preferences (McBride et al., 1995). High AT content, however, is not the whole story. The second factor is selection for translation efficiency, resulting in a bias for specific codons (Ikemura et al.,1985). It has been proposed (Morton, 1993; Morton, 1998) that codon use in plastids is adapted to tRNA levels and that highly expressed genes have a greater bias in codon use as a result of selection for increased translation efficiency. Modification of a transgene to match the codon usage of highly expressed genes should result in even higher levels of expression.
• Rule #1 The primary codon is used, unless conditions met in rules number 2 and 3 are present.
• Rule #3 If the same amino acid is encoded twice with four or fewer intervening amino acids (for example, LXXXL, where L is Leucine and X is any amino acid) the secondary codon is used to encode one of the amino acids (either the first or second L, in the example), being careful to avoid violating Rule #2.
• LXXXL where L is Leucine and X is any amino acid
• Rule #3 If the same amino acid is encoded three times with four or fewer intervening amino acids between the first and third occurence (for example, LLXXL, where L is Leucine and X is any amino acid) the tertiary codon is used to encode one of the amino acids (either the first or second L, in the example), being careful to avoid violating Rule #2.
• the tertiary codon is used to encode one of the amino acids (either the first or second L, in the example), being careful to avoid violating Rule #2.
• the secondary codon is used.
• a synthetic gene was constructed that encoded a polypeptide consisting of the variable region of a murine anti-rotavirus monoclonal antibody fused to the constant region of human IgA2m(2) heavy chain (Chintalacharuvu et al 1994).
• the sequence of this chimeric gene was modified from the native mammalian gene sequences by codon optimization for plastid expression, using the rules in table 2.
• TAA was used as a stop codon.
• Synthesis of the gene was contracted to Entelechon GmbH. The gene was synthesized using the overlap extension PCR method (Rouwendal et al., 1997), but could be synthesized by various methods known to those skilled in the art.
• Another gene, encoding a polypeptide consisting of the variable region of a murine anti-rotavirus monoclonal antibody fused to the constant region of human kappa chain was synthesized by the same method, with codons optimized for plastid expression. Both synthetic genes were cloned into the vector pCR4TOPO (Invitrogen).
• the plasmid containing the heavy chain sequence was cut with Sal I, and the plasmid containing the light chain sequence was cut with Sal I and Xho I.
• a Sal I/Xho I fragment containing the light chain sequence was then isolated and cloned into the Sal I site of the plasmid containing the heavy chain.
• the resulting bacterial clones were screened for a clone with the correct orientation (heavy chain followed by light chain with coding sequences in the same orientation).
• the heavy and light chain genes, with associated ribosome binding sites were then cut out together using Not I and Xba I, and cloned into the pLD vector.
• the sequence between the Not I and Xho I sites of the heavy and light chain cassette is shown in Table 3.
• the pLD vector with codon-opthnized heavy and light chain coding sequences was used to transform tobacco plastids as described in Example 1. Transgenic plants are isolated and shown to contain high levels of human IgA.
• SlgA in plastids is accomplished by the simultaneous integration of four genes, IgA heavy chain, light chain, J chain and secretory component. These genes are expressed on a polycistronic message.
• a plasmid based on pLD, is constructed containing the Guy's 13 heavy and light chains, and the mature-peptide coding regions of human J chain and SC genes, all downstream of the aadA gene and each having a ribosome binding site. The total size of this mRNA is over 4500 nt.
• Tobacco leaves are transformed by particle bombardment and transplastomic plants are selected by regeneration on antibiotic-containing medium by methods similar to those disclosed in Example #1. Appropriate primers are used for PCR analysis.
• J chain and SC are evaluated by western blotting, using antisera specific for human J chain and human secretory component. Detection of a band at ⁇ 370 kDa with anti- IgA, anti-kappa, anti-J and anti-SC antibodies is considered evidence of assembled SlgA.
• Example #4 Expression of SlgA in Plastids with J chain and Secretory Component genes on one vector and Heavy and Light Chain Genes on another vector
• Two plastid expression vectors, one containing heavy and light chain genes, and the other containing the J chain and secretory component genes are constructed by methods similar to those described in Example #1.
• the amino acid sequence of the J chain and secretory component encoded in the second vector are those described in Patent No. 5,959,177 and US Patent No. 6,046,037, incorporated herein by reference.
• the two vectors use different plastid DNA flanking sequences, so that they integrate into the plastid chromosome in different locations.
• Tobacco leaves are transformed by particle bombardment and transplastomic plants are selected by regeneration on antibiotic-containing medium by methods similar to those disclosed in Example #1.
• Appropriate primers are used for PCR analysis.
• Expression of J chain and SC is evaluated by western blotting, using antisera specific for human J chain and human secretory component. Detection of a band at ⁇ 370 kDa with anti-IgA, anti-kappa, anti-J and anti-SC antibodies is considered evidence of assembled SlgA.
• a fragment containing all 5 extracellular Ig-like domains of ICAM-1 is amplified from plasmid pIgAD5 (a gift of T. Springer) using the primers: 5'-AAAATCTAGAGGAGGGATTTATGCAGACATCTGTGTCCCCCTCAAAAGTC-3' and
• the resulting PCR product incorporates a ribosome-binding site utilized by the plastid protein translation machinery, and a methionine codon upstream of the first amino acid of ICAM-1.
• the PCR product is cut with Xba I and Spe I (underlined sequences) and cloned into a vector containing the human IgA2m(2) heavy chain constant region.
• the resulting chimeric gene encodes one continuous protein consisting of 5 domains of ICAM-1 and the constant region of IgA2m(2).
• the mature protein produced from this construct starts with the sequence Met-Gln- Thr-Ser-Val-, and end with the sequence -Lys-Asp-Glu-Leu.
• the sequence of the ICAM gene has been published (Staunton et al., 1988), and is incorporated herein by reference.
• the entire coding sequence of the chimeric gene is cut out with Xba I and cloned into the pLD vector.
• the resulting expression vector is used to transform tobacco plastids.
• the chimeric ICAM-1/IgA protein is expressed in transgenic plastids, and assembles into dimers.
• This multimeric protein comprises an immunoglobulin heavy chain fused to a functional ligand (ICAM-1 domains 1-5), and binds to a site on human rhinoviruses. It is used in a therapeutic manner to prevent rhinovirus colds.
• Renegar KB Jackson GD, Mestecky J (1998) In vitro comparison of the biologic activities of monoclonal monomeric IgA, polymeric IgA, and secretory IgA. Journal of Immunology 160: 1219-23 58. Renegar KB, Parker ASJ (1991) Passive transfer of local immunity to influenza virus infection by IgA antibody. J Immunol, 146:1972-1978.

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