WO2004031362A2 - Multimeric protein engineering - Google Patents
Multimeric protein engineering Download PDFInfo
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- WO2004031362A2 WO2004031362A2 PCT/US2003/031420 US0331420W WO2004031362A2 WO 2004031362 A2 WO2004031362 A2 WO 2004031362A2 US 0331420 W US0331420 W US 0331420W WO 2004031362 A2 WO2004031362 A2 WO 2004031362A2
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
- C07K16/32—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against translation products of oncogenes
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
- C07H21/00—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
- C07H21/04—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids with deoxyribosyl as saccharide radical
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- 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
- C07K16/46—Hybrid immunoglobulins
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- 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
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
- C07K2317/31—Immunoglobulins specific features characterized by aspects of specificity or valency multispecific
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
- C07K2317/55—Fab or Fab'
Definitions
- the present invention relates to the expression and assembly of artificial multimeric proteins, i.e. antibodies and antibody fragments, in eukaryotes, i.e. plants.
- polypeptides can be expressed in a wide variety of cellular hosts.
- a wide variety of genes have been isolated from mammals and viruses, joined to transcriptional and translational initiation and termination regulatory signals from a heterologous source, and introduced into hosts into which these regulatory signals are functional.
- Plants are an important system for the expression of many recombinant proteins, especially those intended for therapeutic purposes.
- Heterologous proteins are reliably made in one of two general ways, either by nuclear transformation of the chromosomal DNA or by infecting the host with viral vector.
- Transgenic plants are created by the stable integration of the foreign DNA into the plant genome, and subsequent genetic recombination by crossing of transgenic plants is a simple method for introducing new genes and accumulating multiple genes into plants.
- viral vectors engineered to carry heterologous genes can be used to transfect the host such that the genes are carried in an episomal manner, parasitizing the host translational machinery to produce the protein of interest.
- plants are attractive hosts because of the opportunity for protein production on an agricultural scale at an extremely competitive cost, but there are also many other advantages.
- the processing and assembly of recombinant proteins in plants may also complement that in mammalian cells, which may be an advantage over the more commonly used microbial expression systems.
- One of the most useful aspects of using a recombinant expression system for antibody production is the ease with which the antibody can be tailored by molecular engineering. This allows the production of antibody fragments, as well as the manipulation of full-length antibodies. For example, a side range of functional recombinant-antibody fragments, such as Fab's, may be generated.
- Fab's functional recombinant-antibody fragments
- the ability of plant cells to produce full-length antibodies can be exploited for the production of antibody molecules with altered Fc- ediated properties. This is facilitated by the domain structure of immunoglobulin chains, which allows individual domains to be "cut and spliced" at the gene level.
- the immunoglobulin molecule is composed of two identical heavy chains and two identical light chains (H 2 L 2 ) where the two chains are present in equimolar ratio and are linked by a disulfide bond.
- the diversity of antibodies created through multiple genes encoding the heavy and light chains, rearrangement of the heavy and light chains, and somatic mutation combined with tight transcription and translational control of maturing antibodies results in a complicated process for B-cell maturation.
- the proper assembly of the expressed antibody is critical to its activity. To address this issue, the secretory machinery of the cell plays a vital role in the proper folding and timing of folding of assembly.
- the two heavy chains are linked together by disulfide bonds such that in any naturally occurring antibody molecule, the two heavy chains and two light chains are identical.
- Proteolytic enzymes such as papain can be used to fragment the Ig molecule into three fragments. Two fragments are identical and contain the antigen binding activity and are referred to as Fab fragments, or Fragment antigen binding, corresponding to the paired light chain with a NH and CHI domains.
- the third fragment contains no antigen binding and is referred to as the Fc or fragment crystallizable which contains the paired, disulfide linked CH2 and CH3 domains.
- the hinge region that links the Fab and Fc portions of the antibody is a flexible tether, allowing independent movement of the two Fab regions which would not be possible if the tether were rigid. Transport of this multimeric complex is dependent on the correct assembly of the component parts, which is controlled, in part, by the association of incompletely assembled Ig heavy chains with the endoplasmic reticulum (ER) chaperone, BiP. (Lee YK, et. al., Mol Biol Cell. 1999 Jul;10(7):2209-19) Although other heavy chain- constant domains interact transiently with BiP, in the absence of light chain synthesis, BiP binds stably to the first constant domain (C H 1) of the heavy chain, causing it to be retained in the ER.
- C H 1 the first constant domain
- BiP and light chain cooperate to ensure that only properly assembled Ig molecules are transported from the ER by controlling the final folding of the heavy chain. Therefore the requirement for presence of both chains in the same cell, in the same sub-cellular organelle, at the same amount at the same time is critical for maximal throughput of mature antibody.
- each chain of the antibody is expressed from an individual gene encoding each chain.
- the transcription, translation and cellular localization or secretion of each chain is controlled independently of its corresponding chain.
- each polypeptide chain of the antibody multimer is controlled by separate promoters and secretory leaders.
- chromosome insertion points Differences in the chromosome insertion points, promoter strength and timing as well as the efficiency of secretory peptides can result in varying levels of each chain being present at a given time in the endoplasmic reticulum (ER), resulting in incomplete or delayed maturation of antibodies because the absence or decreased levels of the counterpart chain. Effects of insertion positions, whether proximal to endogenous promoters or enhancers, differential promoter efficiencies, translocation efficiencies and translational kinetics can result in aberrant accumulation of the recombinant antibody in foreign systems.
- the one gene, one polypeptide rule is occasionally broken for reasons of efficiency, as often is the case with viruses, and proper folding as dictated by the more complex proteins and for temporal control as for otherwise toxic or regulatory molecules such as prohormornes in the form of proproteins.
- each gene encodes a portion of a desired antibody such that the first gene includes a promoter (Pr), a signal peptide (Sp) and a segment that expresses a heavy chain and the second gene includes a promoter, a signal peptide and a segment that expresses a light chain.
- Pr promoter
- Sp signal peptide
- the first gene is inserted into cells of a first plant, and the second gene is inserted into cells of a second plant. Thereafter, the first and second plant are cross pollinated in order to generate progeny that hopefully includes both the first and second genes and will therefore cause expression of a proprotein that will fold to form an antibody of interest.
- the viral vector plant expression system of TMN utilizes endogenous and heterologous viral promoters to drive the expression of foreign genes.
- the vector easily accommodates a single foreign gene, but has more difficulties with additional genes as the size becomes an issue as well as the position effects of additional promoters required to produce an additional polypeptide as is required for antibodies.
- the farther the promoter/gene set is from the 3' end of the genome the lower the transcriptional activity. Therefore the larger the insert the lower the expression as a result of the intervening sequences of the heterologous gene.
- heterodimers as is the case for Fab's, the simultaneous expression of stoichiometric levels of heavy and light chains is essential for secretion.
- BiP has a role in retaining the nascent chain in the oxidizing environment of the ER until the counterpart chain interacts, becomes disulfide linked and subsequently released from the ER resulting in the accumulation of the antibody in the secretory fluid.
- the heavy and light chains must be expressed at comparable levels as the resulting heterodimer contains a one to one ratio of heavy and light chains. Attempts have been made to express one chain from one vector and the second chain form a second vector (Nerch T, et al, J Immunol Methods. 1998 ⁇ ov l;220(l-2):69-75).
- the two vectors were used to super-infect a plant and small amounts of antibodies were recovered. This approach is problematic because of cross-protection of an infected cell with one virus from being infected with a second virus. Typically, only the monolayer of cells present at the confluence of infections are thought to be simultaneously infected with both viruses. In additional an ER retention signal was placed on the chains to facilitate association by retained co-localization of the chains.
- the cleavage of the proprotein occurs at a so-called dibasic site, i.e. a motif consisting of at least two basic amino acids.
- the processing is catalyzed by enzymes located in the Golgi apparatus, the so-called "dibasic processing endoproteases".
- dibasic processing endoproteases There are different "dibasic processing endoproteases" known which are involved in the processing of precursor, for example the mammalian proteases furin, PC2, PCI and PC3, (Barr, Cell 66:1-3, 1991) and the product of the yeast YAP3 gene (Egel- Mitani et al., Yeast 6:127-137 1990) and yeast yscF (also named KEX2 gene product; KEX2p).
- KEX2p is involved in the maturation of the yeast mating pheromone, alpha- mating factor (J. Kurjan and I. Hershkowitz, Cell 30:933-934, 1982).
- the alpha-mating factor is produced as a 165 amino acid precursor which is processed during the transport to the cell surface.
- a 19-amino acid signal sequence pre-sequence
- the alpha-mating factor precursor is also known as alpha factor "leader" sequence.
- a second protease in the Golgi apparatus, i.e. the JKEX1 gene product is responsible for the final maturation of the protein.
- BiP like all hsp70 family members, binds to unfolded nascent polypeptides and is thought to function by recognizing hydrophobic sequences exposed on unfolded or unassembled polypeptides and, by inhibiting intra- or intermolecular aggregation, maintaining them in a state competent for subsequent folding and oligomerization.
- BiP recognizes heptapeptides and prefers those with aliphatic residues (Flynn GC, et al., Nature.
- Plant seed toxins such as Ricin from castor beans utilize a preproprotein expression strategy to mitigate the toxic effects of ricin by having an inactive proprotein.
- the proricin is moved through the ER and Golgi complex to the protein storage vacuoles (PSV) of the bean.
- PSV protein storage vacuoles
- resident proteases mature the protein to produce a highly toxic heterodimer composed of A and B chains linked by a disulfide bond.
- a similar strategy can be envisioned as a useful strategy for the expression of recombinant multimeric proteins that in their mature form would be toxic or otherwise detrimental to the host.
- An antibody that recognized an essential receptor may be such a molecule.
- the expression of the multimeric or heterodimeric protein as an inactive proprotein precursor and delivery of immature proprotein to a organelle such as the PSN followed by the subsequent removal of the propeptide to activate the antibody or other molecule would reduce or eliminate the toxic effects of that molecule.
- Pro-sequence can be any sequence which can act as a molecular chaperone, i.e. a polypeptide which in cis or trans can influence the formation of an appropriate conformation, but is by in large not present in the mature form of the protein.
- proproteins are folded as immature protein intermediates, facilitating proper conformation and disulfide linkages in the ER.
- the propeptide is removed in the Golgi from the proprotein to generate a mature active protein.
- proteins such as insulin, Saccharomyces cerevisiae killer toxin virus (ScN) kl toxin, Kluyveromyces lactis plasmid kl toxin, and the KP6 toxin of Ustilago maydis virus(UmV).
- the insulin C chain is removed to produce the mature, active hormone in newly formed clathrin coated secretory vessicles.
- the Saccharomyces cerevisiae Kl killer toxin precursor is composed of a signal peptide, alpha subunit, a propeptide (gamma subunit), and a beta subunit.
- the secreted precursor protein is folded with inter- and intra-chain disulfide bonds formed with the alpha and beta subunits, and the gamma propeptide is removed by proteolysis.
- the mature Kl toxin is a heterodimeric protein composed of disulfide linked alpha and beta polypeptides.
- the JKP6 toxin consists of two distinct polypeptides, alpha and beta, but differ in that the subunits are not covalently associated, encoded by a 657 base pair double stranded R ⁇ A segment.
- a single transcript produces a 219 amino acid JKP6 preprotoxin, which is then processed to produce the 78 amino acid alpha and the 81 amino acid beta polypeptides.
- processing of the protoxin by Kex2p occurs after the Pro-Arg residues at position 27 and the Lys-Arg residues at 107 to generate alpha and at 139 to generate beta.
- a class of novel, artificial preproproteins has now been designed and engineered which comprise a proprotein, that is, a protein assembly capable of producing a multimeric protein from a single protein comprised of a first peptide, a second peptide and propeptide, where the first peptide and the second peptide associate to assume a biologically functional conformation essentially free of the propeptide.
- a proprotein that is, a protein assembly capable of producing a multimeric protein from a single protein comprised of a first peptide, a second peptide and propeptide, where the first peptide and the second peptide associate to assume a biologically functional conformation essentially free of the propeptide.
- the first peptide and second peptide would be the light and heavy chain of an immunoglobulin molecule, the light chain and a fragment of the heavy chain immunoglobulin molecule, the alpha and beta chain of the T cell receptor, or the alpha and beta chains of hemoglobin.
- propeptide examples include the insulin C chain, Saccharomyces cerevisiae Kl killer toxin propeptide (gamma subunit), Kluyveromyces lactis plasmid kl toxin propeptide or the JKP6 toxin propeptide chain.
- This invention features artificial, proproteins which fold to form a stable intermediate protein containing a propeptide, where the mature multimeric protein has subunits with an associative properties, DNA encoding these proteins prepared by recombinant techniques, host cells harboring these DNAs, and methods for the production of these proteins and DNAs.
- HSP70 heat shock protein 70
- BiP binding protein
- PDI protein disulfide isomerase
- the propeptide is proteolytically removed rendering the mature antibody in its active form, at which time it is transported out of the cell where it accumulates in the extracellular space or apoplast in plants. Proteolytic cleavage at the amino and carboxy termini of the propeptide by proteases results in the release of the propeptide.
- the Kex2 like protease recognition sequence has amino acid residues of lysine at P2 and arginine at PI, using the nomenclature convention of Schechter, I and Berger, A Biochem. Biophys. Res. Com. (1967) 27:157-62.
- the cleavage of the propeptide results in a carboxy terminal Lys-Arg amino acid pair remaining on the first peptide of interest.
- Proline or arginine can also be substituted for Lysine at the P2 position to make a Pro-Arg or Arg-Arg pair.
- the non-native pair may be created by addition of a single amino acid to make the cleavage site.
- a multimeric protein made by the method of the present invention will be characterized by its carboxy terminal lys-Arg, Pro-Arg or Arg-Arg on the first peptide.
- proteases There are many different proteases that occur in different organisms. These proteases have varying specificities. Any amino acid pair that results from proteolytic cleavage of the propeptide is contemplated by this invention.
- the Lys-Arg, Pro-Arg or Arg-Arg pair may be retained or removed.
- a single Arg at the PI position may also be removed without removing the amino acid at the P2 position.
- the derivative proteins made by removal of the amino acid pair are also contemplated by this invention.
- the propeptide facilitates the intersubunit interactions of the multimeric protein, whether the interactions are covalent, as in an antibody or non-covalent, electrostatic forces, hydrogen bonds, or Van der Waals forces and hydrophobic forces as in hemoglobin. Once the associative interaction has occurred the propeptide is then removed to release the desired multimeric protein.
- This patent describes the creation of a chimeric proprotein where the polypeptide subunits of the UmV JKP6 toxin are removed and replaced by polypeptides subunits from a multimeric protein not naturally found as a proprotein, such as immunoglobulin, containing the immunoglobulin light and heavy chains, which directs the synthesis of an artificial proprotein where the proprotein folds to form a stable intermediate and the propeptide is subsequently removed from the proprotein rendering a mature, active multimeric protein essentially free of the propeptide.
- a proprotein such as immunoglobulin, containing the immunoglobulin light and heavy chains
- an artificial proprotein in a first embodiment of the invention includes three peptide sequences, a first peptide, an intermediate propeptide and a second peptide.
- This invention does not include peptides that are naturally bound to a propeptide, such as the insulin molecule.
- the present invention allows us to make proprotein configurations that are not found in nature. These configurations simplify the production of multimeric proteins by allowing them to be placed in a single gene configuration.
- an artificial polynucleotide includes four nucleotide sequences.
- the three-peptide configuration described above is attached to a preceding signal peptide.
- a method of making an artificial polynucleotide includes providing first, second, and third nucleotide sequences each encoding a first peptide of interest, an internal propeptide and a second peptide of interest, respectively.
- the nucleotide sequence that encodes a first peptide of interest can be the same as or different from the nucleotide sequence that encodes a second peptide of interest.
- a method of making an artificial polynucleotide includes providing a first, a second, a third and a fourth nucleotide sequence that encode a signal peptide sequence, a first peptide of interest, a propeptide and a second peptide of interest, respectively.
- the nucleotide sequence that encodes a first peptide of interest can be the same as or different from the nucleotide sequence that encodes a second peptide of interest.
- a method of making an artificial proprotein includes making an artificial polynucleotide that encodes the proprotein; and expressing the artificial polynucleotide in a host organism whereby the proprotein is made.
- a method of making an artificial preproprotein includes making an artificial polynucleotide that encodes the preproprotein; and expressing the artificial polynucleotide in a host organism.
- a method of making and isolating a multimeric protein includes the steps of: providing a first, a second, a third and a fourth nucleotide sequence that encode a signal peptide sequence, a first peptide of interest, a propeptide and a second peptide of interest, respectively; connecting the 3' terminus of the first nucleotide sequence to the 5' terminus of the second nucleotide sequence; connecting the 3' terminus of the second nucleotide sequence to the 5' terminus of the third nucleotide sequence; and connecting the 3' terminus of the third nucleotide sequence to the 5' terminus of the fourth nucleotide sequence, so that an artificial polynucleotide results and is comprised of the four nucleotide sequences, and wherein the nucleotide sequence that encodes a first peptide of interest can be the same as or different from the nucleotide sequence that encodes a second
- the multimeric protein can be any multimeric protein having at least two peptide sequences that are intended to form a multimer but are usually encoded on different gene sequences, or do not naturally have a propeptide sequence between them.
- the peptides can be any set of peptides that are designed by the engineer to form a multimer.
- the host organism can be any host organism. Common host organisms are animal cells, human cells, animal tissues or whole animals, plant cells, plant tissues and whole plants.
- a vector encoding an artificial preproprotein includes a nucleotide sequence necessary for replication of the vector nucleotides and proteins and an artificial polynucleotide inserted into the vector, that comprises a first nucleotide sequence that encodes a signal peptide sequence; a second nucleotide sequence that encodes a first peptide of interest, second nucleotide sequence being connected to the 3' terminus of the first nucleotide sequence; a third nucleotide sequence that encodes a propeptide, third nucleotide sequence being connected to the 3' terminus of the second nucleotide sequence; and a fourth nucleotide sequence that encodes a second peptide of interest, fourth nucleotide sequence being connected to the 3' terminus of the third nucleotide sequence, the artificial polynucleotide inserted into the vector so that the vector can reproduce and, if required, can produce the artificial preproprotein.
- a transgenic cell includes:
- an artificial polynucleotide stably incorporated onto a chromosome comprising: a first nucleotide sequence that encodes a signal peptide sequence; a second nucleotide sequence that encodes a first peptide of interest, second nucleotide sequence being connected to the 3' terminus of the first nucleotide sequence; a third nucleotide sequence that encodes a propeptide, third nucleotide sequence being connected to the 3' terminus of the second nucleotide sequence; and a fourth nucleotide sequence that encodes a second peptide of interest, fourth nucleotide sequence being connected to the 3' terminus of the third nucleotide sequence, the artificial polynucleotide inserted into the vector so that the vector can reproduce and, if required can produce the artificial preproprotein.
- a transgenic plant includes plant cells containing an artificial polynucleotide sequence encoding an artificial preproprotein that artificial preproprotein comprises a) a signal peptide sequence, b) an immunoglobulin heavy chain or light chain peptide, c) a propeptide, and d) an immunoglobulin heavy chain or light chain peptide, wherein the heavy chain can be in either the b or the d position on the preproprotein, and the light chain will be on the other position, wherein the artificial preproprotein contains a signal peptide sequence signal peptide sequence forming a secretion signal containing immunoglobulin molecules encoded by said artificial polynucleotide sequence, wherein said signal peptide sequence signal peptide sequence is cleaved from said artificial preproprotein by proteolytic processing, and wherein said propeptide is cleaved from the heavy chain and
- a method for making a transgenic plant capable of producing immunoglobulin molecules includes:
- an artificial polynucleotide sequence encoding a preproprotein that preproprotein comprises (i) a signal peptide sequence, (ii) an immunoglobulin heavy chain or light chain peptide, (iii) a propeptide, and d) an immunoglobulin heavy chain or light chain peptide, wherein the heavy chain can be in either the b or the d position on the preproprotein, and the light chain will be on the other position;
- the immunoglobulin example is one of many possible examples of a multimeric protein that can be made by a transgenic plant. Any other set of peptides necessary to make a multimeric protein would also be suitable.
- a process for producing an immunoglobulin molecule or an immunologically functional immunoglobulin fragment comprising at least the variable domains of the immunoglobulin heavy and light chains, in a single host cell comprising the steps of: (a) transforming said single host cell with a single DNA sequence encoding at least the variable domain of the immunoglobulin heavy chain, a propeptide and at least the variable domain of the immunoglobulin light chain, and
- a vector in another embodiment of the invention includes a single DNA sequence encoding at least a variable domain of an immunoglobulin heavy chain and at least a variable domain of an immunoglobulin light chain wherein said single DNA sequence is located in said vector at a single insertion site.
- a transformed host cell includes at least two vectors, at least one of said vectors comprising a single DNA sequence encoding at least a variable domain of an immunoglobulin heavy chain and at least the variable domain of an immunoglobulin light chain.
- a method includes:
- step a) inserting the DNA sequence of step a) into a replicable expression vector operably linked to a suitable promoter
- step b) transforming a prokaryotic or eukaryotic microbial host cell culture with the vector of step b);
- a process for producing an immunoglobulin molecule or an immunologically functional immunoglobulin fragment includes at least the variable domains of the immunoglobulin heavy and light chains, in a single host cell, comprising: expressing a single DNA sequence encoding at least the variable domain of the immunoglobulin heavy chain and at least the variable domain of the immunoglobulin light chain so that said immunoglobulin heavy and light chains are produced as a single proprotein molecule in said single host cell transformed with said single DNA sequence.
- a multimeric protein is characterized by a first and second peptides, the first peptide comprising a non-native amino acid pair at the PI and P2 positions of the carboxy terminus.
- a multimeric protein derived from a multimeric protein is characterized by a first and second peptides, the first peptide comprising a non-native amino acid pair at the PI and P2 positions of the carboxy terminus.
- FIG. 1 is a block diagram showing prior art methods for expressing antibodies in plants where two genes are employed initially in two separate plants, the two plants subsequently being cross pollinated to produce progeny that may produce a desired protein in the endoplasmic reticulum.
- FIG. 2 is a flowchart generically showing the basic steps for producing a construct that includes in the following order a promoter sequence, signal peptide, a light chain sequence, a propeptide and a heavy chain sequence, in accordance with the present invention.
- FIG. 3 is a flowchart generically showing the basic steps for producing a construct that includes in the following order a promoter sequence, signal peptide, a heavy chain sequence, a propeptide and a light chain sequence, in accordance with the present invention.
- FIG. 4 is a block diagram representing a one embodiment of the present invention where a construct similar to the construct depicted in FIG. 3, for encoding a preproprotein is introduced into cells.
- the construct includes a short heavy chain is inserted between a signal peptide (Sp) and a propeptide.
- Sp signal peptide
- the signal peptide (Sp) is removed within the endoplasmic reticulum to produce the preproprotein.
- Subsequent maturation within the Golgi of the cell removes the propeptide thereby producing a folded desired antibody fragment or Fab.
- FIG. 5 is a block diagram representing another embodiment of the present invention where a construct encoding a preproprotein is introduced into cells.
- the construct is similar to the construct depicted in FIG. 2 and includes a light chain inserted between the signal peptide (Sp) and the propeptide.
- the signal peptide (Sp) is removed within the endoplasmic reticulum to produce the preproprotein.
- Subsequent maturation within the Golgi of the cell removes the propeptide thereby producing a folded desired antibody fragment or Fab.
- FIG. 6 is a block diagram representing yet another embodiment of the present invention where a single construct encoding a preproprotein is introduced into cells.
- the sequence for encoding a longer heavy chain is inserted between the signal peptide (Sp) and the propeptide.
- the signal peptide (Sp) is removed within the endoplasmic reticulum to produce the preproprotein.
- Subsequent maturation within the Golgi of the cell removes the propeptide thereby producing a folded desired Fab'.
- FIG. 7 is a block diagram representing a further embodiment of the present invention where a single construct encoding a preproprotein is introduced into cells where the construct includes a light chain is inserted between a signal peptide (Sp) and a propeptide with a longer heavy chain attached to the other end of the propeptide.
- Sp signal peptide
- the signal peptide (Sp) is removed within the endoplasmic reticulum to produce the preproprotein.
- Subsequent maturation within the Golgi of the cell removes the propeptide thereby producing a folded desired Fab'.
- FIG. 8 is a block diagram representing a still another embodiment of the present invention where a single construct encoding a preproprotein is introduced into cells where the construct includes a full heavy chain is inserted between the signal peptide (Sp) and the propeptide. After expression, the signal peptide (Sp) is removed within the endoplasmic reticulum to produce the preproprotein. Subsequent maturation within the Golgi of the cell removes the propeptide thereby producing a folded desired antibody.
- Sp signal peptide
- FIG. 9 is a block diagram representing a yet still another embodiment of the present invention where a single construct encoding a preproprotein is introduced into cells.
- the construct includes a light chain between the signal peptide (Sp) and the propeptide with a full heavy chain attached to the other end of the propeptide.
- the signal peptide (Sp) is removed within the endoplasmic reticulum to produce the preproprotein.
- Subsequent maturation within the Golgi of the cell removes the propeptide thereby producing a folded desired antibody.
- FIG. 10 is a block diagram showing various platforms that may be utilized for the production of a polypeptide using a single construct encoding preproprotein construct in accordance with the present invention, where the preproprotein includes a signal peptide (Sp), a light chain attached to the signal peptide, a proprotein attached to the light chain and a heavy chain attached to the proprotein.
- Sp signal peptide
- FIG. 11 is a block diagram similar to FIG. 10, showing various platforms that may be utilized for the production of a polypeptide using a single gene encoding preproprotein construct in accordance with the present invention, where the preproprotein includes a signal peptide (Sp), a heavy chain attached to the signal peptide, a proprotein attached to the heavy chain and a light chain attached to the proprotein.
- Sp signal peptide
- Dicotyledon A flowering plant whose embryos have two seed halves or cotyledons. Examples of dicots are: tobacco; tomato; the legumes including alfalfa; oaks; maples; roses; mints; squashes; daisies; walnuts; cacti; violets; and buttercups.
- Monocotyledon A flowering plant whose embryos have one cotyledon or seed leaf. Examples of monocots are: lilies; grasses; corn; grains, including oats, wheat and barley; orchids; irises; onions and palms.
- Lower plant Any non-flowering plant including ferns, gymnosperms, conifers, horsetails, club mosses, liver warts, hornworts, mosses, red algae, brown algae, gametophytes, sporophytes of pteridophytes, and green algae.
- Eukaryotic hybrid vector A DNA by means of which a DNA coding for a polypeptide (insert) can be introduced into a eukaryotic cell.
- Extrachromosomal ribosomal DNA A DNA found in unicellular eukaryotes outside the chromosomes, carrying one or more genes coding for ribosomal RNA and replicating autonomously (independent of the replication of the chromosomes).
- Palindromic DNA A DNA sequence with one or more centers of symmetry.
- T-DNA A segment of transferred DNA.
- rDNA Ribosomal DNA.
- rRNA Ribosomal RNA.
- Ti-plasmid Tumor-inducing plasmid.
- Ti-DNA A segment of DNA from Ti-plasmid.
- Insert A DNA sequence foreign to the DNA clone it is being inserted into.
- Structural gene A gene coding for a polypeptide and being equipped with a suitable promoter, termination sequence and optionally other regulatory DNA sequences, and having a correct reading frame.
- Signal sequence A DNA sequence coding for a signal peptide attached to the polypeptide.
- Signal peptide A series of amino acids attached to the polypeptide which binds the polypeptide to the endoplasmic reticulum and is essential for protein secretion. This signal may also be referred to herein as a prepeptide.
- signal peptide may also be used to refer to the sequence of amino acids that determines whether a protein will be formed on the rough endoplasmic reticulum or on free ribosomes.
- (Selective) Genetic marker A DNA sequence coding for a phenotypic trait by means of which transformed cells can be selected from untransformed cells.
- Promoter A recognition site on a DNA or RNA sequence or group of DNA or RNA sequences that provide an expression control element for a gene and to which RNA polymerase specifically binds and initiates RNA synthesis (transcription) of that gene.
- Inducible promoter A promoter where the rate of RNA polymerase binding and initiation is modulated by external stimuli. Such stimuli include light, heat, anaerobic stress, alteration in nutrient conditions, presence or absence of a metabolite, presence of a ligand, microbial attack, wounding and the like.
- Viral promoter A promoter with a DNA or RNA sequence substantially similar to the promoter found at the 5' end of a viral gene.
- a typical viral promoter is found at the 5' end of the gene coding for the p21 protein of MMTV described by Huang et al., Cell 27: 245 (1981).
- Synthetic promoter A promoter that was chemically synthesized rather than biologically derived. Usually artificial promoters incorporate sequence changes that optimize the efficiency of RNA polymerase initiation.
- Constitutive promoter A promoter where the rate of RNA polymerase binding and initiation is approximately constant and relatively independent of external stimuli. Examples of constitutive promoters include the cauliflower mosaic virus 35S and 19S promoters described by Poszkowski et al., EMBO J. 3: 2719 (1989) and Odell et al., Nature 313: 810 (1985).
- Temporally regulated promoter A promoter where the rate of RNA polymerase binding and initiation is modulated at a specific time during development. Examples of temporally regulated promoters are given in Chua et al., Science 244: 174- 181 (1989).
- Spatially regulated promoter A promoter where the rate of RNA polymerase binding and initiation is modulated in a specific structure of the organism such as the leaf, stem or root. Examples of spatially regulated promoters are given in Chua et al., Science 244: 174-181 (1989).
- Spatiotemporally regulated promoter A promoter where the rate of RNA polymerase binding and initiation is modulated in a specific structure of the organism at a specific time during development.
- a typical spatiotemporally regulated promoter is the EPSP synthase-35S promoter described by Chua et al., Science 244: 174-181 (1989).
- Chelating agent A chemical compound, peptide or protein capable of binding a metal.
- chelating agents include ethylene diamine tetra acetic acid (EDTA), ethyleneglycol-bis-(beta-aminoethyl ether) N, N, N', N'-tetraacetic acid (EGTA), 2,3-dimercaptopropanel-l-sulfonic acid (DMPS), and 2,3-dimercaptosuccinic acid (DMSA), and the like.
- EDTA ethylene diamine tetra acetic acid
- EGTA ethyleneglycol-bis-(beta-aminoethyl ether) N, N, N', N'-tetraacetic acid
- DMPS 2,3-dimercaptopropanel-l-sulfonic acid
- DMSA 2,3-dimercaptosuccinic acid
- Metal chelation complex A complex containing a metal bound to a chelating agent.
- Immunoglobulin product A polypeptide, protein or multimeric protein containing at least the immunologically active portion of an immunoglobulin heavy chain and is thus capable of specifically combining with an antigen.
- Exemplary immunoglobulin products are an immunoglobulin heavy chain, immunoglobulin molecules, substantially intact immunoglobulin molecules, any portion of an immunoglobulin that contains the paratope, including those portions known in the art as Fab fragments, Fab' fragment, F(ab') 2 fragment and Fv fragment.
- Immunoglobulin molecule A multimeric protein containing the immunologically active portions of an immunoglobulin heavy chain and immunoglobulin light chain associated with each other and capable of specifically combining with antigen.
- Fab fragment A multimeric protein consisting of the portion of an immunoglobulin molecule containing the immunologically active portions of an immunoglobulin heavy chain called the Fd and an immunoglobulin light chain associated with each other and capable of specifically combining with antigen.
- Fab fragments are typically prepared by proteolytic digestion of substantially intact immunoglobulin molecules with papain using methods that are well known in the art.
- 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 well known in the art.
- Fab' fragment An Fab that dimerizes or a dimeric Fab.
- Asexual propagation Producing progeny by regenerating an entire plant from leaf cuttings, stem cuttings, root cuttings, single plant cells (protoplasts) and callus.
- Glycosylated core portion The pentasaccharide core common to all asparagine-linked oligosaccharides.
- the pentasaccharide care has the structure Man ⁇ -1- 3(man ⁇ -l-6) Man ⁇ -l-46LcNAc ⁇ -l-4 6LcNac-(ASN amino acid).
- the pentasaccharide core typically has 2 outer branches linked to the pentasaccharide core.
- N-acetylglucosamine containing outer branches The additional oligosaccharides that are linked to the pentasaccharide core (glycosylated core portion) of asparagine-linked oligosaccharides.
- the outer branches found on both mammalian and plant glycopolypeptides contain N-acetylglucosamine in direct contrast with yeast outer branches that only contain mannose. Mammalian outer branches have sialic acid residues linked directly to the terminal portion of the outer branch.
- Glycopolypeptide multimer A globular protein containing a glycosylated polypeptide or protein chain and at least one other polypeptide or protein chain associated with each other to form a single globular protein. Both heterodimeric and homodimeric glycoproteins are multimeric proteins. Glycosylated polypeptides and proteins are n- glycans in which the C(l) of N-acetylglucosamine is linked to the amide group of asparagine. [00087] Immunoglobulin superfamily molecule: A molecule that has a domain size and amino acid residue sequence that is significantly similar to immunoglobulin or immunoglobulin related domains.
- immunoglobulin gene superfamily contains several major classes of molecules including those shown in Table A and described by Williams and Barclay, in Immunoglobulin Genes, p361, Academic Press, New York, N.Y. (1989).
- MHC Major histocompatibility complex
- MCAM Neural adhesion molecule
- MAG Myelin associated gp
- CEA Carcinoembryonic antigen
- PDGF Platelet-derived growth factor
- CSF1 receptor Colony stimulating factor-1 receptor
- Non-cell surface molecules a ⁇ B-glycoprotein
- Catalytic site The portion of a molecule that is capable of binding a reactant and improving the rate of a reaction. Catalytic sites may be present on polypeptides or proteins, enzymes, organics, organo-metal compounds, metals and the like. A catalytic site may be made up of separate portions present on one or more polypeptide chains or compounds. These separate catalytic portions associate together to form a larger portion of a catalytic site. A catalytic site may be formed by a polypeptide or protein that is bonded to a metal. [00090] Enzymatic site: The portion of a protein molecule that contains a catalytic site. Most enzymatic sites exhibit a very high selective substrate specificity.
- An enzymatic site may be comprised of two or more enzymatic site portions present on different segments of the same polypeptide chain. These enzymatic site portions are associated together to form a greater portion of an enzymatic site. A portion of an enzymatic site may also be a metal.
- Self-pollination The transfer of pollen from male flower parts to female flower parts on the same plant. This process typically produces seed.
- Cross-pollination The transfer of pollen from the male flower parts of one plant to the female flower parts of another plant. This process typically produces seed from which viable progeny can be grown.
- Epitope A portion of a molecule that is specifically recognized by an immunoglobulin product. It is also referred to as the determinant or antigenic determinant.
- Abzyme An immunoglobulin molecule capable of acting as an enzyme or a catalyst.
- Enzyme A protein, polypeptide, peptide RNA molecule, or multimeric protein capable of accelerating or producing by catalytic action some change in a substrate for which it is often specific.
- Light Chain The smaller of two (MWt ca. 23000) of the two types of polypeptide chain in an immunoglobulin monomer and consists of one V and one C domain. There are two classes of light chain known as kappa and lambda.
- V Domain of the immunoglobulin monomer which contains relatively invariant framework regions and hypervariable regions.
- the framework regions provide a protein scaffold for the hypervariable regions that make contact with antigen.
- Constant (C) Domain of the immunoglobulin monomer which is relatively constant in amino acid sequence between different immunoglobulin molecules and determines the particular effector function and the type such as alpha, gamma, delta, epsilon and mu corresponding to the classes IgA, IgG, IgD, IgE and IgM, respectively
- Short Heavy Chain The portion of the heavy chain molecule containing the immunologically active portion of the immunoglobulin heavy chain and consists of one V and one C domain.
- Longer Heavy Chain The Fd portion of the heavy chain molecule containing the immunologically active portion of the immunoglobulin heavy chain and a dimerization domain. One type of dimerization domain is a C domain.
- Heavy Chain A class-specific polypeptide immunoglobulin component (JMWt ca. 50000-70000, depending on Ig class).
- the various types of heavy chain are designated alpha, gamma, delta, epsilon and mu corresponding to the classes IgA, IgG, IgD, IgE and IgM, respectively.
- artificial means an artificial arrangement of peptide or nucleotide domains, one of the domains being a propeptide or propeptide coding sequence, the arrangement having no known analog in nature. The arrangement is not found in nature, because the two domains bonded to the propeptide or propeptide coding sequence are not naturally arranged on a single open reading frame or a single resulting proprotein.
- An artificial nucleotide sequence that encodes a proprotein is an arrangement of nucleotide sequence domains in an open reading frame, wherein one of the domains encodes an internal propeptide, the arrangement having no known analog in nature.
- An artificial proprotein sequence is an arrangement of peptide sequence domains in a proprotein wherein one of the domains is an internal propeptide, the arrangement having no known analog in nature.
- An example of an artificial nucleotide sequence that encodes a proprotein is an arrangement of nucleotide sequence domains in a single open reading frame, wherein one of the domains encodes an internal propeptide and the other two domains encode the heavy and light chains respectively of an antibody or Fab fragment. In nature two separate genes encode the heavy and light chains respectively of the antibody.
- the artificial antibody proprotein sequence is an arrangement of peptide sequence domains.
- One of the domains is an internal propeptide. Flanking the internal propeptide are the light chain on one side of the propeptide and the heavy chain on the other side.
- This arrangement has no known analog in nature. The arrangement will result in a disulfide bonded multimeric protein upon folding and cleavage of the internal propeptide.
- insulin is not an example of an artificial proprotein according to this invention.
- Insulin is a multimer that, in nature, is encoded on a single open reading frame. That open reading frame has three domains that encode a first peptide, a propeptide and a second peptide respectively. The result is an insulin proprotein having an internal propeptide domain.
- An insulin mutein is not an artificial proprotein of the present invention.
- a multimeric antibody proprotein that with one or more
- Propeptide A propeptide is a peptide that occurs between two peptides of interest in a proprotein. The propeptide is thought to assist in forming a conformational and proximational association between the two peptides of interest, which results in a stable intermediate. The two peptides of interest then form a multimeric protein.
- Proprotein is a multimeric protein intermediate, which comprises at least three peptide sequences; a first peptide sequence of interest, an internal propeptide sequence attached to the c-terminus of the first peptide sequence of interest, and a second peptide of interest attached to the c-terminus of the propeptide sequence.
- the proprotein may comprise more than three peptide sequences. Any naturally occurring or non naturally occurring propeptide would conform to the present invention.
- Preproprotein is an arrangement of peptides having a signal peptide that precedes a proprotein in the arrangement.
- Multimeric protein A protein containing more than one polypeptide or protein where the individual polypeptides or proteins are associated with each other to form a single protein. Both heterodimeric and homodimeric proteins are multimeric proteins.
- Polypeptide and peptide A linear series of amino acid residues connected one to the other by peptide bonds between the alpha-amino and carboxy groups of adjacent residues.
- Protein A linear series of greater than about 50 amino acid residues connected one to the other as in a polypeptide.
- a polypeptide or protein "domain” generally refers to a region of a polypeptide chain that is folded in such a way that confers a particular structure and/or biochemical function. (Schulz et al, supra). Domains can be defined in structural or functional terms. A functional domain can be a single structural domain, but may also include more than one structural domain. Such functions can include enzymatic catalytic activity, ligand binding, chelating of an atom or endogenous fluorescence.
- "Template DNA” refers to the DNA that is amplified by "amplification primer pairs" (the population of oligonucleotide primers used in the amplification reaction). This DNA may be produced by biological (recombinant) or artificial (chemical) means. Further, mRNA may be reverse transcribed to form the template DNA that is used in the amplification reaction.
- An "upstream primer” is an oligonucleotide primer, or a mixture of oligonucleotide primers, that anneal(s) to the antisense strand of the template DNA.
- a "downstream primer” is an oligonucleotide primer, or a mixture of oligonucleotide primers, that anneal(s) to the sense strand of the template DNA.
- Amplifying/amplification refers to a reaction wherein the entire template DNA, or portions thereof, are duplicated at least once, preferably many times.
- “Ligatingtligation” refers to covalent coupling of two or more DNA strands (3' end to 5' end) using enzymatic and or chemical methods.
- a "nontemplated endonuclease recognition site” is a sequence within the nontemplated sequence that is recognized by a restriction endonuclease.
- a "library” is a population of nucleic acid molecules produced using the methods described. The number of members contained in the population which differ in nucleotide sequence is determined by the number of sequences contained in the source material.
- constructs such as those depicted generically in FIGS. 2 and 3, for expression of desired multimeric proteins, such as antibodies and antibody fragments.
- desired multimeric proteins such as antibodies and antibody fragments.
- Such constructs include a light chain (Lt) and a heavy chain (Hy) that have been extracted from one or more cells for a desired purpose.
- Lt light chain
- Hy heavy chain
- the light chain and heavy chain may be extracted from the same cell, same type of cell or completely different types of cells depending upon the desired multimeric protein subsequently expressed.
- each of the light chain and heavy chain is provided with a predetermined endonuclease restriction site, such as Rl and R2 depicted in FIGS. 2 and 3. Methods for adding such restriction sites to a gene sequence are well known in the art.
- a predetermined propeptide in accordance is constructed in accordance with methods described in greater detail hereinbelow (for instance, see Example A).
- the propeptide is further provided with Rl and R2 restrictions which are compatible ends or complementary sequences suitable for fusing with the dna fragments (light chain Lt and heavy chain Hy), as shown in FIGS. 2 and 3.
- Rl and R2 restrictions are compatible ends or complementary sequences suitable for fusing with the dna fragments (light chain Lt and heavy chain Hy), as shown in FIGS. 2 and 3.
- the restriction sites enable the construction of the sequences shown in FIGS. 2 and 3 that includes the heavy chain Hy, the propeptide, and the light chain in either of the orientations depicted in FIGS. 2 and 3.
- Hy-propeptide-Lt sequence is cloned into, for example, a virus, such as those used in the GenewareTM system developed by Large Scale Biology Corporation, Vacaville California, adding thereto a signal peptide (Sp) and a promoter (Pr).
- a virus such as those used in the GenewareTM system developed by Large Scale Biology Corporation, Vacaville California, adding thereto a signal peptide (Sp) and a promoter (Pr).
- Sp signal peptide
- Pr promoter
- the final construct is isolated for use in any of a variety of desired expression systems, as is described in greater detail below.
- constructs of the present invention are inserted into cells for expression of a desired protein, proteins, antibody fragments or antibodies. These multimeric proteins may be expressed in the cell by mechanisms within the cell that are described in greater detail below with respect to FIGS. 4-9.
- FIG. 4 is a block diagram representing a one embodiment of the present invention where a single gene encoding a preproprotein is introduced into a cell or cells.
- the construct includes the promoter Pr, the signal peptide Sp, a heavy chain fragment Fd, a propeptide and a short chain Lt.
- the short heavy chain is inserted between a signal peptide (Sp) and the propeptide.
- the construct is introduced into the cell, where after expression, the propeptide (Sp) is removed within the endoplasmic reticulum to produce a folded preproprotein. Subsequent maturation within the Golgi of the cell removes the propeptide thereby producing a folded desired antibody fragment or Fab, which may be extracted by any of a variety of techniques, as is described in greater detail below.
- FIG. 5 is a block diagram similar to FIG. 4, except that positions of the heavy chain fragment Fd and the light chain Lt are reversed such that the construct includes the promoter Pr, the signal peptide Sp, a short chain Lt, a propeptide and a heavy chain fragment Fd.
- the light chain Lt is inserted between a signal peptide (Sp) and the propeptide.
- the construct is introduced into the cell, where after expression, the signal peptide (Sp) is removed within the endoplasmic reticulum to produce a folded preproprotein. Subsequent maturation within the Golgi of the cell removes the propeptide thereby producing a folded desired antibody fragment or Fab, which may be extracted by any of a variety of techniques, as is described in greater detail below.
- FIG. 6 is a block diagram representing another embodiment of the present invention where a single gene encoding a preproprotein is introduced into a cell or cells.
- the construct includes the promoter Pr, the signal peptide Sp, a heavy chain fragment Fd', a propeptide and a short chain Lt. Specifically, the heavy chain fragment Fd' is inserted between a signal peptide (Sp) and the propeptide.
- the construct is introduced into the cell, where after expression, the signal peptide (Sp) is removed within the endoplasmic reticulum to produce a folded preproprotein. Subsequent maturation within the Golgi of the cell removes the propeptide thereby producing a folded desired Fab', which may be extracted by any of a variety of techniques, as is described in greater detail below.
- FIG. 7 is a block diagram similar to FIG. 6, except that positions of the heavy chain fragment Fd' and the light chain Lt are reversed such that the construct includes the promoter Pr, the signal peptide Sp, a short chain Lt, a propeptide and a heavy chain fragment Fd'.
- the light chain Lt is inserted between a signal peptide (Sp) and the propeptide.
- the construct is introduced into the cell, where after expression, the signal peptide (Sp) is removed within the endoplasmic reticulum to produce a folded preproprotein. Subsequent maturation within the Golgi of the cell removes the propeptide thereby producing a folded desired Fab', which may be extracted by any of a variety of techniques, as is described in greater detail below.
- FIG. 8 is a block diagram representing yet another embodiment of the present invention where a single gene encoding a preproprotein is introduced into a cell or cells.
- the construct includes the promoter Pr, the signal peptide Sp, a full length heavy chain Hy, a propeptide and a short chain Lt.
- the construct is introduced into the cell, where after expression, the signal peptide (Sp) is removed within the endoplasmic reticulum to produce a folded preproprotein. Subsequent maturation within the Golgi of the cell removes the propeptide thereby producing a folded desired antibody, which may be extracted by any of a variety of techniques, as is described in greater detail below.
- FIG. 9 is a block diagram similar to FIG. 8, except that positions of the heavy chain Hy and the light chain Lt are reversed such that the construct includes the promoter Pr, the signal peptide Sp, a short chain Lt, a propeptide and a heavy chain Hy. Specifically, the light chain Lt is inserted between a signal peptide (Sp) and the propeptide.
- the construct is introduced into the cell, where after expression, the signal peptide (Sp) is removed within the endoplasmic reticulum to produce a folded preproprotein. Subsequent maturation within the Golgi of the cell removes the propeptide thereby producing a folded desired antibody.
- FIG. 10 is a block diagram showing various platforms that may be utilized for the production of an antibody fragment, Fab, Fab' or a full antibody using a single gene encoding preproprotein construct in accordance the construct depicted in FIG. 2.
- the construct of FIG. 2 may be introduced into mammalian cells, yeast cells, transgenic plant cells, baculovirus or plant viral vectors, such as those used in GenewareTM developed by Large Scale Biology Corporation.
- FIG. 11 is a block diagram showing various platforms that may be utilized for the production of an antibody fragment, Fab, Fab' or a full antibody using a single gene encoding preproprotein construct in accordance the construct depicted in FIG. 3.
- the construct of FIG. 3 may be introduced into mammalian cells, yeast cells, transgenic plant cells, baculovirus or plant viral vectors, such as those used in GenewareTM developed by Large Scale Biology Corporation.
- a class of novel, artificial proproteins has now been designed and engineered which comprise a multimeric proprotein, that is, a protein assembly capable of producing a multimeric protein from a single protein comprised of a first peptide, a second peptide and propeptide, where the first peptide and the second peptide associate to assume a biologically functional conformation essentially free of the propeptide.
- the first peptide and second peptide would be the light and heavy chain of an immunoglobulin molecule, the light chain and a fragment of the heavy chain immunoglobulin molecule, the alpha and beta chain of the T cell receptor, or the alpha and beta chains of hemoglobin.
- propeptide examples include the insulin C chain, Saccharomyces cerevisiae Kl killer toxin propeptide (gamma subunit), Kluyveromyces lactis plasmid kl toxin propeptide and the KP6 toxin propeptide chain.
- This invention features an artificial, proprotein which folds to form a stable intermediate protein containing a propeptide, where the mature multimeric protein has subunits with associative properties, DNA encoding these proteins prepared by recombinant techniques, host cells harboring these DNAs, and methods for the production of these proteins and DNAs.
- the design of artificial proprotein is based on the observation that multimeric proteins often have a requirement for involvement of folding chaperones to complete their complex folding and assembly requirements.
- the proproteins are designed to comprise a molecular chaperon in the form of a propeptide to facilitate the proper folding of multimeric proteins.
- the artificial proproteins are further designed to increase the availability of chaperones, increased local concentration, proper cellular localization, temporal and stochiometric expression of the protein subunits (among others) in order to increase the accumulation of the properly assembled, mature and active multimer.
- the propeptide influences the spatial distribution of the subunits by bringing them into close proximity, such that the relative molar concentration of each subunit is high acilitating the folding performed by BiP, PDI and other associative forces such as disulfide linkages, electrostratic and hydrophobic interactions between and within subunits.
- This invention requires recombinant production of multimeric proproteins have the ability to form a stable intermediate and be further matured to create a multimeric protein.
- This technology has been developed and is disclosed herein.
- persons skilled in recombinant DNA technology, protein design, and protein chemistry can produce such preproproteins which will result in a biologically active mature protein.
- the artificial protein comprises a multimeric protein preproprotein, that is, a protein assembly capable of producing a multimeric protein from a single protein comprised of a signal peptide, a first peptide, propeptide, and a second peptide, where the first peptide and the second peptide associate to assume a biologically functional conformation essentially free of the propeptide and signal peptide.
- a signal peptide would be the kappa light leader or the alpha amylase signal peptide.
- the proprotein is derived in part from a Fab fragment consisting of a portion of a immunoglobulin heavy chain and a immunoglobulin light chain.
- the immunoglobulin heavy chain fragment and light chains are associated with each other and assume a conformation having an antigen binding site for a predetermined or preselected antigen.
- the antigen binding site on a Fab fragment has a binding affinity or avidity similar to the antigen binding site on an immunoglobulin molecule.
- the proprotein is derived form a multimeric immunoglobulin molecule comprised of an immunoglobulin heavy chain and an immunoglobulin light chain .
- the immunoglobulin heavy and light chains are associated with each other and assume a conformation having an antigen binding site specific for, as evidenced by its ability to be competitively inhibited, a preselected or predetermined antigen.
- the proprotein is derived from a ligand binding polypeptide (receptor) that forms a ligand binding site which specifically binds to a preselected ligand to form a complex having a sufficiently strong binding between the ligand and the ligand binding site for the complex to be isolated.
- a ligand binding polypeptide receptor
- the proprotein is derived from a multimeric protein where that protein is an enzyme that binds to a substrate and catalyzes the formation of a product from the substrate. While the topology of the substrate binding site (ligand binding site) of the catalytic multimeric protein is probably more important for its activity than its affinity for the substrate, there is a binding requirement.
- novel multimeric or heterodimers would also fit in this class. Interaction of polypeptides with other polypeptides to produce stable multimeric forms not occurring in nature could be produced with this technology.
- An example of such an artificial interaction would be LJTN-2,7 (L27) heterodimers where each subunit is derived from different species.
- the invention thus provides a family of recombinant molecules expressed form a single piece of DNA, all of which have the capacity to be processed into multiple polypeptide that have an associative property.
- an antibody or antibody fragment is modified to improve desired characteristics as demonstrated in Carter, et al, (1992)Proc.Nat. Acad. Sci. vol. 89 (4285-4289).
- the identified or presumed influential positions are randomized to contain preferred amino acids for optimal structural organization as well as preferred non- immunogenic human sequences. Using any appropriate DNA shuffling method, multiple influential positions containing varied amino acids residues at any one position, are re- assorted to create a population of sequences that contain different combinations of amino acids at these influential sites.
- the population of antibody sequences created by DNA shuffling are cloned as described in EXAMPLE 2 to create a population of preproprotein sequences that are cloned into viral vectors using restriction independent cohesive end cloning or another cloning method known in the art.
- Infectious transcripts are generated and then encapsidated in vitro.
- the encapsidated transcripts are used to infect plants.
- Expressed proteins are subsequently harvested from the interstitial fluid or from a tissue homogenate.
- the extracts are assayed for a desired activity (e.g., antigen binding) as determined by ELIS A or other suitable assay. Additionally, it is preferred if the activity assay has a quantitative aspect.
- the samples are furthered evaluated to determine the quantity of the antibody present by ELISA or with other suitable assay.
- Viral vectors containing improved antibodies can be used to inoculate larger quantities of plants to obtain purified antibody for further characterization, pre- clinical evaluation, and process development.
- the expression system is scaled up to produce sufficiently large-scale quantities. This may involve the creation of a plant line stably transformed with the preferred proprotein or antibody encoding genes.
- Genes useful in practicing this invention include genes coding for polypeptide contained in immunoglobulin products, immunoglobulin molecules, Fab fragments, enzymes, receptors, chemokines, cytokines, blood products, diagnostic, analytical and therapeutic compounds. Particularly preferred are genes coding for polypeptides that associate to form multimeric complexes.
- Genes coding for a polypeptide subunit of a multimeric protein can be isolated from either the genomic DNA containing the gene expressing the polypeptide or the messenger RNA (mRNA) which codes for the polypeptide.
- mRNA messenger RNA
- the difficulty in using genomic DNA is in juxtaposing the sequences coding for the polypeptide where the sequences are separated by introns.
- the DNA fragment(s) containing the proper exons must be isolated, the introns excised, and the exons spliced together in the proper order and orientation. For the most part, this will be difficult so the alternative technique employing mRNA will be the method of choice because the sequence is contiguous (free of introns) for the entire polypeptide.
- the polypeptide coding genes isolated above are assembled into a proprotein and typically operatively linked to an expression vector.
- Expression vectors compatible with the host cells are used to express the genes of the present invention.
- Typical expression vectors useful for expression of genes in various hosts are well known in the art and include vectors derived from with recombinant virus expression vectors (e.g., baculovirus) containing antibody coding sequences; plant cell systems infected with recombinant virus expression vectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) or transformed with recombinant plasmid expression vectors (e.g., Ti plasmid) containing antibody coding sequences; or mammalian cell systems (e.g., COS, CHO, BHK, 293, 3T3 cells) harboring recombinant expression constructs containing promoters derived from the genome of mammalian cells (e.g., metallothionein promoter) or from mammalian viruses
- the expression vectors described above contain expression control elements including the promoter.
- the polypeptide coding genes are operatively linked to the expression vector to allow the promoter sequence to direct RNA polymerase binding and synthesis of the desired polypeptide coding gene.
- Useful in expressing the polypeptide coding gene are promoters which are inducible, viral, synthetic, constitutive, temporally regulated, spatially regulated, and spatiotemporally regulated.
- the choice of which expression vector and ultimately to which promoter a polypeptide coding gene is operatively linked depends directly, as is well J nown in the art, on the functional properties desired, e.g.
- an expression vector useful in practicing the present invention is at least capable of directing the replication, and preferably also the expression of the polypeptide coding gene included in the DNA segment to which it is operatively linked.
- eukaryotic cells especially for the expression of whole recombinant antibody molecule, are used for the expression of a recombinant antibody molecule.
- mammalian cells such as Chinese hamster ovary cells (CHO)
- CHO Chinese hamster ovary cells
- a vector such as the major intermediate early gene promoter element from human cytomegalovirus is an effective expression system for antibodies (Foecking et al., Gene 45:101 (1986); Cockett et al., Bio/Technology 8:2 (1990)).
- a number of viral-based expression systems may be utilized.
- the antibody coding sequence of interest may be ligated to an adenovirus transcription/translation control complex, e.g., the late promoter and tripartite leader sequence.
- This chimeric gene may then be inserted in the adenovirus genome by in vitro or in vivo recombination. Insertion in a non-essential region of the viral genome (e.g., region El or E3) will result in a recombinant virus that is viable and capable of expressing the antibody molecule in infected hosts, (e.g., see Logan & Shenk, Proc.
- Specific initiation signals may also be required for efficient translation of inserted antibody coding sequences. These signals include the ATG initiation codon and adjacent sequences. Furthermore, the initiation codon must be in phase with the reading frame of the desired coding sequence to ensure translation of the entire insert. These exogenous translational control signals and initiation codons can be of a variety of origins, both natural and synthetic. The efficiency of expression may be enhanced by the inclusion of appropriate transcription enhancer elements, transcription terminators, etc. (see Bittner et al., Methods in Enzymol. 153:51-544 (1987)).
- a host cell strain may be chosen which modulates the expression of the inserted sequences, or modifies and processes the gene product in the specific fashion desired. Such modifications (e.g., glycosylation) and processing (e.g., cleavage) of protein products may be important for the function of the protein.
- Different host cells have characteristic and specific mechanisms for the post-translational processing and modification of proteins and gene products. Appropriate cell lines or host systems can be chosen to ensure the correct modification and processing of the foreign protein expressed.
- eukaryotic host cells which possess the cellular machinery for proper processing of the primary transcript, glycosylation, and phosphorylation of the gene product may be used.
- Such mammalian host cells include, but are not limited to, CHO, VERO, BHK, Hela, COS, JMDCK, 293, 3T3, WI38, and in particular, breast cancer cell lines such as, for example, BT483, Hs578T, HTB2, BT20 and T47D, and normal mammary gland cell line such as, for example, CJRL7030 and Hs578Bst.
- cell lines which stably express the antibody molecule may be engineered.
- host cells can be transformed with DNA controlled by appropriate expression control elements (e.g., promoter, enhancer, sequences, transcription terminators, polyadenylation sites, etc.), and a selectable marker.
- appropriate expression control elements e.g., promoter, enhancer, sequences, transcription terminators, polyadenylation sites, etc.
- engineered cells may be allowed to grow for 1-2 days in an enriched media, and then are switched to a selective media.
- the selectable marker in the recombinant plasmid confers resistance to the selection and allows cells to stably integrate the plasmid into their chromosomes and grow to form foci which in turn can be cloned and expanded into cell lines.
- This method may advantageously be used to engineer cell lines which express the antibody molecule.
- Such engineered cell lines may be particularly useful in screening and evaluation of compounds that interact directly or indirectly with the antibody molecule.
- a number of selection systems may be used, including but not limited to the herpes simplex virus thymidine kinase (Wigler et al., Cell 11:223 (1977)), hypoxanthine-guanine phosphoribosyltransferase (Szybalska & Szybalski, Proc. Natl. Acad. Sci. USA 48:202 (1992)), and adenine phosphoribosyltransferase (Lowy et al., Cell 22:817 (1980)) genes can be employed in tk-, hgprt- or aprt-cells, respectively.
- antimetabolite resistance can be used as the basis of selection for the following genes: dhfr, which confers resistance to methotrexate (Wigler et al., 1980, Natl. Acad. Sci. USA 77:357; O'Hare et al., Proc. Natl. Acad. Sci. USA 78:1527 (1981)); gpt, which confers resistance to mycophenolic acid (Mulligan & Berg, Proc. Natl. Acad. Sci.
- the expression levels of an antibody molecule can be increased by vector amplification (for a review, see Bebbington and Hentschel, "The use of vectors based on gene amplification for the expression of cloned genes in mammalian cells," in DNA Cloning, Vol. 3. (Academic Press, New York, 1987)).
- vector amplification for a review, see Bebbington and Hentschel, "The use of vectors based on gene amplification for the expression of cloned genes in mammalian cells," in DNA Cloning, Vol. 3. (Academic Press, New York, 1987)).
- a marker in the vector system expressing antibody is amplifiable
- increase in the level of inhibitor present in culture of host cell will increase the number of copies of the marker gene. Since the amplified region is associated with the antibody gene, production of the antibody will also increase (Crouse et al., Mol. Cell. Biol. 3:257 (1983)).
- Expression of the desired multimeric protein can be identified by assaying for the presence of the biologically multimeric protein using assay methods well l ⁇ iown in the art. Such methods include Western blotting, immunoassays, binding assays, and any assay designed to detect a biologically functional multimeric protein. See, for example, the assays described in Immunology: The Science of Self-Nonself Discrimination, Klein, John Wiley and Sons, New York, N.Y. (1982).
- Preferred screening assays are those where the biologically active site on the multimeric protein is detected in such a way as to produce a detectible signal.
- This signal may be produced directly or indirectly and such signals include, for example, the production of a complex, formation of a catalytic reaction product, the release or uptake of energy, and the like.
- a host containing an antibody molecule produced by this method may be processed in such a way to allow that antibody to bind its antigen in a standard immunoassay such as an ELIS A or a radio-immunoassay similar to the immunoassays described in Antibodies: A Laboratory Manual, Harlow and Lane, eds., Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. (1988).
- a further aspect of the present invention is a method of producing a proprotein comprised of a first and a second polypeptide and a propeptide.
- the method combines the elements of propagating or culturing a host of the present invention, and harvesting the host cell or cells that was cultivated to produce the desired multimeric protein.
- the host of the present invention containing the desired multimeric protein precursor comprised of a first polypeptide and a second polypeptide and a propeptide is propagated or cultured using methods well known to one skilled in the art. Any of the recombinant hosts of the present invention may be cultured or propagated to isolate the desired multimeric protein they contain.
- the recombinant host is harvested to recover the produced multimeric protein.
- This harvesting step may consist of harvesting the entire host, or isolating specific organelles or extracts such as the media or secreted fraction which facilitate further purification.
- this harvesting step further comprises the steps of:
- this harvesting step further comprises the steps of:
- the multimeric protein is isolated from the solution produced above using methods that are well known to those skilled in the art of protein isolation. These methods include, but are not limited to, immuno-affinity purification and purification procedures based on the specific size, electrophoretic mobility, biological activity, and or net charge of the multimeric protein to be isolated.
- the contemplated recombinant hosts contain a multimeric protein.
- This multimeric protein may be an immunoglobulin product described above, an enzyme, a receptor capable of binding a specific ligand, or an abzyme.
- An enzyme of the present invention is a proprotein derived at least two polypeptide chains. This proprotein is encoded by a gene introduced into the recombinant host by the method of the present invention.
- Useful enzymes include aspartate transcarbamylase and the like.
- the proprotein is derived from a receptor capable of binding a specific ligand.
- this receptor is made up of a proprotein encoded by a gene introduced into the recombinant host by a method of the present invention.
- Examples of such receptors and their respective ligands include hemoglobin, O.sub.2 ; protein kinases, cAJMP; and the like.
- the immunoglobulin product present is an abzyme constituted by either an immunoglobulin heavy chain and its associated variable region, or by an immunoglobulin heavy chain and an immunoglobulin light chain associated together to form an immunoglobulin molecule, a Fab or a substantial portion of an immunoglobulin molecule.
- abzymes include those described by Tramontano et al., Science, 234: 1566-1570 (1986): Pollack et al., Science, 234: 1570-1573 (1986): Janda et al., Science, 241: 1188-1191 (1988); and Janda et at., Science, 244: 437-440 (1989).
- proproteins of the present invention contain at least two polypeptides and the propeptide; however, more than two peptides can also be present. Each of these polypeptides is separated by a propeptide such that they fold and are processed into a multimeric protein.
- the polypeptide subunits are associated with one another to form a multimeric protein by disulfide bridges, by hydrogen bonding, or like mechanisms.
- multimeric proteins there are numerous examples of multimeric proteins that could be made in this way.
- the following list comprises several multimeric proteins that are not naturally made with a propeptide.
- the list is intended to be exemplary.
- hemoglobin 2 ⁇ 2
- IL-12 p35 and p40
- TCR JMHC class II heterodimer
- cc ⁇ CD8 heterodimer
- CD3 ⁇
- CD3 ⁇
- CD22 ⁇
- CD41 GP ⁇ ba CD61
- JAK and STAT signal transducers and activators of transcription
- heterodimers IgM heavy chain with I chain, or VpreB and lambda 5 (I chain)
- Ig ⁇ and Ig Integrins such as T-cell integrin LFA-1 ( L ⁇ 2 ), CD152(CTLA-4), JfL-2 rece ⁇ tor(heterotrimer) E -2R( ⁇ c), IL- 15( ⁇ ), Rhematopoietin receptor family (D -3R, GM-CSJFR are a few), TNF- ⁇ (LT- and LT- ⁇ ), IL12R( ⁇ l ⁇ 2), IgM (H 2 L
- propeptide it is preferred to remove the propeptide to obtain the mature multimeric protein, essentially free of foreign sequences which are potentially destabilizing or could interfere with the active site or antigen binding region and potentially be adversely immunogenic. It may be beneficial to engineer the proprotein such that small foreign regions remain after the removal of the propeptide sequence such that these additional sequences would be useful for purification or confer other biological function such as immuno-regulation.
- a few amino acid spacer is inserted between the polypeptide domains and is designed to transition from one domain to another.
- a di-glycine spacer functions to buffer the joint of the heterologous polypeptides and facilitate proper folding of juxtaposed domains and minimize and enhance the transition of one domain to another.
- Other amino acids may be used to further improve the folding and chaperone activity of the propeptide, further optimizing the propeptide folding.
- Novel multimeric proteins which have polypeptide subunits with associative properties but are not naturally found associated would also fit in this class. Interaction of proteins with other proteins to produce stable multimeric forms not occurring in nature could be produced with this technology. Additionally, naturally occurring proteins that do not interact as a result of production in two different organisms, organelles, are temporally or otherwise separated proteins that would interact if produced in the presence of the other. An example of such an artificial interaction would be LJTN- 2,7 (L27) heterodimers where each subunit is derived from different species.
- a domain may be isolated by any of a number of techniques.
- a nucleic acid sequence encoding a polypeptide (or RNA) domain of interest is cloned from an appropriate cDNA library or a genomic DNA library based on hybridization with a oligonucleotide probe that represents the domain.
- nucleic acids and proteins are mammalian, more preferably human sequences.
- the DNA is isolated by amplification techniques using oligonucleotide primers starting with a DNA or RNA template.
- oligonucleotide primers Starting with a DNA or RNA template.
- These primers can be used to amplify either a full length coding sequence or a partial sequence that could constitute a probe (ranging in length up to about several thousand nucleotides).
- the resultant probe sequence is then used to screen a mammalian library for the full-length nucleic acid of interest.
- Use of synthetic oligonucleotide primers and amplification of an RNA or DNA template is described in U.S.
- Methods such as PCR and ligase chain reaction (LCR) can be used to amplify nucleic acid sequences of domains directly from mRNA, from cDNA, or from genomic or cDNA libraries.
- LCR ligase chain reaction
- Degenerate oligonucleotides can be designed to amplify domain homologues using the known sequences that encode the domain. Restriction endonuclease sites can be incorporated into the primers.
- Genes amplified by the PCR reaction can be purified on agarose gels and cloned into an appropriate vector.
- nucleic acids are isolated from expression libraries using as a probe an antibody (or other binding partner) specific for an epitope of the expressed polypeptide.
- Polyclonal or monoclonal antibodies (mAbs) can be raised by immunization with one or more peptide fragments of the domain being cloned.
- Nucleic acid probes preferably oligonucleotides are used under preferably stringent hybridization conditions to screen libraries in order to isolate polymorphic variants or alleles of the genes that encode the polypeptide domain of interest.
- antibody-based expression cloning permits cloning of polymorphic or allelic variants or interspecies homologues.
- DNA extracted from a tissue may be mechanically sheared or enzymatically digested to yield fragments of about 12-20 kb that are separated by gradient centrifugation and inserted into appropriate expression vectors. These vectors are packaged into phage in vitro. Recombinant phage are analyzed by plaque hybridization (Benton et al, Science 196:180-182 (1977). Colony hybridization is carried out, for example, as generally described by Grunstein et al, Proc. Natl. Acad. Sci. USA., 72:3961-3965 (1975).
- Synthetic oligonucleotides can be used to construct recombinant "genes" for use as probes or for expression of the domain polypeptides.
- Oligonucleotides can be chemically synthesized using solid phase phosphoramidite triester methods (Beaucage et al, Tetrahedron Letts. 22:1859-1862 (1981)) using an automated synthesizer (Van Devanter et al, Nucleic Acids Res. 12:6159- 6168 (1984)). Purification of oligonucleotides is typically by native acrylamide gel electrophoresis or by anion-exchange JHPLC (Pearson et al, J. Chrom. 255:137-149 (1983)).
- the nucleic acid encoding the desired polypeptide is typically cloned into an intermediate vector before transformation or transfection of prokaryotic or eukaryotic cells for replication and/or expression of the nucleic acid.
- intermediate vectors e.g., plasmids or shuttle vectors, are typically for use in prokaryotic cells.
- a number of transformation methods permit expression of heterologous proteins in plants. Some involve the construction of a transgenic plant by integrating DNA sequences encoding the protein of interest into the plant genome. The time it takes to obtain transgenic plants may be too long for the rapid production certain embodiments such as a tumor vaccine polypeptide.
- An attractive solution is transient transfection of plants with expression vectors. Both viral and non-viral vectors capable of such transient expression are available (Kumagai, M.H. et al. (1993) Proc. Nat. Acad. Sci. USA 90:427-430; Shivprasad, S. et al. (1999) Virology 255:312-323; Turpen, T.H.
- a viral vector that expresses heterologous proteins in plants preferably includes (1) a native viral subgenomic promoter (Dawson, W.O. et al. (198$>)Phytopathology 78:783-789 and French, R. et al. (1986) Science 231:1294-1297), (2) preferably, one or more non-native viral subgenomic promoters (Donson, J. et al. (1991) Proc. Nat. Acad. Sci. USA 88:7204- 7208 and Kumagai, M.H. et al (1993) Proc. Nat.
- Vectors that include only non-native subgenomic promoters may also be used.
- the minimal requirement for the present vector is the combination of a replicase gene and the coding sequence that is to be expressed, driven by a native or non- native subgenomic promoter.
- the viral replicase is expressed from the viral genome and is required to replicate extrachromosomally.
- the subgenomic promoters allow the expression of the foreign or heterologous coding sequence and any other useful genes such as those encoding viral proteins that facilitate viral replication, proteins required for movement, capsid proteins, etc.
- the viral vectors are encapsidated by the encoded viral coat proteins, yielding a recombinant plant virus.
- This recombinant virus is used to infect appropriate host plants.
- the recombinant viral nucleic acid can thus replicate, spread systemically in the host plant and direct RNA and protein synthesis to yield the desired heterologous protein in the plant.
- the recombinant vector maintains the non- viral heterologous coding sequence and control elements for periods sufficient for desired expression of this coding sequence.
- the recombinant viral nucleic acid is prepared from the nucleic acid of any suitable plant virus, though members of the tobamovirus family are preferred.
- the native viral nucleotide sequences may be modified by known techniques providing that the necessary biological functions of the viral nucleic acid (replication, transcription, etc.) are preserved.
- one or more subgenomic promoters may be inserted. These are capable of regulating expression of the adjacent heterologous coding sequences in infected or transfected plant host.
- Native viral coat protein may be encoded by this RNA, or this coat protein sequence may be deleted and replaced by a sequence encoding a coat protein of a different plant virus ("non-native" or "foreign viral").
- a foreign viral coat protein gene may be placed under the control of either a native or a non-native subgenomic promoter.
- the foreign viral coat protein should be capable of encapsidating the recombinant viral nucleic acid to produce functional, infectious virions.
- the coat protein is foreign viral coat protein encoded by a nucleic acid sequence that is placed adjacent to either a native viral promoter or a non-native subgenomic promoter.
- the nucleic acid encoding the heterologous protein, e.g., an immunogenic polypeptide to be expressed in the plant is placed under the control of a native subgenomic promoter.
- An important element of this invention, that is responsible in part for the proper folding and copious production of the heterologous protein is the presence of a signal peptide sequence that directs the newly synthesized protein to the plant secretory pathway.
- the sequence encoding the signal peptide is fused in frame with the DNA encoding the polypeptide to be expressed.
- a preferred signal peptide is the ⁇ -amylase signal peptide.
- RNA or DNA plant viruses are suitable for use as expression vectors.
- the DNA or RNA may be single- or double-stranded.
- Single-stranded RNA viruses preferably may have a plus strand, though a minus strand RNA virus is also intended.
- the recombinant viral nucleic acid is prepared by cloning in an appropriate production cell.
- Conventional cloning techniques for both DNA and RNA are well known.
- an origin of replication compatible with the production cell may be spliced to the viral DNA.
- RNA virus With an RNA virus, a full-length DNA copy of the viral genome is first prepared by conventional procedures: for example, the viral RNA is reverse transcribed to form +subgenomic pieces of DNA which are rendered double-stranded using DNA polymerases. The DNA is cloned into an appropriate vector and inserted into a production cell. The DNA pieces are mapped and combined in proper sequence to produce a full- length DNA copy of the viral genome.
- Subgenomic promoter sequences with or without a coat protein gene, are inserted into nonessential sites of the viral nucleic acid as described herein. Non-essential sites are those that do not affect the biological properties of the viral nucleic acid or the assembled plant virion.
- cDNA complementary to the viral RNA is placed under control of a suitable promoter so that (recombinant) viral RNA is produced in the production cell. If the RNA must be capped for infectivity, this is done by conventional techniques.
- promoters examples include the lac, lacuv ⁇ , trp, tac, Ipl and ompF promoters.
- a preferred promoter is the phage SP6 promoter or T 7 RNA polymerase promoter.
- Production cells can be prokaryotic or eukaryotic and include Escherichia coli, yeast, plant and mammalian cells.
- viruses are (a) those from the tobacco mosaic virus (TMV) group, such as TMV, tobacco mild green mosaic virus (TMGMV), cowpea mosaic virus (CMV), alfalfa mosaic virus (AJMV), Cucumber green mottle mosaic virus - watermelon strain (CGMMV-W), oat mosaic virus (OMV),
- TMV tobacco mosaic virus
- TMV tobacco mild green mosaic virus
- CMV cowpea mosaic virus
- AJMV alfalfa mosaic virus
- CGMMV-W Cucumber green mottle mosaic virus - watermelon strain
- OMV oat mosaic virus
- viruses from the brome mosaic virus (BMV) group such as BMV, broad bean mottle virus and cowpea chlorotic mottle virus,
- viruses such as rice necrosis virus (RNV), gemini viruses such as Tomato Golden Mosaic virus (TGMV), Cassava Latent virus (CLN) and Maize Streak virus (MSN).
- RMV rice necrosis virus
- TGMV Tomato Golden Mosaic virus
- CLN Cassava Latent virus
- MSN Maize Streak virus
- a preferred host is Nicotiana benthamiana.
- the host plant as the term is used here, may be a whole plant, a plant cell, a leaf, a root shoot, a flower or any other plant part.
- the plant or plant cell is grown using conventional methods.
- a preferred viral vector for use with N. benthamiana is a modified TTO1A vector containing a hybrid fusion of TMN and tomato mosaic virus (ToMV) (Kumagai, MH. et al. (1995) Proc. Natl. Acad. Sci. USA 92:1679-1683).
- the inserted subgenomic promoters must be compatible with TMV nucleic acid and capable of directing transcription of properly situated (e.g., adjacent) nucleic acids sequences in the infected plant.
- the coat protein should permit the virus to systemically infect the plant host. TMV coat protein promotes systemic infection of N. benthamiana.
- Infection of the plant with the recombinant viral vector is accomplished using a number of conventional techniques known to promote infection. These include, but are not limited to, leaf abrasion, abrasion in solution and high velocity water spray.
- the viral vector can be delivered by hand, mechanically or by high pressure spray of single leaves.
- the multimeric protein produced is preferably recovered and purified using standard techniques. Suitable methods include homogenizing or grinding the plant or the producing plant parts in liquid nitrogen followed by extraction of protein. If for some reason it is not desirable to homogenize the plant material, the polypeptide can be removed by vacuum infiltration and centrifugation followed by sterile filtration. Protein yield may be estimated by any acceptable technique. Polypeptides are purified according to size, isoelectric point or other physical property. Following isolation of the total secreted proteins from the plant material, further purification steps may be performed. Immunological methods such as immunoprecipitation or, preferably, affinity chromatography, with antibodies specific for epitopes of the desired polypeptide may be used.
- Various solid supports may be used in the present methods: agarose®, Sephadex®, derivatives of cellulose or other polymers.
- staphylococcal protein A (or protein L) immobilized to Sepharose® can be used to isolate the target protein by first incubating the protein with specific antibodies in solution and contacting the mixture with the immobilized protein A which binds and retains the antibody-target protein complex.
- the polypeptide is purified from the plant material to a purity of greater than about 50%, more preferably greater than about 75%, even more preferably greater than about 95%.
- a preferred reagent to be used in determining proper folding is a specific ligand, preferably an antigen, which (1) is bound by the multimeric protein when the chains are correctly folded but (2) does not bind when the chains are denatured.
- the antigen is employed in any of a number of immunological assays, including dot blot, western blot, immunoprecipitation, radioimmunoassay (RIA), and enzyme immunoassays (EIA) such as an enzyme-linked immunosorbent assays (ELISA).
- Western blots and ELIS As are employed to verify correct folding of the relevant parts of the multimeric polypeptide produced in the plant. Additional Analysis of the Multimeric Protein
- DNA encoding the proprotein can be sequenced, yielding a deduced amino acid sequence of its encoded product. If the DNA molecule has been subcloned, it can be excised from the vector with a restriction enzyme and the resulting fragments analyzed on agarose gels to determine the size of the fragments.
- a DNA molecule encoding a proprotein is first expressed. If desired, the
- DNA can be additionally modified to include sequences that will permit or optimize expression in an appropriate host or in an in vitro transcription/translation system.
- the multimeric polypeptide is then subjected to appropriate functional assays, e.g., measurement of enzymatic activity (of either domain). Also the quantity and physical properties of the multimeric polypeptide can be determined, e.g., by SDS-PAGE. If a domain has binding activity, or other functions as have been described above, this can also be measured by conventional means.
- the UmV JKP6 propeptide region containing amino acids 106-138 was codon optimized for viral expression and assembled using overlapping synthetic oligonucleotides.
- the PCR reaction was amplified at 94°C for 30 seconds, 25 cycles of 94°C for 10 seconds, 48°C for 15 seconds, 72°C for 15 seconds, and 7 minutes at 72°C.
- flanking primers which incorporates the coding sequence of a diglycine spacer at the 5' end and KP6 toxin amino acids 139-141 and a diglycine spacer to the 3' end of the synthetic JKP6 propeptide sequence.
- the PCR reaction was amplified at 94°C for 1 minute, 25 cycles of 94°C for 30 seconds, 55°C for 30 seconds, 72°C for 30 seconds, and 7 minutes at 72°C.
- telomere sequence The amplification of the desired approximately 120 bp KP6 propeptide encoding sequence was confirmed by agarose gel electrophoresis.
- the PCR fragment from the above reaction was cloned into pCR4Blunt-TOPO (Invitrogen) following the manufacturers directions to create plasmid pLSBC1731 (Seq ID No: 75). Briefly, 1 ⁇ L of PCR product, 1 ⁇ L vector, 1 ⁇ L of salt solution and 3 ⁇ L of water were mixed, incubated at room temperature for 5 minutes. The ligation was placed on ice and 25 ⁇ L of chemically competent Top 10 cells was added to the ligation and the mix was incubated on ice for 10 minutes.
- the transformation reaction was heat shocked by incubating at 42°C for 30 seconds and immediately placed on ice and 250 ⁇ L of SOC was added. The transformation was allowed to recover by incubating at 37°C, 200 rpm shaking for 20 minutes. The transformation was plated out on LB plates containing ampicillin and grown overnight at 37°C. Individual colonies were used to inoculate 1.0 mL Super Broth (SB) containing 100 ⁇ g/mL ampicillin in 96 well 2.0 mL flat-bottom blocks and grown overnight at 37°C and 400 rpm. Plasmid was purified from turbid cultures using the QIAprep 96 Turbo Miniprep kits (QIAGEN, Valencia, CA).
- the cells were pelleted by centrifugation at 3 K rpm for 15 minutes in a plate centrifuge. The supernatant was drained from the cell pellets and the cells resuspended in 250 ⁇ L PI Buffer by vortexing. 250 ⁇ L of P2 was added to the cells, mixed by inverting and incubated for 5 minutes to lyse the cells. 350 ⁇ L of N3 was added to the cell lysates, mixed by inverting and transferred to the Turbo Filter plate. A vacuum was applied to the Turbo Filter, which filtered the sample into the QIAprep plate. A vacuum was then applied to the QIAprep plate pulling the sample through the plate and bound the plasmid to the plate membrane.
- the QIAprep plate was washed using vacuum force with 0.9 mL of PB, followed by two washes with 0.9 mL of PE and vacuum dried. 100 ⁇ L EB buffer was added to the purified plasmid, incubated for 1 minute, and subsequently centrifuged for 3 minute at 6K rpm to elute the purified plasmid.
- the purified pLSBC1731 (Seq ID No: 75) plasmid was subjected to nucleic acid sequencing using standard methods to verify the JKP6 propeptide sequence.
- RNA messenger RNA
- mRNA messenger RNA
- mRNA messenger RNA
- dT Dynabeads Oligo 25 (Dynal, Oslo, Norway).
- the RNA was pelleted by centrifugation at 15 K rpm, 4°C for 15 minutes, the supernatant removed and 1 mL of 70 % ethanol added.
- the sample was centrifuged at 15 K rpm, 4°C for 15 minutes, the supernatant removed and the pellet resuspended in 150 ⁇ L nuclease free water (Ambion, Austin, TX).
- RNA 5 ⁇ g of the above prepared total RNA was incubated at 65°C for 2 minutes, immediately placed on ice for 3 minutes, and then applied to 20 ⁇ L of magnetic beads in binding buffer (20 mM Tris-HCl (pH 7.5), 1.0 M LiCl, 2 mM EDTA) where the beads were prepared by washing with 50 ⁇ L of binding buffer. The RNA and bead mixture were incubated for 5 minutes with constant rotating. The supernatant containing unbound material was removed and the beads were washed with 100 ⁇ L washing buffer (10 mM Tris-HCl (pH 7.5), 0.15 M LiCl, 1 mM EDTA) followed by the addition of 40 ⁇ L nuclease free water.
- binding buffer 20 mM Tris-HCl (pH 7.5), 1.0 M LiCl, 2 mM EDTA
- cDNA Complementary DNA
- Tris HC1 pH 8.3
- 75 mM KC1 3 mM MgCl 2
- 10 mM DTT 2 Units RNasin
- 20 Units Superscript II Invitrogen, Carlsbad, CA
- 0.5 mM dATP 0.5 mM dCTP
- 0.5 mM dGTP 0.5 mM dTTP
- oligo dT bound RNA from above.
- the cDNA reaction was incubated at 42°C for 60 jninutes with constant rotation.
- PCR reactions were set up as follows to amplify the gamma VH3 heavy chain Fd (V H - C H 1) regions or the kappa 1 light chains (V L -C L ) including the kappa leader from the synthesized cDNA.
- the 100 ⁇ L PCR reactions contained IX Taq Reaction buffer with MgCl 2 , 0.2 mM dATP, 0.2 mM dCTP, 0.2 mM dGTP, 0.2 mM dTTP, 10 Units Taq Polymerase (Stratagene, La Jolla, CA) l ⁇ M upstream primer, l ⁇ M downstream primer and 1 ⁇ L prepared cDNA.
- the reaction contained the 5230 (Seq JfD No: 29) upstream and 5235 downstream primers.
- the 5230 upstream primer was designed to amplify approximately 13 of the 16 different kappa 1 V-gene segments including the leader sequences.
- the 5230 primer incorporated a Pac I site upstream of the translation start site for subsequent cloning.
- the 5235 downstream primer anneals to the 3' end of the kappa C L OJRF, removing the termination codon, incorporates the coding sequence for a diglycine spacer fused to the 5' end of the KP6 propeptide coding sequence.
- the reaction contained the 5236 (Seq JfD No: 32) upstream and 5233 (Seq JfD No: 30) downstream primers.
- the 5236 upstream primer was designed to amplify approximately 14 of the 18 different VH3 V-gene segments with out the leader sequence.
- the 5236 primer incorporates the coding sequence for a diglycine spacer fused to the 3' end of the KP6 propeptide coding sequence.
- the 5233 downstream primer anneals to the 3' end of the gamma C H I OJRF, and incorporates a termination codon and a Not I site downstream of the terminator for subsequent cloning.
- PCR reactions were amplified at 97°C for 1 minute, 25 cycles of 94°C for 30 seconds, 55°C for 30 seconds, 72°C for 1 minute, and 7 minutes at 72°C.
- the amplification of the desired approximately 700 bp kappa light chains and the approximately 700 bp gamma Fd regions were confirmed by agarose gel electrophoresis.
- the KP6 sequence of pLSBC1731 was PCR amplified for Fab cloning.
- the PCR reaction was amplified at 97°C for 1 minute, 25 cycles of 94°C for 30 seconds, 55°C for 30 seconds, 72°C for 30 seconds, and 7 minutes at 72°C.
- the PCR reaction was amplified at 97°C for 30 seconds, 4 cycles of 94°C for 30 seconds, 50°C for 1 minute, 72°C for 1 minute.
- the fragment was cut from the gel and purified from the agarose slice using QIAquick gel extraction kit following the manufacturers instructions. Briefly, 900 ⁇ L of QG buffer was added to the gel fragment, the mixture was incubated at 65°C for 10 minutes with occasional agitation. The dissolved gel slice was applied to the QIAquick column and centrifuged at 14K rpm for 1 minute. The column was washed with 750 ⁇ L PE and the purified fragment eluted in 50 ⁇ L EB. To assemble the Fab, the KP6-heavy chain Fd PCR fragment from above was fused to the 5230-5235 (Seq ID No: 31) primer amplified kappa leader-light chain from above by SOE.
- the PCR reaction was amplified at 94°C for 2 minutes, 10 cycles of 94°C for 30 seconds, 55°C for 30 seconds, 72°C for 1 minute and finally 72°C for 5minutes.
- the PCR reaction was applied to the QIAquick spin column and centrifuged 14K rpm for 1 minute, washed with 500 ⁇ L PB, washed with twice with 750 ⁇ L PE and spun dry.
- the purified PCR product was eluted with 50 ⁇ L EB.
- the purified 1.5 Kb PCR product was subject to restriction endonuclease digestion with Pac I and Not I to produce cohesive ends for cloning.
- the 200 ⁇ L restriction digest contained 50 ⁇ L of the above purified PCR product, 100 Units Pac 1, 100 Units Not 1, 100 ⁇ g/mL BSA, 50 mM NaCl, 10 mM Tris-HCl (pH 7.9), 10 mM MgCl 2 , 1 mM DTT.
- the reaction was incubated at 37°C for 2 hours and subsequently electrophoresed on a 1.5 % agarose gel with TAE and 0.5 ⁇ g/mL ethidium bromide.
- the 1.5 Kb Pac I and Not I digested fragment was cut from the gel and purified from the agarose slice using QIAquick gel extraction kit following the manufacturers instructions.
- the p5PNCAP plasmid was subject to restriction endonuclease digestion with Pac I and Not I to produce cohesive ends for cloning.
- the 200 ⁇ L restriction digest contained 2.5 ⁇ g of p5PNCAP plasmid DNA, 50 Units Pac I, 50 Units Not I, 100 ⁇ g/mL BSA, 50 mM NaCl, 10 mM Tris-HCl (pH 7.9), 10 mM MgCl 2 , 1 mM DTT.
- the digest was incubated at 37°C for 3.5 hours, and electrophoresed on a 0.8% agarose gel with TAE and 0.5 ⁇ g/mL ethidium bromide to separate the approximately 9.7 Kb fragment from the 0.6 Kb fragment.
- the 9.7 Kb Pac I and Not I digested fragment was isolated in gel using a scalpel blade.
- the fragment was purified away from the agarose using QIAquick gel extraction kit following the manufacturers instructions. Briefly, 1.32 mL of QG buffer was added to the gel fragment, the mixture was incubated at 65 °C for 10 minutes with occasional agitation.
- Bacterial transformation was performed with a Gene Pulser electroporator (BioRad, Hercules, CA) following manufacturer recommendations. Briefly, 40 ⁇ L of electro-competent JM109 cells were mixed with 2 ⁇ L of ligation and transferred to a cold 0.2 cm cuvette. The mixture was pulsed at 2.5 KV, 200 ohms, 25 ⁇ FD. After pulsing, 150 ⁇ L of SOC was added and the cells allowed to recover for 20 minutes at 37°C. Cells were plated on LB plates containing 100 ⁇ g/mL ampicillin and grown overnight at 37°C.
- Infectious transcripts were synthesized in-vitro from each clone using the mMessage mMachine T7 kit (Ambion, Austin, TX) following the manufacturers directions. Briefly, a 5.5 ⁇ L reaction containing 1 ⁇ L 10X Reaction buffer, 2.5 ⁇ L 2X NTP/CAP mix, 1 ⁇ L Enzyme mix and 3.5 ⁇ L plasmid was incubated at 37°C for 2 hours.
- the synthesized transcripts were encapsidated in a 40 ⁇ L reaction containing 0.1 M Na 2 HPO 4 -NaH 2 PO 4 (pH 7.0), 0.5 mg/mL purified Ul coat protein (LSBC, Vacaville, CA) which was incubated overnight at room temperature.
- 40 ⁇ L of FES 0.1 M Glycine, 60 mM K 2 HPO 4 , 22 mM Na 2 P 2 O 7 , 10 g/L Bentonite, 10 g/L Celite 545) was added to each encapsidated transcript.
- the encapsidated transcript from an each individual clone was used to inoculate a 20 day post sow Nicotiana benthamiana plant (Dawson, WO et al. (1986) Proc. Natl. Acad Sci. USA 83:1832-1836). High levels of subgenomic RNA species were synthesized in virus-infected plant cells (Kumagai, JMH. et al (1993) Proc. Natl Acad. Sci. USA 90:427-430), and serve as templates for the translation and subsequent accumulation of Fab protein.
- the IF fraction is recovered into a 96-well microplate by centrifugation for 10 minutes at 3K rpm in a plate centrifuge.
- 30 ⁇ L of each IF sample was prepared for SDS-PAGE analysis by the addition of 5 ⁇ L 5X tris-glycine sample dye containing 10 % 2-mercaptoethanol for reducing gels and no 2-mercaptoethanol for non-reducing gels and the mixture was boiled for 2 minutes.
- Samples were separated on a 15 % Criterion gel (Bio-Rad) and the proteins were detected by Coomassie R-250 Brilliant blue staining. Protein banding in the reducing gel at approximately 25 KDa indicates the presence of the desired 25 KDa heavy chain Fd and the 25 KDa light chain.
- a corresponding protein at approximately 50 Da under non-reducing conditions as seen in samples HuFab A9, HuFab D5, and HuFab H2 are evidence of a assembled, disulfide linked Fab heterodimer consisting of the heavy chain Fd and the kappa light chain.
- the samples were subjected to western blot analysis to verify the presence of the heavy Fd and light chain polypeptides.
- the IF samples were diluted 1:10 in IX tris-glycine sample dye containing 10 % 2-mercaptoethanol.
- the blots were washed three times in TBST and the labeled proteins detected with the ECL+plus Western Blotting Detection System (Amersham Biosciences, Buckinghamshire, England).
- the anti kappa sera detected an approximately 25 KDa proteins in the HuFab A9, HuFab D5 and HuFab H2 samples and a corresponding approximately 25 KDa protein was detected with the anti gamma sera indicating that both the heavy Fd and kappa chains were expressed and secreted.
- Mouse hybridoma line Myc 1-9E10.2 expresses a murine monoclonal antibody (IgGl) that recognizes a human c-myc epitope of amino acid sequence EQKLISEEDL (G.I Evans et al., Molec. Cell. Biol. 5: 3610-3616, 1985).
- Cells were obtained from ATCC (CRL-1729) and cultured under standard conditions. 2 x 10 6 cultured cells were spun and washed to remove excess culture media and lysed with 600 ⁇ L RLT buffer containing 1 % 2-mercaptoethanol (Qiagen, Valencia, CA). Total RNA was purified using the QIAshredder and R EASY column per manufacturers directions.
- the cell lysate was applied to the QIAshredder column and spun in a centrifuge for 2 minutes at 14K rpm. The flow through was collected and diluted with an equal volume of 70% ethanol. The mixture was transferred to a RNeasy column and centrifuged for 15 seconds at 10K rpm until all sample was processed through the column. The RNA bound to the column was washed with 700 ⁇ L RW1 followed by a wash with 500 ⁇ L RPE and subsequently dried. The purified RNA was eluted in 50 ⁇ L RNASE free water by centrifugation for 1 minute at 10K rpm.
- First strand cDNA was synthesized from 0.8 ⁇ g total RNA using a SMART 3' RACE kit (BD Biosciences Clontech, Palo Alto, CA) with 1 ⁇ L 3' CDS primer in 5 ⁇ L.
- the RNA primer mix was heated to 70°C for 2 minutes and placed on ice for an additional 2 minutes.
- To the RNA and primer mix was brought to 10 ⁇ L containing IX First strand buffer, 2 mM DTT,1 mM dATP, 1 mM dCTP, ImM dGTP, 1 mM dTTP and 1 ⁇ L Powerscript Reverse Transcriptase.
- the reaction was incubated at 42°C for 90 minutes and then 100 ⁇ L of Tricine-EDTA Buffer (10 mM Tricine-KOH, pH 8.5, 1 mM EDTA) was added and the reaction heated to 72°C for 7 minutes.
- Tricine-EDTA Buffer (10 mM Tricine-KOH, pH 8.5, 1 mM EDTA) was added and the reaction heated to 72°C for 7 minutes.
- the 9E10 kappa light chain was PCR amplified in a 50 L reaction containing IX Advantage 2 PCR Buffer, 0.2 mM dATP, 0.2 mM dCTP, 0.2 mM dGTP, 0.2 mM dTTP, IX Advantage 2 Polymerase Mix, 2.5 ⁇ L of prepared cDNA, IX UPM, and 0.2 ⁇ M 9E10kl5' (Seq JTD No: 50).
- the 9E10kl5' primer was designed to anneal to the murine kappa light leader sequence from germline sequence V-21C9.5KB'CL.
- the reaction was cycled 5 times at 94°C for 5 seconds, 72°C for 3 minutes followed by 5 times at 94°C for 5 seconds, 70°C for 10 seconds, 72°C for 3 minutes and 25 cycles at 94°C for 5 seconds, 67°C for 10 seconds, 72°C for 3 minutes.
- the amplification of the desired approximately 900 bp fragment was confirmed by agarose gel electrophoresis.
- the 9E10 heavy chain was PCR amplified in a 50 L reaction containing IX Advantage 2 PCR Buffer, 0.2 mM dATP, 0.2 mM dCTP, 0.2 mM dGTP, 0.2 mM dTTP, IX Advantage 2 Polymerase Mix, 2.5 ⁇ L of prepared cDNA, IX UPM, and 0.4 ⁇ M 9E10gfw5' (Seq ID No: 51).
- the 9E10gfw5' primer was designed to anneal to the murine heavy chain variable FRl sequence identified from germline sequence Vh7183(Vh69.1).
- the reaction was cycled 5 times at 94°C for 5 seconds, 70°C for 3 minutes followed by 5 times at 94°C for 5 seconds, 68°C for 10 seconds, 72°C for 3 minutes and 25 cycles at 94°C for 5 seconds, 64°C for 10 seconds, 72°C for 3 minutes.
- the amplification of the desired approximately 1.6 Kb fragment was confirmed by agarose gel electrophoresis.
- the transformations were allowed to recover by incubating at 37°C, 200 rpm shaking for 20 minutes.
- the transformations were plated out on LB plates containing ampicillin and grown overnight at 37°C. Individual colonies were used to inoculate 1.0 mL Super Broth (SB) containing 100 ⁇ g/mL ampicillin in 96 well 2.0 mL flat-bottom blocks and grown overnight at 37°C and 400 rpm. Plasmid was purified from turbid cultures using the QIAprep 96 Turbo Miniprep kits (QIAGEN, Valencia, CA). Briefly, the cells were pelleted by centrifugation at 3 K rpm for 15 minutes in a plate centrifuge.
- the supernatant was drained from the cell pellets and the cells resuspended in 250 ⁇ L PI Buffer by vortexing.
- 250 ⁇ L of P2 was added to the cells, mixed by inverting and incubated for 5 minutes to lyse the cells.
- 350 ⁇ L of N3 was added to the cell lysates, mixed by inverting and transferred to the Turbo Filter plate.
- a vacuum was applied to the Turbo Filter, which filtered the sample into the QIAprep plate.
- a vacuum was then applied to the QIAprep plate pulling the sample through the plate and bound the plasmid to the plate membrane.
- the QIAprep plate was washed using vacuum force with 0.9 mL of PB, followed by two washes with 0.9 mL of PE and vacuum dried. 100 ⁇ L EB buffer was added to the purified plasmid, incubated for 1 minute, and subsequently centrifuged for 3 minute at 6K rpm to elute the purified plasmid. The presence of the approximately 1.2 Kb insert for p9E10Hy- TOPO (Seq ID No: 77) and 700 bp for p9E10Lt-Topo (Seq ID No: 79) was verified with EcoRI restriction digest and agarose gel electrophoresis. The purified p9E10Hy-TOPO and p9E10Lt-TOPO plasmids were subjected to nucleic acid sequencing using standard methods to verify the 9E10 heavy chain and kappa chain sequences.
- the K J P6 sequence of pLSBC1731 was PCR amplified for Fab cloning.
- the PCR reaction was amplified at 97°C for 1 minute, 25 cycles of 94°C for 30 seconds, 55°C for 30 seconds, 72°C for 30 seconds, and 7 minutes at 72°C.
- the amplification of the desired approximately 120 bp KP6 propeptide encoding sequence was confirmed by agarose gel electrophoresis.
- the 9E10 kappa light chain was amplified with primers 6056 (Seq ID No: 41) and 2228 from ⁇ 9E10Lt-TOPO and the 9E10 heavy Chain Fd (V H C H 1) was amplified with 2225 (Seq ID No: 39) and 6055 (Seq ID No: 40) from p9E10Hy-TOPO for Fab proprotein cloning.
- Each primer set incorporated additional regions encoding the termini of the KP6 propeptide coding sequence pLSBC1731 at either the 5' or 3' end, as well as a restriction site for cloning into the appropriate expression vector, (either Sphl at the 5' end of the heavy chain fragment or Avr ⁇ at the 3' end of the light chain fragment).
- a 100 ⁇ L PCR reaction containing 1 ⁇ M upstream , 1 ⁇ M downstream, IX Expand High Fidelity Buffer with MgCl 2 , 0.2 mM dATP, 0.2 mM dCTP, 0.2 mM dGTP, 0.2 mM dTTP, 3.5 Units Expand JHigh Fidelity and l ⁇ L plasmid.
- the PCR reaction was amplified at 97°C for 1 minute, 25 cycles of 94°C for 30 seconds, 55°C for 30 seconds, 72°C for 30 seconds, and 7 minutes at 72°C.
- the amplification of the desired approximately 700 bp kappa chain encoding sequence was confirmed by agarose gel electrophoresis.
- the light chain fragment was fused to the JKP6 PCR fragment by sequence overlap extension (SOE).
- the PCR reaction was amplified at 94°C for 1 minute, 25 cycles of 94°C for 30 seconds, 55°C for 30 seconds, 72°C for 1 minute and 5 minutes at 72°C.
- 9 ⁇ L of lO ⁇ M 5228 upstream primer, 9 ⁇ l of lO ⁇ M 2228 downstream primer, were added to the PCR reaction which was cycled at 25 cycles of 94°C for 30 seconds, 55°C for 30 seconds, 72°C for 1 minute and 5 minutes at 72°C.
- the amplification of the desired approximately 0.8 Kb JKP6 and light chain encoding sequences were confirmed by agarose gel electrophoresis.
- the KP6-light chain PCR fragment from above was fused to the amplified 9E10 heavy chain Fd from above by SOE.
- the PCR reaction was amplified at 97°C for 2 minutes, 10 cycles of 94°C for 30 seconds, 55°C for 30 seconds, 72°C for 1 minute and finally 72°C for 5 minutes.
- the PCR reaction was applied to the QIAquick spin column and centrifuged 14K rpm for 1 minute, washed with 500 ⁇ L PB, washed with twice with 750 ⁇ L PE and spun dry.
- the purified PCR product was eluted with 50 ⁇ L EB.
- the purified 1.5 Kb PCR product was subject to restriction endonuclease digestion with Sph I and Avr JH to produce cohesive ends for cloning.
- the 50 ⁇ L restriction digest contained 25 ⁇ L of the above purified PCR product, 8 Units Sph I, 8 Units Avr ⁇ , 100 ⁇ g/mL BSA, 50 mM NaCl, 10 mM Tris-HCl (pH 7.9), 10 mM MgCl 2 , 1 mM DTT.
- the reaction was incubated at 37°C for 2 hours and subsequently electrophoresed on a 1 % agarose gel with TAE and 0.5 ⁇ g/mL ethidium bromide.
- the 1.5 Kb Sph I and Avr JQJ digested fragment was cut from the gel and purified from the agarose slice using QIAquick gel extraction kit following the manufacturers instructions.
- the ligation was precipitated with 3 volumes ethanol and 0.3 volumes 10 M NH ⁇ Acetate, spun and washed with 70% ethanol. The pellets were resuspended in 20 ⁇ L 10 mM Tris-HCL (pH 8.0). Bacterial transformations were performed with a Gene Pulser electroporator (BioRad, Hercules, CA) following manufacturer recommendations. Briefly, 40 ⁇ L of electro-competent JM109 cells were mixed with 4 ⁇ L of ligation and transferred to a cold 0.2 cm cuvette. The mixture was pulsed at 2.5 KV, 200 ohms, 25 ⁇ FD. After pulsing, 120 ⁇ L of SOC was added and the cells allowed to recover for 20 minutes at 37°C.
- pLSBC1736 (Seq JDD No: 85) clones were confirmed to contain the 1.5 Kb insert and the 9.7 Kb vector fragments by restriction enzyme mapping with Sph I and Avr II followed by agarose gel electrophoresis.
- the 9E10 Fab proprotein was sequenced using standard methods to verify the sequence.
- Infectious transcripts were synthesized in-vitro from the pLSBC1736 clone using the mMessage mMachine T7 kit (Ambion, Austin, TX) following the manufacturers directions. Briefly, a 5.5 ⁇ L reaction containing 1 ⁇ L 10X Reaction buffer, 2.5 ⁇ L 2X NTP/CAP mix, 1 ⁇ L Enzyme mix and 3.5 ⁇ L plasmid was incubated at 37°C for 2 hours.
- the synthesized transcripts were encapsidated in a 40 ⁇ L reaction containing 0.1 M Na 2 HPO 4 -NaH 2 PO 4 (pH 7.0), 0.5 mg/mL purified Ul coat protein (LSBC, Vacaville, CA) which was incubated overnight at room temperature. 20 ⁇ L of FES (0.1 M Glycine, 60 mM K 2 HPO 4 , 22 mM Na 2 P 2 O 7 , 10 g/L Bentonite, 10 g L Celite 545) was added to each encapsidated transcript.
- FES 0.1 M Glycine, 60 mM K 2 HPO 4 , 22 mM Na 2 P 2 O 7 , 10 g/L Bentonite, 10 g L Celite 545
- the encapsidated transcript from an each individual clone was used to inoculate a 19 day post sow Nicotiana benthamiana plant (Dawson, WO et al. (1986) Proc. Natl. Acad Sci. USA 83:1832-1836). High levels of subgenomic RNA species were synthesized in virus-infected plant cells (Kumagai, MH. et al. (1993) Proc. Natl. Acad. Sci. USA 90:427-430), and serve as templates for the translation and subsequent accumulation of Fab protein.
- Interstitial fluid from infected leaves of each plant was harvested 8 days post inoculation and screened by ELISA. Systemically infected upper leaves from individual plants were harvested. The secreted protein fraction, or interstitial fluid (IF) was extracted and analyzed for presence of recombinant protein.
- the leaf tissue was placed in a GF/B 0.8 mL Unifilter (Whatman, Clifton, NJ), covered with 20 mM Tris-HCl (pH 7.0) and subjected to 760 mmHg vacuum for 30 seconds. The vacuum is released and re-applied three times to completely infiltrate the tissue with buffer. The residual buffer is discarded and the tissue dried by centrifugation at 400 rpm in a plate centrifuge for 30 seconds. The IF fraction is recovered into a 96-well microplate by centrifugation for 10 minutes at 3K rpm in a plate centrifuge.
- the blots were washed three times in TBST and the labeled proteins detected with the ECL+plus Western Blotting Detection System (Amersham Biosciences, Buckinghamshire, England).
- the anti kappa sera detected an approximately 25 KDa proteins on the reduced sample and an approximately 50 JKD band on the non-reduced indicating the presence of interchain disulfide bridges and an assembled 9E10 Fab.
- the anti gamma sera detected an approximately 25 KDa proteins on the reduced sample and a approximately 50 JKD band on the non-reduced indicating the presence of interchain disulfide bridges and an assembled 9E10 Fab.
- the membrane was probed with a 1:3000 dilution of Goat anti-mouse kappa- HRP labeled sera (Southern Biotechnology, Birmingham, AL) for 1 hour at room temperature.
- the blots were washed three times in TBST and the labelled proteins detected with the ECL+plus Western Blotting Detection System (Amersham Biosciences, Buckinghamshire, England).
- a band was visualized in the lane containing myc tagged GFP corresponding to the expected size of 53 KDa, and there were no detected proteins in the untagged GFP control lane. There were no bands visualized in lanes which were probed with JEF obtained from healthy plants.
- the specific recognition of the myc-tagged GFP protein with JJTF from pLSBC1736 infected Nicotiana banthamiana plants containing the 9E10 Fab demonstrates the proper activity of the disulfide linked heteromultimeric protein.
- a 9E10 monoclonal antibody artificial proprotein was assembled by fusing the 9E10 kappa light chain to the KP6 propeptide region of pLSBC1731, which was fused to the 9E10 gamma heavy chain. This fusion will result in a first domain light chain, the second domain propeptide and the third domain the complete heavy chain sequence.
- the 9E10 kappa light chain was PCR amplified from plasmid p9E10Lt-TOPO with upstream primer 2230 (Seq JED No: 4) and downstream primer 6057.
- the 9E10 gamma heavy chain was PCR amplified from plasmid p9E10Hy-TOPO and with upstream primer 6058 (Seq ID No: 14) and downstream primer 2227. Separate 100 ⁇ L PCR reactions containing 1 ⁇ M upstream, 1 ⁇ M downstream, IX Expand High Fidelity Buffer with MgCl 2 , 0.2 mM dATP, 0.2 mM dCTP, 0.2 mM dGTP, 0.2 mM dTTP, 3.5 Units Expand High Fidelity and l ⁇ L plasmid template were amplified at 94°C for 1 minute, 30 cycles of 94°C for 30 seconds, 55°C for 30 seconds, 72°C for 1 minute, and a final step of 7 minutes at 72°C.
- the PCR reaction was purified using the JMinElute PCR purification kit (Qiagen) following the manufacturers instructions. Briefly, 5 volumes of PB buffer was added to the reaction, mixed, applied to the column and centrifuged at 14K rpm for 1 minute. The column was washed with 750 ⁇ L Buffer PE and the purified fragment eluted in 10 ⁇ L EB.
- a 50 ⁇ L reaction containing 5 ⁇ L purified PCR product, 50 mM potassium acetate, 20 mM Tris-Acetate pH 7.9, 1 mM DTT, 10 mM magnesium acetate, 20 Units Sphl and 8 Units Avr II was incubated at 37°C for 1 hour and electrophoresed on a 1.0% agarose gel with TAE and 0.5 ⁇ g/mL ethidium bromide to separate the approximately 2.3 Kb 9E10 MAb proprotein encoding sequence.
- the 2.3 Kb MAb proprotein encoding sequence was purified using the QIAquick gel extraction kit (Qiagen) following manufacturers recommendations and eluted with 50 ⁇ L EB Buffer.
- the murine monoclonal antibody S1C5 recognizes the idiotope of the surface immunoglobulin of the murine B cell tumor 38C13 (Maloney et al, Hybridoma. 4:191-209, 1985). Cells were cultured under standard techniques. The heavy chain and kappa light chain genes were isolated by PCR amplification of cDNA produced from hybridoma mRNA. Briefly, 1 xlO 6 cultured cells were spun and washed to remove excess culture media and lysed with 600 ⁇ L RLT buffer containing 1 % 2-mercaptoethanol (Qiagen, Valencia, CA). Total RNA was purified using the QIAshredder and RNEAS Y column per manufacturers directions.
- the cell lysate was applied to the QIAshredder column and spun in a centrifuge for 2 minutes at 15K rpm. The flow through was collected and diluted with an equal volume of 70% ethanol. The mixture was transferred to a RNeasy column and centrifuged for 15 seconds at 10K rpm until all sample was processed through the column. The RNA bound to the column was washed with 700 ⁇ L RW1 followed by a wash with 500 ⁇ L RPE and subsequently dried. The purified RNA was eluted in 50 ⁇ L RNASE free water by centrifugation for 1 minute at 10K rpm.
- RNA 5 ⁇ g of the above prepared total RNA was incubated at 70°C for 2 minutes and then applied to 20 ⁇ L of magnetic beads in binding buffer (20 mM Tris-HCl (pH 7.5), 1.0 M LiCl, 2 mM EDTA) where the beads were prepared by washing with 50 ⁇ L binding buffer. The RNA and bead mixture were incubated at room temperature for 5 minutes with constant rotating. The supernatant containing unbound material was removed and the beads were washed with 100 ⁇ L washing buffer (10 mM Tris-HCl (pH 7.5), 0.15 M LiCl, 1 mM EDTA) followed by the addition of 40 ⁇ L nuclease free water.
- binding buffer 20 mM Tris-HCl (pH 7.5), 1.0 M LiCl, 2 mM EDTA
- cDNA Complementary DNA
- 60 mM Tris HC1 pH 8.3
- 90 mM KC1 4 mM MgCl 2 , 12 mM DTT
- 240 Units RNasin Promega, Madison, WI
- 2400 Units Superscript II Invitrogen, Carlsbad, CA
- 0.6 mM dATP 0.6 mM dCTP
- 0.6 mM dGTP 0.6 mM dGTP
- 0.6 mM dTTP 2400 Units Superscript II
- the reaction was incubated at 42°C for 90 minutes with constant rotation. The supernatant was removed from the magnetic beads.
- the beads were then washed with 50 ul 50 mM potassium acetate, 20 mM Tris-Acetate pH 7.9, 1 mM DTT, 10 mM magnesium acetate and resuspended in 220 ⁇ L 50 mM potassium acetate, 20 mM Tris-Acetate pH 7.9, 1 mM DTT, 10 mM magnesium acetate, 2.5 mM CoCl 2 , 0.2 mM dGTP and 44 Units terminal transferase (New England BioLabs). The reaction mixture was incubated for 40 minutes at 37 °C.
- the S1C5 kappa light chain was PCR amplified with upstream primer C- anchor (Seq ID No: 1), which anneals to the poly-G leader and downstream primer 2228 (Seq ID No: 5), which anneals to the 3' end of the kappa light constant chain and incorporates an Avr II site downstream of the termination codon for subsequent cloning.
- the PCR reactions were amplified at 97°C for 1 minute, 30 cycles of 94°C for 30 seconds, 50°C for 1 minute, 72°C for 1 minute, and a final 7 minute incubation at 72°C.
- the PCR amplified product were electrophoresed on a 1% agarose gel with TAE and 0.5 ⁇ g/mL ethidium bromide.
- the 0.7 Kb band was excised and purified using the QIAquick gel extraction kit as previously described and eluted with 50 ⁇ L elution buffer.
- the amplified S1C5 kappa light chain fragment was cloned into pCR4-TOPO (Invitrogen) following the manufacturers directions to create plasmid pLSBC1757. Briefly, 1 ⁇ L of PCR product, 1 ⁇ L vector, 1 ⁇ L of salt solution and 3 ⁇ L of water were mixed, incubated at room temperature for 5 minutes.
- the ligation was placed on ice and 25 ⁇ L of chemically competent Top 10 cells was added to the ligation and the mix was incubated on ice for 10 minutes.
- the transformation reaction was heat shocked by incubating at 42°C for 30 seconds and immediately placed on ice and 250 ⁇ L of SOC was added.
- the transformation was allowed to recover by incubating at 37°C, 200 rpm shaking for 20 minutes.
- the transformation was plated out on LB plates containing ampicillin and grown overnight at 37°C. Individual colonies were used to inoculate 1.0 mL Super Broth (SB) containing 500 ⁇ g/mL ampicillin in 96 well 2.0 mL flat-bottom blocks and grown overnight at 37°C and 400 rpm.
- SB Super Broth
- Plasmid was purified from turbid cultures using the QIAprep 96 Turbo Miniprep kits (QIAGEN) as previously described. Clones were digested with EcoRl and screened for the presence of a 0.7 Kb insert band. The purified pLSBC1757 plasmid was subjected to nucleic acid sequencing using standard methods.
- the S1C5 heavy chain was PCR amplified with degenerate upstream primers 5'MH1 and 5'MH2 described by Wang et. al., J. of nm. Methods, 233: 167-177 (2000), and downstream primer 2227 (Seq ID No: 3), which anneals to the 3' end of the gamma constant chain and incorporates an Avr II site downstream of the termination codon for subsequent cloning.
- the PCR reactions were amplified at 94°C for 3 minutes, 30 cycles of 94°C for 1 minute, 45°C for 1 minute, 72°C for 2 minutes, and a final 10 minute incubation at 72°C.
- the PCR amplified product was electrophoresed on a 1% agarose gel with TAE and 0.5 ⁇ g/mL ethidium bromide.
- the 1.3 Kb band was excised and purified using the QIAquick gel extraction kit as previously described and eluted with 50 ⁇ L elution buffer.
- the amplified S1C5 heavy chain fragment was cloned into pCR2.1-TOPO (Invitrogen) following the manufacturers directions to create plasmid pLSBC2523 (Seq ID No: 117). Briefly, 1 ⁇ L of PCR product, 1 ⁇ L vector, 1 ⁇ L of salt solution and 3 ⁇ L of water were mixed, incubated at room temperature for 5 minutes.
- the ligation was placed on ice and 25 ⁇ L of chemically competent Top 10 cells was added to the ligation and the mix was incubated on ice for 10 minutes.
- the transformation reaction was heat shocked by incubating at 42°C for 30 seconds and immediately placed on ice and 250 ⁇ L of SOC was added.
- the transformation was allowed to recover by incubating at 37°C, 200 rpm shaking for 20 minutes.
- the transformation was plated out on LB plates containing ampicillin and grown overnight at 37°C. Individual colonies were used to inoculate 1.0 mL Super Broth (SB) containing 500 ⁇ g/mL ampicillin in 96 well 2.0 mL flat-bottom blocks and grown overnight at 37°C and 400 rpm.
- SB Super Broth
- Plasmid was purified from turbid cultures using the QIAprep 96 Turbo JMiniprep kits (QIAGEN) as previously described. Clones were digested with EcoRl and screened for the presence of a 1.3 Kb insert band. The purified pLSBC2523 plasmid was subjected to nucleic acid sequencing using standard methods.
- the S1C5 heavy chain Fd (V H C H 1) region was amplified with primers 7660 (Seq ID No: 8) and 7662 (Seq ID No: 9) from plasmid pLSBC2523 for Fab proprotein cloning.
- the 7660 upstream primer anneals to the 5' end of the S1C5 VH region and fuses it in frame to the 3' end of the JKP6 propeptide coding sequence and the 7662 downstream primer anneals to the 3' end of the C H 1 domain including a translational termination codon followed by an Avr II site for subsequent cloning.
- the amplification of the desired approximately 700 bp S1C5 kappa light chain and 700 bp S1C5 Fd region was confirmed by agarose gel electrophoresis.
- the amplified S1C5 kappa light chain, the pLSBC1731 KP6 PCR fragment, and the amplified S1C5 Fd fragment were fused by sequence overlap extension (SOE).
- the PCR reaction was amplified at 97°C for 1 minute, 15 cycles of 94°C for 30 seconds, 55°C for 2 minutes, 72°C for 90 seconds, and a final step of 72°C for 5 minutes.
- the PCR amplified product was electrophoresed on a 1% agarose gel with TAE and 0.5 ⁇ g/mL ethidium bromide.
- the 1.4 Kb band was excised and purified using the QIAquick gel extraction kit as previously described and eluted with 50 ⁇ L elution buffer.
- the amplified S1C5 Fab proprotein encoding sequence was cloned into pCR4-TOPO (Invitrogen) following the manufacturers directions to create plasmid pLSBC1786.
- Plasmid pLSBC1786 was subjected to restriction endonuclease digestion with NgoMIV.
- a 50 ⁇ L reaction containing 5 ⁇ L plasmid DNA, 50 mM potassium acetate, 20 mM Tris-Acetate pH 7.9, 1 mM DTT, 10 mM magnesium acetate, 20 Units NgoMIV was incubated at 37°C for 2.5 hours and electrophoresed on a 1.0% agarose gel with TAE and 0.5 ⁇ g mL ethidium bromide to separate the approximately 3.6 Kb S1C5 Fab proprotein encoding sequence.
- the 3.6 Kb Fab proprotein encoding sequence was purified using the QIAquick gel extraction kit (Qiagen) ollowing manufacturers recommendations and eluted with 50 ⁇ L EB Buffer. The purified fragment was subjected to restriction endonuclease digestion with Avr ⁇ .
- a 60 ⁇ L reaction containing 50 ⁇ L purified fragment, 50 mM NaCl, 100 mM Tris-HCl pH 7.9, 10 mM MgCl 2 , 1 mM DTT, 12 Units Avr II was incubated at 37°C for 35 minutes and electrophoresed on a 1.0% agarose gel with TAE and 0.5 ⁇ g/mL ethidium bromide to separate the approximately 1.5 Kb S1C5 Fab proprotein encoding sequence.
- the 1.5 Kb Fab proprotein encoding sequence was purified using the QIAquick gel extraction kit (Qiagen) following manufacturers recommendations and eluted with 50 ⁇ L EB Buffer. The presence of the approximately 1.5 Kb NgoJMIV and Avr JJTI purified fragment of pLSBC1786 was verified by gel electrophoresis.
- Bacterial transformations with DH5 ⁇ competent cells (Invitrogen) were performed according to manufacturer recommendations. Cells were plated on LB plates containing 100 ⁇ g/mL ampicillin and grown overnight at 37°C.
- a S 1C5 monoclonal antibody artificial proprotein was assembled by fusing the pLSBC1757 S1C5 kappa light chain to the KP6 propeptide region of pLSBC1731, which was fused to the S1C5 gamma heavy chain of pLSBC2523. This fusion will result in a first domain light chain, the second domain propeptide and the third domain the complete heavy chain sequence to create pLSBC1798.
- the S1C5 kappa light chain was PCR amplified from plasmid pLSBC1757 with upstream primer 7659 and downstream primer 6057 (Seq JED No: 6).
- the S1C5 gamma heavy chain was PCR amplified from plasmid pLSBC2523 with upstream primer 7660 and downstream primer 2227. Separate 100 ⁇ L PCR reactions containing 1 ⁇ M upstream , 1 ⁇ M downstream, IX Expand High Fidelity Buffer with MgCl 2 , 0.2 mM dATP, 0.2 mM dCTP, 0.2 mM dGTP, 0.2 mM dTTP, 3.5 Units Expand JHigh Fidelity and l ⁇ L plasmid template were amplified at 94°C for 1 minute, 30 cycles of 94°C for 30 seconds, 55°C for 30 seconds, 72°C for 1 minute , and a final step of 7 minutes at 72°C.
- the PCR reaction was purified using the MinElute PCR purification kit (Qiagen) following the manufacturers instructions. Briefly, 5 volumes of PB buffer was added to the reaction, mixed, applied to the column and centrifuged at 14K rpm for 1 minute. The column was washed with 750 ⁇ L Buffer PE and the purified fragment eluted in 10 ⁇ L EB.
- a 50 ⁇ L reaction containing 5 ⁇ L purified PCR product, 50 mM potassium acetate, 20 mM Tris-Acetate pH 7.9, 1 mM DTT, 10 mM magnesium acetate, 20 Units NgoMIV and 8 Units Avr DJ was incubated at 37°C for 1 hour and electrophoresed on a 1.0% agarose gel with TAE and 0.5 ⁇ g/mL ethidium bromide to separate the approximately 2.3 Kb S1C5 MAb proprotein encoding sequence.
- the 2.3 Kb MAb proprotein encoding sequence was purified using the QIAquick gel extraction kit (Qiagen) following manufacturers recommendations and eluted with 50 ⁇ L EB Buffer.
- Clones of pLSBC1798 were confirmed to contain the 2.3 Kb insert and the 9.7 Kb vector fragments by restriction enzyme mapping with NgoMJTV and Avr II followed by agarose gel electrophoresis.
- the S1C5 MAb proprotein was sequenced using standard methods to verify the sequence.
- Infectious transcripts were synthesized in-vitro from pLSBC1799 (9E10) and pLSBC1798 (S1C5) clones using the mMessage mMachine T7 kit (Ambion, Austin, TX) following the manufacturers directions. Briefly, a 324 ⁇ L reaction for each plasmid containing 32 ⁇ L 10X Reaction buffer, 162 ⁇ L 2X NTP/CAP mix, 32 ⁇ L Enzyme mix and 5 ⁇ g plasmid was incubated at 37°C for 2 hours.
- the synthesized transcripts were encapsidated in a 7 mL reaction containing 0.1 M Na 2 HPO 4 -NaH 2 PO (pH 7.0), 0.5 mg/mL purified Ul coat protein (LSBC, Vacaville, CA) which was incubated overnight at room temperature. 18 mL of FES (0.1 M Glycine, 60 mM K 2 HPO 4 , 22 M Na 2 P 2 O 7 , 10 g/L Bentonite, 10 g/L Celite 545) was added to each encapsidated transcript.
- FES 0.1 M Glycine, 60 mM K 2 HPO 4 , 22 M Na 2 P 2 O 7 , 10 g/L Bentonite, 10 g/L Celite 545
- the encapsidated transcript from an each individual clone was used to inoculate 25 day post sow Nicotiana benthamiana expressing the TMV 30K movement protein driven by the CaMV 35S promoter and containing the NOS terminator as a transgene was made by standard transformation techniques. JHigh levels of subgenomic RNA species were synthesized in virus-infected plant cells (Kumagai, JMH. et al. (1993) Proc. Natl. Acad. Sci. USA 90:427-430), and serve as templates for the translation and subsequent accumulation of MAb protein.
- Interstitial fluid from infected leaves of each plant was harvested 8 days post inoculation. Systemically infected upper leaves from each of the infected plants was harvested. The secreted protein fraction, or interstitial fluid (JEF) was extracted and analyzed for presence of recombinant protein.
- the leaf tissue was covered with 50 mM Tris-HCl (pH 7.3), 50 mM NaCl, 2 mM EDTA and subjected to 760 mmHg vacuum for 2 minutes. The vacuum is released and re-applied three times to completely infiltrate the tissue with buffer. The IF fraction was recovered by centrifugation for 20 minutes at 4K rpm.
- the recovered IF was adjusted to 1 mM PMSF and clarified by centrifugation at 6K rpm for 10 minutes. The supernatant was adjusted to pH 7.5 and 150 mM NaCl, and loaded onto Protein A HiTrap (Amersham Pharmacia) column equilibrated with 150 mM Tris-HCl (pH 7.3), 50 mM NaCl. Bound MAb was eluted with 100 mM Glycine-HCl, pH 3.0 and JMAb containing fractions were concentrated approximately 10-fold in Microcon- 10 (Amicon) concentrators and diafiltered with phosphate buffered saline (PBS).
- PBS phosphate buffered saline
- Infectious transcripts were synthesized in-vitro from pLSBC1736 (9E10) and pLSBC1792 (S1C5) clones using the mMessage mMachine T7 kit (Ambion, Austin, TX) following the manufacturers directions. Briefly, a 100 ⁇ L reaction for each plasmid containing 10 ⁇ L 10X Reaction buffer, 50 ⁇ L 2X NTP/CAP mix, 10 ⁇ L Enzyme mix and 1.4 ⁇ g plasmid was incubated at 37°C for 2 hours.
- the synthesized transcripts were encapsidated in a 2 mL reaction containing 0.1 M Na 2 HPO -NaH 2 PO 4 (pH 7.0), 0.5 mg/mL purified Ul coat protein (LSBC, Vacaville, CA) which was incubated overnight at room temperature. 5 mL of JFES (0.1 M Glycine, 60 mM K 2 HPO 4 , 22 mM Na 2 P 2 O 7 , 10 g/L Bentonite, 10 g L Celite 545) was added to each encapsidated transcript. The encapsidated transcript from each individual clone was used to inoculate 26 day post sow Nicotiana benthamiana.
- RNA species were synthesized in virus-infected plant cells (Kumagai, MH. et al. (1993) Proc. Natl. Acad. Sci. USA 90:427-430), and serve as templates for the translation and subsequent accumulation of MAb protein.
- interstitial fluid from infected leaves of each plant was harvested 12 days post inoculation. Systemically infected upper leaves from each of the infected plants was harvested. The secreted protein fraction, or interstitial fluid (IF) was extracted and analyzed for presence of recombinant protein.
- the leaf tissue was covered with 50 mM Tris-HCl (pH 7.3), 50 mM NaCl, 2 mM EDTA and subjected to 760 mmHg vacuum for 2 minutes. The vacuum is released and re-applied three times to completely infiltrate the tissue with buffer. The IF fraction was recovered by centrifugation for 20 minutes at 4K rpm.
- the recovered JfF was adjusted to 1 mM PMSF and clarified by centrifugation at 6K rpm for 10 minutes. The supernatant was adjusted to pH 5.2 and then concentrated using a 10 kDa membrane and diafiltered prior to loading on a SP Sepharose JFF (Amersham Pharmacia) column, equilibrated with 25 mM JJtmidazole Buffer, pH 6.0. Bound Fab protein was eluted using a linear gradient of 250-500 mM NaCl in 25 mM Imidazole Buffer, pH 6.0. Eluted fractions were pooled and dialyzed with 10 mM KPO 4 Buffer, pH 6.0 and loaded onto Hydroxyapatite Type I resin (BioRad).
- Bound protein was eluted using a linear gradient of 10-200 mM KPO 4 Buffer, pH 6.0 and flow through fractions containing purified Fab were pooled together, concentrated and diafiltered into Phosphate Buffered Saline (PBS), pH 7.4.
- PBS Phosphate Buffered Saline
- interstitial fluid from infected leaves of each plant was harvested 12 days post inoculation. Systemically infected upper leaves from each of the infected plants was harvested. The secreted protein fraction, or interstitial fluid (JJDF) was extracted and analyzed for presence of recombinant protein.
- the leaf tissue was covered with 50 mM Tris-HCl (pH 7.3), 50 mM NaCl, 2 mM EDTA and subjected to 760 mmHg vacuum for 2 minutes. The vacuum is released and re-applied three times to completely infiltrate the tissue with buffer. The IF fraction was recovered by centrifugation for 20 minutes at 4K rpm.
- the recovered IF was adjusted to 1 mM PMSF and clarified by centrifugation at 6K rpm for 10 minutes. The supernatant was adjusted to pH 5.2 and then concentrated using a 10 kDa membrane and diafiltered prior to loading on a SP Sepharose JFF (Amersham Pharmacia) column, equilibrated with 25 mM Imidazole Buffer, pH 6.0. Bound Fab protein was eluted using a linear gradient of 250-500 mM NaCl in 25 mM Imidazole Buffer, pH 6.0.
- Bound protein was eluted using a linear gradient of 10-200 mM JKPO Buffer, pH 6.0 and flow through fractions containing purified Fab were pooled together, concentrated and diafiltered into Phosphate Buffered Saline (PBS), pH 7.4.
- PBS Phosphate Buffered Saline
- the first membrane was probed with a 1:4000 dilution of Goat anti-mouse kappa-HRP labeled sera and the second membrane was probed with 1:4000 dilution of Goat anti-mouse IgG-HRP labeled sera (Southern Biotechnology, Birmingham, AL) for 1 hour at room temperature.
- the blots were washed three times in TBST and the labeled proteins detected with the ECL+plus Western Blotting Detection System (Amersham Biosciences, Buckinghamshire, England).
- the anti kappa sera detected an approximately 150 JKD band under non-reducing conditions indicating the presence of assembled 9E10 MAb protein containing interchain disulfide bridges.
- the anti gamma sera detected an approximately 150 JKD band under non-reducing conditions indicating the presence of assembled 9E10 MAb protein containing interchain disulfide bridges.
- the antigen was the fusion protein from ⁇ LSBC2268 (Seq ED No: 95), which contains the c-myc epitope fused to the amino terminus of TMN-U1 coat protein.
- the plates were blocked with 2.5% BSA in IX TBST buffer for 1 hour at room temperature. Duplicate samples were tested for the MAb dilutions and Fab dilutions, which were added to the plates and incubated for an hour at room temperature. Plates were washed with TBST, and bound antibody detected with goat anti-mouse kappa JHRP (Southern Biotech). Samples were detected with Turbo-TMB ELISA, 1-STEP (Pierce) and the reaction was stopped with the addition of IN H 2 SO following manufacturers instructions.
- the ELISA assay demonstrates the LSBC 1736 Fab and LSBC 1799 MAb recognize and bind to the c-myc antigen, and this activity is comparable to the hybridoma produced control MAb.
- the first membrane was probed with a 1:4000 dilution of Goat anti-mouse kappa-HRP labeled sera and the second membrane was probed with 1:4000 dilution of Goat anti-mouse IgG-HRP labeled sera (Southern Biotechnology, Birmingham, AL) for 1 hour at room temperature.
- the blots were washed three times in TBST and the labeled proteins detected with the ECL+plus Western Blotting Detection System (Amersham Biosciences, Buckinghamshire, England).
- the anti kappa sera detected an approximately 25 KDa proteins and a corresponding approximately 50 KDa protein was detected with the anti gamma sera indicating that both the kappa light and gamma heavy chains were expressed, processed and secreted.
- the first membrane was probed with a 1:4000 dilution of Goat anti-mouse kappa-HRP labeled sera and the second membrane was probed with 1:4000 dilution of Goat anti-mouse IgG-HRP labeled sera (Southern Biotechnology, Birmingham, AL) for 1 hour at room temperature.
- the blots were washed three times in TBST and the labeled proteins detected with the ECL+plus Western Blotting Detection System (Amersham Biosciences, Buckinghamshire, England).
- the anti kappa sera detected an approximately 150 KD band under non-reducing conditions indicating the presence of assembled S1C5 MAb protein containing interchain disulfide bridges.
- the anti gamma sera detected an approximately 150 KD band under non-reducing conditions indicating the presence of assembled S1C5 MAb protein containing interchain disulfide bridges.
- the ELISA assay demonstrates the LSBC1792 Fab and LSBC1798 MAb recognize and bind to the 38C13 antigen, and this activity is comparable to the ascites produced control Fab and MAb.
- the murine monoclonal antibody mumAb4D5 is directed against the extracellular domain of HER-2/neu gene product pl85 HER2 and it specifically inhibits the growth of cells of the breast cancer cell line SK-BR-3 (ATCC HTB 30) in 6 day culture. Such treatment sensitizes these cells to chemotherapeutic agents (US 5,677,171). The process of Example 4 is repeated using the Ig heavy Fd region and the light chain of the mumAb4D5. The variable gene sequences of the immunoglobulin coding sequences are described in Carter et al, PNAS 9:4285-4289 (1992). [000259] Mouse hybridoma line A-JHER2 expressing murine monoclonal antibody
- the cell lysate was applied to the QIAshredder column and spun in a centrifuge for 2 minutes at 14K rpm. The flow through was collected and diluted with an equal volume of 70% ethanol. The mixture was transferred to a RNeasy column and centrifuged for 15 seconds at 10K rpm until all sample was processed through the column. The RNA bound to the column was washed with 700 ⁇ L RW1 followed by a wash with 500 ⁇ L RPE and subsequently dried. The purified RNA was eluted in 50 ⁇ L RNASE free water by centrifugation for 1 minute at 10K rpm.
- RNA 4 ⁇ g of the above prepared total RNA was incubated at 65°C for 2 minutes, immediately placed on ice for 3 minutes, and then applied to 20 ⁇ L of magnetic beads in binding buffer (20 mM Tris-HCl (pH 7.5), 1.0 M LiCl, 2 mM EDTA) where the beads were prepared by washing with 50 ⁇ L of binding buffer. The RNA and bead mixture were incubated for 5 minutes with constant rotating. The supernatant containing unbound material was removed and the beads were washed with 100 ⁇ L washing buffer (10 mM Tris-HCl (pH 7.5), 0.15 M LiCl, 1 mM EDTA) followed by the addition of 40 ⁇ L nuclease free water.
- binding buffer 20 mM Tris-HCl (pH 7.5), 1.0 M LiCl, 2 mM EDTA
- cDNA was synthesized in 60 ⁇ L reactions containing 50 mM Tris HC1 (pH 8.3), 75 mM KC1, 3 mM MgCl 2 , 10 mM DTT, 2 Units RNasin (Promega, Madison, WI), 20 Units Superscript II (Invitrogen, Carlsbad, CA), 0.5 mM dATP, 0.5 mM dCTP, 0.5 mM dGTP, 0.5 mM dTTP, and the oligo dT bound RNA from above.
- the cDNA reaction was incubated at 42°C for 60 minutes with constant rotation.
- Heavy chain Fd genes were PCR amplified using a gene specific upstream primer which anneals to the 5 'end of the framework 1 region (FRl) of heavy chain gene 4D5 HySph5' (Seq ID No: 42) and a C H 1 specific 3' downstream primer 4D5 Hy Avr3' (Seq JTD No: 52).
- the kappa light chain gene was PCR amplified in a separate reaction with a gene specific upstream primer which anneals to the 5 'end of the framework 1 region (FRl) of kappa light chain gene 4D5 LtSph5' (Seq ID No: 43) and a C L specific 3' downstream primer 4D5 Lt Avr3' (Seq JfD No: 53).
- FRl framework 1 region
- PCR reactions contained IX Expand High Fidelity buffer with MgCl 2 , 0.2 mM dATP, 0.2 mM dCTP, 0.2 mM dGTP, 0.2 mM dTTP, 3.5 Units Expand JHigh Fidelity Polymerase, 0.4 ⁇ M upstream primer, 0.4 ⁇ M downstream primer and 2 ⁇ L prepared cDNA. PCR reactions were amplified at 97°C for 1 minute, 30 cycles of 94°C for 30 seconds, 48°C for 30 seconds, 72°C for 30 seconds, and 5 minutes at 72°C.
- the amplification of the desired approximately 700 bp kappa light chain and the approximately 700 bp gamma Fd region were confirmed by agarose gel electrophoresis.
- the above PCR reactions were precipitated with 3 volumes ethanol and 0.3 volumes 10M NHUAcetate, spun and washed with 70% ethanol. The pellets were resuspended in 20 ⁇ L 10 mM Tris-HCL (pH 8.0).
- the transformations were allowed to recover by incubating at 37°C, 200 rpm shaking for 20 minutes.
- the transformations were plated out on LB plates containing ampicillin and grown overnight at 37°C. Individual colonies were used to inoculate 1.0 mL Super Broth (SB) containing 100 ⁇ g/mL ampicillin in 96 well 2.0 mL flat-bottom blocks and grown overnight at 37°C and 400 rpm.
- Plasmid was purified from turbid cultures using the QIAprep 96 Turbo JMiniprep kits (QIAGEN, Valencia, CA). Briefly, the cells were pelleted by centrifugation at 3 K rpm for 15 minutes in a plate centrifuge.
- the supernatant was drained from the cell pellets and the cells resuspended in 250 ⁇ L PI Buffer by vortexing.
- 250 ⁇ L of P2 was added to the cells, mixed by inverting and incubated for 5 minutes to lyse the cells.
- 350 ⁇ L of N3 was added to the cell lysates, mixed by inverting and transferred to the Turbo Filter plate.
- a vacuum was applied to the Turbo Filter which filtered the sample into the QIAprep plate.
- a vacuum was then applied to the QIAprep plate pulling the sample through the plate and bound the plasmid to the plate membrane.
- the QIAprep plate was washed using vacuum force with 0.9 mL of PB, followed by two washes with 0.9 mL of PE and vacuum dried. 100 ⁇ L EB buffer was added to the purified plasmid, incubated for 1 minute, and subsequently centrifuged for 3 minute at 6K rpm to elute the purified plasmid. The presence of the approximately 700 bp insert for each plasmid was verified with Sph I and Avr TI restriction digest and agarose gel electrophoresis.
- the purified p4D5Hy-TOPO (Seq ID No: 81) and p4D5Lt-TOPO (Seq ID No: 83) plasmids were subjected to nucleic acid sequencing with the Ml 3 forward and reverse primers using standard methods to verify the mum4D5 Fd and kappa chain sequences.
- the JKP6 sequence of pLSBC 1731 was PCR amplified for Fab cloning.
- the PCR reaction was amplified at 97°C for 1 minute, 15 cycles of 94°C for 30 seconds, 55°C for 30 seconds, 72°C for 30 seconds, and 5 minutes at 72°C.
- the amplification of the desired approximately 120 bp KJP6 propeptide encoding sequence was confirmed by agarose gel electrophoresis.
- the 4D5 Fd region (VHC H I) was PCR amplified from plasmid p4D5HyFd with upstream primer 4D5 HySph5' which contains a Sph I site compatible for cloning into vector pl324-MBP which contains the alpha-amylase signal peptide and downstream primer 4D5HyKp63' (Seq ID No: 44) which contains sequence coding for the 3' end of the C H I fused to the 5' end of the JKP6 propeptide sequence amplified from pLSBC1731.
- pl324-MBP a modified 30B vector (Shivprasad, S. et al.
- a TJMV coat protein subgenomic promoter is located upstream of the insertion site of the 4D5 Fab proprotein sequence. Following infection, this TMV coat protein subgenomic promoter directs initiation of the 4D5 Fab proprotein RNA synthesis in plant cells at the transcription start point ("tsp").
- the rice amylase signal peptide (O'Neill, SD et al. (1990) Mol. Gen. Genet.
- 4D5 Fab proprotein sequence encodes a 31 residue polypeptide which targets proteins to the secretory pathway (Firek, S. et al. (1994) Transgenic Res. 3:326-33 1), and is subsequently cleaved off between the C-terminal Gly of the signal peptide and the N-terminal Met of the expressed 4D5 Fab proprotein.
- the sequence encoding 4D5 Fab proprotein has been introduced between the 30K movement protein and the TMGMV-U5 coat protein (Tcp) genes.
- Tcp TMGMV-U5 coat protein
- the Sph I site joins the signal peptide to the FRl of the 4D5 variable region of the Fd and directs the secretion of the artificial proprotein to the ER.
- the 4D5 light chain (V L C L ) was PCR amplified from the plasmid p4D5Lt with downstream primer 4D5Lavstp3' (Seq JTD No: 49) which contains a translation termination codon at the 3' end of the C L coding sequence followed by an Avr II site compatible for cloning into vector pl324-JMBP and upstream primer 4D5LtKp65' (Seq ID No: 45) which contains sequence coding for the 3' end of the JKP6 propeptide sequence amplified from pLSBC1731 fused to the 5' end of the FRl region of the V coding sequence.
- the 4D5 Fd and light chain regions were PCR amplified in separate 25 ⁇ L PCR reactions containing 0.8 ⁇ M upstream primer, 0.8 ⁇ M downstream, IX Expand High Fidelity Buffer with MgCl 2 , 0.2 mM dATP, 0.2 mM dCTP, 0.2 mM dGTP, 0.2 mM dTTP, 1.8 Units Expand High Fidelity Polymerase and 0.03 ⁇ L plasmid template.
- the PCR reaction was amplified at 97°C for 1 minute, 15 cycles of 94°C for 30 seconds, 55°C for 30 seconds, 72°C for 30 seconds, and 5 minutes at 72°C.
- the PCR reaction was amplified at 97°C for 1 minute, 15 cycles of 94°C for 30 seconds, 55°C for 2 minutes, 72°C for 30 seconds, and 5 minutes at 72°C.
- the amplification of the desired approximately 1.4 Kb 4D5 Fab proprotein was verified by agarose gel electrophoresis.
- a phenol chloroform extraction series was performed on the PCR amplified product to remove the thermostable polymerase prior to restriction digestion.
- 5 ⁇ L of the prepared fragment was digested with Sph I and Avr H in a 25 uL reaction containing 2.5 Units Sph 1, 2 Units Avr H, 50 mM NaCl, 10 mM Tris-HCl (pH 7.9), 10 mM MgCl 2 , 1 mM DTT.
- the digest was incubated at 37°C for 2 hours, and electrophoresed on a 1.0% agarose gel to separate the approximately 1.4 Kb fragment.
- the nucleic acids were stained with GelStar (Cambrex Bio Science) and the approximately 1.4 Kb fragment was isolated.
- the fragment was purified away from the agarose using QIAquick gel extraction kit following the manufacturers instructions. The recovery of the Sph I/Avr JTI digested fragment was verified by gel electrophoresis. The 1.4 Kb Sph I and Avr II 4D5 Fab proprotein was cloned into the Sphl and Avr II prepared pl324-MBP plasmid to create pLSBC1740 (Seq JfD No: 71).
- the 4D5 Fab proprotein was sequenced using standard methods to verify the sequence.
- JTD No: 71 JTD No: 71 clone using the mMessage mMachine T7 kit (Ambion, Austin, TX) following the manufacturers directions. Briefly, a 5.5 ⁇ L reaction containing 1 ⁇ L 10X Reaction buffer, 2.5 ⁇ L 2X NTP/CAP mix, 1 ⁇ L Enzyme mix and 3.5 ⁇ L plasmid was incubated at 37°C for 2 hours. The synthesized transcripts were encapsidated in a 40 ⁇ L reaction containing 0.1 M Na 2 HPO 4 -NaH 2 PO 4 (pH 7.0), 0.5 mg/mL purified Ul coat protein (LSBC, Vacaville, CA) which was incubated overnight at room temperature.
- mMessage mMachine T7 kit Anambion, Austin, TX
- encapsidated transcript 40 ⁇ L of FES (0.1 M Glycine, 60 mM K 2 HPO 4 , 22 mM Na 2 P 2 O 7 , 10 g/L Bentonite, 10 g/L Celite 545) was added to each encapsidated transcript.
- the encapsidated transcript from an each individual clone was used to inoculate a 19 day post sow Nicotiana benthamiana plant (Dawson, WO et al. (1986) Proc. Natl Acad Sci. USA 83:1832-1836).
- High levels of subgenomic RNA species were synthesized in virus-infected plant cells (Kumagai, MH. et al. (1993) Proc. Natl. Acad. Sci. USA 90:427-430), and serve as templates for the translation and subsequent accumulation of Fab protein.
- Interstitial fluid from infected leaves of each plant was harvested 9 days post inoculation and screened by ELISA. Systemically infected upper leaves from individual plants were harvested. The secreted protein fraction, or interstitial fluid (IF) was extracted and analyzed for presence of recombinant protein.
- the leaf tissue was placed in a GF/B 0.8 mL Unifilter (Whatman, Clifton, NJ), covered with 20 mM Tris-HCl (pH 7.0) and subjected to 760 mmHg vacuum for 30 seconds. The vacuum is released and re-applied three times to completely infiltrate the tissue with buffer.
- the residual buffer is discarded and the tissue dried by centrifugation at 400 rpm in a plate centrifuge for 30 seconds.
- the IF fraction is recovered into a 96-well microplate by centrifugation for 10 minutes at 3K rpm in a plate centrifuge. Each sample was analyzed by ELISA in triplicate. 6 ⁇ L of IF is adjusted to 50 mM Na 2 CO 3 pH 9.6 in 100 ⁇ L and applied to a 96 well plate (Maxisorb, Nunc) and incubated overnight at 4°C. Plates were blocked with 200 ⁇ L of 1% BSA in PBS for 30 minutes at 37°C followed by washing four times with 150 mM NaCl, 0.05% Trition X-100.
- Kb alpha-amylase signal peptide, 4D5 Fab proprotein including the viral 3' end A 50 ⁇ L reaction containing 2 ⁇ L of plasmid, 10 Units Pac 1, 10 Units Kpn 1, 10 mM Bis-Tris Propane-HCl (pH 7.0), 10 mM MgCl 2 , 1 mM DTT.
- the digest was incubated at 37°C for 2 hours, and electrophoresed on a 1.0% agarose gel to separate the approximately 2.7 Kb fragment.
- the nucleic acids were stained with GelStar (Cambrex Bio Science) and the approximately 2.7 Kb fragment was isolated. The fragment was purified away from the agarose using QIAquick gel extraction kit following the manufacturers instructions.
- Bacterial transformation was performed with a Gene Pulser electroporator (BioRad, Hercules, CA) following manufacturer recommendations with 40 ⁇ L of electro-competent JM109 cells as previously described. Cells were plated on LB plates containing 50 ⁇ g/mL ampicillin and grown overnight at 37°C. Individual colonies were picked and used to inoculate 1 mL Super Broth (SB) containing 500 ⁇ g/mL ampicillin in 96 well 2.0 mL flat-bottom blocks and grown overnight at 37°C and 400 rpm.
- SB Super Broth
- Plasmid was purified from turbid cultures using the QIAprep 96 Turbo Miniprep kits (QIAGEN, Valencia, CA) as previously described and plasmid eluted with 100 ⁇ L EB buffer. The presence of a 1.4 Kb insert was verified by restriction mapping with Sph I and Avr JH followed by agarose gel electrophoresis.
- Infectious transcripts were synthesized in-vitro from 300 ng template plasmid in an 11 ⁇ L reaction using the mMessage mMachine T7 kit (Ambion, Austin, TX) and the transcripts were encapsidated with purified Ul coat protein as above. Transcripts were used to inoculate and systemically infect 20 day old Nicotiana benthamiana plants and the IF protein fraction was isolated at 8 and 11 days post inoculation by vacuum infiltration and centrifugation as previously described.
- V L C L The 4D5 light chain (V L C L ) was PCR amplified from plasmid p4D5Lt with upstream primer 4D5LtSphI5' which contains a Sph I site compatible for cloning into vector pl324-JMBP which contains the alpha-amylase signal peptide and downstream primer 4D5LtKp63' (Seq ID No: 46) which contains sequence coding for the 3' end of the C L fused to the 5' end of the JKP6 propeptide sequence amplified from pLSBC1731.
- the Sph I site joins the signal peptide to the FRl of the 4D5 variable region of the light chain and directs the secretion of the artificial proprotein to the ER.
- the 4D5 Fd heavy chain (V H C H I) was PCR amplified from the plasmid p4D5HyFd with downstream primer 4D5Havstp3' which contains a translation termination codon at the 3' end of the C H I coding sequence followed by an Avr JDJ site compatible for cloning into vector pl324-MBP and upstream primer 4D5HyKp65' (Seq ID No: 47) which contains sequence coding for the 3' end of the KP6 propeptide sequence amplified from pLSBC1731 fused to the 5' end of the FRl region of the N H coding sequence.
- the 4D5 Fd and light chain regions were PCR amplified in separate 25 ⁇ L PCR reactions containing 0.8 ⁇ M upstream primer, 0.8 ⁇ M downstream, IX Expand High Fidelity Buffer with MgCl 2 , 0.2 mM dATP, 0.2 mM dCTP, 0.2 mM dGTP, 0.2 mM dTTP, 1.8 Units Expand JHigh Fidelity Polymerase and 0.03 ⁇ L plasmid template.
- the PCR reaction was amplified at 97°C for 1 minute, 15 cycles of 94°C for 30 seconds, 55°C for 30 seconds, 72°C for 30 seconds, and 5 minutes at 72°C.
- the PCR reaction was amplified at 97°C for 1 minute, 15 cycles of 94°C for 30 seconds, 55°C for 2 minutes, 72°C for 30 seconds, and 5 minutes at 72°C.
- the amplification of the desired approximately 1.4 Kb 4D5 Fab proprotein was verified by agarose gel electrophoresis.
- a phenol chloroform extraction series was performed on the PCR amplified product to remove the thermostable polymerase prior to restriction digestion.
- 5 ⁇ L of the prepared fragment was digested with Sph I and Avr II in a 25 uL reaction containing 2.5 Units Sph I, 2 Units Avr II, 50 mM NaCl, 10 mM Tris-HCl (pH 7.9), 10 mM MgCl 2 , 1 mM DTT.
- the digest was incubated at 37°C for 2 hours, and electrophoresed on a 1.0% agarose gel to separate the approximately 1.4 Kb fragment.
- the nucleic acids were stained with GelStar (Cambrex Bio Science) and the approximately 1.4 Kb fragment was isolated.
- the fragment was purified away from the agarose using QIAquick gel extraction kit following the manufacturers instructions. The recovery of the Sph I/Avr II digested fragment was verified by gel electrophoresis. The 1.4 Kb Sph I and Avr II 4D5 Fab proprotein was cloned into the Sphl and Avr II prepared pl324-JMBP plasmid to create pLSBC1741 (Seq JTD No: 73).
- Bacterial transformation was performed with a Gene Pulser electroporator (BioRad, Hercules, CA) following manufacturer recommendations with 40 ⁇ L of electro-competent JTM109 cells as previously described. Cells were plated on LB plates containing 50 ⁇ g/mL ampicillin and grown overnight at 37°C. Individual colonies were picked and used to inoculate 1 mL Super Broth (SB) containing 500 ⁇ g/mL ampicillin in 96 well 2.0 mL flat-bottom blocks and grown overnight at 37°C and 400 rpm.
- SB Super Broth
- Plasmid was purified from turbid cultures using the QIAprep 96 Turbo Miniprep kits (QIAGEN, Valencia, CA) as previously described and plasmid eluted with 100 ⁇ L EB buffer. Clones were confirmed to contain the 1.4 Kb insert and the 9.7 Kb vector fragments by restriction enzyme mapping with Sph I and Avr ⁇ followed by agarose gel electrophoresis. The 4D5 Fab proprotein was sequenced using standard methods to verify the sequence.
- Infectious transcripts were synthesized in-vitro from pLSBC1741 clones using the mMessage mMachine T7 kit (Ambion, Austin, TX) following the manufacturers directions. Briefly, a 5.5 ⁇ L reaction containing 1 ⁇ L 10X Reaction buffer, 2.5 ⁇ L 2X NTP/CAP mix, 1 ⁇ L Enzyme mix and 3.5 ⁇ L plasmid was incubated at 37°C for 2 hours.
- the synthesized transcripts were encapsidated in a 40 ⁇ L reaction containing 0.1 M Na 2 HPO 4 -NaH 2 PO 4 (pH 7.0), 0.5 mg mL purified Ul coat protein (LSBC, Vacaville, CA) which was incubated overnight at room temperature.
- 40 ⁇ L of FES 0.1 M Glycine, 60 mM K 2 HPO 4 , 22 mM Na 2 P 2 O 7 , 10 g/L Bentonite, 10 g/L Celite 545 was added to each encapsidated transcript.
- the encapsidated transcript from an each individual clone was used to inoculate a 19 day post sow Nicotiana benthamiana plant (Dawson, WO et al. (1986) Proc. Natl. Acad Sci. USA 83:1832-1836). High levels of subgenomic RNA species were synthesized in virus-infected plant cells (Kumagai, MH. et al. (1993) Proc. Natl. Acad. Sci. USA 90:427-430), and serve as templates for the translation and subsequent accumulation of Fab protein.
- Interstitial fluid from infected leaves of each plant was harvested 9 days post inoculation and screened by ELISA. Systemically infected upper leaves from individual plants were harvested. The secreted protein fraction, or interstitial fluid (IF) was extracted and analyzed for presence of recombinant protein.
- the leaf tissue was placed in a GF/B 0.8 mL Unifilter (Whatman, Clifton, NJ), covered with 20 mM Tris-HCl (pH 7.0) and subjected to 760 mmHg vacuum for 30 seconds. The vacuum is released and re-applied three times to completely infiltrate the tissue with buffer.
- the residual buffer is discarded and the tissue dried by centrifugation at 400 rpm in a plate centrifuge for 30 seconds.
- the JTF fraction was recovered into a 96-well microplate by centrifugation for 10 minutes at 3K rpm in a plate centrifuge. Each sample was analyzed by ELISA in triplicate. 6 ⁇ L of JTF is adjusted to 50 mM Na 2 CO 3 pH 9.6 in 100 ⁇ L and applied to a 96 well plate (Maxisorb, Nunc) and incubated overnight at 4°C. Plates were blocked with 200 ⁇ L of 1%) BSA in PBS for 30 minutes at 37°C followed by washing four times with 150 mM NaCl, 0.05% Trition X-100.
- Plates were incubated with 100 ⁇ L of a 1:4000 dilution of goat anti-mouse kappa serum conjugated with horseradish peroxidase(Southern Biotechnology) in PBS and incubated for at room temperature for 1 hour. Plates were washed 4 times with PBST and incubated for 20 minutes at room temperature with 100 ⁇ L of Turbo-TMB ELISA, 1-STEP (Pierce). The reaction was stopped with the addition of 50 ⁇ L IN H 2 SO and read at 450 nm by an absorbance plate reader (Molecular Devices) and the data was analyzed with SoftMax software(Molecular Dynamics). Samples with a reading greater than 0.13 were further analyzed.
- the pLSBC1741 clone was digested with Pac I and Kpn I to isolate the 2.7 Kb alpha-amylase signal peptide, 4D5 Fab proprotein including the viral 3' end.
- the digest was incubated at 37°C for 2 hours, and electrophoresed on a 1.0% agarose gel to separate the approximately 2.7 Kb fragment.
- the nucleic acids were stained with GelStar (Cambrex Bio Science) and the approximately 2.7 Kb fragment was isolated. The fragment was purified away from the agarose using QIAquick gel extraction kit following the manufacturers instructions. The recovery of the Pac I and Kpn I digested fragment was verified by gel electrophoresis. The 2.7 Kb Pac I and Kpn I fragment of ⁇ LSBC1741 was cloned into the Pac I and Kpn I prepared pll77MP5 plasmid 8.0 Kb fragment to create pLSBC1767 (Seq ID No: 91).
- the ligation was ethanol precipitated and used to transform electrocompetent JM109 as previously described. Cells were plated on LB plates containing 50 ⁇ g/mL ampicillin and grown overnight at 37°C.
- Ampicillin resistant colonies were cultured in blocks and plasmid purified using the QIAprep 96 Turbo JMiniprep kits (QIAGEN, Valencia, CA) as above. The presence of a 1.4 Kb insert was verified by restriction mapping with Sph I and Avr II followed by agarose gel electrophoresis.
- Infectious transcripts were synthesized in-vitro from 300 ng template plasmid in an 11 ⁇ L reaction using the mMessage mMachine T7 kit (Ambion, Austin, TJX) and the transcripts were encapsidated with purified Ul coat protein as above. Transcripts were used to inoculate and systemically infect 20 day old Nicotiana benthamiana plants and the JDF protein fraction was isolated at 8 and 11 days post inoculation by vacuum infiltration and centrifugation as previously described.
- a 4D5 monoclonal antibody artificial proprotein was assembled by fusing the pLSBC1767 4D5 Fab proprotein to the murine gamma 1 immunoglobulin constant domains C H 2 and C H 3. This fusion will result in a first domain light chain, the second domain propeptide and the third domain the complete heavy chain sequence.
- the cloned murine IgGl heavy chain sequence was derived from the previously described p9E10Hy- TOPO clone.
- the murine IgGl constant domains genes are conserved within heavy chain genes of the same isotype, therefore the 9E10 CH2 and CH3 are expected to be the same for the 4D5 and 9E10 antibodies as they are both murine IgGl.
- Primers were designed to amplify the pLSBC1767 fragment using a 5696s (Seq ID No: 54) upstream primer which anneals to vector sequence and the 4D5fAb3' (Seq ID No: 55) downstream primer which anneals to the CHI region of pLSBC1767 and removes the translation termination signal.
- the 4D5fAb3' downstream primer is designed to anneal to the 3' end of the pLSBC1767 C H I region such that treatment with the 3' to 5' exonuclease activity of T4 DNA polymerase will result in a "GG" 5' extension where "G" is guanine.
- a 9E10Fc5' (Seq ID No: 56) upstream primer was designed which anneals to the 5' end of the C H 2 domain such that treatment with the 3' to 5' exonuclease activity of T4DNA polymerase will result in a "CC" 5' extension where "C” is cytosine.
- the 9E10Havr3' downstream primer anneals to the 3' end of the CH3 domain including a translational termination codon followed by an Avr II site for subsequent cloning.
- PCR reaction Separate 25 ⁇ L PCR reaction were set up to amplify the 4D5 Fab and the 9E10 C H 2C H 3 domain which contained 0.8 ⁇ M 5' primer, 0.8 ⁇ M 3'primer, IX Expand High Fidelity Buffer with MgCl 2 , 0.16 mM dATP, 0.16 mM dCTP, 0.16 mM dGTP, 0.16 mM dTTP, 1.8 Units Expand High Fidelity Polymerase and 0.03 ⁇ L plasmid template.
- the PCR reaction was amplified at 95°C for 2 minutes, 15 cycles of 95°C for 30 seconds, 55°C for 30 seconds, 72°C for 1 minute, and 7 minutes at 72°C.
- the amplification of the desired approximately 1.6 Kb 4D5 sequence and the approximately 500 bp 9E10 C H 2C H 3 were confirmed by agarose gel electrophoresis.
- the PCR amplified 1.6 Kb 4D5 sequence and the 500 bp 9E10 C H 2C H 3 were digested with Dpn I. 5 Units Dpn I was added to each PCR reaction and incubated at 37°C for 1 hour followed by 80°C for 20 minutes. A phenol chloroform extraction series was performed on the PCR amplified product to remove the thermostable polymerase and the fragment were ethanol precipitated as described earlier and resuspended in 20 ⁇ L 10 mM Tris-HCl pH 8.
- the purified PCR amplified fragments were ligated together in a 30 ⁇ L ligation reaction containing 6 ⁇ L 4D5 Fab PCR fragment, 2 ⁇ L 9E10 C H 2C H 3 PCR fragment, 50 mM Tris- HCl (pH 7.5), 10 mM MgCl 2 , 25 ⁇ g/mL BSA, 10 mM DTT, 0.2 mM dTTP, 0.2 mM dATP, 1 mM ATP, 0.6 Units T4 DNA Polymerase, 1.2 Units T4 DNA Ligase, 1.2 Units T4 Polynucleotide Kinase.
- the reaction was incubated at 23 °C for 1 hour and then heat killed at 75°C for 15 minutes.
- the reaction was phenol chloroform extracted to remove the enzymes and the fragment were ethanol precipitated as described earlier and resuspended in 25 ⁇ L of 50 mM potassium acetate, 20 mM Tris-Acetate pH 7.9, 1 mM DTT, 10 mM magnesium acetate, 10 Units NgoMIV and 4 Units Avr II.
- the restriction digestion will create compatible ends for cloning the 4D5 MAb proprotein into pLSBC1767.
- the reaction was incubated at 37°C for 2 hours and the 2.1 Kb fragment was gel isolated using the QIAquick Gel Extraction kit as described earlier.
- the recovery of the NgoJMIV and Avr II digested fragment was verified by gel electrophoresis.
- the approximately 9.7 Kb NgoMIV and Avr II digested p LSBC1767 fragment was prepared similar to above and the 9.7 Kb fragment was verified by agarose gel electrophoresis.
- the 2.1 Kb NgoMIV and Avr II 4D5 MAb proprotein was cloned into the NgoMIV and Avr II prepared pLSBC1767 plasmid to create pLSBC1773 (Seq ID No: 93).
- Infectious transcripts were synthesized in-vitro from pLSBC1741 clones using the mMessage mMachine T7 kit (Ambion, Austin, TX) following the manufacturers directions. Briefly, a 5.5 ⁇ L reaction containing 1 ⁇ L 10X Reaction buffer, 2.5 ⁇ L 2X NTP/CAP mix, 1 ⁇ L Enzyme mix and 3.5 ⁇ L plasmid was incubated at 37°C for 2 hours.
- the synthesized transcripts were encapsidated in a 40 ⁇ L reaction containing 0.1 M Na 2 HPO 4 -NaH 2 PO 4 (pH 7.0), 0.5 mg/mL purified Ul coat protein (LSBC, Vacaville, CA) which was incubated overnight at room temperature.
- 40 ⁇ L of FES 0.1 M Glycine, 60 mM K 2 HPO 4 , 22 mM Na 2 P 2 O 7 , 10 g/L Bentonite, 10 g/L Celite 545) was added to each encapsidated transcript.
- the encapsidated transcript from an each individual clone was used to inoculate a 26 to 27 day post sow Nicotiana benthamiana expressing the TMV 30K movement protein driven by the CaMN 35S promoter and containing the ⁇ OS terminator as a transgene was made by standard transformation techniques. High levels of subgenomic R ⁇ A species were synthesized in virus-infected plant cells (Kumagai, JMH. et al. (1993) Proc. Natl. Acad. Sci. USA 90:427-430), and serve as templates for the translation and subsequent accumulation of MAb protein.
- Interstitial fluid from infected leaves of each plant was harvested 6 days post inoculation and screened by western blot analysis. Systemically infected upper leaves from individual plants were harvested. The secreted protein fraction, or interstitial fluid (IF) was extracted and analyzed for presence of recombinant protein.
- the leaf tissue was placed in a GF/B 0.8 mL Unifilter (Whatman, Clifton, ⁇ J), covered with 20 mM Tris-HCl (pH 7.0) and subjected to 760 mmHg vacuum for 30 seconds. The vacuum is released and re-applied three times to completely infiltrate the tissue with buffer.
- the residual buffer is discarded and the tissue dried by centrifugation at 400 rpm in a plate centrifuge for 10 seconds.
- the IF fraction is recovered into a 96-well microplate by centrifugation for 10 minutes at 3K rpm in a plate centrifuge.
- the samples were subjected to western blot analysis to verify the presence of the 4D5 heavy chain and light chain polypeptides and run under reducing and nonreducing conditions to determine the presence of expected interchain disulfide bonding.
- 20 ⁇ L JEF sample was adjusted to IX tris-glycine sample dye with and without 10% 2-mercaptoethanol.
- 20 ⁇ L of each sample was loaded on two separate 10-20 % Novex Tris glycine gel and subsequently transferred to Nitrocellulose membrane.
- the membranes were blocked overnight in PBST containing 2.5 % powdered skim milk and 2.5 % BSA.
- One membrane was probed with a 1:3000 dilution of Goat anti-mouse kappa-HRP labeled sera and the second membrane was probed with 1:3000 dilution of Goat anti-mouse IgG-HRP labeled sera (Southern Biotechnology, Birmingham, AL) for 1 hour at room temperature.
- the blots were washed three times in PBST and the labelled proteins detected with the ECL+plus Western Blotting Detection System (Amersham Biosciences, Buckinghamshire, England).
- the anti kappa sera detected an approximately 25 KDa proteins on the reduced sample and a approximately 150 KD band on the non-reduced indicating the presence of interchain disulfide bridges and an assemble 4D5 monoclonal antibody.
- the anti gamma sera detected an approximately 50 KDa proteins on the reduced sample and a approximately 150 KDa band on the non-reduced indicating the presence of interchain disulfide bridges and an assemble 4D5 monoclonal antibody.
- the presence of a disulfide linked 4D5 MAb heterodimer consisting of the gamma heavy chain and the kappa light chain was shown.
- RNA messenger RNA
- mRNA messenger RNA
- mRNA messenger RNA
- dT Dynabeads Oligo 25 (Dynal, Oslo, Norway).
- the RNA was pelleted by centrifugation at 15 K rpm, 4°C for 15 minutes, the supernatant removed and 1 mL of 70 % ethanol added.
- the sample was centrifuged at 15 K rpm, 4°C for 15 minutes, the supernatant removed and the pellet resuspended in nuclease free water (Ambion, Austin, TX) at a concentration of 1 mg/mL.
- RNA 4 ⁇ g of the above prepared total RNA was adjusted to 20 ⁇ L with nuclease free water and incubated at 65°C for 2 minutes and immediately applied to 20 ⁇ L of magnetic beads in binding buffer (20 mM Tris-HCl (pH 7.5), 1.0 M LiCl, 2 mM EDTA). The RNA and bead mixture were incubated for 5 minutes with constant rotating. The supernatant containing unbound material was removed and the beads were washed with 100 ⁇ L washing buffer (10 mM Tris-HCl (pH 7.5), 0.15 M LiCl, 1 mM EDTA).
- cDNA Complementary DNA
- the cDNA reaction was incubated at 42°C for 60 minutes with constant rotation.
- the human heavy chain gamma constant region (C H 1 C H 2C H 3) was PCR amplified with upstream primer hCH15'sr (Seq JED No: 19), which anneals to the 5' end of the gamma constant chain such that treatment with the 3' to 5' exonuclease activity of T4DNA polymerase will result in a "GC” 5' extension where "G” is guanine and "C” is cytosine, and downstream primer hCH3avr3' (Seq JTD No: 20) which anneals to the 3' end of the gamma constant chain and incorporates an Avr II site downstream of the termination codon for subsequent cloning.
- the PCR reactions were amplified at 97°C for 1 minute, 30 cycles of 94°C for 30 seconds, 48°C for 30 seconds, 72°C for 45 seconds, and a 5 minute incubation at 72°C.
- the amplification of the desired approximately 1.0 Kb fragment was verified by agarose gel electrophoresis.
- the amplified human heavy chain constant domain was cloned into pCR4-TOPO (Invitrogen) following the manufacturers directions to create plasmid phCHTOPO (Seq ID No: 57). Briefly, 1 ⁇ L of PCR product, 1 ⁇ L vector, 1 ⁇ L of salt solution and 2 ⁇ L of water were mixed, incubated at room temperature for 5 minutes. The ligation was placed on ice and 25 ⁇ L of chemically competent Top 10 cells was added to the ligation and the mix was incubated on ice for 10 minutes.
- the transformation reaction was heat shocked by incubating at 42°C for 30 seconds and immediately placed on ice and 250 ⁇ L of SOC was added. The transformation was allowed to recover by incubating at 37°C, 200 rpm shaking for 20 minutes. The transformation was plated out on LB plates containing ampicillin and grown overnight at 37°C. Individual colonies were used to inoculate 4.0 mL Luria Broth (LB) containing 100 ⁇ g/mL ampicillin in 14 mL culture tubes and grown overnight at 37°C and 300 rpm. Plasmid was purified from turbid cultures using the QIAspin JMiniprep kits (QIAGEN, Valencia, CA).
- the cells were pelleted by centrifugation at 3 K rpm for 15 minutes in a plate centrifuge. The supernatant was drained from the cell pellets and the cells resuspended in 250 ⁇ L PI Buffer by vortexing. 250 ⁇ L of P2 was added to the cells, mixed by inverting and incubated for 5 minutes to lyse the cells. 350 ⁇ L of N3 was added to the cell lysates, mixed by inverting and spun in centrifuge for 10 minutes at 15 K rpm. The supernatant was transferred to QIAspin column and spun in a centrifuge for 1 minute at 14 K rpm.
- the columns were washed with 0.75 mL of PB, followed by two washes with 0.75 mL of PE and dried. 100 ⁇ L EB buffer was added to the purified plasmid, incubated for 1 minute, and subsequently centrifuged for 1 minute at 15K rpm to elute the purified plasmid.
- the purified phCHTOPO plasmid was subjected to nucleic acid sequencing using standard methods to verify the human gamma IgGl heavy chain constant sequence.
- the KP6 propeptide encoding sequence was PCR amplified from plasmid pLSBC1731 with upstream primer KP6vl5'sr, which was designed to anneal to the 5' end of the JKP6 propeptide encoding sequence such that treatment with the 3' to 5' exonuclease activity of T4DNA polymerase will result in a "GCG” 5' extension where "G” is guanine and "C” is cytosine, and downstream primer KP6vl3'sr (Seq ED No: 24), which was designed to anneal to the 3' end of the KP6 propeptide encoding sequence such that treatment with the 3' to 5' exonuclease activity of T4 DNA polymerase will result in a "CC” 5' extension where "C” is cytosine.
- the KP6 propeptide encoding sequence was PCR amplified from plasmid pLSBC1731 with upstream primer KP6vl5'sr and downstream primer KP6v23'sr (Seq ID No: 15), which was designed to anneal to the 3' end of the JKP6 propeptide encoding sequence such that treatment with the 3' to 5' exonuclease activity of T4 DNA polymerase will result in a "CC" 5' extension where "C” is cytosine.
- the human kappa light chain constant domain (C L ) sequence was PCR amplified from plasmid huscFabmlA ⁇ (Seq JED No: 59) with upstream primer HuCL5'sr (Seq JED No: 21), which anneals to the 5' end of the (C L ) domain such that treatment with the 3' to 5' exonuclease activity of T4DNA polymerase will result in a "CG” 5' extension where "G” is guanine and "C” is cytosine, and downstream primer HuCL3'sr (Seq ID No: 22), which is designed to anneal to the 3' end of the (C L ) domain such that treatment with the 3' to 5' exonuclease activity of T4 DNA polymerase will result in a "CGC” 5' extension where "G” is guanine and "C” is cytosine.
- the human kappa light chain constant domain (C L ) sequence was PCR amplified from plasmid huscFabmlA ⁇ (Seq ID No: 59) with upstream primer HuCL5'sr and downstream primer HuCLv23'sr (Seq ID No: 16), which is designed to anneal to the 3' end of the (C L ) domain such that treatment with the 3' to 5' exonuclease activity of T4 DNA polymerase will result in a "CGC” 5' extension where "G” is guanine and "C” is cytosine.
- the 9E10 light chain variable domain (V L ) was PCR amplified from plasmid pLSBC1736 with upstream primer 9E10Lngo5' (Seq JED No: 10) which contains a Ngo MJTV site compatible for cloning into vector pLSBC1767, which contains the alpha- amylase signal peptide, and downstream primer 9E10L3'sr (Seq ID No: 11), which is designed to anneal to the 3' end of the (V L ) domain such that treatment with the 3' to 5' exonuclease activity of T4 DNA polymerase will result in a "GC” 5' extension where "G” is guanine and "C” is cytosine.
- the Ngo MTV site joins the signal peptide to the FRl of the 9E10 variable region of the light chain and directs the secretion of the artificial proprotein to the ER.
- the 9E10 heavy chain variable domain (V H ) was PCR amplified from the plasmid pLSBC1736 with upstream primer 9E10H5'srs (Seq JED No: 12), which anneals to the 5' end of the C sequence such that treatment with the 3' to 5' exonuclease activity of T4 DNA polymerase will result in a "GG" 5' extension where "G" is guanine, and downstream primer 9E10H3'sr (Seq JED No: 13) which anneals to the 3' end of the V H coding sequence such that treatment with the 3' to 5' exonuclease activity of T4DNA polymerase will result in a "CG" 5' extension where "G” is guanine and "C” is cytosine.
- the human heavy chain gamma constant region (CH1C H C H 3) was PCR amplified from plasmid phCHTOPO with upstream primer hCH15'sr and downstream primer hCH3avr3'.
- the PCR reactions were amplified at 97°C for 1 minute, 25 cycles of 94°C for 30 seconds, 55°C for 15 seconds, 72°C for 20 seconds, and 2 minutes at 72°C.
- the amplification of the desired approximately 350 bp 9E10 V L sequence, 380 bp 9E10 V H sequence and 1.0 Kb human gamma constant sequence were confirmed by agarose gel electrophoresis.
- the amplified KP6 propeptide encoding sequences, human kappa C L sequences, 9E10 V L sequence, 9E10 V H sequence and the human gamma constant sequence were purified using the Strataprep PCR Purification JJKit (Stratagene, La Jolla, California) following manufacturers recommendations. Briefly, an equal volume of DNA- binding solution was added to the PCR product, mixed and transferred to the spin column. The column was centrifuged for 30 seconds at 14 K rpm. The column was washed two times with 750 ⁇ L of wash buffer and centrifuged for 30 seconds to dry. 50 ⁇ L elution buffer was added to the column and the PCR fragment eluted with by centrifugation at 14 K rpm for 30 seconds.
- the purified PCR amplified fragments were ligated together in separate 20 ⁇ L ligation reactions.
- the first reaction contained 0.3 ⁇ L 9E10 V L PCR fragment, 1 ⁇ L HuCL3'sr primed human kappa C L PCR fragment, 1 ⁇ L KP6vl3'sr primed KP6 propeptide PCR fragment , 50 mM Tris-HCl (pH 7.5), 10 mM MgCl 2 , 25 ⁇ g/mL BSA, 10 mM DTT, 0.2 mM dTTP, 0.2 mM dATP, 1 mM ATP, 0.6 Units T4 DNA Polymerase, 1.2 Units T4 DNA Ligase and 1.2 Units T4 Polynucleotide Kinase.
- the second reaction contained 0.3 ⁇ L 9E10 V L PCR fragment, 1 ⁇ L HuCLv23'sr primed human kappa C L PCR fragment, 1 ⁇ L KP6v23'sr primed KP6 propeptide PCR fragment , 50 mM Tris-HCl (pH 7.5), 10 mM MgCl 2 , 25 ⁇ g/mL BSA, 10 mM DTT, 0.2 mM dTTP, 0.2 mM dATP, 1 mM ATP, 0.6 Units T4 DNA Polymerase, 1.2 Units T4 DNA Ligase and 1.2 Units T4 Polynucleotide JKinase. The reactions were incubated at room temperature for 1 hour.
- the first reaction was PCR amplified with upstream primer 9E10Lngo5' and downstream primer KP6vl3'sr and the second reaction was PCR amplified with upstream primer 9E10Lngo5' and downstream primer KP6v23'sr in separate 50 ⁇ L PCR reaction 0.4 ⁇ M upstream primer, 0.4 ⁇ M downstream primer, IX Expand JHigh Fidelity Buffer with MgCl 2 , 0.2 mM dATP, 0.2 mM dCTP, 0.2 mM dGTP, 0.2 mM dTTP, 3.5 Units Expand High Fidelity Polymerase and 1 ⁇ L template plasmid.
- the PCR reactions were amplified at 97°C for 1 minute, 25 cycles of 94°C for 30 seconds, 55°C for 15 seconds, 72°C for 20 seconds, and a final step of 2 minutes at 72°C.
- the reactions were electrophoresed on a 1 % agarose gel with TAE and 0.5 ⁇ g/mL ethidium bromide.
- the 800 bp PCR amplified 9E10 V L -human C -KP6 fragments was cut from the gel and purified from the agarose slice using the JMinElute gel extraction kit following the manufacturers instructions. Briefly, 3 volumes of QG buffer was added to each of the gel fragments, the mixture was incubated at 50°C for 10 minutes with occasional agitation.
- a volume of isopropanol equal to the gel slice volume was added to the dissolved gel slice, mixed, applied to the column and centrifuged at 14K rpm for 1 minute.
- the column was washed with 500 ⁇ L Buffer QB followed by a wash with 750 ⁇ L PE and the purified fragment eluted in 10 ⁇ L EB.
- the ligation was electrophoresed on a 1 % agarose gel with TAE and 0.5 ⁇ g/mL ethidium bromide.
- the 1.4 Kb ligated 9E10 V ⁇ -human gamma constant fragment was cut from the gel and purified from the agarose slice using the JMinElute gel extraction kit following the manufacturers instructions as describe previously and the purified fragment eluted in 10 ⁇ L EB.
- the purified 9E10 V L -human C L -KP6 9E10Lngo5'-KP6v23'sr amplified fragment and the 9E10 V ⁇ -human gamma constant fragment were ligated together in a 20 ⁇ L reaction containing 7 ⁇ L 9E10 V L -human C L - KP6 9E10Lngo5' and KP6v23'sr amplified fragment, 6 ⁇ L 9E10 V H -human gamma constant fragment, 50 mM Tris-HCl (pH 7.5), 10 mM MgCl 2 , 25 ⁇ g/mL BSA, 10 mM DTT, 0.2 mM dTTP, 0.2 mM dATP, 1 mM ATP, 0.6 Units T4 DNA Polymerase, 1.2 Units T4 DNA Ligase, 1.2 Units T4 Polynucleotide Kinase.
- the reactions were incubated at room temperature for 1 hour.
- the reactions were PCR amplified in separate 50 ⁇ L reactions with upstream primer 9E10Lngo5' and downstream primer hCH3avr3' containing 0.4 ⁇ M upstream primer, 0.4 ⁇ M downstream primer, IX Expand High Fidelity Buffer with MgCl 2 , 0.2 mM dATP, 0.2 mM dCTP, 0.2 mM dGTP, 0.2 mM dTTP, 3.5 Units Expand High Fidelity Polymerase and 1 ⁇ L template plasmid.
- the PCR reactions were amplified at 97°C for 1 minute, 15 cycles of 94°C for 30 seconds, 55°C for 15 seconds, 72°C for 60 seconds, and a final step of 2 minutes at 72°C.
- the amplification of the desired approximately 2.1 Kb ligation products was confirmed by agarose gel electrophoresis.
- the PCR amplified products were purified using the Strataprep PCR Purification Kit (Stratgene) following manufacturers recommendations as described previously and eluted in 30 ⁇ L water.
- the PCR amplified product from the ligation of the 9E10 V L -human C L - JKP69E10Lngo5'-KP6v23'sr amplified fragment and the 9E10 V H -human gamma constant fragment was cloned into pCR4-TOPO (Invitrogen) following the manufacturers directions to create plasmid p9E10chimericv2-l (Seq ID No: 63). Briefly, 0.5 ⁇ L of PCR product, 1 ⁇ L vector, 1 ⁇ L of salt solution and 2.5 ⁇ L of water were mixed, incubated at room temperature for 5 minutes.
- the ligations were placed on ice and 25 ⁇ L of chemically competent Top 10 cells was added to the ligations and the mix was incubated on ice for 10 minutes.
- the transformation reaction was heat shocked by incubating at 42°C for 30 seconds and immediately placed on ice and 250 ⁇ L of SOC was added.
- the transformation was allowed to recover by incubating at 37°C, 200 rpm shaking for 20 minutes.
- the transformation was plated out on LB plates containing ampicillin and grown overnight at 37°C. Individual colonies were used to inoculate 4.0 mL Luria Broth (LB) containing 100 ⁇ g/mL ampicillin in 14 mL culture tubes and grown overnight at 37°C and 300 rpm.
- LB Luria Broth
- Plasmid was purified from turbid cultures using the QIAspin JMiniprep kits (QIAGEN) as previously described and eluted in 50 ⁇ L EB buffer.
- the purified p9E10chimericvl-l and p9E10chimericv2-l plasmids was subjected to nucleic acid sequencing using standard methods.
- the chimeric 9E10 V -human C L -JKP6- 9E10 V H encoding sequences were PCR amplified from plasmid p9E10chimericvl-l and p9E10chimericv2-l in separate reactions with upstream primer 9E10Lngo5' and downstream primer 9E10H3'sr.
- the human heavy chain gamma constant region (C H 1C H 2C H 3) was PCR amplified from plasmid phCHTOPO with upstream primer hCH15'sr and downstream primer hCH3avr3'.
- the amplification of the desired approximately 1.1 Kb 9E10 VL-human C L -KP6- 9E10 V H encoding sequences and 1.0 Kb human gamma constant sequence were confirmed by agarose gel electrophoresis.
- the PCR amplified products were electrophoresed on a 1% agarose gel with TAE and 0.5 ⁇ g/mL ethidium bromide.
- the 1.1 Kb 9E10 V -human C L -JKP6- 9E10 V H encoding sequences and 1.0 Kb human gamma constant sequence were cut from the gel and purified from the agarose slice using the MinElute gel extraction kit following the manufacturers instructions as describe previously and the purified fragment eluted in 10 ⁇ L EB.
- the purified 1.1 Kb 9E10 VL- human C L -KP6- 9E10 V H encoding sequences amplified from plasmid p9E10chimericvl- 1 and 1.0 Kb human gamma constant sequence fragment were ligated together in a 20 ⁇ L ligation reaction containing 1.5 ⁇ L each fragment, 50 mM Tris-HCl (pH 7.5), 10 mM MgCl 2 , 25 ⁇ g/mL BSA, 10 mM DTT, 0.2 mM dTTP, 0.2 mM dATP, 1 mM ATP, 0.6 Units T4 DNA Polymerase, 1.2 Units T4 DNA Ligase, 1.2 Units T4 Polynucleotide Kinase and incubated at room temperature for 2 hours.
- the purified 1.1 Kb 9E 10 V L -human C L -KP6- 9E10 VH encoding sequences amplified from plasmid p9E10chimericv2-l and 1.0 Kb human gamma constant sequence fragment were ligated together in a 20 ⁇ L ligation reaction containing 1.5 ⁇ L each fragment, 50 mM Tris-HCl (pH 7.5), 10 mM MgCl 2 , 25 ⁇ g/mL BSA, 10 mM DTT, 0.2 mM dTTP, 0.2 mM dATP, 1 mM ATP, 0.6 Units T4 DNA Polymerase, 1.2 Units T4 DNA Ligase, 1.2 Units T4 Polynucleotide Kinase and incubated at room temperature for 2 hours.
- the reaction was incubated for 15 minutes at 75°C to inactivate enzymes.
- the 20 ⁇ L ligation reactions were adjusted to 50 mM potassium acetate, 20 mM Tris-Acetate pH 7.9, 1 mM DTT, 10 mM magnesium acetate, and subsequently digested for 2 hours at 37°C with 10 Units NgoJMIV, 4 Units Avr II and 10 Units Dpn I.
- the restriction digestions will create compatible ends for cloning the 9E10 chimeric MAb proproteins into pLSBC1767.
- the reactions were gel isolated using the MinElute Gel Extraction kit as described earlier. The recovery of the NgoMIV and Avr II digested fragments was verified by gel electrophoresis.
- the approximately 2.1 Kb NgoJMIV and Avr II digested fragment from pLSBC1767 was prepared similar to above and the 2.1 Kb fragment was verified by agarose gel electrophoresis.
- Clones were confirmed to contain the 2.1 Kb insert and the 9.7 Kb vector fragments by restriction enzyme mapping with NgoJMIV and Avr II followed by agarose gel electrophoresis.
- the 9E10 chimeric MAb proprotein in pLSBC2500 and pLSBC2502 were sequenced using standard methods to verify the sequence.
- the 9E10 chimeric Fab proprotein encoding sequence was PCR amplified from plasmid p9E10chimericv2-l with upstream primer 9E10Lngo5' and downstream primer hCHC2avr3' (Seq ID No: 25), which anneals to the 3' end of the C H I coding sequence and incorporates a termination codon followed by an Avr II site compatible for cloning into vector pLSBC1767.
- the 9E10 chimeric Fab proprotein encoding sequence from p9E10chimericv2-l was PCR amplified in a 50 ⁇ L reactions containing 0.8 ⁇ M upstream primer, 0.8 ⁇ M downstream primer, IX Expand JHigh Fidelity Buffer with MgCl 2 , 0.2 mM dATP, 0.2 mM dCTP, 0.2 mM dGTP, 0.2 mM dTTP, 3.5 Units Expand JHigh Fidelity Polymerase and 0.05 ⁇ L template plasmid.
- the PCR reactions were amplified at 97°C for 1 minute, 25 cycles of 94°C for 30 seconds, 55°C for 15 seconds, 72°C for 30 seconds, and a final step of 2 minutes at 72°C.
- the amplification of the desired approximately 1.4 Kb 9E10 chimeric Fab proprotein encoding sequence was confirmed by agarose gel electrophoresis.
- the PCR amplified products were electrophoresed on a 1% agarose gel with TAE and 0.5 ⁇ g/mL ethidium bromide and the amplified 1.4 Kb 9E10 chimeric Fab proprotein encoding sequence was purified using the Strataprep PCR Purification Kit (Stratagene) following manufacturers recommendations as previously described.
- the prepared PCR amplified product was each digested in with NgoM IV and Avr II.
- a 20 uL reactions containing 3 ⁇ L prepared PCR fragment, 10 Units NgoM IV, 4 Units Avr II, 50mM potassium acetate, 20 mM Tris-acetate, 10 mM magnesium acetate, 1 mM DTT were incubated at 37°C for 2 hours.
- the digested product was electrophoresed on a 1% agarose gel with TAE and 0.5 ⁇ g/mL ethidium bromide.
- the 1.4 Kb 9E10 chimeric Fab encoding sequence was cut from the gel and purified from the agarose slice using the MinElute gel extraction kit following the manufacturers instructions as describe previously and the purified fragment eluted in 10 ⁇ L EB.
- Bacterial transformations into electro-competent JM109 cells was performed with a Gene Pulser electroporator (BioRad) as described previously. Plasmids were purified from turbid cultures using the QIAprep Spin Miniprep Kit (QIAGEN) as described previously and eluted with 50 ⁇ L Buffer EB.
- Clones were confirmed to contain the 1.4 Kb insert and the 9.7 Kb vector fragments by restriction enzyme mapping with NgoMIV and Avr II followed by agarose gel electrophoresis.
- the 9E10 chimeric Fab proprotein in pLSBC2505 was sequenced using standard methods to verify the sequence.
- the 9E10 chimeric Fab proprotein encoding sequence was PCR amplified from plasmid pLSBC2500 with upstream primer 9E10Lngo5' and downstream primer chlCtavr3' (Seq ID No: 18), which anneals to the 3' end of the C H I coding sequence and incorporates a termination codon followed by an Avr II site compatible for cloning into vector pLSBC1766 (Seq ID No: 89).
- the 9E10 chimeric Fab proprotein encoding sequence was PCR amplified from plasmid pLSBC2505 with upstream primer 9E10Lngo5' and downstream primer chlCtavr3'.
- the 9E10 chimeric Fab proprotein encoding sequences from pLSBC2500 and pLSBC2505 were PCR amplified in separate reactions containing 0.8 ⁇ M upstream primer, 0.8 ⁇ M downstream primer, IX Expand High Fidelity Buffer with MgCl 2 , 0.2 mM dATP, 0.2 mM dCTP, 0.2 mM dGTP, 0.2 mM dTTP, 1.8 Units Expand High Fidelity Polymerase and 0.03 ⁇ L template plasmid.
- the PCR reactions were amplified at 97°C for 1 minute, 15 cycles of 94°C for 30 seconds, 55°C for 30 seconds, 72°C for 30 seconds, and a final step of 5 minutes at 72°C.
- the amplification of the desired approximately 1.4 Kb 9E10 chimeric Fab proprotein encoding sequences of pLSBC2500 and pLSBC2505 were confirmed by agarose gel electrophoresis.
- a phenol-chloroform extraction series and ethanol precipitation was performed on the PCR amplified products as previously described.
- the prepared ⁇ LSBC2500 and ⁇ LSBC2505 PCR amplified products were each digested in with NgoM IV and Avr II.
- the 1.4 kb NgoM IV/Avr II prepared 9E10 chimeric Fab proprotein from pLSBC2500 was cloned into the NgoM IV/Avr II prepared pLSBC1766 plasmid to create pLSBC2511 (Seq ID No: 65).
- the 1.4 kb NgoM IV/Avr II prepared 9E10 chimeric Fab proprotein from pLSBC2505 was cloned into the NgoM IV/Avr II prepared pLSBC1766 plasmid to create pLSBC2512 (Seq JED No: 67).
- the KP6 propeptide encoding sequence was PCR amplified from plasmid pLSBC2500 with upstream primer KP6vl5'sr (Seq JED No: 23) and downstream primer natKp6Ct 3' (Seq ID No: 28) which was designed to anneal to the 3' end of the JKP6 propeptide encoding sequence such that treatment with the 3' to 5' exonuclease activity of T4DNA polymerase will result in a "GCC” 5' extension where "G” is guanine and "C” is cytosine.
- the 9E10 chimeric light chain was PCR amplified from plasmid pLSBC2500 with upstream primer 9E10Lngo5' and downstream primer NatKp6Nt3' (Seq ID No: 26) which was designed to anneal to the 5' end of the KP6 propeptide encoding sequence such that treatment with the 3' to 5' exonuclease activity of T4DNA polymerase will result in a "CGC" 5' extension where "G” is guanine and "C” is cytosine.
- the NgoM IV site joins the signal peptide to the FRl of the 9E10 variable light region and directs the secretion of the artificial proprotein to the ER.
- the 9E10 chimeric Fd heavy chain (V H Q H I) was PCR amplified from the plasmid pLSBC2500 with downstream primer chlCtavr3' and upstream primer NatKp6Ct5' (Seq JED No: 27) which was designed to anneal to the 5' end of the JKP6 propeptide encoding sequence such that treatment with the 3' to 5' exonuclease activity of T4DNA polymerase will result in a "CGG" 5' extension where "G” is guanine and "C” is cytosine.
- the JKP6 propeptide encoding sequence, 9E10 chimeric heavy chain Fd sequence and 9E10 chimeric kappa light chain sequences were PCR amplified in separate 25 ⁇ L PCR reactions containing 0.8 ⁇ M upstream primer, 0.8 ⁇ M downstream, IX Expand JHigh Fidelity Buffer with MgCl 2 , 0.2 mM dATP, 0.2 mM dCTP, 0.2 mM dGTP, 0.2 mM dTTP, 1.8 Units Expand High Fidelity Polymerase and 0.03 ⁇ L plasmid template.
- the PCR reactions were amplified at 95°C for 2 minute, 15 cycles of 95°C for 30 seconds, 55°C for 30 seconds, 72°C for 1 minute, and a final step of 7 minutes at 72°C.
- the amplification of the desired approximately 100 bp JKP6 propeptide fragment, 700 bp 9E10 chimeric Fd fragment and 700 bp 9E10 chimeric light chain fragment were verified by agarose gel electrophoresis.
- the PCR amplified JKP6 propeptide encoding fragment, 9E10 chimeric heavy chain Fd fragment and 9E10 chimeric kappa light chain fragment were digested with Dpn I. 5 Units Dpn I was added to each PCR reaction and incubated at 37°C for 1 hour followed by 80°C for 20 minutes. The Dpn I digested PCR fragments were phenol- chloroform extracted followed by ethanol precipitation. The pellets were resuspended in 20 ⁇ L 10 mM Tris-HCL (pH 8.0).
- the purified PCR amplified fragments were ligated together in a 20 ⁇ L ligation reaction.
- the reaction contained 18ng JKP6 propeptide fragment, 126ng 9E10 chimeric Fd fragment, 126ng 9E10 chimeric light fragment, 50 mM Tris-HCl (pH 7.5), 10 mM MgCl 2 , 25 ⁇ g/mL BSA, 10 mM DTT, 0.2 mM dTTP, 0.2 mM dATP, 1 mM ATP, 0.6 Units T4 DNA Polymerase, 1.2 Units T4 DNA Ligase, and 1.2 Units T4 Polynucleotide Kinase.
- the reaction was incubated at 23°C for 1.5 hours and then heat killed at 75°C for 15 minutes.
- the ligation of the desired approximately 1.4kb 9E10 chimeric Fab proprotein fragment was verified by agarose gel electrophoresis.
- a phenol chloroform extraction series was performed on the ligation product followed by ethanol precipitation.
- the pellet was digested in a 25 uL reaction containing 10 Units NgoM IV, 4 Units Avr EJ, 50mM potassium acetate, 20 mM Tris-acetate, 10 mM magnesium acetate, 1 mM DTT.
- the digest was incubated at 37°C for 2 hours, and electrophoresed on a 1.0% agarose gel to separate the approximately 1.4 Kb fragment.
- the gel was stained with GelStar (Cambrex Bio Science) and the approximately 1.4 Kb fragment was isolated. The fragment was purified away from the agarose using QIAquick gel extraction kit following the manufacturers instructions. The recovery of the NgoM IV/Avr II digested fragment was verified by gel electrophoresis.
- the prepared 1.4 kb NgoM IV/Avr II prepared 9E10 chimeric Fab proprotein from pLSBC2500 was cloned into the NgoM IV/Avr II prepared pLSBC1766 plasmid to create pLSBC2514 (Seq JfD No: 69).
- Clones were confirmed to contain the 1.4 kb insert and the 9.7 kb vector fragments by restriction enzyme mapping with NgoM IV and Avr II followed by agarose gel electrophoresis.
- the 9E10 chimeric Fab proproteins were sequenced using standard methods to verify the sequence.
- Infectious transcripts were synthesized in-vitro from pLSBC2511 , pLSBC2512, and pLSBC2514 clones using the mMessage mMachine T7 kit (Ambion, Austin, TX) following the manufacturers directions. Briefly, a 20 ⁇ L reaction containing 2 ⁇ L 10X Reaction buffer, 10 ⁇ L 2X NTP/CAP mix, 2 ⁇ L Enzyme mix and 4 ⁇ L plasmid was incubated at 37°C for 1 hour.
- the synthesized transcripts were encapsidated in a 200 ⁇ L reaction containing 0.1 M Na 2 HPO -NaH 2 PO (pH 7.0), 0.5 mg/mL purified Ul coat protein (LSBC, Vacaville, CA) which was incubated overnight at room temperature. 200 ⁇ L of FES (0.1 M Glycine, 60 mM K 2 HPO 4 , 22 mM Na 2 P 2 O 7 , 10 g/L Bentonite, 10 g/L Celite 545) was added to each encapsidated transcript.
- FES 0.1 M Glycine, 60 mM K 2 HPO 4 , 22 mM Na 2 P 2 O 7 , 10 g/L Bentonite, 10 g/L Celite 545
- the encapsidated transcript from an each individual clone was used to inoculate four 22 day post sow Nicotiana benthamiana expressing the TMV 30K movement protein driven by the CaMV 35S promoter and containing the NOS terminator as a transgene was made by standard transformation techniques. JHigh levels of subgenomic RNA species were synthesized in virus-infected plant cells (Kumagai, JMH. et al. (1993) Proc. Natl. Acad. Sci. USA 90:427-430), and serve as templates for the translation and subsequent accumulation of Fab protein.
- Interstitial fluid from infected leaves of each plant was harvested 7 days post inoculation and screened by Coomassie stained protein gels. Systemically infected upper leaves from each of the four individual plants were harvested. The secreted protein fraction, or interstitial fluid (IF) was extracted and analyzed for presence of recombinant protein.
- the leaf tissue was placed in a GF/B 0.8 mL Unifilter (Whatman, Clifton, NJ), covered with 20 mM Tris-HCl (pH 7.0) and subjected to 760 mmHg vacuum for 30 seconds. The vacuum is released and re-applied three times to completely infiltrate the tissue with buffer.
- the residual buffer is discarded and the tissue dried by centrifugation at 400 rpm in a plate centrifuge for 1 minute.
- the JJEF fraction was recovered in a 96-well microplate by centrifugation for 10 minutes at 3K rpm in a plate centrifuge.
- 20 ⁇ L of each JJTF sample was prepared for SDS-PAGE analysis by the addition of 5 ⁇ L 5X tris-glycine sample dye containing 10 % 2-mercaptoethanol, for reducing gels, and then boiled for 2 minutes. Samples were separated on a 10-20 % gradient Criterion gel (Bio-Rad) and the proteins were detected by Coomassie R-250 Brilliant blue staining. Protein banding in the reducing gel at approximately 25 KDa and 27 KDa indicates the presence of the desired 25 KDa heavy chain Fd and the 27 KDa light chain.
- a FAb construct of 9E10 from pLSBC 1736 is introduced into a T-DNA vector derived from pBI121 (Jefferson, R.A. et al., EMBO J 6 (1987) 3901-3907) using P d and Avrll restriction enzymes wherein the GUS gene is replaced by the FAb sequence such that expression is driven by the 35S promoter.
- the T-DNA construct is transformed into Agrobacterium strain C58C1 carrying pCH32 (Hamilton, CM.; et al., Proc Natl Acad Sci U S A 93 (1996) 9975-9) by electroporation.
- the Agrobacterium is grown into a culture and used to agroinfiltrate (Scofield, S.R. et al., Science 274 (1996) 2063-5, Tang, X.et al., Science 274 (1996) 2060-3, Bendahmane, A., et. al, Plant Cell 11 (1999) 781-791) leaves of Nicotiana benthamiana. After two days proteins are extracted from the leaves and the resulting extracts are analyzed, for instance, by SDS-PAGE and Western blot or by reverse phase HPLC analysis to analyze the expression of the desired gene product.
- the Agrobacterium strain carrying the T-DNA construct from Example 17 is used to transform leaf disks of Nicotiana tabacum, and transgenic plants are regenerated (Horsch, R.B., et al., Science 227 (1985) 1229-1231). Leaves from the transgenic plants are extracted to yield the FAb. The resulting extracts are analyzed, for instance, by SDS- PAGE and Western blot or by reverse phase HPLC analysis to analyze the expression of the desired gene product.
- a MAb construct of 4D5 from pLSBC1773 is introduced into a T-DNA vector derived from pBI121 (Jefferson, R.A. et al., EMBO J 6 (1987) 3901-3907) using Pad and Avrll restriction enzymes wherein the GUS gene is replaced by the FAb sequence such that expression is driven by the 35S promoter.
- the T-DNA construct is transformed into Agrobacterium strain C58C1 carrying pCH32 (Hamilton, CM., et al., Proc Natl Acad Sci U S A 93 (1996) 9975-9) by electroporation.
- the Agrobacterium is grown into a culture and used to agroinfiltrate (Scofield, S.R.
- the Agrobacterium strain carrying the T-DNA construct from Example 19 is used to transform leaf disks of Nicotiana tabacum, and transgenic plants are regenerated (Horsch, R.B., et al., Science 227 (1985) 1229-1231). Leaves from the transgenic plants are extracted to yield the FAb. The resulting extracts are analyzed, for instance, by SDS- PAGE and Western blot or by reverse phase HPLC analysis to analyze the expression of the desired gene product. ill
- the vector pC4 is used for the expression of 4D5 JMAb pre-proprotein.
- Plasmid ⁇ C4 is a derivative of the plasmid ⁇ SV2-dhfr (ATCC Accession No. 37146).
- the plasmid contains the mouse DHFR gene under control of the S V40 early promoter.
- Chinese hamster ovary- or other cells lacking dihydrofolate activity that are transfected with these plasmids can be selected by growing the cells in a selective medium (alpha minus MEM, Life Technologies) supplemented with the chemotherapeutic agent methotrexate.
- MTX methotrexate
- Plasmid pC4 contains for expressing the gene of interest the strong promoter of the long terminal repeat (LTR) of the Rous Sarcoma Virus (Cullen et al., Molec. Cell. Biol. 5:438-447 (1985)) plus a fragment isolated from the enhancer of the immediate early gene of human cytomegalovirus (CMV) (Boshart et al., Cell 41:521-530 (1985)). Downstream of the promoter are BamJHI, JJXbal, and Asp718 restriction enzyme cleavage sites that allow integration of the genes. Behind these cloning sites the plasmid contains the 3' intron and polyadenylation site of the rat insulin gene.
- LTR long terminal repeat
- CMV cytomegalovirus
- high efficiency promoters can also be used for the expression, e.g., the human beta.-actin promoter, the SV40 early or late promoters or the long terminal repeats from other retro viruses, e.g., HIV and JHTLVI.
- Clontech 's Tet-Off and Tet-On gene expression systems and similar systems can be used to express the 4D5 MAb pre-proprotein in a regulated way in mammalian cells (Gossen, M., & Bujard, H., Proc. Natl. Acad. Sci. USA 89: 5547-5551 (1992)).
- other signals e.g., from the human growth hormone or globin genes can be used as well.
- Stable cell lines carrying a gene of interest integrated into the chromosomes can also be selected upon co-transfection with a selectable marker such as gpt, G418 or hygromycin. It is advantageous to use more than one selectable marker in the beginning, e.g., G418 plus methotrexate.
- the plasmid pC4 is digested with the restriction enzymes BamJHI and
- the DNA sequence encoding the complete 4D5 MAb pre-proprotein gene including its leader sequence is amplified using PCR oligonucleotide primers corresponding to the 5' and 3' sequences of the gene.
- the 5' primer has a sequence containing the BamHI restriction enzyme site followed by an efficient signal for initiation of translation in eukaryotes, as described by Kozak, M., J. Mol. Biol. 196:947-950 (1987), and 17 bases of the sequence of 4D5 MAb pre-proprotein.
- the 3' primer has a sequence containing the Asp718I restriction site followed by nucleotides complementary to the 3 'terminus of the 4D5 MAb pre-proprotein gene.
- the amplified fragment is digested with the endonucleases BamHI and
- Chinese hamster ovary cells lacking an active DHFR gene are used for transfection. 5 .mu.g of the expression plasmid pC4 is cotransfected with 0.5 .mu.g of the plasmid pSV2-neo using lipofectin (Feigner et al., Proc. Natl. Acad. Sci. USA 84:7413- 7417 (1987)).
- the plasmid pSV2neo contains a dominant selectable marker, the neo gene from Tn5 encoding an enzyme that confers resistance to a group of antibiotics including G418.
- the cells are seeded in alpha minus JMEM supplemented with 1 mg ml G418.
- the cells are trypsinized and seeded in hybridoma cloning plates (Greiner, Germany) in alpha minus JMEM supplemented with 10, 25, or 50 ng/ml of metothrexate plus 1 mg/ml G418. After about 10-14 days single clones are trypsinized and then seeded in 6-well petri dishes or 10 ml flasks using different concentrations of methotrexate (50 nM, 100 nM, 200 nM, 400 nM, 800 nM).
- methotrexate 50 nM, 100 nM, 200 nM, 400 nM, 800 nM.
- Clones growing at the highest concentrations of methotrexate are then transferred to new 6-well plates containing even higher concentrations of methotrexate (1 mu.M, 2 .mu.M, 5 .mu.M, 10 .mu.M, 20 .mu.M). The same procedure is repeated until clones are obtained which grow at a concentration of 100-200 mu.M. Expression of the desired gene product is analyzed, for instance, by SDS- PAGE and Western blot or by reverse phase HPLC analysis.
- the identified or presumed influential positions are randomized to contain preferred amino acids for optimal structural organization as well as preferred non- immunogenic human sequences.
- DNA shuffling multiple influential positions containing varied amino acids residues at any one position, are re-assorted to create a population of sequences which contain all combinations or many combinations of amino acids at these influential sites.
- a series of computer controlled robots, data based tracking and information management systems are used to pick colonies, prepare plasmid clones, sequence, transcribe and encapsidated infectious transcripts in a high through-put (JHTP) process.
- JHTP high through-put
- the encapsidated transcripts are used to infect plants which are subsequently harvested and extracted in a JHTP manner such as leaf punches followed by JHTP IF extraction or tissue homogenization.
- the extracts are assayed in a HTP manner for a preferred activity such as antigen bind as determined by ELISA or other suitable assay. Additionally, it is preferred if the activity assay has a quantitative aspect.
- the samples are furthered evaluated to determine the quantity of the antibody present. This can be done with an ELISA to detect total antibody or with other suitable assays.
- Identified targets can be immediately used to inoculate larger quantities of plants to obtain purified the antibody for further characterization, pre-clinical evaluation, and process development.
- the expression system is scaled up to produce sufficiently large scale quantities for manufacturing. This may involve the creation of a plant line stably transformed with the preferred proprotein or antibody encoding genes. Plasmid, virus and seed are generated in large scale to accommodate the needs of the manufacturing process.
- Human follicle-stimulating hormone is a disulfide linked, heterodimeric protein containing the glycoprotein hormones alpha subunit and the follicle stimulating hormone beta subunit.
- the follicle stimulating hormone beta subunit was assembled from overlapping synthetic oligonucleotides in a 50 ⁇ L PCR reaction containing 0.1 ⁇ M JJKP509 (Seq ID No: 98), 0.1 ⁇ M KP510 (Seq ID No: 99), 0.1 ⁇ M KP511 (Seq ID No: 100), 0.1 ⁇ M JKP512 (Seq ID No: 101), 0.1 ⁇ M KP513 (Seq ID No: 102), 0.1 ⁇ M JKP514 (Seq ID No: 103), 0.1 ⁇ M KP517 (Seq ID No: 106), 0.1 ⁇ M KP518 107, 0.1 ⁇ M KP519 (Seq ID No: 108), 0.1 ⁇ M J
- the above PCR product was re-amplified in a 50 ⁇ L PCR reaction containing 0.5 ⁇ M KP515 (Seq ID No: 104), 0.5 ⁇ M JKP522 (Seq ID No: 111), 1 ⁇ L PCR product, IX Pfu Buffer, 1 mM dATP, 1 mM dCTP, 1 mM dGTP, 1 mM dTTP, 3.5 Units Pfu DNA Polymerase (Stratgene) was amplified at 98°C for 3 minutes, 20 cycles of 95°C for 30 seconds, 50°C for 30 seconds, 74°C for 30 seconds and a final step of 74°C for 7 minutes.
- Pfu DNA Polymerase (Stratgene) was amplified at 98°C for 3 minutes, 20 cycles of 95°C for 30 seconds, 50°C for 30 seconds, 74°C for 30 seconds and a final step of 74°C for 7 minutes.
- the PCR reaction was purified using the JMinElute PCR purification kit (Qiagen) following the manufacturers instructions.
- the PCR fragment from the above reaction was cloned into pCRHBlunt-TOPO (Invitrogen) following the manufacturers directions to create plasmid pLSB2622.
- the glycoprotein hormones alpha subunit was PCR amplified from a human cDNA clone derived from human mRNA.
- the PCR reaction was purified using the MinElute PCR purification kit (Qiagen) following the manufacturers instructions.
- the PCR fragment from the above reaction was cloned into pCRE-Blunt-TOPO (Invitrogen) following the manufacturers directions to create plasmid pLSB2620.
- the pLSB2622 and pLSB2620 ligations were used to transform chemically competent Top 10 cells following the manufacturers directions.
- the transformations were plated out on LB plates containing antibiotic and grown overnight at 37°C Individual colonies were used to inoculate 1.0 mL Super Broth (SB) containing antibiotic in 96 well 2.0 mL flat-bottom blocks and grown overnight at 37°C and 400 rpm.
- Plasmid was purified from turbid cultures using the QIAprep 96 Turbo Miniprep kits (QIAGEN) as previously described.
- the purified ⁇ LSB2622 and ⁇ LSB2620 plasmids were subjected to nucleic acid sequencing using standard methods.
- the beta subunit from clone pLSB2622 was amplified with upstream primer KP515 which anneals to the 5' end of the beta subunit mature protein and contains a Ngo MIV site compatible for cloning into vector (pLSBC1767), and KP552 downstream primer anneals to the 3' end of the beta subunit, removes the termination codon and fuses the subunit in frame to the 5' end of the KP6 propeptide coding sequence.
- the PCR reaction was purified using the MinElute PCR purification kit (Qiagen) following the manufacturers instructions.
- glycoprotein hormones alpha subunit was amplified with from plasmid pLSB2620 was amplified with upstream primer KP551 which anneals to the 5' end of the alpha subunit and fuses it in frame to the 3' end of the KP6 propeptide coding sequence and KP523 downstream primer which anneals to the 3' end of the alpha subunit including a translational termination codon followed by an Avr II site for subsequent cloning.
- the PCR reaction was purified using the MinElute PCR purification kit (Qiagen) following the manufacturers instructions.
- the above amplified fragments from pLSB2620 and pLSB2622 were fused by sequence overlap extension (SOE).
- SOE sequence overlap extension
- the PCR reaction was purified using the MinElute PCR purification kit (Qiagen) following the manufacturers instructions.
- a 50 ⁇ L reaction containing 10 ⁇ L purified PCR product, 50 mM potassium acetate, 20 mM Tris-Acetate pH 7.9, 1 mM DTT, 10 mM magnesium acetate, 10 Units NgoMIV and 4 Units Avr JH was incubated at 37°C for 3 hours and reaction was purified using the MinElute PCR purification kit (Qiagen) following the manufacturers instructions.
- the 0.7 Kb NgoMIV and Avr II digested follicle stimulating hormone proprotein encoding sequence was ligated into pLSBC1767 to create pLSB2634 (Seq ID No: 96).
- a 21 ⁇ L ligation reaction containing 50 ng NgoMIV and Avrll prepared pLSBC1767, 0.2 ⁇ L purified NgoMIV and Avr II digested PCR fragment, IX Quick Ligation Buffer (New England Biolabs) and 1 ⁇ L Quick T4 DNA Ligase (New England Biolabs) was incubated at 25°C fro 5 minutes.
- Bacterial transformations with DH5 competent cells were performed according to manufacturer recommendations.
- Infectious transcript was synthesized in-vitro from pLSB2634 using the mMessage mMachine T7 kit (Ambion, Austin, TX) following the manufacturers directions. Briefly, a 10 ⁇ L reaction for each plasmid containing 1 ⁇ L 10X Reaction buffer, 5 ⁇ L 2X NTP/CAP mix, 1 ⁇ L Enzyme mix and 0.5 ⁇ g plasmid was incubated at 37°C for 2 hours.
- the synthesized transcripts were encapsidated in a 50 ⁇ L reaction containing 0.1 M Na 2 HPO 4 -NaH 2 PO 4 (pH 7.0), 0.5 mg/mL purified Ul coat protein (LSBC, Vacaville, CA) which was incubated overnight at room temperature.
- 0.1 mL of FES 0.1 M Glycine, 60 mM K 2 HPO 4 , 22 mM Na 2 P 2 O 7 , 10 g/L Bentonite, 10 g L Celite 545
- the encapsidated transcript from an each individual clone was used to inoculate 23 day post sow Nicotiana benthamiana.
- JHigh levels of subgenomic RNA species were synthesized in virus-infected plant cells (Kumagai, JMH. et al. (1993) Proc. Natl. Acad. Sci. USA 90:427-430), and serve as templates for the translation and subsequent accumulation of follicle stimulating hormone protein.
- Interstitial fluid from infected leaves of each plant was harvested 8 days post inoculation. Systemically infected upper leaves from each of the infected plants was harvested. The secreted protein fraction, or interstitial fluid (IF) was extracted and analyzed for presence of recombinant protein.
- IF interstitial fluid
- the leaf tissue was covered with 50 mM Acetate (pH 5.0), 400 mM NaCl, 0.04% sodium metabisulfite and subjected to 760 mmHg vacuum for 2 minutes. The vacuum is released and re-applied three times to completely infiltrate the tissue with buffer. The IF fraction was recovered by centrifugation for 20 minutes at 4K rpm.
- the blots were washed three times in TBST and probed with a 1:2000 dilution of goat anti-rabbit-HRP labeled polyclonal sera for 1 hour at room temperature.
- the blots were washed three times in TBST and the labeled proteins detected with the ECL+plus Western Blotting Detection System (Amersham Biosciences, Buckinghamshire, England).
- the anti-follicle stimulating hormone sera detected an approximately 17 KDa beta protein and a 15 KDa alpha protein indicating that both the alpha and beta subunits were expressed, processed and secreted.
- JTL-12 is a disulfide linked heterdimeric protein, composed of a 35 JKDa subunit (p35) and a 40 KDa subunit (p40), and enhances the cytotoxicity of JNK cells, induces PBL's to produce interferon gamma and stimulates the proliferation of PBL's.
- the IL-12 p35 subunit is PCR amplified from a cDNA clone with an upstream primer containing a NgoJMIV site in frame with the mature protein coding sequence suitable for cloning in frame with the alpha amylase signal peptide of pLSBC1767 and downstream primer which removes the translational termination codon of p35 and fuses the 3' end of the p35 sequence to the 5' end of the JKP6 propeptide sequence amplified from pLSBC1731.
- the IL-12 p40 subunit is PCR amplified from a cDNA clone with an upstream primer which fuses the 3' end of the KP6 propeptide encoding sequence in frame with the 5' end of the mature p40 coding sequence and downstream primer which anneals to the 3' end of the p40 coding sequence and introduces an Avr II site following the translational termination codon suitable for cloning into pLSBC1767.
- the PCR amplified p35 subunit, KP6 propeptide encoding sequence of pLSBC1731 and the p40 subunit are assembled together to create the IL-12 proprotein coding sequence by sequence overlap extension (SOE).
- the resulting fragment is restriction enzyme digested and cloned into prepared pLSBC1767 vector.
- the ligation is used to transform competent E. coli cells and tranformants grown and plasmid DNA purified using standard techniques.
- the resultant IL-12 proprotein assembly in the viral vector is used to synthesize infectious transcript in-vitro using the mMessage mMachine T7 kit (Ambion, Austin, TX) following the manufacturers directions.
- the synthesized transcripts are encapsidated with purified Ul coat protein (LSBC, Vacaville, CA) and mixed with JFES.
- the encapsidated transcript from an each individual clone was used to inoculate Nicotiana benthamiar ⁇ a.
- RNA species were synthesized in virus-infected plant cells (Kumagai, JMH. et al. (1993) Proc. Natl. Acad. Sci. USA 90:427-430), and serve as templates for the translation and subsequent accumulation of JJE -12 protein.
- Infected plant tissue is harvested and proteins are extracted and the resulting extracts are analyzed, for instance, by SDS-PAGE and Western blot or by reverse phase HPLC analysis to analyze the expression of the IL-12 gene product.
- MAb monoclonal antibodies
- polyclonal antibodies in the treatment of cancer and infectious disease is well established. These products exert their beneficial effects by binding to specific targets on the surface of malignant or pathogen cells, to mark these pathogenic cells for immune recognition and destruction.
- the constant region of antibodies can also serve effector functions that help modulate the type, magnitude and duration of the immune response.
- Antibodies can also be fused, either at the gene level or post-translationally, to additional molecules such as toxins or radioisotopes, with the goal of increasing the therapeutic action against the unwanted cell.
- Such bifunctional immunotherapeutics thus consist of a targeting moiety provided by the antibody and a toxic payload provided by the toxin, enzyme, or radioisotope, which may play the major role in destroying the unwanted target cell.
- antibody fragments in this category include Fab, scFv, diabodies, tetrabodies, etc, each having a different conformation and binding functionality.
- the product is used to control B-cell non-Hodgkin's lymphoma (NHL), and rituximab works well by getting rid of malignant as well as non-malignant B cells from the patient.
- NHL non-Hodgkin's lymphoma
- rituximab works well by getting rid of malignant as well as non-malignant B cells from the patient.
- B-cell NHL is a clonal disease
- the healthy B cell arm of the patient's immune system is also destroyed as a consequence, leaving the patient temporarily immunocompromized until his bone marrow can generate new B cells.
- Immunoproteins can also be used to target individual target proteins or structures on the surface of only some subpopulation on target cells. For example, if an antibody could be made against a tumor-specific marker on a malignant cell, that cell population would be targeted and the healthy cells of the same lineage spared. This is an example of highly selective immunotherapy compared to the example for rituximab, in which a panreactive cell-type antigen is targeted.
- Tumor- or pathogen-specific antibodies can be full-size MAb or polyclonal antibodies, or antibody fragments such as scFv, Fab, and other compositions described in the art.
- an antibody or antibody fragment can be selected to target a marker on a clonal tumor, such as NHL. All B cells project an immunoglobulin molecule on their surface, such as IgM. Because each B-cell line, or clone, produces a unique antibody, the antibody sequence can be used as a tumor-specific marker of a malignant B- cell clone in NHL.
- An antibody, or antibody fragment, targeted to bind to the unique immunoglobulin sequence on the malignant B-cell tumor's surface can be expected to bind to, and help destroy, only the malignant clone of B cells while ignoring all other B- cell clones and thus sparing the healthy B cell arm of the patient's immune system.
- Such selectivity would have obvious advantages over the wholesale deletion of the B-cell arm of the immune system, such as is observed with rituximab, as no or minimal humoral immunosuppression would be expected. Because each tumor-specific marker is individual to that patient's specific B cell, the therapeutic antibody to be administered would be expected to show efficacy only in that patient and thus this therapy is considered patient-specific immunotherapy.
- a patient's B-cell, NHL biopsy would be obtained and the exposed IgM (or any other tumor-specific antigen) is used to generate either a full-size antibody or a fragment of an antibody binding specifically to that antigen.
- the generation of a high- affinity antibody or antibody fragment can be achieved by methods known to those skilled in the art, and include immunization of an animal, panning of phage-display libraries, and the like.
- For human therapy it would be preferable to use either a fully human antibody or antibody fragment, or a humanized animal-derived antibody or antibody fragment, to prevent potential concerns over immunogenicity with long-term use of the product.
- An artificial open reading frame encoding the antibody or antibody fragment can be constructed, and the antibody or antibody fragment can be made and isolated by the methods shown in previous examples.
- the antibody or antibody fragment to be used in such patient-specific immunotherapy can be used neat, or as a component of bifunctional agents consisting of the antibody-mediated targeting end linked to either toxins, enzymes or radioisotopes to confer a more effective toxic payload.
- bifunctional immunotherapeutic consisting of a toxin conjugated antibody, one could fuse at the gene level the gene sequence encoding the antibody or antibody fragment to one encoding a toxin or enzyme.
- the translated protein would consist of the target-binding heavy and light variable regions of the Ig, and the toxin-or enzyme-linked antibody constant regions.
- the toxin or enzyme Upon establishing highly specific targeting of the combined moiety by virtue of the antibody-mediated reaction, the toxin or enzyme would act on the surface of the target cell, or be internalized to destroy the cell from within, depending on its characteristics and mode of action.
- Toxins that could be used in this mode of therapy include cholera, diphtheria, ricin, etc.
- a radioisotope or toxin or enzyme could be chemically conjugated to the Ig to produce essentially the same bifunctional agent.
- Radioisotopes that can be used in this therapy include Iodine, Yttrium, etc. Both toxins and radioisotopes with medical utility and approved for use by the regulatory agencies are known to those skilled in the art.
- the neat Ig or bifunctional Ig or Ig fragment would be administered to the patient with the defined affliction, probably by intravenous infusion, so that the drug could target and destroy very specifically only the pathogen or malignant cell population, while sparing the non-target or healthy cells and tissues.
- PEG-Intron [Schering-Plough], Pegasys, [Roche]
- enzymes L-asparaginase, eg. ONCASPAR; adenosine deaminase, eg. ADAGEN [Enzon Pharmaceuticals]
- PEG acts as an inert coat to protect drugs, especially proteins, from immune-mediated and other natural removal mechanisms.
- the Fab and scFv versions of patient-specific antibody fragments could be PEGylated as well, to impart longer circulating half-lives, possibly lowering the required dose (potentially lowering the cost of the therapy), and making administration less frequent, while maintaining the advantages of capillary and tissue penetration of the Ig drug enabled by the lower JMW and lower size of the fragments relative to the whole Ig.
- PEGylation is accomplished by chemically grafting PEG chains, which would be linear or branched, permanent or releasable, and of various MW, onto the Ig, Ig fragment, or Ig-fragment bifunctional conjugate.
- the chemistry for effecting PEGylation has been described and is well known to those skilled in the art. CONCLUSIONS
- An artificial preproprotein comprising four peptide sequences:
- the artificial preproprotein of conclusion 1 that comprises an antibody light chain peptide and an antibody heavy chain peptide, wherein the first peptide is either a heavy chain of the antibody or a light chain of the antibody, and wherein the second peptide is either a heavy chain of the antibody or a light chain of the antibody, but the first peptide is different from the second peptide.
- the artificial preproprotein of conclusion 1 that comprises an antibody light chain peptide and an antibody heavy chain peptide, wherein the first peptide is either a heavy chain of the antibody or a light chain of the antibody, and wherein the second peptide is either a heavy chain of the antibody or a light chain of the antibody.
- the artificial preproprotein of conclusion 1 that comprises a Fab fragment light chain peptide and an Fab fragment heavy chain peptide, wherein the first peptide is either a heavy chain of the Fab fragment or a light chain of the Fab fragment, and wherein the second peptide is either a heavy chain of the Fab fragment or a light chain of the Fab fragment, but the first peptide, but the first peptide is different from the second peptide.
- the artificial preproprotein of conclusion 1 that comprises a Fab fragment light chain peptide and an Fab fragment heavy chain peptide, wherein the first peptide is either a heavy chain of the Fab fragment or a light chain of the Fab fragment, and wherein the second peptide is either a heavy chain of the Fab fragment or a light chain of the Fab fragment.
- the artificial preproprotein of conclusion 1 that comprises a light chain peptide and a heavy chain peptide of a Fab fragment derivative or an antibody derivative, wherein the first peptide is either a heavy chain of the Fab fragment or Antibody derivative or a light chain of the Fab fragment or Antibody derivative, and wherein the second peptide is either a heavy chain of the Fab fragment or Antibody derivative or a light chain of the Fab fragment or Antibody derivative but the first peptide is different from the second peptide.
- the artificial preproprotein of conclusion 1 that comprises a light chain peptide and a heavy chain peptide of a Fab fragment derivative or an antibody derivative, wherein the first peptide is either a heavy chain of the Fab fragment or antibody derivative or a light chain of the Fab fragment or antibody derivative, and wherein the second peptide is either a heavy chain of the Fab fragment or antibody derivative or a light chain of the Fab fragment or antibody derivative.
- An artificial polynucleotide comprising four nucleotide sequences: a first nucleotide sequence that encodes a signal peptide sequence; a second nucleotide sequence that encodes a first peptide of interest, second nucleotide sequence being connected to the 3' terminus of the first nucleotide sequence; a third nucleotide sequence that encodes a propeptide, third nucleotide sequence being connected to the 3' terminus of the second nucleotide sequence; and a fourth nucleotide sequence that encodes a second peptide of interest, fourth nucleotide sequence being connected to the 3' terminus of the third nucleotide sequence.
- the artificial polynucleotide of conclusion 14 that encodes a polypeptide that comprises an antibody light chain peptide and an antibody heavy chain peptide, wherein the first peptide is either a heavy chain of the antibody or a light chain of the antibody, and wherein the second peptide is either a heavy chain of the antibody or a light chain of the antibody, but the first peptide is different from the second peptide.
- the artificial polynucleotide of conclusion 14 that encodes a polypeptide that comprises an antibody light chain peptide and an antibody heavy chain peptide, wherein the first peptide is either a heavy chain of the antibody or a light chain of the antibody, and wherein the second peptide is either a heavy chain of the antibody or a light chain of the antibody.
- the artificial polynucleotide of conclusion 14 that encodes a polypeptide that comprises a Fab fragment light chain peptide and an Fab fragment heavy chain peptide, wherein the first peptide is either a heavy chain of the Fab fragment or a light chain of the Fab fragment, and wherein the second peptide is either a heavy chain of the Fab fragment or a light chain of the Fab fragment, but the first peptide, but the first peptide is different from the second peptide.
- the artificial polynucleotide of conclusion 14 that encodes a polypeptide that comprises a Fab fragment light chain peptide and an Fab fragment heavy chain peptide, wherein the first peptide is either a heavy chain of the Fab fragment or a light chain of the Fab fragment, and wherein the second peptide is either a heavy chain of the Fab fragment or a light chain of the Fab fragment.
- 21 The artificial polynucleotide of conclusion 20 wherein the first peptide and the second peptide are both heavy chain peptides.
- the artificial polynucleotide of conclusion 14 that encodes a polypeptide that comprises a light chain peptide and a heavy chain peptide of a Fab fragment derivative or an antibody derivative, wherein the first peptide is either a heavy chain of the Fab fragment derivative or antibody derivative or a light chain of the Fab fragment or antibody derivative, and wherein the second peptide is either a heavy chain of the Fab fragment or antibody derivative or a light chain of the Fab fragment or antibody derivative but the first peptide is different from the second peptide.
- the artificial polynucleotide of conclusion 14 that encodes a polypeptide that comprises a light chain peptide and a heavy chain peptide of a Fab fragment derivative or an antibody derivative, wherein the first peptide is either a heavy chain of the Fab fragment derivative or antibody derivative or a light chain of the Fab fragment or antibody derivative.
- a method of making an artificial polynucleotide of conclusion 14, comprising: providing a first, a second, a third and a fourth nucleotide sequence that encode a signal peptide sequence, a first peptide of interest, a propeptide and a second peptide of interest respectively; connecting the 3' terminus of the first nucleotide sequence to the 5' terminus of the second nucleotide sequence; connecting the 3' terminus of the second nucleotide sequence to the 5' terminus of the third nucleotide sequence; and connecting the 3' terminus of the third nucleotide sequence to the 5' terminus of the fourth nucleotide sequence, wherein the nucleotide sequence that encodes a first peptide of interest can be the same as or different from the nucleotide sequence that encodes a second peptide of interest.
- the artificial polynucleotide encodes a polypeptide that comprises a light chain peptide and a heavy chain peptide of a Fab fragment derivative or an antibody derivative
- the second nucleotide sequence encodes either a heavy chain of the Fab fragment or Antibody derivative or a light chain of the Fab fragment or Antibody derivative
- the fourth nucleotide sequence encodes either a heavy chain of the Fab fragment or Antibody derivative or a light chain of the Fab fragment or Antibody derivative but the second nucleotide sequence is different from the fourth nucleotide sequence
- the artificial polynucleotide encodes a polypeptide that comprises a light chain peptide and a heavy chain peptide of a Fab fragment derivative or an antibody derivative
- the second nucleotide sequence encodes either a heavy chain of the Fab fragment or Antibody derivative or a light chain of the Fab fragment or Antibody derivative
- the fourth nucleotide sequence encodes either a heavy chain of the Fab fragment or Antibody derivative or a light chain of the Fab fragment or Antibody derivative.
- a method of making an artificial preproprotein comprising: making an artificial polynucleotide that encodes the preproprotein; and expressing the artificial polynucleotide in a host organism whereby the preproprotein is made.
- a method of making a multimeric protein comprising: providing a first, a second, a third and a fourth nucleotide sequence that encode a signal peptide sequence, a first peptide of interest, a propeptide and a second peptide of interest respectively; connecting the 3' terminus of the first nucleotide sequence to the 5' terminus of the second nucleotide sequence; connecting the 3' terminus of the second nucleotide sequence to the 5' terminus of the third nucleotide sequence; and connecting the 3' terminus of the third nucleotide sequence to the 5' terminus of the fourth nucleotide sequence, so that an artificial polynucleotide results and is comprised of the four nucleotide sequences, and wherein the nucleotide sequence that encodes a first peptide of interest can be the same as or different from the nucleotide sequence that encodes a second peptide of interest; introducing the resulting artificial polynucle
- a vector encoding an artificial preproprotein comprising: a nucleotide sequence necessary for replication of the vector nucleotides and proteins and the artificial polynucleotide of conclusion 14 inserted into the vector.
- the vector of conclusion 44 that is a plasmid or a viral vector.
- a transiently transformed cell comprising:
- a vector encoding an artificial preproprotein comprising: a nucleotide sequence necessary for replication of the vector nucleotides and for expression of proteins; an artificial polynucleotide encoding an artificial preproprotein of claim 14 inserted into the vector, a promoter capable of directing expression of the artificial preproprotein; and the artificial preproprotein encoded by the artificial polynucleotide.
- the artificial preproprotein comprises an antibody light chain peptide and an antibody heavy chain peptide, wherein the first peptide is either a heavy chain of the antibody or a light chain of the antibody, and wherein the second peptide is either a heavy chain of the antibody or a light chain of the antibody, but the first peptide is different from the second peptide.
- a transgenic cell comprising:
- the artificial preproprotein encoded by the artificial polynucleotide (c) The artificial preproprotein encoded by the artificial polynucleotide. [000385] 56. The cell of conclusion 55 wherein The artificial preproprotein comprises an antibody light chain peptide and an antibody heavy chain peptide, wherein the first peptide is either a heavy chain of the antibody or a light chain of the antibody, and wherein the second peptide is either a heavy chain of the antibody or a light chain of the antibody, but the first peptide is different from the second peptide.
- a transgenic or transiently transformed plant comprising:
- the artificial preproprotein contains a signal peptide sequence signal peptide sequence forming a secretion signal;
- a method for making a transgenic plant capable of producing immunoglobulin molecules comprising:
- an artificial polynucleotide sequence encoding a preproprotein that preproprotein comprises (i) a signal peptide sequence, (ii) an immunoglobulin heavy chain or light chain peptide, (iii) a propeptide, and (iv) an immunoglobulin heavy chain or light chain peptide, wherein the heavy chain can be in either the b or the d position on the preproprotein, and the light chain will be on the other position;
- the transgenic plant of conclusion 64 wherein The artificial preproprotein further comprises a promoter directing expression of said artificial polynucleotide.
- the transgenic plant of conclusion 80 the promoter is a constitutive promoter.
- the artificial proprotein of conclusion 83 further comprising a signal peptide sequence attached to the N-terminus of the first peptide sequence of interest.
- a process for producing an immunoglobulin molecule or an immunologically functional immunoglobulin fragment comprising at least the variable domains of the immunoglobulin heavy and light chains, in a single host cell comprising the steps of: (a) transforming said single host cell with a single DNA sequence encoding at least the variable domain of the immunoglobulin heavy chain, a propeptide and at least the variable domain of the immunoglobulin light chain, and
- a vector comprising a single DNA sequence encoding at least a variable domain of an immunoglobulin heavy chain and at least a variable domain of an immunoglobulin light chain wherein said single DNA sequence is located in said vector at a single insertion site.
- a transformed host cell comprising at least two vectors, at least one of said vectors comprising a single DNA sequence encoding at least a variable domain of an immunoglobulin heavy chain and at least the variable domain of an immunoglobulin light chain.
- a method comprising:
- step a) inserting the DNA sequence of step a) into a replicable expression vector operably linked to a suitable promoter; (c) transforming a prokaryotic or eukaryotic microbial host cell culture with the vector of step(b);
- the transgenic plant of conclusion 80 the promoter is an inducible promoter.
- a multimeric protein comprising first and second peptides, the first peptide comprising a non-native amino acid pair at the PI and P2 positions of the carboxy terminus.
- a multimeric protein derived from a multimeric protein comprising a first and second peptides, the first peptide comprising a non-native amino acid pair at the PI and P2 positions of the carboxy terminus.
- Plasmid DNA: ⁇ 5PNCAP is Patent Deposit PTA-4742 Deposited October 3, 2002
- Plasmid DNA:pll77MP5 is Patent Deposit PTA-4743 Deposited October 3, 2002
- Plasmid DNA:pl324-JMBP is Patent Deposit PTA-4744 Deposited October 3, 2002
- Plasmid DNA:pLSBC1798 is Patent Deposit Deposited October 2, 2003
- Plasmid DNA:pLSBC2634 is Patent Deposit Deposited October 2, 2003
- Plasmid DNA:Hu Fab A9 is Patent Deposit Deposited October 2, 2003
- Plasmid DNA Human Fab D5 is Patent Deposit Deposited October 2, 2003
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- Chemical & Material Sciences (AREA)
- Health & Medical Sciences (AREA)
- Organic Chemistry (AREA)
- Molecular Biology (AREA)
- Life Sciences & Earth Sciences (AREA)
- Biochemistry (AREA)
- Immunology (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Medicinal Chemistry (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Biophysics (AREA)
- Engineering & Computer Science (AREA)
- Biotechnology (AREA)
- Oncology (AREA)
- Peptides Or Proteins (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Breeding Of Plants And Reproduction By Means Of Culturing (AREA)
Abstract
Description
Claims
Priority Applications (4)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CA002499891A CA2499891A1 (en) | 2002-10-03 | 2003-10-03 | Multimeric protein engineering |
EP03774550A EP1556403A4 (en) | 2002-10-03 | 2003-10-03 | Multimeric protein engineering |
AU2003282667A AU2003282667A1 (en) | 2002-10-03 | 2003-10-03 | Multimeric protein engineering |
JP2004541668A JP2006506056A (en) | 2002-10-03 | 2003-10-03 | Multimeric protein manipulation |
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US41594002P | 2002-10-03 | 2002-10-03 | |
US60/415,940 | 2002-10-03 |
Publications (3)
Publication Number | Publication Date |
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WO2004031362A2 true WO2004031362A2 (en) | 2004-04-15 |
WO2004031362A8 WO2004031362A8 (en) | 2004-06-10 |
WO2004031362A3 WO2004031362A3 (en) | 2004-12-02 |
Family
ID=32069923
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
PCT/US2003/031420 WO2004031362A2 (en) | 2002-10-03 | 2003-10-03 | Multimeric protein engineering |
Country Status (7)
Country | Link |
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US (2) | US20040110930A1 (en) |
EP (1) | EP1556403A4 (en) |
JP (1) | JP2006506056A (en) |
AU (1) | AU2003282667A1 (en) |
CA (1) | CA2499891A1 (en) |
WO (1) | WO2004031362A2 (en) |
ZA (1) | ZA200502572B (en) |
Cited By (2)
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WO2014058389A1 (en) | 2012-10-12 | 2014-04-17 | Agency For Science, Technology And Research | Optimised heavy chain and light chain signal peptides for the production of recombinant antibody therapeutics |
US12044680B2 (en) | 2016-05-13 | 2024-07-23 | Mbl International Corp. | Peptide exchange system and method |
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KR100447530B1 (en) * | 2001-08-14 | 2004-09-08 | 한국과학기술원 | A Method for Extracellular Production of Target Proteins Employing OmpF in E. coli |
WO2006065273A2 (en) * | 2004-05-24 | 2006-06-22 | The Regents Of The University Of California | New human parvovirus |
CN100460508C (en) * | 2004-11-03 | 2009-02-11 | 马延高 | Secretory expression for human insulin gene in methyl alcohol yeast |
WO2007115230A2 (en) * | 2006-03-30 | 2007-10-11 | University Of Medicine And Dentistry Of New Jersey | A strategy for homo- or hetero-dimerization of various proteins in solutions and in cells |
EP1997830A1 (en) | 2007-06-01 | 2008-12-03 | AIMM Therapeutics B.V. | RSV specific binding molecules and means for producing them |
US8709424B2 (en) * | 2009-09-03 | 2014-04-29 | Merck Sharp & Dohme Corp. | Anti-GITR antibodies |
KR20200110476A (en) * | 2011-11-16 | 2020-09-23 | 베링거 인겔하임 인터내셔날 게엠베하 | Anti il-36r antibodies |
BR112014014549A2 (en) | 2011-12-19 | 2020-09-24 | The Rockefeller University | non-sialylated popipeptide anti-inflammatory |
WO2014124457A1 (en) * | 2013-02-11 | 2014-08-14 | University Of Louisville Research Foundation, Inc. | Methods for producing antibodies |
US11155615B2 (en) | 2015-05-18 | 2021-10-26 | Eureka Therapeutics, Inc. | Anti-ROR1 antibodies |
AU2016318158B2 (en) * | 2015-09-04 | 2022-09-15 | Health Research, Inc. | Anti-survivin antibodies for cancer therapy |
WO2019049111A1 (en) | 2017-09-11 | 2019-03-14 | R. J. Reynolds Tobacco Company | Methods and compositions for increasing expression of genes of interest in a plant by co-expression with p21 |
EA202192405A1 (en) | 2019-03-08 | 2022-01-13 | Бёрингер Ингельхайм Интернациональ Гмбх | COMPOSITIONS OF ANTIBODIES AGAINST IL-36R |
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US6403371B1 (en) * | 1996-02-08 | 2002-06-11 | Institut für Pflanzengenetik und Kulturpflanzenforschung | Cassettes for the expression of storable proteins in plants |
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US5959177A (en) * | 1989-10-27 | 1999-09-28 | The Scripps Research Institute | Transgenic plants expressing assembled secretory antibodies |
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US6046037A (en) * | 1994-12-30 | 2000-04-04 | Hiatt; Andrew C. | Method for producing immunoglobulins containing protection proteins in plants and their use |
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2003
- 2003-10-03 AU AU2003282667A patent/AU2003282667A1/en not_active Abandoned
- 2003-10-03 EP EP03774550A patent/EP1556403A4/en not_active Withdrawn
- 2003-10-03 WO PCT/US2003/031420 patent/WO2004031362A2/en active Application Filing
- 2003-10-03 CA CA002499891A patent/CA2499891A1/en not_active Abandoned
- 2003-10-03 JP JP2004541668A patent/JP2006506056A/en active Pending
- 2003-10-03 US US10/679,620 patent/US20040110930A1/en not_active Abandoned
-
2005
- 2005-03-30 ZA ZA200502572A patent/ZA200502572B/en unknown
- 2005-05-17 US US11/132,143 patent/US20050207977A1/en not_active Abandoned
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US6403371B1 (en) * | 1996-02-08 | 2002-06-11 | Institut für Pflanzengenetik und Kulturpflanzenforschung | Cassettes for the expression of storable proteins in plants |
Non-Patent Citations (2)
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SHU ET AL: 'Secretion of a single-gene-encoded immunoglobulin from myeloma cells' PNAS USA vol. 90, September 1993, pages 7995 - 7999, XP002982148 * |
Cited By (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2014058389A1 (en) | 2012-10-12 | 2014-04-17 | Agency For Science, Technology And Research | Optimised heavy chain and light chain signal peptides for the production of recombinant antibody therapeutics |
EP2906599A4 (en) * | 2012-10-12 | 2016-04-27 | Agency Science Tech & Res | Optimised heavy chain and light chain signal peptides for the production of recombinant antibody therapeutics |
US10066019B2 (en) | 2012-10-12 | 2018-09-04 | Agency For Science, Technology And Research | Optimised heavy chain and light chain signal peptides for the production of recombinant antibody therapeutics |
US12044680B2 (en) | 2016-05-13 | 2024-07-23 | Mbl International Corp. | Peptide exchange system and method |
Also Published As
Publication number | Publication date |
---|---|
US20050207977A1 (en) | 2005-09-22 |
EP1556403A2 (en) | 2005-07-27 |
EP1556403A4 (en) | 2006-06-28 |
WO2004031362A8 (en) | 2004-06-10 |
JP2006506056A (en) | 2006-02-23 |
WO2004031362A3 (en) | 2004-12-02 |
ZA200502572B (en) | 2006-11-29 |
AU2003282667A1 (en) | 2004-04-23 |
CA2499891A1 (en) | 2004-04-15 |
US20040110930A1 (en) | 2004-06-10 |
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