AU773602B2 - Methods for producing immunoglobulins containing protection proteins in plants and their use - Google Patents

Description

S&F Ref: 3834401)1

AUSTRALIA

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Name and Address of Applicants: 111, t~trs D 4 c Actual Inventor(s): Address for Service: Invention Title: Planet Biotechnology, Inc.

Suite 102, 8445 Camnino Santa Fe San Diego California 92121 United States of America K~in is (Olec~ Lordo(\

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11 uld M! ia1 4 1 d 9 3J 100 1 3 eof 4uyQ and UUNed-Kingdu2rm- Andrew C. Hiatt, Julian Ma, Thomas Lehner kAt Z Spruson Ferguson St Martins Tower 31 Market Street Sydney NSW 2000 Methods For Producing Immunoglobulins Containing Protection Proteins In Plants And Their Use The following statement is a full description of this invention, including the best method of performing it known to me/us:- 5845c

DESCRIPTION

Methods for Producing Immunoqlobulins Containing Protection Proteins in Plants and Their Use Field of the Invention The present invention relates to expression of immunoglobulins in plants that contain a protection protein as well as to transgenic plants that express such immunoglobulins. The therapeutic use of these immunoglobulins is also contemplated.

,o Background to the Invention Monoclonal antibodies have great potential for numerous therapeutic purposes. The advantages of monoclonal antibody therapeutics over conventional pharmaceuticals include their exquisite selectivity, multiple effector functions, and ease of molecular manipulation such as radio-isotope labelling and other types of conjugation. A wide variety of target antigens have been used to generate specific monoclonal antibodies.

See for example Therapeutic Monoclonal Antibodies, C. A.

K. Borrebaeck and J.W. Larrick eds., Stockton Press, New York, 1990, and The Pharmacology of Monoclonal Antibodies, M. Rosenberg and G.P. Moore eds., Springer-Verlag, Berlin, 1994.

One therapeutic application of monoclonal antibodies 2 IS is passive immunotherapy in which the exogenously produced immunoglobulins are administered directly to the animal :being treated by injection or by ingestion. To be successful, passive immunotherapy must deliver an appropriate amount of an immunoglobulin to the animal, 30 because passive immunotherapy does not rely on an immune *e o *OO_ °o response in the animal being treated. The immunoglobulins administered must be specific for the pathogen or molecule desired to effect treatment. One advantage of passive immunotherapy is the speed at which the antibody can be contacted with the target compared to a normal immune response. Passive immunotherapy can also be used as a prophylaxis to prevent the onset of diseases or infections.

A major potential use of passive immunotherapy is in combating bacterial infections. Recent emergence of antibiotic resistant bacteria make treatment of bacterial infections with passive immunotherapy desirable.

Antibiotic treatment targeted to a single pathogen often *involves eradication of a large population of normal 15 microbes, and this can have undesired side effects. An alternative approach has been to utilize the inherent specificity of immunoglobulins to inhibit a specific pathogenic function in very specific microbial populations. In this strategy, purified immunoglobulins 20 of the appropriate specificity would be administered in order to provide a passive barrier to pathogen invasion.

In addition, the immunoglobulins used for passive immunotherapies for example, for oral administration of o* immunoglobulins must meet certain requirements. First, the immunoglobulin must be functional in very harsh ~environments, such as the gastrointestinal tract. Second, the immunoglobulin must be resistant to the actions of proteases so that it will not be degraded prior to inactivating the target.

Certain types of cells, including epithelial cells and hepatocytes, are capable of assembling immunoglobulin molecules which have been specifically adapted to function in harsh environments. These immunoglobulins are referred to as secretory immunoglobulins (SIg) and include both secretory IgA (SIgA) and secretory IgM (SIgM). The protection provided by endogenous secretory immunoglobulins have been demonstrated. Several mechanisms for protection from bacterial infection by secretory immunoglobulins have been proposed, including, but not limited to, direct killing, agglutination, inhibition of epithelial attachment and invasion, inactivation of enzymes and toxins, opsonization, and complement activation. In an animal, endogenously produced SIgA are exposed to very harsh environments where numerous proteases, such as intestinal and bacterial enzymes are extremely active and denaturants, such as stomach acid, are also present.

One component of secretory immunoglobulins, the secretory component, helps to protect the immunoglobulin against these inactivating agents thereby increasing the biological effectiveness of secretory immunoglobulin.

15 The mechanism of synthesis and assembly of these secretory immunoglobulins, such as SIgA or SIgM is extremely complex. In animal cells, secretory immunoglobulins are assembled in a process involving different cell types. Each secretory immunoglobulin is 20 made up of immunoglobulin heavy and light chains, joining chain (J chain) and a secretory component. The immunoglobulin producing B cells make and assemble the immunoglobulin heavy and light chain together with J chain to produce dimeric or polymeric IgM or IgA. The secretory 25 component is produced by a second type of cell, either epithelial cells or hepatocytes, and secretory immunoglobulin is assembled in and secreted from these cells. The mechanism by which these cells assemble and secrete the secretory immunoglobulin is extremely complex and requires a unique microenvironment provided, for example, by mucosal tissues. The microenvironment places the B cells that produce the polymeric immunoglobulin near the cells that assemble and secrete secretory immunoglobulin onto the mucosal surface of an animal.

The epithelial cells have a receptor, the polyimmunoglobulin receptor (pIgR), that specifically recognizes and binds polymeric immunoglobulin/containing J chain, internalizing it and transporting it through the epithelial cell. Expressed on the basolateral cell surface, the pIgR has an N-terminal signal peptide of 18 amino acids, an extracellular polyimmunoglobulin binding portion of 629 amino acids, a membrane spanning segment of 23 hydrophobic residues, and a cytoplasmic tail of 103 amino acids. The extracellular portion contains five immunoglobulin-like domains of 100-111 amino acids each and constitutes the secreted form of the molecule. See for example, Mostov, Ann. Rev. Immol., 12: 63-84 (1994) The site at which the polyimmunoglobulin receptor is cleaved to generate mature secretory component has not been accurately determined.

The polyimmunoglobulin receptor is located on the S. 15 basolateral surface of epithelial cells in animals.

Polymeric, J chain-containing immunoglobulins produced in B cells interact with and are bound by the receptor resulting in vesicularization, transport across the epithelial cell, and ultimate secretion to the mucosal surface.

Transepithelial transport also involves proteolysis and phosphorylation to produce the mature SIg containing the secretory component. The close association of the S"required cells found in the mucosal microenvironment, specifically the B lymphocytes and epithelial cells, is 25 required for secretory immunoglobulin assembly.

The targeting of the production of immunoglobulins in transgenic organisms, such as mice, is extremely difficult and transgenic organisms made from fungus or plants do not contain the proper cell types and mucosal microenvironment to produce secretory immunoglobulins. The production of large amounts of secretory immunoglobulins in transgenic organisms and cell culture has, before this invention, been impossible. One desiring to produce a secretory immunoglobulin in cell culture or a transgenic organism must express the immunoglobulin heavy chain, the immunoglobulin light chain, and J chain in a B lymphocyte.

To mimic the proper mucosal microenvironment a cell having the pIgR receptor on its surface would also have to be present and be in close association with that B lymphocyte to even attempt to assemble a functional secretory immunoglobulin.

This elaborate process required for natural secretory immunoglobulin assembly is extremely difficult to duplicate in cell culture or transgenic organisms.

Production of SIg in cell culture or transgenic organisms would require coupling the functions of cells producing immunoglobulin with the functions of epithelial cells in artificial (in vitro) systems. Moreover, if the desired transgenic organism is a fungus, a bacterium, or a plant, the cell types and pathways of receptor-mediated cellular internalization, transcytosis, and secretion simply are 15 not present. Those organisms lack epithelial cells and the required mucosal microenvironment.

To date only the assembly of immunoglobulins having light, heavy and J chain within the same cell has been reported. See Carayannopoulos et al. Proc. Nat Acad.

20 Sci., 91:8348-8352 (1994). However, the assembly of an immunoglobulin having the additional protein component, secretory component, within a single cell has not been described.

The present invention discloses a novel method for the assembly of these complex molecules. Rather than assemble the tetrameric complex at the epithelial cell surface by the interaction of a membrane bound polyimmunoglobulin receptor with immunoglobulin, we have assembled secretory immunoglobulin composed of alpha, J, and kappa immunoglobulin chains associated with a protection protein derived from pIgR. This invention produces transgenic plants that assemble secretory immunoglobulins with great efficiency. The present invention makes passive immunotherapy economically feasible.

26. FEB. 2004 16:11 SPRUSON AND FERGUSON 61292615486 NO. 7547 P. 6 Summary of the invention The present invention contemplates a new type of immunoglobulin molecule.

Immunoglobulins of the present invention contain a protection protein in association with an immunoglobulin derived heavy chain having at least a portion of an antigen binding domain. In other embodiments, the immunoglobulin of the present invention further comprise an immunoglobulin derived light chain having at least a portion of an antigen binding domain associated with the immunoglobulin derived heavy chain.

Herein disclosed is an immunoglobulin produced from a single eukaryotic cell, cell culture thereof, or organism derived therefrom comprising a protection protein in association with an immunoglobulin derived heavy chain having at least a portion of an antigen binding domain.

The protection proteins of the present invention give the immunoglobulins containing these proteins useful properties including resistance to chemical and enzymatic degradation and resistance to denaturation. These protection proteins enhance the resistance of the immunoglobulins to environmental conditions.

The protection proteins of the proteins of the present invention comprise at least a segment of amino acid residues 1 to 606 of native polyimmunoglobulin receptor (pIgR) of any species. Other useful protection proteins include protection proteins that contain portions of the plgR molecule. For example, the protection protein may comprise all or part of: amino acids 1-118 (domain I of rabbit pIgR), amino acids 1 to 223 (domains I and II of rabbit pIgR); amino acids 1 to 332 (domains I, II, III of rabbit pIgR); amino acids I to 441 (domains I, II, III, and IV of rabbit pIgR); amino acids 1 to 552 (domains I, II, III, IV and V of rabbit pIgR); and amino acids 1 to 606 or 1 to 627 ofplgR. Additional amino acids, derived either from the pIgR sequence 653-755, or from other sources, may be 25 included so long as they do not constitute a functional transmembrane spanning segment.

Thus, according to one embodiment of the invention, there is provided an ***immunoglobulin recombinantly produced by a single eukaryotic cell, cell culture thereof, or organism derived therefrom comprising a protection protein that does not constitute a functional transmembrane spanning domain in association with an immunoglobulin derived heavy chain having at least a portion of an antigen binding domain, said protection protein comprising at least a portion of amino acid residues 1 to 627 and 653 to o 755 of a native polyimmunoglobulin receptor (pIgR).

In other preferred embodiments, the immunoglobulins of the present invention have a protection protein which has a first amino acid sequence which substantially 35 corresponds to at least a portion of the amino acid residues 1 to 606 or 1 to 627 of the rabbit S*LBAA62 LIBA.oZ3DI COMS ID No: SMBI-00637806 Received by IP Australia: Time 16:18 Date 2004-02-26 polyimmunoglobulin receptor and has a second amino acid residue sequence contiguous with said first amino acid sequence, wherein said second amino acid residue sequence does not have an amino acid residue sequence corresponding to the transmembrane segment of the rabbit polyimmunoglobulin receptor.

In more preferred embodiments, the second amino acid residue sequence has at least a portion of an amino acid sequence which corresponds to amino acid residues 655 to 755 of a polyimmunoglobulin receptor. In other preferred embodiments, the second amino acid residue is at least a portion of one or more of the following: an intracellular domain of a polyimmunoglobulin molecule, a domain of a member of the immunoglobulin gene superfamily, an enzyme, 15 a toxin, or a linker.

The present invention contemplates protection proteins which do not have an amino acid residue corresponding to the transmembrane segment of rabbit polyimmunoglobulin receptor but may have amino acid 20 residues corresponding to the intracellular domain of the rabbit polyimmunoglobulin receptor and this are deletion mutants of the receptor.

The present invention also contemplates immunoglobulins containing protection proteins which have an amino acid sequence which does not contain amino acid residues of a polyimmunoglobulin receptor from a species which are analogous to amino acid residues 288 to 755 of the rabbit immunoglobulin receptor, but does contain at least a portion of the amino acid residues or the domains from a polyimmunoglobulin receptor of a species which are analogous to one or more of these amino acid segments: Amino acids corresponding to amino acid residues 20-45 of the rabbit polyimmunoglobulin receptor; amino acids corresponding to or analogous to amino acid residues 1 to 120 of the rabbit polyimmunoglobulin receptor: amino acids corresponding to or analogous to amino acid residues numbers 120 230 of the rabbit immunoglobulin receptor; amino acids corresponding to or analogous to amino acid residues numbers 230 340 of the rabbit polyimmunoglobulin receptor; amino acids corresponding to or analogous to amino acid residues 340 456 of the rabbit polyimmunoglobulin receptor; amino acids corresponding to or analogous to amino acid residues numbers 450 550 to 570 of the rabbit polyimmunoglobulin receptors; amino acids corresponding to or analogous to amino acid residues 550 to 570 606 to 627 of the rabbit polyimmunoglobulin receptor.

The protection proteins of the present invention may be derived from many species and include protection proteins derived from mammals, rodents, humans, bovine, porcine, ovine, fowl, caprine, mouse, rat, guinea pig, 15 chicken or other bird and rabbit.

In preferred embodiments, the immunoglobulins of the present invention contain two or four immunoglobulin derived heavy chains having at least a portion of an antigen binding domain associated with the protection 20 protein and two or four immunoglobulin derived light chains having at least a portion of an antigen binding S.domain bound to the each of the immunoglobulin derived heavy chains.

In other preferred embodiments, the immunoglobulins 25 of the present invention further comprise immunoglobulin SJ chain bound to at least one of the immunoglobulin derived heavy chains. In preferred embodiments, the component parts of the immunoglobulins of the present invention are bound together by hydrogen bonds, disulfide bonds, covalent bonds, ionic interactions or combinations of said bonds. In other preferred embodiments, the immunoglobulin of the present invention contain protection proteins and/or immunoglobulin derived heavy, light or J chains that are free from N-linked and/or O-linked oligosaccharides.

The immunoglobulins of the present invention may be used as therapeutic immunoglobulins against, for example, 26 FEB. 2004 16:12 SPRUSON AND FERGUSON 61292615486 NO. 7547 P. 21 9 mucosal pathogen antigens. In preferred embodiments, the immunoglobulins of the present invention are capable of preventing dental caries by binding to an antigen from S.

mutans serotypes c, e and f; and S. sobrinus serotypes d and g, using older nomenclature S. mutans a, c, d, e, f, g and h.

The present invention also contemplates a eukaryotic cell, including a plant cell containing an immunoglobulin of the present invention. Eukaryotic cells, including plant cells, containing a nucleotide sequence encoding a protection protein and a nucleotide sequence encoding an immunoglobulin derived heavy chain having at least a portion of an antigen binding domain is also contemplated. Eukaryotic cells, including plant cells, that additionally contain a nucleotide sequence encoding an immunoglobulin derived light chain having at least a portion of an antigen binding domain is also contemplated. In preferred embodiments, the eukaryotic cells, including plant cells, of the present invention contain nucleotide sequences that encode immunoglobulins that have an antigen binding domain which is capable of binding an antigen from S, mutans serotypes a, c, d, e, f, g, and h mutans serotypes c, e and f and S. sobrinus serotypes d and g under new nomenclature). The nucleotide sequences include RNA and appropriate DNA molecules arranged for expression.

Thus, according to an embodiment of the invention, there is provided a eukaryotic cell containing a recombinantly expressed protection protein that does not constitute a functional transmembrane spanning domain and which also contains at least one additional recombinantly expressed molecule selected from the group consisting of: an immunoglobulin derived heavy chain having at least a portion of an antigen binding domain, an immunoglobulin derived light chain having at least a portion of an antigen binding domain, or an immunoglobulin J chain, said protection protein comprising at least a portion of amino acid residues 1 to 627 and 653 to 755 of a native polyimmunoglobulin receptor (pIgR).

According to another embodiment of the invention, there is provided a plant cell containing a nucleotide sequence encoding a protection protein that does not constitute a functional transmembrane spanning domain and a nucleotide sequence encoding an s30 immunoglobulin derived heavy chain having at least a portion of an antigen binding domain, said protection protein comprising at least a portion of amino acid residues 1 to 627 and 653 to 755 of a native polyimmunoglobulin receptor (pIgR).

Also disclosed herein is a eukaryotic cell, typically a plant cell, containing a protection protein.

35 Also disclosed herein is a transgenic plant or plant cell expressing a multimeric protein that is heterologous to the plant cell, wherein said plant cells are characterised by adjacent integration of multiple expression cassettes, each expression cassette encoding less than all of the polypeptide components ot the multimeric protein, and said multiple expression cassettes encoding all of the polypeptide components of the multimeric protein 40 The present invention also contemplates methods of producing eukaryotic cells according to the invention.

aaA LBAA62DI COMS ID No: SMBI-00637806 Received by IP Australia: Time 16:18 Date 2004-02-26 26. FEB. 2004 16:12 SPRUSON AND FERGUSON 61292615486 NO. 7547 P. 22 9a Thus, according to an embodiment of the invention, there is provided a method of transforming a eukaryotic cell to express an assembled immunoglobulin molecule having heavy, light and J chains and a protection protein comprising introducing into a eukaryotic cell nucleotide sequences operably linked for expression encoding: i) an immunoglobulin derived heavy chain having at least a portion of an antigen binding domain, ii) an immunoglobulin derived light chain having at least a portion of an antigen binding domain, iii) an immunoglobulin J chain, and iv) a protection protein that does not constitute a fimunctional transmembrane spanning domain, said protection protein comprising at least a portion of amino acid residues 1 to 627 and 653 to 755 of a native polyimmunoglobulin receptor (pIgR).

According to the invention, there is also provided a method of transforming a plant cell to express an immunoglobulin comprising a protection protein that does not constitute a functional transmembrane spanning domain in association with an immunoglobulin derived heavy chain having at least a portion of an antigen binding domaifi, said method comprising the steps of: introducing into a plant cell an expression vector containing a nucleotide sequence encoding a protection protein operably linked to a transcriptional promoter, said protection protein comprising at least a portion of amino acid residues 1 to 627 and 653 to 755 of a native polyimmunoglobulin receptor (pIgR); and introducing into said plant cell an expression vector containing a nucleotide sequence encoding an immunoglobulin derived heavy chain having at least a portion of an antigen binding domain operably linked to a transcriptional promoter.

25 Also disclosed herein is a method of transforming a plant cell to express a multimeric protein, wherein the multimeric protein is heterologous to the plant cell, the method comprising transforming a plant cell with a plurality of plasmids, each plasmid encoding less than all of the polypeptide components of the multimeric protein, and said plurality encoding all of the polypeptide components of the polypeptide components of 30 the multimeric protein.

In preferred embodiments, the plant cells of the present invention are part of a plant such as a whole plant. The present invention contemplates the use of all types of plants.

both dicotyledonous and monocotyledonous including alfalfa, tobacco and Lenna gibba The present invention also contemplates compositions comprising an -"immunoglobulin of the present invention and plant macromolecules derived from one of the plants useful in practicing the present invention. Particularly contemplated are compositions containing ribulose bisphosphate carboxylase, light harvesting complex, pigments, secondary metabolites or chlorophyll and an immunoglobulin of the UBAA423ItI COMS ID No: SMBI-00637806 Received by IP Australia: Time 16:18 Date 2004-02-26 present invention. Preferred compositions have an immunoglobulin concentration of between 0.001% and 99.9% mass excluding water. In more preferred embodiments, the immunoglobulin concentrations present in the composition is between 0.1% and 99%. Other preferred compositions have plant macromolecules present in a concentration of between 1% and 99% mass excluding water.

The present invention also contemplates methods for making an immunoglobulin of the present invention comprising introducing into a plant cell an expression vector having a nucleotide sequence encoding a protection protein dperably linked to a transcriptional promoter; and introducing into the same plant cell an expression vector containing a nucleotide sequence encoding an S. 15 immunoglobulin derived heavy chain having at least a portion of an antigen binding domain, operably linked to a transcriptional promoter. Other methods that further include the step of introducing into the same plant cell an expression vector containing a nucleotide sequence 20 encoding an immunoglobulin derived light chain having at least a portion of an antigen binding domain, operably linked to a transcriptional promoter. Other preferred methods include also introducing into a plant cell an expression vector containing a nucleotide sequence 25 encoding an immunoglobulin J chain operably linked to a transcriptional promoter.

The present invention also contemplates methods for producing assembled immunoglobulins having heavy, light and J chains and a protection protein by introducing into a eukaryotic cell nucleotide sequences operatively linked for expression to encode an immunoglobulin derived heavy chain having at least a portion of an antigen binding domain, an immunoglobulin light chain having at least a portion of an antigen binding domain, and immunoglobulin J chain, and a protection protein. The method further comprises maintaining the eukaryotic cell under conditions allowing the production and assembly of the immunoglobulin 26. FEB. 2004 16:13 SPRUSON AND FERGUSON 61292615486 NO. 7547 P. 23 11 derived heavy and light chains together with the immunoglobulin J chain and the protection protein to form an immunoglobulin containing a protection protein.

Thus, also herein disclosed is a method for producing a multimeric protein in' a plant cell wherein the multimeric protein is heterologous to the plant cell, the method s comprising the steps of: transforming a plant cell with a plurality of naked plasmids, each plasmid encoding less than all of the polypeptide components of the multimeric protein, and said plurality encoding all of the polypeptide components of the polypeptide components of the multimeric protein; and culturing the plant cell under conditions suitable for protein expression, thereby producing the multimeric protein.

Another embodiment of the invention provides a method for producing an assembled immunoglobulin molecule having heavy, light and J chains and a protection protein comprising the steps of: Is a) introducing into a eukaryotic cell nucleotide sequences operably linked for expression encoding: i) an immunoglobulin derived heavy chain having at least a portion of an antigen binding domain, ii) an immunoglobulin derived light chain having at least a portion of an antigen binding domain, iii) an immunoglobulin J chain, and iv) a protection protein that does not constitute a functional transmembrane spanning domain, said protection protein comprising at least a portion of amino acid residues 1 to 627 and 653 to 755 of a native polyimmunoglobulin receptor (pIgR); and b) maintaining said cell under conditions allowing production and assembly of •said immunoglobulin derived heavy and light chains, said immunoglobulin J chain and said protection protein into an immunoglobulin molecule.

Yet another embodiment of the invention provides a method for producing an assembled immunoglobulin molecule having heavy, light and J chains and a protection protein by maintaining under conditions allowing protein production and immunoglobulin assembly, a eukaryotic cell containing nucleotide sequences operably linked for recombinant expression encoding: i) an immunoglobulin derived heavy chain having at least a portion of an antigen binding domain, UBA6A23DI COMS ID No: SMBI-00637806 Received by IP Australia: Time 16:18 Date 2004-02-26 26. FEB. 2004 16:13 SPRUSON AND FERGUSON 61292615486 NO. 7547 P. 24 Ila ii) an immunoglobulin derived light chain having at least a portion of an antigen binding domain, iii) an immunoglobulin J chain, and iv) a protection protein that does not constitute a functional transmembrane s spanning domain, said protection protein comprising at least a portion of amino acid residues 1 to 627 and 653 to 755 of a native polyimmunoglobulin receptor (pIgR).

The present invention also contemplates methods of making an immunoglobulin resistant to various environmental conditions (more stable) and harsh conditions by operatively linking a nucleotide sequence encoding at least a portion of a desirable to antigen binding domain derived from an immunoglobulin heavy chain to a nucleotide sequence encoding at least one domain derived from an immunoglobulin p or a (IgM or IgA) heavy chain (or and other immunoglobulin having increased stability in the environment) to form a nucleotide sequence encoding a chimeric immunoglobulin heavy chain and expressing that nucleotide sequence in a eukaryotic cell which also contains at 13 least one molecule from the following list: a protection protein, an immunoglobulin derived light chain having at least a portion of an antigen binding domain and an immunoglobulin J chain. The method further comprises allowing the chimeric immunoglobulin heavy chain to assemble with the other molecule present in the same cell to form an immunoglobulin which is resistant to environmental conditions and more stable.

Thus, according to a further embodiment of the invention, there is provided a method of transforming a eukaryotic cell to express an immunoglobulin resistant to environmental conditions comprising the steps of: introducing into a eukaryotic cell an expression vector comprising a nucleotide sequence encoding a chimeric immunoglobulin heavy chain, wherein a nucleotide sequence encoding at least a portion of the antigen binding domain derived from an immunoglobulin heavy chain is operably linked to a nucleotide sequence encoding at least one domain derived from an immunoglobulin alpha heavy chain; and introducing into the eukaryotic cell one or more expression vectors 30 comprising a nucleotide sequence encoding at least one other protein selected from the ****group: ii) an immunoglobulin derived light chain having at least a portion of an antigen binding domain, iii) an immunoglobulin J chain, or LUDAA622DI COMS ID No: SMBI-00637806 Received by IP Australia: Time 16:18 Date 2004-02-26 26. FEB. 2004 16:13 SPRUSON AND FERGUSON 61292615486 NO. 7547 P. llb iv) a protection protein that does not constitute a functional transmembrane spanning domain, said protection protein comprising at least a portion of amino acid residues 1 to 627 and 653 to 755 of a native polyimmunoglobulin receptor (pIgR); wherein if more than one expression vector comprising a nucleotide sequence s encoding said at least one other protein is introduced, these may comprise the same or different nucleotide sequences encoding said at least one other protein.

The invention accordingly also provides a method of producing an immunoglobulin resistant to environmental conditions comprising the steps of: operably linking a nucleotide sequence encoding at least a portion of the 1o antigen binding domain derived from an immunoglobulin heavy chain to a nucleotide sequence encoding at least one domain derived from an immunoglobulin alpha heavy chain to form a nucleotide sequence encoding a chimeric immunoglobulin heavy chain; expressing said nucleotide sequence encoding said chimeric immunoglobulin heavy chain to produce said chimeric immunoglobulin heavy chain in a eukaryotic cell is which also contains at least one other molecule selected from the group consisting of: a protection protein that does not constitute a functional transmembrane spanning domain, said protection protein comprising at least a portion of amino acid residues 1 to 627 and 653 to 755 of a native polyimmunoglobulin receptor (pIgR); an immunoglobulin derived light chain having at least a portion of an antigen binding domain; and an immunoglobulin J chain; and thereby allowing the chimeric immunoglobulin heavy chain to assemble with said at least one other molecule to form said immunoglobulin resistant to said environmental Sconditions.

fee. The large scale production of immunoglobulins of the present invention is contemplated by growing the plants of the present invention and extracting the immunoglobulins from those plants. In preferred embodiments, the method of producing therapeutic immunoglobulin compositions containing plant macromolecules includes the step of shearing under pressure a portion of a plant of the present invention to produce a 0. pulp containing a therapeutic immunoglobulin and plant macromolecules in an liquid derived from the apoplast or symplast of the plant and solid plant derived material.

Further processing steps are contemplated which include separating the solid plant .derived material from the liquid and using a portion of the plant including a leaf, stem, root, tuber, flower, LIBAM IfJi COMS ID No: SMBI-00637806 Received by IP Australia: Time 16:18 Date 2004-02-26 fruit, seed or entire plant. The present invention contemplates the use of a mechanical device or enzymatic method which releases liquid from the apoplast or symplast of said plant followed optionally by separating using centrifugation, settling, flocculation or filtration.

The present invention contemplates immunoglobulins that are chimeric and thus they contain immunoglobulin domains derived from different immunoglobulin molecules.

Particularly preferred are immunoglobulins containing domains from IgG, IgM and IgA.

The present invention contemplates immunoglobulins where the immunoglobulin derived heavy chain is comprised of immunoglobulin domains from two different isotopes of immunoglobulin. In preferred embodiments, the 15 immunoglobulin domains used include at least the CHl, C,2, or C, 3 domain of mouse IgG, IgG1, IgG2a, IgG2b, IgG3, IgA, IgE, or IgD or the Cvar domain. In other preferred embodiments, the immunoglobulin heavy chain is comprised of at least the Cg1, Cu2, CA3 or Cp4 domain of mouse 20 IgM.

The present invention also contemplates immunoglobulin derived heavy chains made up of immunoglobulin domains include at least the C,1, CH 2 or CH 3 domain of a human IgG, IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, or IgD; or 25 least the Cl1, Cx2, Ci3 or Cj4 domain of human IgM; or the Cvar domain. The use of immunoglobulin domains derived from mammals, animals or rodents including any IgG isotype, any IgA isotype, IgE, IgM or IgD is contemplated.

The present invention also contemplates tetratransgenic organisms which are comprised of cells containing four different transgenes each encoding a different polypeptide of a multipeptide molecule wherein at least one of those peptides is associated together to form a multipeptide molecule. The transgenic organisms contemplated by the present invention include transgenic organisms which contain as one of the four transgenes present a transgene encoding a protection protein. The 26. FEB. 2004 16:14 SPRUSON AND FERGUSON 61292615486 NO. 7547--P. 26 13 protection protein present in the transgenic organism's cells is able to assemble together with immunoglobulin heavy chains when present to form immunoglobulins which contain the protection protein.

Thus, according to another embodiment of the invention, there is provided a s tetratransgenic non-human organism comprised of cells containing four different transgenes each encoding a different polypeptide of a multipeptide molecule wherein at least one of each of said different polypeptides is associated together in said multipeptide molecule, wherein at least one of said four transgenes is a transgene encoding a protection protein that does not constitute a functional transmembrane spanning domain, said protection protein comprising at least a portion of amino acid residues 1 to 627 and 653 to 755 of a native polyimmunoglobulin receptor (pIgR).

In preferred transgenic organisms, the cells of the organism express four transgenes which encode an immunoglobulin derived heavy chain having at least a portion of an antigen binding domain, an immunoglobulin derived light chain having at least a portion is of an antigen binding domain, an immunoglobulin J chain, and a protection protein. In other preferred transgenic organisms, the cells contain a transgene which encodes a chimeric immunoglobulin heavy chain, an immunoglobulin heavy chain derived from an IgA heavy chain, an immunoglobulin derived from an IgM heavy chain or an immunoglobulin derived from some other isotype of heavy chain.

20 Also disclosed herein is a set of vectors, each vector encoding less than all of the 'polypeptide components of a multimeric protein, and said set encoding all of the polypeptide components of the multimeric protein.

ooo The vectors may be in the form of naked plasmids, or be contained in a suitable transformation vector such as an Agrobacterium species.

Also described herein are microparticles coated with a plurality of plasmids, each plasmid encoding less than all of the polypeptide components of a multimeric protein, and S. said plurality encoding all of the polypeptide components of the multimeric protein.

Preferably, the microparticles are tungsten or gold.

In the most preferred embodiment, the transgenic organisms of the present 30 invention are plants. Various types and species of plants are contemplated by the present LIBAAO223D COMS ID No: SMBI-00637806 Received by IP Australia: Time 16:18 Date 2004-02-26 23. FEB. 2004 15:33 SPRUSON FERGUSON NO. 7273 P. 43 13a invention. In addition, the present invention also contemplates mammals which are transgenic organisms containing the various molecules of the present invention.

Mammalian transgenic organisms are contemplated by the present invention and include mammalian transgenic organisms which contain four transgenes encoding different polypeptides.

Compositions, including therapeutic agents and compositions comprising immunoglobulins of the invention are also provided by the invention.

According to another embodiment of the invention, there is provided a method of treating or preventing a patient suffering from a condition indicating or preventable by administration of passive immunotherapy, said method comprising administering to said .*-;patient a therapeutically and/or immunologically effective amount of an immunoglobulin S Soo; or composition according to the invention.

:According to another embodiment of the invention, there is provided the use of an immunoglobulin or composition according to the invention, for the manufacture of a medicament for treating or preventing a patient suffering from a condition indicating, or preventable by passive immunotherapy.

According to another embodiment of the invention, there is provided a therapeutic *0 agent comprising an immunoglobulin or composition according to the invention, when used for treating or preventing a patient suffering from a condition indicating, or *S S* 20 preventable by administration of passive immunotherapy.

0*

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Brief Description of the Drawings The drawings will first briefly be described. FIGURE 1 illustrates synthetic oligonucleotides J1-J5 (SEQ ID NOs: 15 to 19 respectively) restriction enzyme sites are underlined) that were used to amplify DNA fragments for Guy's 13 and alpha chain domains in the construction of hybrid IgG/A heavy chains. The relative positions of the areas encoded by each oligonucleotide are shown diagrammatically. The resulting LIB AA0223DI COMS ID No: SMBI-00631115 Received by IP Australia: Time 15:44 Date 2004-02-23 recombinant heavy chains produced by combining various DNA fragments expressed in plants are also shown.

Detailed Description of the Invention A. Definitions Dicotyledon (dicot): 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 (monocot): 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.

15 Lower plant: Any non-flowering plant including ferns, gymnosperms, conifers, horsetails, club mosses, •liver warts, hornworts, mosses, red algaes, brown algaes, gametophytes, sporophytes of pteridophytes, and green algaes.

.0.0 20 Eukarvotic hybrid vector: A DNA by means of which a O..o DNA coding for a polypeptide (insert) can be introduced into a eukaryotic cell.

Extrachromosomal ribosomal DNA (rDNA) 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.

DNA: Deoxyribonucleic acid.

T-DNA: A segment of transferred DNA.

rDNA: Ribosomal DNA.

RNA: Ribonucleic acid.

rRNA: Ribosomal RNA.

Ti-plasmid: Tumor-inducing plasmid.

Ti-DNA: A segment of DNA from Ti-plasmid.

Insert: A DNA sequence foreign to the rDNA, consisting of a structural gene and optionally additional DNA sequences.

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 an amino acid sequence attached to the polypeptide which binds the polypeptide to the endoplasmic reticulum and is essential for protein secretion.

(Selective) Genetic marker: A DNA sequence coding for a phenotypical trait by means of which transformed *cells can be selected from untransformed cells.

o. 15 Promoter: A recognition site on a DNA sequence or group of DNA sequences that provide an expression control element for a gene and to which RNA polymerase specifically binds and initiates RNA synthesis oee (transcription) of that gene.

•eg 20 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

O*-

25 attack, wounding and the like.

Viral promoter: A promoter with a DNA sequence substantially similar to the promoter found at the 5' end of a viral gene. A typical viral promoter is found at the end of the gene coding for the p21 protein of MMTV described by Huang et al., Cell, 27:245 (1981). Other examples include the promoters found in the 35S transcript of the cauliflower mosaic virus as described by Benfey et al., Science, 250:959 (1990).

Synthetic promoter: A promoter that was chemically synthesized rather than biologically derived. Usually synthetic 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 3:2719 (1989) and Odell et al., Nature, 313:810 (1985).

Regulated promoter: A promoter where the rate of RNA polymerase binding and initiation is modulated at a specific time during development, or in a specific structure of an organism or both of these types of modulation. Examples of regulated promoters are given in Chua et al., Science, 244:174-181 (1989).

Single-chain antigen-bindinc protein: A polypeptide 15 composed of an immunoglobulin light-chain variable region amino acid sequence tethered to an immunoglobulin heavy-chain variable region amino acid sequence by a peptide that links the carboxyl terminus of the V, sequence to the amino terminus of the V, sequence. Generally any 20 combination of the heavy chain and light chain antigen binding domains into the same polypeptide using a linker polypeptide to allow the binding domains to assume a useful conformation. Such combinations include VH-Linker- VL, VH-Linear-Light chain, or V,-Linear-Fd.

Single-chain antigen-binding protein-coding gene: A recombinant gene coding for a single-chain antigen-binding protein.

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 amino acid residues connected one to the other as in a polypeptide.

Immunoglobulin product: A polypeptide, protein or 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.

Immunolobulin molecule: A protein containing the immunologically active portions of an immunoglobulin heavy chain and immunoglobulin light chain covalently coupled together and capable of specifically combining with antigen.

Immunoglobulin derived heavy chain: A polypeptide that contains at least a portion of the antigen binding 15 domain of an immunoglobulin and at least a portion of a variable region of an immunoglobulin heavy chain or at least a portion of a constant region of an immunoglobulin heavy chain. Thus, the immunoglobulin derived heavy chain has significant regions of amino acid sequence homology 20 with a member of the immunoglobulin gene superfamily. For example, the heavy chain in an Fab fragment is an immunoglobulin derived heavy chain.

Immunoglobulin derived light chain: A polypeptide that contains at least a portion of the antigen binding 25 domain of an immunoglobulin and at least a portion of the variable region or at least a portion of a constant region of an immunoglobulin light chain. Thus, the immunoglobulin derived light chain has significant regions of amino acid homology with a member of the immunoglobulin gene superfamily.

Antigen binding domain: The portion of an immunoglobulin polypeptide that specifically binds to the antigen. This antigen is typically bound by antigen binding domains of the immunoglobulin heavy and light chain. However, antigen binding domains may be present on a single polypeptide.

J chain: Is a polypeptide that is involved in the polymerization of immunoglobulins and transport of polymerized immunoglobulins through epithelial cells.

See, The Immunoglobulin Helper: The J Chain in Immunoglobulin Genes, at pg. 345, Academic Press (1989).

J chain is found in petameric IgM and dimeric IgA and typically attached via disulphide bonds. J chain has been studied in both mouse and human.

Fab fragment: A protein consisting of the portion of an immunoglobulin molecule containing the immunologically active portions of an immunoglobulin heavy chain and an immunoglobulin light chain covalently coupled together and capable of specifically combining with 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.

"However an Fab fragment may also be prepared by expressing in a suitable host cell the desired portions of g* immunoglobulin heavy chain and immunoglobulin light chain 20 using methods well known in the art.

F~ fragment: A protein consisting of the immunologically active portions of an immunoglobulin heavy chain variable region and an immunoglobulin light chain variable region covalently coupled together and capable of 25 specifically combining with antigen. F, fragments are typically prepared by expressing in suitable host cell the desired portions of immunoglobulin heavy chain variable region and immunoglobulin light chain variable region using methods well known in the art.

Asexual propagation: Producing progeny by regenerating an entire plant from leaf cuttings, stem cuttings, root cuttings, single plant cells (protoplasts) or callus.

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.

Chimeric immunoqlobulin heavy chain: An immunoglobulin derived heavy chain having at least a portion of its amino acid sequence derived from an immunoglobulin heavy chain of a different isotype or subtype or some other peptide, polypeptide or protein.

Typically, a chimeric immunoglobulin heavy chain has its amino acid residue sequence derived from at least two Sdifferent isotypes or subtypes of immunoglobulin heavy chain.

Transqene: A gene that has been introduced into the germ line of an animal. The gene may be introduced into 20 the animal at an early developmental stage. However, the gene could be introduced into the cells of an animal at a later stage by, for example, a retroviral vector.

Multiple molecule: A molecule comprised of more than one peptide or polypeptide associated together by any 25 means including chemical bonds.

B. Immunoglobulins Containing Protection Proteins The present invention provides novel methods for producing immunoglobulin molecules containing protection proteins. The immunoglobulins contain a protection protein in association with an immunoglobulin derived heavy chain that has at least a portion of an antigen binding domain.

The protection proteins of the present invention have an amino acid sequence substantially corresponding to or analogous to at least a portion of residues 1 to 627 of the amino acid residue sequence of the rabbit polyimmunoglobulin receptor and is derived from a precursor protein that does not contain the amino acid residue sequence greater than amino acid residue 627 or analogous to amino acid residue 627 of the rabbit polyimmunoglobulin receptor. The nucleotide sequence and the amino acid sequence of the rabbit polyimmunoglobulin receptor are now and have been described by the Mostov et al., Nature, 308:37 (1984) and EMBL/Gene Bank K01291. The nucleotide sequence of the polyimmunoglobulin receptor is SEQ ID NO. 1 and the corresponding amino acid residue sequence is SEQ ID NO. 2.

The polyimmunoglobulin receptors from any species may be used as a protection protein and these protection proteins do not contain and are derived from a precursor 15 protein that does not contain amino acids having numbers greater than the amino acid number analogous to amino acids 1-627 of the rabbit immunoglobulin sequence. In preferred embodiments, the protection protein is derived from any species and precursor protein that contains amino acids analogous to at least a portion of amino acids 1-606 of the rabbit polyimmunoglobulin receptor and does not contain amino acid residues analogous to residues 607-755 of the rabbit polyimmunoglobulin receptor.

The human polyimmunoglobulin receptor sequence has 25 been determined and reported by Krajci et al., Eur. J.

Immunol., 22:2309-2315 (1992) and Krajci et al., Biochem.

Biophys. Res. Comm., 158:783-789 (1989) and EMBL/Gene Bank Accession No. X73079. The nucleotide sequence of the human polyimmunoglobulin receptor is SEQ ID NO. 3 and the corresponding amino acid residue sequence is SEQ ID NO. 4.

The human polyimmunoglobulin receptor shows extensive sequence homology and has an analogous domain structure to that of the rabbit polyimmunoglobulin receptor. See, Kraehenbuhl et al., Trends in Cell Biol., 2:170 (1992).

The portions of the human polyimmunoglobulin receptor which are analogous to the domains and/or amino acid residues sequence of the rabbit polyimmunoglobulin receptor are shown in Table 1.

The rat polyimmunoglobulin receptor sequence has been determined and reported by Banting et al., FEBS Lett., 254:177-183 (1989) and EMBL/Gene Bank Accession No.

X15741. The nucleotide of the rat polyimmunoglobulin receptor nucleotide sequence is SEQ ID NO. 9 and the corresponding amino acid residue sequence is SEQ ID NO The rat polyimmunoglobulin receptor shows extensive sequence homology and has an analogous domain structure to that of the rabbit and human polyimmunoglobulin receptor.

See, Kraehenbuhl et al., T. Cell Biol., 2:170 (1992). The portions of the rat polyimmunoglobulin receptor which are analogous to the domains and/or amino acid residue 15 sequence of the rabbit polyimmunoglobulin receptor are shown in Table 1.

The bovine polyimmunoglobulin receptor sequence has been determined and reported in EMBL/Gene Bank Accession No. X81371. The bovine polyimmunoglobulin receptor 20 nucleotide sequence is SEQ ID NO.5 and the corresponding amino acid residue sequence is SEQ ID NO. 6. The bovine polyimmunoglobulin receptor shows extensive sequence homology and has an analogous domain structure to that of the rabbit and human polyimmunoglobulin receptor. The 25 portions of the bovine polyimmunoglobulin receptor which are analogous to the domains and/or amino acid residues sequence of the rabbit polyimmunoglobulin receptor are shown in Table 1.

The mouse polyimmunoglobulin receptor sequence has been determined and reported by Piskurich et al., J.

Immunol., 150:38 (1993) and EMBL/Gene Bank U06431. The mouse polyimmunoglobulin receptor nucleotide is SEQ ID NO.

7 and the corresponding amino acid residue sequence is SEQ ID NO. 8. The mouse polyimmunoglobulin receptor shows extensive sequence homology and has an analogous domain structure to that of the rabbit and human polyimmunoglobulin receptor. The portions of the mouse 22 polyimmunoglobulin receptor which are analogous to the domains and/or amino acid residue sequence of the rabbit polyimmunoglobulin receptor are shown in Table 1.

In addition to the above-identified nucleic acid and corresponding amino acid residue sequences of the polyimmunoglobulin receptor from a variety of species, the present invention contemplates the use of a portion of a polyimmunoglobulin receptor from any species. The conserved domain structure of the polyimmunoglobulin receptor between species allows the selection of analogous amino acid residue sequences within each polyimmunoglobulin receptor from different species. The present invention contemplates the use of such analogous amino acid residue sequences from any polyimmunoglobulin 15 receptor. The analogous sequences from several polyimmunoglobulin receptor amino acid sequences is as shown in Table 1.

S

.6. S S S S S S S S S S S .5 Table 1 Analogous Regions of the Amino Acid Residue Sequence of The Polyimmunoglobulin Receptor of Several Species. The nucleotide sequence coordinates approximately define the boundaries of the domains of molecules.

Immunoglobulin Binding Residues of Domain I domain I domain II domain III Rabbit (SEQ ID NO. 2) 21 43 1 118 119 223 224 332 333 441 442 552 Bovine (SEQ ID NO. 6) -13 45 1 120 110 230 210 340 320 450 440 570 Human (SEQ ID NO. 4) -13 45 Rat (SEQ ID NO. 10) -13 45 1 120 1 120 domain IV domain V 110 230 210 340 320 450 440 550 550 606 550 627 625 660 650 end 110 230 210 340 320 450 440 550 550 606 550 627 Mouse (SEQ ID NO. 8) -13 1 120 110 230 210 340 320 450 440 550 550 606 550 627 External Portions of 553 606 domain VI 553 627 550'- 606 550 627 transmembrane segment intracellular portion 630 652 653 755 625 660 650 end 625 660 653 end 625 660 653 end The protection proteins of the present invention may contain substantially less than the entire amino acid residue sequence of the polyimmunoglobulin receptor. In preferred embodiments the protection protein contains at least a portion of the amino acid residues 1 to 606 of the native polyimmunoglobulin receptor of rabbit. Unlike the native polyimmunoglobulin receptor, the protection proteins of the present invention are derived from precursor proteins that do not contain the entire amino acid residue sequence greater than the amino acid residue 627 derived from the native polyimmunoglobulin receptor and thus may contain more amino acids or fewer amino acids than secretory components. In preferred embodiments, the protection proteins of the present invention do not 15 contain the entire amino acid residue sequence greater than amino acid residue 606 of the native polyimmunoglobulin receptor of rabbit. The present invention contemplates using only portions of the native polyimmunoglobulin receptor sequence as a protection 20 protein. In other embodiments, it is contemplated that the protection protein may end at any amino acid between amino acid residue 606 to 627, including every amino acid position between 606 and 627, such as 607, 608, 609, 610, 611, 612, 613, 614, 615, 616, 617, 618, 619, 620, 621, 622, 623, 624, 625, 626.

In preferred embodiments, a protection protein of the present invention has an amino acid sequence which corresponds to one or more of the following amino acid segments: 1) amino acids (AA) corresponding to AA 21-43 of domain I of the rabbit polyimmunoglobulin receptor; 2) amino acids (AA) corresponding to AA 1 118 of domain I of the rabbit polyimmunoglobulin receptor; 3) amino acids (AA) corresponding to AA 119 223 of domain II of the rabbit polyimmunoglobulin receptor; 4) amino acids (AA) corresponding to AA 224 332 of domain III of the rabbit polyimmunoglobulin receptor; amino acids (AA) corresponding to AA 333 441 of domain IV of the rabbit polyimmunoglobulin receptor; 6) amino acids (AA) corresponding to AA 442 552 of domain V of the rabbit polyimmunoglobulin receptor; 7) amino acids (AA) corresponding to AA of 553 to 606 or 553 to 627 of domain VI of the rabbit polyimmunoglobulin receptor; and does not contain amino acid residues corresponding to AA residues 607 to 755 or 628 to 755 of the rabbit polyimmunoglobulin receptor.

It should be noted the exact boundary of a domain may vary within approximately 20 amino acids. However, the domain structure and boundaries will be understood by one skilled in the art.

In addition, the present invention contemplates 15 protection protein ending at the following amino acid :residues of the rabbit polyimmunoglobulin receptor or at an amino acid residue which corresponds to the following residues but is in the polyimmunoglobulin receptor of another species: 580 605.

20 In other preferred embodiments, a protection protein has an amino acid sequence which corresponds to the amino acid sequence of a polyimmunoglobulin receptor for a particular species and which is analogous to the following amino acid segments: 25 1) amino acids (AA) corresponding to AA 21 43 of domain I of the rabbit polyimmunoglobulin receptor; 2) amino acids (AA) corresponding to AA 1 118 of domain I of the rabbit polyimmunoglobulin receptor; 3) amino acids (AA) corresponding to AA 119 223 of domain II of the rabbit polyimmunoglobulin receptor; 4) amino acids (AA) corresponding to AA 224 332 of domain III of the rabbit polyimmunoglobulin receptor; amino acids (AA) corresponding to AA 333 441 of domain IV of the rabbit polyimmunoglobulin receptor; 6) amino acids (AA) corresponding to AA 442 552 of domain V of the rabbit polyimmunoglobulin receptor; 7) amino acids (AA) corresponding to AA of 553 606 or 553 627 of domain VI of the rabbit polyimmunoglobulin receptor; and does not contain amino acid residues analogous to amino acid residues 607 755 or 630 755 of the rabbit polyimmunoglobulin receptor.

In other preferred embodiments, the protection protein comprises domains I, IV, V and AA 550 606 or 550 -627 of domain VI of the rabbit polyimmunoglobulin receptor or the amino acid sequence from analogous domains and regions of a polyimmunoglobulin receptor from a different species.

In other embodiments, a protection protein of the present invention has an amino acid residue sequence which substantially corresponds to at least a portion of the 15 amino acid residues from the polyimmunoglobulin receptor of a species which are analogous to amino acid residues 1- 627 of the rabbit polyimmunoglobulin receptor. This portion of the amino acid sequence would correspond to at least a portion of the extracellular domains of the 20 receptor of that-species.

In preferred embodiments, a protection protein of the present invention has an amino acid sequence which substantially corresponds to at least a portion of the amino acid residues from the polyimmunoglobulin receptor 25 of a species which are analogous to amino acid residues 1- 606 of the rabbit polyimmunoglobulin receptor.

In other preferred embodiments, a protection protein of the present invention has an amino acid residue sequence which substantially corresponds to or is analogous to (if from a species other than rabbit) at least a portion of the following amino acid residue sequences: 1) amino acids (AA) corresponding to AA 21 43 of domain I of the rabbit polyimmunoglobulin receptor; 2) amino acids (AA) corresponding to AA 1 118 to of domain I of the rabbit polyimmunoglobulin receptor; 3) amino acids (AA) corresponding to AA 119 223 of domain II of the rabbit polyimmunoglobulin receptor; 4) amino acids (AA) corresponding to AA 224 332 of domain III of the rabbit polyimmunoglobulin receptor; 5) amino acids (AA) corresponding to AA 333 441 of domain IV of the rabbit polyimmunoglobulin receptor; 6) amino acids (AA) corresponding to AA 442 552 of domain V of the rabbit polyimmunoglobulin receptor; 7) amino acids (AA) corresponding to AA of 553 606 or 553 627 of domain VI of the rabbit polyimmunoglobulin receptor; and does not contain amino acid residues corresponding to AA 628 to 755 of the rabbit polyimmunoglobulin receptor.

SIn other preferred embodiments, the immunoglobulins 15 of the present invention have a protection protein which has a first amino acid sequence which substantially corresponds to at least a portion of the amino acid residues 1 to 606 or 1 to 627 of the rabbit polyimmunoglobulin receptor and has a second amino acid 20 residue sequence contiguous with said first amino acid sequence, wherein said second amino acid residue sequence does not have an amino acid residue sequence corresponding to the transmembrane segment of the rabbit polyimmunoglobulin receptor.

25 In more preferred embodiments, the second amino acid residue sequence has at least a portion of an amino acid sequence which corresponds to amino acid residues 655 to 755 of a polyimmunoglobulin receptor. In other preferred embodiments, the second amino acid residue is at least a portion of one or more of the following: an intracellular domain of a polyimmunoglobulin molecule, a domain of a member of the immunoglobulin gene superfamily, an enzyme, a toxin, or a linker.

The present invention contemplates protection proteins which do not have an amino acid residue corresponding to the transmembrane segment of rabbit polyimmunoglobulin receptor but may have amino acid residues corresponding to the intracellular domain of the rabbit polyimmunoglobulin receptor and this are deletion mutants of the receptor.

In other embodiments, protection proteins of the present invention have an amino acid sequence which substantially corresponds to at least one of the extracellular domains of polyimmunoglobulin receptor of a particular species. The protection protein may have an amino acid sequence of which a segment of that amino acid sequence which substantially corresponds to an extracellular domain of the polyimmunoglobulin receptor of one species, and a different segment of that amino acid sequence may be from a second species and substantially correspond to an extracellular domain from a different 15 species. This invention contemplates embodiments in which a protection protein has an amino acid sequence which has ~one amino acid sequence segment which corresponds to the amino acid sequence of the polyimmunoglobulin receptor from one species and has a second amino acid sequence 20 within the same domain which corresponds to the amino acid and sequence of the polyimmunoglobulin receptor of a different species. Thus, the protection protein may have individual domains or portions of a particular domain that are comprised of amino acid sequences which correspond to 25 the polyimmunoglobulin receptor from different species.

Other embodiments are contemplated in which protection protein has portions of its amino acid sequence derived from a molecule which is a member of the immunoglobulin superfamily. See, Williams and Barclay, "The Immunoglobulin Superfamily." In Immunoglobulin Genes, p. 361, Academic Press (Honjo Alt and Rabbits Eds.

1989). These derived portions may include amino acid sequences encoding peptides, domains or multiple domains from an immunoglobulin superfamily molecule.

The present invention also contemplates a nucleotide sequence encoding a protection protein which has a first nucleotide sequence encoding at least a portion of amino acids 1-606 or 1-627 of the rabbit polyimmunoglobulin receptor nucleotide sequence and which does not have a nucleotide sequence which encodes a functional transmembrane segment 3' of the first nucleotide sequence.

Further preferred embodiments include a second nucleotide sequence located 3' of the first nucleotide sequence which encodes the amino acids 1-606 or 1-627 of the rabbit polyimmunoglobulin receptor sequence. This second nucleotide sequence may encode a variety of molecules including portions of the intracellular domain of rabbit polyimmunoglobulin receptor or another polyimmunoglobulin receptor or a portion of an immunoglobulin superfamily molecule. In addition, embodiments are contemplated in which this second nucleotide sequence encodes various 15 effector molecules, enzymes, toxins and the like.

Preferred embodiments include a second nucleotide sequence which encodes amino acid residues which correspond to amino acid residues 655 to 775 of the rabbit polyimmunoglobulin receptor or polyimmunoglobulin receptor 20 from another species.

The present invention also contemplates expression vectors containing a nucleotide sequence encoding a protection protein which has been operatively linked to for expression. These expression vectors place the 25 nucleotide sequence to be expressed in a particular cell 3' of a promoter sequence which causes the nucleotide sequence to be transcribed and expressed. The expression vector may also contain various enhancer sequences which improve the efficiency of this transcription. In addition, such sequences as terminators, polydenylation (poly A) sites and other 3' end processing signals may be included to enhance the amount of nucleotide sequence transcribed within a particular cell.

In preferred embodiments, the protection protein is part of an immunoglobulin that is in association with an immunoglobulin derived heavy chain having at least a portion of an antigen binding domain. Immunoglobulin derived heavy chains containing at least a portion of an antigen binding domain are well known in the art and have been described, for example, by Huse et al., Science, 246:1275 (1989), and by Lerner and Sorge, PCT Application WO 90/14430, published November 29, 1990. The disclosure of these documents are hereby incorporated by reference.

In other embodiments, the immunoglobulins of the present invention contain a protection protein and immunoglobulin derived heavy chain and immunoglobulin derived light chain that contain at least a portion of an antigen binding site in association with the immunoglobulin derived heavy chain. Immunoglobulin light chains having at least a portion of an antigen binding domain are well known in the art and are described in 15 available sources. See, for example, Early and Hood, Genetic Engineering, Setlow Hollaender, Vol. 3, Plenum Publishing Corp., New York (1981), pages 157-188; and Kabat et al., Sequences of Immunologic Interest, National Institutes of Health, Bethesda, Maryland (1987) 20 The disclosures of all references cited herein are hereby incorporated by reference.

The immunoglobulin components of the complex (alpha, J, kappa or lambda) can contain all or part of the full length polypeptide. Parts of these chains may be used to 25 substitute for the whole chain. For instance, the entire immunoglobulin alpha heavy chain may be replaced by the variable region and only a portion of the alpha constant region sufficient to enable assembly with the other components. Likewise, a truncated kappa or lambda chain, containing only a small section of constant region can replace the full length kappa or lambda chains. The prerequisite of any complex is the ability to bind the protection protein.

In addition to truncated components, the present invention contemplates the combination of different types of immunoglobulins. For example, a heavy chain constant region comprising the CHl and C 2 regions of IgG followed by the C 2 and C,3 regions derived from an IgA will form a stable complex containing the protection protein. This is specifically described as an example.

The immunoglobulins containing the protection proteins of the present invention preferably contain at least a portion of an IgM or IgA heavy chain which allows that immunoglobulin heavy chain to bind to immunoglobulin J chain and thereby bind to the protection protein. It is contemplated that the immunoglobulin heavy chain of the present invention may be comprised of individual domains selected from the IgA heavy chain or the IgM heavy chain or from some other isotype of heavy chain. It is also contemplated that an immunoglobulin domain derived from an immunoglobulin heavy chain other than IgA or IgM may be S15 molecularly engineered to bind immunoglobulin J chain and thus may be used to produce immunoglobulins of the present invention.

One skilled in the art will understand that immunoglobulins consist of domains which are approximately 20 100-110 amino acid residues. These various domains are well known in the art and have known boundaries. The removal of a single domain and its replacement with a domain of another antibody molecule is easily achieved with modern molecular biology. The domains are globular structures which are stabilized by intrachain disulfide bonds. This confers a discrete shape and makes the domains a self-contained unit that can be replaced or interchanged with other similarly shaped domains. The heavy chain constant region domains of the immunoglobulins confer various properties known as antibody effector functions on a particular molecule containing that domain.

Example effector functions include complement fixation, placental transfer, binding to staphyloccal protein, binding to streptococcal protein G, binding to mononuclear cells, neutrophils or mast cells and basophils. The association of particular domains and particular immunoglobulins isotopes with these effector functions is well known and for example, described in Immunoloyv, Roitt et al., Mosby St. Louis, Missouri (1993 3rd Ed.) The immunoglobulins of the present invention may, in addition to the protection protein, contain immunoglobulin heavy chains, immunoglobulin light chains, or immunoglobulin J chain bound to the immunoglobulin derived heavy chains. In preferred embodiments, the immunoglobulin of the present invention comprises two or four immunoglobulin derived heavy chains, together with two or four immunoglobulin light chains and an immunoglobulin J chain bound to at least one of the immunoglobulin derived heavy chains. The immunoglobulin J chain is described and known in the art. See, for example, M. Koshland, The Immunoglobulin Helper: The J Chain, in Immunoglobulin Genes, Academic Press, London, Pg. 345, (1989) and Matsuuchi et al., Proc. Natl. Acad.

Sci. 83:456-460 (1986). The sequence of the immunoglobulin J chain is available on various data bases in the United States.

20 The immunoglobulin of the present invention has a protection protein associated with at least an immunoglobulin derived heavy chain. This association may occur by hydrogen bonds, disulfide bonds, covalent bonds, ionic interactions or combinations of these various bonds.

Typically, immunoglobulin molecules are held together by disulfide bonds between the immunoglobulin heavy chains and immunoglobulin light chains. The interaction of the protection protein with the immunoglobulin is by noncovalent or disulfide bonding.

The immunoglobulins of the present invention containing the protection protein, the immunoglobulin derived heavy chain and optionally an immunoglobulin derived light chain, and J chain are typically bonded together by one of the following: hydrogen bonds, disulfide bonds, covalent bonds, ionic interactions or combinations of these bonds. The present invention contemplates molecules in which the required portions of the immunoglobulin heavy, light and/or J chain have been placed into a single polypeptide and function to bind antigen and protection protein. Examples of such proteins are single-chain antigen-binding proteins.

The present invention contemplates a method of assembling a multimeric immunoglobulin comprising the steps of: introducing into an organism a DNA segment encoding all or part of an immunoglobulin J chain, and a DNA segment encoding all or part of an immunoglobulin alpha chain, and a DNA segment encoding all or part of either an immunoglobulin kappa chain or an immunoglobulin lambda chain; and introducing into the same organism a protection protein, said protection protein comprising at least a segment of the amino acid residues 1 to residue 606 of the 15 rabbit polyimmunoglobulin receptor (pIgR) amino acid residue sequence or analogous amino acid residues from other species such that the segment is derived from a precursor protein that does not contain the amino acid residues comprising a functional membrane spanning region 20 nor is the -segment derived from a precursor protein in which the sequence of amino acid residues from the beginning of the membrane spanning region (approximately residue 630 of rabbit polyimmunoglobulin receptor) to the carboxyl end of the protein (approximately residue 755 of the rabbit polyimmunoglobulin receptor) are fully intact.

In preferred embodiments the precursor protein does not contain amino acid residues greater than 606 of the rabbit polyimmunoglobulin receptor or analogous amino acid residues from other species.

As is understood by those of ordinary skill in the art, a membrane spanning region or functional transmembrane segment consists of a contiguous section of amino acid residues containing from about 20 to about amino acids in which none of the residues is charged, virtually all of the residues are hydrophobic or non-polar, and the segment forms an alpha helix. A functional transmembrane segment is capable of spanning a biomembrane. Membrane spanning regions can be bounded by charged residues. An example of a membrane spanning region of pIgR is residues 630 to 653 of the polyimmunoglobulin receptor amino acid residue sequence of rabbit.

The chains that comprise the immunoglobulin containing the protection protein may be derived from precursors containing a signal sequence at the amino terminal of the protein. Each component can thereby be synthesized into an endomembrane system where assembly occurs. In addition to a signal sequence, the various components of the complex may or may not contain additional signals for N terminal glycosylation or for Svarious other modifications which can affect the structure of the complex. In one embodiment of the invention, the signals for glycosylation asparagine-X-serine or threonine or the signals for O-linked glycosylation) are not present or present in more or less places within the nucleotide sequence. The resulting antibody therefore 20 would contain no carbohydrate, which may be advantageous for applications in which carbohydrates elicit an immune response.

In preferred embodiments, the immunoglobulin of the present invention contains a protection protein associated with an immunoglobulin derived heavy chain and the protection protein is free from N-linked and/or O-linked oligosaccharides. One skilled in the art will understand that a gene coding for a polypeptide having within its amino acid residue sequence the N-linked glycosylation signal asparagine-X-serine/threonine where X can be any amino acid residue except possibly proline and aspartic acid, when introduced into a plant cell would be glycosylated via oligosaccharides linked to the asparagine residue of the sequence (N-linked). See, Marshall, Ann.

Rev. Biochem., 41:673 (1972) and Marshall, Biochem. Soc.

Svmp., 40:17 (1974) for a general review of the polypeptide sequences that function as glycosylation signals. These signals are recognized in both mammalian and in plant cells. One skilled in the art will understand that the N-linked glycosylation signal may be easily removed using common mutagenesis procedures to change the DNA sequence encoding the protection protein of the present invention. This mutagenesis typically involves the synthesis of oligonucleotide having the Nlinked glycosylation signal deleted and then preparing a DNA strand with that oligonucleotide sequence incorporated into it. Such mutagenesis procedures and reagents are commercially available from many sources such as Stratagene (La Jolla, CA.).

Assembly of the individual polypeptides that form a multi-peptide molecule (for example immunoglobulin) may be obtained by expressing in a single cell by directly introducing all the transgenes encoding the individual polypeptides into that cell either sequentially or all at once. The transgenes encoding the polypeptides may be present on individual constructs or DNA segments or may be 20 contained in a DNA segment or construct together with one or more other transgenes.

Assembly of these components can be by cross pollination as originally described by Mendel to produce .a population of segregants expressing all chains.

Previous disclosures have demonstrated this to be an adequate method for the assembly and co-segregation of multimeric glycoconjugates. The disclosure of U.S. Patent No. 5,202,422 is hereby incorporated by reference and describes these methods. In a preferred embodiment of the present invention, the antibody molecules contain a reduced number of glycans and antibody molecules with no glycans are contemplated.

The immunoglobulins of the present invention containing the protection protein, the immunoglobulin derived heavy chain and optionally an immunoglobulin derived light chain, and J chain may contain a protection protein that is free from N-linked oligosaccharides.

The immunoglobulins of the present invention that contain the protection protein are preferably therapeutic immunoglobulins that are useful in preventing a disease in an animal. In preferred embodiments, the immunoglobulins of the present invention are therapeutic immunoglobulins which are capable of binding to mucosal pathogen antigens.

In other preferred embodiments, the therapeutic immunoglobulins of the present invention are capable of preventing dental caries. In the most preferred embodiment, the immunoglobulin of the present invention containing the protection protein contains an antigen binding domain that is capable of binding to an antigen from S. mutans serotypes a, c, d, e, f, g and h mutans c, e and f and S. sobrinus serotypes d and g under new nomenclature). Such antigen binding domains are known in the art and include, for example, the binding domains described in U.S. Patent 5,352,446, J. K-C. Ma et al., Clin. Exp. Immunol. 77:331 (1989); and J. K-C. Ma et al., Eur. J. Immunol. 24:131-138 (1994); U.S. Patent 5,352,446; 20 U.S. Patent 4,594,244; and European Patent Publication 371 017 Bl. The disclosures of these documents are hereby incorporated by reference. In preferred embodiments, the immunoglobulins of the present invention are part of a composition that has a therapeutic activity on either animals or humans. Examples of therapeutic immunoglobulins are numerous, however, we envision the most appropriate therapeutic effect to be prophylaxis for mucosal and enteric pathogens by direct oral administration of the composition derived from an edible plant.

Administration of the therapeutic composition can be before or after extraction from the plant or other transgenic organism. Once extracted the immunoglobulins may also be further purified by conventional techniques such as size exclusion, ion exchange, or affinity chromatography. In the preferred embodiment, the transgenic organism is an edible plant and administration of the complex is by ingestion after partial purification.

Plant molecules may be co-administered with the complex.

The present invention also contemplates that the relative proportion of plant-derived molecules and animalderived molecules can vary. Quantities of specific plant proteins, such as RuBisCo, or chlorophyll may be as little as 1% of the mass or as much as 99.9% of the mass of the extract, excluding water.

The present invention also contemplates the use of the therapeutic plant extract containing immunoglobulins having a protection protein directly without any further purification of the specific therapeutic component, e.g.

the antibody. Administration may be by topical application, oral ingestion or any other method 15 appropriate for delivering the antibody to the mucosal target pathogen. This form of administration is distinct oo o S• from parenteral applications involving direct injection or comingling of the therapeutic plant extract with the blood stream.

20 The present invention also contemplates the use of ••co the therapeutic plant extract containing immunoglobulins having a protection protein after manipulating the taste or texture of the extract. Appropriate quantities of gelling substances or flavorings could be added to enhance the contact of the antibody with the target pathogen in, .for example, direct oral applications.

In preferred embodiments, the immunoglobulins of the present invention are used to passively immunize an animal against a preselected ligand by contacting a composition comprising an immunoglobulin containing a protection protein of the present invention that is capable of binding a preselected ligand with a mucosal surface of an animal. Passive immunization requires large amounts of antibody and for wide-spread use this antibody must be inexpensive.

Immunoglobulin molecules containing protection proteins that are capable of binding a preselected antigen can be efficiently and economically produced in plant cells. In preferred embodiments, the immunoglobulin molecule is either IgA, IgM, secretory IgM or secretory IgA or an immunoglobulin having a chimeric immunoglobulin heavy or light chain.

The immunoglobulins containing protection proteins are more resistant to proteolysis and denaturation and therefore are desirable for use in harsh environments.

Contemplated harsh environments include acidic environments, protease containing environments, high temperature environments, and other harsh environments.

For example, the gastrointestinal tract of an animal is a harsh environment where both proteases and acid are present. See, Kobayashi et al., Immunochemistry, 10:73 15 (1973).

Passive immunization of the animal using these more resistant immunoglobulins of the present invention is produced by contacting the immunoglobulin containing the protection protein with a mucosal surface of the animal.

20 Animals have various mucosal surfaces including the lungs, the digestive tract, the nasopharyngeal cavity, the urogenital system, and the like. Typically, these mucosal surfaces contain cells that produce various secretions including saliva, lacrimal fluid, nasal fluid, tracheobronchial fluid, intestinal fluid, bile, cervical fluid, and the like.

In preferred embodiments the immunoglobulins that contain the protection protein are immunospecific for a preselected antigen. Typically, this antigen is present on a pathogen that causes a disease that is associated with the mucosal surface such as necrotizing enterocolitis, diarrheal disease, ulcers, and cancer caused by carcinogen absorption in the intestine. See McNabb and Tomasi, Ann. Revl. Microbiol., 35:477 (1981) and Lawrence et al., Science, 243:1462 (1989).

Typical pathogens that cause diseases associated with a mucosal surface include both bacterial and viral pathogens, such as E. coli., S. typhimurium, V. cholera, H. pylori, and S. mutans. See also, European Patent Application 484, 148 Al, published 5/6/92 and hereby incorporated by reference. The immunoglobulins of the present invention are capable of binding to these pathogens and preventing them from causing mucosal associated diseases.

Immunoglobulins capable of binding to S. mutans and preventing dental caries have been described in European Patent Specification 371,017 which is hereby incorporated by reference. The disclosure of U.S. Patent No. 5,352,440 is also hereby incorporated by reference.

Therapeutic immunoglobulins of the present invention oo.. that contain protection proteins that would be effective against bacterial infection or carcinomas are contemplated. Monoclonal antibodies with therapeutic activity have been described in U.S. Patents 4,652,448, 4,443,549 and 5,183,756 which are hereby incorporated by reference.

20 In preferred embodiments, the immunoglobulin of the invention are part of a composition which is contacted with the animal mucosal surface comprises plant material and an immunoglobulin of the present invention that is capable of binding a preselected ligand. The plant material present may be plant cell walls, plant organelles, plant cytoplasms, intact plant cells, viable plants, and the like. This plant cell material is present in a ratio from about 10,000 grams of plant material to about 100 nanograms of immunoglobulin to about 100 nanograms of plant material for each 10 grams of immunoglobulin present. In more preferred embodiments, the plant material is present in a ratio from about 10,000 grams of plant material for each 1 gram of immunoglobulin present to about a ratio of 100 nanograms of plant material present for each gram of immunoglobulin present.

In other preferred embodiments, the plant material is present in a ratio from about 10,000 grams of plant material for each milligram of immunoglobulin present to about 1 milligram of plant material present for each 500 milligram of immunoglobulin present.

In preferred embodiments, the composition containing the immunoglobulins of the present invention is a therapeutic composition. The preparation of therapeutic compositions which contain polypeptides or proteins as active ingredients is well understood in the art.

Therapeutic compositions may be liquid solutions or suspensions, solid forms suitable for solution in, or suspension in a liquid prior to ingestion may also be prepared. The therapeutic may also be emulsified. The active therapeutic ingredient is typically mixed with o oo inorganic and/or organic carriers which are pharmaceutically acceptable and compatible with the active ingredient. The carriers are typically physiologically oo acceptable excipients comprising more or less inert substances when added to the therapeutic composition to confer suitable consistencies and form to the composition.

oe..

S. 20 Suitable carriers are for example, water, saline, dextrose, glycerol, and the like and combinations thereof.

In addition, if desired the composition can contain minor amounts of auxiliary substances such as wetting or emulsifying agents and pH buffering agents which enhance the effectiveness of the active ingredient. Therapeutic compositors containing carriers that have nutritional value are also contemplated.

In embodiments in which a composition containing an immunoglobulin having a protection protein of the present invention is applied to the tooth or mouth of a mammal, any convenient method may be used. Methods for applying such a composition to the teeth are well known and utilize various materials for a variety of purposes. For example, the composition may be directly applied to the tooth by painting the surface of the tooth with that composition.

Alternatively, the composition of the present invention may be included in a toothpaste, mouthwash, chewing gum, lozenge or gel that will result in it being applied to the teeth. In some formulations, it may be desirable to provide for a formulation that prolongs the contact of the composition and therefore the immunoglobulin having the protection protein with the tooth surface. Formulations for this purpose are well known and include such formulations that may be placed in various dental trays that are used to cover the tooth and other dental apparatuses that are used in adjusting various conditions with the teeth.

The exact amount of a composition that must be applied to the teeth during any particular application is not critical because such treatment may be easily repeated at a given interval. For example, compositions present in S* 15 toothpaste would be applied to the teeth each time that toothpaste is used, typically twice per day. For example, *the order of 10 to 100 micrograms of an immunoglobulin having a protection protein can be applied to each tooth on each occasion the composition is applied to the teeth.

20 However, this in no way should be taken as a limitation on a range that may be applied during any particular application as applications of a composition having more or less immunoglobulin of the present invention may be used without detrimental effect. The use of much lower concentrations of an immunoglobulin of the present invention would result in, at some point, a reduction in the protection provided by such formulation.

The exact formulation for the composition of the present invention may vary and will depend on the method of application to be used and the frequency of that application. In general, it may be any formulation which has an appropriate pH and which is free of material which would render the immunoglobulin having the protection protein of the present invention ineffective. For example, the compositions of the present invention may be applied as a simple aqueous solution in which the composition is disbursed at anywhere from 0.1 to milligrams of immunoglobulin per 100 microliters of that solution. Generally, such a solution would be applied during dental surgery at a rate of approximately 1 to microliters of the solution per tooth.

The formulations of the compositions of the present invention which are designed to be self-administered may vary and will be formulated taking in to account the frequency of application of the particular product in which is it used.

In preferred embodiments, a composition containing an immunoglobulin of the present invention comprises an immunoglobulin molecule that is immunospecific for a pathogen antigen. Pathogens are any organism that causes ~a disease in another organism. Particularly preferred are immunoglobulins that are immunospecific for a mucosal pathogen antigen. A mucosal pathogen antigen is present on a pathogen that invades an organism through mucosal tissue or causes mucosal associated diseases. Mucosal pathogens include lung pathogens, nasal pathogens, 20 intestinal pathogens, oral pathogens, and the like. For a general discussion of pathogens, including mucosal pathogens, see, Davis et al., Microbioloqv, 3rd ed., Harper and Row, Hagerstown, MD (1980).

Antibodies immunospecific for a pathogen may be produced using standard monoclonal antibody production techniques. See, Antibodies: A Laboratory Manual, Harlow et al., eds., Cold Spring Harbor, NY (1988). The genes coding for the light chain and heavy chain variable regions can then be isolated using the polymerase chain reaction and appropriately selected primers. See, Orlandi et al., Proc. Natl. Acad. Sci., 86:3833 (1989) and Huse et al., Science, 246:1275 (1989). The variable regions are then inserted into plant expression vectors, such as the expression vectors described by Hiatt et al., Nature, 342:76-78 (1989).

In a preferred embodiment, the immunoglobulin of the present invention is immunospecific for an intestinal pathogen antigen. Particularly preferred are immunoglobulins immunospecific for intestinal pathogens such as bacteria, viruses, and parasites that cause disease in the gastrointestinal tract, such as E. coli, Salmonellae, Vibrio cholerae, Salmonellae typhimurium, Shigella and H. pylori.

In other preferred embodiments, the immunoglobulin containing the protection protein present in the composition is an immunoglobulin molecule that is immunospecific for a dental pathogen such as Streptococcus mutans and the like. Particularly preferred are immunoglobulins immunospecific for a Streptococcus mutans antigen such as the immunoglobulin produced by hybridoma ~15B2 (ATCC No. HB 8510); the hybridoma deposited as European Collection of Animal cells Deposit No. 86031901; and the Guy's 13 monoclonal antibody described by Ma et Eur. J. Immunol., 24:131 (1994) and Smith and Lehner, Oral Micro. Immunol., 4:153 (1989).

The present invention contemplates producing passive 20 immunity in an animal, such as-vertebrate. In preferred embodiments, passive immunity is produced in fish, birds, reptiles, amphibians, or insects. In other preferred embodiments passive is produced in an mammal, such as a human, a domestic animal, such as a ruminant, a cow, a pig, a horse, a dog, a cat, and the like. In particularly preferred embodiments, passive immunity is produced in an adult or child mammal.

In preferred embodiments, passive immunity is produced in an animal, such as a mammal that is weaned and therefore no longer nurses to obtain milk from its mother.

Passive immunity is produced in such an animal by administering to the animal a sufficient amount of composition containing an immunoglobulin containing a protection protein immunospecific for a preselected ligand to produce a prophylactic concentration of the immunoglobulin within the animal. A prophylactic concentration of an immunoglobulin is an amount sufficient to bind to a pathogen present and prevent that pathogen from causing detectable disease within the animal. The amount of composition containing the immunoglobulin of the present invention required to produce a prophylactic concentrations will vary as is well known in the art with the size of the animal, the amount of pathogen present, the affinity of the particular immunoglobulin for the pathogen, the efficiency with which the particular immunoglobulin is delivered to its active location within the animal, and the like.

C. Eukaryotic Cells Containing ImmunoQlobulins Having A Protection Protein The present invention contemplates eukaryotic cells, 15 including plant cells, containing immunoglobulins of the present invention. The present invention also *contemplates plant cells that contain nucleotide sequences encoding the various components of the immunoglobulins of the present invention. One skilled in the art will 4c 20 understand that the-nucleotide sequences that encode the protection protein and the various immunoglobulin heavy and light chains and J chain will typically be operably linked to a promoter and present as part of an expression -vector or cassette.

After the immunoglobulin heavy and light chain genes, and J chain genes are isolated, they are typically operatively linked to a transcriptional promoter in an expression vector.

Expression of the components in the organism of choice can be derived from an independently replicating plasmid, or from a permanent component of the chromosome, or from any piece of DNA which may transiently give rise to transcripts encoding the components. Organisms suitable for transformation can be either prokaryotic or eukaryotic. Introduction of the components of the complex can be by direct DNA transformation, by ballistic delivery into the organism, or mediated by another organism as for example by the action of recombinant Agrobacteria on plant cells. Expression of proteins in transgenic organisms usually requires co-introduction of an appropriate promoter element and polyadenylation signal. In one embodiment of the invention, the promoter element potentially results in the constitutive expression of the components in all of the cells of a plant. Constitutive expression occurring in most or all of the cells will ensure that precursors can occupy the same cellular endomembrane system as might be required for assembly to occur.

Expression vectors compatible with the host cells, preferably those compatible with plant cells are used to Sexpress the genes of the present invention. Typical 0 15 expression vectors useful for expression of genes in plants are well known in the art and include vectors derived from the tumor-inducing (Ti) plasmid of Aqrobacterium tumefaciens described by Rogers et al., Meth. in Enzvmol., 153:253-277 (1987). However, several other expression vector systems are known to function in plants. See for example, Verma et al., PCT Publication No. W087/00551; and Cocking and Davey, Science, 236:1259- 1262 (1987).

The expression vectors described above contain expression control elements including the promoter. The genes to be expressed are operatively linked to the *Vso expression vector to allow the promoter sequence to direct RNA polymerase binding and synthesis of the desired polypeptide coding gene. Useful in expressing the genes are promoters which are inducible, viral, synthetic, constitutive, and regulated. The choice of which expression vector and ultimately to which promoter a nucleotide sequence encoding part of the immunoglobulin of the present invention is operatively linked depends directly, as is well known in the art, on the functional properties desired, e.g. the location and timing of protein expression, and the host cell to be transformed, 46 these being limitations inherent in the art of constructing recombinant DNA molecules. However, 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.

In preferred embodiments, the expression vector used to express the genes includes a selection marker that is effective in a plant cell, preferably a drug resistance selection marker. A preferred drug resistance marker is the gene whose expression results in kanamycin resistance, the chimeric gene containing the nopaline synthase promoter, Tn5 neomycin phosphotransferase II and nopaline synthase 3' nontranslated region described by Rogers et al., in Methods For Plant Molecular BioloQy, a Weissbach and H. Weissbach, eds., Academic Press Inc., San Diego, CA (1988). A useful plant expression vector is commercially available from Pharmacia, Piscataway, NJ.

20 Expression vectors and promoters for expressing foreign proteins in plants have been described in U.S.

Patent Nos. 5,188,642; 5,349,124; 5,352,605, and 5,034,322 which are hereby incorporated by reference.

A variety of methods have been developed to operatively link DNA to vectors via complementary cohesive termini. For instance, complementary homopolymer tracks :can be added to the DNA segment to be inserted and to the vector DNA. The vector and DNA segment are then joined by hydrogen bonding between the complementary homopolymeric tails to form recombinant DNA molecules.

Alternatively, synthetic linkers containing one or more restriction endonuclease sites can be used to join the DNA segment to the expression vector. The synthetic linkers are attached to blunt-ended DNA segments by incubating the blunt-ended DNA segments with a large excess of synthetic linker molecules in the presence of an enzyme that is able to catalyze the ligation of blunt- 47 ended DNA molecules, such as bacteria phage T4 DNA ligase.

Thus, the products of the reaction are DNA segments carrying synthetic linker sequences at their ends. These DNA segments are then cleaved with the appropriate restriction endonuclease and ligated into an expression vector that has been cleaved with an enzyme that produces termini compatible with those of the synthetic linker.

Synthetic linkers containing a variety of restriction endonuclease sites are commercially available from a number of sources including New England BioLabs, Beverly,

MA.

The nucleotide sequences encoding the protection protein and any other of the immunoglobulins of the present invention are introduced into the same plant cell S: 15 either directly or by introducing each of the components into a plant cell and regenerating a plant and crosshybridizing the various components to produce the final plant cell containing all the required components.

Any method may be used to introduce the nucleotide sequences encoding the components of the immunoglobulins of the present invention into a eukaryotic cell. For example, methods for introducing genes into plants include Aarobacterium-mediated plant transformation, protoplast transformation, gene transfer into pollen, injection into reproductive organs and injection into immature embryos.

Each of these methods has distinct advantages and .disadvantages. Thus, one particular method of introducing genes into a particular eukaryotic cell or plant species may not necessarily be the most effective for another eukaryotic cell or plant species.

Aqrobacterium tumefaciens-mediated transfer is a widely applicable system for introducing genes into plant cells because the DNA can be introduced into whole plant tissues, bypassing the need for regeneration of an intact plant from a protoplast. The use of Aqrobacteriummediated expression vectors to introduce DNA into plant cells is well known in the art. See, for example, the methods described by Fraley et al., Biotechnology, 3:629 (1985) and Rogers et al., Methods in Enzymology, 153:253- 277 (1987). Further, the integration of the Ti-DNA is a relatively precise process resulting in few rearrangements. The region of DNA to be transferred is defined by the border sequences and intervening DNA is usually inserted into the plant genome as described by Spielmann et al., Mol. Gen. Genet., 205:34 (1986) and Jorgensen et al., Mol. Gen. Genet., 207:471 (1987).

Modern Agrobacterium transformation vectors are capable of replication in Escherichia coli as well as Aqrobacterium, allowing for convenient manipulations as described by Klee et al., in Plant DNA Infectious Agents, T. Hohn and J.

Schell, eds., Springer-Verlag, New York (1985) pp. 179- 15 203. Further recent technological advances in vectors for Aarobacterium-mediated gene transfer have improved the arrangement of genes and restriction sites in the vectors to facilitate construction of vectors capable of expressing various polypeptide coding genes. The vectors 20 described by Rogers et al., Methods in Enzymoloqy, 153:253 (1987), have convenient multi-linker regions flanked by a promoter and a polyadenylation site for direct expression of inserted polypeptide coding genes and are suitable for present purposes.

Acrobacterium-mediated transformation of leaf disks and other tissues appears to be limited to plant species that Aqrobacterium tumefaciens naturally infects. Thus, Agrobacterium-mediated transformation is most efficient in dicotyledonous plants. However, the transformation of Asparagus using Agrobacterium can also be achieved. See, for example, Bytebier, et al., Proc. Natl. Acad. Sci., 84:5345 (1987).

In those plant species where Agrobacterium-mediated transformation is efficient, it is the method of choice because of the facile and defined nature of the gene transfer. However, few monocots appear to be natural hosts for Agrobacterium, although transgenic plants have been produced in asparagus using Acrobacterium vectors as described by Bytebier et al., Proc. Natl. Acad. Sci.

84:5345 (1987). Therefore, commercially important cereal grains such as rice, corn, and wheat must be transformed using alternative methods. Transformation of plant protoplasts can be achieved using methods based on calcium phosphate precipitation, polyethylene glycol treatment, electroporation, and combinations of these treatments. See, for example, Potrykus et al., Mol. Gen.

Genet., 199:183 (1985); Lorz et al., Mol. Gen. Genet., 199:178 (1985); Fromm et al. Nature, 319:791 (1986); Uchimiya et al., Mol. Gen. Genet., 204:204 (1986); Callis et al., Genes and Development, 1:1183 (1987); and Marcotte et al., Nature, 335:454 (1988).

Application of these systems to different plant species depends upon the ability to regenerate that particular plant species from protoplasts. Illustrative methods for the regeneration of cereals from protoplasts are described in Fujimura et al., Plant Tissue Culture Letters, 2:74 (1985); Toriyama et al., Theor App1. Genet., 73:16 (1986); Yamada et al., Plant Cell Rep., 4:85 (1986); Abdullah et al., Biotechnology, 4:1087 (1986).

To transform plant species that cannot be successfully regenerated from protoplast, other ways to introduce DNA into intact cells or tissues can be utilized. For example, regeneration of cereals from immature embryos or explants can be effected as described by Vasil, Biotechnolocv, 6:397 (1988). In addition, "particle gun" or high-velocity microprojectile technology can be utilized as well. Using such technology, DNA is carried through the cell wall and into the cytoplasm on the surface of small (0.525 um) metal particles that have been accelerated to speeds of one to several hundred meters per second as described in Klein et al., Nature, 327:70 (1987); Klein et al., Proc. Natl. Acad. Sci.

85:8502 (1988); and McCabe et al., Biotechnology, 6:923 (1988). The metal particles penetrate through several layers of cells and thus allow the transformation of cells within tissue explants. Metal particles have been used to successfully transform corn cells and to produce fertile, stably transformed tobacco and soybean plants. Transformation of tissue explants eliminates the need for passage through a protoplast stage and thus speeds the production of transgenic plants.

DNA can be introduced into plants also by direct DNA transfer into pollen as described by Zhou et al., Methods in Enzymoloqc, 101:433 (1983); D. Hess, Intern Rev.

Cytol., 107:367 (1987); Luo et al., Plant Mol. Biol.

Reporter, 6:165 (1988). Expression of polypeptide coding genes can be obtained by injection of the DNA into reproductive organs of a plant as described by Pena et al., Nature, 325:274 (1987). DNA can also be injected directly into the cells of immature embryos and the rehydration of desiccated embryos as described by Neuhaus et al., Theor. Apl. Genet., 75:30 (1987); and Benbrook et al., in Proceedings Bio Expo 1986, Butterworth, Stoneham, 20 MA, pp. 27-54 (1986) The regeneration of plants from either single plant protoplasts or various explants is well known in the art.

See, for example, Methods for Plant Molecular Biology, A.

q Weissbach and H. Weissbach, eds., Academic Press, Inc., San Diego, CA (1988) This regeneration and growth process includes the steps of selection of transformant cells and shoots, rooting the transformant shoots and growth of the plantlets in soil.

The regeneration of plants containing the foreign gene introduced by Agrobacterium tumefaciens from leaf explants can be achieved as described by Horsch et al., Science, 227:1229-1231 (1985). In this procedure, transformants are grown in the presence of a selection agent and in a medium that induces the regeneration of shoots in the plant species being transformed as described by Fraley et al., Proc. Natl. Acad. Sci. 80:4803 (1983). This procedure typically produces shoots within two to four weeks and these transformant shoots are then transferred to an appropriate root-inducing medium containing the selective agent and an antibiotic to prevent bacterial growth. Transformant shoots that rooted in the presence of the selective agent to form plantlets are then transplanted to soil to allow the production of roots. These procedures will vary depending upon the particular plant species employed, such variations being well known in the art.

The immunoglobulins of the present invention may be produced in any plant cell including plant cells derived from plants that are dicotyledonous or monocotyledonous, solanaceous, alfalfa, legumes, or tobacco.

Transgenic plants of the present invention can be 15 produced from any sexually crossable plant species that can be transformed using any method known to those skilled in the art. Useful plant species are dicotyledons including tobacco, tomato, the legumes, alfalfa, oaks, and maples; monocotyledons including grasses, corn, grains, 20 oats, wheat, and barley; and lower plants including gymnosperms, conifers, horsetails, club mosses, liver warts, horn warts, mosses, algaes, gametophytes, sporophytes of pteridophytes.

The plant cells of the present invention may in addition to the protection protein and the immunoglobulin derived heavy chain also contains a nucleotide sequence encoding an immunoglobulin derived light chain having at least a portion of an antigen binding domain.

The plant cells of the present invention may have an antigen binding domain that is capable of binding an antigen from S. mutans serotypes a, c, d, e, f, g, and h mutans serotypes c, e, and f; and S. sobrinus serotypes d and g under new nomenclature) on the immunoglobulin derived heavy and light chains. T h e antigen binding domain present in these plant cells also can be able to bind to the responsible mucosal pathogens and prevent dental caries.

The plant cells of the present invention may be part of a plant and make up one of the following types of plants: dicotyledonous, monocotyledonous, solanaceous, alfalfa, tobacco or other type of plant.

D. Compositions Containing Immunoglobulins Having Protection Proteins The present invention contemplates compositions of matter that comprise immunoglobulins of the present invention and plant macromolecules. Typically these plant macromolecules are derived from any plant useful in the present invention. The plant macromolecules are present together with an immunoglobulin of the present invention for example, in a plant cell, in an extract of a plant 15 cell, or in a plant. Typical plant macromolecules associated with the immunoglobulins of the present invention in a composition are ribulose bisphosphate carboxylase, light harvesting complex, (LH6) pigments, secondary metabolites or chlorophyll. The compositions of 20 the present invention have an immunoglobulin of the present invention present in a concentration of between 1% and 99% mass excluding water. Other preferred compositions include compositions having the immunoglobulins of the present invention present at a concentration of between 1% and 50% mass excluding water.

Other preferred compositions include immunoglobulins at a concentration of 1% to 25% mass excluding water.

The compositions of the present invention contain plant macromolecules at a concentration of between 1% and 99% mass excluding water. Typically the mass present in the composition will consist of plant macromolecules and immunoglobulins of the present invention. When the immunoglobulins of the present invention are present at a higher or lower concentration the concentration of plant macromolecules present in the composition will vary inversely. In preferred embodiments the composition of plant macromolecules are present in a concentration of between 50% and 99% mass excluding water. In the most preferred compositions, the plant macromolecules are present in a concentration of between 75% and 99% mass excluding water.

The present invention contemplates a composition of matter comprising all or part of the following: an IgA heavy chain, a kappa or lambda chain, a J chain. These components form a complex and are attached to the protection protein as defined earlier. The composition also contains molecules derived from a plant. This composition may also be obtained after an extraction process yielding functional antibody and plant-derived molecules.

The extraction method comprises the steps of applying 15 a force to a plant containing the complex whereby the apoplastic compartment of the plant is ruptured releasing said complex. The force involves shear, in dyn/cm2, as the primary method of releasing the apoplastic liquid.

The whole plant or plant extract contains an admixture of antibody and various other macromolecules of the plant. Among the macromolecules contained in the admixture is ribulose bisphosphate carboxylase (RuBisCo) or fragments of RuBisCo. Another macromolecule is LHCP.

Another molecule is chlorophyll.

Shear force is a useful component of the overall force applied to the plant for disruption of apoplastic spaces. Other types of force may also be included to optimize the effects of shear. Direct pressure, for example, measured in lbs/in2, may enhance the effects of the apparatus used to apply shear. Commonly used homogenization techniques which are not appropriate for antibody extraction involve the use of high speed blades or cylinders which explosively destroy all plant structures.

The compositions of the present invention may contain an immunoglobulin of the present invention and plant molecules that are derived from a dicotyledonous, monocotyledonous, solanaceous, alfalfa, tobacco or other plant. The plant molecules present in the compositions of the present invention can be ribulose bisphosphate carboxylase, light harvesting complex, pigments, secondary metabolites, chlorophyll or other plant molecules.

Other useful methods for preparing composition containing immunoglobulins having protection protein include extraction with various solvents and application of vacuum to the plant material. The compositions of the present invention may contain immunoglobulins of the present in a concentration of between 1% and 99% mass excluding water. The compositions of the present invention may contain plant macromolecules in a concentration of between 1% and 99% mass excluding water.

15 Therapeutic compositions containing immunoglobulins of the present invention and plant macromolecules may be produced by processing a plant of the present invention by shearing under pressure a portion of that plant to produce a pulp containing the therapeutic immunoglobulin and plant *20 macromolecules in a liquid derived from the apoplast or symplast of the plant which also contains the solid plant derived material. Further processing may be accomplished by separating the solid plant derived material from the plant derived liquid containing the immunoglobulins of the present invention. The starting material for such a S. process may include plant leaves, stem, roots, tubers, seeds, fruit or the entire plant. Typically, this processing is accomplished by a mechanical device which releases liquid from the apoplast or symplast of the plant. Additional processing steps may include separation of the solid plant derived material from the liquid using centrification settling flocculation or filtration. One skilled in the art will understand that these separation methods result in removing the solid plant derived material from the liquid including the immunoglobulins of the present invention. The methods of the present invention may produce immunoglobulins containing a protection protein and an immunoglobulin derived heavy chain that is comprised of domains or portions of immunoglobulin alpha chain and immunoglobulin gamma chain.

The methods of the present invention may produce immunoglobulins containing a protection protein and an immunoglobulin derived light chain that is comprised of domains or portions of immunoglobulin kappa or lambda chain.

The methods of the present invention are operable on plant cells or part of a plant. The methods of the present invention may also included methods that further comprise growing the plant. The methods of the present invention may be applied to any plant including dicotyledonous, monocotyledonous, solanaceous, leguminous, 15 alfalfa or tobacco plant. The methods of the present *invention may be used to extract immunoglobulins from a Sportion of the plant such as a leaf, stem, root, tuber, seeds, fruit or entire plant. The methods of the present invention may use a mechanical device to shear the plants 20 to release liquid from the apoplast or symplast of the plant. The plant pulp of the present invention may be separated to remove the solid plant material using one of o*o the following methods: centrifugation, settling, flocculation or filtration.

E. Methods of Producing Immunoqlobulins Containing Protection Proteins The present invention contemplates methods of producing an immunoglobulin containing a protection protein comprising the steps of: Introducing into the plant cell an expression vector containing a nucleotide sequence encoding a protection protein operatively linked to a transcriptional promoter; and Introducing into the same plant cell an expression vector containing a nucleotide sequence encoding an immunoglobulin derived heavy chain having at least a portion of an antigen binding domain operatively linked to a transcriptional promoter.

The methods of the present invention optionally include introducing into the plant cell containing the expression vector with the nucleotide sequences for the protection protein and the immunoglobulin derived heavy chain a nucleotide sequence encoding an immunoglobulin derived light chain at least having a portion of an antigen binding domain operatively linked to a transcriptional promoter. Methods are also contemplated that introduce into a cell that already contains nucleotide sequences and promoters operatively linked to encode a protection protein and an immunoglobulin heavy chain and an immunoglobulin light chain, a promoter 15 operatively linked to a nucleotide sequence encoding J chain. This results in a cell containing the nucleotide sequences operatively linked to promoters for an immunoglobulin heavy chain and an immunoglobulin light chain, J chain and a protection protein.

20 The plant cells of the present invention may be present as part of a plant that is capable of growth.

Particularly useful plants for this invention include dicotyledonous, monocotyledonous, solanaceous, legumes, alfalfa, tomato, and tobacco plants.

The methods of the present invention include producing an assembled immunoglobulin having heavy, light and J chains and a protection protein within a eukaryotic cell. This eukaryotic cell is produced by introducing into that cell nucleotide sequences operatively linked for expression encoding an immunoglobulin derived heavy chain having at least a portion of an antigen binding domain, an immunoglobulin derived light chain having at least a portion of an antigen binding domain, an immunoglobulin J chain, and a protection protein. These nucleotide sequences are operatively linked for expression by attaching appropriate promoters to each individual nucleotide sequence or to more than one nucleotide sequence thereby placing two nucleotide sequences encoding various molecules in tandem.

The eukaryotic cell produced by the present methods which contains these nucleotide sequences encoding the immunoglobulin heavy, light and J chains and the protection protein is maintained under conditions which allow those molecules to reproduce and assemble into an immunoglobulin which contains the protection proteins of the present invention.

The present invention also contemplates methods for making a particular immunoglobulin or antigen binding domain or domains of an immunoglobulin resistant to environmental conditions and more stable by operatively linking a nucleotide sequence encoding at least a portion 15 of an antigen binding domain derived from an immunoglobulin heavy chain to a nucleotide sequence encoding at least one domain derived from an immunoglobulin a or j heavy chain to form a nucleotide sequence encoding a chimeric immunoglobulin heavy chain.

20 That nucleotide sequence encoding the chimeric immunoglobulin heavy chain is expressed in a eukaryotic cell which also contains at least one other molecule such as a protection protein, an immunoglobulin derived light chain having at least a portion of an antigen binding domain and an immunoglobulin J chain. In preferred embodiments, the cell contains all of the molecules including an immunoglobulin derived light chain having an antigen binding domain which is complementary to the antigen binding domain present on the immunoglobulin derived heavy chain. This method allows the chimeric immunoglobulin heavy chain to assemble with at least one other molecule, for example, the immunoglobulin derived light chain having the complementary antigen binding domain and an immunoglobulin J chain and the protection protein to form an immunoglobulin containing the protection protein which is resistant to environmental conditions.

These immunoglobulins are resistant to environmental conditions and thus more stable when subjected to elevated or reduced temperatures, high or low pH, high ionic or low ionic concentrations proteolytic enzymes and other harsh conditions. Such harsh conditions are typically found in the environment within natural water sources, within the human body, for example within the gut and on mucosal surfaces, and on the surface of an animal such as a mammal.

F. Chimeric Immunoqlobulins Containing Protection Proteins The present invention contemplates immunoglobulins containing a protection protein in which the 15 immunoglobulin domains comprising the heavy and light chain are derived from different isotopes of either heavy or light chain immunoglobulins. One skilled in the art will understand that using molecular techniques these domains can be substituted for a similar domain and thus 20 produce an immunoglobulin that is a hybrid between two different immunoglobulin molecules. These chimeric immunoglobulins allow immunoglobulins containing S..protection proteins to be constructed that contain a variety of different and desirable properties that are conferred by different immunoglobulin domains.

The present invention also contemplates chimeric immunoglobulins, including heavy, light and J chain which contain less than an entire domain derived from a different molecule. The same molecular techniques may be employed to produce such chimeric immunoglobulins.

In preferred embodiments, the immunoglobulins of the present invention contain at least the CHl, C,2, C,3, domain of mouse IgG, IgG1, IgG2A, IgG2B, IgG3, IgA, IgE, or IgD.

Other preferred embodiments of the present invention contain immunoglobulin domains that include at least the CAl, Cy2, CA3, or Ci4 domain of mouse IGM. Preferred immunoglobulins include immunoglobulins that contain the domains of Ce2, CE3, and CE4 of mouse immunoglobulin IGE.

The present invention also contemplates chimeric immunoglobulins derived from human immunoglobulins. These chimeric immunoglobulins contain domains from two different isotopes of human immunoglobulin. Preferred immunoglobulins include immunoglobulins that contain immunoglobulin domains including at least the CH1, CH 2 or C,3 of human IgG, IgG1, IgG2, IgG3, IgG4, IgAl, IgA2, IgE, or IgD. Other preferred immunoglobulins include immunoglobulins that contain domains from at least the C,1, CH2, Cy3, or C,4 domain of human IgM or IgE. The present invention also contemplates immunoglobulins that contain immunoglobulin domains derived from at least two different 15 isotopes of mammalian immunoglobulins. Generally, any of the mammalian immunoglobulins can be used in the preferred embodiments, such as the following isotopes: any isotype of IgG, any isotype of IgA, IgE, IgD or IgM. The immunoglobulins of the present invention contained at 20 least one of the constant region domains from two different isotopes of mammalian immunoglobulin.

The present invention also contemplates immunoglobulins that contain immunoglobulin domains derived from two different isotopes of rodent immunoglobulin. The isotopes of rodent immunoglobulin are 0. well known in the art. The immunoglobulins of the present invention may contain immunoglobulin derived heavy chains that include at least one of the following immunoglobulin domains: the CHl, C,2, or C, 3 domain of a mouse IgG, IgG1, IgG2a, IgG2b, IgG3, IgA, IgE, or IgD; the CH,, CH2, CH3, C4C domain of mouse IgE or IgM; the Cl,, CH 2 or CH3 domain of a human IgG, IgG1, IgG2, IgG3, IgG4, IgAl, IgA2, or IgD; the CHI, CH2, C,3, C,4 domain of human IgM or IgE; the CH1,

C,

2 or C,3 domain of an isotype of mammalian IgG, an isotype of IgA, IgE, or IgD; the C,1, C,2, CH 3 C,4 domain of a mammalian IgE or IgM; the C,1, CH2, or C,3 domain of an isotype of rodent IgG, IgA, IgE, or IgD; the C,1, CH2,

C,

3

C,

4 domain of a rodent IgE or IgM; the CHl, C, 2 or C,3 domain of an isotype of animal IgG, an isotype of IgA, IgE, or IgD; and the Cl,, CH 2

CH

3

C,

4 domain of an animal IgE or IgM. The present invention also contemplates the replacement or addition of protein domains derived from molecules that are members of the immunoglobulin superfamily. The molecules that belong to the immunoglobulin superfamily have amino acid residue sequence and nucleic acid sequence homology to immunoglobulins. The molecules that are part of the immunoglobulin superfamily can be identified by amino acid or nucleic acid sequence homology. See, for example, p.

361 of Immunoglobulin Genes, Academic Press (1989).

Tetratranscenic Organisms: 15 The present invention also contemplates a tetratransgenic organism which is comprised of cells having incorporated into the nucleic acid of that cell or plant within the cell four different transgenes, each encoding a different polypeptide. These transgenes are different in that the messenger RNA and polypeptides produced from that transgene are different from the messenger RNA and polypeptides produced from the other of the four transgenes. Thus, the number of transgenes referred to in the present invention does not include multiple copies of the same transgene as is commonly found in transgenic organisms. The present invention is directed to transgenic organisms having four transgenes which are not identical copies of other transgenes. The present invention does not exclude the possibility that each of the four different transgenes may be present in multiple copies. However, at least four separate transgenes that are different are present within the cells of the transgenic organism.

In addition, the present invention contemplates that four different transgenes are related in that the transgenes encode a polypeptide that is part of a multipolypeptide molecule. Therefore, the present 61 invention contemplates that each individual polypeptide chain of a multipeptide molecule would be present on a transgene within a cell of the transgenic organism. The expression of each individual different polypeptide of the multipeptide molecule allows the different polypeptides to associate together to form the multipeptide molecule within the transgenic animal's cells. Thus, the present invention does not include within the four different transgenes in each individual cell, transgenes which encode polypeptides which do not associate together to perform a multipeptide molecule. Examples of such transgenes encoding molecules that do not associate together are polypeptides for antibiotic resistance such as kanamycin or neomycin or thymidine kinase.

15 In preferred embodiments, the transgenes present within a transgenic organism of the present invention encode the following four different polypeptides: a protection protein; an immunoglobulin derived heavy chain having at least a portion of an antigen binding domain; an 20 immunoglobulin derived light chain having at least a •co portion of an antigen binding domain; and an immunoglobulin J chain. In other preferred embodiments, one of the transgenes present in the transgenic organism encodes a chimeric immunoglobulin heavy, light or J chain.

In other preferred embodiments, a transgene of the transgenic organisms of the present invention encode either an immunoglobulin heavy chain derived at least in part from an IgA or a IgM immunoglobulin. Other preferred embodiments include transgenic organisms containing transgenes which encode at least a portion of the amino acid sequence derived from an immunoglobulin heavy chain derived from either an IgA or IgM immunoglobulin heavy chain.

The present invention contemplates transgenic organisms including mammals, plants, rodents, reptiles, insects, amphibians, fishes or other organisms. In preferred embodiments, the transgenic organism of the present invention is a plant or a mammal. Methods of producing such organisms are well known. See, U.S.

Patents 4,736,866; 4,607,388; 4,870,009 and 4,873,191 which are hereby incorporated by reference.

The present invention also contemplates immunoglobulin that contain immunoglobulin derived heavy or immunoglobulin derived light chains that contain immunoglobulin domains which have been engineered to make those domains less immunogenic in a particular species.

Typically, the immunoglobulin molecule is engineered as to be "humanized" in that it appears to be a human immunoglobulin even though derived from various other species.

15 Examples The following examples illustrate the disclosed invention. These examples in no way limit the scope of the claimed invention.

1. Construction of DNA Vectors For Expression of 20 Antibodies in Plants.

a. Isolation of the Nucleotide Sequences Encoding the Guy's 13 Immunoglobulin Molecular cloning of the gamma and kappa chains of the Guy's 13 anti-S. mutans antibody was done by the 25 procedures described in Ma et al., Eur. J. Immunol., 24:131 (1994). Briefly, mRNA was extracted from the Guy's 13 hybridoma cell line and converted to the cDNA by standard procedures. The cDNA was then amplified with the use of a pair of oligonucleotides specifically complementary to either the gamma or kappa cDNA.

Amplification was catalyzed by Tag 1 polymerase using a thermal cycler as described. The amplified cDNAs were then digested with the appropriate restriction endonucleases and ligated into the corresponding restriction site in a standard plant expression vector.

Numerous examples of such vectors have been reported in the literature and are generally available. An example of one vector that may be used is pBIN19.

In a related series of experiments, the cDNAs were cloned into the bacterial vector bluescript. Using this construct, the sequence of the gamma and kappa cDNAs was determined using the methods of Maxam and Gilbert.

Procedures for cloning antibody cDNAs involving PCR techniques or by construction of cDNA libraries followed by ligation of the obtained cDNAs into appropriate vectors are commonplace techniques which are familiar to one of ordinary skill in the art.

b) Hybrid cDNAs encoding the Guy's 13 heavy chain variable region, a part of the gamma chain constant region S* 15 and a part of an alpha chain constant region.

These constructs were synthesized as described in Ma •et al., Eur. J. Immunol., 24:131 (1994) and ligated into the appropriate plant expression vectors as described above. The final construct had the structure: Guy's 13 variable region (IgG1 CHI) (IgGl CH2) (IgA CH2) (IgA

CH

3 referred to as IgG2A heavy chain, and Guy's 13 variable region (IgG1CH) (IgACH2) (IgACH3).

c) The Protection Protein and J chain.

The cloned rabbit polyimmunoglobulin receptor (pIgR) cDNA was described by Mostov, Nature, 308:37 (1984) and shown in SEQIDNO:1. The protection protein portion was obtained by PCR amplification of a portion of the nucleotide sequence coding for the (pIgR) and ligation into appropriate plant expression vectors as described above. The protection protein portion of the pIgR used in these constructs included the codon for amino acid number 1 to the codon for amino acid number 606. The method to accomplish this construction are well known in the art and the oligonucleotides can be selected using the pIgR nucleic acid sequence.

64 d) cDNAs encoding aglycosvlated derivatives of heavy-chain constant regions.

Mutagenesis procedures were performed either according to Stratagene protocols. In each case (i.e.

alpha constant region, or protection protein) the codon for the asparagine utilized as the attachment site for carbohydrates, was changed to a codon for histidine.

2. Production of Transqenic Plants Expressing Therapeutic Antibodies.

Plants and plant cells containing immunoglobulins having a protection protein were produced in the following manner.

a) Transfer of vectors to Aqrobacterium 15 tumefaciens.

Plant transformation was accomplished by using Agrobacterium tumefaciens. E. coli DH5c bearing the recombinant pMON530 plant expression vector were mated with Agrobacterium in the presence of a helper strain (pRK2013) to provide transfer functions. Alternatively, pMON530 plasmid DNA was introduced into Agrobacteria by *direct transformation. In this procedure, the Agrobacterium strain was first grown overnight at 280 C in YEP medium. 2 ml of the overnight culture was used to 25 inoculate 50 ml of YEP and was grown to an OD 6 00 Of 1.0. The cells were then chilled to 40 C, pelletted by centrifugation and resuspended in 1 ml of ice cold 20 mM CaC12. About 1 fg of DNA was added to aliquots of 0.1 ml of ice cold cells. The cells were then rapidly frozen by immersion in liquid nitrogen or in a dry ice ethanol bath.

The cells were thawed by incubation at 370 C for 5 minutes followed by the addition of 1 ml YEP medium. The cells were allowed to incubate for 2-4 hours with gentle shaking. Individual colonies carrying the recombinant vector were isolated by incubation on YEP agar plates containing the appropriate antibiotic.

Agrobacteria containing pMON530 were grown in media containing kanamycin, spectinomycin and chloramphenicol.

Small segments of tobacco leaf were then co-cultivated with the Agrobacterium for 2 days after which the leaf segments were transferred to plates containing carbenicillin to kill the Agrobacterium. Regeneration of transformed leaf cells into whole plants was allowed to proceed in the presence of kanamycin selection until the plants were competent for growth in soil.

b) Regeneration of transformed tobacco and petunia plants.

Leaves from greenhouse grown tobacco or petunia plants were sterilized in 20% (by volume) Chlorox bleach, 15 0.1% sodium dodecyl sulfate at room temperature for 8 minutes. The leaves were then briefly rinsed in ethanol and allowed to dry in sterile Petri plates.

Leaf discs of approximately 0.5 cm diameter were removed with a sterile hole puncher and placed on agar 20 plates containing MS10 medium.(MS10 medium per liter: 4.4 e g Murashige and Skoog basal salts with minimal organics [Sigma #M68991, 30 g sucrose, 0.2 mg naphthalene acetic acid, 2 mg benzylaminopurine, 0.1 mg nicotinic acid, 0.1 mg pyridoxin, 0.1 mg thiamine, 10 g agar, pH 5.7 with

KOH)

A 2 ml aliquot of a suspension of Agrobacterium in LB (approximately 1 x 108 Agrobacteria per ml) was then added to the leaf pieces. All surfaces of the leaf discs were contacted with Agrobacteria, excess liquid was poured off the plate, and the discs were co-cultivated with the bacteria for 2 days at room temperature. The discs were then transferred to agar plates containing MS10 medium, gg/ml kanamycin and 250 jg/ml carbenicillin Regeneration was allowed to proceed with weekly transfer of discs to fresh MS10-KC plates until regenerating shoots were visible. Shoots were then transferred to agar plates containing MSO-KC medium (MSO-KC per liter: 4.4 g Murashige and Skoog basal salts with minimal organics [Sigma #M68991, 30 g sucrose, 1 mg nicotinic acid, 1 mg pyridoxin, 0.1 mg thiamine, 50 g/ml kanamycin and 250 Ag/ml carbenicillin, 10 g agar, pH 5.7 with KOH).

After root formation, plantlets were transferred to soil and grown to maturity.

c) Regeneration of transformed alfalfa plants.

Alfalfa trifoliates were cut from a greenhouse grown plant and sterilized in 20% Chlorox bleach, 0.1% sodium dodecyl sulfate at room temperature for 8 minutes.

The trifoliates were then briefly rinsed in 70% ethanol and allowed to dry in sterile Petri plates.

Leaf pieces of approximately 1 cm X 4 mm were cut oo. 15 with a sterile scalpel and placed on agar plates containing B5H medium (B5H medium per liter: 3.1 g Gamborg's powdered medium (Sigma #G5893), 500 mg KN03, 250 mg MgS04 7H20, 30 g sucrose, 500 mg proline, 1 mg 2,4dichlorophenoxyacetic acid, 100 gg kinetin, 100 mg inositol, 1 mg nicotinic add, 1 mg pyridoxin, 10 mg thiamine, 10 g agar, 30 ml stock amino acids, pH 5.7 with KOH; stock amino acids consist of 26.6 g L-glutamine, 3.32 g serine, 16.8 mg adenine, 333 mg glutathione per liter and are added after autoclaving when the medium is •25 approximately 500 C).

To the leaf pieces was then added 2 ml of a suspension of Agrobacterium in LB (approximately 1 x 108 Agrobacteria per ml). All surfaces of the leaf were contacted with Agrobacteria, excess liquid was poured off the plate, and the leaves were co-cultivated with the bacteria for 2 days at room temperature. The leaf pieces were then transferred to agar plates containing B5H medium, 25 pg/ml kanamycin and 250 jg/ml carbenicillin Regeneration was allowed to proceed with weekly transfer of leaf pieces to fresh B5H-KC plates until somatic embryos were visible. Embryos were then transferred to agar plates containing BI02Y-KC medium (BI02Y-KC per liter: 25 ml macronutrients, 10 ml micronutrients, 25 ml iron, 1 ml vitamins, 1 ml aminos, 2 g yeast extract, 100 mg myo-inositol, 30 g sucrose, 10 g agar, 25 mg kanamycin, 250 mg carbenicillin, pH 5.9 with KOH; macronutrients consist of 40 g KN03, 40 g NH4N03, 13.88 g Ca(N03)2-4FUO, 1.4 g MgSO4-7H20,2.6 g KC1, 12 g Kh2P04 per liter yielding a 40X stock; vitamins consist of 100 mg thiamine HC1, 500 mg nicotinic acid, 100 mg pyridoxin-HCl per liter yielding a 1000X stock; aminos consists of 2 g per liter glycine yielding a 1000X stock; micronutrients consist of 580 mg MnSO4-4H20, 1550 mg ZnSO4-7H20, 160 mg H3B03, 80 mg KI per liter yielding a 100X stock; iron consists of 1.28 g NaFeEDTA per liter yielding a 40X stock) After root formation, plantlets were transferred to .4 soil and grown to maturity.

d) Regeneration of Transformed Tomato Plants.

Cotyledons from 7 day old tomato seedlings were sterilized in 20% Chlorox bleach, 0.1% sodium 20 dodecyl sulfate at room temperature for 8 minutes. The leaves were then briefly rinsed in 70% ethanol and allowed to dry in sterile Petri plates.

Cotyledon pieces of approximately 0.5 cm diameter were cut with a sterile scalpel and placed on agar plates containing MS4 medium (MS4 medium per liter: 4.4 g Murashige and Skoog basal salts with minimal organics [Sigma #M68991, 30 g sucrose, 2 mg zeatin riboside, 5 mg nicotinic acid, 0.5 mg pyridoxin, 0.5 mg thiamine, 1 mM acetosyringone, 10 g agar, pH 5.7 with KOH) To the leaf pieces was then added 2 ml of a suspension of Agrobacterium in LB (approximately 1 x 108 Agrobacteria per ml). All surfaces of the leaf discs were contacted with Agrobacteria, excess liquid was poured off the plate, and the discs were co-cultivated with the bacteria for 2 days at room temperature. The discs were then transferred to agar plates containing MS4 medium minus acetosyringone containing 50 ig/ml kanamycin and 250 pg/ml carbenicillin (MS4-KC). Regeneration was allowed to proceed with weekly transfer of discs to fresh MS4-KC plates until regenerating shoots were visible. Shoots were then transferred to agar plates containing MSO-KC medium (MSO-KC per liter: 4.4 g Murashige and Skoog basal salts with minimal organics [Sigma #M68991, 30 g sucrose, 1 mg nicotinic acid, 1 mg pyridoxin, 10 mg thiamine, pg/ml kanamycin and 250 pg/ml carbenicillin, 10 g agar, pH 5.7 with KOH).

After root formation, plantlets were transferred to soil and grown to maturity.

e) Regeneration of Transformed ArabidoDsis Plants.

Intact roots derived from Arabidopsis thalliana 15 plants grown in sterile culture were first pretreated on callus inducing medium (CIM) for 3 days at 280 C in the dark (CIM medium per liter: 3.1 g Gamborg's powdered omedium (Sigma #G5893), 30 g sucrose, 1 mg 2,4dichlorophenoxyacetic acid, 100 pg kinetin, 1 mg inositol, 0.1 mg nicotinic acid, 0.1 mg pyridoxin, 0.1 mg thiamine, 8 g agar, pH 5.7 with KOH).

To the intact roots was then added 2 ml of a suspension of Agrobacterium in LB (approximately 1 x 108 Agrobacteria per ml). All surfaces of the roots were con- 25 tacted with Agrobacteria and excess liquid was poured off the plate. The intact roots were then cut into 5 mm segments and were co-cultivated with the Agrobacteria for 2 days at 280 C on CIM plates. The root pieces were then transferred to agar plates containing shoot inducing medium (SIM) containing 50 pg/ml kanamycin and 250 pg/ml carbenicillin (SIM medium per liter: 3.1 g Gamborg's powdered medium (Sigma #G5893), 30 g sucrose, 5 mg N 6 isopentenyl) adenine, 150 pg indole-3-acetic acid, 1 mg inositol, 0.1 mg nicotinic acid, 0.1 mg pyridoxin, 0.1 mg thiamine, 8 g agar, pH 5.7 with KOH).

Regeneration was allowed to proceed with weekly transfer of root pieces to fresh SIM plates until green regenerating shoots were visible. Shoots were then transferred to agar plates containing EM medium (MSO-KC per liter: 4.4 g Murashige and Skoog basal salts with minimal organics [Sigma #M6899], 10 g sucrose, 1 mg indole-3-butyric acid 1 mg nicotinic acid, 0.1 mg pyridoxin, 0.1 mg thiamine, 250 Lg/ml carbenicillin, 8 g agar, pH 5.7 with KOH).

After root formation, plantlets were transferred to soil and grown to maturity.

3. Identification of Transgenic Plants.

Kanamycin resistant- transformants expressing individual immunoglobulin chains were identified by ELISA as described. Further analysis of the transformants in- 15 cluded evaluation of RNA by Northern blotting and evaluation of immunoglobulin polypeptides by Western blotting, both as described in Maniatis et al.

0 For each immunoglobulin chain, antigenic material, RNA or protein were detected by the respective assays.

20 Transformants identified as having the highest levels of immunoglobulin chains were used in cross pollination protocols.

0 4. Assembly of Antibodies by Cross Pollination of 6 Transformants.

Cross pollinations were performed in order to obtain plants co-expressing the various components of the desired antibodies. These crosses yielded alfalfa, tomato, tobacco and Arabidopsis plants containing the following assembled components, all of which also contained the Guy's 13 antigen binding domain.

Type of Antibody Immunoqlobulin Components 1 Gi heavy chain, kappa light chain 2 G2/A heavy chain, kappa light chain 3 G2/A heavy chain, kappa light chain, J chain 4 Gl/A heavy chain, kappa light, J chain, protection protein Gl/A heavy chain Kappa light chain 5. Extraction and Evaluation of Guy's 13 Type 1, 2 and 3 4 Antibodies From Transqenic Plants.

a) Extraction and enrichment of antibody contained in leaf.

Leaf pieces were chopped into approximately 1 cm 2 pieces. The pieces were then added to a cold solution of TBS having 10g/ml leupeptin (1 ml TBS per gram of leaf) contained in a chilled porcelain mortar both at approximately 40 C. Plant liquid was extracted by pulver- Sizing the pieces with a cold pestle using a circular 20 motion and hand pressure. Pulverizing was continued until the pieces became a nearly uniform pulp (approximately 3 minutes of pulverizing). The pulp was centrifuged at C and approximately 50,000 X g to yield a supernatant devoid of solid plant pieces. Alternatively, the pulp was 25 filtered through a plastic mesh with a pore size of approximately 100 microns.

Depending on the titer of antibody contained in the particular plant, the supernatant was either directly suitable for exposure to antigen or required enrichment to 30 a suitable concentration. Yields of IgGl's or IgG/A's in the crude extract were routinely less than 10 pg/ml and averaged approximately 5 ug/ml. For applications of a Guy's 13 antibody to mucosal surfaces, enrichment to a concentration of 1 to 4 mg/ml may be required. As a Type 1, 2 or 3 construct, Guy's 13 antibody required a ten to forty-fold enrichment to yield the desired concentration.

This was accomplished either by affinity adsorption (utilizing either Protein A or Protein or by lyophilization to remove water. Size exclusion chromatography was also used for enrichment but required complete fractionation of the crude extract to yield an antibody of the required concentration. By ELISA assay and by polyacrylamide gel electrophoresis, the coexpressed chains assembled into a complex of approximately 180-200 k daltons for types 1 2 and approximately 400 k daltons for type 3. Crude extracts were routinely obtained containing approximately of 5-10 pg/ml.

A dramatic increase in antibody accumulation was observed when the protection protein was crossed into a plant containing Type 3 antibody yielding a plant containing a Type 4 antibody. By ELISA assay and by polyacrylamide gel electrophoresis, the co-expressed chains assembled into a complex of approximately 470,000 daltons. Crude extracts were routinely obtained containing in excess of 200 g/ml with an average of approximately 250 ug/ml. Therefore, the SIgA construct of 20 the Guy's 13 antibody required minimal enrichment to achieve the target concentration. This enrichment could be accomplished by the techniques described above.

Alternatively, it was found that the antibody is readily S* separated from the majority of plant molecules by a one S 25 ultrafiltration step using membrane with a molecular exclusion of 200,000 d.

b. Functionality of the Guy's 13 Type 4 Antibody.

Functional antibody studies were carried out by 30 ELISA. All plants expressing antibody light ard heavy chains assembled functional antibody that specifically recognized streptococcal antigen (SA I/II). The levels of binding and titration curves were similar to those of mouse hybridoma cell supernatants. No SA I/II binding was detected with plants expressing only J chain or only protection protein. Likewise, wild-type plants expressing no immunoglobulin showed no detectable levels of binding.

In a similar set of experiments, binding of antibody to immobilized purified streptococcal antigen or native antigen on the bacterial cell surface was detected using an anti-secretory component antiserum. In these assays, only the Type 4 antibody binding was detected. The functional Type 1, 2 or 3 antibodies did not bind the anti-secretory component antiserum. These results confirm that the protection protein was assembled with antibody in the plants expressing Type 4 constructs and in a manner which did not interfere with antigen binding.

6. Expression of Chimeric Immunoclobulins.

The genes encoding the heavy and light chains of a murine monoclonal antibody (mAb Guy's 13) have been cloned and expressed in Nicotiana tabacum. Transgenic plants have been 'regenerated that secrete full-length Guy's 13 antibody. By manipulation of the heavy chain gene sequence, constant region domains from an immunoglobulin alpha heavy chain have been introduced, and plants secreting Guy's 13 mAb with chimeric gamma/alpha heavy chains have also been produced. For each plant antibody, light and heavy chains have been detected by Western blot analysis and the fidelity of assembly confirmed by demonstrating that the antibody is fully functional, by 25 antigen binding studies. Furthermore, the plant antibodies retained the ability to aggregate streptococci, which confirms that the bivalent antigen-binding capacity of the full length antibodies is intact.

30 a. Cloning of heavy and light chain genes Messenger RNA was purified from the Guy's 13 and a murine IgA (MOPC315) hybridoma cell line, using an acid guanidiniumthiocyanate-phenol-chloroform extraction.

Complementary DNA was made using Moloney murine leukemia virus reverse transcriptase (Promega, GB). DNA encoding the gamma and kappa chains of Guy's 13 were amplified by polymerase chain reaction (PCR). The degenerate 23. FEB. 2004 15:33 SPRUSON FERGUSON NO. 7273 P. 44 73 oligonucleotides used in the PCR were designed to incorporate a 5' terminal XhoI, and a 3'-terminal EcoRI restriction site in the amplified DNA fragments. Following restriction enzyme digestion, the immunoglobulin chain encoding DNA was ligated into a constitutive plant expression vector (pMON 530), which contains a mouse s immunoglobulin leader sequence upstream of the cloning site. The recombinant vector was used to transform E. colt (DH5-a, Gibco BRL) and screening was by Southern blotting, using radiolabeled DNA probes derived from the original PCR products. Plasmid DNA was purified from positive transformants and introduced into Agrobacterium tumefaciens.

A similar approach was used to construct two forms of a hybrid Guy's 13 heavy chain. The synthetic oligonucleotides shown in Fig. 1 were used in PCR to amplify the regions: Guy's 13 signal sequence to the 3' end of CTI domain (Jl-JS), (b) Guy's 13 signal sequence to the 3' end of Cr2 domain (J1-J2), and 5' end of Ca2 domain to the 3' terminus of DNA from the MOPC 315 hybridoma (J3-J4). The 15 fragments were purified (Geneclean II, Bio 101, La Jolla, CA) and digested with HindIII for I h at 37°C. The Guy's 13 fragments were ligated to the MOPC 315 fragment with T4 DNA ligase (Gibco, BRL), at 16 0 C for 16 h, and an aliquot of the reaction mixture was used as template DNA for a further PCR, using the 5' terminal S. oligonucleotide for Guy's 13 (J1) and the 3' terminal oligonucleotide for MOPC 315 20

J

Amplified DNA fragments were purified and ligated into the pMON 530 vector as described above. The vector used in this procedure did not have a previously inserted mouse leader sequence, as in this case, the DNA encoding the gamma chain, represented by SEQ ID NOs: 13 and 14, and kappa chain, represented by SEQ ID NOs: 11 and 12, of Guy's 13 were amplified by polymerase chain reaction (PCR).

b. Plant transformation and regeneration Leaf discs, about 6 mm in diameter, were cut from surface-sterilized tobacco leaves (Nicotiana tabacum, var.

LIBAA6223DI COMS ID No: SMBI-00631115 Received by IP Australia: Time 15:44 Date 2004-02-23 74 xanthii) and incubated overnight at 28 0 C, with a culture of the recombinant A. tumefaciens, containing immunoglobulin cDNA inserts. The discs were transferred to culture plates containing a medium that induces regeneration of shoots, supplemented with kanamycin (200 mg/l) and carbenicillin (500 mg/l). Shoots developing after this stage were excised and transplanted onto a root-inducing medium, supplemented with kanamycin (200 mg/) Rooted plantlets were transplanted into soil as soon as possible after the appearance of roots. Plants were screened for expression of immunoglobulin chains as described below. Those that expressed heavy chains were crossed with those expressing light chains, by crosspollination. The resulting seeds were sown in soil and allowed to germinate. Twenty-two transgenic plants were regenerated from transformations with light or heavy chain constructs, as determined by ELISA. Crossing of light and heavy chain-secreting plants resulted in 3/10 Fl progeny plants expressing kappa and gamma chains together, 4/17 20 plants expressing both kappa and the plant G1/A heavy chain and 3/8 plants expressing both kappa and the plant ~G2/A heavy chain together.

The three different forms of Guy's 13 monoclonal antibody expressed in plants, therefore, all contain the 25 identical light (kappa) chain, but different heavy chains.

These will be abbreviated throughout this report as follows (Fig. Guy's 13 IgG1 with original gamma heavy chain, plant G13, Guy's 13 with IgG/IgA hybrid heavy chain consisting of var-T1-T2-a2-a3 domains, plant G2/A. The 30 Guy's 13 hybridoma cell culture supernatant used as a positive control will be abbreviated to Mouse G13.

Negative control plants were those that had been transformed with pMON 530 vector containing an insert that encodes an irrelevant mouse protein.

c. Antibody chain detection Production of either gamma, kappa or the gamma/ alpha chain hybrids was detected by ELISA. Microtiter wells were coated with a goat anti-mouse heavy or light chainspecific IgG (Fisher, USA; Sigma, GB; Nordic Pharmaceuticals, GB) in 150 mM NaC1, 20 mM Tris-HCl (pH 8) (TBS). Blocking was with 5% non-fat dry milk in TBS at overnight. Plant leaves were homogenized in TBS with leupeptin (10 pg/ml) (Calbiochem, USA). The supernatant was added in serial twofold dilutions to the microtiter plate and incubation was at 4 0 C overnight. After washing with TBS with 0.05% Tween 20, bound immunoglobulin chains were detected with the appropriate goat anti-mouse heavy or light chain-specific antibody, conjugated with horseradish peroxidase (Fisher; Sigma; Nordic Pharmaceuticals), for 2 h at 37 0 C. Detection was with 2.2'-azino-di-(3-ethyl-benzthiazoline-sulfonate) (Boehringer, FRG).

A similar assay was used to determine the concentra- 20 tions of the murine and plant Guy's 13 antibodies. These were compared with a mouse IgG1 mAb (MOPC 21), and a mouse IgA mAb (TEPC 21) used at known concentrations (Sigma) ELISA plates were coated with an anti-mouse kappa antiserum. After blocking, bound antibody was detected 25 with horseradish peroxidase-labeled anti-mouse gamma or alpha antiserum. Antibody concentration was determined by comparison of binding curves for each antibody.

ELISA was also used to detect the binding function of the assembled antibody. Binding to SA I/II was detected 30 using microtiter plates that had been coated with purified SA I/II at an optimized concentration of 2 yg/ml. The ELISA procedure was as described above. The ability to bind S. mutans or E. coli cells was detected using intact cells (strains Guy's c, S. mutans and DH5-a, E. coli) that had been grown to stationary phase, for 18 h at 370C and fixed in 10% formalin. All the antibody solutions were adjusted to an initial concentration of 1.5 Ag/ml and used in serial twofold dilutions. Extracts from plants expressing wither Guy's 13 heavy or light chain singly were also included in these assays, to determine if the single immunoglobulin chains exhibited any antigen-binding activity. Antibodies bound to either cells or purified SA I/II were detected using a horseradish peroxidaseconjugated goat anti-mouse light or heavy chain antiserum (Nordic Pharmaceuticals). The results are expressed as mean standard deviation of duplicate results from three separate assays.

Competition ELISA was performed on microtiter plates coated with purified SA I/II as above. The plates were incubated with plant extracts of Guy's 13 hybridoma supernatant at 1.5 kg/ml and serial twofold dilutions at 37 0 C for 1 h and 4 0 C overnight. After washing, 12I-labeled mouse Guy's 13 was added and left to incubate for 2 h at 37 0 C. The plates were washed again and the bound radioactivity was counted in a gamma counter (Hydragamma 16, Innotec, GB). The results are expressed as 20 inhibition of labeled mouse Guy's 13 binding, in which 100% is the radioactive count from wells to which no blocking solution had been added.

d. Western blot analysis 25 Aliquots of 10l1 of leaf homogenates were boiled with mM Tris-HCl (pH 2% SDS, under reducing and nonreducing conditions. SDS-PAGE in 10% acrylamide was performed, and the gels were blotted onto nitrocellulose.

The blots were incubated for 16 h in TBS with 0.05% Tween S. 30 20 and 1% non-fat dry milk, followed by goat anti-mouse IgG1, kappa (Nordic Pharmaceuticals) or alpha chainspecific antisera (Sigma), and incubated for 2 h at 37 0

C.

After washing, the second-layer antibody, an alkaline phosphatase-conjugated rabbit anti-goat IgG (Sigma) was applied for 2 hours at 37 0 C. Antibody binding was detected by incubation with 300 Ag/ml nitroblue tetrazolium and 1 5 p ig/ml 5-bromo-4-chloro-3-idolyl phosphate (Promega).

e. DNA seauencing The DNA sequence of each cloned immunoglobulin gene insert confirmed that no mutations had occurred during PCR amplification or the cloning procedures. The introduction of the HindIII site in the X/y hybrid heavy chains resulted in the predicted addition of the leucine residue between the Cy2 and Co'2 domains in Plant G2/A and leucinelysine between the Cyl and Co2 domains in Plant G1/A. The additional Cy2 domain in the Plant G2/A construct is predicted to increase the length of the heavy chain by 141 amino acid residues (approximately 12000 Da). The plant Gl/A heavy chain in predicted to be slightly larger than the native Guy's 13 heavy chain, by 33 amino acids, approximately 3000 Da.

Plasmid DNA that was purified from positive transformants in E. coli was sequenced. The immunoglobulin gene 20 inserts were excised and sub-cloned into Bluescript (Stratagene, USA). The DNA sequence was determined by a di-deoxy termination procedure (Sequenase, USB, USA) f. Expression of assembled antibody 25 Western blot analysis on extracts from three representative F1 progeny plants was performed and reported in Figure 2 of Ma et al., Eur. J. Immunol., 24:131-138 (1994). Samples run under reducing conditions demonstrate the presence of light (kappa) chain at approximately 30 Kd, in the mouse Guy's 13, as well as in the three transgenic plants, but not in the control plant. Guy's 13 heavy (gamma) chain was also detected in plant G13 at approximately 57 Kd, but not in the control plant extract.

A single protein species was detected, unlike the hybridoma producing the Guy's 13 antibody cell culture supernatant, in which a two protein species was a consistent finding. The difference in the molecular size of the mouse heavy chains is probably due to glycosylation differences, and the result suggests that in plants the two heavy chains may be glycosylated in the same way.

The heavy chains of plant G1/A and G2/A were detected with an anti-alpha chain antiserum. Compared with the mouse Guy's 13 heavy chain, (approximately 57 Kd), the heavy chain of plant Gl/A has a slightly higher relative molecular mass (approximately 60 Kd) and the plant G2/A heavy chain is much larger (approximately 70 Kd) This is consistent with the molecular weights predicted by sequence analysis. Several other protein species were detected in the transgenic plant extracts. These are likely to be proteolytic fragments of either light/heavy chain complexes, or of the heavy chain, as no bands were detected in the extract from the control transgenic plant.

The anti-alpha chain antiserum did not cross-react with the mouse Guy's 13, which only contains gamma chain domains.

Samples were also run under nonreducing conditions to 20 confirm the assembly of heavy and light chains into an immunoglobulin molecule and reported in Figure 3 of Ma et al., Eur. J. Immunol., 24:131-138 (1994). Detection was with a labeled anti-kappa antiserum, and all three transgenic plants had assembled immunoglobulin at the 25 correct M, of above 150 Kd for full-length antibody. The plant G13 antibody has the same Mr as the mouse G13, but the plant G2/A and plant Gl/A antibodies have higher Mr as predicted. A number of smaller proteolytic fragments were also detected, which is consistent with previous findings 30 and the fact that a number of proteases are released by plants during the antibody extraction procedure. That S"these are antibody fragments, is confirmed by the absence of any detectable bands in the control plant extract.

g. Antigen binding Ten plants which were producing immunoglobulin were made in total, and the concentration of immunoglobulin in plant extracts varied between 1 and 10 gg/ml (mean jg/ml) For the murine antibody and the representative plants used in this study, the concentrations estimated by ELISA were: mouse IgG-15.4 jg/ml, plant IgG-7.7 pg/ml, plant Gl/A-1.5 jg/ml and plant G2/A-2.1 ug/ml. The concentrations determined for plant antibodies containing hybrid heavy chains are possibly underestimated, as they do not carry all of the constant region determinants, as compared with the standard mAb IgA used.

Titration curves for extracts from the three representative transgenic plants binding to SA I/II were generated and reported in Figure 4 of Ma et al., Eur. J.

Immunol., 24:131-138 (1994). Specific antibody was detectable in all three transgenic plant extracts, and the titration curves were similar to that of the murine hybridoma cell culture supernatant, used at the same concentration. The binding of the plant Gl/A antibody appeared to be slightly lower than the other antibodies, 99 *.e Salthough the titration curve followed a similar pattern.

20 No SA I/II binding activity was detected in the negative control plant nor did extracts from plants individually expressing light or heavy chains have binding activity towards purified SA I/II. These findings demonstrate that the transgenic plants expressing both light and heavy S* 25 chains have assembled the antibody molecule correctly to form a functional antigen binding site and that single light or heavy chains are not capable of binding the antigen.

The plant antibodies also recognized native antigen 30 on the surface of streptococcal cells as shown in Figure 5 of Ma et al., Eur. J. Immunol., 24:131-138 (1994) (S.

mutans serotype which further confirms the integrity of the antigen-binding site in the plant antibodies.

There were no significant differences between the binding of the different antibodies. Neither extracts from control plants, nor plants expressing only heavy or light chains showed any binding to S. mutans cells. There was no binding to E coli cells by any of the plant extracts, at concentrations of 1.0 and 0.5 gg/ml.

The plant antibodies competed with the original mouse Guy's 13 mbAb for binding to SA I/II. Up to inhibition of 1 5 I-labeled mouse Guy's 13 mAb binding to SA I/II was demonstrated using the plant antibodies as shown in Figure 6 of Ma et al., Eur. J. Immunol., 24:131-138 (1994). As before, the inhibition titration curves of the plant antibodies were similar to each other, and comparable to that of the mouse Guy's 13, whereas the control plant extract gave no inhibition.

h. Aggregation of S. mutans The action of the immunoglobulin produced in plants having the Guy's 13 antigen binding region on bacteria was determined and reported in Figure 7 of Ma et al., Eur. J.

Immunol., 24:131-138 (1994). Plant extracts were sterilized by filtration through a 0.22 gm pore size filter and diluted tenfold with Todd Hewitt broth. The 20 samples were inoculated with 0.05 vol of an overnight S.

mutans culture and incubated at 370C overnight. The samples were Gram stained and examined under oil immersion microscopy. S. mutans grown in the presence of mouse Guy's 13, plant Guy's 13, plant Gl/A or plant G2/A became 25 aggregated and cell clumping was evident. However, the control plant extract had no effect on S. mutans growth.

None of the plant mAb appeared to affect S. mutans rate of growth, as determined by culture of viable organisms at 8, 12 and 16 h. This result demonstrates not only that the plant antibodies have correctly assembled antigen-binding regions, but also that the antibody molecules bind antigen bivalently.

Example 7. Production of Immunoglobulins Containing Protection Proteins Four transgenic Nicotiana tabacum plants were generated to express a murine monoclonal immunoglobulin kappa chain having the antigen binding site of the Guy's 13 light chain, a hybrid IgA/G murine immunoglobulin heavy chain containing Cy and Co chain domains and the antigen binding site of the Guy's 13 heavy chain, a murine J chain and protection protein comprised of amino acids 1-606 of rabbit polyimmunoglobulin receptor and did not contain amino acids 627-675 of the rabbit polyimmunoglobulin receptor.

See, Example 1. Successive sexual crosses between these plants resulted in simultaneous expression of all four protein chains in the progeny plants. In some cases, back crossing was used to produce homozygous plants. The four recombinant polypeptides were assembled into a functional, high molecular weight immunoglobulin containing a protection protein of approximately 470,000 Kd. The assembly of the protection protein with the immunoglobulin was dependent on the presence of a J chain, as no association of the protection protein was detected when plants expressing antibody alone were crossed with those expressing the protection protein. Microscopic evaluation of plants expressing the immunoglobulins containing the protection protein demonstrated co-incident expression of protection protein and immunoglobulin heavy chains in single cells. Single cells are able to produce immunoglobulin having a protection protein in transgenic 30 plants, whereas two cells are required for natural "VO ~production of secretory immunoglobulin in mammals. The results demonstrate that sexual crossing of transgenic plants expressing recombinant sub-units is suitable for large scale production of immunoglobulin containing a protection protein for passive immunotherapy, as well as for expressing other complex protein molecules.

82 The immunoglobulin which contains the protection protein has the heavy and light chain antigen binding domains from the Guy's 13 monoclonal antibody that specifically recognize the cell surface adhesion molecule SA 1/11 of an oral streptococcus as shown by Smith, R. Lehner, T. Oral Microbiol. Immunol. 4, 153-158 (1989) Transgenic immunoglobulin of this type containing only heavy and light chains has been generated in Nicotiana tabacum plants as described in Example 6. A mouse J chain construct containing the coding length cDNA was amplified using synthetic oligonucleotide primers corresponding to the N terminus.MKTHLL and the C terminus SCYPD of mouse J chain as described by Matsuuchi, Cann, G. M. Koshland, M.E. PNAS 83, 456-460 (1986). This amplified 15 nucleotide sequence was ligated into a constitutive plant expression vector, pMON 530, that includes the promoter from Cauliflower Mosaic Virus and has been 'o described by Rogers, S. Klee, H. Horsch, R. B. Fraley, R. T. Meth. Enzymol. 153, 253-276 (1987). Tobacco 20 leaf tissue was transformed using agrobacterium containing the recombinant plasmid as described in the previous Examples. Regenerated plants were screened for the production of messenger RNA encoding J chain and positive transformants were self fertilized in order to generate 25 homozygous progeny. The J chain expressing plants were crossed initially with those expressing the chimeric immunoglobulin heavy chain and kappa chain. Western blot analysis of the plant extract from plants expressing the chimeric immunoglobulin heavy chain with anti-kappa antiserum under non-reducing conditions, revealed a protein species of approximately 210 Kd, which is consistent with the presence of the extra constant region domains present in the chimeric immunoglobulin heavy chain, as compared with the original IgGl antibody. The progeny from the cross between the plant expressing the immunoglobulin and a J chain plant resulted in the appearance of a major immunoglobulin band at approximately twice the relative molecular mass of approximately 400 Kd, demonstrating that assembly of the 3 polypeptides had occurred to form dimeric immunoglobulin (dlgA/G).

The protection protein construct consisted of a coding length cDNA amplified using synthetic oligonucleotide primers corresponding to the N terminus MALFLL and AVQSAE at amino acids 601-606 of the C terminus of rabbit polyimmunoglobulin receptor. The nucleotide sequence of the rabbit polyimmunoglobulin receptor was reported by Mostov, K. Friedlander, M. Blobel, G.

Nature 308, 37-43 (1984). The protection protein was generated in transgenic plants as described above and positive transformants expressing the protection protein were identified by Western blot analysis.

15 Plants expressing J chain assembled with the immunoglobulin having the IgA/G heavy chains to form dimers were then crossed with a homozygous plant expressing the protection protein. The progeny plants expressing the immunoglobulin having the protection 20 protein contained a higher molecular weight protein species at approximately 470 Kd as determined by Western blot analysis under non-reducing conditions. This molecular size was consistent with that expected for an immunoglobulin containing a protection protein. This high molecular weight protein contained the protection protein Sas confirmed by Western blotting, using antiserum that specifically recognized the protection protein. The plant extracts also contained a protein species of approximately 400 Kd corresponding to the dimers of IgA/G and a protein species of approximately 210 Kd corresponding to the immunoglobulin with the chimeric heavy chain, but these were only detected by anti-kappa antiserum and not the anti-protection protein antiserum. In the transgenic plant producing the protection protein alone, there was no evidence that the protection protein assembled with endogenous plant proteins or formed multimers, as no high molecular weight proteins were detected in Western blotting under non-reducing conditions. Western blot analysis demonstrated that extracts from the plants expressing immunoglobulin heavy chain (IgA/G, dimeric IgA/G and the immunoglobulin containing a protection protein), but not the plants containing only the protection protein or J chain or wild-type plants, contained identical immunoglobulin derived heavy and light chains. Furthermore, only the plants containing protection proteins and the plants containing the IgG/A immunoglobulin having the protection protein expressed proteins that were recognized by the antiserum that specifically recognized the protection protein. No cross reacting proteins were detected in extracts from the wildtype control plant.

15 In mammals, the assembly of secretory component with :the immunoglobulin requires the presence of J chain as described by Brandtzaeg, P. Prydz, H. Nature 311, 71-73 (1984). Plants expressing immunoglobulins containing a chimeric heavy chain (IgA/G) were crossed with plants 20 expressing protection protein. None of the 10 resulting progeny that expressed immunoglobulin and the protection protein without J chain produced assembled complexes as compared with the 10/10 plants that co-expressed J chain dimerized immunoglobulin and the protection protein without J chain, which assembled the M, 470 Kd immunoglobulin containing the protection protein. This confirms that J chain is required for the protection protein association with immunoglobulin as found in mammals. Only the approximately 210 Kd monomeric form of the immunoglobulin was recognized by anti-kappa antiserum, and the antisera that specifically bound the protection protein, recognized free protection protein, but no immunoglobulin heavy or light chains proteins.

Functional studies were carried out using the immunoglobulin produced in the 5 plant constructs using ELISA. All plants expressing immunoglobulin light and heavy chains, assembled functional immunoglobulin that specifically recognized streptococcal antigen (SA I/II).

The levels of binding and titration curves were similar to those of the native mouse hybridoma cell supernatant. No SA I/II binding was detected in plants expressing only J chain or only protection protein or in wildtype plants.

Binding of the immunoglobulins to immobilized purified streptococcal antigen or to native antigen on the bacterial cell surface was also detected using the antiserum which specifically binds the protection protein.

In these assays, the binding of the immunoglobulin containing the protection protein to the streptococcal antigen was specifically detected. These results confirmed that the protection protein was assembled with the immunoglobulin to produce an immunoglobulin containing 15 a protection protein in a manner which did not interfere with antigen binding.

The assembly of heavy and light chains into functional immunoglobulin molecules in plants is very efficient as shown by Hiatt, A. Cafferkey, R. 20 Bowdish, K. Nature 342, 76-78 (1989). A signal peptide must be present on both heavy and light chain constructs to direct the recombinant proteins to the endoplasmic reticulum antibody for assembly to take place in plants as was previously shown by Hiatt, A. Cafferkey, R. Bowdish, K. Nature 342, 76-78 (1989). This study has demonstrated the fidelity of immunoglobulin assembly which includes dimerization of monomeric antibody by J chain in the transgenic plants. These results demonstrated that in plants the dimeric immunoglobulin population represents a major proportion (approx. 57%) of the total antibody.

These results also demonstrate the production of an assembled immunoglobulin containing a protection protein which binds the corresponding antigen as well as the parent murine monoclonal antibody, which makes up a major proportion of the total antibody when the protection protein is incorporated (approximately 86 Co-expression of dimeric immunoglobulin with the protection protein in plants has led to assembly of a functional immunoglobulin containing a protection protein.

All four transgenes for this complex protein were introduced into plants with the identical pMON530 expression cassette and native leader sequences. This vector contains a promoter sequence derived from the transcript of the cauliflower mosaic virus which directs expression of transgenes in a variety of cell types of most plant organs as has been described by Benfey, P. N.

Chua, N-H. Science 250, 959-966 (1990); and Barnes, W.

M. PNVAS 87,9183-9187 (1990). Directing expression of all four transgenes with the same promoter maximized the likelihood of coincidental expression in a common plant 15 cell. Microscopic observation of plants expressing an immunoglobulin containing a protection protein revealed that many cell types of the leaves contain the individual protein components that make up the immunoglobulin. These proteins accumulated at highest concentration in bundle 20 sheath cells and were confined by the cell walls of these and other cells, but were not found in intercellular spaces. Restriction of the largest immunoglobulin components, the protection protein and the chimeric immunoglobulin heavy chain, within the confines of a protoplastic or apoplastic compartment of individual cells would constrain the assembly of the secretory immunoglobulin to those cells in which all the component molecules are synthesized. The subcellular site(s) and mechanism of assembly remain to be determined, assembly of IgG heterotetramers in plants requires targeting of both proteins to the endomembrane system as has been previously shown by Hiatt, A. Cafferkey, R. Bowdish, K. Nature 342, 76-78 (1989); and Hein, M. Tang, McLeod, D.

Janda, K. D. Matt, A. C. Biotechnol Prog. 7, 455-461 (1991). In addition, we have demonstrated that a protection protein derived from mature secretory component devoid of signals for membrane integration, transcytosis or subsequent proteolysis can be assembled with chimeric immunoglobulin heavy chain containing immunoglobulin gamma and alpha protein domains. These results demonstrate that the inherent functions of IgG constant regions (protein A binding, complement fixation, Fc receptor activity) may be maintained in a dimeric immunoglobulin, capable of binding to a protective protein. These additional capabilities may be employed to enhance the function of an immunoglobulin used for passive immunotherapy and the development of plants capable of generating a functional immunoglobulin containing a protection protein will have significant implications in passive immunotherapy. The level of expression of the immunoglobulin containing a 15 protection protein is high and the production can be scaled up to agricultural proportions, to allow economical production of monoclonal antibodies.

Methods 20 The following methods were used to prepare and analyze the Immunoglobulin of this Example.

i) Antibody assembly in transgenic Nicotiana tabacum.

Leaf segments were homogenized in 150mM NaC1 25 Tris-HCI (pH8) (TBS), with leupeptin (10og/ml). The extracts were boiled for 3 minutes, in 75mM Tris-HCI (pH6.8), 2% SDS, under non-reducing conditions and SDS- PAGE in 4% acrylamide was performed. The gels were blotted onto nitrocellulose. The blots were incubated for 2 hrs in TBS with 0.05% Tween 20 and 1% non-fat dry milk, followed by the appropriate antiserum and incubated for 2 hrs at 37 0 C. After washing, the second layer alkaline phosphatase conjugated antibody was applied for 2 hrs at 37 0 C. Antibody binding was detected by incubation with 300mg/ml nitroblue tetrazolium and 150mg/ml 5-bromo-4chloro 3-indolyl phosphate.

88 These extracts were analyzed using western analysis to determine whether the immunoglobulins were assembled into immunoglobulin molecules by analyzing Western blots of plant extracts prepared under non-reducing conditions, were with anti-kappa antiserum (Bradsure, UK) and an antiserum which specifically recognizes protection protein. The immunoglobulins produced in the plants were compared to the monoclonal IgGl Guys 13 immunoglobulin described by Smith, R. Lehner, T. Oral Microbiol.

Immunol. 4, 153-158 (1989).

ii) Western Analysis.

Western analysis was performed on each of the plant extracts prepared under reducing conditions to identify individual protein components of the immunoglobulin.

15 Samples of the various plant extracts were prepared as described previously, but with the addition of 5% 3mercaptoethanol. SDS-PAGE in 10% acrylamide was performed and the protein in the gels transferred to nitrocellulose.

Individual proteins were detected using anti-mouse yl heavy chain (Sigma, UK); anti-mouse kappa chain (Bradsure, UK) or an antiserum that specifically recognized the protection protein, followed by the appropriate alkaline S"phosphatase conjugated antibody.

iii) Western Analysis to Show Production of Immunoglobulin ~Having a Protection Protein Western analysis of transgenic plant extract was performed as described in ii) above. The plant extracts from plants expressing the immunoglobulin containing the protection protein were subjected to SDS-PAGE under both non-reducing and reducing conditions and the proteins transferred to nitrocellulose. The immunoglobulin components were detected with an anti-kappa antiserum or with a sheep antiserum which specifically recognized the protection protein followed by an appropriate alkaline phosphatase labeled 20 antibody.

iv) Expression of Antigen-Specific Immunoglobulin Containing a Protection Protein in transqenic Nicotiana tabacum.

To demonstrate that the plants were producing antigen-specific immunoglobulin, plant extract binding to purified streptococcal antigen (SA) I/II, detected with horseradish peroxidase labeled anti-kappa chain antiserum was determined. The presence of a protection protein in the antigen-specific immunoglobulin was demonstrated by plant extract binding to purified streptococcal antigen I/II and streptococcal cells detected with a sheep antiserum immunospecific for a protection protein, followed by alkaline phosphatase labeled donkey anti-sheep antiserum. These tests for antigen-specific 15 immunoglobulin were carried out in microtitre plates that were coated with purified SA I/II (2ug/ml) in TBS, or log phase growth Strep, mutans (NCTC 10449), in bicarbonate buffer (pH Blocking was with 5% non-fat dry milk in TBS at room temperature for 2 hours. Plant leaves were 20 homogenized in TBS with 1Oug/ml leupeptin (Calbiochem, USA). Mouse Guy's 13 hybridoma cell culture supernatant (IgG) was used as a positive control. The supernatants were added in serial two-fold dilutions to the microtitre plate and incubation was at room temperature for 2 hours.

25 After washing with TBS with 0.05% Tween 20, bound immunoglobulin chains were detected with either a goat anti-mouse light chain specific antibody, conjugated with horseradish peroxidase (Nordic Pharmaceuticals, UK), or a sheep anti-SC antiserum, followed by an alkaline phosphatase labeled donkey anti-sheep antibody for 2 hours at room temperature. Detection was with 2.2'-azino-di-[3ethyl-benzthiazolin-sulphonate (Boehringer, W. Germany) for HRPO conjugated antibody or disodium p-nitrophenyl phosphate (Sigma, UK) for alkaline phosphatase conjugated antibody.

v) Localization of Immunoqlobulin Components in Plants Photomicrographs of transgenic plants expressing immunoglobulins containing protection proteins and control Nicotiana tabacum leaf were prepared using immunogold detection of murine alpha chain. Briefly, leaf blades were cut into 2mm x 10mm segments and fixed in 3% (w/v) paraformaldehyde, 0.5% glutaraldehyde, 5% (w/v) sucrose in lOOmM sodium phosphate (pH After dehydration in anhydrous ethanol, leaf segments were infiltrated with xylene, embedded in paraffin and cut into 3mm sections and mounted on glass slides for immunochemical staining. The leaf sections were incubated with primary antibodies, affinity purified rabbit antimouse alpha chain (which reacts with the A/G hybrid heavy 15 chain) or sheep anti-rabbit SC, and then with secondary antibody; goat anti-rabbit-lOmn gold or rabbit anti-sheep- 10mn gold. The immunogold signal was intensified by silver enhancement. The plants were visualized using both Phase contrast and bright field microscopy on the same leaf cross section. Immunolocalization of the protection protein on serial sections was used to show the same cellular localization for heavy chain as immunoglobulin.

The analysis was carried out on the following cells and cell compartments: spongy mesophyll cells, epidermal cells, intercellular spaces, palisade parenchyma cells, and vascular bundles.

SFurther analysis of the exact localization of immunoglobulin components was carried out by analyzing serial sections of Nicotiana tabacum vascular bundle and control Nicotiana tabacum vascular bundle with immunogold detection for each of the components of the immunoglobulin. Serial sections of a transgenic plant leaves from plants expressing secretory immunoglobulin were incubated with an antibody that specifically recognizes the protection protein or with anti-IgA antibody followed by the appropriate gold-labeled secondary antibody. A control leaf section from a transgenic plant that did not contain any immunoglobulin coding sequences was also incubated with anti-IgA antibody, followed by gold-labeled goat anti-rabbit antiserum, or with the gold-labeled secondary antibodies alone and confirmed the specificity of staining. Both Phase contrast illumination of a minor vascular bundle and Bright field illumination of the same field were used to show immunogold localization of the protection protein.

Bright field illumination of a serial leaf cross section of the vascular bundle demonstrated the same immunogold localization of the immunoglobulin heavy chain as was shown for the protection protein.

Example 8. Production of a Useful Plant Extract 15 Containing Immunoqlobulins Having a Protection Protein Plant pieces (either leaf, stem, flower, root, or combinations) from plants producing immunoglobulins containing a protection protein were mixed with homogenization buffer (2 milliliter buffer per gram of 20 plant material; homogenization buffer: 150 mM NaC1, 20 mM Tris-Cl, pH homogenized into a pulp using a Waring blender and centrifuged at 10,000 X g to remove debris.

The supernatant was then extracted with an equal volume of HPLC-grade ethyl acetate by shaking at room temperature, followed by centrifugation at 10,000 X g. The aqueous ~phase was transferred to another container, remaining ethyl acetate was removed from the aqueous phase by placing the solution under vacuum. The resulting crude extract consistently contained 100 Ag immunoglobulin having a protection protein per ml. This method is useful for any plant containing an immunoglobulin having a protection protein.

A number of methods for homogenization have been used including a mortar and pestle or a Polytron and can be performed either in the cold or at room temperature.

The extract may be further purified by delipidation, by extraction with hexane or other organic solvents.

Delipidation is not essential for deriving a useful product from the plant extract but is advantageous in cases where the final product is a purified immunoglobulin having a protection protein. In many instances the crude extract will contain a sufficiently high quantity of immunoglobulin having a protection protein 100 pg/mL) to be useful without any further purification or enrichment. For an oral application, the extract would be mixed with commonly used flavorings and stabilizers. For a dental application, the extract would in addition be mixed with a gelling reagent to maintain contact of the extract with teeth. For a gastric application, the flavored extract could be swallowed directly.

15 Example 9. Stability of an Immunoqlobulin Containing a Protection Protein.

Two sets of crude plant extracts were prepared as described above. The first extract was derived from a plant expressing an IgG1 antibody and the second extract 20 was derived from a plant expressing an immunoglobulin containing a protection protein. Crude plant extracts of this type from plants are known to contain a variety of proteolytic enzymes. Prolonged incubation of extracts at room temperature or at 370 C therefore constitutes a proteolytic digestion.

Using ELISA the quantity of gamma-kappa complexes in the two extracts was determined as a function of time at both room temperature and 370 C. In these assays, an anti-kappa chain antibody was used to coat the plate followed by incubation with the plant extract at--37 0 C for 1 hour. An anti-gamma chain antibody conjugated to HRPO was used for detection of immunoglobulin derived from the plant. The quantity of immunoglobulin having a protection protein contained in the extract immediately after the extract was prepared was taken to be 100%. After 3 hours at room temperature, the IgG1 contained 40% apd the immunoglobulin containing the protection protein contained After 6 hours, the remaining IgG1 antibody was and the immunoglobulin containing the protection protein abundance was still After 12 hours, there was no detectable IgG1 whereas -90% of the immunoglobulin containing the protection protein remained. A significant decrease (to in the abundance of protected antibody was not observed until 48 hours after the extract was prepared.

Example 10. Eukarvotic Tetratransgenic Cells ExDressing Immunoglobulins Containing Protection Protein.

The four chains comprising the immunoglobulin containing a protection protein can also be expressed in other cell types either in in vitro (cell cultures) or in 15 vivo (transgenic animals). See, Manipulating the Mouse Embryo; A Laboratory Manual, B. Hogan et al., Cold Spring Harbor Laboratory (1986) In the case of transgenic animals, purified preparations of appropriate vector DNAs are adjusted to a final concentration of 2 ng/pl in 10 mM Tris, 0.2 mM EDTA, pH 7.4. Pronuclear injections are performed using zygotes prepared from inbred animals.

Injected eggs are then transferred to pseudopregnant females using standard techniques. Live born animals are then screened for the presence of transgenes using any of a number of commonly used techniques such as PCR and ELISA. Members of the pedigree expressing different .components of the immunoglobulin containing the protection protein are then mated to produce multi-transgene animals.

Progeny from these crosses are then screened to identify those that express all four chains. Depending on the type of vector used for zygotic injections various cell types can be identified in the transgenic animals which assemble the complete immunoglobulin containing a protection protein. These vector DNAs can consist of specific promoter elements which allow transcription of the transgene in particular cell types or tissues. Each vector could express a single component of the protected antibody (IgG/A, J chain, protection protein, or kappa chain) or could potentially express more than one component. In this instance, the vector would contain an appropriate number of promoter regions and restriction sites to allow for transcription of each transgene.

Expression of all four chains in a cell culture system can be achieved using a DNA vector from which each component can be individually promoted. This would require four expression cassettes (containing promoter, multiple cloning site, and polyadenylation region) on the same vector DNA. Alternatively, individual cell lines can be sequentially transfected with individual vectors expressing single chains so long as each vector confers a selective resistance onto the cell line.

15 Commonly available vectors, such as pMAMneo (Clontech) can be adapted either for multiple expression or as a series of vectors expressing distinct selectable markers.

Transfection of any eukaryotic cells, such as 20 fibroblasts, is done by conventional techniques. Briefly, cells are split 1:20 the day before transfection and are transfected at approximately 30% confluency using 125 mM CaC12, 140 mM NaC1, 25 mM Hepes, 0.75 mM NaHPO4, pH 7.05, and 5 ig DNA 10 cm dish. After 16 hours of DNA incubation, cells are shocked by 10% dimethyl sulfoxide for 3 minutes. Forty eight hours after transfection, S" cells are subjected to selection by growth in the appropriate medium containing an antibiotic or other cytotoxic reagent.

The resulting cells produce all the components for the immunoglobulin containing the protection protein.

These components are properly assembled to produce a functional immunoglobulin containing a protection protein.

Example 11. Engineering A Protection Protein Fused to A Portion of the Cvtoplasmic Domain of the Rabbit Polvimmunoglobulin Receptor.

The construction of DNA segments encoding a protection protein fused to a segment encoding a segment of the cytoplasmic domain of the rabbit polyimmunoglobulin receptor is produced as follows. Protection protein cDNA encoding from the first amino acid of the signal sequence (METI,,) to GLU 6 0 6 is ligated into any plant expression vector, such as the pMON530 vector (digested with Bgl II and Xho I) as a Bgl II Xho I fragment. This protection protein derivative is obtained by PCR amplification using the appropriate oligonucleotide primers containing either 0 40 Sa Bgl II or Xho I recognition sequence which are also 15 complementary to DNA encoding residues -18 to -13 and residues 601 to 606 of the rabbit polyimmunoglobulin ~receptor respectively. The same procedure is performed in order to obtain a protection protein cDNA encoding from

MET-

8 to ALA, 6 2 except that the oligonucleotide containing 20 an Xho site is also complementary to the protection protein cDNA encoding residues 623 to 628.

~The cDNA encoding the rabbit polyimmunoglobulin receptor cytoplasmic domain fragment is obtained, also by PCR amplification, as a Xho I fragment. The 25 oligonucleotides employed are complementary to DNA encoding from ARG 6 s 3 to ALA 7 both containing Xho I a recognition sequences. This fragment is then ligated into the pMON530 vectors which contain the either of the protection protein cDNAs described above. The appropriate orientation of the cytoplasmic domain cDNA is determined by restriction digestions and by sequence analysis of plasmids obtained from transformed bacterial colonies.

The oligonucleotides employed for PCR amplification contain the appropriate number of nucleotides to ensure that the resulting cDNAs are in frame and capable of being translated as a continuous fusion protein containing both protection protein and cytoplasmic domain.

96 The resulting constructs in the appropriate orientation encode a protection protein fused directly to the polyimmunoglobulin receptor cytoplasmic domain with no functional transmembrane segment, operably linked to a DNA segment (promoter) enabling expression in a plant cell.

The constructs encode two additional amino acids (SER TRP) which are derived from introduction of the Xho I restriction site and which serve as a linker between the protection protein and the cytoplasmic domain.

These vectors are then used to transform Agrobacterium as previously described which in turn is used to transform plant cells. The same techniques described in the above Examples are used to produce a plant expressing this protein as part of an 15 immunoglobulin.

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34, 613 212/127 (6 19) 552 -840 0 (619) 552-0159 67-3510 a 99 SEQUENCE LISTING INFORMATION FOR SEQ ID NO: 1: SEQUENCE CHARACTERISTICS: LENGTH: 3517 base pairs 0 TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear DESCRIPTION: Rabbit polyimmunoglobulin receptor (ix) FEATURE: NAME/KEY: Coding Sequence LOCATION: 124....2445 0 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1: GGCCGGGGTT ACGGGCTGGC CAGCAGGCTG TGCCCCCGAG TCCGGTCAGCAGGAGGGGAA GAAGTGGCCT AAAATCTCTC CCGCATCGGC AGCCCAGGCC TAGTGCCCTA CCAGCCACCA GCC ATG GCT CTC TTC TTG CTC ACC TGC CTG CTG GCT GTC TTT TCA GCG Met Ala Leu Phe Leu Leu Thr Cys Leu Leu Ala Val Phe Ser Ala r

GCC

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Phe

AAG

Lys

GTG

Val 130

CAA

Gin

AAT

Asn

ACC

Thr

AGC

Ser

TAC

Tyr

GTG

Val

TGT

Cys 115

CTG

Leu

TTA

Leu

GAA

Glu

ACC

Thr

TGC

Cys 70

AGA

Arg

GTT

Val

GGA

Gly

CAG

Gin

TTG

Leu

GGC

Gly

CGG

Arg 55

GTG

Val

GGC

Gly

GAC

Asp

GTC

Val

AAG

Lys 135

GGT

Gly

GAC

Asp 40

CAC

His

ACG

Thr

AAG

Lys

CAA

Gln

AAC

Asn 120

CCA

Pro

CCC

Pro

TCG

Ser

AGC

Ser

CTT

Leu

CTC

Leu

CTC

Leu 105

GGC

Gly

GAG

Glu 10 AGC TCC Ser Ser GTG TCC Val Ser CGG AAG Arg Lys GCC TCG Ala Ser ACC GAC Thr Asp 90 ACC CAG Thr Gin CGT GGC Arg Gly CCT GAT Pro Asp

ATA

Ile

ATC

Ile

TTC

Phe

ACC

Thr

TTC

Phe

AAC

Asn

CTG

Leu

GAC

Asp 140

TTT

Phe

ACA

Thr

TGG

Trp

GGC

Gly

CCT

Pro

GAC

Asp

GAC

Asp 125

GTT

Val

GGT

Gly

TGC

Cys

TGC

Cys

TAC

Tyr

GAT

Asp

TCA

Ser 110

TTC

Phe

GTT

Val

CCC

Pro

TAC

Tyr

CGG

Arg

ACG

Thr

AAA

Lys

GGG

Gly

GGT

Gly

TAC

Tyr 120 168 216 264 312 360 408 456 504 552 600 AAA CAA TAT GAG AGT TAT ACA GTA ACC ATC ACC TGC CCT TTC ACA TAT Lys Gin Tyr Glu Ser Tyr Thr Val Thr Ile Thr Pro Phe Thr Tyr 100 GCG ACT AGG CAA Ala Thr Arg Gin CTA AAG AAG TCC TTT TAC AAG GTG GAA Val Glu Leu Lys Lys Ser Phe Tyr 160

CTT

Leu

TAT

Tyr

TTC

Phe

GTC

Val

CTC

Leu 240

GTG

Val

TCC

Ser

ACA

Thr

AAC

Asn

GGG

Gly 320

CCC

Pro

CGC

50 Arg

CC

Arg

CTC

Leu

GGG

Gly 400

GTA

Val1

AAG

Lys

ACA

Thr

TGC

Cys 225

CGA

Arg

ACC

Thr

TTG

Leu

ATA

Ile

GGC

Gly 305

AAC

Asn

ACC

Thr

AGC

Ser

TGC

Cys

TGG

Trp 385

CTG

CTC

Leu

GC

Gly

GTC

Val 210

CAG

Gin

CTG

Leu

TTT

Phe

CGC

Arg

GAT

Asp 290

CAC

His

TAT

Tyr

CAG

Gin

CCC

Pro

CCC

Pro 370

GAA

Giu

GTT

ATC

Ile

AGA

Arg 195

ACC

Thr

ACT

Ser

CTA

Leu

CAA

Giu

CAC

Gin 275

CCA

Pro

TTC

Phe

CTC

Leu

CTT

Leu

CCT

Pro 355

TAC

Tyr

CCC

Gly

CAC

ATT

Ile 180

ATA

Ile

ATC

Ile

CCA

Gly

ACT

Thr

TGT

Cys 260

CTT

Val

CC

Ala

ACT

Ser

TC

Cys

CCC

Arg 340

CTC

Val

AAC

Asn

ACT

Ser

AAA

CAT

Asp

ACC

Thr

AAC

Lys

AC

Ser

CCT

Pro 245

CC

Al a

ACC

Arg

TTC

Phe

GTA

Val

GA

Gly 325

CAA

Gin

TTC

Leu

CCC

Pro

CAA

Gin

GAC

TCC ACC ACT AAC Ser Ser Ser Lys 185 TTC CAC ATC CAA Leu Gin Ile Gin 200 CAT TTC CAC CTC His Leu Gin Leu 215 GAC CCC ACT CCT Asp Pro Thr Ala 230 CCT CTG CTC TAT Cly Leu Leu Tyr CTC CAC TCT GAA Leu Asp Ser Ciu 265 CCT CCC AAT CTC Gly Cly Asn Val 280 GAG CCC AGG ATC Clu Gly Arg Ile 295 CTC ATC CCA GCC Val Ile Ala Gly 310 CTC CAC TCC AAT Val Gin Ser Asn CTC TTC GTC AAT Leu Phe Val Asn 345 AAG CCC TTT CCA Lys Gly Phe Pro 360 AAG, ACA ACC CAC Lys Arg Ser Asp 375 ACC CCC CAT CTC Thr Arg His Leu 390 TAC ACA CCC AGG Tyr Thr Gly Arg Lys 170

GAG

Clu

ACT

Ser

AAT

Asn

CAA

Ciu

CCA

Cly 250

GAC

Asp

GTC

Val

CTC

Leu

CTG

Leu

CCT

Gly 330

CAA

Glu

CGA

Cly

AGC

Ser

CTG

Leu'

CTG

Leu 410

GCA

Al a

ACC

Thr

CAT

Asp

CAA

Ciu 235

AAC

Asn

CCA

Al a

ATT

Ile

TTC

Phe

ACC

Arg 315

CAG

Gin

GAG

Giu

GCC

Cly

CAC

His

CTC

Val 395

CC

Ala

AAC

Lys

ACA

Thr

GCT

Al a 220

CAC

Gin

CTC

Leu

AAC

Asra

GAC

Asp

ACC

Thr 300

AAC

Lys

TCT

Ser

ATC

Ile

TCC

S er

CTC

Leu 380

GAC

Asp

CTG

CAC

Asp

CCA

Al a 205

CCC

Cly

AAC

Asra

CCC

Cly

CC

Al a

AC

Ser 285

AAC

Lys

GAA

Clu

CCC

Gly

GAC

Asp

GTC

Val 365

CAC

Gin

AC

Ser

TTC

CCC

Pro 190

AAA

Lys

CAC

Gin

GTT

Val1

GC

Cly

CTA

Val 270

CAG

Gin

CCT

Al a

GAC

Asp

GAT

Asp

GTG

Val 350

ACC

Thr

CTG-

Leu

GC

Gly

GAA

175

AGC

Arg

CAA

Glu

TAT

Tyr

CAC

Asp

TCC

Ser 255

CCA

Ala

CCC

Gly

GAC

Ciu

ACA

Thr

CCC

Gly 335

TCC

Ser

ATA

Ile

TAT

Tyr

GAG

Ciu

GAG

Ciu 415 CAC CCC GAA Asp Cly Giu 648 696 744 792 840 888 936 984 1032 1080 1128 1176 1224 1272 1320 1368 Leu Val Gin Lys Asp 405 Leu Phe Giu 101 CCT GGC AAT GGC Pro Gly Asn Gly ACC TTC TCA GTC GTC CTC AAC CAG CTC Thr Phe Ser Val Val Leu Asn Gin Leu 420 425 GAT GAA GGC TTC TAC TGG TGT GTC Asp Giu Giy Phe Tyr Trp Cys Val 435

AGC

Ser 440 GAT GAC GAT GAG Asp Asp Asp Glu ACT GCC GAG Thr Ala Glu 430 TCC CTG ACG Ser Leu Thr 445 CCC ACG ATC Pro Thr Ile ACT TCG GTG Thr Ser Vai 450 GAC AAG TTC Asp Lys Phe 465 AAG CTC CAG ATC Lys Leu Gin Ile GAC GGA GAA CCA Asp Gly Giu Pro ACT GCT GTG Thr Ala Val GGA GAG CCT GTT Gly Giu Pro Val ATC ACC TGC CAC Ile Thr Cys His

TTC

Phe 480 CCA TGC AAA TAC Pro Cys Lys Tyr TCC TCC GAG AAG Ser Ser Glu Lys

TAC

Tyr 490 TGG TGC AAG TGG Trp Cys Lys Trp GAC CAT GGC TGC Asp His Gly Cys GAG GAC CTG CCC ACT AAG CTC AGC TCC AGC GGC GAC Glu Asp Leu Pro Thr Lys Leu Ser Ser Ser Giy Asp 500 505 510 CTT GTG AAA Leu Val Lys GTC AGC GAA Val Ser Glu 530 CAC GAG TTT His Giu Phe 545 AAC AAC AAC CTG Asn Asn Asn Leu

GTC

Vai 520 CTC ACC CTG Leu Thr Leu ACC TTG GAC TCG Thr Leu Asp Ser 525 GAT GAC GAG GGC Asp Asp Giu Gly TAC TGG TGT GGC Tyr Trp Cys Gly AAA GAC GGG Lys Asp Gly 1416 1464 1512 1560 1608 1656 1704 1752 1800.

1848 1896 1944 1992 2040 2088 2136 GAA GAG GTT Glu Giu Val GCC GTC AGG GTG Ala Vai Arg Vai CTG ACA GAG CCA Leu Thr Giu Pro

GCC

Ala 560 AAG GTA GCT GTC Lys Vai Ala Val CCA GCC AAG GTA Pro Ala Lys Val

CCT

Pro 570 GTC GAC CCA GCC Val Asp Pro Ala GCA GCC CCC GCG Ala Ala Pro Ala GCT GAG GAG AAG Ala Giu Giu Lys

GCC

Ala 585 AAG GCG CGG TGC Lys Aia Arg Cys CCA GTG Pro Val 590 CCC AGG AGA Pro Arg Arg TGT CCA GAA Cys Pro Glu 610 CAG TGG TAC CCA Gin Trp Tyr Pro TCA AGG AAG CTG Ser Arg Lys Leu AGA ACA AGT Arg Thr Ser 605 CAG AGT GCG Gin Ser Ala CCT CGG CTC CTT GCG GAG GAG GTA GCA Pro Arg Leu Leu Ala Giu Glu Val Ala 615 GAA GAC Glu Asp 625 CCA GCC AGT GGG Pro Ala Ser Gly

AGC

Ser 630 AGA GCG TCT GTG Arg Ala Ser Val GCC AGC ACT GCT Ala Ser Ser Ala GGA CAA AGC GGG AGT GCC AAA GTA CTG Gly Gin Ser Gly Ser Ala Lys Val Leu

ATC

Ile 650 TCC ACC CTG GTG Ser Thr Leu Val

CCC

Pro 655 TTG GGG CTG GTG Leu Gly Leu Val

CTG

Leu 660 GCA GCG GGG Ala Ala Gly GCC ATG GCC GTG GCC ATA Ala Met Ala Val Ala Ile 665 GCC AGA Ala Arg 670 102 GCC CGG CAC Ala Arg His ACA GAC ATT Thr Asp Ile 690 ATT GAC AAC Ile Asp Asn 705 GGA AAG GAT Gly Lys Asp 720 GAG CCC AAG Giu Pro Lys TAC TCA GCT Tyr Ser Ala AGG AAC Arg Asn AGC ATG TCA Ser Met Ser CCA AGC GCC Pro Ser Ala GTG GAC CGA GTT TCC ATC Val Asp Arg Val. Ser Ile 680 GAC TTG GAG AAC TCC AGG Asp Leu Glu Asn Ser Arg 695 TGC CCC GAT GCC CGG GAG Cys Pro Asp Ala Arg Glu 710 715 ACG GCC ACC GAG AGC ACC Thr Ala Thr Glu Ser Thr 730 CGG TCA TCC AAG GAA GAA Arg Ser Ser Lys Giu Glu 745 GGA AGC TAC AGG Gly Ser Tyr Arg 685 GAG TTC GGA GCC Glu Phe Gly Ala 700 ACG GCC CTC GGA Thr Ala Leu Gly GTG GAG ATT GAG Val Glu Ile Giu 735 GCC GAC CTG GCC Ala Asp Leu Ala 750 GCT GAG CAC CAA Ala Giu His Gin 765 GAG TTA Giu Leu AAG GCA Lys Ala 740 TTC CTG Phe Leu 755

GCG

Al a 725

AAA

Lys CTC CAA TCC AAC ACC ATA GCT Leu Gin Ser Asn Thr Ilie Ala 760 25 GAT GGC CCC Asp Gly Prc 77C

CGCCGCCGCC

GCCGGGGGCT

GCCCAGAGGT

GGGGTGGGGG

CCAGGTCCTG

TGGATGGGAG

CCTTCATTCA

TCGAAGCCGT

TCACTCAGGC

CCCTGGGCGT

ACTGAGGTTG

TGCTGTCCTC

TTTAGAACAT

ACGGGGGAAA

GTGCCCCGTG

CGGGGAGGGC

CCAGCACCTC

TGTGATTGCC

AAG GAG GCC Lys Giu Ala ACCTGTGAAA AT] CAACCGCCCT GC GTGCTGGTCC CC TGTGAGGGGT TC AGGGAGGGGC CT] GCCAGAGGCG CT] AAAGTCCCAG TG TGTGCAAACA TC ATCCTGTCCT CC GTCTGCAAGT CA TACTCAAGGG CA TTGGCCAGAG GI GGAAGAAGAA GA AGGCCCCCTC CT TGTCTGAAGA GI CTGATCCCCA GA CGGGCTGACC CT

ACTGGGA

TAG GCACAGCCGG CCACCGCCGC CGCCGCCACC GCCGC

'CACCTTCC

ACCCCCCA

TCCTCCAC

CTACTTGC

*CTCGAAGG

TTCCGGCC

~GCTGCTGC

ACTGGAGG

CCAGTATC

CCCCAGAC

~CCCTGCGA

'CTCTCCAC

~GGGGGATG

'TTTCTGTC

TCCCAGTG

~CAGCTGAA

TGCTAACA

AGAATCACGT TGATCCTCGG TGTCCCCACC ACCTAAACTT GGCATCCAGG CCTGGCTCAA AGCCCGGTTC TCCCGAGAGA CAGACAGACC AGAGAGGGGG ACCCCCTCCC TCCCTGCCCC CTAGGGTCCA GGCGCTGGCC AAGCCAGGGC TCCTCCCGGG AGGAGATGTC AAGCGTCTGA ACATGTTCTC GCCATTTTAC GATGGAGCAA CAGCAAACTA AGGAGCCCCT GCCCCTGTAG GCCCTGGACG CTGACCTCTC ACTCTCGGGG ACCTGCGGAG GAAAGAAGAA AAGAGGGTGT GTTTAAGGTC CTTGTCCCTG CAT CAGAAAT GTGATTTAAT

GGTCCCCAGA

CCCTACCTGT

TGTTCCCGTT

AGCTAAGGAT

GAGGAGCCCT

CACCCTCCTT

GCACGCCTCC

CTGTGTATCC

AGGCTGTGTG,

AGATGAGAAC

GATGGGCTTC

GAAGCAGAGT

C CAAGCC CC C

TTGAGCATTC

TTGTCAGTGC

TGAGCTTTAA

CATTAAACAT

2184 2232 2280 2328 2376 2424 2480 2540 2600 2660 2720 2780 2840 2900 2960 3020 3080 3140 3200 3260 3320 3380 3440 3500 3517 103 INFORMATION FOR SEQ ID NO: SEQUENCE CHARACTERISTICS: LENGTH: 773 amino acids TYPE: amino acid STRANDNESS: single TOPOLOGY: linear DESCRIPTION: Rabbit polyimmunoglobulin receptor (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2: Met Ala Leu Phe Leu Leu Th 1 5 Thr Ala Gin Ser Ser Leu Le r r r r r Glu Pro 25 Glu Gin Glu Tyr Asn Gin 145 Thr Val Lys Thr Cys 225 Arg Val Thr Glu Glu Phe Lys Val 130 Tyr Arg Leu Gly Val 210 Gin Leu SAsn Val 35 SThr Ser Ser Gly Tyr Ser Val Val 100 Cys Gly 115 Leu Val Glu Ser Gin Leu Ile Ile 180 Arg lie 195 Thr Ile Ser Gly Leu Thr Val Arg Gly Thr Val Ser Tyr Lys 165 Asp Thr Lys Ser Pro 245 Ala Thr Cys 70 Arg Val Gly Gin Thr 150 Lys Ser Leu His Asp 230 Gly Leu Arg 55 Val Gly Asp Val Lys 135 Val Ser Ser Gin Leu 215 Pro Leu Asp Leu Glu Gly r Cys Leu Leu Ala 10 i Gly Pro Ser Ser 25 I Asp Ser Val Ser 40 His Ser Arg Lys SThr Leu Ala Ser 75 Lys Leu Thr Asp 90 Gin Leu Thr Gn 105 Asn Gly Arg Gly 120 Pro Glu Pro Asp Thr Ile Thr Cys 155 Phe Tyr Lys Val 170 Ser Lys Glu Ala I 185 Ile Gin Ser Thr J 200 Gin Leu Asn Asp 2 Thr Ala Glu Glu G 235 Leu Tyr Gly Asn L 250 Ser Glu Asp Ala A 265 Val Ile Ile Phe Thr Phe Asn Leu Asp 140 Pro 3lu Lys rhr %la l1n 5eu sn SPhe Ser Ala Phe Gly Pro Thr Cys Tyr Trp Cys Arg Gly Tyr Thr Pro Asp Lys Asp Ser Gly 110 Asp Phe Gly 125 Val Val Tyr Phe Thr Tyr Asp Gly Glu 175 Asp Pro Arg 190 Ala Lys Glu 205 Gly Gin Tyr Asn Val Asp I Gly Gly Ser 255 Ala Val Ala S 270 Ala Gly Tyr Glu Ser Gly Ser Val Lys Ala 160 Leu Tyr Phe Val ,eu 240 al er

I

Thr Phe Glu Cys 260 Leu Arg Gin 275 Val Arg Gly Gly Asn 280 Val Val Ile Asp Ser Gln Gly Thr 285 104 Ile Asp 290 Pro Ala Phe Glu Gly Arg 295 Ile Leu Phe Thr Lys Ala Glu Asn 300 4 .4 4 Gly 305 Asn Thr Ser Cys Trp 385 Leu Gly Glu Ser Lys 465 Pro 40 His Val Ser Glu 545 Lys Ala Arg His Tyr Gin Pro Pro 370 Glu Val Asn Gly Val 450 Phe Cys Gly Lys Glu 530 Phe Val Pro Arg Phe Leu Leu Pro 355 Tyr Gly Gin Gly Phe 435 Lys Thr Lys Cys Cys 515 Asp Glu Ala Ala Arg 595 Ser Cys Arg 340 Val Asn Ser Lys Thr 420 Tyr Leu Ala Tyr Glu 500 Asn Asp Glu Val Pro 580 Gin Val Gly 325 Gin Leu Pro Gin Asp 405 Phe Trp Gin Val Phe 485 Asp Asn Glu Val Glu 565 Ala Trp Val 310 Val Leu Lys Lys Thr 390 Tyr Ser Cys Ile Gln 470 Ser Leu Asn Gly Ala 550 Pro Glu Tyr Leu Ser 630 Ile Gin Phe Gly Arg 375 Arg Thr Val Val Val 455 Gly Ser Pro Leu Trp 535 Ala Ala Glu Pro Ala 615 Ala Ser Val Phe 360 Ser His Gly Val Ser 440 Asp Glu Glu Thr Val 520 Tyr Val Lys Lys Leu 600 Glu Gly Asn Asn 345 Pro Asp Leu Arg Leu 425 Asp Gly Pro Lys Lys 505 Leu Trp Arg Val Ala 585 Ser Glu' Leu Gly 330 Glu Gly Ser Leu Leu 410 Asn Asp Glu Val Tyr 490 Leu Thr Cys Va1 Pro 570 Lys Arg Val Arg 315 Gin Glu Gly His Val 395 Ala Gin Asp Pro Glu 475 Trp Ser Leu Gly Glu 555 Val Ala Lys Ala Asp 635 Lys Ser Ile Ser Leu 380 Asp Leu Leu Glu Ser 460 Ile Cys Ser Thr Ala 540 Leu Asp Arg Leu Val 620 Glu Gly Asp Val 365 Gin Ser Phe Thr Ser 445 Pro Thr Lys Ser Leu 525 Lys Thr Pro Cys Arg 605 Gin Asp Asp Va1 350 Thr Leu Gly Glu Ala 430 Leu Thr Cys Trp Gly 510 Asp Asp Glu Ala Pro 590 Thr Ser Thr Gly 335 Ser Ile Tyr Glu Glu 415 Glu Thr Ile His Asn 495 Asp Ser Gly Pro Lys 575 Val Ser Ala Gly 320 Pro Arg Arg Leu Gly 400 Pro Asp Thr Asp Phe 480 Asp Leu Val His Ala 560 Ala Pro Cys Glu Pro Giu Pro Arg Leu 610 Asp 625 Pro Ala Ser Gly Arg Ala Ser Val Ala Ser Ser Ala Ser 640 105 Gly Gln Ser Gly Ser Ala Lys Val Leu Ile Ser Thr Leu Val Pro Leu 645 650 655 Gly Leu Val Leu Ala Ala Gly Ala Met Ala Val Ala Ile Ala Arg Ala 660 665 670 Arg His Arg Arg Asn Val Asp Arg Val Ser Ile Gly Ser Tyr Arg Thr 675 680 685 Asp Ile Ser Met Ser Asp Leu Glu Asn Ser Arg Glu Phe Gly Ala Ile 690 695 700 Asp Asn Pro Ser Ala Cys Pro Asp Ala Arg Glu Thr Ala Leu Gly Gly 705 710 715 720 Lys Asp Glu Leu Ala Thr Ala Thr Glu Ser Thr Val Glu Ile Glu Glu 725 730 735 Pro Lys Lys Ala Lys Arg Ser Ser Lys Glu Glu Ala Asp Leu Ala Tyr 740 745 750 Ser Ala Phe Leu Leu Gin Ser Asn Thr Ile Ala Ala Glu His Gin Asp 755 760 765 Gly Pro Lys Glu Ala 770 INFORMATION FOR SEQ ID NO: 3: SEQUENCE CHARACTERISTICS: LENGTH: 2919 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear DESCRIPTION: Human polyimmunoglobulin Receptor (ix) FEATURE: NAME/KEY: Coding Sequence LOCATION: 235....2472 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3: AGAGTTTCAG TTTTGGCAGC AGCGTCCAGT GCCCTGCCAG TAGCTCCTAG AGAGGCAGGG GTTACCAACT GGCCAGCAGG CTGTGTCCCT GAAGTCAGAT CAACGGGAGA GAAGGAAGTG GCTAAAACAT TGCACAGGAG AAGTCGGCCT GAGTGGTGCG GCGCTCGGGA CCCACCAGCA ATGCTGCTCT TCGTGCTCAC CTGCCTGCTG GCGGTCTTCC CAGCCATCTC CACG AAG Lys 1 AGT CCC ATA TTT GGT CCC GAG GAG GTG AAT AGT GTG GAA GGT AAC TCA Ser Pro Ile Phe Gly Pro Glu Glu Val Asn Ser Val Glu Gly Asn Ser 10 GTG TCC ATC ACG TGC TAC TAC CCA CCC ACC TCT GTC AAC CGG CAC ACC Val Ser Ile Thr Cys Tyr Tyr Pro Pro Thr Ser Val Asn Arg His Thr 25 CGG AAG TAC TGG TGC CGG CAG GGA GCT AGA GGT GGC TGC ATA ACC CTC Arg Lys Tyr Trp Cys Arg Gin Gly Ala Arg Gly Gly Cys Ile Thr Leu 40 120 180 237 285 333 381 106 TCC TCG GAG GGC Ser Ser Giu Gly GTC TCC AGC AAA Val Ser Ser Lys GCA GGC AGG GCT Ala Gly Arg Ala CTC ACC AAC TTC CCG GAG AAC GGC ACA TTT GTG GTG AAC ATT Leu Thr Asn Phe Pro Giu Asn Gly Thr Phe Val Val Asn Ile GCC CAG Ala Gin CTG AGC CAG Leu Ser Gin GAC TCC GGG CGC Asp Ser Giy Arg AAG TGT GGC CTG Lys Cys Gly Leu GGC ATC AAT Gly Ile Asn AGC CGA GGC Ser Arg Giy 100 CTG TCC TTT GAT Leu Ser Phe Asp

GTC

Vai 105 AGC CTG GAG GTC AGC CAG GGT CCT Ser Leu Giu Vai Ser Gin Gly Pro 110 GGG CTC Giy Leu 115 CTA AAT GAC ACT Leu Asn Asp Thr

AAA

Lys 120 GTC TAC ACA GTG Val Tyr Thr Val CTG GGC AGA ACG Leu Gly Arg Thr ACC ATC AAC TGC Thr Ile Asn Cys TTC AAG ACT GAG AAT GCT CAA AAG AGG Phe Lys Thr Giu Asn Ala Gin Lys Arg 140 TCC TTG TAC AAG Ser Leu Tyr Lys ATA GGC CTG TAC Ile Giy Leu Tyr

CCT

Pro 155 GTG CTG GTC ATC Val Leu Val Ile GAC TCC Asp Ser 160 AGT GGT TAT 30 Ser Gly Tyr CAG GGT ACT Gin Gly Thr 180 AAT CCC AAC TAT Asn Pro Asn Tyr

ACA

Thr 170 GGA AGA ATA CGC Gly Arg Ile Arg CTT GAT ATT Leu Asp Ile 175 CAA CTC AGG Gin Leu Arg GGC CAG TTA CTG Gly Gin Leu Leu AGC GTT GTC ATC Ser Val Val Ile CTC AGC Leu Ser 195 GAT GCT GGG CAG Asp Ala Giy Gin CTC TGC CAG GCT Leu Cys Gin Aia

GGG

Gly 205 GAT GAT TCC AAT Asp Asp Ser Asn

AGT

Ser 210 AAT AAG AAG AAT Asn Lys Lys Asn GAC CTC CAA GTG Asp Leu Gin Val AAG CCC GAG CCC Lys Pro Giu Pro CTG GTT TAT GAA Leu Val Tyr Giu CTG AGG GGC Leu Arg Gly TCA GTG Ser Val 235 AAA TTT Lys Phe 250 ACC TTC CAC TGT Thr Phe His Cys GCC CTG Ala Leu 240 GGC CCT GAG GTG GCA AAC GTG GCC Gly Pro Giu Val Ala Asn Val Ala 245 CTG TGC CGA Leu Cys Arg CAG AGC AGT Gin Ser Ser 255 AGG GCC CCA Arg Ala Pro GGG GAA AAC Gly Giu Asn 260 TGT GAC GTG GTC Cys Asp Val Val AAC ACC CTG GGG Asn Thr Leu Gly 1005 1053 i101 1149 GCC TTT Ala Phe 275 GAG GGC AGG ATC Glu Gly Arg Ilie

CTG

Leu 280 CTC AAC CCC CAG Leu Asn Pro Gin AAG GAT GGC TCA Lys Asp Gly Ser

TTC

Phe 290 AGT GTG GTG ATC Ser Val Val Ile GGC CTG AGG AAG Gly Leu Arg Lys GAT GCA GGG CGC Asp Ala Giy Arg

TAC

Tyr 305 107 CTG TGT GGA GCC CAT TCG GAT GGT CAG CTG CAG GAA GGC TCG CCT Leu Cys Gly Ala His Ser Asp Gly Gin Leu Gin Glu Gly Ser Pro 310 320 *fl*

CAG

Gin

CCC

Pro

CCC

Pro

GAA

Giu 370

TGG

Trp

GGC

Gly

GCC

Ala

ACC

Thr

GGG

Gly 450

TTT

Phe

AAC

Asn

GCC

Ala

AAC

Asn

CAG

Gin 530

GCC

Al a

ACT

Thr

TAC

Tyr 355

GGG

Gi y

GTT

Val

AAC

Asn

GGC

Gly

GTG

Val 435

AAT

Asn

CCA

Pro

ACG

Thr

TTC

Phe

CTG

Leu 515

GGC

Gly

TGG

Trp,

GTG

Val 340

AAC

Asn

GCC

Al a

AAG

Lys

GGC

Gly

TTC

Phe 420

GAG

Glu

GTC

Val

TGC

Cys

GGC

Gly

GTG

Val 500

GTG

Val

CAC

His

CAA

Gin 325

GTG

Val1

CGT

Axg

CAG

Gin

GCC

Al a

ACC

Thi- 405

TAC

Tyr

ATC

Ile

ACG

Thi-

AAA

Lys

TGC

Cys 485

AAC

Asn

ACC

Thr

TTC

Phe

CTC

Leu

AAG

Lys

AAG

Lys

AAT

Asn

CAG

Gin 390

TTC

Phe

TGG

Ti-p

AAG

Lys

GCT

Al a

TTC

Phe 470

CAG

Gin

TGT

Cys

AGG

Arg

TAT

Tyr

TTC

Phe

GGG

Gly

GAA

Giu

GGC

Gly 375

TAC

Tyr

ACT

Thr

TGT

Cys

ATT

Ile

GTG

Val 455

TCC

Ser

GCC

Al a

GAC

Asp

GCT

Ala

GGA

Gly 535

GTC

Val1

GTG

Val

AGC

Ser 360

CGC

Arg

GAG

Glu

GTC

Val

CTG

Leu

ATC

Ile 440

CTG

Leu

TCG

Ser

CTG

Leu

GAG

Glu

GAT

Asp 520

GAG

Glu

AAT

Asn

GCA

Ala 345

AAA

Lys

TGC

Cys

GGC

Gly

ATC

Ile

ACC

Thr 425

GAA

Giu

GGA

Gly

TAC

Tyr

CCC

Pro

AAC

Asn 505

GAG

Giu

ACT

Thr

GAG

Glu 330

GGA

GJly

AGC

Ser

CCC

Pro

CGC

Arg

CTC

Leu 410

AAC

Asn

GGA

Gly

GAG

Glu

GAG

Glu

AGC

Ser 490

AGC

Ser

GGC

Gly

GCA

Al a

GAG

*Giu

AGC

Ser

ATC

Ile

CTG

Leu

CTC

Leu 395

AAC

Asn

GGC

Gly

GAA

Giu

ACT

Thi-

AAA

Lys 475

CAA

Gin.

CGG

Arg

TGG

T-p,

GCC

Ala

GTC

Val

TCC

Ser

TCT

Ser

AAG

Lys

CTG

Leu 380

TCC

Ser

CAG

Gin

GAT

Asp

CCA

Pro

CTC

Leu 460

TAC

Tyr

GAC

Asp

CTT

Leu

TAC

Tyr

GTC

VJal 540

ACG

Thr

GTG

Val

TAC

Tyr 365

GTG

Val

CTG

Leu

CTC

Leu

ACT

Thi-

AAC

Asn 445

AAG

Lys

TGG

Ti-p

GAA

Glu

GTC

Val

TGG

Ti-p 525

TAT

Tyr

ATT

Ile

GCC

Al a 350

TGG

Ti-p

GAC

Asp

CTG

Leu

ACC

Thi-

CTC

Leu 430

CTC

Leu

GTC

Val1

TGC

Cys

GGC

Gly

TCC

Ser 510

TGT

Cys

GTG

Val

CCC

Pro 335

GTG

Val

TGT

Cys

AGC

Ser

GAG

Giu

AGO

Ser 415

TGG

Ti-p

AAG

Lys

CCC

Pro

AAG

Lys

CCC

Pro 495

CTG

Leu

GGA

Gly

GCA

Ala

CGC

Axg

CTC

Leu

CTC

Leu

GAG

Giu

GAG

Giu 400

CGG

Arg

AGG

Arg

GTA

Val

TGT

Cys

TGG

Trp, 480

AGC

Ser

ACC

Thr

GTG-

Val

GTT

Val

AGC

Ser

TGC

Cys

TGG

Ti-p

GGG

Gly 385

CCA

Pro

GAC

Asp

ACC

Thi-

CCA

Pro

CAC

His 465

AAT

Asn

AAG

Lys

CTG

Leu

AAG

Lys

GAA

Giu 545 1197 1245 1293 1341 1389 1437 1485 1533 1581 1629 1677 1725 1773 1821 1869 1917 GAG AGG AAG GCA GCG GGG Glu Arg Lys Ala Ala Gly 550 TCC CGC GAT Ser Arg Asp AGC CTA GCG AAG GCA GAC Ser Leu Ala Lys Ala Asp 108 GCT GCT CCT GAT Ala Ala Pro Asp 565 GAG AAG GTG Glu Lys Val CTA GAC TCT Leu Asp Ser 570 AGG CTT TTT Arcr Leu Phe GGT TTT CGG GAG ATT GAG Gly Phe Arg Glu Ile Glu 575 GCA GAG GAA AAG GCG GTG Ala Glu Glu Lys Ala Val AAC AAA GCC Asn Lys Ala 580 GCA GAT ACA Ala Asp Thr ATT CAG GAT CCC Ile Gln Asp Pro 590

TCT

S er AGA GAT CAA Arg Asp Gln

GCC

Al a 600 GGG AGC AGA Gly Ser Arg GTG GAT TCC Val Asp Ser 595

AGC

Ser

GGC

Gly 610 TCT GAG GAA Ser Glu Glu GGT GGA AGC TCC Gly Gly Ser Ser

AGA

Arg 620 CTG GTC TCC Leu Val Ser CTG GTG CCC CTG Leu Val Pro Leu

GGC

Gly 630 CTG GTG CTG GCA Leu Val Leu Ala

GTG

Val 635

GTC

Val1 GGA GCC GTG GCT Gly Ala Val Ala GTG GCC AGA Val Ala Arg CGG CAC AGG AAG Arg His Arg Lys GAC CGA GTT Asp Arg Val 0* Se

S

S

S

S

S. S S 4.~S

S

S.

25 AGC TAC AGG Ser Tyr Arg 660 TTT GGA GCC 30 Phe Gly Ala 675 TCC CTC GGA Ser Leu Gly ACA GAC ATT AGC Thr Asp Ile Ser GAC TTC GAG Asp Phe Glu TCA ATC AGA Ser Ile Arg 655 TCC AGG GAA Ser Arg Glu CAG GAG ACA Gln Glu Thr 1965 2013 2061 2109 2157 2205 2253 2301 2349 2397 2445 AAT GAC AAC Asn Asp Asri GGA GCC TCT TCG Gly Ala Ser Ser GGA AAA Gly Lys 690

GAG

Glu

GAA

Glu 695

AAG

Lys TTT GTT GCC Phe Val Ala ACC ACT Thr Thr 700 GAG AGC ACC Glu Ser Thr

ACA

Thr 705 ACC AAA GAA Thr Lys Glu

CCC

Pro 710 AAG GCA AAA AGG TCA TCC AAG GAG GAA GCC Lys Ala Lys Arg Ser Ser Lys Glu Glu Ala 715 720

S

GAG ATG GCC Glu Met Ala

TAC

Tyr 725 AAA GAC TTC CTG Lys Asp Phe Leu CAG TCC AGC ACC Gln Ser Ser Thr GTG GCC GCC Val Ala Ala 735 GAG GCC CAG GAC GGC CCC CAG Glu Ala Gln Asp Gly Pro Gln 740 TAGACGGTGT CGCCGCCTGC TCCCTGCA 2500

CCCATGACAA

CACTCCCTGC

CCTCCTCAGT

GGAGGTCCCA

GTCCTCATGG

GAGACGTGCA

ACTCCATCCC

TCACCTTCAG

TCTAACACCT

GACATCAAAG

CTTGCAACTT

GTCCCTTGAA

GCGCCCCTCT

TCCCTCCCGT

AATCATGTCG

GCCTAGGTTT

CCTGGCCTAA

CTTTCTGTTG

GGAAGAGGGA

GCACCCTTAT

CCTTCCCCTC

ATCCTGGGGG

TTCCTACTGT

TTGTTCCTAT

AGAGAACCTC

CCAGGGTGGG

CATGGGATGT

TTCTTCTTTC

CCCTCAGCTC

CCTCAGAGGC

TGGGGATGAG

AGGTACGGAG

AGAGCTGATT

CAACAGAATT

CTTACCATCA

CTGGGGACCC

GTGCTGGTCC

GGTGGCATGA

AAGAATAGAG

GCAGAAAGGA

TTTTCCCTCC

AAAGATGTA

2560 2620 2680 2740 2800 2860 2919 109 INFORMATION FOR SEQ ID NO: 4: SEQUENCE CHARACTERISTICS:

LENGTH:

TYPE:

746 amino acids amino acid single linear

STR

TOP

DESCRIPTION:

(xi) SEQUENCE Pro Ile Phe Gly 5

ANDNESS:

OLOGY:

Human Polyimmunogibulin Receptor RIPTION: SEQ ID NO: 4

DESC

Lys 1 Ser Pro Giu Glu Val Asn Ser Val Giu Gly Asn 10 Ser Ile Thr sea$ 6 0000 5 s es a :0 6 00*4 0

S

S.

S

**SS

0 0*00 S S 0 6004

S

0.60

S

Thr Arg Lys Leu Ile Ser Asn Leu Thr 25 65 Gin Leu Ser 30 Asn Ser Arg Pro Giy Leu 115 Thr Val Thr 130 Lys Ser Leu 145 Ser Ser Gly Ile Gin Gly Arg Leu Ser 195 Asn Ser Asn 210 Glu Leu Vai 225 Leu Gly Pro Ser Gly Glu Tyr Trp Ser Glu Asn Phe Gin Asp 85 Gly Leu 100 Leu Asn Ile Asn Tyr Lys Tyr Val 165 Thr Gly 180 Asp Ala Lys Lys Tyr Glu Glu Val 245 Asn Cys Cys Cys Gly Pro 70 Asp Ser Asp Cys Gin 150 Asn Gin Gly Asn Asp 230 Ala Asp Tyr Arg Tyr 55 Glu Ser Phe Thr Pro 135 Ile Pro Leu Gin Ala 215 Leu Asn Val Tyr Gin 40 Val Asn Gly Asp Lys 120 Phe Gly Asn Leu Tyr 200 Asp Arg Val Val Pro 25 Gly Ser Gly Arg Val 105 Val Lys Leu Tyr Phe 185 Leu Leu Gly kla Val Pro Ala Ser Thr Tyr 90 Ser Tyr Thr Tyr Thr 170 Ser Cys Gin Ser Lys 250 Asn Lys Phe 75 Lys Leu Thr Glu Pro 155 Gly Val Gin Val Val 235 Phe Thr Tyr Val Cys Glu Val Asn 140 Val Arg Val Ala Leu 220 Thr Leu Leu Ala Vai Gly Val Asp 125 Ala Leu Ile Ile Gly 205 Lys Phe Cys Gly Arg Gly Gly Cys Gly Asn Leu Ser 110 Leu Gin Val Arg Asn 190 Asp Pro His Arg Lys 270 Thr Ser Val Asn Arg His Ile Arg Ile Gly Gin Gly Lys Ile Leu 175 Gln Asp Glu Cys Gin 255 krg Thr Ala Ala Ile Gly Arg Arg Asp 160 Asp Leu Ser Pro Ala 240 Ser Ala 260 265 Pro Ala Phe 275 Glu Gly Arg Ile Leu Asn Pro Gin Asp 285 Lys Asp Gly 110 r r Ser Tyr 305 Ile Ser Cys Trp Gly 385 Pro Asp Thr Pro His 465 Asn Lys Leu Lys Glu 545 Asp Glu Val Ser Phe 290 Leu Gin Pro Pro Glu 370 Trp Gly Ala Thr Gly 450 Phe Asn Ala Asn Gin 530 Glu Ala Asn Ala Gly 610 Ser Cys Ala Thr Tyr 355 Gly Val Asn Gly Val 435 Asn Pro Thr Phe Leu 515 Gly Arg Ala Lys Asp 595 Ser Val Val Gly Ala Trp Gin 325 Val Val 340 Asn Arg Ala Gin Lys Ala Gly Thr 405 Phe Tyr 420 Glu Ile Val Thr Cys Lys Gly Cys 485 Val Asn 500 Val Thr His Phe Lys Ala Pro Asp 565 Ala Ile 580 Thr Arg Ser Glu Ile His 310 Leu Lys Lys Asn Gin 390 Phe Trp Lys Ala Phe 470 Gin Cys Arg Tyr Ala 550 Glu Gin Asp Glu Gly 630 Thr 295 Ser Phe Gly Glu Gly 375 Tyr Thr Cys Ile Val 455 Ser Ala Asp Ala Gly 535 Gly Lys Asp Gin Gin 615 Gly Leu Asp Gly Val Asn Val Ala 345 Ser Lys 360 Arg Cys Glu Gly Val Ile Leu Thr 425 Ile Glu 440 Leu Gly Ser Tyr Leu Pro Glu Asn 505 Asp Glu 520 Glu Thr Ser Arg Val Leu Pro Arg 585 Ala Asp 600 Gly Gly Arg Gin Glu 330 Gly Ser Pro Arg Leu 410 Asn Gly Glu Glu Ser 490 Ser Gly Ala Asp Asp 570 Leu Gly Ser Lys Leu 315 Glu Ser Ile Leu Leu 395 Asn Gly Glu Thr Lys 475 Gin Arg Trp Ala Val 555 Ser Phe Ser Ser Glu 300 Gin Ser Ser Lys Leu 380 Ser Gin Asp Pro Leu 460 Tyr Asp Leu Tyr Val 540 Ser Gly Ala Arg Arg 620 Asp Glu Thr Val Tyr 365 Val Leu Leu Thr Asn 445 Lys Trp Glu Val Trp 525 Tyr Leu Phe Glu Ala 605 Ala Gly Ser Pro 335 Val Cys Ser Glu Ser 415 Trp Lys Pro Lys Pro 495 Leu Gly Ala Lys Glu 575 Lys Val Val Arg Pro 320 Arg Leu Leu Glu Glu 400 Arg Arg Val Cys Trp 480- Ser Thr Val Val Ala 560 Ile Ala Asp Ser Val 640 Thr Leu Val Pro Leu 625 Leu Val Leu Ala Val Gly Ala Val Ala Gly Arg Glu Thr Thr 705 Ala Ala Val Ser Phe Ser 690 Glu Glu Glu Arg Ala 645 Arg Thr 660 Ala Asn Gly Gly Lys Glu Ala Tyr 725 Gln Asp 740 His Ile Asn Glu 695 Lys Asp Pro Arg Ser Met 680 Glu Lys Phe Gln Lys Met 665 Gly Phe Ala Leu Glu 745 11l Asn Val 650 Ser Asp Ala Ser Val Ala Lys Arg 715 Leu Gln 730 Ala Asp Arg Val Phe Glu Asn 670 Ser Ile Thr 685 Thr Thr Glu 700 Ser Ser Lys Ser Ser Thr Ser 655 Ser Gln Ser Glu Val 735 INFORMATION FOR SEQ ID NO: 25 SEQUENCE CHARACTERISTICS: LENGTH: 3630 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear DESCRIPTION: Bovine Polyimmunoglobulin Receptor (ix) FEATURE: NAME/KEY: Coding Sequence LOCATION: 152....2425 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: GATCTCCTCG GAGGGTCGTG CAGCGGCCCT GGGTCCCTGC CGGCACCAGT ACTTGCGCGT GTGCTCCCAA AGCTGACGGG ATAGGAGGAA GGAGCTCAAA CAACCACACA GGACGGTGGC TGGCGGCAGA GACCCGCGGG AGCCCCCAGC G ATG TCG CGC CTG TTC CTC GCC 45 Met Ser Arg Leu Phe Leu Ala 1 TGC CTG CTG GCC ATC TTC CCA GTG GTC TCC ATG AAG AGT CCC ATC TTC Cys Leu Leu Ala Ile Phe Pro Val Val Ser Met Lys Ser Pro Ile Phe 10 15 GGT CCC GAG GAG GTG AGC AGC GTG GAA GGC CGC TCA GTG TCC ATC AAG Gly Pro Glu Glu Val Ser Ser Val Glu Gly Arg Ser Val Ser Ile Lys 30 TGC TAC TAC CCG CCC ACC TCC GTC AAC CGG CAC ACG CGC AAG TAC TGG Cys Tyr Tyr Pro Pro Thr Ser Val Asn Arg His Thr Arg Lys Tyr Trp 45 50 TGC CGG CAG GGA GCC CAG GGC CGC TGC ACG ACC CTC ATC TCC TCG GAG Cys Arg Gln Gly Ala Gln Gly Arg Cys Thr Thr Leu Ile Ser Ser Glu 65 GGC TAC GTC TCC GAC GAC TAC GTG GGC AGA GCC AAC CTC ACC AAC TTC Gly Tyr Val Ser Asp Asp Tyr Val Gly Arg Ala Asn Leu Thr Asn Phe 80 120 172 220 268 316 364 412 112 CCG GAG AGC GGC ACG TTT GTG GTG GAC ATC AGC CAT CTC ACC CAT AAA Pro Glu Ser Gly Thr Phe Val Val Asp Ile Ser His Leu Thr His Lys

GAC

Asp

AAC

Asn 120

CAT

His

TGC

Cys

AAG

Lys 25 AGC Ser

ACA

30 Thr 200

GGG

Gly 35

AAC

Asn

GAC

Asp

GCA

Al a

AAT

Asn 280

AGG

Arg

ACC

Thr TCA GGG Ser Oly 105 TTC GAT Phe Asp GCC CAC Ala His CCT TTC Pro Phe ACA ATC Thr Ile 170 AAC AGC Asn Ser 185 TTA GTG Leu Val ATG TAT Met Tyr ATT GAC Ile Asp TTG AGG Leu Arg 250 AAT GTG Asn Val 265 GTA GTC Val Val ATC OTG Ile Val AGC CTG Ser Leu

CGC

Ar g

GTG

Val

GTC

Val1

ACO

Thr 155

CAG

Gin

TAT

Tyr

TTC

Phe

OTC

Val

CTC

Leu 235

AGC

Ser

CCC

Pro

ATC

Ile

TCC

Ser

AGG

Arg 315

TAC

Tyr

AGC

Ser

TAC

Tyr 140

COT

Arg

GAC

Asp

AAA

Lys

AOC

Ser

TGC

Cys 220

CAG

Gin

TCG

Ser

AAA

Lys

AAC

Asn

OTO

Val 300

AAA

Lys

AAG

Lys

CTG

Leu 125

ACT

Thr

OCO

Ala

TOT

Cys

GAC

Asp

OTT

Val 205

CAG

Gin

GTO

Val

OTO

Val

TTT

Phe

ACG

Thr 285

CCC

Pro

GAG

Oiu

TOT

Cys 110

GAO

Glu

ATA

Ile

AAT

Asn

TTC

Phe

AGA

Arg 190

GTC

Val

OCT

Al a

CTO

Leu

ACC

Thr

CTO

Leu 270

TTG

Leu

AAO

Lys

GAC

Asp 95

GOC

Oly

GTC

Val

GAC

Asp

TCT

Ser

CAA

Gin 175

OCA

Al a

ATC

Ile 000 Oly

GAO

Oiu

TTT

Phe 255

TOC

Cys 000 Gly

GAC

Asp G CA Ala

CTO

Leu

AOC

Ser

CTO

Leu

GAG

Giu 160

OTT

Val

CAT

His

AAC

Asn

GAC

Asp

CCT

Pro 240

GAC

Asp

CAG

Gin

AAG

Lys

AAT

Asn 000 Oly 320

GC

Oly

CAA

Gin

GOC

Gly 145

AAO

Lys

GTC

Val

ATC

Ile

COA

Arg

OAT

Asp 225

GAG

Giu

TOT

Cys

AAG

Lys

AG

Lys

GOT

Gly 305

COC

Arg

ATT

Ile

OAT

Asp 130

AGO

Arg

AGA

Arg

GAC

Asp

AOT

Ser

OTC

Val 210 0CC Ala

CCT

Pro

TCC

Ser

AAG

Lys

OCT

Ala 290

OTC

Val1

TAC

Tyr

AOC

Ser 115

CCT

Pro

ACT

Thr

AAA

Lys

TCC

Ser

ATC

Ile 195

AAO

Lys

AAA

Lys

GAO

Glu

CTG

Leu

AAT

Asn 275

CAG

Gin

TTC

Phe

OTO

Val

AOC

Ser

OCA

Al a

OTO

Val

TCC

Ser

ACC

Thr 180

CTA

Leu

CTC

Leu 0CC Al a

CTO

Leu

GOC

Oly 260 000 Gly

GAC

Asp

AOT

Ser

TOC

Cys

COT

Arg

CAG

Gin

ACC

Thr

TTG

Leu 165 000 Gly

GOT

Oly

AOT

Ser

OAT

Asp

OTT

Val1 245

CCC

Pro

OGA

Oly

TTC

Phe

OTO

Val 000 Gly 325

GOC

Gly

OCA

Al a

ATC

Ile 150

TOC

Cys

TAT

Tyr

ACC

Thr

OAT

Asp

AAA

Lys 230

TAT

Tyr

GAO

Glu

OCT

Ala

CAG

Gin

CAC

His 310 0CC Ala

CTT

Leu

AOT

Ser 135

AAC

Asn

AAO

Lys

OTG

Val

AAC

As n

OCT

Al a 215

ATC

Ile

OGA

Gly

GTO

Val

TGC

Cys

GGC

Gly 295

ATT

Ile

CAG

Gin 100 460 508 556 604 652 700 748 796 844 892 940 988 1036 1084 1132 1180 CCT GAO GOT GAG Pro Oiu Gly Glu 330 CCC CAG GAC Pro Gin Asp GOC TOG CCT OTO CAG GCC TOO CAA CTC Gly Trp Pro Val Gin Ala Trp, Gin Leu 335 340 113 TTC GTC AAT GAA GAG ACG GCA ATC CCC GCA AGC CCC TCC GTG GTG AAA 1228 Phe Val Asn Giu Giu Thr Ala Ile Pro Ala Ser Pro Ser Val Val Lys 345 350 355 GGT GTG AGG GGA GGC TCT GTG ACT GTA TCT TGC CCC TAC AAC CCT AAG 1276 Gly Val Arg Gly Gly Ser Val Thr Val Ser Cys Pro Tyr Asn Pro Lys 360 365 370 375 GAT GCC AAC AGC GCG AAG TAC TGG TGT CAC TGG GAA GAG GCT CAA AAC 1324 Asp Ala Asn Ser Ala Lys Tyr Trp Cys His Trp Glu Glu Ala Gin Asn 380 385 390 GGC CGC TGC CCG CGG CTG GTG GAG AGC CGG GGG CTG ATG AAG GAG CAG 1372 Gly Arg Cys Pro Arg Leu Val Giu Ser Arg Gly Leu Met Lys Giu Gin 395 400 405 TAC GAG GGC AGG CTG GTG CTG CTC ACC GAG CCG GGC AAC GGC ACC TAC 1420 Tyr Giu Gly Arg Leu Val Leu Leu Thr Glu Pro Gly Asn Gly Thr Tyr 410 415 420 ACC GTC ATC CTC AAC CAG CTC ACC GAT CAG GAC GCC GGC TTC TAC TGG 1468 Thr Val Ile Leu Asn Gin Leu Thr Asp Gin Asp Ala Gly Phe Tyr Trp 425 430 435 TGC GTG ACC GAC GGC GAC ACG CGC TGG ATC TCC ACA GTG GAG CTC AAG 1516 Cys Val Thr Asp Gly Asp Thr Arg Trp Ile Ser Thr Val Glu Leu Lys 440 445 450 455 GTT GTC CAA GGA GAA CCA AGC CTC AAG GTA CCC AAG AAC GTC ACG GCT 1564 30 Val Val Gin Gly Glu Pro Ser Leu Lys Val Pro Lys Asn Val Thr Ala 460 465 470 TGG CTG GGA GAG CCC TTA AAG CTC TCC TGC CAC TTC CCC TGC AAA TTC 1612 Trp Leu Gly Glu Pro Leu Lys Leu Ser Cys His Phe Pro Cys Lys Phe 475 480 485 TAC TCC TTT GAG AAG TAC TGG TGT AAG TGG AGC AAC AGA GGC TGC AGC 1660 Tyr Ser Phe Giu Lys Tyr Trp Cys Lys Trp Ser Asn Arg Gly Cys Ser 490 495 500 GCC CTG CCC ACC CAG AAC GAC GGC CCC AGC CAG GCC TTT GTG AGC TGC 1708 Ala Leu Pro Thr Gin Asn Asp Gly Pro Ser Gin Ala Phe Vai Ser Cys 505 510 515 GAC CAG AAC AGC CAG GTC GTC TCC CTG AAC CTG GAC ACA GTC ACC AAG 1756 Asp Gin Asn Ser Gin Val Val Ser Leu Asn Leu Asp Thr Val Thr Lys 520 525 530 535 GAG GAT GAA GGC TGG TAC TGG TGT GGA GTG AAG GAA GGC CCC CGA TAC 1804 Glu Asp Giu Gly Trp Tyr Trp Cys Giy Val Lys Giu Gly Pro Arg Tyr 540 545 550 GGG GAG ACG GCG GCT GTC TAC GTG GCA GTG GAG AGC AGG GTG AAG GGG 1852 Gly Giu Thr Ala Ala Val Tyr Val Ala Vai Giu Ser Arg Val Lys Gly 555 560 565 TCC CAA GGC GCC AAG CAA GTG AAA GCT GCC CCT GCG GGG GCG GCA ATA 1900 Ser Gin Gly Ala Lys Gin Val Lys Ala Ala Pro Ala Giy Ala Ala Ile 570 575 580 CAG TCG AGG GCC GGG GAG ATC CAG AAC AAA GCC CTT CTG GAC CCC AGC 1948 Gin Ser Arg Ala Gly Glu Ile Gin Asn Lys Ala Leu Leu Asp Pro Ser 585 590 595 114 TTT TTC GCA AAG GAA AGT GTG AAG GAC GCT GCT GGT GGA CCC GGA Gly Gly Pro Gly Phe 600

CCT

Pro Phe Ala Lys Glu Ser Val 605 Lys Asp Ala Al a 610

GCA

Al a 615 GCA GAT CCT Ala Asp Pro

CGC

Arg

GTG

Val CCT ACA GGA TAC Pro Thr Gly Tyr 625 CCC CTG GCC CTG Pro Leu Ala Leu AGC GGG AGC TCC Ser Gly Ser Ser AAA GCA Lys Ala 630 CTG GTC TCC Leu Val Ser GTC CTG GTC Val Leu Val GTG GCG ATC Val Ala Ile 650 GGG GTG GTC CGA Gly Val Val Arg CAC AGG AAG His Arg Lys

AAC

Asn 660

TCA

Ser GCA GGG GTC Ala Gly Vai 645 GTC GAC CGG Val Asp Arg GAC TTT GAG Asp Phe Glu ATT TCA Ile Ser 665 AAC TCC Asn Ser

ATC

Ile AGG AGC TAC Arg Ser Tyr GAT ATC AGC Asp Ile Ser AGG GAT TTT Arg Asp Phe GGA CGT GAC AAC Gly Arg Asp Asn GGA GCC TCT CCA Gly Ala Ser Pro 680

GCC

Ala CAA GAG ACG Gln Glu Thr

TCT

Ser 700

GAG

Glu CTC GGA GGG AAG Leu Gly Gly Lys

GAC

Asp 705

AAG

Lys GAG TTT GCC ACC Giu Phe Ala Thr ACT ACC Thr Thr 710 GAG GAC ACC 30 Glu Asp Thr AAG GAG GAA Lys Giu Glu 730

GTG

Val 715

GCC

Al a AGC AAA GAA Ser Lys Giu

CCC

Pro 720 AAG GCA AAG AGG TCG TCC Lys Ala Lys Arg Ser Ser 725 TTC CTC CTC CAG GCC AAA Phe Leu Leu Gin Ala Lys 740 GAC GAG GCC Asp Glu Ala ACC ACC Thr Thr 1996 2044 2092 2140 2188 2236 2284 2332 2380 2431 2491 2551 2611 2671 2731 2791 2851 2911 2971 3031 3091 3151 3211 AAC CTG GCC TCC GCC GCA ACC CAG AAC GGC CCG Asn Leu Ala Ser Ala Ala Thr Gln Asn Gly Pro 745 750

CCCTGGGCGC

GGCCCTCAGC

45 TGTCCTCAGA.

TATTGGGGGT

AAGGTGTGGA

GGAGACAACC

AGTGGAATCC

CTCTTCTTCT

TGCTAGACAC

GAGCCCCTGG

GGAGAAATCA

ACACAAGGCA

6 5 AGGTGGAGAG

CCCTTCCCTC

TCGGGGGGCT

GGGTGTGCTG

GAGGTGGTAC

GGAGAATTAA

GCAGAAAGGG

'TCCCTTCCAC

TTCCCTCATT

TGGGATAGGT

AGCCCACAGC

TAAAGGGTCT

CCATCAACAC

GTTTGCTCAG

CGCACGTGGC

CCACTGCCTG

GTTCCTTCTT

GAGGAGTTCC

GATCGCAGAG

GGCCATTCAG

CCCATCTCTG

AAAAATGTGC

AGGCCGCAAT

ATCTCTTCAC

GCAGCCCTGA

ATTCTTACCA

AGTCAGCAAG

AATCACGCTC

CACTCACACC

GGTGGCATCC

CACCTGCAGC

GGGCCTCTCA

CGCTTCCCTG

CACCTCTCCA

ATTTGGTTAC

CCCAGGCGGC

GTGTACACTC

GGCCTTAGGG

TTTCACAGGT

TGAGATGTAC

ACA GAA GCC Thr Glu Ala 755 CGAATCACGC IJ CCGCCTAGGC IJ AAGCCTGGCT I TTATTCGAAC C

GAAAGAAAAGC

TCCCCTTATT I TCCCCACTCC .7 TCACTAGATT C

AGCCTTCCGC

ACTGACCTCT C ATTATGTAAC z~ GAGAAAGCCG GAGTCTCAAG C TAG ACGGAG

~GATCCTCAG

'TCTCCTGTC

~ACTTGTTCC

;AGAGAACTA

xAGTGGGTGG

~GGGGATGTC

~TTCCATCTT

CAGGGACTC

LAACATCAAG

CCTCTGCTG

LCAGGCATAC

LGGTCCTGAG

~TAAAGATTT

115 GACACCTGCT GTCCCTACAG AGGAGAAGAA AAATGTAAAT

CCTTTCCCTG

CCCCCGAATG

AGGGAGGGGC

TTCTAAGCTC

GTGAATTAAT

TCTGTCACTC

TGAAGAGTTC

TGATCTCCAA

TGCACTTCAA

TAATAATTAA

GAGGGCCTCC

AAGACTGGTC

ACAGAGACCT

TAAGTGGAAA

AGAACTAAGG

CTAGCATCTA

AGACCATGAT

TCTCTCCAGA

TTTCACAGGC

AATAGGATAA

GGGAGGAAAA

TTTAAGTTTT

TGAGCTGGCA.

TTCCTCCAA-A

TGAGACAGCA TTCCATAGGA CCCACATCAG GGAAGATACC GAGAATGGTC AACACTCAAA AGGGGGGATT TGATGGTGCC TTTGTTTTTT TTTTTCCTTC CTTGCTAACA AATCAAAAAT 3271 3331.

3391.

3451 3511 3571 3630 INFORMATION FOR SEQ ID NO: 6: SEQUENCE CHAR-ACTERISTICS: LENGTH: 757 amino acids TYPE: amino acid STRANDNESS: single TOPOLOGY: linear DESCRIPTION: Bovine Polyimmunoglobulin Receptor (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 6: Met 30 1 Ser 35 Gly Ser Arg Leu Phe 5 Met Lys Ser Pro Arg Ser Val Ser Leu Ala Cys Ile Phe Gly Leu Leu Ala Ile Phe Pro Val Val 10 Ile Lys Pro 25 Tyr A-rg Glu Glu Val Ser Tyr Pro Pro Thr Gln Gly Ala Gln Ser Val Glu Ser Val Asn Gly Arg Cys 35 Arg His Thr Arg Lys Tyr Thr Leu Ile Ser Glu Gly Tyr Val Ser Asp Asp Tyr Gly Thr Phe Val Ala Asn Leu Phe Pro Glu Ser Gly Val Gly Val Asp Ile Ser His Gly Ile Ser 115 Gln Asn Pro His Lys Asp Ser 105 Phe Arg Tyr Lys Arg Gly Leu Asp Val Ser Cys Gly Leu 110 Glu Val Ser Ile Asp Leu Ala Gln Ala Ala His Val Gly 145 Lys Arg Thr Val Thr Ile 150 Asn Cys Pro Phe Thr Arg Ala 155 Asn Ser Arg Lys Ser Leu 165 Gly Val Asp Ser Cys Lys Lys Thr Tyr Val Ser Asn 185 Thr Asn Thr Leu 200 Gln Tyr Asp Cys Phe Gln Val 175 Lys Asp Arg Ala His 190 Ser Val Val Ile Asn 205 Ile Ser Ile Leu Gly 195 Val Phe 116 Arg Val Lys Leu Ser Asp Ala Gly Met Tyr Val 210 215 Cys Gin Ala Gly Asp 220 Asp 225 Glu Cys Lys Lys Gly 305 Arg 25 Pro Ala Ser His 385 Arg Glu Gin Ile Val 465 Cys Trp Ser Asn Ala Pro Ser Lys Ala 290 Val Tyr Val Ser Cys 370 Trp Gly Pro Asp Ser 450 Pro His Ser Gin Leu 530 Lys Glu Leu Asn 275 Gin Phe Val Gin Pro 355 Pro Glu Leu Gly Ala 435 Thr Lys Phe Asn Ala 515 Asp Ala Leu Gly 260 Gly Asp Ser Cys Ala 340 Ser Tyr Glu Met Asn 420 Gly Val Asn Pro Arg 500 Phe Thr Asp Val 245 Pro Gly Phe Val Gly 325 Trp Val Asn Ala Lys 405 Gly Phe Glu Val Cys 485 Gly Val Val Lys 230 Tyr Glu Ala Gin His 310 Ala Gin Val Pro Gin 390 Glu Thr Tyr Leu Thr 470 Lys Cys Ser Thr Ile Gly Val Cys Gly 295 Ile Gin Leu Lys Lys 375 Asn Gin Tyr Trp Lys 455 Ala Phe Ser Cys Lys 535 Asn Asp Ala Asn 280 Arg Thr Pro Phe Gly 360 Asp Gly Tyr Thr Cys 440 Val Trp Tyr Ala Asp 520 Glu Ile Leu Asn 265 Val Ile Ser Glu Val 345 Val Ala Arg Glu Val 425 Val Val Leu Ser Leu 505 Gin Asp Asp Arg 250 Val Val Val Leu Gly 330 Asn Arg Asn Cys Gly 410 Ile Thr Gin Gly Phe 490 Pro Asn Glu Leu 235 Ser Pro Ile Ser Arg 315 Glu Glu Gly Ser Pro 395 Arg Leu Asp Gly Glu 475 Glu Thr Ser Gly Gin Ser Lys Asn Val 300 Lys Pro Glu Gly Ala 380 Arg Leu Asn Gly Glu 460 Pro Lys Gin Gin Trp 540 Val Val Phe Thr 285 Pro Glu Gin Thr Ser 365 Lys Leu Val Gin Asp 445 Pro Leu Tyr Asn Val 525 Tyr Leu Thr Leu 270 Leu Lys Asp Asp Ala 350 Val Tyr Val Leu Leu 430 Thr Ser Lys Trp Asp 510 Val Trp Glu Phe 255 Cys Gly Asp Ala Gly 335 Ile Thr Trp Glu Leu 415 Thr Arg Leu Leu Cys 495 Gly Ser Cys Pro 240 Asp Gin Lys Asn Gly 320 Trp Pro Val Cys Ser 400 Thr Asp Trp Lys Ser 480 Lys Pro Leu Gly Val 545 Lys Glu Gly Pro Tyr Gly Glu Thr Ala Ala Val Tyr Val Ala 555 560 117 Gly Ala Lys Gin Val 570 Val Glu Ser Arg Val Lys Gly Ser Gin Lys Ala 575 Ala Lys Ala Tyr 625 Leu His Ile 2.5 ::n Pro Ala Ala 610 Ser Val Arg Ser Met 690 Glu Ala Leu 595 Gly Gly Leu Lys Met 675 Gly Phe Gly 580 Leu Gly Ser Val Asn 660 Ser Ala Ala Ala Asp Pro Ser Ala 645 Val Asp Ser Thr Ala Pro Gly Lys 630 Gly Asp Phe Pro Thr 710 Ile Ser Ala 615 Ala Val Arg Glu Glu 695 Thr Gin Phe 600 Pro Leu Val Ile Asn 680 Ala Glu Ser 585 Phe Ala Val Ala Ser 665 Ser Gin Asp Glu Arg Ala Asp Ser Ile 650 Ile Arg Glu Thr Glu 730 Ala Lys Pro Thr 635 Gly Arg Asp Thr Val 715 Ala Gly Glu Gly 620 Leu Val Ser Phe Ser 700 Glu Asp Glu Ser 605 Arg Val Val Tyr Glu 685 Leu Ser Glu Ile 590 Val Pro Pro Arg Arg 670 Gly Gly Lys Ala Gin Asn Lys Asp Thr Gly Leu Ala 640 Ala Arg 655 Thr Asp Arg Asp Gly Lys Glu Pro 720 Phe Thr 735 L Ala Lys Arg 725 Ser Ser Lys Ala Lys Asn Th .mly Leu Gin 740 Glu Ala Leu Ala 745 Ser Ala Ala Thr Gin Asn 750 ';:FORMATION FOR SEQ ID NO: 7: SEQUENCE CHARACTERISTICS: LENGTH: 3095 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear DESCRIPTION: Mouse Polyimmunoglobulin Receptor (ix) FEATURE: NAME/KEY: Coding Sequence LOCATION: 85....2400 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 7: TCACCTGGAG AGAAGGAAGT AGCTAAAACA TTCTCATACA AGAAGCCAAC CTGAGCGGCA CAGCCCCCCT GGAAGCCACA AGCA ATG AGG CTC TAC TTG TTC ACG CTC TTG Met Arg Leu Tyr Leu Phe Thr Leu Leu 1 GTA ACT GTC TTT TCA GGG GTC TCC ACA AAA AGC CCC ATA TTT GGT CCC Val Thr Val Phe Ser Gly Val Ser Thr Lys Ser Pro Ile Phe Gly Pro 111 159 118 a CAG GAG GTG AGT AGT ATA GAA Gin Giu Val Ser Ser Ile Giu 30 TAC CCA GAC ACC TCT GTC AAC Tyr Pro Asp Thr Ser Val Asn CAA GGA GCC AGC GGC ATG TGC Gin Gly Ala Ser Gly Met Cys CTC TCC AAG GAG TAT TCA GGC Leu Ser Lys Giu Tyr Ser Gly 80 AAC AAC ACA TTT GTG ATT AAC Asn Asn Thr Phe Vai Ile Asn 95 GGG AGC TAC AAG TGT GGC CTG Gly Ser Tyr Lys Cys Gly Leu 25 110 GAT GTC AGC CTG GAG GTC AGC Asp Vai Ser Leu Giu Val Ser 125 CAC GTC TAC ACA AAG GAC ATA His Val Tyr Thr Lys Asp Ile 140 TTC AAA AGG GAG AAT GTT CCC Phe Lys Arg Giu Asn Val Pro 155 160 A.AC CAG TCC TGC GAA CTT GTC Asn Gin Ser Cys Giu Leu Val 170 175 AGC TAT ATA GGC AGA GCA AAA Ser Tyr Ile Gly Arg Ala Lys 45 190 GTA TTC TAT GTC AAC ATT AGT Val Phe Tyr Val Asn Ile Ser 205 TAC ATC TGC CAA GCT GGA GAA Tyr Ile Cys Gin Ala Gly Giu 220 GAC CTC CAG GTG CTA GCG CCT Asp Leu Gin Val Leu Ala Pro 235 240 AGG TCC TCA GTG ACT TTT GAA Arg Ser Ser Val Thr Phe Glu 250 255 GAG GCC AAA TAT CTG TGC CGG Giy

CGG

Arg

ACA

Thr 65

AGA

Arg

ATT

Ile

GGT

Gly

CAG

Gin

GGC

Gly 145

AGC

Ser

ATT

Ile

CTT

Leu

CAC

His

GGT

Gly 225

GAG

Glu

TGT

Cys Asp

CAC

His 50

ACG

Thr

GCC

Ala

GAG

Giu

ACC

Thr

GTT

Vai 130

AGA

Arg

AAG

Lys

GAC

Asp

TTT

Phe

CTA

Leu 210

CCT

Pro

CCA

Pro

GAC

Asp Ser 35

ACC

Thr

CTC

Leu

AAC

Asn

CAG

Gin

AGT

Ser 115

CCT

Pro

A.AT

Asn

AAA

Lys

TCT

Ser

ATG

Met 195

ACG

Thr

AGT

Ser

GAG

Glu

CTG

Leu

AAG

Lys 275 GGC GAC TCT GTT TCC Val Ser CGG AAA Arg Lys ATC TCT Ile Ser CTC ATC Leu Ile CTC ACC Leu Thr 100 AAC CGA Asn Arg GAG TTG Glu Leu GTG ACC Val Thr TCC CTG.

Ser Leu 165 ACT GAG Thr Giu 180 AAA GGG Lys Gly CAC AAT His Asn GCT GAT Ala Asp CTG CTT Leu Leu 245 GGC CGT Gly Arg 260 GAA ACC

ATC

Ile

TAC

Tyr

TCA

Ser

AAC

Asn

CAG

Gin

GGC

Gly

CCG

Pro

ATT

Ile

TGT

Cys

AAG

Lys

ACC

Thr

GAT

Asp

AAG

Lys 230

TAT

Tyr

GAG

Glu

TGT

ACO

Thr

TGG

Trp

AAT

Asn

TTC

Phe

GAC

Asp

CTG

Leu

AGT

Ser 135

GAA

Giu

AAG

Lys

GTG

Val

GAC

Asp

GCT

Ala 215

AAG

Lys

AAA

Lys

GTG

Val

GAT

TGC

Cys

TGC

Cys

GGC

Gly

CCA

Pro

GAC

Asp

TCC

Ser 120

GAC

Asp

TGC

Cys

AAG

Lys

AAC

Asn

CTA

Leu 200

GGG

Gly

AAT

Asn

GAC

Asp

GCA

Al a

GTG

TAC

Tyr

CGA

Arg

TAC

Tyr

GAG

Giu

ACT

Thr 105

TTC

Phe

ACC

Thr

CCT

Pro

ACA

Thr

CCC

Pro 185

ACT

Thr

CTG

Leu

GTT

Val1

CTG

Leu

AAC

Asn.

265

ATC

207 255 303 351 399 447 495 543 591 639 687 735 783 831 879 927 ATG AAT Giu Ala Lys Tyr Leu Cys Arg Met Asn Glu Thr Cys Asp Val Ile 280 119 ATT AAC ACC GTG GGG AAG AGG GAT CGA GAC TTT GAG GGC AGG ATC CTG 9. 9* 9 9 .9 99 9 9*9* 9 9 999999 999999 9 9*99 9 999999 Ile

ATA

Ile

CTG

Leu

GGT

Gly 330

AAT

Asn

ACA

Thr 25 AGC Ser

TGC

30 cys

GGC

Gly 410

ATC

Ile

ACC

Thr 45 GAA Giu

GCA

Ala

TTC

Phe 490

GAG

His rn Asn

ACC

Thr

AGG

Arg 315

TTG

Leu

GAA

Glu

GGA

Gly

AGC

Ser

CCC

Pro 395

CGA

Arg

CTC

Leu

AAT

Asn

GCT

Ala

GTA

Val 475

TAC

Tyr

ATC

Ile Thr

CCC

Pro 300

AAG

Lys

CCT

Pro

GAG

Glu

GGC

Gly

CTC

Leu 380

GCG

Al a

CTG

Leu

AAC

Asn

GGT

Gly

AGA

Thr 460

CTA

Leu

TCC

Ser

CTG

Leu Leu 285

AAG,

Lys

GAG

Glu

CAA

Gin

TCT

Ser

TCT

Ser 365

AAG

Lys

CTT

Leu

GGA

Al a

GAG

Gin

GAG

Asp 445

AGG

Arg

GGA

Gly

GAG

Gin

GCA.

Pro Gly

GAT

Asp

GAT

Asp

GAA

Giu

ACC

Thr 350

GTG

Val

TAG

Tyr

GTG

Val

GTG

Leu

GTC

Leu 430

TCT

Ser

GAG

Giu

GAG

Giu

GAG

Giu

AGG

Ser 510 Lys

GAG

Asp

GGA

Al a

GGG

Gly 335

ATT

Ile

GGG

Ala

TGG

Trp,

GGG

Gly

TTT

Phe 415

ACG

Thr

GGC

Arg

GGA

Pro

AGG

Thr

AAA

Lys 495

GAT

His Arg

AAT

Asn

GGG

Gly 320

TGG

Trp,

GGG

Pro

ATG

Ile

TGT

Gys

AGG

Thr 400

GAT

Asp

ACG

Thr

TGG

Trp

AAG

Asn

TTG

Phe 480

TAG

Tyr

GAG

Asp Asp

GGC

Gly 305

GAG

His

CCC

Pro

AAT

Asn

GCC

Ala

GGC

Arg 385

GAG

Gin

GAG

Gin

GAG

Giu

AGA

Arg

GTT

Leu 465

ACC

Thr

TGG

Trp

GAA

Glu Pro Asp 290 COC TTC Arg Phe TAG GAG Tyr Gin ATC GAG Ile Gin CGT GG Arg Arg 355 TGT CG Cys Pro 370 TGG GAA Trp Giu GGC GAG Ala Gin CGA GGG Pro Gly GAT GCT Asp Ala 435 ACC ACA Thr Thr 450 GAG GTG Glu Val GTT TCG Val Ser TGC AAG Cys Lys GGT GCG Gly Ala 515

AGT

Ser

TGT

Gys

ACT

Thr 340

TCT

Ser

TAT

Tyr

GGG

Gly

GTG

Val1

AAT

Asn 420.

GGG

Gly

ATA

Ile

AG

Thr

TGG

Cys

TGG

Trp 500

GG

Arg

GTG

Val

GGA

Gly 325

TGG

Trp

GTT

Vai

AAG

Asn

GAC

Asp

GAA

Gin 405

GGT

Gly

TTC

Phe

GAA

Giu

CGA

Pro

GAG

His 485

AGG

Ser

CAA

Gin

TTG

Leu 310

GCG

Al a

GAA

Gin

GTG

Vai

CC

Pro

OGA

Gly 390

GAA

Glu

AGT

Thr

TAT

Tyr

GTC

Leu

GAG

Gin 470

TAT

Tyr

AAG

Asn

TCT

Ser Phe Giu Gly Arg 295

ATG

Ile

GAG

His

GTG

Leu

AAG

Lys

AAG

Lys 375

AAT

Asn

GAG

Glu

TAG

Tyr

TGG

Trp

GAG

Gin 455

AAC

Asn

CG

Pro

AAG

Lys

TCT

Ser Ile

AGA

Thr

AGT

Ser

TTT

Phe

GGA

Gly 360

GAA

Giu

GGA

Gly

TAT

Tyr

AGT

Thr

TGT

Cys 440

GTT

Vai

GGA

Ala

TGC

Gys

GGT

Gly

GTG

Val 520 Leu

GGG

Gly

TGT

Ser

GTG

Val 345

GTC

Val

AGC

Ser

GAT

His

GAA

Giu

GTC

Val 425

CTT

Leu

GCC

Al a

ACA

Thr

AAA

Lys

TGC

Cys 505

AGG

Ser 975 1023 1071 1119 1167 1215 1263 1311 1359 1407 1455 1503 1551 1599 1647 1695 TGG GAC GAG AGC AGG GAG Cys Asp Gin Ser Ser Gin GTG GTG TGC ATG ACG GTG AAG CGG GTC AGT Leu Val Ser Met Thr Leu Asn Pro Val Ser 525 530 120 TGG TGT GGG G Trp Cys Gly V~ AAG GAA GAT GAA GGC TGG TAC Lys Glu Asp 540 Giu Gly Trp, Tyr rA AAG CAA GGC CAG al Lys Gin Gly Gin 545 550 *Go* :fee., #0040: 6.000 *9 06..

0:.9f

TAT

Tyr

GGG

Gly 570

GCT

Al a

AAA

Lys

AAT

Asn 25 AGT Ser

ACC

30 Thr 650

TGG

Trp

AGC

Ser

GAT

Asp 45 ACA Thr

GCT

Ala 730

GCT

Ala

GCA

GGA

Gly 555

TCA

Ser

CTG

Leu

GCC

Ala

GTG

Val1

GCT

Ala 635

CTG

Leu

GTG

Val

AGC

Ser

TTG

Leu

GTC

Val 715

GAG

Giu

GAC

Asp

CAG

GAA

Giu

TCC

Ser

GAA

Giu

ATT

Ile

AGA

Arg 620

GAT

Asp

GTG

Vai

GCC

Al a

TAC

Tyr

GGA

Gly 700

ATC

Ile

CCA

Pro

ATG

Met

GTC

ACT

Thr

CAT

His

GAA

Giu

CCA

Pro 605

GAC

Asp

GGA

Gly

CCC

Pro

AGA

Arg

AGG

Arg 685

GGC

Gly

GAA

Giu

GAA

Giu

GCC

Ala

CAC

ACC

Thr

GTC

Vai

GAG

Giu 590

AAT

Asn

CAA

Gin

CAA

Gin

CTG

Leu

GTC

Val 670

ACA

Thr

AAT

Asn

GGA

Gly

GAA

Giu

TAC

Tyr 750

GAT

GCC

Ala

AAC

Asn 575

GTA

Vai

CCC

Pro

GCT

Al a

AGC

Ser

GGT

Gly 655

CGA

Arg

GAC

Asp

GAC

Asp

AAA

Lys

TCC

Ser 735

TCG

Ser

GGT

ATC

Ile 560

CCA

Pro

GTG

Vai

GGG

Gly

CAG

Gin

AGG

Arg 640

CTG

Leu

CAT

His

ATT

Ile

AAC

Asn

GAT

Asp 720

AAG

Lys

GCA

Al a

CCC

TAT

Tyr

ACA

Thr

GAC

Asp

CCT

Pro

GAG

Glu 625

AGC

Ser

GTG

Val

CGG

Arg

AGC

Ser

ATG

Met 705

GAA

Giu

AAA

Lys

TTC

Phe

CAG

ATA

Ile

GAT

Asp

TCC

S er

TTT

Phe 610

AAC

Asn

TCC

Ser

CTG

Leu

AAG

Lys

ATG

Met 690

GGG

Gly

ATC

le

GCA

Ala

CTG

Leu

GAA

GCA GTT Al1a Vai GCA AAT Ala Asn 580 TCC ATC Ser Ile 595 GCC AAC Ala Asn AGA GCA Arg Ala AGC TCC Ser Ser GCA GTG Ala Val 660 AAT GTA Asn Val 6'75 GCA GAC Ala Asp GCC TCT Ala Ser GTG ACT Val Thr AAA AGG Lys Arg 740 CTT CAG Leu Gin 755 GCC TAG

GAA

Giu 565

GCA

Aila

AGT

Ser

GAA

Giu

TCT

Ser

AAA

Lys 645

GGT

Gly

GAC

Asp

TTC

Phe

CCA

Pro

ACC

Thr '725

TCA

Ser

TCC

GAG

Giu

CGT

Arg

GAA

Giu

AGA

Arg

GGG

Giy 630

GTG

Val

GCT

Aila

CGC

Arg

AAG

Lys

GAC

Asp 710

ACG

Thr

TCC

Ser

AGC

AGG

Arg

GCC

Aila

AAA

Lys

GAG

Glu 615

GAT

Asp

CTG

Leu

ATA

Ile

ATG

Met

AAC

Asn 695

ACA

Thr

GAG

Giu

AAG

Lys

ACC

ACC

Thr

AAA

Lys

GAG

Glu 600

ATA

Ile

GCT

Aia

TTC

Phe

GCT

Al a

TCA

Ser 680

TCC

Ser

CAG

Gin

TGC

Cys

GAG

Giu

ATA

AGA

Arg

GTC

Val 585

AAC

Asn

CAG

Gin

GGC

Gly

TCC

Ser

GTG

Val 665

ATC

Ile

AGA

Arg

CAA

Gin

ACC

Thr

GAA

Glu 745

GCT

1743 1791 1839 1887 1935 1983 2031 2079 2127 2175 2223 2271 2319 2367 2420 2480 2540 2600 Aia Gin Vai His Asp Gly Pro Gin Giu Al1a 765 770 TTGCCTGTGA CAATCAACTT GAGAATCACA CTGATCCGCT CACCTCCGCT CTTCCCTCCT GTCCTCAGAG GTGTGCTGGT CCTGGCCTAG TTACGCCTGT TTAGGAGAGA GTGTGAGGCG Ser Ser Thr Ile Ala 760 GCAGTGCTGA CCACCCACCC CGCAGCCCAC ACTCACCCAT TCCTTCCTCG GCCATGGAAG TTCTTTTCTC TATGAAGAGA 121 GTGAGGTGGA AATGAGGAGG

AATAGGGGCT

ATGTCAGCGT

ACATCTGAGA

TTCTATAAAT

TAAGACATC.A

AAGGGCATCA

CTAGCGTCAA

CGTTTCAGGA

AACTCTTCTC

ACTCACTAGG

ATTACTGGAA

TTACCAGGCA

TGATTTTCAG

ACCAGATGTG

AGGTGAACCT

GAAAAGGCCA

TCCTCCATCT

CTTCAGTGCC

GAGATGCCAT

TACCTCCTGC

ATGAGAAGAG

GCAACTCCTG

GAGAGACATC TCTGGAGGAA TTTGAATCTT CTTTATAACC CTCCTTTCCT ATCCTCTTGA TACTAAATGC TGAGAGCCAG CTCCTCCCAG ATTCTGTCTT CTCTGTGCCT CATAGGCATA ATGTTTCTCA. AGAGTGCCTA GCTC!TTGGCC TACGATCTGT

GAGGGTTGAG

ATATGATAGG

TTCAAACAAC

GCCACAATCT

TTCATTALAGA

CACAAGCCAT

GTGAGATAGA

CTTCAAGAAA

2660 2720 2780 2840 2900 2960 3020 3080 3095 0 0000 0@06 S. 0 S S 56. 0 0 eq eq..

0 INFORMATION FOR SEQ ID NO: 8: SEQUENCE CHARACTERISTICS: LENGTH: 771 amino acids TYPE: amino acid STRANDNESS: single TOPOLOGY: linear DESCRIPTION: Mouse Polyimmunogiobulin Receptor (xi) SEQUENCE DESCRIPTION: SEQ ID NO: Met Ser Arg Leu Tyr Leu Phe Thr Leu Leu Thr Lys Ser Asp Ser Val Pro Ile Phe Gly Val Thr Val Gln Glu Val Tyr Pro Asp Phe Ser Gly Val 0 400000

S

0S00*0 0

S

ego...

C

Gly Ser Ile Thr Ser Ser Ile Giu Thr Ser Val Asn Ser Gly Met Cys 35 Arg His 45 Thr Thr Thr Arg Lys Tyr Cys Arg Gin Gly Leu Ile Ser Asn Giy Tyr Leu Lys Glu Tyr Ser Arg Aia Asn Leu Phe Pro Giu Asn Thr Phe Val Ile Asn Ile Giu Gln Leu 100 Gln Asp Asp Ser Tyr Lys Gly Thr Ser 115 Gln Vai Pro 130 Asn Arg Giy Leu Glu Leu Pro Ser 135 Ser 120 Asp Asp Vai Ser Cys Giy Leu 110 Giu Val Ser Lys Asp Ile Thr His Val Giy 145 Arg Asn Val Thr Giu Cys Pro Phe Lys Arg Giu Asn Vai Pro 155 160 Lys Lys Thr Asn Gin Ser Cys Giu Leu Val 170 175 Ser Lys Lys Ser 122 Ile Asp Ser Thr Giu Lys Val Asn Pro Ser Tyr Ile Gly Arg Ala Lys 190 180 185 0 00. 0 0 0 0 0 0 Leu His Gly 225 Glu Cys Met Asp Gly 305 His Pro Asn 35 Ala 40 Arg 385 Gin Gin Glu Arg 55 Leu 465 Thr Trp Glu Phe Leu 210 Prc Pro Asp Asn Pro 290 Arg Tyr Ile Arg Cys 370 Trp Ala Pro Asp Thr 450 Glu Val Cys Gly Met 195 Thr Ser Glu Leu Lys 275 Asp Phe Gin Gin Arg 355 Pro Glu Gin Gly Ala 435 Thr Val Ser Lys Ala 515 Lys His Ala Leu Gly 260 Glu Phe Ser Cys Thr 340 Ser Tyr Gly Val Asn 420 Gly Ile Thr Cys Irp 500 Arg Gly Asn Asp Leu 245 Arg Thr Glu Va1 Gly 325 Trp Val Asn Asp Gin 405 Gly Phe Glu Pro His 485 Ser Gin Thr Asp Lys 230 Tyr Glu Cys Gly Leu 310 Al a Gin Val Pro Gly 390 Glu Thr Tyr Leu G1n 470 Tyr Asn Ser Asp Ala 215 Lys Lys Val Asp Arg 295 Ile His Leu Lys Lys 375 Asn Glu Tyr Trp Gln 455 Asn Pro Lys Ser Leu 200 Gly Asn Asp Ala Va1 280 Ile Thr Ser Phe Gly 360 Glu Gly Tyr Thr Cys 440 Va1 Ala Cys Gly Val 520 Thr Leu Val Leu Asn 265 Ile Leu Gly Ser Val 345 Val Ser His Glu Val 425 Leu Ala Thr Lys Cys 505 Ser Val Tyr Asp Arg 250 Glu Ile Ile Leu Gly 330 Asn Thr Ser Cys Gly 410 Ile Thr Glu Ala Phe 490 His Cys Phe Ile Leu 235 Ser Ala Asn Thr Arg 315 Leu Glu Gly Ser Pro 395 Arg Leu Asn Ala Va1 475 Tyr Ile Asp Tyr Cys 220 Gln Ser Lys Thr Pro 300 Lys Pro Glu Gly Leu 380 Ala Leu Asn Gly Thr 460 Leu Ser Leu Gln Val 205 Gln Vai Va1 Tyr Leu 285 Lys Glu Gin Ser Ser 365 Lys Leu Ala Gin Asp 445 Arg Gly Gin Pro Ser 525 Asn Ala Leu Thr Leu 270 Gly Asp Asp Glu Thr 350 Val Tyr Vai Leu Leu 430 Ser Glu Glu Glu Ser 510 Ser Ile Gly Ala Phe 255 Cys Lys Asp Ala Gly 335 Ile Ala Trp Gly Phe 415 Thr Arg Pro Thr Lys 495 His 1 Gin Ser Glu Pro 240 Glu Arg Arg Asn Gly 320 Trp Pro Ile Cys Thr 400 Asp Thr rrp Asn Phe 180 Tyr ksp .eu 123 Val Ser Met Thr Leu Asn Pro Val Ser Lys Glu 530 535 Asp Glu Gly Trp Tyr 540 Trp Cys 545 Tyr Ile Thr Asp Asp Ser Pro Phe 610 Glu Asn 625 Ser Ser Val Leu Arg Lys Ser Met 690 Met Gly 705 Glu Ile Lys Ala Phe Leu Gin Glu Gly Ala Ala Ser 595 Ala Arg Ser Ala Asn 675 Ala Ala Val Lys Leu 755 Ala Val Val Asn 580 Ile Asn Ala Ser Val 660 Val Asp Ser Thr Arg 740 Gin Lys Glu 565 Ala Ser Glu Ser Lys 645 Gly Asp Phe Pro Thr 725 Ser Gin Gly 550 Glu Arg Arg Ala Glu Lys Arg Glu 615 Gly Asp 630 Val Leu Ala Ile Arg Met Lys Asn 695 Asp Thr 710 Thr Glu Ser Lys Thr Tyr Arg Gly 570 Val Ala 585 Asn Lys Gin Asn Gly Ser Ser Thr 650 Val Trp 665 Ile Ser Arg Asp Gin Thr Thr Ala 730 Glu Ala 745 Gly 555 Ser Leu Ala Val Ala 635 Leu Val Ser Leu Val 715 Glu Asp Glu Ser Glu Ile Arg 620 Asp Val Ala Tyr Gly 700 Ile Pro Met Thr Thr His Val Glu Glu 590 Pro Asn 605 Asp Gin Gly Gin Pro Leu Arg Val 670 Arg Thr 685 Gly Asn Glu Gly Glu Glu Ala Tyr 750 Ala Ile 560 Asn Pro 575 Val Val Pro Gly Ala Gin Ser Arg 640 Gly Leu 655 Arg His Asp Ile Asp Asn Lys Asp 720 Ser Lys 735 Ser Ala Ser Ser Thr Ile Ala Ala Gin Val His Asp Gly Pro INFORMATION FOR SEQ ID NO: 9: SEQUENCE CHARACTERISTICS: LENGTH: 3269 base pairs TYPE: nucleic acid 55 STRANDEDNESS: single TOPOLOGY: linear DESCRIPTION: Rat Polyimmunoglobulin Receptor (ix) FEATURE: NAME/KEY: Coding Sequence LOCATION: 74....2383 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 9: GGCAACGAAG GTACCATGGA TCTTATACAA GAAGTGAACC AACATGCCGC AACCTCCTTG 124 GAAGCCACAA GCG ATG AGG CTC TCC TTG TTC GCC CTC TTG GTA ACT GTC Met Arg Leu Ser Leu Phe Ala Leu Leu Val. Thr Val 1 S

TTC

Phe

AGT

Ser

ACC

Thr 45

AAC

Asn

GAG

Giu

TTT

Phe

AAG

Lys

CTG

Leu 35 125

ACA

Thr

GGG

Gly 45 TGC Cys

GAC

Asp

GTC

Val 205

CAA

Gin

GTG

TCA GGG Ser Gly AGT ATT Ser Ile TCT GTC Ser Vai GGC TAC Gly Tyr TAT TCA Tyr Ser GTG ATT Val Ile TGT GGT Cys Gly 110 GAG GTC Giu Vai AAG GAC Lys Asp AAT GCT Asn Ala GAA GTT Giu Val 175 AGA GCA Arg Ala 190 AAC ATT Asn Ile GCT GGA Ala Gly CTA GAG

GTC

Val

GAA

Giu

AAC

Asn

TGC

Cys

GGC

Gly

AAC

Asn

CTG

Leu

AGC

Ser

ATA

Ile

CAT

His 160

GTC

Val

ATC

Ile

AGC

Ser

GAA

G1u

CCT

TCC ACA Ser Thr GGT AAC Gly Asn CGG CAC Arg His 50 GCA ACC Ala Thr AGA GCC Arg Ala ATT GCA Ile Ala GGT ACC Gly Thr CAG GTT Gin Val 130 GGC AGA Gly Arg 145 AGC AAG Ser Lys ATC GAC Ile Asp CTT TTT Leu Phe CAC CTA His Leu 210 GGC CCC Gly Pro 225 GAG CCA Gin

TCG

Ser 35

ACC

Thr

CTC

Leu

AGC

Ser

CAT

His

ACT

Thr 115

CCT

Pro

ACT

Thr

AAA

Lys

TCT

Ser

ATG

MIet 195

ATA

Ile

AGT

Ser Ser 20

GTC

Val1

CGG

Arg

ATC

Ile

CTC

Leu

CTC

Leu 100

AAC

Asn

GAG

Glu

GTG

Val

TCC

Ser

ACT

Thr 180

AAA

Lys

CCC

Pro

GCT

Ala Pro

TCC

Ser

AAA,

Lys

TCT

Ser

ATC

Ile 85

ACC

Thr

CGA

Arg

TTC

Phe

ACC

Thr

CTG

Leu 165

GAG

Giu

GGG

Gly

AGT

Ser

GAT

Asp Ile Phe ATC ACG Ile Thr TAC TGG Tyr Trp, 55 TCA AAT Ser Asn 70 A.AC TTC Asn Phe CAG GAG Gin Glu GGC CTG Gly Leu CCA AAT Pro Asn 135 ATC GAA Ile Giu 150 TGT AAG Cys Lys TAC GTG Tyr Vai ACC AGC Thr Ser GAT GCT Asp Ala 215 AAA AAT Lys Asn 230 TAT AAA Tyr Lys Gly Pro TGC TAC Cys Tyr TGC CGA Cys Arg GGC TAC Gly Tyr CCA GAG Pro Giu GAC ACT Asp Thr 105 TTT TTC Phe Phe 120 GAC ACC Asp Thr TGC CGT Cys Arg AAG AGA Lys Arg GAC CCC Asp Pro 185 CGC GAT Arg Asp 200 GGA CTG Gly Leu AAT GCT Asn Ala Gin Asp

TAC

Tyr

CAA

Gin

CTC

Leu

AAT

Asn

GGG

Gly

GAT

Asp

CAT

His

TTC

Phe

GGA

Gly 170

AGC

Ser

ATA

Ile

TAT

Tyr

GAC

Asp

CCA

Pro

GGA

Gly

TCG

Ser

AGC

Ser

AGC

Ser

GTC

Val1

GTC

Val

AAA

Lys 155

GAG

Giu

TAT

Tyr

TTC

Phe

GTT

Val.

CTC

Leu 235 CAA AGC CCC ATA TTT GGT CCC CAG GAT

GTG

Val

GAC

Asp

GCC

Al a

AAG

Lys

ACA

Thr

TAC

Tyr

AGC

Ser

TAC

Tyr 140

GAG

Giu

GCC

Al a

AAG

Lys

TAT

Tyr

TGC

Cys 220

CAG

Gin 157 205 253 301 349 397 445 493 541 589 637 685 733 781 829 GAG CTG CTT Val Leu Giu Pro Giu Pro Glu Leu Leu 240 245 GAC CTG AGG TCC TCA Asp Leu Arg Ser Ser 250 125 GTG ACT TTT GAA TGT GAC CTG GGC CGT GAA Val Thr Phe Glu Cys Asp Leu Gly Arg Giu

TAT

Tyr

CTG

Leu 285

AGG

Arg

GAG

Glu

CAA

Gin

TCC

S er

TCT

Ser 365

AAG

Lys

CTC

Leu

GCA

Ala

CAG

Gin

GAC

50 Asp 445

AAG

Lys

GGA

Gly

CAG

Gln

CTG

Leu 270

GGG

Gly

GAT

Asp

GAT

Asp

GAA

Glu

ACG

Thr 350

GTG

Val1

TAC

Tyr

GTG

Val

CTG

Leu

CTC

Leu 430

TCT

Ser

AAG

Lys

GAG

Giu

GAG

Giu

TGT

Cys

AAG

Lys

GAC

Asp

GCA

Ala

GGC

Gly 335

ATT

Ile

GCC

Al a

TGG

Trp

GG

Gly

TTC

Phe 415

ACC

Thr

CGC

Arg

CCA

Pro

ACC

Thr

AAA

Lys 495

CGG

Arg

AGA

Arg

AAT

Asn

GGG

Giy 320

TGG

Trp

CCC

Pro

ATC

Ile

TGT

Cys

ACC

Thr 400

GAT

Asp

ACC

Thr

TGG

Trp

GAC

Asp

TTC

Phe 480

TAC

Tyr

AAG

Lys

GAT

Asp

GGC

Gly 305

CAC

His

CCC

Pro

AAT

Asn

GTC

Val

CAC

His 385

CAG

Gin

CAG

Gin

CAG

Gin

AGA

Arg

CTT

Leu 465

ACA

Thr

TGG

Trp

AAC

Asn

CCA

Pro 290

CGC

Arg

TAC

Tyr

GTC

Val

AGT

Ser

TGT

Cys 370

TGG

Trp

GCC

Al a

CCG

Pro

GAT

Asp

ACC

Thr 450

GAG

Giu

ATC

Ile

TGC

Cys

AAG

Lys 275

GCC

Aila

TTC

Phe

CAG

Gin

CAG

Gin

CGC

Arg 355

CCC

Pro

GAA

Giu

CTG

Leu

GGC

Gly

TCT

Ser 435

ACG

Thr

GTG

Vai

TCC

Ser

AAG

Lys

GAA

Glu

TTT

Phe

AGT.

Ser

TGT

Cys

GCT

Al a 340

TCT

Ser

TAT

Tyr

GCC

Al a

GTG

Vai

AGT

Ser 420

GGC

Giy

ATA

Ile

ACA

Thr

TGC

Cys

TGG

Trp, 500

ACC

Thr

GAA

Giu

GTG

Val

GGA

Giy 325

TGG

Trp

GTT

Val

AAC

Asn

GAC

Asp

CAA

Gin 405

GGC

Giy

TTC

Phe

GAA

Giu

CCA

Pro

CAC

His 485

AGC

Ser

TGT

Cys

GGC

Giy

TTG

Leu 310

GCG

Aila

CAA

Gin

GTG

Val

CCC

Pro

GAG

Giu 390

GAA

Giu

GCC

Al1a

TAC

Tyr

CTG

Leu

CAG

Gin.

470

TAT

Tyr

AAC

Asn GTG GCA Vai Aia GAT GTC Asp Vai 280 AGG ATC Arg Ilie 295 ATC ACA Ile Thr CAC AGT His Ser CTC TTT Leu Phe A-AG GGT Lys Gly 360 A-AG GAA Lys Giu 375 vLAT GGA ksn Gly GGA TAT Giy Tyr rAC ACT 1'yr Thr IrGG TGT E'rp Cys 440 AG GTT 31n Vai 155 .AC GCG Lsn Ala 'CG TGC ?ro Cys AC GGC sp Gly

ATC

Ile

CTG

Leu

GGC

Gly

TCT

Ser

GTC

Val 345

GTC

Vai

AGC

Ser

CGC

Arg

GAA

Giu

GTC

Val 425

CTT

Leu

GCT

Ala

ACC

Thr

~AA

Lys

TGC

Cys 505 AAT GAT GCC AAA Asn Asp Ala Lys 265

ATC

Ile

CTA

Leu

CTG

Leu

GGT

Giy 330

AAT

Asn

ACA

Thr

AGC

Ser

TGC

Cys

GGC

Giy 410

ATC

Ile

ACC

Thr

GAA

Giu

GCG

Aia

TTC

Phe 490

CAC

His

AAC

Asn

ACC

Thr

AGG

Arg 315

TTG

Leu

GAA

Giu

GGA

Gly

AGC

Ser

CCG

Pro 395

CGA

Arg

CTC

Leu

GAT

Asp

GCT

Al a

GTG

Val.

475

TAC

Tyr

ATC

Ile

ACC

Thr

CCC

Pro 300

AAG

Lys

CCT

Pro

GAG

Glu

GGC

Gly

CTC

Leu 380

GTG

Vai

CTG

Leu

AAC

Asn

GGT

Gly

ACA

Thr 460

ATA

Ile

TCC

Ser

CTG

Leu 877 925 973 1021 1069 1117 1165 1213 1261 1309 1357 1405 1453 1501 1549 1597 00*000 0 126 CCG AGC Pro Ser 510 CAT GAT GAA GGT His Asp Giu Gly GCC CGC Ala Arg 515 CAG TCC TCT Gin Ser Ser AGC TGT GAC CAG Ser Cys Asp Gin AGC AGC Ser Ser 525 CAG ATC GTC TCC ATG ACC CTG AAC Gin Ile Val Ser Met Thr Leu Asn GTC AAA AAG GAA Val Lys Lys Giu GAA GGC TGG TAC Glu Gly Trp Tyr TGT GGG GTA AAA Cys Gly Val Lys

GAA

Giu 550 GGT CAG GTC TAT Gly Gin Val Tyr GGA GAA Gly Glu 555 ACT ACA Thr Thr CAC ATC His Ile GAA GAG Giu Glu 590

GCC

Al a TAT GTA GCA GTT Tyr Val Ala Val GAG AGG ACC AGA Giu Arg Thr Arg GGG TCA CCC Gly Ser Pro 570 GCT CCA GAG Ala Pro Glu AAC CCG ACA GAT Asn Pro Thr Asp 575 GCA ATG GAA TCC Ala Met Glu Ser GCA AAC Ala Asn 580 GCA CGT GCA AAA Ala Arg Ala Lys GTC AGG GAG GAT Val Arg Giu Asp AAC AAG GCC AAT Asn Lys Ala Asn

CTG

Leu 605 GAC CCC AGG CTT Asp Pro Arg Leu GCA GAC GAA AGA Ala Asp Glu Axg ATA CAG AAT GCG Ile Gin Asn Ala GAC CAA GCT CAG GAG AAC AGA GCA TCT.GGG AAT GCT GGC AGT GCT GGT Asp Gin Ala Gin Giu Asn Arg Ala Ser Gly Asn Ala Gly Ser Ala Giy 625 630 635 1645 1693 1741 1789 1837 1885 1933 1981 2029 2077 2125 2173 2221 2269 2317 2365 GGA CAA AGC Gly Gin Ser GGT TTG GTG Gly Leu Val 655 AGC TCC AAA GTC Ser Ser Lys Val TTC TCC ACC CTG Phe Ser Thr Leu GTG CCC CTG Val Pro Leu 650 GCC AGA GTC Ala Axg Val CTG GCA GTG GGT Leu Ala Val Gly

GCT

Ala 660 GTG GCT GTG TGG Vai Ala Val Trp CGA CAT Arg His 670 CGG AAG AAT GTA Arg Lys Asn Val

GAC

Asp 675 CGC ATG TCA ATC Arg Met Ser Ile AGC TAC AGG ACA Ser Tyr Arg Thr

GAC

Asp 685 ATT AGC ATG GGA Ile Ser Met Gly TTC AGG AAC TCC Phe Arg Asn Ser GAT TTG GGA GGC Asp Leu Gly Gly

AAT

Asn 700 GAC AAC ATG GGC Asp Asn Met Gly ACT CCA GAC ACA Thr Pro Asp Thr

CAA~

Gin 710 GAA ACA GTC CTC Giu Thr Val Leu GAA GGA Giu Gly 715 AAA GAT GAA Lys Asp Giu TCC AAG AAA Ser Lys Lys 735 GAG ACT ACC ACC Glu Thr Thr Thr TGT ACC ACC GAG Cys Thr Thr Giu CCA GAG GAA Pro Giu Giu 730 GCA AAA AGG TCA Ala Lys Arg Ser TCC AAG GAG GAA GCT GAC ATG GCC TAC Ser Lys Glu Glu Ala Asp Met Ala Tyr 740 745 TCA GCA Ser Ala 750 TTC CTG TTT CAG Phe Leu Phe Gin AGC ACA ATA GCT Ser Thr Ile Ala

GCG

Al a 760 CAG GTC CAT GAT Gin Val His Asp 127 GGT CCC CAG GAA GCC TAG GCAGTGCTGA CCACCTACCC CTGCCTGTGA CAATCAACT Gly Pro Gin Giu Ala 765 TGAGAATCAC ATTGATCCAC TC!GCAGCCCA CCCTCGCCCA TCACCCAGGC TCTTCCCTCC

TGTTCTCAGA

TTTAGGAGAG

GCCCAAGAGG

GCCATTTGAA

CTCTCCTTTC

GTCTACTAAA

CACCTCGTCC

GCCTCTGCAC

GCCATAAGGG

ATAGACTAGT

AGATCTCTGC

TACATGGGCA

GGTGTGCTGG

AGCGTGAGGA

TGTCTCTGAG

GCCTCTTTAT

TTCTCTTCTT

TGCTGAGAGT

CAGATTCTGT

CTCATAGGCA

CACCACGAGA

GTCAAGCCAG

TCTTATTAGA

TGGTGGTGTG

TTCCTCCCTC

GTTCTTTTTG

AGACGAGGGT

ACACATATGC

GATTCAGACA

CAGGCCACAG

CTTTTCCCTA

ACAAAAGAAA

CTCAGATGAG

ATGGGGCAAC

GAAAGAACTT

CTCCTGCAAT

AGTCGTGGAA

CTGTTAAAGA

TCAGAGCAGG

TAGGATGTCA

ACAGATCCGA

CCTTTCTATA

AGCTATCAAT

CATAAGTCCT

AAGAGATTTT

TCCTGGCTCT

TAGCATGAGG

GCCTGGCCTA

GTAAGGTGGA

GGCTCATTTC

GGATAGCTCT

AAACTCACTA

AACATCACTG

CATTACCGGG

GCAGTCTAAG

TCTCCAGAGT

TGGCCTGGGA

AAAAGTAAGA

CTTATGCCTG

AATGAGTTGA

AGGAGGAAGA

TCTCCTCCAT

GGCTTCCGGT

GAAGAGACAC

GATTCCCTTT

GCATACCCAA

ACTCAGTGAG

CTTGTCTTCA

GAAAACAAGT

AAATAGGACC

2422 2482 2542 2602 2662 2722 2782 2842 2902 2962 3022 3082 3142 3202 3262 3269 CCCAATATTA AGAGGTTAA-A 0 0

AGAAGTTTAA

AGTTGGT

AGTAATCCTT GGCTACCTAG TGAGTGTAAG GCCAGCCTGG AATCAATAAG INFORMATION FOR SEQ ID NO: SEQUENCE CHARACTERISTICS: LENGTH: '770 amino acids TYPE: amino acid STRANDNESS: single TOPOLOGY: linear DESCRIPTION: Rat Polyimmnunogiobuiin Receptor (xi) SEQUENCE DESCRIPTION: SEQ ID NO: Met Arg Leu Ser Leu Phe Ala Leu Leu Val Thr Val Phe Ser Gly Val 1 5 10 Ser Thr Gin Ser Pro Ile Phe Giy Pro Gin Asp Vai Ser Ser Iie-.Giu 25 Giy Asn Ser Val Ser Ile Thr Cys Tyr Tyr Pro Asp Thr Ser Val Asn 40 A-rg His Thr Arg Lys Tyr Trp Cys Arg Gin Gly Ala Asn Gly Tyr Cys 55 Al a Arg Thr Leu Ile Ser Ser Asn Gly Tyr Leu Ser Lys Giu Tyr Ser Gly 70 75 Ala Ser Leu Ile Asn Phe Pro Glu Asn Ser Thr Phe Val Ile Asn 90 Ile Ala His Leu Thr Gin Giu Asp Thr 100 105 Gly Gin Gly 145 Ser Ile Leu His Gly 225 Glu Cys Lys Asp Gly 305 His Pro Asn Val His 385 Gin Gin Gin Thr Vai 130 Arg Lys Asp Phe Leu 210 Pro Pro Asp Asn Pro 290 Arg Tyr Vai Ser Cys 370 Trp Ala Pro Asp Thr 115 Pro Thr Lys Ser Met 195 Ile Ser Glu Leu Lys 275 Ala Phe Gin Gin Arg 355 Pro Glu Leu Gly Ser 435 Asn Glu Vai Ser Thr 180 Lys Pro Ala Leu Gly 260 Glu Phe Ser Cys Ala 340 Ser Tyr Ala Vai Ser 420 Gly Arg Phe Thr Leu 165 Glu Gly Ser Asp Leu 245 Arg Thr Glu Vai Gly 325 Trp Val Asn Asp Gin 405 Gly Phe Gly Pro Ile 150 Cys Tyr Thr Asp Lys 230 Tyr Glu Cys Gly Leu 310 Ala Gin Val Pro Glu 390 Glu Ala Tyr Leu Asn 135 Glu Lys Val Ser Ala 215 Asn Lys Val Asp Arg 295 Ile His Leu Lys Lys 375 Asn Gly Tyr Trp Phe 120 Asp Cys Lys Asp Arg 200 Gly Asn Asp Ala Val 280 Ile Thr Ser Phe Gly 360 Glu Gly Tyr Thr Cys 440 Phe Thr Arg Arg Pro 185 Asp Leu Ala Leu Asn 265 Ile Leu Gly Ser Val 345 Val Ser Arg Glu Val 425 Leu 128 Gly Ser Asp Val His Val Phe Lys 155 Gly Glu 170 Ser Tyr Ile Phe Tyr Val Asp Leu 235 Arg Ser 250 Asp Ala Ile Asn Leu Thr Leu Arg 315 Gly Leu 330 Asn Glu Thr Gly Ser Ser Cys Pro 395 Gly Arg 410 Ile Leu Thr Asp Tyr Lys Cys Gly Leu Leu 125 Thr Gly Cys Asp Va1 205 Gin Val Val Tyr Leu 285 Arg Glu Gin Ser Ser 365 Lys Leu Ala Gin Asp 445 110 Glu Lys Asn Glu Arg 190 Asn Ala Leu Thr Leu 270 Gly Asp Asp Glu Thr 350 Val Tyr Val Leu Leu 430 Ser Val Asp Ala Val 175 Ala Ile Gly Glu Phe 255 Cys Lys Asp Ala Gly 335 Ile Ala Trp Gly Phe 415 Thr Arg Ser Ile His 160 Vai Ile Ser Glu Pro 240 Glu Arg Arg Asn Gly 320 Trp Pro Ile Cys Thr 400 Asp Thr Trp 129 Arg Thr Thr Ile Giu Leu Gin Val Ala Giu Ala Thr Lys Lys Pro Asp Leu 465 Thr Trp Glu Val Trp 545 Tyr Thr Glu Leu Glu 35 625 Ser Ala Asn Gly Ala 705 Glu 450 Glu Ile Cys Gly Ser 530 Cys Val Asp Ser Phe 610 Asn Ser Va1 Va1 Asp 690 Thr Thr Vai Ser Lys Ala 515 Met Gly Ala Ala Ser 595 Ala Arg Lys Gly Asp 675 Phe Pro Thr Thr Cys Trp 500 Arg Thr Va1 Val Asn 580 Val Asp Ala Val Ala 660 Arg Arg Asp rhr Pro His 485 Ser Gin Leu Lys Glu 565 Ala Arg Glu Ser Leu 645 Val Met Asn Thr Glu 725 Lys Gin 470 Tyr Asn Ser Asn Glu 550 Glu Arg Glu Arg Gly 630 Phe Ala Ser Ser Gin 710 Cys Glu 455 Asn Pro Asp Ser Pro 535 Gly Arg Ala Asp Glu 615 Asn Ser Val Ile Arg 695 Glu Thr Glu Ala Cys Gly Val 520 Va1 Gin Thr Lys Glu 600 Ile Al a Thr Trp Ser 680 Asp Thr Thr Ala Thr Lys Cys 505 Ser Lys Val Arg Asp 585 Asn Gin Gly Leu Val 665 Ser Leu Val Glu Asp 745 Ala Phe 490 His Cys Lys Tyr Gly 570 Ala Lys Asn Ser Val 650 Ala Tyr Gly Leu Pro 4 730 Met Val 475 Tyr Ile Asp Glu Gly 555 Ser Pro Ala Ala Ala 635 Pro Arg Arg Gly Glu 715 Glu kla Ile Ser Leu Gin Asp 540 Glu Pro Glu Asn Gly 620 Gly Leu Val Thr Asn 700 Gly Glu Tyr 460 Gly Gin Pro Ser 525 Glu Thr His Glu Leu 605 Asp Gly Gly Arg I Asp 685 Asp Lys 2 Ser I Ser 1 Gly 1 765 Glu Glu Ser 510 Ser Gly Thr Ile Glu 590 Asp Gin ,ln Leu His 670 lie ksn ksp -ys la Thr Lys 495 His Gin Trp Ala Asn 575 Ala Pro Ala Ser Val 655 Arg Ser Met Glu Lys 735 Phe Phe 480 Tyr Asp Ile Tyr Ile 560 Pro Met Arg Gin Gly 640 Leu Lys Met Gly Ile 720 Ala Leu *5 a a Lys Arg Ser Ser 740 Phe Gin Ser Ser Thr lie Ala Ala Gin Vai His Asp 755 760 Ala ,ro Gin Glu 130 INFORMATION FOR SEQ ID NO: 11: SEQUENCE CHARACTERISTICS:

LENGTH:

TYPE:

STRANDEDNESS:

TOPOLOGY:

DESCRIPTION: Guy's 322 base pairs nucleic acid single linear 13 Kappa (ix) FEATURE: NAME/KEY: Coding Sequence LOCATION: 8. 320 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 11: CTCGAGC GAC ATT GTG ATG ACC CAG TCT CCA GCA ATC ATG TCT GCA TCT Asp Ile Val Met Thr Gin Ser Pro Ala Ile Met Ser Ala Ser GGG GAG AAG GTC Gly Glu Lys Val ATA ACC TGC AGT Ile Thr Cys Ser

GCC

Ala AGC TCA AGT GTA Ser Ser Ser Val

AGT

Ser TAC ATG CAC TGG Tyr Met His Trp CAG CAG AAG CCA GGC ACT TCT CCC AAA Gln Gln Lys Pro Gly Thr Ser Pro Lys CTC TGG Leu Trp CTT TAT AGC ACA TCC AAC CTG GCT Leu Tyr Ser Thr Ser Asn Leu Ala GGA GTC CCT GCT Gly Val Pro Ala CGC TTC AGT Arg Phe Ser CGA ATG GAG Arg Met Glu GGC AGT GGA 35 Gly Ser Gly TCT GGG ACC TCT Ser Gly Thr Ser TCT CTC ACA ATC Ser Leu Thr Ile 193 241 289 GCT GAA GAT GCT GCC ACT TAT TAC TGC CAT CAA AGG ACT AGT TAC CCG Ala Glu Asp Ala Ala Thr Tyr Tyr Cys His Gin Arg Thr Ser Tyr Pro 80 85

TAC

Tyr ACG TTC GGA Thr Phe Gly GGG GGG Gly Gly 100 ACC AAG CTC Thr Lys Let GAA A TA I Glu Ile 105 12: INFORMATION FOR SEQ ID NO: SEQUENCE CHARACTERISTICS:

LENGTH:

TYPE:

STRANDNESS:

TOPOLOGY:

DESCRIPTION: Guy's 105 amino acids amino acid single linear 13 Kappa (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 12: Asp Ile Val Met Thr Gln Ser Pro Ala Ile Met Ser Ala Ser Pro Gly 1 5 10 Glu Lys Val Thr Ile Thr Cys Ser Ala Ser Ser Ser Val Ser Tyr Met 25 His Trp Phe Gln Gln Lys Pro Gly Thr Ser Pro Lys Leu Trp Leu Tyr 40 131 Ser Thr Ser Asn Leu Ala Ser Gly Val Pro Ala Arg Phe Ser Gly Ser 55 Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser Arg Met Glu Ala Glu 65 70 75 Asp Ala Ala Thr Tyr Tyr Cys His Gin Arg Thr Ser Tyr Pro Tyr Thr 90 Phe Gly Gly Gly Thr Lys Leu Glu Ile 100 105 INFORMATION FOR SEQ ID NO: 13: SEQUENCE CHARACTERISTICS: LENGTH: 402 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear DESCRIPTION: Guy's 13 Gamma 1 (ix) FEATURE: NAME/KEY: Coding Sequence LOCATION: 7...402 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 13: CTCGAG ATG GAA TGG ACC TGG GTT TTT CTC TTC CTC CTG TCA GGA ACT 48 ,Met Glu Trp Thr Trp Val Phe Leu Phe Leu Leu Ser Gly Thr 1 5 GCA GGC GTC CAC TCT GGG GTC CAG CTT CAG CAG TCA GGA CCT GAC CTG 96 Ala Gly Val His Ser Gly Val Gin Leu Gin Gin Ser Gly Pro Asp Leu 15 20 25 40 GTG AAA CCT GGG GCC TCA GTG AAG ATA TCC TGC AAG GCT TCT GGA TAC 144 Val Lys Pro Gly Ala Ser Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr 40 ACA TTC ACT GAC TAC AAC ATA CAC TGG GTG AAG CAG AGC CGT GGA AAG 192 Thr Phe Thr Asp Tyr Asn Ile His Trp Val Lys Gln Ser Arg Gly Lys 55 AGC CTT GAG TGG ATT GGA TAT ATT TAT CCT TAC AAT GGT AAT ACT TAC 240 Ser Leu Glu Trp Ile Gly Tyr Ile Tyr Pro Tyr Asn Gly Asn Thr Tyr 65 70 TAC AAC CAG AAG TTC AAG AAC AAG GCC ACA TTG ACT GTA GAC AAT TCC 288 Tyr Asn Gln Lys Phe Lys Asn Lys Ala Thr Leu Thr Val Asp Asn Ser 85 TCC ACC TCA GCC TAC ATG GAG CTC CGC AGC CTG ACA TCT GAG GAC TCT 336 Ser Thr Ser Ala Tyr Met Glu Leu Arg Ser Leu Thr Ser Glu Asp Ser 100 105 110 GCA GTC TAT TAC TGT GCA ACC TAC TTT GAC TAC TGG GGC CAA GGC ACC 384 Ala Val Tyr Tyr Cys Ala Thr Tyr Phe Asp Tyr Trp Gly Gin Gly Thr 115 120 125 ACT CTC ACA GTC TCC TCA 402 Thr Leu Thr Val Ser Ser 130 132 INFORMATION FOR SEQ ID NO: 14: SEQUENCE CHARACTERISTICS: LENGTH: 132 amino acids TYPE: amino acid STRANDNESS: single TOPOLOGY: linear DESCRIPTION: Guy's 13 Gamma 1 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 14: Met Glu Trp Thr Trp Val Phe Leu Phe Leu Leu Ser Gly Thr Ala Gly 1 5 10 Val His Ser Gly Val Gln Leu Gin Gin Ser Gly Pro Asp Leu Val Lys 20 25 Pro Gly Ala Ser Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Thr Phe 40 Thr Asp Tyr Asn Ile His Trp Val Lys Gin Ser Arg Gly Lys Ser Leu 55 Glu Trp Ile Gly Tyr Ile Tyr Pro Tyr Asn Gly Asn Thr Tyr Tyr Asn 70 75 Gin Lys Phe Lys Asn Lys Ala Thr Leu Thr Val Asp Asn Ser Ser Thr 85 90 Ser Ala Tyr Met Glu Leu Arg Ser Leu Thr Ser Glu Asp Ser Ala Val 100 105 110 Tyr Tyr Cys Ala Thr Tyr Phe Asp Tyr Trp Gly Gln Gly Thr Thr Leu 115 120 125 40 Thr Val Ser Ser 130 45 INFORMATION FOR SEQ ID NO: SEQUENCE CHARACTERISTICS: LENGTH: 31 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: ACCAGATCTA TGGAATGGAC CTGGGTTTTT C 31 INFORMATION FOR SEQ ID NO: 16: SEQUENCE CHARACTERISTICS: LENGTH: 30 base pairs TYPE: nucleic acid STRANDEDNESS: single 133 TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 16: CCCAAGCTTG GTTTTGGAGA TGGTTTTCTC INFORMATION FOR SEQ ID NO: 17: SEQUENCE CHARACTERISTICS: LENGTH: 31 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 17: GATAAGCTTG GTCCTACTCC TCCTCCTCCT A INFORMATION FOR SEQ ID NO: 18: SEQUENCE CHARACTERISTICS: LENGTH: 30 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 18: 35 AATCTCGAGT CAG-TAGCAGA TGCCATCTCC INFORMATION FOR SEQ ID NO: 19: SEQUENCE CHARACTERISTICS: LENGTH: 30 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 19: 50 GGAAAGCTTT GTACATATGC AAGGCTTACA

GOOS

0 6 Ot t *055 0040 0 4.

0406

C

S

.55.

0 e

S

Claims (156)

1. 26. FEB. 2004 16:14 SPRUSON AND FERGUSON 61292615486 NO. 7547 P. 27 134 The claims defiing the invention are as follows: 1. An immunoglobulin produced recombinantly by a single eukaryotic cell, cell culture thereof, or organism derived therefrom comprising a protection protein that does not constitute a functional transmembrane spanning domain in association with an s immunoglobulin derived heavy chain having at least a portion of an antigen binding domain, said protection protein comprising at least a portion of amino acid residues 1 to 627 and 653 to 755 of a native polyimmunoglobulin receptor (plgR). 2. An immunoglobulin according to claim 1 wherein said immunoglobulin derived heavy chain contains at least a portion of an IgM or IgA heavy chain of any subtype. 3. An immunoglobulin according to claim I or claim 2 wherein said immunoglobulin derived heavy chain is comprised of immunoglobulin domains from two different isotopes of immunoglobulin. 4. An immunoglobulin according to any one of claims 1 to 3 wherein said is immunoglobulin domains are selected from the group consisting of: a) the C 1 l of a mouse IgG1 and the C 2 and CH3 of mouse IgA; and b) the C 1 and Cn2 of a mouse IgGI and the CH2 and C3 of mouse IgA. An immunoglobulin according to any one of claims 1 to 4 wherein said antigen binding domain substantially corresponds to the Guy's 13 heavy chain variable region. 6. An immunoglobulin according to any one of claims 1 to 5 further comprising an immunoglobulin derived light chain having at least a portion of an antigen binding domain associated with said immunoglobulin derived heavy chain. 7. An immunoglobulin according to claim 6 wherein said antigen binding domain substantially corresponds to the Guy's 13 light chain variable region. 8. An immunoglobulin according to any one of claims 1 to 7 further comprising a second immunoglobulin derived heavy chain having at least a portion of an antigen S. binding domain associated with said protection protein. S. 9. An immunoglobulin according to claim 8 further comprising a second 0s immunoglobulin derived light chain having at least a portion of an antigen binding domain bound to said second immunoglobulin derived heavy chain. 10. An immunoglobulin according to any one of claims 1 to 9 further comprising an immunoglobulin J chain bound to at least one of said immunoglobulin derived heavy chains. S 3 5 11. An immunoglobulin according to any one of claims 1 to 10 that is a therapeutic immunoglobulin. 12. An immunoglobulin according to claim 11 wherein said therapeutic immunoglobulin binds to mucosal pathogen antigens. 13. An immunoglobulin according to claim 12 that is capable of preventing dental caries. LUBAASl2D COMS ID No: SMBI-00637806 Received by IP Australia: Time 16:18 Date 2004-02-26 135 14. An immunoglobulin according to any one of claims 1 to 13 wherein said antigen binding domain is capable of binding an antigen from S. mutans serotypes c, e and for S. sobrinus serotypes d and g. An immunoglobulin according to any one of claims 1 to 14 wherein said protection protein has an amino acid sequence which substantially corresponds to at least a portion of the amino acid residues 1 to 627 of the rabbit polyimmunoglobulin receptor and does not have an amino acid residue sequence corresponding to amino acid residues 628 to 755 of the rabbit polyimmunoglobulin receptor. 16. An immunoglobulin according to any one of claims 1 to 14 wherein said protection protein has an amino acid sequence which substantially corresponds to at least a portion of the amino acid residues 1 to 606 of the rabbit polyimmunoglobulin receptor and does not have an amino acid sequence corresponding to amino acid residues 628 to 755 of the rabbit polyimmunoglobulin receptor. 17. An immunoglobulin according to claim 15 or claim 16 wherein said s5 protection protein has an amino acid sequence which does not contain amino acid residues corresponding to amino acid residues 628 to 755 of the rabbit polyimmunoglobulin receptor and which does contain amino acid residues which correspond to one or more of the following amino acid segments: a) amino acids corresponding to amino acid residues 21-43 of the rabbit 20 polyimmunoglobulin receptor; b) amino acids corresponding to amino acid residues 1-118 of the rabbit polyimmunoglobulin receptor; c) amino acids corresponding to amino acid residues 119-223 of the rabbit polyimmunoglobulin receptor; d) amino acids corresponding to amino acid residues 224-332 of the rabbit polyimmunoglobulin receptor; e) amino acids corresponding to amino acid residues 333-441 of the rabbit polyimmunoglobulin receptor; f) amino acids corresponding to amino acid residues 442-552 of the rabbit polyimmunoglobulin receptor; g) amino acids corresponding to amino acid residues 553-606 or 553-627 of the rabbit polyimmunoglobulin receptor. 18. An immunoglobulin according to any one of claims 1 to 14 wherein said protection protein has an amino acid sequence which does not contain amino acid residues of a polyimmunoglobulin receptor of a species which are analogous to amino acid residues 628 to 755 of the rabbit polyimmunoglobulin receptor and which does contain amino acid residues from a polyimmunoglobulin receptor of a species which are analogous to one or more of the following amino acid segments: LIBAA6223D1 136 a) amino acids corresponding to amino acid residues 21-43 of the rabbit polyimmunoglobulin receptor; b) amino acids corresponding to amino acid residues 1-118 of the rabbit polyimmunoglobulin receptor; c) amino acids corresponding to amino acid residues 119-223 of the rabbit polyimmunoglobulin receptor; d) amino acids corresponding to amino acid residues 224-332 of the rabbit polyimmunoglobulin receptor; e) amino acids corresponding to amino acid residues 333-441 of the rabbit polyimmunoglobulin receptor; f) amino acids corresponding to amino acid residues 442-552 of the rabbit polyimmunoglobulin receptor; g) amino acids corresponding to amino acid residues 553-606 or 553-627 of the i rabbit polyimmunoglobulin receptor. 15 19. An immunoglobulin according to claim 18 wherein said species is human. An immunoglobulin according to any one of claims 1 to 14 wherein said protection protein includes the amino acid sequence of at least one of the domains selected from the group consisting of the following portions of the rabbit polyimmunoglobulin receptor: domain I, domain II, domain III, domain IV, domain V, 20 and amino acid residues 553 to 627 of domain VI; and does not have an amino acid sequence corresponding to amino acid residues 628-755 of the rabbit polyimmunoglobulin receptor. 21. An immunoglobulin according to any one of claims 1 to 14 wherein said protection protein does not have any amino acid sequence which corresponds to or is analogous to amino acid residues 628-755 of the rabbit polyimmunoglobulin receptor and which does include: a) at least one domain which is from the polyimmunoglobulin receptor of a first animal and which is analogous to at least a portion of the following amino acid segments of the rabbit polyimmunoglobulin receptor: domain I, domain II, domain III, domain IV, domain V, and amino acid residues 553 to 627 of domain VI; b) at least one domain which is from the polyimmunoglobulin receptor of a second animal and which corresponds to or is analogous to the following amino acid residue segments of the rabbit polyimmunoglobulin receptor: domain I, domain II, domain III, domain IV, domain V, and amino acid residues 553 to 627 of domain VI. 22. An immunoglobulin according to any one of claims 1 to 14 wherein said protection protein does not have any amino acid sequence which corresponds to or is analogous to amino acid residues 628-755 of the rabbit polyimmunoglobulin receptor and which does include: LIBAA6223DI 137 a) at least one amino acid segment which is from the polyimmunoglobulin receptor of a first animal and which is analogous to at least a portion of the following amino acid residue segments of the rabbit polyimmunoglobulin receptor: domain I, domain II, domain III, domain IV, domain V, and amino acid residues 553 to 627 of domain VI; b) at least one amino acid segment which is from the polyimmunoglobulin receptor of a second animal and which is analogous to at least a portion of the following amino acid residue segments of the rabbit polyimmunoglobulin receptor: domain I, domain II, domain III, domain IV, domain V, and amino acid residues 553 to 627 of domain VI. 23. An immunoglobulin according to claim 21 wherein said first animal is a mammal and said second animal is a rabbit. 24. An immunoglobulin according to claim 22 wherein said first animal is a human and said second animal is a rabbit. 15 25. An immunoglobulin according to any one of claims 1 to 14 wherein said protection protein has a first amino acid sequence which substantially corresponds to at least a portion of the amino acid residues 1 to 606 or 1 to 627 of the rabbit polyimmunoglobulin receptor and has a second amino acid residue sequence contiguous with said first amino acid sequence, wherein said second amino acid residue sequence 20 does not have an amino acid residue sequence corresponding to the functional transmembrane segment of the rabbit polyimmunoglobulin receptor. 26. An immunoglobulin according to claim 25 wherein said second amino acid residue sequence has an amino acid sequence which corresponds to amino acid residues 655 to 755 of a polyimmunoglobulin receptor.
2. 27. An immunoglobulin according to claim 25 wherein said second amino acid residue sequence is a portion of one or more of the following: an intracellular domain of a *I polyimmunoglobulin molecule, a domain of a member of the immunoglobulin gene superfamily, an enzyme, a toxin, or a linker.
3. 28. An immunoglobulin according to claim 1 wherein said immunoglobulin derived heavy chain contains an immunoglobulin domain from one of the following immunoglobulin heavy chains: IgG, IgA, IgM, IgE, IgD; and also contains a protection protein-binding domain from IgA or IgM.
4. 29. An immunoglobulin according to claim 28 wherein said immunoglobulin heavy chains are human, rodent, rabbit, bovine, ovine, caprine, fowl, canine, feline or primate immunoglobulin heavy chains. An immunoglobulin according to claim 28 or claim 29 wherein said protection protein-binding domain is from the IgA of a human, rodent, rabbit, bovine, ovine, canine, feline or primate. LIBAA6223D1 26. FEB. 2004 16:14 SPRUSON AND FERGUSON 61292615486 NO. 7547 P. 28 138 3 1. An immunoglobulin according to any one of claims 28 to 30 wherein said chimeric immunoglobulin heavy chain is comprised of immunoglobulin chains of mouse IgGI and said protection protein-binding domain is from mouse IgA or IgM.
5. 32. An immunoglobulin according to any one of claims 28 to 30 wherein said s chimeric immunoglobulin heavy chain is comprised of immunoglobulin domains of a human IgG, IgM, IgD or IgE and said protection protein-binding domain is from a human IgA or IgM.
6. 33. An immunoglobulin produced from a single eukaryotic cell, cell culture thereof, or organism derived therefrom comprising a protection protein, substantially as to hereinbefore described with reference to any one of the examples.
7. 34. A eukaryotic cell containing an immunoglobulin according to any one of claims I to 33. A eukaryotic cell according to claim 34, wherein said eukaryotic cell is a plant cell.
8. 36. A plant cell according to claim 35 wherein said plant cell is part of a plant.
9. 37. A eukaryotic cell according to any one of claims 34 to 36 containing a nucleotide sequence encoding and capable of producing a protection protein.
10. 38. A eukaryotic cell according to claim 37 which also contains a second nucleotide sequence encoding at least one of the molecules selected from the group consisting of: an immunoglobulin derived heavy chain having at least a portion of an antigen binding domain, an immunoglobulin derived light chain having at least a portion of an antigen binding domain, or an immunoglobulin J chain.
11. 39. A eukaryotic cell according to claim 38 wherein said second nucleotide sequence encodes an immunoglobulin derived heavy chain having at least a portion of an antigen binding domain; and which also contains a third nucleotide sequence encoding an immunoglobulin derived light chain having at least a portion of an antigen binding 0.domain.
12. 40. A eukaryotic cell according to claim 39 which also contains a fourth *nucleotide sequence encoding an immunoglobulin J chain. S. 30 41. A eukaryotic cell according to any one of claims 38 to 40, wherein said eukaryotic cell is a plant cell.
13. 42. A plant cell containing a nucleotide sequence encoding a protection protein that does not constitute a functional transmembrane spanning domain and a nucleotide sequence encoding an immunoglobulin derived heavy chain having at least a portion of an 35 antigen binding domain, said protection protein comprising at least a portion of amino -acid residues 1 to 627 and 653 to 755 of a native polyimrnunoglobulin receptor (pIgR).
14. 43. A eukaryotic cell containing a recombinantly expressed protection protein that does not constitute a functional transmembrane spanning domain and which also contains at least one additional recombinantly expressed molecule selected from the group consisting of: an immunoglobulin derived heavy chain having at least a portion of an antigen binding domain, an MBAA*3223DI COMS ID No: SMBI-00637806 Received by IP Australia: Time 16:18 Date 2004-02-26 23. FEB. 2004 15:34 SPRUSON FERGUSON NO. 7273 P. 47 139 immunoglobulin derived light chain having at least a portion of an antigen binding domain, or an immunoglobulin J chain, said protection protein comprising at least a portion of amino acid residues 1 to 627 and 653 to 755 of a native polyimmunoglobulin receptor (pIgR).
15. 44. A eukaryotic cell according to claim 43 wherein said additional molecule is an immunoglobulin derived heavy chain having at least a portion of an antigen binding domain, and which also contains an immunoglobulin derived light chain having at least a portion of an antigen binding domain. A eukaryotic cell according to claim 43 or claim 44 which also contains an immunoglobulin J chain.
16. 46. A eukaryotic cell according to any one of claims 43 to 45, wherein said eukaryotic cell is a plant cell.
17. 47. A plant cell according to any one of claims 35, 41, 42 and 46 wherein said S plant cell is derived from a dicotyledonous or monocotyledonous plant. 5 48. A plant cell according to any one of claims 35, 41,42, 46 and 47 wherein said plant cell is derived from a solanaceous plant.
18. 49. A plant cell according to any one of claims 35,41, 42,46 and 47 wherein said plant cell is an alfalfa cell.
19. 50. A plant cell according to any one of claims 35, 41, 42, and 46 to 48 wherein said plant cell is derived from a tobacco plant.
20. 51. A plant cell according to any one of claims 35, 41, 42, and 46 to 50 wherein 'said plant cell is part of a plant.
21. 52. A transformed eukaryotic cell containing an immunoglobulin comprising a protection protein, said cell being substantially as hereinbefore described with reference ~25 to any one of the examples. .ito 53. A composition comprising an immunoglobulin according to any one of claims 1 to 33 and plant macromolecules.
22. 54. A composition according to claim 53 wherein the plant molecules are derived from a dicotyledonous, monocotyledonous, solanaceous, alfalfa or tobacco plant.
23. 55. A composition according to claim 53 or claim 54 wherein said plant molecules are ribulose bisphosphate carboxylase, light harvesting complex, pigments, secondary metabolites or chlorophyll. S56. A composition according to any one of claims 53 to 55 wherein said immunoglobulin is present in a concentration of between 0.001% and 99% mass excluding water.
24. 57. A composition according to any one of claims 53 to 56 wherein said plant macromolecules are present in a concentration of between 1% and 99% mass excluding water. LIBAA623DI SCOMS ID No: SMBI-00631115 Received by IP Australia: Time 15:44 Date 2004-02-23 23. FEB. 2004 15:34 SPRUSON FERGUSON NO. 7273 P. 48 140
25. 58. A method of producing an immunoglobulin according to any one of claims 1 to 33 comprising the steps of: introducing into a plant cell an expression vector containing a nucleotide sequence encoding a protection protein operably linked to a transcriptional promoter; and s introducing into said plant cell an expression vector containing a nucleotide sequence encoding an immunoglobulin derived heavy chain having at least a portion of an antigen binding domain operably linked to a transcriptional promoter,
26. 59. A method according to claim 58 further comprising the step of: introducing into said plant cell an expression vector containing a nurleotide sequence encoding an imlunoglobulin derived light chain having at least a portion of an antigen binding domain operably linked to a transcriptional promoter. A method according to claim 58 OT claim 59 further comprising the step of introducing into said plant cell an expression vector containing a nucleotide sequence encoding an immunoglobulin J chain operably linked to a transcriptional promoter. 15 61. A method according to any one of claims 58 to 60 wherein said imnunoglobulin derived heavy chain is immunoglobulin alpha chain and said 00000." immunoglobulin derived light chain is an innrunoglobulin kappa or lambda chian.
27. 62. A method according to any one of claims 58 to 60 wherein said 0. ".inmunoglobulin derived heavy chain is comprised of portions of immunoglobulin alpha chain and immunoglobulin gamma chain.
28. 63. A method according to any one of claims 5 8 to 62 wherein the plant cells are part of a plant.
29. 64. A method according to claim 63 further comprising growing said plant. A method according to claim 63 or claim 64 wherein said plant is a 00.. 25 dicotyledonous or a monocotyledonous plant,
30. 66. A method according to claim 65 wherein said plant is solanaceous or :leguminous. o
31. 67. A method according to claim 66 wherein said plant is an alfalfa or a tobacco plant.
32. 68. A method according to any one of claims 58 to 67 wherein said immunoglobulin derived heavy chain is a chimeric immnunoglobulin heavy chain.
33. 69. A method of producing a therapeutic immunoglobulin composition containing plant macromolecules, said method comprising the step of shearing under pressure a portion of a plant comprising plant cells according to claim 36 or claim 51 to produce a pulp containing a therapeutic immunoglobulin and plant macromolecules in a liquid derived from the apoplast or symplast of said plant and solid plant derived material. A method according to claim 69 further comprising the step of separating said solid plant derived material from said liquid. LIMAA6223DL COMS ID No: SMBI-00631115 Received by IP Australia: Time 15:44 Date 2004-02-23 26. FEB. 2004 16:15 SPRUSON AND FERGUSON 61292615486 NO. 7547 P. 29 141
34. 71. A method according to claim 69 or claim 70 wherein said portion of said plant is a leaf, stem, root, tuber, fruit or entire plant.
35. 72. A method according to any one of claims 69 to 71 wherein said shearing is accomplished by a mechanical device which releases liquid from the apoplast or symplast s of said plant.
36. 73. A method according to claim 70 wherein said separation is by centrifugaion, settling, flocculation or filtration.
37. 74. A method for producing an assembled immunoglobulin molecule having heavy, light and J chains and a protection protein comprising the steps of: a) introducing into a eukaryotic cell nucleotide sequences operably linked for expression encoding: i) an immunoglobulin derived heavy chain having at least a portion of an antigen binding domain, ii) an inmunoglobulin derived light chain having at least a portion of an antigen binding domain, iii) an immunoglobulin J chain, and iv) a protection protein that does not constitute a functional transmembrane spanning domain, said protection protein comprising at least a portion of amino acid residues 1 to 627 and 653 to 755 of a native polyimmunoglobulin receptor (pIgR); and b) maintaining said cell under conditions allowing production and assembly of said immunoglobulin derived heavy and light chains, said immunoglobulin J chain and said protection protein into an immunoglobulin molecule, A method for producing an assembled immunoglobulin molecule having heavy, light and J chains and a protection protein by maintaining under conditions 25 allowing protein production and innunoglobulin assembly, a eukaryotic cell containing nucleotide sequences operably linked for recombinant expression encoding: S.:i i) an immunoglobulin derived heavy chain having at least a portion of an antigen binding domain, ii) an immunoglobulin derived light chain having at least a portion of an antigen 30 binding domain, S"iii) an immunoglobulin J chain, and iv) a protection protein that does not constitute a functional transmembrane spanning domain, said protection protein comprising at least a portion of amino acid *residues I to 627 and 653 to 755 of a native polyimmunoglobulin receptor (pIgR).
38. 76. A method according to claim 74 or claim 75, wherein said eukaryotic cell is a plant cell.
39. 77. An immunoglobulin comprising a protection protein produced by a method according to any one of claims 58 to 77. LAA* **D IBAA622DI COMS ID No: SMBI-00637806 Received by IP Australia: Time 16:18 Date 2004-02-26 26. FEB. 2004 16:15 SPRUSON AND FERGUSON 61292615486 NO. 7547 P. 142
40. 78. An immunoglobulin according to any one of claims 1 to 33 or 77, wherein said eukaryotic cell is a plant cell.
41. 79. A tetratransgenic non-human organism comprised of cells containing four different transgenes each encoding a different polypeptide of a multipeptide molecule s wherein at least one of each of said different polypeptides is associated together in said multipeptide molecule, wherein at least one of said four transgenes is a transgene encoding a protection protein that does not constitute a functional transmembrane spanning domain, said protection protein comprising at least a portion of amino acid residues 1 to 627 and 653 to 755 of a native polyimmunoglobulin receptor (pIgR).
42. 80. A transgenic organism according to claim 79 wherein at least one of said four transgenes is a transgene encoding an immunoglobulin derived heavy chain having at least a portion of an antigen binding domain.
43. 81. A transgenic organism according to claim 79 or claim 80 wherein at least one of said four transgenes is a transgene encoding an immunoglobulin derived light chain having at least a portion of an antigen binding domain.
44. 82. A transgenic organism according to any one of claims 79 to 81 wherein at least one of said four transgenes is a transgene encoding an immunoglobulin J chain,
45. 83. A transgenic organism according to any one of claims 79 to 82 wherein at least one of said four transgenes is a transgene encoding a chimeric immunoglobulin heavy chain.
46. 84. A transgenic organism according to any one of claims 79 to 83, wherein said transgenic organism is a plant. A process for preparing a composition for passive immunotherapy, said process comprising combining an immunoglobulin according to any one of claims 1 to 33 2s or 77 or a composition according to any one of claims 53 to 57 with a pharmaceutically acceptable carrier and optionally a pharmaceutically acceptable flavour, 86, A composition comprising an immunoglobulin prepared by a process according to claim
47. 87. A method of treating or preventing a patient suffering from a condition indicating or preventable by administration of passive immunotherapy, said method comprising administering to said patient a therapeutically and/or immunologically effective amount of an immunoglobulin according to any one of claims 1 to 33 or 77, or a composition according to any one of claims 53 to 57 or 86. S.88. A method according to claim 87, wherein the condition indicating, or s3 preventable by passive immunotherapy involves mucosal or enteric pathogens.
48. 89. A method according to claim 87 or claim 88, wherein the condition is dental caries.
49. 90. A method according to any one of claims 87 to 89, wherein the immunoglobulin binds to an antigen from S. mutans serotypes c, e or f or S. sobrinus 40 serotypes d or g. LIBAA6222DI COMS ID No: SMBI-00637806 Received by IP Australia: Time 16:18 Date 2004-02-26 26. FEB. 2004 16:16 SPRUSON AND FERGUSON 61292615486 N0. 7547 P. 31 143
50. 91. A therapeutic agent comprising an Inummunoglobulin according to any one of claims 1 to 33 or 77, or a composition according to any one of claims 53 to 57 or 86, when used for treating or preventing a patient suffering from a condition indicating, or preventable by administration of passive immunotherapy.
51. 92. A therapeutic agent according to claim 91, wherein the condition indicating, or preventable by passive immunotherapy involves mucosal or enteric pathogens.
52. 93. A therapeutic agent according to claim 91 or claim 92, wherein the condition is dental caries.
53. 94. A therapeutic agent according to any one of claims 91 to 93, wherein the immunoglobulin binds to an antigen from 5. mutans serotypes c, e or f or S. sobrinus serotypes d or g. Use of an immunoglobulin according to any one of claims 1 to 33 or 77, or a composition according to any one of claims 53 to 57 or 86, for the manufacture of a medicament for treating or preventing a patient suffering from a condition indicating, or Is preventable by passive immunotherapy,
54. 96. A use according to claim 95, wherein the condition indicating, or preventable by passive immunotherapy involves mucosal or enteric pathogens.
55. 97. A use according to claim 95 or claim 96, wherein the condition is dental caries, 98, A use according to any one of claims 95 to 97, wherein the immunoglobulin binds to an antigen from S. mutans serotypes c, e or f or S. sobrinus serotypes d or g,
56. 99. A medicament manufactured by a use according to any one of claims 95 to 98,
57. 100. A method of transforming a plant cell to express an immunoglobulin 25 comprising a protection protein that does not constitute a functional transmembrane spanning domain in association with an inmmunoglobulin derived heavy chain having at least a portion of an antigen binding domain, said method comprising the steps of: introducing into a plant cell an expression vector containing a nucleotide sequence encoding a protection protein operably linked to a transcriptional promoter, said 30 protection protein comprising at least a portion of amino acid residues I to 627 and 653 to 755 of a native polyimnimunoglobulin receptor (pIgR); and introducing into said plant cell an expression vector containing a nucleotide sequence encoding an immunoglobulin derived heavy chain having at least a portion of an antigen binding domain operably ilianked to a transcriptional promoter.
58. 101. A method according to claim 100 further comprising the step of: introducing into said plant cell an expression vector containing a nucleotide 9 sequence encoding an immunoglobulin derived light chain having at least a portion of an antigen binding domain operably linked to a transcriptional promoter. S S 9@ LIBAAGI2'D COMS ID No: SMBI-00637806 Received by IP Australia: Time 16:18 Date 2004-02-26 23. FEB. 2004 15:36 SPRUSON FERGUSON NO. 7273 P. 52 144
59. 102. A method according to claim 100 or claim 101 further comprising the step of introducing into said plant cell an expression vector containing a nucleotide sequence encoding an immunoglobulin J chain operably linked to a transcriptional promoter.
60. 103. A method according to claim 101 or claim 102, wherein said immunoglobulin s derived heavy chain is immunoglobulin alpha chain and said immunoglobulin derived light chain is an immunoglobulin kappa or lambda chain.
61. 104. A method according to any one of claims 100 to 102, wherein said immunoglobulin derived heavy chain is comprised of portions of immunoglobulin alpha chain and immunoglobulin gamma chain.
62. 105. A method according to any one of claims 100 to 104, wherein said immunoglobulin derived heavy chain is a chimeric immunoglobulin heavy chain.
63. 106. A method according to any one of claims 100 to 105, wherein said plant is a dicotyledonous or a monocotyledonous plant.
64. 107. A method according to claim 106, wherein said plant is solanaceous or is leguminous,
65. 108. A method according to claim 107, wherein said plant is an alfalfa or a tobacco plant.
66. 109. A method according to claim 105, wherein said plant is Lenna gibba
67. 110. A method according to any one of claims 100 to 109, wherein said expression 20 vector is introduced into said plant cell by direct contact of said cell with said vector *under conditions permitting uptake of the vector by said cell.
68. 111. A method according to claim 110, wherein said conditions comprise chemically enhanced permeabilisation.
69. 112. A method according to claim 110, wherein said conditions comprise 25 temperature perturbation of the cellular membrane.
70. 113. A method according to any one of claims 100 to 109, wherein said expression vector is introduced into said plant cell by means of transfection with an Agrobacterium species containing said vector.
71. 114. A method according to any one of claims 100 to 109, wherein said expression vector is introduced into said plant cell by means of biolistic transformation utilising microparticles coated with said vector.
72. 115. A method according to claim 114, wherein said microparticles are made of tungsten or gold.
73. 116. A method according to any one of claims 100 to 109, wherein one or more of said expression vectors is introduced into said plant cell by means of fusion with another cell containing said one or more expression vectors.
74. 117. A method according to claim 116, wherein said fusion is protoplast fusion.
75. 118. A method according to claim 116, wherein said fusion comprises cross- fertilisation. LIsAA223DI COMS ID No: SMBI-00631115 Received by IP Australia: Time 15:44 Date 2004-02-23 23. FEB.2004 15:36 SPRUSON FERGUSON NO. 7273 P. 53 145
76. 119. A method of transforming a plant cell to express an immunoglobulin comprising a protection protein in association with an immunoglobulin derived heavy chain having at least a portion of an antigen binding domain, said method being substantially as hereinbefore described with reference to any one of the examples. s 120. A plant cell transformed by a method according to any one of claims 100 to 119.
77. 121. A transformed plant cell expressing an immunoglobulin comprising a protection protein in association with an immunoglobulin derived heavy chain having at least a portion of an antigen binding domain, substantially as hereinbefore described with to reference to any one of the examples.
78. 122. A method for producing a transgenic plant expressing an immunoglobulin comprising a protection protein in association with an immunoglobulin derived heavy chain having at least a portion of an antigen binding domain, said method comprising generating a whole plant from a transformed cell according to claim 120 or claim 121.
79. 123. A transgenic plant produced by a method according to claim 122.
80. 124. A transgenic plant comprising a plurality of cells according to claim 120 or claim 121.
81. 125. A transgenic plant expressing an immunoglobulin comprising a protection protein in association with an immunoglobulin derived heavy chain having at least a portion of an antigen binding domain, substantially as hereinbefore described with S' reference to any one of the examples.
82. 126. A method of producing a therapeutic immunoglobulin composition containing plant macromolecules, said method comprising the step of shearing under pressure a portion of a plant according to any one of claims 123 to 125, or cells according to claim S 25 120 or 121, to produce a pulp containing a therapeutic immunoglobulin and plant macromolecules in a liquid derived from the apoplast or symplast of said plant and solid splant derived material.
83. 127. A method according to claim 126 further comprising the step of separating said solid plant derived material from said liquid. 30 128, A method according to claim 127, wherein said separation is by centrifugation, settling, flocculation or filtration.
84. 129. A method according to any one of claims 126 to 128, wherein said portion of said plant is a leaf, stem, root, tuber, fruit or entire plant.
85. 130. A method according to any one of claims 126 to 129, wherein said shearing is accomplished by a mechanical device which releases liquid from the apoplast or symplast of said plant/cells.
86. 131. A therapeutic immunoglobulin composition produced by a method according to any one of claims 126 to 130. LBAA6223DI COMS ID No: SMBI-00631115 Received by IP Australia: Time 15:44 Date 2004-02-23 26. FEB.2004 16:16 SPRUSON AND FERGUSON 61292615486 NO. 7547 P. 32 146
87. 132. A method of transforming a eukaryotic cell to express an assembled immunoglobulin molecule having heavy, light and I chains and a protection protein comprising introducing into a eukaryotic cell nucleotide sequences operably linked for expression encoding: s i) an immunoglobulin derived heavy chain having at least a portion of an antigen binding domain, ii) an immunoglobulin derived light chain having at least a portion of an antigen binding domain, iii) an immunoglobulin J chain, and iv) a protection protein that does not constitute a functional transmembrane spanning domain, said protection protein comprising at least a portion of amino acid residues 1 to 627 and 653 to 755 of a native polyimmunoglobulin receptor (pIgR).
88. 133. A method according to claim 132, wherein said immunoglobulin derived heavy chain is immunoglobulin alpha chain and said immunoglobulin derived light chain is is an immunoglobulin kappa or lambda chain.
89. 134. A method according to claim 132, wherein said immunoglobulin derived heavy chain is comprised of portions of immunoglobulin alpha chain and immunoglobulin gamma chain.
90. 135. A method according to any one of claims 132 to 134, wherein said immunoglobulin derived heavy chain is a chimeric immunoglobulin heavy chain.
91. 136. A method according to any one of claims 132 to 135, wherein said expression vector is introduced into said cell by direct contact of said cell with said vector under conditions permitting uptake of the vector by said cell.
92. 137. A method according to claim 136, wherein said conditions comprise 25 chemically enhanced permeabilisation.
93. 138. A method according to claim 136, wherein said conditions comprise *temperature perturbation of the cellular membrane.
94. 139. A method according to any one of claims 132 to 135, wherein said expression vector is introduced into said cell by means of transfection with an Agrobacterium species 30 containing said vector.
95. 140. A method according to any one of claims 132 to 135, wherein said expression vector is introduced into said cell by means of biolistic transformation utilising microparticles coated with said vector.
96. 141. A method according to claim 140, wherein said microparticles are made of tungsten or gold,
97. 142. A method according to any one of claims 132 to 135, wherein one or more of said expression vectors is introduced into said cell by means of fusion with another cell containing said one or more expression vectors.
98. 143. A method according to claim 142, wherein said fusion is protoplast fusion. LIBAM223DI COMS ID No: SMBI-00637806 Received by IP Australia: Time 16:18 Date 2004-02-26 26, FEB. 2004 16:16 SPRUSON AND FERGUSON 61292615486 NO. 7547 P. 33 147
99. 144. A method according to claim 142, wherein said fusion comprises cross- fertilisation.
100. 145. A method according to any one of claims 132 to 144, wherein said eukaryotic cell is a plant cell. s 146. A method according to claim 145, wherein said plant is a dicotyledonous or a monocotyledonous plant.
101. 147. A method according to claim 146, wherein said plant is solanaceous or leguminous.
102. 148. A method according to claim 147, wherein said plant is an alfalfa or a tobacco plant.
103. 149. A method according to claim 145, wherein said plant is Lenna gibba
104. 150. A method according to any one of claims 132 to 134, wherein said expression vector is introduced into said cell by pronuclear injection.
105. 151. A method of transforming a eukaryotic cell to express an assembled inmmunoglobulin molecule having heavy, light and J chains and a protection protein, substantially as hereinbefore described with reference to any one of the examples.
106. 152. A eukaryotic cell expressing an assembled immunoglobulin molecule having heavy, light and J chains and a protection protein, transformed by a method according to any one of claims 132 to 151.
107. 153. A transformed eukaryotic cell expressing an assembled immunoglobulin molecule having heavy, light and J chains and a protection protein, substantially as hereinbefore described with reference to any one of the examples.
108. 154. A eukaryotic cell according to claim 152 or claim 153 which is a plant cell.
109. 155. A method for producing a transgenic non-human organism expressing an assembled immunoglobulin molecule having heavy, light and J chains and a protection protein, said method comprising generating a whole organism from a transformed cell 9 according to any one of claims 152 to 154. 0
110. 156. A transgenic organism produced by a method according to claim 155.
111. 157. A transgenic organism comprising a plurality of cells according to any one of 30 claims 152 to 154. 0
112. 158. A transgenic organism according to claim 156 or claim 157 which is a plant
113. 159. A transgenic organism expressing an assembled immunoglobulin molecule having heavy, light and J chains and a protection protein, substantially as hereinbefore described with reference to any one of the examples.
114. 160. A method of producing a composition containing an assemnbled immunoglobulin having heavy, light and J chains and a protection protein, said method oo@e comprising the step of shearing under pressure an organism according to any one of claims 156 to 159, or a portion thereof, or cells according to any one of claims 152 to 154, to produce a pulp containing an assembled immunoglobulin having heavy, light and J 0: LIBAAA22SDI COMS ID No: SMBI-00637806 Received by IP Australia: Time 16:18 Date 2004-02-26 26. FEB. 2004 16:17 SPRUSON AND FERGUSON 61292615486 .NO, 7547' P. 34 148 chains and a protection protein in a liquid derived from the apoplast or symplast of said organism/cells and solid derived material.
115. 161. A method according to claim 160 further comprising the step of separating said solid derived material from said liquid.
116. 162. A method according to claim 161, wherein said separation is by centrifugation, settling, flocculation or filtration.
117. 163. A method according to any one of claims 160 to 162, wherein said shearing is accomplished by a mechanical device which releases liquid from the apoplast or symplast of said organism/cells.
118. 164. A method of producing an assembled immunoglobulin having heavy, light and J chains and a protection protein, said method being substantially as hereinbefore described with reference to any one of the examples.
119. 165. A method of transforming a eukaryotic cell to express an immunoglobulin resistant to environmental conditions comprising the steps of: is introducing into a eukaryotic cell an expression vector comprising a nucleotide sequence encoding a chimeric immunoglobulin heavy chain, wherein a nucleotide sequence encoding at least a portion of the antigen binding domain derived from an immunoglobulin heavy chain is operably linked to a nucleotide sequence encoding at least one domain derived from an immunoglobulin alpha heavy chain; and introducing into the eukaryotic cell one or more expression vectors comprising a nucleotide sequence encoding at least one other protein selected from the group: ii) an immunoglobulin derived light chain having at least a portion of an antigen binding domain, 25 iii) an immunoglobulin J chain, or iv) a protection protein that does not constitute a functional transmembrane spanning domain, said protection protein comprising at least a portion of amino acid residues 1 to 627 and 653 to 755 of a native polyirnmunoglobulin receptor (plgR); C wherein if more than one expression vector comprising a nucleotide sequence 30 encoding said at least one other protein is introduced, these may comprise the same or different nucleotide sequences encoding said at least one other protein.
120. 166. A method according to claim 165, wherein said other molecule is a protection protein and said eukaryotic cell also contains an inmmunoglobulin derived light chain having at least a portion of an antigen binding domain and an immunoglobulin J chain,
121. 167. A method according to claim 165 or claim 166, wherein said immunoglobulin CC. *derived heavy chain is immunoglobulin alpha chain and said immunoglobulin derived light chain is an immunoglobulin kappa or lambda chain. C C C. 9* C. LSBA2Z1DI COMS ID No: SMBI-00637806 Received by IP Australia: Time 16:18 Date 2004-02-26 23. FEB. 2004 15:38 SPRUSON FERGUSON NO. 7273 P. 57 149
122. 168. A method according to any one of claims 165 to 167, wherein said expression vectors are introduced into said cell by direct contact of said cell with said vector under conditions permitting uptake of the vector by said cell.
123. 169. A method according to claim 168, wherein said conditions comprise s chemically enhanced permeabilisation.
124. 170. A method according to claim 168, wherein said conditions comprise temperature perturbation of the cellular membrane.
125. 171. A method according to any one of claims 165 to 167, wherein said expression vectors are introduced into said cell by means of transfection with an Agrobacterium species containing said vector.
126. 172. A method according to any one of claims 165 to 167, wherein said expression vector is introduced into said cell by means of biolistic transformation utilising microparticles coated with said vector.
127. 173. A method according to claim 172, wherein said microparticles are made of tungsten or gold. ~174. A method according to any one of claims 165 to 167, wherein one or more of said expression vectors is introduced into said cell by means of fusion with another cell containing said one or more expression vectors. o*
128. 175. A method according to claim 174, wherein said fusion is protoplast fusion.
129. 176. A method according to claim 174, wherein said fusion comprises cross- fertilisation.
130. 177. A method according to any one of claims 165 to 176, wherein said eukaryotic cell is a plant cell. ~178. A method according to claim 177, wherein said plant is a dicotyledonous or a 25 monocotyledonous plant. ".179. A method according to claim 178, wherein said plant is solanaceous or S leguminous.
131. 180. A method according to claim 179, wherein said plant is an alfalfa or a tobacco plant.
132. 181. A method according to claim 178, wherein said plant is Lenna gibba
133. 182. A method according to any one of claims 165 to 176, wherein said expression vector is introduced into said cell by pronuclear injection.
134. 183. A method of transforming a eukaryotic cell to express an immunoglobulin resistant to environmental conditions, substantially as hereinbefore described with reference to any one of the examples.
135. 184. A eukaryotic cell expressing an immnioglobulin resistant to environmental conditions, transformed by a method according to any one of claims 165 to 183. LrAAZlIDI COMS IDNo:SMBI-00631115 Received by IP Australia: Time 15:44 Date 2004-02-23 26, FEB. 2004 16:1-7 SPRUSON AND FERGUSON 61292615486 NO. 7547- 1 P. 35 u.. 150
136. 185. A transformed eukaryotic cell expressing an immunoglobulin resistant to environmental conditions, substantially as hereinbefore described with reference to any one of the examples.
137. 186. A eukaryotic cell according to claim 184 or claim 185 which is a plant cell. 3
138. 187. A method for producing a transgenic non-human organism expressing an immunoglobulin resistant to environmental conditions, said method comprising generating a whole organism from a transformed cell according to any one of claims 184 to 186,
139. 188. A transgenic organism produced by a method according to claim 187,
140. 189. A transgenic organism comprising a plurality of cells according to any one of claims 184 to 186,
141. 190. A transgenic organism according to claim 188 or claim 189 which is a plant
142. 191. A transgenic organism expressing an inummunoglobulin resistant to environmental conditions, substantially as hereinbefore described with reference to any one of the examples, 192, A method of producing an immunoglobulin resistant to environmental conditions comprising the steps of: operably linking a nucleotide sequence encoding at least a portion of the antigen binding domain derived from an immunoglobulin heavy chain to a nucleotide sequence encoding at least one domain derived from an immunoglobulin alpha heavy chain to form a nucleotide sequence encoding a chimeric immunoglobulin heavy chain; expressing said nucleotide sequence encoding said chimeric immunoglobulin heavy chain to produce said chimeric immunoglobulin heavy chain in a eukaryotic cell which also contains at least one other molecule selected from the group consisting of: a protection protein that does not constitute a functional transmembrane spanning domain, said protection protein comprising at least a portion of amino acid residues 1 to 627 and :653 to 755 of a native polyimmunoglobulin receptor (pIgR); an immunoglobulin derived light chain having at least a portion of an antigen binding domain; and an immunoglobulin J chain; and :thereby allowing the chimeric immunoglobulin heavy chain to assemble with said at 30 least one other molecule to form said immunoglobulin resistant to said environmental conditions.
143. 193. A method according to claim 192, wherein said other molecule is a protection protein and said eukaryotic cell also contains an immunoglobulin derived light chain having at least a portion of an antigen binding domain and an immunoglobulin J chain. 3
144. 194. A method according to claim 192 or claim 193, wherein said eukaryotic cell is a cell according to any one of claims 184 to 186. Sc
145. 195. A method according to claim 192 or claim 193, wherein said eukaryotic cell is part of an organism according to any one of claims 188 to 191, or aportion thereof. 196, A method according to any one of claims 192 to 195, said method further comprising the step of shearing under pressure a plurality of said cells, to produce a pulp containing an immunoglobulin resistant to environmental conditions in a liquid derived from the apoplast or symplast of said organism/cells and solid derived material. LIBAA622DI COMS ID No: SMBI-00637806 Received by IP Australia: Time 16:18 Date 2004-02-26 23. FEB 2004 15:39 SPRUSON FERGUSON NO 7273 P. 59 151
146. 197. A method according to claim 196, wherein said shearing is accomplished by a mechanical device which releases liquid from the apoplast or symplast of said organism/cells.
147. 198. A method according to claims 196 or claim 197 further comprising the step of s separating said solid derived material from said liquid.
148. 199. A method according to claim 198, wherein said separation is by centrifugation, settling, flocculation or filtration.
149. 200. A method of producing an immunoglobulin resistant to environmental conditions, substantially as hereinbefore described with reference to any one of the i0 examples.
150. 201. An immunoglobulin resistant to environmental conditions made by a method according to any one of claims 192 to 200.
151. 202. A process for producing an irnmunoglobulin resistant to environmental conditions by maintaining under conditions allowing protein production and is immunoglobulin assembly a cell according to any one of claims 184 to 186, or an organism according to any one of claims 188 to 191, or a portion thereof. 0@S.
152. 203. A process for making an immunoglobulin resistant to environmental conditions, substantially as hereinbefore described with reference to any one of the examples.
153. 204. An immunoglobulin resistant to environmental conditions produced by a *too&; process according to claims 202 or claim 203. 205, A transgenic plant comprising an immunoglobulin as defined in claim 1, comprising a plurality of cells according to any one of claims 35, 36, 41, 42, 46 to 51, 120, 121, 154, or 186. 0S 25.
154. 206. A transgenic plant comprising an immunoglobulin as defined in claim 1, regenerated from a cell according to any one of claims 35, 36, 41, 42, 46 to 51, 120, 121, '41 154, or 186. s.c
155. 207. Transgenic seed of a plant according to any one of claims 84, 123 to 125, 158, 190, 205 or 206. 30 208. Transgenic progeny of a plant according to any one of claims 84, 123 to 125, 158, 190, 205 or 206,
156. 209. A transgenic portion of a plant according to any one of claims 84, 123 to 125, 158, 190, 205 or 206, or of the transgenic progeny according to claim 208, wherein said portion of said plant is a leaf, stem, root, tuber, fruit or entire plant, Dated 23 February, 2004 Planet Biotechnology, Inc. King's College London Regents of the University of California Patent Attorneys for the Applicant/Nominated Person SPRUSON FERGUSON LMU6223D COMS ID No: SMBI-00631115 Received by IP Australia: Time 15:44 Date 2004-02-23