Neth. J. P1. Path. 98 (1992) Supplement 2:183-191

'Plantibodies': a flexible approach to design resistance against pathogens

A. SCHOTS 1, J. DE BOER 2, A. SCHOUTEN 1,2, J. ROOS1EN 1,2, J.F. ZILVERENTANT j, H. POMP 1, L. BOUWMAN-SMITS 2, H. OVERMARS 2, F.J. GOMMERS 2, B. VISSER 3, W.J. STIEKEMA 3 and J. BAKKER 2.

i Laboratory for Monoclonal Antibodies, P.O. Box 9060, 6700 GW Wageningen 2 Department of Nematology, Wageningen Agricultural University, P.O. 8123, 6700 ES Wageningen 3 Center for Plant Breeding and Reproduction Research, P.O. Box 16, 6700 AA Wageningen.

Accepted 9 January 1992

Abstract

Engineering resistance against various diseases and pests is hampered by the lack of suitable genes. To overcome this problem we started a research program aimed at obtaining resistance by transfec- ring plants with genes encoding monoclonal antibodies against pathogen specific proteins. The idea is that monoclonal antibodies will inhibit the biological activity of molecules that are essential for the pathogenesis. Potato cyst nematodes are chosen as a model and it is thought that monoclonal anti- bodies are able to block the function of the saliva proteins of this parasite. These proteins are, among others, responsible for the induction of multinucleate transfer cells upon which the nematode feeds. It is well documented that the ability of antibodies to bind molecules is sufficient to inactivate the function of an antigen and in view of the potential of animals to synthesize antibodies to almost any molecular structure, this strategy should be feasible for a wide range of diseases and pests. Antibodies have several desirable features with regard to protein engineering. The antibody (IgG) is a Y-shaped molecule, in which the domains forming the tips of the arms bind to antigen and those forming the stem are responsible for triggering effector functions (Fc fragments) that eliminate the antigen from the animal. Domains carrying the antigen-binding loops (Iv and Fab fragments) can be used separately from the Fc fragments without loss of affinity. The antigen-binding domains can also be endowed with new properties by fusing them to toxins or enzymes. Antibody engineering is also facilitated by the Polymerase Chain Reaction (PCR). A systematic comparison of the nucleotide sequence of more than 100 antibodies revealed that not only the 3Lends, but also the 5'-ends of the antibody genes are relatively conserved. We were able to design a small set of primers with restric- tion sites for forced cloning, which allowed the amplification of genes encoding antibodies specific for the saliva proteins of Globodera rostochiensis. Complete heavy and light chain genes as well as single chain Fv fragments (scFv), in which the variable parts of the light (VL) and heavy chain (VH) are linked by a peptide, will be transferred to potato plants. A major challenge will be to establish a correct expression of the antibody genes with regard to three dimensional folding, assembly and int- racellular location.

Additional keywords: Monoclonal antibodies, single chain antibodies, scFv, potato cyst nematodes

Introduction

Monoclonal antibodies (MAbs) play an increasingly important role in medicine. Monoclonal antibodies conjugated to drugs, isotopes, or toxins are being utilized as the tar- geting components of diagnostic and therapeutic agents for cancer, cardiovascular and

183

other diseases. However, a major drawback of murine monoclonal antibodies is their immunogenicity in man. Various efforts have been directed towards making the antibodies more human-like, smaller in size or both. In the last few years these efforts have been very successful and the ability to manipulate antibody genes has expanded enormously. Recently, antibody genes or fragments thereof have been expressed in bacteria, yeast, mammalian cells and recently also in plants. In view of these advances we started a research program aimed at engineering resistance against potato cyst nematodes using monoclonal antibodies. To obtain resistance, genes encoding monoclonal antibodies, rai- sed against essential proteins from the potato cyst nematode, will be transferred to potato. In theory this strategy should be applicable to a wide range of plant pathogens. To illustrate the feasibility of this approach a brief overview is given with regard to antibodies as inhi- bitors and the flexibility to manipulate antibodies.

Inhibiting antibodies

Blocking the biological activity of an antigen with monoclonal antibodies is a common way to study the topography of active sites (Table 1). For example, Pfeiffer et al. (1987) explored the topographical separation of the catalytic sites of asparagine synthetase with MAbs. Two different classes of inhibiting MAbs were found. Inhibition of the catalytic activity of human pancreatic elastase 2, probably involved in diseases lille emphysema and atherosclerosis, by MAbs was observed by Shirasu et al. (1988). One MAb reacted with the activation peptide of the enzyme, while another inhibited its enzymatic activity. Inhibition of enzymatic activity is not necessarily due to binding of a MAb to the catalytic site itself. This was shown with a MAb against lysozyme. Crystallographic studies revealed that the inhibiting MAb bound to the enzyme at a region remote from the cataly- tic site. Indeed, in an enzymatic assay using Micrococcus leisodeikticus as substrate, lyso- zyme was enzymatically inactive. However, no inhibition was observed if oligomers of N- acetylglucosamine were used. (Kenett et al., 1987). Apparently, inhibition was due to steric hindrance which could be avoided by using a smaller substrate.

Antibodies are also able to inhibit biological processes in situ. A profound effect on cell morphology (Feramisco et al., 1985) and DNA synthesis (Riabowol et al., 1988) was found by blocking oncogene products through intracellular micro-injection of antibodies.

Table 1. Examples of inhibition of protein function by monoclonal antibodies (MAbs). For each of the protein antigens, the number of raised MAbs and the number of inhibiting MAbs are shown. Note that in many cases a relatively high percentage of the raised MAbs interfered with the function of the antigen.

Protein antigen Number of Number of References MAbs inhibiting raised MAbs

asparagine synthetase 11 4 human pancreatic elastase 2 3 1 terminal deoxynucleotidyl transferase 4 3 RNA polymerase II 5 3 [3-1actamase 9 5

Pfeiffer et al., 1987 Shirasu et al., 1988 Smith & Bauingarten, 1987 Dahmus et al., 1988 Bibi and Laskov, 1990

184 Neth. J. PI. Path. 98 (1992) Supplement 2

Similarly, chromosome condensation was prevented by intranuclear injection of anti-actin antibodies into Xenopus oocytes (Rungger et al., 1979).

It is noteworthy, that when raising MAbs against a protein antigen a relatively high pro- portion of the MAbs interferes with its function (Table 1). A possible explanation is that the antigenicity of protein regions is determined by their accessibility (Nov6tny et al., 1986, 1987), flexibility (Tainer et al., 1984; Getzoff et al., 1987; Geysen et al., 1987) or both (Berzofsky, 1984). Since active sites are among the most accessible and flexible regions of proteins, this might explain the high incidence of inhibiting MAbs.

Antibody engineering

In 1975 K6hler and Milstein developed the hybridoma technology which made it possible to develop antibodies with defined properties and specificity. In the 1980's this technology was revolutionized by gene technology. Until then only few functional properties of mono- clonal antibodies could be changed, for instance by switching heavy chain constant regions (Kipps, 1985) or by making bispecific antibodies (Milstein and Cuello, 1983). Now, anti- body genes can be taken from hybridoma cell lines, cloned into plasmid vectors and expressed in bacteria, yeast, mammalian cells and plants. An antibody (IgG) is a Y-shaped molecule (Fig. 1), in which the domains forming the tips of the arms bind to antigen (Fv and Fab fragments) and those forming the stem (Fc fragment) are responsible for triggering effector functions that eliminate the antigen from the animal. An IgG molecule consists of four polypeptide chains, two heavy (H) and two light (L) chains. Homologous domains of

Antibody

CL cCL~H3 N ~ ' Fig. 1. Schematic drawing of an antibody molecule (IgG). The four domains are drawn and the dif- ferent fragments indicated. Abbreviations used: CH1 - CH3, constant domains 1 - 3 of the heavy chain; C L, constant domain of the light chain; V H and V L, variable domains of the heavy respective- ly light chain; Fv, variable fragment; Fab, antigen binding fragment; Fc, constant fragment.

Neth. J. Pl. Path. 98 (1992) Supplement 2 185

1

- . . . , F ( a b ) 2 v.

v, c m v.

H1

)

ciAb m.r.u Fig. 2. The domain structure of the antibody molecule facilitates antibody engineering. The diffe- rent possibilities are indicated. F(ab) 2 and Fab fragment can be obtained through proteotytic cleava- ge of antibody molecules or through gene technology. To obtain Fv-fragments, single chain Fv-frag- ments (both polypeptide chains linked by a 15-mer peptide) or single domain antibodies (dAbs) gene technology must be used. A minimal recognition unit (m.r.u.) is a synthetic peptide resembling the amino acid sequence of a hypervariable region in the heavy or light chain V-region.

the light and heavy chains are paired in the Fab regions as are the CH3 domains of the IgG heavy chains. The CH2 domains tend to be separated by carbohydrate moieties. Each domain consists of two g-sheets which pack together to form a sandwich, with exposed loops at the end of the strands.

The domain structure makes antibodies particularly accessible for protein engineering (Winter and Milstein, 1991; Fig. 2): - The domains carrying the antigen-binding loops (Fv and Fab fragments) can be used separately from the Fc-fragments without loss of affinity. This can be achieved using PCR for forced cloning of Fv and Fab fragments and single chain Fvs (scFvs). In the latter the variable parts of the light (V L) and heavy chain (V H) are linked by a peptide. Fv-fragments, Fab-fragments and scFvs have been successfully expressed in bacteria, yeast and murine myeloma cell lines. - The antigen-binding domains can be endowed with new properties by fusing them to toxins or enzymes. Immunotoxins have been constructed as Fv fusion proteins (Batra et al., 1990; Chaudhary et al., 1990) and expressed in Escherichia coli.

- Chimeric antibodies can be made by attaching variable domains of murine antibodies to the constant domains of human IgG isotypes followed by expression in myeloma cells (Orlandi et al., 1989).

186 Neth. J. Pl. Path. 98 (1992) Supplement 2

Engineering of antibodies is not restricted to the use of whole domains. An example is the humanization of rodent antibodies. By replacing the antigen-binding loops in a V- region framework of a human antibody by those of a rodent antibody, the antigen binding- site was transferred (Jones et al., 1986).

Antibody engineering is facilitated by the polymerase chain reaction (PCR, Orlandi et al., 1989). A comparison of the nucleotide sequence of more than 100 antibodies revealed that not only the constant domains, but also the 5Lends of the variable domains are rela- tively conserved. In our laboratory we were able to develop a small set of primers which can be used to amplify over 95% of the IgG heavy and light chains.

Expression of antibodies in heterologous systems

Successful expression of whole antibodies and antibody fragments has been described for yeast (Wood et al., 1985, Horwitz et al., 1988), myeloma celt lines (Orlandi et al., 1989; Shin and Morisson, 1990), plants (Hiatt et al., 1989; Dfiring et al., 1990), algae (Stieger et al., 1991) and bacteria (Pltickthun, 1991). A nice example is a bacteriophage )~ vector expression system to express a combinatorial library of Fab fragments of the mouse anti- body repertoire (Huse et al., 1989). This Fab expression library was constructed, using PCR, from mRNA isolated from stimulated mouse spleen cells. Immunoglobulin heavy chain fragments and light chains were randomly combined in a bacteriophage )~ vector expression system. With this procedure several million Fab fragments can be constructed and examined for antigen binding. These results invite speculation concerning the produc- tion of antibodies without the use of animals.

Promoter and leader sequences have a large influence on the expression of antibody fragments in E. coli (Plt~ckthun, 1991). The promoter determines the expression rate which must be commensurate with the rates for the transport, folding and assembly steps. If not, the protein will accumulate as insoluble material in either the periplasm or the cytoplasm. Transport to the periplasm will only take place if the appropriate leader sequence is pre- sent.

Also, chaperon proteins may be of importance to obtain functional antibodies in hetero- logous expression systems. Molecular chaperons are defined as proteins that mediate the correct assembly of other polypeptides but that are not components of the functional assembled structures (Ellis and Hemmingsen, 1989). Inside the endoplasmic reticulum of lymphoid cells the assembly of immunoglobulin heavy and light chains is mediated by the heavy chain binding protein (BiP). BiP belongs to the heat-shock protein 70 (hsp 70) family of stress proteins which occur in prokaryotes and in several compartments of euka- ryotic cells (Ellis and Van der Vies, 1991). It can therefore be expected that chaperones related to BiP might mediate assembly of immunoglobulin light and heavy chains in various heterologous expression systems.

Expression of antibodies in plants

The production of antibodies in plants was first described by Hiatt et al. (1989). Constructs of coding-length cDNAs of the 7- and ~:-chain, with and without immunoglobulin leader sequences, were ligated into a constitutive plant expression vector using the 35 S promo- ter. Plasmids containing the gene for either the heavy or the light chain were used to trans- form tobacco plants. The transformants expressing individual immunoglobulin chains

Neth. J. Pl. Path. 98 (1992) Supplement 2 187

were then sexually crossed to produce progeny expressing both chains. Only plants expres- sing immunoglobulin chains with the original leader peptide contained assembled gamma- kappa complexes. Functional immunoglobulins, assessed using ELISA, were reported to accumulate up to 1.3% of the total leaf protein. However, nothing was reported on correct assembly and the in situ localization of assembled antibody molecules.

Also, D~iring et al. (1990) described the synthesis of antibody in tobacco. However, they constructed a vector containing both heavy and light chain genes and the barley aleurone a-amylase leader peptide coding sequence under control of the pT R and pNOS promoters. The barley aleurone c~-amylase leader peptide is thought to direct proteins to the inter- -cellular spaces. The in situ localization and correct assembly of functional antibodies was studied using anti-idiotype antibodies. Assembled antibodies were detected in the endo- plasmic reticulum (ER) and surprisingly also inside chloroplasts. Nothing was reported on the presence of antibodies in the intercellular spaces.

If resistance against pathogens is the final goal it is apparent that several aspects con- cerning antibody expression in plants need optimization. A striking difference is observed in the amount of accumulated antibody found by Hiatt et al. (1989) and Dtiring et al. (1990). The former reports up to 1.3% of total leaf protein, while the latter found much lower amounts. This discrepancy can be the result of different promoters (35 S versus pT R and pNOS) used by the two groups possibly in combination with different leader peptides. Both aspects have to be studied in more detail. A third point of interest is the ability to assemble functional immunoglobulins in the cytoplasm. Transient expression of immu- noglobulin ~- and ~t-chains was demonstrated in the alga Acetabularia mediterranea (Stieger et al., 1991). Plasmids containing the gene for either the heavy or the light chain, without leader sequence, were injected in nuclei of the alga. Using anti-idiotype antibodies it was shown that antibodies containing a functional binding site were formed. Since the immunoglobulins did not contain a leader sequence assembly must have taken place in the cytoplasm. A fourth important aspect for future research to optimize the production of anti- bodies in plants is the aforementioned role of chaperone proteins.

Engineering durable resistance against potato cyst nematodes

Sedentary plant parasitic nematodes like Globodera sp., Heterodera sp., and Meloidogyne sp. induce specialized feeding cells in the roots of their host plant upon infection. Depending on their ontogeny, these feeding cells are named either giant cells or syncytia. The feeding cells are the result of a redifferentiation of existing root cells, in which the met- abolism has been changed to produce so-called transfer cells. Saliva proteins from the nematode, which are injected via the stylet into the root cells are thought to play an impor- tant role in both the induction and the exploitation of the feeding cells. Blocking of the function of these saliva proteins may prevent the nematodes from creating a feeding site. For this purpose, we have raised MAbs against proteins from the salivary glands of G. ros- tochiensis, The genes encoding for these MAbs will be transferred to potato plants. It is expected that expression Of these MAbs in the feeding cells will inhibit the saliva proteins and thus induce resistance.

Resistance obtained in this way is most likely durable. This can be inferred from expe- rience with the H 1 resistance gene against G. rostochiensis, a natural gene from Solanum tuberosum ssp, andigena. Durability of a resistant cultivar depends on the number of viru- lent individuals present in a population before growth of the resistant cultivar. In Great

188 Neth. J. Pl. Path. 98 (1992) Supplement 2

Britain H 1 resistance is very effective and durable. No virulent G. rostochiensis popula- tions have been found after 20 years of repeated growth of potato cultivars with the H 1 resistance gene. Apparently no virulent individuals of G. rostochiensis have been introdu- ced from South America. This shows that the reproduction capacity of potato cyst nemat- odes is too low to break resistance by mutation and therefore holds the promise that anti- body-mediated resistance is durable.

Conclusion

Advances in gene technology have offered various novel routes to improve the disease resistance of crops. This has resulted in resistance against a number of insect species using genes encoding for protease inhibitors (Hilder et al., 1987; Johnson et al., 1989) and 8- endotoxin of Bacillus thuringiensis (Vaeck et al., 1987; Delannay et al., 1989). Also resi- stance against a number of viruses was obtained by expressing genes encoding for the viral coat protein, applying the principle of cross-protection (Powell Abel et al., 1986; Cuozzo et al., 1988; Hoekema et al., 1989).

Suitable genes to engineer resistance against potato cyst nematodes have not been found yet. In this paper we presented an appealing approach to design resistance against potato cyst nematodes. In view of the vast immune repertoire of animals this strategy should be applicable to a wide range of plant pathogens.

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