WO1989001037A1 - Production of ricin toxins in a baculovirus-insect cell expression system - Google Patents

Abstract

Cloning and expression of ricin toxin, muteins of ricin toxin having reduced galactose binding activity, or subunits thereof is described wherein these molecules are cloned into novel baculovirus transfer vectors and co-infected into insect cells with baculovirus thereby effecting recombination with the transfer vectors producing baculovirus expression vectors capable of infecting insect cells and expressing ricin toxins, muteins, or subunits thereof.

Description

^•Production of ricin toxins in a baculovirus-insect cell expression system.

The present invention relates to the field of molecular biology and proteins. In particular, the invention relates to the achievement of expression of ricin toxin proteins using an insect cell/baculovirus expression system.

Numerous different expression systems are now available to the genetic engineer who wishes to express a cloned gene of interest. Generally the goal is the production of high levels of biologically active material. The requirements for the expression system chosen to accomplish this goal are dependent upon the nature of the protein to be expressed.

The value of utilizing prokaryotic host vector systems for the synthesis of desirable eukaryotic proteins is diminished by certain limitations inherent in such systems. For instance, the mRNA transcript or protein product of such systems may be unstable in the prokaryote. In addition, before a protein will be synthesized within a prokaryotic cell, the optimal DNA sequence introduced into the microorganism must be free of intervening DNA sequences, nonsense sequences, and initial or terminal sequences which encode for polypeptide sequences which do not comprise the active eukaryotic protein. Further, some eukaryotic proteins require modification after synthesis (e.g., glycosylation and all membrane associated processing) to become biologically active, and prokaryotic cells are generally incapable of such modifications.

Various nonviral eukaryotic host vector systems are also available for the expression of heterologous proteins. Certain limitations are inherent in each of these systems as well. For example, high levels of expression are frequently difficult to obtain in yeast systems where autonomously replicating vectors may be unstable. Additionally, glycosylation patterns in yeast differ from those in higher euk^'aryotes. Limitations encountered with mammalian host vector systems include difficulties in host cell cultun^'ng and its scale-up. The expense of mammalian cell culture media as well as a requirement for serum often restricts its use on a large scale and complicates the use of these systems for production of parenterally administered pharmaceuticals. Furthermore, levels of expression in these systems are generally substantially lower than that obtained in prokaryotic or viral expression systems.

The use of viruses in eukaryotic host-vector systems has been the object of much speculation. However, some viral vector systems also suffer from significant disadvantages and limitations which diminish their utility. For example, a number of eukaryotic viral vectors are either tumorgenic or oncogenic in mammalian systems and create potential health and safety problems associated with resultant gene products and accidental infection.

The baculovirus expression vector system involved in the instant invention overcomes many of the above-mentioned limitations. Baculoviruses are insect pathogenic viruses which, until recently, were studied mostly for their potential use as viral insecticides for control of agriculturally important insect pests. Because certain baculoviruses are highly virulent for pest insects, some of the most promising have been commercially developed and are used as biological pesticides ( iltenburger and rieg 1984 Bioinsecticides:II: Baculoviridae. Adv. Biotechnol. Processes _3:291; Granados, R.R. and Feden^'ci, B.A. eds. The Biol. of Baculov ruses Vol II, Boca Raton, FL: CRC Press, Inc. 1986). Baculoviruses are very stable and are able to persist for longer times in the environment than other animal viruses. This unusual biological stability is the result of a unique association of the infectious virus particles and a viral occlusion that is a crystalline assembly of a viral encoded structural protein called polyhedrin. Late in viral replication, baculovirus particles become embedded in a protein occlusion composed of the polyhedrin protein. Insects acquire a baculovirus disease by ingesting the occluded virus (OV) which contaminate their food supply. The polyhedrin matrix protects the virus particles in the environment and during their passage through the foregut of the insect. In the insect midgut, the alkaline pH activates the dissolution of the polyhedrin crystalline matrix resulting in the release of many viruses. The virus become absorbed by the midgut epithelial cells and initiate the infection process.

There is a second infectious form of nuclear polyhedrosis viruses (NPVs), known as the extracellular or nonoccluded virus (NOV) form, which is generated by the budding of viral nucleocapsids through the plasma membrane of the infected cells. NOVs are responsible for spreading a secondary infection via the hemolymph of the insect. It is the NOV form of the virus which is infectious in insect cell cultures; the occluded (OV) form is not infectious in cell cultures since dissolution of the crystalline matrix occurs only at high alkaline pH (i.e., pH 10.5). The formation of NOVs and OVs occurs in a biphasic manner during the infection process. NOVs are abundantly produced before occlusion is initiated. During a typical synchronous infection of fully permissive cell lines, the majority of NOVs are produced between 12 and 24 hr post-infection (p.i.). The synthesis of polyhedrin is initiated at 20 hr p.i. and does not reach maximal levels until _.48 to 72 hr p.i. The significance of this temporal regulation with respect to the expression vector system is that foreign gene products that may have adverse effects on the cell should not diminish the production of progeny NOVs to be used for further infection. Of the 450-500 species of known baculoviruses, practically all encode a polyhedrin protein. As previously discussed, the viral occlusion is a paracrystalline assembly of a polyhedrin monomer which, . for most viruses, has an average molecular weight of 28,000-30,000 (Summers, M.D. and Smith., G.E., 1978 Virology 84:390). Baculoviruses are unique among animal viruses, not only in the protective function of the viral occlusion in the viral life cycle but also because the. polyhedrin gene is the most highly expressed eucaryotic virus gene known. The polyhedrin protein can accumulate to greater than 1 mg/ml of infected cultured insect cells (70-75% of the total cellular protein) or can comprise up to 25% of the total protein of an infected insect. Although wery highly expressed, neither the polyhedrin gene nor its protein is essential for viral infection or replication in cultured insect cells or insects, thus making the polyhedrin gene an ideal target for genetic manipulation.

The most extensively studied baculovirus is the Autographa cal forn ca nuclear polyhedrosis virus (AcNPV). The Autographa californica host for AcNPV is a moth commonly referred to as the alfalfa looper. Studies of the physical and functional organization of the AcNPV genome have resulted in the mapping, cloning, and sequencing of the AcNPV polyhedrin gene and its regulatory sequences

(Iddekinge et al. 1983 Virology 131:561; Smith et al . 1983 J. Virol.

_46_:584). Not only does the polyhedrin gene exhibit a strong promoter, but expression can continue late in infection well beyond the point of repression of nearly all other baculovirus and host genes.

The genetic engineering of the baculovirus polyhedrin gene for high level expression of a heterologous protein, in this case, recombinant human s-interferon was first reported by Smith et al . (Mol. Cell. Biol. 3(12):2156-2165 (1983)). Since then, human interleukin 2 has been expressed in insect cells by a baculovirus expression vector as described by Smith et al. (Proc. Natl. Acad. Sci. USA 82:8404-8408 (1985)). Recently, the synthesis of functional human T-cell leukemia virus Type I p40^x protein using a baculovirus expression vector has been reported (Jeang, K.T., et al., J. Virol. _61_:708-713 (1987). Other heterologous proteins that have been expressed in this system are summarized in Summers et al . ("Genetic Engineering of the Genome of the Autographa californica nuclear polyhedrosis virus," Banbury Report: Genetically Altered Viruses in the Environment," _22_:319-339 Cold Spring Harbor Laboratory (1985)). The baculovirus expression system has several advantages for the expression of foreign genes in comparison to other prokaryotic, yeast or mammalian cell expression vector systems. First, high levels of expressed proteins are possible. Greater than 1.0 mg per ml of polyhedrin protein is normally produced in infected cells. Another non-essential occlusion-related viral protein, plO, is also abundantly produced and its promoter has reportedly been used to drive foreign gene expression (D.W. Miller et al . in Genetic Engineering Principles & Methods _8_:277-298, Setlow and Hollaender, eds. New York: Plenum Press, 1986). Using the polyhedrin gene promoter, heterologous gene expression levels never reach polyhedrin levels but are usually in the range of tens to hundreds of micrograms per ml (Summers et al., 1985, p.321, supra). Secondly, in contrast to those produced in bacterial cells, recombinant proteins produced in insect cells may be co- and post-translationally processed in a manner similar to what occurs in mammalian cells. In at least one case, glycosylation of IFN-β in infected insect cells has been reported (G.E. Smith et al . (1983) supra). Whereas about 40% of the natural IL-2 produced in human Jurkat cells is not glycosylated, there was no evidence of any glycosylation of the recombinant IL-2 produced in insect cells (G.E. Smith et al., (1985) supra). In addition, correct cleavage of mammalian secretory signal peptides has been observed (G.E. Smith et al., (1983) supra ; G.E. Smith et al., (1985) supra; D.W. Miller et al., (1986) supra). The expression of naturally occurring toxins which exert their toxic effects by inactivating the large subunit of eukaryotic ribosomes presumably requires either temporal regulation of expression or modified processing so as to protect the cell against potential toxicity in order not to adversely effect the host eukaryotic cells. The baculovirus expression vector system has been tested as an alternative system to produce whole ricin toxin, ricin B chain, and products containing ricin A chain.

The potent ricin toxin and the Ricin communis agglutinin (RCA) are two major lectins found in the beans produced by the castor oil plant (Ricinus communis). These proteins have been extensively studied, including determination of the complete amino acid sequence for one form of ricin toxin (for review, see Olsnes, S. and Pihl, A. (1982a) in Molecular Action of Toxins and Viruses, pp. 51-105, Cohen and vanHeyningen (eds.), Elsevier Biomedical Press; Olsnes, S. and Pihl, A. (1982b) Pharmac. Ther. 15, 355-381). Each of these lectins contains two different glycosylated subunits (A and B), each of approximate molecular weight 30,000, linked via a disulfide bridge. The toxin contains one subunit of each type, whereas the agglutinin contains two of each. The A subunit acts to catalytically inactivate eukaryotic ribosomes, whereas the B subunit binds to cell surface galactose-containing structures and facilitates entry of the A subunit into the cytoplasm.

In recent years, both of the toxin subunits have been used extensively as components in hybrid toxins targeted to specific cells (Olsnes, S. et al . (1982b) supra; Moller, G. (ed.) (1982) Immunol . Rev., 62, Vitetta, E.S. et al. (1985) Cell, _41_:653). Whereas the A subunit provides the essential catalytic cytotoxicity to these molecules, the B subunit may also be used to enhance membrane transport (Vitetta, E. , et al . (1983) Proc. Nat! . Acad. Sci. (USA) 80, 6332).

Ricin subunits have been individually expressed in bacterial hosts. In general, the ribosomes of prokaryotic cells are resistant to enzymatic inactivation by ricin toxin A and intact ricin comprising ricin toxin A and B chains. However, it is important to note that ricin fragments are thought to be toxic in E. coli. ^" European Patent Publication No. 237,676, published September 27, 1987, discloses the expression of the ricin A subunit in E^ coli. European Patent Application No. 86301227.4, filed February 20, 1986 and PCTW0/88/00593, filed February 24, 1988 describe the expression of ricin B subunit or its muteins in E. coli. M. O_.'Hare et al. (Febs Letts. (1987) 216:73) more recently reported the expression of ricin A subunit in E. coli. There are no known disclosures of full length ricin toxin expression in prokaryotic hosts.

There are no reports known to the inventors at the time of this filing of the expression of full length toxin in a eukaryotic system. This is not surprising since upon internalization, ricin A functions to catalytically inactivate the large subunits of eukaryotic ribosomes thereby causing cell death (Olsnes, S. et al. (1982a) supra; Olsnes, S. et al . (1982b), supra). Vitetta et al . (Proc. Nat! . Acad. Sci. (in press), 1987) have described the expression of ricin B chain as a secreted product from monkey C0S-M6 cells, although the amount of ricin B produced is extremely low.

The invention relates, in one respect, to methods for producing by recombinant DNA technology biologically active ricin toxin, ricin toxin subunits or isotoxins thereof and proteins having an amino acid sequence substantially equivalent to these molecules, including Ricin communis agglutinin, and expressing these molecules using recombinant baculovirus expression vectors in suitable host insect cells. Accordingly, one aspect of the invention relates to growing the infected insect cells under suitable conditions to produce the desired recombinant ricin toxin, subunits thereof or related molecules and recovering the biologically active polypeptide, dimerized polypeptide or subunits thereof from the culture medium. A second aspect of the invention is directed to recombinant baculovirus transfer and expression vectors for producing active muteins of ricin isotoxins D, E and El, or their subunits, and Ricin communis agglutinin, having reduced galactose binding activity.

In another aspect, the invention is directed to recombinant baculovirus expression vectors which are capable of affecting the expression of ricin toxin, ricin toxin subunits or proteins having an amino acid sequence substantially equivalent to that of ricin toxin or to the host insect cells infected .with such vectors, and to cultures thereof. One aspect of the invention concerns recombinant baculovirus expression vectors in which ricin toxin, ricin toxin subunits and related molecules are expressed under the transcriptional control of a baculovirus promoter. In one aspect of the invention the baculovirus promoter is the polyhedrin gene promoter. Another aspect of the invention concerns recombinant baculovirus expression vectors in which the DNA encoding ricin toxin, ricin toxin subunits or proteins having an amino acid sequence substantially equivalent to that of ricin toxin is in proper translational reading frame with the DNA encoding a heterologous secretory signal peptide. Also, aspects of the invention are the recombinant baculovirus transfer vectors which are used to transfer the desired recombinant gene into the baculovirus genome.

Figure 1 is a ribbon representation of the ricin backbone. The A chain is in the upper right and the B chain at lower left. The two lactose moieties bound to the B chain are each represented as pairs of discs. The chains have been separated slightly to facilitate viewing. The disulfide bond linking the chains is indicated in the lower right portion of the molecule. Figure 2A shows the position and sequence of oligodeoxyribonucleotide primers used to create Sail and PvuII sites in the amino terminal region of the ricin B sequence.

Figure 2B shows the position and sequence of oligodeoxyribonucleotide primers used to create an Xbal and SacII site in the carboxyl terminal region of the ricin B sequence. The numbers at the right of the figures are nucleotide numbers in the complete ricin sequence. Amino acids are designated by the single letter observations approved by the IUPAC-IUB Commission on Biochemical Nomenclature. Figure 3 shows the DNA sequence of ricin toxin D.

Figure 4 shows the DNA sequence comparison between the recombinant baculovirus transfer vectors pAcCl-C5. The carrots represent restriction endonuclease cleavage sites.

Figure 5 shows the vector constructs used to produce ricin B muteins.

Figure 6 shows the vector constructs used to produce the ricin B double construct-46/255.

Figure 7 shows cytotoxicity assays of supernatants of baculovirus expression system products. Figure 8 shows a Western blot analysis -of insect cell expression products after infection with a recombinant baculovirus containing full length ricin toxin gene sequences. Figure 9 shows a Western blot analysis of insect cells expression products after infection with recombinant baculovirus containing ricin B gene sequences.

Figure 10 shows the binding of native ricin B to asialofetuin coated on the wells of 96-well dishes in the presence or absence of lactose.

Figure 11 shows the binding of ricin B mutein 255 at different concentrations to asialofetuin in the presence or absence of lactose.

Modes for Carrying Out the Invention

A. Definitions for Ricin Toxin

Ricin toxin is best defined by describing what is known in the scientific literature. Ricin toxin (RT or ricin) is a naturally occurring toxin composed of an enzyrπatically active, cytotoxic "A" amino acid sequence or subunit, and a "B" sequence or subunit, which is presumed to be responsible both for attaching the "A" subunit to a target cell to be killed, and to aid in the translocation or transport of A subunit into the cytoplasm (see Olsnes, S. et al . (1982a&b) supra). Other examples of such toxins include diphtheria toxin and the exotoxin from Pseudomonas aeruginosa. Other toxic proteins, such as, for example, those derived from Phytolacca americana (PAPI, PAPII, and PAP-S) and gelonin show in vitro activities comparable to the "A" subunits of the above toxins, but are much less active _rn_ vivo, presumably due to the absence of a "B" subunit. The "ricin"_, peptides of the present invention are derived from the seeds of Ricinus communis, commonly known as castor beans. Two similar proteins (often called lectins) are extractable from these seeds: the above-mentioned ricin and Ricin communis agglutinin (RCA). Both proteins contain A and B portions; however, the A and B portions do not comprise a single peptide. The A portions or these moieties are- capable of catalytically inactivating the large subunit of ribosomes J_n_ vitro and the mechanism of ricin for jm_ vivo cytotoxicity is believed to reside in this capacity for π^'bosome inactivation. Ricin and RCA appear to be highly homologous (Cawley, D. B., et al, Arch. Biochem. Biophys. (1978) 190:744) but differences exist. RCA is dramatically less toxic, and appears to exhibit some characteristics corresponding to those expected of a dimer of ricin. Careful fractionation of castor bean extracts shows the presence of several ricin isotoxins. The distinction between ricins D and E has been previously disclosed (Mise, et al., Agric Bio! Chem (1977) _41_:2041-2046; Wei, et al., J Biol Chem (1978) 253:2061-2066; Lin, et al., Eur J Biochem (1980) 105:453-459; Genaud, et al., J_ Immunol Meth (1982) _49_:323-332). Ricin D has a pi near 7.4 and a high affinity for agarose; ricin E has a pi near 8.8 and a low affinity for agarose. There are several reports of purported isotoxins which have been shown to be more acidic forms of ricin D (Olsnes, et al., J Biol Chem (1974) _249_:803-810; Ishiguro, et al., Toxicon (1976) _H:157-165; Cawley et al., Arch Biochem Biophys (1978) 190:744-755).

The differences in properties between ricins D and E seem to reside in the B chain (Funatsu et al., Agric Biol Chem (1978) 42:851- 859, Araki, T. et al. (1987) Biochem Biophys. Acta, 911:191). The RTA chains from ricins D and E are identical in composition, pi, and apparent molecular weight. In the ricin toxins there are two distinct RTA species, RTA1 and RTA2. These isoenzymes differ in molecular weight by SDS-PAGE and in carbohydrate content, and can be resolved by ion exchange chro atography with a very shallow salt gradient (Olsnes, et al., Biochemistry (1973) _12_:3121-3126, Foxwell, B.M.J., et al . (1985) Bioch. Biophys. Acta, 840:193).

The ribotoxin most similar to a ricin E isotoxin preparation was designated as ricin El and the novel ribotoxin was designated ricin E2. Ricin E2 has a pi identical to that of ricin El. Compared to ricin El, it is 1% as toxic to mice and 2-4% as toxic to cultured cell lines, is bound to agarose more tightly at moderate to high ionic strength, and is approximately 2 kD larger by SDS-PAGE.

The components of ricin and of RCA have been well characterized on the basis of the extracted materials, and their properties extensively reviewed: Olsnes, S., Perspectives in Toxicology, A.W. Bernheimer, Ed (1977) J. Wiley & sons, NY, pp. 122- 147; Olsnes, S., et al., Molecular Action of Toxins and Viruses, Cohen, et al., Ed (1982) Elsevier, Amsterdam, pp. 51-105. Ricin has an apparent molecular weight of 58,000 daltons and consists of the A chain with a molecular weight of 32,000 daltons and a B chain of molecular weight of 34,700 daltons. RCA is a tetramer which has two A subunits of molecular weight 32,000, and two B subunits of molecular weight 36,000 each. In their native environments, the A and B chains are generally glycosylated. The A and B subunits of both ricin and RCA are linked only by a single disulfide bond, and not by peptide linkage unlike, for example, diphtheria toxin which is found as a single chain peptide. It is also known that both ricin and RCA, though having separate peptides for A and B portions, are each derived from a single chain precursor in each case (Butterworth, H.E., et al., Eur J Biochem (1983) 137:57). This precursor- was shown to contain a sequence of 12 amino acids between the A chain (amino terminal) and B chain (carboxy terminal) sequence; PCTUS/88/00197 filed February 7, 1985. European Patent Appplication No. 86308877.9 filed November 13, 1988 shows the ricin A sequence to contain 265 amino acids preceded by ^' a 35 amino acid leader (signal) peptide. It is assumed that upon excision of the dodecameric intervening peptide, the A and B chains remain linked through the single disulfide bond. The cDNA insert in pRT17 corresponds to the composite between the ricin toxin B chain encoded in the DNA disclosed in PCT/US88/00197 (supra) and the ricin A encoding sequences disclosed in European Patent Publication No. 237,676 (supra). This is the DNA, then, encoding the precursor for ricin D. In addition, the cDNA sequence for the ricin toxin precursor and RCA has recently been reported (Lamb, F.I., Roberts, L.M., and Lord, J.M. (1985) Eur J Biochem 148, 265-270; European Patent Application Publication No. 0145,111 to Lord, J.M. et al., June, 1985).

As is the case for all proteins, the precise chemical structure of ricin toxin, its muteins or subunits, depends on a number of factors. As ionizable amino and carboxyl groups are present in the molecule, a particular protein may be obtained as an acidic or basic salt, or in neutral form. All such preparations which retain their activity when placed in suitable environmental conditions are included in the definition. Further, the primary amino acid sequence may be augmented by derivatization using sugar moieties (glycosylation) or by other supplementary molecules such as lipids, phosphate, acetyl groups and the like, more commonly by conjugation with saccharides. The primary amino acid structure may also aggregate to form complexes. Certain aspects of such augmentation are accomplished through post- trans!ational processing systems of the producing host; other such modification may be introduced j_n_ vitro. In any event, such modifications are included in the definition so long as the activity of the protein, as defined above, is not destroyed. It is expected, of course, that such modifications may quantitatively or qualitatively affect the activity, either by enhancing or diminishing the activity of the protein in the various assays. Further, individual amino acid residues in the chain may be modified by oxidation, reduction, or other derivatization, and the protein may be cleaved to obtain fragments which retain activity. Such alterations which do not destroy activity do not remove the protein sequence from the definition.

Modifications to the primary structure of ricin toxin, its muteins or subunits, by deletion, addition, or alteration of the amino acids incorporated into the sequence during translation can be made without destroying the activity of the protein. Native ricin and ficin A exist in a number of homologous but not exactly identical forms depending on the plant variety used as source, and even proteins derived from a single plant may exhibit more than one primary structure. Such substitutions, alterations or variations result in proteins having an amino acid sequence which falls within the definition of proteins "having an amino acid sequence substantially equivalent to that of ricin toxin."

In summary, "ricin" refers to proteins having cytotoxic activity which contain both A and B chains, as set forth herein.

Conventionally, as described above, ricin is distinguished from RCA in the art. Both ricin D and ricin E contain A and B chains; it appears that the differences in these proteins lies in the B portions. Mutei ns

"Ricin B muteins" according to the invention are defined to be substantially similar forms of ricin B or isotoxins thereof according to the invention in that they fulfill the functional definition of facilitating the intracellularization of an associated toxin molecule. The alterations of the galactoside binding sites of the ricin B muteins decrease the affinity of the ricin B muteins according to the invention for galactosides, yet retain, at least partial functional ability to facilitate the intracellularization of an associated toxin molecule. The precise mechanism whereby ricin B or ricin B muteins facilitates translocation of ricin A is unknown.

Operably Linked

"Operably linked" refers to juxtaposition such that the normal function of the components can be performed. Thus, a coding sequence "operably linked" to control sequences refers to a configuration wherein the coding sequence can be expressed under the control of these sequences.

Control Sequences

"Control sequences" refers to DNA sequences necessary for the expression of an operably linked coding sequence in a particular host organism. Eukaryotic cells including the insect cells of the instant invention appear to utilize promoters and polyadenylat on signals.

Expression System "Expression system" refers to DNA sequences containing a desired coding sequence and control sequences in operable linkage, so that hosts transformed with these sequences are capable of producing the encoded proteins. These DNA sequences may also direct the synthesis of the encoded proteins in an j_n_ vitro cellular environment. In order to effect transformation, the expression system may be included on a transfer vector; however, the relevant DNA may then also be integrated into the viral chromosome to result in a recombinant viral genome.

Cell, Cell Line, Cell Culture As used herein "cell", "cell line", and "cell culture" are used interchangeably and all such designations include progeny. Thus "transformants" or "transformed cells" includes the primary subject cell and cultures derived therefrom without regard for the number of transfers. It is also understood that all progeny may not be precisely identical in DNA content, due to deliberate or inadvertent mutations. Mutant progeny which have the same functionality as screened for in the originally transformed cell are included. Where distinct designations are intended, it will be clear from the context.

Infection "Infection" as used herein refers to the invasion of cells by pathogenic viral agents where conditions are favorable for their replication and growth. "Transfection" refers to a technique for infecting cells with purified nucleic acids by adding calcium chloride to solutions of DNA containing phosphate or other appropriate agents such as dextran sulfate thereby causing the DNA to precipitate and be taken up into the cells.

Recombinant Transfer Vector

"Recombinant transfer vector" refers to a plasmid containing a "heterologous" gene under the control of a functional promoter (e.g., polyhedrin or plO promoter) and flanked by viral sequences. The "recombinant expression vector" is formed after cotransfection of the recombinant transfer vector and wild-type baculovirus DNA into host insect cells whereupon homologous recombination occurs between the viral sequences flanking the heterologous gene and the homologous sequences in the wild-type viral DNA. This results in the replacement of wild-type sequences in the virus with the transfer vector sequences between the crossover points. The recombinant expression vector is the recombinant viral DNA containing the desired heterologous gene. Biologically Active

"Biologically active" refers to retaining the enzymatic or other biological behavior which typifies the function of the protein in its native state. The biological activity of ricin A refers in one aspect to enzymatic activity, i.e., its ability to inhibit protein synthesis in a rabbit reticulocyte j_n_ vitro translation system (a commercially available system obtainable, e.g., from Bethesda Research Laboratories, Rockville, MD). In addition to being enzymatically active, soluble preparations of ricin A toxin are also capable of exhibiting specific cytotoxic activity when associated with specific binding portions, for example, immunoglobulins, to form immunotoxins or with the ricin B subunit to reconstitute ricin toxin activity. The biological activity of ricin B refers to its ability to facilitate the intracellularization of an associated toxin molecule via cell surface _ binding to galactose-containing receptors.

Secretory Signal Peptide

"Secretory signal peptide" refers to a sequence of amino acids that functions to transport a protein expressed in insect cells, such as ricin toxin or subunits thereof, outside the cell. A "heterologous secretory signal peptide" is an amino acid sequence not naturally found in association with the protein to be secreted.

B. General Description

B.l.a. Cloning of the Ricin A Coding Sequence

The approach followed to obtain recombinant ricin A has been described in European Publication No. 215,658 published May 25, 1987 and European Publication No. 237,676 published September 23, 1987. It is, briefly, as described below.

A cDNA library was constructed by isolating mRNA from maturing castor bean seeds, and preparing the corresponding cDNA by, in general, conventional methods. The oligonucleotide 5'-GACCATTTCGAC CTACG-3' which complements the mRNA encoding the N-terminal region of the B chain (which is thus just downstream from the A chain codons) was used as primer in synthesizing the single stranded copy; and an oligo dC homopolymeric tail was added to the 3¹ end to permit oligo dG to be used as primer in double stranding. The resulting double stranded cDNA fragments were then inserted into the PstI site of the_t cloning vector, pBR322, by annealing homopolymeric oligo dC tails provided by standard tailing methods to the cDNA with the oligo dG tails which are also thus provided on the cleaved vector. The ligation mixture was transformed into E. coli. About 5000 successful 0 transformants were screened for hybridization with probe.

The olignonucleotide mixture δ'-GCATCTTCTTGGTTGTCNGGATGAAA GAAATAGGC-3¹ (wherein N is A, T, G, or C) was used as a probe. This sequence was initially predicted based on the amino acid sequence described in the review by Olsnes, S., et al., supra, and verified. 5 Positive colonies were analyzed by restriction and showed two pattern types—one predicted to be found from ricin A, and the other presumed to be associated with agglutinin A, since it was significantly different from that obtained from ricin A. A colony was obtained which contained the entire sequence for ricin A, as confirmed 0 by sequencing and comparison of the deduced amino acid sequence to that of native ricin A. Plasmid DNA isolated from this colony was designated pRAI23, and given number CMCC 2108 in the assignee's culture collection. pRA123 was deposited with the ATCC on 14 August 1984, and has accession No. 39799.

25 It should be noted that the procedures of the foregoing paragraphs need not now be repeated in order to obtain the desired ricin A encoding sequences. Using methods known in the art, the appropriate sequence spanning approximately 750 nucleotides may be synthesized. (See, for example, Edge, M.D., et al., Nature (1981)

30 292:256; Nambiar, K.P., et al., Science (1984) _223_:1299; or Jay, Ernest, et al., J. Biol. Chem (1984) 259:6311.) Desired sequence modifications useful in obtaining the desired portions of the ricin A sequence or appended sequences for the construction of expression vectors may be made using site-specific mutagenesis in a manner analogous to that described for the construction of expression vectors below.

The cDNA insert in pRA123, which contained the coding sequence for the entire ricin A chain, was modified by primer directed mutagenesis to place a HindiII site in front of a newly constructed ATG start codon preceding the RTA sequence, and to place a stop signal at the C-terminus. The properly terminating coding sequence for the ricin A chain could then be removed as a Hindlll/BamHI cassette and ligated into appropriate transfer vectors.

B.2.a. Cloning of the Ricin B Coding Sequence

The approach followed to obtain recombinant ricin B has been described in European Application No. 86301227.4 (supra) and PCTW0/8800593 (supra). It is, briefly, as described below.

The sequence published for naturally-occurring ricin B showed the presence of the amino acid sequence, Trp-Met-Phe-Lys-Asn-

Asp-Gly, which is associated with minimal codon redundancy. A mixture of all oligonucleotide sequences encoding this sequence was constructed as a probe.

A cDNA library was constructed by isolating mRNA from castor bean seeds, and preparing the corresponding cDNA by, in general, conventional methods. However, during the construction, appropriate linkers were ligated to the ends of the cDNA so as to obtain inserts bounded by EcoRI/Sal I sites. EcoRI/Sal I inserts were then ligated into the cloning vector, pUC13, and transformed into _E^_ coli. Successful transformants capable of hybridizing with the probe were selected and sequenced.

Colonies were obtained which contained large portions of the ricin B and agglutinin B sequences. In addition, a colony was obtained which contained the sequences for a portion of the putative peptide precursor of both RCA and ricin which was thus shown to contain a twelve amino acid bridging peptide. The cDNA insert contained a sequence which began in the A portion and overlapped into the B region of each. The plasmids derived from the foregoing colonies are designated pRTB5, pRTB4, and pRTAllδ, respectively. The cDNA insert in pRTB5, which contained the coding sequence for the entire ricin B chain except for the 11 N-terminal amino acids, was excised and placed in the correct orientation with respect to the lac promoter by insertion into pUC8, to give pRTBlδl. pRTBlδl was modified by the procedure described in Section B.2.b. below to add the appropriate coding sequences, a start codon, and a conveniently placed upstream Hindlll site to give pRTB601. The cloning vector used to obtain the cDNA library contains a HindiII site immediaely downstream of the Sail site used for ligation into the vector, and thus the entire coding sequnce including the start codon can be excised by treatment of the modified vector with Hindlll.

B.2.b. Construction of the Ricin B Coding Sequence as a Hindlll- Cassette - pRTB601

Ten μg of pRTBlδl DNA was digested to completion with EcoRI, dissolved in 60 μl SI buffer and digested for four minutes at room temperature under conditions which remove about 1 base pair of duplex DNA per minute. DNA recovered from the foregoing buffer was dissolved in 60 μl exonuclease III buffer and digested for four minutes at room temperature. Subsequent analysis showed that the plasmid DNA had lost approximately 120 bp from each 3' end, leaving 5' ends available for hybridization. DNA recovered from the exonuclease III buffer was dissolved in 50 μl water and 20 μl used in the ligation/repair reaction below.

Thus, 20 μl sample (2 pmoles) was mixed with 20 p oles each of the synthetic ol gonucleotides:

Oligo 2 5'-GACCATGATAAGCTTATGGCTGATGTTTGTATGGATCC and

HindllI 3'TACCTAGGACTCGGGTATCACGCATAGCATCC-5'

Oligo 1 which have complementary sequnces as shown, and wherein 01igo-2 encodes a Hindlll site upstream of an ATG start codon. The 5' end of Oligo-l is complementary to 15 bases at the 5' end of the pRTBlδl cDNA sequence as there shown and is complementary to the contiguous missing codons of the ricin B sequence. The 5' end of 01igo-2 is complementary to the 5' sticky end of the vector residue of the exonuclease III treated pRTBlδl.

The mixture was heated to 60°C for five minutes in order to denature completely completion of single-stranded DNA, cooled to 37°C for five minutes to hybridized complementary strands, and then chilled on ice. The solution brought to polymerase I ( lenow) buffer conditions and reacted for two hours at 12°C in the presence of the δO μM each of the 4 dNTPs, 0.1 mM NAD, 0.3 units/μl Klenow, and 0.08 units/μl E. coli DNA ligase. The ligation mixture was used directly to transform competent E^ coli MM294 and several thousand Amp^ colonies found. Several hundred of these were replicated and grown on nitrocellulose filters and subjected to standard colony hybridization using P kinased 01igo-2 as probe. Two clones which hybridized with the probe were analyzed by restriction analysis and sequenced, and a correct construction designated pRTB601. pRTB601 thus contains the ricin B coding sequence as a Hindlll cassette. The upstream Hindlll site is introduced immediately upstream of the ATG codon in 01igo-2; the downstream Hindlll site arises from the pUC8 vector plasmid.

B.2.c. Identification of Galactose Binding Sites on Ricin B - Muteins of Ricin B

Ricin B has two functional characteristics, it first plays a role in binding to galactoside on the surface of cells and then participates in the internalization of ricin toxin A chain into the cell. The muteins of ricin B according to the invention have amino acid sequences that are specifically altered from those described sequences herein for ricin toxin B chain. The alterations are made in amino acids that comprise the galactoside binding sites of ricin B, and most preferably in amino acids that affect the binding of ricin B chain to galactosides, e.g., lactose. The muteins of ricin B of the present invention are altered in these amino acids to decrease the binding of ricin B to galactoside. B.2.d. Identification of Galactoside Binding Sites of Ricin B

B.2.d.l. General Description of the Structure of Ricin B

The three dimensional structure of the ricin B molecule has been determined to a resolution of 2.8 Angstroms A by Robertus et al., and a two dimensional representation of the ricin B chain structure is shown in Figure 1.

The representation shows two domains within the B chain of ricin and each domain has a galactoside binding region. Each domain of the ricin B chain has two disulfide loops, and each domain has a single galactoside binding site. The two domains have folding patterns that are similar and can be classified for purposes of the invention as an amino terminal domain encompassing amino acid residues 1-135 which includes the amino galactoside binding site, and a carboxyl terminal domain encompassing amino acids 136-267 which includes the carboxyl galactoside binding site.

The amino galactoside binding site is defined by two regions of the amino acid sequences, residues 22-28 (Asn22, Val23, Arg24, Asp25, Gly26, Arg27, and Phe28) and residues 35-46 (Gln3δ Leu36 Trp37 Pro38 Cys39 Lys40 Ser41 Asn42 Thr43, Asp44 AIa4δ and Asn46). Amino acids that can potentially interact with galactose are contained within the residues 22-28 and 3δ-46. The carboxyl galactoside binding site may be defined by three regions of amino acid sequences, residues 197-200 (Argl97, Glul98, Thrl99 and Val200) residues 233-239 (Leu233 Asp234 Val23δ Arg236 Ala238 Ser238 Asp239) and residues 244-256 (Gln244, Ile24δ, Ile246, Leu247, Tyr248, Pro249, Leu2δ0, His251, Gly252, Asp253, Pro2δ4, Asn255 and G!n256). Amino acids that can potentially interact with galactose are contained within the residues 233-239 and 244-256. Not all of the residues described above however are considered to bind or contact to lactose.

A computer analysis of the 2.8 Angstrom (A) crystal!ographic Structure of the ricin B chain was carried out using Mogli protein modeling program on an Evans and .Sutherland Graphics System both of which are commercially available from Evans and Sutherland, Salt Lake

City, Utah, U.S.A. Table 1, generated using this system, shows the distances in Angstroms of particular amino acid residues of the amino galactoside binding site of ricin B to some part of the lactose residue bound therein.

Table 1

From these data, residues • withi n 3 Angstroms of an atom in lactose are: From these data, residues within 3 Angstroms of an atom in lactose are:

Asp22 Arg24 Asn46 Gln35 Lys40

Further analysis was carried out to determine the part of the structures of the amino acids of the amino galactoside binding site and lactose bound within the site that fall within specified distances of one another. The results of the analysis, in Table 2, showed that the indicated parts of the following amino acid side chains were within the specified distance of some part of galactose.

Table 2

The same computer analysis was carried out on the carboxyl galactoside binding site. Table 3 shows the distances of particular amino acids residues of the carboxyl galactoside binding site of ricin B to some part of the lactose residue bound therein.

From these data, residues within 3 Angstroms of an atom in lactose are:

Ile246 Asn25δ His2δl

The same analysis used to generate the data in Table 2 showed that the indicated parts of the structure amino acid residues of the carboxyl galactoside binding site of Table 4 are within the specified distances of a part of galactose.

Table 4

B.2.e. Alterations in Ricin B Amino Acids Involved in Bonding to Galactoside

B.2.e.l. Alteration of Hydrogen Bonding Amino Acids

All of the amino acids in Tables 2 and 4 have proximities close enough to galactose to be involved in the binding of galactoside in the respective amino or the carboxy galactoside binding sites.

Asp22, Gln3δ, Lys40, and Asn46 all are within 3.2δ A of at least one atom of galactose. The nature of the side groups of the amino acids and galactose that are within 3.7δ A of one another suggest that they are hydrogen bonded. The approximate bond lengths of biologically important hydrogen bonds range from to 3.lO± 0.13 A and below. (See Molecular Biology of the Gene, Watson ed., W. A. Benjamin Inc., New York, 2nd Edition (1970). A hydrogen bond can be considered to be an intermediate stage of transfer of a proton from an acid to a base. The strength of a hydrogen bond increases with the acidity (ability to donate a proton) of the proton donor and with the basicity (ability to accept a proton) of the proton acceptor. Hydrogen bonds can arise between covalently bound hydrogen atoms having a positive charge and negatively charged covalently bound, acceptor atoms, e.g., C=0^", or between groups of atoms having a unit charge. By changing the charge of the side groups of the amino acids forming hydrogen bonds, or by chemical der vatization of the side group involved, or by substitution of the amino acid with a different amino acid that does not form a hydrogen bond, for example, one that has uncharged or oppositely charge side groups, or lastly, by deleting the amino acid involved in hydrogen bonding, the binding of galactoside by the galactose binding site is decreased.

Within the scope of ^'the invention are alterations in amino acids that form hydrogen bonds with the galactoside. Such alterations include amino acid derivatives, amino acid substitutions and deletions that result in a decrease in binding of galactoside to the galactoside binding site or sites. Amino acid residues to which such alterations may be carried out are those that form hydrogen bonds with the galactoside, and amino acids stabilizing amino acids that form 26 hydrogen bonds with the galactoside. With respect to the amino galactoside bonding site, residues Asp22, Arg24, Gln35, Lys40 and Asn46 of the ricin B chain are in positions that indicate potential formation of hydrogen bonds with galactoside. With respect to the carboxyl galactoside bonding site, residues His251 and Asn2δδ are in positions that indicate potential formation of hydrogen bonds with galactoside. Furthermore, Asp234 also is in a position that suggests hydrogen bond formation.

Amino acid residues that stabilize an amino acid which is in a position to form a hydrogen bond with galactoside are Asp22 of the amino galactoside binding site, and Asp234 of the carboxy galactoside binding site. Both of these residues may also participate in hydrogen bonding to the galactoside. Intervening water molecules between amino acid residues, such as Asn2δδ, may hydrogen bond to galactose. Muteins of ricin B according to the invention may be formed by deletion or substitution of at least one of the amino acids that form hydrogen bonds with galactoside or stabilize amino acids that form hydrogen bonds with galactoside. Such substituting amino acids that do not form hydrogen bonds will generally have either a side group that lacks charge, such as glycine, alanine, valine, isoleucine, leucine. Substituting amino acids with no side chain (glycine) or short side chain are generally preferred. Also preferred are amino acids having side chains that are oppositely charged from side chain of the amino acid for which it substitutes. Thus, when aspartic acid or glutamic acid are the residues in the native ricin B chain to be substituted, both of which have negatively charged carboxyl side groups, lysine and arginine, which have terminal amino side groups are preferred in the ricin B mutein. Conversely when lysine or arginine are the residues in the native ricin B chain to be replaced, aspartic or glutamic acid residues are used as replacements in the ricin B chain mutein. In general, it is preferred to replace hydrogen bonding or stabilizing amino acids with those having small uncharged side groups, such as glycine and alanine. Such amino acids are sufficiently small that binding of the galactoside through the weak interaction of Van der Waals forces is not expected. The following Tables 5 and 6 show the substitutions for particular residues in decending order of preference. The most preferred are at the top of the list and the least preferred substitutions are at the bottom of the list. Amino acids in the middle of the list are placed only in approximate relative preference. Each substitution may be made singly independent of substitution of any of the other replaced amino acids. Multiple amino acids in the native sequence may be replaced.

Table 5 Amino Galactoside Binding Site

As 22 Ar 24 As 2δ Gln3δ Tr 37 L s40 Asn46

Both the amino and carboxyl galactoside binding sites have one site that 1s formed by an aromatic amino acid residue, Trp37 and Tyr248 1n the amino and carboxyl galactoside binding sites, respectively. The positioning of the aromatic side chain of these two amino adds in the respective galactoside binding site 1s substantially parallel to the ring of the lactose moiety 1n the 2.8 A resolution crystal structure of ricin B. As set out in Tables 2 and 4, the aromatic side chain of both of these amino acids is about 5 A from the lactose residue and substantial portions of each amino acid are within 4 A of the lactose residue. The distances of the side chains from the lactose ring are such that strong nonspecific attractive forces or Van der Waals interactions are indicated. Van der Waals interactions may occur over distances such as those Indicated above between the aromatic ring of the amino adds and the ring structure of lactose.

In the muteins of rldn B, according to the invention, the aromatic amino adds are deleted or are substituted with amino acids that do not lead to stabilization of nonspecific attractive forces such as Van der Waals^' invention. In general, substitutions will be made with amino acids that do not have aromatic or heterocyclic side chains. Thus, substitution with tryptophan, phenylalanine, tyrosine and histidine are not desirable for either Trp37 or Tyr248. The substitutions are preferably made with amino acids that have small side chains. Most preferred are those that do not have significant charge separation and therefore do not have the potential for formation of stabilizing hydrogen bonds. Glycine and alanine are particularily preferred. Not desirable are large uncharged side chains such as those of leucine and isoleucine which, because of their extended uncharged structure, may have sufficient proximity to the lactose residue to stabilize the bonding thereto by Van der Waals interaction.

B.2.e.3. Double Alterations: Substitutions

Of course, combinations of substitutions at more than one residue of either or both galactoside binding sites are within the scope of the invention. To decrease binding to galactose by both the amino and carboxyl galactoside binding sites, it 1s preferable to alter at least one amino acid 1n each of the galactoside binding sites. Also within the scope of the Invention are deletions of more than one residue of either or both galactoside binding site. Furthermore, combinations of at least one substitution and at least one deletion^' at amino add residues of either or both galactoside binding site are within the scope of the invention. The following Tables 7 and 8 list the preferred double substitutions 1n the amino and carboxy galactoside binding sites. Of course one or both galactoside binding sites may be double substituted or on galatoslde binding site may be singly substituted while the other is doubly substituted.

Table 7

Table 8

The following Tables 9 and 10 indicate substitutions embodying modifications which increase the side chain size on one group and eliminate hydrogen bonding or Van der Waals interactions:

Table 9

Asp234 Leu Asn255 Gly B.2.e.4. Substitutions with Cystelne

A cystelne residue or residue may be inserted into one or both of the gal actoside binding sites of ricin B chain. The thi ol group of cystelne reacts quickly under mild conditions with Iodoacetate, Iodoacetaralde, N-ethylmalelmide and other reagents that are specific, or can be made specific, for thiol groups.

By repl acing a side chain of an amino acid that contacts galactose 1n the binding site with cystelne, a site is provided for easy manipulation. After ricin B chain is folded, 8 thiol groups have formed 4 disulfide bonds. The ninth thiol group, Cys4, remains free. This can be either left and chemically modified, or it can be removed by site-specifi c modifi cati on (changed to a serine or other residue). If the molecule being modified is ricin, then Cys4 would be left and it would be linked to the Interchain thiol group of ricin A chain to form a disulfide bond. In such a molecule the only thi ol that can react with sulfhydryl reagents would be the cysteines inserted into the galactose binding pockets. The cysteine at 171 of, ricin A chain has been shown to be unavailable for reaction as it is deeply situated 1n a hydrophobic_. region of the molecule. The substitution of a cysteine residue for an amino acid in either or both of the gal actoside binding sites may be sufficient to decrease or el iminate the binding of gal actosi de to ricin B. In additi on to the extent that the binding of galactoside is not decreased by the above-menti oned cysteine substituti on , the cysteine residue may be derivatized with thiol specific groups such as alkylating agents to yield a cysteine derivative that interferes with gal actoside binding.

The size of the thiol specific reagent may. be increased if lodoacet mide, iodoacetate or N-ethylmaleimide did not prevent galactose binding. For example, the carboxyl group of iodoacetate may be l inked in an amide bond to glycine. It could l ink to the ami no group of cysteine 1n which the thiol group was blocked by a disulfide, such as with 5-thio-2-nitrobenzoic acid (TNB). After reaction with the recombinant ricin B chai n, the thi ol coul d be exposed by gentl e reduction under conditions that did not reduce the disul fides on the protein. The thiol group could be modified with iodoacetate, lodoacet amide or N-ethylmaleimide. Various means for chemically derivatizing the cystelne residue placed In the gal actoside binding site are possible and are considered within the scope of the invention to the extent that the Hdn B mutein shows decreased binding to galactoside while retaining the ability to aid in translocation of the toxin molecule.

The following residues may be modified by substitution with cysteine:

Asp 22 and Asn 46 for the amino galactoside binding site and

Asp 234 and Asn 255 for the carboxyl gal actoside binding site are the preferred residues for substitution with cysteine.

The nucleic acid and ami no acid sequences in the amino terminal site are shown in Fi gure 2A. Residues 22 (Asp ) and 46 (Asn ) in the amino terminal site can be modified using the following ol igonucleotides for site specific modification:

B '-CGAAATGGTCTATGTGTπGCGTTAGGGATGGAAGATTCC-S' Asp 22 to Cys

^' CGAAATGGTCTATGTGTTGATGTTAGGGATGGAAGATTCC . ri cin B sequence ArgAsnGlyLeuCysVal AspVal ArgAspGlyArgPheHi s

22

S'-TGCAAGTCTAATACAGATGCAAATTGCCTCTGGCCATGCAAGTCT-S' Gin 46 to Cys TGCAAGTCTAATACAGATGCAAATCAGCTCTGGCCATGCAAGTCT ricin B sequence CysLysSerAsanThrAspAlaAsnGlnLeuTrpProCysLysSer

46 The nucleic acid and amino acid sequence of the carboxyl terminal site are shown in Figure 2B. Amino acids 234 (Asp) and 255 (Asn) in the carboxyl galactoside binding sites can be modified using the fol lowing ol igonucleotides:

5'-GTGGGTTGGTGπATGCGTGAGGGCATCGGATCC-3 ' Asp234 to Cys GTGGGTTGGTGTTAGATGTGAGGGCATCGGATCC ri cin B sequence

G L V L D V R A S D P 234

5 ' -CCTCTCCATGGTGACCCATGCCAAATATGGTTACC-3 * Asn255 to Cys CCTCTCCATGGTGACCCAAACCAAATATGGTTACC ri cin B sequence P L H G D P N Q I W L P

255.

B.2.e.5. Methods for Carrying out Alterations of the Amino and Carboxy Galactoside Binding Sites of RicinT

A number of known methods may be used to carry out the desired modifications to form the ricin B muteins according to the invention.

Ricin B protein havinq the desired amino acid replacement or deletion may be made by conventional Merrifield synthesis as is known in the art. However, Merrifield synthesis of a complete ricin B molecule is undesirably complicated. .

Substitutions and deletions may be accomplished by digesting to completion DNA encoding the native ricin B protein with specified endonucleases that cut in the region of the DMA surrounding the amino acid to be altered, removing the DNA fragment which encodes the amino acid residue or residues of the native ricin B galactoside binding site to be altered, and Ugating, either under blunt ended or sticky ended conditions as appropriate, a double stranded DNA made of complementary chemically synthesized oligonucleotides that encode the desired amino acid alteration. The means for making such oligonucleotides are known and Include commercially available automated DNA synthesizers such as that made by Biosearch, San Rafael, California.

Site-specific mutagenesis may also be used to carry out alterations to the DNA encoding specific amino acid. In general, the DNA encoding ricin B chain in the region to-be altered is cut using an appropriate endonuclease, the fragment carrying the specific ridn region is removed, ligated into an appropriate vector such as an M13 vector and is mutagenized using a single-stranded oligodeoxyribo¬ nucleotide primer synthesized to insert, change, or delete nucleotides from the fragment after replication with an appropriate DNA polymerase.

To obtain the DNA fragment encoding the desired ricin B region, endonuclease restriction sites that are found in the native ricin B chain sequence may be used, or unique restriction sites on either side of the areas of interest are made in the DNA sequence of ricin B.

In a preferred embodiment, a new site for cleavage by Sail in the area of the amino galactoside binding site is made using site- specific mutagenesis to modify the sequence at Val21 and Asp22. Another site is created for cleavage by PvuII using the same technique to modify the base sequences around Gln47 and Leu48. Both modifications may be made without changing the amino acid sequence of ri in B. Figure 2A illustrates the position of the Sail and PvuII sites that can be created and the oligonucleotide sequences that can be used to mutagenize the sequence of ricin B and retain the amino acid sequence.

Also in a preferred embodiment, a new site for cleavage by Xbal in the area of the carboxyl galactoside binding site is constructed by site-specific mutagenesis by modifying the sequence at Val232, Leu233 and Asp234. Another unique site 1s created for cleavage downstream of the carboxyl terminal galactoside binding site of the ricin B chain by SacII. Figure 2B illustrates the position of the Xbal and SacII sites, and the oligonucleotide sequences that are used to mutagenize the sequence of ricin B and retain the amino acid sequence.

The unique restriction sites introduced into the ricin B sequence are produced by site-specific mutagenesis using conventional means. The above mentioned restriction sites are preferred because they do not alter the amino acid sequence -of ricin B. Other unique restriction sites may be Introduced as long as the amino add sequence of ricin B is not changed, or 1f changed, the new sequence does not affect the essential biological properties of ricin B that relate to trans!ocation. As mentioned above, various methods may be used to Introduce changes 1n the DNA sequence encoding amino acids of the amino terminal and carboxyl terminal galactoside binding sites. Double stranded oligodeoxyrlbonucleotides having "sticky ends" compatable with the unique restriction site engineered Into the ricin B sequence by site- specific mutagenesis may be used. Such oligonucleotides may be made by conventional comrnercially available oligonucleotide synthesizers.

Table 11 shows the double-stranded oligodeoxyribonucleotide spanning the unique Sail to PvuII site engineered into the amino terminal region of the ricin B chain that encompasses the amino galactoside binding site. Table 12 shows the double-stranded oligodeoxyribonucleotide spanning the unique Xbal to SacII sites in the carboxyl region of ricin B chain that encompasses the carboxyl galactoside binding site. Each table shows the nucleotide changes required for the substitution of various amino acids. As mentioned above, the nucleotide change may be made to single or multiple amino acids in this region of the ricin B molecule. In addition, any of the changes may be made independently of all other changes.

Table 11

Amino Add # 22 35 37 40 46

22 35 37 40 46 TCGACGTTAGGGATGGAAGATTCCACAACGGAAACGCAATACAGTTGTGGCCATGCAAGTCTAATACAGATGCAAATCAG Asp Gin Trp Lys Asn ACAATCCCTACCTTCTAAGGTGTTGCCTTTGCGTTATGTCAACACCGGTACGTTCAGATTATGTCTACGTTTAGTC Gly Gly Gly Gly

. Ala Ala Ala Ala

Asp Ser Leu Asp

Glu Thr He Gin

Asn Val Asp Gin

Leu

Amino Add #

234 248 251 255

234248251255 CTAGATGTGAGGGCATCGGATCCGAGCCTTAAACAAATCATTCTTTACCCTCTCCATGGTGACCCAAACCAAATATGGTTACCATTATTTTGATAGACC Asp Tyr His Asn TACACTCCCGTAGCCTAGGCTCGGAATTTGTTTAGTAAGAAATGGGAGAGGTACCACTGGGTTTGGTTTATACCAATGGTAATAAAACTATCTGG Gly Gly Gly Gly GGT

CA Ala Ala Ala Ala GCG

GC Gly Ser Asp Glu GAA

TT

Glu Gin Lys Asp

TTT CTA

Deletions of amino acids may be made using essentially the same method, however, instead of changing the nucleotide sequence to encode a substituted amino add, the complete trinucleotlde codon encoding the amino acid to be deleted 1s removed. Such deletions are particularly desirable 1f they do not change the comformation of the protein, though not necessarily preferred for Trp37 and Tyr248.

Modifications of the DNA sequence encodinq Asp22 may be made using the double-stranded break and repair method of Mandeckl, Proc. Natl. Acad. Sci. USA 7_: 177-7181 (1986). Briefly, Sail cleaves the sequence 5'GTCGAC-3' at a position immediately 3* of the 5' G of this sequence, and the GAC portion codes for Asp22 in ricin B chain. Briefly, the plas id comprising the ricin B sequence is cleaved at the Sail site, inserted as described above, to convert the circular structure to a linear one. An oligonucleotide containing sequences at either end that are identical to the ricin B chain DNA sequence with the site at residue 22 mutated to the desired amino acid is mixed with the linear plasmid. After heating and annealing, the DNA is used to transform cells rendered competent by calcium chloride treatment and incubation on ice followed by incubation at 37C for a short time. Transformed cells containing the

32 desired mutation are then detected by using P-labelled oligonucleotide probes.

The sequence flanking and including the Sail site is as follows (the gap is to illustrate the Sail site): Sail cleavage site

5' Aspέέ

CCCATAGTGCGTATCGTAGGTCGAAATGGTCTATGTG TCGACGTTAGGGATGGAA

GGGTATCACGCATAGCATCCAGCTTTACCAGATACACAGCT GCAATCCCTACCTT 3'

3' GATTCCACAACGGAAACGCAATACAGTTGTGGCCATGCAAGTCTAATACAGATGCAAATCAG CTAAGGTGTTGCCTTTGCGTTATGTCAACACCGGTACGTTCAGATTATGTCTACGTTTAGTC

5'

The oligonucleotides to be used to repair the strand break and insert new amino acids substituting for Asp22 are shown in Table 13. Modifications to the DNA sequence of the carboxyl galactoside binding site encoding Asp234 are made 1n essentially the same manner as described for the modifications of Asn22.

The sequence flanking and Including the Xbal site is as follows (the gap 1s to Illustrate the Xbal site):

5' Xbal cleavage site

GGAACCATTTTAAATTTGTATAGTGGGTTGGTT ΕTXGATGTGAGGGCATCGGATCCG . CCTTGGTAAAATTTAAACATATCACCCAACCAAGATC TACACTCCGGTAGCCTAGGC 3'

3' AGCCTTAAACAAATCATTCTTTACCCTCTCCATGGTGACCCAAACCAAATATG TCGGAATTTGTTTAGTAAGAAATGGGAGAGGTACCACTGGGTTTGGTTTATAC

5'

Gly, Ala and Glu codons are accomodated with retention of the Xbal restriction sequence as shown above 1n connection with Table

13. The repair oligonucleotides to introduce codons for amino acids that change the Xbal restriction sequence for Gin and Asn are shown in

Table 14.

Table 13

Amino Acid #

22 22 Asp GCGTATCGTAGGTCGAAATGGTCTATGTGTCGACGTTAGGGATGGAAGATTCGACAACAACGG

Gly GGT

Ala GCG

Asn AAC

Glu GAA Gin CAG

Table 14

Amino Acid #

234 234

Asp CCAHTTAAATTTGTATAGTGGGTTGGTTCTAGATGTGAGGGCATCGGATCCGAGCC Gin CAG

Asn AAC In general, cloning and expression vectors used In conjunction with the rldn B sequence Into which the unique restriction sites have been introduced, will lack the unique restriction sites. If necessary, restriction sites In the vector that 5 are the same as the unique restriction sites may be removed by site- specific mutagenesis as 1s known in the art.

B.3. Cloning of idn B isotoxins D. and E and muteins thereof

As mentioned above, there are several known isotoxins of ridn B and these include ricins D, and E. Furthermore, ricin E has

10 recently been shown to consist of at least two molecular species, El and E2. It will be appreciated by those skilled in the art that cloning of these molecules is achievable using the materials and methods described in the preceding sections that permit cloning of ridn B. It will also be appreciated by those skilled in the art that

15 muteins of the isotoxins having reduced galactose binding are realizable, using the materials and methods shown for constructing muteins of ricin B, because the amino acid sequences of the isotoxins are, to a large degree, similar to ricin B. This is particularly true of the amino acids that compose the galactose binding regions.

20 Butterworth, R. and Lord, M. Dur. J. Biochem. 137, 57 (1983), Hailing et al., Nucleic Acid Res., U, 8019 (1985) O'Hare, et a!., FEB. 216, 73 (1987), and Araki , T. and Funatsu, G., Bioochemica et Biophysica Acta , 191 (1987).

Thus, the isotoxins D and E and muteins thereof are capable 25 of being cloned and expressed by the Instant Invention.

B.4. Cloning of FullrLenqth Ridn and RCA Encoding Clones

The full-length sequences encoding ricin D, putative ricin E, and RCA in precursor form were obtained, using the messenger RNA prepared as described above for ricin A, to obtain a cDNA library, and 30_.then probing the library to retrieve the desired cDNA inserts. The library was prepared using the method of Okayama and Berg (Mol . and Cell Biol. (1983) 3ι 230-289) and was probed using the same 35-mer used for ricin A-encoding sequences. Out of several thousand transformants with cloning vector, a number of positively hybridizing clones were obtained.

• Positively hybridizing colonies were subjected to restriction analysis and showed restriction patterns corresponding to ridn D and to RCA, and a third type which corresponded to neither. The cDNA inserts from representative clones of each of the three types were sequenced. The results of the sequence Information for ricin D are shown 1n Figure 3. The sequence for ricin D 1s used in the construction of the transfer vectors described below. However, related sequences such as those cross-hybridizing to the probe may also be used in the transfer vectors disclosed. As stated for ridn A above, the procedures set forth herein to isolate the sequences need not be repeated, as synthetic methods are available so that the DNA sequences shown in the figures can be constructed using chemical and enzymatic means in vitro.

In addition, the DNA sequence coding for a plant toxin of the ricin type has been published. (Lamb, F.I. et al., supra; Lord, J.M. et al., supra). The inserts described above can be placed into expression vectors in a manner analogous to that described for ricin A. For the straightforward expression of the coding sequences contained in the isolated inserts, the inserts are subcloned into M13 vectors for site- directed mutagenesis to place an ATG start codon and a Hindlll site at the beginning of the mature protein, in a manner analogous to that set forth for ricin A above, or to place a Hindlll site immediately prior t^'o the ATG of the secretory leader sequence where appropriate. The mutagenized DNAs can be retrieved from the M13 vectors by cleaving with PstI. blunt-ending with Klenow, digestion with Hindlll at the newly created site, and isolation of the appropriate length sequence.

To facilitate conversion of the precursors to either RCA or the ricin toxins, modifications may be made, in particular in the linker portion, to provide suitable means for detaching the A and B portions. A variety of strategies are possible. Two convenient ones are: 1) construction of a trypsin cleavage site by creating an "arg- argⁿ form of the linker wherein the prollne following the arginine residue already present 1s replaced by another arginine; and 2) Insertion of a stop and a start codon in the linker region so that the A and B regions are separately but simultaneously produced.

C. Methods Employed

C.l. Transformations and Transfectlons

Transformation of t±. coli cells was done according to procedures set forth in T. Maniatis, E.F. Fritsch and J. Saπbrook Molecular Cloning: A Laboratory Manual (1982) Cold Spring Harbor Press.

Transfections of Sf9 Spodoptera frugiperda cells are accomplished using a modification of the calcium phosphate precipitation technique (Graham, F.L. et al., 1973, Virology 52:456) as modified for insect cells (Burand, J.P. et al. (1980) Virol ., pl_:286; Carstens, E.B. et al . (1980) Virol ., _101_:311) and further described by Summers, M.D, and Smith, G.E. (A Manual of Methods for Baculovirus Vectors and Insect Cell Culture Procedures, Texas ASM Press, 1987).

C.2. Vector Construction

Construction of suitable vectors containing the desired coding and control sequences employs standard Hgation and restriction techniques which are well understood in the art and are described in Maniatis, T. et al., supra. Isolated plasmids, DNA sequences, or synthesized oligonucleotides are cleaved, tailored, and religated in the form desired.

Site specific DNA cleavage is performed by treating with the suitable restriction enzyme (or enzymes) under conditions which are generally understood in the art, and the particulars of which are specified by the manufacturer of these commercially available restriction enzymes. See, e.g., New England Biolabs, Product Catalog. In general, about 1 yg of plasmid or DNA sequence is cleaved by one unit of enzyme in about 20 ul of buffer solution; in the examples herein, typically, an excess of restriction enzyme is used to Insure complete digestion of the DNA substrate. Incubation times of about one hour to two hours at about 37^βC are workable, although variations can be tolerated. After each Incubation, protein is removed by extraction with phenol/chloroform, and may be followed by ether extraction, and the nucleic add recovered from aqueous fractions by precipitation with ethanol. If desired, size separation of the cleaved fragments may be performed by polyacrylamide gel or agarose gel electrophoresis using standard techniques. A general description of size separations is found in Methods in Enzymology (1980) _,65:499-560.

Restriction cleaved fragments may be blunt ended by treating with the large fragment of E. coli^" DNA polymerase I (Klenow) in the presence of the four deoxynucleotide triphosphates (dNTPs) using incubation times of about 15 to 25 minutes at 20 to 25^βC in 50 mM Tris pH 7.6, 5 mM MgCl₂, 10 mM dithiothrei ol (DTT) and about 50 y of each dNTP. The Klenow fragment fills in at 5¹ sticky ends but chews back protruding 3¹ single strands, even though the four dNTPs are present. If desired, selective repair can be performed by supplying only one of the, or selected, dNTPs within the limitations dictated by the nature of the sticky ends. After treatment with Klenow, the mixture is extracted with pehnol/chloroform and ethanol preci itated. Treatment under appropriate conditions with SI nuclease results 1n hydrolysis of any single-stranded portion.

Synthetic oligonucleotides may be prepared by the triester method of Matteucci, et al., J. Am. Chem. Soc. (1981) 103:3185-3191 or . using automated synthesis methods. Kinasing of single strands prior to annealing or for labeling is achieved using an excess, e.g., approximately 10 units of polynucleotide kinase to 1 nM substrate in the presence of 50 M Tris, pH 7.6, 5 mM MgCl₂, 10 mM DTT, 1-2 irM ATP. If kinasing is for labeling or probe, the ATP will contain high specific activity 32γP. Ligations are performed in 15-30 yl volumes under the following standard conditions and temperature: 50 mM Tris-Cl pH 7.5,

5 mM MgCl₂, 10 mM DTT, 100 yq/ml BSA, 1 mM ATP, 0.3-0.6 (Weiss) units

T4 DNA ligase at 14^βC Ligations are usually performed at 33-100 ug/ml total DNA concentrations (5-100 nM total end concentration).

In vector construction employing "vector fragments", the vector fragment 1s commonly treated with bacterial alkaline phosphatase (BAP) in order to remove the 5* phosphate and prevent religation of the vector. BAP digestions are conducted at pH 8.5 1n approximately 50 mM Tris, 5mM MgCl₂, using 0.1-1 unit of BAP per yg of vector at 37-55°C for about one hour. Alternatively, religation can be prevented in vectors which have been double digested by additional restriction enzyme digestion of the unwanted fragments.

C.3. Modification of DNA Sequences For portions of vectors derived from cDNA or genoπic DNA which require sequence modifications, site specific primer directed mutagenesis 1s used. This technique is now standard in the art, and 1s conducted using a synthetic oligonucleotide primer complementary to a single stranded phage DNA to be mutagenized except for limited mismatching, representing the desired mutation. Briefly, the synthetic oligonucleotide is used as a primer to direct synthesis of a strand complementary to the phage, and the resulting double-stranded DNA is transformed into a phaqe-supporting host bacterium. Cultures of the transformed bacteria are plated in top agar, permitting plaque formation from single cells which harbor the phage.

Theoretically, 50% of the new plaques will contain the phage having, as a single strand, the mutated form; 50% will have the original sequence. The plaques are hybridized with kinased synthetic primer at a temperature which permits hybridization of an exact match, but at which the mismatches with the orignal strand are sufficient to prevent hybridization. Plaques which hybridize with the probe are then picked, cultured, and the DNA recovered. Details of site specific mutation procedures are described below in specific examples. C.4. Verification of Construction

In the constructions set forth below, correct ligations for plasmid construction are confirmed by first transforming E__j_ coli strain W294, or other suitable host with the 11gation mixture. Miniprep DNA was prepared according to Ish-Horowicz, D. et al. (Nucl. Adds Res. (1981) _,9:2989) and screened by restriction analysis. DNA may be further analyzed by restriction and/or sequenced by the dideoxy method of Sanger, F., et al., Proc. Natl. Acad. Sci. (USA) (1977) 74:5463 as further described by Messing, et al., Nucleic Acids Res. (1981) _,9:309, or by the method of Maxam, et al., Methods in Enzymology

C.5. Transfer Vector Construction

Because the genome of AcNPV is so large (125kb), there are too many restriction sites to allow site-specific Insertion of heterologous genes. Therefore, it is necessary to derive recombinant virus, containing the gene to be expressed, through homologous recombination between viral DNA and genetically engineered chiπeric plasmids called transfer vectors.

The transfer vectors which have been described by Smith, G.E., et al. ((1983), supra) were originally constructed by cloning the AcNPV EcoRI-1 fragment containing the polyhedrin gene into the

EcoRI site of E. coli plasmid pUC8 (Vieira, J., et al., Gene 19:259-

268 (1982)). A series of plasmids or transfer vectors having single

BamHI cloning sites at various locations in the polyhedrin gene were created as^' described (Smith et al., 1983, supra). One of these, pAc373, has a single BamHI site 50 bp downstream from the polyhedrin cap site I.e., 8 bp before the polyhedrin ATG translation initiation codon (Smith et al., (1985) supra). The transfer vectors, pAc610 and pAcδll have the polyϋnker from M13mpl0 and M13τnpll, respectively, Inserted at this BamHI site of pAc373 (Summers, M.D. et al., personal communication). Partial nucleotide sequence of pAc401 and pAc436 transfer vectors for the production of polyhedrin/foreign gene fusion proteins is also reported (Summers, M.D. et al., 1987, p. 53, supra). C.6. Preparation and Isolation of Recombinant AcNPV Virus

Detailed methods for the generation of recombinant virus can be found in European Patent Application No. 0127839 to G.E. Smith and M.D. Summers of the Texas A S M University System published December 12, 1984. In general, 2 μg of genetically engineered transfer vector DNA and 1 yg of AcNPV viral DNA are cotransfected onto onolayer culture cells of Spodoptera frugiperda. The Infected cells usually show viral occlusions by day 3 or 4, with 10-90% of the cells being Infected. The virus tlter of the medium is expected to be 10⁷ pfu/ml and 0.1%-0.5% are expected to be recombinant virus.

Several methods for the detection of recombinant virus are known in the art. Visual detection of the plaques is best achieved using a low power dissecting microscope and observing the plaques on inverted plates with a black background and illumination from the side. More unequivocal methods for detecting recombinants are plaque hybridization using DNA probes to the cloned gene. Antibody probes to he product of the cloned gene may also be employed.

Isolation of the recombinant virus is achieved through plaque purification of serially infected monolayer cells overlayed with soft agar. After two or three cycles the recombinant virus would be seen as separate plaques showing the characteristic occlusion- negative morphology. The plaques, containing about 10,000 pfu of virus, are picked using a sterile Pasture pipet and transferred to 2 ml of medium.

C.7. Insect Cell Culture

Methods for Insect cell cultures are well known in the art and detailed procedures for their cultivation can be found in Summers, M.D. et al. (1987, supra) or in EPO 127,839 to G.E. Smith et al . The insect expression host of the current invention, Spodoptera frugiperda (Sf9) is well suited to the production of heterologous proteins because of its ability to grow in either monolayer or suspension culture. As monolayer cultures, Spodoptera frugiperda cells will divide every 18-24 hours depending on the culture media. The cells do not require carbon dioxide to maintain the pH of the medium and they will grow well at temperatures between 25-30°C. Subculturing is done 2 or 3 times a week when the cells are confluent. Because insect cells are loosely adherent they are easily resuspended without the need of proteases.

Suspension culture conditions will vary depending on the medium and culture volume and should be determined empirically. Subculturing is required when the cell density reaches 2x10° cells/ml by replacing 80% or more of the culture with an equal volume of fresh medium. With suspension cultures larger than 500 ml it becomes necessary to aerate by either bubbling or diffusion.

Preferred media and culture conditions can be found in Summers, M.D. et al., A Manual of Methods for Baculovirus Vectors and Insect Cell Culture Procedures, Texas Agricultural Experiment Station Bulletin No. 1555.

C.8. Electrophoretic Analysis of Expression Products

In order to concentrate expression products, culture supernatants were incubated with Protein A-Sepharose CL-4B beads cross-linked to antibody to either ricin A (αRTA) or ricin B (αRTB) for 1 hour. The beads were then pelleted and the supernatants reserved. The beads were washed once in 0.5 ml buffer containing 1M

LiCl, 20 mM Tris-HCl pH 8.0 and 0.5% NP40, three times in O.δ ml buffer containing 150 mM NaCl , 20 mM Tris-HCl pH8 and 0.5% NP40 and then suspended in sample loading buffer lacking reducing agent. The samples were then heated to 37°C for δ minutes, pelleted and the supernatant removed to new tubes These supernatants were adjusted to contain 0.14 M beta-mercaptoethanol or 20 mM DTT, heated to 100°C for 4 min. and run on SDS-PAGE.

SDS-PAGE was performed essentially according to the procedure of Laemmli (Nature (1970) 227:680-686). Immunoblotting procedures have been described in European Publication No. 219,286 published April 22, 1987, essentially as described below. I munoblotting of the gel onto nitrocellulose (Schleicher and Schuell, 0.45 y eter) was performed in a Bio-rad Trans-blot cell at 35 V for one hour at room temperature essentially according to published methods (Towbin et al. (1979) Proc. Natl. Acad. of Sci.,

5. _USA_, _76_:4350-4364; Bittner et al. (1980) Anal. Biochem., 102:459-0471; Burnette et al. (1981) Anal. Biochem., 112_,:196-203). Following transfer, nonspecific antibody binding sites on the nitrocellulose were blocked by incubation for 30 minutes at room temperature with gentle agitation in 250 ml of 0.1% Tween 20 in phosphate buffered

10 saline (PBS). Then the blot was washed three times with gentle agitation at room temperature for five minutes each in 250 ml volumes of 0.1% nonfat dry milk, 0.1% ovalbumin in PBS, and incubated with gentle agitation for three hours at room temperature in 5 ml of a 1/500 to 1/1000 dilution in the preceding buffer of rabbit antiserum

15 against ricin A subunit or ricin B subunit (supplied by BABCO). After washing three times as described above, the blot was incubated for one hour at room temperature with 5 ml of a 1/2000 dilution of goat anti- rabbit IgG conjugated to horseradish peroxidase (this conjugate supplied by Zymed) and washed again three times as above.

20 In order to visualize the immobilized horseradish peroxidase i munoconjugate, the immunoconjugate-treated blot was soaked at room temperature without agitation for five minutes in 50 ml of 10 M Na citrate, 10 mM EDTA, pH δ and then for 16 minutes in 60 ml of freshly prepared 10 mM Na citrate, 10 mM EDTA and 1% dextran sulfate. In a

25 glass container the blot was next agitated for 16 min in 60 ml citrate buffer (above) containing a 0.1 mg/ml tetramethylbenzidine (TMB added from a 2 mg/ml stock solution in ethanol; Miles Laboratories) then H₂0₂ was added to 0.0016%.^' When the pattern had reached the desired degree of contrast between specifically stained bands and background,

30 the blot was soaked for 30-60 minutes in 50 ml of ice cold water and the blot was photographed. E.9. Cytotoxicity Assay

Culture supernatants from Sf-9 cells were dil uted 1: 10 and 1:100 1n culture medium and applied to RAT-2 cells (a flbroblast line transformed by Harvey murine sarcoma virus DNA; Clark, R. et al . (1985) Proc Natl . Acad. Sd ., _,82:5280). One series of 1:10 diluted samples were adjusted first to contain 50 mM lactose which is known to specifically block binding of native ricin toxin to the cell surface (Olsnes, S. et al . (1982) supra). These samples become the negative control . For positive controls, various concentrations of ridn toxin are prepared in Sf-9 cel l culture medium. After 4 hours at 37^βC, the samples were repl aced with fresh medium and incubation was continued for 20 hours. The cel l onol ayers were then stained with crystal violet and visually analyzed for viability.

E.10. Assays for Binding Ricin B Chain Muteins The abil ity of ricin B chain muteins according to the invention to bind to gal actose is determined by^' measuring the binding of ricin B chain to gal actose-containing resins or surfaces coated with gal ctose-containing molecules or membranes which contain gal actose residues . Agarose-containing resins such as Sepharose, treated with dil ute acid to expose gal actose residues, Bio-Gel A resins , resins such as Sepharose or Bi o-Gel A coupl ed to saccharides such as fetuin, are suitable for binding ricin with an intact B chain or ri cin B chain alone. Ricin or ricin B chain in whi ch the galactoside binding site or sites thereof has not been modified according to the invention when passed through these columns binds to the column material . If the galactoside-binding site has been altered to produce a ridn B mutein according to the invention, binding to galactoside will be measurably diminished or el iminated.

The abil ity of the ricin B mutein to bind galactose is further determined by equil ibrium dialysis using 3H-l abeled gal actose. Fluorescent polarization techniques using methyl u bell if ery! galactose may be used to measure the associati on of the gal actose derivati ve with ri cin B chain. When ricin B muteins according to the Invention are produced Independently of ricin A chain, the abil ity of the ricin B mutein to interact with ricin A chain is determined by adding the ridn B mutein to a concentration of ricin A chain, deglycosylated ridn A chain, or recombinant ridn A chain that does not by Itself Inhibit protein synthesis in cells such as MCF-7 or HSB-2 cells. In order for the added ridn B chain to convert ricin A chain into a toxin, the galactose binding sites on ricin B chain must be capable of binding gal actose-containing receptors on the surface of the target cel l and the two chain must Interact, either covalently through a disulfide bond or non-covalently. The ability of ricin B chain muteins according to the invention encoded by DNA containing alterations in the gal actose binding regions as outl ined above, to convert ricin A chain into a toxin is substantially decreased or absent. The abil ity of ricin B chai n muteins according to the invention to interact with ricin A chain is measured by a competition assay. Native ricin B chain, having intact gal actose bindinq sites , interacts with ricin A chain and when the ricin A-ricin B complex is added to cel l s , protein synthesi s is prevented. Ri cin B chain muteins accordi ng to the Invention made from DNA in whi ch the gal actose binding residues have been modified as outl ined above is added to ricin A chain before or at the same time as native ricin B chain. The ricin B chain muteins according to the invention reduce the amount of measurable protein synthesis inhibition because the ri cin B mutein displaces native ricin . B chain in the complex. The extent of reduction 1n protein synthesis 1s proportional to the concentration of the competing mutant ricin B chain.

If the cystelne at position 4 in ridn B chain 1s capable of reacting with the cysteine In ricin A chain that forms the di sul fide bridge between the two chains, gel electrophoresi s in non-reducing polyacryl amide gels shows the presence of a 65,000 molecular weight species. Ricin A chain has a molecular weight of about 30,000 and ricin B chain has a molecul ar weight of about 33,000 (both nati ve species ) . Since the nati ve species are qlycosyl ated , thei r apparent size will be larger than the recombinant molecules, if they are made in non-glycosyl ating prokaryotic cel ls. Also within the scope of the invention are conjugates of ricin in which the B chain thereof 1s a mutein which has reduced binding to galactosides. Ricin is covalently bound to a binding moiety that can bind to a selected target cell or tissue and which can be Internalized by such target cell or tissue. Such binding moieties may be selected from a vast number of substance that bind to specific cells or tissues and include lymphokines such as Inter!eukin-1, 2 and 3 and Interferon α* β and γ; cytoklnes such as tumor necrosis factor and colony stimulating factors such as, CSF-1, G-CSF and GM-CSF; hormones that bind to specific hormone receptors associated with specific tissues such as the reproductive hormones that bind to ovarian tissue, e.g., leutinizing hormone; cell growth factors such as transferrin and epidermal growth factor and antibodies that bind specifically to a desired target cell or which bind to an epitope that is expressed at high level on a target cell as compared to other cell or normal cells. Such antibodies may be polyclonal or monoclonal antibodies.

EXAMPLE I A. Construction of New Baculovirus Transfer Vectors Λ.l. Construction of pAcCl pAcCl is similar to pAc401 (described previously in Section C.5.) except that the recognition site for EcoRI endonuclease has been removed. To accomplish this, pAc401 was digested to completion with EcoRI and the ends were made blunt using Klenow fragment. After 11gation and transformation, candidates were screened for the absence of an EcoRI site.

A.2. Construction of pAcC2 pAcC2 is similar to pAc436 (described previously in Section C.5.) except that the recognition site for EcoRI endonuclease has been removed. To accomplish this, pAc436 was digested to completion with EcoRI and the ends were made blunt using Klenow fragment. After ligation and transformation, candidates were screened for the absence of an EcoRI site. A.3. Construction of pAcC3 pAcC3 differs from pAcC2 in that an Ncol restriction site has been introduced at the ATG trans! ational start of the polyhedrin gene. To accomplish this the new transfer vector, pAcC2, was digested to completion with Smal endonuclease. Following phenol extracton and ethanol precipitation, S al digested pAcC2 was dissolved 1n TE buffer (10 mM Tris-HCl pH 7.4; 1 M EDTA). In a 50 μl volume of ExoIII buffer (50 mM Tris-HCl pH 8.0; 5 mM MgCl₂; 10 mM β- mercaptoethanol ), 10 μg of Smal digested pAcC2 was treated with 50 units of E. coll Exonuclease III (ExoIII) at 30^βC for 5 minutes. The sample was phenol extracted and ethanol precipitated twice. Then 50 pmoles of a primer EK85, 5'-AACCTATAAACCATGGCGGCCCGG-3', was kinased with cold ATP 1n a 20 μl reaction volume (50 mt1 Tris-HCl pH 7.8; 10 M MgCl ; 10 mM β-mercaptoethanol ) . To 5 yg of ExoIII treated pAcC2 was added 10 pmoles of kinased EK85 in a final volume of 20 ml NET (100 mM NaCl; lO-rrM Tris-HCl pH 7.5; 1 mM EDTA) buffer. To anneal the plasmid and primer, the reaction was heated to 65^βC for 10 minutes, incubated at 37°C for 10 minutes, and placed on ice. The extension reaction was performed by adding 20 μl 2 x Klenow buffer (40 mM Tris-HC! pH 7.5; 20 M MgC12; 2 M β-mercaptoethanol) containing 1 μl 10 M dNTPs, 1 ul 10 mMATP, 1 μl (about 2 units) Klenow fragment and 1 μl (about 1-2 units) T4 DNA ligase. The reaction was incubated at 16^eC for about 4 hours and then transformed into MM294. Minilysates were screened by analyzing for the presence of an Ncol site. Miniprep DNA was then used to retransform and obtain the desired pure clone.

A.4 Construction of pAcC4 and pAcC5 pAcC4 and pAcCδ are derivatives of pAcC3 containing a polyl inker sequence at the Smal site. The polyl inker contains recognition sites for restriction endonucleases Smal, Kpnl, PstI, Ball I, Xbal (cleavable when DNA is unmethylated), EcoRI, BamHI and Bell. pAcC4 contains the sequence in one orientation while pAcCδ contains the polylinker in the opposite orientation (see Figure 4). To construct these vectors pAcC3 was digested to completion with Xmal endonuclease and Ugated with two complementary self-annealed oligomers having the sequence: 5'-CCGGGTACCTGCAGATCTAGAATTCGGATCCTGATCA-S' 3*- CATGGACGTCTAGATCTTAAGCCTAGGACTAGTGGCC-5'

After transformation of MM294, miniprep DNAs of transformants were analyzed for the presence of restriction sites in the polylinker sequence.

EXAMPLE II

Sensitivity of insect cells to ricin toxin

The insect host cell line, Sf-9, and ribosomes isolated from this cell line were tested for resistance to ricin toxin and ricin A chain, respectively.

The Sf-9 host cells were found to be unaffected by a 4 hour exposure to ricin D at concentrations of up to 10 μg/ml. After washing, the Sf-9 cells were incubated an additional 20 hours and remained unaffected. A derivative of the mouse 3T3 cell line called psi-2 (Mann, R. et al. (1983) Cell, _33_:153), however, was almost completely inhibited/killed by a 4 hour exposure to ricin D at a concentration of 1 ng/ l. The ID50 (concentration at which 50% of the cells are inhibited/killed) for ricin on a sensitive cell line is typically 1-10 ng/ml. The observed resistance of insect cells to ricin is apparently not due to the lack of toxin binding to the cell surface. In a sandwich-type binding assay using anti-ricin A sera for detection, a strong signal for ridn binding to the insect cells was observed. The cell surface moiety to which ricin A is apparently bound has not been characterized.

Ribosomes from Sf-9 cells appear to be resistant to ricin A chain. Sf-9 ribosomes were prepared according to Palmiter, R.D. (Biochem (1974) 13_:3606) and tested in an in vitro translation assay (Cawley, D.B. et al. Biochem (1979) 12:2648) for sensitivity to added ricin A chain. At a concentration of recombinant ricin A of about 10 yg/ml, translation activity was depressed about 50%. Typically, 1-10 ng/ml of recombinant ricin A inhibits the rabbit reticulocyte translation system by 50%. EXAMPLE III

Expression of Recombinant Ricin Toxin and Its Subunits

A. Construction of Reco binant Baculovirus Transfer Vectors Con¬ taining Full Length Ricin Toxin Sequences The ricin sequence was obtained as a HindUI-Hindlll fragment from a vector in which a Hindlll site had been created at the ATG trans!atlonal start at the beginning of the secretory signal peptide sequence as previously described. The Hindlll site after the ricin gene can be derived from M13 or other suitable vectors carrying polylinker sequences. The Hindlll fragment containing the ricin gene was made blunt ended with Klenow fragment and ligated to Smal digested pAcC3. Recombinants containing the ricin sequence in the correct orientation for expression under polyhedrin promoter control were identified by restriction analysis. Two correct constructs were selected for transfection into baculovirus and were designated oBRTδ and pBRTlδ.

The sequence across the fusion junction and the expected amino acid sequence of the translation product are shown below. polyhedrin 5¹ leader +1 preproricin sequence δ'-AACCTATAAACC ATG GCG GCC CAG CTT ATG AAA CCG GGA-

Met Ala Ala Gin Leu Met Lys Pro Gly-

The native sequence in this construction is expected to be expressed with additional residues on Its amino-terminus (in this case five) but these would be eliminated along with the signal peptide upon secretion.

B. Construction of Recombinant Baculovirus Transfer Vectors for Ricin B Subunit

1. As an Intracellular Product

The sequence encoding ricin B subunit was taken from pRTB601 on a Hindlll-Hi'ndlll fragment. The fragment was treated with Klenow enzyme and al! 4 dNTP's to blunt repair the sticky ends and was subsequently ligated into Smal-digested pAcC3. Two recombinants containing the ricin B chain sequence in the correct orientation were identified and designated pBIB4 and pBIB24. The expected fusion sequence for expression 1s shown below. polyhedrin 5' leader +1 ricin B chain sequence δ'-AACCTATAAACC ATG GCG GCC CAG CTT ATG GCT GAT

Met Ala Ala Gin Leu Met Ala Asp

It was hoped that the six additional residues on the amino- terminus would not affect ridn B chain activity and that by keeping this short vector sequence at the beginning of the ricin B sequence, the efficiency of translational initiation would less likely be affected.

2. As a Secreted Product

The sequence encoding ricin B was taken from pRTB601 on a Hindlll-HindlH fragment. After cloning this fragment into M13, site- specific mutagenesis using the primer, 5'-GTGCCAAGCTTTGCGCAGATGTTTGT- 3' was carried out to introduce an Fspl restriction site (underlined in primer) including the amino-terminal alanine codon (GCA) of the B subunit coding sequence. The B subunit coding sequence was then excised as an FspI-PstI fragment using the PstI site from the M13 vector at the 3' end of the B subunit insert.

The 97-mer encodes the CSF-1 secretory signal peptide (Kawasaki, E. et al., 1985, supra) except for four modifications at nucleotides 5, 10, 82 and 97 which were third position codon changes to create restriction recognition sites. Only the change at position 5 resulted in an amino acid coding change (Thr to Ala). The 97-mer having the sequence 5'-CATGGCCGCCCCGGGCGCCGCCGGGCGCTG CCTCCCACGACATGG CTGGGCTCCCTGCTGTTGTTGGTCTGTCTCCTGGCCAGCAGGAGTWTCACG-3^, and its complement when annealed have a 5' Ncol sticky end for insertion Into The transfer vector and a 3¹ blunt end for fusion to the Fspl blunt end at the 5' end of the B subunit coding sequence. The transfer vector, pAcCδ, was doubly digested with Ncol and PstI. A three fragment ligation resulted in a baculovirus transfer vector containing a CSF-1 secretory peptide fused to the ricin B subunit. This vector is designated pBSBl. C. Construction of Recombinant Baculovirus Transfer Vectors for Ricin B Muteins

Native ridn B has asparagine at positions 46 and 255. Muteins were generated wherein glydne was substituted for asparagine at either one or both of these positions. Standard M13 cloning techniques were used to realize the 255 mutein, and the techniques described by Mandecki, W., Proc. Natl. Acad. Sci. USA 83. 7177 (1986) were used to produce the 46 mutein. The oligomer used to mutagenize asparagine at position 255 to glydne was GGTGACCCAGGTCAAATATGGTTACC, while the oligomer used to mutagenize the asparagine at position 46 to glycine was GTGGCCATGCAAGTCTAATACAGATGCCGGCCAGCTCTGGACTTTGAAA. In order to perform this mutagenesis it was previously necessary to introduce a unique restriction site near position 46 using standard mutagenesis techniques and the oligomer GCAAATCAGCTCTGGACTTTG.

1. Production of Transfer Vector pBG255

Briefly, RB was obtained from pRTB601 as a Hindlll cassette, and using the oligomer shown above, asparagine at position 255 was converted to glycine in M13MP18. After mutagensis and confirmation of the mutagenized insert as described below, M13MP18 containing the mutagenized RTB DNA sequence was digested using Hindlll, and the mutagenized sequence ligated into plasmid pPL231, thereby producing plasmid pPL231-RTB/AE82. The transfer vector, pBG255 was then constructed as shown in Figure 5 using the plasmid pBSBl, and pPL231- RTB/AE82. The plasmid pBSBl was treated with the following enzymes

PstI, Klenow enzyme, BglH. and bacterial alkaline phosphatase. This treatment results in the excision of a ricin B encoding fragment and vector sequences, downstream of the fragment. The remaining large fragment, which exhibits ricin B sequences uøtream of the BglH site, was Ugated to a BglH-HgiAI fragment which contains sequences of ridn B wherein the asparagine amino add at position 255 was site- specific mutagenized in M13 to Glycine. The Bglll-HgiAI fragment was derived from the plasmid pPL231-RTB/AE82 by subjecting the plasmid to HgiAl, Klenow, and BglH treatment, and by gel purifying the BglH- HgiAI fragment. Ligation of the large fragment obtained from pBSBl to the Bglll-HgiAI fragment yields the plasmid, pBG255 as shown 1n Figure 5.

A transfer vector, pBG46/255, containing a RB insert having asparagines at positions 46 and 255 mutated to glydne was constructed as shown in Figure 6. First, a RB construct was generated wherein asparagine at position 46 was mutated to glydne which consisted of inserting RB removed from pRTB601 as a Hindlll cassette Into pDGHl. Next, the "oligomer overlay" technique of Mandeckl, referred to above, was used to perform the mutagensis employing the oligomer also shown above. This procedure requires a restriction site in the vicinity of the sequence to be mutagenized .such that sequences which flank the sequence are complementary to the oligomer. Because there is a unique PvuII site near position 46 of RB, and because pDG141 does not have any PvuII restriction sites, it was possible to perform the overlay technique which produced plasmid pDG141 harboring the asparagine to glycine mutation. Next, pDG141 was digested with Hindlll thereby removing the RB cassette containing glycine at position 46. The cassette was inserted into pPL231 thereby providing pPL231-RTB/AE98.

2. Production of Transfer Vector pBG46/255 and pBG46 pBG255 described above was restricted with BaπHI , and treated with bacteria alkaline phosphatase. This treatment results in the excision of the middle portion of ricin B from the vector, while leaving associated with the large vector fragment ridn sequences on the upstream and downstream side of the removed fragment. Downstream of the fragment 1s the amino acid 255 which was mutagenized from asparagine to glycine. The large fragment resulting from BamHI digestion was ligated to a BamHI-BamHI fragment obtained from plasmid pPL231-RTB/AE98. This plasmid, which contains ridn B having had the asparagine amino add at position 46 mutagenized to glycine, was ligated to the large fragment resulting from BamHI digestion of pBG255 thereby yielding the plasmid pBG46/255 having full length ricin B wherein the amino acids at positions 46 and 255 have been mutagenized from asparagine to glycine. Details of the construction of pBG46/255 are shown in Figure 6. Transfer vector pBG46 was constructed using the same general materials and methods used to produce pBG46/25δ with the exception that pBSBl was substituted for pBG2Sδ.

It will be appreciated that details regarding some of the vectors described above are shown in European Publication No. 196,762, published October 8, 1986.

D. Construction of Recombinant Baculovirus Transfer Vectors for Ricin A Subunit

The sequence encoding ricin A subunit is contained on a Hindlll/BamHI restriction fragment as described earlier. This fragment was Klenow repaired and ligated into Smal digested pAcC3 analogous to the construction in Section B.l.a. Transformants of E. coli MM294 were screened by restriction analysis of inilysates.

EXAMPLE- IV - Expression of Ricin Toxin and its Subunits in Insect Cells

A. Expression of Full Length Toxin pBRTδ and pBRTlδ were each cotransfected into Sf9 cells with wild-type baculovirus DNA. Recombinant viruses were selected and plaqued a second time. The data discussed below are for second-round plaqued viruses. Each virus plaque was infected into two separate wells of cells. One was Sf-9 cells grown in standard medium containing 10% fetal calf serum. This infection was for virus propagation and to assay for secreted activity. The second was Sf-9 cells adapted to and grown in protein-free medium. This infection was to produce samples for SDS-PAGE and Western blot analyses on the medium. After 4-δ days, the culture supernatants were collected and assayed. Cytotoxicity assays were run on the first set of supernatants. Aliquots were diluted 1:10 and 1:100 into medium and placed onto RAT-2 cells (this is a tk^" variant of the rat cell line, RAT-1) for 4 hours, replaced with fresh medium, and examined 20 hours later. The results are shown in Figure 7. The infected cell supernatants from the BRT15 plaques, 6-1 and 6-2, show definite cytoxicity at both 1:10 and 1:100 dilutions that can be blocked by the addition of 50 mM lactose. Lactose is able to block specific binding of native ricin. Support for this being ricin-like activity comes from the Western blot analysis of the second set of infected supernatants. As shown in Figure 8, immunoreacti e proteins -are clearly present in the BRT15, 6-1 and 6-2 samples (lanes 7 and 8), and are much less, if at all, present in the other samples. Lane 5 is the cell supernatant from Sf9 cells infected with wild-type baculovirus. Compared with native ricin standards (lane 1), each of the two supernatants (lanes 7 and 8) contain a product that migrates close to the position of intact ricin that is detected by both the ant i -RTA and anti-RTB sera. This may correspond to secreted product that is not processed further into the A and B subunits. ^■ It represents perhaps 60-70% of the total detected products. The anti-RTA sera also detects two products which migrate approximately with native ricin Al and A2 chains and likely are analogous to those forms of ricin A chain. The anti-RTB sera detects additionally a product that migrates approximately with native ricin B chain. These smaller products collectively comprise the remainder of the 30-40% of the detected material. These results demonstrate that the baculovirus expression vector system can express, secrete and process into its subunits, active ricin toxin.

B. Expression of Ricin B

1. pBIB4 and pBIB24 DNAs were each cotransfected into Sf9 cells with wild type baculovirus DNA.

The initial transfected cells and cells infected with the subsequently plaque-purified viruses were assayed for ricin B chain expression to aid in selecting recombinant viruses. The data discussed below was taken from experiments in which either once or twice plaque-purified virus was used. Infected cells were harvested 4-6 days after infection, when the cultures showed a high percentage of l te -infected cells. By Western blot analysis of several pBIB4 and pBIB24 fi rst- round plaqued vi ruses, 1t was clear that a ricin B chain protein was expressed In most, but not all , pl aque-pool infections. "Fal se" recombinant viruses may be attributed to a natural loss of the polyhedrin phenotype as there Is no selection for 1t 1n vitro. The ricin B chain product expressed has a molecular weight of 29 kD (see Figure 9, lane 5), consistent with that expected for a non-secreted, non-glycosyl ated product. In other analyses (data not shown) of second-round plaqued vi ruses, the same product was noted by Western blot but could not be visualized in Coomassie stained gels. From a comparison of signal strengths , it was estimated that the ridn B chain is produced at about 2 mi cro gram/ml . Cells were disrupted by soπication and upon low speed centrifugation this product distributed approximately equally between the supernatant and pel let fractions . 2. The transfer vector, pBSBl, containing the CSF-1 secretory pepti de fused to ricin B was transfected with bacul ovi rus DNA into Sf-9 cel l s and recombinant vi rus was selected. Ricin B was . detected by Western bl ot analysis as described by Towbin et al . , (1979) Proc . Nat! . Acad. Sci . USA, _76_:4350. Rabbit anti -sera to ricin B-chain was util ized. It was observed that ricin B chain is expressed as two cel l associated proteins, one having a molecul ar wei ght of about 32,000. The 32,000 and 36,000 species accumul ated to si gnificant level s in the range of about 10 μg/ml and 2 mg/πl , respecti vely. Of this , approximately 10 nanograms/μl was acti ve B chai n. Additional ly, it was observed that ricin B chain was al so expressed as a secreted protein having a molecular weight of about 36,000. This molecule accumulates to significant level in the range of about 2 mg/ml . Of this, approximately 10 nanograms/per ml was active B chain. Additional ly, a thi rd form of B chain was detected in the culture supernatant. It has an apparent molecul ar weight of 32,000 and was present in the culture supernatant at about 10 nanogram/per ml or greater levels. C. Expression of Ricin B Muteins

The materials and methods used to express ricin B muteins was essentially similar to that described for the expression of ricin

B above, with the exception that generally higher levels of the mutein was observed in culture supernatant and were in the range of at least

20 nanogra s/per ml.

D. Expression of Ricin A

After transfection of the recombinant transfer vector and baculovirus DNA into Sf-9 cells, recombinant viruses were selected. There is no evidence of ricin A production by either bioactivity assay or Western blot analysis.

This invention has been described in detail including the preferred embodiments thereof. However, it will be appreciated that those skilled in the art, upon consideration of this disclosure, may make modifications and improvements within the scope of this invention.

EXAMPLE V Purifi cation of Ri cin B Muteins A. Puri fi cati on of Ri dn B Chai n Doubl e Mutant The ricin B chai n doubl e mutant described in Exampl e VI . C above was purified from Sf9 culture medium. Approximately 5.5 l iters of culture medium in whi ch Sf9 contai ning the baculovi rus expressi on vecttor pBG46/255 cell were grown was adjusted to a pH of about 7.2 and appl ied to a Zetaprep-QAE cartridge, obtainabl e from AMF Molecul ar Separations , Catal og No. 1600 1501 OOQA. Prior to applying the culture media to the cartridge, the cartridge was acti vated by fi rst passing 0.1 mol ar tribasic sodium phosphate, pH 9, through the cartridge, and then 0.1 mol ar sodi um acetate, pH 3. Next the cartridge was washed with 0.1 mol ar sodi um phosphate, pH 7.2 until the pH fluid eninating from the cartridge was 7.2, at whi ch time it was further washed with 10 mM sodi um phosphate,. pH 7.2, with 40 mM NaCl until the conductivity of buffer entering and leaving the cartridge was about 4 mmisiemens/cm.

The culture medium was passed through the cartridge and the cartridge then washed with 10 mM sodium phosphate, pH 7.2, containing 40 mM NaCl until such time that the absorbance reading at 280 nm was 0. Next, the protein which bound to the cartridge was eluted in a single step gradient with 10 M sodium phosphate, pH 7.2, containing 1 M NaCl. Those fractions containing the mutein were Identified by immunoblots using affinity purl fied-anti body raised against naturally occurring ricin B chain. It was calculated that the concentration of the ricin B chain mutein was about 240 nanogra s/per ml.

EXAMPLE VI A. Galactose Binding Activity of Ricin B Muteins

In order to ascertain whether the substitution of glycine for asparagine at positions 46 and/or 255 of ricin B reduced galactose binding activity, an assay was employed wherein binding of the muteins was compared to binding of native ricin B chain. The assay consisted of determining 1f these molecules bound to asialofetuin which was absorbed to the bottom of a 96-well plastic tissue culture plate. Asialofetuin was obtained from Sigma Corporation, and was dissolved at 1 mg/per ml in phosphate buffered saline containing 0.5% bovine serum albumin, 0.05% Tween-20, and the preservative thiπersal at 0.01%. More specifically, the procedure involved diluting the asialofetuin solution to a concentration of 5 μg per ml with 0.05 molar sodium carbonate, pH 9.6, and 100 microHters of this solution was added to wells 1n the 96 well plate. The solution was allowed to incubate overnight at 4^βC so as to provide maximum time for asialofetuin to adhere to the tissue culture plates. Next, the plates were washed with phosphate buffered saline to remove unattached asialofetuin, and then areas on the culture tissue wells which did not bind asialofetuin were blocked with bovine serum albumin in phosphate buffered saline, Tween-20 and thi erisal for one hour at room temperature. The plates were washed a second time with phosphate buffered saline containing Tween-20, and subsequently 100 microl iters of either native ricin B, or the mutein was added to each well . The samples were diluted into phosphate buffered saline containing 0.5% bovine serum albumin before addition to the wells, and where necessary, lactose was also present. The samples were incubated for two hours with moderate shaking at 21 ^βC and the plates were washed with phosphate buffered saline containing Tween-20 to remove unbound reactants. Subsequently, 100 microliters of polyclonal rabbit anti -ricin B chain antibody, conjugated to horseradish peroxidase, previously diluted in the range of 1:1000 to 1:5000 was added to each of the wells and incubated for two hours with shaking at 21^βC. The wells were further washed with

PBS contai ning Tween-20 and 100 microl iters of a solution containing horseradish peroxi dase substrate, ortho-phenylenedi amine, was added

• and al l owed to react for 15 minutes prior to stopping the reaction with 100 microliters of two normal sulfuri c acid. The substrate was made by dissolving a 30 g tablet (Sigma lot. 36F-8815 No. P-8412) in 50 ml of sodium phosphate-citrate buffer/urea peroxide. The amount of antibody bound to either native ricin or the mutein was ascertained by measuring the absorbance of the solution at 490 and 405 nm. Figure 10 shows that nati ve ri cin B chain at three different concentrations , 10, 50, and 101 ng per ml , bi nds effi caciously to asialofetuin. It further shows the expected result: as lactose concentration is increased the amount of native ri ci n B chai n displaced from asial ofetuin is reduced consi derably and this i s dependent on the amount of native ricin B chain initially present in the reacti on solution. In contrast, Figure 11 shows that ricin B chain mutein having the asparagine at position 255 mutated to glycine exhibits markedly reduced binding to asialofetuin, and that considerably less lactose is required to prevent the binding of the single amino acid mutein. It was further observed, that the ricin B chai n double mutein having amino acids at positions 46 and 255 changed from aspargine to glycine exhibits no binding, or at least binding which is so low as not to be detectable by the assay uti l ized. This Invention has been described in detail including the preferred embodiments thereof. However, 1t will be appreciated that those skilled in the art, upon consideration of this disclosure, may make modifications and improvements within the scope of this invention.

Deposits

The materials listed below were deposited with the American Type Culture Collection, Rockville, MD, USA (ATCC). The deposits were made under the provisions of the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure and the Regulations thereunder (Budapest Treaty). Maintenance of a viable culture is assured for 30 years from date of deposit. The organisms will be made available by ATCC under the terms of the Budapest Treaty, and subject to an agreement between Applicants and ATCC which assures unrestricted availability upon issuance of the pertinent U.S. patent. Availability of the deposited strains is not to be construed as a license to practice the invention in contravention rights granted under the authority of any government in accordance with its patent laws.

Claims

WHAT IS CLAIMED IS:

1. A recombinant bacul ovirus transfer vector capable of introducing a ricin toxin gene or portion thereof into a baculovirus genome said transfer vector comprising a portion of the baculovi rus genome, a bacterial replication origin and a DNA sequence encoding a ridn toxin, subunit thereof or an amino acid sequence substantially equivalent to that of ricin toxin under the transcriptional control of a bacul ovirus promoter.

2. A recombinant bacul ovi rus transfer vector according to cl aim 1 wherein said DNA sequence encoding a ri cxin toxin or a subunit thereof is sel ected from the group of pl asmids consi sting of pRT17, pRA123 or pRTB601.

3. A recombinant bacul ovi rus transfer vector according to claim 1 wherein said bacul ovi rus promoter is the polyhedrin gene promoter.

4. A recombinant baculovirus transfer vector according to claim 3 wherein said DNA sequence encoding a ricin toxin or a subunit thereof is selected from the group of plasmids consisting of pRT17, pRA123 or pRTBδOl.

5. A recombinant baculovirus transfer vector according to claim 3 wherein a Ncol restriction endonuclease recognition site has been introduced at the ATG trans!ational initiation codon of the polyhedrin gene.

6. A recombinant baculovirus transfer vector accordinq to claim 3 wherein said ridn toxin gene or portion thereof is in proper trans!ational reading frame with a heterologous secretory signal peptide.

7. pBRT8.

8. pBRTlδ.

9. pBIB24.

10. pBSBl.

11. A recombinant baculovirus transfer vector according to claim 1 wherein said subunit of ricin toxin is a mutein of ricin B comprising an alteration in at least one amino acid residue forming a part of at least one of the ricin B binding sites for galactosides wherein said amino acid forming at least a part^" of one galactoside binding site of ricin B comprises those amino acids that are within about δ Angstroms of the galactoside in the 2.8 Angstrom resolution crystal structure of ricin, and are selected from the group consisting of Asp22, Arg24, Asp2δ, Gly26, Gln3δ, Trp37, Lys40, Asp44, Asn46, Asp234, Arg236, Ala237, Ile246, Tyrl48, His2δl, Asn2δδ and Gln2δ6.

12. A recombinant baculovirus transfer vector according to claim II wherein said amino acid alteration comprises deletion or substitution of at least one amino acid of the galactoside binding site that participates in hydrogen bonding to galactoside with a substituting amino acid that does not participate in hydrogen bonding to galactoside, wherein said at least one amino acid of the galactoside binding site participating in hydrogen bonding to galactoside is an amino acid selected from the group consisting of Arg24, Asp25, Gly26, Gln3δ, Lys40, Asp44, Asn46, Arg236, Ala237, Ile246, His2δl, Asn2δδ and Gln2δ6, and said substituting amino acid that does not participate in hydrogen bonding of galactoside is selected from the group consisting of Gly and Ala.

13. A recombinant baculovirus transfer vector according to claim 11 wherein said amino acid alteration comprises deletion of an

^• amino acid of the galactoside binding site that stabilizes an amino acid that participates in hydrogen bonding to galactoside or the substitution of an amino acid of the galactoside binding site that stabilizes an amino acid that participates in hydrogen bonding to galactoside, with an amino acid that does not stabilize an amino acid that participates in hydrogen bonding to galactoside wherein said stabilizing amino acid is selected from the group consisting of Asp22 and Asp234, and said amino acid that substitutes for a stabilizing amino acid is selected from the group consisting of Gly, Ala and Ser.

14. A recombinant baculovirus transfer vector having a mutein of ricin B comprising at least one of Asp22, Arg24, Asp25,

Gly26, Gln3δ, Trp37, Lys40, Asp44, Asp234, Arg236, Ala237, Ile246, Tyr248, His2δl, Asn2δδ and Gln2δ6 substituted by an amino acid selected from the group consisting of Gly, Ala, Ser, Lys or Arg when the substituted amino acid is Asp or Gin, and Gin when the substituted amino acid is lys or Arg, said mutein having a lower affinity for galactoside than the corresponding unsubstituted form of ricin B.

15. A recombinant baculovirus transfer vector having a mutein of ricin B comprising at least one of Asp22, Arg24, Asp25, Gly26, Gln3δ, Trp37, Lys40, Asp44, Asn46, Asp234, Arg236, Ala237, Ile246, Tyr248, His2δl, Asn25δ and Gln266 deleted, said mutein having a lower affinity for galactoside than the corresponding unsubstituted form of ricin B.

16. A recombinant baculovirus transfer vector having a mutein of ricin B comprising at least one of Asp22, Arg24, Asp2δ, Gly26, Gln35, Trp37, Lys4Q, Asp44, Asn46,- Asp234, Arg236, Ala237, Ile246, Tyr248, His2δl, Asn2δδ and Gln2δ6 substituted by an amino acid selected from the group consisting of Gly, Ala, Ser₃ Lys or Arg when the substituted amino acid is Asp or Gin, and Asp or Glu when the substituted amino acid is Lys or Arg, and at least one of the remaining unsubstituted amino acids deleted, said mutein having a lower affinity for galactoside than the corresponding undeleted unsubstituted form of ricin B.

17. . A recombinant baculovirus transfer vector having a mutein of ricin B, said ricin B comprising an amino and a carboxyl galactoside binding site, said mutein comprising the substitution of two amino acids of at least one of said galactoside binding sites, said two amino acids of the amino binding site selected from the group consisting of residues 22, 26, 3δ, 37 and 46, said two amino acids of the carboxyl binding site selected from the group consisting of residues 234, 248, 261 and 255 wherein said substitution is either Gly or Ala.

18. A recombinant baculovirus transfer vector having a mutein of ricin B, said ricin B comprising an amino and a carboxyl galactoside binding site, said mutein comprising the substitution of one amino acid of at least one of said galactoside binding sites, said one amino acid of the amino binding site selected from the group consisting of residues 22, 25, 26, and 46, said amino acid of the carboxyl binding site selected from the group consisting of residues 234 and 256, wherein said substitution is either Leu or He.

19. A recombinant baculovirus transfer vector having a mutein of ricin B, said ricin B comprising an amino and a carboxyl galactoside binding site, said mutein comprising the substitution of two amino acids of at least one of said galactoside binding sites, said two amino acids of the amino binding site selected from the group consisting of residues 22, 25, 26, 34 and 46, said amino acid of the carboxyl binding site selected from the group consisting of 234, 248, 251 and 255 wherein when the substitution at amino acids selected from the group consisting of 22, 25, 26, 34 and 46 is either Leu or He, the other substitution of the selected amino acids of the amino binding site is Gly or Ala and when the substitution- at amino acids selected from the group consisting of 234 and 255 is either Leu or He, the other substitution of the selected amino acids of the carboxyl binding site consisting of 234, 243, 251 and 255 is Gly ofr Ala.

20. A recombinant baculovirus transfer vector having a mutein of ricin toxin or the precursor thereof wherein the ricin B chain thereof comprises the ricin B mutein of claims 11, 12, 13, 14,

15, 16, 17, 18 and 19.

21. A recombinant baculovirus transfer vector having a DNA sequence encoding the mutein of ricin B of claims 11, 12, 13, 14, 15,

16, 17, 18, or 19.

22. A recombinant baculovirus transfer vector having a DNA sequence encoding the mutein of ricin toxin or the precursor thereof of claim 20.

23. A recombinant baculovirus expression vector for the production of biologically active ricin toxins, ricin toxin subunits or proteins having an amino acid sequence substantially equivalent to that of ricin toxin, said expression vector comprising a recombinant baculovirus genome and a DNA sequence substantially equivalent to that of ricin toxin under the transcriptional control of a baculovirus promoter, wherein said DNA sequence encoding said ricin toxin or subunit thereof is selected from the group of plasmids consisting of pRT17, pRA123 or pRTBδOl, and said baculovirus promoter is the polyhedrin gene promoter.

24. A recombinant baculovirus expression vector having a mutein of ricin B comprising at least one of Asp22_s Arg24, Asp25, Gly26, Gln3δ, Trp37, Lys40, Asp44, Asn46, Asp234, Arg236, Ala237, Ile246, Tyr248, His2δl, Asn2δδ and Gln2δ6 substituted by an amino acid selected from the group consisting of Gly, Ala, Ser, Lys or Arg when the substituted amino acid is Asp or Gin, and Asp or Gin when the substituted amino acid is Lys or Arg, said mutein having a lower affinity for galactoside than the corresponding unsubstituted form of ricin B.

25. A recombinant baculovirus expression vector having a mutein of ricin B comprising at least one of Asp22, Arg24, Asp25, Gly26, Gln3δ, Trp37, Lys40, Asp44, Asn46, Asp234, Arg236, Ala237, Ile246, Tyr248, His251, Asn2δδ and Gln2δ6 deleted, said mutein having a lower affinity for galactoside than the corresponding unsubstituted form of ricin B.

26. A recombinant baculovirus expression vector having a mutein of ricin B comprising at least one of Asp22, Arg24, Asp2δ, Gly26, Gln35, Trp37, Lys40, Asp44, Asn46, Asp234, Arg236, Ala237, Ile246, Tyr248, His2δl, Asn2δδ and Gln256 substituted by an amino acid selected from the group consisting of Gly, Ala, Ser, Lys or Arg when the substituted amino acid is Asp or Gin, and Asp or Glu when the substituted amino acid, is Lys or Arg, and at least one of the remainaing unsubstituted amino acids deleted, said mutein having a lower affinity for galactoside than the corresponding undeleted unsubstituted form of ricin B.

27. A recombinant baculovirus expression vector having a mutein of ricin B, said ricin B comprising an- amino and a carboxyl galactoside binding site, said mutein comprising the substitution of two amino acids of at least one of said galactose binding sites, said two amino acids of the amino binding site selected from the group consisting of residues 22, 25, 36, 37 and 46, said two amino acids of the carboxyl binding site selected from the group consisting of residues 234, 248, 251 and 256 wherein said substitution is either Gly or Ala.

28. A recombinant baculovirus expression vector having a mutein of ricin B, said ricin B comprising an amino and a carboxyl galactoside binding site, said mutein comprising the substitution of one amino acid of at least one of said galactoside binding sites, said one amino acid of the amino binding site selected from the group consisting of residues 22, 25, 26 and 46, said amino acid of the carboxyl binding site selected from the group consisting of 234 and 255, wherein said substitution is either Leu or He.

29. A recombinant baculovirus expression having a mutein of ricin B, said ricin B comprising an amino and a carboxyl galactoside binding site, said mutein comprising the substitution of two amino acids of at least one of said galactoside binding sites, said two amino acids of the amino binding site selected from the group consisting of residues 22, 25, 26, 34 and 46, said amino acid of the carboxyl binding site selected from the group consisting of 234, 248, 251 and 255, wherein when the substitution at amino acids selected from the group consisting of 22, 25, 26 and 46 is either Leu or He, the other substitution of the selected amino acids of the amino binding site is Gly or Ala and when the substitution at amino acids selected from the group consisting of 234 and 256 is either Leu or lie, the other substitution of the selected amino acids of the carboxyl binding site consisting of 234, 248, 251 and 256 is Gly or Ala.

30. pBG46.

31. pBG25δ.

32. pBG46/2δδ.

33. Proteins encoded by said baculovirus transfer vectors of claim 1.

34. Proteins encoded by said baculovirus transfer vectors selected from the group consisting of pBG46, pBG25δ and pBG46/25δ.

36. Proteins encoded by the transfer vector pBG46/25δ.

36. Proteins produced by said baculovirus expression vector of claim 23.

37. Proteins produced by a baculovirus expression vectors having the ricin B mutein DNA sequences of plasmids selected from the group consisting of pBG46, pBG25δ and pBG46/25δ.

38. Proteins produced by a baculovirus expression vector having the ricin B mutein DNA sequence of plasmid pBG46/25δ.

39. A method for producing a biologically active ricin toxin, ricin toxin subunits, or muteins or proteins thereof, having amino acid sequences substantially equivalent to that of ricin toxin, said subunits, muteins, or proteins comprising infecting a susceptible host insect cell with a recombinant baculovirus expression vector wherein said expression vector is a recombinant baculovirus genome comprising at least one DNA sequence encoding said ricin toxin, subunits, muteins or proteins thereof under the transcriptional control of a baculovirus promoter, growing said infected insect cells under suitable conditions and recovering said polypeptide from the culture medium.

40. The method of claim 39 wherein the recombinant baculovirus expression vector containing a gene encoding ricin toxin or one of its subunits is produced by recombination with a transfer vector selected from the group consisting of pBRT8, pBRTlδ, pBIB24, pBSBl, pBG46, pBG2δδ, and pBG46/25δ.