ANTIVIRAL RICIN-LIKE PROTEINS

Abstract: The present invention provides a protein having an A chain of a ricin-like toxin, a B chain of a ricin-like toxin and a heterologous linker amino acid sequence, linking the A and B chains. The linker sequence contains a cleavage recognition site for a retroviral protease such as HIV or an HTLV protease. The invention also relates to a nucleic acid molecule encoding the protein and to expression vectors incorporating the nucleic acid molecule. Also provided is a method of inhibiting or destroying mammalian cells infected with a retrovirus utilizing the proteins of the invention; and pharmaceutical compositions for treating HIV infections and human T-cell leukemias involving HTLV.

Description: Title: Antiviral Ricin-Like Proteins FIELD OF THE INVENTION The invention relates to protems havmg A and B chains of a πcin-hke toxin, linked by a linker sequence which is specifically cleavable by a retroviral protease to release the active A chain The mvention also relates to a nucleic acid molecule encoding the protein and to expression vectors incorporating the nucleic acid molecule Also provided is a method of inhibiting or destroying mammalian cells infected with a retrovirus utilizing the protems of the mvention and pharmaceutical compositions for treating HIV mfection BACKGROUND OF THE INVENTION Bacteria and plants are known to produce cytotoxic protems which may consist of one, two or several polypeptides or subunits Those protems havmg a smgle subunit may be loosely classified as Type I protems Manv of the cytotoxms which have evolved two subunit structures are referred to as type II ρroteιns(Saehnger, C B m Trafficking of Bacterial Toxins (eds Saelmger, C B ) 1-13 (CRC Press Ine , Boca Raton, Florida, 1990) One subunit, the A cham, possesses the toxic activity whereas the second subunit, the B chain, binds cell surfaces and mediates entry of the toxin mto a target cell A subset of these toxins kill target cells by inhibiting protein biosynthesis For example, bacterial toxins such as diphtheria toxm or Pseudomonas exotoxm inhibit protein synthesis by inactivating elongation factor 2 Plant toxins such as πcin work by directly mactivatmg πbosomes [Olsnes, S & Phil, A m Molecular action of toxins and viruses (eds Cohen, P & vanHeyningen, S ), 51-105 (Elsevier Biomedical Press, Amsterdam, 1982] Ricin, derived from the seeds of Rtcinus communis (castor oil plant), is the most potent of the plant toxms It is estimated that a smgle πcm A cham is able to mactivate ribosomes at a rate of 1500 πbosomes /minute Consequently, a smgle molecule of πcm is enough to kill a cell (Olsnes, S & Phil, A in Molecular action of toxins and viruses (eds Cohen, P & vanHeyningen, S ) 51-105 (Elsevier Biomedical Press, Amsterdam, 1982) The πcm toxm is a glycosylated heterodimer with A and B cham molecular masses of 30,625 Da and 31,431 Da respectively The A cham of ricin has an N-glycosidase activity and catalyzes the excision of a specific adenine residue from the 28S rRNA of eukaryotic πbosomes (Endo, Y, & Tsurugi, K J Biol Chem 262 8128 (1987)) The B cham of ncm, although not toxic in itself, promotes the toxicity of the A cham bv bmdmg to galactose residues on the surface of eukaryotic cells and stimulating receptor-mediated endocytosis of the toxm molecule (Simmons et al Biol Chem 261 7912 (1986)) Protein toxins are initially produced in an inactive, precursor form Ricm is initially produced as a smgle polypeptide (preproncm) with a 35 ammo acid N-terminal presequence and 12 amino acid linker between the A and B chains. The pre-sequence is removed during translocation of the ricin precursor into the endoplasmic reticulum (Lord, J.M. Eur. J. Biochem. 146:403-409 (1985) and Lord, J.M. Eur. ]. Biochem. 146:411-416 (1985)). The proricin is then translocated into specialized organelles called protein bodies where a plant protease cleaves the protein at a linker region between the A and B chains (Lord, J.M. et al, FΛSΛB Journal 8:201-208 (1994)). The two chains, however, remain covalently attached by an interchain disulfide bond (cysteine 259 in the A chain to cysteine 4 in the B chain) and mature disulfide linked ricin is secreted from the plant cells. The A chain is inactive in the proricin (O'Hare, M., et al. FEBS Lett. 273:200-204 (1990)) and it is inactive in the disulfide-linked mature ricin(Richardson, P.T., et al. FEBS Lett. 255:15-20 (1989)). The ribosomes of the castor bean plant are themselves susceptible to inactivation by ricin A chain; however, as there is no cell surface galactose to permit B chain recognition the A chain cannot re-enter the cell. The exact mechanism of A chain release and activation in target cell cytoplasm is not known (Lord, J.M. et al., FASAB Journal 8:201-208 (1994)). However, it is known that for activation to take place the disulfide bond between the A and B chains must be reduced and, hence, the linkage between subunits broken. The ricin gene has been cloned and sequenced, and the X-ray crystal structures of the A and B chains have been described (Rutenber, E., et al. Proteins 10:240-250 (1991); Weston et al., Mol. Bio. 244:410-422, 1994; Lamb and Lord Eur. J. Biochem. 14:265 (1985); Hailing, K., et al. Nucleic Acids Res. 13:8019 (1985)). Due to its extreme toxicity there has been much interest in making ricin-based immunotoxins as therapeutic agents for destroying or inhibiting target cells or organisms (Vitetta et al., Science 238:1098- 1104(1987)). An immunotoxin is a conjugate of a specific cell-binding component, such as a monoclonal antibody or growth factor and the toxin in which the two protein components are covalently linked. Generally, the components are chemically coupled. However, the linkage may also be a peptide or disulfide bond. The antibody directs the toxin to cell types presenting a specific antigen thereby providing a specificity of action not possible with the natural toxin. Immunotoxins have been made both with the entire ricin molecule (i.e. both chains) and with the ricin A chain alone ( Spooner et al. Mol. Immunol. 31:117- 125, (1994)). Immunotoxins made with the ricin dimer (IT-Rs) are more potent toxins than those made with only the A chain (IT-As). The increased toxicity of IT-Rs is thought to be attributed to the dual role of the B chains in binding to the cell surface and in translocating the A chain to the cytosolic compartment of the target cell (Vitetta et al., Science 238:1098-1104(1987); Vitetta & Thorpe Seminars in Cell Biology 2:47-58 (1991)). However, the presence of the B chain in these conjugates also promotes the entry of the lmmunotoxm mto nontarget cells. Even small amounts of B cham may override the specificity of the cell-binding component as the B cham binds nonspecifically to N- glycosylated galactose, present on most cells IT-As are more specific and safer to use than IT-Rs However, m the absence of the B cham the A cham has greatly reduced toxicity. A number of immunotoxins have been designed to recognize antigens on the surfaces of tumour cells A major problem with the use of ITs is that often the target antigen is also found on non-tumour cells (Vitetta et al , Immunology Today 14 252-259 (1993)) Also, due to the reduced potency of IT-As as compared to ITRs, large doses of IT-As must be administered to patients. The large doses frequently cause immune responses and production of neutralizing antibodies in patients (Vitetta et al , Science 238.1098- 1104(1987)). IT-As and IT-Rs both suffer from reduced toxicity as the A cham is not released from the conjugate mto the target cell cytoplasm The insertion of intramolecular cleavage sites between the cytotoxic and cell- binding components of a toxm can mimic the way that the natural toxm is activated European patent application no 466,222 describes the use of maize-derived pro-protems which can be converted mto active form by cleavage with extracellular blood enzymes such as factor Xa, thrombm or collagenase Westby et al (Bioconjugate Chem., 3 375-381, 1992) documented fusion proteins which have a specific cell binding component and proπcm with a protease sensitive cleavage site specific for factor Xa within the lmker sequence. O'Hare et al (FEBS Lett. 273.200-204, 1990) also describe a recombinant fusion protein of RTA and staphylococcal protein A joined by a trypsin-sensitive cleavage site. In view of the prevalence of the extracellular proteases utilized in these approaches, such artificial activation of the toxm precursor or immunotoxin do not confer a mechanism for intracellular toxin activation, and the problems of target specificity and adverse immunological reactions to the cell-binding component of the immunotoxin remain In view of the extreme toxicity of protems such as πcm, the lack of specificity of the immunotoxins may severely limit their usefulness as therapeutics for the treatment of cancer and infectious diseases The preparation of a suitable specific cell binding component may be problematic For example, antigens specific for the target cell may not be available and many potential target cells and infective organisms can alter their antigenic make up rapidly to avoid immune recognition The potential of bacterial and plant toxms for inhibiting mammalian retroviruses, particularly AIDS, has been investigated. Bacterial toxins such as Pseudomonas exotoxin-A and subunit A of diphtheria toxm; dual chain ribosomal inhibitory plant toxms, such as πcm and smgle cham ribosomal inhibitory protems such as tπchosanthm and poke weed antiviral protein have been used for the elimination of HIV mfected cells (Olson et al 1991, AIDS Res. and Human Retroviruses 7 1025-1030) The high toxicity of these toxms for mammalian cells, combined with a lack of specificity of action poses a major problem to the development of pharmaceuticals mcorporatmg the toxms, such as immunotoxins Immunotoxins are designed such that their specificity of action is determmed solely by the antibody component, antigen presentmg cells are preferentially destroyed by the drug (Pastan et al., Annals New York Academy of Sciences 758 345-353 (1995)) The toxm protem of immunotoxin conjugates does not give the therapeutic any additional specificity of action, it will bring about the destruction of any cell it is delivered to SUMMARY OF THE INVENTION The present inventors have prepared novel recombinant toxic protems which are specifically toxic to cells infected with retroviruses and which do not depend for their specificity of action on a specific cell-binding component The recombinant protems of the mvention have an A chain of a ricin-like toxm linked to a B cham by a lmker sequence, which may be specifically cleaved by a retroviral protease within infected cells to activate the toxic A cham In one aspect, the present mvention provides a purified and isolated nucleic acid having a nucleotide sequence encodmg an A cham of a ricin-like toxm, a B cham of a πcin- hke toxm and a heterlogous lmker ammo acid sequence, linking the A and B chains The lmker sequence is not a lmker sequence of a πcin-hke toxm, but rather the heterologous lmker sequence contains a cleavage recognition site for a retroviral protease The A and or the B cham may be those of πcm In an embodiment, the cleavage recognition site is the cleavage recognition site for an HIV protease In a particular embodiment, the lmker ammo acid sequence comprises VSQNYPIVQNFN, SKARVLAEAMSN, or SIRKILFLDGIN In further particular embodiments, the nucleic acid has the nucleotide sequence shown in Figure 8, Figure 9 or Figure 10 In another embodiment, the cleavage recognition site is the cleavage recognition site for a human T-cell leukemia virus protease In a particular embodiment, the lmker ammo acid sequence comprises SAPQVLPVMHPN or SKTKVLVVQPKN cleaved by a human T-cell leukemia virus-I (HTLV-I) protease, or, SKTKVLVVQPRN or STTQCFPILHPN cleaved by a human T-cell leukemia virus-II (HTLV-II) protease The present mvention further provides a plasmid mcorporatmg the nucleic acid of the mvention In an embodiment, the plasmid has the restriction map as shown in Figure IA, 2A, 3A, 16A, 17A, 18A, or 19A In another embodiment, the present invention provides a baculovirus transfer vector mcorporatmg the nucleic acid of the invention In particular embodiments, the invention provides a baculovirus transfer vector havmg the restriction map as shown m Figures 5, 6, 7, 16C, 17C, 18C, or 19C or havmg the DNA sequence as shown in Figure 11 In a further aspect, the present invention provides a recombmant protein comprising an A cham of a ricin-like toxm, a B chain of a ricin- like toxm and a heterologous lmker ammo acid sequence, linking the A and B chams, wherem the lmker sequence contams a cleavage recognition site for a retroviral protease The A and or the B cham may be those of ricin In another aspect, the invention provides a method of inhibiting or destroymg mammalian cells mfected with a retrovirus having a protease, comprismg the steps of preparmg a recombmant protem of the mvention havmg a heterologous lmker sequence which contams a cleavage recognition site for the retrovirus protease and mtroducmg the recombmant protem mto the cells In an embodiment, the retrovirus is HIV The present mvention also relates to a method of treatmg a mammal infected with HIV by administering the recombmant protems of the mvention to the mammal Also provided is a process for preparmg a pharmaceutical for treatmg a mammal mfected with a retrovirus havmg a protease comprismg the steps of preparmg a purified and isolated nucleic acid havmg a nucleotide sequence encoding an A cham of a ricin-like toxm, a B cham of a πcm-like toxin and a heterologous linker ammo acid sequence, linking the A and B chams, wherem the lmker sequence contains a cleavage recognition site for the protease, mtroducmg the nucleic acid mto a host cell, expressing the nucleic acid m the host cell to obtam a recombmant protem comprismg an A cham of a ricin-like toxm, a B cham of a ricin-like toxm and a heterlogous lmker ammo acid sequence, linking the A and B chams wherein the lmker sequence contams the cleavage recognition site for the protease, and suspending the protein in a pharmaceutically acceptable carrier, diluent or excipient In an embodiment, a process is provided for preparmg a pharmaceutical for treatmg a mammal mfected with a retrovirus havmg a protease comprismg the steps of identifying a cleavage recognition site for the protease, preparmg a recombmant protem comprising an A chain of a ricin-like toxin, a B chain of a ricin-like toxm and a heterlogous lmker ammo acid sequence, lmkmg the A and B chains wherem the lmker sequence contams the cleavage recognition site for the protease and suspending the protem m a pharmaceutically acceptable carrier, diluent or excipient In a further aspect, the invention provides a pharmaceutical composition for treatmg a retroviral mfection, such as HIV, m a mammal comprising the recombmant protem of the mvention and a pharmaceutically acceptable carrier, diluent or excipient The mvention also contemplates a method for treating cancer cells contammg an HTLV protease comprismg (a) preparmg a recombmant protem of the mvention havmg a heterologous lmker sequence which contams a cleavage recognition site for an HTLV protease; an (b) introducing the recombinant protein into the cells. The method can be used to treat a mammal with human T-cell leukemias involving HTLV. Compositions for treating human T-cell leukemias involving HTLV comprising the recombinant protein of the invention having a heterologous linker sequence which contains a cleavage recognition site for an HTLV protease, and a pharmaceutically acceptable carrier , diluent, or excipient are also provided. Other objects, features and advantages of the present invention will become apparent from the following detailed description. It should be understood, however, that the detailed description and the specific examples while indicating preferred embodiments of the invention are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description. DESCRIPTION OF THE DRAWINGS The invention will be better understood with reference to the drawings in which: Figure IA summarizes the cloning strategy used to generate the ρAP-146 construct; Figure IB shows the nucleotide sequence of the HIV-A linker region of pAP-146; Figure 2A summarizes the cloning strategy used to generate the pAP-147 construct; Figure 2B shows the nucleotide sequence of the HIV-B linker region of pAP-147; Figure 3A summarizes the cloning strategy used to generate the pAP-148 construct; Figure 3B shows the nucleotide sequence of the HIV-H linker region of pAP-148; Figure 4 shows the amino acid sequences of the wild type ricin linker, the pAP-146 linker, the pAP-147 linker and the pAP-148 linker; Figure 5 shows the subcloning of the HIV-A linker variant into a baculovirus transfer vector; Figure 6 shows the subcloning of the HIV-B linker variant into a baculovirus transfer vector; Figure 7 shows the subcloning of the HIV-H linker variant into a baculovirus transfer vector; Figure 8 shows the DNA sequence of the pAP-190 insert; Figure 9 shows the DNA sequence of the ρAP-196 insert; Figure 10 shows the DNA sequence of the pAP-197 insert; Figure 11 shows the DNA sequence of the baculovirus transfer vector pVL1393; Figure 12 is a diagram of the vector pSB2; Figure 13 shows a Western Blot of a pAP-190 proricin variant; Figure 14 is a blot showing cleavage of a pAp 190 proricin variant by HIV protease; Figure 15 is a blot showing activation of pAP-190 proricin variant by HIV protease, Figure 16A is a diagram summarizing the cloning strategy used to generate the pAP-205 construct, Figure 16B shows the nucleotide sequence of the HTLV-I-A lmker regions of pAP-205, Figure 16C is a diagram showmg the subclonmg of the HTLV-I-A lmker variant mto a baculovirus transfer vector, Figure 16D shows the DNA sequence of the pAP-206 insert contammg πcm and the HTLV-I-A lmker, Figure 17A is a diagram summaπzmg the cloning strategy used to generate the pAP-207 construct, Figure 17B shows the nucleotide sequence of the HTLV-I-B linker regions of pAP-207, Figure 17C is a diagram summarizing the subclonmg of the HTLV-I-B lmker variant mto a baculovirus transfer vector, Figure 17D shows the DNA sequence of the pAP-208 insert contammg πcm and the HTLV-I-B lmker, Figure 18A is a diagram summarizing the cloning strategy used to generate the pAP-209 construct, Figure 18B shows the nucleotide sequence of the HTLV-II-A lmker regions of pAP-209, Figure 18C is a diagram summanzmg the subcloning of the HTLV-II-A linker variant mto a baculovirus transfer vector, Figure 18D shows the DNA sequence of the pAP-210 insert contammg ricin and the HTLV-II-A lmker, Figure 19A is a diagram summanzmg the cloning strategy used to generate the pAP-211 construct, Figure 19B shows the nucleotide sequence of the HTLV-II-B lmker regions of pAP-211, Figure 19C is a diagram summanzmg the subcloning of the HTLV-II-B lmker variant mto a baculovirus transfer vector, Figure 19D shows the DNA sequence of the pAP-212 msert containmg πcm and the HTLV-II-B lmker, and Figure 20 shows the ammo acid sequences of the wild type ricin lmker and HTLV protease-sensitive ammo acid lmkers contained m linkers pAP-205 to pAP-212 DETAILED DESCRIPTION OF THE INVENTION Nucleic Acid Molecules of the Invention The present inventors have cloned and expressed novel nucleic acid molecules having a nucleotide sequence encoding an A chain of a ricin-like toxin, a B chain of a ricin- like toxin and a heterologous linker amino acid sequence, linking the A and B chains. The heterologous linker sequence contains a cleavage recognition site for a retroviral protease such as a cleavage recognition site for HIV or a human T-cell leukemia virus protease. The term "isolated and purified" as used herein refers to a nucleic acid substantially free of cellular material or culture medium when produced by recombinant DNA techniques, or chemical precursors, or other chemicals when chemically synthesized. An "isolated and purified" nucleic acid is also substantially free of sequences which naturally flank the nucleic acid (i.e., sequences located at the 5' and 3' ends of the nucleic acid) from which the nucleic acid is derived. The term "nucleic acid" is intended to include DNA and RNA and can be either double stranded or single stranded. The term "linker sequence" as used herein refers to an intemal amino acid sequence within the protein encoded by the nucleic acid molecule of the invention which contains residues linking the A and B chain so as to render the A chain incapable of exerting its toxic effect, for example catalytically inhibiting translation of a eukaryotic ribosome. By heterologous is meant that the linker sequence is not a sequence native to the A or B chain of a ricin-like toxin or precursor thereof. However, preferably, the linker sequence may be of a similar length to the linker sequence of a ricin-like toxin and should not interfere with the role of the B chain in cell binding and transport into the cytoplasm. When the linker sequence is cleaved the A chain becomes active or toxic. The nucleic acid molecule of the invention was cloned by subjecting a preproricin cDNA clone (pAP-144) to site-directed mutagenesis in order to generate a series of variants differing only in the sequence between the A and B chains (linker region). Oligonucleotides, corresponding to the extreme 5' and 3' ends of the preproricin gene were synthesized and used to PCR amplify the gene. Using the cDNA sequence for preproricin (Lamb et al., Eur. J. Biochem., 145:266-270, 1985), several oligonucleotide primers were designed to flank the start and stop codons of the preproricin open reading frame. The preproricin cDNA was amplified using the upstream primer Ricin-99 (or Ricin-109 may be used) and the downstream primer Ricinl729C with Vent DNA polymerase (New England Biolabs) using standard procedures (Sambrook et al., Molecular Cloning: A Laboratory Manual, Second Edition, (Cold Spring Harbor Laboratory Press, 1989)). The use of the upstream primer Ricin-109 circumvents the subcloning step into vector pSB2. The purified PCR fragment encoding the preproricin cDNA was then ligated into an Eco RV-digested pBluescript II SK plasmid (Stratagene), and used to transform competent XLl-Blue cells (Stratagene). The cloned PCR product containing the putative preproricin gene was confirmed by DNA sequencmg of the entire cDNA clone (pAP-144) The sequences and location of oligonucleotide primers used for sequencmg are shown m Table 1 The preproricin cDNA clone (pAP-144) was subjected to site-directed mutagenesis m order to generate a series of variants differing only in the sequence between the A and B chams (lmker region) The wild-type preproricin lmker region was replaced with the three lmker sequences, pAP-146, pAP-147 and pAP-148 shown in Figure 4 The lmker regions of the vaπants encode an HIV protease cleavage recognition sequence (Slalka et al., Cell, 56:911-913, 1989). The mutagenesis and cloning strategy used to generate the linker variants pAP-146, pAP-147 and pAP-148 are summarized in Figures IA and IB, 2A and 2B and 3A and 3B respectively The first step mvolved a DNA amplification usmg a set of mutagemc primers (HIVA 1, 2, HIVB 1, 2, HIVH 1, 2) m combmation with the two flanking primers Rιcιn-99Eco and Rιcιnl729Xba Restriction digested PCR fragments were gel purified and then ligated with PBluescript SK which had been digested with Eco RI and Xba I Ligation reactions were used to transform competent XLl-Blue cells (Stratagene) Recombmant clones were identified by restriction digests of plasmid miruprep DNA and the mutant linker sequences were confirmed by DNA sequencmg Recombmant clones were subcloned mto vector pSB2. The three constructs obtamed were pAP-151, pAP-159, and pAP-163, with each havmg the mutant lmker found m pAP- 146, pAP-147, and pAP-148 respectively The clonmg strategy described above may also be applied to the preparation of recombmant clones contammg a cleavage recognition site for a human T-cell leukemia virus protease For example, recombmant clones pAP-205, pAP-207, pAP-209, and pAP- 211 were prepared usmg a method similar to the one described above The nucleic acid molecule of the mvention has sequences encoding an A cham of a non-like toxm, a B chain of a ricm-like toxin and a heterologous linker sequence contammg a cleavage recognition site for a retroviral protease, such as an HIV protease or an HTLV protease The nucleic acid may be expressed to provide a recombmant protem havmg an A cham of a ricin-like toxm, a B cham of a ricin-like toxm and a heterologous linker sequence contammg a cleavage recognition site for a retroviral protease, such as an HIV protease or an HTLV protease The nucleic acid molecule may comprise the A and /or B cham of πcm The πcm gene has been cloned and sequenced, and the X-ray crystal structures of the A and B chains are published (Rutenber, E., et al Protems 10 240-250 (1991), Weston et al , Mol Bio 244:410-422, 1994; Lamb and Lord Eur J Biochem 14.265 (1985), Hailing, K , et al Nucleic Acids Res 13:8019 (1985)) It will be appreciated that the mvention mcludes nucleic acid molecules encodmg truncations of A and B chams of ricin-like protems and analogs and homologs of A and B chams of ricin-like protems and truncations thereof (i.e ,πcιn-lιke proteins), as described herein. It will further be appreciated that variant forms of the nucleic acid molecules of the invention which arise by alternative splicing of an mRNA corresponding to a cDNA of the invention are encompassed by the invention. Another aspect of the invention provides a nucleotide sequence which hybridizes under high stringency conditions to a nucleotide sequence encoding the A and /or B chains of a ricin-like protein. Appropriate stringency conditions which promote DNA hybridization are known to those skilled in the art, or can be found in Current Protocols in Molecular Biology, John Wiley & Sons, N.Y. (1989), 6.3.1-6.3.6. For example, 6.0 x sodium chloride /sodium citrate (SSC) at about 45°C, followed by a wash of 2.0 x SSC at 50°C may be employed. The stringency may be selected based on the conditions used in the wash step. By way of example, the salt concentration in the wash step can be selected from a high stringency of about 0.2 x SSC at 50°C. In addition, the temperature in the wash step can be at high stringency conditions, at about 65°C. The nucleic acid molecule may comprise the A and /or B chain of a ricin-like toxin. Methods for cloning ricin-like toxins are known in the art and are described, for example, in E.P. 466,222. Sequences encoding ricin or ricin-like A and B chains may be obtained by selective amplification of a coding region, using sets of degenerative primers or probes for selectively amplifying the coding region in a genomic or cDNA library. Appropriate primers may be selected from the nucleic acid sequence of A and B chains of ricin or ricin- like toxins. It is also possible to design synthetic oligonucleotide primers from the nucleotide sequences for use in PCR. Suitable primers may be selected from the sequences encoding regions of ricin-like proteins which are highly conserved, as described for example in U.S. Patent No 5,101,025 and E.P. 466,222. A nucleic acid can be amplified from cDNA or genomic DNA using these oligonucleotide primers and standard PCR amplification techniques. The nucleic acid so amplified can be cloned into an appropriate vector and characterized by DNA sequence analysis. It will be appreciated that cDNA may be prepared from mRNA, by isolating total cellular mRNA by a variety of techniques, for example, by using the guanidinium- thiocyanate extraction procedure of Chirgwin et al., Biochemistry, 18, 5294-5299 (1979). cDNA is then synthesized from the mRNA using reverse transcriptase (for example, Moloney MLV reverse transcriptase available from Gibco /BRL, Bethesda, MD, or AMV reverse transcriptase available from Seikagaku America, Inc., St. Petersburg, FL). It will be appreciated that the methods described above may be used to obtain the coding sequence from plants, bacteria or fungi, preferably plants, which produce known ricin-like proteins and also to screen for the presence of genes encoding as yet unknown ricin-like proteins. A sequence containing a cleavage recognition site for a retroviral protease may be selected based on the retrovirus which is to be targeted by the recombmant protem The cleavage recognition site may be selected from sequences known to encode a cleavage recognition site for the retrovirus protease Sequences encodmg cleavage recognition sites may be identified by testmg the expression product of the sequence for susceptibility to cleavage by a retroviral protease An assay to identify peptides having cleavage recognition sites for HIV protease is described m PCT/US88/01849 The HIV protease encoded by the pl7 gene of HIV and has the highly conserved Asp-Thr-Gly sequence characteristic of the active site of cellular aspartyl proteases The HIV protease may be prepared by methods known m the art and used to test suspected cleavage recognition sites For example, a polypeptide contammg the suspected cleavage recognition site may be mcubated with the protease and the amount of cleavage product determmed (Dilanrut, 1990, J Biol Chem 285 17345-17354) Substrates for HIV proteases are described m U S Patent No 5,235,039 The mvention is not restricted to protems mcludmg the cleavage recognition site for HIV proteases, but includes recognition sites of other retroviral proteases, mcludmg proteases of members of the subfamilies oncovirmae, lentiviπnae and spumavirmae for example from HTLV, AMV, RSV, BLV, FeLV and MMTV Examples of retroviral proteases and conserved sequences thereof are provided, for example, in Katoh et al , (Nature 329654-656) A sequence contammg a cleavage recognition site for an HTLV protease may be selected usmg the conventional methods described herein The preparation of human T- cell leukemia virus proteases, their substrates and enzymatic activity assay methodology have been described by Petit, S C et al (J Biol Chem 266 14539-14547 (1991)) and Blaha, I et al (FEBS Lett 309389-393 (1992)) In an embodiment, the cleavage recognition site is the cleavage recognition site for an HIV protease In a particular embodiment, the lmker ammo acid sequence comprises VSQNYPIVQNFN, SKARVLAEAMSN, or SIRKILFLDGIN In further particular embodiments, the nucleic acid has the nucleotide sequence shown m Figure 8, Figure 9 or Figure 10 In another embodiment, the cleavage recognition site is the cleavage recognition site for a human T-cell leukemia virus protease In a particular embodiment, the lmker ammo acid sequence comprises SAPQVLPVMHPN or SKTKVLVVQPKN cleaved by a human T-cell leukemia virus-I (HTLV-I) protease, or, SKTKVLVVQPRN or STTQCFPILHPN cleaved by a human T-cell leukemia virus-II (HTLV-II) protease The nucleic acid molecule of the mvention may be prepared by site directed mutagenesis For example, the cleavage site of a retroviral protease may be prepared by site directed mutagenesis of the homologous lmker sequence of a proπcin-like toxm Procedures for cloning proncin-hke genes, encodmg a lmker sequence are described in EP 466,222. Site directed mutagenesis may be accomplished by DNA amplification of mutagenic primers in combination with flanking primers. Suitable procedures usmg the mutagenic primers HIVA1, HIVB1 and HIVH1 are shown in Figures IA to 3B, and Figures 16A, 16B, 17A, 17B, 18A, 19A and 19B. The nucleic acid molecule of the invention may also encode a fusion protein. A sequence encoding a heterologous linker sequence containing a cleavage recognition site for a retroviral protease may be cloned from a cDNA or genomic library or chemically synthesized based on the known sequence of such cleavage sites. The heterologous linker sequence may then be fused in frame with the sequences encoding the A and B chains of the ricin-like toxin for expression as a fusion protein. It will be appreciated that a nucleic acid molecule encoding a fusion protein may contain a sequence encoding an A chain and a B chain from the same ricin-like toxin or the encoded A and B chains may be from different toxins. For example, the A chain may be derived from ricin and the B chain may be derived from abrin. A protein may also be prepared by chemical conjugation of the A and B chains and linker sequence using conventional coupling agents for covalent attachment. An isolated and purified nucleic acid molecule of the invention which is RNA can be isolated by cloning a cDNA encoding an A and B chain and a linker into an appropriate vector which allows for transcription of the cDNA to produce an RNA molecule which encodes a protein of the invention. For example, a cDNA can be cloned downstream of a bacteriophage promoter, (e.g. a T7 promoter) in a vector, cDNA can be transcribed in vitro with T7 polymerase, and the resultant RNA can be isolated by standard techniques. Recombinant Protein of the Invention As previously mentioned, the invention provides novel recombinant proteins which incorporate the A and B chains of a ricin-like toxin linked by a heterologous linker sequence containing a cleavage recognition site for a retroviral protease, such as an HIV protease or an HTLV protease. It is an advantage of the recombinant proteins of the invention that they are non-toxic until the A chain is liberated from the B chain by specific cleavage of the linker by the retroviral protease, such as an HIV protease or an HTLV protease. Thus the protein may be used to specifically target cells infected with the retrovirus in the absence of additional specific cell-binding components to target infected cells. It is a further advantage that the retroviral protease cleaves the heterologous linker intracellularly thereby releasing the toxic A chain directly into the cytoplasm of the infected cell. As a result, infected cells are specifically targeted and non-infected cells are not directly exposed to the activated free A chain. Ricin is a plant derived ribosome inhibiting protein which blocks protein synthesis in eukaryotic cells. Ricin may be derived from the seeds of Ricinus communis (castor oil ρlant).The πcm toxm is a glycosylated heterodimer with A and B cham molecular masses of 30,625 Da and 31,431 Da respectively The A cham of ricin has an N- glycosidase activity and catalyzes the excision of a specific adenine residue from the 28S rRNA of eukaryotic πbosomes (Endo, Y, & Tsurugi, K J Biol Chem 262.8128 (1987)) The B cham of ricin, although not toxic in itself, promotes the toxicity of the A chain by binding to galactose residues on the surface of eukaryotic cells and stimulating receptor- mediated endocytosis of the toxm molecule (Simmons et al Biol Chem 261-7912 (1986)) Protem toxms are initially produced in an inactive, precursor form Ricm is initially produced as a smgle polypeptide (preproπcm) with a 35 ammo acid N-termmal presequence and 12 ammo acid lmker between the A and B chams The pre-sequence is removed dunng translocation of the ricm precursor mto the endoplasmic reticulum (Lord, J.M Eur J Biochem 146 403-409 (1985) and Lord, J M Eur J Biochem 146 411-416 (1985)) The proπcm is then translocated mto specialized organelles called protem bodies where a plant protease cleaves the protem at a lmker region between the A and B chams (Lord, J.M. et al., FASAB Journal 8 201-208 (1994)) The two chams, however, remain covalently attached by an interchain disulfide bond (cysteme 259 in the A chain to cysteme 4 in the B cham) and mature disulfide Imked ricm is secreted from the plant cells. The A cham is mactive m the proπcm (O'Hare, M , et al FEBS Lett 273 200-204 (1990)) and it is inactive m the disulhde-linked mature πcιn(Rιchardson, P T , et al FEBS Lett 255:15-20 (1989)) The πbosomes of the castor bean plant are themselves susceptible to inactivation by ricm A cham, however, as there is no cell surface galactose to permit B chain recognition the A cham cannot re-enter the cell Ricin-like protems mclude bacterial, fungal and plant toxms which have A and B chams and mactivate πbosomes and inhibit protein synthesis The A cham is an active polypeptide subunit which is responsible for the pharmacologic effect of the toxm In most cases the active component of the A cham is an enzvme The B cham is responsible for binding the toxm to the cell surface and is thought to facilitate entry of the A cham mto the cell cytoplasm The A and B chams m the mature toxms are lmked by disulfide bonds The toxms most similar m structure to ricm are plant toxms which have one A cham and one B cham Examples of such toxms mclude abπn which may be isolated from the seeds of Abrus precatortus, ricm which may be isolated from the seeds of castor beans Ricinus commums, and modeccm Ricm-like bacterial proteins include diphtheria toxm, which is produced by Corynebacterium dtphtheriae, Pseudomonas enterotoxin A and cholera toxin It will be appreciated that the term ricin-like toxms is also mtended to mclude the A chain of those toxms which have only an A cham The recombmant protems of the mvention could mclude the A cham of these toxms conjugated to, or expressed as, a recombmant protem with the B cham of another toxm Examples of plant toxins havmg only an A cham mclude tπchosanthin, MMC and pokeweed antiviral proteins, dianthin 30, dianthin 32, crotin II, curcm II and wheat germ inhibitor Examples of fungal toxms havmg onlv an A cham mclude alpha-sarcin, restπctocin, mitogillm, enomycin, phenomycin. Examples of bacterial toxms havmg only an A cham mclude cytotoxm from Shigella dysenteriae and related Shiga-hke toxms. Recombmant tπchosanthin and the coding sequence thereof is disclosed m U.S. Patents Nos. 5,101,025 and 5,128,460 In addition to the entire B or A chams of a πcin-like toxm, it will be appreciated that the recombinant protem of the invention may contain only that portion of the A chain which is necessary for exerting its cytotoxic effect. For example, the first 30 ammo acids of the ricm A cham may be removed resultmg m a truncated A cham which retams toxic activity. The truncated ncm or ricin-like A cham may be prepared by expression of a truncated gene or by proteolytic degradation, for example with Nagarase (Funmatsu et al. , 1970, Jap. J Med. Sci. Biol 23:264-267) Similarly, the recombinant protein of the invention may contam only that portion of the B cham necessary for galactose recognition, cell bmdmg and transport mto the cell cytoplasm Truncated B chains are described for example m EP 145,111. The A and B chams may be glycosylated or non-glycosylated Glycosylated A and B chams may be obtamed by expression m the appropriate host cell capable of glycosylation. Non-glycosylated chams may be obtamed by expression m nonglycosylatmg host cells or by treatment to remove or destroy the carbohydrate moieties The protems of the mvention may be prepared usmg recombmant DNA methods. Accordmgly, the nucleic acid molecules of the present mvention may be incorporated m a known manner mto an appropriate expression vector which ensures good expression of the protem Possible expression vectors mclude but are not limited to cosmids, plasmids, or modified viruses (e.g. replication defective retroviruses, adenoviruses and adeno- associated viruses), so long as the vector is compatible with the host cell used The expression vectors are "suitable for transformation of a host cell", means that the expression vectors contam a nucleic acid molecule of the invention and regulatory sequences selected on the basis of the host cells to be used for expression, which is operatively lmked to the nucleic acid molecule Operatively lmked is mtended to mean that the nucleic acid is lmked to regulatory sequences in a manner which allows expression of the nucleic acid The mvention therefore contemplates a recombinant expression vector of the mvention containmg a nucleic acid molecule of the mvention, or a fragment thereof, and the necessary regulatory sequences for the transcription and translation of the inserted protem-sequence Suitable regulatory sequences mav be derived from a variety of sources, mcludmg bacterial, fungal, viral, mammalian, or insect genes (For example, see the regulatory sequences described m Goeddel, Gene Expression Technology Methods m Enzymology 185, Academic Press, San Diego, CA (1990) Selection of appropriate regulatory sequences is dependent on the host cell chosen as discussed below, and may be readily accomplished by one of ordmary skill m the art Examples of such regulatory sequences mclude a transcriptional promoter and enhancer or RNA polymerase bmdmg sequence, a ribosomal bmdmg sequence, including a translation initiation signal Additionally, depending on the host cell chosen and the vector employed, other sequences, such as an ongm of replication, additional DNA restriction sites, enhancers, and sequences conferrmg inducibihty of transcription may be incorporated into the expression vector. It will also be appreciated that the necessary regulatory sequences may be supplied by the native A and B chams and /or its flanking regions The recombmant expression vectors of the mvention may also contam a selectable marker gene which facilitates the selection of host cells transformed or transfected with a recombmant molecule of the mvention Examples of selectable marker genes are genes encodmg a protem such as G418 and hygromycin which confer resistance to certain drugs, β-galactosidase, chloramphenicol acetyltransferase, firefly luciferase, or an immunoglobulin or portion thereof such as the Fc portion of an immunoglobulin preferably IgG Transcription of the selectable marker gene is monitored by changes in the concentration of the selectable marker protem such as β-galactosidase, chloramphenicol acetyltransferase, or firefly luciferase If the selectable marker gene encodes a protein conferrmg antibiotic resistance such as neomycin resistance transformant cells can be selected with G418 Cells that have incorporated the selectable marker gene will survive, while the other cells die This makes it possible to visualize and assay for expression of recombinant expression vectors of the invention and in particular to determine the effect of a mutation on expression and phenotype It will be appreciated that selectable markers can be introduced on a separate vector from the nucleic acid of mterest The recombmant expression vectors may also contam genes which encode a fusion moiety which provides increased expression of the recombinant protein, increased solubility of the recombinant protein, and aid in the purification of the target recombmant protem by actmg as a ligand in affinity purification For example, a proteolytic cleavage site may be added to the target recombinant protein to allow separation of the recombmant protem from the fusion moiety subsequent to purification of the fusion protem Typical fusion expression vectors include pGEX (Amrad Corp , Melbourne, Australia), pMAL (New England Biolabs, Beverly, MA) and pRIT5 (Pharmacia, Piscataway, NJ) which fuse glutathione S-transferase (GST), maltose E bmdmg protem, or protem A, respectively, to the recombmant protem Recombmant expression vectors can be mtroduced mto host cells to produce a transformant host cell The term "transformant host cell" is intended to include prokaryotic and eukaryotic cells which have been transformed or transfected with a recombinant expression vector of the invention The terms "transformed with", "transfected with", "transformation' and 'transfection ' are intended to encompass introduction of nucleic acid (e g a vector) mto a cell by one of many possible techniques known in the art Prokaryotic cells can be transformed with nucleic acid by, for example, electroporation or calcium-chloride mediated transformation Nucleic acid can be mtroduced mto mammalian cells via conventional techniques such as calcium phosphate or calcium chloride co-precipitation, DEAE-dextran-mediated transfection, hpofectm, electroporation or πucroinjection. Suitable methods for transforming and transfecting host cells can be found in Sambrook et al (Molecular Clonmg. A Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory press (1989)), and other laboratory textbooks Suitable host cells mclude a wide variety of prokaryotic and eukaryotic host cells For example, the protems of the mvention may be expressed m bacterial cells such as E. colt, msect cells (usmg baculovirus), yeast cells or mammalian cells Other suitable host cells can be found m Goeddel, Gene Expression Technology Methods m Enzymology 185, Academic Press, San Diego, CA (199 1) More particularly, bacterial host cells suitable for carrymg out the present mvention mclude E coli, B subtilis, Salmonella typhimunum, and various species within the genus' Pseudomonas, Streptomyces, and Staphylococcus, as well as many other bacterial species well known to one of ordinary skill in the art Suitable bacterial expression vectors preferably comprise a promoter which functions in the host cell, one or more selectable phenotypic markers, and a bacterial origin of replication Representative promoters mclude the β-lactamase (penicillinase) and lactose promoter system (see Chang et al., Nature 275 615, 1978), the trp promoter (Nichols and Yanofsky, Meth in Enzymology 101.155, 1983) and the tac promoter (Russell et al , Gene 20 231, 1982) Representative selectable markers include various antibiotic resistance markers such as the kanamycin or ampicillin resistance genes Suitable expression vectors mclude but are not limited to bacteriophages such as lambda derivatives or plasmids such as pBR322 (see Bohvar et al , Gene 2.9S, 1977), the pUC plasmids pUC18, pUC19, pUCllδ, pUC119 (see Messmg, Meth m Enzymology 101.20-77, 1983 and Vieira and Messing, Gene 19 259-268, 1982), and pNH8A, pNH16a, pNH18a, and Bluescπpt M13 (Stratagene, La Jolla, Calif ) Typical fusion expression vectors which may be used are discussed above, e g pGEX (Amrad Corp , Melbourne, Australia), pMAL (New England Biolabs, Beverly, MA) and pRIT5 (Pharmacia, Piscataway, NJ) Examples of inducible non-fusion expression vectors mclude pTrc (Amann et al., (1988) Gene 69:301-315) and pET lid (Studier et al , Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, California (1990) 60-89) Yeast and fungi host cells suitable for carrying out the present invention include, but are not limited to Saccharomyces cerεvisae, the genera Pichia or Kluyveromyces and various species of the genus Aspergillus Examples of vectors for expression m yeast S. cerwisae mclude pYepSecl (Baldaπ. et al., (1987) Embo J 6 229-234), pMFa (Kur j an and Herskowitz, (1982) Cell 30.933-943), pJRY88 (Schultz et al , (1987) Gene 54-113-123), and pYES2 (Invitrogen Coφoration, San Diego, CA) Protocols for the transformation of yeast and fungi are well known to those of ordmary skill m the art.(see Hinnen et al , PNAS USA 75:1929, 1978; Itoh et al., J. Bacteriology 153:163, 1983, and Cullen et al. (Bio/Technology 5:369, 1987). Mammalian cells suitable for carrying out the present mvention include, among others: COS (e.g., ATCC No. CRL 1650 or 1651), BHK (e.g , ATCC No CRL 6281), CHO (ATCC No. CCL 61), HeLa (e.g., ATCC No. CCL 2), 293 (ATCC No 1573) and NS-1 cells. Suitable expression vectors for directing expression m mammalian cells generally include a promoter (e.g., derived from viral material such as polvoma, Adenovirus 2, cytomegalovirus and Simian Virus 40), as well as other transcriptional and translational control sequences. Examples of mammalian expression vectors mclude pCDM8 (Seed, B., (1987) Nature 329:840) and pMT2PC (Kaufman et al. (1987), EMBOJ 6:187-195) Given the teachmgs provided herein, promoters, terminators, and methods for mtroducmg expression vectors of an appropriate type mto plant, avian, and insect cells may also be readily accomplished. For example, within one embodiment, the protems of the invention may be expressed from plant cells (see Sinkar et al , J Biosci (Bangalore) 11:47-58, 1987, which reviews the use of Agrobacterium rhizogenes vectors; see also Zambryski et al., Genetic Engmeermg, Principles and Methods, Hollaender and Setlow (eds.), Vol. VI, pp. 253-278, Plenum Press, New York, 1984, which describes the use of expression vectors for plant cells, including, among others, pAS2022, pAS2023, and pAS2034). Insect cells suitable for carrying out the present mvention mclude cells and cell lines from Bombyx or Spodotera species. Baculovirus vectors available for expression of protems in cultured msect cells (SF 9 cells) include the pAc series (Smith ct al , (1983) Mol. Cell Biol. 3:2156-2165) and the pVL series (Lucklow, V A , and Summers, M.D., (1989) Virology 170:31-39). Some baculovirus-insect cell expression systems suitable for expression of the recombinant protems of the mvention are described in PCT/US/02442. Alternatively, the protems of the mvention may also be expressed in non-human transgenic animals such as, rats, rabbits, sheep and pigs (see Hammer et al. (Nature 315:680-683, 1985), Palmiter et al. (Science 222.809-814, 1983), Bπnster et al (Proc Natl. Acad. Sci USA 82:44384442, 1985), Palmiter and Brmster (Cell 41 343-345, 1985) and U.S Patent No. 4,736,866) The protems of the mvention may also be prepared by chemical synthesis usmg techniques well known m the chemistry of proteins such as solid phase synthesis (Memfield, 1964, J Am Chem Assoc. 85.2149-2154) or synthesis in homogenous solution (Houbenweyl, 1987, Methods of Organic Chemistry, ed F Wansch, Vol 15 I and II, Thieme, Stuttgart). The present mvention also provides protems comprismg an A cham of a ricm-like toxm, a B cham of a ricin-like toxm and a heterologous lmker ammo acid sequence lmkmg the A and B chams, wherem the lmker sequence contams a cleavage recognition site for a retroviral protease, such as an HIV protease or an HTLV protease Such a protem could be prepared other than by recombinant means, for example by chemical synthesis or by conjugation of A and B chains and a lmker sequence isolated and purified from their natural plant, fungal or bacterial source Such A and B chains could be prepared havmg the glycosylation pattern of the native πcin-hke toxm N-termmal or C-termmal fusion proteins comprising the protein of the mvention conjugated with other molecules, such as protems may be prepared by fusing, through recombinant techniques The resultant fusion protems contam a protem of the mvention fused to the selected protem or marker protein as described herein The recombmant protem of the mvention may also be conjugated to other protems bv known techniques For example the protems may be coupled usmg heterobifunctional thiol-containing linkers as described in WO 90/10457, N-succιnιmιdyl-3-(2-pyπdyldιthιo-proprιonate) or N- succmmudyl-5-thιoacetate Examples of proteins which may be used to prepare fusion protems or conjugates mclude cell bmdmg protems such as immunoglobulins, hormones, growth factors, lectms, msulm, low density lipoprotein, glucagon, endorphms, transferrin, bombesin, asialoglycoprotein glutathione-S-transferase (GST), hemagglutinin (HA), and truncated myc. Utility of the Nucleic Acid Molecules and Proteins of the Invention The protems of the invention mav be used to specifically inhibit or destroy mammalian cancer cells or mammalian cells mfected with a retrovirus. It is an advantage of the recombmant protems of the invention that they have specificity tor the infected cells without the need for a cell binding component The πcin-hke B cham of the recombinant proteins recognize galactose moieties on the cell surface and ensure that the protem is taken up by the cell and released into the cytoplasm When the protem is released mto a non-infected cell, the A cham will remam inactive bound to the B cham. However, when the protem is released mto a cell mfected with a retrovirus or contammg an HTLV or HIV protease, the retroviral protease will cleave the cleavage recognition site in the linker, releasing the toxic A chain. The specificity of a recombinant protein of the invention may be tested by treating the protein with the retroviral protease, such as HIV protease or HTLV protease which is thought to be specific for the cleavage recognition site of the linker and assaying for cleavage products. Retroviral proteases such as HIV protease or HTLV protease may be isolated from infected cells or may be prepared recombinantly, for example following the procedures in Darket et al. (1988, J. Biol. Chem. 254:2307-2312). The cleavage products may be identified for example based on size, antigenicity or activity. The toxicity of the recombinant protein may be investigated by subjecting the cleavage products to an in vitro translation assay in cell lysates, for example using Brome Mosaic Virus mRNA as a template. Toxicity of the cleavage products may be determined using a ribosomal inactivation assay (Westby et al. 1992, Bioconjugate Chem. 3:377-382). The effect of the cleavage products on protein synthesis may be measured in standardized assays of in vitro translation utilizing partially defined cell free systems composed for example of a reticulocyte lysate preparation as a source of ribosomes and various essential cofactors, such as mRNA template and amino acids. Use of radiolabelled amino acids in the mixture allows quantitation of incorporation of free amino acid precursors into trichloroacetic acid precipitabie proteins. Rabbit reticulocyte lysates may be conveniently used (O'Hare, FEBS Lett. 1990, 273:200-204). The ability of the recombinant proteins of the invention to selectively inhibit or destroy mammalian cells infected with a retrovirus such as cancer cells associated with HTLV or cells associated with HIV may be readily tested in vitro using mammalian cell cultures infected with the retrovirus of interest, or cancer lines. The selective inhibitory effect of the recombinant proteins of the invention may be determined by demonstrating the selective inhibition of viral antigen expression in mammalian cells, or selective inhibition of cellular proliferation in cancer cells or infected cells. For example, a selective inhibitory effect may be demonstrated by the selective inhibition of viral antigen expression in HIV-infected mononuclear phagocytic lineage cells; selective inhibition of cellular proliferation as measured against protein and DNA synthesis levels in treated, noninfected T cells and; selective loss of T cell viability. For example, the below-noted culture systems may be used to test the ability of recombinant proteins having a heterlogous linker sequence containing a cleavage recognition site for the HIV protease to selectively inhibit HIV infected cells. The term HIV refers to a CD4+ dependent human immunodeficiency retrovirus, such as HIV-1 and variants thereof. Normal human T lymphocytes may be prepared from peripheral blood samples and cultured in vitro, as generally described in U.S. Patent No. 4,869,903. HIV infected cells may also be obtained from AIDS patients. The cells may be infected in vitro with HIV derived from an AIDS patient. The toxicity of the recombinant protein for infected and non-infected cultures may then be compared. HIV-infected T cells express HIV envelope protein on the cell surface, in particular the proteins gpl20 and gp41. The abihty of the recombinant protein of the invention to inhibit the expression of these viral antigens may be an important indicator of the ability of the protein to inhibit viral replication. Toxicity may be measured based upon cell death or lysis, or by a reduction in the expression of HIV antigens, such as the major envelope proteins gpl20 and gp41 or the HIV core protein antigen p24. Levels of these antigens may be measured in assays using labelled antibodies having specificity for the antigens. Inhibition of viral antigen expression has been correlated with inhibition of viral replication (U.S. Patent No. 4,869,903). Similar assays may be carried out using other suitable mammalian cells which can be cultured in vitro and which are capable of maintaining retroviral replication. Examples of suitable cells include mononuclear phagocytic lineage cells. Toxicity may also be assessed based on a decrease in protein synthesis in target cells, which may be measured by known techniques, such as incorporation of labelled amino acids, such as [3H] leucine (O'Hare et al. 1990, FEBS Lett. 273:200-204). Infected cells may also be pulsed with radiolabelled thymidine and incoφoration of the radioactive label into cellular DNA may be taken as a measure of cellular proliferation. In the models of viral infection and replication for confirming the activity of the recombinant proteins of the invention, suitable mammalian cells used as hosts are those cells which can be cultured in vitro and which are capable of maintaining viral replication. Examples of suitable cells can be human T lymphocytes or mononuclear phagocytic lineage cells. Normal human T lymphocytes may be prepared from peripheral blood samples and cultured in vitro, as generally described in U.S. Patent No. 4,869,903. Virally infected cells may also be obtained from the blood of infected patients. The toxicity of the recombinant protein for infected and non-infected cultures may then be compared. The ability of the recombinant protein of the invention to inhibit the expression of these viral antigens may be an important indicator of the ability of the protein to inhibit viral replication. Levels of these antigens may be measured in assays using labelled antibodies having specificity for the antigens. Inhibition of viral antigen expression has been correlated with inhibition of viral replication (U.S. Patent No. 4,869,903). Toxicity may also be assessed based on a decrease in protein synthesis in target cells, which may be measured by known techniques, such as incorporation of labelled amino acids, such as [3H] leucine (O'Hare et al. 1990, FEBS Lett. 273:200-204). Infected cells may also be pulsed with radiolabelled thymidine and incorporation of the radioactive label into cellular DNA may be taken as a measure of cellular proliferation. In addition, toxicity may be measured based on cell viability, for example the viability of infected and non-infected cell cultures exposed to the recombinant protein may be compared. Cell viability may be assessed by known techniques, such as trypan blue exclusion assays. Although, the specificity of the proteins of the invention for retrovirally infected cells is mediated by the specific cleavage of the cleavage recognition site of the linker, it will be appreciated that specific cell binding components may optionally be conjugated to the proteins of the invention. Such cell binding components may be expressed as fusion proteins with the proteins of the invention or the cell binding component may be physically or chemically coupled to the protein component. Examples of suitable cell binding components include antibodies to retroviral proteins, or to cancer cell proteins. Antibodies having specificity for a cell surface protein may be prepared by conventional methods. A mammal, (e.g., a mouse, hamster, or rabbit) can be immunized with an immunogenic form of the peptide which elicits an antibody response in the mammal. Techniques for conferring immunogenicity on a peptide include conjugation to carriers or other techniques well known in the art. For example, the peptide can be administered in the presence of adjuvant. The progress of immunization can be monitored by detection of antibody titers in plasma or serum. Standard ELISA or other immunoassay procedures can be used with the immunogen as antigen to assess the levels of antibodies. Following immunization, antisera can be obtained and, if desired, polyclonal antibodies isolated from the sera. To produce monoclonal antibodies, antibody producing cells (lymphocytes) can be harvested from an immunized animal and fused with myeloma cells by standard somatic cell fusion procedures thus immortalizing these cells and yielding hybridoma cells. Such techniques are well known in the art, (e.g., the hybridoma technique originally developed by Kohler and Milstein (Nature 256, 495-497 (1975)) as well as other techniques such as the human B-cell hybridoma technique (Kozbor et al., Immunol. Today 4, 72 (1983)), the EBV-hybridoma technique to produce human monoclonal antibodies (Cole et al. Monoclonal Antibodies in Cancer Therapy (1985) Allen R. Bliss, Inc., pages 77-96), and screening of combinatorial antibody libraries (Huse et al., Science 246, 1275 (1989)]. Hybridoma cells can be screened immunochemically for production of antibodies specifically reactive with the peptide and the monoclonal antibodies can be isolated. The term "antibody" as used herein is intended to include fragments thereof which also specifically react with a cell surface component. Antibodies can be fragmented using conventional techniques and the fragments screened for utility in the same manner as described above. For example, F(ab')2 fragments can be generated by treating antibody with pepsin. The resulting F(ab')2 fragment can be treated to reduce disulfide bridges to produce Fab' fragments. Chimeric antibody derivatives, i.e., antibody molecules that combine a non- human animal variable region and a human constant region are also contemplated within the scope of the invention. Chimeric antibody molecules can include, for example, the antigen binding domain from an antibody of a mouse, rat, or other species, with human constant regions. Conventional methods may be used to make chimeric antibodies containing the immunoglobulin variable region which recognizes a cell surface antigen (See, for example, Morrison et al., Proc. Natl Acad. Sci. U.S.A. 81,6851 (1985); Takeda et al., Nature 314, 452 (1985), Cabilly et al., U.S. Patent No. 4,816,567; Boss et al., U.S. Patent No. 4,816,397; Tanaguchi et al., European Patent Publication EP171496; European Patent Publication 0173494, United Kingdom patent GB 2177096B). It is expected that chimeric antibodies would be less immunogenic in a human subject than the corresponding non-chimeric antibody. Monoclonal or chimeric antibodies specifically reactive against cell surface components can be further humanized by producing human constant region chimeras, in which parts of the variable regions, particularly the conserved framework regions of the antigen-binding domain, are of human origin and only the hypervariable regions are of non-human origin. Such immunoglobulin molecules may be made by techniques known in the art, (e.g., Teng et al., Proc. Natl. Acad. Sci. U.S.A., 80, 7308-7312 (1983); Kozbor et al., Immunology Today, 4, 7279 (1983); Olsson et al, Meth. Enzymol., 92, 3-16 (1982)), and PCT Publication WO92/06193 or EP 0239400). Humanized antibodies can also be commercially produced (Scotgen Limited, 2 Holly Road, Twickenham, Middlesex, Great Britain.) Specific antibodies, or antibody fragments, reactive against cell surface components may also be generated by screening expression libraries encoding immunoglobulin genes, or portions thereof, expressed in bacteria with cell surface components. For example, complete Fab fragments, VH regions and FV regions can be expressed in bacteria using phage expression libraries (See for example Ward et al., Nature 341, 544-546: (1989); Huse et al., Science 246, 1275-1281 (1989); and McCafferty et al. Nature 348, 552-554 (1990)). Alternatively, a SCID-hu mouse, for example the model developed by Genpharm, can be used to produce antibodies, or fragments thereof. The proteins of the invention may be formulated into pharmaceutical compositions for adminstration to subjects in a biologically compatible form suitable for administration in vivo . By "biologically compatible form suitable for administration in vivo" is meant a form of the substance to be administered in which any toxic effects are outweighed by the therapeutic effects The substances may be administered to living organisms mcludmg humans, and animals Administration of a therapeutically active amount of the pharmaceutical compositions of the present invention is defined as an amount effective, at dosages and for periods of time necessary to achieve the desired result For example, a therapeutically active amount of a substance may vary accordmg to factors such as the disease state, age, sex, and weight of the individual, and the ability of antibody to elicit a desired response in the individual Dosage regime may be adjusted to provide the optimum therapeutic response For example, several divided doses may be administered daily or the dose may be proportionally reduced as indicated by the exigencies of the therapeutic situation The active substance may be admmistered in a convenient manner such as by in j ection (subcutaneous, mtravenous, etc ), oral administration, inhalation, transdermal application, or rectal administration Depending on the route of administration, the active substance may be coated m a material to protect the compound from the action of enzymes, acids and other natural conditions which may inactivate the compound The compositions described herein can be prepared by per se known methods for the preparation of pharmaceutically acceptable compositions which can be administered to subjects, such that an effective quantity of the active substance is combmed in a mixture with a pharmaceutically acceptable vehicle Suitable vehicles are described, for example, m Remington's Pharmaceutical Sciences (Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, Pa , USA 1985) On this basis, the compositions mclude, albeit not exclusively, solutions of the substances in association with one or more pharmaceutically acceptable vehicles or diluents, and contained in buffered solutions with a suitable pH and iso-osmotic with the physiological fluids The pharmaceutical compositions may be used in methods for treatmg mammals, mcludmg humans, mfected with a retrovirus It is anticipated that the compositions will be particularly useful for treating patients infected with HIV-1, HIV-2 or cancers mvolvmg retroviruses, such as human T-cell leukemias involving HTLV The efficacy of such treatments may be monitored by assessing the health of the patient treated and by measuring the percentage of HIV positive monocytes in treated patients The dose of the recombmant protem to be administered will depend on a variety of factors which may be readily monitored in human subjects Such factors include HIV antigen levels associated with HIV infected T cells or mononuclear phagocytes, HIV antigen levels in the bloodstream, reverse transcriptase activ ity associated with HIV- mfected T cells or mononuclear phagocytes, and the ratio of viable HIV infected cells to unmfected cells HIV antigen levels m plasma, for example, may be readily determined usmg an ELISA assay The followmg non-limiting examples are illustrative of the present mvention EXAMPLES EXAMPLE 1 Cloning and Expression of Proricin Variants Activated by HIV Proteases Isolation of total RNA The preproπcm gene was cloned from new foliage of the castor bean plant Total messenger RNA was isolated according to established procedures (Maniatis et al , Molecular Cloning A Lab Manual (Cold Sprmg Harbour Press Cold Sprmg Harbour, (1989)) and cDNA generated usmg reverse transcriptase cDNA Synthesis Oligonucleotides, correspondmg to the extreme 5 and 3 ends of the preproπcm gene were synthesized and used to PCR amplify the gene Usmg the cDNA sequence for preproricin (Lamb et al., Eur J Biochem , 145 266-270, 1985), several oligonucleotide primers were designed to flank the start and stop codons of the preproricin open readmg frame The oligonucleotides were synthesized using an Applied Biosystems Model 392 DNA/ RNA Synthesizer First strand cDNA synthesis was primed using the oligonucleotide Rιcιnl729C (Table 1) Three micrograms of total RNA was used as a template for oligo Rιcιnl729C primed synthesis of cDNA using Superscript II Reverse Transcriptase (BRL) followmg the manufacturer's protocol DNA Amplification and Clonmg The first strand cDNA synthesis reaction was used as template for DNA amplification by the polymerase chain reaction (PCR) The preproricin cDNA was amplified using the upstream primer Rιcιn-99 and the downstieam primer Rιcιnl729C with Vent DNA polymerase (New England Biolabs) usmg standai d procedures (Sambrook et al., Molecular Clonmg A Laboratory Manual, Second Edition, (Cold Spring Harbor Laboratory Press, 1989)) Amplification was carried out in a Biometra thermal cycler (TRIO-Thermalcycler) using the followmg cycling parameters denaturation 95 C C for 1 mm., annealing 52°C for 1 mm , and extension 72°C for 2 mm , (33 cycles), followed by a fmal extension cycle at 72°C for 10 mm The 1846bp amplified product was fractionated on an agarose gel (Sambrook et al , Molecular Cloning A Laboratory Manual, Second Edition, (Cold Sprmg Harbor Laboratory Press, 1989), and the DNA purified from the gel slice usmg Qiaex resin (Qiagen) following the manufacturer's protocol The purified PCR fragment encodmg the preproncm cDNA was then ligated (Sambrook et al , Molecular Clonmg A Laboratory Manual, Second Edition, (Cold Spring Harbor Laboratory Press, 1989)) mto an Eco RV-digested pBluescript II SK plasmid (Stratagene), and used to transform competent XLl-Blue cells (Stratagene) Positive clones were confirmed by restriction digestion of purified plasmid DNA Plasmid DNA was extracted usmg a Qiaprep Spin Plasmid Mmiprep Kit (Qiagen) DNA Sequencmg The cloned PCR product contammg the putative preproπcm gene was confirmed by DNA sequencmg of the entire cDNA clone (pAP-144) Sequencing was performed using an Applied Biosystems 373A Automated DNA Sequencer, and confirmed by double-stranded dideoxy sequencmg by the Sanger method using the Sequenase kit (USB) The oligonucleotide primers used for sequencing were as follows Rιcιn267, Rιcιn486, Rιcιn725, Rιcιn937, Rιcmll51, Rιcιnιl399, Rιcml627, T3 primer (5ΑATTAACCCTCACTAAAGGG-3') and T7 primer (5'GTAATACGACTCACTATAGGGC-3) Sequence data was compiled and analyzed usmg PC Gene software package (intelligenetics) The sequences and location of oligonucleotide primers is shown m Table 1 Mutagenesis of Preproπcm Lmker The preproπcm cDNA clone (pAP-144) was subjected to site-directed mutagenesis m order to generate a series of variants differmg only m the sequence between the A and B chains (lmker region) The wild-type preproricin lmker region was replaced with the three lmker sequences, pAP-146, pAP-147 and pAP-148 displayed in Figure 4 The lmker regions of the variants encode an HIV protease cleavage recognition sequence (Slalka et al , Cell, 56 911-913, 1989) The mutagenesis and clonmg strategy used to generate the lmker variant pAP-146 is summarized m Figures IA and IB The mutagenesis and clonmg strategy used to generate the linker variant pAP-147 is summarized in Figures 2A and 2B The mutagenesis and clonmg strategy used to generate the linker variant pAP-148 is summarized m Figures 3A and 3B The first step involved a DNA amplification usmg a set of mutagenic primers (HIVA1, HIVB1, HIVH1) in combination with the two flankmg primers Rιcιn-99Eco and Rιcιnl729Xba The PCR protocol and conditions used were the same as descnbed above PCR products from each mutagenesis reaction were gel purified then restriction digested with either Eco Rl for the A-chain encoding fragment, or Xba I for the B cham encodmg fragment Restriction digested PCR fragments were gel purified and then ligated with PBluescript SK which had been digested with Eco RI and Xba I Ligation reactions were used to transform competent XLl-Blue cells (Stratagene) Recombmant clones were identified by restriction digests of plasmid mmiprep DNA and the mutant linker sequence were confirmed by DNA sequencmg Subclonmg Preproπcm Mutants into Vector pSB2 Full length preproπcm cDNA was created from clones pAP-146, pAP-147, and pAP-148, which lack the first three nucleotides of the signal sequence (Hailing et al, Nucleic Acids Research, 13 8019-8033, 1985) The missing ATG (stait codon) was mtroduced mto each mutant by site-directed mutagenesis using primers Ricin-109 and Rιcml729C The DNA template for each reaction was pAP-146, pAP-147, or pAP-148, and the PCR conditions were the same as described above PCR products were gel purified and then ligated with Sma I-digested pSB2 (see Figure 12) Recombinant clones were identified by restriction digests of plasmid mmiprep DNA, and the 5' and 3' j unctions confirmed by DNA sequencmg The three constructs obtained were pAP-151, ρAP-159, and pAP-163, with each havmg the mutant lmker found in pAP-146, pAP-147 and pAP-148 respectively Subclonmg Preproricin Mutants mto Vector pVL1393 Preproπcm variants were subcloned mto the baculovirus transfer vector pVL1393 (PharMmgen, sequence shown in Figure 11) The subclonmg strategy for the HIV-A lmker variant is summarized m Figure 5 The subclonmg strategy for the HIV-B lmker variant is summarized in Figure 6 The subcloning strategy for the HIV-H linker variant is summarized m Figure 7 The 1315 bp Eco RI/Kpn I fragment cncodmg the ricm A-cham and each mutant linker was isolated from each of the variant clones in pSB2 (pAP-151, pAP-159, and pAP-163) Each of these purified fragments was ligated with a 564 bp Kpnl/PStl fragment obtamed from pAP-144, and with Eco Rl/Pst I cleaved pVL1393 Recombmant clones were identified by restriction digests of plasmid minφrep DNA and the 5' and 3' j unctions confirmed by DNA sequencmg The three constructs obtamed were pAP-190, pAP-196, and pAP-197, each having the mutant linker found in pAP-146, pAP- 147, and pAP-148, respectively Isolation of Recombmant Baculoviruses Insect cells S frugiperda (Sf9), and Tnchoplusia m (Tn368 and BTI-TN-581-4 (High Five)) were mamtamed on TMN-FH medium supplemented with 10% total calf serum (Summers et al , A Manual of Methods of Baculovims Vectors and Insect Cell Culture Procedures, (Texas Agricultural Experiment Station, 1987)) Two micrograms of recombmant pVL1393 DNA (pAP-190, pAP-196, or pAP-197) was co-transfected with 0 5 microgram of BaculoGold AcNPV DNA (Pharmingen) into 2 x 10 6 Tn368 insect cells followmg the manufacturer's protocol (Gruenwald et al , Baculovirus Expression Vector System Procedures and Methods Manual, 2nd Edition, (San Diego, CA, 1993)) On day 5 post-transfection, media were centrifuged and the supernatants tested in limiting dilution assays with Tn368 cells (Summers et al , A Manual of Methods of Baculovπ us Vectors and Insect Cell Culture Procedures, (Texas Agricultural Experiment Station, 1987)) Recombmant viruses in the supernatants were then amplified by infecting Tn368 cells at a multiplicity of mfection (moi) of 0 1, followed by collection of day 7 supernatants A total of three rounds of amplification were performed for each recombinant following established procedures (Summers et al , A Manual of Methods of Baculovirus Vectors and Insect Cell Culture Procedures, (Texas Agricultural Expeπment Station, 1987 and Gruenwald et al., Baculovirus Expression Vector System: Procedures and Methods Manual, 2nd Edition, (San Diego, CA, 1993)). Expression of Mutant Proricin Recombinant baculoviruses (pAP-190baculo, pAP-196baculo, and pAP-197-baculo) were used to infect 2 x 10 5 Tn368 or sf9 cells of an moi of 5 in EX-CELL400 media (JRH Biosciences) with 25mM α-lactose in spinner flasks. Media supernatants containing mutant proricins were collected on day 6 post-infection. Purification of Mutant Proricin Media supernatants were ultracentrifuged at 100,000g for 1 hour. After the addition of 1 mM phenylmethylsulfonyl fluoride, the supernatants were concentrated using an Amicon 8050 Ultrafiltration Cell fitted with a Diaflo XM50 membrane. Supernatants were then dialysed extensively against 137 mN NaCl, 2.2 mM KCl, 2.6 mM KH 2 P0 4 , and 8.6 mM Na 2 HP0 4 , pH 7.4 containing 1 mM dithiothreitol (dialysis buffer). Recombinant proricin proteins were purified by affinity chromatography using lactose agarose (Sigma) as previously described for recombinant ricin-B chain (Ferrini et al., Eur. J. Biochem., 233:772-777, 1995). Fractions containing recombinant proricin were identified using SDS/PAGE, (Sambrook et al., Molecular Cloning: A Laboratory Manual, Second Edition, (Cold Spring Harbor Laboratory Press, 1989) and by Western blot analysis using anti-ricin antibodies (Sigma). In Vitro HIV Protease Digestion of Proricin Variants Affinity -purified mutant proricin was treated with HIV protease to confirm specific cleavage in the linker region. Proricin variants were eluted from the lactose- agarose matrix in protease digestion buffer (50mM NaCl, 50mM Na-acetate, pH 5.5, ImM dithiothreitol) containing lOOmM lactose. Proricin substrate was then incubated at 37°C for 60 minutes with 400 ng/ml recombinant HIV protease (BACHEM Biosciences Inc.). The cleavage products of proricin (ricin A and B chains) were identified using SDS/PAGE (Sambrook et al., Molecular Cloning: a Laboratory Manual, 2nd. ed., Cold Spring Harbor Press, 1989), followed by Western blot analysis using anti-ricin antibodies (Sigma). In Vitro Translation Assay The activity of protease-treated proricin variants was monitored using a rabbit reticulocyte lysate in a non-radioactive (Amersham, ECL system) in vitro translation assay. Protease-treated proricin was added to a standard 50 μl translation reaction mix containing Brome Mosaic Virus mRNA as template (following the manufacturer's protocol). Active ricin variants inhibit the in vitro translation reaction by inactivating ribosomes. Therefore, in the presence of an active ricin variant, no viral proteins are synthesized. EXAMPLE 2 Harvesting and affinity column purification of pro-ricin variants Protem samples were harvested three days post transfection The cells were removed by centrifuging the media at 1465 g for ten minutes using a SLA-1500 (Sorvall) centrifuge rotor. The supernatant was further clarified by centrifuging at 7970g for fifteen minutes Protease inhibitor phenylmethyl-sulfonyl fluoride (Sigmn) was added to a final concentration of 1% The samples were concentrated (five-fold) and dialyzed (four times five-fold) mto dialysis buffer (IX baculo buffer (8 6 mM Na 2 HP0 4 , 26 mM KH 2 P0 4 , 137 mM naCl and 2.6 mM KCl, pH 7.4) contammg 2 5 mM lactose, and 0 02% NaN 3 ) usmg a MINITAN concentrator (Millipore) with 30kDa NMWL plates Dithiothreitol (DTT) was then added to a final concentration of 1 mM, and the samples were centrifuged at 37000g for one hour Followmg centrifugation, dialysis buffer containing 1 mM DTT was added to the samples to a final volume of 500 mL The samples were degassed and applied overnight at 4°C to an ASF-sepharose affinity column (prepared according to Pharmacia protocol) m a 10 mL chromatography column (Biorad) The column was washed with 300 mL of wash buffer (100 mM NaOAc, pH 5.2, ImM DTT, and 002% NaN 3 ) Elution of pro-πcm variant was performed by applymg 500 mL of elution buffer (100 mM NaOAc, pH 5 2, 250 mM lactose, and 5 mM DTT) The eluate was concentrated using an Amicon 8050 concentrator (Amicon) with a YM10 176 mm membrane, utilizing argon gas to pressurize the chamber The samples were further concentrated and dialyzed into IX Baculo buffer usmg Ultrafree-15 Biomax (Millipore) 10 kDa NMWl filter devices, which were spun in a Beckman S4180 rotor (Beckman) at 2000g Samples were flash frozen m dry ice and stored at -20°C Purification of pAP 190 by gel filtration chromatography In order to purify the pro-ricin variant from processed mateπal produced durmg fermentation, the protem was applied to a SUPERDEX 75 (16/60) column and SUPERDEX 200 (16/60) column (Pharmacia) connected in series equilibrated with 50 mM Tns, lOOmM NaCl, pH 7 5 contammg 100 mM Lactose and 0 1% β-mercaptocthanol (βME) The flow rate of the column was 0 15 mL/mrn and fractions were collected cverv 25 mmutes The UV (280 nm) trace was used to determine the approximate location of the purified pAP 190 and thus determine the samples for Western analysis Western analysis of column fractions Fractions eluted from the SUPERDEX columns (Pharmacia) were analyzed for purity usmg standard Western blottmg techniques An aliquot of lOμL from each fraction was boiled m IX sample buffer (626 mM Tπs-Cl, pH 6 8, 44% βME, 2% sodium dodecyl sulfate (SDS), 5% glycerol (all from Sigma) and 0 002% bromophenol blue (Biorad)) for five minutes. Denatured samples were loaded on 12% Tns-Glycine Gels (Biorad) along with 50 ng of RCA 60 (Sigma) and 5 μL of kaleidoscope prestained standards (Biorad). Electrophoresis was carried out for ninety minutes at 100V m 25 mM Tπs-Cl, pH 8.3, 0.1% SDS, and 192 mM glycine using the BioRad Mini Protean II cells (Biorad). Following electrophoresis gels were equilibrated in transfer buffer (48 mM Tris, 39 mM glycine, 0.0375% SDS, and 20% Methanol) for a few mmutes PVDF Biorad membrane was presoaked for one minute in 100% methanol and two minutes in transfer buffer. Whatman paper was soaked briefly m transfer buffer. Five pieces of Whatman paper, membrane, gel, and another five pieces of Whatman paper were arranged on the bottom cathode (anode) of the Pharmacia Novablot transfer apparatus (Pharmacia) Transfer was for one hour at constant current (2 mA/cm 2 ) Transfer was confirmed by checking for the appearance of the prestained standards on the membrane. Non-specific sites on the membrane were blocked by incubating the blot for thirty minutes in IX Phosphate Buffered Salme (IX PBS; 137 mM NaCl, 2.7 mM KCl, 8 mM Na 2 HP0 4 , 1.5 mM KH 2 P0 4 , pH 7.4) with 5% skim milk powder (Carnation). Primary antibody (Rabbit α-πcin, Sigma) was diluted 1:3000 in IX PBS containmg 0.1% Tween 20 (Sigma) and 2 5% skim milk and incubated with blot for forty five minutes on a orbital shaker (VWR) Non-specifically bound primary antibody was removed by washing the blot for ten mmutes with IX PBS containing 0.2% Tween 20. This was repeated four times. Secondary antibody donkey anti-rabbit (Amersham) was incubated with the blot under the same conditions as the primary antibody. Excess secondary antibody was washed as described above Blots were developed with the ECL Western Blottmg detection reagents according to the manufacturer's instructions Blots were exposed to Medtec's Full Speed Blue Film (Medtee) or Amersham's ECL Hyperfilm (Amersham) for three to fifteen mmutes. Film was developed in a KODAK Automatic Developer. Determination of lectin binding ability of pro-ricin variant An Immulon 2 place (VDVR) was coated with 100 μl per well of lOμg/ml of asialofetuin and left overnight at 4°C. The plate was washed with 3X 300 μL per well with ddH 2 0 usmg an automated plate washer (BioRad). The plate was blocked for one hour at 37°C by addmg 300 μL per well of PBS contammg 1% ovalbumin. The plate was washed again as above. Pro-ricin variant pAP 190 was added to the plate in various dilutions in IX Baculo. A standard curve of RCA 60 (Sigma) from 1 -10 ng was also mcluded. The plate was incubated for 1 h at 37°C The plate was washed as above Anti-ricin monoclonal antibody (Sigma) was diluted 1.3000 in IX PBS containmg 0.5% ovalbumm and 0.1% tween-20, added at 100 μL per well and incubated for 1 h at 37°C. The plate was washed as above Donkey-anti rabbity polyclonal antibody was diluted 1 3000 in IX PBS contammg 05% ovalbumm, 0 1% Tween-20, and added at lOOμL per well and mcubated for 1 h at 37°C The plate was given a final wash as described above Substrate was added to plate at lOOμL per well (1 mg/ml o-phenylenediamine (Sigma), 1 μL/ml H 2 0 7 , 25 μL of stop solution (20% H 2 S0 4 ) was added and the absorbance read (A490nm-A630nm) usmg a SPECTRA MAX 340 plate reader (Molecular Devices) Determination of pAP 190 activity using the rabbit reticulocyte assay Ricm sample were prepared for reduction A) RCA 6ϋ = 3,500 ng/ μL of RCΛ^ + 997 μL lxFndo buffer (25mM Tris, 25mM KCl,5mM MGC1 2 , pH 7 6) Reduction = 95 μL of lOng/μL + 5 μL β-mucaptoethanol B ) Ricm variants Reduction = 40 μL variant + 2 μL β-mercaptoethanol The ricm standard and the variants were incubated for 30 mmutes at room temperature Ricin - Rabbit Reticulocyte lysate reaction The required number of 0 5 mL tubes were labelled (2 tubes for each sample, + and - animlme) To each of the sample tubes 20 μL of IX endo buffei w as added, and 30 μL of buffer was added to the controls To the sample tubes either 10 μL of lOng/μL Ricm or lOμL of variant was added Finally, 30μL of rabbit reticulocyte lvsate was added to all the tubes The samples were mcubated for 30 minutes at 30°C using the thermal block Samples were removed from the eppendorf tube and contents added into a 1 5 mL tube contammg 1 mL of TRIZOL (Gibco) Samples were mcubated ior 15 minutes at room temperature After the mcubation, 200 μL of chloroform was added, and the sample was vortexed and spun at 12,000 g for 15 minutes at 4°C The top aqueous layer from the samples was removed and contents added to a 1 mL tube containing 500 μL of isopropanol Samples were mcubated for 15 mmutes at room temperature and then centrifuged at 12,000 for 15 mmutes at 4°C Supernatant was removed and the pellets w ere washed with 1 mL of 70% ethanol Centrifugation at 12,000 g foi 5 mmutes at 4°C precipitated the RNA All but approximately 20 μL of the supernatant was removed and the remaining liquid evaporated using the speed vacuum machine The control samples (-aniline) were dissolved m 10 μL of 0 1 X E buffer (36 mM Ti is, 30 mM NaH 2 P0 4 1 mM EDTA, pH 78) and stored at -70°C or on dry ice until latei Pellets from the other samples (+anιlιne samples) were dissolved in 20 μL of DEPC treated ddH 2 0 An 80 μL aliquot of 1 M aniline (distilled) with 2 8 M acetic acid was added to these RNA samples and transfeired to a fresh 0 5 mL tube The samples were incubated m the dark for 3 mmutes at 60°C RNA was precipitated by addmg 100 μL of 95% ethanol and 5μL of 3M sodium acetate, pH 5 2 to each tube and centπfugmg at 12,000 g for 30 mmutes at 4°C Pc llets were washed with 1 mL 70% ethanol and centrifuged again at 12,000g for 5 minutes at 4°C to precipitate RNA The supernatant was removed and excess liquid evaporated using the speed vacuum machme These pellets (+ anilme samples) were dissolved m lOμL of O I X E buffer To all samples (+ and - anilme), 10 μL of formamide loading dye was added The RNA ladder (8 μL of ladder + 8 μL of loading dye) was also included Samples were mcubated for 2 minutes at 70°C on the thermal block Electrophoresis was carried out on the samples usmg 1 2% agarose, 50% formamide gels in 0 IX E buffer + 0 2% SDS The gel was run for 90 mmutes at 75 watts RNA was visualized by staining the gel in 1 μg/μL ethidium bromide m running buffer for 45 minutes The gel was exammed on a 302 nm UV box and photographed usmg the gel documentation system Results: Protem Expression Yields Aliquots were taken at each stop of the harvesting /purification and tested Yields of functional ricm variant were determined by ELISA Typical results on an 800 mL prep of mfected T nt cells are given below Afrqwt μgpAP 190 Before concentration and dialysis 648 5 After concentration and dialysis 364 4 ASF column flow through 62 1 ASF column eluαon 300 7 Yield 3007/6485 = 464% Purification of pAP 190 and Western Analysis of column fractions Partially purfied pAP 190 was applied to Superdex 75 and 200 (16/60) columns connected m series m order to remove the contaminating non-specifically processed pAP 190 Eluted fractions were tested via Western analysis and the fractions containing the most pure protem were pooled, concentrated and re-applied to the column The variant was applied a total of three times to the column The final purified pAP 190 has less than 1% processed variant Figure 13 shows that the purified pAP 190 is in three fractions and the processed mateπal eluted in two separate fractions The purified pAP 190 was tested tor susceptibility to cleavage by HIV protease and for activation of the A-chain of the pro-πcin variant (inhibition of protem synthesis) PAP 190 was mcubated with and without HIV protease for a specified time period and then electrophoresed and blotted Cleaved pAP 190 will run as two 30 kDa protems (B is slightly larger) under reducing (SDS-PAGE) conditions Unprocessed pAP 190, which contams the lmker region, will run at 60 kDa HIV protease was able to cleave the pAP 190 (shown m Figure 14) Lanes B and D show untreated, while lanes C and E to G show HIV protease treated pAP 190 Activation of pAP 190 variant with HIV protease Activation of HIV protease treated pAP 190 190, based on the method of May et al (EMBO Journal 8. 301-8, 1989) was demonstrated in Figure 15 The appearance of the 390 based pair product is observed in lane B, which is the positive control, and not observed in lane C, the negative control Lanes D-G show that there was no N- glycosidase activity m the pAP 190 variant as predicted Lanes H K show that processed pAP 190 possesses N-glycosidase activity as predicted The pAP 190 variant has been expiessed in msect cells purified to greater than 99%, and activation of the variant has been demonstrated by cleavage with HIV protease Example 3 Cloning and Expression of Proricin Variants Activated by HTLV Isolation of total RNA The preproπcm gene is cloned from new foliage of the castor bean plant Total messenger RNA is isolated according to established procedures (Maniatis et al , Molecular Cloning A Lab Manual (Cold Spring Harbour Press, Cold Sprmg Harbour, (1989)) and cDNA generated usmg reverse transcriptase cDNA Synthesis Oligonucleotides, corresponding to the extreme 5 and 3 ends of the preproricm gene are synthesized and used to PCR amplify the gene Using the cDNA sequence for preproricm (Lamb et al , Eur J Biochem 145 266-270 (1985)), several oligonucleotide primers are designed to flank the start and stop codons of the preproricin open readmg frame The oligonucleotides are synthesized using an Applied Biosystems Model 392 DNA/RNA Synthesizer First strand cDNA synthesis is primed using the oligonucleotide Rιcιnl729C (Table 1) Three micrograms oi total RNA is used as a template for oligo Rιcιnl729C primed synthesis of cDNA using Superscript II Reverse Transcriptase (BRL) followmg the manufacturer's protocol DNA Amplification and Clonmg The first strand cDNA synthesis reaction is used as template for DNA amplification by the polymerase chain reaction (PCR) The preproricin cDNA is amplified usmg the upstream primer Ricin-109 and the downstream primer Rιcml729C with Vent DNA polymerase (New England Biolabs) using standard procedures (Sambrook et al., Molecular Cloning: A Laboratory Manual, Second Edition, (Cold Spring Harbor Laboratory Press, 1989)). Amplification is carried out in a Biometra thermal cycler (TRIO-Thermalcycler) using the following cycling parameters: denaturation 95°C for 1 min., annealing 52°C for 1 min., and extension 72 °C for 2 min., (33 cycles), followed by a final extension cycle at 72°C for 10 min. The 1846bp amplified product is fractionated on an agarose gel (Sambrook et al., Molecular Cloning: A Laboratory Manual, Second Edition, (Cold Spring Harbor Laboratory Press, 1989), and the DNA purified from the gel slice using Qiaex resin (Qiagen) following the manufacturer's protocol. The purified PCR fragment encoding the preproricin cDNA is then ligated (Sambrook et al., Molecular Cloning: A Laboratory Manual, Second Edition, (Cold Spring Harbor Laboratory Press, 1989)) into an Eco Rl-digested pBluescript II SK plasmid (Stratagene), and used to transform competent XLl-Blue cells (Stratagene). Positive clones are confirmed by restriction digestion of purified plasmid DNA. Plasmid DNA is extracted using a Qiaprep Spin Plasmid Miniprep Kit (Qiagen). DNA Sequencing: The cloned PCR product containing the putative preproricin gene is confirmed by DNA sequencing of the entire cDNA clone (pAP-144). Sequencing is performed using an Applied Biosystems 373A Automated DNA Sequencer, and confirmed by double-stranded dideoxy sequencing by the Sanger method using the Sequenase kit (USB). The oligonucleotide primers used for sequencing are as follows: Ricin267, Ricin486, Ricin725, Ricin937, Ri cinll51, Ricin 1399, Ri cinl627, T3 pri mer (5 ΑATTAACCCTCA CTA A Λ GGG- 3') a nd T7 p ri me r (5'GTAATACGACTCACTATAGGGC-3). Sequence data is compiled and analyzed using PC Gene software package (intelligenetics). The sequences and location of oligonucleotide primers is shown in Table 1. Mutagenesis of Preproricin Linker: The preproricin cDNA clone (pAP-144) is subjected to site directed mutagenesis in order to generate a series of variants differing only in the sequence between the A and B chains (linker region). The wild-type preproricin linker region is replaced with the linker sequences displayed in Figure 20. The linker regions of the variants encode a disease-specific protease cleavage recognition sequence (Slalka et al., Cell, 56:911-913, 1989). The mutagenesis and cloning strategy used to generate the HTLV protease- sensitive linker variants is summarized in Figures 16A, 17A, 18A and 19A. The first step involves a DNA amplification using a set of mutagenic primers encoding for the disease-specific protease-sensitive linker in combination with the two flanking primers Ricin-109Eco and Ricinl729Xba. The PCR protocol and conditions used are the same as described above PCR products from each mutagenesis reaction are gel purified then restriction digested with either Eco Rl for the A-cham encodmg fragment, or Xba I for the B cham encoding fragment Restriction digested PCR fragments are gel purified and then ligated with pBluescript SK which has been digested with Eco RI and Xba I Ligation reactions are used to transform competent XLI -Blue cells (Stratagene) Recombmant clones are identified by restriction digests of plasmid mmiprep DNA and the mutant linker sequence are confirmed by DNA sequencmg Subclonmg Preproricm Mutants mto Vector pVL1393 Preproricin variants are subcloned into the baculovirus transfer vector pVL1393 (PharMingen) The subclonmg strategy for the HTLV protease-sensitive lmker variants is summarized m Figures 16C, 17C, 18C, and 19C The 1315 bp hco RI/Kpn I fragment encodmg the ricm A-chain and each mutant linker is isolated from pAP-205, pAP-207, pAP-209 or pAP-211 Each of these purified tragments is ligated with a 564 bp Kpnl/PStl fragment obtamed from pAP-144, and w ith Leo Rl/Pst I cleaved pVL1393 Recombinant clones are identified by restriction digests of plasmid mmiprep DNA and the 5 and 3 junctions confirmed by DNA sequencmg Isolation of Recombinant Baculoviruses Insect cells S frugψerda (Sf9), and Triclwplusia m (Tn368 and BTI TN-581-4 (High Five)) are maintained on TMN-FH medium supplemented with 10% total calf serum (Summers et al , A Manual of Methods of Baculovirus Vectors and Insect Cell Culture Procedures, (Texas Agricultural Experiment Station, 1987)) Two miciograms of recombmant pVL1393 DNA (pAP-190, pAP-196, or pAP-197) is co-transfected with 05 microgram of BaculoGold AcNPV DNΛ (Pharmingen) into 2 x 106 Tn368 insect cells following the manufacturer s protocol (Grucnwald ct al , Baculo\ uus Expression Vector System Procedures and Methods Manual, 2nd Edition, (San Diego, CA, 1993)) On day 5 post-transfection, media are centrifuged and the supernatants tested in limiting dilution assays with Tn368 cells (Summers et al , A Manual of Methods of Baculovirus Vectors and Insect Cell Culture Procedures, (Texas Agricultural Experiment Station, 1987)) Recombmant viruses in the supernatants are then amplified by infecting Tn368 cells at a multiplicity of mfection (moi) of 0 1, followed by collection of day 7 supernatants A total of three rounds of amplification are performed for each lecombinant following established procedures (Summers et al , Λ Manual of Methods of Baculovirus Vectors and Insect Cell Culture Procedures, (Texas Agricultural Experiment Station, 1987 and Gruenwald et al , Baculovirus Expression Vector System Procedures and Methods Manual, 2nd Edition, (San Diego, CA, 1993)) Expression of Mutant Proricin: Recombinant baculoviruses (pAP-206-baculo, pAP-208-baculo, pAP-210-baculo, and pAP-212-baculo) are used to infect 2 x 105 Tn368 or sf9 cells of an moi of 5 in EX-CELL400 media (JRH Biosciences) with 25mM a-lactose in spinner flasks. Media supernatants containing mutant proricins are collected on day 6 post-infection. Purification of Mutant Proricin: Media supernatants are ultracentrifuged at 100,000g for 1 hour. After the addition of 1 mM phenylmethylsulfonyl fluoride, the supernatants are concentrated using an Amicon 8050 Ultrafiltration Cell fitted with a Diaflo XM50 membrane. Supernatants are then dialysed extensively against 137 mN NaCl, 2.2 mM KCl, 2.6 mM KH 2 P0 4 , and 8.6 mM Na 2 HP0 4 , pH 7.4 containing 1 mM dithiothreitol (dialysis buffer). Recombinant proricin proteins are purified by affinity chromatography using lactose agarose (Sigma) as previously described for recombinant ricin-B chain (Ferrini et al., Eur. J. Biochem. 233:772-777 (1995)). Fractions containing recombinant proricin are identified using SDS/PAGE, (Sambrook et al., Molecular Cloning: A Laboratory Manual, Second Edition, (Cold Spring Harbor Laboratory Press, 1989) and by Western blot analysis using anti-ricin antibodies (Sigma). In Vitro Protease Digestion of Proricin Variants: Affinity-purified proricin variant is treated with individual disease-specific proteases to confirm specific cleavage in the linker region. Ricin-like toxin variants are eluted from the lactose-agarose matrix i n μ->rotease digestion buffer (50mM NaCl, 50mM Na-acetate, pH 5.5, ImM dithiothreitol) containing lOOmM lactose. Proricin substrate is then incubated at 37°C for 60 minutes with a disease-specific protease. The cleavage products consisting ricin A and B chains are identified using SDS/PAGE (Sambrook et al., Molecular Cloning: a Laboratory Manual, 2nd. ed., Cold Spring Harbor Press, 1989), followed by Western blot analysis using anti-ricin antibodies (Sigma). HTLV proteases may be obtained from Bachem Bioscience. Cathepsin B may be obtained from Medcor or Calbiochem. In Vitro Translation Assay: The activity of protease-treated ricin-like toxin variants is monitored using a rabbit reticulocyte lysate in a non-radioactive (Amersham, ECL system) in vitro translation assay. Protease-treated proricin is added to a standard 50 ml translation reaction mix containing Brome Mosaic Virus mRNA as template (following the manufacturer's protocol). Active ricin variants inhibit the in vitro translation reaction by inactivating ribosomes. Therefore, in the presence of an active ricin variant, no viral proteins are synthesized. In Vitro Yeast Protein Synthesis Assay The activity of protease-treated proπcin-hke toxms mav also be assessed by a yeast protem synthesis assay For example, Murakami, S et al , Mol , Cel Biol 2 588- 592, 1982, teaches a yeast protein synthesis assay to determine ricin-like toxicity which is as sensitive as mammalian cell assays Six five mL cultures of Sacchai omyces cerevisiase (Y235 cells and 2 cell wall mutants) in YPD medium (10 g/L yeast extract, 20 g/L peptone) are started by inoculating 800 uL of medium with 1 colony of Sacchai omyces cerevisiase, vortexing, then addmg 100 uL of this suspension to 5 mL of medium Cultures are grown overnight at 30°C with gentle agitation Cells are expanded by inoculating 100 uL of YPD medium with one or more of the 5 mL overnight cultures and are grown at 30°C with gentle agitation until a concentration of 1 x 10 5 cells/mL Cells are washed with sterile double-distilled water, centrifuged at 1,200 g for 3 mmutes and concentrated 3-fold in ZSM buffer(l M sorbitol, 10 mM Tns-Cl, pH 7 5, 50 mM dithiotheitol (DDT)) Samples are incubated with gentle shaking for 10 minutes at 30°C, centrifuged at 1,200 g for 3 mmutes and resuspended m ZSM buffer such that the cell concentration was 1 x 10 8 cells/ml Cell walls are disrupted by addmg 1 mL of beta-glucuronidase (Sigma, St Louis, MO) to the samples and incubatmg for 1 hour at room temperature with gentle agitation Cells aie washed 3 times with ZSM and protoplast cells resuspended m tegeneration medium (0 17% yeast nitiogen base without ammo acids (Difco, Detroit, Michigan), 2 Dropout + all (essential amino acids), 10 mM Tns-Cl, pH 7.5, 2% glucose, IM sorbitol) to a fmal concentration of 1 x 10 h cells/mL An activated proricin variant which has been dialysed in sterile IX baculo buffer (0 137 M) NaCl, 2.7 mM KCl, 2 6 mM KH 2 P0 4 pH 7 4) is added to one half of the protoplast, while sterile 1 X baculo buffer alone is added to the other half of the protoplasts as control Both sets of samples are incubated at room temperature with gentle agitation At time periods of 0, 1, 2, and 3 hours, an aliquot of each culture is lemoved The cells are diluted serially from 10 "4 to 10' 8 in ZSM and plated on soft agar (1 1 ZSM YPD, 15% agar) Simultaneously, dilutions are made from 10 2 to 10 4 in sterile double-distilled water and 50 uL aliquots are plated onto YPD medium with 20% agar Plates are incubated for 2 days at 30°C after which times colonies were counted A plot of cell count vs time is used to compare the ricm test culture vs the control culture with no ricin The activated proπcm-like toxin variant inhibits in vitro protein synthesis through ribosomal inactivation The rate of cell growth of the treatment group is expected to be substantially lower than that of the control group frJ-Glycosidase Activity of Proricin Variants on rRNA Oligonucleotides Ricin-like toxms inhibit ribosomal function by hydrolysmg the N-glycosidic bond between the nucleotide base and the nbose at position A4319 m eukaryotic 28S ribosomal RNA (rRNA). The ability of the activated ricin-like toxins to inhibit ribosomal RNA (rRNA) function may be examined m an m vitro ribonucleotide catalysis assay using a synthetic oligoribonucleotide possessing the secondary structure of the natural RNA hydrolytic cleavage domain. A synthetic 32-nucleotide RNA oligomer (University of Calgary, DNA Core Services) that mimics the 28S rRNA toxin active site is used to test the N-glycosidase activity of proricin variants. The sequence of oligonucleotide and the general methodology are substantially as described in Gluck, A. and Wool I.G., / Mol. Biol. 256:838-848, 1996. A labelling reaction is set up to mclude- 50 pmol of oligonucleotide, 20 units of T4 polynucleotide kinase (PNK; Gibco-BRL, Gaithersburg, MA), 25 pmol of γ- 32 P (Amersham, Arlington, IL), IX T4 PNK buffer in a fmal volume of 50 uL The samples are incubated for 30 minutes at 37°C and them for 20 minutes at 65°C The labelled oligonucleotide is precipitated with 95% ethanol an dried using a thermal cycler. A second ethanol precipitation step can be repeated to remove further trace contaminants. The RNA was resuspended to a final concentration of 1 ng/uL in 10 mM Tns-Cl (pH 7.6) and 50 mM NaCl (5 ng of oligonucleotide is used per sample). Activated proricin variant is reduced in 1 X baculo buffer with 1% beta- mercaptoethanol for 30 minutes at room temperature prior to use. The oligonucleotides are heated at 90°C for 1 minute in 10 mM Tns-Cl (pH 7.6), 50 mM NaCl and allowed to renature at 0°C. CaCl 2 , EGTA and water are added to the renatured RNA to give the following concentrations: 3 mM Tπs-HCl (pH 7.6), 15 mM NaCl, 5 mM CaCl 2 , and 5 mM EGTA. An activated proricin variant or ricm A-chain (Sigma, St Louis, MO) is added to each tube. The concentration of the ricin ranged from 1-10 uM and the proricin variant 10- fold greater. The tubes are incubated at 35 C for 20 mmutes and the reaction is stopped by the addition of sodium dodecylsulfate (SDS) at a fmal concentration of 0.5% (w/v). The oligonucleotide and 15 ug of added carπei tRNA (yeast tRNA, Gibco-BRL Gaithersburg, MA) are precipitated with 300 mM NaCl and 2.5 volumes of 95% ethanol The pellets are washed once with 70% ethanol and dried on a CENTRIVAP (Labconco, Kansas City, MO). The RNA is dissolved in 5 uL of water, 25 uL of a solution of anilme and acetic acid (1 and 2.8 mM respectively) is added and the sample is mcubated for 10 minutes at 40°C. The aniline-treated RNA is precipitate with ethanol and 300 mM NaCl, washed once in 70% ethanol and dried on the CENTRIVAP The pellets are dissovled in 10 uL of DEPC- treated double-distilled water and 10 uL of 2X loadmg dy (178 mM Tris-HCl (pH 8.3), 178 mM boric acid, 5 mM EDTA, 0.05% (w/v) bromophenol blue and 14 M urea), and are electrophoresed for 3 hours at 50 watts in 10% (w/v) polyacrylamide gel containing 7 M urea in 1 X TBE buffer (89 mM Tris-HCl (pH 8.3), 89 mM boric acid, 2.5 mM boric acid, 2.5 mM EDTA). Gels are exposed to KODAK full speed blue X-ray film and left at -70°C. After 2 days, film was developed in a KODAK automatic film processor. When proricin variant activated with a disease-specific protease is added to the oligoribonucleotide, hydrolysis of the N- glycosidic bond at position 20 (depurination of adenosine) would occur and appearance of two bands on the autograph is expected. Proricin variant without pretreatment with the disease-specific protease would not cleave the RNA oligonucleotide and would result in a single band on the autoradiograph. In Vitro Cytotoxicity Assay: Human ovarian cancer cells (e.g. MA148) are seeded in 96-well flat-bottom plates and are exposed to ricin-like toxin variants or control medium at 37°C for 16 h. The viability of the cancer cells is determined by measuring [ 35 S]methionine incorporation and is significantly lower in wells treated with the toxin variants than those with control medium. In Vivo Tumour Growth Inhibition Assay: Human breast cancer (e.g. MCF-7) cells are maintained in suitable medium containing 10% fetal calf serum. The cells are grown, harvested and subsequently injected subcutaneously into ovariectomized athymic nude mice. Tumour size is determined at intervals by measuring two right-angle measurements using calipers. In Vivo Tumour Metastasis Assay: The metastasis study is performed substantially as described in Honn, K.V. et al. (Biochem. Pharmacol. 34:235-241 (1985)). Viable B16a melanoma tumour cells are prepared and injected subcutaneously into the left axillary region of syngeneic mice. The extent of tumour metastasis is measured after 4 weeks. The lungs are removed from the animals and are fixed in Bouin's solution and macroscopic pulmonary metastases are counted using a dissecting microscope. In general without therapeutic intervention, injection of 10 5 viable tumour cells forms approximately 40-50 pulmonary metastases. Having illustrated and described the principles of the invention in a preferred embodiment, it should be appreciated to those skilled in the art that the mvention can be modified in arrangement and detail without departure from such principles. We claim all modifications coming within the scope of the following claims. All publications, patents and patent applications referred to herein are mcorporated by reference in their entirety to the same extent as if each individual publication, patent or patent application was specifically and mdividually indicated to be mcorporated by reference m its entirety TABLE 1 Table 1 - Sequence and Location of Oligonucleotide Primers Name of Primer Sequence t Corresponds to Primer proricin nucleotide numbers (see Figures 8-10) Ricin-109 5 • -GGAGATGAAACCGGGAGGAAATACTATTGTAAT-3 • 27 to 59 Ricin-99Eco 5 ' - GCGGAATTCCGGGAGGAAATACTATTGTAAT - 3' 37 to 59 Ricin 267 5' - ACGGTTTAΓΠTAGTTGA - 3' 300 to 317 Ricin486 5" - ACTTGCTGGTAATCTGAG - 3' 519 to 536 Ricin 725 5' - AGAATAGTTGGGGGAGAC - 3' 758 to 775 Ricin937 5" - AATGCTGATGTTTGTATG - 3' 970 to 987 Ricin1151 5' - CGGGAGTCTATGTGATGA - 3' 1184 to 1201 Ricin 1399 5' - GCAAATAGTGGACAAGTA - 3' 1432 to 1449 Ricin1627 5'-GGATTGGTGTTAGATGTG-3' 1660 to 1677 Ricin1729C 5"-ATAACTTGCTGTCCTTTCA-3' 1864 to 1846 Ricin1729C 5" - CGCTCTAGATAACTTGCTGTCCTTTCA - 3' 1864 to 1846 Xba t underlined sequences inserted for subcloning purposes and not included in final preproricin sequences

Claims: WE CLAIM: 1. A purified and isolated nucleic acid having a nucleotide sequence encoding an A chain of a ricin-like toxin, a B chain of a ricin- like toxin and a heterologous linker amino acid sequence, linking the A and B chains, wherein the linker sequence contains a cleavage recognition site for a retroviral protease. 2. The nucleic acid of claim 1 wherein the A chain is ricin A chain. 3. The nucleic acid of claim 1 wherein the B chain is ricin B chain. 4. The nucleic acid of claim 1 wherein the cleavage recognition site is the cleavage recognition site for an HIV protease. 5. The nucleic acid of claim 1 wherein the linker amino acid sequence comprises VSQNYPIVQNFN; SKARVLAEAMSN; or SIRKILFLDGIN. 6. The nucleic acid of claim 1 having the nucleotide sequence shown in Figure 8, Figure 9 or Figure 10. 7. The nucleic acid of claim 1 wherein the cleavage recognition site is the cleavage recognition site for a human T-cell leukemia virus (HTLV) protease. 8. The nucleic acid of claim 7 wherein the linker amino acid sequence comprises SAPQVLPVMHPN; SKTKVLWQPKN; SKTKVLVVQPRN or STTQCFPILHPN. 9. A plasmid incorporating the nucleic acid of claim 1. 10. A plasmid as claimed in claim 7 having the restriction map as shown in Figure IA, 2A, 3A, 16A, 17A or 18A. 11. A baculovirus transfer vector incorporating the nucleic acid of claim 1. 12. A baculovirus transfer vector as claimed in claim 11 having the restriction map as shown in Figure 5, 6, 7, 16C, 17C, or 18C. 13. A baculovirus transfer vector as claimed in claim 11 having the DNA sequence as shown in Figure 11. 14. A recombinant protein comprising an A chain of a ricin-like toxin, a B chain of a ricin-like toxin and a heterologous linker amino acid sequence, linking the A and B chains, wherein the linker sequence contains a cleavage recognition site for a retroviral protease. 15. The recombinant protein of claim 14 wherein the A chain is ricin A chain. 16. The recombinant protein of claim 14 wherein the B chain is ricin B chain. 17. The recombinant protein of claim 14 wherein the cleavage recognition site is the cleavage recognition site for an HIV protease. 18. The recombinant protein of claim 14 wherein the linker amino acid sequence comprises VSQNYPIVQNFN; SKARVLAEAMSN; or SIRKILFLDGIN. 19. A recombinant protein comprising an A chain of a ricin-like toxin, a B chain of a ricin- like toxin and a heterologous linker amino acid sequence, linking the A and B chains, wherein the linker sequence contains a cleavage recognition site for a HTLV protease 20. The recombinant protein of claim 19 wherein the linker amino acid sequence comprises SAPQVLPVMHPN; SKTKVLWQPKN; SKTKVLVVQPRN or STTQCFPILHPN. 21. A method of inhibiting or destroying mammalian cells infected with a retrovirus having a protease, comprising the steps of preparing a recombinant protein of claim 14 wherein the linker sequence contains a cleavage recognition site for the retrovirus protease and administering the fusion protein to the cells. 22. A method as claimed in claim 21, wherein the retrovirus is HIV. 23. A method as claimed in claim 21 wherein the mammalian cells are human cells. 24. A method of treating a mammal infected with HIV, comprising the steps of preparing a recombinant protein of claim 14 and administering the protein to the mammal. 25. A process for preparing a pharmaceutical for treating a mammal infected with a retrovirus having a protease comprising the steps of preparing a purified and isolated nucleic acid having a nucleotide sequence encoding an A chain of a ricin-like toxin, a B chain of a ricin-like toxin and a heterologous linker amino acid sequence, linking the A and B chains, wherein the linker sequence contains a cleavage recognition site for the protease; introducing the nucleic acid into a host cell; expressing the nucleic acid in the host cell to obtain a recombinant protein comprising an A chain of a ricin-like toxin, a B chain of a ricin-like toxin and a linker amino acid sequence, linking the A and B chains wherein the linker sequence contains the cleavage recognition site for the protease, and suspending the protein in a pharmaceutically acceptable carrier, diluent or excipient. 26. A process for preparing a pharmaceutical for treating a mammal infected with a retrovirus having a protease comprising the steps of identifying a cleavage recognition site for the protease; preparing a recombinant protein comprising an A chain of a ricin- like toxin, a B chain of a ricin-like toxin and a heterologous linker amino acid sequence, linking the A and B chains, wherein the linker sequence contains the cleavage recognition site for the protease and suspending the protein in a pharmaceutically acceptable carrier, diluent or excipient. 27. A pharmaceutical composition for treating a retroviral infection in a mammal comprising the recombinant protein of claim 14 and a pharmaceutically acceptable carrier, diluent or excipient. 28. A pharmaceutical composition for treating HIV infection in a mammal comprising the recombinant protein of claim 14 and a pharmaceutically acceptable carrier, diluent or excipient.

Inventors: BORGFORD THOR, BORGFORD, THOR
Applicants: BORGFORD THOR, NOVO ENZYME CORP DE, BORGFORD, THOR, DE NOVO ENZYME CORPORATION
Application year: 1997
Main classification: C12N15
IPC codes: A61K38, C07K14, C12N15