Antiviral ricin-like proteins

Details Antiviral ricin-like proteins Antiviral ricin-like proteins

2000-04-14

2003-03-11

Stucker, Jeffrey (Department: 1648)

Drug, bio-affecting and body treating compositions

Enzyme or coenzyme containing

Stabilized enzymes or enzymes complexed with nonenzyme

C424S094100, C530S350000, C530S370000, C514S002600, C536S023200, C536S023400, C536S023600, C435S320100

Reexamination Certificate

active

06531125

ABSTRACT:

FIELD OF THE INVENTION
The invention relates to proteins having A and B chains of a ricin-like toxin, linked by a linker sequence which is specifically cleavable by a retroviral protease to release the active A chain. 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 infection.
BACKGROUND OF THE INVENTION
Bacteria and plants are known to produce cytotoxic proteins which may consist of one, two or several polypeptides or subunits. Those proteins having a single subunit may be loosely classified as Type I proteins. Many of the cytotoxins which have evolved two subunit structures are referred to as type II proteins(Saelinger, C. B. in
Trafficking of Bacterial Toxins
(eds. Saelinger, C. B.) 1-13 (CRC Press Inc., Boca Raton, Fla., 1990). One subunit, the A chain, possesses the toxic activity whereas the second subunit, the B chain, binds cell surfaces and mediates entry of the toxin into a target cell. A subset of these toxins kill target cells by inhibiting protein biosynthesis. For example, bacterial toxins such as diphtheria toxin or Pseudomonas exotoxin inhibit protein synthesis by inactivating elongation factor 2. Plant toxins such as ricin work by directly inactivating ribosomes [Olsnes, S. & Phil, A. in
Molecular action of toxins and viruses
(eds. Cohen, P. & vanHeyningen, S.); 51-105 (Elsevier Biomedical Press, Amsterdam, 1982].
Ricin, derived from the seeds of
Ricinus communis
(castor oil plant), is the most potent of the plant toxins. It is estimated that a single ricin A chain is able to inactivate ribosomes at a rate of 1500 ribosomes/minute. Consequently, a single molecule of ricin 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 ricin toxin is a glycosylated heterodimer with A and B chain molecular masses of 30,625 Da and 31,431 Da respectively. The A chain of ricin has an N-glycosidase activity and catalyzes the excision of a specific adenine residue from the 28S rRNA of eukaryotic ribosomes (Endo, Y; & Tsurugi, K. J.
Biol. Chem.
262:8128 (1987)). The B chain 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 toxin molecule (Simmons et al.
Biol. Chem.
261:7912 (1986)).
Protein toxins are initially produced in an inactive, precursor form. Ricin is initially produced as a single polypeptide (preproricin) with a 35 amino 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. J. 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.,
FASAB 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); Halling, 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)).
Class 2 ribosomal inhibitory proteins (RIP-2) constitute other toxins possessing distinct functional domains for cytotoxicity and cell binding/toxin translocation which include abrin, modeccin, volkensin, (Sandvig, K. et al.,
Biochem. Soc. Trans.
21:707-711 (1993)) and mistle toe lectin (viscumin) (Olsnes, S. & Phil, A. in Molecular action of toxins and viruses (eds. Cohen, P. & vanHeyningen, S.) 51-105 Elsevier Biomedical Press, Amsterdam, 1982; Fodstad et al. Canc. Res. 44: 862 (1984)).
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 immunotoxin into nontarget cells. Even small amounts of B chain may override the specificity of the cell-binding component as the B chain binds nonspecifically to N-glycosylated galactose, present on most cells. IT-As are more specific and safer to use than IT-Rs. However, in the absence of the B chain the A chain 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 chain is not released from the conjugate into the target cell cytoplasm.
The insertion of intramolecular cleavage sites between the cytotoxic and cell-binding components of a toxin can mimic the way that the natural toxin is activated. European patent application no. 466,222 describes the use of maize-derived pro-proteins which can be converted into active form by cleavage with extracellular blood enzymes such as factor Xa, thrombin or collagenase. Westby et al. (Bioconjugate Chem., 3:375-381, 1992) documented fusion proteins which have a specific cell binding component and proricin with a protease sensitive cleavage site specific for factor Xa within the linker sequence. O'Hare et a

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