JPH02502287A - Improved ricin molecules and ricin toxin conjugates - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

[Detailed description of the invention] Improved research on ricin molecules and ricin toxin conjugates This invention provides novel lysine molecules with lysine-type activity and these novel lysine molecules. The present invention relates to a conjugate in which the child is attached to a selected ligand. difference More specifically, this invention relates to the use of recombinant DNA technology, the production of syn-type substances, and and conjugates of these novel lysine molecules bound to selected ligands. It is related to the therapeutic use of

The plant toxin ricin consists of two polypeptides called A and B chains or subunits. It is a known molecule composed of tide subunits. A chain has catalytic (toxic) activity It is thought that the B chain exhibits cell surface binding affinity (lectin activity) and a transport activity that can move the A chain of lysine through the cell membrane and into the cytosol. It is thought that it exhibits both genders. [For example, R.J. Neil and Tori; M.

Neville, Journal of Biological Chemistry, 257.159B -1601 (1982) reference. The toxicity is determined by the liposome 60S support of liposome particles. Enzyme action on 28S rRNA in the protein block inhibits protein synthesis. considered to be harmful. [For example, Y., Endo et al., Journal of Biologica See Le Chemistry, 262°5908-5912 (1987)]. cell binding The region is comprised of galactosylated glycoproteins and glycolipids that are present on the surface of many cell types. Ricin, like many toxins, is not cell-specific because it recognizes molecule residues.

Properties of two different forms of ricin, known as ricin D and ricin E The test was carried out [T., Araki and G., Komoto, “Biochimica et biophysics”. Power/Actor” World, 191-200 (1987) and B, F, Love-In, etc., “P "Lant Molecular Biology"-2-1287-295 (1987)] . Although both are equally toxic to animals, only ricin D toxin Binding to the containing support. Their amino acid sequences were compared to closely related agglutinin sites. In comparison with the case of This appears to be the result of genetic recombination between the ricin D and agglutinin genes. egg In all isoforms of white matter, the A and B chains are composed of a single relatively stable disulfide connected by fido bonds. Initially, they were putative signal peptides and adjacent approximately 550 amino acids with a short fragment connecting the A and B chains. It is formed as a prepro-like molecule consisting of amino acid protein. - Maturation from next gene product During processing into proteins, interdisulfide bonds link two subunits. It is thought that four intradisulfide bonds are formed in the B chain. During proteolysis, the signal peptide and amino acid linker are removed. [F, I.

Lam et al., “European Journal of Biochemistry”: 2 65-270 (1985)].

Comparing the primary protein sequence and X-ray crystal structure of the B chain subunit of ricin D , between this subunit [E., Ruthenbar et al., “Nature Journal Of Biological Chemistry J, 262:5398-5403 (198 7) Two. The B chain unit is thought to be composed of two regions, and in this specification, is domain I (amino acids 1-135) and domain ■ (amino acids 136-26 2). Furthermore, as a result of experiments on the -order and tertiary structure, each domain has 4 It was shown that it can be divided into two homologous subdomains or subregions. these 1λ (amino acids 1-16), 1α (amino acids 17-17) in domain I. 59), lβ (amino acids 6O-100) and lλ (amino acids 101-135) and 2λ (amino acids 136-147) and 2α (amino acids 1 48-183), 2β (amino acids 184-226) and 2γ (amino acids 227 −262).

Ricin and other toxins have been shown to contain ligands such as growth factors, such as growth factors. or in conjugates formed by binding to antibodies. the latter is a cell Give specificity. [For example, U.S. Pat. No. 4,340,535, U.S. Pat. 59457 and 4664911]. Consists of toxin moiety and antibody Conjugates are better known in the art as "immunotoxins" or ITs. ing. [For example, 1. Pasta et al. Cell Sat 7:641-648 (1986) reference]. These conjugated molecules may be useful in the treatment of various forms of cancer. can be used as target or cell-specific therapeutics. Reconstitution of toxin conjugates The Gund moiety binds to its receptor or antigenic determinant on the cell surface and The conjugate is taken up into cells, eg, by endocytosis. Once thin Once inside the vesicle, the toxin moiety is transferred from the cytosol to endocytic vesicles or receptors. It is thought to be released into the tosome.

The non-specific cell binding properties of ricin B chain can be altered or removed by various means. . In the case of U.S. Pat. No. 4,340,535, the toxin portion of the conjugate is Since it is composed only of the A chain of ricin, the B chain is completely removed. united states of america In the case of Patent No. 4359457, the lysine/galactose site is replaced by lactose. Gallostose residues present on the surface of many cell types are blocked. Non-specific binding to the group is prevented. Alternatively, the B chain can be added to the ricin A chain immunosorbent. the constant region of the antibody portion in the syn, or directed against the same antigenic determinant on the target cell. There is a method of binding it to antibodies. [E,, S.

Vitetta et al., “Journal of Experimental Medicine”, ed. :341-346 (1984) and E, S., Bitetta et al., PNAS once-0: 6332-6335 (1983).

Many of these conjugate molecules known in the art suffer from significant shortcomings. can face. For example, those conjugates containing only the A chain can be used to create intact toxin molecules. may be more specific but less active.

It appears to be the result of loss or reduction of transport function associated with the B chain. [example Beil-Hillman et al., Cancer Research 45:132B-1336. (1985) Reference Ko. Conjugation between toxin and ligand In the case of conjugates carried out by double bond formation, other problems may arise. child types of disulfide bonds are generally stable in blood and other tissue fluids. As such, it can be destroyed before reaching its intended target. [N, L, Letvin et al. , “Di 7-984 (1986)].Furthermore, the released target component is then released on the cell surface. Can compete with intact conjugate for surface markers.

Summary of the invention A new lysine molecule has been discovered. This new molecule is based on the amino acid sequence of the natural D-lysine molecule. It has an amino acid sequence substantially similar to that of the lectin binding domain of the B chain. Modification or elimination of non-specific cell binding mechanisms through decoration or “engineering” has been done. However, since the modification does not change the transport activity of the B chain, The novel toxin conjugates of this invention containing decorated B chains exhibit both activity and specificity. it is conceivable that. In each embodiment described herein, the amino acid sequence is identical to the sequence of natural ricin D modified by the specific changes noted at that location. Well, L is substantially the same. Specific modifications are described later in the “Description (of the invention)” section. There is.

The gluesin molecule of this invention can be modified with various modifications or combinations of modifications disclosed in this specification. It includes analogues of ricin characterized by A DNA sequence in which the DNA (before the modification of this invention) encodes lysine under strict conditions other variants, such as allelic variants, as long as they can hybridize with or additional deletion(s) or substitution(s) of amino acids that retain lysine-type activity ( (or insertion(s)). ET, Maniatis et al. "Reular Cloning" (A Laboratory Manual), Cold Steel See Pring Harbor Laboratory (1982) pp. 387-389].

Natural ricin B chain contains two N-linked glycosylation sites. Furthermore, the expression system Select mutagenesis or site mutagenesis techniques to remove the hydrocarbon composition of these sites. Modifications may favor in vivo clearance of this molecule or its derivatives. It is conceivable that this could have a positive impact.

Furthermore, this invention provides a method for treating cells transfected with the ricin B chain gene. Modified ricin B chain produced in a more homogeneous form than the modified B chain that was not produced It is characterized by

Another aspect of this invention is a DN encoding the amino acid sequence of the ricin molecule of this invention. Vectors containing these sequences in operably accompanied by A sequences and expression control sequences includes.

The lysin molecule of the present invention can be used in host cells transformed with the above-mentioned DNA. Produced by expression of a DNA molecule encoding a amino acid sequence. these host cells can be mammals, yeasts, insects, fungi or bacteria.

Furthermore, this invention provides the modified ricin B chain of this invention in which the ricin A chain is tri-linked. and targeted toxin conjugates containing selected ligands. preferred Potential ligands include antibodies, growth factors, hormones, and other cell surface binders. , but are not limited to these. This conjugate can be used with selected ligands. crosses the membrane of the cell carrying the receptor, antigen or tumor gene, and within the cell. This action can destroy the cells. Modified ricin B of this invention Conjugates containing the chains can be produced by gene fusion or the methods described below.

Another aspect of this invention provides a pharmaceutical composition containing an effective amount of said lysine conjugate. provide something. These pharmaceutical compositions, depending on the selected ligand, can be used to treat diseases including cancer. Can be used in the treatment of certain medical conditions. The selected ligand has a composition The composition targets the receptor, antigen or tumor gene or selector. It acts by binding to other recognition sites on the Gantt, and crosses the cell membrane. It delivers ricin, where the toxin destroys cells. The toxin conjugate is It is stable in other tissue fluids, and only appears once the conjugate enters the cytosol. It is thought that the toxin moiety is released.

Accordingly, another aspect of this invention is to provide a conjugate in combination with a suitable pharmaceutical carrier. cancer and ligand-directed medicine by administering to a patient a therapeutically effective amount of It relates to a method of treating a medical condition.

The vectors and transformed cells of this invention are novel vectors and transformed cells of the recombinant ricin molecule of this invention. Used in manufacturing methods. This production method is based on the selected The process involves culturing the cells to obtain a conditioned medium and purifying the molecules therefrom.

Another aspect of this invention provides a novel method for producing targeted lysine conjugates. . This method is extremely stable in vivo, and It is believed that this provides a conjugate that exhibits similar activity. [For example, A , J., Kamber et al., Methods in Enzymology Jl 12:207 225 (1985).

This method involves binding a peptide cross-linking agent to the modified ricin B fraction molecule of the present invention, and The structure consists of a step of reforming the holotoxin by forming a disulfide bond with the A chain of the protein. will be accomplished. The B chain of the modified holotoxin is then covalently linked to the selected ligand. (described below), a toxin conjugate is produced. The B chain consists of several It can be covalently attached to a ligand by conjugation chemistry. In a preferred embodiment, the modification The B chain of holotoxin was converted to N-succinimidyl-5-acetylthiopropionate. The holotoxin molecule is produced by treatment with A chain and reassociation with the A chain.

Treatment of holotoxin with hydroxylamine exposes free hydrosulfur groups, which reacts with maleimide-containing protein ligands. [1, M. Klobb and R.E. , Heiny, Archives of Biochemistry and Biophys. Jitsukusuni 96:606-612 (1962, >2° The ligand may be an integral hydrosulfate group, such as free cysteine or a peptide or carbonized Additional hydrogen sulfate groups may be included attached to the hydrogen moiety. In another embodiment, engineered The reassociated holotoxin containing the B chain was purified by N-succinimidyl-3-(2-pyridyldi thio)propionate and then to protein ligands by standard methods. crosslink. Alternatively, by using other cross-linking agents, the modified B chain can be linked to selected ligands. can be covalently bonded to

Other embodiments of the invention will be apparent from consideration of the following detailed description of the invention, including the Examples. The advantages and benefits are obvious.

Brief description of the drawing Figure 1 is extracted from castor seeds (Ricinus communis, Zanivariensis var.) found in the EcoRI-H1ndIII fragment of the genomic DNA The DNA and amino acid sequences of the ricin D gene are shown.

FIG. 2 shows the amino acid sequence of B chain SynD polypeptide.

Figure 3 shows the 4.2kb EeoRI-H1ndllll file containing the lysine gene. A restriction map of the fragment is shown.

Description of the invention 1. Modified lysine molecule This invention features modified lysine molecules with deficient or reduced cell binding affinity. It is a color. These molecules include novel analogs, derivatives and mutants of natural ricin D. mutants, in which case the B chains are proteins involved in cell-binding functions. Their structure differs from natural molecules in that they contain modifications in their structural regions. Furthermore, this invention features a ricin D1B chain molecule. These modifications retain transport function. It is expected to alter or eliminate cell-binding functions while For each embodiment The amino acid sequence is the native 92 amino acid sequence modified by the specific changes described therein. It is characterized by the same or substantially the same amino acid sequence as 20 molecules.

As used herein, "identical or substantially identical to the amino acid sequence of a natural molecule" The phrase "characterized by the same amino acid sequence" does not apply to strict hybridization conditions. by a DNA sequence that can hybridize under the conditions with the DNA sequence of natural lysine. means the encoded amino acid sequence. That is, the glue molecule of this invention is or characterized by various modifications or combinations of modifications disclosed herein. The DNA encoding these proteins (of this invention) (before modification), hybridization with a DNA sequence encoding lysine under strict conditions. Other variants, e.g. Additional deletion(s), substitution(s) or insertion(s) of amino acids in ) may be included. [T. Maniatis et al., Molecular Cloning (A Lab. Laboratory Manual), Cold Spring Harbor Laboratory (1 982), pp. 387-389].

The amino acid sequence of the B chain is shown in standard three letter code form and numbered 1-262. Referring to FIG. 2, the subdomain is 1λ in domain I. (amino acids 1-16), 1α (amino acids 17-59), lβ (amino acids 6O-1 00) and 1λ (amino acids 10l-135) and 2λ (in domain ■) amino acids 136-147), 2α (amino acids 148-183), 2β, (amino acids Acid 184-. 226) and 2γ (amino acids 227-262).

- In embodiments, the protein sequence of ricin B is such that in the amino acids of the subdomain lα It is characterized by a reorganization. A preferred embodiment is the deletion of amino acid 40 or deletion of amino acid 40 other than lysine. Natural amino acids and preferably arginine, leucine or another uncharged amino acid characterized by its replacement by . In yet another embodiment, amino acids 20 and/or or 39 is deleted or preferably substituted by serine. Furthermore, this The mutant has two cysteines at positions 20 and 3 within amino acids 1-10. It can be modified by insertion into either position 9.

Another modification is cysteine at either position 20 or 39. Substitutions of naturally occurring amino acids within the first ten amino acids to.

In another embodiment, the protein sequence of ricin B comprises amino acids in subdomain 2γ. It is characterized by a reorganization. In a preferred embodiment, positions 229, 237, 247 At least one of the amino acids at positions 248, 250, 253 and 254 are respectively Asn, Arg, Val, His, Phe or Val, As The modification to n and Leu matches the sequence of ricin E. Another aspect In this case, the last 8 to 34 amino acids at the carboxyl terminus are deleted.

In yet another aspect, the protein sequence of ricin B comprises subdomain lα and/or is characterized by a rearrangement in the subdomain 2γ. - In embodiments, amino acids 46 and and/or amino acid 255 is deleted or replaced by a natural amino acid other than asparagine. Replace. In a preferred embodiment, amino acids 46 and/or 255 are Substituted by amine, leucine, aspartic acid, serine or lysine.

In another preferred embodiment, the modified lysine has amino acids 37 and/or 248 Deletion or phenylalanine and/or alanine, histidine or other non- It is characterized by modifications such as substitution with aromatic amino acids.

In another aspect, amino acids 22 and/or 23 in the ricin molecule of the invention 4 is deleted or replaced with a natural amino acid other than aspartic acid. preferred condition , amino acids 22 and/or 234 are substituted with asparagine, glutamic acid, Substitution by alanine or another uncharged amino acid.

In another embodiment, the tripeptide sequence at positions 22-24 and 234-236 Deletion of at least one amino acid of lagic acid, valine and arginine, or In another embodiment, substitution. Another preferred embodiment includes the prolyte at positions 38 and 249. amino acids in which at least one of the amino acids is deleted or replaced by a different naturally occurring amino acid; Contains arrays.

Another embodiment includes 1-5 in the region extending from amino acids 20-49 and 234-258. insertion of additional prolines, extending from amino acids 20-49 and 234-258 Substitution of amino acids 1 to 5 with proline in the region or amino acids 20 to 49 or and 1-10 amino acids in the region extending from 234 to 258, such as leucine and It is characterized by substitution with branched chain amino acids such as and isoleucine.

Another embodiment provides tripeptides at positions 35-37, 47-49 and 256-258. Minor of the following sequence: glutamine, leucine or isoleucine and tryptophan. At least one amino acid is deleted or substituted.

In yet another embodiment, the method comprises converting amino acids from homologous subdomains. do. For example, amino acids 17-59 can be replaced with amino acids 148-183. Can be done. In another aspect, amino acids 227-262 are amino acids 101-135 Replace with

In yet another aspect, two common N-linked glycosylation sites on the B chain of the lysine molecule at least one of them is modified other than the common N-linked glycosylation site. It is characterized by

In other embodiments, the following amino acid changes, Leu150 to Met, Glul58 to Lys, I lel 61 as Leu, 5erl 65 as Thr, 5erl 93 is ThrSSerf 95 is AlaSArgl 98 is Lys, Glu l　99 as Guy, A1a210 as Val, 5er229 as Asn and A1a 237 and at least one of Arg and 10 or less.

In yet another aspect, one or more of the aspects described above may be modified or modified according to the invention. Incorporated into the B chain of the molecule. The above-mentioned mutations may include one or more additions It is used in conjunction with a specific mutation.

■Production method of modified lysine molecule The DNA sequence of the ricin gene was cloned and the PolyA selection mRNA Rossiedinges of the National Academy of Sciences, Dan, 5640-5644 (1987)] or from genomic DNA [rK, C, Haring, “Nucleitsk Acids Research”, 13, 8061-8033 ( (1985)], described in Example I below and characterized as described in Example I below.

The present invention provides DNA sequences encoding each mutation of the present invention, which are allelic variants, analogs, derivatives and stringent hybridization procedures. In Figure 1, including (but not limited to) DNA sequences that can hybridize under the conditions The normal part of the DNA sequence encoding the ricin B chain, analog or variant shown. It can be produced by position-directed mutagenesis [see Mania Nice, supra. This space One example of trigent hybridization conditions is hybridization at 65°C 4x SCC. followed by washing with 0.1x SCC at 65°C.

The t: o N A sequence encoding prolysin can also be used [F , 1. See Lamb et al., supra]. Methods for such mutations include existing restriction sites or mutations. Schiller and Smith, “Nuclear "Methods Research", Retired Emperor, 6487-6500 (1982), "Methods Research", Retired Emperor, 6487-6500 (1982) 3.479-488 (1984), Kunne, T.A. Le, Proceedings of the National Academy of Sciences, 8. Phenotypic selection method of L488-492 (1985), B. A. Austra et al. Hetero double-stranded DNA of ``Char'', Yeongdo, 456-459 (1983), or S. D. Porter and M. Smith, Nature, 320.766-768. (1986) and M, D., Matsuterashii and H. L., Heinekel, “Nucle. "Itsuku Assists Research", 11.3113-3121 (1983) Contains "cassette mutation".

Oligopeptide 1 used for direct mutation in the above method, denatured version and DNA A sequence is defined and one or more defined mutant sequences B It is assumed that the chain is obtained. Of course, each of the gluesin molecules of the present invention encodes The obtained DNA is isolated using appropriately selected oligonucleotides (one or more). They can also be produced by those skilled in the art by directed mutagenesis.

Modification of one or both glycosylation sites may result in asparagine-linkages present in the sequence. Synthesis is carried out by substitution or deletion of amino acids at the glycosylation recognition site. aspa Lagin-linked glycosylation recognition sites are specific to appropriate cellular glycosylation sites. It consists of a tripeptide sequence that is recognized by The above tripeptide sequence is asparagus Gyne-X-threonine or asparagine-X-serine, where X is It is always any amino acid. First or third amino acid position of glycosylation recognition site various amino acid substitutions or deletions in one or both of the amino acid deletion) results in non-glycosylation at the modified tripeptide sequence.

The present invention provides vectors for use in a novel method for expressing synthetic molecules. of the present invention To express the modified phosphorus B chain, apply DNA encoding the modified molecule. host cells transfected with the appropriate expression vector and selected by standard genetic engineering techniques. to be introduced.

More preferably, the vector encodes the novel synmolecule of the present invention. The description is completely new and includes NA. For example, replicators, selection genes, enhancers, Other components of the vector, such as the promoter etc., may be obtained from natural sources or Synthesized using techniques within the knowledge of the manufacturer. In addition, the vector beam B chain polype Also included are appropriate expression control sequences capable of expressing the peptide sequence. The vector is , also includes DNA sequences encoding polypeptide bre or prebro sequences. can secrete new ricin molecules from suitable host cells into the medium. [See M.S. Chan, supra]. The vector replicates and a selection control operably linked to a DNA encoding sequence of the invention capable of directing expression. Can contain arrays. Restriction sequences useful in the vectors described above are known to those skilled in the art. and is generally selected based on the type of host cell. Such a choice is also conventional Therefore, it does not form part of the present invention. Useful chain lysine expression vectors include M, S , Chan et al., supra.

Those skilled in the art will appreciate that the DNA sequences of the invention and, for example, pCD [Okayama et al., "Molecula - and Cellular Biology J, 2.161-170 (1982)] and pJL 3, pJL4 [Go et al., “Eurovia 645-653 (1985 )] The mammalian expression vector used in the present invention is You can build a vector. Transformation of these vectors into suitable host cells, e.g. The s-x cell line produces expression of the inventive resin molecule.

Those skilled in the art will appreciate that flanking the coding sequence involves leaving out the mammalian control sequences or removing the bacterial control sequences. intracellular or intracellular by bacterial cells by manipulating the DNA sequence with sequence substitutions. Bacterial vectors can be created that are expressed extravesicularly. For example, coding sequences are It can be further engineered for bacterial expression by methods known to those skilled in the art. Preferably this The sequence allows bacterial expression, secretion and processing of the mature mutant protein by methods known to those skilled in the art. A nucleotide sequence encoding a secretory leader peptide capable of function, in a frame connected within. Compounds expressed in bacterial host cells are recovered by known methods. , purification and/or physicochemical, biochemical and/or clinical parameters. Characterized in terms of data. Sequences are from T. Taniguchi et al. National Academy of Sciences, 77.5230-5 233 (1980) using known bacterial vectors. can be inserted into. This exemplary bacterial vector transforms bacterial host cells. and thereby express the ricin B chain.

Similar operations can be performed using insect vectors [European published patent application] for expression in insect cells. Reference is made to the manufacturing method described in Application No. 155,476.

Regulation of yeast for intracellular or extracellular expression of the molecules of this invention by yeast cells The sequences can be used to construct yeast vectors. [Reference: e.g. PCT Application Publication W O36100639 and European Patent Application EPA 123,289 Method].

The method for producing high levels of the molecules of this invention from mammalian cells involves the production of multiple copies of the gene. This includes the construction of cells containing -. Amplifiable markers, e.g. Kaufman and Sharpe, Journal of Molecular Biology, vol. 159, 601-629 (1982) (2). cells can be selected for reproduction by increasing the concentration of methotrexate (MTX). by combining the dihydrofolate reductase (DHPR) gene with a heterologous gene. obtain. This method can use many different cell types. For example, Kaufman et al., Molecular and Cellular Biochemistry, vol. 5, p. 1750. (1983), other plasmid sequences capable of carrying out the expression. column, and DHPR expression plasmid pAdA26SV(A)3 [Kaufman and Sharp, Molecuy and Cellular Biochemistry, Volume 2, 1 304 (1982)] and its derivatives. A plasmid containing the DNA sequence of the syn molecule was introduced into DHPR-deficient CHO cells, DT It can be simultaneously introduced into JKX-BI and then MTX (0,02,02,1,0 and 5uM-MTX Selection is made for growth by growth at increasing concentrations (in the sequence step).

Transformants were cloned and analyzed using a similar assay system as described by Chan et al., supra. to observe biologically active and syn B chain expression. Increase the level of MTX tolerance It is expected that lysine expression will increase with increasing age.

The invention also provides methods for producing ricin molecules. This method uses known Using the DNA sequence encoding the glue molecule of this invention under the control of the nodal sequence It involves culturing the appropriate transformed cells or cell lines. New part Suitable cells and cell lines for pup expression are Chinese hamster ovary Cells (C) 10), monkey C09-1 cells are like CV-1 cells. It can be a celloid. Suitable for mammalian host cells and transformation, culture, amplification, screening. Selection and purification methods of leaning and product production are those known in the art. . [See, eg, Gething and Sambruch, Nature, vols. 2-3, 62 Pages 0-625 (1981) Alternatively, Kaufman et al., Molecular and Cellular Biochemistry, Volume 5(7), Pages 1750-1759 ( (1985) Howley et al., U.S. Pat. No. 4,419,446. another dawn Mammalian host cells are particularly suitable for primate cell lines, including transformed cell lines, and mammalian cell lines. Contains rodent cell lines. In vitro culture of normal diploid cells and primary tissues Also suitable are cell lines and primary transplants obtained from. Other suitable mammalian cells Ins were obtained from HeLa, mouse-929 cells, Switzerland. T3 lines, Balb-c or NIH mice, BHK or HaK hummus It includes, but is not limited to, Tercel lines.

Bacterial cells are suitable hosts. For example, various strains of Escherichia coli (e.g. For example, HBIOI, MC1061) are used as host cells in the field of biotechnology. It is publicly known. Various species such as Bacillus subtilis, Pseudomonas, and other bacilli Strains may also be used in this method.

Many strains of yeast cells for which techniques are known in the art can also be treated with the polypeptides of this invention. It is possible as a host for the expression of the following species. Furthermore, if desired, insect cells can be It can be used as a host in the method of the invention.

[References: e.g. Miller et al., Genetech Engineering, Vol. 8, 277-2 98 (Bremham Press, 1986) and references cited herein.

Test stable transformants for product expression by standard immunological or functional group analysis. Screen about. All expressed compounds were collected and purified using known methods. , and/or physicochemical, biochemical and/or clinical parameters. characterized by.

The lectin binding properties of ricin B chain variants can be tested in several ways. For example, from the subject culture medium or purified material, free in solution or on a solid support, e.g. Asialofetuin bound to Sepharose or acid-treated Sepharose The lack of the mutant's ability to bind to Mission (reduction) can be used to analyze exclusion [M. Niss Chan et al. Teaching of the National Academy of Sciences of the United States Nailed State of Amelia, Vol. 84, pp. 5640-5644 (19 1987) and T. Mize et al., Agriculture and Biological Chemistry, Vol. 41, pp. 2041-2046, (1977)].

Additionally, a mutant form of ricin B chain on ricin A chain containing a toxin conjugate Thus, toxicity promoting effects can be tested in two formats. The first is A-chain conduit. By addition of excess mutant B chain to target cells in the presence of Y. Neal et al., Journal of Biological Chemistry, 257 Vol., pp. 1598-1601, (1982) and D.B. Matsukintola. Schuller, Federated Genes of European Biochemical Society Letters, Vol. 164, pp. 17-20, (1982) Ko. Alternatively, The variant B chain is raised with an antibody against the ligand of the ricin A chain toxin conjugate, or with A E.R.J. Luton et al., Journal of Immunology, vol. 136, pp. 3103-3109. (1986)]. Similarly, the modified B chain can be directly linked to the ricin A chain and the selected ligand. can be bound and tested on target cells.

IIl. Production of toxin conjugates The ricin B chain of this invention is expressed and isolated, and then toxin conjugates are produced. choice A peptide crosslinker is attached to the isolated ricin B chain.

One preferred crosslinking agent is S-acetylated protected thiopropionic acid. then restart Adding ricin A chain to ricin B chain regenerates holotoxin through disulfide bond formation. Form. Without being limited thereby, the A chain may include recombinant A chain, naturally occurring A, natural variant, chemically deglycosylated or with SynA or other variant A chain could be. [See, eg, US Pat. No. 4,689,401]. Then the selected ligand Covalently attach the modified holotoxin.

Holotoxin-conjugated ligands are not limited by this invention. Conjuge The ligand is chosen depending on the target to which the target is to be directed. Ligands are 11-α, β , 11-2.11-3.11-4.11-5.11-6, M-CSF, G-CS Composed of growth factors such as F, GM-CSFSFGF, TGFα, β and TNF. can be obtained. Ligands include those present on tumor cells, viruses, fungi or bacteria. contains monoclonal antibodies directed against various epitopes on the target site. It could also be an antibody. The above antibodies are NR-Co 1-5 and Medicinal for rectal cancer. Including, but not limited to, R24 of ranoma.

In a preferred method for the production of toxin conjugates, N-succinimidyl S - Treating the B chain with acetylthiopropionate [N.Fujii, Chem. Palm Paiol, vol. 33, p. 362 (1985)]. Then ricin A chain , recombines with the functionalized B chain to form holotoxin. Then hydroxylamine Cleavage of the S acetylated protecting group using chlorine or hydrazine [Clob and Ha Inei, supra]. Thereafter, integral sulfhydryl groups or further peptides are added if desired. Added maleimide or sulfhydryl attached to a hydrogen or carbohydrate moiety A protein ligand containing the group is linked to the B chain of the holotoxin.

In another embodiment, 3-(2-pyridyl treated with dithiothreitol) Treat the B chain with N-succinimidyl dithio)furopionate. after that, Add the A chain and ligand as described by the method steps above. too Of course, treatment with other standard crosslinking agents is within the scope of this invention.

In other embodiments, when the prolysin molecule is modified, prolysin can be converted into a protein. Decomposition treatment yields the A chain which can be released under reducing conditions. Then restart as described above. The gund is coupled to a lysine molecule.

The ricin toxin conjugates of this invention are used in many clinical conditions. the disease Depending on the situation, an appropriate ligand that directs the lysine moiety to an appropriate site is selected. Li The Gund confers specificity to the conjugate molecule. The toxin conjugate of this invention A possible use of these drugs is to use antibody ligands directed to the tumor cell surface. Includes treatment of cancer using the conjugates. For example, malignant tumors, lymphoma and lung The conjugate can be used to treat localized cancers such as carcinomas. release lysine moiety If the conjugate is introduced into the cell where it is thought to destroy the cell, Insert. Types of specific antibodies and growth factors and conjugates available There are several possible uses for other ligands that can consist of Gantt moieties.

It is therefore another object of this invention to provide therapeutic and and compositions. The composition comprises a therapeutically effective amount of at least one This invention consists of a ricin toxin conjugate. These condensers according to this invention The sgate can be mixed with a pharmaceutically acceptable medium or matrix to form therapeutic compositions. can exist inside. Additionally, the therapeutic methods and compositions of this invention provide for lower therapeutic amounts. A therapeutic amount of Norisinco of this invention together with one other Norisin conjugate of this invention. conjugates. Furthermore, the glue-sin conjugates of the present invention or The conjugate combinations of this invention may be co-administered with other agents useful in the treatment. p ) The above-mentioned physiologically acceptable protein having reasonable isotonicity, stability, etc. Methods for making the compositions are within the concave state of the art.

The treatment method involves administering the conjugate to the subject in a mixture with a pharmaceutically acceptable carrier. Including giving. Of course, upon administration, therapeutic compositions for use in this invention is in a pyrogen-free, physiologically acceptable form. Additionally, the composition may desirably be encapsulated or injected in a viscous delivery form.

The mode of administration depends on various factors that modify the action of a particular conjugate, such as the treatment. type of condition, age, sex and diet of the patient, severity of infection, time of administration and The decision will be made by the attending physician after considering other clinical factors. Addition of other factors to the final composition may also affect dosage.

Hereinafter, the method of carrying out the present invention in the production of ricin molecules and its implementation will be explained by examples. The toxin conjugates containing the following are described.

Example 1 Production of modified ricin B chain.

E. L. Jelton, “Mize for Biological Research J (W. F. , edited by Sheridan University Press, Grand Forks, 197- 202 (1982)), castor bean (Ricinus communis, Genomic DNA is extracted from seedlings. Oligonucleotide probes mentioned below Whole genome digested with Hindll and/or EcoRI using Southern analysis of DNA confirmed the presence of 4 and 2 kb fragments.

Using the restriction enzymes EcoR] and Hindlll, R.D. Nichols et al. Kraitsk Acid Research” Vol. 569-7, 57B (1985) ), the 4.2kb E containing the ricin gene was extracted using the restriction fragment enrichment method. The eoRl-H1ndl11 fragment [K, C, Hering et al. Ta. pUc18 digested with the same enzymes was obtained from the 4 and 2 kb size range. The resulting DNA was cloned. Host JMI O7mcrB-(ER1451, After transforming the DNA into the ricin gene (New England Biolabs), The sequences previously published in EK, C, Hering et al., supra and F, 1. rum et al., supra] by oligonucleotides #1 and =2 (Table 1). I screened the library. These oligonucleotides are A and Each hybridizes specifically to the portion of the ricin gene that encodes the B chain. Ru. One double-positive clone was isolated from a library of approximately 30,000 transformants. A sample was obtained, which was named pRICB.

Figure 3 shows 4.2kb EcoRI-, Hindll!, containing the ricin gene. centre A restriction map of the fragment is shown. In the following description, the information in parentheses is shown in Figure 3. matches the name you want to use. In particular, I) in Figure 3 is the restriction fragment described below. represents. ----- represents a deleted sequence.

Furthermore, ~~ represents the ptJc18 sequence. Oligonus cited in the following description Creotides are listed in Table 1.

Referring to Figure 3, the "parent" plasmid, PRAB, is intended for subsequent mutations. as a method to facilitate the cloning of B chain gene fragments into various expression systems. It is constructed as follows. This plasmid contains the KpnI-BamHI flag of pRICB. (rK-BH, Figure 3, Fragment A) with two oligonucleos #3 and #4, re-forming the two restriction sites and Codon Pst! Constructed by introducing a [P] site. Then the above program In the lasmid (pRI CB 5'), NCoI-H1ndll[ The N-H3 fragment (Fig. 3, fragment B) was inserted into two oligonucleotides. #5 and #6 to re-form the above site and create a stop codon (TAG ) of the Xba1 site [Xb3 and Pstl[P co and Xhol [X] site pRAB is generated.

Table 1 #6'r)N λ　　　　　　　　　　　　　C #9 #lOαDingNincludeαλ exposedS #u 　 　 　 　 　 　 　 　 　 　 　 　 　 　 Nigo#L! Y48~Lal^234-236 For mutation purposes, the Pstl-Xbal EP-Xb'H flag of pRAB was fragment (Fig. 3, fragment C) with the same <Pstl and Xbal. Cloned into the restricted Rf form of M13mp18. Subsequent cloning stages To facilitate the process, Av The aI[A3 site was introduced between the Pstl and BamH1 sites to yield mp18B and L (Figure 3).

The mammalian expression plasmid pSHvB contains the Fono-Billebrand factor (VWF). ) [e.g., PCT Publication W08610609 6], which then contains the coding sequence for the ricin B chain.

This plasmid contains an oligonucleotide encoding the prepolypeptide of vWF. Pstl-Xbal fragment from ap18B in the presence of #8 and #9 (Pstl cohesive ends are removed by large fragments of DNA polymerases. ) was restricted by PstJ-Xbal [pSHIL-3-1 was introduced in 1987 February 24th, American Type Culture Collection with accession number 67326. Action (1230 I Parklawn Drive, Rockville, MD, AL) Co., Ltd. deposited in the United States of America). This deposit is a microorganism intended for patent proceedings. Requirements of the Budapest Treaty on the International Recognition of Deposits and the provisions contained therein satisfy. [Refer again to PCT Publication WO 100598.

As mentioned above, the mutant contains the entire DNA sequence encoding the ricin B chain. or by conventional site mutagenesis in some parts.

For example, the method of Schiller and Smith (ibid.) Extraction using a mutagenesis system devised by National (UK) Austra et al. (supra) heteroduplex method and limitations applied by Tine There is fragment replacement. Unless otherwise specified, the DNA described in this specification All operations are performed by T6P, Maniatis, etc., Molecular Cloning: A Lab. This is based on the Laboratories Manual (mentioned above).

The large fragment obtained from pSHVB digested with Kpnl-Xbal was , obtained from pRAB in the presence of oligonucleotides #10 and 311. It was ligated to a 70 bp Kpn1-BamHI fragment. As a result, the last 22 amino acids at the carboxyl terminus to match the sequence found in protein E. A DNA sequence encoding the ricin B chain was obtained, in which the ricin B chain was modified.

Using the M13 system with oligonucleotides #12 and #13, Tyr Codon 248 was changed to Leu and amino acids 247-257 were deleted, respectively. Repair Decorative DNA is restricted by Aval-Xbal, and a fragment encoding ricin B chain is generated. The expression plasmid pSHvB (the wild-type sequence of the ricin B chain is expressed as Aval-Xba (removed by l digestion and purification of the large fragment). This base By using the appropriate oligonucleotides in this procedure, the methods listed in Table 1 can be achieved. A natural mutant was obtained.

Ricin B chain and mutant forms were transiently transfected in CO5-1 monkey cells. Chinese hamster ovary (CH can be expressed by stable transformation of O). H. Rutman et al., “Nukreitsk ・Acid Research” Volume 1 = 1295-1308 (1983) and Tori, M , Manpilak et al., rPNAsJ78Nia 575-7578 (1981) according to pSHvB containing any one of the mutants listed in Table 1. COS monkey cells were transfected. Rabbit poly for ricin B chain A, J., Toner et al., “Journal of Cells,” using clonal antibodies. Radioactive labeling according to "Biology" published by: 2665-2674 (1987) and tested the expression of the mutants in CO5-1 cells by immunoprecipitation. [ S., Ramakrisnan, et al., “Biochimica Et Biophysic Power Acta J7↓9: 341-348 (1982)]. 40-70 hours after transfection, CO5-1 cells were treated with [”S]-methionine, 100 μci/xf21 = 15 Label for 3 minutes and then chase for 3 hours in medium containing 0.1M D-galactose. I did it. The lysed medium and cell extracts were immunoprecipitated in the same manner as above. 12% Immunoprecipitation studies in 5DS-PAGE showed that 3 One major band at 4-36 kDa as well as at 28-30 kDa in cell extracts. A small band appeared.

Example 2 Purification of modified ricin B chain.

Conditioned media containing engineered or SynB were diluted with water and diluted with 50 mmol Na phosphate. Applied to an ion exchange membrane cartridge that has been equilibrated in a buffer solution (pH 7, 5). Ru. The bound protein was washed with the same buffer containing 0.1M galactose, and Na Dilute with C]. Load the eluent into a lentil-lectin affinity column and wash with loading buffer. Alpha-methylmannopyranoside specific binding protein Elute with High molecular weight species are removed by means of highly separated gel filtration columns. do.

The preferred embodiments of this invention have been described in detail above. Expertise in the industry If a person considers these statements, he will make many modifications and variations in his practice. It is expected that it will be possible. Those modifications and variations are included within the scope of the following claims. It is considered that

Example 3 Preparation of M-CSF-ricin toxin conjugate.

The M-CSF-engineered ricin conjugate according to the present invention is produced as follows. be done. For example, similar to the preparation and separation in Example 1, 100 mmol Na Engineered ricin B chain 119 (30 5ATP (130 micromoles) in dimethylformamide (DMF) mol). The reaction is allowed to proceed for 5 hours at 4°C. phosphate buffered NaCl In the process, the derivatized B chain is newly reduced and then by reaction with Ellman's reagent. EG, L, Elma reacted with activated ricin A chain (130 micromoles) [Archives of Biochemistry and Biophysics] J82 Near 0-77 (1959) Ko. Functionalized holotoxin Gel™ TSK-3000 High Pressure Liquid Chromatography Column Gel Purify by filtration.

50 mmol NaHtPO, (pH 7,0)/150 mmol N a in C1, Is it produced in mammalian cells as described in PCT publication WO37106954? = M-CSF (35 micromol) was added to succinimidyl 4-(N-male). imidomethyl)cyclohexa-1-carboxylate (1750 micromol) and react for 1 hour at room temperature. Excess crosslinker is removed by gel filtration. Argon Spray an equal volume of 20 mL of 5ATP-functionalized holotoxin in phosphate/NaCl buffer. Treat with molar hydroxylamine for 30 min at 4 °C and quickly remove the gel filtration column. and then immediately reacted with maleimide-functionalized M-CSF in the same buffer. 4 After reacting for 16 hours at °C, the desired M-CS F-engineered lysine conjugate was This was subjected to gel filtration using a TSK-4000 high pressure liquid chromatography column. Purify by filtration.

The preferred embodiments of this invention have been described in detail above. Experts in the industry Consideration of these statements will lead to many modifications and variations in their practice. It is expected that Those modifications and variations are included within the scope of the following claims. It is considered that

Figure 1 Figura l (Con’t) Figura l (CCn’t) rigura l (canlt) Figure 1 (Can’t) 253fu 　　　　　　　　　　2552 2567'l'GG GCT CTT T-in T GCA GAT GGT　TCA　ATA　CGT　CcT　CAG　C-in A@　AAC TrP Ala　Lau　Tyr　Enter 1a　Asp　Gly　Sar　ENG 1 * Arg Pro Gln Gln Asn2612 262F 2642CGA T GG ATG TTCAAG AAT GAT GCA λCCATT TTA input AT TTG TλT input rg Trp MET Phe L ys Asn Asp Gly Thr Zla Lau Asn L@ u Tyr2702 2717 27 pieces 2λG’i’ GGG TTG GTG TTA G AT GTG AGG GCA TCG GA’! ’　CCG　入GCCTTS @r　Gly　Lau　vaI　Lau　Asp　Val　Arg Ala Ser Asp Pro Sar @Leu 2F92 2B05 2815 2825λTX TGG’! 'TA CCA TTA TTT TGA TAGACAG ATTACTCTCT TGCλGTGTGT Engineering la Trp Lau Pro Lau PheATGTCCTGCCATGAAAATAG ATGGCTTAAA TAAAAAAGGACATTGTAAATTFicp ra　　　　　(Coh’t)λATATGTTTT　TTC input λ input CTTA TAAλλCTATG λλTGATATG included AT included T included TGCG Fort 32 JL Asp Van C% Trial Asp Pro Cdu Pw ne Va l　A-”i　na　Val　Gly　jeg〒Gly　fLea　Cys VILLAsp Veal)xg Asp Gly Arg’Rwa Pi s)m Gly krr a na cln Iaz; drip ow Cys island S 50 6: l5er 15’l’Ihr Asp-)s*Gln’Lm;p total’Lieu 'Lys L7 Asp) m total s Lg AStt:) s* Gly island SCF ≧ meter) τGly%τ at 2°C y electric current) τ electric residual tko1) τAspC ysso mAsn' r! x to - to a’! hr)sp Ala’Ihr)xg Thp Qn n comic pA%) Asn Gly total na na■P m n.

bkg Sar carp - total ssr Gly Hato Sz Gly total 1 total 13o 　　　　　　　　　　　　　　　　　　　　　　　　　　　　 40Gln Asn’X1m Ty Ala Vhl Ssr Glrr Gly　;p stain u　≧fuTi　Asn　Asn　’Ik　Mr. Gi@Pw　’E ks Asp　GILy″ThrT1m　’Liu　Asn　-’X%　Ser　Gl yXsx Val-Asp Veal)q Ala St Chichi@Asp’Er rs c2 −≧Cao international search report m'#ImdMI　A##ca1mN6-,,,,,,C,mouth/IIA F1 negative

Claims (38)

[Claims] (1) A configuration of natural ricin D in which the lectin-binding function of the B chain has been removed or reduced. A ricin molecule having an amino acid sequence substantially identical to a sequence. (2) Amino acids of subdomain 1α are changed or removed, according to claim 1. lysine molecule. (3) Deletion of amino acid at position 40 or substitution with a natural amino acid other than lysine 3. The ricin molecule according to claim 2, wherein the ricin molecule has the following characteristics. (4) Natural amino acids are a group consisting of arginine, leucine, and uncharged amino acids. The ricin molecule according to claim 3, selected from: (5) One or both cysteine residues at positions 20 and 39 are deleted or natural 3. The ricin molecule according to claim 2, which is substituted by an amino acid. (6) A claim in which one or both cysteine residues are substituted with serine. 5. The ricin molecule according to 5. (7) Furthermore, a. Within 1-10 amino acid molecular distance of either position 20 or 39 inserting two cysteines, and b. For either 20th or 39th place From substitution of natural amino acids by cysteine within molecular distance of 1-10 amino acids Ricin molecule according to claim 5, characterized by a modification selected from the group consisting of: (8) Subdomain 27ν amino acids are changed or removed, according to claim 1. lysine molecule. (9) Amino acid 229 was replaced by asparagine, 237 was replaced by argenine. 247 replaced by valine, 248 replaced by histidine, 250 replaced by feline. Substitution by nilalanine or valine, substitution of 253 by asparagine, and and the substitution of leucine for 254. The ricin molecule according to claim 8. (10) The last 8 to 34 amino acids at the carboxyl terminus are the lysine E B chain subunit. Amino acids have been substituted or deleted to match those found in 9. The ricin molecule according to claim 8. (11) Amino acids in subdomain 1δ and/or subdomain 2ν are changed. 2. The ricin molecule of claim 1, wherein the ricin molecule has been removed. (12) At least one of the amino acids at positions 46 and 255 is deleted or naturally occurring. 12. The lysine molecule of claim 11, which is substituted by a amino acid. (13) Natural amino acids include glutamine, leucine, aspartic acid, serine and 13. The lysine molecule of claim 12, wherein the lysine molecule is selected from the group consisting of lysine and lysine. (14) At least one of the amino acids at position 37 or 248 is deleted or selected from the group consisting of nilalanine, alanine, histidine and non-aromatic amino acids. 12. The ricin molecule according to claim 11, which is substituted with a discovered amino acid. (15) Deletion of amino acid at position 22 and/or 234 or aspartic acid 12. Lysine molecule according to claim 11, characterized by substitution by natural amino acids other than . (16) Natural amino acids include asparagine, glutamic acid, alanine and uncharged 16. The ricin molecule of claim 15, selected from the group consisting of amino acids. (17) Tripeptide sequence asparagine at positions 22-24 and 234-236 At least one amino acid among acid, valine and arginine is deleted or substituted. 12. The ricin molecule according to claim 11, wherein the ricin molecule is (18) At least one of prolines at positions 38 and 249 is deleted or 12. The ricin molecule of claim 11, wherein is substituted with a different natural amino acid. (19) 1 to 5 amino acids in the region extending from amino acids 20 to 49 and 234 to 258; 12. The lysine molecule of claim 11, wherein the amino acid is substituted by proline. (20) Amino acids 20-49 and amino acids 234- by branched chain amino acids A claim characterized by substitutions of 1 to 10 amino acids in the region surrounding No. 258. 12. The ricin molecule according to 11. (21) The branched chain amino acid is selected from the group consisting of leucine and isoleucine. 21. The ricin molecule of claim 20. (22) Tripeptide sequences at positions 35-37, 47-49 and 256-258 Less of the column glutamine, leucine or isoleucine and tributophane The ricin molecule according to claim 1, wherein at least one amino acid is deleted or substituted. . (23) Amino acids from one subdomain are from homologous subdomains 2. The ricin molecule of claim 1, wherein the ricin molecule is substituted with (24) amino acids 17-59 are replaced by amino acids 148-183, 24. Ricin molecule according to claim 23. (25) Amino acids 227-262 are replaced by amino acids 101-135. 24. The ricin molecule of claim 23. (26) Amino acids 227-262 are replaced by amino acids 101-135, Claim 23, wherein amino acids 17-59 are replaced by amino acids 148-183. Lysine molecule described. (27) The following amino acid changes, Leu150 to Met, Gln158 to Lys, I Le161 is Leu, Ser165 is Thr, Ser193 is Thr, Ser1 95 is Ala, Arg198 is Ly8, Glu199 is Gly, Ala210 is Val, Ser229 is Asn, and Ala237 is Arg. 2. The ricin molecule of claim 1, comprising one and up to ten. (28) at least one of the common N-linked glycosylation sites on the B chain of the ricin molecule; 2. wherein one of the N-linked glycosylation sites is modified at a site other than the common N-linked glycosylation site. ricin molecule. (29) Amino acids in the region extending from amino acids 13 to 143 (including these) are missing. 2. The ricin molecule according to claim 1, wherein the ricin molecule is missing. (30) The amino acids in the region extending from amino acids 134 to 260 (including these) are 2. The ricin molecule of claim 1, wherein the ricin molecule is deleted. (31) DNA encoding the ricin molecule according to claims 1-30. (32) A host selected from the group consisting of mammalian, yeast, insect, fungal, and bacterial cells. Non-specific cells produced by expression of the DNA molecule according to claim 30 in cells. Ricin molecules with reduced or reduced vacuole-binding affinity. (33) Effectiveness of the ricin molecule according to claims 1-30 bound to the selected ligand. A therapeutic composition comprising an amount. (34) The therapeutic composition according to claim 33, wherein the ligand is a growth factor. (35) The growth factors are II-Iα, β, II-2, II-3, II-4, II- 5, II-6, M-CSF, GM-CSF, G-CSF, FGF, TGFα, β 35. The therapeutic composition of claim 34, selected from the group consisting of and TNF. (36) The therapeutic composition according to claim 33, wherein the ligand is an antibody or a part thereof. . (37) (a) bonding a peptide crosslinking agent to the B chain molecule according to claims 1-30; The ricin A chain molecule is attached to the B chain molecule by rufido bond formation to form a modified ricin molecule. (c) modifying the B chain of the holotoxin with a selected protein ligand; covalently bond to A method for producing a ricin conjugate, comprising the steps. (38) The crosslinking agent is N-succinimidyl S-acetylthiopropionate. (a) The B chain of the modified holotoxin, (a) (b) to attach a hologram to a ligand containing a maleimide group or other hydrosulfur-reactive moiety. coupling toxins 38. The method of claim 37, wherein the method comprises the step of: