CA1217156A - Hybrid proteins - Google Patents

Abstract

A hybrid protein including protein fragments joined together by peptide bonds, the hybrid protein including, in sequential order, beginning at the amino terminal end of the hybrid protein, a) the enzymatically active Fragment A of diphtheria toxin, b) a fragment including the cleavage domain l1 adjacent Fragment A, c) a fragment including at least the portion of Fragment B of diphtheria toxin encoded by the portion of the Fragment B encoding gene fragment of the tox operon between l1 and the position about 90 base pairs upstream from the position on the tox operon of the NRU I site of the tox 228 allele, and d) a fragment including a portion of a cell-specific polypeptide ligand, such portion including at least a portion of the binding domain of the polypeptide ligand, such portion of the binding domain being effective to cause the hybrid protein to bind selectively to a predetermined class of cells to be attacked by enzymatically active Fragment A.

Description

his invention relates to the use of recombinant DNA techniques to make hybrid protein molecules, and to the use of such molecules in the treatment of medical disorders.
The literature contains man~y examples of fused genes which code for hybrid proteins. For example,-Villa-IComaroff et al. (1978) P.N.A.S. U.S.A. 75, 3727-3731 describes a fused gene made up of a eukaryotic ; structural gene fused to a non-cytoplasmic bacterial gene. The fused gene codes for a hybrid protein which is transported out of the cytoplasm.
Hybrid proteins have been also made by methods, e.g. the coupling of two different protein molecules, which do not involve recombinant DNA techniques. For example, it has been proposed to form, by coupling, therapeutic hybrid proteins consisting of a toxin coupled to a ligand capable of hinding specifically to a selected class of cells. One attempt to make such a hybrid protein, reported in Chang et al. (1977) J. Biol.
Chem. 252, 1515-1522, resulted in a hybrid consisting of the diphtheria to~in A chain coupled to human placental lactogen hormone by cross-linking through a disulfide bond. The hybrid protein, although it bound to cells containing lactogen receptors, did not inhibit protein synthesis in those cells.

~ `
.`
~: :
,:1 ,,.
: :

.
, ~2~

A hybrid protein consisting of ricin A toxin coupled to the ~ chain of human chorionic gonadotropin hormone by similarly cross-linking through a disulfide bond has also been reported; although said to havespeclfic~ty its binding capacity has not been reported, and extremely high concentrations were required to significantly inhibit protein synthesis in rat Leydig tumor cells, making it difficult to distinguish between "non-specific" entry caused by endocytosis and "specific" entry caused by transport of the toxic portion of the hybrid across the cytoplasmic membrane of the target cells. Oeltman et al. (1979) J. Biol. Chem., 254, 1028-1032.
The same shortcoming was found in a hybrid consisting of diphtheria A coupled to insulin using cystamine as the cross-linking agent. Miskimins et al. (1979) Biochem. Biophys. Res.
Commun., 91, 143-151. A hybrid consisting of ricin A coupled to epiderma] growth fac~or (EGF) by means of a heterobifunction-al cross-linker has also been made, bu-t the binding character-istics provided by the EGF are not limited to specific cells, but encompass a wide variety of cell types. Cawley et al.
(1980) Cell, 22, 563-570.
It has now been found that a superior diphtheria toxin/hormone hybrid protein can be made in which the protein is synthesized as a single unit; i.e., fragments are joined together not by cross-linking but by peptide bonds.
According to one aspect of the present invention there is provided a method for preparing a hybrid protein which comprises formirlg a fused gene coding for said hybrid pro-tein and expressing said fused gene, wherein said fused gene comprises a fragment of a gene coding for diphtheria : ,' .}

~7~

- 2a -toxin fused to a fragment of a gene coding for a cell-specific polypeptide ligand, said fragment of said diphther.ia toxin gene including in sequential order beginning at the 5' terminal end of said fragment a) the fragment coding for the hydrophobic leader signal sequence of said diphtheria toxin, b) the fragment coding for the enzymatically active Fragment A of said diphtheria toxin c) the fragment coding for protease sensi-tive loop 1, adjacent said Fragment A of said diphtheria toxin, and d) a fragment encoding at least a portion of the hydrophobic domain of Fragment B of diphtheria toxin and not including the generalized eukaryotic binding site of Fragment B, and said fragment of a gene coding for said cell-specific polypeptide ligand encoding a portion of a cell-specific polypep~ide ligand, said portion including at least a portion of the binding domain of said polypeptide ligand~ said portion of said binding domain being effective to cause said hybrid protein to bind selectively to a predetermined class of cells to be attacked by said enzymatically active Fragment A.
According ~o another aspect of the present invention ~here is provided a hybrid protein comprising protein ragments joined toge~her by peptide bonds9 said hybrid protein compris-~7~
- 2b -ing, in sequential order, beginning at the amino terminal end of said hybrid protein, a) the enzymatically active Fragment A of diphtheria toxin, b) a fragment including the cleavage domain 1 adjacent said Fragment A of diphtheria toxin, c) a fragment comprising at least a portion of the hydrophobic domain of Eragment B of diphtheria toxin and not including the generalized eukaryotic binding site of Fragment B, and d) a fragment comprisiny a portion of a cell-specific polypeptide ligand, said portion including at least a portion of the binding domain of said polypeptide ligand, said portion of said binding domain being effective ~o cause said hybrid protein to bind selectively to a predetermined class of cells to be attacked by said enzymatically active Fragment A whenever prepared by the above process.
~ ccording to a further aspect of the present invention there is provided a fused gene comprising a ~ragment of the gene coding for diphtheria toxin fused to a fragment of a gene coding for a cell-specific polypeptide ligand, said fused gene codin~ for a hybrid protein, said fragment of said diphtheria toxin gene including in sequential order, beginning at the 5' terminal end of said ,, . I
.",i,~

- 2c -fragment, a) the fragment coding for the hydrophobic leader signal sequence of said diphtheria toxin, b) the region coding for the enzyrna-tically active Fragment A of said diphtheria toxin, c) the region coding for protease sensitive loop 11 adjacent said Fragment A of said diphtheria toxin, and d) at least a por-tion of the hydrophobic domain of Fragment B and not including the generalized eukariotic binding site of Fragment B, and said fragment of a gene coding for said polypeptide ligand encoding a portion of said ligand effective to cause said hybrid protein to bind selectively to a predetermined class of cells to be attacked by said enzymatlcally active Fragment A~

The invention will be best understood by referring to the drawings, in which Figure 1 is a diagra~natic representation of the diphtheria toxin molecule;

~ L7~

; Fig. 2 is a diagrammatic representation of a hybrid protein molecule of the invention;
Fig. 3 is a restriction map showing the location and orientation of the diphtheria tox operon on the 3.9 kb BamH-I restriction fragment of corynephage ~toY.~r (including a site, ~RU I, not found on the wild-type tox allele, but only on the mutant tox 228 allele, as will be explained in more detail below), and Fig. 4 is a diagrammatic representation of a fused gene of the invention, encoding a hybrid protein of the invention (the gene fragments are labeled in terms of the encoded protein fragments).
Referrin~ to Fig~ 1, the diphtheria toxin molecule consists of several functional "domains" which can be characterized, starting at the amino terminal end of the molecule, as hydrophobic leader signal sequence 5, enzymatically active Fragment A, -the fourteen amino acid exposed protease sensitive disulfide loop (DSL) 11, containing a cleavage domain, and Fragment B, ~hich includes hydrophobic do~ain h, DSL 12, and carboxy terminal end.
The process by which diphtheria toxin intoxicates sensitive eukaryotic cells involves at least the ~ollowing steps: (i) diphtheria toxin binds to - 25 specific receptors on the surface of a sensitive cell;
(ii) while bound to its receptor, the toxin molecule is internalized in an endocytic vesicle; ~iii) either prior to internalization, or within the endocytic vesicle, the toxin ~olecule is cleaved (or processed) at a site in the re~ion of 47,000 daltons from the N-terminal end:

~ -(iv) as the pH of the endocytic vesicle decreases to below 6, the structural intermediate form of toxin, while still bound to its receptor, is inserted into the membrane; (v) once embedded in the membrane the -hydrophobic domain h forms a pore; (vi) a proteolyticcleavage in 11, between Fragment A and B, occurs, (vii) thereafter, Fragment A, or a polypeptide containing Fragment A, is released into the cytosol;
(viii) the catalytic activity of Fragment A, i.e., the nicotinamide adenine dinucleotide - adenosine diphosphate ribosylation of Elongation Factor 2, causes -the death of the intox~cated cell. It is apparent that a single molecule of Fragment A introduced into the cytosol is sufficient to kill the cell.
~he hybrid proteins of the invention include, in sequential order, beginning at the amino terminal end of the hybrid protein, the following peptide -fragments, joined together by peptide bonds:
a) the enæymatically active Fragment A of diphtheria toxin (without the leader Fragment s, which is clipped during secretion of the protein), b) a fragment including the cleavage domain 11 adjacent said Fragment A of diphtheria toxin, c) a fragment comprisirlg at least the portion of Fragment B of diphtheria tox~n ~ncoded by the portion of the Fragment B encoding ~e~ion of tox operon between 11 and the position about 90 base pairs upstream from the position on the tox operon of the NRU
I site of the tox 228 allele, and ~ ' ~
r d) a fragment comprising a portion of a cell-specific polypeptide ligand, the portion including at least a portion oE the binding domain of the polypeptide ligand, the portion of the binding domain being effective to cause the hybrid protein to bind selectively to a predetermined class of cells to be attacked by enzymatically active Fragment A of diphtheria toxin. mhe necessary portion of the toxin molecule included is depicted as the portion of the molecule between lines y and x in Fig. 1. Preferably, the hybrid protein also includes protease sensitive DSL
12 of the toxin molecule, i.e. the portion of the molecule between lines x and z is also included. Line z is preferably at th~ point 47 amino acids from the carboxy terminal end of B', i.e;, at the end of 12, not closer, to ensure that the generalized eukaryotic binding site of Fragment B is excluded, so that binding will be controlled by the binding domain of the cell~specific ligand. It has been demonstrated that a little more than one-half of Fragment B must be provided for the molecule to act as an effective toxi~n.
Referring to Fig. 2, a diagrammatici representation of a hybrid protein molecule of the invention, the y-z portion of the diphtheria toxin molecule is joined, by a peptide bond, to Fragment r of a cell-specific polypeptide ligand, i.e. a polypeptide which selectively binds to a predetermined class of cells which are to be attacked by enzymatically active ~2~5~

Fragment A of the diphtheria toxin molecule. Fragment r can consist of the entire ligand, or a portion of the ligand which includes the entire binding domain of the ligand, or an effective portion of the binding domain.
~hen the ligand being used is large, it is desirable that as little of the non-binding portion as possible of the ligand be included, so that the binding domain of the molecule is positioned close to the hydrophobic domain h of Fragment B. It is also desirable to include all or most of the binding domain of the ligand molecule. Tn the case of a melanocyte stimulating hormone (M5H), which is a small peptide of thirteen amino acids, ox ~MSH, which contains seventeen amino acid~s, the portion of the molecule consisting of nine amino acids at the carbo~y terminal end of the molecule can be used, or the entire molecule can be used.
The regions within cell-specific ligands in which the binding domain is located are now known for a number of such ligands. Furthermore, recent advances in solid phase polypeptide synthesis can enable those skilled in this technology to determine the ~ nding ' domain of practically any such ligand, by synthesizing various fragments of the ligand and testing them for the ability to bind to the class of cells to be attacked.
The hybrid protein molecules of the invention, are virtually non-toxic to all mammalian cells except the cells of the specific class to which the ligand J ~ .
"~

, . . .

~2~

- binding domain bindsO Thus, the hybrid proteins of the invention are much more specific than many other therapeutic agents, e.g., general cytotoxic anti-cancer drugs.
The hybrid proteins of the invention, in which fragments are joined via peptide bonds, are also superior to cross-linked hybrids because the proteins of the invention can be provided in a homogeneous sample, in which all of the identical molecules are effective and selective for a particular class of cells.
The specific class of cells which are bound and attacked by the hybrid proteins of the invention is determined by the specific polypeptide ligand which supplies the binding domain of the hybrid molecule. Any cell-specific polype~ptide ligand can be used which has a binding domain which is specific for a particular class of cells which are to be attacked. Polypeptide hoxmones are useful such ligands. Hybrids made using a portion of the binding domain of ~ or ~ ~ISH, for example, selectively bind to melanocytes, rendexing the hybrids useful in the treatment of melanoma. Other ligands provide different specificities; e.g., the b~nding domain of substance P recognizes receptors on the surfaces of neurons involved in the transmission of pain, so that hybrids made using substance P can be used to destroy such neurons to relieve chronic pain. These hybrids can also be used to map areas of the nervous system containing substance P receptorsO Other specific-binding ligands which can be used include somotostatin, interleukin I, interleukin II, and ;

Z~7~

interleukin III. Interleu]cin II is of particular importance because of its role in alleryic reactions and autoimmune diseases such as lupus, involving activated T
cells. In addition, since all of the-interleukins are specific for T cells, hybrids made with them could be used to treat cancers involving the immune system, and to inhibit the rejection of transplanted organs. Other useful polypeptide ligands having cell-specific binding domains are follicle stimulating hormone (specific for ovarian cells); luteinizing hormone (speci~c for ovarian cells), thyroid stimulating hormone (specific for th~roid cells); vasopressin ~speciic for uterine cells, as ~ell as bladder and intestinal cells); prolactin (specific for breast cells); and growth hormone (specific for certain bone cells).
The hybrid proteins of the invention are preferably prepared using recombinant DNA techniques involving forming the desired fused gene coding for the hybrid protein, and then expressing the fused gene, using conventional procedures. Referring to Fig. 3, the ; location and orientation of the diphtheria tox operon on the 3.9 kb BamH-I restriction fragment of corynephage ~ to~
allows the tox operon to be cIeaved at a desired location, and the desired portion of the operon to be fused with the desired portion o~ the gene for a selected polypeptide ligand. A more detailed description of the tox operonS and a description of the cloning of Fragment A, are contained in Leong et al.
(1983) Science 220, 515.
Fragment A, cloned as described therein to make plasmid pDT201, was deposited in the American Type Culture Collection, Rockville, MD on May 11, 1983, and has been given ATCC Acccession No. 39359 .

~2~

Referring to Figs. 3 and 4, the portion of the diphtheria to; operon (Fig. 3) used to make the fused gene (Fig. 4) encoding the hybrid proteins of the invention is pre erably the portion indicated by the dotted lines delinea~ing Fragment D; i.e. the portion of the tox gene from the first Sau 3~I site to the SPH I
site. Fragment D thus includes the 831 base pair (bp)-gene fragment encoding Fragment A (including the 177 bp sequence ahea~ of the structural gene which includes the promoter and the portion encoding the signal sequence, S
in Fig. l); the portion of the gene fragment encoding the hydrophobic domain of Fragment B; and the gene fragment encoding DSL 12.
As shown in Fig. 3, the first 177 bp of the ~5 gene fragment encoding Fragment A includes, ahead of the tox promoter, some DNA which is not part of the tox operon. mhis DNA is irrelevant and is not transcribed;
it is incLuded only because the Sau 3AI site at the start of the Fragment A encoding gene fragment is the most convenient restriction site near the tox promoter (which is depicted as the line just to the left of Hind III).
It should be possible to obtain Fragment D by simply excising it from the tox operon via cleavage at Sau 3AI and SPH I. Alternatively, gene fragments can be fused together as follows. First the gene fxagment encoding Fragment A is obtained. Next, the gene fragment encoding most of Fragment B, from Sau 3AI to Sau 3~I (B' of Fig. 3) is obtained. The gene fragment encoding Fragment B' has been cloned in plasmid pUC8, to make plasmid pDT301, and was deposited in the American Type Culture Coll~ection~ Rockville, MD on May 11, lg83, and was given ATCC Accession No. 39360 _. Fragment B' ' is cut back, using enzyme Bal31, about 200 bp, to the position of the ~RU I site on tox 22~ (the wild-type tox allele does not have an NRU I site), to give 3'' (Fig. 3). Fragments A and B'' are fused, a fragment encoding 12 is fused to B'', and a fragment encoding the desired portion of the polypeptide ligand is fused to 12.
In the above scheme, the Fragment B' encoding gene fragment is preferably cut back to the precise location of NRU I, but can also be cut back to any location between the location 90 bp ahead of NRU I, and the amino te~ninal end of the 12 encoding region, (i.e., within the 208 bp region between the location 90 bp ahead of NPU I, and the beginning o~f the 12 encoding region). Or, the B' encoding fragment could be cut back to the carboxy terminal end of the 12 encoding region (129 bp downstream from NRU I ), in which case fusion of a synthetic 12 encoding region is unnecessary. (It should be evident that, when the B' encoding region is cut back and a synthetic 12 encoding -fragment is fused to it, there generally will be a small number of base pairs normally found between NP~U I and SPH I which will not be present in the fused gene, so that, strictly speaking, not all of D of Fig. 3, or all o' y-z of Figs.
1 and 2, will necessarily be included.) An alternative method, less preferred than the scheme above, is to fuse the ligand-encoding gene frag~ent directly to B'', without employing the 12 encoding fragment.
~he fused gene, either including or omitting the 12-encoding fragment, can alternatively be made using the B'' encoding gene fragment from the mu~ant 5~

tox 228 allele, rather than the wild-type allele. The tox 228 allele, containing the NRU I site, is easily processed to yield the B'' encoding fragment. The tox allele is described in Uchi~a et al. (1973) Jour. Biolog. Chem. 248, 383-8.

In more detail, fused genes encoding hybrid proteins of the invention can be made as follows.
Vectors The preferred vectors are plasmids pUC7, pUC8, pUC9, and pBR322. The pUC plasmids, described in Viera et al. (1982) Gene 19, 259 are particularly well-suited for double dlgest cloning, a procedure which permits the fused genes to be made unarnbiguously.
~ :
Fused ~
Below is a flow chart for constructing diphtheria toxin-MSI~ fused genes containing the protease sensitive loop 12 between the tox sequences and the ligand (in this case, MSH) sequences.
(i) pDT201 Sau3Al ~ purify Sau3Al-2 digest ' (Fragment A) -(ii) p~T301 HindIII Bal31 Pstl ~ Sau3Al digest > cutback > linkers ~ digest ~
reclone Select blue colonies purify Sau3Al-Pstl Sau3Al-Pstl ~ on X-G for proper ~ (Fragment B'') in pUC8 reading frame (iii) in vitro s~esis of gene fragment fox clone into Pstl-EcoRl protease sensitive loop ~ sites on pUC9 ~ purify 12 (Pstl-loop-EcoRl) fragment (iv) clone (ii) Sau3Al-Pstl and (iii) Pstl-Eco ~1 into the BamH]-EcoRl sl-tes on pUC~ (Sau3Al-Fragment B''-loop-1) ,~

1~.,`.

~2~

(v) clone (iv) Sau3Al-EcoRl and MSH-encoding sequence into the BamHa site ~n p ~ (Sau3Al-Fragment B''-loop-MSH-BamHl) (vi) clone (i) Sau3Al-Fragment A-Sau3Al and (v) Sau3Al-Frag~ent B''-loop-r~SH-BamHl into the BamHl site on pUC8 (Sau3Al-Fragment A-Fragment B'' loop-MSH-BamHl) Referring to the above flow chart, in step (i), the Fragment A encoding gene fragment is first obtained and purified, as described in Science, Id. In step (ii) the Fragment B'' encoding fragment is obtained by cutting back a larger Fragment B' encoding region using the enzyme Bal31.
As shown in Fig, 3 the gene fragment encoding Fragment B' is the 1,023 bp region between two Sau3AI
sites, the first of which is at the DNA sequence encoding the third a~ginine in 11, and the second of which is 49 bp before the end of the tox structural gene. The gene fragment encoding B' has, as is mentioned above, been cloned in pUC8. mhe plasmid carrying this fragment in the same orietation as the lac z gene has been designated pDT301; (the lac Z and B' genes are out of frame on pDT301).
Referring again to the above flow ch~rt, pDT301 is opened via HindIII digestion and then the B`' encoding . .
fragment is cut back by exposure to the exonuclease Bal31 for varying time periods. The ends of the resulting shortened gene fragments (of varying lengths) are then blunt-end ligated with EcoRl and Pstl linkers and the fragments are then digested withSau3Al. This results in a heterogeneous (in terms of size) population 30 of gene fragments encoding part of B'.
These fragments are then recloned in either the BamHl~Pstl or the BamHl-EcoRl sites of pUC8. Cloning in these sites allows selection only of clones having BamHl (Sau3AI) on one end and Pstl or EcoP~l on the other.
~ .

~2~

Also, cloning in double digested pUC8 allows for only one fragment orientation, and selection of blue colonies (those expressing lac Z) on X-G plates verifes an in-frame junction between the shortened B' encoding region and lac ~. The preferred clones are those in which the B' encoding region is 830 bp long; i.e. the gene has been cut back 200 bp, to the position of NRU I
on tox 2 8.
~ he next step (iii) is the in vitro synthesis o~ the gene fragment encoding the protease sensitive loop 12. This is carried out by means of conventional solid phase oligonucleotide synthesis. The sequence of this fragment, including the Pstl and EcoRl linkers which are attached after synthesis, is shown below:
Cys-Arg-Ala-Ile-Asp-Gly-Asp-Val-Thr-Phe-Cys TGC-AGA-GCT-ATA-GAG-GG~-GAT-GTA-ACT-TTT-TGC
Pstl EcoRl linker linker The next step (iv) is to clone the B'' encoding fragment from step (ii) and the 12 encoding fragment from step (iii) into BamHl-EcoRl sites on pU~8.
Next, the desired -Eragment encoding a portion of the cell-specific ligand is provided, e~;~ther from a natural source or by oligonucleotide synthesis. For example, gene fragments encoding specific binding portions of ~ and ~ MSH can be synthesized via conventional solid phase oligonucleotide synthesis.
The DNA sequences of those gene fragments, along with the appropriate linkers, are shown below:
~-MSH:
Ser-Tyr-Ser-Met-Glu-His-Phe-Arg-Trp-Gly-Lys-Pro-Val _ AGC-TAT-AGC-ATG-GAA-GAT-TTT-AGA-TGG-GGX-AAA-CCX-GTX _ T C T G C C G G
EcoRl stop codon &
Linker BamHl linker r -. .

- 3 -~IS~I
Asp-Glu-Gly-Pro-Tyr-~et-Glu-His-Phe-GAT--GAA--GGT-CCA--TAT--AmG--GAG--CAC--TTT
C G X X C A T C
Arg-Txp-Gly~Ser-Pro-Pro-Lys-Asp AGA-TGG-GGT-TCT-CCG-CCG-AAA-GAT
G X X X X TC C
EcoRl stop codon &
lin~;er BamE~l linker The unique Pstl and EcoRl sites of pUC8 permit the subcloning of either of the above synthetic ~ISH
sequences, downstream from the 12 encoding fragment.
Finally (step vi) the Fragment A encoding gene fragment is fused to the gene fragment encoding B''-12-MSH, to complete the gene fusion as C
illustrated in Fig. 4 (labeled in terms of encoded protein fragments), ~hich codes for a hybrid protein which selectively binds to and attaclcs a selected class of cells (in this case, melanocytes).
Another example of a suitable polypeptide ligand is substance P, the utility of which is described above. A fused gene containing the substance P gene, rather than the a or ~ MSH gene, is made basically as outlined above. The substance P gene is synthesized ; using conventional solid phase oligonucleotide synthesis. The substance P gene sequence is:
CGTCCTAAACCTCAGCAGTTCTmCGGTCTGATG.
As is clear from the above, the portion of the genetic sequence for the polypeptide ligand must be sufficient to enable the corresponding amino acid sequence to cause the hybrid protein to bind to the predetermined class of cells. Preferably, the gene for the polypeptide hormone will include all or mos~ of the qenetic sequence for the binding domain of the ligand.

:~ ~

~2~

Generally, as in the above examples, the manipulative operations are carried out usin~ cloning vectors, e.g., phages or plasmids. The genetic material coding for the binding domain of the polypeptide ligand can be either cloned DNA or a synthetic oligonucleotide sequence, whichever is more convenient for the particular ligand gene employed. Generally the fused gene will reside on a cloning vector, e.g., a plasmid or a phage, which is used to trans~orm cultured microorganisms. The hybrid protein is then harvested from the culture media of the cells using conventional techniques.
~ he hybrid proteins of the invention are administered to a mammal, e.g., a human, suf~ering from a medical disorder, e.g., cancer, characterized by the presence of a class of unwanted cells to which a polypeptide ligand can selectively bind. The amount of protein administered will vary with the type o~ disease, extensiveness of the disease, and size and species of ~the mammal suf~ering from the disease. Generally, amounts will be in the range of those used for other cytotoxic agents used in the treatment of cancer, ; although in certain instances lower amounts will be needed because of the specificity of the hybrid proteins.
The hybrid proteins can be administered using any conventional m~thod; e.g., via injection, or via a timed-release implant. In the case of MSH hybrids, topical creams can be used to kill primary cancer cells, and injections or implants can be used to kill metastatic cells. The hybrid proteins can be combined with any non to~ic, yharmaceutically-acceptable carrier sub.stance.

Claims (23)

1. A method for preparing a hybrid protein which comprises forming a fused gene coding for said hybrid protein and expressing said fused gene, wherein said fused gene comprises a fragment of a gene coding for diphtheria toxin fused to a fragment of a gene coding for a cell-specific polypeptide ligand, said fragment of said diphtheria toxin gene including in sequential order beginning at the 5' terminal end of said fragment a) the fragment coding for the hydrophobic leader signal sequence of said diphtheria toxin b) the fragment coding for the enzymatically active Fragment A of said diphtheria toxin, c) the fragment coding for protease sensitive loop l1, adjacent said Fragment A of said diphtheria toxin, and d) a fragment encoding at least a portion of the hydrophobic domain of Fragment B of diphtheria toxin and not including the generalized eukaryotic binding site of said Fragment B, and said fragment of a gene coding for said cell-specific polypeptide ligand encoding a portion of a cell-specific polypeptide ligand, said portion including at least a portion of the binding domain of said polypeptide ligand, said portion of said binding domain being effective to cause said hybrid protein to bind selectively to a predetermined class of cells to be attacked by said enzymatically active Fragment A.

2. A method according to claim 1 wherein said fragment d) is a portion of the Fragment B encoding region between l1 and the position of the NRU I site.

3. A method according to claim 1 wherein said fused gene further comprises a fragment coding for the protease sensitive loop l2 between the fragment coding for said diphtheria toxin and the fragment coding for said cell-specific polypeptide ligand.

4. A method according to claim 1 wherein said fragment of a gene coding for said cell-specific polypeptide ligand is the fragment coding for at least a portion of a polypeptide hormone.

5. A method according to claim 4 wherein said portion of said polypeptide hormone is a portion of or .beta. melanocyte stimulating hormone effective to cause said hybrid protein to bind to malignant melanocyte cells.

6. A method according to claim 4 wherein said portion of said polypeptide hormone is a portion of substance P
effective to cause said hybrid protein to bind to pain receptor neurons.

7. A method according to claim 4 wherein said portion of said polypeptide hormone is a portion of interleukin I
effective to cause said hybrid protein to bind to T cells.

8. A method according to claim 4 wherein said portion of said polypeptide hormone is a portion of interleukin II
effective to cause said hybrid protein to bind to T cells.

9. A method according to claim 4 wherein said portion of said polypeptide hormone is a portion of interleukin III
effective to cause said hybrid protein to bind to T cells.

10. A method according to claim 1 wherein said fragment of said diphtheria toxin is obtained by excising a complete said fragment from the tox operon.

11. A method according to claim 1 wherein said fragment of said diphtheria toxin is obtained by fusing together suitable fragments.

12. A hybrid protein comprising protein fragments joined together by peptide bonds, said hybrid protein comprising, in sequential order, beginning at the amino terminal end of said hyrid protein, a) the enzymatically active Fragment A of diphtheria toxin, b) a fragment including the cleavage domain l1 adjacent said Fragment A of diphtheria toxin, c) a fragment comprising at least a portion of the hydrophobic domain of Fragment B of diphtheria toxin and not including the generalized eukaryotic binding site of said Fragment B, and d) a fragment comprising a portion of a cell-specific polypeptide ligand, said portion including at least a portion of the binding domain of said polypeptide ligand, said portion of said binding domain being effective to cause said hybrid protein to bind selectively to a predetermined class of cells to be attacked by said enzymatically active Fragment A, whenever prepared by a process according to claim l or by an obvious chemical equivalent thereof.

13. A hybrid protein as defined in claim 12 wherein said fragment comprising a portion of a cell-specific polypeptide ligand is at least a portion of said ligand effective to cause said hybrid protein to bind selectively to a predetermined class of cells whenever prepared by a process according to claim 1 or by obvious chemical equivalent thereof.

14. A hybrid protein as defined in claim 12 wherein said fragment c) is encoded by the portion of the Fragment B
encoding region between l1 and said position of the NRU I

site, whenever prepared by a process according to claim 2 or by an obvious chemical equivalent thereof.

15. A hybrid protein as defined in claim 12 further comprising fragment l2 between fragments c) and d), whenever prepared by a process according to claim 3 or by an obvious chemical equivalent thereof.

16. A hybrid protein as defined in claim 12 wherein said polypeptide ligand is at least a portion of a polypeptide hormone whenever prepared by a process according to claim 5 or by an obvious chemical equivalent thereof.

17. A hybrid protein as defined in claim 16 wherein said portion of said polypeptide hormone is a portion of or .beta.
melanocyte stimulating hormone effective to cause said hybridprotein to bind to malignant melanocyte cells, whenever prepared by a process according to claim 5 or by an obvious chemical equivalent thereof.

18. A hybrid protein as defined in claim 16 wherein said portion of said polypeptide hormone is a portion of substance P effective to cause said hybrid protein to bind to pain receptor neurons, whenever prepared by a process according to claim 6 or by an obvious chemical equivalent thereof.

19. A hybrid protein as defined in claim 16 wherein said portion of said polypeptide hormone is a portion of interleukin I effective to cause said hybrid protein to bind to T cells, whenever prepared by a process according to claim 7 or by an obvious chemical equivalent thereof.

20. A hybrid protein as defined in claim 15 wherein said portion of said polypeptide hormone is a portion of interleukin II effective to cause said hybrid protein to bind to T cells, whenever prepared by a process according to claim or by an obvious chemical equivalent thereof.

21. A hybrid protein as defined in claim 16 wherein said portion of said polypeptide hormone is a portion of interleukin III effective to cause said hybrid protein to bind to T cells whenver prepared by a process according to claim 9 or by an obvious chemcial equivalent thereof.

22. A fused gene comprising a fragment of the gene coding for diphtheria toxin fused to a fragment of a gene coding for a cell-specific polypeptide ligand, said fused gene coding for a hybrid protein, said fragment of said diphtheria toxin gene including in sequential order beginning at the 5' terminal end of said fragment, a) the fragment coding for the hydrophobic leader signal sequence of said diphtheria toxin, b) the region coding for the enzymatically active Fragment A of said diphtheria toxin, c) the region coding for protease sensitive loop 11 adjacent said Fragment A of said diphtheria toxin, and d) at least a portion of the hydrophobic domain of Fragment B of diphtheria toxin and not including the generalized eukaryotic binding site of said Fragment B, and said fragment of a gene coding for said polypeptide ligand encoding a portion of said ligand effective to cause said hybrid protein to bind selectively to a predetermined class of cells to be attacked by said enzymatically active Fragment A.

23. The fused gene of claim 22 wherein said polypeptide ligand is .alpha. or .beta. melanocyte stimulating hormone.