CA2103258A1

CA2103258A1 - Cytokine receptor targeted molecules for treatment of neoplastic cell growth

Info

Publication number: CA2103258A1
Application number: CA002103258A
Authority: CA
Inventors: Cory A. Waters; Louis R. Poisson
Original assignee: Individual
Current assignee: Seragen Inc
Priority date: 1991-05-17
Filing date: 1992-05-15
Publication date: 1992-11-18
Also published as: WO1992020364A1; JPH06507900A; EP0584251A4; EP0584251A1

Abstract

The invention features a method for treating meoplastic cell growth in a patient, wherein the meoplastic cell is of non-lymphoid and non-monocytic origin. The method includes administering to the patient a molecule which is capable of specifically binding to a cytokine receptor expressed on the meoplastic cell, which molecule is capable of decreasing the viability of the meoplastic cell.
The cytokine receptor is a receptor normally expressed on cells of lymphoid or monocytic origin.

Description

W092/20~PCT/US92/~093 ~! 3 2 5 8 CYTOKINE RECEPTOR TARGETED MOLECULES FOR
TREATMENT NEOPLASTIC CELL GROWTH

Backaround of the I~vention 5This invention relates to the treatment of neoplastic cell growth, e.g., cancer.
There have been many attempts to limit neoplastic cell growth using drugs targeted to neoplastic cells.
Both monoclonal antibodies and receptor ligands have been 10 suggested as targeting agents.
Murphy ~United States Patent 4,675,382) suggests that a cytotoxin/interleukin-2 hybrid protein could be used for treatment of cancers involving the immune system.
LeMaistre et al. (Lancet 337:1124, 1991) describe the treatment of a patient with chronic ly,mphocytic leukemia using a diphtheria toxin/interleukin-2 hybrid molecule.

ummary of the Invention In general, the invention features a method for treating neoplastic cell ,growth in a patient, wherein the neoplastic cell is of non-lymphoid and non-monocytic origin. The method includes administering to the patient a molecule whirh is capable of specifically binding to a 25 cytokine receptor expressed on the neoplastic cell, which molecule is capable of decreasing the viability of the neoplastic cell. The cytokine receptor is a receptor normally expressed on cells of lymphoid or monocytic origin.
In various preferred embodiments, the neoplastic cell is not of hematopoietic stem cell origin; the neoplastic cell is a sarcoma; the neoplastic cell is a W092/203~ PCT/US92~ ~3 r n ~J ~

osteosarcoma; the neoplastic cell is a fibrosarcoma; the neoplastic cell is a leiomyosarcoma; the neoplastic cell is a carcinoma; and the neoplastic cell is a rhabdomyosarcoma. In more preferred embodiments, the 5 carcinoma is a lung carcinoma; and the carcinoma is hepatocellular aarcinoma.
In a preferred embodiment, the cytokine receptor is an interleukin receptor. In more preferred embodiments, the interleukin receptor is an interleukin-10 2 receptor; the interleukin receptor is an interleukin-4 receptor; and the interleukin receptor is an interleukin-6 receptor. In an even more preferred embodiment, the interleukin-2 receptor is a high affinity interleukin-2 receptor. In another preferred embodiement, the cytokine 15 receptor is not expressed on non-neoplastic cells of non-lyphoid and non-monocytic origin.
In still other preferred embodiments, the molecule kills cells bearing the cytokine receptor; the molecule is a hybrid molecule includes a first and a second 20 portion joined together covalently, the first portion includes a molecule capable of decreasing cell viability and the second portion includes a molecule capable of specifically binding to the cytokine receptor. In more preferred embodiments, the second portion includes all or 25 a binding portion of an antibody specific for the cytokine receptor; the second portion includes all or a binding portion of a ligand for the cytokine receptor;
and the first portion includes a cytotoxin; and the antibody is a complement activating antibody. In an even 30 more preferred embodiment, the cytokine receptor is an interleukin receptor. In a yet more preferred embodiment, the ligand is an interleukin.
In another preferred embodiment, the cytotoxin is a fragment of a peptide toxin which is enzymatically ~ 35 active but which does not possess generalized eukaryotic ~092/20~ 2 `1 0 3 2 5 8 PCT/US92/~093 receptor binding activity. In a more preferred embodiment, the fragment of a peptide toxin includes fragment A of diphtheria toxin and enough of fragment B
of diphtheria toxin to facilitate translocation into the 5 cytosol.
In other preferred embodiments, the molecule is DAB389IL-2; the molecule is DAB389IL-4; the molecule is DAB389IL-6; the molecule is DAB~86IL-2; the molecule is DAB486IL-4; and the molecule is DAB~86IL-6.
By "non-lymphoid and non-monocytic origin" is meant all cells that are not descended from lymphoid stem cells or monocytes, e.g., cells other than T lymphocytes, B lymphocytes, and macrophages. By " lymphoid and monocytic origin" is meant cells descended from lymphoid 15 stem cells or monocytes, e.g., T lymphocyte~, B lymphocytes, plasma cells, and macrophages. By "specifically binding" is meant that the molecule does not substantially bind to other cell surface receptors.
By "not of hematopoietic stem cell origin" is meant all ;~; 20 cell are not descended from pluripotent stem~cells.
Other features and advantages of the invention will be apparent from the following description of the preferred embodiments thereof, and from the claims.

Detailed Descri~tion 25 DAB389IL-4 and DAB486IL-2 Reduce the Viability of Several Cancer Cell Ty~es Our in vitro toxicity experiments have demonstrated that certain cancer cells are sensitive to interleukin-4 or interleukin-2 receptor targeted 30 cytotoxins. Accordingly these interleukin receptor-targeted cytotoxins, described in detail below, can provide a means by which to decrease the viability of : ~
~ cancer cells.

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W092/~0~ PCT/US92/04093 2 5 ~ - 4 -In the experiments described below various cancer cell lines are shown to be sensitive to a fusion protein (DAB389IL-4) in which the receptor binding domain of diphtheria toxin (a single molecule of which, when 5 present intracellularly, can block protein synthesis) has been replaced by a portion of human interleukin-4.
Various cell lines were exposed to DAB389IL-4 and the level of protein synthesis was measured as described below. The results of this analysis are presented in 10 Table 1 where the IC50 is defined as the concentration of drug required to cause a 50% decrease in protein synthesis. Cancer cell lines from striated muscle, smooth muscle, liver, bone, and lung are sensitive to DAB389IL-4; the IC50 for these cell lines was found to be 15 lO lOM to 10 8M.

T~ble 1: Sensitivity of Cancer Cells to DAB389IL-4 Ce~ ATCC No. C~sssi~ication IC50 ~ 2 Hep G2 ~ HB 8065 Human Hepaeocel~ular Carcinoma, 2.7 x 10 10 . Hepatitis B
NT-1080 CCL 121 Human F~brosarcoma 3.9 x 10-9 ~673 CR~ 1598 Human Rhabdomyos~rcoma 2.7 ~ 10 9 A549 CCL 185 Human Lung Carcinoma 2.1 x 10 2 5 U-2 05 HTB 96 Human Ostec~enic Sarcoma, bone primary 2.6 x 10 SK-ES-1 HTB 86 Human ~nap~as~ic Osteo~arcoma, 2.2 x 10 bone primary S~-LMS-1 HTB 88 Human Leiomyosarcoma, vulva 1.5 x 10 3 0 .. ~merican ~ype Culture Co~ection (Bethesda, YD) accession number.
The concentration of DAB3891L-4 ~hich leads to a 50X decreae in protein synthesis.

The sensitivity of these cancer cell lines is comparable to that of cells, such as HTLV-1 transformed T-lymphocytes and phytohemagglutinin activated T-3 5 lymphocytes, which are expected to express high numbersof interleukin-4 receptors (Table 2). In contrast, activated rat T-lymphocytes, which express an interleukin-4 receptor that is not recognized by human V092/20~ 2 l~a3,1~58 PCT/US92/04093 5 _ interleukin-4, are not nearly as sensitive to DAB389IL-4, even though they are sensitive to diphtheria toxin (Table -2). This result demonstrates that DAB389IL-4 selectively intoxicates interleukin-4 receptor expressing cells.

S T~bl- 2: DAB389IL-4 Sensitivity of Normal and Neoplastic Cells Derived from Lymphocytes and Monocytes Celt or Cel~ Line Classification IC50 (M) T r,ell origin HUn 102/~TG Human, CTCL, HTLV I~ 2.9 X 10-11 C91~PL Human, HTLV-I~, transformed 6.3 X 10 1 5 ~ r,ell origin ~ji Human, 8urkitt's lymphoma E8V~ ?.2 X 10-1 ~yel _ cle r r,ell tr~37 Human, histiocytic lymphora 2.0 X 10 9 l ~C
2 0 PHA ctivated T cells Human 1.6 X 10 10 ~onrpri _ te Con A-activated norma~
splenic T cells Rat >10-7 ' 2 5 This is the concentration of drug which leads to a 50X dbcresse in protein synthesis.

A similar set of experiments demonstrated that human rhabdomyosarcoma cell line A673 (ATCC Accession No.
CRL 1598) is sensitive to a fusion protein (DAB4a6-IL-2) in which the receptor binding domain of diphtheria toxin has 30 been replaced by a portion of human interleukin-2.
: DAB486IL-2 at 7.6 x 10 8M decreased protein synthesis by 50%.
Assay for Sensitivity of Neo~lastic Cells to Interleukin Receptor Taraeted Cvtotoxins Cytotoxicity was evaluated by measuring protein synthesis~ tl~C]leucine incorporation in the presence and absence of DAB389IL-4 (or DAB~86IL-2) as follows. Cells were plated 96 well microtiter plates in growth medium appropiate to the cell type. DAB389IL-4 ((or DABq86IL~2) ~: .

W092/20~ PCT/US92/~ ~3 r ,~ 3 ~

was added at various concentrations and the cultures were incubated for 20 hours prior to pulse labelling with ~l~C]leucine. Cells were then trypsinized, harvested onto glass fiber filter mats and counted. The IC50 is the 5 concentration of cytotoxin which leads to a 50% decre~se in tl~C]leucine incorporation.
The sensitivity of other cell types, including cells isolated from a tissue sample (e.q., a biopsy) obtained from a patient, can be evaluated as described 10 above. Viability can also be measured using any standard viability assay appropriate to the cell type being studied. For example, viability can be determined using a trypan blue dye exclusion assay (Kruse et al., eds. Tissue Culture: Methods and Applications, Academic ; 15 Press, 1989).
Molecules Useful in the Method of the Invention The molecules useful in the method of the invention are tarageted to a cytokine receptor. In general, there are three ways in which the molecules 20 useful in the invention can act: (1) the molecule can kill a cell because the molecule has a cytotoxic domain;
(2) the molecule (an antibody) can cause cell lysis by inducing complement fixation; and (3) the molecule; can block binding or uptake of receptor's ligand. In all 25 three cases the molecule must be targeted to receptor bearing cells; this is accomplished by including the receptor's ligand (or a portion or derivative thereof~ or an anti-receptor antibody as part of the molecule.
Interleukin-2 (IL-2) receptor targeted molecules 30 provide examples of each of these three approaches. A
fusion molecule which includes the IL-2 receptor binding portion of IL-2 and a cytotoxin can be used to kill neoplastic cells bearing the interleukin-2 receptor.
Likewise, the second type of molecule described above, a 35 complement fixing antibody, in this instance directed ~092/20~ 5 ~ PCT/US92/04093 against the IL-2 receptor, can eliminate IL-2 receptor-bearing cells. In this example, the third type of molecule could be a molecule that blocks binding of IL-2 to its receptor. This molecule would prevent neoplastic 5 cells that bear the interleukin-2 receptor from receiving a proliferation signal from IL-2.
Molecules useful for treating neoplastic cell growth can take a number of forms. When IL-2 itself is the targeting agent, the molecule can be a cytotoxic 10 hybrid molecule in which IL-2 is fused to a toxin molecule, preferably a polypeptide toxin. Derivatives of IL-2 which bind to IL-2R, lack IL-2 activity and block binding and/or uptake of bona fide IL-2 are useful in the method of the invention because they will prevent IL-2-15 induced proliferation of IL-2R bearing cells. When an anti-IL-2R antibody is the targeting agent, a cytotoxic hybrid molecule can be formed by fusing all or part of the antibody to a cytotoxin. The effectiveness of such an antibodyjtoxin hybrid, like that of an IL-2/toxin 20 hybrid, depends on the hybrid molecule being taken up by cells to which it binds. Anti-IL-2R antibodies which block binding and/or uptake of IL-2 are also useful in - the method of the invention. Lytic anti-IL-2R antibodies are useful in the invention because they can cause 25 complement-mediated lysis of IL-2R-bearing cells.
Some of the molecules can be hybrid molecules formed by the fusion of all or part ~f two or more molecules. The hybrid molecule can be a hybrid protein encoded by a recombinant DNA molecule, in which case the 30 two domains are joined (directly or through an intermediary domain) by a peptide bond. Alternatively, two domains can be produced separately and joined by a covalent bond in a separate chemical linkage step. In some cases, the cytotoxic domain of a hybrid molecule may 35 itself be derived from two separate molecules.
;

W092/20~ PCT/US92/~09~

~ 0 3.?58 - 8 -Interleukin-2 as a Taraetina Aaent Interleukin-2 (IL-2) or any IL-2 receptor binding derivative thereof can be used as a targeting agent for a cytotoxin. The DNA and amino acid sequences of IL-2 are 5 known (Tadatsugu et al., Nature 302:305, 1983), and its structure has been predicted by x-ray crystallography (Brandhuber et al., Science ~38 : 1707, 1987) . Analysis of genetically engineered variants of IL-2 has provided some information concerning which residues are important for 10 IL-2R binding (Collins et al., Proc. Natl. Acad. Sci. USA
85:7709, 1988) and bioactivity (Cohen et al. Science 234 : 349, 1989 ; Collins et al., supra) . Variants of IL-2 which are useful in the invention include deletion mutants (Genbauffe et al., USSN 388,557, hereby 15 incorporated by reference) which lack one or more amino acid residues in the region between residue 74 and residue 79 (numbering according to Williams et al., Nucl.
Acids Res. 16:1045, 1988). These mutants effectively target toxins to IL-2R-bearing cells (Genbauffe et al., 20 supra). Generally, IL-2 variants useful for targeting a cytotoxin must efficiently bind IL-2R and be endocytosed.
The ability of various derivatives to bind to the IL-2 receptor can be tested with an IL-2R binding assay described below. ~
In designing molecules targeted to cells bearing the IL-2 receptor it must be recognized that the IL-2 receptor, like other receptors, has several forms; and it - may be desirable to target cells bearing one form and not another. The human interleukin-2 receptor has a high-, 30 an intermediate-, and a low-affinity form. The high affinity receptor has an apparent Kd of -10 lOM and is composed of two subunits, p55 and p75 (also called p70).
When expressed on the cell surface, both the p75 and pS5 subunits are capable of binding IL-2. The p75 subunit 35 corresponds to the intermediate affinity receptor (Kd ~

-`'092/20~ PCT/US92/~093 8.2 x 10 lOM), and p55 subunit corresponds to the low affinity receptor (Kd ~ 1-3 x 10 8M). The p75 subunit is expressed on the surface of resting T cells, natural killer cells, monocytes/macrophages, and lymphokine-5 activated killer (LAK) cell precursors, while the highaffinity receptor is expressed on activated T- and B-cells.
In the method of the invention it may be desirable to target only cells bearing the high affinity receptor.
10 In these circumstances useful molecules will eliminate or neutralize cells bearing the high affinity IL-2 receptor at a concentration which leaves cells bearing the intermediate or low affinity receptor largely unaffected.
When the molecule, like IL-2 itself, has affinity for all 15 three classes of IL-2 receptor, selectivity can be accomplished by administering the molecule at a concentration which does not permit significant binding to cells bearing lower affinity receptors. A hybrid molecule may have altered receptor affinities compared to 20 IL-2. Such hybrid molecules may be more or less selective for cells bearing the high affinity IL-2 receptor. For example, cells bearing the high-affinity receptor are 500-1000 times more sensitive to DAB~86IL-2, a fusion protein consisting of part of diphtheria toxin 25 and part of IL-2, than are cells bearing the intermediate- affinity receptor (Waters et al., Eur. J.
Immunol . ~0: 785, 1990).
A cytotoxin can be attached to an IL-2 derivative in a number of ways. Preferably, an IL-2/toxin hybrid is 30 a hybrid protein produced by the expression of a fused gene. Alternatively, the cytotoxin and the IL-2 derivative can be produced separately and later coupled by means of a non-peptide covalent bond. Linkage methods are described below.

W092/203~ PCT/US92/040 S ~3 Useful cytotoxins are preferably significantly cytotoxic only when present intracellularly and are substantially excluded from any given cell in the absence of a targeting domain. Peptide toxins fulfill both of 5 these criteria and are readily incorporated into hybrid molecules. A mixed cytotoxin, a cytotoxin composed of all or part of two or more toxins, can also be used.
Several useful toxins are described in more detail below.
Interleukin-4 and Interleukin-6 as a Taraetina Aaents Interleukin-4 (IL-4) is a cytokine which acts on a variety of cell types. Its receptor is expressed on a number of cell types, including CD4~ T cells and monocytes. IL-4 can act as a T cell growth factor and it is thought to have an influence on IL-2 induced 15 lymphocyte proliferation.
A cytotoxin directed against IL-4 receptor-bearing cells or IL-6 receptor-bearing cells may enhance the effectiveness of molecules directed against IL-2R-bearing cells. The protein and DNA sequence of IL-4 and IL-6 are 20 known ~Lee et al., J. Biol . Chem. 263:10817,~1988; Hirano et al., Nat~re 324:73, 1986). These lymphokines can be used to create hybrid lymphokine/toxin molecules similar , to IL-2/toxin hybrid molecules.
Monoclonal Antibodies as Taraeting Agents Monoclonal antibodies directed against the lymphokine receptor of choice can be used to direct toxins to cells bearing that receptor. These antibodies or antibody fragments can be fused to a cytotoxin either by virtue of the toxin and the antibody being encoded by 30 a fused gene which encodes a hybrid protein molecule, or by means of a non-peptide covalent bond which is used to join separately produced ligand and toxin molecules.
Several useful toxins are described below.
Antibody/toxin hybrids can be tested for their 35 ability to kill receptor bearing cells using a toxicity -~092/203~ ~ 1~J 3 q ~ 8 PCT/US92/~093 assay similar to that which is described below for IL-2R
bearing cells.
Monoclonal antibodies useful in the method of the invention can be made by immunizing mice with human IL-5 2R or cultured T-lymphocytes, fusing the murine splenocytes with appropriate myeloma cells, and screening the antibodies produced by the resultant hybridoma lines for the requisite IL-2R binding properties by means of an ELISA assay. Antibody production and screening can be 10 performed according to Uchiyama et al. (J. Immunol.
126:1393, 1981). Alternatively, useful antibodies may be isolated from a combinatorial library produced by the method of Huse et al. (Science 246:1275, 1989).
The invention can employ not only intact 15 monoclonal or polyclonal antibodies, but also an immunologically-active antibody ~ragment, for example, a Fab or (Fab)2 fragment; an antibody heavy chain, an antibody light chain; a genetically engineered single-chain Fv molecule (Ladner 20 et al., U.S. Patent No. 4,94~,778); or a chimeric antibody, for example, a "humanized" antibody which contains the binding specificity of a murine antibody, but in which most or all of the remaining portions are of human origin (Reichman et al., Nature 332:323, 1988).
25 Toxins The toxin molecules useful in the method of the invention are preferably toxins, such as peptide toxins, which are significantly cytotoxic only when present intracellularly. Of course, under these circumstances 30 the molecule must be able to enter a cell bearing the targeted receptor. This ability depends on the nature of the molecule and the nature of the cell receptor. For example, cell receptors which naturally allow uptake of a ligand are likely to provide a means for a ~olecule which 35 includes a toxin to enter a cell bearing that receptor.

W092/20~ PCT/US92/04~3 ~ - `J 3 ;~ S 8 Preferably, a peptide toxin is fused to an IL-2R binding domain by producing a recombinant DNA molecule which encodes a hybrid protein molecule. Such an approach ensures consistency of composition.
Many peptide toxins have a generalized eukaryotic receptor binding domain; in these instances the toxin must be modified to prevent intoxication of non-receptor bearing cells. Any such modifications must be made in a manner which preserves the cytotoxic functions of the 10 molecule (see U.S. Department of Health and Human Services, U.S. Serial No. 401,412). Potentially useful toxins include, but are not limited to: cholera toxin, ricin, 0-Shiga-like toxin (SLT-I, SLT-II, SLT IIV), LT
toxin, C3 toxin, Shiga toxin, pertussis toxin, tetanus 15 toxin, Pseudomonas exotoxin, alorin, saporin, modeccin, and gelanin.
Diphtheria Toxin-based Molecules Diphtheria toxin can be used to produce molecules useful in the method of the in~ention. Diphtheria toxin, 20 whose sequence is known, is described in detail in Murphy U.S. Patent 4,675,382, hereby incorporated by reference.
The natural diphtheria toxin molecule secreted by Corynebacterium diphtheriae consists of several functional domains which can be characteri~ed, starting 25 at the amino terminal end of the molecule, as enzymatically-active Fragment A (amino acids Glyl -Arg~93) and Fragment B (amino acids Serlg~ - Ser535), which includes a translocation domain and a generalized cell binding domain (amino acid residues 475 through 535).
The process by which diphtheria toxin intoxicates sensitive eukaryotic cells involves at least the following steps: (i) the binding domain of diphtheria toxin binds to specific receptors on the surface of a sensitive cell; (ii) while bound to its receptor, the 35 toxin molecule is internalized into an endocytic vesicle;

~0 92/20364 PCI /IJS92/04093 ~r~
~ ~ J ~ h~

(iii) either prior to internalization, or within the endocytic vesicle, the toxin molecule undergoes a proteolytic cleavage between fragments A and B; (iv) as the pH of the endocytic vesicle decreases to below 6, the 5 toxin crosses the endosomal membrane, facilitating the delivery of Fragment A into the cytosol; (v) the catalytic activity of Fragment A (i.e., the nicotinamide adenine dinucleotide - dependent adenosine diphosphate (ADP) ribosylation of thè eukaryotic protein synthesis 10 factor termed "Elongation Factor 2") causes the death of the intoxicated cell. It is apparent that a single molecule of Fragment A introduced into the cytosol is sufficient to block down the cell's protein synthesis machinery and kill the cell. The mechanism of cell 15 killing by Pseudomonas exotoxin A, and possibly by certain other naturally-occurring toxins, is very similar.
DAB486IL-2, a fusion protein in which the receptor binding domain of diphtheria toxin has been replaced by a 20 portion of human IL-2 (Williams et al., J. Biol. Chem.
35:20673, 1990; see also Williams et al., Protein Eng.
1:493, 1987), is an example of a molecule useful in the method of the invention. This molecule selectively kills IL-2R-expressing tumor cells and lymphocytes (Waters et 25 al., Eur. J. Immunol. 20:785, 1990; Kiyokawa et al., Cancer Res. 49:4042, 1989). Because of its ability to kill activated lymphocytes, DAB486IL-2 has been used to control graft rejection (Pankewycz et al., Transplantation 47:318, 1989; Kickman et al., 30 Transplantation 47:327, 1989) and to treat certain autoimmune disorders (Forte et al., 2nd International Symposium on ~mmunotoxins, 1990).
DAB~86IL-2 is a chimeric molecule consisting of Met followed by amino acid residues 1 throuqh 485 of the 35 mature diphtheria toxin fused to amino acid residues 2 4 PCI/US92/04~3 iJ ~ 2 ~ 8 through 133 of IL-2. Thus, DAB~86IL-2 includes all of diphtheria toxin fraqment A, which encodes the enzymatically active portion of the molecule, and a portion of fragment B. The portion of fragment B present 5 in DAB~86IL-2 does not include the generalized receptor binding domain but does include the translocation domain which facilitates delivery of the enzymatically active portion into the cytosol.
Pre~aration of DAB486IL-2 and DAB389IL-2 DAB~86IL-2 was produced in E. coli harboring the DA8~,86IL-2 encoding plasmid, pDW24 (Williams et al., J.
Biol. Chem. 265:20673, 1990, except ampr is replaced by kanr). The protein was purified by in~nunoaffinity chromatoqraphy and high pressure liquid chromatography 15 (Williams et al., supra) . DAB389IL-2 can be prepared as described below for DAB389IL-4 by substituting IL-2 for IL-4.
Preparation of DAB389IL-4 and DAB486IL-2 A synthetic gene encoding human interleukin-4 was io synthesized (Milligen/Biosearch i500 DNA sy~thesizer).
The IL-4 sequence (Yodota et al., Proc Nat ' l Acad Sci .
USA, 83:58994, 1986) was modified to incorporate E. coli-preferred codon usage (deBoer et al., in Maximizing Gene Expression , Reznikioff et al., eds., 1986, Butterworths, 25 Boston), and restriction endonuclease cleavage sites were added to facilitate subsequent cloning steps. IL-4 coding sequence (Hisl to Serl29) was inserted into pDW27 plasmid. pDW27 is derived from pDW24 (Williams et al., J. Biol. Chem. 265:11885, 1990) by deleting DNA
30 corresponding to amino acids 388 to 485 OI native diphtheria toxin. DAB~86IL-4 can be prepared as described above for DAB~86IL-2 by substituting IL-4 for IL-2.
CYtotoxicitY of DAB389IL-4 ~092/203~ ,~ A~ r 8 PCT/US92/04093 ~_ J~

The ability of DAB389IL-4 to reduce viability of various cell types was measured using an inhibition of protein synthesis-assay; the results of this assay are presented in Table 3. ICso (M) is the concentration of 5 DAB389IL-4 required for a 50% decrease in protein synthesis. The rat, Con A-activated, normal splenic lymphocytes were far less sensitive to DAB389IL-4 than any of the other cells or cell lines. Since the rat interleukin-4 receptor does not bind human interleukin-10 4, this result demonstrates the specificity of DAB389IL-4. These rat cells are sensitive to a diphtheria toxin/rat interleukin-2 hybrid molecule.
Preparation of DAB389IL-6 and DAB~86IL-6 A synthetic gene encoding human interleukin-6 was 15 synthesized (Nilligen/Biosearch 7500 DNA synthesizer).
The IL-6 sequence (Revel et al., EPA 8611404.9) was modified to incorporate E. Coli preferred codon usage (deBoer et al., supra ), and restriction endonuclease cleavage sites were added to facilitate subsequent 20 cloning steps. The entire IL-6 coding sequence was inserted into pDW27 plasmid as described above for DA8389IL-4. DAB~86IL-6 can be produced as described above~
for DABq86IL~2 by substituting IL-6 for IL-2.
Mixed Toxins 2S The cytotoxic portion of some molecules useful in the invention can be provided by a mixed toxin molecule.
A mixed toxin molecule is a molecule derived from two different polypeptide toxins. Generally, as discussed above in connection with diphtheria toxin, polypeptide 30 toxins have, in addition to the domain responsible for generalized eukaryotic cell binding, an enzymatically active domain and a translocation domain. The binding and translocation domains are required for cell recognition and toxin entry respectively. The W092/20~ PCT/US92/04~93 ~ ~ f~ r~ ~
d enzymatically active domain is the domain responsible for cytotoxic activity once the molecule is inside a cell.
Naturally-occurring proteins which are known to have a translocation domain include diphtheria toxin, 5 Pseudomonas exotoxin A, and possibly other peptide toxins. The translocation domains of diphtheria toxin and Pseudomonas exotoxin A are well characterized (see, e.g., Hoch ~t al., Proc. Natl. Acad. Sci. USA 82:1692, 1985; Colombatti et al., J. Biol. Chem. 261:3030, 1986;
10 and Deleers et al., FEBS Le~t. 160:82, 1983), and the existence and location of such a domain in other molecules may be determined by methods such as those employed by Hwang et al., Cell 48:129, 1987; and Gray et al., Proc. Natl. Acad. Sci. USA 81:2645, 1984).
One useful IL-2/mixed toxin hybrid molecule is formed by ~using the enzymatically active A subunit of E.
coli Shiga-like toxin (Calderwood et al.j Proc. Natl.
Acad. Sci. USA 84:4364, 1987) to the translocation domain (amino acid residues 202 through 460) of diphtheria 20 toxin, and to IL-2. This three-part bybrid molecule, SLT-A/DTB'/IL-2, is useful in the method of the invention in the same way as DAB~86IL-2 described above. The IL-2 portion of the three-part hybrid causes the molecule to attach specifically to IL-2R-bearing cells, and the 25 diphtheria toxin translocation portion acts to insert the enzymatically active A subunit of the Shiga-like toxin into the targeted cell. The enzymatically active portion of Shiga-like toxin, like diphtheria toxin, acts on the protein synthesis machinery of the cell to prevent 30 protein synthesis, thus killing the cell. The difference between these two types of hybrid toxins is the nature of their enzymatic activities: the enzymatic portion of DAB486IL-2 catalyzes the ADP-ribosylation by nicotinamide adenine dinucleotide of Elongation Factor 2, thereby 35 inactivating this factor which is necessary for protein W092/203~ 2 1 0 ~ 2 5 ~ PCT/US92/~093 synthesis, while the enzymatic portion of SLT-A/DTB'/IL-2 is a ribonuclease capable of cleaving ribosomal RNA at a critical site, thereby inactivating t~e ribosome. SLT-A/DTB'/IL-2 hybrid would therefore be useful as a 5 treatment for the same indications as DAB~86IL-2, and could be substituted or used in conjunction with it.
Linkaae of Toxins to Bindina Liaands The binding ligand and the cytotoxin of useful hybrid molecules can be linked in several ways. If the 10 hybrid molecule is produced by expression of a fused gene, a peptide bond serves as the link between the cytotoxin and the binding ligand. Alternatively, the toxin and the binding ligand can be produced separately and later coupled by means of a non-peptide covalent 15 bond.
For example, the covalent linkage may take the form of a disulfide ~ond. In this case, if the IL-2R
binding ligand is a protein, e.g., IL-2, the DNA encoding IL-2 can be engineered to contain an extra cysteine codon 20 as described in Murphy et al. U.S. Serial No. 313,599, hereby incorporated by reference~ The cysteine must be positioned so as to not interfere with the IL-2R binding activity of the molecule. For example, the cysteine codon can be inserted just upstream of the DNA encoding 25 pro2 of the mature form of IL-2. The toxin molecule must be derivatized with a sulfhydryl group reactive with the cysteine the modified IL-2. In the case of a peptide toxin this can be accomplished by inserting a cysteine codon into the DNA sequence encoding the toxin.
30 Alternatively, a sulfhydryl group, either by itself or as part of a cysteine residue, can be introduced using solid phase polypeptide techniques. For example, the introduction of sulfhydryl groups into peptides is described in Hiskey (Peptides 3:137, 1981).
35 Derivatization can also be carried out according to the ~ .

W092/20~ PCT/US92/~093 ~lJ~258 18 -method described for the derivatization of a peptide hormone in Bacha et al. U.S. Patent No. 4,468,382, hereby incorporated by reference. Similarly, proteins can be derivatized at the DNA or protein chemistry level. The 5 introduction of sulfhydryl groups into proteins is described in Maasen et al. (Eur. J. Bioch~m. 134:32, 1983). The cytotoxin and the IL-2R binding ligand are then produced and purified, and the disulfide bond between the purified molecules formed by reducing both 10 sulfur groups, mixing toxin and ligand, in a ratio of about 1:5 to 1:20, and allowing disulfide bond formation to proceed to completion (generally 20 to 30 minutes) at room temperature. The mixture is then dialyzed against phosphate buffered saline to remove unreacted ligand and 15 toxin molecules. Sephadex chromatography or the like is then carried out to separate on the basis of size the desired toxin-ligand conju~ates from toxin-toxin and ligand-ligand conjugates.
~ssays for IL-2 Receptor Bindina and IL-4 Receptor 20 Bindina The IL-2R binding ability of various molecules can be measured using an IL-2R assay for high affinity (Ju et al., J. Biol . Chem. 262:5723, 1987) or intermediate affinity receptors (Rob et al., Proc. Natl . Acad.- Sci .
25 USA 84:2002, 1987~. The IL-4R binding activity of various molecules can be measured using the assay described by Park et al. (J. Exp. ~ed. 166:176, 1984) or the assay described by Foxwell et al. (Eur. J. Immunol.
19:1637, 1989).
30 Assays for ToxicitY
Molecules of the invention (both antibodies and hybrid molecules) can be screened for the ability to decrease viability of cells bearing the targeted receptor by mèans of assays such as those described below.

~ 092/203~ 2 1 u 3 2 5 8 PCT/US92/04093 Toxicity towards IL-2R bearing cells can be tested as follows. Cultured HUT 102/6TG (Tsudo et al., Proc.
Natl . Acad . Sci . USA 83:9694, 1986) or YT2C2 (Teshigiwari et al., J . Exp. Med . 165:223, 1987) cells are maintained 5 in RPMI 1640 me~ium (Gibco, Grand Island, NY~
supplemented with 25 mM HEPES (pH 7.4), 2mM l-glutamine, 100 U/ml penicillin, 100 ~g/ml streptomycin, and 10%
fetal calf serum (Hazelton, Lenexa, KS). Cells are seeded in 96-well V-bottomed plates (Linbro-FIow 10 Laboratories, McLean, VA) at a concentration of 1 x 105 per well in complete medium. Putative toxins are added to varying c~ncentrations (10 12M to 10 6M) and the cultures are incubated for 18 hrs. at 37C in a 5% C02 atmosphere. Following incubation, the plates are 15 centrifuged for 5 min. at 170 x g, and the medium removed and replaced with 100 ~1 leucine-free medium (MEM, Gibco) containing 8 ~Ci/ml (3H-leucine; New England Nuclear, Boston, MA). After an additional 90 min. at 37C, the plates are centrifuged for 5 min. at 170 x g, the medium 20 is removed, and the cells are collected on glass fiber filters using a cell harvester (Skatron, Sterling, VA).
Filters are washed, dried, and counted according to standard methods. Cells cultured with medium alone serve as the control.
Toxicity towards cells bearing IL-4R may be tested by an assay similar to that described above for IL-2R
bearing cells, but utilizing MLA144 cells (Rabin et al.
J. Immunol . 127 :1852,-1981) or HUT 102/6TG cells, seeded at 1 x 105 cells per well and incubated for 40 hours.
30 Therapv Generally, the molecules of the invention will be administered by intravenous infusion. They may also be administered subcutaneously. Dosages of molecules useful in the methods of the invention will vary, depending on 35 factors such as whether the substance is a cytotoxin, a W092/20~ PCT/USg2/~3 2 i ~ 8 - 20 -lytic antibody, or an IL-2R blocking molecule. In the case of toxic molecules that act intracellularly, the extent of cell uptake is an important factor; less permeable molecules must be administered at a higher S dose.
More than 60 patients have received DAB~86IL-2 in Phase I/II clinical protocols. The maximum tolerated dose (MTD) established by transient asymptomatic hepatic transaminase elevations in about 30% of patients treated 10 at the NTD. Anti-tumor effects have been seen in approximately 40% of patients; responses were noted in B-cell leukemias and lymphomas, cutaneous T-cell lymphoma and Hodgkin's disease (LeMaistre et al., Blood 360à:abstract 1429, 1990; Woodworth et al., Fourth 15 International Conference on Human Retrovirology, 1991).
Serum concentrations of 10 8M DAB~86IL-2 have been achieved in patients with IL-2 receptor expressing malignancies. Anti-tumor effects have been observed in highly refractory leukemia/lymphoma patients and these 20 effects have occurred despite the presence o~f elevated soluble IL-2R levels in all patients. This observation is consistent with data which suggest that soluble IL-2R , does not interfere with binding of IL-2 to the high affinity interleukin-2 receptor. Animal and human 25 studies have demonstrated that DAB486IL-2 has no general immunosuppressive effect (LeMaistre et al., supra ;
Woodworth et al., supra ) .
- Experiments indicate that binding and internalization of DAB~86IL-2 by cells bearing the high 30 affinity IL-2 receptor occurs within 30 minutes of exposure, resulting in maximal inhibition of protein synthesis within several hours. Therefore, the molecule should be effective even if the serum half-life is rather short.

~.~VO 92/20364 PC~r/US92/04093 t3 U

Generally, drugs targeted to IL-2 receptor will be administered immediately after (e.g., within several minutes or less) vascular injury. Preferably, treatment begins before the accumulation of platelets and 5 leukocytes. Animal models of denuding balloon catheter injury have been used to show that platelet aggregation and thrombus formation occurs immediately after injury and that leukocyte adhesion begins within several hours.
Autopsy of a patient who died S days after angioplasty 10 revealed that proliferating smooth muscle cells were invading the dilated region (Austin et al., J. Amer.
Coll . Cardiol . 6:369, 1985).
Once initiated, endothelial regeneration is complete within one to two weeks. Since re-15 endothelialization of the vessel wall appears to inhibitsmooth muscle cell proliferation (Ip et al., J. Amer.
Coll . Cardiol . 15:1667, 1990), treatment may need to last for only a few weeks. Accordingly, it is desirable to administer the compounds of the invention periodically 20 over a period adequate to allow regeneration of the endothelium.
The hybrid molecule can be administered as an unmodified molecule or in the form of a pharmaceutically acceptable salt, admixed with a therapeutically -25 acceptable carrier, e.g., saline. Examples of preferredsalts are therapeutically acceptable organic acids, e.g., acetic, lactic, maleic, citric, or salicylic. For example, the hybrid molecule may be purified and sterile filtered using 2 micron filters and suspended in sterile 30 phosphated buffer saline (0.15M NaCl; 0.02M phosphate buffer, pH 7.2).

Other Embodiments Derivatives of IL-2 which block utilization of endogenous IL-2 are useful for preventing proliferation W092/20~ PCT/US92/04~93 ~ i ù ~, 2 ~ 8 of IL-2R bearing cells. Activated cells deprived of IL-2 fail to proliferate and, in the absence of the essential anabolic stimulus provided by IL-2, will eventually die. The ability of a given IL-2 derivative 5 to interfere with IL-2 function can be tested in an IL-2 bioactivity assay such as the one described by Ju et al.
(J. Biol . Chem. 262: 5723, 1987). IL-2R/toxin hybrids in which the toxin has been rendered inactive can be also used to block IL-2 receptors. A non-toxic mutant 10 diphtheria toxin molecule has been described (Uchida et al. J. Biol. Chem. 248:3838, 1973), and this molecule can be used to produce a non-toxic IL-2/diphtheria toxin hybrid. See Svrluga et al. U.S. Serial No. 590,113, hereby incorporated by reference, for an example of such 15 a hybrid molecule.
~-~ Monoclonal antibodies which interfere with the binding and/or uptake of IL-2 are useful in the method of the invention because IL-2R bearing cells deprived of IL-2 fail to proliferate. Blocking monoclonal antibodies 20 can be tested for their ability to interfere with IL-2 ~ bioactivity using the method of Ju et al.,(supra) .
`~ Nonoclonal antibodies which induce complement can be used to destroy IL-2R-bearing cells. Complement inducing antibodies are generally those of the Ig~l, 25 IgG2, IgG3, and IgM isotypes. Monoclonal anti-IL-2R
antibodies can be screened for those able to induce complement using a complement-dependent cytotoxicity test, as follows.
Human T-lymphocytes and EBV transformed B-30 lymphocytes are labeled with 51Cr sodium chromate and used as target cells; these cells are incubated with hybridoma culture supernatants and with complement, and then the supernatants are collected and counted with a gamma counter. Those supernatants exhibiting toxicity against 35 activated T-lymphocytes, but not resting T- or B-~092/20~ 2 1 '~ 3 2 5 8 PCT/US92/04093 . - 23 -lymphocytes, are selected, and then subjected to a further screening step to select those supernatants containing antibody which precipitates (i.e., is specifically reactive with) the 50 kd glycoprotein IL-2 5 receptor (described in detail in Leonard et al. (Proc.
Natl . Ac~d . Sci . USA 80:6957, 1983). The desired anti-IL-2 rèceptor antibody is purified from the supernatants using conventional methods.

Claims

WO 92/20364 PCT/US92/04???

Claims

1. A method of making a medicament for treating a sarcoma characterized by growth of neoplastic cells of non-lymphoid and non-monocytic origin, said method comprising admixing a pharmaceutically acceptable carrier and a molecule which is capable of specifically binding to a cytokine receptor expressed on said neoplastic cell, wherein said cytokine receptor is normally expressed on cells of lymphoid or monocytic origin, said molecule being capable of decreasing the viability of said neoplastic cell.

2. The method of claim 1 wherein said neoplastic cell is not of hematopoietic stem cell origin.

3. The method of claim 1 wherein said sarcoma is an osteosarcoma.

4. The method of claim 1 wherein said sarcoma is a fibrosarcoma.

5. The method of claim 1 wherein said sarcoma is a leiomyosarcoma.

6. The method of claim 1 wherein said sarcoma is a rhabdomyosarcoma.

7. A method of making a medicament for treating a lung carcinoma characterized by growth of neoplastic cells of non-lymphoid and non-monocytic origin, said method comprising admixing a pharmaceutically acceptable carrier and a molecule which is capable of specifically binding to a cytokine receptor expressed on said neoplastic cell, wherein said cytokine receptor is normally expressed on cells of lymphoid or monocytic origin, said molecule being capable of decreasing the viability of said neoplastic cell.

8. The method of claim 1 wherein said cytokine receptor is an interleukin receptor.

9. The method of claim 8 wherein said interleukin receptor is an interleukin-2 receptor.

10. The method of claim 9 wherein said interleukin-2 receptor is a high affinity interleukin-2 receptor.

11. The method of claim 8 wherein said interleukin receptor is an interleukin-4 receptor.

12. The method of claim 8 wherein said interleukin receptor is an interleukin-6 receptor.

13. The method of claim 1 wherein said molecule kills cells bearing said cytokine receptor.

14. The method of claim 1 wherein said molecule is a hybrid molecule comprising a first and a second portion joined together covalently, said first portion comprising a molecule capable of decreasing cell viability and said second portion comprising a molecule capable of specifically binding to said cytokine receptor.

15. The method of claim 14 wherein said second portion comprises all or a binding portion of an antibody specific for said cytokine receptor.

WO 92/20364 PCT/US92/040?3

16. The method of claim 14 wherein said second portion comprises all or a binding portion of a ligand for said cytokine receptor.

17. The method of claim 16 wherein said cytokine receptor is an interleukin receptor.

18. The method of claim 16 wherein said ligand is an interleukin.

19. The method of claim 14 wherein said first portion comprises a cytotoxin.

20. The method of claim 19 wherein said cytotoxin is a fragment of a peptide toxin which is enzymatically active but which does not possess generalized eukaryotic receptor binding activity.

21. The method of claim 20 wherein said fragment of a peptide toxin comprises fragment A of diphtheria toxin and enough of fragment B of diphtheria toxin to facilitate translocation into the cytosol.

22. The method of claim 21 wherein said molecule is DAB389IL-2.

23. The method of claim 21 wherein said molecule is DAB389IL-4.

24. The method of claim 21 wherein said molecule is DAB389IL-6.

25. The method of claim 21 wherein said molecule is DAB486IL-2.

26. The method of claim 21 wherein said molecule is DAB486IL-4.

27. The method of claim 21 wherein said molecule is DAB486IL-6.

28. The method of claim 15 wherein said antibody is a complement activating antibody.

29. The method of claim 1 wherein said cytokine receptor is not expressed on non-neoplastic cells of non-lymphoid and non-monocytic origin.