CA2035868A1

CA2035868A1 - Hybrid molecules

Info

Publication number: CA2035868A1
Application number: CA002035868A
Authority: CA
Inventors: Richard C. Svrluga; Cory A. Waters
Original assignee: Individual
Current assignee: Seragen Inc
Priority date: 1989-07-06
Filing date: 1990-07-06
Publication date: 1991-01-07
Also published as: JPH04501808A; EP0433442A4; EP0433442A1; WO1991001004A1

Abstract

Abstract of the Disclosure A hybrid molecule containing a hematopoietin receptor superfamily cytokine (or a fragment or analog thereof capable of binding to the high-affinity receptor for such cytokine) covalently bonded, at its amino terminal end, to a chemical entity which causes the hybrid molecule to be processed via a lower affinity receptor for such cytokine to a degree less than the degree of processing of the cytokine by the lower affinity receptor, provided that the chemical entity cannot be a fragment of diptheria toxin which includes the translocation domain of that toxin.

Description

6, r~

HYBRID MOLECULES
Backqround of the Invention The field of the invention is cytokine hybrid molecules.
The hematopoietin receptor superfamily (Idzerda et al., J. Exp. Med. 171:861-873, 1990) is a family of cytokine receptors, the extracellular domains of which exhibit a significant degree of amino acid homology with each other. These receptors, which include the Interleukin 2 (IL-2) p75 receptor, are located on the surfaces of hematopoietic cells, including both lymphoid cells and myeloid cells, and are capable of binding and mediating the cellular proliferat~ve effects of such cytokines as IL-2, Interleukin 3 (IL-3), Interleukin 4 (IL-4), Interleukin 6 (IL-6), erythropoietin (EP0), and prolactin. The cytokines which bind to members of the hematopoietin receptor superfamily are herein collectively termed "hematopoietin receptor superfamily cytokines".
The ability of IL-2 to bind specifically to IL-2-receptor-bearing cells, such as allograft-activated human T-cells and certain leukemic lymphocytes, has inspired the construction of toxin-IL-2 hybrid molecules capable of targeting their toxic effect specifically on cells displaying these IL-2 receptors ("IL-2R") on their surfaces. Although some of these hybrid molecules have been created by chemical conjugation of IL~2 with a toxin (Greenfield et al., Science, pp. 238, 536 (1987), more recently, recombinant DNA methodologies have been applied to the genetic assembly of hybrids such as diphtheria toxin/IL-2 hybrid (U.5. Patent No. 4,675,382) and Pseudomonas exotoxin AIIL-2 hybrid (Lorberboum-Galski et al., Proc. Natl. Acad. Sci. USA 85:1922-1926, 1988), in which IL-2 replaces the endogenous cell-specific receptor binding domain of the toxin protein. Both ~)3~?~

native diphtheria toxin and native Pseudomonas toxin kill cells by binding to certain receptors on the surface of a susceptible cell, passing through the cell membrane, and, once inside the cell, shutting down protein synthesis by inactivating a crucial protein synthesis factor. The mechanism of cell killinq by recombinant toxin-IL-2 hybrids is the same, except that the toxin's own cell-binding doma~n is replaced with IL-2, so that the hybrid toxin binds to IL-2-receptor-bearing cells exclusively.
The nature of these IL-2-specific receptors has been the object of several studies (e.g., Robb et al., J.Exp.Med. 154:1455-1464, 1981; Tsudo et al., Proc. Natl.
Acad. Sci. USA 83:9694-9698, 1986; and Teshigawara et al., J.Exp.Med. 165:223-234, 1987). Two separate IL-2-bindlng receptor molecules, a 55 kilodalton glycoprotein ("p55" or "Tac") and a 75 kilodalton glycoprotein ("p75"), have been reported on human lymphocytes; each of these binds IL-2 alone or to~ether in a heterodimeric receptor complex (Yagita et al., Cancer Res. 49, in press, 1989). Where the IL-2-binding molecule is p55 alone, IL-2 binds with relatively low affinity (Kd = 10 8M); p75 alone binds IL-2 with an intermediate affinity (Xd = 10 9M); and the heterodimeric form ("p75 + p55n) binds with relatively high affinity to IL-2 (~d = 10 llM). The N-terminal amino acid residues of IL-2, particularly Asp20, are essential for binding of IL-2 to the p75 receptor molecule (Collins et al., Proc. Natl.
Acad. Sci. USA 85:7709-7713, 1988).
It has been shown that picomolar quantities of a diphtheria toxin/IL-2 recombinant hybrid are sufficient to inhibit protein synthesis by 50% in cells bearing the heterodimeric p75+p55 ("high affinity") type of IL-2 receptor, while cells which express only the p55 ("lowest affinity") or the p75 ("intermediate affinity") type of receptor are resistant to the protein-synthesis-~` ~i c~

inhibiting effect of the same diphtheria toxin/IL-2 hybrid (Waters et al., Eur. J. Immunology 20:785-791, 1990) .
In contrast, ~seudomonas exotoxin AIIL-2 recombinant hybrid has been shown to be internalized by the low and intermediate affinity receptor subunits as well as by the heterodimeric high affinity receptor (Lorberbaum-Galski et al., J. Biol. Chem. 263:18650-18656, 1988). In these constructions, the Pseudomonas exotoxin i5 linked to IL-2 through IL-2's carboxy-terminus.
Recently, all of the hematopoietin receptor superfamily cytokines have been demonstrated to have multiple receptors composed of distinct subunits of different binding affinities (cf. Itoh et al., Science 247:324-327, 1990; and Sawyer, Clinical Biotechnology

2:77-85, 1990~.
Summary of the Invention In general, the invention features a hybrid molecule containing a hematopoietin receptor superfamily cytokine (or a fragment or analog thereof capable of binding to the high-affinity receptor for such cytokine) covalently bonded, at its amino terminal end, to a chemical entity which causes the hybrid molecule to be processed via a lower affinity receptor for such cytokine to a degree less than the degree of processing of the cytokine by the lower affinity receptor, provided that the chemical entity cannot be a fragment of diphtheria toxin which includes the translocation domain of that toxin, as such domain is described in John R. Murphy U.S.
Patent No. 4,675,382, hereby incorporated by reference.
A "lower affinity receptor" for a given cytokine is any receptor capable of processing that cytokine, other than the high-affinity receptor for that cytokine.
"Processing" is a term which i5 used to mean the binding of a cytokine by one of that cytokine's receptors and internalization of the receptor-bound cytokine into an endocytic veslcle or into the cell. (For those hybrld molecules of the inven~ion which are not internalized, the term "processing" means simply the binding of the cytokine by one of that cytokine's r~eceptors.) Thus, a cytok~ne molecule is said to be ~processed via a lower affinity receptor for such cytokine" when the cytokine molecule has bound to a low or intermediate-affinity receptor for that cytokine and has also been internaliz~d by the cell (for example, in an endocytic vesicle) as a ligand/ receptor complex. The degree of such processing of a hybrid molecule of the invention can be quantitatively compared to the degree of processing of a naturally-occurring cytokine molecule by, for example, measuring the amount of each of such molecule which is internalized by cells bearing one type of receptor. The ability of a hybrid molecule to distinguish between high and lower affinity receptors is determined by comparing the amount of the molecule internalized by cells bearing only high-affinity receptors to the amount internalized by cells bearing only the lower affinity receptors:
those hybrids which are capable of distinguishing between receptor types to a significant degree are useful for the purposes cited herein.
A "fragment" of a cytokine molecule is a polypeptide having an amino acid sequence exactly corresponding to some portion of the naturally-occurring molecule that is less than all of that naturally-occurring molecule, and can be generated, for example, byproteolytic digestion of the naturally-occurring molecule, by chemical synthesis, or by recombinant DNA
techniques. An "analog" of a cytokine molecule is a polypeptide which differs from the naturally-occurring cytokine in that it has substitutions, additions, andlor Cl ,( A ~

deletions of one or more amino-acids at one or more locations: for example, an analog of a given cytokine would include those polypeptides which contain a contiquous segment at least half as long as the naturally-occurring cytokine and which has at least 80%
sequence homology with some portion of the naturally-occurr~ng molecule. The amino acid substitutions may be either conservative or non-conservative, and may be designed, for example, to remove proteolytically sensitive sites from the polypeptide. [By conservative is meant that the substituted amino acyl residue is chemically similar (e.g., acidic, basic, hydrophobic, aromatic) to the residue for which it is substituted:
for example, substitution of a valine for a leucine.]
Such analogs may be conveniently obtained by recombinant DNA techniques, or by any other method known to those in the field. Once generated, any such fragments or analogs can be tested for their ability to be processed via the high-affinity and the lower affinity receptors of the cytokine from which they are derived. Those which behave comparably to the naturally-occurring cytokine can be incorporated into the hybrid molecules of the invention.
In preferred embodiments, the cytokine is selected from the group consisting5 of IL-2, IL-3, IL-4, IL-6, EPO, and prolactin;
the chemical entity interferes with the binding by the cytokine portion of the hybrid molecule to the lower-affinity receptor;
the chemical entity is an X-ray-opaque moiety, a0 fluorescent moiety, a radioactive moiety, a polypeptide (herein defined as two or more amino acids linked by peptide bonds) such as an antibody molecule, ricin, streptavidin, gelanin, or ferritin, or an enzymatically-active fragment or analog of such a polypeptide;
the covalent bond is a peptide bond; and i'~J b, ~

the hybrid molecule is capable of affecting the rate of proliferation of cells bearing high-affinity receptors.
The hybrid molecule of the invention may be used in a method for selectively binding a moiety to a cell having a high-affinity receptor for the cyto~ine of interest, by exposing the cells to hybrid molecule which contains the moiety covalently bonded to the amino terminui of cytokine (or a fragment or analog thereof capable of binding to the high-affinity receptor). In preferred embodiments, the moiety includes a label and the method is used for imaging high-affinity receptors or cells bearing high-affinity receptors; or the moiety is a molecule complexed with iron and the method is used for separatlng cells having a relatively high number of the high-affinity receptors from other cells having a lower number of such high-affinity receptors; or the hybrid molecule i~ capable of decreasing the rate of proliferation of cells to which it binds, ~nd the method 2Q is used for therapeutic treatment of a condition characterized by overproduction of cells having high-affinity receptors.
The hybrid molecule of the invention can also be used in a method for selectively isolating cells having hiyh-affinity receptors for the cytokine of interest, out of a population of cells some of which lack such high-affinity receptors, which method involves (1) immobilizing the hybrid molecule, (2) causing the population of cells to contact the immobilized hybrid molecules under conditions permitting binding of high-affinity receptors to the immobilized hybrid molecules, and (3~ separating unbound cells from bound cells. This method may be used to remove, from bodily fluids or tissues, cells having such high-affinity receptors;
alternatively, the method includes the additional step of eluting the bound cells from the immobilized hybrid molecules, and i5 used for isolating, from bodily fluids or tissues, cells bearing high-affinity receptors.
The hybrid molecule in which the chemical entity 5 i8 a polypeptide may be made, for example, by providing a recombinant DNA molecule containing a DNA sequence encoding that polypeptide fused to a DNA sequence encoding the cytokine of interest (or a high affinity-receptor-binding fragment or analog thereof), introducing the recombinant DNA molecule into an appropriate expression system, and expressing the recombinant DNA
molecule.
The streptavidin-containing hybrid molecule of the invention may be used in a method for imaging high-affinity receptors specific for the cytokine of interest,or, in a population of cells, a cell bearing such high-affinity receptors, by (a) exposing the high-affinity receptors to an amount of the hybrid molecule sufficient su~stantially to maximize the difference between (1) the amount of the hybrid molecule which binds to the high-affin~ty receptors and (2) the amount which binds to all lower-affinity receptors on the population of cells; (b) exposing the receptor-bound hybrid molecules to a labelled probe covalently linked to biotin; and (c) detecting the labelled probe which binds to the receptor-bound hybrid molecules.
~ he invention takes advantage of the ability of each of the hematopoietin receptor superfamily cytokines, when linked at its N-terminus to a chemical entity, to be processed via its lower-affinity receptors to a lesser degree than is the cytokine itself, resulting in enhanced discrimination between high- and lower-affinity receptors. This enhanced discrimination between receptor types by the hybrid molecule of the invention permits selective labelling, isolation, removal, or ~'r~

killing of cells bearing the high-affinity receptor, to the virtual exclusion of cells lacking this receptor, and thus presents an important tool for the study, diagnosis, and therapeutic treatment of various conditions in which myeloid or lymphoid cells are implicated as critical elements. For example, a hybrid molecule of the invention having IL-2 as the cytokine portion would target the high-affinity receptors on activated T cells, and so would be useful for applications involvlng transplant rejection, autoimmune disease, and certain lymphatic cancers.
Other features and advantages of the invention will be apparent from the following description of the preferred embodlments thereof and from the claims.

Description of the Preferred Embodiments The drawings will first briefly be described.
~rawinas Fig. 1 is a representation of the DNA coding 6equence, and the corresponding amino acid sequence, for the IL-2 gene portion of plasmid pDW15, following ~EhI
digestion of the plasmid.
Fig. 2 is a diagram illustrating the stepwise construction of plasmid PSI130 bearing the CRM197/IL-2 recombinant gene.
Fig. 3~a) is an illustration of a recombinant DNA
plasmid intermediate in the construction of a ricin A/IL-2 hybrid gene~
Fig. 3(b) is an illustration of a recombinant DNA
vector bearing the ricin A/IL-2 hybrid gene and suitable for expression in E.coli.
Fig. 4(a) is an illustration of a recombinant DNA
plasmid intermediate in the construction of a streptavidin/IL-2 hybrid gene.

Fig. 4 (b) is an illustration of a recombinant DNA
vector bearing the streptavidin/IL-2 hybrid gene and suitable for expression in E.coli.
Discrimination Amona IL~2 Rece~tors ~y IL-2 HYbrid And Native IL-2.
The invent~on is based upon the observation that attachment of a chemical entity to the N-terminal region of IL-2 1nterferes with processing of the IL-2 portion of the hybrid via intermediate-affinity (p75) receptors on T-cells, possibly due to structural constraints imposed on the chemical entity portion of the hybrid by the p75 receptor. Using an assay comparing (1) the molar concentration of a diphtherial2t5Oxin/ IL-2 hybrid molecule necessary to displace 50% of iodine-labelled-IL-2 from binding sites on three cell lines, each of which displays only one of the three types of IL-2 receptors, with (2) the molar concentration of IL-2 necessary to effect the ~ame displacement, it was found that, on a cell line displaying only the p55 receptor molecule, 10-fold more IL-2 hybrid was required to displace the labelled IL-2, while for cells d~splaying only the p75 receptor, the hybrid mo~ecule had to be present at a concentration 100-fold higher than the concentration of IL-2 necessary to effect 50% displace~ent of labelled IL-2. Cell~
displaying the high-affinity (p75+p55) receptor required approximately a 100-fold higher concentration of IL-2 hybrid than of IL-2 to effect 50% displacement. Thus, (a) the affinity of hybrid-IL 2 for the p55 receptor is within an order of magnitude of the affinity of IL-2 for that receptor, ~b) the affinity of hybrid-IL-2 for the p75 receptor is about 1% of the affinity of IL-2 for that receptor, and (c) the affinity of hybrid-IL-2 for the high-affinity (p75+p55) receptor is about 1% of the affinity of IL-2 for that receptor.

While these binding data suggest a slight binding differential between IL-2 and IL-2 hybrid for cells bearing only high-affinity receptors, and those bearing only the lower-affinity p75 receptors, the net result of attaching the chemical entity to the N-terminus of IL-2 i5 a 300-lOOO fold alteration in processing of the hybrid ligand, such that processing, in this instance measured as an IC50 for half-maximal inhibition of protein synthesis, is 300-1000-fold more efficient for the high-affinity-receptor-bearing cells than for lower-affinity-receptor-bearing cells.
The experimental results given above indicate that the ma~or alteration in the ligand/receptor interaction caused by attaching a chemical entity to the amino terminus of IL-2 takes place with respect to the p75 receptor. The fact that the p75 receptor shares a high sequence homology with certain subunits of the receptors for all of the hemopoietin receptor superfamily cytokines suggests that placing a chemical entity such as a polypeptide at the N-terminus of any of these cytoklnes will inhibit, probably by steric hindrance, the processing of ~i.e., the binding to and internalization of) the hybrid molecule by the subunit of the cytokine's receptor which is homologous with the p75 subunit of the IL-2 receptor, permitting the hybrid molecules of the invention to discriminate between their high-affinity receptors and their lower-affinity receptors to a significantly greater degree than does either the native cytokine or a cytokine hybrid constructed without introducing such steric hindrance near the binding domain analogous to the IL-2 p75 binding domain. This enhancement of the level of discrimination can be exploited in a number of ways, some of which are described below.

Example 1. Con~truction an~ expresslo~ of CRM197/IL-2 reco~binant fu3ed gene.
The IL-2 gene used for these fusions (Fig. 1) was o~tained from plasmid pDW15 (Fig. 2) (Williams et al., Nucleic Acids Res. 16:10453-10467, 1988) containing a synthetic form of the IL-2 gene which, when cloned into E.coli JM101, expresses IL-2 protein at a rate about 16 times that of the native cDNA sequence cloned into the same strain of E.coli.
Standard DNA cloning techniques were employed.
Plasmids were introduced into E.coli by CaCl2 transformation, isolated by the alkaline lysis procedure, and purified by CsCl density gradient centrifugation (Maniatis et al., Molecular Cloning: a laboratory manual. Cold Spring Harbor Laboratory, Cold Sprinq Harbor, N.Y., 1982). The genetic fusion was made at the ~hI site of pDW15 so that the IL-2 domain of the fused gene would encode 133 amino acids of IL-2, plus one additional amino acid on its amino terminus encoded by the Sph site (Fiq. 1).
The construction methodology is illustrated in Fig. 2. Plasmid pABM6508, containing a gene cod~ng for the N-terminal 48S amino acids of diphtheria toxin ~oined to -melanocyte-stimulating hormone (Bishai et al., J.
Bacteriol. 169:5140-5151, 1987), was digested co~pletely with ~in~III and partially with ~hI; a 6kB SphI -HindIII vector fragment was gel-purified and ligated to the 0.5 k8 SphI - HindIII IL-2-gene-containing fragment from pD~15, resulting in a plasmid designated pABI6508. A 5.7 kB vector fragment resulting from AccI and XmnI digestion of pABI6508 was gel-purified and ligated to a 0.8kB
~ccI - XmnI fragment of plasmid p~197 (Bishai et al.), yielding a plasmid that is referred to as pSI130. p~197 carries the gene for CRM197, which is a full-length (535 amino acids) Gly52- Glu52 missense mutant form of diphtheria toxin that is devoid of ADP-ribosyl transferase activity and thus is nontoxic (Uchida et al., J. Biol. Chem. 248:3838-3844, 1973). The missPnse mutation occurs within the 0.8kB ~ç~ mnI fragment of pB197, and ~o appears in pSI130 as well.
Expression of pSI130 in ~.coli was induced a~
described by Bishai (JOBacteriol. 1987); the CRM197/IL-2 gene product was purified using affinity chromatography (Williams et al. 1988) followed by HPLC size exclusion chromatography. (Methods in Enzymology 91: 137-190; 1983) Example ~. Biologic~ s~y for IL-2 receptor dl~ar~min~tlon.
Fifty micrograms of recombinant human IL (rIL-2) was enzymatically iodinated with enzymobeads (Bio-Rad Laboratorie~, Richmond, CA) in accordance with the manufacturer 1 8 instructions. One millicurie Nal25I
(DuPont NEN, Boston, MA) was used in the reaction; this represented a limiting concentration of iodine; one atom of iod~ne to eight molecules of rIL-2. The reaction was allowed to proceed for five minutes at room temperature, and was quenched by the addition of NaN3 and NaI. Yetal calf serum (FCS; Hyclone Laboratories, Logan, UT) was added to a final concentration of 10% and the mixture chromatographed through one milliliter of Sephadex G-10 which had been equilibrated with RPMI 1640 medium supplemented with 25 mM HEPES, (pH 7.4) (GIBC0, Grand Island, NY), 2 mM glutamine (GI~CO), 100 units/ml penicillin, 100 ~g/ml streptomycin (GIBCO), and 10S FCS
(Hyclone).
The radiolabeled IL-2 binding assay was performed essentially as described by Smith and co-workers (Robb et al.; Teshigawara et al.). Cells were harvested and washed three times with RPMI 1640 medium containing 10%
FCS. To determine total IL-2 binding, 5 x lO5 cells were exposed to a known concentration of l25I-rIL-2 (generally n 1 x 10 ' ~) for 30 minutes at 37C in RPMI 1640 medium supplemented with 25 mM HEPES (pH 7.4), (GIBCO, Grand Island, NY), 2 mM glutamine (GIBC0), 100 units/ml penicillin and 100 ~g/ml streptomycin (GIBCO), and 10%
FCS (Hyclone)(total volume 150 ~l). To determlne the amount of rIL-2 or PT/IL-2 required to di~place 125I-rIL-2 from its receptor, increasing concentrations of unlabeled rIL 2 or PT/IL-2 (0.5- to 2000-fold molar excess) were added to each tube in the presence of 1 x lO 9 M 125I-rIL-2 and the percentage of total bindin~ at each concentration of cold comp~titor determined. The binding reaction was terminated by microcentrifuging (Beckman Instruments, Irvine, CA) the cells through an oil mixture of 80% 550 phenyl fluid (Dexter ~ysol, Olean, NY) and 20% paraffin oil (Sigma, St. Louis, MO)(final density 1.03 gm/ml) in 400 mlcroliter microcentrifuge tubes (Beckman Instruments~. The cell pellet was cut from the microcentrifuge tube leaving the oil and medium containing free ligand in the tube. Cell pellets representing bound radioactivity and the oil/medium supernatants representing ~ree ligand were counted in a Beckman gamma counter, and the percentage of the total radioactivity that pelleted with the cells was calculated. All assays were performed in triplicate.
Example 3. Con~truction and expression of Ricin A~ 2 reco~blnant fu~ed gene, an~ u~e of the re~ultnnt hybrid protein.
Standard procedures for DNA cloning, cell transformation and plasmid isolation (as described, for example, by Maniatis et al.~, could be employed to carry out the following construction:

~} ~

Ric~n cDNA is prepared as descr~bed by Halling et al. (Nucleic Acids Res. 13:8019-8033~ 1985) or Lamb et al. (Eur. J. ~iochem. 148:265-270, 1985). Following digestion with ~mHI and HaeIII, an 856-bp ~HI - HaeIII
fragment of ricin cDNA (encoding the 5' end of the ricin gene, the signal sequence and ricin amino acids 1 through 272) is isolated by agarose gel electrophoresis.
An unphosphorylated synthetic DNA oligomer, comprising 5' AAGCGTCGGCATG 3'

3' TTCGCAGCC 5' and coding ~or a protease-sensitive region (Lys Arg) and one-half of a ~I site (CATG~, is synthesized by standard B-cyanoethyl phosphoramidite chemistry on a Milligen 7500 DAN synthesizer according to manufacturer' 8 instructions. The synthetic oligonucleotides are purified by gel electrophoresis, complementary strands are annealed and ligated to the ~3~III blunt end of the ricin gene fragment. The sequence of this oligomer was selected (1) to allow fusion of the ricin gene fragment to an IL-2 gene having the matching half of a ~hI site, and (2) to allow release of enzymatically-active ricin A
peptide from the ricin A/IL-2 polypeptide by proteolytic cleavage at the added protease-sensitive region upon internalization of the toxin molecule by a cell.
In order to insert a second half-~hI site at the appropriate spot upstream of the ricin coding sequence, the above construct is next digested with FnuDII (or ~haI) and the 740 bp FnuDII - (HaeIII/SphI) fragment that results is lsolated and ~ '`",1 r~

lin~ed (at the FnuDII blunt end) to the following unphosphorylated synthetic oligonucleotide:
5~CC ATC GCT ATA TTC CCC AAA CAA TAC CCA ATT
ATA-5 3'GTACGG TAC CGA TAT AAG GGG TTT GTT ATG GGT TAA
TAT-encoding:f~et Al~ Ile Phe Pro Lys Gl~ Tyr ~ro Ile Ile-(cont.): -AAC TTT ACC ACA GCG GGT GCC ACT GTG CAA AGC TAC ACA

-TT~ AAA TGG TGT CGC CCA CGG TGA CAC GTT TCG ATG TGT
TTG--Asn Phe Thr Thr Ala Gly Ala Tbr Val Gln 8er Tyr Thr A9n--(cont.): -m ATC AGA GCT GTT CG 3' -AAA TAG TCT CGA CAA GC 5' -Phe Ile Arg Ala V~l Arg This DNA sequence, which has a half of an S~hI site at each end, can then be ligated into SphI partially-20 digested (linearized) plasmid pDW15 as described inExample 1. This plasmid is then digested with ~coI
and HindIII, yielding a 1.3 kb DNA fragment coding for all of the following: mature ricin A, part of the ricin A linker, the synthetic protease-sensitive site, 25 and IL-2.
Thi~ 1.3 kb NcoI - HindIII fragment is isolated and cloned onto ~çQI - HindIII digested pKK233-2 vector (Fig. 3(b)) (Pharmacia; Piscataway, NJ; Armann et al., Gene 40:183-190, 1985) for 30 expression in E.~oli. Expression of the ricin A/IL-2 protein is induced by isopropyl-B-D-thiogalactoside (IPTG). The protein may be purified by immunoaffinity chromatography followed by HPLC size exclusion chromatography.

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ExamRle 4. Construction an~ expresslon of streptavidi~/IL-2 recomb~ant fusea qene, an~ u~e of th~ re~ult~nt bybri~ proteln in imagi~q h~gh-~f~inity receptors.
Standard procedures for DNA cloning, cell transformation, and plasmid isolation would be employed to carry out the following construction The streptavidin gene is isolated from S~re~omYces avidinii a~ described by Argarana et al., 10 Nucleic Acids Res. 14:1871-1882, 1986. Following ~
digestion of the gene, a ~433 bp fragment is isolated and further digested with TaaI. A -414 bp NaeI ~
fragment i6 thereupon isolated and its 5' ~I blunt end i8 ligated with the following synthetic DNA
15 oligomer (synthesized on a Milligen 7500 DNA
synthesizer according to manufacturer instructions):
5' CC ATG GAC CCC TCC AAG GAC TCG AAG GCC CAG
GTC-3' GTACGG TAC CTG GGG AGG TTC CTG AGC TTC CGG GTC

ç~codina: fMet A~p Pro Ber ~y8 Asp Ber Lys Ala ~ln Val-(cont.): -TCG GCC GCC GAG GCC 3' -AGC CGG-CGG CTC CGG 5' -Ber Ala Ala alu Ala The following synthetic oligomer is ligated onto the (3') l~a~I end of the fragment:
5' C GAC GCG GCG AAG AAG GCC GGC GTC AAC AAC
GGC-5 3' TG CGC CGC TTC TTC CGG CCG CAG TTG TTG
CCG-encodinq: Asp Ala Ala Lys Ly~ Ala Gly ~al Asn A~n Gly-(cont.): -AAC CCG CTC GAC GCC GTT CAG CAG CAT G
10 3' -TTG GGC GAG CTG CGG CAA GTC GTC
5' -~sn ~ro ~eu Asp Ala V~l Gln Gln His The synthetic sequences so added replace all of the 15 streptavidin coding sequences removed in the course of the aeI and TaqI digestions, and add one half of a ~hI site at each end of the fragment. This fragment can then be ligated into SphI partially-digested linearized pDW15 (see Example 1~ and transfor~ed into 20 E.coli. The appropriate product of the ligation, which can be identified by plasmid restriction site mapping of DNA obtained from single colonies resulting from the transformation, is illustrated in Fig. 4(a).
Following NcoI - ~n~III digestion of this plasmid, a 25 1.0 Xb NcoI -HindIII fragment containing the entire coding region for the streptavidin/IL-2 fusion protein is isolated and cloned onto NçoI - HindIII-digested pKK233-2 vector for expression in E.coli. The final form of this streptavidin/IL-2-encoding vector ~s 30 illustrated in Fig. 4(b). E.coli calls are transformed and the streptavidin/IL-2 polypeptide is isolated and purified by immunoaffinity chromatography followed by HPLC size exclusion chromatography, and used, for example, to assay for r ~ 18 ~
the presence of high-affinity IL-2 receptors on lymphoma cells.
Frozen or paraffin sections of a human lymphoma biopsy are prepared on micro;cope slides by 5 standard techniques. Prior to staining, frozen sections are first fixed with acetone or another suitable fixative, then washed with a buffer such as .lm Tris, pH 7.2. Unlabeled hiotin and/or free fluorochrome is then added to saturate all available 10 nonspecific binding sites. After removal of unbound biotin by rinsing in buffer, the slides are treated with streptavidin/IL-2 hybrid protein and then rinsed in buffer to remove unbound streptavidin/IL-2. The slide~ are then treated with a solution containing a 15 fluorochrome-biotin conjugate. The slides are again rinsed in buffer to remove unbound labeled biotin, and observed under a fluorescence mlcroscope illuminated at an appropriate wavelength of light. The presence of a high proportion of fluorochrome-labeled cells in 20 a biopsy of a lymphoma would be an indicator of that tumor's potential susceptibility to chemotherapeutic treatment with diphtheria toxin/IL-2 hybrid or another IL-2 N-terminus-linked toxic hybrid.
Other Embodiments Other embodiments are within the following claims. For example, the cytokine portion of the hybrid molecule could be contributed by any of the hematopoietin receptor superfamily cytokines, or fragments or analogs thereof (prepared by standard 30 techniques). The chemical entity could be ferritin, for use in separating cells bearing high affinity cytokine receptors from those which do not; an inert moiety, the function of which is simply to enhance the cytokine's discrimination between receptor types; or an antigen, the function of which is to induce a host-mediated immunological attack on the host's own high-affinity-receptor-bearing cells. Alternatively, the chemical entity could be an enzyme, a carbohydrate, a 5 lipid, a synthetic polymer, a viral particle, or an inorganic molecule. The linkage between the cytokine and the chemical entity can be a peptide bond or any other type of covalent bond. By using a chemical entity each molecule of which is capable of being 10 linked to the N-termini of multiple cytokine molecules, a multivalent hybrid would be created which could bind simultaneously to two or more high-affinity-receptor-bearing cells, causing the cells to agglutinate and thus be cleared from a mixed 15 lymphocyte suspension. This procedure would be useful as an assay or as a therapeutic technique.
The hybrid protein could be synthesized by utilizing recombinant DNA techniques, beginning with either a synthetic DNA sequence or a cloned natural 20 gene, or could be assembled by chemically conjugating a chemical entity with the cytokine. After synthesis, the hybrid may be modified enzymatically or chemically (as by the addition of fluorescent or radioactive label). The polypeptide portion of any hybrid could 25 be synthesized in a system incorporating one or more radioact~ve amino acid~, so that the resulting hybrid protein i8 intrinsically labelled.
Any fragment or analog of the cytokine of interest which is capable of binding to the high-30 affinity receptor of that cytokine could substitutefor the intact cytokine. An IL-2-containing hybrid could be used as a drug delivery device to deliver a lethal dosage of, for ~xample, a radioactive isotope or a toxin such as ricin or gelanin specifically to 35 high-affinity-receptor-bearing T-cells, which are implicated in certain cancers of the lymphatic system, and which play a crucial role in cases of transplant rejection. Alternatively, the hybrid could be used to image high-affinity receptors, either ln vivQ or in 5 vitro, py selecting an appropriate chemical entity to attach to the cytokine portion of the hybrid.
Examples would include the treatment of a frozen section of lymphoma tissue with an IL-2 hybrid containing a radioactive isotope such as 125Iodins, 10 coupled with autoradiographic analysis of the labeled tissue, and administration of an IL-2 hybrid containing an X-ray-opaque entity to a transplant patient, coupled with a CAT-scan of the transplanted organ to monitor transplant rejection. Attaching a 15 hybrid of the invention to an appropriate solid support material would create an efficient and reusable means of ~eparating high-affinity-receptor-bearing T-cells from cellc lacking such receptors, permitting the collection of either fraction of cells0 for therapeutic or other use.
What is claimed is:

Claims

1. A hybrid molecule comprising a hematopoietin receptor superfamily cytokine, or a fragment or analog of said cytokine capable of binding to said cytokine's high affinity receptor, covalently bonded, at its amino terminal end, to a chemical entity which causes said hybrid molecule to be processed via a lower affinity receptor of said cytokine to a lesser degree than is said cytokine, provided that said chemical entity does not comprise the translocation domain of diphtheria toxin.

2. The hybrid molecule of claim 1, wherein said cytokine is selected from the group consisting of IL-2, IL-3, IL-4, IL-6, EPO, and prolactin.

3. The hybrid molecule of claim 1, wherein said chemical entity sterically hinders the binding of the cytokine portion of said hybrid molecule to said lower-affinity receptor, compared to the binding of said cytokine to said lower-affinity receptor.

4. The hybrid molecule of claim 1, wherein said chemical entity comprises a polypeptide.

5. The hybrid molecule of claim 4, wherein said covalent bond is a peptide bond.

6. The hybrid molecule of claim 4, wherein said polypeptide comprises an antibody molecule.

7. The hybrid molecule of claim 4, wherein said polypeptide comprises ricin or a fragment or analog thereof.

8. The hybrid molecule of claim 7, wherein said polypeptide comprises an enzymatically-active fragment or analog of ricin.

9. The hybrid molecule of claim 4, wherein said polypeptide comprises streptavidin or a fragment or analog thereof.

10. The hybrid molecule of claim 4, wherein said polypeptide comprises ferritin.

11. The hybrid molecule of claim 1, wherein said chemical entity comprises an X-ray-opaque moiety.

12. The hybrid molecule of claim 1, wherein said chemical entity comprises a fluorescent moiety.

13. The hybrid molecule of claim 1, wherein said chemical entity comprises a radioactive moiety.

14. The hybrid molecule of claim 1, wherein said hybrid molecule is capable of affecting the rate of proliferation of cells bearing said high-affinity receptors.

15. A method for selectively binding a moiety to a cell bearing a high-affinity receptor for a cytokine, said method comprising exposing said cell to the hybrid molecule of claim 1, provided that said chemical entity comprises said moiety.

16. The method of claim 15, wherein said moiety comprises a label and said method is used for imaging said cell or said high affinity receptor.

17. The method of claim 15, wherein said moiety comprises a molecule complexed with iron and said method is used for separating cells having a relatively high number of said high-affinity receptors from other cells having a lower number of said high-affinity receptors.

18. The method of claim 15, wherein said hybrid molecule is capable of decreasing the rate of proliferation of cells to which it binds, and said method is used for therapeutic treatment of a condition comprising overproduction of cells having said high-affinity receptors.

19. A method for using the hybrid molecule of claim 1 for selectively isolating cells having said high-affinity receptors out of a population of cells some of which lack said high-affinity receptors, said method comprising, immobilizing said hybrid molecule, causing said population of cells to contact said immobilized hybrid molecule under conditions permitting binding of said high-affinity receptors to said immobilized hybrid molecule, and separating unbound cells of said population of cells from cells bound via said high-affinity receptors to said immobilized hybrid molecule.

20. The method of claim 19, wherein said method is used to remove, from bodily fluids or tissues, cells having said high-affinity receptors.

21. The method of claim 19, said method additionally comprising the step of eluting from said immobilized hybrid molecule any cell which was bound to said immobilized hybrid molecule.

22. A method of making the hybrid molecule of claim 4, said method comprising providing a recombinant DNA molecule comprising a DNA sequence encoding said polypeptide fused to a DNA sequence encoding said cytokine or said fragment or analog thereof, introducing said recombinant DNA molecule into an appropriate expression system, and expressing said recombinant DNA molecule.

23. A method of using the streptavidin-containing hybrid molecule of claim 9 for imaging said high-affinity receptors, or for imaging, in a population of cells, a cell bearing said high-affinity receptors, said method comprising exposing said high-affinity receptors to an amount of said hybrid molecule sufficient substantially to maximize the difference between (1) the amount of said hybrid molecule which binds to said high-affinity receptors, and (2) the amount which binds to all other receptors on said population of cells;
exposing receptor-bound hybrid molecules to a labelled probe, said labelled probe being covalently linked to biotin; and detecting said labelled probe which binds to said receptor-bound hybrid molecules.

24. The hybrid molecule of claim 4, wherein said polypeptide comprises an enzymatically-active fragment or analog of gelanin.