WO1993021308A1

WO1993021308A1 - Mutant cytokines having increased receptor affinity

Info

Publication number: WO1993021308A1
Application number: PCT/US1993/003613
Authority: WO
Inventors: Fadi Lakkis; John R. Murphy
Original assignee: The University Hospital
Priority date: 1992-04-17
Filing date: 1993-04-16
Publication date: 1993-10-28
Also published as: AU4289193A

Abstract

A variant of a naturally-occurring cytokine having a Phe-Leu or Tyr-Leu sequence in a helical domain and a negatively charged amino acid within two amino acids immediately upstream or downstream from said Phe-Leu or Tyr-Leu sequence, said variant having an increased affinity for the receptor of said naturally-occurring cytokine by virtue of a neutral or basic amino acid having been substituted for a said negatively charged amino acid.

Description

MUTANT CYTOKINES HAVING INCREASED RECEPTOR AFFINITY

Background of the Invention The invention relates to mutant cytokines having an altered receptor affinity.

PCT Application PCT/US86/02464 discloses native and mutant forms of interleukin-4 from a variety of mammalian sources.

Taniguchi et al. (U.S. Patent No. 4,738,927) discloses a gene encoding interleukin-2.

Murphy et al. (U.S. Patent No. 4,675,392) disclose cytotoxic fusion proteins that include a portion of interleukin-2.

Bazan (Immunology Today 11:350, 1990) describes structural models of cytokine receptor interactions. Cunningham et al., (Science 244:1081, 1989) describe alanine substitution mutants of human growth hormone, including a substitution at Glu¹⁷⁴ which increases affinity. Summary of the Invention

The invention features variants of naturally- occurring cytokines containing a Phe-Leu or Tyr-Leu sequence in an alpha helical domain and a negatively- charged amino acid (Glu or Asp) within two amino acids immediately upstream or downstream from the Phe-Leu or Tyr-Leu sequence; the variants have an increased affinity for the receptor of the naturally-occurring cytokine by virtue of a neutral (Gly, Ala, Val, lie, Leu, Phe, Pro, Met, Ser, Thr, Tyr, Trp, Asn, Gin, Lys) amino acid which is substituted for the negatively-charged amino acid which is within two amino acids immediately upstream or downstream of the Phe-Leu or Tyr-Leu sequence.

In general, the invention features a variant of a naturally-occurring cytokine having a Phe-Leu or Tyr-Leu sequence in a alpha helical domain and a negatively charged amino acid within two amino acids immediately upstream or downstream from the Phe-Leu or Tyr-Leu sequence, the variant having an increased affinity for 5 the receptor of the naturally-occurring cytokine by virtue of a neutral amino acid having been substituted for the negatively charged amino acid. Receptor affinity can be measured using standard receptor binding assays. In preferred embodiments, the negatively charged

10 amino acid is aspartic acid or glutamic acid; and the naturally occurring cytokine is an interleukin. In a more preferred embodiment, the interleukin is IL-4. In a even more preferred embodiment, the IL-4 is human IL-4.^" In a related aspect, the invention features a

15. hybrid molecule which includes a first and a second portion joined together covalently, the first portion includes a receptor-binding portion of the above- described variant cytokine and the second portion includes a molecule having enzymatic activity. In a

20 preferred embodiment, the enzymatic activity is capable of decreasing cell viability. By "reduces viability" is meant kills or inhibits proliferation.

In a preferred embodiment, the second portion includes a cytotoxin. In a more preferred embodiment,

25 the cytotoxin is a fragment of a peptide toxin which is enzymatically active but which does not possess generalized eukaryotic receptor binding activity. In an even more preferred embodiment, the fragment of a peptide toxin includes fragment A of diphtheria toxin and enough

30 of fragment B of diphtheria toxin to form a pore in a cell membrane.

The variants of the invention generally are based on cytokines which, like human IL-4, have the sequence Phe-Leu-or Tyr-Leu in an alpha helix; the alpha helix

35 generally is within the carboxy half of the cytokine and is usually the most carboxy-terminal alpha helix. Cytokines having the aforementioned characteristics are referred to as human IL-4 related cytokines. Examples of human IL-4 related cytokines include: human prolactin, human interleukin-2, human interleukin-6, and human interleukin-8. Generally, the alpha helices of human IL- 4 related cytokines encompassing the above-described Phe- Leu (or Tyr-Leu) sequence are amphiphilic. With regard to protein structure an alpha helix is a structure in which the backbone of the peptide chain forms a helix. In such a helix there are roughly 3.6 amino acids per turn of the helix. An amphiphilic alpha helix has a charged face and an uncharged face.

A cytokine has characteristics which permit its classification as a human IL-4 related cytokine if two criteria are meet. First, there must be an Phe-Leu (or Tyr-Leu) sequence in the carboxy terminal half of the cytokine. Second, the Phe-Leu or Tyr-Leu sequence must be within an alpha helix. While extensive structural analysis, e.g., x-ray crystallography or NMR analysis, may be required to make such a determination with complete certainty, for the purposes of the identifying human IL-4 related cytokines a determination that the Phe-Leu or Tyr-Leu sequence lies within a region which has the characteristics of an alpha helix forming sequence is sufficient. Such secondary structure predictions can be made using well-known techniques (Chothia et al., J. Biol . Chem . 145:215, 1981; Cohen et al. , J. Mol . Biol . 132:275, 1979; Cohen et al. , Proteins 7:1, 1990; Cornette et al., J. Mol . Biol . 195:659, 1987; Bazan et al., Proc . Natl . Acad . Sci . USA 85:7872, 1988; Richardson et al., Science 240:1648, 1988). Secondary structure prediction can also be made using a computer program such as PCGENE™ (IntelliGenetics, Mountain View, CA) . If such analysis indicates that the Phe-Leu (or Tyr-Leu) sequence lies within an alpha helix, then the cytokine is a human IL-4 related cytokine. In general, the alpha helix; containing the Phe-Leu or Tyr-Leu sequence is the most carboxy-terminal alpha helix. The finding that the alpha helix is amphipathic gives added assurance that the cytokine is a human IL-4 related cytokine, but is not required to fulfill the criteria for inclusion. Bazan (supra) describes structural features of cytokines. The increased affinity cytokine variants of the invention can be used in any application currently employing the naturally occurring cytokine. Of particular importance are hybrid toxin molecules in which the cytokine or a receptor binding portion thereof replaces the generalized eukaryotic binding domain of toxins such as diphtheria toxin to form a hybrid molecule capable of selectively targeting an unwanted class of cells, e.g., IL-2 receptor- or IL-4 receptor-bearing cells involved in human disease such as T-cell lymphoma. The increased affinity of the variants of the invention for the receptor will render the hybrid molecules more toxic than hybrids made using the corresponding naturally-occurring cytokine, and thus smaller dosages will be required to achieve the same therapeutic effect.

Other features and advantages of the invention will be apparent from the following detailed description, and from the claims.

Detailed Description. The drawings are first briefly described.

Fig. 1 is a graph illustrating the effect of various molecules on binding of [¹²⁵I]-mIL-4 bound to P815 cells. The percentage of the maximum amount [¹²⁵I]-mIL-4 bound is plotted as a function of the concentration (M) of mIL-4 (O) , DAB₃₈₉-mIL-4(D⁴⁹⁵ →N) (Δ) , DAB₃₈₉-mIL-4 (O) , DAB₃₈₉-mIL-4(F⁴⁹⁶ →A) (♦), or DAB₃₈₉-mIL-4(F⁴⁹⁶ →P) (1).

Fig. 2 is a graph illustrating the effect of DAB₃₈₉-mIL-4 and DAB₃₈₉-mIL-4 variants on incorporation of [¹⁴C]-leucine into P815 murine mastocytoma cell. Percent incorporation (relative to untreated cells) is plotted as a function of the concentration (M) of DAB₃₈₉-mIL-4 (O) , DAB₃₈₉-mIL-4(D⁴⁹⁵ →N) (•) , DAB₃₈₉-mIL-4(F⁴⁹⁶ →A) (♦), or DAB₃₈₉-mIL—4(F⁴⁹⁶ →P) (1). Cytokine Structure and Receptor Binding

A number of cytokines have similar structural elements. In particular, several that are known to bind to one or another member of the hematopoietin cytokine receptor superfamily are predicted to have a carboxy terminal alpha helix. In many cases the alpha helix is substantially amphiphilic in nature. Importantly, each has a highly conserved Phe-Leu (or Tyr-Leu) sequence within the amphipathic helix. Murine and human interleukin-4 are examples of cytokines having the above- described conserved structure.

Described below are a series of experiments employing DAB₃₈₉-mIL-4, a murine interleukin-4 diphtheria toxin-related fusion protein. These experiments demonstrate the importance of certain structural elements to receptor binding. DAB₃₈₉-mIL-4 is created by replacing the generalized cell binding domain (deletion of 97 amino acids Thr³⁸⁷ to His⁴⁸⁵ of diphtheria toxin with murine interleukin-4 (mIL-4) . Thus, the amino terminus of mIL-4 is linked by a peptide bond to a carboxy-terminal portion of diphtheria toxin. This molecule is selectively toxic to cells bearing the mIL-4 receptor. Using site directed and in-frame deletion mutagenesis to alter the mIL-4 portion of DAB₃₈₉-mIL-4, we have found that deletion of the carboxy-terminal 15 amino acids of mIL-4, substitution of Phe⁴⁹⁶ with either Pro, Ala, or Tyr, or substitution of Leu⁴⁹⁷ with either Ala or Glu decreases binding to the mIL-4 receptor and cytotoxicity. In contrast, the substitution of the negatively changed residue Asp⁴⁹⁵ with Asn results in a four-fold increase in cytotoxic potency and binding affinity to mIL-4 receptor bearing cells in vitro . DAB_3B9mIL-4 Variants

DAB₃₈₉mIL-4 is a fusion protein in which the receptor binding domain of native diphtheria toxin has been replaced by murine interleukin-4 (mIL-4) . DAB₃₈₉mIL- 4 selectively binds to eukaryotic cells expressing the murine IL-4 receptor (mIL-4R) , causing them to internalize the DAB-derived portion of the molecule which inhibits protein synthesis in the cells, causing cell death. DAB₃₈₉mIL-4 cytotoxicity is inhibited by excess mIL-4 or antibodies directed against mIL-4, demonstrating that entry of DAB₄₈₉mIL-4 into target cells is mediated by the mIL-4 receptor.

We have modified the mIL-4 portion of DAB₃₈₉mIL-4 to create variants with altered toxicity towards mIL-4R bearing cells. Since the change is in the mIL-4 portion of the molecule, it follows that altered toxicity is caused by altered affinity of the molecule for the mIL-4 receptor. Thus, these same mutations, when introduced into a related fusion protein, should alter its affinity for its receptor in an analogous fashion.

Described below are several DAB₃₈₉mIL-4 variants created by site directed mutagenesis. The ability of each variant to bind and intoxicate cells bearing the mIL-4 receptor was tested. The results permit prediction of the effect of mutation on IL-4 related cytokines including human IL-2, human IL-6, human IL-8.

Several in-frame deletions and point mutations were introduced into the mIL-4 segment of the structural gene encoding DAB₃₈₉mIL-4 carried on plasmid pFL389 (Lakkis et al., Eur . J . Immunol . 21:2253, 1991). Briefly, oligonucleotide site-directed mutagenesis was performed using a T7-Gen in vitro mutagenesis kit (United States Biochemical, Cleveland, OH) based on the previously published work (Vandeyar et al. , Gene 65:129, 1988) . The mIL-4 cDNA obtained from plasmid pFL389 was cloned into the Sphl-Hindlll sites of M13(mpl9) viral vector. The single stranded viral DNA served as the template in all mutagenesis reactions. Oligonucleotide primers were synthesized on an Applied Biosystems 391A DNA synthesizer. Correct base substitution was confirmed by DNA sequencing (Sanger et al., 1977). The mIL-4 cDNA fused to a truncated diphtheria toxin gene on the expression plasmid pFL389 was then substituted with the respective mutated mIL-4 cDNAs. In-frame deletion mutations were constructed by utilizing convenient restriction endonuclease sites in the fusion gene and by using oligonucleotide linkers. E. Coli JM101 (BRL/GIBCO, Bethesda, MD) was used throughout. Recombinant DAB₃₈₉mIL-4 was prepared from recombinant E. coli grown in 10 liters of M9 minimal medium (ImM MgS0₄, O.lmM CaCl_2/ 0.0005% thiamine, 0.5% glycerol) supplemented with 1% Casamino acids (Difco) and 100 μg/ l ampicillin in a New Brunswick Microferm. Cultures were incubated at 30°C and sprayed with air at 10 L/min. When the absorbance (A_590nm) of the culture reached 0.3 - 0.5, expression of the chimeric tox gene was induced by the addition of 1 gm isopropyl-0-D- thiogalactopyranoside (IPTG) (United States Biochemicals, Cleveland, OH) . Ninety min after induction, bacteria were harvested by centrifugation, resuspended in lysis buffer (50mM KH₂P0₄, lOmM EDTA, 750mM NaCl, 0.1% Tween 20, pH 8.0) and disrupted by sonication (Branson). The bacterial lysate was centrifuged at 2,500 x g for 20 min at 4°C to remove whole bacteria and debris, and the clarified lysate was applied to an anti-diphtheria toxin immunoaffinity column. Bound proteins were eluted from the immunoaffinity column with elution buffer (4M guanidine hydrochloride, lOOmM H₂P0₄, 0.1% Tween 20, pH 7.2). Recombinant DAB₃₈₉-mIL-4 fusion toxins were further purified by high pressure liquid chromatography on a 7.5 x 600mm G4000PW column (TosoHass) . Prior to use the fusion toxins were exhaustively dialyzed against phosphate buffered saline (PBS, pH 7.4) . Protein concentration was determined by the Bradford method (Pierce Chemical Co. , Rockford, IL) .

Table l summarizes the single amino acid substitutions introduced into DAB₃₈₉mIL-4. In addition, two deletion mutants were expressed and purified: DAB₃₈₉-mIL-4(Δ495-509) is a variant of DAB₃₈₉-mIL-4 that lacks the 15 carboxy-terminal amino acids, while DAB₃₈₉-mIL-4(Δ390-475) consists of the first 389 amino acids of diphtheria toxin fused directly to the fif h cysteine residue in mIL-4 by a His-Ala encoding oligonucleotide linker. Analysis of DAB₃₈₉-mIL-4 and mutant fusion toxins on coo assie blue stained 12% SDS- polyacrylamide gels indicated that the proteins were highly purified. Their electrophoretic mobilities corresponded to their respective molecular weights as deduced from DNA sequence analysis. However, DAB₃₈₉-mIL- 4(F⁴⁹⁶→P), in which Phe⁴⁹⁶ is replaced with a Pro residue, had slightly aberrant electrophoretic mobility.

TABLE 1 : DAB₃₈₉ mIL-4 Variants

PLASMID TOX GENE PRODUCT

pFL389 DAB₃₈₉-mIL-4

PFL389 ( 15 ) DAB₃₈₉-mIL-4 ( Δ495-509 ) pFL389 ( 86 ) DAB₃₈₉-mIL-4 ( Δ390-475 ) pFL389 (F→P) DAB₃₈₉-mIL-4 (F⁴⁹⁶→P) pFL389 ( F→A) DAB₃₈₉-mIL-4 ( F⁴⁹⁶→A) pFL389 (F→Y) DAB₃₈₉-mIL-4 (F⁴⁹⁶→Y) pFL389 (L→A) DAB₃₈₉-mIL-4 (L⁴⁹⁷→A) pFL389 (L→E) DAB₃₈₉-mIL-4 (L 497_ >E) pFL389 (D→N) DAB₃₈₉-mIL-4 (D⁴⁹⁵→N) pFL389 (K→A) DAB₃₈₉-mIL-4 (K ⁹⁴→A) pFL389 (K→L) DAB₃₈₉-mIL-4 ( ⁸⁹→L) pFL389 (C→G) DAB₃₈₉-mIL-4 (C 4*7'6⁰.→G)

In vitro cytotoxic activity of DAB_3Bg-mIL-4 and related variants on mIL-4 receptor bearing cells

The cytotoxic action of DAB₃₈₉-mIL-4 requires binding to the mIL-4 receptor, receptor-mediated endocytosis, passage through an acidic compartment, and delivery of the ADP-ribosyltransferase component of the fusion toxin into the cytosol (Lakkis et al., supra) . In order to investigate the effect of mutations in the mIL-4 component of DAB₃₈₉-mIL-4 on the function of this fusion protein, we have tested the in vitro cytotoxic potency of DAB₃₈₉-mIL-4 and related variant fusion toxins on the P815 murine mastocytoma cell line by means of a [¹ C]-leucine uptake assay. Briefly, P815 murine mastocytoma cells (American Type Culture Collection, Rockville, MD) , maintained in RPMI 1640 medium supplemented with 10% fetal bovine serum (Cellect, BRL/GIBCO, Bethesda, MD) , 2 mM glutamine, and penicillin and streptomycin to 50 IU and 50 μg/ml_r respectively. For cytotoxicity assays, cells were seeded in 96-well plates (Linbro-Flow laboratories, McLean, VA) at a concentration of 1 x 10⁴ per well. Fusion toxins were added in varying concentrations and the cultures were incubated for 40 hours at 37°C in a 5% C0₂ incubator. Following incubation, the cells were pulsed with [¹ C]-leucine and radioactivity incorporated into proteins was measured essentially as described earlier by Williams et al. (J. Biol . Chem . 265:11885, 1990). All assays were performed in quadruplicate. Dose response curves (Fig. 1) compare the percent incorporation of [¹⁴C]-leucine by the fusion toxin treated cultures relative to untreated controls. The results of the cytotoxicity assays are presented in Table 2, where IC₅₀ is the concentration of fusion toxin required for a 50% reduction in protein synthesis. Values are means of 3-5 independent experiments. Standard deviations were within 25% of the respective means.

TABLE 2: Cytotoxicity and receptor binding affinity of DAB₃₈₉-mIL-4 and variants.

Fusion Toxin IC₅₀ (M) K_L (M) =

DAB₃₈₉-mIL-4 1 x 10~⁹ 2.1 x 10^"7

DAB₃₈₉-mIL-4(Δ495-509) > 1 x 10^"7 > 1.0 X 10^"6

DAB₃₈₉-mIL-4(Δ390-475) > 1 X 10~⁷ > 1.0 X 10^"6

DAB₃₈₉-mIL-4(F⁴⁹⁶→P) > 1 X 10^"7 > 1.0 X 10^"6 DAB₃₈₉-mIL-4(F⁴⁹⁶→A) 5 X 10^"8 > 1.0 X 10^"6

DAB₃₈₉-mIL-4(F⁴⁹⁶→Y) 3 X 10^"8 > 1.0 X 10^"6

DAB₃₈₉-mIL-4 (L⁴⁹⁷→A) 3 X 10^"8 > 1.0 X 10^"6

DAB₃₈₉-mIL-4 (L⁴⁹⁷→E) > 1 x 10^"7 > 1.0 X 10^"6

DAB₃₈₉-mIL-4(D⁴⁹⁵→N) 4 X 10^"10 5.2 X 10^"8 DAB₃₈₉-mIL-4 (K⁴⁹⁴→A) 1 X 10^"9 4.0 x 10^"7

DAB₃₈₉-mIL-4 (K⁴⁸⁹→L) 2 X 10^"9 3.8 X 10^"7

DAB₃₈₉-mIL-4(C⁴⁷⁶→G) > 1 x 10^"7 > 1.0 X 10"⁶

Deletion of the carboxy-terminal 15 amino acids of DAB₃₈₉-mIL-4 results in a complete loss of cytotoxic activity in the mutant DAB₃₈₉-mIL-4 (Δ495-509) (Table l) . This deletion encompasses residues Asp⁴⁹⁵-Met⁵⁰⁴ and indicates that the C-terminal region of IL-4 is required for the cytotoxic activity of the fusion toxin. In order to examine whether this segment alone was capable of targeting a diphtheria toxin-based fusion protein into the cytosol of mIL-4 receptor expressing cells, we then constructed a deletion mutant of DAB₃₈₉-mIL-4 in which Thr³⁸⁹ was fused to Cys⁴⁷⁶ through an oligonucleotide linker. As shown in Table 1, DAB₃₈₉-mIL-4 (Δ390-475) is also devoid of cytotoxic activity. These observations suggest that other regions of the mIL-4 component of DAB₃₈₉-mIL-4, acting alone or in concert, are required for biological activity of the fusion toxin.

We next examined two DAB₃₈₉mIL-4 mutants in which Phe⁴⁹⁶ is replaced with either Pro or Ala. Fig. 1, in which percent incorporation of [¹⁴C]-leucine by fusion toxin treated cultures relative to untreated controls is plotted as a function of fusion toxin concentration (M) , shows that both mutations DAB₃₈₉-mIL-4(F⁴⁹⁶→P) (filled squares) and DAB₃₈₉mIL-4(F⁴⁹⁶→A) (filled diamonds) result in a marked reduction in cytotoxic potency to P815 cells. To further analyze the role of Phe⁴⁹⁶ in the cytotoxic process, the activity of DAB₃₈₉-mIL-4(F ⁹⁵→Y) , in which . Tyr was substituted for Phe⁴⁹⁶, was tested. Since tyrosine has a hydroxyl group on carbon-4 in the phenyl ring, position 496 in DAB₃₈₉-mIL-4(F ⁹⁶→Y) is occupied by a more polar residue which is otherwise structurally similar to phenylalanine. As shown in Table 2, this mutation, DAB₃₈₉-mIL-4(F ⁹⁶→Y) , causes a 13-fold decrease in cytotoxic potency.

Since a Phe-Leu pair is almost invariably found in the carboxy terminal helices of human IL-4-related cytokines, we have also examined the role of Leu⁴⁹⁷ in the cytotoxic activity of DAB₃₈₉-mIL-4. Substitution of Leu⁴⁹⁷ with either an Ala or Glu [DAB₃₈₉-mIL-4(L⁴⁹⁷→A) and DAB₃₈₉-mIL-4(L⁴⁹⁷→E) , respectively] results in a marked loss of cytotoxic potency (Table 2) .

In contrast to the negative effect of the above substitutions on the biological activity of DAB₃₈₉-mIL-4, we have found that mutations involving some of the hydrophilic residues in the region surrounding the conserved Phe-Leu pair result in either no change or an increase in cytotoxicity. For example, substitution of Lys⁴⁹⁴ with Ala does not alter the cytotoxic potency of the resulting fusion toxin (Table 2) . DAB₃₈₉-mIL- 4(D⁴⁹⁵→N⁴⁹⁵) , in which Asp⁴⁹⁵ is replaced with an uncharged Asn, is approximately four-fold more cytotoxic than the parental fusion toxin, DAB₃₈₉-mIL-4 (Table 2) . In addition, substitution of Asp⁴⁹⁵ with an Ala also results in four-fold increase in cytotoxic potency. It is of interest to note that Asp⁴⁹⁵ is adjacent to the Phe-Leu pair. Mutations which increase the relative hydrophobicity of this region of the helix have resulted in an increase in the biological activity of the fusion toxin.

Although predicted carboxy-terminal alpha helix on mIL-4 displays significant amphiphilic character, the hydrophobic face of the helix is interrupted by a charged residue, Lys⁴⁸⁹. We, therefore, tested whether a Lys⁴⁸⁹ to a Leu mutation would enhance the amphiphilicity of this segment and possibly improve the cytotoxic potency of the fusion toxin (Table 2) . Interestingly, DAB₃₈₉-mIL- 4(Lys⁴⁸⁹→Leu) demonstrates 2-3 fold less cytotoxicity than the parental DAB₃₈₉-mIL-4 form of the fusion toxin (Table 2) . Finally, substitution of Cys⁴⁷⁶ with a Gly residue led to total loss of cytotoxic activity. Apparent affinities of DAB_3S0-mIL and related variant fusion toxins to the m-IL-4 receptor

The biological activity of DAB₃₈₉-mIL-4 has been shown to be mediated through the mIL-4 receptor on target cells since cytotoxicity could be specifically blocked with either excess mIL-4 or antibody to mIL-4 (Lakkis et al., supra) . Since we have introduced mutations into the mIL-4 component of the fusion toxin, it is likely that the observed changes in the cytotoxicity of the DAB₃₈₉- mIL-4 variants are due to altered binding to the mIL-4 receptor. To test this hypothesis, we conducted a series of competitive displacement experiments using [¹²⁵I]- labeled recombinant mIL-4. (Recombinant murine IL-4, Sterling Drug Inc. , Malvern, PA was radioiodinated using the Enzymobead reagent, BioRad, Richmond, GA) .

Briefly, P815 cells were washed with tissue culture medium, resuspended in PBS (pH 7.4) containing 3 mg/ml BSA at 1 x 10⁶ cells reaction tube, and incubated with 175 pM [¹²⁵I]-mIL-4 in the presence or absence of increasing concentrations of unlabeled recombinant mIL-4 (Sterling Drug Inc. , Malver, PA) or DAB₃₈₉-mIL-4 fusion toxins for 25 minutes at 37°C under 5% C0₂. The reaction was overlaid on a mixture of 80% 550 fluid (Accumetric Inc. , Elizabethtown, N) and 20% mineral oil (Sigma, St. Louis, MO) and microcent ifuged for 2 minutes. The aqueous phase and pellet of each sample, representing free and bound ligand respectively, were then counted in a Beckman Gamma 5500 counter. Inhibition constants, K_if were calculated from the Cheng-Prusoff equation (Cheng et al., Biochem. Pharm . 22:3099, 1973).

As shown in Fig. 2 , where the percentage [¹²⁵I]- labeled recombinant mIL-4 bound is plotted as a function of fusion toxin concentration (M) , there is a direct correlation between the cytotoxic potency of a given fusion toxin and its affinity to the mIL-4 receptor. While neither DAB₃₈₉-mIL-4(F⁴⁹⁶→P) (filled squares) nor DAB ₃₈₉-mIL-4(F⁴⁹⁶→A) (filled diamonds) significantly displaced [¹²⁵I]-mIL-4 bound to P815 cells, DAB₃₈₉-mIL- 4(D⁴⁹⁵→N) (open diamonds) appears to have a four-fold higher affinity than parental DAB₃₈₉-mIL-4 (K = 5.2 x 10" ⁸M versus 2.1 x 10^~7 respectively). Displacement of mIL-4 (filled circles) was measured as a control. The K^ and IC₅₀ values for DAB₃₈₉-mIL-4 and each of the mutant fusion toxins are summarized in Table 2. The inhibition constants were calculated following the Cheng-Prusoff equation using 129 ± 5 pM (mean ± SD of 3 independent experiments) for the radioligand equilibrium dissociation constant. Values are means of 3-5 independent experiments. Standard deviations were within 25% of the respective means. Taken together, the data presented in Table 1 demonstrates that mutations in the C-terminus which alter cytotoxic potency also modify the affinity of the respective fusion toxin for the mIL-4 receptor. Structural characteristics of DAB_3Sg-mIL-4 and related variant fusion toxins

In an attempt to explore the mechanism by which mutations in the carboxy-ter inal segment of the mIL-4 component influence binding of the fusion toxin to the mIL-4 receptor, we have examined the electrophoretic mobilities of the parental and mutant fusion toxins under native, non-denaturing conditions. These studies demonstrated that these proteins are predominantly monomeric in non-denaturing buffer. These data imply that the loss in cytotoxic activity and binding affinity observed in DAB₃₈₉-mIL-4 variants is not due to increased aggregation of the respective fusion toxins. We then investigated the possibility that marked reduction in affinity could be due to altered tertiary structure of the mIL-4 component of the fusion toxin. Non-denatured fusion proteins were therefore transferred to nitrocellulose paper and im unoblotted (Towbin et al., Proc . Natl . Acad . Sci . USA, 76:4350, 1979) with a conformationally sensitive monoclonal antibody to mIL-4 (11B11, Genzyme, Boston, MA) . DAB₃₈₉-mIL-4 is immunoblot positive when probed with 11B11 only following electrophoresis under non-denaturing conditions (Lakkis et al., supra). This analyzing demonstrated that DAB₃₈₉- mIL-4, as well as those variants with similar or higher cytotoxic activity and binding affinity, are strongly immunoreactive with 11B11. Conversely DAB₃₈₉-mIL-4(Δ495- 509), DAB₃₈₉-mIL-4(Δ390-475) , DAB₃₈₉-mIL-4(C⁴⁷⁶→G) , DAB₃₈₉- mIL-4(F⁴⁹⁶→P) and DAB₃₈₉-mIL-4(L₄₉₇→E) which are non-toxic and fail to bind to the mIL-4 receptor are not recognized by the anti-mIL-4 monoclonal antibody. Conservative substitutions of Phe⁴⁹⁶ or Leu⁴⁹⁷, on the other hand, are immunoblot positive but with lower intensity than DAB₃₈₉- mIL-4. DAB₃₈₉-mIL-4(L₄₉₇→A) , for example, is 13-fold less cytotoxic than DAB₃₈₉-mIL-4, has a markedly reduced affinity for the mIL-4 receptor yet is significantly reactive with 11B11. Other Variant Human Growth Hormone-Related Cytokines

Human IL-4-related cytokines include human IL-6, human IL-8, and human IL-2. In each case the predicted amino acid sequence indicates the presence of an acidic (negatively charged) amino acid one or two amino acids upstream of a Phe-Leu (or Tyr-Leu) sequence within the carboxy-terminal portion of the molecule. Residues which could be changed to a neutral residue include position

126 of hIL-4 (U.S. Patent 5,017,691); 117 of hIL-6 (EPA 0 261 625) ; the E within the sequence RWEKFLKRA of hIL-8 (Matsushima et al., J^". Exp. Med . 167:1883, 1988); and 136 of hIL-2 (Taniguichi et al. , Nature 302:305, 1987). These negatively charged residues may be replaced by any neutral residue as described above for IL-4. The affinity of each variant may then be tested using standard binding assays. Hybrid Molecules

A variant cytokine of the invention can be linked to other molecules (e.g., a molecule with enzymatic activity) to create hybrid molecules. These hybrid molecules will bind to cells bearing the relevent cytokine receptor. This provides a convenient method for targeting molecules to cytokine receptor bearing cells. Because the variant cytokines of the invention bind their receptors with higher affinity than the related naturally occurring cytokines, the hybrid molecules will be particularly useful for directing molecules cells bearing the relevent cytokine receptor. Hybrid cytotoxins are a useful class of hybrid molecule which can be created using the variant cytokines of the invention. Such molecules can be used to reduce the viability of cells bearing particular cytokine receptors. In these molecules a cytotoxin is linked to the variant cytokine. Hybrid cytotoxins are described by Murphy et al. (U.S. Patent 4,675,392). Other hybrid cytotoxins are described by Murphy (PCT/US90/07619) . Methods for constructing such cytotoxins are well known (Murphy et al., U.S. Patent 4,675,392; Murphy,

(PCT/US90/07619) ; Lakkis et al., supra ; Williams et al., J. Biol . Chem. 265:20673, 1990; Williams et al., J^". Biol . Chem. 265:11885, 1990). Toxicity can be tested using standard assays. Other useful hybrid molecules are those in which a non-cytotoxic, enzymatically active molecule is linked to a variant cytokine of the invention. Such molecules, when taken up by the targeted cell, can be used to correct an enzyme deficiency or generate other molecules within the cell by enzymatic activity. It is also possible to link a detectable label to the variant cytokines of the invention for the purpose of detectably labeling receptor-bearing cells. Use The variant cytokines of the invention can be used for the same therapeutic purposes of the related naturally occurring molecule. In addition, truncated variant cytokines which bind receptor but do not elicit the normal biological response can be used to inhibit action of the naturally occurring cytokine. Because of their higher receptor affinities, such molecules employing the variant cytokines of the invention will generally be more potent than molecules employing the related naturally occurring cytokine. SEQUENCE LISTING

(1) GENERAL INFORMATION: (i) APPLICANT: The University Hospital (ii) TITLE OF INVENTION: MUTANT CYTOKINES HAVING INCREASED RECEPTOR AFFINITY

(ill) NUMBER OF SEQUENCES: (iv) CORRESPONDENCE ADDRESS:

(A) ADDRESSEE: Fish & Richardson

(B) STREET: 225 Franklin Street

(C) CITY: Boston

(D) STATE: Massachusetts

(E) COUNTRY: U.S.A.

(F) ZIP: 02110-2804

(v) COMPUTER READABLE FORM:

(A) MEDIUM TYPE: 3.5" Diskette, 1.44 Mb

(B) COMPUTER: IBM PS/2 Model 50Z or 55SX

(C) OPERATING SYSTEM: MS-DOS (Version 5.0)

(D) SOFTWARE: WordPerfect (Version 5.1)

(vi) CURRENT APPLICATION DATA:

(A) APPLICATION NUMBER:

(B) FILING DATE:

(C) CLASSIFICATION:

(vii) PRIOR APPLICATION DATA:

(A) APPLICATION NUMBER: 07/870,500

(B) FIIrING DATE: April 17, 1992

(viii) ATTORNEY/AGENT INFORMATION:

(A) NAME: Paul T. Clark

(B) REGISTRATION NUMBER: 30,162

(C) REFERENCE/DOCKET NUMBER: 00563/053001

(ix) TELECOMMUNICATION INFORMATION:

(A) TELEPHONE: (617) 542-5070

(B) TELEFAX: (617) 542-8906

(C) TELEX: 200154 (2) INFORMATION FOR SEQUENCE IDENTIFICATION NUMBER:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 576

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: Is

TTAGCATCTG TTGATAAACT TAATTGTCTC TCGTCACTGA CGCACAGAGC TATTG 55

AAC CCC CAG CTA GTT GTC ATC CTG CTC TTC TTT CTC GAA TGT ACC AGG 103 Asn Pro Gin Leu Val Val lie Leu Leu Phe Phe Leu Glu Cys Thr Arg 1 5 10 15

AGC CAT ATC CAC GGA TGC GAC AAA AAT CAC TTG AGA GAG ATC ATC GGC 151 Ser His lie His Gly Cys Asp Lys Asn His Leu Arg Glu lie lie Gly 20 25 30

ATT TTG AAC GAG GTG ACA GGA GAA GGG ACG CCA TGC ACG GAG ATG GAT 199 He Leu Asn Glu Val Thr Gly Glu Gly Thr Pro Cys Thr Glu Met Asp 35 40 45

GTG CCA AAC GTC CTC ACA GCA ACG AAG AAC ACC ACA GAG AGT GAG CTC 247 Val Pro Asn Val Leu Thr Ala Thr Lys Asn Thr Thr Glu Ser Glu Leu 50 55 60

GTG TGT AGG GCT TCC AAG GTG CTT CGT ATA TTT TAT TTA AAA CAT GGG 295 Val Cys Arg Ala Ser Lys Val Leu Arg He Phe Tyr Leu Lys His Gly 65 70 75 80

AAA ACT CCA TGC TTG AAG AAG AAC TCT AGT GTT CTC ATG GAG CTG CAG 343 Lys Thr Pro Cys Leu Lys Lys Asn Ser Ser Val Leu Met Glu Leu Gin

85 90 95

AGA CTC TTT CGG GCT TTT CGA TGC CTG GAT TCA TCG ATA AGC TGC ACC 391 Arg Leu Phe Arg Ala Phe Arg Cys Leu Asp Ser Ser He Ser Cys Thr 100 105 110 ATG AAT GAG TCC AAG TCC ACA TCA CTG AAA GAC TTC CTG GAA AGC CTA 439 Met Asn Glu Ser Lys Ser Thr Ser Leu Lys Asp Phe Leu Glu Ser Leu 115 120 125

AAG AGC ATC ATG CAA ATG GAT TAC TCG 466

Lys Ser He Met Gin Met Asp Tyr Ser 130 135

TAGTACTGAG CCACCATGCT TTAACTTATG AATTTTTAAT GGTTTTATTT TTAATATTTA 526

TATATTTATA ATTGATAAAA TAAAATATTT GTATAATGTA ACAGAAAAAA 576

Claims

1. A variant o'f a naturally-occurring cytokine having a Phe-Leu or Tyr-Leu sequence in a alpha helical domain and a negatively charged amino acid within two amino acids immediately upstream or downstream from said Phe-Leu or Tyr-Leu sequence, said variant having an increased affinity for the receptor of said naturally- occurring cytokine by virtue of a neutral amino acid having been substituted for a said negatively charged amino acid.

2. The variant cytokine of claim 1 wherein a said negatively charged amino acid is aspartic acid or glutamic acid.

3. A hybrid molecule comprising a first and a second portion joined together covalently, said first portion comprising a receptor-binding portion of the variant cytokine of claim 1 and said second portion comprising a molecule having enzymatic activity.

4. The hybrid molecule of claim 3 wherein said molecule decreases cell viability.

5. The hybrid molecule of claim 4 wherein said second portion comprises a cytotoxin.

6. The hybrid molecule of claim 5 wherein said cytotoxin is a fragment of a peptide toxin which is enzymatically active but which does not possess generalized eukaryotic receptor binding activity.

7. The hybrid molecule of claim 6 wherein said fragment of a peptide toxin comprises fragment A of diphtheria toxin and enough of fragment B of diphtheria toxin to form a pore in a cell membrane.

8. The variant of claim 1 wherein said naturally occurring cytokine is an interleukin.

9. The variant of claim 8 wherein said interleukin is IL-4.

10. The variant of claim 9 wherein said IL-4 is human IL-4.