WO1987007617A1

WO1987007617A1 - A novel protein, neuroleukin

Info

Publication number: WO1987007617A1
Application number: PCT/US1987/001323
Authority: WO
Inventors: Mark E. Gurney; John L. Knopf
Original assignee: Genetics Institute, Inc.
Priority date: 1986-06-05
Filing date: 1987-06-04
Publication date: 1987-12-17
Also published as: KR880701249A; AU7518987A; EP0308424A4; EP0308424A1; JPH01502747A

Abstract

A novel protein, neuroleukin, which is capable of extending the survival of in vitro cultured spinal neurons and sensory neurons. The protein is further characterized by the ability to activate immunoglobulin secretion by peripheral blood lymphocytes.

Description

A NOVEL PROTEIN, NEUROLEUKIN

The present invention relates to a novel protein which we call neuroleukin. Among biological properties of this material is its ability to extend the survival of sensory ganglia, brain cells and spinal neurons in culture. This protein also may be useful in the diagnosis and treatment of certain diseases, especially involving the immune system. Unlike most polypeptide growth factors, neuroleukin consists of a single polypeptide chain. It has an apparent molecular weight of approximately 56,000 + 2000 daltons as determined by sodium dodecylsulfate polyaσrylamide gel electrophoresis (SDS-PAGE) . Its isoelectriσ point is about pH 8.5 + 0.5 as determined by isoelectric focusing. Neuro¬ leukin has an amino acid sequence as shown in Tables I and II below. It displays a bioactivity in a neurotrophic assay for maintaining one-half maximal survival of cultured spinal neurons or sensory neurons. Neuroleukin also displays ability to stimulate immunoglobin secretion by peripheral blood mononuclear cells.

We originally derived neuroleukin from salivary gland and muscle; however, its tissue distribution is widespread. We have also detected it in, inter alia, skeletal tissue, brain, heart, kidney, liver and serum, bone marrow, and human cell lines of lymphoma-leukemia tumor origin. Neuroleukin has demonstrated the ability to maintain the survival of subpopulations of spinal neurons and dorsal root ganglion sensory neurons in vitro. This factor is also apparently involved in spinal motor neuron growth and regeneration ia vivo. We have previously reported its presence in other tissue, absent however a method for its isolation. [M.E. Gurney, Nature. 307; 546-548 (1984) and M. E. Gurney et al. New Enσl. J. Med.. 311; 933-939 (1984) ] . The molecular cloning and expression of neuroleukin, its activity as a neurotrophic factor for spinal and sensory neurons and its ability to stimulate immunoglobin secretion by cultured peripheral blood mononuclear cells have also been reported. [M. E. Gurney et al Science 234; 566-574 (1986) and M. E. Gurney et al Science 234; 574-581 (1985)]

Another aspect of the present invention is a process for producing neuroleukin. The method involves culturing cells transformed with a vector containing one of several nucleotide sequences identified as encoding a neuroleukin polypeptide. The sequence is inserted in the vector under the control of an appropriate regulatory (e.g. promoter) sequence. The regulatory sequence may be selected from a variety of well-known, published sequences. Such selection is well within the abilities of one skilled in the art of protein expression. The particular sequences selected would naturally depend upon the host cells selected for the ultimate expression system e.g., mammalian cells, bacterial cells, yeast cells and viruses. Preparation of many such expression systems has been amply described in the scientific literature and is well within the skill of the art.

The neuroleukin coding sequence can encompass the two specific DNA sequences identified herein, as well as sequences which are capable of hybridizing to the identified sequences and which, on expression, code for polypeptides which demon¬ strate neuroleukin bioactivity. Also included herein are DNA sequences which differ from those of Tables I and II below due to the degeneracy of the genetic code (i.e. more than one codon can code for the same amino acid) . The resulting neuroleukin protein can be characterized by the amino acid sequences specifically identified below, and analogs thereof.

Slight variations in the sequences of Tables I and II which are caused by point mutations, naturally occurring allelic genes and induced modifications should not change the functional protein for which the sequence code on expression. Such variations may be expected to enhance the activity or production of neuroleukin. Such modifictions to the sequences, including those due to the degeneracies of the genetic code, are encompassed in the invention. Nucleotide modifications can be deliberately engineered into the DNA sequences employed in this method, whichmodifications can be made by one skilled in the art using known techniques. Such modifications can include the deletion, insertion or substitution of amino acids. Mutageniσ techniques for such replacement or deletion are well known to one skilled in the art.

The biologically active neuroleukin produced by the expression of the neuroleukin sequence in accordance with the present invention can be used as a culture additive in a manner similar to other known nerve growth factors which are commonly employed to extend the survival of various neural cells in vitro. [See, e.g., P.I. Baccaglini et al., Proc. Natl. Acad. Sci. USA. 0: 594-598 (1983)]. Unlike other presently available nerve growth factors, however, neuroleukin is capable of prolonging the viability of sensory ganglia, spinal neurons, and brain cells in vitro. Thus, neuroleukin fills a need in the field of tissue culture additives for extending the life of these neuronal cells. As with other presently available nerve growth factors, the amount of neuroleukin added to a culture medium can be easily experimentally determined, and will depend on the amounts and kinds of other medium supple¬ ments, the number and types of tissue cells to be cultured and the size of the culture. Such determinations are simple and within the abilities of one skilled in the art.

The protein of the present invention may be further characterized by its ability to stimulate immunoglobulin secretion by peripheral blood lymphocytes in a lymphokine assay. It may therefore also be useful as an assay reagent in studies of B-cell function because it appears to mimic the effect of pokeweed mitogen to elicit B-cell differenti¬ ation. Thus, neuroleukin may supplement media for, e.g., the culturing of bone marrow cells. Again the amount of neuroleukin added to the media will depend on such factors enumerated above and will involve simple experimentation well within the skill of the art.

As yet a further characteristic of this novel protein growth factor, we have discovered that a portion of the DNA sequence of neuroleukin has significant sequence homology to a portion of the HTLV III/LAV envelope (env) protein gene. The retrovirus HTLV-III/LAV is the causative agent of acquired immune deficiency syndrome (AIDS) . Retroviruses of the HTLV/LAV-type are now known as the human immuno¬ deficiency virus (HIV) . A computerized search by the National Biomedical Service, Washington, D.C., illustrated that four published HTLV III env protein sequences scored the most homologous to our novel neuroleukin sequence. Specifically the region of the neuroleukin sequence in Table II extending from codon 403 to codon 447 is significantly homologous to the HTLV III/LAV/ARC sequence from nucleotide 6514 to 6648 as set out in M. Muesing et al.. Nature. 313;450-458 (1985). The homologous portion of the HTLV III sequence is in the GP120 protein portion of the env protein. We have preliminarily determined that viral preparations including GP120 inhibit the bioactivity of neuroleukin.

Such homology indicates that neuroleukin or a portion of the neuroleukin sequence may have utility as an in vivo therapeutic treatment for, or as a vaccine against, AIDS. More generally, it may have utility as a treatment for patients infected with HIV. A pharmaceutical formulation of the present invention for use as a therapeutic treatment or vaccine, will generally comprise active neuroleukin as above described, together with one or more pharmaceutically acceptablecarriers therefore andoptionallyothertherapeutic ingredients. The amount of active ingredient will, of course, depend upon the severity of the condition being treated, the route of administration chosen, and the specific activity of the active neuroleukin. The active neuroleukin may be systematically administered by any route appropriate, such as parenterally, i.e. by direct injection into the bloodstream. Dosage of the neuroleukin would be determined by a physician and vary according to the stage of the disease, age, physical condition, time and mode of treatment.

Neuroleukin was originally purified from mouse salivary gland as detailed by the following Examples 1 through 3. However, recombinant methods employing selected expression vector systems into which the cDNA sequence of Table I or II below may be conveniently inserted and expressed can provide a more efficient route of neuroleukin production. Any number of known expression systems can be conveniently employed to express the neuroleukin protein coding sequence of the present invention. An exemplary mammalian expression system is described in Example 4 below. Techniques for constructing such expression systems are well-known and plentifully published, e.g., providing restriction endo- nuclease enzyme linkers onto the termini of the coding sequence and inserting the sequence into a vector under operative association with an expression control sequence (i.e., promoter/regulator or appropriate host cell signal sequences) . Such manipulations, linkers and expression sequences are known to those skilled in the art.

The following examples are illustrative only and therefore are not considered to limit the scope of the invention. EXAMPLE 1 Purification of Neuroleukin from Murine Salivary Gland

Approximately 800 ug of neuroleukin was purified from 200 salivary glands obtained from male BALB/c mice (retired breeders) . The glands were homogenized in a buffer containing NaH₂P0 /EDTA/EGTA/leupeptin/PMSF and the homogenate was clarified by centrifugation. A 100,000 x g supernatant was precipitated with polyethylene glycol (PEG) , passed over a dye-ligand matrix affinity column (Red agarose, Amicon) , and the unbound material was re-precipitated with PEG. Chromatography over Procion Red HE3B-agarose resulted in recovery of the protein in the column flowthrough. This step required dye loading of the agarose according to the procedures of Lowe and Pearson, "Affinity Chromatography on Immobilized Dyes" in Methods in Enzymolocry. 104; 97-113 (1984).

A second PEG precipitation was followed by gel fil¬ tration over AcA54 (LKB, Inc.). The factor eluted from AcA54 with an apparent molecular weight of 55-60 kd. Following gel filtration, the preparation was chromatographed over hydroxylapatite (Biogel HT, BioRad) eluted with a gradient of sodium phosphate, and finally chromatographed over a quarternary ammonium anion exchange HPLC Column (Q300, Synchrom, Inc.) from which it eluted isocratically in 5mM sodium phosphate (pH 7.5). No binding to a cation exchange HPLC column (CM103, Synchrom, Inc.) was observed at this pH and ionic strength. Neuroleukin behaved as an homogeneous species in each chromatographic fractionation.

Purified neuroleukin focuses as a sharp band on isoelectric focusing at about pH 8.5. Thus, it is a mono- meric, weakly basic protein. The protein was purified further by chromatography over a C-18 Vydac reverse phase HPLC column (Separations Group) using a 0-95% acetonitrile gradient in 0.1% trifluroacetic acid (TFA) over 100 min. Neuroleukin eluted in the gradient at approximately 60% acetonitrile.

The sequence of neuroleukin was determined for both the mouse and human proteins as follows.

EXAMPLE 2 Sequence Analysis of Murine Neuroleukin

To obtain peptide fragments from the 56kd factor for sequence analysis, 200ug of the purified neuroleukin was reduced with dithiothreitol, alkylated with iodoacetamide, and then digested to completion with TPCK-treated trypsin (Worthington) (2% w/w/enzyme/substrate) for 18 hr at 37°C. The tryptic digest then was subjected to reverse-phase HPLC using the conditions described above, and the absorbance at both 280 nm and 214 nm was monitored on-line. The wel.1 separated peaks indicated on the chromatogram of the tryptic peptides were evaporated to near dryness and subjected directly to N-terminal sequence analysis.

Sequences were determined for eleven different peptides obtained from the mouse neuroleukin. A particularly long sequence, designated T-36 was chosen for the synthesis of oligonucleotide probes. A 33 er was prepared from the T-36 sequence (5'd CTCCATGTCACCCTGCTGGAAGTAGGCAGCAAA) using an Applied Biosystem Model 380A DNA synthesizer.

A cDNA library was prepared in lambda gtlO using oligo(dT) primed double-stranded cDNA synthesized from male BALB/cJ salivary gland poly A+ mRNA according to U. Gubler et al.. Gene. 25; 163-269 (1983), and linker ligation into the EcoRl site of the vector described in J. J. Toole et al.. Nature. 312; 342-347 (1984). The salivary gland cDNA library was screened using a modification of the in situ amplification protocol described originally by S.L.C. Woo et al., Proc. Natl. Acad. Sci. U.S.A.. 7_5_; 3688-3691 (1978) using the T-36 oligonucleotide labeled at its 5' end using polynucleotide kinase (New England Biolabs) and -P-32 ATP (NEN) .

Approximately 100,000 phage were screened and eleven independent phage were found to hybridize to the probe, five of which were found to contain inserts of roughly equivalent size. DNA from each of the five phage was digested with EcoRl and subcloned into M13 for DNA sequence analysis using the dideoxy chain termination method [See, e.g. Sanger et al, Proc. Natl. Acad. Sci. U.S.A., 74; 5463-5467 (1977)]. The sequence of two clones (designated C-2 and C-19) was determined completely and revealed a single open reading frame which precisely codes for all of the tryptic peptides sequenced from the mouse neuroleukin.

The sequence contains 2,063 nucleotides which terminate in a 3' poly A+ tract. The length of the cDNA agrees well with the length estimated for the message represented by these clones. Primer extension with the oligonucleotide 33 er from the 5' end of the message failed to reveal significant extension of the sequence in the 5' direction. Although the long open reading frame encoding the protein extends to the 5' end of the sequence, the first ATG in this reading frame is at nucleotide 50 and is embedded in a canonical sequence for eukaryotic translation initation sites (CCA/GCCAUG(G) ) [See M. Kozak, Nucl. Acids Res.. 12.:857-872 (1984)]. We presently believe that translation initiates at this codon. From the ATG at nucleotide 50, the open reading frame continues until terminated by a stop codon beginning at nucleotide 1,750.

The first sequence in the cDNA established by peptide data is at codon 13 and the other peptide sequences occur throughout the sequence until the last is reached at codons 498-503. Thus, the established protein sequence begins very near the amino terminal of the deduced amino acid sequence and extends to within 46 amino acids of the carboxy terminal of the deduced sequence. Three potential N-linked glycosylation sites (Arg-X-Thr or Arg-X-Ser) are predicted by the deduced amino acid sequence, however, no biochemical evidence indicating that glycosylation of the factor occurs has been obtained. The sequence encodes a protein of 558 amino acids. The complete DNA and amino acid sequence of the murine neuroleukin is shown in Table I below.

Table I 10 20 30 40 50

CAATΓOCGCΓ TCCGAGCACG TCXΠGCTOCG TC-TACCTCTC GGCTCOCTCG OC ATG GCT GCG

MET Ala Ala

67 82 97 112

CTC AOC OGG AAC CCG CAG TIC CAG AAG CTC CTGGAGTGGCACOGCGCGAACTCT Leu Thr Arg Asn Pro Gin Phe Gin Lys Leu Leu Glu Trp His Arg Ala Asn Ser

127 142 157

GGC AAC CTC AAG CTGCGCGAACTrTITGAGGCGGAT CCG GAG CGC TTC AAC AAC Ala Asn Leu Lys Leu Arg Glu Leu Phe Glu Ala Asp Pro Glu Arg Phe Asn Asn

172 187 202 217

TTC AGC TIG AAC CTC AAC ACC AAC CAT GGG CAT ATT CTG GG GAC TAG TOC AAG

Phe Ser Leu Asn Leu Asn Thr Asn His Gly His ie leu Val Asp Tyr Ser Lys

232 247 262 277

AAC CTΓ GTG AAC AAG GAG GTCATG CBGATG CTG GTCGAGCEGGCCAAGTCC AGA Asn Leu Val Asn Lys Glu Val MET Gin MET Leu Val Glu Leu Ala Lys Ser Arg

292 307 322

GGC GTG GAG GCT GCA GGG GAC AAC ATG TTC ACT GCT TOC AAG ATC AAC TAC ACC Gly Val Glu Ala Ala Arg Asp Asn MET Phe Ser Gly Ser Lys lie Asn Tyr Thr

337 352 367 382

GAG GAT CX-GGCGGTG CTG C^ GTG GCC CTT CGGAAC CGGT∞ ACA CCC ATC

Glu Asp Arg Ala Val Leu His Val Ala Leu Arg Asn Arg Ser Asn Thr Pro lie

397 412 427

AAG GTG GAC GGC AAA GAT CTG ATG CCG GAG CTG AAC AGG GIT CTG GAC AAG ATG Lys Val Asp Gly Lys Asp Val MET Pro Glu Val Asn Arg Val Leu Asp Lys MET

442 457 472 487

AAG TCT TTC TGC CAG OGG CTC CGG ACT GCT GAC TGG AAA GGG TAC ACT GGC AAA

Lys Ser Phe Cys Gin Arg Val Arg Ser Gly Asp Trp Lys Gly Tyr Thr Gly Lys

502 517 532 547

TCC ATC ACG GAC ATC ATC AAC ATC GGC ATC GGG GGC TCT GAC CTG GGA CCC CTC Ser lie Thr Asp lie lie Asn lie Gly lie Gly Gly Ser Asp Leu Gly Pro Leu

562 577 592

ATG GTGACTGAAGCTCTCAAGCCTTACTOGAAAGGAGCT CX^ MET Val Thr Glu Ala Leu Lys Pro Tyr Ser Lys Gly Gly Pro Arg Val Trp Phe

607 622 637 652

CTC TCT AAC ATT GAT GGG ACC CAC ATT GCC AAA ACA CTG GCC AGC TIG TCC CCT Val Ser Asn lie Asp Gly Thr His lie Ala Lys Thr Leu Ala Ser Leu Ser Pro

667 682 697

GAGACTTCC OETITATAATC GCCT∞AAGA∞TTCA∞ Glu Thr Ser Leu Phe lie lie Ala Ser Lys Thr Phe Thr Thr Gin Glu Thr lie 712 727 742 757

ACCAAT GCAGAGACAGCAAAG GAGTGGTIT CTCGAAGOG

Thr Asn Ala Glu Thr Ala Lys Glu Trp Phe Leu Glu Ala Ala Lys Asp Pro Ser

772 787 802 817

GCAGTTGCAAAGCACTITGTCGCC CrcTCTACGAACACGGCCAAAGTC Ala Val Ala Lys His Phe Val Ala Leu Ser Thr Asn Thr Ala Lys Val Lys Glu

832 847 862

TIT GGA ATT GAC CCT CAA AAC ATG TICGAGTICT∞GATTGGCTAGGTGGC CGC Phe Gly lie Asp Pro Gin Asn MET Phe Glu Phe Trp Asp Trp Val Gly Gly Arg

877 892 907 922

TAT TOG CTG TGG TCA GCC ATT GGA CIT TOC ATT GCT CTG CAT GTA GCT TIT GAC Tyr Ser Leu Trp Ser Ala lie Gly Leu Ser lie Ala Leu His Val Gly Phe Asp

937 952 967

CΑCTICGAGCS^CIGCTGTCCGGGGCTCACTGG ATG GAC CAG CAC TTC CTC AAG His Phe Glu Gin Leu Leu Ser Gly Ala His Trp MET Asp Gin His Phe Leu Lys

982 997 1012 1027

ACG CCC CTG GAG AAG AAT GOC ∞CGTC CTGCTGGCTCTACTGGGC ATC TGG TAC

Thr Pro Leu Glu Lys Asn Ala Pro Val Leu Leu Ala Leu Leu Gly lie Trp Tyr

1042 1057 1072 1087

ATC AAC TGC TAC GGC TCT GAG ACC CAC GCC ITS CTG CCC TAT GAC CAG TAC ATG lie Asn Cys Tyr Gly Cys Glu Thr His Ala Leu Leu Pro Tyr Asp Gin Tyr MET

1102 1117 1132

CΑC CGCTIT GCTGCCTAT Trc <^ CMGCTGACATGG

His Arg Phe Ala Ala Tyr Phe Gin Gin Gly Asp MET Glu Ser Asn Gly Lys Tyr

1147 1162 1177 1192

ATCACCAAGTCCGGGGCC OCTGTGGAC CACCAG ACA GGC CCC ATC CTG TGG GGG lie Thr Lys Ser Gly Ala Arg Val Asp His Gin Thr Gly Pro lie Val Trp Gly

1207 1222 1237

GAA CCA GGG ACC AAT GCT CAA CAT GGA TTC TAC CAG CTC ATC CAC CAA GGC ACC Glu Pro Gly Thr Asn Gly Gin His Ala Phe Tyr Gin Leu lie His Gin Gly Thr

1252 1267 1282 1297

AAGATGATA<XX: TCTGACTrT CTCATC CCTCTC CMACr C3^ C^ CCC ATA CGG Lys MET lie Pro Cys Asp Phe Leu lie Pro Val Gin Thr Gin His Pro lie Arg

1312 1327 1342 1357

AAA GCT CIG C^ C-ACAAGATC CTC<^GCTAACTTCTTGGCC<^ACT Lys Gly Leu His His Lys lie Leu Leu Ala Asn Phe Leu Ala Gin Thr Glu Ala

1372 1387 1402

CTGATGAAGGGGAAGTIG

Leu MET Lys Gly Lys Leu Pro Glu Glu Ala Arg Lys Glu Leu Gin Ala Ala Gly

1417 1432 1447 1462

AAG AGC CCA GAA GAC TIG GAG AAA CTC TIG CCA CAC AAG CTC TIT GAA GGA AAC Lys Ser Pro Glu Asp Leu Glu Lys Leu Leu Pro His Lys Val Phe Glu Gly Asn

1477 1492 1507

GGC CCG ACC AAC TCT ATT GTG TIT ACC AAG CTG ACA CCC TTC ATT CTG GGG GCC Arg Pro Thr Asn Ser lie Val Phe Thr Lys Leu Thr Pro Phe lie Leu Gly Ala

1522 1537 1552 1567

TIG ATT GCC ATG TAT GAG CAC AAG ATC TTT CTT CAG GGC ATC ATG TGG GAC ATC

Leu lie Ala MET Tyr Glu His Lys lie Phe Val Gin Gly lie MET Trp Asp lie

1582 1597 1612 1627

AACAGCTICGAC CAGTGGGGAGTGGAG CIGGGGAAG CAG CTGGCC Asn Ser Phe Asp Gin Trp Gly Val Glu Leu Gly Lys Gin Leu Ala Lys Lys lie

1642 1657 1672

GAG CCG GAGCTCGAGGGCAGCTCT GCT GTGACCT∞ CAT G

Glu Pro Glu Leu Glu Gly Ser Ser Ala Val Thr Ser His Asp Ser Ser Thr Asn

1687 1702 1717 1736

GGA CTGATCAGC TICATCAAG CAA CAG CGGGACAOC AAA CTA GAA TAACTCCAGC Gly Leu lie Ser Phe lie Lys Gin Gin Arg Asp Thr Lys Leu Glu

1746 1756 1766 1776 1786 1796 1806

C-GCGGCCCΓA CK_ΑCTGGTC CTCCGTGTCC CTICTCACCA TATGC.ACTGC ATGCTCCTGC CCCTCCCTGC

1816 1826 1836 1846 1856 1866 1876

CCAGAGCGCA CCACCGGTAG TTCGCCIGGA CTACAAGGCT CTIGGGAGAA GCTGCTCTGG AACTGCCATC

1886 1896 1906 1916 1926 1936 1946

CACCCACTAC GCACCCTCCC TTTGAAGCT GATGGAAGGG CTTTGAOSTG TCaTGTTCTT CTGACCICTA

1956 1966 1976 1986 1996 2006

TITCACACCC ⁽ΩGCTAGAAT AAAGACACCT AGAGGAGGCA AAAAAAAAAA AAAAAAAAAA AAA

EXAMPLE 3 Production of Human Neuroleukin

To obtain human neuroleukin the 33-mer prepared from the T-36 murine sequence (see Example 2 above) was employed as an oligonucleotide probe.

A human cDNA library was prepared in lambda gtlO using oligo (dT) primed double-stranded cDNA of 2 kb and greater synthesized from human muscle poly A+ mRNA from patients suffering fromamyotrophic lateral schlerosis (ALS) , according to the method of U. Gubler et al., (1983) supra. Linkers were ligated into the EcoRl site of the vector described in J. J. Toole et al., (1984) supra. The human muscle cDNA library was screened using a modification of the in situ amplification protocol described originally by S.L.C. Woo et al., (1978) supr . using the T-36 oligonucleotide labeled at the 5' end using polynucleotide kinase (New England Biolabs) and -P-32 ATP (NEN) .

Approximately 10⁵ phage were screened and independent phage were found to hybridize, 4 of which were found to contain inserts of roughly equivalent size. DNA from each of the 4 phage was digested with EcoRl and subcloned into M13 for DNA sequence analysis using the dideoxy chain termination method of Sanger et al., (1977) supra. The sequence of one clone (designated H510) was determined completely and revealed a single open reading frame.

The complete DNA and amino acid sequence for human neuroleukin is shown in Table II below. The mouse and human nucleotide sequences of Tables I and II respectively are homologous up to the first AUG at the mouse nucleotide 50. After that point, the sequences diverge, as expected for the 5¹ untranslated regions. To date, no bioactivity has been detected for the human sequence. Table II 10 30 45

CTCGAGAGCT CCGCC ATG GCC GCTCTCACC CGGGAC CCC CftGTIC CAG AAG CG CAG MET Ala Ala Leu Thr Arg Asp Pro Gin Phe Gin Lys Leu Gin

60 75 90 105

CAA TGG TAC CGC GAG CAC CGC TCC GAG CTG AAC CTG CGC CGC CTC TTC GAT GCC Gin Trp Tyr Arg Glu His Arg Ser Glu Leu Asn Leu Arg Arg Leu Phe Asp Ala

120 135 150 165

AAC AAG GAC CGC TTC AAC CAC TTC AGC TIG ACC CTC AAC ACC AAC CAT GGG CAT Asn Lys Asp Arg Phe Asn His Phe Ser Leu Thr Leu Asn Thr Asn His Gly His

180 195 210

ATC CTG GTG GAT TAC TCC AAG AAC CTG CTG ACG GAG GAC GTG ATG OGG ATG CTG lie Leu Val Asp Tyr Ser Lys Asn Leu Val Thr Glu Asp Val MET Arg MET Leu

225 240 255 270

GTG GAC TTC GCC AAG TCC AGG GGC GTG GftG GCC GCC CGGGAG CGGATG TTCAAT Val Asp Leu Ala Lys Ser Arg Gly Val Glu Ala Ala Arg Glu Arg MET Phe Asn

285 300 315

GCT GAG AAG ATC AAC TAC ACC GAGGCT O.ΑG0C CTG CTG CAC GTG GCT CTG CGG Gly Glu Lys lie Asn Tyr Thr Glu Gly Arg Ala Val Leu His Val Ala Leu Arg

330 345 360 375

AAC GGG TCA AAC ACA CCC ATC CIG GTA GAC GGC AAG GAT GTG ATG CCA GAG CTC

Asn Arg Ser Asn Thr Pro lie Leu Val Asp Gly Lys Asp Val MET Pro Glu Val

390 405 420 435

AAC AAG GTT CTG GAC AAG ATG AAG TCTTTC TGC CAG CCT CTC CGGAGC GCT GAC Asn Lys Val Leu Asp Lys MET Lys Ser Phe Cys Gin Arg Val Arg Ser Gly Asp

450 465 480

TGG AAG GGG TAC ACA GGCAAGACCATCACGGAC GTCATCAACATT GGCATT CTC Trp Lys Gly Tyr Thr Gly Lys Thr lie Thr Asp Val lie Asn lie Gly lie Val

495 510 525 540

GGC TCC GAC CIG GGA CCC CTC ATG GTG ACT GAA GCC CTT AAG CCA TAC TCT TCA Gly Ser Asp Leu Gly Pro Leu MET Val Thr Glu Ala Leu Lys Pro Tyr Ser Ser

570 585

GGA GGT CCC CGCGTCTGGTATCTCTCC AAC ATT GAT GGA ACT CAC ATT GCC AAA Gly Gly Pro Arg Val Trp Tyr Val Ser Asn He Asp Gly Thr His He Ala Lys

600 615 630 645

ACC CTCGCC <-M CrcAACCπ3GAGTCCTCE CTCTTC

Thr Leu Ala Gin Leu Asn Pro Glu Ser Ser Leu Phe He lie Ala Ser Lys Thr

660 675 690 705

TTT ACT ACC CAG GAG ACC ATC ACG AAT GCA GAG ACG GOG AAG GAG TGG TTT CTC Phe Thr Thr Gin Glu Thr He Thr Asn Ala Glu Thr Ala Lys Glu Trp Phe Leu 720 735 750

CAG GOG GCC AAG GAT CCT TCT GCA GTG GCG AAG CAC TTT GTT GCC CTG TCT ACT Gin Ala Ala Lys Asp Pro Ser Ala Val Ala Lys His Phe Val Ala Leu Ser Thr

765 780 795 810

AACACAA02AAA GTCAAG GAG TTTGGAATT GAC (XT σAAACATGTTC Asn Thr Thr Lys Val Lys Glu Phe Gly He Asp Pro Gin Asn MET Phe Glu Phe

825 840 855

TGG GAT TGG GTG GGAGGA CX^TAC TCG CTGTGGTCG GCC ATC GGA CTC TCC ATT Trp Asp Trp Val Gly Gly Arg Tyr Ser Leu Trp Ser Ala He Gly Leu Ser He

870 885 900 915

GCC CTG CS^GTGGCTTIT GACAACTICGAG CΪβ CTG σCT∞

Ala Leu His Val Gly Phe Asp Asn Phe Glu Gn Leu Leu Ser Gly Ala His Trp

930 945 960 975

ATG GAC CAG C-AC TIC CGCACGACG CCC CTG GAGAAGAACG∞ MET Asp Gin His Phe Arg Thr Thr Pro Leu Glu Lys Asn Ala Pro Val Leu Leu

990 1005 1020

GCC CTG CTG GCT ATC TGG TAC ATC AAC TGC TTT GGG TCT GAG ACA CAC GCC ATG Ala Leu Leu Gly He Trp Tyr He Asn Cys Phe Gly Cys Glu Thr His Ala MET

1035 1050 1065 1080

CTG CCC TAT GAC C-AG TAC CTG CAC CGC TIT GCT G^ Leu Pro Tyr Asp Gin Tyr Leu His Arg Phe Ala Ala Tyr Phe Gin Gin Gly Asp

1095 U10 1125

ATG GAG TCC AAT GGG AAA TACATCACCAAATCTGGAACC CCT GTG GAC CAC CAG MET Glu Ser Asn Gly Lys Tyr He Thr Lys Ser Gly Thr Arg Val Asp His Gin

1140 1155 1170 1185

ACA GGC CCCATT GTCT∞GGGG^CCΆGGGACCAΆT GGC CAG CΪCT GCT TTΓTAC

Thr Gly Pro lie Val Trp Gly Glu Pro Gly Thr Asn Gly Gin His Ala Phe Tyr

1200 1215 1230 1245

CAG CICATC C^ CAAGGCACCAAGATGATA CXC TCT GAC TTC Gin Leu He His Gin Gly Thr Lys MET He Pro Cys Asp Phe Leu He Pro Val

1260 1275 1290

CAG ACC CAG CAC CCC ATA OGG AAG GCT CTG CAT CAC AAG ATC CTC CTG GCC AAC Gin Thr Gin His Pro He Arg Lys Gly Leu His His Lys He Leu Leu Ala Asn

1305 1320 1335 1350

TTC TTG GCC CAG ACA GAGGCC CIGATGAGGGGAAAA CGAOG GAGGAG GCC CGA Phe Leu Ala Gn Thr Glu Ala Leu MET Arg Gly Lys Ser Thr Glu Glu Ala Arg

1365 1380 1395

AAG GAG GI£ C2G GCT GQ3 GGC AAG ACT 0C& G&G GAC CTΓ GAG A∞ Lys Glu Leu Gin Ala Ala Gly Lys Ser Pro Glu Asp Leu Glu Arg Leu Leu Pro

1410 1425 1440 1455

CAT AAG CTC TIT GAA GGA AAT CGC CCA ACC AAC TCT ATT CTG TTC ACC AAG CTC

His Lys Val Phe Glu Gly Asn Arg Pro Thr Asn Ser He Val Phe Thr Lys Leu

1470 1485 0500 1515

ACA CCA TTC ATG CTT GGA GCC TIG CTC GCC ATG TAT GAG CAC AAG ATC TTC GTT Thr Pro Phe MET Leu Gly Ala Leu Val Ala MET Tyr Glu His Lys lie Phe Val

1530 1545 1560

CAG GGC ATC ATC TGG GAC ATC AAC AGC TT GAC O^ TGG GGA GTG GAG CTS GGA Gin Gly He He Trp Asp lie Asn Ser Phe Asp Gin Trp Gly Val Glu Leu Gly

1575 1590 1605 1620

AAG CAG CTG GCT AAG AAA ATA GAG CX^ GAG CTT Grø GGC ACT GCT C^ Lys Gin Leu Ala Lys Lys He Glu Pro Glu Leu Asp Gly Ser Ala Gin Val Thr

1635 1650 1665

TCT CAC GAC GCT TCT ACC AAT GGG CTC ATC AAC TTC ATC AAG CAG CAG CGC GAG Ser His Asp Ala Ser Thr Asn Gly Leu He Asn Phe lie Lys Gin Gin Arg Glu

1680 1699 1709 1719 1729 1739

GCC AGA CTC CAA TAAACTCGTG CTCATCTGCA GCCTCCTCIG TGACTCCCCT TTCTCTTCTC Ala Arg Val Gin

1749 1759 1769 1779 1789 1799 1809

GTCCCTCCTC CCCGGAGCCG GCACTGCATG TTCCTGGACA CCACCCAGAG CAC0CTCTGG TIGTGGGCTT

1819 1829 1839 1849 1859 1869 1879

GGACCACGAG CCCITAGCAG GGAAGGCIGG TCTOOCOCAG CCTAACCCCC AGCCCCTCCA TCTCTATGCT

1889 1899 1909 1919 1929 1939 1949

CCCTCTGTCT TAGAAITGGC TGAACTCTTT TTGTGCAGCT GACTTTTCTG ACXX-ATGTTC ACCTTGTTCA

1959 1969 1979 1989 1999 2009 2019

CATCCCATGT AGAAAAACAA AGATGCCACG GAGGAGGTAA AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA

2029 AAAAAAAAAA AAAAAA EXAMPLE 4 Expression of Murine Neuroleukin

The murine neuroleukin sequence identified in Example 2, was inserted into a vector and expressed in mammalian cells as follows: The expression vector p91023(B) described by G. G. Wong et al.. Science. 228: 810-815 (1985) vector contains the adenovirus major late promoter, a simian virus 40 (SV40) polyadenylation sequence, an SV40 enhancer and origin of replication and the adenovirus virus-associated gene. The mouse neuroleukin sequence identified in Table I above was inserted into the EcoRl site of the p91023(B) vector downstream of the adenovirus major late promoter. This construct was transfected into COS-1 cells using DEAE-dextran-mediated DNA transfeetion with the addition of chloroquin treatment as described by R. J. Kaufman et al., Mol. Cell Biol. £: 1304 (1982) . Thereafter an immunoprecip- itable 56kd polypeptide was secreted into the culture supernatant. Control cultures transfected with the p91023(B) vector alone did not produce detectable 56kd factor.

EXAMPLE 5 Biological Assay of Murine Neuroleukin

The biological activity of the neuroleukin expressed in Example 4 was assessed using three bioassays — two assays for neurotrophic activity in spinal neurons and in sensory neurons and an assay for lymphokine activity.

A. One neurotrophic assay employs cultured chick spinal neurons which are dissociated from 5 day embryonic chick spinal cord using 0.25% trypsin and cultured at 10,000 cells per 16 mm well on a substrate of poly-ornithine coated with la inin (5 ug/ l, Bethesda Research Labs.). The cells are cultured in L-15 containing 10% zeta Sera-D (processed adult bovine serum, AMF) , 6 mg/ml glucose. lOOU/ml penicillin and 100 ug/ml streptomycin at 37C and 5% C0₂ in a humidified incubator. After 24 hrs. in culture, 50% of the neurons plated initially die in the absence of added neuroleukin. Thus, the biological activity of the neuroleukin polypeptide can be quantitated by determining the amount of transfected cell supernatant required to maintain one-half maximal survival of the cultured spinal neurons. The purified neuroleukin maintains one-half maximal survival at a concentration of 1.25 x 10"¹¹ M. Neurons are scored microscopically as cells with neurites.

B. To study the effect of neuroleukin on sensory neurons from 10-day and 16-day chick embryo dorsal root ganglia, sensory ganglia are dissected under sterile con¬ ditions, incubated in Ca, Mg-free Hank's balanced salt solution (CMF-HBSS) for 20 minutes at 37^βC, and digested in 0.10% trypsin in CMF-HBSS for 10 minutes at 37'C. Trypsin- treated ganglia are washed in the culture medium, i.e., Dulbecco's modified Eagle medium (DMEM) with 10% heat- inactivated horse serum (v/v%) and 1.5mg% added glucose, and dissociated into single-cell suspensions by trituration through a glass Pasteur pipette. To enrich for neurons, cell suspensions are preplated on tissue culture plastic for 3 hours at 37*C. Neurons are then seeded at 5,000 cells per 16mm tissue culture well. Each well has been treated with 0.3 ml of 100 ug/ml poly-L-ornithine (hydro- bromide, M.W. 30,000-70,000, Sigma) overnight at 4*C followed by 0.3 ml of 10 ug/ml purified laminin (Bethesda Research Labs) for 3 hrs at 37*C. Neuroleukin expressed in Example 4 was added to experimental cultures in the form of serum-free conditioned medium. A control is provided by conditioned medium collected from COS-1 cells transfected with the p91023(B) vector only. Cultures are maintained in a 5% C0₂:95% air, humidified atmosphere at 37"C for 48 hours, at which time they are examined for cells with neurites which are scored as neurons. Maximal stimulation by neuroleukin supports the survival of approximately 55% of the neurons cultured from 10-day chick embryo dorsal root ganglia. In the control culture medium alone, approximately 15% of the neurons survive for 48 hours. Thus, neuroleukin promotes survival of 40% of the sensory neurons initially plated. On the 48 hour survival of neurons in dissociated cell cultures from 16-day chick embryo dorsal root ganglia, stimulation by neuroleukin also supports the survival of approximately 50% of the neurons cultured.

To demonstrate thatneuroleukin is capable of supporting the survival of the responsive neurons in long-term culture, 10-day chick embryo dorsal root ganglion neurons are cultured with the culture medium being changed every three days. After 1 week in culture, a 1:25 dilution of neuroleukin supports the survival of 33% of neurons cultured. In addition, the surviving neurons have extensive neurite outgrowth. In control cultures there are no surviving neurons.

Addition of neuroleukin to cultures of neonatal rat superior cervical ganglion (SCG) neurons had no effect on neuronal survival. Few SCG neurons survive when cultured in mediuim alone or with added neuroleukin whereas SCG neurons experience a high survival rate when treated with NGF. Thus the biological activity of the murine neuroleukin is distinct from that of previously known neurotrophic factors, e.g., nerve growth factor.

C. To assay for ly phokine activity, freshly drawn peripheral venous blood is obtained from consenting young adults (age <50 yr) known to be high responders to pokeweed mitogen (>1000 ng/ml lg) . Mononuclear cells (MNCs) are isolated on a Ficoll-Hypaque gradient by following standard protocols. Cells then are suspended in Hank's Balanced Salt Solution, washed ten times at 4 C, and then resuspended in culture medium at 10⁶ cells per ml. The medium is RPMl 1640 supplemented with 10% fetal bovine serum, 4 nmol glutamine and 0.1 ng/1 gentamycin. The pokeweed mitogen control is used at a 1:100 final concentration from stock (GIBCO) . For assay of neuroleukin stimulated Ig secretion, 0.2 ml of the MNC cell suspension is cultured with neuro¬ leukin expressed in Example 4 above in round-bottom 96 wellplates (Costar) for 7 days at 37°C in a 5% C0₂ humidified incubator. Supernatants of quadruplicate cultures then are pooled and assayed by ELISA for Ig content using anti-human IgG (H + L) and biotinylated anti-human (Vectastain) as previously described. Parallel cultures of unseparated MNCs are similarly cultured for 7 days with pokeweed mitogem as a control. Both neuroleukin and pokeweed mitogen produce comparable induction of immunoglobulin secretion. Mock serum-free culture supernatant collected from COS-1 cells that were transfected with the expression vector alone did not contain neuroleukin and did not induce Iq secretion. Neuroleukin dependent Ig induction is apparently both monocyte and T-cell dependent. Removal of monocytes from mononuclear cells reduces Ig production in response to both PWM and neuroleukin. Upon removal of T-cells and monocytes the resultant B-cell subset did not differentiate into Ig- secreting cells when cultured with neuroleukin.

The amount of neuroleukin produced by transfected COS-1 cells can be quantitated by determining a dose-response relationship between the amount of transfected cell supernatant added to the assay well and the content of Ig produced in the assay well. Approximately one biological unit of neuroleukin can be defined as the amount of neuroleukin required to produce one-half maximal stimulation of Ig synthesis.

Fractionation of bulk stimulated MNCs into T-cell and B-cell subsets reveals that neuroleukin is a PWM-induced, T-cell secretory product. Neuroleukin production can be induced in mononuclear cells freshly isolated from peripheral blood by PWM. MNL's stimulated with PWM are fractionated into subsets ofT-cells, B-cells and monocytes to determine whether neuroleukin is a T-cell, B-cell, or monoσyte product. In each experiment, PWM-stimulated T-cell subsets produced neuroleukin, whereas the B-cell subset failed to produce neuroleukin.

Numerous modifications and variations in practice of the invention are expected to occur to those skilled in the art upon consideration of the foregoing description. For example, one skilled in the art may construct analogs of the sequences identified above, to produce a related "family" of neuroleukins. Similarly, therapeutic compositions or vaccines for use in treating AIDS may employ only a portion of the sequences identified herein. Such modifications will be encompassed in the scope of the appended claims.

Claims

WHAT IS CLAIMED IS:

1. A neuroleukin protein substantially free from associ¬ ation with other naturally oσcuring proteins characterized by: a) a single polypeptide chain; b) an amino acid sequence selected from the group consisting of the sequence of Table I and the sequence of Table II; and c) an apparent molecular weight of about 56,000 + 2000 daltons as determined by SDS-PAGE.

2. The protein of Claim 1, further characterized by d) the ability to maintain one-half maximal survival of spinal or sensory neurons cultured in vitro at a neuroleukin concentration of approximately 1.25 x 10"¹¹ M.

3. The protein according to Claim 1, further characterized by e) the ability to activate immunoglobulin secretion by peripheral blood lymphocytes.

4. A method for producing a neuroleukin protein comprising culturing a cell transformed with a vector comprising a DNA sequence selected from the group consisting of

(a) DNA sequence of Table I

(b) DNA sequence Table II

(c) DNA sequences which hybridize to any of the foregoing DNA sequences and which code on expression for a polypeptide that is substantially immunologically equivalent to a neuroleukin polypeptide; said DNA sequence being operatively linked to a regulatory sequence therefore.

5. A tissue culture medium for culturing neural cells comprising an effective amount of a neuroleukin protein according to Claim 1.

6. A tissue culture medium for culturing bone marrow cells comprising an effective amount of a neuroleukin protein according to Claim 1.

7. A pharmaceutical composition comprising an effective amount of neuroleukin protein.

8. A therapeutic composition for treating patients infected with human immunodeficiency virus comprising an effective amount of the protein of claim 1, 2 or 3.

9. A method for treating a patient infected with human immunodeficiency virus comprising administering an effective amount of the protein of claim 1 or 2 or 3.