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• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • C07K14/475—Growth factors; Growth regulators
  • C07K14/48—Nerve growth factor [NGF]
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
  • A61P31/12—Antivirals
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides

Definitions

• the present invention relates to a novel protein which we call neuroleukin.
• 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.
• neuroleukin consists of a single polypeptide chain. It has an apparent molecular weight of approximately 56,000 + 2000 daltons as determined by sodium dodecylsulfate polya ⁇ rylamide gel electrophoresis (SDS-PAGE) . Its isoelectri ⁇ 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.
• 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.
• 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.
• 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.
• 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.
• 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) .
• HAV human immuno ⁇ deficiency virus
• 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.
• 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.
• 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 NaH 2 P0 /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).
• 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.
• neuroleukin The sequence of neuroleukin was determined for both the mouse and human proteins as follows.
• 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.
• 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..
• 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.
• 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)].
• CCA/GCCAUG(G) canonical sequence for eukaryotic translation initation sites
• 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.
• 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.
• 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
• ATC CTG TGG GGG lie Thr Lys Ser Gly Ala Arg Val Asp His Gin Thr Gly Pro lie Val Trp Gly
• 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
• 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) .
• Table II 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 5 1 untranslated regions. To date, no bioactivity has been detected for the human sequence.
• AAG CAG CTG GCT AAG AAA ATA GAG CX ⁇ GAG CTT Gr ⁇ GGC ACT GCT C ⁇ Lys Gin Leu Ala Lys Lys He Glu Pro Glu Leu Asp Gly Ser Ala Gin Val Thr
• 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
• 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 4 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.
• 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% C0 2 in a humidified incubator. After 24 hrs. in culture, 50% of the neurons plated initially die in the absence of added neuroleukin.
• zeta Sera-D processed adult bovine serum
• 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.
• CMF-HBSS Mg-free Hank's balanced salt solution
• 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.
• DMEM Dulbecco's modified Eagle medium
• v/v% heat- inactivated horse serum
• v/v% heat- inactivated horse serum
• Example 4 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% C0 2 :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.
• neuroleukin 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.
• SCG neonatal rat superior cervical ganglion
• 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 10 6 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) .
• GIBCO a 1:100 final concentration from stock
• 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% C0 2 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.
• 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 mono ⁇ yte product.
• PWM-stimulated T-cell subsets produced neuroleukin, whereas the B-cell subset failed to produce neuroleukin.

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