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WO1992006112A1 - Activated ctap and analogs thereof and their use - Google Patents

Activated ctap and analogs thereof and their use Download PDF

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Publication number
WO1992006112A1
WO1992006112A1 PCT/US1991/007556 US9107556W WO9206112A1 WO 1992006112 A1 WO1992006112 A1 WO 1992006112A1 US 9107556 W US9107556 W US 9107556W WO 9206112 A1 WO9206112 A1 WO 9206112A1
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Prior art keywords
iii
ctap
peptide
des
connective tissue
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PCT/US1991/007556
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French (fr)
Inventor
Paul H. Johnson
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Sri International
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Publication of WO1992006112A1 publication Critical patent/WO1992006112A1/en

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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
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/575—Hormones
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • This invention relates to peptides. More particularly, this invention concerns analogs of human connective tissue-activating peptides (CTAPs) , in particular analogs of the human connective tissue- activating peptide CTAP-III.
  • CTAPs connective tissue-activating peptides
  • CTAPs- are a group of naturally occurring polypeptides that are capable of activating connective tissue cells. These peptides are present in platelets and leukocytes and stimulate mitogenesis, glycosaminoglycan and hyaluronic acid synthesis, prostaglandin E 2 and cyclic AMP formation, plasminogen activator secretion, fibroblast chemotaxis, glucose transport and glycolysis. CTAPs are being investigated as pharmaceuticals for regenerating connective tissue (e.g., would healing). Castor, C. ., et al., PNAS (USA) (1983) 80.765-769 reports the amino acid sequence of o,ne CTAP, known as CTAP-III, and the biological characteristics of CTAP- III.
  • CTAP- III Connective tissue activating peptide III is a human platelet granule-derived growth factor found in 1000 times the quantity of other growth factors presently known to be in platelets.
  • CTAP-III stimulates synthesis of DNA, hyaluronic acid (HA) , sulfated glycosaminoglycan (GAG) chains, proteoglycan monomer and proteoglycan core protein in human synovial fibroblast cultures (1-6) . These references are listed below under “References.”
  • HA hyaluronic acid
  • GAG sulfated glycosaminoglycan
  • proteoglycan monomer proteoglycan monomer
  • proteoglycan core protein in human synovial fibroblast cultures
  • CTAP-III isolated by immunoaffinity chromatography showed significant molecular size heterogeneity by SDS PAGE when visualized by silver. stains and/or Western blotting (11) .
  • CTAP-III purified by immunoaffinity methods appeared heterogeneous by analytical isoelectric focusing (IEF) (12) .
  • This heterogeneity was detectable immediately after extraction from platelet -granules freshly obtained from individual donors as well as in pooled outdated blood bank platelets.
  • Such microheterogeneity was thought likely to have biologic significance since fractions of CTAP-III isolated by preparative IEF had varying specific activities in stimulating DNA and glycosaminoglycan synthesis in human synovial cell cultures (13) .
  • CTAP-III Two NH 2 - terminal cleavage products were identified: CTAP-III (des 1-13) and CTAP-III (des 1-15).
  • CTAP-III (des 1-13) had a pi of 8.6 and was a stable proteolytic cleavage product that retained the capacity to stimulate [ C]GAG synthesis in human synovial cell cultures.
  • CTAP-III (de 1-15) was an elastase and chymotrypsin cleavage product identical to NAP-2, an entity thought to have neutrophil activating properties (14) .
  • CTAP-III connective tissue activating peptide
  • CTAP-I lymphocyte
  • CTAP- III platelet
  • CTAP-III connective tissue activating peptide III
  • CTAP-III connective tissue activating peptide III
  • CTAP-I lymphocyte
  • CTAP- III platelet
  • CTAP-III connective tissue activating peptide-III
  • this invention concerns a peptide having human connective tissue-activating activity and the peptide sequence of human connective tissue-activating peptide III but with from 10 to 19 amino acids deleted from the NH- end.
  • this invention provides these 1-10 des through 1-19 des CTAP-IIIs with the 21- position methionine replaced with an acyclic side-chain hydrophobic amino acid such as leucine.
  • this invention provides pharmaceutical compositions for connective tissue activation made up of one or more of these peptides and the use of these compositions to bring about connective tissue activation.
  • Figure 1 is a graph showing the results of preparative IEF over a range of pH 3-10 separated CTAP-III into four major peaks, each possessing the capacity to stimulate [ 3H]DNA synthesis.
  • Figure 2 is a silver stained SDS-PAGE gel which shows two CTAP-III preparations.
  • Lane 1 contains a typical CTAP-III preparation eluted from a heparin affinity column with 0.3M NaCl.
  • Band A shows the CTAP- III sequence on icrosequencing
  • band B contains variable mixtures of small isoforms (see text) .
  • Lane 2 contains molecular weight markers
  • Lane 3 shows CTAP-III forms which on microsequencing of Immobilon blots revealed the structural alterations (CTAP-III [Asp-1] , CTAP-III [des 1-14]) as labeled in the figure.
  • Figure 3 is a pair of graphs.
  • the top panel plots the mitogenic activity (mean ⁇ S.E.M.) of the CTAP- III (des 1-14) isofor and CTAP-III at different concentrations; the lower panel records the GAG stimulating activity of the two forms.
  • the mitogenic activity of the des 1-14 isoform is not significantly different from CTAP-III.
  • the apparent reduction of GAG stimulating activity of the des 1-14 isoform was significant (P ⁇ 0.01).
  • Figure 4 is a pair of graphs in which immunoaffinity purified CTAP-III is compared to an aliquot cleaved to the des 1-15 form with porcine elastase.
  • the heparin affinity column used to separate elastase from the isoforms yielded two affinity forms of CTAP-III (des l-15)/NAP-2, (0.3M and 0.5M) .
  • Biologic activity (mean ⁇ SEM) is plotted versus increasing concentrations of the peptides. Both affinity forms had mitogenic activity similar to the parental form (P ⁇ 0.15).
  • the 0.3M form showed a significant increase in GAG stimulating activity (P ⁇ 0.01); the 0.5M form appeared similar to the parental CTAP-III in activity.
  • FIG. 5 is a pair of graphs which show the biologic activities (mean ⁇ S.E.M.) of rCTAP-III-Leu-21 and its elastase cleavage products, des 1-15, 0.3M and 0.5M heparin affinity forms plotted as a function of concentration.
  • the upper panel shows that rCTAP-III- Leu-21 had no mitogenic activity for human synovial cells; the lower panel shows that the intact recombinant molecule had very little GAG stimulating activity.
  • the GAG stimulating capacity of the des 1-15 forms was significantly enhanced compared to the intact molecule (P ⁇ 0.001). In the mitogenic assay both des 1-15 forms were significantly more active than the parent molecule (for the 0.3M isoform, P ⁇ 0.02; for the 0.5M isoform, P ⁇ 0.001) .
  • Figure 6 is a schematic representation of CTAP- III.
  • the single letter code identifies the amino acid residues in this schematic representation of CTAP-III; cleavage sites giving rise to the isoforms studied in this report are indicated.
  • CTAP-III is a peptide material of known sequence.
  • Figure 6 shows its structure.
  • the peptides of interest herein differ from CTAP-III by having at least their first 10 amino acids and at most the first 19 amino acids deleted from the NH 2 end. These materials are known as des 1-10 CTAP-III through des 1-19 CTAP-III.
  • the peptides can also have their "21" position methionine replaced with a leucine or with another acyclic side-chain, hydrophobic amino acid.
  • the parent CTAP-III material can be obtained from natural sources as known in the art or produced recombinantly.
  • the recombinant method and its application to produce native or 21-substituted materials are fully set forth in incorporated U.S. patent no. 4,897,348.
  • the products of the invention can be attained by incubating the CTAP-III or 21-substituted CTAP-III with a suitable proteolytic enzyme such as chymotrypsin or porcine elastase, or the like. Thereafter the cleavage products can be separated and isolated.
  • a suitable proteolytic enzyme such as chymotrypsin or porcine elastase, or the like.
  • the peptides of the invention have CTAP-III activity and thus can be administered, typically by injection.
  • a peptide of this invention or a pharmaceutical composition containing the same is administered to the subject in need of such treatment.
  • peptide compositions may be administered by any of a variety of routes depending upon the specific end use, including particularly parenterally (including subcutaneous, intramuscular, and intravenous administration) .
  • the materials can be administered to mammals such as humans, monkeys, dogs, rodents, and the like.
  • the compositions generally include a pharmaceutical diluent such as injectable saline, mineral oil or the like.
  • the compound or composition may also be administered by means of slow-release, depot, or implant formulations, as is well known in the art.
  • the polypeptides described herein are usually administered in amounts of 0.001 to 1000 micrograms per kg of body weight, particularly in amounts of 1-500 micrograms per kg of body weight, although higher or lower amounts may be used.
  • CTAP-III was isolated from platelet pellets by extraction into acid/ethanol and precipitation with cold acetone (1,2,4).
  • 50g of pelleted platelets were added to 500 ml of acid ethanol (5 ml 1.25 N HCl/95 ml ethanol); this was stirred slowly at 4°C for 16 hr, centrifuged (15,000g, 10 min) , and the supernatant fluid was added to 1500 ml cold (4°C) acetone.
  • Glycosaminoglycan polysulfate (Arteparon, Luitpold Werke, Jupiter, Fla.), a proteinase inhibitor, was then added at a concentration of 100 ⁇ g/ l. This resulted in prompt flocculation of several protein species including essentially all of the CTAP-III.
  • the turbid preparation was allowed to settle for 2 hr at 4H°C and then centrifuged at 12,000g; the resultant pellet was dispersed in PBS, pH 7.5, allowed to stand overnight at 4°C and then centrifuged at 12,003g and the CTAP-III rich supernatant fluid was recovered for further processing.
  • the isolation process was continued by heparin affinity and/or immunoaffinity chromatography.
  • Heparin affinity columns were made by coupling heparin (Sigma crude unbleached heparin, Sigma Chemical Co., St. Louis, MO) to Affigel 15 (Bio-Rad Laboratories, Richmond, CA) as directed by the manufacturer (15) .
  • Partially purified CTAP-III was applied to a heparin affinity column in phosphate-buffered saline, pH 7.0; CTAP-III was eluted with 0.3M sodium chloride and dialyzed against PBS.
  • Monospecific immunoaffinity isolated anti-CTAP-III IgG was prepared by passing polyclonal rabbit anti-CTAP-III antisera over an antigen column of highly purified CTAP- .
  • Protein measurement and antisera development Protein was measured by a colorimetric method (18) and/or UV absorption (19) . Antisera to both platelet-derived and recombinant CTAP-III-Leu-21 were raised in rabbits. Eight- to 10-week old male New Zealand white rabbits were immunized with 50 ⁇ g of CTAP-III or rCTAP-III in 0.15M NaCl in Freund's complete adjuvant. Booster injections were given at 6 and 12 weeks with antigens in incomplete Freund's adjuvant. Animals were bled at 4 weeks and then at biweekly intervals after initial immunization. Measurement of CTAP-III by radial immunodiffusion (RID) utilized filtered, heat inactivated rabbit anti-human CTAP-III (20) .
  • RID radial immunodiffusion
  • Analytic polyacrylamide gel electrophoresis Highly purified CTAP-III and its isoforms were analyzed by sodium dodecyl sulfate-polyacryia-iti ⁇ e gel electrophoresis (SDS-PAGE) in 8M urea/8% total acrylamide and by analytical IEF in ampholyte gradients pH 3-10 (15,21). Proteins separated by SDS-PAGE were detected by silver stain and on IEF by both silver and Coomassie Brilliant Blue R-250 staining (15,22,23).
  • Proteins were prepared for sequencing by blotting onto Immobilon-P using a semi-dry blotter (Polyblot, American Bionetics, Inc., Hayward, CA 94545) and identified by Coomassie Brilliant Blue R-250 or immunostaining with antisera to recombinant CTAP-III (15,20,24) .
  • Western blots of proteins following electrophoretic separation and immobilization in a nitrocellulose membrane were accomplished as described (15) .
  • Membrane-bound antigens were probed with antisera to CTAP-III or rCTAP-III (1:500) and the complexes detected with a Bio-Rad Immunoblot (GAR-HRP) assay kit (BioRad Laboratories, Richmond, CA) .
  • GAR-HRP Bio-Rad Immunoblot
  • Preparative isoelectric focusing of CTAP-III Platelet preparations were fractionated using the Pharmacia Flat Bed Apparatus FBE 3000 and the Electrophoresis Constant Power Supply ECPS 3000/150. Sephadex G-75M was washed with 2 x deionized water and dried. A stable pH gradient was achieved using a system of amphoteric and nonamphoteric buffers according to the method of Prestidge and Hearn (25) . The gel bed was then cut into 26 separate segments and the pH of each was measured. The protein focused in individual segments was eluted with PBS pH 7.0, concentrated, dialyzed against PBS, and stored frozen until assay.
  • Carbohydrate analysis Carbohydrate analyses were performed by methods previously described in detail (26,27). Three different acid hydrolyses were used for each sample aliquot. Peptides were incubated 1 hr at 80°C in 0.1N sulfuric acid to release sialic acid residues for assay. For analyses of neutral sugars, samples were hydrolyzed in 4N trifluoroacetic acid at 100°C for 2 hr. Analysis of amino sugars required hydrolyses in 6N HC1 at 100°C for 3 hr.
  • Sialic acid was measured by the thiobarbituric acid method; neutral sugars were converted to the corresponding glycamines by reductive amination with 0.2M sodium cyanoborohydride in 1M ammonium sulfate (100°C, 90 min) . Amino sugars and neutral sugar glycamines were separated and quantitated as described for amino acids, by cation exchange on a Kratos automated amino acid analyzer using post-column o- phthalaldehyde derivatization and fluorometric detection.
  • fibroblasts Normal human fibroblastic cells (synovium and cartilage) were developed from explants obtained at amputation or arthrotomy; fibroblasts from dermis were obtained • following reduction mammoplasty as described earlier (1,2). Cells were grown as monolayer cultures in T-75 flasks in CMRL 1066 (Gibco, Grand Island, NY) medium supplemented with 5% human serum and 15% fetal calf serum (FCS) , sodium bicarbonate, L-glutamine, 0.02M Hepes buffer, penicillin, streptomycin and genta icin. Trypsin dispersal was performed to facilitate cell propagation, study, and preparation for cold storage.
  • FCS fetal calf serum
  • rCTAP-III Recombinant CTAP- III-Leu-21, an analogue containing a leucine substitution for methi'onine at position 21, was produced from a synthetic gene using an E. coli expression system, pNP6, based on the genetic regulatory elements of the colicin El operon (30,31) . .
  • Six liter shake-flask cultures were grown to an optical density (650 nm) of 0.4 and gene expression was induced by addition of mitomycin C to the culture at a final concentration of 0.5 ⁇ g/ml for 4 hr.
  • the protein reaction mixture was dialyzed against 0.1N acetic acid and partially purified by gel filtration chromatography using Sephacryl-200 (Pharmacia) . Fractions containing rCTAP-III-Leu-21 were identified by polyacrylamide gel electrophoresis, pooled and lyophilized.
  • the protein was dissolved in 50 mM Tris buffer, pH 8.5 containing 6M guanidinium hydrochloride at a concentration of 0.2 mg per ml. Protein folding was initiated by the addition of a redox agent to a final concentration of 2 mM oxidized glutathione and 1 mM reduced glutathionine and dialyzed against 100 volumes of buffer without GndHCl for 12 hr at room temperature.
  • rCTAP-III- Leu-21 refolded to the native conformation as verified by analysis using analytical reverse-phase HPLC and purified platelet-derived CTAP-III as a control. Finally, the rCTAP-III-Leu-21 was purified to greater than 95% purity by heparin affinity chromatography using sodium chloride gradient elution.
  • Isoelectric point (pi) microheterogeneity Highly purified, biologically active CTAP-III was separated by preparative IEF. Fractions were examined for total protein content, CTAP-III content by RID, purity by SDS-PAGE and biologic activity was measured in human synovial cell cultures. Mitogenic activity of the IEP (isoelectric point) variants of CTAP-III is shown in Figure 1; previously we showed a similar biologic activity profile by measuring the incorporation of [ C]glucosamine into [ C]HA (12) . An analytical IEF gel (inset, Figure 1) suggests that the preparative fractions are mixtures of charge isomers. Data summarized in Figure 1 suggest that the specific biologic activity of CTAP-III varies with the IEP of the different fractions. Three additional preparative IEF experiments generated similar data. The relative magnitude of two CTAP-III anabolic activities, measured as enhanced GAG and DNA synthesis, were concordant in the different fractions.
  • CTAP-III Glycosylation of CTAP-III: To explain some of the IEP heterogeneity we examined immunoaffinity isolated CTAP-III for covalently linked carbohydrate before and following acid hydrolysis. A mixture of CTAP-III variants not separated by IEF was studied. Unhydrolyzed CTAP-III (4.76 nmole based on amino acid composition) contained 0.6 nmole glucose; acid hydrolyzed CTAP-III had 10.0 nmole glucose/4.76 nmole CTAP-III. Therefore, this mixture of CTAP-III IEP variants contained 1.97 nmole of covalently linked glucose/nmole CTAP-III. Only glucose was detected; no galactose or amino sugars were found.
  • CTAP-III Seven additional biologically active samples, including four IEP species of CTAP-III (from two separate preparative IEF studies) , were examined by the same procedures (see Table 1) . Glucose and lysine content of the four separate CTAP-III charge-isomers is shown. The amino acid composition of CTAP-III charge-isomers was examined prior to and following treatment with sodium borohydride to stabilize sugar-lysyl residue Schiff base adducts.
  • Lysyl residues not determined by virtue of borohydride treatment are an estimate of sugar-lysyl adducts and are labeled "% lysine glycosylated.”
  • bound glucose ranged from 1.97 to 2.81 nmole glucose/nmole CTAP-III.
  • Glycosylation of CTAP-III occurs commonly, varies with the preparation and was not clearly related to pi or biologic activity. Glycosylation was not required for biologic activity; one nonglycosylated preparation (PI861 S 5-I M) stimulated [ C]HA synthesis by over 900%.
  • CTAP-III Amino-terminal Deamidation, (CTAP-III [Asp- 1]): CTAP-III (Asp-1) was detected by microsequencing the band from an Immobilon blot of an analytical IEF gel at the pi 7.0 locus, as well as in the SDS-PAGE gel shown in Figure 2.
  • CTAP-III was detected by microsequencing the band from an Immobilon blot of an analytical IEF gel at the pi 7.0 locus, as well as in the SDS-PAGE gel shown in Figure 2.
  • CTAP-III was detected by microsequencing the band from an Immobilon blot of an analytical IEF gel at the pi 7.0 locus, as well as in the SDS-PAGE gel shown in Figure 2.
  • Our earlier studies showed asparagine to be the amino terminal residue of CTAP-III (3,4); further, the gene for human CTAP-III was recently shown to code for an amino terminal asparagine residue (32) .
  • CTAP-III (des 1-13) was identified in purified preparations of CTAP-III recovered from prolonged cold storage (11) . SDS-PAGE gels showed a single silver stained band which immunostained with anti- rCTAP-III in a Western blot. The apparent molecular weight of the isoform was 6200 Da, the pi was 8.6 and NH 2 -terminal sequencing showed:
  • CTAP-III (des 1-13) was the same as that for intact CTAP-III.
  • CTAP-III (des 1-13) stimulated synthesis of [ 14C]HA in human synovial cell cultures with a specific activity similar to that attributed to the parent molecule; interestingly, no mitogenic activity was detected.
  • CTAP-III (des 1-14) was identified for the first time during the present study as an isoform which failed to bind to a heparin column. After isolation from an immunoaffinity column, an aliquot of this material was separated by SDS-PAGe. A Western blot of CTAP-III antigen using antik-rCTAP-III antibody then showed the major reactive species to have a molecular weight of 6500-7000 Da; a minor fraction had a molecular weight of 9300 Da ( Figure 2) . Amino terminal sequencing of the bands from an Immobilon blot showed that the larger protein, representing 25% of the total, was CTAP-III (Asp-1) . The 6500 Da fragment was sequenced through 10 cycles which showed:
  • CTAP-III Asp-l
  • CTAP-III Asp-l
  • CTAP-III (des 1-14) stimulated by 50-60 percent the incorporation of [ 35 S0 4 ] into [ 35 S]GAG formed in human chondrocyte cultures.
  • the 75%/25% mixture of CTAP- III (des 1-14) /CTAP-III (Asp-l) contributes some degree of ambiguity to the activity studies reported here.
  • CTAP-III (Asp-l) to be responsible for the observed activity, its specific activity would have to be much greater than the parent (control) molecule.
  • CTAP-III (Asp-l) appears to have "normal” specific biologic activity; therefore it seems unlikely that this minor component could account for all of the biologic activity shown in Figure 3. Consequently, we attribute the major portion of the biologic activity of this preparation to CTAP-III (des 1-14) .
  • CTAP-III (des 1-15) was detected in platelet- derived CTAP-III preparations as a small isoform (11) .
  • NH 2 -terminal sequencing of the electrophoretically- separated band blotted onto Immobilon showed:
  • CTAP-III (des 1-10) was detected when microsequencing a small isoform band blotted onto Immobilon. This band contained approximately 20 picomoles of CTAP-III (des 1-14) , 20 pico oles of CTAP- III (des 1-15) and 10 picomoles of CTAP-III (des 1-10) .
  • the (des 1-10) variant should probably be considered a naturally-occurring form in platelet extracts. it has not yet been possible to test the biologic activity of this form as a single entity.
  • CTAP-III node 1-14
  • des 1-10 the present application provides evidence for several naturally-occurring isoforms of CTAP-III not previously known, including CTAP-III (des 1-14) , and des 1-10. Further, these studies show that CTAP-III (des 1- 14) and CTAP-III (des 1-15) retain or enhance the anabolic biologic properties of uncleaved CTAP-III. Recombinant CTAP-III-Leu-21 clearly acquired increased specific activity with respect to stimulating DNA and GAG synthesis after cleavage to the des 1-15 form.
  • the structural relationships of the CTAP-III cleavage isoforms to CTAP-III and ⁇ -TG are illustrated in Figure 6 (4,33). The carboxyterminus of these CTAP-III isoforms described in detail was found to be intact.
  • CTAP-III and its isoforms cannot be avoided during organic extraction and conventional molecular sieve chromatography. Further, most of the isoforms bind to heparin affinity columns and all bind to immunoaffinity columns. The fact that all of the isoforms discussed in this report react with polyvalent antisera raised against CTAP-III has one unfortunate consequence: published studies of plasma CTAP-III/ ⁇ -TG antigen levels measured by RIA or ELISA have much less specificity than once thought.
  • CTAP-III and CTAP-III (des l-15)/NAP-2 have recently been separated by reverse-phase HPLC with a gradient of acetonitrile in 0.1% trifluoroacetic acid (34) .
  • HPLC offers an attractive approach to separating CTAP-III from its isoforms if it is accomplished without modifying the biologic activities of the proteins. This might allow separate testing of the platelet-derived CTAP-III and its isoforms and permit the "activation by cleavage experiment" described above for rCTAP-III-Leu- 21.
  • Immunoaffinity isolated CTAP-III exhibits significant microheterogeneity as determined by SDS-PAGE and IEF analytical methods.
  • Table 3 shows that the calculated IEPs of known and hypothetical members of the CTAP-III family range from about 7 to 9.3; measured values agree reasonably well with the predicted IEPs. These data account for a substantial portion of the observed IEP heterogeneity and identify some of the cationic forms possessing increased specific biologic activity. Nonenzymatic glycosylation of lysine in CTAP- III was considered as a cause for IEP heterogeneity.
  • Table 3 shows that pi values computed for hypothetical glycosylated CTAP-III isoforms appear to support this hypothesis. Experimentally we found that six of eight CTAP-III preparations contained modest amounts of covalently-bound glucose.
  • PF-4 platelet ⁇ -granule protein
  • PF-4 has extensive homology and different actions; it is noted for its heparin neutralizing properties, chemotactic activity, and has been shown to be an immunoregulator which reverses immunosuppression in mice (35) . It is pertinent that the immunoregulatory activity of PF-4 is protease-induced immediately after platelet aggregation.
  • a monocyte-derived protein (MDNCF/NAP-1/I1-8) has extensive homology with CTAP-III and is a potent neutrophil chemotactic agent (36) .
  • Chick fibroblasts, activated by serum or Rous sarcoma virus (RSV) are induced to form the 9E3 protein which is homologous to CTAP-III (37,38) .
  • RSV-transformed cells form copious amounts of hyaluronic acid, as do human fibroblasts stimulated by CTAP-III.
  • a tumorigenic hamster cell line produces increased amounts of mRNA coding for a protein (CHEF-GRO) with homology to CTAP-III (39) .
  • Recombinant gamma-interferon treated human fibroblasts, monocytes and endothelial cells exhibit induction of a gene which codes for a protein, gamma-IP-10, that also shows homology to CTAP-III and PF-4 (40) .
  • MGSA Melanoma growth stimulatory activity isolated from Hs294T melanoma cells shows striking homology to CTAP-III and is a potent mitogen which exists in two molecular weight forms (41) .
  • These 10 to 19-des CTAP-III amino acid sequence homologies reflect a family of materials which may play roles in inflammation, wound healing and growth (37) , and may be derived from a common ancestral gene.
  • Numbering of residues is from (4) .

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Abstract

Peptides having human connective tissue activating activity are disclosed. These peptides differ from native CTAP-III by having the first from 10 to 19 amino acids deleted from their NH2 end. In addition they may optionally be substituted at the position corresponding to the 21-methionine of CTAP-III. The peptides are more active than CTAP-III.

Description

ACTIVATED CTAP AND ANALOGS THEREOF AND THEIR USE
Background of the Invention
1. Technical Field
This invention relates to peptides. More particularly, this invention concerns analogs of human connective tissue-activating peptides (CTAPs) , in particular analogs of the human connective tissue- activating peptide CTAP-III.
2. Background Art Human connective tissue-activating peptides
(CTAPs-) are a group of naturally occurring polypeptides that are capable of activating connective tissue cells. These peptides are present in platelets and leukocytes and stimulate mitogenesis, glycosaminoglycan and hyaluronic acid synthesis, prostaglandin E2 and cyclic AMP formation, plasminogen activator secretion, fibroblast chemotaxis, glucose transport and glycolysis. CTAPs are being investigated as pharmaceuticals for regenerating connective tissue (e.g., would healing). Castor, C. ., et al., PNAS (USA) (1983) 80.765-769 reports the amino acid sequence of o,ne CTAP, known as CTAP-III, and the biological characteristics of CTAP- III. United States Patent 4,897,348 issued Jan. 30, 1990 to Johnson et al. discloses that analogs of CTAP- III in which the 21 position methionine is replaced with leucine or a like acyclic side chain hydrophobic amino acid have CTAP-III activity with enhanced stability. This patent also discloses genetic engineering techniques for producing CTAP-III and its 21-position analogs and is incorporated herein by reference.
Connective tissue activating peptide III (CTAP- III) is a human platelet granule-derived growth factor found in 1000 times the quantity of other growth factors presently known to be in platelets. CTAP-III stimulates synthesis of DNA, hyaluronic acid (HA) , sulfated glycosaminoglycan (GAG) chains, proteoglycan monomer and proteoglycan core protein in human synovial fibroblast cultures (1-6) . These references are listed below under "References." CTAP-III also stimulates glucose transpo'rt, formation of prostaglandin E~, HA synthetase activity and plasminogen activator activity (7-10) . CTAP-III isolated by immunoaffinity chromatography showed significant molecular size heterogeneity by SDS PAGE when visualized by silver. stains and/or Western blotting (11) . Further, CTAP-III purified by immunoaffinity methods appeared heterogeneous by analytical isoelectric focusing (IEF) (12) . This heterogeneity was detectable immediately after extraction from platelet -granules freshly obtained from individual donors as well as in pooled outdated blood bank platelets. Such microheterogeneity was thought likely to have biologic significance since fractions of CTAP-III isolated by preparative IEF had varying specific activities in stimulating DNA and glycosaminoglycan synthesis in human synovial cell cultures (13) . Evidence for microhetero- geneity due to proteolytic processing of CTAP-III from human platelets was recently reported (11) . Two NH2- terminal cleavage products were identified: CTAP-III (des 1-13) and CTAP-III (des 1-15). CTAP-III (des 1-13) had a pi of 8.6 and was a stable proteolytic cleavage product that retained the capacity to stimulate [ C]GAG synthesis in human synovial cell cultures. CTAP-III (de 1-15) was an elastase and chymotrypsin cleavage product identical to NAP-2, an entity thought to have neutrophil activating properties (14) . The data reported now demonstrate that the molecular size and charge heterogeneity of CTAP-III is due, in part, to deamidation, nonenzymatic glycosylation and proteolytic cleavage; only proteolytic cleavage clearly influences biologic activities measured in vitro.
3. References
The following references are cited herein by their reference numbers:
1. Castor, C.W. et al., connective tissue activation. XI . Stimulation of glycosaminoglycan and DNA formation by a platelet factor. Arthritis Rheum. (1977) 20:859-68.
2. Castor, C.W. et al. , Connective tissue activation XIV. Composition and actions of a human platelet autocoid mediator. Arthritis Rheum. (1979) 2_2:260-72.
3. Walz, D.A. et al., Connective Tissue Activation: Structural Studies on a Human Platelet Mitogen [Abstract]. Clin. Res. (1979) 27:649.
4. Castor, C.W. et al., structural and biological characteristics of connective tissue activating peptide (CTAP-III) , a major human platelet- derived growth factor. Proc. Natl. Acad. Sci. USA (198 80:765-9.
5. Castor, C.W. et al., connective tissue activation XXV. Regulation of proteoglycan synthesis i human synovial cells. Arthritis Rheum. (1983) 26:522-7
6. Castor, C.W. et al., connective tissue activation XXI. Regulation of glycosaminoglycan metabolism by lymphocyte (CTAP-I) and platelet (CTAP- III) growth factors. In Vitro (1981) 17:777-85.
7. Castor, C.W. et al., connective tissue activation XXIX: Stimulation of glucose transport by connective tissue activating peptide-III. Biochemistry (1985) 24.:1762-7.
8. Castor, C.W. et al., connective tissue activation XX. Stimulation of prostaglandin secretion mediators from lymphocytes (CTAP-I) and platelets (CTA III) . Arthritis Rheum. (1981) 24:504-9.
9. Sisson J.C. et al., connective tissue activation XVIII. Stimulation of hyaluronic acid synthetase activity. J. Lab Clin. Med. (1980) 96:189- 97.
10. Ragsdale, C.G. et al., connective tissu activating peptide-III. Induction of synthesis and secretion of plasminogen activator by synovial fibroblasts. Arthritis Rheum. (1984) 27: 663-7.
11. Castor, C.W. et al., connective tissue activation XXXIII: Biologically active cleavage produ of CTAP-III from human platelets. Bioche . Biophys. R Commu . (1989) 163:1071-8.
12. Green, M.S. et al., connective tissue activation XXX: Isoelectric point icroheterogeneity CTAP-III, a human platelet derived growth factor. Proc. Soc. Exp. Biol. Med. (1986) 181:555-9.
13. Castor, C.W. et al., connective tissue activation: evidence for molecular heterogeneity of a platelet derived growth factor (CTAP-III) [Abstract]. Clin. Res. (1983) 31:784.
14. Walz, A. et al., a novel cleavage product of β-thromboglobulin formed in cultures of stimulated mononuclear cells activates human neutrophils. Biochem. Biophys. Res. Commun. (1989) 159:969-75.
15. Castor, C.W. , connective tissue activating peptide III (CTAP-III) and its isoforms. In: Barnes D. , ed. Methods in Enzymology, Peptide Growth Factors, Part C. Orlando, FL: Academic Press (in press) .
16. Castor, C.W. et al. , connective tissue activating peptide III (CTAP-III) : Characteristics of a biosynthetic form expressed in E. coli [Abstract] . Arthritis Rheum. (1986) 29:S43.
17. Schneider, C. et al., a one-step purification of membrane proteins using a high efficiency immunomatrix. J. Biol. Che . (1982) 257:10766-9.
18. Oya a, V.I. et al., measurement of cell growth in tissue culture with a phenol reagent (Folin- Ciocalteau) . Proc. Soc. Exp. Biol. Med. (1956) 91:305- 7.
19. Waddell, W.J. et al., a simple ultraviolet spectrophotometric method for the determination of protein. J. Lab. Clin. Med. (1956) 48:311-4.
20. Castor, C.W. et al., Connective Tissue Activating Peptides. In: Sabato g. , ed. Methods in Enzymology, Orlando, Florida: Academic Press, (1988) 731-48. 21. Anderson, B.L. et al. , a sodium dodecyl sulfate-polyacrylamide gel electrophoresis system that separates peptides and proteins in the molecular weight range of 2500 to 90,000. Anal. Biochem. (1983) 132:365- 75.
22. Morrissey, J.H. , silver stain for proteins in polyacrylamide gels: a modified procedure with enhanced uniform sensitivity. Analyt. Biochem. (1981) 117:307-10.
23. Heukeshoven, J. et al. , simplified method for silver staining of proteins in polyacrylamide gels and the mechanism of silver staining. Electrophoresis (1985) 6.:103-12.
24. Matsudiara, P., sequence from picomole quantities of proteins electroblotted onto polyvinylidene difluoride membranes. J. Biol. Chem. (1987) 262:10035- 8.
25. Prestidge, R.L. et al., preparative flatbed electrofocusing in granulated gels with natural pH gradients generated from simple buffers. Anal. Biochem. (1979) 97:95-102.
26. Seetharama, A. et al., reversability of the ketoamine linkages of aldoses with proteins.
27. Cole, L.A. et al., the structures of the serine-linked sugar chains on human chorionic gonadotropin. Biochem. Biophvs. Res. Co m. (1985) 126:333-9.
28. Hewick, R.M. et al. , a gas-liquid solid phase peptide protein sequenator. J. Biol. Chem. (1981) 256:7990-7.
29. Castor, C.W. et al., connective tissue activation XXI. Regulation of glycosaminoglycan metabolism by lymphocyte (CTAP-I) and platelet (CTAP- III) growth factors. In Vitro (1981) 17:777-85.
30. Johnson, P.H. et al. , connective tissue activating peptide-III (CTAP-III) : cloning the synthetic gene and characterization of the protein expressed in E coli [Abstract]. Fed. Proc. (1986) 4j5:l790.
31. Waleh, N.S., et al., structural and functional organization of the colicin El operon. Proc. Natl. Acad. Sci. USA (1985) 82:8389-93.
32. Wenger, R.H. et al., cloning of cDNA coding for connective tissue activating peptide III from a human platelet-derived λgtll expression library. Blood (1989) 71:1498-1503.
33. Begg, G.S. et al., complete covalent structure of human β-thromboglobulin. Biochemistry (1978) 17:1739-44.
34. Walz, A.R. et al. , effects of the neutrophil-activating peptide NAP-2, platelet basic protein, connective tissue activating peptide III, and platelet factor 4 on human neutrophils. J. Exp. Med. (1989) 170:1745-50.
35. Katz, I.R. et al., protease-induced immunoregulatory activity of platelet factor 4. Proc. Natl. Acad. Sci. USA (1986) 83:3491-5.
36. Matsushi a, K. et al., Molecular cloning of a human monocyte-derived neutrophil chemotactic factor (MDNCF) and the induction of MDNCF mRNA by Interleukin 1 and tumor necrosis factor. J. Ex . Med. (1988) 167:1883- 93.
37. Sugano, S. et al., transformation by Rous sarcoma virus induces a novel gene with homology to a mitogenic platelet protein. Cell (1987) 49:321-8. 38. Bedard, P.A. et al. , constitutive expression of a gene encoding a polypeptide homologous to biologically active human platelet protein in Rous sarcoma virus-transformed fibroblasts. Proc. Natl. Acad. Sci. USA (1987) 84:6715-9.
39. Anisowicz, A. et al. , constitutive overexpression of a growth-related gene in transformed Chinese hamster and human cells. Proc. Natl. Acad. Sci. USA (1987) 84:7188-92.
Statement of the Invention
We now have discovered that NH--terminal cleavage products of CTAP-III and 21-modified analogs thereof have CTAP-III activity often at enhanced levels as compared to the natural material.
More particularly, we have found that the deletion of the first 10 to 19 amino acids from the NH2 end of CTAP-III or from CTAP-III substituted at the 21 position yields peptides with enhanced connective-tissue activating properties.
Thus in one aspect this invention concerns a peptide having human connective tissue-activating activity and the peptide sequence of human connective tissue-activating peptide III but with from 10 to 19 amino acids deleted from the NH- end.
In another aspect, this invention provides these 1-10 des through 1-19 des CTAP-IIIs with the 21- position methionine replaced with an acyclic side-chain hydrophobic amino acid such as leucine. In other aspects, this invention provides pharmaceutical compositions for connective tissue activation made up of one or more of these peptides and the use of these compositions to bring about connective tissue activation.
Brief Description of the Invention
Brief Description of the Drawings
In this application reference will be made to the accompanying drawings in which Figure 1 is a graph showing the results of preparative IEF over a range of pH 3-10 separated CTAP-III into four major peaks, each possessing the capacity to stimulate [ 3H]DNA synthesis.
Figure 2 is a silver stained SDS-PAGE gel which shows two CTAP-III preparations. Lane 1 contains a typical CTAP-III preparation eluted from a heparin affinity column with 0.3M NaCl. Band A shows the CTAP- III sequence on icrosequencing, band B contains variable mixtures of small isoforms (see text) . Lane 2 contains molecular weight markers, Lane 3 shows CTAP-III forms which on microsequencing of Immobilon blots revealed the structural alterations (CTAP-III [Asp-1] , CTAP-III [des 1-14]) as labeled in the figure.
Figure 3 is a pair of graphs. The top panel plots the mitogenic activity (mean ± S.E.M.) of the CTAP- III (des 1-14) isofor and CTAP-III at different concentrations; the lower panel records the GAG stimulating activity of the two forms. The mitogenic activity of the des 1-14 isoform is not significantly different from CTAP-III. The apparent reduction of GAG stimulating activity of the des 1-14 isoform was significant (P<0.01). Figure 4 is a pair of graphs in which immunoaffinity purified CTAP-III is compared to an aliquot cleaved to the des 1-15 form with porcine elastase. The heparin affinity column used to separate elastase from the isoforms yielded two affinity forms of CTAP-III (des l-15)/NAP-2, (0.3M and 0.5M) . Biologic activity (mean ± SEM) is plotted versus increasing concentrations of the peptides. Both affinity forms had mitogenic activity similar to the parental form (P<0.15). The 0.3M form showed a significant increase in GAG stimulating activity (P<0.01); the 0.5M form appeared similar to the parental CTAP-III in activity.
Figure 5 is a pair of graphs which show the biologic activities (mean ± S.E.M.) of rCTAP-III-Leu-21 and its elastase cleavage products, des 1-15, 0.3M and 0.5M heparin affinity forms plotted as a function of concentration. The upper panel shows that rCTAP-III- Leu-21 had no mitogenic activity for human synovial cells; the lower panel shows that the intact recombinant molecule had very little GAG stimulating activity. The GAG stimulating capacity of the des 1-15 forms was significantly enhanced compared to the intact molecule (P<0.001). In the mitogenic assay both des 1-15 forms were significantly more active than the parent molecule (for the 0.3M isoform, P<0.02; for the 0.5M isoform, P<0.001) .
Figure 6 is a schematic representation of CTAP- III. The single letter code identifies the amino acid residues in this schematic representation of CTAP-III; cleavage sites giving rise to the isoforms studied in this report are indicated. The Peptides and Their Preparation
CTAP-III is a peptide material of known sequence. Figure 6 shows its structure. The peptides of interest herein differ from CTAP-III by having at least their first 10 amino acids and at most the first 19 amino acids deleted from the NH2 end. These materials are known as des 1-10 CTAP-III through des 1-19 CTAP-III.
The peptides can also have their "21" position methionine replaced with a leucine or with another acyclic side-chain, hydrophobic amino acid.
The parent CTAP-III material can be obtained from natural sources as known in the art or produced recombinantly. The recombinant method and its application to produce native or 21-substituted materials are fully set forth in incorporated U.S. patent no. 4,897,348.
The products of the invention can be attained by incubating the CTAP-III or 21-substituted CTAP-III with a suitable proteolytic enzyme such as chymotrypsin or porcine elastase, or the like. Thereafter the cleavage products can be separated and isolated.
Use of the Peptides
The peptides of the invention have CTAP-III activity and thus can be administered, typically by injection.
In the practice of the medical and veterinary methods of this invention an effective amount of a peptide of this invention or a pharmaceutical composition containing the same is administered to the subject in need of such treatment. These peptide compositions may be administered by any of a variety of routes depending upon the specific end use, including particularly parenterally (including subcutaneous, intramuscular, and intravenous administration) .
The materials can be administered to mammals such as humans, monkeys, dogs, rodents, and the like. In such uses, the compositions generally include a pharmaceutical diluent such as injectable saline, mineral oil or the like. The compound or composition may also be administered by means of slow-release, depot, or implant formulations, as is well known in the art. The polypeptides described herein are usually administered in amounts of 0.001 to 1000 micrograms per kg of body weight, particularly in amounts of 1-500 micrograms per kg of body weight, although higher or lower amounts may be used.
This invention will be further explained by the following experimental description which is intended to illustrate the practice of the invention but not limit its scope.
Experi enta1
Methods
Preparation of CTAP-III: Outdated platelets were obtained from local blood banks and the Southeast Michigan Red Cross Blood Services. Quality control studies on eight separate platelet packs five days after bleeding showed 0.84 x 10 platelets per pack and 3.2 x
10 8 ± 2.9 x 108 leukocytes per pack; that is, there are approximately 1-2 leukocytes/1000 platelets. Platelet- rich plasma was centrifuged at 4°C and the pellet preparation was stored at -20°C.
CTAP-III was isolated from platelet pellets by extraction into acid/ethanol and precipitation with cold acetone (1,2,4). In practice, 50g of pelleted platelets were added to 500 ml of acid ethanol (5 ml 1.25 N HCl/95 ml ethanol); this was stirred slowly at 4°C for 16 hr, centrifuged (15,000g, 10 min) , and the supernatant fluid was added to 1500 ml cold (4°C) acetone. After 1 hr at 4°C the supernatant fluid was removed by aspiration and centrifugation; the precipitated protein was dissolved in 30-50 ml of 0.5M acetic acid and dialyzed overnight against 0.2M acetate buffer, pH 4.0, containing protease inhibitors (0.001M Benzamadine and 0.005M EACA) . The dialyzed preparation was applied to a Sephacryl S-200 HR column (5x72 cm) and eluted with the same buffer and inhibitors in 15 ml fractions at a flow rate of 4.0 ml/ in. Fractions containing CTAP-III were identified by immunodiffusion with anti-CTAP-III antisera and pooled. Glycosaminoglycan polysulfate (Arteparon, Luitpold Werke, Jupiter, Fla.), a proteinase inhibitor, was then added at a concentration of 100 μg/ l. This resulted in prompt flocculation of several protein species including essentially all of the CTAP-III. The turbid preparation was allowed to settle for 2 hr at 4H°C and then centrifuged at 12,000g; the resultant pellet was dispersed in PBS, pH 7.5, allowed to stand overnight at 4°C and then centrifuged at 12,003g and the CTAP-III rich supernatant fluid was recovered for further processing. The isolation process was continued by heparin affinity and/or immunoaffinity chromatography. Heparin affinity columns were made by coupling heparin (Sigma crude unbleached heparin, Sigma Chemical Co., St. Louis, MO) to Affigel 15 (Bio-Rad Laboratories, Richmond, CA) as directed by the manufacturer (15) . Partially purified CTAP-III was applied to a heparin affinity column in phosphate-buffered saline, pH 7.0; CTAP-III was eluted with 0.3M sodium chloride and dialyzed against PBS. Monospecific immunoaffinity isolated anti-CTAP-III IgG was prepared by passing polyclonal rabbit anti-CTAP-III antisera over an antigen column of highly purified CTAP- . III/BTG or rCTAP-III-Leu-21 coupled to activated CH- Sepharose 4B (16) . Bound monospecific anti-CTAP-III IgG was eluted at low pH, neutralized and cross-linked to protein A Sepharose with dimethylpimelimidate using a modification of the method of Schneider (17) . CTAP-III was applied to the immunoaffinity column, the column was washed with PBS and CTAP-III was eluted with 0.1M acetic acid (pH 2.8). To reduce growth factor binding to plastic and glass, either polyethylene glycol or highly purified human albumin was added before lyophilization in amounts sufficient to make their concentrations 0.01% in final preparations.
Protein measurement and antisera development: Protein was measured by a colorimetric method (18) and/or UV absorption (19) . Antisera to both platelet-derived and recombinant CTAP-III-Leu-21 were raised in rabbits. Eight- to 10-week old male New Zealand white rabbits were immunized with 50 μg of CTAP-III or rCTAP-III in 0.15M NaCl in Freund's complete adjuvant. Booster injections were given at 6 and 12 weeks with antigens in incomplete Freund's adjuvant. Animals were bled at 4 weeks and then at biweekly intervals after initial immunization. Measurement of CTAP-III by radial immunodiffusion (RID) utilized filtered, heat inactivated rabbit anti-human CTAP-III (20) .
Analytic polyacrylamide gel electrophoresis: Highly purified CTAP-III and its isoforms were analyzed by sodium dodecyl sulfate-polyacryia-itiάe gel electrophoresis (SDS-PAGE) in 8M urea/8% total acrylamide and by analytical IEF in ampholyte gradients pH 3-10 (15,21). Proteins separated by SDS-PAGE were detected by silver stain and on IEF by both silver and Coomassie Brilliant Blue R-250 staining (15,22,23).
Proteins were prepared for sequencing by blotting onto Immobilon-P using a semi-dry blotter (Polyblot, American Bionetics, Inc., Hayward, CA 94545) and identified by Coomassie Brilliant Blue R-250 or immunostaining with antisera to recombinant CTAP-III (15,20,24) . Western blots of proteins following electrophoretic separation and immobilization in a nitrocellulose membrane were accomplished as described (15) . Membrane-bound antigens were probed with antisera to CTAP-III or rCTAP-III (1:500) and the complexes detected with a Bio-Rad Immunoblot (GAR-HRP) assay kit (BioRad Laboratories, Richmond, CA) .
Preparative isoelectric focusing of CTAP-III: Platelet preparations were fractionated using the Pharmacia Flat Bed Apparatus FBE 3000 and the Electrophoresis Constant Power Supply ECPS 3000/150. Sephadex G-75M was washed with 2 x deionized water and dried. A stable pH gradient was achieved using a system of amphoteric and nonamphoteric buffers according to the method of Prestidge and Hearn (25) . The gel bed was then cut into 26 separate segments and the pH of each was measured. The protein focused in individual segments was eluted with PBS pH 7.0, concentrated, dialyzed against PBS, and stored frozen until assay.
Carbohydrate analysis: Carbohydrate analyses were performed by methods previously described in detail (26,27). Three different acid hydrolyses were used for each sample aliquot. Peptides were incubated 1 hr at 80°C in 0.1N sulfuric acid to release sialic acid residues for assay. For analyses of neutral sugars, samples were hydrolyzed in 4N trifluoroacetic acid at 100°C for 2 hr. Analysis of amino sugars required hydrolyses in 6N HC1 at 100°C for 3 hr. Sialic acid was measured by the thiobarbituric acid method; neutral sugars were converted to the corresponding glycamines by reductive amination with 0.2M sodium cyanoborohydride in 1M ammonium sulfate (100°C, 90 min) . Amino sugars and neutral sugar glycamines were separated and quantitated as described for amino acids, by cation exchange on a Kratos automated amino acid analyzer using post-column o- phthalaldehyde derivatization and fluorometric detection.
Amino acid analyses: Amino acid compositions were determined as previously described (27) . One aliquot of CTAP-III was reduced with sodium borohydride prior to acid hydrolysis in order to preserve glucosyl- lysine linkages (26) . Briefly, CTAP-III, with and without borohydride reduction, was subjected to 6N HC1 hydrolysis (3 hr 100°C) , dried, and applied to a Kratos automated amino acid analyzer where the residues were separated by cation exchange, reacted with hypochlorite and an o-phthalaldehyde reagent and then detected with a Kratos FS950 fluorometer. Effluent peaks were compared with standards using a Hewlett-Packard 3390A integrator.
Amino acid sequence determination: Stained protein bands on Immobilon-P were cut out and destained in 100% methanol and subjected to NH2-terminal sequencing using an Applied Biosystems 470A Gas Phase Protein Sequencer interfaced with an Applied Biosystems 120A Analyzer (28) . Alternatively, aqueous protein solutions were applied to a glass fiber filter pretreated with 2.0 mg of Biobrene and sequenced as described above. The authentic carboxyterminal peptide of CTAP-III (residues 78-85) was isolated by HPLC Clg chromatography; its sequence was subsequently determined (data not shown) . The presence of the authentic CTAP-III carboxyterminus in the isoforms was confirmed by comparing the HPLC profile of their tryptic digests with the native CTAP-III HPLC digest profile.
Cell culture methods: Normal human fibroblastic cells (synovium and cartilage) were developed from explants obtained at amputation or arthrotomy; fibroblasts from dermis were obtained •following reduction mammoplasty as described earlier (1,2). Cells were grown as monolayer cultures in T-75 flasks in CMRL 1066 (Gibco, Grand Island, NY) medium supplemented with 5% human serum and 15% fetal calf serum (FCS) , sodium bicarbonate, L-glutamine, 0.02M Hepes buffer, penicillin, streptomycin and genta icin. Trypsin dispersal was performed to facilitate cell propagation, study, and preparation for cold storage.
Isotope incorporation studies: Measurement of [ C]glucosamine incorporation into [ C]GAG, primarily [ C]HA, was accomplished by plating cells in 96-well icrotiter plates at a density of 10 cells/well in 200 μl semisynthetic medium (Leibovit.: medium, L-15, Hazelton Biologies, Inc., Lenexa, Kansas) , containing 1% FCS, L- glutamine, penicillin, gentamicin, streptomycin, sodium carbonate, and 0.02M Hepes buffer, pH 7.6 (15,29) .
Identification of [ 14C]HA was accomplished by incubating labeled media with hyaluronidase (ex.Streptomyces) in an appropriate buffer and then subjecting digested and undigested samples to the CPC-fixation-waεh procedure (6,15).
3
To measure [ Hlthymid e incorporation into fibroblast DNA, cells were plated, 10 cells/microtiter well in 100 μl of ESM medium supplemented with 3% FCS, antibiotics, L-glutamine and Hepes buffer and incubated in a humidified chamber at 35-37°C (15,20) . After the preliminary 20 hr incubation period, test samples or vehicles were added (5-15 μl/well) and incubation
3 continued for 24 hr at which time [ H]-methylthymidme (1.5 μCi/15 μl ESM/well) was added and incubation resumed for the final 24 hr. Medium was aspirated, discarded, and the cell sheets were washed twice, each separately, with phosphate-buffered saline (pH 7.0) , 5% trichloroacetic acid and absolute methanol. After air drying (at 35°C) , the cells were lysed for 1 hr at 37°C with 50 μl of 0.3N sodium hydroxide. Fifty μl of cell lysate was pipetted into a counting vial containing Ecolume cocktail and radioactivity was measured in a scintillation counter.
Preparation of rCTAP-III: Recombinant CTAP- III-Leu-21, an analogue containing a leucine substitution for methi'onine at position 21, was produced from a synthetic gene using an E. coli expression system, pNP6, based on the genetic regulatory elements of the colicin El operon (30,31) . . Six liter shake-flask cultures were grown to an optical density (650 nm) of 0.4 and gene expression was induced by addition of mitomycin C to the culture at a final concentration of 0.5 μg/ml for 4 hr. Cells were collected by centrifugation and suspended in 25 mM Tris, 10 mM EDTA, 50 mM glucose, pH 8.0, containing 1M guanidinium hydrochloride. Cells were lysed by sonication and the insoluble material was removed by centrifugation. The resulting soluble material containing rCTAP-III-Leu-21 was dialyzed against 0.1N acetic acid, lyophilized and dissolved in 70% formic acid and incubated with a 50-200 molar excess of cyanogen bromide at room temperature for 12 hr. This treatment removed a short leader sequence connected to CTAP-III- Leu-21 by a single methionine residue and resulted in generating the native amino-terminal end. The protein reaction mixture was dialyzed against 0.1N acetic acid and partially purified by gel filtration chromatography using Sephacryl-200 (Pharmacia) . Fractions containing rCTAP-III-Leu-21 were identified by polyacrylamide gel electrophoresis, pooled and lyophilized. The protein was dissolved in 50 mM Tris buffer, pH 8.5 containing 6M guanidinium hydrochloride at a concentration of 0.2 mg per ml. Protein folding was initiated by the addition of a redox agent to a final concentration of 2 mM oxidized glutathione and 1 mM reduced glutathionine and dialyzed against 100 volumes of buffer without GndHCl for 12 hr at room temperature. Under these conditions, rCTAP-III- Leu-21 refolded to the native conformation as verified by analysis using analytical reverse-phase HPLC and purified platelet-derived CTAP-III as a control. Finally, the rCTAP-III-Leu-21 was purified to greater than 95% purity by heparin affinity chromatography using sodium chloride gradient elution.
Results
Isoelectric point (pi) microheterogeneity: Highly purified, biologically active CTAP-III was separated by preparative IEF. Fractions were examined for total protein content, CTAP-III content by RID, purity by SDS-PAGE and biologic activity was measured in human synovial cell cultures. Mitogenic activity of the IEP (isoelectric point) variants of CTAP-III is shown in Figure 1; previously we showed a similar biologic activity profile by measuring the incorporation of [ C]glucosamine into [ C]HA (12) . An analytical IEF gel (inset, Figure 1) suggests that the preparative fractions are mixtures of charge isomers. Data summarized in Figure 1 suggest that the specific biologic activity of CTAP-III varies with the IEP of the different fractions. Three additional preparative IEF experiments generated similar data. The relative magnitude of two CTAP-III anabolic activities, measured as enhanced GAG and DNA synthesis, were concordant in the different fractions.
Glycosylation of CTAP-III: To explain some of the IEP heterogeneity we examined immunoaffinity isolated CTAP-III for covalently linked carbohydrate before and following acid hydrolysis. A mixture of CTAP-III variants not separated by IEF was studied. Unhydrolyzed CTAP-III (4.76 nmole based on amino acid composition) contained 0.6 nmole glucose; acid hydrolyzed CTAP-III had 10.0 nmole glucose/4.76 nmole CTAP-III. Therefore, this mixture of CTAP-III IEP variants contained 1.97 nmole of covalently linked glucose/nmole CTAP-III. Only glucose was detected; no galactose or amino sugars were found. Seven additional biologically active samples, including four IEP species of CTAP-III (from two separate preparative IEF studies) , were examined by the same procedures (see Table 1) . Glucose and lysine content of the four separate CTAP-III charge-isomers is shown. The amino acid composition of CTAP-III charge-isomers was examined prior to and following treatment with sodium borohydride to stabilize sugar-lysyl residue Schiff base adducts. Lysyl residues not determined by virtue of borohydride treatment are an estimate of sugar-lysyl adducts and are labeled "% lysine glycosylated." In the two IEF preparations with isoelectric points from 6.89 to 9.32, bound glucose ranged from 1.97 to 2.81 nmole glucose/nmole CTAP-III. In two of the eight preparations there was no evidence for glycosylation. Glycosylation of CTAP-III, then, occurs commonly, varies with the preparation and was not clearly related to pi or biologic activity. Glycosylation was not required for biologic activity; one nonglycosylated preparation (PI861S5-I M) stimulated [ C]HA synthesis by over 900%.
Amino-terminal Deamidation, (CTAP-III [Asp- 1]): CTAP-III (Asp-1) was detected by microsequencing the band from an Immobilon blot of an analytical IEF gel at the pi 7.0 locus, as well as in the SDS-PAGE gel shown in Figure 2. Our earlier studies showed asparagine to be the amino terminal residue of CTAP-III (3,4); further, the gene for human CTAP-III was recently shown to code for an amino terminal asparagine residue (32) . In this context it was interesting that several recent CTAP-III preparations from outdated platelet packs which had been stored frozen showed 100% aspartic acid at the NH2~ terminus. Other preparations revealed 40% Asp/60% Asn. Data illustrating a measure of biologic activity attending partial or complete deamidation of the amino terminal asparagine of CTAP-III are shown in Table 2. It is clear that neither partial nor complete NH2 _terminal deamidation abolishes the capacity of CTAP-III to stimulate [ C]GAG synthesis by synovial cella; in fact the range of activities seen hers is similar to that found in preparations without evidence of deamidation.
In [ 3H] thymid e assays, NH2-termm. al deamidated CTAP- III (P-1890 and P-1892) stimulated DNA synthesis by 146% and 86% in synovial cell cultures.
Amino terminal sequence cleavage isoforms of CTAP-III, isolation, molecular characteristics and biologic activities: CTAP-III (des 1-13) was identified in purified preparations of CTAP-III recovered from prolonged cold storage (11) . SDS-PAGE gels showed a single silver stained band which immunostained with anti- rCTAP-III in a Western blot. The apparent molecular weight of the isoform was 6200 Da, the pi was 8.6 and NH2-terminal sequencing showed:
NH2-Leu-Tyr-Ala-Glu-Leu-Arg-Cys-Met-Cys-Ile-Lys-Thr.
Carboxyterminal analysis of CTAP-III (des 1-13) was the same as that for intact CTAP-III. CTAP-III (des 1-13) stimulated synthesis of [ 14C]HA in human synovial cell cultures with a specific activity similar to that attributed to the parent molecule; interestingly, no mitogenic activity was detected.
CTAP-III (des 1-14) was identified for the first time during the present study as an isoform which failed to bind to a heparin column. After isolation from an immunoaffinity column, an aliquot of this material was separated by SDS-PAGe. A Western blot of CTAP-III antigen using antik-rCTAP-III antibody then showed the major reactive species to have a molecular weight of 6500-7000 Da; a minor fraction had a molecular weight of 9300 Da (Figure 2) . Amino terminal sequencing of the bands from an Immobilon blot showed that the larger protein, representing 25% of the total, was CTAP-III (Asp-1) . The 6500 Da fragment was sequenced through 10 cycles which showed:
NH2-Tyr-Ala-Glu-Leu-Arg-Cys-Met-Cys-Ile-Lys- COOH This analysis corresponds to CTAP-III (des 1-14) ; subsequent studies have detected CTAP-III (des 1-14) in material which had bound to heparin, hence it is clear that this isoform may occur with or without a modified heparin binding domain. Biologic activity measurements of CTAP-III (des 1-14) and its accompanying larger form showed that the isoform stimulated DNA synthesis with a specific activity similar to that shown by a typical preparation of the parent molecule (Figure 3) . The des 1-14 isoform appeared slightly less active than the parent molecule in stimulating GAG synthesis. The significance of this finding is uncertain in view of the presence of 25% CTAP-III (Asp-l) in the preparation. In addition, CTAP-III (des 1-14) stimulated by 50-60 percent the incorporation of [35S04] into [35S]GAG formed in human chondrocyte cultures. The 75%/25% mixture of CTAP- III (des 1-14) /CTAP-III (Asp-l) contributes some degree of ambiguity to the activity studies reported here. For CTAP-III (Asp-l) to be responsible for the observed activity, its specific activity would have to be much greater than the parent (control) molecule. As noted earlier, CTAP-III (Asp-l) appears to have "normal" specific biologic activity; therefore it seems unlikely that this minor component could account for all of the biologic activity shown in Figure 3. Consequently, we attribute the major portion of the biologic activity of this preparation to CTAP-III (des 1-14) .
CTAP-III (des 1-15) was detected in platelet- derived CTAP-III preparations as a small isoform (11) . NH2-terminal sequencing of the electrophoretically- separated band blotted onto Immobilon showed:
NH2-Ala-Glu-Leu-Arg-Cys-Met-Cys-Ile-Lys-Thr. We noted earlier that incubation of the parent molecule with porcine elastase or chymotrypsin resulted in cleavage to the CTAP-III (des 1-15) isoform (11) .
In the present study, we incubated 28.5 μg porcine elastase with 2.5 mg of CTAP-III for 17 hr at 21°C in order to generate this isoform for biologic activity measurements. Following enzymatic cleavage, the digestion mixture was eluted from a heparin affinity column with increasing concentrations of NaCl applied in a stepwise manner. The elastase was removed in the unbound fraction and CTAP-III (des 1-15) was eluted at both 0.3M and 0.5M NaCl. Both salt fractions were sequenced and shown to be CTAP-III (des 1-15) . An HPLC comparison of the tryptic digest of native CTAP-III with CTAP-III (des 1-15) . An HPLC comparison of the tryptic digest of native CTAP-III with CTAP-III (des 1-15) (both the 0.3 and 0.5M eluates) as well as CTAP-III (des 1-14) revealed that the carboxyterminal peptide (residues 78- 85) was identical (intact) in each (data not shown) . The anabolic biologic activity of CTAP-III (des l-15)/NAP-2 is illustrated in Figure 4. It is clear that the 0.3M salt fraction of CTAP-III (des 1-15) stimulates glycosaminoglycan synthesis with a specific activity greater than that of the parent molecule; the 0.5M salt fraction, however, was not significantly more active than CTAP-III. The mean specific activity of the des 1-15 isoforms with respect to DNA synthesis, while generally greater than the parent molecule was not significantly increased.
CTAP-III (des 1-10) was detected when microsequencing a small isoform band blotted onto Immobilon. This band contained approximately 20 picomoles of CTAP-III (des 1-14) , 20 pico oles of CTAP- III (des 1-15) and 10 picomoles of CTAP-III (des 1-10) . The (des 1-10) variant should probably be considered a naturally-occurring form in platelet extracts. it has not yet been possible to test the biologic activity of this form as a single entity.
Biologic activity studies of rCTAP-III-Leu-21 and rCTAP-III-Leu-21 (des 1-15) : Recombinant CTAP-III- Leu-21 was subjected to the elastase cleavage procedure described above for platelet-derived CTAP-III; digestion products were applied to a heparin affinity column and fractions were eluted with 0.3M and 0.5M NaCl. The cleaved rCTAP-III fractions had a MW of approximately 7,000 Da by SDS-PAGE and microsequencing confirmed the des 1-15 structure. The marked increase in biologic activity following cleavage to the des 1-15 form is demonstrated in Figure 5. Both isoforms of rCTAP-III- Leu-21 (des 1-15) stimulated [ C]GAG synthesis far more than the parent molecule; the specific activity was increased by 200-700 percent. The recombinant cleavage isoforms stimulated [ 3H1DNA synthesis markedly compared to the parental molecule which actually appeared slightly inhibitory.
Discussion
The present application provides evidence for several naturally-occurring isoforms of CTAP-III not previously known, including CTAP-III (des 1-14) , and des 1-10. Further, these studies show that CTAP-III (des 1- 14) and CTAP-III (des 1-15) retain or enhance the anabolic biologic properties of uncleaved CTAP-III. Recombinant CTAP-III-Leu-21 clearly acquired increased specific activity with respect to stimulating DNA and GAG synthesis after cleavage to the des 1-15 form. The structural relationships of the CTAP-III cleavage isoforms to CTAP-III and β-TG are illustrated in Figure 6 (4,33). The carboxyterminus of these CTAP-III isoforms described in detail was found to be intact. The "activation" of rCTAP-III-Leu-21 by removal of the amino terminal 15 residues raises the possibility that platelet-derived CTAP-III owes much or all of its biological activity to trace amounts of small isoforms coisolated with intact CTAP-III. Current evidence from comparing platelet-derived CTAP-III and CTAP-III (des 1- 15) does not support the idea that all biologic activity resides in t he cleaved form. The most degraded preparation of platelet-derived CTAP-III contained a maximum of 20% (determined at sequencing) as small isoforms. If one postulates that all of the biologic activity derives from a CTAP-III (des 1-15) component representing one fifth of the total protein, then the specific activity of the isolated des 1-15 form would be expected to be five times that of an 80%/20% mixture of CTAP-III and CTAP-III (des 1-15) . Measurements show, however, that increases in specific activity fall short of that prediction; thus it is premature to ascribe all of the biologic activity to the cleaved forms.
Coisolation of CTAP-III and its isoforms cannot be avoided during organic extraction and conventional molecular sieve chromatography. Further, most of the isoforms bind to heparin affinity columns and all bind to immunoaffinity columns. The fact that all of the isoforms discussed in this report react with polyvalent antisera raised against CTAP-III has one unfortunate consequence: published studies of plasma CTAP-III/β-TG antigen levels measured by RIA or ELISA have much less specificity than once thought.
CTAP-III and CTAP-III (des l-15)/NAP-2 have recently been separated by reverse-phase HPLC with a gradient of acetonitrile in 0.1% trifluoroacetic acid (34) . HPLC offers an attractive approach to separating CTAP-III from its isoforms if it is accomplished without modifying the biologic activities of the proteins. This might allow separate testing of the platelet-derived CTAP-III and its isoforms and permit the "activation by cleavage experiment" described above for rCTAP-III-Leu- 21.
Immunoaffinity isolated CTAP-III exhibits significant microheterogeneity as determined by SDS-PAGE and IEF analytical methods. Table 3 shows that the calculated IEPs of known and hypothetical members of the CTAP-III family range from about 7 to 9.3; measured values agree reasonably well with the predicted IEPs. These data account for a substantial portion of the observed IEP heterogeneity and identify some of the cationic forms possessing increased specific biologic activity. Nonenzymatic glycosylation of lysine in CTAP- III was considered as a cause for IEP heterogeneity. Table 3 shows that pi values computed for hypothetical glycosylated CTAP-III isoforms appear to support this hypothesis. Experimentally we found that six of eight CTAP-III preparations contained modest amounts of covalently-bound glucose. It is not known whether glycosylation occurs in vivo or in vitro. It is also clear that glycosylation is not required for biologic activity, and that the degree of glycosylation of CTAP- III does not explain variations in biologic activity. At least seven mammalian proteins with extensive homology to CTAP-III (see Tables 4 and 5) are now known. CTAP-III and/or its isoforms are potent stimulators of DNA and GAG synthesis, glucose transport, glycolysis, PGE2 formation and plasminogen activator secretion. Further, it is reported to be a chemoattractant for neutrophils after cleavage to the des 1-15/NAP-2 form (34) . There has been little clear evidence of biologic activity for PBP and β-TG. Another platelet α-granule protein, PF-4, has extensive homology and different actions; it is noted for its heparin neutralizing properties, chemotactic activity, and has been shown to be an immunoregulator which reverses immunosuppression in mice (35) . It is pertinent that the immunoregulatory activity of PF-4 is protease-induced immediately after platelet aggregation.
A monocyte-derived protein (MDNCF/NAP-1/I1-8) has extensive homology with CTAP-III and is a potent neutrophil chemotactic agent (36) . Chick fibroblasts, activated by serum or Rous sarcoma virus (RSV) are induced to form the 9E3 protein which is homologous to CTAP-III (37,38) . RSV-transformed cells form copious amounts of hyaluronic acid, as do human fibroblasts stimulated by CTAP-III. A tumorigenic hamster cell line produces increased amounts of mRNA coding for a protein (CHEF-GRO) with homology to CTAP-III (39) . Recombinant gamma-interferon treated human fibroblasts, monocytes and endothelial cells exhibit induction of a gene which codes for a protein, gamma-IP-10, that also shows homology to CTAP-III and PF-4 (40) . Melanoma growth stimulatory activity (MGSA) isolated from Hs294T melanoma cells shows striking homology to CTAP-III and is a potent mitogen which exists in two molecular weight forms (41) . These 10 to 19-des CTAP-III amino acid sequence homologies reflect a family of materials which may play roles in inflammation, wound healing and growth (37) , and may be derived from a common ancestral gene. This plethora of structurally-related molecules, viewed in the context of the complexity of the inflammatory response, argues the importance of relating specific molecular forms to specific biologic activities. The data reported here indicate that the biologic activities of CTAP-III are generated mainly by selective NH~-terminal proteolytic processing.
Table 1
Glucose Content of CTAP-III Charge-Isomers
Figure imgf000032_0001
* = % Expected lysine not found, presumably due to glycosylation.
** = Not performed.
*** = Isolated by immunoaffinity chromatography.
Table 2
Effect of Amino Terminal Deamidation on [ 14C]glucosamme Incorporation
Platelet
Preparation [ C]GAG Synthesis
Number %Asn/%Asp Exper/control Ratio
P-1908 44/56 4.0 P-1882 40/60 20.3 P-1890 0/100 2.57 P-1895 0/100 34.6
* The platelets from which P-1908 were prepared were collected recently; the other platelets had been stored for an extended period.
Figure imgf000034_0001
Table 3
Isoelectric Points of CTAP-III and Its Isoforms
Residues Synonym pi Ref.
Calculated Observed
1-85 CTAP-III 8.29 8.5 (2)
1-85 CTAP-III (Asp-l) 7.14 7.0
1-85 rCTAP-III-Leu-^l 8.29 8.2
5-85 CTAP-III (des 1-4) /β-TG 7.14 7.0 (42)
14-85 CTAP-III (des 1-13) 9.24 8.6
15-85 CTAP-III (des 1-14) 9.24 8.7 .
16-85 CTAP-III (des 1-15) 9.27 9.2 *f
1-85** CTAP-III glycosylated at 71 7.14
1-85*** CTAP-III glycosylated at 71 and 76 6.99
# Isoelectric points were calculated using the IBI Pustell Sequence Analysis
Programs, version 2.02.
*
Numbering of residues is from (4) .
** Random glycosylation of a single lysine, here residue 71, was assumed; for calculation of pi, glycine was substituted in the amino acid sequence.
Random multiple glycosylation sites, here at positions 71 and 76, were assumed. No differences were observed when alternative lysine residues (4, 6, 24,

Claims

WHAT IS CLAIMED IS:
1. A peptide having human connective tissue- activating activity and the peptide sequence of human connective tissue-activating peptide III but with a number of from 10 to 19 amino acids deleted from the NH2 end.
The peptide of claim 1 wherein the number is 10.
3. The peptide of claim 1 wherein the number is 11.
4. The peptide of claim 1 wherein the number is 12.
5. The peptide of claim 1 wherein the number is 13
6. The peptide of claim 1 wherein the number is 14
7. The peptide of claim 1 wherein the number s 15.
8. The peptide of claim 1 wherein the number s 16,
9. The peptide of claim 1 wherein the number s 17
10. The peptide of claim 1 wherein the number is 18.
11. The peptide of claim 1 wherein the number is 19
12. The peptide of claim 1 in substantial isolation from other peptides.
13. The peptide of claim 1 in substantial isolation from the peptide comprising the 10 to 19 amino acids deleted from the NH2 end.
14. A peptide having human connective tissue- activating activity and having the peptide sequence of human connective tissue- activating peptide III but with the methionme position 21 replaced by an amino acid having an acyclic side chain and hydrophobic properties similar to methionine and with a number of from 10 to 19 amino acids deleted from the NH2 end.
15. The peptide of claim 14 wherein the amino acid replacing the methionine at position 21 is leucine.
16. The peptide of claim 14 wherein the number is 10.
17. The peptide of claim 14 wherein the number is 11.
18. The peptide of claim 14 wherein the number is 12
19. The peptide of claim 14 wherein the number is 13.
20. The peptide of claim 14 wherein the number is 14,
21. The peptide of claim 14 wherein the number is 15,
22. The peptide of claim 14 wherein the number is 16,
23. The peptide of claim 14 wherein the number is 17.
24. The peptide of claim 14 wherein the number is 18,
25. The peptide of claim 14 wherein the number is 19.
26. A pharmaceutical preparation comprising an injectable carrier in combination with a pharmaceutically effective level of the peptide of claim 1.
27. A pharmaceutical preparation comprising an injectable carrier in combination with a pharmaceutically effective level of the peptide of claim 13.
28. A method for achieving connective tissue cell activation in a patient in need thereof comprising administering to said patient an effective dose of the composition of claim 26.
29. A method for achieving connective tissue cell activation in a patient in need thereof comprising administering to said patient an effective dose of the composition of claim 27.
PCT/US1991/007556 1990-10-09 1991-10-09 Activated ctap and analogs thereof and their use WO1992006112A1 (en)

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Cited By (1)

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WO1996038559A1 (en) * 1995-05-31 1996-12-05 Dana Farber Cancer Institute Chemokine n-terminal deletion mutations

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US4897348A (en) * 1983-08-25 1990-01-30 Sri International Recombinant materials and methods for producing human connective tissue-activating peptide-III and analogs thereof
US4939135A (en) * 1988-10-03 1990-07-03 Alcon Laboratories, Inc. Pharmaceutical compositions and methods of treatment to prevent and treat corneal scar formation produced by laser irradiation
US4962091A (en) * 1986-05-23 1990-10-09 Syntex (U.S.A.) Inc. Controlled release of macromolecular polypeptides

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Publication number Priority date Publication date Assignee Title
US4897348A (en) * 1983-08-25 1990-01-30 Sri International Recombinant materials and methods for producing human connective tissue-activating peptide-III and analogs thereof
US4962091A (en) * 1986-05-23 1990-10-09 Syntex (U.S.A.) Inc. Controlled release of macromolecular polypeptides
US4939135A (en) * 1988-10-03 1990-07-03 Alcon Laboratories, Inc. Pharmaceutical compositions and methods of treatment to prevent and treat corneal scar formation produced by laser irradiation

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Title
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS, Volume 159, No. 3, issued 31 March 1989, A. WALZ et al., "A Novel Cleavage Product of B-Thromboglobulin Formed in Cultures of Stimulated Mononuclear Cells Activates Human Neutrophils", pages 969-975. *
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS, Volume 163, No. 2, issued 15 September 1989, C.W. CASTOR et al., "Connective Tissue Activation XXXIII. Biologically Active Cleavage Products of CTAP-III From Human Platelets", pages 1071-1078. *
EUROPEAN JOURNAL OF IMMUNOLOGY, Volume 20, issued September 1990, J. VAN DAMME et al., "The Neutrophil-activating proteins interleukin 8 and B-thromboglobulin: in vitro and in vivo comparison of NH2 -terminally processed forms", pages 2113-2118. *

Cited By (5)

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Publication number Priority date Publication date Assignee Title
US5705360A (en) * 1993-11-12 1998-01-06 Dana-Farber Cancer Institute Chemokine N-terminal deletion mutations
US5739103A (en) * 1993-11-12 1998-04-14 Dana-Farber Cancer Institute Chemokine N-terminal deletion mutations
US5854412A (en) * 1993-11-12 1998-12-29 Dana-Farber Cancer Institute Chemokine N-terminal deletion mutations
WO1996038559A1 (en) * 1995-05-31 1996-12-05 Dana Farber Cancer Institute Chemokine n-terminal deletion mutations
EP1647597A1 (en) * 1995-05-31 2006-04-19 Dana Farber Cancer Institute Chemokine N-terminal deletion mutations

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