WO1987004723A1 - Facteur de croissance des cellules b humaines obtenu par adn recombinant - Google Patents

Facteur de croissance des cellules b humaines obtenu par adn recombinant Download PDF

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WO1987004723A1
WO1987004723A1 PCT/US1987/000268 US8700268W WO8704723A1 WO 1987004723 A1 WO1987004723 A1 WO 1987004723A1 US 8700268 W US8700268 W US 8700268W WO 8704723 A1 WO8704723 A1 WO 8704723A1
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bcgf
recombinant
protein
cells
dna
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PCT/US1987/000268
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English (en)
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Abby L. Maizel
Shashikant R. Mehta
Surendra Sharma
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Board Of Regents, The University Of Texas System
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Publication of WO1987004723A1 publication Critical patent/WO1987004723A1/fr

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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
    • C07K14/52—Cytokines; Lymphokines; Interferons
    • 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 the use of recombin ⁇ ant DNA technology to clone and sequence genes which code for proteins exhibiting human B-cell growth factor activity and, more particularly, the use of these cloned genes in the production of such proteins.
  • lymphokines and onokines are secreted by specific cells of lymphoid and monocytoid derivation.
  • T-cell growth factor T-cell growth factor
  • IL-2 interleukin-2
  • TCGF T-cell growth factor
  • IL-2 interleukin-2
  • BCGF B cell growth factors have been identified which stimulate B-lymphocyte proliferation. It has been observed that BCGF's exhibit acivities that are roughly analogous to various activities exhibited by TCGF. Although no BCGF species has yet been isolated to sufficient purity and in sufficient quantity for confir ⁇ mation, it is thought that BCGF may play a similarly significant role in treating certain human diseases such as cancer.
  • BCGF BCGF that would be free of interfering cytokine activity.
  • the availability of an anti-BCGF antibody would provide a reasonably reliable probe for the isolation of a recombinant-BCGF producing cellular clone.
  • BCGF has been isolated on an analytical level from lectin-activated peripheral blood mononucleocytes (Maizel et al. (1982), Proc. Natl. Acad. Sci. U.S.A., 79: 5998), reseachers have been unable to produce such an anti-BCGF antibody.
  • the present invention discloses a DNA segment comprising a DNA sequence which codes for a protein exhibiting human B-cell growth factor activity.
  • DNA segment may conveniently be provided in the form of a recombinant DNA vector which includes the DNA sequence.
  • Recombinant DNA vectors made in accordance with the present invention and when introduced into an appropriate host, provide such host with the genetic information necessary for the production of an active recombinant human B-cell growth factor.
  • the present invention discloses techniques suitable for the construction of recombinant DNA vectors which contain a DNA sequence which codes for a protein exhibiting human BCGF activity.
  • the present invention discloses a method for producing genetically engineered human BCGF utilizing recombinant DNA sequences which code for a human BCGF. These sequences will generally be referred to as a recombinant human BCGF gene.
  • Genetically engineered BCGF is produced by providing a cellular clone containing a recombinant DNA expression vector, wherein the expression vector contains a DNA sequence which codes for human BCGF, and culturing the cellular clone under suitable conditions to promote the transcription and translation of the recombinant human BCGF gene into a human BCGF.
  • a recombinant DNA expression vector as used herein refers to a cloning vector, such as the pUCq vector described herein, which has been particu- larly adapted for expressing the protein product coded for by the inserted recombinant DNA sequence.
  • a cell which expresses human BCGF is provided by first, preparing at least one first nucleic acid sequence which is complementary to at least one second nucleic acid sequence wherein the second nucleic acid sequence codes for human BCGF, followed by transforming an appropriate host cell with the first nucleic acid sequence so prepared.
  • a cellular clone actively expressing human BCGF can then be selected from the group of cells so trans ⁇ formed.
  • BCGF may be provided in the above manner by providing the recombinant expres ⁇ sion vector directly, such protein may similarly be provided from recombinant DNA trasfer vectors, such as pBR322, and which DNA sequences include a code for human BCGF.
  • a recombinant expression vector containing the recombinant BCGF gene DNA may then be constructed using the recombinant BCGF gene DNA sequence contained within such recombinant transfer vector.
  • a recombinant DNA transfer vector as used herein refers to a cloning vector which may be used in transferring cloned recombinant DNA sequences from one host to another and are particularly useful for preparing recombinant clone banks. Examples of transfer vectors include pBR322 and its numerous derivations. It should be noted that transfer vectors can be used for expressing a cloned gene, however such vectors are generally not as efficient as the expression vectors for such purposes.
  • Recombinant DNA vectors as used herein may be either recombinant DNA transfer vectors or recombinant DNA expression vectors.
  • recombinant DNA vectors include but are not limited to plasmids (which are extrachrom- asomally replicating circular DNA's), bacteriophage (such as the lambda phage), and human viruses such as SV-40 which have been adapted for both transfer and expression of recombinant DNA sequences (see, e.g., Okayama and Berg, infra. ) .
  • One method for preparing at least one nucleic acid sequence which is complementary to at least one second nucleic acid sequence which codes for human BCGF entails enzymatically copying total human messenger RNA (mRNA) , preferably RNA from activated T-lymphocytes, into complementary DNA (cDNA) sequences. In this manner, at least one of the cDNA sequences so obtained should contain the necessary genetic sequence information to code for a human BCGF mRNA.
  • mRNA total human messenger RNA
  • cDNA complementary DNA sequences
  • the preferred enzyme for enzymatically copying human mRNA into complementary nucleic acid sequences is the enzyme reverse trascriptase.
  • nucleic acid polymerizing enzymes such as DNA polymerase I, may be useful.
  • Nucleic acid sequences capable of coding for a human BCGF may be prepared in other ways as well, includ ⁇ ing synthetic prepartion of the appropriate DNA sequence, using the nucleic acid sequence or amino acid sequence of Figure 5 as a guide. Synthetically prepared BCGF gene sequences should function identically with BCGF gene sequences which are enzymatically prepared.
  • Fig. 1A exhibits the enrichment of BCGF specific mRNA by methyl mercuric hydroxide gels. Symbols represent activity for: ( ) BCGF; ( __ ) TCGF. The left sided ordinate represents thymidine incorporation in the BCGF assay; the right sided ordinate represents incorporation in the TCGF assay.
  • Fig. IB exhibits the translation products of hybrid selected BCGF mRNA's in Xenopus laevis. Results are represented in terms of 3H-Tdr incorporation in 12 x 103 long term cultured B cells. Symbols represent DNA from: ( ⁇ ) pUC 9; ( > ) pARJ 43; ( a__ ) pARJ 45; ( __. ) pARJ 55; ( • ) pARJ 36.
  • Fig. 2A demonstrates the biological activity of recombinant BCGF. Results are represented in terms of
  • Fig. 2B demonstrates specificity on anti-u activated B cells. Fractions from panel A were also assayed on anti-u activated B cells at 1.2% final concentration essentially as described for panel A, except incubation in the microtiter assay continued for 96 hr before addition ooff 3 HH--TTddrr aatt 772 hrs. Symbols represent: ( # ) pUC 9; ( A ) pARJ 43.
  • Fig. 3 is a representative restriction map of pARJ 43.
  • a pR designates the location of the ampicillin resistence gene.
  • Lac Z indicates the location of the B- galactosidase structural gene.
  • Figs. 4A and B together display the DNA sequence of the BCGF insert in pARJ43 along with the corresponding derived amino acid sequence.
  • the numbers refer to nucleotide sequence with respect to the first BCGF-coding nucleotide. Also displayed are the various endonuclease restriction sites.
  • Figures 4A and 4B together illustrate one continuous nucleotide and protein sequence, they will be referred to throughout the present disclosure as simply Figure 4. From the nucleotide numbering system shown in Figures 4A and 4B, it will be readily apparent to those of skill in the art that the leftmost nucleotides of Figure 4B should be aligned to the right to the rightmost nucleotides of Figure 4A to display the entire continuous sequence.
  • BCGF Human B-cell growth factor
  • T-cell derived lymphokine under normal circumstances, that has been shown to be involved in the proliferation of activated B lymphocytes.
  • Biochemical characterization of human BCGF has revealed that it is a heat-and protease-sensitive protein which exhibits an approximate molecular weight of 12,000 to 14,000 with a major isoelectric point of 6.5.
  • BCGF may also belong to a cascade of signals that are involved in the initial activation of cells of this lineage.
  • BCGF is predominately released by normal human T cell lymphocytes following antigen and/or lectin stimulation of such cells. Similarly, BCGF activity has been shown to be released from certain granular lymphocytes and "immorta- lized" human B cells. Moreover, multiple BCGF species of different molecular weights have been detected in super- natants of T-cell hybridomes, malignant T cells and normal lectin activated T cells costimulated with PMA. The numbers and variations of BCGF's suggest the existence of multiple BCGF species. Unfortunately, in all of the foregoing cases, the molecules exhibiting BCGF activation activity are not produced in sufficient quantities to render the isolation of significant amounts of BCGF practical.
  • the present invention takes advantage of recombinant DNA technology to provide cellular clones which produce a protein having BCGF activity.
  • protein molecules which exhibit BCGF activity will be referred to generally as BCGF.
  • the production of recominant human BCGF is achieved by, first, selective enrichment for BCGF-specific messenger RNA (mRNA) ; second, preparation of complementary DNA (cDNA) segments which correspond to the BCGF-enriched mRNA fraction and; thirdly, inserting the resultant BCGF-enriched cDNA's into an appropriate expression host/vector cloning system.
  • mRNA messenger RNA
  • cDNA complementary DNA
  • Such DNA expression vectors are well known to those of skill in the art to be useful in both the cloning of recombinant gene segments and in transforming their hosts to produce the corresponding gene segment protein.
  • the pUC-9 vector as described by Heidecker et al. (1983), Nucl. Acad. Res. , 11: 4891, is utilized to take advantage of insertion of the cDNA sequences into the B-galacto- sidase gene which it possesses.
  • the Okayama vector permits expression of proteins in selected eukaryotic cells. Numerous other appropriate host/vector systems are known to the art and are contem ⁇ plated to be within the scope of the present invention.
  • Such cell lines provide a convenient system to sensitively assay either full length or fragmented BCGF molecules produced by recombinant methods. However, because it is a biological assay, it selects for only those clones which produce a biologically active position. The utilization of the biological assay additionally provides for lymphokine specificity in selections for BCGF activity.
  • full advantage of the present invention may be realized by preparing such BCGF dependent target B cell lines which are continuously selected for a demonstrated reactivity to BCGF in the essential absence of proliferative reactivity to other cytokines such as interleukin-1, interleukin-2, or gamma- interferon.
  • Such "closely-selected" BCGF-target cells therefore provide for the identification of BCGF activity without identifying other cytokine activities.
  • BCGF-dependent B cell lympho ⁇ cytes described in section c. below, is therefore of particular importance in the development of a sufficiently BCGF-specific bioassay. Additionally, such cells are necessary to provide an assay that is sufficiently BCGF- sensitive when used in conjunction with mRNA-primed Xenopus oocyte translations. Thus, to provide sufficient- ly BCGF-dependent B cell lymphocytes, it is generally preferrable to select such cells in the presence of highly purified quantities of BCGF. The following techniques provide such purification.
  • CM culture supernatants
  • the CM is then concentrated 20-fold using a Pellicon (Millipore) concentrator containing a 10,000 Dalton m.w. cut-off membrane.
  • the concentrated material is subse ⁇ quently diafiltered by using 10 vol. of lOmM Tris, pH 8.0. After centrifugation at 10,000 x G for 10 min. the clarified sample is loaded onto a previously equilibrated Pellicon (Millipore) concentrator containing a 10,000 Dalton m.w. cut-off membrane.
  • the concentrated material is subse ⁇ quently diafiltered by using 10 vol. of lOmM Tris, pH 8.0. After centrifugation at 10,000 x G for 10 min. the clarified sample is loaded onto a previously equilibrated
  • the concentrated sample (equivalent to 1 liter of crude CM) is then loaded onto a hydroxylapatite column (1-mm column with 30-ml bed volume) previously equilibrated with 10 mM sodium phosphate buffer. The column is subsequently washed with 0.04 M sodium phosphate until absorbance at 280 nm is reduced' to base line. The nonbound material is collected, whereas bound material is eluted with 150 mM sodium phosphate, pH 6.8. The flow-through and bound eluants are bioassayed for both BCGF and TCGF activities as described below.
  • This column chromatographic step of purification provides relatively good recovery (>24% yield) of BCGF biologic activity and generally yield BCGF essentially free of other important contaminating cytokine activities.
  • the hydroxylapatite-purified BCGF activity may next be chromatographed on an analytical HPLC-DEAE anion- exchange column (Protein Pak 5PW; Waters Associates) equilibrated in 15 mM phosphate buffer, pH 8.0. :
  • the loosely bound BCGF bioactivity eluted at 0.07 to 0.10 M salt concentration.
  • the recovery of the eluted BCGF bioactivity showed approximately 65% of the bioactivity contained in 1% of the total protein content.
  • the column was regenerated for repeat use after increasing the gradient concentration to 1 M salt to elute bound contaminating proteins and then equilibrating to the starting phosphate buffer containing no salt.
  • a highly reproducible elution pattern permitted the collection of about 10 consecutive runs of BCGF within a narrow gradient concentration or salt without any further loss of bioactivity or excessive contamination of closely spaced protein peaks.
  • BCGF activity isolated in a manner as described above may be futher purified on HPLC size exclusion columns.
  • a sample of reconstituted material (0.5 ml) obtained after affinity HPLC and lyophilization was loaded onto gel filtration columns (1-60 + 1-125 + I- 60; Waters Associates) in series that were previously equilibrated in 20 mM phosphate buffer, pH 7.0, at a flow rate of 0.5 ml/min.
  • a molecular weight calibration was performed by using bovine serum albumin (67,000 dalton) and myoglobin, (18,000 dalton) as standards, the BCGF bioactivity eluted as a sharp peak at a m.w. of 12,000 to 14,000, as determined by calibration of the column with the m.w. standards.
  • These fractions displayed BCGF activity when assayed with either anti-u-activated human B cells or BCGF-dependent long-term B cells lines. The recovery of the biologic ativity from the gel filtration was >35% of the applied BCGF activity.
  • the presently disclosed purification scheme involves multiple procedures, yet incorporates a single step for the removal of TCGF by hydroxylapatite chromatography. The remainder of the procedure after this separation step was analytically directed toward eliminating other contaminating proteins, an anion-exchange (DEAE-HPLC) and gel filtration on HPLC were used. The complete procedure yielded purification of BCGF with a specific activity of 10 5 to 10 6 U/mg of protein.
  • B cells which are BCGF- dependant for proliferation
  • B cells are first isolated from human peripheral blood.
  • B-cells are isolated essentially by negayive selection and are derived from the non-adherant E-rosette negative population of cells. Lymphocytes isolated in this manner are usually greater than are equal to 85% slg positive containing less than 2% E-rosette positive cells.
  • B-cells Following the preparation of B-cells from human peripheral blood, the isolated B-cells are activated with known B cell activators such as dextran sulfate staph protein A or anti-immunoglobulin, mu-chain specific. Such activation procedures are well known to those of skill in the art.
  • B cell activators such as dextran sulfate staph protein A or anti-immunoglobulin, mu-chain specific.
  • the activated B cells are grown in the presence of highly purified BCGF (free of TCGF activity) for approximately one month to select for BCGF-dependant B cells.
  • T lymphocytes sometimes found to grow out during the initial phases of culture are routinely removed by E- rosette dependant procedures.
  • the medium containing BCGF is replenished every 48-72 hours dependant upon cell growth. After approximately 30 days culture, if cells are proliferating, the cultures are maintained on BCGF and cell proliferation is continually monitored in terms of density and surface marker profile.
  • TCGF bioactivity 30- to 45- day-old T cell blasts, grown in. the present of TCGF, were washed free of factor and were used in culture at a final concentration of 1 x 10 /0.2 ml of culture volume with and without growth factor at 37°C for 72 hr (6). 3 H-Tdr was added during the final 16 hr. and incorporation was measured after cell harvesting.
  • BCGF-specific mRNA To enrich for BCGF-specific mRNA, relative size determinations for BCGF mRNA were made by sizing total poly A mRNA in association with in, vitro translation. Normal human T cells (500x10 ), in the presence of an obligate number of monocytes (5%) were stimulated with PHA (o.75%) for 20 hr at a density of 2 x 10 cells/mo. Total RNA was isolated using the RNase inhibior guanidinium isothiocyanate (cite), and CsCl gradient centrifugation. Poly A mRNA was obtained by one cycle elution of total RNA through an oligo-dT cellulose column. 300 ug of total RNA routinely contributed 10 ug of poly A RNA.
  • RNA was size fractionated on a 1% low melting agarose gel in the presence of 10 mM methyl-mercury hydroxide (cite); the gel was subsequently sliced. The RNA was eluted from each sliced gel fraction and injected into Xenopus laevis oocytes. Supernatants conditioned by the injected oocytes were assayed for BCGF and TCGF activity at multiple dilutions. Shown in Fig. 1A is a representative microtiter culture assay on long term cultured B and T cells. TCGF activity coded for by the 12 S mRNA served as an internal marker for size as well as for in vitro translation.
  • Two micrograms of 16-18 S mRNA was used as a template to synthesize double stranded cDNA using reverse transcriptase for insertion into a Pst I digested and oligo-dT tailed pUC 9 vector essentially as described by Heideker et al. (1983), supra.
  • the pUC 9 vector was selected with the prior assumption that the promoter of the B-galactosidase gene would direct the efficient expression of partial or full length cDNA molecules inserted in the correct sense orientation. Recombinant proteins would thus be translated as a fused product to a portion of the amino terminus of the B-galactosidase protein.
  • fusion proteins created between the aminoterminal end of B-galactosidase and peptides encoded by BCGF cDNA molecules might be biologically active for BCGF activity.
  • cDNA containing colonies were therefore grown and pooled into groups of 17 colonies each. 10 mis of bacteria in each pool were grown and cell extracts were made by sonication of cells followed by high speed centrifugation. Crude cell extracts containing 0.2 mM PMSF were then dialyzed against RPMI media. The dialzyed cell extracts were screened for BCGF activity using the microassay and the data demonstrated that only two pools consistently produced the lymphokine activity when compared to extracts from bacteria containing only the parental vector pUC 9.
  • each pool here found to contain only a single colony (the colony # denoted in parentheses) which exhibited BCGF activity. Plasmid DNA's were isolated and characterized for size. cDNA inserts in plasmids pARJ 45 (pool 4) and pARJ 43 (pool 11) were of approximately 400 bp and 700 bp respectively.
  • plasmids pARJ 45 and pARJ 43 contained BCGF specific cDNA inserts
  • these plasmids were used to hybrid select BCGF specific mRNA as described by Parnes et al. (1981), Proc. Natl. Acad, Sci. U.S.A., 78: 2253.
  • This assay serves to identify clones bearing BCGF- coding DNA sequences by the ability of the clonal DNA to select BCGF mRNA by DNA/RNA hybridization. Following such "hybrid selection" of mRNA, the mRNA is analyzed for its ability to prime the translation of proteins exhibiting BCGF activity. In particular, DNA was prepared from colonies containing these plasmids and was bound to nitrocellulose filters.
  • the filters were hybridized to mRNA from activated T cells.
  • pUC 9 and two other insert-containing plasmids (pARJ 36 and pARJ 55; inserts not related to the BCGF gene) were analyzed.
  • mRNA that hybridized to the various plasmid DNAs was injected into Xenopus oocytes and secreted products were assayed for BCGF activity.
  • Figure IB shows that plasmids pARJ 45 and pARJ 43 specifically hybridized to BCGF mRNA. Briefly, plasmid
  • DNAs (2 ug) were isolated and hybridized to poly A RNA (400 ug) from activated T cells as described by Fung et al. (1984) Nature, 307: 233. Bound mRNA was eluted by boiling for 1 minute and microinjected in Xenopus oocytes.
  • Cell extracts (supernatants) were obtained by centrifuga ⁇ tion at 25000 rpm for 30 min at 4 C and were immediately dialyzed overnight against 20 mM NaP0 4 (pH 7.5) buffer containing 0.2 mM PMSF. Dialyzed cell extracts were loaded onto a Sephadex G-50 gel filtration column. Proteins were eluted with 20 mM phosphate buffer (pH 7.0) and fractions were assayed for the BCGF activity using long term cultured B cells at various concentrations.
  • the extract from bacteria containing pUC 9 was negative for BCGF activity, whereas BCGF activity was found in the extracts from bacteria harboring pARJ 45 and pARJ 43.
  • the BCGF containing biologically active extracts may represent truncated proteins of approximately 14kD (pARJ 43) and 8-10 kD (pARJ 45).
  • the size estimations were made by direct comparison of the cell extracts with mature BCGF calibrated on the same gel filtration column. The size estimate with and respect to pARJ 43 was confirmed by DNA sequence techniques (see Example II).
  • Plasmid pARJ 43 was also tested by northern analysis for its specifi ity of interaction with BCGF mRNA using total mRNA from PHA activated T cells.
  • Northern blot anaysis was carried out by electrophoresis of 8 ug and 24 ug of poly A mRNA from PHA activated T cells (20 hr) on a 1% glyoxal-agarose gel. RNA samples were denatured with glyoxal for 1 hr at 50°C before gel electrophoresis. After elecrophoresis, the RNA was transferred to a nitro ⁇ cellulose paper and the blots were baked for 3 hrs at
  • BCGF cDNA specifically hybridized to a 17 S mRNA species confirming our earlier prediction of mRNA size leading to the use of 16-18S mRNA for cDNA library construction.
  • High molecular weight RNA species can be attributed to the presence of nuclear precursors.
  • the BCGF probe also detected a 11 s mRNA species .
  • This northern analysis was done under low-stringency conditions (5 x o standard saline citrate, 42 C) .
  • northern analysis using mRNA from a cell line devoid of BCGF secretory activity i.e. monocytes
  • Figure 3 depicts the restriction map of recombinant clone pARJ 43.
  • the DNA segment insert of pARJ 43 which bears the sequence coding for BCGF activity is slightly greater than 700 base pairs in length and is transcribed in the indicated direction.
  • the coding sequence of the BCGF is presented in Figure 4.
  • the corresponding BCGF amino acid sequence shown therein was derived from a consideration of the start, stop and amino-acid specifying codons found in this particular BCGF sequence. It will be appreciated that the DNA and amino acid sequences which precede the zero point (designated by negative numbers above the sequence segment) are derived from the B-galactosidase structural gene.
  • BCGF biological functional equivalents of BCGF may be obtained by substitution of amino acids having similar hydropathic values.
  • a biological functional equivalent of BCGF is defined as a protein that is functionally equivalent to BCGF in terms of biological activity.
  • isoleucine which has a hydropathic index of +4.5
  • valine (+4.2) or leucine (+3.8) can be substituted for valine (+4.2) or leucine (+3.8)
  • lysine -3.9
  • arginine arginine
  • amino acids can be successfully substituted where such amino acid has a hydropathic score of within about +/- 1 hydropathic index unit of the replaced amino acid.
  • E. coli SB229 (rec A ⁇ , JM 103, Tet ) bearing the preferred recombinant plasmid pARJ43 was deposited with the American Type Culture Collection, 12301 Parklawn Drive, Rockville, Maryland on April 18, 1986 in accordance with the Budapest Treaty and assigned ATCC accession number 67099. h. Purification of Recombinant BCGF
  • the recombinant cells are cultured in an acceptable bacterial culture medium or broth under conditions which allow for the transcription and
  • the cells After the recombinant cells have been cultured to a selected density, for example, an optical density of about 1.0 at 600 nm has been found to provide sufficient quantities of the recombinant BCGF protein, the cells are
  • the cells are then removed
  • the cells are hypotonically shocked, the intact cells removed by centrifugation or other similar means and the supernatant retained as a starting source for protein purification.
  • the recombinant BCGF cell culture mixture defined herein as any culture mixture which includes a recombinant microorganism producing recombinant BCGF, or extracts, lysates or sonicates of such microorganisms, is used for further purification of recombinant derived BCGF.
  • the supernatants are rendered devoid of insoluble cellular material and subjected to molecular fractionation to obtain a fraction which includes the recombinant BCGF in a relatively purified form.
  • relatively purified recombinant BCGF is defined as recombinant BCGF that is relatively devoid of contaminating biological or immunological activities. Numerous molecular fractionation techniques may be devised by those of skill in the art to provide a relatively purified fraction, however, the classical scheme for the relative purification of natural BCGF from conditioned media
  • BCGF protein may be synthesized by one of the various protein or DNA synthesizing techniques well known to those of skill in the art, from a consideration of the amino acid or nucleic acid sequence information provided by the present disclosure.
  • Both approaches will incorporate the advantages of the present invention by allowing the production of a BCGF preparation that will be devoid of contaminating cytokine and related activities. For simplicity sake, all such synthetically derived species are intended to be included by reference to recombinant derived BCGF.
  • the recombinant molecule may be formulated into a pharmaceutical composition for clinical application.
  • the composition is formulated to include effective concentrations of the BCGF protein together with an acceptable pharmaceutical diluent or excipient.
  • the compositions typically include various salts, buffers and/or stabilizing and preservative agents.
  • solutions of the novel compound may include sesame or peanut oil, aqueous propylene glycol, or the like, together in an aqueous sterile solution.
  • aqueous solutions should be suitably buffered if necessary and the liquid diluent first rendered isotonic with sufficient saline or glucose.
  • Such solutions are particularly suitable for intravenous, intramuscular, subcutaneous and intraperitoneal administration.
  • the sterile aqueous media employed are all readily available by standard techniques known to those skilled in the art.
  • Effective concentrations of recombinant BCGF will generally be determined based on the particular clinical application envisioned. However, treatment regimens employing BCGF typically require from about 10 5 to 107 units BCGF per application. Therefore, it will typically be desirable to provide formulations which include BCGF in concentrations sufficient to allow the administration of such dose units by whatever route is employed.
  • Human BCGF as described in the present application has been shown to be involved in the clonal expansion of normal mature human b-cells. This effector function will therefore likely possess utility both in those clinical situations of idiopathic immunodeficiencies and in those clinical situations where a B cell immunodeficiency state is induced by pharmocologic agents.
  • this growth factor may play a role in further understanding the B cell receptor for the growth factor.
  • the receptor has recently been demonstrated to be present on several neoplasms of B cell origin. Therefore, the described growth factor may help our understanding of receptor binding sites and thereby provide a means for the development of anti-receptor reagents.

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Abstract

Sont décrites l'isolement et la séquence d'acide nucléique d'un insert de vecteur recombinant d'ADNc qui contient la séquence de codage pour une protéine du facteur de croissance des cellules B humaines (BCGF). La séquence d'acides aminés prédite d'un BCGF humain est également décrite. Le vecteur recombinant portant l'ADNc a été sélectionné dans une banque de clones recombinants humains réalisée dans le vecteur pUC9. Les clones cellulaires recombinants réalisés selon les procédés ci-décrits produisent de manière active une protéine présentant une activité BCGF.
PCT/US1987/000268 1986-02-11 1987-02-10 Facteur de croissance des cellules b humaines obtenu par adn recombinant WO1987004723A1 (fr)

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US82838886A 1986-02-11 1986-02-11
US94490587A 1987-01-05 1987-01-05
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US828,388 1992-01-30

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US5013824A (en) * 1985-11-19 1991-05-07 Schering Corporation Human interleukin-4 peptides and conjugates thereof
US5700915A (en) * 1985-11-19 1997-12-23 Schering Corporations Human interleukin immunopurification processes
US5807996A (en) * 1985-11-19 1998-09-15 Schering Corporation Fused polypeptides comprising interleukin-4 polypeptide fragments
US5912136A (en) * 1985-11-19 1999-06-15 Schering Corporation Monoclonal antibodies against human interleukin-4 and hybridomas producing the same
US5955315A (en) * 1985-11-19 1999-09-21 Schering Corporation Nucleic acids encoding human interleukin-4
WO1999054469A1 (fr) * 1998-04-20 1999-10-28 Incyte Pharmaceuticals, Inc. Proteine liee au facteur de croissance des lymphocytes b

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PT83761B (pt) * 1985-11-19 1989-06-30 Schering Biotech Corp Metodo para a producao de interleuquina-4 de mamifero

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US5912136A (en) * 1985-11-19 1999-06-15 Schering Corporation Monoclonal antibodies against human interleukin-4 and hybridomas producing the same
US5955315A (en) * 1985-11-19 1999-09-21 Schering Corporation Nucleic acids encoding human interleukin-4
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