# US 5,346,988 A · Process for obtaining cellular protein having anti-HIV activity > Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt - Number: US 5,346,988 A - Document ID: US5346988A - Status: Granted - Kind: A - Application number: 07/659,564 - Filed: 1991-02-22 - Granted: 1994-09-13 - Earliest claimed: 1990-06-20 - Assignees: Tampa Bay Research Institute - Inventors: Meihan Nonoyama; Akiko Tanaka; Patrick K. Lai - Web page: https://patentgrep.com/patent/US5346988A ## CPC - C07K 14/47: from mammals - C12N 5/0694: {Cells of blood, e.g. leukemia cells, myeloma cells} - A61K 38/00: Medicinal preparations containing peptides (peptides containing beta-lactam rings A61K31/00; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, A61K31/00; ergot alkaloids of the cyclic peptide type A61K31/48; containing macromolecular compounds having statistically distributed amino acid units A61K31/74; medicinal preparations containing antigens or antibodies A61K39/00; medicinal preparations characterised by the non-active ingredients, e.g. peptides as drug carriers, A61K47/00) - C07K: PEPTIDES (peptides in foodstuffs A23; obtaining protein compositions for foodstuffs, working-up proteins for foodstuffs A23J; preparations for medicinal purposes A61K; peptides containing beta-lactam rings C07D; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, C07D; ergot alkaloids of the cyclic peptide type C07D519/02; macromolecular compounds having statistically distributed amino acid units in their molecules, i.e. when the preparation does not provide for a specific; but for a random sequence of the amino acid units, homopolyamides and block copolyamides derived from amino acids C08G69/00; macromolecular products derived from proteins C08H1/00; preparation of glue or gelatine C09H; single cell proteins, enzymes C12N; genetic engineering processes for obtaining peptides C12N15/00; compositions for measuring or testing processes involving enzymes C12Q; investigation or analysis of biological material G01N33/00) - C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00) - A61K: PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES (devices or methods specially adapted for bringing pharmaceutical products into particular physical or administering forms A61J3/00; chemical aspects of, or use of materials for deodorisation of air, for disinfection or sterilisation, or for bandages, dressings, absorbent pads or surgical articles A61L; soap compositions C11D) ## Abstract A process for obtaining cellular protein having potent anti-HIV activity comprises culturing CD4-positive T cells or myeloid cells in a tissue culture medium under conditions in which the cells are induced to produce and secrete a protein having anti-HIV activity, separating the cells from the culture medium containing the protein and then recovering the protein from the medium, wherein the molecular weight of the protein having anti-HIV activity, as measured by gel filtration analysis, is within the range of about 7,000 to about 12,000 daltons. ## Claims ### Claim 1 A method for obtaining a cellular protein having anti-HlV activity which comprises: (a) culturing CD4-positive T cells in a tissue culture medium under conditions in which the cells are induced to produce and secrete at least one protein soluble in the culture medium having anti-HIV activity, (b) separating the cells from the culture medium containing the protein having anti-HIV activity, and (c) recovering the protein from the medium, wherein the protein is characterized by a molecular weight of about 7,000 to about 12,000 daltons as determined by gel filtration analysis. ### Claim 2 (depends on 1) The method of claim 1, wherein the tissue culture medium comprises an agent to activate the cells. ### Claim 3 (depends on 2) The method of claim 2, wherein the activating agent comprises PC6, alkaline lignin, de-alkaline lignin or lignin sulfonate. ### Claim 4 (depends on 2) The method of claim 2, wherein the CD.sub.4 -positive T cells are CEM cells. ### Claim 5 (depends on 3) The method of claim 3, wherein the cells are seeded in the culture medium at a concentration of about 1.times.10.sup.5 cells/ml to about 6.times.10.sup.5 cells/ml and the activating agent is provided at a concentration of from about 1 .mu.g/ml to about 30 .mu.g/ml. ### Claim 6 (depends on 5) The method of claim 5, wherein the cells are grown for about 1-7 days. ### Claim 7 (depends on 2) The method of claim 2, wherein the culture medium further comprises 5% v/v bovine serum, 100 units/ml penicillin and 100 .mu.g/ml streptomycin. ### Claim 8 A method for obtaining a cellular protein having anti-HIV activity which comprises: (a) culturing myeloid cells in a tissue culture medium comprising PC6, alkaline lignin, de-alkaline lignin or lignin sulfonate under conditions in which the cells are induced to produce and secrete at least one protein soluble in the culture medium having anti-HIV activity; (b) separating the cells from the culture medium containing the protein having anti-HIV activity; and (c) recovering the protein from the medium, wherein the protein is characterized by a molecular weight of about 7,000 to about 12,000 daltons as determined by gel filtration anaylsis. ### Claim 9 (depends on 8) The method of claim 8, wherein the myeloid cells are U937 or THP-1 cells. ### Claim 10 (depends on 8) The method of claim 8, wherein the protein having anti-HIV activity is recovered from the culture medium by (a) passing the medium containing the anti-HIV protein through a 30 kDa molecular sieve and retaining that portion of the medium which passed through the sieve and (b) passing the retained portion of the medium through a 10 kDa molecular sieve and collecting that part of the retained portion which does not pass through the sieve. ### Claim 11 (depends on 10) The method of claim 10, wherein the anti-HIV protein is purified by adjusting the pH of the collected part of the retained portion of the medium to be within the range of about 6.8 to about 7.5, then loading it onto a basic ion exchange. column, eluting the column with an elution buffer containing a linear gradient of 50 mM to 1M sodium chloride, collecting the resulting eluate in a series of fractions, and recovering the fraction or fractions obtained with 200 mM to about 350 mM sodium chloride eluant. ### Claim 12 (depends on 10) The method of claim 10, wherein the anti-HIV protein is purified by adjusting the pH of the collected part of the retained portion of the medium to be within the range of about 6.8 to about 7.5, then loading it onto a basic ion exchange column, eluting the column with an elution buffer containing a linear gradient of 50 mM to 1M sodium chloride, collecting the resulting eluate in a series of fractions, and recovering the fraction or fractions obtained with 600 mM sodium chloride eluant. ### Claim 13 (depends on 1, 8) The method of claim 1 or 8, wherein the protein having anti-HIV activity is recovered from the culture medium by (a) passing the medium containing the anti-HIV protein through a 30 kDa molecular sieve and retaining that portion of the medium which passed through the sieve and (b) passing the retained portion of the medium through a 10 kDa molecular sieve and collecting that part of the retained portion which does not pass through the sieve. ### Claim 14 (depends on 13) The method of claim 13, wherein the anti-HIV protein in the collected part of the retained portion of the medium is precipitated from the medium by adding a supersaturated aqueous solution of ammonium sulfate to the medium. ### Claim 15 (depends on 9) The method of claim 9, wherein the anti-HIV protein is purified by adjusting the pH of the collected part of the retained portion of the medium to be within the range of about 6.8 to about 7.5, then loading it onto a basic ion exchange column, eluting the column with an elution buffer containing a linear gradient of 50 mM to 1M sodium chloride, collecting the resulting eluate in a series of fractions, and recovering the fraction or fractions obtained with 200 mM to about 350 mM sodium chloride eluant. ### Claim 16 (depends on 13) The method of claim 13, wherein the anti-HIV protein is purified by adjusting the pH of the collected part of the retained portion of the medium to be within the range of about 6.8 to about 7.5, then loading it onto a basic ion exchange column, eluting the column with an elution buffer containing a linear gradient of 50 mM to 1M sodium chloride, collecting the resulting eluate in a series of fractions, and recovering the fraction or fractions obtained with 600 mM sodium chloride eluant. ### Claim 17 A cellular protein having anti-HIV activity produced by (a) culturing CD4 positive T cells or myeloid cells in a tissue culture medium comprising PC6 to activate the cells; (b) incubating said tissue culture medium for a period of time sufficient to induce said cells to produce a protein having anti-HIV activity; (c) separating the cells from the culture medium containing the protein having anti-HIV activity; and (d) recovering the protein from the medium, by passing the medium containing the anti-HIV protein through a 30 kDa molecular sieve and retaining that portion of the medium which passed through the sieve, wherein the protein is characterized by a molecular weight of about 7,000 to about 12,000 daltons as determined by gel filtration analysis. ### Claim 18 (depends on 17) The cellular protein of claim 17, wherein the cells being cultured are CEM cells. ### Claim 19 (depends on 17) The cellular protein of claim 17, wherein the cells being cultured are U937 cells.