# US 10,308,716 B2 · Method for producing polyclonal antibodies using an antigenic composition comprising protein-containing membrane vesicles > Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt - Number: US 10,308,716 B2 - Document ID: US10308716B2 - Status: Granted - Kind: B2 - Application number: 15/027,775 - Filed: 2014-09-24 - Published: 2016-09-01 - Granted: 2019-06-04 - Earliest claimed: 2013-10-15 - Assignees: Fraunhofer-Gesellschaft zur Foerderung der angewandten Forschung e. V. - Applicants: Fraunhofer-Gesellschaft zur Foerderung der angewandten Forschung e. V. - Inventors: Marlitt Stech; Katja Hanack; Katrin Messerschmidt; Stefan Kubick - Web page: https://patentgrep.com/patent/US10308716B2 ## CPC - C07K 16/2863: {against receptors for growth factors, growth regulators} - A61K 39/39: characterised by the immunostimulating additives, e.g. chemical adjuvants - C07K 14/43563: {from insects} - C07K 16/18: against material from animals or humans - C07K 16/30: from tumour cells - A61K 2039/505: {comprising antibodies} - A61K 2039/54: {characterised by the route of administration} - A61K 2039/572: {cytotoxic response} - A61K 2039/585: {wherein the target is cancer} - C07K 2317/10: characterized by their source of isolation or production - C12N 2710/24133: Use of viral protein as therapeutic agent other than vaccine, e.g. apoptosis inducing or anti-inflammatory - 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) - 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) - C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00) ## Abstract The invention relates to a method for producing polyclonal antibodies against an antigenic target protein which comprises inducing an immune response in a host by applying an immunogenic composition comprising membrane vesicles incorporating said antigenic target protein to said host and obtaining antibodies against said target protein from the host's serum. A more specific embodiment of this method comprises at least the following steps: a) synthesizing the target protein my means of an in vitro translation reaction in a reaction medium comprising a nucleic acid template coding for the target protein and a cell lysate which contains membrane vesicles; b) separating membrane vesicles comprising the synthesized target protein from the medium; c) providing the membrane vesicles comprising the synthesized target protein in a physiologically compatible medium; d) applying the membrane vesicles of step c) to a host; e) testing a serum sample of the host for a specific immune response against said target protein; and f) obtaining specific antibodies against said target protein from the serum of a host exhibiting a specific immune response against said target protein. ## Claims ### Claim 1 A method for producing polyclonal antibodies against an antigenic target protein which comprises inducing an immune response in a host by applying an immunogenic composition comprising membrane vesicles incorporating said antigenic target protein to said host and obtaining antibodies against said target protein from a serum of the host, wherein the membrane vesicles are ER-derived and the antigenic target protein is de novo synthesized in a eukaryotic cell-free system. ### Claim 2 (depends on 1) The method according to claim 1 comprising at least the following steps: a) synthesizing the target protein by an in vitro translation reaction in a reaction medium comprising a nucleic acid template coding for the target protein and a cell lysate which contains membrane vesicles; b) separating membrane vesicles comprising the target protein from the medium; c) providing the membrane vesicles comprising the target protein in a physiologically compatible medium; d) applying the membrane vesicles of step c) to a host; e) testing a serum sample of the host for a specific immune response against said target protein; and f) obtaining specific antibodies against said target protein from the serum of a host exhibiting a specific immune response against said target protein. ### Claim 3 (depends on 2) The method according to claim 2 wherein the cell lysate is a eukaryotic cell lysate. ### Claim 4 (depends on 3) The method according to claim 3 wherein the cell lysate is a member selected from the group consisting of wheat germ lysates, insect cell lysates, reticulocyte lysates, keratinocyte lysates, cell extracts from CHO cells, HeLa cells, myeloma cells, hybridoma cells and cultivated lymphoma cells. ### Claim 5 (depends on 2) The method according to claim 2, wherein the membrane vesicles and the cell lysate are derived from the same cell line. ### Claim 6 (depends on 2) The method according to claim 2, wherein the membrane vesicles comprising the target protein are separated from the medium by centrifugation. ### Claim 7 (depends on 2) The method according to claim 2, wherein the in vitro translation reaction in step a) is effected under conditions which promote enrichment of the target protein in the membrane vesicles. ### Claim 8 (depends on 7) The method according to claim 7, wherein step b) is followed by an additional step b′) which comprises transferring the separated membrane vesicles into a secondary reaction medium comprising a nucleic acid template coding for the target protein and a cell lysate which does not contain membrane vesicles, performing an in vitro translation reaction in said secondary reaction medium, and separating membrane vesicles comprising an increased amount of the synthesized target protein from the secondary medium, wherein step b′ may be repeated one or more times. ### Claim 9 (depends on 7) The method according to claim 7, wherein the in vitro translation reaction in step a) is performed by a continuous dialysis-based method involving the addition and/or discharge of reactants or products in a course of the translation reaction. ### Claim 10 (depends on 9) The method according to claim 9, wherein the in vitro translation reaction in step a) is performed in a presence of a caspase inhibitor. ### Claim 11 (depends on 1) The method according to claim 1, wherein the immunogenic composition does not contain any immunostimulatory adjuvant additional to the membrane vesicles incorporating the antigenic target protein. ### Claim 12 (depends on 1) The method according to claim 1, wherein the host is a non-human mammal. ### Claim 13 A method for accumulating a target protein in vesicles of a membrane vesicle preparation, comprising at least the following steps: a) synthesizing the target protein by an in vitro translation reaction in a reaction medium comprising a nucleic acid template coding for the target protein and a cell lysate which contains membrane vesicles; b) separating membrane vesicles comprising the target protein from the medium to provide separated membrane vesicles; c) transferring the separated membrane vesicles into a secondary reaction medium comprising a nucleic acid template coding for the target protein and a cell lysate which does not contain membrane vesicles, performing an in vitro translation reaction in said secondary reaction medium, and separating membrane vesicles comprising an increased amount of the target protein from the secondary medium, wherein step c) may be repeated one or more times. ### Claim 14 (depends on 4) The method of claim 4, wherein the cell lysate is an Sf21 cell lysate.