Claims for Patent: 9,487,773

Summary for Patent: 9,487,773

Title:	Cell-based methods for coupling protein interactions and binding molecule selection
Abstract:	The invention relates to cell-based methods for diversifying, expressing and selecting binding molecules, e.g., antibodies, and target molecules to which they bind, all of which are expressed in the same cell. The target molecule can be a member of a ligand binding pair comprising a cell-surface expressed ligand binding receptor molecule and its cognate ligand, which interact within the cell. The methods provide retaining either the antibody or its target in a cell organelle as the site of binding and interaction. By performance of the methods, the binding or non-binding of the antibody to its target molecule within the cell produces a cell phenotype that is detectable at the cell surface via high throughput assays, e.g., flow cytometry. The methods are particularly useful for generating, recovering and providing antibodies that have optimal target molecule binding properties or activities for potential therapeutic use. Methods for generating diversity in such antibodies are also provided.
Inventor(s):	Goncalves; Joao Manuel Braz (Lisbon, PT), da Silva; Frederico Nuno Castanheira Aires (Lisbon, PT), Corte-Real; Sofia Volker (Cruz Quebrada, PT), Santiago de Oliveira; Soraia Rafaela (Lisbon, PT)
Assignee:	TECHNOPHAGE, INVESTIGACAO E DESENVOLVIMENTO EM BIOTECNOLOGIA, SA (Lisbon, PT)
Application Number:	14/193,679
Patent Claims:	1. A method of selecting a binding molecule which blocks or disrupts an interaction between a cell surface expressed receptor protein and its cognate ligand within a cell, comprising: (a) expressing in the cell a receptor protein which is expressible on the surface of the cell; (b) expressing in the cell a cognate ligand of the receptor protein, said cognate ligand being molecularly tagged with a peptide for retaining the ligand in an intracellular organelle under conditions allowing for the retention of the ligand in the organelle and the interaction of the receptor protein and the cognate ligand in the organelle; wherein the intracellular organelle is selected from the group consisting of endoplasmic reticulum (ER), Golgi, cellular plasma membrane; mitochondrion; nucleus; and peroxisome; (c) introducing into the cell a binding molecule, or a binding fragment or portion thereof, which specifically binds either the receptor protein or the ligand protein retained in the intracellular organelle; and (d) detecting the level of the receptor protein expressed on the cell surface; wherein, if the binding molecule, or a binding fragment or portion thereof, binds to either the receptor protein or the ligand and blocks or disrupts their interaction in the organelle, the receptor protein is expressed and detectable on the cell surface, and wherein the binding molecule, or a binding fragment or portion thereof, which blocks or disrupts the receptor protein and ligand interaction is selectable. 2. The method according to claim 1, further comprising recovering or isolating the selected binding molecule, or a binding fragment or portion thereof, from the cell. 3. The method according to claim 1, wherein the binding molecule, or a binding fragment or portion thereof, is selected from an antibody, or a binding fragment or portion thereof, a genetically diversified antibody, or a binding fragment or portion thereof, a member of an antibody library, or a binding fragment or portion thereof, a member of a genetically diversified antibody library, or a binding fragment or portion thereof, a single domain antibody, a member of a single domain antibody library, a member of a genetically diversified single domain antibody library, a V.sub.H domain, a genetically diversified V.sub.H domain, a V.sub.L domain, or a genetically diversified V.sub.L domain, or a complementarity determining region (CDR). 4. The method according to claim 1, wherein the binding molecule binds to the receptor protein or to the cognate ligand. 5. The method according to claim 1, wherein the cognate ligand is molecularly tagged with a peptide for retaining the ligand in the endoplasmic reticulum (ER). 6. The method according to claim 5, wherein the ER retention sequence is set forth in SEQ ID NO: 1. 7. The method according claim 3, wherein the antibody, or a binding fragment or portion thereof, is selected from a Fab fragment, a Fab' fragment, a F(ab')2 fragment, an Fd fragment, an Fv fragment, a single-chain variable fragment (scFv), a diabody, or a domain antibody (dAb). 8. The method according to 3, wherein the antibody binding fragment or portion thereof is selected from (i) a V.sub.L domain or a genetically diversified V.sub.L domain; or (ii) a V.sub.H domain or a genetically diversified V.sub.H domain. 9. The method according to claim 1, wherein the binding molecule is a non-antibody molecule. 10. The method according to claim 1, wherein the binding molecule binds (i) to the receptor protein, or (ii) to the cognate ligand. 11. The method according to claim 1, wherein the cognate ligand is molecularly tagged with a peptide for retaining the ligand in the Golgi and wherein the Golgi retention signal peptide sequence is set forth in SEQ ID NO: 2. 12. A method of detecting whether a binding molecule, or a binding fragment or portion thereof, specifically binds to a cell surface expressed target protein within a cell, comprising: (a) expressing in a cell a target protein which is expressible on the surface of the cell; (b) expressing in the cell the binding molecule, or a binding fragment or portion thereof, fused to a sequence for retaining the binding molecule in an intracellular organelle selected from the group consisting of endoplasmic reticulum (ER), Golgi, cellular plasma membrane; mitochondrion; nucleus; and peroxisome; wherein, if the binding molecule, or a binding fragment or portion thereof, specifically binds to the target protein in the cell, the target protein is retained in the intracellular organelle bound to the binding molecule, or the binding fragment or portion thereof, thereby preventing expression of the target protein on the cell surface; and (c) detecting the level of the target protein expressed on the cell surface, such that a non-detectable or low level of the target protein detected on the cell surface indicates binding of the binding protein, or a binding fragment or portion thereof, to the target molecule in the cell. 13. The method according to claim 12, wherein the binding molecule, or a binding fragment or portion thereof, is molecularly tagged with a peptide for retaining the ligand in the endoplasmic reticulum (ER). 14. The method according to claim 13, wherein the ER retention sequence is set forth in SEQ ID NO: 1. 15. The method according to claim 12, further comprising recovering or isolating the binding molecule, or a binding fragment or portion thereof, from the cell. 16. The method according to claim 12, wherein the binding molecule, or a binding fragment or portion thereof, is selected from an antibody, or a binding fragment or portion thereof, a genetically diversified antibody, or a binding fragment or portion thereof, a member of an antibody library, or a binding fragment or portion thereof, a member of a genetically diversified antibody library, or a binding fragment or portion thereof, a single domain antibody, a member of a single domain antibody library, a member of a genetically diversified single domain antibody library, a V.sub.H domain, a genetically diversified V.sub.H domain, a V.sub.L domain, or a genetically diversified V.sub.L domain, or a complementarity determining region (CDR). 17. The method according to claim 16, wherein the antibody, or a binding fragment or portion thereof, is selected from a Fab fragment, a Fab' fragment, a F(ab')2 fragment, an Fd fragment, an Fv fragment, a single-chain variable fragment (scFv), a diabody, or a domain antibody (dAb). 18. The method according to 12, wherein the antibody binding fragment or portion thereof is selected from (i) a V.sub.L domain or a genetically diversified V.sub.L domain; or (ii) a V.sub.H domain or a genetically diversified V.sub.H domain. 19. A method of selecting for an antibody, or a binding fragment or portion thereof, that specifically binds to a target protein expressed on the surface of a cell, comprising: (a) expressing the target protein on the surface of cells in a cell line, wherein the cell surface-expressed target protein is detectable by a detectably labeled binding molecule, an antibody or a binding fragment or portion thereof, for selection for binding to the target protein, wherein the antibody, or a binding fragment or portion thereof, is fused to a signal peptide for retaining the antibody or a binding fragment or portion thereof in an intracellular organelle under conditions allowing for the interaction and binding of the antibody, or a binding fragment or portion thereof, and the target protein within the organelle; wherein the intracellular organelle is selected from the group consisting of endoplasmic reticulum (ER), Golgi, cellular plasma membrane; mitochondrion; nucleus; and peroxisome; and (b) detecting the level of the target protein expressed on the cell surface with the detectably labeled binding molecule; wherein, if the antibody, or a binding fragment or portion thereof, specifically binds to the target protein in the organelle, the target protein is bindably retained therein, thereby decreasing or eliminating the level of expression of the target protein on the cell surface; and indicating the retention of a selectable antibody, or a binding fragment thereof, in the cell. 20. The method according to claim 19, further comprising: recovering from the cells the antibody, or a binding fragment or portion thereof, that specifically binds to the target protein. 21. The method according to claim 19, wherein the antibody or a binding fragment or portion thereof is fused to a signal peptide for retaining the antibody, or a binding fragment or portion thereof, in the endoplasmic reticulum (ER) intracellular organelle. 22. The method according to claim 21, wherein the sequence encoding the peptide for retaining the antibody, or a binding fragment or portion thereof, in the ER is set forth in SEQ ID NO:1. 23. The method according to claim 19, wherein the antibody or a binding fragment or portion thereof is fused to a signal peptide for retaining the antibody, or a binding fragment or portion thereof, in the Golgi intracellular organelle. 24. The method according to claim 23, wherein the sequence encoding the peptide for retaining the antibody, or a binding fragment or portion thereof, in the Golgi is set forth in SEQ ID NO:2. 25. The method according to claim 19, wherein the target protein is a receptor protein or a membrane-expressible ligand protein. 26. The method according to claim 19, wherein the antibody or a binding fragment or portion thereof is selected from a member of an antibody library, a member of a genetically diversified antibody library, a member of an antibody single domain library, a member of a genetically diversified antibody single domain library; an antibody V.sub.L domain; a genetically diversified antibody V.sub.L domain; an antibody V.sub.H domain; or a genetically diversified antibody V.sub.H domain; a polyclonal antibody, a monoclonal antibody, a chimeric antibody, a humanized antibody and a fully human antibody. 27. The method according to claim 19, wherein the antibody, or a binding fragment or portion thereof, is selected from a Fab fragment, a Fab' fragment, a F(ab')2 fragment, an Fd fragment, an Fv fragment, a single-chain variable fragment (scFv), a diabody, or a domain antibody (dAb). 28. The method according to claim 19, wherein the level of the target protein expressed on the cell surface is detected by flow cytometry and a detectably labeled antibody. 29. A method of selecting from a plurality of binding molecules a binding molecule which binds a target antigen that is a member of an interacting pair comprising a cell surface-expressed receptor binding protein and a cognate ligand, the method comprising: (a) introducing a nucleic acid molecule encoding one of a plurality of binding molecules into cells expressing the receptor binding protein and the cognate ligand, wherein the cognate ligand is fused to a retention signal peptide that retains the cognate ligand in an intracellular organelle selected from the group consisting of endoplasmic reticulum (ER), Golgi, cellular plasma membrane; mitochondrion; nucleus; and peroxisome; (b) expressing in the cells the nucleic acid molecule of (a), wherein an expressed binding molecule binds to the receptor binding protein or to the cognate ligand target antigen retained in the organelle and disrupts or blocks the interaction between the receptor binding protein and the cognate ligand; and (c) detecting the level of receptor binding protein expressed on the cell surface; wherein, if the binding molecule binds to either the receptor binding protein or to the cognate ligand in the organelle and disrupts or blocks their interaction, the receptor binding protein is expressed and detectable on the cell surface, and wherein the binding molecule that disrupts or blocks the interaction is selectable. 30. The method according to claim 29, wherein the plurality of binding molecules is selected from one or more of: (i) an antibody V.sub.L library or a genetically diversified V.sub.L library; or (ii) an antibody V.sub.H library or a genetically diversified V.sub.H library. 31. The method according to claim 29, further comprising recovering or isolating the selectable binding molecule from the cell. 32. The method according to claim 29, wherein the plurality of binding molecules is selected from an antibody library, a genetically diversified antibody library, a single domain antibody library, a genetically diversified single domain antibody library, a V.sub.L domain library, a genetically diversified V.sub.L domain library, a V.sub.H domain library, or a genetically diversified V.sub.H domain library. 33. The method according to claim 29, wherein the binding molecule is an antibody or a binding fragment or portion thereof. 34. The method according to claim 33, wherein the antibody binding fragment or portion is selected from a Fab fragment, a Fab' fragment, a F(ab')2 fragment, an Fd fragment, an Fv fragment, a single-chain variable fragment (scFv), a diabody, a domain antibody (dAb), a heavy chain variable domain (V.sub.H), a genetically diversified heavy chain variable domain (V.sub.H), a light chain variable domain (V.sub.L), a genetically diversified light chain variable domain (V.sub.L), or a complementarity determining region (CDR). 35. The method according to claim 33, wherein the antibody binding fragment or portion is selected from a V.sub.L domain; a genetically diversified V.sub.L domain; a V.sub.H domain; or a genetically diversified V.sub.H domain. 36. The method according to claim 29, wherein the intracellular organelle is the endoplasmic reticulum (ER) and the cognate ligand is molecularly tagged with an ER retention peptide having the sequence as set forth in SEQ ID NO: 1. 37. The method according to claim 29, wherein the intracellular organelle is the Golgi and the cognate ligand is molecularly tagged with an Golgi retention peptide having the sequence as set forth in SEQ ID NO: 2. 38. A method of selecting from a plurality of binding molecules, a binding molecule, or a binding portion thereof, that binds a cell surface expressed target protein, comprising: (a) introducing into cells a nucleic acid molecule encoding one of a plurality of binding molecules and operably linked to a nucleic acid molecule encoding a intracellular organelle retention signal peptide, wherein the cells express the target protein which is expressible on the cell surface; (b) expressing in the cells one of the plurality of binding molecules comprising the retention signal peptide; wherein, if one of the plurality of binding molecules comprises a binding molecule, or a binding portion thereof, that specifically binds to the target protein, the target protein is retained in the cell organelle through its being bound to the binding molecule, or a binding portion thereof, which is retained in the intracellular organelle, thereby preventing both exit of the target protein from the organelle and expression of the target protein on the cell surface; and (c) detecting the level of the target protein expressed on the cell surface, such that a non-detectable or low level of the target protein detected on the cell surface indicates specific binding of the binding molecule, or the binding portion thereof, to the target molecule in the cell, wherein the binding molecule, or the binding portion thereof, is selectable from the cells. 39. The method according to claim 38, wherein the nucleic acid molecule encoding one of a plurality of binding molecules is introduced into the cells by lentivirus particles harboring the nucleic acid molecule. 40. The method according to claim 38, further comprising recovering or isolating the selected binding molecule from the cell. 41. The method according to claim 38, wherein the plurality of binding molecules is selected from an antibody library, a genetically diversified antibody library, a single domain antibody library, a genetically diversified single domain antibody library, a V.sub.L domain library, a genetically diversified V.sub.L domain library, a V.sub.H domain library, or a genetically diversified V.sub.H domain library. 42. The method according to claim 41, wherein the plurality of binding molecules is selected from one or more of: (i) an antibody V.sub.L library or a genetically diversified V.sub.L library; or (ii) an antibody V.sub.H library or a genetically diversified V.sub.H library. 43. The method according to claim 38, wherein the binding molecule or a binding portion thereof is an antibody or a binding fragment or portion thereof. 44. The method according to 198, wherein the antibody binding fragment or portion is selected from a Fab fragment, a Fab' fragment, a F(ab')2 fragment, an Fd fragment, an Fv fragment, a single-chain variable fragment (scFv), a diabody, a domain antibody (dAb), a heavy chain variable domain (V.sub.H), a genetically diversified heavy chain variable domain (V.sub.H), a light chain variable domain (V.sub.L), a genetically diversified light chain variable domain (V.sub.L), or a complementarity determining region (CDR). 45. The method according to claim 38, wherein the binding molecule, or a binding portion thereof, is selected from one or more of: (i) a V.sub.L domain or a genetically diversified V.sub.L domain; or (ii) a V.sub.H domain or a genetically diversified V.sub.H domain. 46. The method according to claim 38, wherein the intracellular organelle retention signal peptide is the ER signal peptide having the sequence set forth in SEQ ID NO: 1 or the Golgi signal peptide having the sequence set forth in SEQ ID NO: 2. 47. A method of preventing or knocking down cell surface expression of a ligand-binding receptor protein, comprising: (a) expressing in a cell the ligand-binding receptor protein; (b) expressing in the cell a ligand protein capable of being bound by the ligand-binding receptor protein, wherein the ligand protein is fused to exogenous signal peptide for retaining the ligand protein in an intracellular organelle, under conditions permitting the interaction of the ligand-binding receptor protein and the ligand protein in the organelle wherein the intracellular organelle is selected from the group consisting of endoplasmic reticulum (ER), Golgi, cellular plasma membrane; mitochondrion; nucleus; and peroxisome; (c) measuring the level of the ligand-binding receptor protein expressed on the cell surface; wherein the binding of the ligand-binding receptor protein to the ligand protein retained in the organelle concomitantly retains the ligand-binding receptor protein bound to the ligand protein in the organelle, thereby preventing or knocking down the cell surface expression of the ligand-binding receptor protein. 48. A method of selecting from a plurality of binding molecules a binding molecule which binds a target antigen that is a member of an interacting pair comprising a cell surface-expressed receptor binding protein and a cognate ligand, the method comprising: (a) co-expressing in a single cell which expresses the receptor binding protein (i) a nucleic acid molecule encoding one of a plurality of binding molecules, and (ii) a nucleic acid molecule encoding a cognate ligand of the receptor binding protein, wherein either the nucleic acid molecule encoding the binding molecule or the nucleic acid molecule encoding the cognate ligand is operably coupled to a nucleic acid molecule encoding a retention signal peptide for retaining either (i) or (ii) in an intracellular organelle following expression of (i) or (ii) in the cell; under conditions allowing for retention of the expressed binding molecule or the expressed cognate ligand in the intracellular organelle; wherein the intracellular organelle is selected from the group consisting of endoplasmic reticulum (ER), Golgi, cellular plasma membrane; mitochondrion; nucleus; and peroxisome and wherein, if an expressed binding molecule binds to the receptor binding protein or the cognate ligand as target antigen, such binding disrupts, neutralizes, or blocks the natural interaction between the receptor binding protein and the cognate ligand and releases the receptor binding protein from its interaction with the cognate ligand for expression of the receptor binding protein on the cell surface; and (b) detecting the level of receptor binding protein expressed on the cell surface such that a high level of receptor binding protein expression of the cell surface indicates the expression and selection of a binding molecule which binds a target antigen within the cell. 49. The method according to claim 48, wherein the plurality of binding molecules is selected from an antibody library, a genetically diversified antibody library, a single domain antibody library, a genetically diversified single domain antibody library, a V.sub.L domain library, a genetically diversified V.sub.L domain library, a V.sub.H domain library, or a genetically diversified V.sub.H domain library. 50. The method according to claim 48, wherein the binding molecule is an antibody or a binding fragment or portion thereof. 51. The method according to 50, wherein the antibody or a binding fragment or portion thereof is selected from a Fab fragment, a Fab' fragment, a F(ab')2 fragment, an Fd fragment, an Fv fragment, a single-chain variable fragment (scFv), a diabody, a domain antibody (dAb), a heavy chain variable domain (V.sub.H), a genetically diversified heavy chain variable domain (V.sub.H), a light chain variable domain (V.sub.L), a genetically diversified light chain variable domain (V.sub.L), or a complementarity determining region (CDR). 52. The method according to 48, wherein the binding molecule is selected from one or more of: (i) a V.sub.L domain or a genetically diversified V.sub.L domain; or (ii) a V.sub.H domain or a genetically diversified V.sub.H domain. 53. The method according to claim 48, wherein the intracellular organelle is the endoplasmic reticulum (ER) and the cognate ligand is molecularly tagged with an ER retention peptide having the sequence as set forth in SEQ ID NO: 1. 54. The method according to claim 48, wherein the intracellular organelle is the Golgi and the cognate ligand is molecularly tagged with an Golgi retention peptide having the sequence as set forth in SEQ ID NO: 2. 55. The method according to claim 48, wherein the target antigen is selected from growth factor receptors, hormone receptors, enzyme receptors, Fc receptors, neurotransmitter receptors, chemokine receptors, cytokine receptors, lymphokine receptors, interleukin receptors, tumor antigen receptors, cell recognition or stimulatory receptors, receptors in receptor families selected from apolipoprotein receptors, EGFR, ErbB-1R, HER1, HER2, aFGFR, bFGFR, NGFR, VEGFR, FltR, TGFR, TGFR-.alpha.-1, TGFR-.beta., TNFR-.alpha., BDNFR, insulin receptor, insulin-like growth factor receptor (IGFR), PDGFR, HGFR, TRKR, BDNFR, CNTFR, GMFR, NT3R, NT5R, HARPR/pleiotrophinR, TIE receptors, Eph receptors, DDR receptors, ROR receptors, LTK receptors, AXL receptors, RET receptors, or TOLL-like receptors; steroid hormone receptors, thyroid hormone receptors, melatonin receptors, adrenergic receptors; receptors for peptides selected from amylin, angiotensinogen, angiotensin, atrial natriuretic peptide, brain natriuretic peptide, calcitonin, corticotropin, erythropoietin, endothelin, enkephalin, follicle stimulating hormone, gastrin, ghrelin, glucagon, human chorionic gonadotropin, inhibin, leptin, luteinizing hormone, melanocyte stimulating hormone, oxitocin, pancreatic polypeptide, parathyroid hormone, prolactin, prolactin releasing hormone, rennin, secretin, somatostatin, thrombopoietin, thyroid stimulating hormone, or thyrotropin releasing hormone; or GP130 or IL6 receptors. 56. The method according to claim 48, wherein the target antigen is selected from growth factors, hormones, enzymes, metabolic enzymes, neurotransmitters, chemokines, cytokines, lymphokines, interleukin, tumor antigens, tumor suppressor antigens, multidrug resistance proteins, coagulation factors, Factor VII, Factor VIII, Factor IX, trophic factors, cell recognition or stimulatory molecules, apolipoproteins, EGF, ErbB-1, HER1, HER2, aFGF, bFGF, NGF, VEGF, Flt, TGF, TGF-.alpha.-1, TGF-.beta., TNF-.alpha., BDNF, insulin, insulin-like growth factor (IGFR), PDGF, HGF, TRK, BDNF, CNTF, GMF, NT3, NT5, HARP/pleiotrophin, TIE proteins, Eph proteins, DDR proteins, ROR proteins, LTK proteins, AXL proteins, RET proteins, TOLL-like proteins; hormones selected from steroid hormones, thyroid hormones, melatonin; adrenergic protein; peptides selected from amylin, angiotensinogen, angiotensin, atrial natriuretic peptide, brain natriuretic peptide, calcitonin, corticotropin, erythropoietin, endothelin, enkephalin, follicle stimulating hormone, gastrin, ghrelin, glucagon, human chorionic gonadotropin, inhibin, leptin, luteinizing hormone, melanocyte stimulating hormone, oxitocin, pancreatic polypeptide, parathyroid hormone, prolactin, prolactin releasing hormone, rennin, secretin, somatostatin, thrombopoietin, thyroid stimulating hormone, thyrotropin releasing hormone; GP130 or IL6.

Details for Patent 9,487,773

Applicant	Tradename	Biologic Ingredient	Dosage Form	BLA	Approval Date	Patent No.	Expiredate
Ferring Pharmaceuticals Inc.	NOVAREL	chorionic gonadotropin	For Injection	017016	01/15/1974		2033-03-01
Ferring Pharmaceuticals Inc.	NOVAREL	chorionic gonadotropin	For Injection	017016	12/27/1984		2033-03-01
Ferring Pharmaceuticals Inc.	NOVAREL	chorionic gonadotropin	For Injection	017016	02/15/1985		2033-03-01
Ferring Pharmaceuticals Inc.	NOVAREL	chorionic gonadotropin	For Injection	017016	02/16/1990		2033-03-01
Bel-mar Laboratories, Inc.	CHORIONIC GONADOTROPIN	chorionic gonadotropin	Injection	017054	03/26/1974		2033-03-01