Claims for Patent: 6,479,258

Summary for Patent: 6,479,258

Title:	Non-stochastic generation of genetic vaccines
Abstract:	This invention provides methods of obtaining vaccines by use of non-stochastic methods of directed evolution (DirectEvolution.TM.). These methods include non-stochastic polynucleotide site-satuaration mutagenesis (Gene Site Saturation Mutagenesis.TM.) and non-stochastic polynucleotide reassembly (GeneReassembly.TM.). Through use of the claimed methods, vectors can be obtained which exhibit increased efficacy for use as genetic vaccines. Vectors obtained by using the methods can have, for example, enhanced antigen expression, increased uptake into a cell, increased stability in a cell, ability to tailor an immune response, and the like.
Inventor(s):	Short; Jay M. (Rancho Santa Fe, CA)
Assignee:	Diversa Corporation (San Diego, CA)
Application Number:	09/495,052
Patent Claims:	1. A method for obtaining an immunomodulatory polynucleotide that has an optimized modulatory effect on an immune response as compared to the response prior to optimization, or encodes a polypeptide that has an optimized modulatory effect on an immune response as compared to the response prior to optimization, the method comprising: creating a library of non-stochastically generated progeny polynucleotides from a parental polynucleotide set, wherein optimization is achieved by at least one directed evolution method in any combination, permutation and iterative manner. 2. The method of claim 1, wherein said optimized modulatory effect on an immune response is induced by a genetic vaccine vector. 3. A method for obtaining an immunomodulatory polynucleotide that has an optimized modulatory effect on an immune response as compared to the response prior to optimization,or encodes a polypeptide that has an optimized modulatory effect on an immune response as compared to the response prior to optimization, the method comprising: screening a library Qf non-stochastically generated progeny polynucleotides to identify an optimized non-stochastically generated progeny polynucleotide that has, or encodes a polypeptide that has, a modulatory effect on an immune response; wherein the optimized non-stochastically generated polynucleotide or the polypeptide encoded by the non-stochastically generated polynucleotide exhibits an enhanced ability to modulate an immune response compared to a parental polynucleotide from which the library was created. 4. The method of claim 3, wherein said optimized modulatory effect on an immune response is induced by a genetic vaccine vector. 5. A method for obtaining an immunomodulatory polynucleotide that has an optimized modulatory effect on an immune response as compared to the response prior to optimization, or encodes a polypeptide that has an optimized modulatory effect on an immune response as compared to the response prior to optimization, the method comprising: a) creating a library of non-stochastically generated progeny polynucleotides from a parental polynucleotide set; and b) screening the library to identify an optimized non-stochastically generated progeny polynucleotide that has, or encodes a polypeptide that has, a modulatory effect on an immune response induced by a vector; wherein the optimized non-stochastically generated polynucleotide or the polypeptide encoded by the non-stochastically generated polynucleotide exhibits an enhanced ability to modulate an immune response compared to a parental polynucleotide from which the library was created; and whereby optimization is achieved using one or more directed evolution methods in any combination, permutation, and iterative manner. 6. The method of claim 5, wherein said optimized modulatory effect on an immune response is induced by a genetic vaccine vector. 7. The method of any claims 1, 3 or 5, wherein the optimized non-stochastically generated polynucleotide is incorporated into a vector. 8. The method of any of claims 1, 3 or 5, wherein the optimized non-stochastically generated polynucleotide, or a polypeptide encoded by the optimized non-stochastically generated polynucleotide, is administered in conjunction with a vector. 9. The method of any of claims 1, 3 or 5, wherein the library of non-stochastically generated progeny polynucleotides is created by a process selected from gene reassembly or oligonucleotide-directed saturation mutagenesis, and any combination, permutation and iterative manner. 10. The method of any of claims 1, 3 or 5, wherein the optimized non-stochastically generated polynucleotide that has a modulatory effect on an immune response is obtained by: a) non-stochastically reassembling at least two parental template polynucleotide, each of which is, or encodes a molecule that is, involved in modulating an immune response; wherein the first and second parental templates differ from each other in two or more nucleotides, to produce a library of non-stochastically generated polynucleotides; and b) screening the library to identity at least one optimized non-stochastically generated polynucleotide that exhibits, either by itself or through the encoded molecule, an enhanced ability to modulate an immune response in comparison to a parental polynucleotide from which the library was created. 11. The method of claim 10, wherein the method further comprises the steps of: c) subjecting an optimized non-stochastically generated polynucleotide to a further round of non-stochastic reassembly with at least one additional polynucleotide, which is the same or different from the first and second polynucleotides, to produce a further library of recombinant polynucleotides; d) screening the library produced in c) to identify at least one further optimized non-stochastically generated polynucleotide that exhibits an enhanced ability to modulate an immune response in comparison to a parental polynucleotide from which the library was created; and e) optionally repeating c) and d) as necessary, until a desirable further optimized non-stochastically generated polynucleotide that exhibits an enhanced ability to modulate an immune response than a form of the nucleic acid from which the library was created. 12. The method of any of claims 1, 3 or 5, wherein the optimized non-stochastically generated polynucleotide encodes a polypeptide that interacts with a cellular receptor involved in mediating an immune response; wherein the polypeptide acts as an agonist or antagonist of the receptor. 13. The method of claim 12, wherein the cellular receptor is a macrophage scavenger receptor. 14. The method of claim 12, wherein the cellular receptor is selected from the group consisting of a cytokine receptor and a chemokine receptor. 15. The method of claim 14, wherein the chemokine receptor is CCR5 or CCR6. 16. The method of claim 12, wherein the polypeptide mimics the activity of a natural ligand for the receptor but does not induce immune reactivity to said natural ligand. 17. The method of claim 12, wherein the library is screened by: i) expressing the non-stochastically generated progeny polynucleotides so that the encoded polypeptides are produced as fusions with a protein displayed on the surface of a replicable genetic package; ii) contacting the replicable genetic packages with a plurality of cells that display the receptor; and iii) identifying cells that exhibit a modulation of an immune response mediated by the receptor. 18. The method of claim 17, wherein the replicable genetic package is selected from the group consisting of a bacteriophage, a cell, a spore, and a virus. 19. The method of claim 18, wherein the replicable genetic package is an M13 bacteriophage and the protein is encoded by geneIII or geneVIII. 20. The method of claim 12, which method further comprises introducing the optimized non-stochastically generated polynucleotide into a genetic vaccine vector and administering the vector to a mammal, wherein the peptide or polypeptide is expressed and acts as an agonist or antagonist of the receptor. 21. The method of claim 12, which method further comprises producing the polypeptide encoded by the optimized non-stochastically generated polynucleotide and introducing the polypeptide into a mammal in conjunction with a genetic vaccine vector. 22. The method of claim 12, wherein the optimized non-stochastically generated polynucleotide is inserted into an antigen-encoding nucleotide sequence of a genetic vaccine vector. 23. The method of claim 22, wherein the optimized non-stochastically generated polypeptide is introduced into a nucleotide sequence that encodes an M-loop of an HBsAg polypeptide. 24. The method of any of claims 1, 3 or 5, wherein the optimized non-stochastically generated polynucleotide comprises a nucleotide sequence rich in unmethylated CpG. 25. The method of any of claims 1, 3 or 5, wherein the optimized non-stochastically generated polynucleotide encodes a polypeptide that inhibits an allergic reaction. 26. The method of claim 25, wherein the polypeptide is selected from the group consisting of interferon-.alpha., interferon-.gamma., IL-10, IL-12, an antagonist of IL-4, an antagonist of IL-5, and an antagonist of IL-13. 27. The method of 1, wherein the optimized recombinant polynucleotide encodes an antagonist of IL-10. 28. The method of claim 27, wherein the antagonist of IL-10 is soluble or defective IL-10 receptor or IL-20/MDA-7. 29. The method of any of claims 1-6, wherein the optimized non-stochastically generated polynucleotide encodes a co-stimulator. 30. The method of claim 29, wherein the co-stimulator is B7-1 (CD80) or B7-2 (CD86) and the screening step involves selecting variants with altered activity through CD28 or CTLA-4. 31. The method of claim 29, wherein the co-stimulator is CD1, CD40, CD154 (ligand for CD40) or CD150 (SLAM). 32. The method of claim 29, wherein the co-stimulator is a cytokine. 33. The method of claim 32, wherein the cytokine is selected from the group consisting of IL-1, IL-2, IL-3, IL4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-18, GM-CSF, G-CSF, TNF-.alpha., IFN-.alpha., IFN-.gamma., and IL-20 (MDA-7). 34. The method of 33, wherein the library of non-stochastically generated polynucleotides is screened by testing the ability of cytokines encoded by the non-stochastically generated polynucleotides to activate cells which contain a receptor for the cytokine. 35. The method of claim 34, wherein the cells contain a heterologous nucleic acid that encodes the receptor for the cytokine. 36. The method of 33, wherein the cytokine is interleukin-12 and the screening is performed by: growing mammalian cells which contain the genetic vaccine vector in a culture medium; and detecting whether T cell proliferation or T cell differentiation is induced by contact with the culture medium. 37. The method of 33, wherein the cytokine is interferon-.alpha. and the screening is performed by: i) expressing the non-stochastically generated polynucleotides so that the encoded polypeptides are produced as fusions with a protein displayed on the surface of a replicable genetic package; ii) contacting the replicable genetic packages with a plurality of B cells; and iii) identifying phage library members that are capable of inhibiting proliferation of the B cells. 38. The method of claim 33, wherein the immune response of interest is differentiation of T cells to T.sub.H 1 cells and the screening is performed by contacting a population of T cells with the cytokines encoded by the members of the library of recombinant polynucleotides and identifying library members that encode a cytokine that induces the T cells to produce IL-2 and interferon-.gamma.. 39. The method of claim 32, wherein the cytokine encoded by the optimized non-stochastically generated polynucleotide exhibits reduced immunogenicity compared to a cytokine encoded by a non-optimized polynucleotide, and the reduced immunogenicity is detected by introducing a cytokine encoded by the non-stochastically generated polynucleotide into a mammal and determining whether an immune response is induced against the cytokine. 40. The method of claim 29, wherein the co-stimulator is B7-1 (CD80) or B7-2 (CD86) and the cell is tested for ability to costimulate an immune response. 41. The method of any of claims 1, 3, or 5, wherein the optimized recombinant polynucleotide encodes a cytokine antagonist. 42. The method of claim 41, wherein the cytokine antagonist is selected from the group consisting of a soluble cytokine receptor and a transmembrane cytokine receptor having a defective signal sequence. 43. The method of claim 41, wherein the cytokine antagonist is selected from the group consisting of .DELTA.IL-1 OR and .DELTA.IL-4R. 44. The method of any of claims 1, 3, or 5, wherein the optimized non-stochastically generated polynucleotide encodes a polypeptide capable of inducing a predominantly T.sub.H 1 immune response. 45. The method of any of claims 1, 3, or 5 wherein the optimized non-stochastically generated polynucleotide encodes a polypeptide capable of inducing a predominantly T.sub.H 2 immune response. 46. The method of any of claims 1, 3, or 5, wherein said optimized modulatory effect on an immune response is a decrease in an unwanted modulatory effect on an immune response; whereby application of the method can be used to generate a molecule having a decreased ability to elicit an immune response from a host recipient of said molecule, where said recipient can be a human or an animal host; and whereby application of the method can thus be used to generate a molecule having decreased antigenicity with respect to at least one host recipient of said molecule. 47. The method of any of claims 1, 3, or 5, wherein said optimized modulatory effect on an immune response is an increase in a desirable modulatory effect on an immune response; whereby application of the method can. be used to generate a molecule having an increased ability to elicit an immune response from a host recipient of said molecule, where said recipient can be a human or an animal host; and whereby application of the method can thus be used to generate a molecule having increased antigenicity with respect to at least one host recipient of said molecule. 48. The method of any of claims 1, 3, or 5, wherein said optimized modulatory effect on an immune response is both a decrease in a first unwanted modulatory effect on an immune response as well as an increase in a second desirable modulatory effect on an immune response; whereby application of the method can be used to generate a molecule having both a decreased ability to elicit a first immune response from a first host recipient of said molecule as well as a an increased ability to elicit a second immune response from a second host recipient of said molecule; whereby the first and the second recipient hosts can be the same or different; whereby each of the first and the second recipient hosts can be a human or an animal host; and whereby application of the method can thus be used to generate a molecule having both a first decreased antigenicity with respect to at least one host recipient of said molecule and a second decreased antigenicity with respect to at least one host recipient of said molecule. 49. The method of claim 48, wherein said first and said second modulatory effect on an immune response are evolved for respectively a first and a second module on the same multimodule vaccine vector; whereby a module is exemplified by the following modules, as well as by a fragment derivative or analog thereof: an antigen coding sequence, a polyadenylation sequence, a sequence coding for a co-stimulatory molecule, a sequence coding for an inducible repressor or transactivator, a eukaryotic origin or replication, a prokaryotic origin of replication, a sequence coding for a prokaryotic marker, and enhancer, a promoter, and operator, and an intron. 50. The method of any of claims 1, 3, or 5, wherein the optimized modulatory effect on an immune response is comprised of an increase in the stability of the immunomodulatory (IM) polynucleotide or polypeptide encoded thereby; whereby application of the method can be used to generate a molecule having an increased stability ex vivo, thus, for example, increasing shelf-life and/or ease of storage and/or length of time before expiration of activity upon storage; and whereby application of the method can also be used to generate a molecule having an increased stability in vivo upon administration to a host recipient, thus, for example, increasing resistance to digestive acids and/or increasing stability in the circulation and/or any other method of elimination or destruction by the host recipient. 51. The method of any of claims 1, 3, or 5, wherein the immunomodulatory (IM) polynucleotide or polypeptide encoded thereby; has an optimized modulatory effect on an immune response in a human host recipient as compared with prior to optimization; whereby application of the method can thus be used to generate an optimized genetic vaccine for human recipients. 52. The method of any of claims 1, 3, or 5, wherein the immunomodulatory (IM) polynucleotide or polypeptide encoded thereby; has an optimized modulatory effect on an immune response in an animal host recipient as compared with prior to optimization; whereby application of the method can thus be used to generate an optimized genetic vaccine for animal recipients, including animals that are farmed or raised by man, animals that are not fanned or raised by man, domesticated animals, and non-domesticated animals. 53. A method for obtaining an optimized polynucleotide that encodes an accessory molecule that improves the transport or presentation of antigens by a cell, the method comprising: a) creating a library of non-stochastically generated polynucleotides by subjecting to optimization by non-stochastic directed evolution a parental polynucleotide set in which is encoded all or part of the accessory molecule; and b) screening the library to identify an optimized non-stochastically generated progeny polynucleotide that encodes a recombinant molecule that confers upon a cell an increased or decreased ability to transport or present an antigen on a surface of the cell compared to an accessory molecule encoded by template polynucleotides not subjected to the non-stochastic reassembly; whereby application of the method can thus be used to generate an optimized molecule for human recipients &/or animal recipients, including animals that are farmed or raised by man, animals that are not farmed or raised by man, domesticated animals, and non-domesticated animals; whereby optimization can thus be achieved using one or more of the directed evolution methods as described herein in any combination, permutation, and iterative manner; whereby these directed evolution methods include the introduction of point mutations by non-stochastic methods, including by "gene site saturation mutagenesis" as described herein; and whereby these directed evolution methods also include the introduction mutations by non-stochastic polynucleotide reassembly methods as described herein; including by synthetic ligation polynucleotide reassembly as described herein. 54. The method of claim 53, wherein the screening involves: i) introducing the library of non-stochastically generated polynucleotides into a genetic vaccine vector that encodes an antigen to form a library of vectors; introducing the library of vectors into mammalian cells; and ii) identifying mammalian cells that exhibit increased or decreased immunogenicity to the antigen. 55. The method of claim 53, wherein the accessory molecule comprises a proteasome or a TAP polypeptide. 56. The method of claim 53, wherein the accessory molecule comprises a cytotoxic T-cell inducing sequence. 57. The method of claim 56, wherein the cytotoxic T-cell inducing sequence is obtained from a hepatitis B surface antigen. 58. The method of claim 53, wherein the accessory molecule comprises an immunogenic agonist sequence. 59. A method for obtaining an immunomodulatory polynucleotide that has, an optimized expression in a recombinant expression host, the method comprising: creating a library of non-stochastically generated progeny polynucleotides from a parental polynucleotide set; whereby optimization can thus be achieved using one or more of the directed evolution methods as described herein in any combination, permutation and iterative manner; whereby these directed evolution methods include the introduction of mutations by non-stochastic methods, including by "gene site saturation mutagenesis" as described herein; and whereby these directed evolution methods also include the introduction mutations by non-stochastic polynucleotide reassembly methods as described herein; including by synthetic ligation polynucleotide reassembly as described herein. 60. A method for obtaining an immunomodulatory polynucleotide that has an optimized expression in a recombinant expression host, the method comprising: screening a library of non-stochastically generated progeny polynucleotides to identify an optimized non-stochastically generated progeny polynucleotide that has an optimized expression in a recombinant expression host when compared to the expression of a parental polynucleotide from which the library was created. 61. A method for obtaining an immunomodulatory polynucleotide that has an optimized expression in a recombinant expression host, the method comprising: a) creating a library of non-stochastically generated progeny polynucleotides from a parental polynucleotide set; and b) screening a library of non-stochastically generated progeny polynucleotides to identify an optimized non-stochastically generated progeny polynucleotide that has an optimized expression in a recombinant expression host when compared to the expression of a parental polynucleotide from which the library was created; whereby optimization can thus be achieved using one or more of the directed evolution methods as described herein in any combination, permutation, and iterative manner; whereby these directed evolution methods include the introduction of point mutations by non-stochastic methods, including by "gene site saturation mutagenesis" as described herein; and whereby these directed evolution methods also include the introduction mutations by non-stochastic polynucleotide reassembly methods as described herein; including by synthetic ligation polynucleotide reassembly as described herein. 62. The method of any of claims 59-61, wherein the recombinant expression host is a prokaryote. 63. The method of any of claims 59-61, wherein the recombinant expression host is a eukaryote. 64. The method of claim 63, wherein the recombinant expression host is a plant. 65. The method of claim 64, wherein the recombinant expression host is a monocot. 66. The method of claim 64, wherein the recombinant expression host is a dicot. 67. The method of any of claims 1, 3, 5, 53, or 59-61, wherein creating a library of non-stochastically generated progeny polynucleotides from a parental polynucleotide set is comprised of subjecting the parental polynucleotide set to "gene site saturation mutagenesis" as described herein. 68. The method of any of claims 1, 3, 5, 53, or 59-61, wherein creating a library of non-stochastically generated progeny polynucleotides from a parental polynucleotide set is comprised of subjecting the parental polynucleotide set to "synthetic ligation polynucleotide reassembly" as described herein. 69. The method of any of claims 1, 3, 5, 53, or 59-61, wherein creating a library of non-stochastically generated progeny polynucleotides from a parental polynucleotide set is comprised of subjecting the parental polynucleotide set to both "gene site saturation mutagenesis" as described herein, and to "synthetic ligation polynucleotide reassembly" as described herein. 70. The method of claim 1, wherein the directed evolution method is synthetic ligation reassembly. 71. The method of claim 1, wherein the directed evolution method is gene site saturated mutagenesis. 72. The method of claim 1, wherein the directed evolution method is non-stochastic ligation reassembly. 73. The method of claim 1, wherein the directed evolution method is exonuclease-mediated reassembly. 74. The method of claim 1, wherein the directed evolution method is end selection. 75. The method of claim 1, wherein the directed evolution method is shuffling. 76. The method of claim 1, wherein the immunomodulatory polynucleofide encodes a cancer antigen. 77. The method of claim 1, wherein the immunomodulatory polynucleotide encodes a bacterial antigen. 78. The method of claim 1, wherein the immunomodulatory polynucleotide encodes a viral antigen. 79. The method of claim 1, wherein the immunomodulatory polynucleotide encodes a parasite antigen. 80. The method of claim 1, wherein the immunomodulatory polynucleotide encodes a self-antigen. 81. The method of claim 1, wherein the immune response is a humoral immune response. 82. The method of claim 1, wherein the immune response is a cellular immune response. 83. The method of claim 1, wherein the immunomodulatory polynucleotide encodes a cytokine. 84. The method of claim 83, wherein the cytokine is IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL14, IL-15, IL-16, IL-17, IL-18, GM-CSF, G-CSF, TNF-.alpha., IFN-.alpha., IFN-.gamma., or IL-20 (MDA-7). 85. The method as in any of claims 1, 3, or 5, wherein the immune response prior to optimization or following optimization is determined in vitro. 86. The method as in any of claims 1, 3, or 5, wherein the immune response prior to optimization or following optimization is determined in vivo.

Details for Patent 6,479,258

Applicant	Tradename	Biologic Ingredient	Dosage Form	BLA	Approval Date	Patent No.	Expiredate
Merck Sharp & Dohme Corp.	INTRON A	interferon alfa-2b	For Injection	103132	06/04/1986		2015-12-07
Merck Sharp & Dohme Corp.	INTRON A	interferon alfa-2b	For Injection	103132			2015-12-07
Merck Sharp & Dohme Corp.	INTRON A	interferon alfa-2b	Injection	103132			2015-12-07