(1A) 11‘ lLl‘lVAllUlVAL Al’l’LlLAl lUlV I’UDLIDHLU UlVUlLl‘ 111114 I’Al LIV l LUUI’LI‘AIIUI‘ lKLAl I (1’1, 1)
`
`(19) World Intellectual Property Organization
`
`International Bureau
`
`(43) International Publication Date
`21 November 2002 (21.11.2002)
`
`PCT
`
`(10) International Publication Number
`W0 02/092780 A2
`
`(51) International Patent Classification7:
`
`(21) International Application Number:
`
`C12N (74) Agent: EINHORN, Gregory, R; Fish & Richardson RC,
`4350 La Jolla Village Drive, Suite 500, San Diego, CA
`92122 (US).
`
`PCT/US02/15767
`
`(22) International Filing Date:
`
`17 May 2002 (17.05.2002)
`
`(25) Filing Language:
`
`(26) Publication Language:
`
`(30) Priority Data:
`60/300,381
`60/300,907
`
`English
`
`English
`
`17 May 2001 (17.05.2001)
`25 June 2001 (25.06.2001)
`
`US
`US
`
`(63) Related by continuation (CON) or continuation-in-pa rt
`(CIP) to earlier applications:
`3? d
`U18" 0“
`Filed on
`
`203030359867 :55?
`““0 60/380 381'“:ng
`’
`17 May 2001 (17.05.2001)
`
`25 J
`
`(81) Designated States (national): AE, AG, AL, AM, AT, AU,
`AZ, BA, BB, BG, BR, BY, BZ, CA, CH, CN, CO, CR, CU,
`CZ, DE, DK, DM, DZ, EC, EE, ES, FI, GB, GD, GE, GH,
`GM, HR, HU, ID, IL, IN, IS, JP, KE, KG, KP, KR, KZ, LC,
`LK, LR, LS, LT, LU, LV, MA, MD, MG, MK, MN, MW,
`MX, M2, NO, NZ, OM, PH, PL, PT, RO, RU, SD, SE, SG,
`SI, SK, SL, TJ, TM, TN, TR, TT, TZ, UA, UG, US, UZ,
`VN, YU, ZA, ZM, ZW,
`
`(84) Designated States (regional): ARIPO patent (GH, GM,
`KE, LS, MW, MZ, SD, SL, SZ, TZ, UG, ZM, ZW),
`Eurasian patent (AM, AZ, BY, KG, KZ, MD, RU, TJ, TM),
`European patent (AT, BE, CH, CY, DE, DK, ES, FI, FR,
`GB, GR, IE, IT, LU, MC, NL, PT, SE, TR), OAPI patent
`BFBJCFCGCICMGAGNG GWMLMR
`(
`’
`’
`’
`’
`’
`’
`’
`’ Q’
`’
`’
`’
`NE, SN, TD, TG).
`
`(for all designated States except US): DI-
`(71) Applicant
`VERSA CORPORATION [US/US]; 4955 Directors
`Place San Diego CA 92121 (US).
`
`Published=
`7 without international search report and to be republished
`upon receipt of that report
`
`(72) Inventor; and
`For two-letter codes and other abbreviations, refer to the "Guid-
`Jay,
`SHORT,
`(for US only):
`(75) Inventor/Applicant
`M.
`[US/US]; PO. Box 7214, Rancho Santa Fe, CA ance Notes on Codes and Abbreviations " appearing at the begin-
`92067—7214 (US).
`ning ofeach regular issue ofthe PCT Gazette.
`
`2
`
`W002/092780
`
`NOVEL ANTIGEN BINDING MOLECULES FOR THERAPEUTIC, DIAGNOSTIC, PROPHYLACTIC,
`(54) Title:
`ENZYMATIC,
`INDUSTRIAL, AND AGRICULTURAL APPLICATIONS, AND METHODS FOR GENERATING AND
`SCREENING THEREOF
`
`(57) Abstract: The invention is directed to methods for generating sets, or libraries, of nucleic acids encoding antigen—binding sites,
`such as antibodies, antibody domains or other fragments, including single and double stranded antibodies, major histocompatibility
`complex (MHC) molecules, T cell receptors (TCRs), and the like. This invention provides methods for generating variant antigen
`binding sites, e. g., antibodies and Specific domains or fragments of antibodies (e.g., Fab or PC domains), by altering template nucleic
`acids including by saturation mutagenesis, synthetic ligation reassembly, or a combination thereof.
`In one aspect, the invention
`provides methods for generating all human or humanized antibodies and evolving them to achieve optimized properties related to
`stability, duration, expression, production, enzymatic activity, affinity, avidity, localization, and other immunological properties.
`Polypeptides generated by these methods can be analyzed using a novel capillary array platform, which provides unprecedented
`ultra—high throughput screening.
`
`

`

`W0 02/092780
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`PCT/US02/15767
`
`NOVEL ANTIGEN BINDING MOLECULES FOR
`
`THERAPEUTIC, DIAGNOSTIC, PROPHYLACTIC,
`ENZYMATIC, INDUSTRIAL, AND AGRICULTURAL
`APPLICATIONS, AND METHODS FOR GENERATING AND
`SCREENING THEREOF
`
`CROSS-REFERENCES TO RELATED APPLICATIONS
`
`The present application claims the benefit of priority under 35 U.S.C. §119(e) of US.
`
`Provisional Application Nos. 60/300,381, filed May 17, 2001, and 60/300,907, filed June 25,
`
`2001; and is a continuation—in—part
`
`(CIP) of US. Patent Application Serial No.
`
`(USSN)
`
`09/535,754, filed March 27, 2000 (entitled Exonuclease-Mediated Gene Assembly in Directed
`
`Evolution); which is a CIP of USSN 09/522,289, filed March 9, 2000 (entitled End Selection in
`
`Directed Evolution); which is a CIP of USSN 09/498,557, filed February 4, 2000 (entitled Non—
`
`Stochastic Generation of Genetic Vaccines and Enzymes), which is hereby incorporated by
`
`reference; which is a CIP of USSN 09/495,052, filed on January 31, 2000 (entitled Non—
`
`Stochastic Generation of Genetic Vaccines); which is a CIP of USSN 09/276,860, filed on March
`
`26, 1999 (entitled Exonuclease—Mediated Gene Assembly in Directed Evolution); which is a CIP
`
`of USSN 09/267,118, filed on March 9, 1999 (entitled End Selection in Directed Evolution);
`
`which is a continuation-in part of USSN 09/246,178, filed Feb. 4, 1999 (entitled Saturation
`
`Mutagenesis in Directed Evolution, now USPN 6,171,820); which is a continuation of USSN
`
`09/ 185,373 filed on November 3, 1998 (entitled Directed Evolution of Thermophilic Enzymes);
`
`which is a continuation of USSN 08/760,489 filed on Dec. 5, 1996 (entitled Directed Evolution of
`
`Thermophilic Enzymes, now USPN 5,830,696); which claims the benefit of US. provisional
`
`application number 60/008,311 filed on Dec. 07, 1995.
`
`USSN 09/246,178, filed Feb. 4, 1999 (entitled Saturation Mutagenesis in Directed
`
`Evolution) is also a CIP of USSN 08/962,504 filed on October 31, 1997 (entitled Method of DNA
`
`Shuffling); which is a CIP of USSN 08/677,112 filed on July 9, 1996 (entitled Method of DNA
`
`Reassembly by Interrupting Synthesis, now USPN 5,965,408).
`
`“
`
`USSN 09/246,178,
`
`filed Feb. 4, 1999 (entitled Saturation Mutagenesis in Directed
`
`Evolution) is also a CIP of USSN 08/651,568 filed on May 22, 1996 (entitled Production of
`
`Enzymes Having Desired Activities by Mutagenesis, now USPN 5,939,250); which claims the
`l
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`benefit of US. provisional application serial No. 60/008,316, filed December 7, 1995 (entitled
`
`Combinatorial Enzyme Development).
`
`The present application is also a ClP of PCT application No. PCT/USOO/ 16838, filed June
`
`14, 2000 (entitled Synthetic Ligation Reassembly in Directed Evolution, now PCT publication
`
`No. WO 00/77262); which claims the benefit of USSN 09/594,459, filed June 14, 2000 (entitled
`
`Synthetic Ligation Reassembly in Directed Evolution); which is a C]? of USSN 09/332,835, filed
`
`June 14, 1999 (entitled Synthetic Ligation Reassembly in Directed Evolution).
`
`The present application is also a ClP of PCT application No. PCT/US00/O8245, filed
`
`March 27, 2000 (entitled Exonuclease—Mediated Nucleic Acid Reassembly in Directed Evolution,
`
`now PCT publication No. WO 00/58517); which claims the benefit of USSN 09/276,860, filed on
`
`March 26, 1999 (entitled Exonuclease—Mediated Gene Assembly in Directed Evolution); which is
`
`a CIP of USSN 09/267,118, filed on March 9, 1999 (entitled End Selection in Directed
`
`Evolution); which is a continuation—in part of USSN O9/246,178, filed Feb. 4, 1999 (entitled
`
`Saturation Mutagenesis in Directed Evolution, now USPN 6,171,820); which is a continuation of
`
`USSN 09/ 185,373 filed on November 3, 1998 (entitled Directed Evolution of Thermophilic
`
`Enzymes); which is a continuation of USSN 08/760,489 filed on Dec. 5, 1996 (entitled Directed
`
`Evolution of Thermophilic Enzymes, now USPN 5,830,696); which claims the benefit of US.
`
`provisional application number 60/008,311 filed on Dec. 07, 1995.
`
`The present application is also a ClP of PCT application No. PCT/US00/06497, filed
`
`March 9, 1999 (entitled End Selection in Directed Evolution, now PCT publication No. WO
`
`00/53744); which claims the benefit of USSN 09/332,835, filed June 14, 1999 (entitled Synthetic
`
`Ligation Reassembly in Directed Evolution). PCT application No. PCT/USOO/O6497, filed March
`
`9, 1999 (entitled End Selection in Directed Evolution, now PCT publication No. WO 00/53744)
`
`also claims the benefit of USSN O9/267,118, filed on March 9, 1999 (entitled End Selection in
`
`Directed Evolution); which is a continuation-in part of USSN O9/246,178, filed Feb. 4, 1999
`
`(entitled Saturation Mutagenesis in Directed Evolution, now USPN 6,171,820); which is a
`
`continuation of USSN 09/ 185,373 filed on November 3, 1998 (entitled Directed Evolution of
`
`Thermophilic Enzymes); which is a continuation of USSN O8/760,489 filed on Dec. 5, 1996
`
`(entitled Directed Evolution of Thermophilic Enzymes, now USPN 5,830,696); which claims the
`
`benefit of US. provisional application number 60/008,311 filed on Dec. 07, 1995.
`
`PCT application No. PCT/USOO/06497, filed March 9, 1999 (entitled End Selection in
`
`Directed Evolution, now PCT publication No. WO 00/53744) also claims the benefit of USSN
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`
`09/276,860, filed on March 26, 1999 (entitled Exonuclease-Mediated Gene Assembly in Directed
`
`Evolution); which is a CIP of USSN 09/267,118, filed on March 9, 1999 (entitled End Selection
`
`in Directed Evolution); which is a continuation-in part of USSN 09/246,178, filed Feb. 4, 1999
`
`(entitled Saturation Mutagenesis in Directed Evolution, now USPN 6,171,820); which is a
`
`continuation of USSN 09/185,373 filed on November 3, 1998 (entitled Directed Evolution of
`
`Thermophilic Enzymes); which is a continuation of USSN 08/760,489 filed on Dec. 5, 1996
`
`(entitled Directed Evolution of Thermophilic Enzymes, now USPN 5,830,696); which claims the
`
`benefit of US. provisional application number 60/008,311 filed on Dec. 07, 1995.
`
`The present application is also a CIP of USSN 09/594,459, filed June 14, 2000 (entitled
`
`Synthetic Ligation Reassembly in Directed Evolution); which is a CIP of USSN 09/332,835, filed
`
`June 14, 1999 (entitled Synthetic Ligation Reassembly in Directed Evolution).
`
`The present application is also a CIP of PCT application No. PCT/US00/03086, filed
`
`February 4, 2000 (entitled Non-Stochastic Generation of Genetic Vaccines and Enzymes); which
`
`claims the benefit of USSN 09/246,178, filed Feb. 4, 1999 (entitled Saturation Mutagenesis in
`
`Directed Evolution, now USPN 6,171,820); which is a continuation of USSN 09/ 185,373 filed on
`
`Nov. 3, 1998 (entitled Directed Evolution of TherrnOphilic Enzymes); which is a continuation of
`
`USSN 08/760,489 filed on Dec. 5, 1996 (entitled Directed Evolution of Thermophilic Enzymes,
`
`now USPN 5,830,696); which claims the benefit of US. provisional application number
`
`60/008,311 filed on Dec. 07, 1995.
`
`The present application is also a CIP of USSN 09/756,459, filed January 8, 2001 (entitled
`
`Saturation Mutagenesis in Directed Evolution); which is a continuation of USSN 09/246,178,
`
`filed Feb. 4, 1999 (entitled Saturation Mutagenesis in Directed Evolution, now USPN 6,171,820);
`
`which is a continuation of USSN 09/ 185,373 filed on Nov. 3, 1998 (entitled Directed Evolution of
`
`Thermophilic Enzymes); which is a continuation of USSN 08/760,489 filed on Dec. 5, 1996
`
`(entitled Directed Evolution of Thermophilic Enzymes, now USPN 5,830,696); which claims the
`
`benefit of US. provisional application number 60/008,311 filed on Dec. 07, 1995.
`
`The present application is also a CIP of USSN [UNASSIGNED], filed January 9, 2001
`
`(entitled Optimized Directed Evolution System and Method).
`
`The present application is also a CIP of USSN O9/376,727, filed August 17, 1999 (entitled
`
`Method of DNA Shuffling with Polynucleotides Produced by Blocking or Interrupting a Synthesis
`
`or Amplification Process); which is a continuation of USSN 08/677,112, filed July 9, 1996
`
`(entitled Method of DNA Reassembly by Interrupting Synthesis, now USPN 5,965,408).
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`The present application is also a CIP of PCT application No. PCT/US98/22596, filed
`
`October 23, 1998 (entitled Method of DNA Shuffling); which claims the benefit of USSN
`
`09/962,504, filed October 31, 1997 (entitled Method of DNA Shuffling); Which is a CIP of USSN
`
`08/677,112, filed July 9, 1996 (entitled Method of DNA Reassembly by Interrupting Synthesis,
`
`now USPN 5,965,408).
`
`The present application is also a CIP of USSN 09/214,645, filed September 27, 1999
`
`(entitled Method of DNA Shuffling with Polynucleotides Produced by Blocking or Interrupting a
`
`Synthesis or Amplification Process); which is a national phase application of PCT application No.
`
`PCT/US97/ 12239, filed July 9, 1997 (entitled Method of DNA Shuffling with Polynucleotides
`
`Produced by Blocking or Interrupting a Synthesis or Amplification Process, now PCT publication
`
`No. WO 98/01581); which claims the benefit of USSN 08/677,112, filed July 9, 1996 (entitled
`
`Method of DNA Reassembly by Interrupting Synthesis, now USPN 5,965,408).
`
`The present application is also a CIP of USSN 09/790,321, filed February 21, 2001
`
`(entitled Capillary Array-Based Enzyme Screening); which is a divisional of USSN 09/687,219,
`
`filed October 12, 2000 (entitled Capillary Array—Based Sample Screening); which is a CIP of
`
`USSN 09/636,778,
`
`filed August 11, 2000 (entitled High Throughput Screening of Novel
`
`Enzymes); which is a continuation of USSN 09/098,206, filed June 16, 1998 (entitled High
`
`Throughput Screening of Novel Enzymes, now USPN 6,174,673); which is a CIP of USSN
`
`09/876,276, filed June 16, 1997 (entitled High Throughput Screening of Novel Enzymes).
`
`The present application is also a CIP of USSN 09/761,559, filed January 16, 2001
`
`(entitled High Throughput Screening of Novel Enzymes); which is a divisional of USSN
`
`09/098,206, filed June 16, 1998 (entitled High Throughput Screening of Novel Enzymes, now
`
`USPN 6,174,673); which is a CIP of USSN 09/876,276, filed June 16, 1997 (entitled High
`
`Throughput Screening of Novel Enzymes).
`
`The present application is also a CIP of USSN 09/848,185 filed May 3, 2001 (entitled
`
`High Throughput Screening for Novel Enzymes); which is a divisional of USSN 09/636,778, filed
`
`August 11, 2000 (entitled High Throughput Screening of Novel Enzymes); which is. a
`
`continuation of USSN 09/098,206, filed June 16, 1998 (entitled High Throughput Screening of
`
`Novel Enzymes, now USPN 6,174,673); which is a CIP of USSN 09/876,276, filed June 16, 1997
`
`(entitled High Throughput Screening of Novel Enzymes).
`
`The present application is also a CIP of USSN 09/738,871, filed December 14, 2000
`
`(entitled High Throughput Screening for a Bioactivity or Biomolecule); which is a CIP of USSN
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`09/685,432, filed October 10, 2000 (entitled High Throughput Screening for Sequences of
`
`Interest); which is a CIP of USSN 09/444,112, filed November 22, 1999 (entitled Capillary Array-
`
`Based Enzwne Screening); which is a CIP of USSN 09/098,206, filed June 16, 1998 (entitled
`
`High Throughput Screening of Novel Enzymes, now USPN 6,174,673); which is a CIP of USSN
`
`09/876,276, filed June 16, 1997 (entitled High Throughput Screening of Novel Enzymes).
`
`.
`
`The present application is also a CIP of PCT application No. PCT/USOO/32208, filed
`
`November 22, 2000 (entitled Capillary Array—Based Sample Screening); which claims the benefit
`
`of USSN 09/687,219, filed October 12, 2000 (entitled Capillary Based—Based Sample Screening);
`
`which is a CIP of USSN 09/636,778, filed August 11, 2000 (entitled High Throughput Screening
`
`of Novel Enzymes); which is a continuation of USSN O9/O98,206, filed June 16, 1998 (entitled
`
`High Throughput Screening of Novel Enzymes, now USPN 6,174,673); which is a CIP of USSN
`
`09/876,276, filed June 16, 1997 (entitled High Throughput Screening of Novel Enzymes).
`
`The present application is also a CIP of PCT application No. PCT/US98/ 12674, filed June
`
`16, 1998 (entitled High Throughput Screening for Novel Enzymes, now PCT publication No. WO
`
`98/58085); which claims the benefit of USSN O9/876,276, filed June 16, 1997 (entitled High
`
`Throughput Screening of Novel Enzymes).
`
`PCT/USOO/32208, filed November 22, 2000 (entitled Capillary Array—Based Sample
`
`Screening), also claims the benefit of USSN 09/444,112, filed November 22, 1999 (entitled
`
`Capillary Array—Based Enzyme Screening); which is a CIP of USSN 09/098,206, filed June 16,
`
`1998 (entitled High Throughput Screening of Novel Enzymes, now USPN 6,174,673); which is a
`
`CIP of USSN 09/876,276, filed June 16, 1997 (entitled High Throughput Screening of Novel
`
`Enzymes).
`
`These aforementioned applications and patents are explicitly incorporated
`
`herein by reference in their entirety and for all purposes.
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`TECHNICAL FIELD
`
`The present invention is generally directed to the fields of medicine, protein
`
`engineering, immunology and molecular biology.
`
`In one aspect, the invention is directed to
`
`methods for generating sets, or
`
`libraries, of nucleic acids encoding antigen binding
`
`molecules, including, e.g., antibodies and related molecules, such as antigen binding sites and
`
`domains and other antigen binding fragments,
`
`including single and double stranded
`
`antibodies, T cell receptors (TCRs) and Class I and Class 11 major histocompatibility (NIHC)
`
`molecules. This invention also provides methods for generating new or variant antigen
`
`binding polypeptides, e.g., antigen binding sites, antibodies and specific domains or
`
`fragments of antibodies (e.g., Fab or PC domains), TCRs and MHC molecules by altering
`
`template nucleic acids by, e.g., saturation mutagenesis, an optimized directed evolution
`
`system, synthetic ligation reassembly, or a combination thereof.
`
`Polypeptides generated by these methods can be analyzed using any liquid or
`
`solid state screening method, e.g., phage display, ribosome display, using capillary array
`
`platforms, and the like. The polypeptides generated by the methods of the invention can be
`
`used in vitro, e.g., to isolate or identify antigens or in viva, e.g., to treat or diagnose various
`
`diseases and conditions, to modulate, stimulate or attenuate an immune response.
`
`This invention pertains to the field of genetic vaccines.
`
`Specifically, the
`
`invention provides multi—component genetic vaccines that contain components that are
`
`optimized for a particular vaccination goal.
`
`In one aspect, this invention provides methods
`
`for improving the efficacy of genetic vaccines by providing materials that facilitate targeting
`
`of a genetic vaccine to a particular tissue or cell type of interest. The invention also provides
`
`antigen binding molecules, e.g., T cell
`
`receptors and Class I and Class
`
`11 major
`
`histocompatibility (MI-IC) molecules, having an engineered affinity to an antigen,
`
`thus
`
`allowing manipulation of the immune response to the vaccine.
`
`This invention pertains to the field biologic therapeutics by providing
`
`polypeptides comprising antigen binding sites, such as antibodies, with modified (e.g.,
`
`increased or decreased) affinity for antigen. For example, the methods of the invention
`
`provide antibodies of altered or enhanced affinities for an antigen for use, e.g.,
`
`in
`
`immunotherapeutics or diagnostics.
`
`The antibodies generated by the methods of the
`
`invention can be administered therapeutically to slow the growth of or kill cells, such as
`
`cancer cells, or, to stimulate cell division, e.g., for enhancing an immune response or for
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`tissue regeneration, or,
`
`to alter any biological mechanism or response.
`
`For example,
`
`administration of antibodies that bind to immune effector or regulatory cells, or
`
`to
`
`lymphokines or cytokines, can alter, e. g., upregulate, stimulate or attenuate, an humoral or a
`
`cellular immune response.
`
`This invention pertains to the field of modulation of immune responses such
`
`as those induced by genetic vaccines and also pertains to the field of methods for developing
`
`immunogens that can induce efficient immune responses against a broad range of antigens.
`
`This invention pertains to the field of modulation of immune responses by
`
`modifying molecules that are involved in the stimulation and regulation of the immune
`
`response, including, e.g., T cell receptors and Class I and Class II major histocompatibility
`
`(NEE-1C) molecules. Thus, molecules generated by the methods of the invention can have
`
`increased or decreased affinity of binding sites to antigen. For example, by decreasing the
`
`affinity of a T cell receptor for an antigen (which a TCR binds in conjunction with an MHC
`
`molecule, i.e., the MHC “presents” the antigen to the TCR), the methods of the invention can
`
`generate a non-autoreactive variant of an autoreactive TCR.
`
`In another example, by
`
`increasing the affinity of an MI-IC molecule for an antigen, e. g., a pathogenic antigen, the
`
`methods of the invention can generate an enhanced immune response to that pathogen.
`
`Similarly, if the antigen is a self antigen, by decreasing the affinity of the MHC molecule for
`
`the antigen, the methods of the invention can generate an abated or attenuated immune
`
`response to that self antigen.
`
`Thus,
`
`the present
`
`invention also relates generally to novel proteins, and
`
`fragments thereof, as well as nucleic acids which encode these proteins, and methods of
`
`making and using these proteins in diagnostic, prophylactic and therapeutic applications.
`
`In
`
`a particular exemplification, the present invention relates to proteins from the Plasmodium
`
`falciparum erythrocyte membrane protein 1 ("PfEMPl”) gene family and fragments thereof
`
`which are derived from malaria—parasitized erythrocytes.
`
`In particular, these proteins are
`
`derived from the erythrocyte membrane protein of Plasmodium falcipamm parasitized
`
`erythrocytes, also termed "PfEMP1”. The present invention also provides nucleic acids
`
`encoding these proteins, which proteins and nucleic acids are associated with the pathology
`
`of malaria infections, and which may be used as vaccines or other prophylactic treatments for
`
`the prevention of malaria infections, and/or in diagnosing and treating the symptoms of
`
`patients who suffer from malaria and associated diseases.
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`This invention also relates to the field of protein engineering. Specifically,
`
`this invention relates to a directed evolution method for preparing a polynucleotide encoding
`
`a polypeptide. More specifically, this invention relates to a method of using mutagenesis to
`
`generate a novel polynucleotide encoding a novel polypeptide, which novel polypeptide is
`
`itself an altered (“improved”) biological molecule and/or contributes to the generation of
`
`another improved biological molecule. More specifically still, this invention relates to a
`
`method of performing both non—stochastic polynucleotide chimerization and non—stochastic
`
`site—directed point mutagenesis.
`
`Thus, in one aspect, this invention relates to a method of generating a progeny
`
`library, or set, of chimeric polynucleotide(s) by means that are synthetic and non—stochastic.
`
`The design of the progeny polynucleotide(s) is derived by analysis of a parental set of
`
`polynucleotides and/or of the polypeptides correspondingly encoded by the parental
`
`polynucleotides.
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`In another aspect, this invention relates to a method of performing site-
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`directed mutagenesis using means that are exhaustive, systematic, and non-stochastic.
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`Furthermore this invention relates to a step of selecting from among a
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`generated set of progeny molecules a subset comprised of particularly desirable species,
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`including by a process termed end—selection, which subset may then be screened further.
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`This invention also relates to the step of screening a set of polynucleotides for the production
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`of a polypeptide and/or of another expressed biological molecule having a useful property,
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`such as an antibody with increased affinity for an antigen.
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`Novel biological molecules whose manufacture is taught by this invention
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`include genes, gene pathways, and any molecules whose expression is affected thereby,
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`including directly encoded polypetides and /or any molecules affected by such polypeptides.
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`Said novel biological molecules include those that contain a carbohydrate, a lipid, a nucleic
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`acid, and /or a protein component, and specific but non—limiting examples of these include
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`antibiotics, antibodies, TCRs, IVIHC molecules, enzymes, and steroidal and non—steroidal
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`hormones.
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`In one aspect,
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`the present
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`invention relates to enzymes, particularly to
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`thermostable enzymes, and to their generation by directed evolution. More particularly, the
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`present invention relates to thermostable enzymes which are stable at high temperatures and
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`which have improved activity at lower temperatures.
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`BACKGROUND
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`Antigen binding polypeptides, such as antibodies, are increasingly used in a variety of
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`therapeutic applications. For example, in immunotherapy, antibodies are used to directly kill
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`target cells, such as cancer cells. Antigen binding polypeptides are also used as carriers to
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`deliver cytotoxic or imaging reagents. Monoclonal antibodies (mAbs) approved for cancer
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`therapy are now in Phase II and III trials. Certain anti-idiotypic antibodies that bind to the
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`antigen—combining sites of antibodies can effectively mimic the three-dimensional structures
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`and functions of the external antigens and can be used as surrogate antigens for active
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`specific immunotherapy. Bi-specific antibodies combine immune cell activation with tumor
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`cell recognition; thus, tumor cells or cells expressing tumor specific antigens (e. g., tumor
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`vasculature) are killed by pre-defined effector cells. Antibodies can be administered to
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`increase or decrease the levels of cytokines or hormones by direct binding or by stimulating
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`or inhibiting secretory cells. Accordingly, increasing the affinity or avidity of an antibody to
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`a desired antigen, such as a cancer-specific antigen, would result in greater specificity of the
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`antibody to its target, resulting in a variety of therapeutic benefits, such as needing to
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`administer less antibody-containing pharmaceutical.
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`Providing protective immunity even in situations when the pathogens are poorly
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`characterized or cannot be isolated or cultured in laboratory environment.
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`Genetic immunization represents a novel mechanism of inducing protective humoral
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`and cellular immunity. Vectors for genetic vaccinations generally consist of DNA that
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`includes a promoter/enhancer sequence,
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`the gene of interest and a polyadenylation/
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`transcriptional terminator sequence. After intramuscular or intradermal injection, the gene of
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`interest is expressed, followed by recognition of the resulting protein by the cells of the
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`immune system. Genetic immunizations provide means to induce protective immunity even
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`in situations when the pathogens are poorly characterized or cannot be isolated or cultured in
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`laboratory environment.
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`Small improvement in the efficiency of genetic vaccine vectors can result in dramatic
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`increase if the level of immune response
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`The efficacy of genetic vaccination is often limited by inefficient uptake of genetic
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`vaccine vectors into cells. Generally, less than 1% of the muscle or skin cells at the sites of
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`injections express the gene of interest. Even a small improvement in the efficiency of genetic
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`vaccine vectors to enter the cells can result in a dramatic increase in the level of immune
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`response induced by genetic vaccination. A vector typically has to cross many barriers which
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`can result in only a very minor fraction of the DNA ever being expressed.
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`Various limitations to immunogenicity
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`Limitations to immunogenicity include: loss of vector due to nucleases present in
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`blood and tissues; inefficient entry of DNA into a cell; inefficient entry of DNA into the
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`nucleus of the cell and preference of DNA for other compartments; lack of DNA stability in
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`the nucleus (factor limiting nuclear stability may differ from those affecting other cellular
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`and extracellular compartments), and, for vectors that integrate into the chromosome, the
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`efficiency of integration and the site of integration. Moreover, for many applications of
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`genetic vaccines, it is preferable for the genetic vaccine to enter a particular target tissue or
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`cell.
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`Thus, a need exists for genetic vaccines that can be targeted to specific cell and tissue
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`types of interest, and which exhibit an increased ability to enter the target cells.
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`Pathways for immune responses induced by genetic vaccines
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`Elicitation of a desired in viva response by a genetic vaccine generally requires
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`multiple cellular processes in a complex sequence. Several potential pathways exist along
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`which a genetic vaccine can exert its effect on the mammalian immune system. In one
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`pathway, the genetic vaccine vector enters cells that are the predominant cell type in the
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`tissue that receives vaccine (e.g., muscle or epithelial cells). These cells express and release
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`the antigen encoded by the vector. The vaccine vector can be engineered to have the antigen
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`released as an intact protein from living transfected cells (i.e., via a secretion process) or
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`directed to a membrane—bound form on the surface of these cells. Antigen can also be
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`released from an intracellular compartment of such cells if those cells die.
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`The antigen derived from vaccine vector internalization and antigen expression within the
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`predominant cell type in the tissue ends up within APC, which then process the antigen
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`1nternally to prime NIHC Class I and or Class II, essential steps in activation of CD4)r T—
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`helper cells and development of potent specific immune responses.
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`Extracellular antigen derived from any of these situations interacts with antigen
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`presenting cells (APC) either by binding to the cell surface (specifically via IgM or via other
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`non-immunoglobulin receptors) and subsequent endocytosis of outer membrane, or by fluid
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`phase micropinocytosis wherein the APC intemalizes extracellular fluid and its contents into
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`an endocytic compartment. Interaction with APC may occur before or after partial proteolytic
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`cleavage in the extracellular environment. In any case, the antigen derived from vaccine
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`vector internalization and antigen expression within the predominant cell type in the tissue
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`ends up within APC. The APC then process the antigen internally to prime MHC Class I and
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`or Class H, essential steps in activation of CD4+ T—helper cells (THl and/or TH2) and
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`development of potent specific immune responses.
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`The genetic vaccine plasmid enters APC and antigen is proteolflically cleaved in the cell
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`cytoplasm.
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`In a parallel pathway, the genetic vaccine plasmid enters APC (or the predominant
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`cell type in the tissue) and, instead of antigen derived from plasmid expression being directed
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`to extracellular export, antigen is proteolytically cleaved in the cell cytoplasm (in a
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`proteasome dependent or independent process). Often, intracellular processing in such cells
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`occurs via proteasomal degradation into peptides that are recognized by the TAP-1 and TAP—
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`2 proteins and transported into the lumen of the rough endoplasmic reticulum (RER).
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`The peptide fragments are transported into the RER complex, expressed on the cell surface;
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`in the presence of appropriate additional sigpals, can differentiate into functional CTLs.
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`The peptide fragments transported into the RER complex with MHC Class I. Such
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`antigen fragments are then expressed on the cell surface in association with Class I. CD8+
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`cytotoxic T lymphocytes (CTL) bearing specific T cell receptor then recognize the complex
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`and can, in the presence of appropriate additional signals, differentiate into functional CTLs.
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`By virtue of poorly characterized pathwayifor trafficldng_o_f cyt_qplasmically generated
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`peptides into endosomal compartmen_ts,_a ge_netic vaccine vector can lead to CD4+ T cell
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`stimulation.
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`In addition, poorly characterized pathways, which are generally not dominant, exist in
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`APC for trafficking of cytoplasmically generated peptides into endosomal compartments
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`where they can end up complexed with MHC Class II, and thereby act to present antigen
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`peptides to CD4+ THI and THZ cells. Because activation, proliferation, differentiation and
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`immunoglobulin isotype switching by B lymphocytes requires help of CD4)r T cells, antigen
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`presentation in the context of MHC Class II molecules is crucial for induction of antigen—
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`specific antibodies. By virtue of this pathway, a genetic vaccine vector can lead to CD4)r T
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`cell stimulation in addition to the dominant CD8)r CTL activation process described above.
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`This alternative pathway is, hOWever, of little consequence in muscle cells where levels of
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`MHC Class II expression are very low or zero.
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