EP1421203A2 - Neue antigenbindende moleküle für therapeutische, diagnostische, prophylaktische, enzymatische, industrielle und landwirtschaftliche anwendungen und verfahren zur erzeugung und zum screening davon - Google Patents

Neue antigenbindende moleküle für therapeutische, diagnostische, prophylaktische, enzymatische, industrielle und landwirtschaftliche anwendungen und verfahren zur erzeugung und zum screening davon

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Publication number
EP1421203A2
EP1421203A2 EP02747843A EP02747843A EP1421203A2 EP 1421203 A2 EP1421203 A2 EP 1421203A2 EP 02747843 A EP02747843 A EP 02747843A EP 02747843 A EP02747843 A EP 02747843A EP 1421203 A2 EP1421203 A2 EP 1421203A2
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EP
European Patent Office
Prior art keywords
antigen binding
binding site
amino acid
encoding
polypeptide
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EP02747843A
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English (en)
French (fr)
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EP1421203A4 (de
Inventor
Jay M. Short
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BASF Enzymes LLC
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Diversa Corp
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Publication of EP1421203A2 publication Critical patent/EP1421203A2/de
Publication of EP1421203A4 publication Critical patent/EP1421203A4/de
Withdrawn legal-status Critical Current

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C12N15/1034—Isolating an individual clone by screening libraries
    • C12N15/1093—General methods of preparing gene libraries, not provided for in other subgroups
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C12N15/102—Mutagenizing nucleic acids
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C12N15/102—Mutagenizing nucleic acids
    • C12N15/1027—Mutagenizing nucleic acids by DNA shuffling, e.g. RSR, STEP, RPR
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/66—General methods for inserting a gene into a vector to form a recombinant vector using cleavage and ligation; Use of non-functional linkers or adaptors, e.g. linkers containing the sequence for a restriction endonuclease

Definitions

  • Directed Evolution now PCT publication No. WO 00/53744
  • USSN 09/276,860 filed on March 26, 1999
  • USSN 09/267,118 filed on March 9, 1999
  • End Selection in Directed Evolution which is a continuation-in part of USSN 09/246,178, filed Feb. 4, 1999
  • USSN 09/185,373 filed on November 3, 1998
  • USSN 08/760,489 filed on Dec. 5, 1996
  • USPN 5,830,696 claims the benefit of U.S. provisional application number 60/008,311 filed on Dec. 07, 1995.
  • the present application is also a CE? 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 CD? 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 CD? of USSN 09/876,276, filed June 16, 1997 (entitled High Throughput Screening of Novel Enzymes).
  • the present application is also a CD? of USSN 09/761,559, filed January 16, 2001
  • PCT/US00/32208 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 CD? 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 invention is generally directed to the fields of medicine, protein engineering, immunology and molecular biology.
  • 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 U major histocompatibility (MHC) molecules.
  • 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 U major histocompatibility (MHC) molecules.
  • TCRs T cell receptors
  • MHC major histocompatibility
  • 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 Fc 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.
  • antigen binding polypeptides e.g., antigen binding sites, antibodies and specific domains or fragments of antibodies (e.g., Fab or Fc 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 vivo, 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 ⁇ major histocompatibility (MHC) molecules, having an engineered affinity to an antigen, thus allowing manipulation of the immune response to the vaccine.
  • antigen binding molecules e.g., T cell receptors and Class I and Class ⁇ major histocompatibility (MHC) 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.
  • 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 tissue regeneration, or, to alter any biological mechanism or response.
  • 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 (MHC) molecules.
  • 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.
  • the methods of the invention can generate an enhanced immune response to that pathogen.
  • the antigen is a self antigen
  • the methods of the invention can generate an abated or attenuated immune response to that self antigen.
  • 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.
  • 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.
  • these proteins are derived from the erythrocyte membrane protein of Plasmodium falciparum parasitized erythrocytes, also termed "PfEMPl".
  • 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.
  • 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.
  • 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.
  • this invention relates to a method of performing site- directed mutagenesis using means that are exhaustive, systematic, and non-stochastic.
  • this invention relates to a step of selecting from among a generated set of progeny molecules a subset comprised of particularly desirable species, including by a process termed end-selection, which subset may then be screened further.
  • This invention also relates to the step of screening a set of polynucleotides for the production of a polypeptide and/or of another expressed biological molecule having a useful property, such as an antibody with increased affinity for an antigen.
  • Novel biological molecules whose manufacture is taught by this invention include genes, gene pathways, and any molecules whose expression is affected thereby, including directly encoded polypetides and /or any molecules affected by such polypeptides.
  • Said novel biological molecules include those that contain a carbohydrate, a lipid, a nucleic acid, and /or a protein component, and specific but non-limiting examples of these include antibiotics, antibodies, TCRs, MHC molecules, enzymes, and steroidal and non-steroidal hormones.
  • the present invention relates to enzymes, particularly to thermostable enzymes, and to their generation by directed evolution. More particularly, the present invention relates to thermostable enzymes which are stable at high temperatures and which have improved activity at lower temperatures.
  • Antigen binding polypeptides such as antibodies
  • antibodies are increasingly used in a variety of therapeutic applications.
  • antibodies are used to directly kill target cells, such as cancer cells.
  • Antigen binding polypeptides are also used as carriers to deliver cytotoxic or imaging reagents.
  • Monoclonal antibodies (mAbs) approved for cancer therapy are now in Phase Et and trials.
  • Certain anti-idiotypic antibodies that bind to the antigen-combining sites of antibodies can effectively mimic the three-dimensional structures and functions of the external antigens and can be used as surrogate antigens for active specific immunotherapy.
  • Bi-specific antibodies combine immune cell activation with tumor cell recognition; thus, tumor cells or cells expressing tumor specific antigens (e.g., tumor vasculature) are killed by pre-defined effector cells.
  • Antibodies can be administered to increase or decrease the levels of cytokines or hormones by direct binding or by stimulating or inhibiting secretory cells. Accordingly, increasing the affinity or avidity of an antibody to a desired antigen, such as a cancer-specific antigen, would result in greater specificity of the antibody to its target, resulting in a variety of therapeutic benefits, such as needing to administer less antibody-containing pharmaceutical.
  • Genetic immunization represents a novel mechanism of inducing protective humoral and cellular immunity.
  • Vectors for genetic vaccinations generally consist of DNA that includes a promoter/enhancer sequence, the gene of interest and a polyadenylation/ transcriptional terminator sequence. After intramuscular or intradermal injection, the gene of interest is expressed, followed by recognition of the resulting protein by the cells of the immune system. Genetic immunizations provide means to induce protective immunity even in situations when the pathogens are poorly characterized or cannot be isolated or cultured in laboratory environment.
  • Limitations to immunogenicity include: loss of vector due to nucleases present in blood and tissues; inefficient entry of DNA into a cell; inefficient entry of DNA into the nucleus of the cell and preference of DNA for other compartments; lack of DNA stability in the nucleus (factor limiting nuclear stability may differ from those affecting other cellular and extracellular compartments), and, for vectors that integrate into the chromosome, the efficiency of integration and the site of integration. Moreover, for many applications of genetic vaccines, it is preferable for the genetic vaccine to enter a particular target tissue or cell.
  • the genetic vaccine vector enters cells that are the predominant cell type in the tissue that receives vaccine (e.g., muscle or epithelial cells). These cells express and release the antigen encoded by the vector.
  • the vaccine vector can be engineered to have the antigen released as an intact protein from living transfected cells (i.e., via a secretion process) or directed to a membrane-bound form on the surface of these cells. Antigen can also be released from an intracellular compartment of such cells if those cells die.
  • the antigen derived from vaccine vector internalization and antigen expression within the predominant cell type in the tissue ends up within APC. which then process the antigen internally to prime MHC Class I and or Class II, essential steps in activation of CD4 " * " T- helper cells and development of potent specific immune responses.
  • Extracellular antigen derived from any of these situations interacts with antigen presenting cells (APC) either by binding to the cell surface (specifically via IgM or via other non-immunoglobulin receptors) and subsequent endocytosis of outer membrane, or by fluid phase micropinocytosis wherein the APC internalizes extracellular fluid and its contents into an endocytic compartment. Interaction with APC may occur before or after partial proteolytic cleavage in the extracellular environment.
  • the antigen derived from vaccine vector internalization and antigen expression within the predominant cell type in the tissue ends up within APC.
  • the APC then process the antigen internally to prime MHC Class I and or Class Et, essential steps in activation of CD4 + T-helper cells (THI and/or T H 2) and development of potent specific immune responses.
  • the genetic vaccine plasmid enters APC and antigen is proteolytically cleaved in the cell cytoplasm.
  • the genetic vaccine plasmid enters APC (or the predominant cell type in the tissue) and, instead of antigen derived from plasmid expression being directed to extracellular export, antigen is proteolytically cleaved in the cell cytoplasm (in a proteasome dependent or independent process).
  • APC or the predominant cell type in the tissue
  • antigen is proteolytically cleaved in the cell cytoplasm (in a proteasome dependent or independent process).
  • intracellular processing in such cells occurs via proteasomal degradation into peptides that are recognized by the TAP-1 and TAP- 2 proteins and transported into the lumen of the rough endoplasmic reticulum (RER).
  • the peptide fragments are transported into the RER complex, expressed on the cell surface; in the presence of appropriate additional signals, can differentiate into functional CTLs.
  • a genetic vaccine vector can lead to CD4 + T cell stimulation.
  • poorly characterized pathways which are generally not dominant, exist in APC for trafficking of cytoplasmically generated peptides into endosomal compartments where they can end up complexed with MHC Class EC, and thereby act to present antigen peptides to CD4 + T H I and T H 2 cells.
  • MHC Class II molecules Because activation, proliferation, differentiation and immunoglobulin isotype switching by B lymphocytes requires help of CD4 + T cells, antigen presentation in the context of MHC Class II molecules is crucial for induction of antigen- specific antibodies.
  • a genetic vaccine vector can lead to CD4 + T cell stimulation in addition to the dominant CD8 + CTL activation process described above.
  • MHC Class D expression are very low or zero.
  • cytokines are derived not only from processes intrinsic to the interaction of DNA with cells, or specific cell responses to the antigen, but via synthesis directed by the vaccine plasmid.
  • cytokine release can also elicit cytokine release from cells that bind to or take up DNA.
  • So-called immunostimulatory or adjuvant properties of DNA are derived from its interaction with cells that internalize DNA. Cytokines can be released from cells that bind and/or internalize DNA in the absence of gene transcription. Separately, interaction of antigen with APC followed by presentation and specific recognition also stimulates release of cytokines that have positive feedback effects on these cells and other immune cells. Chief among these effects are the direction of CD4 + T H cells to differentiate/ proliferate preferentially to THI or T R 2 phenotypes.
  • cytokines released at the site of DNA vaccination contribute to recruitment of other immune cells from the immediate local area and more distant sites such as draining lymph nodes.
  • some investigators have included the genes for one or more cytokines in the DNA vaccine plasmid along with the target antigen for immunization.
  • cytokines are derived not only from processes intrinsic to the interaction of DNA with cells, or specific cell responses to the antigen, but via synthesis directed by the vaccine plasmid. Movement of immune cells from the blood stream and different sites to the site of immunization and also from the site of immunization to other sites
  • Immune cells are recruited to the site of immunization from distant sites or the bloodstream. Specific and non-specific immune responses are then greatly amplified. Immune cells, including APC, bearing antigen fragments complexed to MHC molecules or even expressing antigen from uptake of plasmid, also move from the immunization site to other sites (blood, hence to all tissues; lymph nodes; spleen) where additional immune recruitment and qualitative and quantitative development of the immune response ensue.
  • Booster immunizations are typically required 3-4 weeks after the primary injection with existing genetic vaccines. Therefore a need exists for improved genetic vaccine vectors and formulations, and methods for development of such vectors.
  • the interactions between pathogens and hosts are results of millions of years of evolution, during which the mammalian immune system has evolved sophisticated means to counterattack pathogen invasions.
  • bacterial and viral pathogens have simultaneously gained a number of mechanisms to improve their virulence and survival in hosts, providing a major challenge for vaccine research and development despite the powers of modem techniques of molecular and cellular biology. Similar to the evolution of pathogen antigens, several cancer antigens are likely to have gained means to downregulate their immunogenicity as a mechanism to escape the host immune system.
  • Efficient vaccine development is also hampered by the antigenic heterogeneity of different strains of pathogens, driven in part by evolutionary forces as means for the pathogens to escape immune defenses.
  • Pathogens also reduce their immunogenicity by selecting antigens that are difficult to express, process and/or transport in host cells, thereby reducing the availability of immunogenic peptides to the molecules initiating and modulating immune responses.
  • the mechanisms associated with these challenges are complex, multivariate and rather poorly characterized. Accordingly, a need exists for vaccines that can induce a protective immune response against bacterial and viral pathogens.
  • cytokines interleukins, interferons, chemokines, hematopoietic growth factors, tumor necrosis factors and transforming growth factors
  • Characteristic features of cytokines are pleiotropy and redundancy; that is, one cytokine often has several functions and a given function is often mediated by more than one cytokine.
  • cytokines have additive or synergistic effects with other cytokines, and a number of cytokines also share receptor components.
  • cytokine networks Due to the complexity of the cytokine networks, studies on the physiological significance of a given cytokine have been difficult, although recent studies using cytokine gene-deficient mice have significantly improved our understanding on the functions of cytokines in vivo.
  • membrane- bound costimulatory molecules play a fundamental role in the regulation of immune responses. These molecules include CD40, CD40 ligand, CD27, CD80, CD86 and CD150 (SLAM), and they are typically expressed on lymphoid cells after activation via antigen recognition or through cell- cell interactions.
  • T helper (T H ) cells key regulators of the immune system, are capable of producing a large number of different cytokines, and based on their cytokine synthesis pattern T H cells are divided into two subsets (Paul and Seder (1994) Cell 76: 241-251).
  • T H 1 cells produce high levels of EL-2 and EFN- ⁇ and no or minimal levels of EL-4, EL-5 and IL-13.
  • T H 2 cells produce high levels of IL-4, IL-5 and E -13, and IL-2 and EFN- ⁇ production is minimal or absent.
  • T H I cells activate macrophages, dendritic cells and augment the cytolytic activity of CD8 + cytotoxic T lymphocytes and NK cells (Id.), whereas T ⁇ t 2 cells provide efficient help for B cells and they also mediate allergic responses due to the capacity of H 2 cells to induce IgE isotype switching and differentiation of B cells into IgE secreting cell (De Vries and Punnonen (1996) In Cytokine regulation of humoral immunity: basic and clinical aspects. Eds. Snapper, CM., John Wiley & Sons, Ltd., West Wales, UK, p. 195- 215). The exact mechanisms that regulate the differentiation of T helper cells are not fully understood, but cytokines are believed to play a major role.
  • EL-4 has been shown to direct H 2 differentiation, whereas EL- 12 induces development of T H I cells (Paul and Seder, supra.).
  • membrane bound costimulatory molecules such as CD80, CD86 and CD150, can direct T H I and/or TH2 development, and the same molecules that regulate T H cell differentiation also affect activation, proliferation and differentiation of B cells into Ig- secreting plasma cells (Cocks et al. (1995) Nature 376: 260-263; Lenschow et al. (1996) Immunity 5: 285-293; Punnonen et al. (1993) Proc. Nat'l. Acad. Sci. USA 90: 3730-3734; Punnonen et al. (1997) / Exp. Med. 185: 993-1004).
  • T H T helper
  • T H 2-like cytokine synthesis profiles have been proposed to protect from AEDS (Maggi et al. (1994) J Exp. Med. 180: 489-495).
  • the survival from meningococcal septicemia is genetically determined based on the capacity of peripheral blood leukocytes to produce
  • TNF- ⁇ and EL- 10 TNF- ⁇ and EL- 10. Individuals from families with high production of EL- 10 have increased risk of fatal meningococcal disease, whereas members of families with high TNF- ⁇ production were more likely to survive the infection (Westendorp et al. (1997) Lancet 349: 170-173).
  • Cytokine treatments can dramatically influence T H 1/T H 2 cell differentiation and macrophage activation, and thereby the outcome of infectious diseases.
  • BALB/c mice infected with Leishmania major generally develop a disseminated fatal disease with a T H phenotype, but when treated with anti-EL-4 mAbs or EL- 12, the frequency of T H I cells in the mice increases and they are able to counteract the pathogen invasion (Chatelain et al. (1992) / Immunol. 148: 1182-1187).
  • EFN- ⁇ protects mice from lethal Herpes Simplex Virus (HSV) infection
  • MCP-1 prevents lethal infections by Pseudomonas aeruginosa or Salmonella typhimurium
  • cytokine treatments such as recombinant EL-2, have shown beneficial effects in human common variable immunodeficiency (Cunningham-Rundles et al. (1994) N. Engl. J Med. 331: 918-921).
  • cytokines and other molecules to modulate immune responses in a manner most appropriate for treating a particular disease can provide a significant tool for the treatment of disease.
  • presently available immunomodulator treatments can have several disadvantages, such as insufficient specific activity, induction of immune responses against, the immunomodulator that is administered, and other potential problems.
  • immunomodulators that exhibit improved properties relative to those currently available. Erythrocytes infected with the malaria parasite P. falciparum disappear from the peripheral circulation as they mature from the ring stage to trophozoites (Bignami and Bastianeli, Reforma Medica (1889) 6:1334-1335).
  • Endothelial cell surface proteins such as CD36, thrombospondin (TSP) and ICAM-1 have been identified as major host receptors for mature PE. See, e.g., Barnwell et al., J. Immunol. (1985) 135:3494-3497;
  • PE sequestration confers unique advantages for P. falciparum parasites (Howard and
  • P. falciparum PE The capacity of P. falciparum PE to express variant forms of PfEMPl contributes to the special virulence of this parasite.
  • Variant parasites can evade variant-specific antibodies elicited by earlier infections.
  • the P. falciparum variant antigens have been defined in vitro using antiserum prepared in Aotus monkeys infected with individual parasite strains (Howard et al, Molec. Biochem. Parasitol. (1988) 27:207-223). Antibodies raised against a particular parasite will only react by PE agglutination, indirect immuno- fluorescence or immuno- electron microscopy with PE from the same strain (van Schravendijk et al, Blood (1991)
  • chabaudi in mice confirmed that antigenic variation at the PE surface is associated with prolonged or chronic infection and the capacity to repeatedly re-establish blood infection in previously infected animals.
  • Studies with cloned parasites demonstrated that antigenic variants can arise with extraordinary frequency, e.g., 2% per generation with P. falciparum (Roberts et al., Nature (1992) 357:689-692) and 1.6 % per generation with P. chabaudi (Brannan et al., supra).
  • PfEMPl was identified as a 125 I-labeled, size diverse protein (200-350 kD) on PE that is lacking from uninfected erythrocytes, and that is also labeled by biosynthetic incorporation of radiolabeled amino acids (Leech et al., J. Exp. Med. (1984) 159:1567-1575; Howard et al., Molec. Biochem. Parasitol. (1988) 27:207-223). PfEMPl is not extracted from PE by neutral detergents such as Triton X-100 but is extracted by SDS, suggesting that it is linked to the erythrocyte cytoskeleton (Aley et al., J. Med. Exp.
  • PfEMPl After addition of excess Triton X-100, PfEMPl is immunoreactive with appropriate serum antibodies (Howard et al., (1988), supra). Mild trypsinization of intact PE rapidly cleaves PfEMPl from the cell surface (Leech et al., J. Exp. Mod. (1984) 159:1567-1575). PfEMPl bears antigenically diverse epitopes since it is immunoprecipitated from particular strains of P. falciparum by antibodies from sera of Aotus monkeys infected with the same strain, but not by antibodies from animals infected with heterologous strains (Howard et al. (1988), supra).
  • Knobless PE derived from parasite passage in splenectomized Aotus monkeys do not express surface PfEMPl and are not agglutinated with sera from immune individuals or infected monkeys (Howard et al. (1988), supra; Howard and Gilladoga, Blood (1989) 74:2603-2618).
  • the adherence of parasitized erythrocytes to endothelial cells is mediated by multiple receptor/counter- receptor interactions, including CD36, thrombospondin and intracellular adhesion molecule-1 (ICAM_1) as the major host cell receptors (Howard and Gilladoga,
  • VCAM-1 Vascular cell adhesion molecule- 1
  • ELAM-1 endothelial leukocyte adhesion molecule- 1
  • the adherence receptors on the surface of PE has not yet been conclusively identified, and several molecules, including AG 332 (Udomsangpetch, et al., Nature (1989) 338:763-765), modified band 3 (Crandall, et al., Proc. Nat'l Acad. Sci. USA (1993) 90:4703-4707), Sequestrin (Ockenhouse, Proc. Nat'l Acad. Sci. USA (1991) 88:3175-3179), and PfEMPl (Howard and Gilladoga, supra, and Pasloske and Howard, supra), have been proposed as candidates.
  • PE adherence is correlated with the expression of PfEMPl on the surface of mature stage PE (Leech, et al., J. Exp. Med. (1984) 159:1567- 1575). Alterations in the adherence phenotype of the PE selected for in vitro are usually associated with the emergence of new forms of PfEMPl (Biggs, et al., J. Immunol.
  • Pfalhesin modified band 3
  • Sequestrin which appears to be homologous to PfEMPl, extracted with TX100 from knobless PE, was shown to bind to immobilized CD36 (Ockenhouse, Proc. Nat'l Acad. Sci. USA (1991) 88:3175-3179).
  • directed evolution of experimentally modifying a biological molecule towards a desirable property, can be achieved by mutagenizing one or more parental molecular templates and by idendifying any desirable molecules among the progeny molecules.
  • technologies in directed evolution include methods for achieving stochastic (i.e. random) mutagenesis and methods for achieving non-stochastic (non-random) mutagenesis.
  • critical shortfalls in both types of methods are identified in the instant disclosure.
  • stochastic or random mutagenesis is exemplified by a situation in which a progenitor molecular template is mutated (modified or changed) to yield a set of progeny molecules having mutation(s) that are not predetermined.
  • a progenitor molecular template is mutated (modified or changed) to yield a set of progeny molecules having mutation(s) that are not predetermined.
  • stochastic mutagenesis reaction for example, there is not a particular predetermined product whose production is intended; rather there is an uncertainty - hence randomness - regarding the exact nature of the mutations achieved, and thus also regarding the products generated.
  • non-stochastic or non-random mutagenesis is exemplified by a situation in which a progenitor molecular template is mutated (modified or changed) to yield a progeny molecule having one or more predetermined mutations. It is appreciated that the presence of background products in some quantity is a reality in many reactions where molecular processing occurs, and the presence of these background products does not detract from the non-stochastic nature of a mutagenesis process having a predetermined product.
  • stochastic mutagenesis is manifested in processes such as error- prone PCR and stochastic shuffling, where the mutation(s) achieved are random or not predetermined.
  • non-stochastic mutagenesis is manifested in instantly disclosed processes such as gene site-saturation mutagenesis and synthetic ligation reassembly, where the exact chemical structure(s) of the intended product(s) are predetermined.
  • Natural evolution has been a springboard for directed or experimental evolution, serving both as a reservoir of methods to be mimicked and of molecular templates to be mutagenized. It is appreciated that, despite its intrinsic process-related limitations (in the types of favored &/or allowed mutagenesis processes) and in its speed, natural evolution has had the advantage of having been in process for millions of years & and throughout a wide diversity of environments. Accordingly, natural evolution (molecular mutagenesis and selection in nature) has resulted in the generation of a wealth of biological compounds that have shown usefulness in certain commercial applications.
  • nucleic acids do not reach close enough proximity to each other in a operative environment to undergo chimerization or incorporation or other types of transfers from one species to another.
  • the chimerization of nucleic acids from these 2 species is likewise unlikely, with parasites common to the two species serving as an example of a very slow passageway for inter-molecular encounters and exchanges of DNA.
  • the generation of a molecule causing self-toxicity or self -lethality or sexual sterility is avoided in nature.
  • the propagation of a molecule having no particular immediate benefit to an organism is prone to vanish in subsequent generations of the organism. Furthermore, e.g., there is no selection pressure for improving the performance of molecule under conditions other than those to which it is exposed in its endogenous environment; e.g. a cytoplasmic molecule is not likely to acquire functional features extending beyond what is required of it in the cytoplasm. Furthermore still, the propagation of a biological molecule is susceptible to any global detrimental effects - whether caused by itself or not - on its ecosystem. These and other characteristics greatly limit the types of mutations that can be propagated in nature. On the other hand, directed (or experimental) evolution - particularly as provided herein
  • the directed evolution invention can provide more wide-ranging possibilities in the types of steps that can be used in mutagenesis and selection processes. Accordingly, using templates harvested from nature, the instant directed evolution invention provides more wide-ranging possibilities in the types of progeny molecules that can be generated and in the speed at which they can be generated than often nature itself might be expected to in the same length of time.
  • the instantly disclosed directed evolution methods can be applied iteratively to produce a lineage of progeny molecules (e.g. comprising successive sets of progeny molecules) that would not likely be propagated (i.e., generated &/or selected for) in nature, but that could lead to the generation of a desirable downstream mutagenesis product that is not achievable by natural evolution.
  • progeny molecules e.g. comprising successive sets of progeny molecules
  • Previous Directed Evolution Methods Are Suboptimal:
  • Mutagenesis has been attempted in the past on many occasions, but by methods that are inadequate for the purpose of this invention.
  • previously described non- stochastic methods have been serviceable in the generation of only very small sets of progeny molecules (comprised often of merely a solitary progeny molecule).
  • a chimeric gene has been made by joining 2 polynucleotide fragments using compatible sticky ends generated by restriction enzyme(s), where each fragment is derived from a separate progenitor (or parental) molecule.
  • Another example might be the mutagenesis of a single codon position (i.e. to achieve a codon substitution, addition, or deletion) in a parental polynucleotide to generate a single progeny polynucleotide encoding for a single site- mutagenized polypeptide.
  • stochastic methods have been used to achieve larger numbers of point mutations and/or chimerizations than non-stochastic methods; for this reason, stochastic methods have comprised the predominant approach for generating a set of progeny molecules that can be subjected to screening, and amongst which a desirable molecular species might hopefully be found.
  • a major drawback of these approaches is that - because of their stochastic nature - there is a randomness to the exact components in each set of progeny molecules that is produced. Accordingly, the experimentalist typically has little or no idea what exact progeny molecular species are represented in a particular reaction vessel prior to their generation. Thus, when a stochastic procedure is repeated (e.g.
  • IC information content
  • Information density is the IC per unit length of a sequence. Active sites of enzymes tend to have a high information density. By contrast, flexible linkers of information in enzymes have a low information density.
  • Error-prone PCR uses low-fidelity polymerization conditions to introduce a low level of point mutations randomly over a long sequence. In a mixture of fragments of unknown sequence, error-prone PCR can be used to mutagenize the mixture.
  • the published error- prone PCR protocols suffer from a low processivity of the polymerase. Therefore, the protocol is unable to result in the random mutagenesis of an average-sized gene. This inability limits the practical application of error-prone PCR.
  • Some computer simulations have suggested that point mutagenesis alone may often be too gradual to allow the large- scale block changes that are required for continued and dramatic sequence evolution. Further, the published error-prone PCR protocols do not allow for amplification of DNA fragments greater than 0.5 to 1.0 kb, limiting their practical application.
  • repeated cycles of error-prone PCR can lead to an accumulation of neutral mutations with undesired results, such as affecting a protein's immunogenicity but not its binding affinity.
  • oligonucleotide-directed mutagenesis In oligonucleotide-directed mutagenesis, a short sequence is replaced with a synthetically mutagenized oligonucleotide. This approach does not generate combinations of distant mutations and is thus not combinatorial. The limited library size relative to the vast sequence length means that many rounds of selection are unavoidable for protein optimization. Mutagenesis with synthetic oligonucleotides requires sequencing of individual clones after each selection round followed by grouping them into families, arbitrarily choosing a single family, and reducing it to a consensus motif. Such motif is re-synthesized and reinserted into a single gene followed by additional selection. This step process constitutes a statistical bottleneck, is labor intensive, and is not practical for many rounds of mutagenesis. Error-prone PCR and oligonucleotide-directed mutagenesis are thus useful for single cycles of sequence fine tuning, but rapidly become too limiting when they are applied for multiple cycles.
  • cassette mutagenesis a sequence block of a single template is typically replaced by a (partially) randomized sequence. Therefore, the maximum information content that can be obtained is statistically limited by the number of random sequences (i.e., library size). This eliminates other sequence families which are not currently best, but which may have greater long term potential.
  • mutagenesis with synthetic oligonucleotides requires sequencing of individual clones after each selection round. Thus, such an approach is tedious and impractical for many rounds of mutagenesis.
  • RNA ligase ribozyme from a random library using many rounds of amplification by error-prone PCR and selection.
  • the evolution of most organisms occurs by natural selection and sexual reproduction.
  • sexual reproduction ensures mixing and combining of the genes in the offspring of the selected individuals.
  • homologous chromosomes from the parents line up with one another and cross-over part way along their length, thus randomly swapping genetic material. Such swapping or shuffling of the DNA allows organisms to evolve more rapidly.
  • the invention provides a method for producing a plurality of, or a library of, nucleic acids encoding a plurality of modified antigen binding sites, wherein the modified antigen binding sites are derived from a first nucleic acid comprising a sequence encoding a first antigen binding site, the method comprising: (a) providing a first nucleic acid encoding a first antigen binding site; (b) providing a set of mutagenic oligonucleotides that encode naturally-occurring amino acid variants at a plurality of targeted codons in the first nucleic acid; and, (c) using the set of mutagenic oligonucleotides to generate a set of antigen binding site-encoding variant nucleic acids encoding a range of amino acid variations at each amino acid codon that was mutagenized, thereby producing a library of nucleic acids encoding a plurality of modified antigen binding sites.
  • step (b) provides a set of mutagenic oligonucleotides that encode all nineteen naturally-occurring amino acid variants for each targeted codon, thereby generating all 19 possible natural amino acid changes at each amino acid codon mutagenized.
  • the method can further comprise expressing the set of variant antigen binding site- encoding nucleic acids such that antigen binding site-encoding polypeptides encoded by the variant nucleic acids are expressed.
  • the set of mutagenic oligonucleotides comprises a 19-fold degenerate mutagenic oligonucleotide for each codon to be mutagenized, wherein each of the 19-fold degenerate mutagenic oligonucleotides comprises a homologous first sequence and a degenerate triplet second sequence.
  • the antigen binding site can comprise a single stranded antigen binding polypeptide, a Fab fragment, an Fc fragment, a Fd fragment, a F(ab') 2 fragment, a Fv fragment or a complementarity determining region (CDR).
  • the antigen binding site polypeptide can further comprise an antibody polypeptide.
  • the antigen binding site polypeptide further comprises an antigen binding site of a T cell receptor (TCR).
  • TCR antigen binding site polypeptide sequence modified by the methods of the invention can include the TCR alpha chain, the TCR beta chain, or both.
  • the antigen binding site polypeptide can further comprise a T cell receptor (TCR).
  • the antigen binding site polypeptide further comprises an antigen binding site of a major histocompatibility complex (MHC) molecule.
  • the antigen binding site polypeptide can further comprise a major histocompatibility complex (MHC) molecule.
  • MHC major histocompatibility complex
  • the major histocompatibility complex (MHC) molecule can comprise a Class I MHC molecule or a Class Et MHC molecule.
  • the MHC antigen binding site polypeptide sequence modified by the methods of the invention can include the MHC Class II alpha chain, the MHC Class Et beta chain, or both.
  • the nucleic acid of step (a) is derived from a nucleic acid encoding a mammalian polypeptide, such as a human polypeptide.
  • the mammalian polypeptide can be an antibody, a T cell receptor (alpha chain and/or beta chain), a Class I MHC molecule or a Class II MHC molecule(alpha chain and/or beta chain).
  • the nucleic acid of step (a) can be derived from a human nucleic acid encoding an antigen binding site.
  • the nucleic acid of step (a) can be derived from a phage comprising a human nucleic acid sequence encoding an antigen binding site, wherein the phage expresses the antigen binding site.
  • the nucleic acid of step (a) can be derived from a non-human mammal comprising a human nucleic acid sequence encoding an antigen binding site, wherein the non-human mammal expresses the antigen binding site.
  • the non-human mammal can be a transgenic non-human mammal, such as a mouse.
  • at least two amino acid codons in the antigen binding site are mutagenized.
  • all the amino acid codons in the antigen binding site are mutagenized, or, all the amino acid codons in the protein, e.g., the antibody, T cell receptor (TCR) or MHC polypeptide are mutagenized.
  • the degenerate mutagenic oligonucleotide comprises a first homologous sequence, a degenerate triplet second sequence, and a third homologous sequence.
  • each degenerate oligonucleotide comprises a first homologous sequence, a plurality of degenerate triplets second sequences, and a third homologous sequence.
  • the method can further comprise screening the expressed antigen binding site polypeptide for its ability to specifically bind an antigen. In one aspect, the method further comprises screening the expressed antigen binding site polypeptide for its ability to specifically bind an antigen capable of being specifically bound by the first antigen binding site polypeptide. In one aspect, the method comprises identifying an antigen binding site variant by its increased antigen binding affinity or antigen binding specificity as compared to the affinity or specificity of the first antigen binding site to the antigen. In one aspect, the method comprises identifying an antigen binding site variant by its decreased antigen binding affinity or antigen binding specificity as compared to the affinity or specificity of the first antigen binding site to the antigen.
  • the method can comprise mutagenizing the first nucleic acid of step (a) by a method comprising an optimized directed evolution system.
  • the method can comprise mutagenizing the first nucleic acid of step (a) by a method comprising a synthetic ligation reassembly.
  • the method can comprise screening the expressed antigen binding site polypeptide for its ability to specifically bind an antigen by a method comprising expression of the expressed antigen binding site polypeptide in a solid phase.
  • the method can comprise screening the expressed antigen binding site polypeptide for its ability to specifically bind an antigen by a method comprising a capillary array.
  • the method can comprise screening the expressed antigen binding site polypeptide for its ability to specifically bind an antigen by a method comprising a double-orificed container, such as a double-orificed capillary array, e.g., a GIGAMATREXTM capillary array.
  • the method can comprise screening the expressed antigen binding site polypeptide for its ability to specifically bind an antigen by a method comprising use of an ELISA.
  • the method also can comprise screening the expressed antigen binding site polypeptide for its ability to specifically bind an antigen by a method comprising phage display of the antigen binding site polypeptide.
  • the method also can comprise screening the expressed antigen binding site polypeptide for its ability to specifically bind an antigen by a method comprising expression of the expressed antigen binding site polypeptide in a liquid phase.
  • the method also can comprise screening the expressed antigen binding site polypeptide for its ability to specifically bind an antigen by a method comprising ribosome display of the antigen binding site polypeptide.
  • the set of progeny antigen binding site-encoding variant nucleic acids is generated by amplifying the nucleic acid of step (a) by a polymerase-based amplification using a plurality of oligonucleotides, such as polymerase chain reaction (PCR).
  • PCR polymerase chain reaction
  • the invention provides a library of nucleic acids encoding a plurality of modified antigen binding sites, wherein the modified antigen binding sites are derived from a first nucleic acid comprising a sequence encoding a first antigen binding site, made by a method comprising the following steps: (a) providing a first nucleic acid encoding a first antigen binding site; (b) providing a set of mutagenic oligonucleotides that encode naturally- occurring amino acid variants at a plurality of targeted codons in the first nucleic acid; and, (c) using the set of mutagenic oligonucleotides to generate a set of antigen binding site- encoding variant nucleic acids encoding a range of amino acid variations at each amino acid codon that was mutagenized, thereby producing a library of nucleic acids encoding a plurality of modified antigen binding sites.
  • the invention provides a method for producing from a library of variant antibodies from a template antibody, the method comprising: (a) providing a first nucleic acid encoding the template antibody; (b) providing a set of mutagenic oligonucleotides that encode naturally-occurring amino acid variants at a plurality of targeted codons in the first nucleic acid; and, c) using the set of mutagenic oligonucleotides to generate a set of antibody-encoding variant nucleic acids encoding a range of amino acid variations at each amino acid codon that was mutagenized, thereby producing a library of nucleic acids encoding a plurality of variant antibodies.
  • step (b) provides a set of mutagenic oligonucleotides that encode all nineteen naturally-occurring amino acid variants for each targeted codon, thereby generating all 19 possible natural amino acid changes at each amino acid codon mutagenized.
  • the antibody can be a polypeptide comprising a Fab fragment, an Fd fragment, an Fc fragment, a F(ab')2 fragment, a Fv fragment or a complementarity determining region (CDR).
  • the plurality of oligonucleotides comprises a degenerate oligonucleotide for each codon to be mutagenized, wherein each of the degenerate oligonucleotides comprises a homologous first sequence and a degenerate triplet second sequence.
  • the set of progeny polynucleotides encoding antibodies can be generated by amplifying the nucleic acid of step (a) using a plurality of oligonucleotides.
  • the invention provides a library of variant antibodies derived from a template antibody made by a method comprising the following steps: (a) providing a first nucleic acid encoding the template antibody; (b) providing a set of mutagenic oligonucleotides that encode naturally-occurring amino acid variants at a plurality of targeted codons in the first nucleic acid; and, (c) using the set of mutagenic oligonucleotides to generate a set of antibody-encoding variant nucleic acids encoding a range of amino acid variations at each amino acid codon that was mutagenized, thereby producing a library of nucleic acids encoding a plurality of variant antibodies.
  • the invention provides a method for producing from a library of variant T cell receptors (TCRs) from a template T cell receptor (TCR), the method comprising: (a) providing a first nucleic acid encoding the template T cell receptor; (b) providing a set of mutagenic oligonucleotides that encode naturally-occurring amino acid variants at a plurality of targeted codons in the first nucleic acid; and, c) using the set of mutagenic oligonucleotides to generate a set of T cell receptor (TCR)-encoding variant nucleic acids encoding a range of amino acid variations at each amino acid codon that was mutagenized, thereby producing a library of nucleic acids encoding a plurality of variant T cell receptors (TCRs).
  • TCRs T cell receptors
  • the invention provides a library of variant T cell receptors (TCRs) derived from a template T cell receptor (TCR) made by a method comprising the following steps: (a) providing a first nucleic acid encoding the template T cell receptor; (b) providing a set of mutagenic oligonucleotides that encode naturally-occurring amino acid variants at a plurality of targeted codons in the first nucleic acid; and, (c) using the set of mutagenic oligonucleotides to generate a set of T cell receptor (TCR)-encoding variant nucleic acids encoding a range of amino acid variations at each amino acid codon that was mutagenized, thereby producing a library of nucleic acids encoding a plurality of variant T cell receptors (TCRs).
  • TCRs T cell receptors
  • the invention provides a method for producing from a library of variant major histocompatibility complex (MHC) molecules from a template major histocompatibility complex (MHC) molecule, the method comprising: (a) providing a first nucleic acid encoding the template major histocompatibility complex (MHC) molecule; (b) providing a set of mutagenic oligonucleotides that encode naturally-occurring amino acid variants at a plurality of targeted codons in the first nucleic acid; and, (c) using the set of mutagenic oligonucleotides to generate a set of major histocompatibility complex (MHC) molecule- encoding variant nucleic acids encoding a range of amino acid variations at each amino acid codon that was mutagenized, thereby producing a library of nucleic acids encoding a plurality of variant major histocompatibility complex (MHC) molecules.
  • MHC major histocompatibility complex
  • the invention provides a library of variant major histocompatibility complex (MHC) molecules derived from a template major histocompatibility complex (MHC) molecule made by a method comprising the following steps: (a) providing a first nucleic acid encoding the template major histocompatibility complex (MHC) molecule; (b) providing a set of mutagenic oligonucleotides that encode naturally-occurring amino acid variants at a plurality of targeted codons in the first nucleic acid; and, (c) using the set of mutagenic oligonucleotides to generate a set of major histocompatibility complex (MHC) molecule- encoding variant nucleic acids encoding a range of amino acid variations at each amino acid codon that was mutagenized, thereby producing a library of nucleic acids encoding a plurality of variant major histocompatibility complex (MHC) molecules.
  • MHC major histocompatibility complex
  • the invention provides a method of making a set of nucleic acids encoding a set of antigen binding site variants comprising the steps of: (a) providing a template nucleic acid encoding an antigen-binding polypeptide; (b) providing a plurality of oligonucleotides that encode all nineteen naturally-occurring amino acid variants at a single amino acid residue of the antigen-binding polypeptide; and, (c) generating a set of progeny antigen binding site- encoding variant nucleic acids encoding a non-stochastic range of single amino acid substitutions at each amino acid codon that was mutagenized, whereby all 19 possible natural amino acid changes are generated at each amino acid codon mutagenized, thereby making a set of nucleic acids encoding a set of antigen binding site variants.
  • the method further comprises expressing the set of progeny antigen binding site- encoding polynucleotides such that antigen binding site-encoding polypeptides encoded by the progeny polynucleotides are expressed.
  • the plurality of oligonucleotides can comprise a set of degenerate oligonucleotides and each of the degenerate oligonucleotides comprises a homologous first sequence and a degenerate triplet second sequence.
  • the antigen binding site-encoding polypeptide comprises a single stranded antigen binding polypeptide.
  • the antigen binding site-encoding polypeptide can comprise an antibody polypeptide.
  • the antigen binding site-encoding polypeptide can comprise an antigen binding site of a T cell receptor (TCR), or, a T cell receptor (TCR).
  • TCR T cell receptor
  • TCR T cell receptor
  • TCR T cell receptor
  • TCR T cell receptor
  • the antigen binding site-encoding polypeptide can comprise an antigen binding site of a major histocompatibility complex (MHC) molecule, or, a major histocompatibility complex (MHC) molecule.
  • MHC major histocompatibility complex
  • the nucleic acid of step (a) can be derived from a nucleic acid encoding a mammalian antibody polypeptide.
  • the nucleic acid of step (a) can be derived from a human nucleic acid.
  • the at least two amino acid codons in the antigen binding site are mutagenized and a set of degenerate oligonucleotides that encode all nineteen naturally- occurring amino acid variants are provided for each amino acid codon mutagenized.
  • all the amino acid codons in the antigen binding site are mutagenized and a set of degenerate oligonucleotides that encode all nineteen naturally-occurring amino acid variants are provided for each amino acid codon mutagenized.
  • all the amino acid codons in the antibody polypeptide can be mutagenized.
  • a degenerate oligonucleotide comprises a first homologous sequence, a degenerate triplet second sequence, and a homologous third sequence.
  • each degenerate oligonucleotide comprises a first homologous sequence, a degenerate triplet second sequence, and a homologous third sequence.
  • the method further comprises mutagenizing the template nucleic acid by a method comprising an optimized directed evolution system and a method comprising a synthetic ligation reassembly.
  • the method further comprises screening an expressed antigen binding site-encoding polypeptide for its ability to specifically bind an antigen.
  • the method can also comprise screening the expressed antigen binding site-encoding polypeptide for its ability to specifically bind an antigen capable of being specifically bound by the first antigen binding site.
  • the method can comprise identifying an antigen binding site variant by its increased or decreased or altered antigen binding affinity or antigen binding specificity to the antigen as compared to the affinity or specificity of the antigen binding site encoded by the nucleic acid of step (a).
  • the method comprises screening the expressed antigen binding site-encoding polypeptide for its ability to specifically bind an antigen in a solid phase or a liquid phase.
  • the method comprises a capillary array, such as a double-orificed capillary array.
  • the method can comprise screening the expressed antigen binding site- encoding polypeptide for its ability to specifically bind an antigen by an ELISA.
  • the set of variant nucleic acids is generated by performing amplification reactions on the nucleic acid of step (a) using the set of oligonucleotides to generate a set of variant nucleic acids encoding nineteen amino acid substitution variants at least one amino acid residue of the antigen-binding polypeptide, or, all of the amino acid residue of the antigen-binding polypeptide.
  • the amplification can comprise a polymerase- based amplification, such as a polymerase chain reaction (PCR), or another equivalent reaction.
  • the set of variant nucleic acids comprises 10 10 members, 10 9 , 10 8 , 10 7 , 10 6 10 5 , 10 4 , 10 3 , 10 2 members.
  • the invention provides a method of making a set (i.e., a library) of antibody variants comprising the steps of: (a) providing a nucleic acid encoding an antibody; (b) providing a plurality of oligonucleotides; (c) generating a non-stochastic range of single amino acid substitutions at each amino acid codon, whereby all 19 possible natural amino acid changes are generated at each amino acid codon mutagenized, thereby generating a set of variant nucleic acids; and, (d) expressing the set of variant nucleic acids such that the antibody variants encoded by the variant nucleic acids are expressed.
  • the antibody can be selected from the group consisting of polypeptides comprising a Fab fragment, a Fd fragment, an Fc fragment, a F(ab') 2 fragment, a Fv fragment and a complementarity determining region (CDR).
  • the plurality of oligonucleotides can comprise a set of degenerate oligonucleotides that encode all nineteen naturally-occurring amino acid variants at a single amino acid residue of the antibody, wherein each of the degenerate oligonucleotides comprises a homologous first sequence and a degenerate triplet second sequence.
  • the method in generating a non-stochastic range of single amino acid substitutions, can comprise performing amplification reactions on the nucleic acid of step (a) using the set of oligonucleotides to generate a set of variant nucleic acids encoding nineteen amino acid substitution variants at a single amino acid residue of the antibody.
  • the invention provides a method of identifying a variant of an antigen binding site comprising the steps of: (a) providing a nucleic acid encoding an antigen binding site; (b) providing a set of oligonucleotides that encode all nineteen naturally- occurring amino acid variants at all residues of the antigen- binding site; (c) incorporating the sequence of the oligonucleotides of step (b) into the nucleic acid of step (a) to generate a set of variant nucleic acids encoding nineteen amino acid substitution variants at each residue of the antigen binding site; (d) expressing each of the variant nucleic acids as polypeptides and measuring the variant's affinity to the antigen; and, (e) identifying a variant of the antigen binding site by its increased or decreased antigen binding specificity as compared to the antigen binding affinity of the antigen binding site encoded by the nucleic acid of step (a).
  • the variant nucleic acids are expressed using in vitro transcription/translation. In alternative aspects, the variant nucleic acids are expressed using phage display, ribosome display, or equivalent methods. In alternative aspects, the method comprises screening the expressed antigen binding site for its ability to specifically bind an antigen in a solid phase or a liquid phase. In one aspect, the screening is accomplished using a double orificed container, such as a using a double orificed capillary array.
  • the set of oligonucleotides comprises a set of degenerate oligonucleotides that encode all nineteen naturally-occurring amino acid variants at at least one amino acid residue of the antibody, wherein each of the degenerate oligonucleotides comprises a homologous first sequence and a degenerate triplet second sequence.
  • the set of oligonucleotides comprises a set of degenerate oligonucleotides that encode all nineteen naturally-occurring amino acid variants at all amino acid residues of the antibody.
  • the method incorporates the sequence of the oligonucleotides of step (b) into the nucleic acid of step (a) is accomplished by an amplification reaction using the oligonucleotides as primers.
  • the antigen binding site comprises an antibody, including a Fab fragment, an Fd fragment, an Fc fragment, a F(ab') 2 fragment, a Fv fragment and a complementarity determining region (CDR).
  • the antigen binding site comprises an antigen binding site of a T cell receptor and a major histocompatibility complex molecule.
  • the antigen binding site-encoding nucleic acids generated by the methods of the invention are further changed or "evolved.”
  • These nucleic acid sequences can be changed by mutagenesis, base residue insertion(s) or base residue deletion(s).
  • Evolution technologies can be used to further engineer these sequences, including, e.g., Gene Site Saturation MutagenesisTM (GSSM) and GeneReassemblyTM (Diversa Corporation, San Diego, CA), as described in further detail herein.
  • these nucleic acid sequences can be changed or "evolved” or “genetically engineered” by, e.g., error-prone PCR, shuffling, oligonucleotide-directed mutagenesis, assembly PCR, sexual PCR mutagenesis, in vivo mutagenesis, cassette mutagenesis, recursive ensemble mutagenesis, exponential ensemble mutagenesis, site-specific mutagenesis, gene reassembly, gene site saturated mutagenesis (GSSM), synthetic ligation reassembly (SLR) and/or a combination thereof.
  • error-prone PCR shuffling
  • oligonucleotide-directed mutagenesis assembly PCR
  • sexual PCR mutagenesis in vivo mutagenesis
  • cassette mutagenesis cassette mutagenesis
  • recursive ensemble mutagenesis recursive ensemble mutagenesis
  • exponential ensemble mutagenesis site-specific mutagenesis
  • gene reassembly gene
  • the modifications, additions or deletions are introduced by, e.g., recombination, recursive sequence recombination, phosphothioate-modified DNA mutagenesis, uracil-containing template mutagenesis, gapped duplex mutagenesis, point mismatch repair mutagenesis, repair-deficient host strain mutagenesis, chemical mutagenesis, radiogenic mutagenesis, deletion mutagenesis, restriction-selection mutagenesis, restriction- purification mutagenesis, artificial gene synthesis, ensemble mutagenesis, chimeric nucleic acid multimer creation and/or a combination thereof.
  • these methods are iteratively repeated until an antigen binding site (e.g., an antibody) having an altered or different activity or an altered or different stability from that of the antigen binding site to be "evolved” is produced.
  • an antigen binding site e.g., an antibody
  • the CDR3 region of the antigen binding molecule- encoding nucleic acid sequence is changed or "evolved.”
  • the instant invention provides non-stochastic means for comprehensively and exhaustively generating all possible point mutations in a parental template.
  • the instant invention further provides means for exhaustively generating all possible chimerizations within a group of chimerizations.
  • the present invention provides multicomponent genetic vaccines that include at least one, or, two or more, genetic vaccine components that confer upon the vaccine the ability to direct an immune response so as to achieve an optimal response to vaccination.
  • the genetic vaccines can include a component that provides optimal antigen release; a component that provides optimal production of cytotoxic T lymphocytes; a component that directs release of an immunomodulator; a component that directs release of a chemokine; and/or a component that facilitates binding to, or entry into, a desired target cell type.
  • a component can confer improved improves binding to, and uptake of, the genetic vaccine to target cells such as antigen- expressing cells or antigen-presenting cells.
  • Additional components include those that direct antigen peptides derived from uptake of an antigen into a cell to presentation on either Class I or Class EL molecules.
  • this invention provides a method for obtaining an immunomodulatory polynucleotide that has an optimized modulatory effect on an immune response, or encodes a polypeptide that has an optimized modulatory effect on an immune response, the method comprising: creating a library of non-stochastically generated progeny polynucleotides from a parental polynucleotide set; wherein 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.
  • this invention provides a method for obtaining an immunomodulatory polynucleotide that has an optimized modulatory effect on an immune response, or encodes a polypeptide that has an optimized modulatory effect on an immune response, the method comprising: screening a library of 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.
  • this invention provides a method for obtaining an immunomodulatory polynucleotide that has an optimized modulatory effect on an immune response, or encodes a polypeptide that has an optimized modulatory effect on an immune response, 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 genetic vaccine 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; whereby optimization can thus be achieved using one or more of the directed evolution methods as described herein in
  • this invention provides 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.
  • this invention provides 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.
  • this invention provides 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
  • this invention provides that the ability to a vaccine, for example a genetic vaccine, or a component of a vaccine, for example a component of a genetic vaccine by optimizing its immunogenicity.
  • the present invention provides for the modification of other properties, including its:
  • the instant invention provides for the modification of molecule's immunogenic properties such as
  • one or more of the genetic vaccine components is obtained by a method that involves: (1) reassembling (&/or subjecting to one or more directed evolution methods described herein) at least first and second forms of a nucleic acid which can confer a desired property upon a genetic vaccine, wherein the first and second forms differ from each other in two or more nucleotides, to produce a library of recombinant nucleic acids; and (2) screening the library to identify at least one optimized recombinant component that exhibits an enhanced capacity to confer the desired property upon the genetic vaccine.
  • the following additional steps can be conducted: (3) reassembling (&/or subjecting to one or more directed evolution methods described herein) at least one optimized recombinant component with a further form of the nucleic acid, which is the same or different from the first and second forms, to produce a further library of recombinant nucleic acids; (4) screening the further library to identify at least one further optimized recombinant component that exhibits an enhanced capacity to confer the desired property upon the genetic vaccine; and (5) repeating (3) and (4), as necessary, until the further optimized recombinant component exhibits a further enhanced capacity to confer the desired property upon the genetic vaccine.
  • the first form of the nucleic acid is a first member of a gene family and the second form of the nucleic acid comprises a second member of the gene family.
  • Additional forms of the module nucleic acid can also be members of the gene family.
  • the first member of the gene family can be obtained from a first species of organism and the second member of the gene family obtained from a second species of organism.
  • the optimized recombinant genetic vaccine component obtained by the methods of the invention can be backcrossed by, for example, reassembling (&/or subjecting to one or more directed evolution methods described herein) the optimized recombinant genetic vaccine component with a molar excess of one or both of the first and second forms of the substrate nucleic acids to produce a further library of recombinant genetic vaccine components; and screening the further library to identify at least one optimized recombinant genetic vaccine component that further enhances the capability of a genetic vaccine vector that includes the component to modulate the immune response.
  • Additional embodiments of the invention provide methods of obtaining a genetic vaccine component that confers upon a genetic vaccine vector an enhanced ability to replicate in a host cell. These methods involve creating a library of recombinant nucleic acids by subjecting to reassembly (&/or one or more additional directed evolution methods described herein) at least two forms of a polynucleotide that can confer episomal replication upon a vector that contains the polynucleotide; introducing into a population of host cells a library of vectors, each of which contains a member of the library of recombinant nucleic acids and a polynucleotide that encodes a cell surface antigen; propagating the population of host cells for multiple generations; and identifying cells which display the cell surface antigen on a surface of the cell, wherein cells which display the cell surface antigen are likely to harbor a vector that contains a recombinant vector module which enhances the ability of the vector to replicate episomally.
  • Genetic vaccine components that confer upon a vector an enhanced ability to replicate in a host cell can also be obtained by creating a library of recombinant nucleic acids by subjecting to reassembly (&/or one or more additional directed evolution methods described herein) at least two forms of a polynucleotide derived from a human papillomavirus that can confer episomal replication upon a vector that contains the polynucleotide; introducing a library of vectors, each of which contains a member of the library of recombinant nucleic acids, into a population of host cells; propagating the host cells for a plurality of generations; and identifying cells that contain the vector.
  • the invention provides methods obtaining a genetic vaccine component that confers upon a vector an enhanced ability to replicate in a human host cell by creating a library of recombinant nucleic acids by subjecting to reassembly (&/or one or more additional directed evolution methods described herein) at least two forms of a polynucleotide that can confer episomal replication upon a vector that contains the polynucleotide; introducing a library of genetic vaccine vectors, each of which comprises a member of the library of recombinant nucleic acids, into a test system that mimics a human immune response; and determining whether the genetic vaccine vector replicates or induces an immune response in the test system.
  • a suitable test system can involve human skin cells present as a xenotransplant on skin of an immunocompromised non-human host animal, for example, or a non-human mammal that comprises a functional human immune system. Replication in these systems can be detected by determining whether the animal exhibits an immune response against the antigen.
  • the invention also provides methods of obtaining a genetic vaccine component that confers upon a genetic vaccine an enhanced ability to enter an antigen- presenting cell. These methods involve creating a library of recombinant nucleic acids by subjecting to reassembly (&/or one or more additional directed evolution methods described herein) at least two forms of a polynucleotide that can confer episomal replication upon a vector that contains the polynucleotide; introducing a library of genetic vaccine vectors, each of which comprises a member of the library of recombinant nucleic acids, into a population of antigen-presenting or antigen-processing cells; and determining the percentage of cells in the population which contain the nucleic acid vector.
  • Antigen- presenting or antigen-processing cells of interest include, for example, B cells, monocytes/macrophages, dendritic cells, Langerhans cells, keratinocytes, and muscle cells.
  • the present invention provides methods of obtaining a polynucleotide that has a modulatory effect on an immune response, including a T cell receptors, major histocompatibility complex (MHC) molecules, antibodies, or those induced by a genetic vaccine, either directly (i.e., as an immunomodulatory polynucleotide) or indirectly (i.e., upon translation of the polynucleotide to create an immunomodulatory polypeptide.
  • MHC major histocompatibility complex
  • the methods of the invention involve: creating a library of experimentally generated (in vitro &/or in vivo) polynucleotides; and screening the library to identify at least one optimized experimentally generated (in vitro &/or in vivo) polynucleotide that exhibits, either by itself or through the encoded polypeptide, an enhanced ability to modulate an immune response than a form of the nucleic acid from which the library was created.
  • Examples include, for example, CpG-rich polynucleotide sequences, polynucleotide sequences that encode a costimulator (e.g., B7-1, B7-2, CD1, CD40, CD154 (ligand for CD40), CD150 (SLAM), or a cytokine.
  • the screening step used in these methods can include, for example, introducing genetic vaccine vectors which comprise the library of recombinant nucleic acids into a cell, and identifying cells which exhibit an increased ability to modulate an immune response of interest or increased ability to express an immunomodulatory molecule.
  • a library of recombinant cytokine-encoding nucleic acids can be screened by testing the ability of cytokines encoded by the nucleic acids to activate cells which contain a receptor for the cytokine.
  • the receptor for the cytokine can be native to the cell, or can be expressed from a heterologous nucleic acid that encodes the cytokine receptor.
  • the optimized costimulators can be tested to identify those for which the cells or culture medium are capable of inducing a predominantly T H 2 immune response, or a predominantly T H I immune response.
  • the polynucleotide that has a modulatory effect on an immune response is obtained by: (1) reassembling (&/or subjecting to one or more directed evolution methods described herein) at least first and second forms of a nucleic acid that is, or encodes a molecule that is, involved in modulating an immune response, wherein the first and second forms differ from each other in two or more nucleotides, to produce a library of experimentally generated (in vitro &/or in vivo) polynucleotides; and (2) screening the library to identify at least one optimized experimentally generated (in vitro &/or in vivo) polynucleotide that exhibits, either by itself or through the encoded polypeptide, an enhanced ability to modulate an immune response than a form of the nucleic acid from which the library was created.
  • the method can further involve: (3) reassembling (&/or subjecting to one or more directed evolution methods described herein) at least one optimized experimentally generated (in vitro &/or in vivo) polynucleotide with a further form of the nucleic acid, which is the same or different from the first and second forms, to produce a further library of experimentally generated (in vitro &/or in vivo) polynucleotides; (4) screening, the further library to identify at least one further optimized experimentally generated (in vitro &/or in vivo) polynucleotide that exhibits an enhanced ability to modulate an immune response than a form of the nucleic acid from which the library was created.; and (5) repeating (3) and (4), as necessary, until the further optimized experimentally generated (in vitro &/or in vivo) polynucleotide exhibits an further enhanced ability to modulate an immune response than a form of the nucleic acid from which the library was created.
  • the library of experimentally generated (in vitro &/or in vivo) polynucleotides is screened by: expressing the experimentally generated (in vitro &/or in vivo) polynucleotides so that the encoded peptides or polypeptides are produced as fusions with a protein displayed on the surface of a replicable genetic package; contacting the replicable genetic packages with a plurality of cells that display the receptor; and identifying cells that exhibit a modulation of an immune response mediated by the receptor.
  • the invention also provides methods for obtaining a polynucleotide that encodes an accessory molecule that improves the transport or presentation of antigens by a cell.
  • These methods involve creating a library of experimentally generated (in vitro &/or in vivo) polynucleotides by subjecting to reassembly (&/or one or more additional directed evolution methods described herein) nucleic acids that encode all or part of the accessory molecule; and screening the library to identify an optimized experimentally generated (in vitro &/or in vivo) polynucleotide that encodes a recombinant accessory 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 the non-recombinant nucleic acids.
  • the screening step involves: introducing the library of experimentally generated (in vitro &/or in vivo) 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 identifying mammalian cells that exhibit increased or decreased immunogenicity to the antigen.
  • the cytokine that is optimized 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.
  • the cytokine is interferon- ⁇ and the screening is performed by expressing the recombinant vector module as a fusion protein which is displayed on the surface of a bacteriophage to form a phage display library, and identifying phage library members which are capable of inhibiting proliferation of a B cell line.
  • Another embodiment utilizes B7-1 (CD80) or B7-2 (CD86) as the costimulator and the cell or culture medium is tested for ability to modulate an immune response.
  • the invention provides methods of using stochastic (e.g. polynucleotide shuffling & interrupted synthesis) and non-stochastic polynucleotide reassembly to obtain optimized recombinant vector modules that encode cytokines and other costimulators that exhibit reduced immunogenicity compared to a corresponding polypeptide encoded by a non- optimized vector module.
  • the reduced immunogenicity can be detected by introducing a cytokine or costimulator encoded by the recombinant vector module into a mammal and determining whether an immune response is induced against the cytokine.
  • the invention also provides methods of obtaining optimized immunomodulatory sequences that encode a cytokine antagonist.
  • suitable cytokine agonists include a soluble cytokine receptor and a transmembrane cytokine receptor having, a defective signal sequence. Examples include sEL-lOR and sEL- 4R, and the like.
  • the present invention provides methods for obtaining a cell-specific binding molecule that is useful for increasing uptake or specificity of a genetic vaccine to a target cell.
  • the methods involve: creating a library of experimentally generated (in vitro &/or in vivo) polynucleotides that by reassembling (&/or subjecting to one or more directed evolution methods described herein) a nucleic acid that encodes a polypeptide that comprises a nucleic acid binding domain and a nucleic acid that encodes a polypeptide that comprises a cell-specific binding domain; and screening the library to identify a experimentally generated
  • Target cells of particular interest include antigen- presenting and antigen-processing cells, such as muscle cells, monocytes, dendritic cells, B cells, Langerhans cells, keratinocytes, and M-cells.
  • the methods of the invention for obtaining a cell- specific binding moiety useful for increasing uptake or specificity of a genetic vaccine to a target cell involve: (1) reassembling (&/or subjecting to one or more directed evolution methods described herein) at least first and second forms of a nucleic acid which comprises a polynucleotide that encodes a nucleic acid binding domain and at least first and second forms of a nucleic acid which comprises a cell-specific ligand that specifically binds to a protein on the surface of a cell of interest, wherein the first and second forms differ from each other in two or more nucleotides, to produce a library of recombinant binding moiety- encoding nucleic acids; (2) transfecting into a population of host cells a library of vectors, each of which comprises: a) a binding site specific for the nucleic acid binding domain and b) a member of the library of recombinant binding moiety-encoding nucleic acids, wherein the re
  • the methods can further involve: (6) reassembling (&/or subjecting to one or more directed evolution methods described herein) at least one optimized recombinant binding moiety-encoding nucleic acid with a further form of the polynucleotide that encodes a nucleic acid binding domain and/or a further form of the polynucleotide that encodes a cell-specific ligand, which are the same or different from the first and second forms, to produce a further library of recombinant binding moiety-encoding nucleic acids; (7) transfecting into a population of host cells a library of vectors that comprise: a) a binding site specific for the nucleic acid binding domain and 2) the recombinant binding moiety-encoding nucleic acids, wherein the recombinant binding moiety is expressed and binds to the binding site to form a vector- binding moiety complex; (8) lysing the host cells under conditions that do not disrupt binding of the vector-binding moiety complex
  • the invention also provides cell-specific recombinant binding moieties produced by expressing in a host cell an optimized recombinant binding moiety-encoding nucleic acid obtained by the methods of the invention.
  • the invention provides genetic vaccines that include: a) an optimized recombinant binding moiety that comprises a nucleic acid binding domain and a cell-specific ligand, and b) a polynucleotide sequence that comprises a binding site, wherein the nucleic acid binding domain is capable of specifically binding to the binding site.
  • a further embodiment of the invention provides methods for obtaining an optimized cell-specific binding moiety useful for increasing uptake, efficacy, or specificity of a genetic vaccine for a target cell by: reassembling (&/or subjecting to one or more directed evolution methods described herein) at least first and second forms of a nucleic acid that comprises a polynucleotide which encodes a non-toxic receptor binding moiety-of an enterotoxin or other toxin, wherein the first and second forms differ from each other in two or more nucleotides, to produce a library of recombinant nucleic acids; (2) transfecting vectors that contain the library of nucleic acids into a population of host cells, wherein the nucleic acids are expressed to form recombinant cell- specific binding moiety polypeptides; (3) contacting the recombinant cell-specific binding moiety polypeptides with a cell surface receptor of a target cell; and (4) determining which recombinant cell-specific binding moiety polypeptides exhibit
  • the present invention also provides methods for evolving a vaccine delivery vehicle, genetic vaccine vector, or a vector component to obtain an optimized delivery vehicle or component that has, or confers upon a vector, enhanced ability to enter a selected mammalian tissue upon administration to a mammal.
  • These methods involve: (1) reassembling (&/or subjecting to one or more directed evolution methods described herein) members of a pool of polynucleotides to produce a library of experimentally generated (in vitro &/or in vivo) polynucleotides; (2) administering to a test animal a library of replicable genetic packages, each of which comprises a member of the library of experimentally generated (in vitro &/or in vivo) polynucleotides operably linked to a polynucleotide that encodes a display polypeptide, wherein the experimentally generated (in vitro &/or in vivo) polynucleotide and the display polypeptide are expressed as a fusion protein which is which is displayed on the
  • the methods of the invention further involve: (4) reassembling (&/or subjecting to one or more directed evolution methods described herein) a nucleic acid that comprises at least one experimentally generated (in vitro &/or in vivo) polynucleotide obtained from a replicable genetic package recovered from the selected tissue with a further pool of polynucleotides to produce a further library of experimentally generated (in vitro &/or in vivo) polynucleotides; (5) administering to a test animal a library of replicable genetic packages, each of which comprises a member of the further library of experimentally generated (in vitro &/or in vivo) polynucleotides operably linked to a polynucleotide that encodes a display polypeptide, wherein the experimentally generated (in vitro &/or in vivo) polynucleotide and the display polypeptide are expressed as a fusion protein which is which is displayed on the surface
  • the invention provides methods for evolving a vaccine delivery vehicle, genetic vaccine vector, or a vector component to obtain an optimized delivery vehicle or component to obtain an optimized delivery vehicle or vector component that has, or confers upon a vector containing the component, enhanced specificity for antigen-presenting cells by: reassembling (&/or subjecting to one or more directed evolution methods described herein) members of a pool of polynucleotides to produce a library of experimentally generated (in vitro &/or in vivo) polynucleotides; producing a library of replicable genetic packages, each of which comprises a member of the library of experimentally generated (in vitro &/or in vivo) polynucleotides operably linked to a polynucleotide that encodes a display polypeptide, wherein the experimentally generated (in vitro &/or in vivo) polynucleotide and the display polypeptide are expressed as a fusion protein which is which is displayed on the surface of the replicable genetic package; (3)
  • the invention provides methods for evolving a vaccine delivery vehicle, genetic vaccine vector, or a vector component to obtain an optimized delivery vehicle or component to obtain an optimized delivery vehicle or vector component that has, or confers upon a vector containing the component, an enhanced ability to enter a target cell by: (1) reassembling (&/or subjecting to one or more directed evolution methods described herein) at least first and second forms of a nucleic acid which encodes an invasin polypeptide, wherein the first and second forms differ from each other in two or more nucleotides, to produce a library of recombinant invasin nucleic acids; (2) producing a library of recombinant bacteriophage, each of which displays on the bacteriophage surface a fusion polypeptide encoded by a chimeric gene that comprises a recombinant invasin nucleic acid operably linked to a polynucleotide that encodes a display polypeptide; (3) contacting the library of recombinant bacteriophage with
  • the optimized recombinant genetic vaccine vectors, delivery vehicles, or vector components obtained using these methods exhibit improved ability to enter an antigen presenting cell.
  • These methods can involve washing the cells after the transfection step to remove vectors which did not enter an antigen presenting cell.; culturing the cells for a predetermined time after transfection; lysing the antigen presenting cells; and isolating the optimized recombinant genetic vaccine vector from the cell lysate.
  • Antigen presenting cells that contain an optimized recombinant genetic vaccine vectors can be identified by, e.g., detecting expression of a marker gene that is included in the vectors.
  • the invention also provides methods of evolving a bacteriophage-derived vaccine delivery vehicle to obtain a delivery vehicle having enhanced ability to enter a target cell. These methods involve the steps of. (1) reassembling (&/or subjecting to one or more directed evolution methods described herein) at least first and second forms of a nucleic acid which encodes an invasin polypeptide, wherein the first and second forms differ from each other in two or more nucleotides, to produce a library of recombinant invasin nucleic acids; (2) producing a library of recombinant bacteriophage, each of which displays on the bacteriophage surface a fusion polypeptide encoded by a chimeric gene that comprises a recombinant invasin nucleic acid operably linked to a polynucleotide that encodes a display polypeptide; (3) contacting the library of recombinant bacteriophage with a population of target cells; (4) removing unbound phage and phage which is bound to the surface
  • the methods can include the further steps of (6) reassembling (&/or subjecting to one or more directed evolution methods described herein) a nucleic acid which comprises at least one recombinant invasin nucleic acid obtained from a bacteriophage which is recovered from a target cell with a further pool of polynucleotides to produce a further library of recombinant invasin polynucleotides; (7) producing a further library of recombinant bacteriophage, each of which displays on the bacteriophage surface a fusion polypeptide encoded by a chimeric gene that comprises a recombinant invasin nucleic acid operably linked to a polynucleotide that encodes a display polypeptide; (8) contacting the library of recombinant bacteriophage with a population of target cells; (9) removing unbound phage and phage which is bound to the surface of the target cells; and (10) recovering
  • the methods of evolving a bacteriophage-derived vaccine delivery vehicle to obtain a delivery vehicle having enhanced ability to enter a target cell can include the additional steps of (12) inserting into the optimized recombinant delivery vehicle a polynucleotide which encodes an antigen of interest, wherein the antigen of interest is expressed as a fusion polypeptide which comprises a second display polypeptide; (13) administering the delivery vehicle to a test animal; and (14) determining whether the delivery vehicle is capable of inducing a CTL response in the test animal.
  • the following steps can be employed: (12) inserting into the optimized recombinant delivery vehicle a polynucleotide which encodes an antigen of interest, wherein the antigen of interest is expressed as a fusion polypeptide which comprises a second display polypeptide; (13) administering the delivery vehicle to a test animal; and (14) determining whether the delivery vehicle is capable of inducing neutralizing antibodies against a pathogen which comprises the antigen of interest.
  • a target cell of interest for these methods is an antigen-presenting cell.
  • the present invention provides recombinant multivalent antigenic polypeptides that include a first antigenic determinant from a first disease-associated polypeptide and at least a second antigenic determinant from a second disease-associated polypeptide.
  • the disease- associated polypeptides can be selected from the group consisting of cancer antigens, antigens associated with autoimmunity disorders, antigens associated with inflammatory conditions, antigens associated with allergic reactions, antigens associated with infectious agents, and other antigens that are associated with a disease condition.
  • the invention provides a recombinant antigen library that contains recombinant nucleic acids that encode antigenic polypeptides.
  • the libraries are typically obtained by reassembling (&/or subjecting to one or more directed evolution methods described herein), at least first and second forms of a nucleic acid which includes a polynucleotide sequence that encodes a disease-associated antigenic polypeptide, wherein the first and second forms differ from each other in two or more nucleotides, to produce a library of recombinant nucleic acids.
  • Another embodiment of the invention provides methods of obtaining a polynucleotide that encodes a recombinant antigen having improved ability to induce an immune response to a disease condition.
  • These methods involve: (1) reassembling (&/or subjecting to one or more directed evolution methods described herein) at least first and second forms of a nucleic acid which comprises a polynucleotide sequence that encodes an antigenic polypeptide that is associated with the disease condition, wherein the first and second forms differ from each other in two or more nucleotides, to produce a library of recombinant nucleic acids; and (2) screening the library to identify at least one optimized recombinant nucleic acid that encodes an optimized recombinant antigenic polypeptide that has improved ability to induce an immune response to the disease condition.
  • These methods optionally further involve: (3) reassembling (&/or subjecting to one or more directed evolution methods described herein) at least one optimized recombinant nucleic acid with a further form of the nucleic acid, which is the same or different from the first and second forms, to produce a further library of recombinant nucleic acids; (4) screening the further library to identify at least one further optimized recombinant nucleic acid that encodes a polypeptide that has improved ability to induce an immune response to the disease condition; and (5) repeating (3) and (4), as necessary, until the further optimized recombinant nucleic acid encodes a polypeptide that has improved ability to induce an immune response to the disease condition.
  • the optimized recombinant nucleic acid encodes a multivalent antigenic polypeptide and the screening is accomplished by expressing the library of recombinant nucleic acids in a phage display expression vector such that the recombinant antigen is expressed as a fusion protein with a phage polypeptide that is displayed on a phage particle surface; contacting the phage with a first antibody that is specific for a first serotype of the pathogenic agent and selecting those phage that bind to the first antibody; and contacting those phage that bind to the first antibody with a second antibody that is specific for a second serotype of the pathogenic agent and selecting those phage that bind to the second antibody; wherein those phage that bind to the first antibody and the second antibody express a multivalent antigenic polypeptide.
  • the invention also provides methods of obtaining a recombinant viral vector which has an enhanced ability to induce an antiviral response in a cell.
  • Methods of obtaining a recombinant genetic vaccine component that confers upon a genetic vaccine an enhanced ability to induce a desired immune response in a mammal are also provided.
  • the invention provides methods of obtaining a recombinant genetic vaccine component that confers upon a genetic vaccine an enhanced ability to induce a desired immune response in a mammal. These methods involve: (1) reassembling (&/or subjecting to one or more directed evolution methods described herein) at least first and second forms of a nucleic acid which comprise a genetic vaccine vector, wherein the first and second forms differ from each other in two or more nucleotides, to produce a library of recombinant genetic vaccine vectors; (2) transfecting the library of recombinant vaccine vectors into a population of mammalian cells selected from the group consisting of peripheral blood T cells, T cell clones, freshly isolated monocytes/macrophages and dendritic cells; (3) staining the cells for the presence of one or more cytokines and identifying cells which exhibit a cytokine staining pattern indicative of the desired immune response; and (4) obtaining recombinant vaccine vector nucleic acid sequences from the cells which exhibit the desired
  • Another embodiment of the invention provides methods of obtaining a recombinant genetic vaccine vector that has an enhanced ability to induce a desired immune response in a mammal upon administration to the skin of the mammal. These methods involve: (1) reassembling (&/or subjecting to one or more directed evolution methods described herein) at least first and second forms of a nucleic acid which comprise a genetic vaccine vector, wherein the first and second forms differ from each other in two or more nucleotides, to produce a library of recombinant genetic vaccine vectors; (2) topically applying the library of recombinant genetic vaccine vectors to skin of a mammal; (3) identifying vectors that induce an immune response; and (4) recovering genetic vaccine vectors from the skin cells which contain vectors that induce an immune response.
  • Methods of inducing an immune response in a mammal by topically applying to skin of the mammal a genetic vaccine vector wherein the genetic vaccine vector is optimized for topical application through use of stochastic (e.g. polynucleotide shuffling & interrupted synthesis) and non-stochastic polynucleotide reassembly.
  • stochastic e.g. polynucleotide shuffling & interrupted synthesis
  • non-stochastic polynucleotide reassembly e.g. polynucleotide shuffling & interrupted synthesis
  • the genetic vaccine is administered as a formulation selected from the group consisting of a transdermal patch, a cream, naked DNA, a mixture of DNA and a transfection-enhancing agent.
  • Suitable transfection-enhancing agents include one or more agents selected from the group consisting of a lipid, a liposome, a protease, and a lipase.
  • the genetic vaccine can be administered after pretreatment of the skin by abrasion or hair removal.
  • Methods of obtaining an optimized genetic vaccine component that confers upon a genetic vaccine containing the component an enhanced ability to induce or inhibit apoptosis of a cell into which the vaccine is introduced are provided.
  • the invention provides methods of obtaining an optimized genetic vaccine component that confers upon a genetic vaccine containing the component an enhanced ability to induce or inhibit apoptosis of a cell into which the vaccine is introduced. These methods involve: (1) reassembling (&/or subjecting to one or more directed evolution methods described herein) at least first and second forms of a nucleic acid which comprise a nucleic acid that encodes an apoptosis- modulating polypeptide, wherein the first and second forms differ from each other in two or more nucleotides, to produce a library of recombinant nucleic acids; (2) transfecting the library of recombinant nucleic acids into a population of mammalian cells; (3) staining the cells for the presence of a cell membrane change which is indicative of apoptosis initiation; and (4) obtaining recombinant apoptosis-modulating genetic vaccine components from the cells which exhibit the desired apoptotic membrane changes.
  • Methods of obtaining a genetic vaccine component that confers upon a genetic vaccine reduced susceptibility to a CTL immune response in a host mammal are provided.
  • inventions provide methods of obtaining a genetic vaccine component that confers upon a genetic vaccine reduced susceptibility to a CTL immune response in a host mammal. These methods can involve: (1) reassembling (&/or subjecting to one or more directed evolution methods described herein) at least first and second forms of a nucleic acid which comprises a gene that encodes an inhibitor of a CTL immune response, wherein the first and second forms differ from each other in two or more nucleotides, to produce a library of recombinant CTL inhibitor nucleic acids; (2) introducing genetic vaccine vectors which comprise the library of recombinant CTL inhibitor nucleic acids into a plurality of human cells; (3) selecting cells which exhibit reduced MHC class I molecule expression; and (4) obtaining optimized recombinant CTL inhibitor nucleic acids from the selected cells.
  • Methods of obtaining a genetic vaccine component that confers upon a genetic vaccine reduced susceptibility to a CTL immune response in a host mammal are provided.
  • the invention also provides methods of obtaining a genetic vaccine component that confers upon a genetic vaccine reduced susceptibility to a CTL immune response in a host mammal. These methods involve: (1) reassembling (&/or subjecting to one or more directed evolution methods described herein) at least first and second forms of a nucleic acid which comprises a gene that encodes an inhibitor of a CTL immune response, wherein the first and second forms differ from each other in two or more nucleotides, to produce a library of recombinant CTL inhibitor nucleic acids; (2) introducing viral vectors which comprise the library of recombinant CTL inhibitor nucleic acids into mammalian cells; (3) identifying mammalian cells which express a marker gene included in the viral vectors a predetermined time after introduction, wherein the identified cells are resistant to a CTL response; and (4) recovering as the genetic vaccine component the recombinant CTL inhibitor nucleic acids from the identified cells.
  • the present invention provides substantially pure polypeptides which have amino acid sequences substantially homologous to the amino acid sequence of a PfEMPl protein, or biologically active fragments thereof.
  • the polypeptides of the present invention are substantially homologous to the amino acid sequence shown, described &/or referenced herein (including incorporated by reference), biologically active fragments or analogues thereof.
  • pharmaceutical compositions comprising these polypeptides.
  • the present invention provides nucleic acids which encode the above-described polypeptides.
  • Exemplary nucleic acids of the invention can be substantially homologous to a part or whole of the nucleic acid sequence shown, described &/or referenced herein (including incorporated by reference) or the nucleic acid encoding for the sequences shown, described &/or referenced herein (including incorporated by reference).
  • the present invention also provides expression vectors comprising these nucleic acid sequences and cells capable of expressing same.
  • the present invention provides antibodies which recognize and bind PfEMPl polypeptides or biologically active fragments thereof. These peptides can recognize and bind PfEMPl proteins associated with infection by more than one variant of P. falciparum.
  • the present invention provides methods of inhibiting the formation of PfEMPl ligand complex, comprising contacting PfEMPl or its ligands with polypeptides of the present invention.
  • the present invention provides methods of inhibiting sequestration of erythrocytes in a patient suffering from a malaria infection, comprising administering to said patient, an effective amount of a polypeptide of the present invention, such administration may be carried out prior to or following infection.
  • the present invention provides a method of detecting the presence or absence of PfEMPl in a sample. The method comprises exposing the sample to an antibody of the invention, and detecting binding, if any, between the antibody and a component of the sample.
  • the present invention provides a method of determining whether a test compound is an antagonist of PfEMPl/ligand complex formation.
  • the method comprises incubating the test compound with PfEMPl or a biologically active fragment thereof, and its ligand, under conditions which permit the formation of the complex.
  • the amount of complex formed in the presence of the test compound is determined and compared with the amount of complex formed in the absence of the test compound. A decrease in the amount of complex formed in the presence of the test compound is indicative that the compound is an antagonist of PfEMPl/ligand complex formation.
  • This invention also relates generally to the field of nucleic acid engineering and correspondingly encoded recombinant protein engineering. More particularly, the invention relates to the directed evolution of nucleic acids and screening of clones containing the evolved nucleic acids for resultant activity(ies) of interest, such nucleic acid activity(ies) &/or specified protein, particularly enzyme, activity(ies) of interest.
  • Mutagenized molecules provided by this invention may have chimeric molecules and molecules with point mutations, including biological molecules that contain a carbohydrate, a lipid, a nucleic acid, &/or a protein component, and specific but non-limiting examples of these include antibiotics, antibodies, enzymes, and steroidal and non-steroidal hormones.
  • This invention relates generally to a method of: 1) preparing a progeny generation of molecule(s) (including a molecule that is comprised of a polynucleotide sequence, a molecule that is comprised of a polypeptide sequence, and a molecules that is comprised in part of a polynucleotide sequence and in part of a polypeptide sequence), that is mutagenized to achieve at least one point mutation, addition, deletion, &/or chimerization, from one or more ancestral or parental generation template(s); 2) screening the progeny generation molecule(s) - in one aspect, using a high throughput method - for at least one property of interest (such as an improvement in an enzyme activity or an increase in stability or a novel chemotherapeutic effect); 3) optionally obtaining &/or cataloguing structural &/or and functional information regarding the parental &/or progeny generation molecules; and 4) optionally repeating any of steps 1) to 3).
  • this progeny generation of polynucleotides there is also generated a set of progeny polypeptides, each having at least one single amino acid point mutation.
  • amino acid site-saturation mutagenesis one such mutant polypeptide for each of the 19 naturally encoded polj ⁇ eptide-forming alpha-amino acid substitutions at each and every amino acid position along the polypeptide.
  • amino acid site-saturation mutagenesis one such mutant polypeptide for each of the 19 naturally encoded polj ⁇ eptide-forming alpha-amino acid substitutions at each and every amino acid position along the polypeptide.
  • this approach is also serviceable for generating mutants containing - in addition to &/or in combination with the 20 naturally encoded polypeptide-forming alpha- amino acids - other rare &/or not naturally-encoded amino acids and amino acid derivatives.
  • this approach is also serviceable for generating mutants by the use of - in addition to &/or in combination with natural or unaltered codon recognition systems of suitable hosts - altered, mutagenized, &/or designer codon recognition systems (such as in a host cell with one or more altered tRNA molecules).
  • this invention relates to recombination and more specifically to a method for preparing polynucleotides encoding a polypeptide by a method of in vivo re- assortment of polynucleotide sequences containing regions of partial homology, assembling the polynucleotides to form at least one polynucleotide and screening the polynucleotides for the production of polypeptide(s) having a useful property.
  • this invention is serviceable for analyzing and cataloguing - with respect to any molecular property (e.g. an enzymatic activity) or combination of properties allowed by current technology - the effects of any mutational change achieved (including particularly saturation mutagenesis).
  • a comprehensive method for determining the effect of changing each amino acid in a parental polypeptide into each of at least 19 possible substitutions.
  • This allows each amino acid in a parental polypeptide to be characterized and catalogued according to its spectrum of potential effects on a measurable property of the polypeptide.
  • the method of the present invention utilizes the natural property of cells to recombine molecules and/or to mediate reductive processes that reduce the complexity of sequences and extent of repeated or consecutive sequences possessing regions of homology. It is an object of the present invention to provide a method for generating hybrid polynucleotides encoding biologically active hybrid polypeptides with enhanced activities.
  • a method for introducing polynucleotides into a suitable host cell and growing the host cell under conditions that produce a hybrid polynucleotide in another aspect of the invention, provides a method for screening for biologically active hybrid polypeptides encoded by hybrid polynucleotides. The present method allows for the identification of biologically active hybrid polypeptides with enhanced biological activities.
  • the invention provides a method for determining the immunogenicity of a test molecule (i.e., a test antigen) comprising the following steps: (a) providing an immunocompromised non-human mammal populated with a plurality of human lymphocytes; (b) providing a test molecule; (c) administering the test molecule to the immunocompromised non-human mammal of step (a); (d) determining the test molecule- specific immune response of the human lymphocytes; and, (e) removing a sample of human lymphocytes from the non-human mammal and testing for their ability to proliferate or produce antibodies in response to challenge by the test molecule.
  • a test molecule i.e., a test antigen
  • the immunocompromised non- human mammal can be any mammal, e.g., a SCED mouse or rat.
  • the non-human mammals can be genetically manipulated to be immuno-compromised (e.g., SCED) or they can be treated with chemicals (drugs) and/or irradiated.
  • the antigen structure or dosage, the route and/or number of administrations, the formulation (e.g., the adjuvant) and/or the non-human animal can be varied and/or manipulated to generate the desired immune response ("immunogenicity"), e.g., the form of response (e.g., humoral or cellular response), isotype of response (e.g., a humoral IgM, IgG,
  • affinity of resultant antibody e.g., high affinity (e.g., about 10 6 or higher) or low affinity
  • the test molecule can be administered two or more times before determining the results of the test molecule-specific immune response, e.g., nature of response, affinity of antibodies, and the like.
  • the test molecule can administered two or more times and the resultant immune response can be determined after each administration.
  • the test molecule can be modified between each round of administration and re-testing of immune response.
  • the test molecule comprises a polypeptide, a peptide, a lipid, a nucleic acid, a small molecule and/or a polysaccharide.
  • the polypeptide can be synthetic, isolated from a natural source or recombinant.
  • the test molecule is structurally modified after each or one or more administrations.
  • the process can be reiterated to generate a desired response. For example, after an initial administration, if the response generate a low humoral response and a high humoral response is desired, the test molecule is structurally modified, re-administered and the resultant immune response is analyzed. In another example, if a T helper response is generated and a T suppressor response is desired, the test molecule is structurally modified, re-administered and retested. This process can be reiterated as many times as necessary to generate a desired response.
  • the structural modification in the test molecule can be combined with other changes in administration or formulation, e.g., dosages, routes of administration and the like.
  • test molecule is a polypeptide (including peptides), it can be modified from its native (e.g., wild type) sequence by modifications, additions or deletions.
  • the modifications can be a change in amino acid residue(s) (e.g., either a conservative change, such as a hydrophobic residue to another hydrophobic residue, or a non-conservative change, e.g., a hydrophobic residue to a hydrophilic residue) or a change in the structure of a residue, e.g., a post-translational change (e.g., phosphorylation, lipidation) or a post-synthetic structural modification in an amino acid residue (e.g., to a cyclodepsipeptide, mycosporine-like amino acid, amidation, oxidation and the like).
  • a conservative change such as a hydrophobic residue to another hydrophobic residue, or a non-conservative change, e.g., a hydrophobic residue to
  • Modifications in the test polypeptide can be introduced by, e.g., error-prone PCR, shuffling, oligonucleotide-directed mutagenesis, assembly PCR, sexual PCR mutagenesis, in vivo mutagenesis, cassette mutagenesis, recursive ensemble mutagenesis, exponential ensemble mutagenesis, site-specific mutagenesis, gene reassembly, gene site saturated mutagenesis (GSSM), synthetic ligation reassembly (SLR) and/or a combination thereof.
  • the modifications, additions or deletions are introduced by, e.g., recombination, recursive sequence recombination, phosphothioate-modified DNA mutagenesis, uracil-containing template mutagenesis, gapped duplex mutagenesis, point mismatch repair mutagenesis, repair- deficient host strain mutagenesis, chemical mutagenesis, radiogenic mutagenesis, deletion mutagenesis, restriction-selection mutagenesis, restriction-purification mutagenesis, artificial gene synthesis, ensemble mutagenesis, chimeric nucleic acid multimer creation and/or a combination thereof.
  • the plurality of human lymphocytes can comprise human peripheral blood lymphocytes.
  • the lymphocytes can be unchallenged ("naive"), pre-challenged (antigen stimulated) or activated (e.g., mitogen-, hormone- or interleukin-activated) cells.
  • the immune response can comprise a humoral response (an antibody based response) or a cellular (white blood cell) response. Ln one aspect, the isotypes of the antibodies generated in the humoral response are characterized.
  • the human lymphocyte can be, e.g., a sample of human lymphocytes comprising T cells, macrophages, monocytes, dendritic cells, B cells and/or plasma cells.
  • the invention provides methods for generating high affinity antibodies comprising the following steps: (a) providing a sample of isolated B lymphocytes; (b) isolating or cloning from the isolated B lymphocytes a nucleic acid encoding an antibody molecule; (c) translating the antibody molecule-encoding nucleic acid and placing the translated polypeptides in conditions wherein VH/VL pairing can occur to form an antigen binding molecule; (d) screening the antigen binding molecule for its ability to selectively bind to an antigen and its affinity for the antigen; (e) isolating the antigen binding molecule-encoding nucleic acid and changing its nucleic acid sequence; and, (f) re-screening the antigen binding molecule for its ability to selectively bind to an antigen by (i) expressing the antibody-encoding nucleic acid isolated in step (e) to generate antigen binding polypeptides, (ii) placing the expressed polypeptides in conditions wherein VH/VL pairing can occur to form antigen binding molecules
  • the B lymphocytes are human or mouse B lymphocytes.
  • the B lymphocytes can be isolated by FACS sorting.
  • the B lymphocytes can be labeled with fluorescent tags before the FACS sorting.
  • the nucleic acid encoding the antibody comprises an mRNA.
  • the nucleic acid encoding an antibody can be isolated by RT-PCR.
  • the B lymphocytes are pooled into separate fractions before the antibody- encoding nucleic acid is isolated or cloned.
  • the B lymphocytes can be pooled into separate fractions of about 1000 cells, 500 cells, 100 cells, 50 cells, 25 cells or 10 cells per fraction.
  • the nucleic acid sequences is changed by mutagenesis, base residue insertion or base residue deletion. Evolution technologies can be used to further engineer these sequences, including, e.g., Gene Site Saturation MutagenesisTM (GSSM) and GeneReassemblyTM (Diversa Corporation, San Diego, CA), as described in further detail herein.
  • GSSM Gene Site Saturation Mutagenesis
  • GeneReassemblyTM Diversa Corporation, San Diego, CA
  • nucleic acid sequences can be changed or "evolved” or “genetically engineered” by, e.g., error-prone PCR, shuffling, oligonucleotide-directed mutagenesis, assembly PCR, sexual PCR mutagenesis, in vivo mutagenesis, cassette mutagenesis, recursive ensemble mutagenesis, exponential ensemble mutagenesis, site-specific mutagenesis, gene reassembly, gene site saturated mutagenesis (GSSM), synthetic ligation reassembly (SLR) and/or a combination thereof.
  • error-prone PCR shuffling
  • oligonucleotide-directed mutagenesis assembly PCR
  • sexual PCR mutagenesis in vivo mutagenesis
  • cassette mutagenesis cassette mutagenesis
  • recursive ensemble mutagenesis recursive ensemble mutagenesis
  • exponential ensemble mutagenesis site-specific mutagenesis
  • gene reassembly gene site
  • the modifications, additions or deletions are introduced by, e.g., recombination, recursive sequence recombination, phosphothioate- modified DNA mutagenesis, uracil-containing template mutagenesis, gapped duplex mutagenesis, point mismatch repair mutagenesis, repair-deficient host strain mutagenesis, chemical mutagenesis, radiogenic mutagenesis, deletion mutagenesis, restriction-selection mutagenesis, restriction-purification mutagenesis, artificial gene synthesis, ensemble mutagenesis, chimeric nucleic acid multimer creation and/or a combination thereof.
  • these methods are iteratively repeated until an antibody having an altered or different activity or an altered or different stability from that of the antibody to be "evolved” is produced.
  • the CDR3 region of the antigen binding molecule-encoding nucleic acid sequence is changed or "evolved.”
  • the invention provides an array (e.g., biochip) comprising a plurality of polypeptides, wherein each polypeptide is immobilized to a discrete and known spot on a substrate surface to form an array of polypeptides, wherein the plurality of polypeptides comprise a sample of (i.e., a subset of), or all of, the antigen binding sites that are isolated from and/or expressed by an individual, or, complementary to antigen binding sites isolated from and/or expressed by the individual.
  • one or more of these antigen binding sites can be an antigen binding site encoded by a nucleic acid modified, or "evolved," by one or more of the methods of the invention, as described herein.
  • one or more of these antigen binding sites can be an antigen binding site encoded by a nucleic acid from a library of the invention (e.g., antigen binding sites encoded by a library of nucleic acids).
  • the polypeptides on the array comprise antigen binding sites isolated from or complementary to antigen binding sites of antibodies expressed by the individual, including secreted or cell-expressed (e.g., cell-bound) antibodies or fragments thereof.
  • antigen binding sites can be isolated from or complementary to antigen binding sites expressed on circulating antibodies expressed by the individual.
  • One or more of these secreted, circulating and/or cell-expressed antigen binding sites can be encoded by a nucleic acid modified, or "evolved,” by one or more of the methods of the invention, as described herein.
  • the cell-bound antibodies comprise B cell-bound, plasma cell-bound or macrophage-bound antibodies.
  • the cell-bound antibodies can be IgG, IgM, IgD, IgA and/or
  • the sample comprises antigen binding sites isolated from or complementary to antigen binding sites expressed on cell-bound and circulating antibodies expressed by the individual. In one aspect, the sample comprises a complete repertoire of the antigen binding sites of antibodies expressed by the individual.
  • the antigen binding site comprises a polypeptide selected from the group consisting of a single stranded antigen binding polypeptide, a Fab fragment, an Fc fragment, a F(ab')2 fragment, a Fv fragment and a complementarity determining region (CDR).
  • the antigen binding site can comprise an antibody polypeptide comprising two light chains and two heavy chains.
  • the sample comprises a complement of antigen binding sites isolated from or complementary to antigen binding sites expressed in a lymph node of the individual.
  • the lymph node can be isolated by, e.g., dissection or biopsy.
  • the cells can be harvested by aspiration or by cell sorting.
  • the sample comprises a complement of antigen binding sites isolated from or complementary to T cell receptors (TCRs) expressed by the individual.
  • the sample can comprise a complement of antigen binding sites isolated from or complementary to T cell receptors (TCRs) and antibodies expressed by the individual.
  • the sample can comprise a complete repertoire of the T cell receptors (TCRs) expressed in the individual.
  • the individual can be any mammal, e.g., a mouse, a rat or a human.
  • the plurality of polypeptides can further comprise a sample comprising antigen binding sites that are structural variations of antigen binding sites expressed by the individual.
  • the structural variations can be made by a method comprising the following steps: (a) providing a template polynucleotide, wherein the template polynucleotide comprises sequence encoding an antigen binding site; (b) providing a plurality of oligonucleotides, wherein each oligonucleotide comprises a sequence homologous to the template polynucleotide, thereby targeting a specific sequence of the template polynucleotide, and a sequence that is a variant of antigen binding site-encoding sequence; (c) generating progeny polynucleotides comprising non-stochastic sequence variations by replicating the template polynucleotide of step (a) with the oligonucleotides of step (b), thereby generating polynucleotides comprising antigen binding site-encoding sequence variations; and
  • the sequence homologous to the template polynucleotide can be x bases long, wherein x is an integer between 10 and 30, or, between 2 and 20.
  • the oligonucleotide of step (b) can further comprises a second sequence homologous to the template polynucleotide and the variant sequence is flanked by the sequences homologous to the template polynucleotide.
  • a codon encoding an amino acid in the antigen binding site can be targeted to be modified, and the plurality of oligonucleotides comprise variant sequences encoding all nineteen naturally-occurring amino acid variants for the targeted codon, thereby generating an antigen binding site polypeptide for all nineteen possible natural amino acid variations at the targeted amino acid.
  • codons encoding all amino acids in the antigen binding site are targeted to be modified.
  • the plurality of oligonucleotides can comprise variant sequences encoding all nineteen naturally-occurring amino acid variants for the targeted codon, thereby generating an antigen binding site polypeptide for all nineteen possible natural amino acid variations at each targeted amino acid.
  • An oligonucleotide of step (b) can further comprise a nucleic acid sequence capable of introducing one or more nucleotide residues into the template polynucleotide, or, deleting one or more residue from the template polynucleotide.
  • Structural variations of antigen binding sites can be made by a method comprising the following steps: (a) providing a template polynucleotide, wherein the template polynucleotide comprises sequence encoding an antigen binding site; (b) providing a plurality of building block polynucleotides, wherein the building block polynucleotides are designed to cross-over reassemble with the template polynucleotide at a predetermined sequence, and a building block polynucleotide comprises a sequence that is a variant of an antigen binding site-encoding sequence and a sequence homologous to the template polynucleotide flanking the variant sequence; (c) combining a block polynucleotide with a template polynucleotide such that the building block polynucleotide cross-over reassembles with the template polynucleotide to generate polynucleotides comprising antigen binding site-encoding sequence variations; and (d) expressing the
  • the building block polynucleotides comprise a sequence homologous to the template polynucleotide x bases long, wherein x is an integer between 10 and 30.
  • the building block polynucleotides can comprise a sequence that is a variant of the template polynucleotide x bases long, wherein x is an integer between 2 and 20.
  • the codon encoding an amino acid in the antigen binding site can be targeted to be modified, and the building block polynucleotides comprise variant sequences encoding all nineteen naturally-occurring amino acid variants for the targeted codon, thereby generating an antigen binding site polypeptide for all nineteen possible natural amino acid variations at the targeted amino acid.
  • the codons encoding all amino acids in the antigen binding site can be targeted to be modified.
  • the plurality of oligonucleotides comprise variant sequences encoding all nineteen naturally-occurring amino acid variants for the targeted codon, thereby generating an antigen binding site polypeptide for all nineteen possible natural amino acid variations at each targeted amino acid.
  • the building block polynucleotide can further comprise a nucleic acid sequence capable of introducing one or more nucleotide residues into the template polynucleotide, or, deleting one or more residue from the template polynucleotide.
  • a variant antigen binding site has a higher affinity for antigen than the template antigen binding site.
  • the methods for modifying antigen binding site structures can further comprise iteratively repeating steps (a) through (d), thereby generating further structural variations of antigen binding sites. In one aspect, the methods further comprising selecting a variant antigen binding site capable of enzymatically catalyzing a reaction.
  • invention provides methods of making anays comprising a plurality of polypeptide antigen binding sites, the methods comprising the following steps: (a) providing a plurality of polypeptides comprising a sample (e.g., a subset) of antigen binding sites that are isolated from or complementary to antigen binding sites expressed by an individual; and, (b) immobilizing to a discrete and known spot on a substrate surface one or more polypeptides each comprising the same antigen binding site, thereby forming an array of antigen binding site polypeptides.
  • the sample comprises antigen binding sites isolated from or complementary to antigen binding sites expressed on secreted antibodies expressed by the individual.
  • the sample can comprise antigen binding sites isolated from or complementary to antigen binding sites expressed on circulating antibodies expressed by the individual.
  • the sample can comprise antigen binding sites isolated from or complementary to antigen binding sites expressed on cell-bound antibodies expressed by the individual.
  • the cell-bound antibodies comprise B cell-bound antibodies.
  • the sample can comprise a complement of antigen binding sites isolated from or complementary to antigen binding sites expressed in a lymph node of the individual.
  • the sample can comprise antigen binding sites isolated from or complementary to antigen binding sites expressed on cell-bound and circulating antibodies expressed by the individual.
  • the sample can comprise a complete repertoire of the antigen binding sites of antibodies expressed by the individual.
  • the sample comprises a complement of antigen binding sites isolated from or complementary to T cell receptors (TCRs) expressed by the individual.
  • TCRs T cell receptors
  • the sample can comprise a complement of antigen binding sites isolated from or complementary to T cell receptors (TCRs) and antibodies expressed by the individual.
  • the sample comprises a complete repertoire of the antigen binding sites expressed in the individual.
  • the antigen binding sites can comprise antibodies comprising a ⁇ , ⁇ , ⁇ 2, ⁇ 3, ⁇ 4, ⁇ , ⁇ , ⁇ l or ⁇ 2 constant region.
  • the antigen binding sites are generated by expression of nucleic acid generated by amplification of nucleic acid from the individual.
  • the amplification can comprise, e.g., polymerase chain reaction (PCR).
  • the nucleic acid can comprise a cDNA library.
  • the cDNA library can be made from nucleic acid isolated from B cells or plasma cells.
  • the cDNA library can be made from nucleic acid isolated from, e.g., a lymph node, a spleen, a thymus, B cells or plasma cells.
  • the antigen binding sites and/or the nucleic acid encoding them can be isolated from, e.g., a lymph node, a spleen, a thymus, a blood or serum sample or a biopsy.
  • the invention provides methods of selecting an antibody capable of selectively binding to an antigen, the methods comprising the following steps: (a) providing an array comprising a plurality of polypeptides, wherein each polypeptide is immobilized to a discrete and known spot on a substrate surface to form an anay of polypeptides, wherein the plurality of polypeptides comprise a sample (e.g., a subset) of antigen binding sites expressed by an individual; (b) contacting the array with an antigen under conditions where the antigen can specifically bind to the antibody; (c) washing unbound antigen off the anay; and, (c) determining which spot has selectively bound the antigen, thereby selecting an antibody capable of selectively binding to the antigen.
  • the antigen is contacted with the anay under varying conditions of increasingly stringent conditions, selecting an antibody having a high affinity to the antigen.
  • the affinity can be selected from the group consisting of about 1 x 105 M-l, about 1 x 10 5 M 1 , about 1 x 10 6 M 1 , about 1 x 10 7 M “1 , about 1 x 10 8 M “1 , about 1 x 10 9 M “1 , about 2 x 10 9 M “1 , about 5 x 10 9 M “1 , about 1 x 10 10 M “1 , about 1 x 10 11 M 1 and greater than 1 x 10 11 M "1 .
  • the antigen comprises a detectable label, such as a fluorescent molecule, e.g., umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin.
  • a detectable label such as a fluorescent molecule, e.g., umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin.
  • the detectable label comprises a radioactive molecule or an enzyme, such as a horseradish peroxidase, beta-galactosidase, luciferase or an alkaline phosphatase.
  • Figure 1 shows the activity of the enzyme exonuclease III. This is an exemplary enzyme that can be used to shuffle, assemble, reassemble, recombine, and/or concatenate polynucleotide building blocks. The asterisk indicates that the enzyme acts from the 3' direction towards the 5' direction of the polynucleotide substrate.
  • Figure 2 illustrates a method of generating a double-stranded nucleic acid building block with two overhangs using a polymerase-based amplification reaction (e.g., PCR).
  • a polymerase-based amplification reaction e.g., PCR
  • F 2 and Ri a first polymerase-based amplification reaction using a first set of primers, F 2 and Ri
  • a blunt-ended product labeled Reaction 1, Product 1
  • a second polymerase-based amplification reaction using a second set of primers, Fi and R 2 is used to generate a blunt-ended product (labeled Reaction 2, Product 2), which is essentially identical to Product B.
  • the product with the 3' overhangs is selected for by nuclease-based degradation of the other 3 products using a 3' acting exonuclease, such as exonuclease Ed.
  • Alternate primers are shown in parenthesis to illustrate serviceable primers may overlap, and additionally that serviceable primers may be of different lengths, as shown.
  • Figure 3 illustrates the point that the number of unique overhangs of each size (e.g. the total number of unique overhangs composed of 1 or 2 or 3, etc. nucleotides) exceeds the number of unique couplings that can result from the use of all the unique overhangs of that size. For example, there are 4 unique 3' overhangs composed of a single nucleotide, and 4 unique 5' overhangs composed of a single nucleotide. Yet the total number of unique couplings that can be made using all the 8 unique single-nucleotide 3' overhangs and single-nucleotide 5' overhangs is 4.
  • Figure 4 illustrates the fact that in order to assemble a total of "n" nucleic acid building blocks, "n-1" couplings are needed. Yet it is sometimes the case that the number of unique couplings available for use is fewer that the "n-1" value. Under these, and other, circumstances a stringent non-stochastic overall assembly order can still be achieved by performing the assembly process in sequential steps. In this example, 2 sequential steps are used to achieve a designed overall assembly order for five nucleic acid building blocks. In this illustration the designed overall assembly order for the five nucleic acid building blocks is: 5'-(#l-#2-#3-#4-#5)-3', where #1 represents building block number 1, etc.
  • Figure 5 Unique Couplings Available Using a Two-Nucleotide 3' Overhang.
  • Figure 5 further illustrates the point that the number of unique overhangs of each size (here, e.g. the total number of unique overhangs composed of 2 nucleotides) exceeds the number of unique couplings that can result from the use of all the unique overhangs of that size. For example, there are 16 unique 3' overhangs composed of two nucleotides, and another 16 unique 5' overhangs composed of two nucleotides, for a total of 32 as shown.
  • Figure 6 Generation of an Exhaustive Set of Chimeric Combinations by Synthetic Ligation Reassembly.
  • Figure 6 showcases the power of this invention in its ability to generate exhaustively and systematically all possible combinations of the nucleic acid building blocks designed in this example. Particularly large sets (or libraries) of progeny chimeric molecules can be generated. Because this method can be performed exhaustively and systematically, the method application can be repeated by choosing new demarcation points and with conespondingly newly designed nucleic acid building blocks, bypassing the burden of re-generating and re-screening previously examined and rejected molecular species. It is appreciated that, codon wobble can be used to advantage to increase the frequency of a demarcation point.
  • nucleic acid building block Ln other words, a particular base can often be substituted into a nucleic acid building block without altering the amino acid encoded by progenitor codon (that is now altered codon) because of codon degeneracy.
  • demarcation points are chosen upon alignment of 8 progenitor templates.
  • Nucleic acid building blocks including their overhangs are then designed and synthesized. In this instance, 18 nucleic acid building blocks are generated based on the sequence of each of the 8 progenitor templates, for a total of 144 nucleic acid building blocks (or double-stranded oligos).
  • double-stranded nucleic acid building blocks are designed by aligning a plurality of progenitor nucleic acid templates.
  • these templates contain some homology and some heterology.
  • the nucleic acids may encode related proteins, such as related enzymes, which relationship may be based on function or structure or both.
  • Figure 7 shows the alignment of three polynucleotide progenitor templates and the selection of demarcation points (boxed) shared by all the progenitor molecules.
  • the nucleic acid building blocks derived from each of the progenitor templates were chosen to be approximately 30 to 50 nucleotides in length.
  • Figure 8 Nucleic acid building blocks for synthetic ligation gene reassembly.
  • Figure 8 shows the nucleic acid building blocks from the example in Figure 7.
  • the nucleic acid building blocks are shown here in generic cartoon form, with their compatible overhangs, including both 5' and 3' overhangs.
  • the ligation synthesis procedure can produce a library of progeny molecules comprised of yield of 3 22 (or over 3.1 x 10 10 ) chimeras.
  • Figure 9 Addition of Introns by Synthetic Ligation Reassembly.
  • Figure 9 shows in generic cartoon form that an intron may be introduced into a chimeric progeny molecule by way of a nucleic acid building block. It is appreciated that introns often have consensus sequences at both termini in order to render them operational. It is also appreciated that, in addition to enabling gene splicing, introns may serve an additional purpose by providing sites of homology to other nucleic acids to enable homologous recombination. For this purpose, and potentially others, it may be sometimes desirable to generate a large nucleic acid building block for introducing an intron.
  • such a specialized nucleic acid building block may also be generated by direct chemical synthesis of more than two single stranded oligos or by using a polymerase-based amplification reaction as shown, described &/or referenced herein (including incorporated by reference).
  • Figure 10 Ligation Reassembly Using Fewer Than All The Nucleotides Of An Overhang.
  • Figure 10 shows that coupling can occur in a manner that does not make use of every nucleotide in a participating overhang. The coupling is particularly lively to survive (e.g. in a transformed host) if the coupling reinforced by treatment with a ligase enzyme to form what may be refened to as a "gap ligation" or a "gapped ligation". It is appreciated that, as shown, this type of coupling can contribute to generation of unwanted background product(s), but it can also be used advantageously increase the diversity of the progeny library generated by the designed ligation reassembly.
  • nucleic acid building blocks can be chemically made (or ordered) that lack a 5' phosphate group (or alternatively they can be remove - e.g. by treatment with a phosphatase enzyme such as a calf intestinal alkaline phosphatase (CLAP) - in order to prevent palindromic self-ligations in ligation reassembly processes.
  • a phosphatase enzyme such as a calf intestinal alkaline phosphatase (CLAP)
  • FIG. 12 Site-directed mutagenesis by polymerase-based extension.
  • Panel A This figure shows one method of site-directed mutagenesis, among many methods of site-directed mutagenesis, that are serviceable for performing site-saturation mutagenesis.
  • Section (1) shows the first and second mutagenic primer annealed to a circular closed double-stranded plasmid. The dot and the open-sided triangle indicate the mutagenic sites in the mutagenic primers. The anows indicate the direction of synthesis.
  • Section (2) shows the newly synthesized (mutagenized) DNA strands annealed to each other.
  • the parental DNA can be treated with a selection enzyme.
  • the mutagenized DNA strands are shown as being annealed to form a double-stranded mutagenized circular DNA intermediate.
  • the dot and the open- sided triangle indicate the mutagenic sites in the experimentally generated progeny (mutagenized) DNA strands.
  • the staggered openings on the mutagenized DNA strands form "sticky" ends.
  • Section (3) shows the first and second mutagenic primer annealed to the mutagenized DNA strands of Section (2).
  • the arrows indicate the direction of synthesis. Note the opening on each of the mutagenized DNA strands (i.e. they have not been ligated).
  • Section (4) shows a "Gapped Product", which is composed of second generation mutagenized DNA strands, synthesized using the mutagenized DNA strands (shown in Step (2)) as a template.
  • the DNA strands of the "Gapped Product” are shown as being annealed to form a double-stranded mutagenized circular DNA intermediate.
  • the dot and the open-sided triangle indicate the mutagenic sites in the mutagenized DNA strands. Note the large gap in each of the mutagenized DNA strands.
  • Section (5) shows the "Gapped Product” annealed to the parental (non-mutated) plasmid, enabling polymerase-based synthesis to occur. The anows indicate the direction of synthesis.
  • Section (6) shows the newly synthesized DNA strands, as being annealed to form a double-stranded mutagenized circular DNA product.
  • the dot and the open-sided triangle indicate the mutagenic sites in the mutagenized DNA strands. Note the staggered openings on the mutagenized DNA strands. Also note the presence of both mutagenic sites on each of the mutagenized DNA strands.
  • Panel B This figure shows two possible molecular structures produced from the amplification steps of Figure 12A. Molecule (A) is shown also in Section (2) of Figure 12 A. Molecule (B) is also shown in Section (6) of Figure 12A.
  • FIG. 13 Site-directed mutagenesis by polymerase-based extension and ligase- based ligation.
  • Panel A This figure shows one method of site-directed mutagenesis, among many methods of site-directed mutagenesis, that are serviceable for performing site- saturation mutagenesis.
  • Section (1) shows the first and second mutagenic primer annealed to a circular closed double-stranded plasmid. The dot and the open-sided triangle indicate the mutagenic sites in the mutagenic primers. The anows indicate the direction of synthesis.
  • Section (2) shows the newly synthesized (mutagenized) DNA strands annealed to each other. The parental DNA can be treated with a selection enzyme.
  • the mutagenized DNA strands are shown as being annealed to form a double-stranded mutagenized circular DNA intermediate.
  • the dot and the open-sided triangle indicate the mutagenic sites in the experimentally generated progeny (mutagenized) DNA strands. Note that the staggered openings on the mutagenized DNA strands form "sticky" ends.
  • Section (3) shows the resultant double-stranded mutagenized circular DNA molecule produced after the double- stranded mutagenized circular DNA intermediate of Section (2) is ligated (e.g. with T4 DNA ligase).
  • Section (4) shows the first and second mutagenic primer annealed to the mutagenized DNA strands of Section (3). The anows indicate the direction of synthesis.
  • Section (5) shows the recently generated (blue) mutagenized DNA strands as being annealed to form a double-stranded mutagenized circular DNA intermediate.
  • the dot and the open- sided triangle indicate the mutagenic sites in the recently generated mutagenized DNA strands (blue).
  • the staggered openings on the mutagenized DNA strands form "sticky ends".
  • the presence of both mutagenic sites on each of the two recently generated mutagenized DNA strands (blue). Note the opening on each of the mutagenized DNA strands (i.e. they have not been ligated).
  • Section (6) shows the resultant double- stranded mutagenized circular DNA molecule produced after the double-stranded mutagenized circular DNA intermediate of Section (5) is ligated (e.g. using T4 DNA ligase).
  • the dot and the open-sided triangle indicate the mutagenic sites in the mutagenized DNA molecules. Again, note the presence of both mutagenic sites on each of the mutagenized DNA strands.
  • Panel B This figure shows two molecular structures produced from the amplification steps of Figure 13A. Molecule (A) is also shown in Section (3) of Figure 13A. Molecule (B) is produced in Section (6) of Figure 13A.
  • Figure 14 Strategy for Obtaining and Using Nucleic Acid Binding Proteins that Facilitate Entry of Genetic Vaccines. Shown here is a strategy for obtaining and using nucleic acid binding proteins that facilitate entry of genetic vaccines, in particular, naked DNA, into target cells. Members of a library obtained by the directed evolution methods described herein are linked to a coding region of M 13 protein VEQ so that a fusion protein is displayed on the surface of the phage particles. Phage that efficiently enter the desired target tissue are identified, and the fusion protein is then used to coat a genetic vaccine nucleic acid.
  • Figure 15 A schematic representation of a method for generating a chimeric, multivalent antigen that has immunogenic regions from multiple antigens.
  • Antibodies to each of the non-chimeric parental immunogenic polypeptides are specific for the respective organisms (A, B, C). After canying out the directed evolution and selection methods of the invention, however, a chimeric immunogenic polypeptide is obtained that is recognized by antibodies raised against each of the three parental immunogenic polypeptides.
  • Figure 16A and Figure 16B Method for Obtaining Non-Stochastically Generated Polypeptides that can induce a Broad-Spectrum Immune Response. Shown here is a schematic for a method by which one can obtain non-stochastically generated polypeptides that can induce a broad-spectrum immune response.
  • wild-type immunogenic polypeptides from the pathogens A, B, and C provide protection against the conesponding pathogen from which the polypeptide is derived, but little or no cross-protection against the other pathogens (left panel).
  • an A/B/C chimeric polypeptide is obtained that can induce a protective immune response against all three pathogen types (right panel).
  • FIG 16B directed evolution is used with substrate nucleic acids from two pathogen strains (A, B), which encode polypeptides that are protective only against the conesponding pathogen. After directed evolution, the resulting chimeric polypeptide can induce an immune response that is effective against not only the two parental pathogen strains, but also against a third strain of pathogen (C).
  • Figure 17 Possible factors for determining whether a particular polynucleotide encodes an immunogenic polypeptide having a desired property. Shown here are some of the possible factors that can determine whether a particular polynucleotide encodes an immunogenic polypeptide having a desired property, such as enhanced immunogenicity and/or cross-reactivity.
  • sequence regions that positively affect a particular property are indicated as plus signs along the antigen gene, while those sequence regions that have a negative effect are shown as minus signs.
  • a pool of related antigen genes are non- stochastically generated using the methods described herein and screened to obtain those evolved nucleic acids that have gained positive sequence regions and lost negative regions. No pre-existing knowledge as to which regions are positive or negative for a particular trait is required.
  • Figure 18 Screening strategy for antigen library screening. Shown here is a schematic representation of the screening strategy for antigen library screening.
  • Figure 19 Strategy for pooling and deconvolution as used in antigen library screening. Shown here is a schematic representation of a strategy for pooling and deconvolution as used in antigen library screening.
  • Figure 20 Exemplary Embodiments of Site-Saturation Mutagenesis.
  • Figure 21 Schematic representation of a multimodule genetic vaccine vector. Shown here is a schematic representation of a multimodule genetic vaccine vector.
  • a typical genetic vaccine vector will include one or more of the components indicated, each of which can be native or optimized using the directed evolution methods described herein.
  • These directed evolution methods can include the introduction of point mutations by stochastic methods &/or by non-stochastic methods, including "gene site saturation mutagenesis" as described herein.
  • These directed evolution methods can also include stochastic polynucleotide reassembly methods, for example by interrupted synthesis (as described in US 5965408).
  • directed evolution methods can also include non-stochastic polynucleotide reassembly methods as described herein, including synthetic ligation polynucleotide reassembly as described herein.
  • the components can be present on the same vaccine vector, or can be included in a genetic vaccine as separate molecules.
  • Figure 22A and Figure 22B Generation of vectors with multiple T cell epitopes.
  • each individual non- stochastically generated epitope-encoding gene is linked to a single promoter, and multiple promoter-epitope gene constructs can be placed in a single vector.
  • the scheme shown, described &/or referenced herein involves linking multiple epitope-encoding genes to a single promoter.
  • Figure 23. Generation of optimized genetic vaccines by directed evolution. Shown here is a diagram of the application of directed evolution to the generation of optimized genetic vaccines.
  • FIG. 24 Recursive application of directed evolution and selection of evolved promoter sequences as an example of flow cytometry-based screening methods. Shown here is a diagram of flow cytometry-based screening methods (FACS) for selection of optimized promoter sequences evolved using recursive applications of the directed evolution methods as described herein. A cytomegalovirus (CMV) promoter is used for illustrative purposes.
  • Figure 25 An apparatus for microinjections of skin and muscle.
  • the apparatus is suitable for microinjection of genetic vaccines and other reagents into tissue such as skin and muscle.
  • the apparatus is particularly useful for screening large numbers of agents in vivo, being based on a 96-well format.
  • the tips of the apparatus are movable to allow adjustment so that the tips fit into a microtiter plate. After obtaining a reagent of interest is obtained from a plate, the tips are adjusted to a distance of about 2-3 min apart, enabling transfer of 96 different samples to an area of about 1.6 cm by 2.4 cm to about 2.4 cm by 3.6 cm.
  • the volume of each sample transfened can be electronically controlled; typically the volumes transfened range from about 2 ul to about 5 ul.
  • Each reagent can be mixed with a marker agent or dye to facilitate recognition of the injection site in the tissue.
  • a marker agent or dye for example, gold particles of different sizes and shaped can be mixed with the reagent of interest, and microscopy and immunohistochemistry used to identify each injection site and to study the reaction induced by each reagent.
  • Figure 26 Polynucleotide reassembly. Shown in Panel A is an example of directed evolution, n different strains of a virus are used in this illustration, but the technique is applicable to any single nucleic acid as well as to any nucleic acid for which different strains, species, or gene families have homologous nucleic acids that have one or more nucleotide changes compared to other homologous nucleic acids.
  • the different variant nucleic acids are experimentally generated, in one aspect, non-stochastically, as described herein, and screened or selected to identify those variants that exhibit the desired property.
  • the directed evolution method(s) and screening can be repeated one or more times to obtain further improvement.
  • Panel B shows that successive rounds of directed evolution can produce progressively enhanced properties, and that the combination of individual beneficial mutations can lead to an enhance improvement compared to the improvement achieved by an individual beneficial mutation.
  • Figure 27 Vector for promoter evolution. Shown here is an example of a vector that is useful for screening to identify improved promoters from a library of promoter nucleic acids evolved using the directed evolution methods as described herein. Experimentally generated putative promoters are inserted into the vector upstream of a reporter gene for which expression is readily detected. For many applications, it is desirable that the product of the reporter gene be a cell surface protein so that cells which express high levels of the reporter gene can be sorted using flow cytometry-based cell sorting using the reporter gene product. Examples of suitable reporter genes include, for example, B7-2 and mAb 179 epitopes. A polyadenylation region is typically placed downstream of the reporter gene (SV40 polyA is illustrated).
  • the vector can also include a second reporter gene an internal control (GFP; green fluorescent protein); this gene is linked to a promoter (SR ⁇ p).
  • GFP green fluorescent protein
  • the vector also typically includes a selectable marker (kanamycin/neomycin resistance is shown), and origins of replication that are functional in mammalian (SV40 ori) and/or bacterial (pUC ori) cells.
  • Figure 28 Iterative evolution of inducible promoters using directed evolution and flow cytometry-based selection. Shown here is a diagram of a scheme for iterative evolution of inducible promoters using the directed evolution methods as described herein and flow cytometry-based selection.
  • a library of experimentally generated (i.e. produced by one or more directed evolution methods as descried herein) promoter nucleic acids present in appropriate vectors is transfected into the cells, and those cells which exhibit the least expression of marker antigen when grown under uninduced conditions are selected.
  • the vectors (&/or cells containing them) are recovered, and the vectors are introduced into cells (if not contained therein already), and grown under inducing conditions. Those cells that express the highest level of marker antigen are selected.
  • Figure 29 Iterative evolution of inducible promoters using directed evolution and flow cytometry-based selection. Shown is a diagram of a scheme for iterative evolution of inducible promoters using the directed evolution methods as described herein and flow cytometry-based
  • Evolving a genetic vaccine vector for Oral, Intravenous, Intramuscular, Intradermal, Anal, Vaginal, or Topical Delivery Illustrated is a strategy for screening of M13 libraries (e.g. generated experimentally using directed evolution as descried herein) for desired targeting of various tissues.
  • the particular example shown here is a schematic diagram of a method for evolving a genetic vaccine vector for improved oral delivery. This may comprise selecting for stability under the acidic conditions of the stomach, and resistance to other degradatory factors of the digestive tract.
  • the particular example illustrated relates to screening for improved oral delivery, but the same principle applies to libraries administered by other routes, including intravenously, intramuscularly, intradermally, anally, vaginally, or topically.
  • the Ml 3 phage (or a product thereof) is recovered from the tissue of interest. The procedure can be repeated to obtain further optimization.
  • Figure 30 An alignment of the nucleotide sequences of two human CMV strains and one monkey strain. Shown here is an alignment of the nucleotide sequences of two human cytomegalovirus (CMV) strains and one monkey (Rhesus) strains. This alignment is serviceable for performing non-stochastic polynucleotide reassembly. Nucleotide sequences shared by 2 sequences are in blue lettering & nucleotide sequences shared by 3 sequences are in red lettering to illustrate exemplary but non-limiting examples of reassembly points.
  • CMV cytomegalovirus
  • Rhesus monkey
  • Figure 31 An alignment of EL-4 nucleotide sequences from 3 species (human, primate, and canine). Shown here is an alignment of the EL-4 nucleotide sequences of human, dog and primate strains. This alignment is serviceable for performing non-stochastic polynucleotide reassembly. Nucleotide sequences shared by 2 sequences are in blue lettering & nucleotide sequences shared by 3 sequences are in red lettering to illustrate exemplary but non-limiting examples of reassembly points.
  • Figure 32 Evolution of polypeptides by synthesizing (in vivo or in vitro) conesponding deduced polynucleotides and subjecting the deduced polynucleotides to directed evolution and expression screening subsequently expressed polypeptides.
  • Figure 33 Non-stochastic Reassembly of oligo-directed CpG knock-outs. Shown here is a schematic representation of the use of the non-stochastic methods described herein to generate promoter sequences in which unnecessary CpG sequences are deleted, potentially useful CpG sequences are added, and non-replaceable CpG sequences are identified. Additionally, other sequences (aside from the CpG sequences) can be substituted into, added to, &/or deleted from working polynucleotides.
  • FIG. 34 An Example of a CTIS obtained from HbsAg polypeptide (PreS2 plus S regions). Shown here is an example of a cytotoxic T-cell inducing sequence (CTIS) obtained from HBsAg polypeptide (PreS2 plus S regions).
  • CTIS cytotoxic T-cell inducing sequence
  • FIG. 35 A CTIS Having Heterologous Epitopes Attached to the Cytoplasmic Portion. Shown here is a CTIS having heterologous epitopes attached to the cytoplasmic portion.
  • FIG. 36 Method for preparing immunogenic agonist sequences (IAS). Shown here is a method for preparing immunogenic agonist sequences (IAS). Wild-type (WT) and mutated forms of nucleic acids encoding a polypeptide of interest are assembled and subjected to non-stochastic reassembly to obtain a nucleic acid encoding a poly-epitope region that contains potential agonist sequences.
  • WT Wild-type
  • mutated forms of nucleic acids encoding a polypeptide of interest are assembled and subjected to non-stochastic reassembly to obtain a nucleic acid encoding a poly-epitope region that contains potential agonist sequences.
  • FIG. 37 Improving Lmmunostimulatory Sequences (ISS) Using Directed Evolution. Shown here is a scheme for improving immunostimulatory sequences by the directed evolution methods described herein. Oligonucleotide building blocks (e.g. synthetically generated), oligos with known ISS, CpG containing hexamers &/or oligos containing CpG containing hexamers, poly A, C, G, T, etc... can be assembled. The resultant molecule(s) can then by subjected to 1 or more directed evolution methods as described herein.
  • Oligonucleotide building blocks e.g. synthetically generated
  • CpG containing hexamers &/or oligos containing CpG containing hexamers poly A, C, G, T, etc...
  • the resultant molecule(s) can then by subjected to 1 or more directed evolution methods as described herein.
  • FIG. 38 Screening to identify EL-12 genes that encode recombinant EL-12 having an increased ability to induce T Cell proliferation. Shown here is a diagram of a procedure by which experimentally generated molecules, e.g. non-stochastically generated libraries of human EL-12 genes can be screened to identify evolved EL-12 genes that encode evolved forms of EL-12 having increased ability to induce T cell proliferation.
  • FIG 39 Model of induction of T cell activation or anergy by genetic vaccine vectors encoding different CD80 and/or CD86 variants. Shown here is a model of how T cell activation or anergy can be induced by genetic vaccine vectors that encode different B7-1 (CD80) and/or B7-2 (CD86) variants.
  • Figure 40 Screening of CD80/CD86 variants that have improved capacity to induce
  • T cell activation or anergy Shown here is a method for using directed evolution as described herein to obtain CD80/CD86 variants that have improved capacity to induce T cell activation or anergy.
  • Figure 41 An alignment of two CMV-derived nucleotide sequences from human and primate species. Shown here is an alignment of two CMV-derived nucleotide sequences of human and primate strains. This alignment is serviceable for performing non-stochastic polynucleotide reassembly. Nucleotide sequences shared by 2 sequences are in red lettering to illustrate exemplary but non-limiting examples of reassembly points.
  • Figure 42 An alignment of the EFN-gamma nucleotide sequences from human, cat, rodent species. Shown here is an alignment of the EFN-gamma nucleotide sequences from human, cat, and rodent species. This alignment is serviceable for performing non-stochastic polynucleotide reassembly. Nucleotide sequences shared by 2 sequences are in blue lettering & nucleotide sequences shared by 3 sequences are in red lettering to illustrate exemplary but non-limiting examples of reassembly points.
  • Figure 43 is a schematic summarizing exemplary applications of the novel capillary array of the invention, e.g., GIGAMATREXTM, Diversa Corporation, San Diego, CA.
  • Figure 44 is a schematic showing use of paramagnetic beads with the methods of the invention.
  • Figure 45 is a schematic showing an exemplary use of paramagnetic beads with the methods of the invention.
  • Figure 46 is a schematic summarizing exemplary applications of the novel capillary anay of the invention, e.g., GIGAMATREXTM, Diversa Corporation, San
  • Figure 48 is a schematic summarizing exemplary applications of the novel GENE- REASSEMBLYTM method of the invention, as described in detail, below.
  • Figure 49 is a schematic summarizing exemplary applications of the novel GENE-
  • Figure 50 is a schematic summarizing an exemplary application of the novel GENE- REASSEMBLYTM method of the invention, as described in detail, below.
  • Figure 51 is a schematic summarizing an exemplary application of the novel GENE- REASSEMBLYTM method of the invention.
  • Figure 52 is a schematic summarizing an exemplary application ("dehalogenase reassembly") of the novel GENE-REASSEMBLYTM method of the invention.
  • Figure 53 is a schematic summarizing the novel TUNEABLE-GENE- REASSEMBLYTM method of the invention, as described, below.
  • Figure 54 is a schematic summarizing the DNACARPENTERTM reassembly control software that can be used with the methods of the invention.
  • Figure 55 is a schematic summarizing an exemplary gene family reassembly method of the invention.
  • Figure 56 is a schematic summarizing an exemplary gene family reassembly method of the invention.
  • Figure 57 is a schematic summarizing exemplary methods of the invention as described in detail, below.
  • Figure 58 is a schematic summarizing cunent deficiencies in antibody generation as discussed in detail, below.
  • Figure 59 is a schematic summarizing antibodies generated by the methods of the invention, e.g., NATUBODEESTM, as described in detail, below.
  • Figure 60 is a schematic summarizing a bivalent human antibody structure, as discussed in detail, below.
  • Figure 61 is a schematic summarizing exemplary synthetic human antibodies generated by the methods of the invention, as described in detail, below.
  • Figure 62 is a schematic summarizing an antibody V-region structure and variability, as discussed in detail, below.
  • Figure 63 is a schematic summarizing antibody variable region structure, as discussed in detail, below.
  • Figure 64 is a schematic summarizing exemplary synthetic human antibodies, particularly re-engineered CDR regions, generated by the methods of the invention, as described below.
  • Figure 65 is a schematic summarizing exemplary synthetic de novo antibody libraries generated by the methods of the invention, as described in detail, below.
  • Figure 66 is a schematic summarizing exemplary methods for generating and screening synthetic human antibodies by the methods of the invention, as described below.
  • Figure 67 is a schematic summarizing an exemplary method of the invention for screening antibodies, as described below.
  • Figure 68 is a schematic summarizing an exemplary method for generating antibodies by the methods of the invention, including affinity maturation by a combination of methods of the invention, as described below.
  • Figure 69 is a schematic summarizing an exemplary application of the novel GENE- REASSEMBLYTM method of the invention.
  • the invention provides methods for generating variant antigen binding sites, antibodies and specific domains or fragments of antibodies (e.g., Fab or Fc domains) by altering template nucleic acid by saturation mutagenesis, an optimized directed evolution system, synthetic ligation reassembly, or a combination thereof.
  • Polypeptides generated by these methods can be analyzed, e.g., screened for a binding activity (e.g., to an antigen), using a novel capillary array platform of the invention.
  • the invention provides for the modification (e.g., mutagenesis) of Fc domains.
  • this invention provides for mutagenizing a percentage, including at least every integer value (i.e. at least 1%, at least 2%, at least 3%, . . . , to at least 99%, or, 100%) of an Fc region or of an Fc-region containing molecule, or fragment, domain or subsection thereof.
  • a nucleic acid encoding an Fc domain can be modified such that it gains, loses or acquires a modified function (e.g., a binding property) or property (e.g., solubility or antigenicity), or has a modified (e.g., higher or lower) binding affinity.
  • a modified function e.g., a binding property
  • property e.g., solubility or antigenicity
  • the ability (including, e.g., affinity) of an Fc to bind a particular cell surface receptor e.g., an Fc receptor, or FcR on, e.g., a B cell, T cell, macrophage, monocyte, mast cell, basophil, dendritic cell, Langerhan cell and the like, or a complement protein or receptor
  • a particular cell surface receptor e.g., an Fc receptor, or FcR on, e.g., a B cell, T cell, macrophage, monocyte, mast cell, basophil, dend
  • the Fc domain can be changed to bind a different cell surface receptor (e.g., changed to bind a B cell FcR when the wild type Fc bound to a macrophage FcR or a mast cell FcR), an additional receptor (added function) or fewer receptors (loss of function).
  • the Fc domain can be changed such that it binds to a different complement polypeptide (e.g., changing specificities) or an added specificity or an altened affinity to a complement protein.
  • any protein with an antigen binding site including antibodies, T cell receptors and Fc domains
  • their crystal structures can be used to predict which residues may be desirable for targeting.
  • nucleic acid residues encoding solvent exposed residues e.g., those involved in protei protein binding
  • the saturation mutagenesis methods of this invention provide mutagenizing (e.g. saturation mutagensis, GSSM) solvent-exposed amino acids of a region (e.g. Fc); e.g., the mutagenesis (e.g.
  • GSSM saturation mutagenesis
  • this invention provides a method for making (as well as the product of the method) a library of variants (e.g. generated by saturation mutgenesis) of an antibody comprising a human immunoglobulin (lg) Fc region (including IgG, IgE, IgA, IgM, IgD).
  • a library of variants e.g. generated by saturation mutgenesis
  • an antibody comprising a human immunoglobulin (lg) Fc region (including IgG, IgE, IgA, IgM, IgD).
  • the invention provides variants comprising from at least 2 amino acid substitutions (and every integer value including up to all 19 naturally-occurring amino acid substitutions) at amino acid position 329, or at two or all of amino acid positions 329, 331 and 322 of the human IgG Fc region, where the numbering of the residues in the IgG Fc region is that of the EU index as in Kabat (see also U.S. Patent Nos. 6,242,195 and 6,194,551; and WO 99/51642) and wherein the variant retains the ability to bind antigen.
  • an antibody comprising a human IgG Fc region is an antibody comprising a human IgGl Fc region.
  • this invention provides a method for modifying an antibody comprising a human IgG Fc region, the method comprising making (including making and screening) a library of variants having amino acid substitutions at amino acid position (or residue) 329, or at two or all of amino acid positions 329, 331 and 322 of the human IgG Fc region, where the numbering of the residues in the IgG Fc region is that of the EU index as in Kabat, and wherein the variant retains the ability to bind antigen.
  • the screening criterion is the selection for a variant that does not activate complement.
  • a screening criterion is the selection for a variant that binds an FcR.
  • a screening criterion is the selection for a variant that binds an FcR, such as FcRI, FcRII, FcRLTJ or FcRn.
  • a screening criterion is the selection for a variant of an antibody comprising a human IgG Fc region, which variant does not activate complement and comprises an amino acid substitution at amino acid position 322 or amino acid position 329, or both amino acid positions of the human IgG Fc region, where the numbering of the residues in the IgG Fc region is that of the EU index as in Kabat, and wherein the variant retains the ability to bind antigen.
  • a composition of the invention and a physiologically acceptable carrier for example, a composition of the invention comprising any variant described herein and a physiologically acceptable carrier.
  • nucleic acid refers to a deoxyribonucleotide (DNA) or ribonucleotide (RNA) in either single- or double-stranded form.
  • DNA deoxyribonucleotide
  • RNA ribonucleotide
  • the term encompasses nucleic acids containing known analogues of natural nucleotides.
  • the term encompasses mixed oligonucleotides comprising an RNA portion bearing 2'-O-alkyl substituents conjugated to a DNA portion via a phosphodiester linkage, see, e.g., U.S. Patent No. 5,013,830.
  • the term also encompasses nucleic-acid-like structures with synthetic backbones.
  • DNA backbone analogues provided by the invention include phosphodiester, phosphorothioate, phosphorodithioate, methylphosphonate, phosphoramidate, alkyl phosphotriester, sulfamate, 3'-thioacetal, methylene(methylimino), 3'-N-carbamate, morpholino carbamate, and peptide nucleic acids (PNAs); see Oligonucleotides and Analogues, a Practical Approach, edited by F. Eckstein, ERL Press at Oxford University Press (1991); Antisense Strategies, Annals of the New York Academy of Sciences, Volume 600, Eds. Baserga and Denhardt (NYAS 1992); Milligan (1993) J. Med. Chem.
  • PNAs contain non-ionic backbones, such as N-(2-aminoethyl) glycine units. Phosphorothioate linkages are described, e.g., by U.S. Patent Nos. 6,031,092; 6,001,982; 5,684,148; see also, WO 97/03211; WO 96/39154; Mata (1997) Toxicol. Appl. Pharmacol. 144:189-197.
  • Other synthetic backbones encompassed by the term include methyl-phosphonate linkages or alternating methylphosphonate and phosphodiester linkages (see, e.g., U.S. Patent No.
  • nucleic acid is used interchangeably with gene, polynucleotide,
  • polypeptide include compositions of the invention that also include “analogs,” or “conservative variants” and “mimetics” or “peptidomimetics” with structures and activity that substantially conespond to the polypeptide from which the variant was derived.
  • saturatedation mutagenesis includes a method that uses degenerate oligonucleotide primers to introduce point mutations into a polynucleotide, as described in detail, below.
  • optical directed evolution system or “optimized directed evolution” includes a method for reassembling fragments of related nucleic acid sequences, e.g., related genes, and explained in detail, below.
  • This invention provides methods for generating variant antigen binding sites, antibodies and specific domains or fragments of antibodies (e.g., Fab or Fc domains) by mutagenizing a template nucleic acid by an optimized directed evolution system.
  • SLR synthetic ligation reassembly
  • antibody includes a peptide or polypeptide derived from, modeled after or substantially encoded by an immunoglobulin gene or immunoglobulin genes, or fragments thereof, capable of specifically binding an antigen or epitope, see, e.g. Fundamental immunology, Third Edition, W.E. Paul, ed., Raven Press, N.Y. (1993); Wilson (1994) J. Immunol. Methods 175:267-73; Yarmush (1992) J. Biochem. Biophys. Methods 25:85-97.
  • antibody-encoding nucleic acids and polypeptides may be isolated or synthesized de novo either chemically or by utilizing recombinant DNA methodology.
  • antibody includes antigen-binding portions, i.e., "antigen binding sites,” (e.g., fragments, subsequences, complementarity determining regions (CDRs)) that retain capacity to bind antigen, including (i) a Fab fragment, a monovalent fragment consisting of the VL, VH, CL and CHI domains; (ii) a F(ab') 2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) an Fd fragment consisting of the VH and CHI domains; (iv) an Fv fragment consisting of the VL and VH domains of a single arm of an antibody, (v) a dAb fragment (Ward et al., (1989) Nature 341:544-546), which consists of a VH domain; and (vi) an isolated complementarity determining region (CDR).
  • antigen binding sites e.g., fragments, subseque
  • the two domains of the Fv fragment, VL and VH are coded for by separate genes, they can be joined, using recombinant methods, by a synthetic linker that enables them to be made as a single protein chain in which the VL and VH regions pair to form monovalent molecules; also known as single chain Fv (scFv); see e.g., Bird (1988) Science 242:423-426; Huston (1988) Proc. Natl. Acad. Sci. USA 85:5879- 5883).
  • Single chain antibodies are also included by reference in the term "antibody.” Fragments can be prepared by recombinant techniques or enzymatic or chemical cleavage of intact antibodies.
  • the term also includes multivalent antigen-binding proteins, see, e.g., 6,027,725.
  • the term antibody also includes “chimeric” antibodies either produced by the modification of whole antibodies or those synthesized de novo using recombinant DNA methodologies. Such chimeric antibodies can be "humanized antibodies,” i.e., where the epitope binding site is generated from an immunized mammal, such as a mouse, and the structural framework is human. Methods for making chimeric, e.g., "humanized,” antibodies are well known in the art, see e.g., U.S. Patent Nos.
  • the term also includes human antibody nucleic acids and polypeptides generated by transgenic non-human animals (e.g., mice) capable of producing human antibodies, as described by, e.g., U.S. Patent Nos. 5,939,598; 5,877,397; 5,874,299; 5,814,318.
  • antibody also includes epitope binding polypeptides generated using phage display libraries, and variations thereof, as described by, e.g., U.S. Patent Nos. 5,855,885; 6,027,930. See also, discussion below.
  • major histocompatibility molecule or "MHC molecule” as used herein includes all Class I and Class U molecules, including alpha and beta chains of class II molecules and beta-2 microglobulin of Class I chains. Human MHC molecules can also be refened to as "Human Leukocyte Antigens" or HLA.
  • Class II MHC molecules are heterodimers displayed on the cell surface of antigen processing/ presenting cells (APCs), that include, e.g., macrophages, monocytes, activated endothelial cells, human B cells.
  • APCs antigen processing/ presenting cells
  • the methods of the invention include modification of any part or all of these polypeptides to, e.g., modify expression, their association with other molecules, such as antigens or co-stimulatory molecules or T cell receptors, and the like.
  • the structures of, and the isolating, making and using MHC molecules are well known in the art, see, e.g., Fundamental Immunology, Third Edition, Paul (ed) Raven Press, Ltd., New York; and, U.S. Patent Nos.
  • T cell receptor or "TCR” as used herein includes all antigen specific T cell receptor molecules.
  • TCRs are heterodimers (alpha and beta chains, or, gamma and delta chains) displayed on the cell surface of T lymphocytes.
  • the TCR binds to antigenic peptides presented in the binding pocket of an MHC molecule.
  • the methods of the invention include modification of any part or all of these polypeptides to, e.g., modify their expression, their association with other molecules, such as antigenic peptides or co-stimulatory molecules or an MHC molecule, and the like.
  • the invention provides anays comprising samples of (i.e., subsets of), or all of, the antigen binding sites that are isolated from and/or expressed by an individual, or, complementary to antigen binding sites isolated from and/or expressed by the individual.
  • the invention also provides anays comprising nucleic acids encoding these antigen binding sites.
  • the present invention can be practiced with any known “anay,” also refened to as a "microarray” or "DNA anay” or “nucleic acid anay” or “polypeptide anay” or “biochip,” or variation thereof.
  • agent is used herein to denote a chemical compound, a mixture of chemical compounds, an array of spatially localized compounds (e.g., a VLSEPS peptide anay, polynucleotide anay, and/or combinatorial small molecule anay), biological macromolecule, a bacteriophage peptide display library, a bacteriophage antibody (e.g., scFv) display library, a polysome peptide display library, or an extract made form biological materials such as bacteria, plants, fungi, or animal (particular mammalian) cells or tissues.
  • a chemical compound e.g., a VLSEPS peptide anay, polynucleotide anay, and/or combinatorial small molecule anay
  • biological macromolecule e.g., a VLSEPS peptide anay, polynucleotide anay, and/or combinatorial small molecule anay
  • Agents are evaluated for potential activity as anti-neoplasties, anti-inflammatories or apoptosis modulators by inclusion in screening assays described hereinbelow.
  • Agents are evaluated for potential activity as specific protein interaction inhibitors (i.e., an agent which selectively inhibits a binding interaction between two predetermined polypeptides but which doe snot substantially interfere with cell viability) by inclusion in screening assays described hereinbelow.
  • An "ambiguous base requirement" in a restriction site refers to a nucleotide base requirement that is not specified to the fullest extent, i.e. that is not a specific base (such as, in a non-limiting exemplification, a specific base selected from A, C, G, and T), but rather may be any one of at least two or more bases.
  • HSPs high scoring sequence pairs
  • the word hits are then extended in both directions along each sequence for as far as the cumulative alignment score can be increased. Cumulative scores are calculated using, for nucleotide sequences, the parameters M (reward score for a pair of matching residues; always > 0) and N (penalty score for mismatching residues; always ⁇ 0). For amino acid sequences, a scoring matrix is used to calculate the cumulative score. Extension of the word hits in each direction are halted when: the cumulative alignment score falls off by the quantity X from its maximum achieved value; the cumulative score goes to zero or below, due to the accumulation of one or more negative-scoring residue alignments; or the end of either sequence is reached.
  • the BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment.
  • W wordlength
  • E expectation
  • W wordlength
  • E expectation
  • BLOSUM62 scoring matrix see Henikoff & Henikoff (1989) Proc. Natl. Acad. Sci. USA 89:10915.
  • the BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, e.g., Karlin & Altschul (1993) Proc. Nat'l. Acad. Sci.
  • BLAST algorithm One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability by which a match between two nucleotide or amino acid sequences would occur by chance.
  • P(N) the smallest sum probability
  • a nucleic acid is considered similar to a reference sequence if the smallest sum probability in a comparison of the test nucleic acid to the reference nucleic acid is less than about 0. 1, or less than about 0.0 1, or less than about 0.001.
  • Another indication that two nucleic acid sequences are substantially identical is that the two molecules hybridize to each other under stringent conditions.
  • hybridizing specifically to refers to the binding, duplexing, or hybridizing of a molecule only to a particular nucleotide sequence under stringent conditions when that sequence is present in a complex mixture (e.g., total cellular) DNA or RNA.
  • Bod(s) substantially refers to complementary hybridization between a probe nucleic acid and a target nucleic acid and embraces minor mismatches that can be accommodated by reducing the stringency of the hybridization media to achieve the desired detection of the target polynucleotide sequence.
  • amino acid refers to any organic compound that contains an amino group (-NH 2 ) and a carboxyl group (-COOH); in one aspect, either as free groups or alternatively after condensation as part of peptide bonds.
  • the "twenty naturally encoded polypeptide-forming alpha-amino acids” are understood in the art and refer to: alanine (ala or A), arginine (arg or R), asparagine (asn or N), aspartic acid (asp or D), cysteine (cys or C), gluatamic acid (glu or E), glutamine (gin or Q), glycine (gly or G), histidine (his or H), isoleucine (ile or I), leucine (leu or L), lysine (lys or K), methionine (met or M), phenylalanine (phe or F), proline (pro or P), serine (ser or S), threonine (thr or T
  • antibody refers to intact immunoglobulin molecules, as well as fragments of immunoglobulin molecules, such as Fab, Fab', (Fab') 2 , Fv, and SCA fragments, that are capable of binding to an epitope of an antigen.
  • Fab fragments of immunoglobulin molecules
  • Fab' fragments of immunoglobulin molecules
  • Fv fragments of an antigen.
  • SCA fragments of immunoglobulin molecules
  • These antibody fragments which retain some ability to selectively bind to an antigen (e.g., a polypeptide antigen) of the antibody from which they are derived, can be made using well known methods in the art (see, e.g., Harlow and Lane, supra), and are described further, as follows.
  • An Fab fragment consists of a monovalent antigen-binding fragment of an antibody molecule, and can be produced by digestion of a whole antibody molecule with the enzyme papain, to yield a fragment consisting of an intact light chain and a portion of a heavy chain.
  • An Fab' fragment of an antibody molecule can be obtained by treating a whole antibody molecule with pepsin, followed by reduction, to yield a molecule consisting of an intact light chain and a portion of a heavy chain. Two Fab' fragments are obtained per antibody molecule treated in this manner.
  • An (Fab') 2 fragment of an antibody can be obtained by treating a whole antibody molecule with the enzyme pepsin, without subsequent reduction.
  • a (Fab') 2 fragment is a dimer of two Fab' fragments, held together by two disulfide bonds.
  • An Fv fragment is defined as a genetically engineered fragment containing the variable region of a light chain and the variable region of a heavy chain expressed as two chains.
  • An single chain antibody (“SCA”) is a genetically engineered single chain molecule containing the variable region of a light chain and the variable region of a heavy chain, linked by a suitable, flexible polypeptide linker.
  • SCA single chain antibody
  • AME Applied Molecular Evolution
  • a molecule that has a "chimeric property" is a molecule that is: 1) in part homologous and in part heterologous to a first reference molecule; while 2) at the same time being in part homologous and in part heterologous to a second reference molecule; without 3) precluding the possibility of being at the same time in part homologous and in part heterologous to still one or more additional reference molecules.
  • a chimeric molecule may be prepared by assemblying a reassortment of partial molecular sequences.
  • a chimeric polynucleotide molecule may be prepared by synthesizing the chimeric polynucleotide using plurality of molecular templates, such that the resultant chimeric polynucleotide has properties of a plurality of templates.
  • the term "cognate” as used herein refers to a gene sequence that is evolutionarily and functionally related between species.
  • the human CD4 gene is the cognate gene to the mouse 3d4 gene, since the sequences and structures of these two genes indicate that they are highly homologous and both genes encode a protein which functions in signaling T cell activation through MHC class II-restricted antigen recognition.
  • a “comparison window,” as used herein, refers to a conceptual segment of at least 20 contiguous nucleotide positions wherein a polynucleotide sequence may be compared to a reference sequence of at least 20 contiguous nucleotides and wherein the portion of the polynucleotide sequence in the comparison window may comprise additions or deletions (i.e., gaps) of 20 percent or less as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences.
  • Optimal alignment of sequences for aligning a comparison window may be conducted by the local homology algorithm of Smith (Smith and Waterman, Adv Appl Math, 1981; Smith and Waterman, J Teor Biol, 1981; Smith and Waterman, / Mol Biol, 1981; Smith et al, J Mol Evol, 1981), by the homology alignment algorithm of Needleman (Needleman and Wuncsch,
  • complementarity-determining region and "CDR” refer to the art-recognized term as exemplified by the Kabat and Chothia CDR definitions also generally known as supervariable regions or hypervariable loops (Chothia and Lesk, 1987; Clothia et al, 1989; Kabat et al, 1987; and Tramontano et al, 1990).
  • Variable region domains typically comprise the amino-terminal approximately 105-115 amino acids of a naturally- occurring immunoglobulin chain (e.g., amino acids 1-110), although variable domains somewhat shorter or longer are also suitable for forming single-chain antibodies.
  • Constant amino acid substitutions refer to the interchangeability of residues having similar side chains.
  • a group of amino acids having aliphatic side chains is glycine, alanine, valine, leucine, and isoleucine
  • a group of amino acids having aliphatic- hydroxyl side chains is serine and threonine
  • a group of amino acids having amide-containing side chains is asparagine and glutamine
  • a group of amino acids having aromatic side chains is phenylalanine, tyrosine, and tryptophan
  • a group of amino acids having basic side chains is lysine, arginine, and histidine
  • a group of amino acids having sulfur-containing side chains is cysteine and methionine.
  • Exemplary conservative amino acids substitution groups are : valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, and asparagine-glutamine.
  • Consatively modified variations of a particular polynucleotide sequence refers to those polynucleotides that encode identical or essentially identical amino acid sequences, or where the polynucleotide does not encode an amino acid sequence, to essentially identical sequences. Because of the degeneracy of the genetic code, a large number of functionally identical nucleic acids encode any given polypeptide. For instance, the codons CGU, CGC, CGA, CGG, AGA, and AGG all encode the amino acid arginine. Thus, at every position where an arginine is specified by a codon, the codon can be altered to any of the conesponding codons described without altering the encoded polypeptide.
  • nucleic acid variations are "silent variations," which are one species of “conservatively modified variations.” Every polynucleotide sequence described herein which encodes a polypeptide also describes every possible silent variation, except where otherwise noted.
  • each codon in a nucleic acid except AUG, which is ordinarily the only codon for methionine
  • each "silent variation" of a nucleic acid which encodes a polypeptide is implicit in each described sequence.
  • a polynucleotide sequence is homologous (i.e., is identical, not strictly evolutionarily related) to all or a portion of a reference polynucleotide sequence, or that a polypeptide sequence is identical to a reference polypeptide sequence.
  • the term “complementary to” is used herein to mean that the complementary sequence is homologous to all or a portion of a reference polynucleotide sequence.
  • TATAC corresponds to a reference "TATAC” and is complementary to a reference sequence "GTATA.”
  • cytokine includes, for example, interleukins, interferons, chemokines, hematopoietic growth factors, tumor necrosis factors and transforming growth factors. In general these are small molecular weight proteins that regulate maturation, activation, proliferation and differentiation of the cells of the immune system.
  • degrading effective amount refers to the amount of enzyme which is required to process at least 50% of the substrate, as compared to substrate not contacted with the enzyme. In one aspect, at least 80% of the substrate is degraded.
  • defined sequence framework refers to a set of defined sequences that are selected on a non-random basis, generally on the basis of experimental data or structural data; for example, a defined sequence framework may comprise a set of amino acid sequences that are predicted to form a ⁇ -sheet structure or may comprise a leucine zipper heptad repeat motif, a zinc-finger domain, among other variations.
  • a “defined sequence kernal” is a set of sequences which encompass a limited scope of variability.
  • a completely random 10-mer sequence of the 20 conventional amino acids can be any of (20) 10 sequences
  • a pseudorandom 10-mer sequence of the 20 conventional amino acids can be any of (20) 10 sequences but will exhibit a bias for certain residues at certain positions and/or overall
  • a defined sequence kernal is a subset of sequences if each residue position was allowed to be any of the allowable 20 conventional amino acids (and/or allowable unconventional amino/imino acids).
  • a defined sequence kernal generally comprises variant and invariant residue positions and/or comprises variant residue positions which can comprise a residue selected from a defined subset of amino acid residues), and the like, either segmentally or over the entire length of the individual selected library member sequence.
  • sequence kernels can refer to either amino acid sequences or polynucleotide sequences.
  • sequences (NNK) 10 and (NNMT o, wherein N represents A, T, G, or C; K represents G or T; and M represents A or C are defined sequence kernels.
  • “Digestion” of DNA refers to catalytic cleavage of the DNA with a restriction enzyme that acts only at certain sequences in the DNA.
  • restriction enzymes used herein are commercially available and their reaction conditions, cofactors and other requirements were used as would be known to the ordinarily skilled artisan.
  • typically 1 ⁇ g of plasmid or DNA fragment is used with about 2 units of enzyme in about 20 ⁇ l of buffer solution.
  • DNA fragments for plasmid construction typically 5 to 50 ⁇ g of DNA are digested with 20 to 250 units of enzyme in a larger volume. Appropriate buffers and substrate amounts for particular restriction enzymes are specified by the manufacturer. Incubation times of about 1 hour at 37°C are ordinarily used, but may vary in accordance with the supplier's instructions. After digestion the reaction is electrophoresed directly on a gel to isolate the desired fragment.
  • Directional ligation refers to a ligation in which a 5' end and a 3' end of a polynuclotide are different enough to specify an exemplary ligation orientation.
  • an otherwise untreated and undigested PCR product that has two blunt ends will typically not have an exemplary ligation orientation when ligated into a cloning vector digested to produce blunt ends in its multiple cloning site; thus, directional ligation will typically not be displayed under these circumstances.
  • directional ligation will typically displayed when a digested PCR product having a 5' EcoR I-treated end and a 3' BamH I-is ligated into a cloning vector that has a multiple cloning site digested with EcoR I and BamH I.
  • DNA shuffling is used herein to indicate recombination between substantially homologous but non-identical sequences, in some embodiments DNA shuffling may involve crossover via non-homologous recombination, such as via cer/lox and/or flp/frt systems and the like.
  • epitope refers to an antigenic determinant on an antigen, such as a phytase polypeptide, to which the paratope of an antibody, such as an phytase-specific antibody, binds.
  • Antigenic determinants usually consist of chemically active surface groupings of molecules, such as amino acids or sugar side chains, and can have specific three-dimensional structural characteristics, as well as specific charge characteristics.
  • epitopope refers to that portion of an antigen or other macromolecule capable of forming a binding interaction that interacts with the variable region binding body of an antibody. Typically, such binding interaction is manifested as an intermolecular contact with one or more amino acid residues of a CDR.
  • heterologous DNA segment is one that originates from a source foreign to the particular host cell, or, if from the same source, is modified from its original form.
  • a heterologous gene in a host cell includes a gene that is endogenous to the particular host cell, but has been modified. Modification of a heterologous sequence in the applications described herein typically occurs through the use of stochastic (e.g. polynucleotide shuffling & interrupted synthesis) and non- stochastic polynucleotide reassembly.
  • the terms refer to a DNA segment which is foreign or heterologous to the cell, or homologous to the cell but in a position within the host cell nucleic acid in which the element is not ordinarily found. "Exogenous" DNA segments are expressed to yield exogenous polypeptides.
  • genes are used broadly to refer to any segment of DNA associated with a biological function. Thus, genes include coding sequences and/or the regulatory sequences required for their expression. Genes also include nonexpressed DNA segments that, for example, form recognition sequences for other proteins. Genes can be obtained from a variety of sources, including cloning from a source of interest or synthesizing from known or predicted sequence information, and may include sequences designed to have desired parameters.
  • the source polynucleotides or polypeptides from which the different nucleic acid or amino acid sequences are derived are sometimes homologous (i.e., have, or encode a polypeptide that encodes, the same or a similar structure and/or function), and are often from different isolates, serotypes, strains, species, of organism or from different disease states, for example.
  • fragment when referring to a reference polypeptide comprise a polypeptide which retains at least one biological function or activity that is at least essentially same as that of the reference polypeptide. Furthermore, the terms “fragment”, “derivative” or “analog” are exemplified by a "pro-form” molecule, such as a low activity proprotein that can be modified by cleavage to produce a mature enzyme with significantly higher activity.
  • a method for producing from a template polypeptide a set of progeny polypeptides in which a "full range of single amino acid substitutions" is represented at each amino acid position.
  • “full range of single amino acid substitutions” is in reference to the naturally encoded 20 naturally encoded polypeptide- forming alpha-amino acids, as described herein.
  • gene means the segment of DNA involved in producing a polypeptide chain; it includes regions preceding and following the coding region (leader and trailer) as well as intervening sequences (introns) between individual coding segments (exons).
  • Genetic instability refers to the natural tendency of highly repetitive sequences to be lost through a process of reductive events generally involving sequence simplification through the loss of repeated sequences. Deletions tend to involve the loss of one copy of a repeat and everything between the repeats.
  • heterologous means that one single-stranded nucleic acid sequence is unable to hybridize to another single-stranded nucleic acid sequence or its complement.
  • areas of heterology means that areas of polynucleotides or polynucleotides have areas or regions within their sequence which are unable to hybridize to another nucleic acid or polynucleotide. Such regions or areas are for example areas of mutations.
  • homologous or “homeologous” means that one single-stranded nucleic acid nucleic acid sequence may hybridize to a complementary single-stranded nucleic acid sequence.
  • the degree of hybridization may depend on a number of factors including the amount of identity between the sequences and the hybridization conditions such as temperature and salt concentrations as discussed later. In one aspect the region of identity is greater than about 5 bp, or, the region of identity is greater than 10 bp.
  • An immunoglobulin light or heavy chain variable region consists of a "framework" region interrupted by three hypervariable regions, also called CDR's.
  • the extent of the framework region and CDR's have been precisely defined; see “Sequences of Proteins of Immunological Interest” (Kabat et al, 1987).
  • the sequences of the framework regions of different light or heavy chains are relatively conserved within a specie.
  • a "human framework region” is a framework region that is substantially identical (about 85 or more, usually 90-95 or more) to the framework region of a naturally occurring human immunoglobulin.
  • the framework region of an antibody that is the combined framework regions of the constituent light and heavy chains, serves to position and align the CDR's.
  • the CDR's are primarily responsible for binding to an epitope of an antigen.
  • nucleic acid sequences have the same sequence or a complementary sequence.
  • areas of identity means that regions or areas of a polynucleotide or the overall polynucleotide are identical or complementary to areas of another polynucleotide or the polynucleotide.
  • nucleic acid or polypeptide sequences refer to two or more sequences or subsequences that are the same or have a specified percentage of amino acid residues or nucleotides that are the same, when compared and aligned for maximum correspondence, as measured using one of the following sequence comparison algorithms or by visual inspection.
  • sequence comparison typically one sequence acts as a reference sequence to which test sequences are compared.
  • test and reference sequences are input into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated.
  • sequence comparison algorithm then calculates the percent sequence identity for the test sequence(s) relative to the reference sequence, based on the designated program parameters.
  • nucleic acid sequences or polypeptides are substantially “identical” is that the polypeptide encoded by the first nucleic acid is immunologically cross reactive with, or specifically binds to, the polypeptide encoded by the second nucleic acid.
  • a polypeptide is typically substantially identical to a second polypeptide, for example, where the two peptides differ only by conservative substitutions.
  • isolated means that the material is removed from its original environment (e.g., the natural environment if it is naturally occurring).
  • a naturally-occurring polynucleotide or enzyme present in a living animal is not isolated, but the same polynucleotide or enzyme, separated from some or all of the coexisting materials in the natural system, is isolated.
  • Such polynucleotides could be part of a vector and/or such polynucleotides or enzymes could be part of a composition, and still be isolated in that such vector or composition is not part of its natural environment.
  • isolated when applied to a nucleic acid or protein, denotes that the nucleic acid or protein is free of at least one other cellular components with which it is associated in the natural state. It can be substantially is free of at least one other cellular components with which it is associated in the natural state. It can be in a homogeneous state although it can be in either a dry or aqueous solution. Purity and homogeneity are typically determined using analytical chemistry techniques such as polyacrylamide gel electrophoresis or high performance liquid chromatography. A protein which is the predominant species present in a preparation is substantially purified. In particular, an isolated gene is separated from open reading frames which flank the gene and encode a protein other than the gene of interest.
  • isolated nucleic acid is meant a nucleic acid, e.g., a DNA or RNA molecule, that is not immediately contiguous with the 5' and 3' flanking sequences with which it normally is immediately contiguous when present in the naturally occurring genome of the organism from which it is derived.
  • the term thus describes, for example, a nucleic acid that is incorporated into a vector, such as a plasmid or viral vector; a nucleic acid that is incorporated into the genome of a heterologous cell (or the genome of a homologous cell, but at a site different from that at which it naturally occurs); and a nucleic acid that exists as a separate molecule, e.g., a DNA fragment produced by PCR amplification or restriction enzyme digestion, or an RNA molecule produced by in vitro transcription.
  • the term also describes a recombinant nucleic acid that forms part of a hybrid gene encoding additional polypeptide sequences that can be used, for example, in the production of a fusion protein.
  • ligand refers to a molecule, such as a random peptide or variable segment sequence, that is recognized by a particular receptor.
  • a molecule or macromolecular complex
  • the binding partner having a smaller molecular weight is refened to as the ligand and the binding partner having a greater molecular weight is refened to as a receptor.
  • Ligase refers to the process of forming phosphodiester bonds between two double stranded nucleic acid fragments (Sambrook et al, 1982, p. 146; Sambrook, 1989). Unless otherwise provided, ligation may be accomplished using known buffers and conditions with 10 units of T4 DNA ligase ("ligase”) per 0.5 ⁇ g of approximately equimolar amounts of the DNA fragments to be ligated.
  • ligase T4 DNA ligase
  • linker refers to a molecule or group of molecules that connects two molecules, such as a DNA binding protein and a random peptide, and serves to place the two molecules in an exemplary configuration, e.g., so that the random peptide can bind to a receptor with minimal steric hindrance from the DNA binding protein.
  • a "molecular property to be evolved” includes reference to molecules comprised of a polynucleotide sequence, molecules comprised of a polypeptide sequence, and molecules comprised in part of a polynucleotide sequence and in part of a polypeptide sequence.
  • Particularly relevant - but by no means limiting - examples of molecular properties to be evolved include enzymatic activities at specified conditions, such as related to temperature; salinity; pressure; pH; and concentration of glycerol, DMSO, detergent, &/or any other molecular species with which contact is made in a reaction environment.
  • Additional particularly relevant - but by no means limiting - examples of molecular properties to be evolved include stabilities - e.g. the amount of a residual molecular property that is present after a specified exposure time to a specified environment, such as may be encountered during storage.
  • a “multivalent antigenic polypeptide” or a “recombinant multivalent antigenic polypeptide” is a non-naturally occurring polypeptide that includes amino acid sequences from more than one source polypeptide, which source polypeptide is typically a naturally occurring polypeptide. At least some of the regions of different amino acid sequences constitute epitopes that are recognized by antibodies found in a mammal that has been injected with the source polypeptide.
  • the source polypeptides from which the different epitopes are derived are usually homologous (i.e., have the same or a similar structure and/or function), and are often from different isolates, serotypes, strains, species, of organism or from different disease states, for example.
  • mutants includes changes in the sequence of a wild-type or parental nucleic acid sequence or changes in the sequence of a peptide. Such mutations may be point mutations such as transitions or trans versions. The mutations may be deletions, insertions or duplications. A mutation can also be a "chimerization", which is exemplified in a progeny molecule that is generated to contain part or all of a sequence of one parental molecule as well as part or all of a sequence of at least one other parental molecule. This invention provides for both chimeric polynucleotides and chimeric polypeptides.
  • N,N,G/T nucleotide sequence represents 32 possible triplets, where "N” can be A, C, G or T.
  • nucleic acid refers to deoxyribonucleotides or ribonucleotides and polymers thereof in either single- or double-stranded form. Unless specifically limited, the term encompasses nucleic acids containing known analogues of natural nucleotides which have similar binding properties as the reference nucleic acid and are metabolized in a manner
  • nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (e.g. degenerate codon substitutions) and complementary sequences and as well as the sequence explicitly indicated.
  • degenerate codon substitutions may be achieved by generating sequences in which the third position of one or more selected (or all) codons is
  • nucleic acid is used interchangeably with gene, cDNA, and mRNA encoded by a gene.
  • Nucleic acid derived from a gene refers to a nucleic acid for whose synthesis the
  • an mRNA, a cDNA reverse transcribed from an mRNA, an RNA transcribed from that cDNA, a DNA amplified from the cDNA, an RNA transcribed from the amplified DNA, etc. are all derived from the gene and detection of such derived products is indicative of the presence and/or abundance of the original gene and/or gene transcript in a sample.
  • nucleic acid molecule is comprised of at least one base or one base pair, depending on whether it is single-stranded or double-stranded, respectively. Furthermore, a nucleic acid molecule may belong exclusively or chimerically to any group of nucleotide-containing molecules, as exemplified by, but not limited to, the following groups of nucleic acid molecules: RNA, DNA, genomic nucleic acids, non-genomic nucleic acids,
  • nucleic acids 25 naturally occurring and not naturally occurring nucleic acids, and synthetic nucleic acids.
  • nucleic acid molecule may contain in part one or more non- nucleotide-based components as exemplified by, but not limited to, amino acids and sugars.
  • a ribozyme that is in part nucleotide-based and in part protein-based is considered a "nucleic acid molecule”.
  • nucleic acid molecule that is labeled with a detectable moiety, such as a radioactive or alternatively a non-radioactive label, is likewise considered a "nucleic acid molecule”.
  • nucleic acid sequence coding for or a "DNA coding sequence of or a “nucleotide sequence encoding” a particular enzyme - as well as other synonymous terms - refer to a DNA sequence which is transcribed and translated into an enzyme when placed under the control of appropriate regulatory sequences.
  • a "promotor sequence” is a DNA regulatory region capable of binding RNA polymerase in a cell and initiating transcription of a downstream (3' direction) coding sequence. The promoter is part of the DNA sequence. This sequence region has a start codon at its 3' terminus. The promoter sequence does include the minimum number of bases where elements necessary to initiate transcription at levels detectable above background.
  • RNA polymerase binds the sequence and transcription is initiated at the start codon (3' terminus with a promoter)
  • transcription proceeds downstream in the 3' direction.
  • a transcription initiation site (conveniently defined by mapping with nuclease SI) as well as protein binding domains (consensus sequences) responsible for the binding of RNA polymerase.
  • nucleic acid encoding an enzyme (protein) or “DNA encoding an enzyme (protein)” or “polynucleotide encoding an enzyme (protein)” and other synonymous terms encompasses a polynucleotide which includes only coding sequence for the enzyme as well as a polynucleotide which includes additional coding and/or non-coding sequence.
  • a "specific nucleic acid molecule species” is defined by its chemical structure, as exemplified by, but not limited to, its primary sequence.
  • a specific "nucleic acid molecule species” is defined by a function of the nucleic acid species or by a function of a product derived from the nucleic acid species.
  • a “specific nucleic acid molecule species” may be defined by one or more activities or properties attributable to it, including activities or properties attributable its expressed product.
  • the instant definition of "assembling a working nucleic acid sample into a nucleic acid library” includes the process of incorporating a nucleic acid sample into a vector-based collection, such as by ligation into a vector and transformation of a host. A description of relevant vectors, hosts, and other reagents as well as specific non-limiting examples thereof are provided hereinafter.
  • the instant definition of "assembling a working nucleic acid sample into a nucleic acid library” also includes the process of incorporating a nucleic acid sample into a non-vector-based collection, such as by ligation to adaptors.
  • the adaptors can anneal to PCR primers to facilitate amplification by PCR.
  • a "nucleic acid library” is comprised of a vector-based collection of one or more nucleic acid molecules.
  • a "nucleic acid library” is comprised of a non-vector-based collection of nucleic acid molecules.
  • a "nucleic acid library” is comprised of a combined collection of nucleic acid molecules that is in part vector-based and in part non- vector-based.
  • the collection of molecules comprising a library is searchable and separable according to individual nucleic acid molecule species.
  • the present invention provides a "nucleic acid construct” or alternatively a “nucleotide construct” or alternatively a "DNA construct”.
  • construct is used herein to describe a molecule, such as a polynucleotide (e.g., a phytase polynucleotide) may optionally be chemically bonded to one or more additional molecular moieties, such as a vector, or parts of a vector.
  • a nucleotide construct is exemplified by a DNA expression DNA expression constructs suitable for the transformation of a host cell.
  • oligonucleotide refers to either a single stranded polydeoxynucleotide or two complementary polydeoxynucleotide strands which may be chemically synthesized. Such synthetic oligonucleotides may or may not have a 5' phosphate. Those that do not will not ligate to another oligonucleotide without adding a phosphate with an ATP in the presence of a kinase. A synthetic oligonucleotide will ligate to a fragment that has not been dephosphorylated.
  • a "32-fold degenerate oligonucleotide that is comprised of, in series, at least a first homologous sequence, a degenerate N,N,G/T sequence, and a second homologous sequence" is mentioned.
  • homologous is in reference to homology between the oligo and the parental polynucleotide that is subjected to the polymerase-based amplification.
  • a nucleic acid is "operably linked” when it is placed into a functional relationship with another nucleic acid sequence. For instance, a promoter or enhancer is operably linked to a coding sequence if it increases the transcription of the coding sequence.
  • operably linked refers to a linkage of polynucleotide elements in a functional relationship.
  • a nucleic acid is “operably linked” when it is placed into a functional relationship with another nucleic acid sequence.
  • a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the coding sequence.
  • Operably linked means that the DNA sequences being linked are typically contiguous and, where necessary to join two protein coding regions, contiguous and in reading frame.
  • enhancers generally function when separated from the promoter by several kilobases and intronic sequences may be of variable lengths, some polynucleotide elements may be operably linked but not contiguous.
  • a coding sequence is "operably linked to" another coding sequence when RNA polymerase will transcribe the two coding sequences into a single mRNA, which is then translated into a single polypeptide having amino acids derived from both coding sequences.
  • the coding sequences need not be contiguous to one another so long as the expressed sequences are ultimately processed to produce the desired protein.
  • parental polynucleotide set is a set comprised of one or more distinct polynucleotide species. Usually this term fis used in reference to a progeny polynucleotide set which can be obtained by mutagenization of the parental set, in which case the terms “parental”, “starting” and “template” are used interchangeably.
  • physiological conditions refers to temperature, pH, ionic strength, viscosity, and like biochemical parameters which are compatible with a viable organism, and/or which typically exist intracellularly in a viable cultured yeast cell or mammalian cell.
  • intracellular conditions in a yeast cell grown under typical laboratory culture conditions are physiological conditions.
  • Suitable in vitro reaction conditions for in vitro transcription cocktails are generally physiological conditions.
  • in vitro physiological conditions comprise 50-200 mM NaCl or KC1, pH 6.5-8.5, 20- 45DC and 0.001-10 mM divalent cation (e.g., Mg 4"1" , Ca “1””1” ); or about 150 mM NaCl or KC1, pH 7.2-7.6, 5 mM divalent cation, and often include 0.01-1.0 percent nonspecific protein
  • a non-ionic detergent (Tween, NP-40, Triton X-100) can often be present, usually at about 0.001 to 2%, typically 0.05-0.2% (v/v).
  • Particular aqueous conditions may be selected by the practitioner according to conventional methods. For general guidance, the following buffered aqueous conditions may be applicable: 10-250 mM NaCl, 5-50 mM Tris HCl, pH 5-8, with optional addition of divalent cation(s) and/or metal chelators and/or non- ionic detergents and/or membrane fractions and/or anti-foam agents and/or scintillants. Standard convention (5' to 3') is used herein to describe the sequence of double standed polynucleotides.
  • population means a collection of components such as polynucleotides, portions or polynucleotides or proteins.
  • a molecule having a "pro-form” refers to a molecule that undergoes any combination of one or more covalent and noncovalent chemical modifications (e.g. glycosylation, proteolytic cleavage, dimerization or oligomerization, temperature-induced or pH-induced conformational change, association with a co-factor, etc.) en route to attain a more mature molecular form having a property difference (e.g. an increase in activity) in comparison with the reference pro-form molecule.
  • covalent and noncovalent chemical modifications e.g. glycosylation, proteolytic cleavage, dimerization or oligomerization, temperature-induced or pH-induced conformational change, association with a co-factor, etc.
  • a property difference e.g. an increase in activity
  • the referemce precursor molecule may be termed a "pre-pro-form" molecule.
  • nucleic acid or protein gives rise to essentially one band in an electrophoretic gel. Particularly, it means that the nucleic acid or protein is at least about 50% pure, or at least about 85% pure, or at least about 99% pure.
  • Quadsi-repeated units refers to the repeats to be re-assorted and are by definition not identical. Indeed the method is proposed not only for practically identical encoding units produced by mutagenesis of the identical starting sequence, but also the reassortment of similar or related sequences which may diverge significantly in some regions. Nevertheless, if the sequences contain sufficient homologies to be reassorted by this approach, they can be refened to as "quasi-repeated" units.
  • random peptide library refers to a set of polynucleotide sequences that encodes a set of random peptides, and to the set of random peptides encoded by those polynucleotide sequences, as well as the fusion proteins contain those random peptides.
  • random peptide sequence refers to an amino acid sequence composed of two or more amino acid monomers and constructed by a stochastic or random process.
  • a random peptide can include framework or scaffolding motifs, which may comprise invariant sequences.
  • receptor refers to a molecule that has an affinity for a given ligand.
  • Receptors can be naturally occurring or synthetic molecules. Receptors can be employed in an unaltered state or as aggregates with other species. Receptors can be attached, covalently or non-covalently, to a binding member, either directly or via a specific binding substance.
  • receptors include, but are not limited to, antibodies, including monoclonal antibodies and antisera reactive with specific antigenic determinants (such as on viruses, cells, or other materials), cell membrane receptors, complex carbohydrates and glycoproteins, enzymes, and hormone receptors.
  • Recombinant when used with reference to a cell indicates that the cell replicates a heterologous nucleic acid, or expresses a peptide or protein encoded by a heterologous nucleic acid.
  • Recombinant cells can contain genes that are not found within the native (non-recombinant) form of the cell.
  • Recombinant cells can also contain genes found in the native form of the cell wherein the genes are modified and re-introduced into the cell by artificial means.
  • the term also encompasses cells that contain a nucleic acid endogenous to the cell that has been modified without removing the nucleic acid from the cell; such modifications include those obtained by gene replacement, site-specific mutation, and related techniques.
  • Recombinant enzymes refer to enzymes produced by recombinant DNA techniques, i.e., produced from cells transformed by an exogenous DNA construct encoding the desired enzyme.
  • Synthetic enzymes are those prepared by chemical synthesis.
  • a "recombinant expression cassette” or simply an “expression cassette” is a nucleic acid construct, generated recombinantiy or synthetically, with nucleic acid elements that are capable of effecting expression of a structural gene in hosts compatible with such sequences. Expression cassettes include at least promoters and optionally, transcription termination signals.
  • the recombinant expression cassette includes a nucleic acid to be transcribed (e.g., a nucleic acid encoding a desired polypeptide), and a promoter.
  • an expression cassette can also include nucleotide sequences that encode a signal sequence that directs secretion of an expressed protein from the host cell. Transcription termination signals, enhancers, and other nucleic acid sequences that influence gene expression, can also be included in an expression cassette.
  • related polynucleotides means that regions or areas of the polynucleotides are identical and regions or areas of the polynucleotides are heterologous.
  • Reductive reassortment refers to the increase in molecular diversity that is accrued through deletion (and/or insertion) events that are mediated by repeated sequences.
  • sequence relationships between two or more polynucleotides are used to describe the sequence relationships between two or more polynucleotides: “reference sequence,” “comparison window,” “sequence identity,” “percentage of sequence identity,” and “substantial identity.”
  • a "reference sequence” is a defined sequence used as a basis for a sequence comparison; a reference sequence may be a subset of a larger sequence, for example, as a segment of a full-length cDNA or gene sequence given in a sequence listing, or may comprise a complete cDNA or gene sequence. Generally, a reference sequence is at least 20 nucleotides in length, frequently at least 25 nucleotides in length, and often at least 50 nucleotides in length.
  • two polynucleotides may each (1) comprise a sequence (i.e., a portion of the complete polynucleotide sequence) that is similar between the two polynucleotides and (2) may further comprise a sequence that is divergent between the two polynucleotides
  • sequence comparisons between two (or more) polynucleotides are typically performed by comparing sequences of the two polynucleotides over a "comparison window" to identify and compare local regions of sequence similarity.
  • Repetitive Index is the average number of copies of the quasi- repeated units contained in the cloning vector.
  • restriction site refers to a recognition sequence that is necessary for the manifestation of the action of a restriction enzyme, and includes a site of catalytic cleavage.
  • a site of cleavage may or may not be contained within a portion of a restriction site that comprises a low ambiguity sequence (i.e. a sequence containing the principal determinant of the frequency of occurrence of the restriction site).
  • relevant restriction sites contain only a low ambiguity sequence with an internal cleavage site (e.g. G/AATTC in the EcoR I site) or an immediately adjacent cleavage site (e.g. /CCWGG in the EcoR Et site).
  • relevant restriction enzymes e.g. the Eco57 I site or CTGAAG(16/14)] contain a low ambiguity sequence (e.g.
  • an enzyme e.g. a restriction enzyme
  • cleave a polynucleotide
  • the restriction enzyme catalyzes or facilitates a cleavage of a polynucleotide.
  • screening describes, in general, a process that identifies optimal antigens.
  • properties of the antigen can be used in selection and screening including antigen expression, folding, stability, immunogenicity and presence of epitopes from several related antigens.
  • Selection is a form of screening in which identification and physical separation are achieved simultaneously by expression of a selection marker, which, in some genetic circumstances, allows cells expressing the marker to survive while other cells die (or vice versa).
  • Screening markers include, for example, luciferase, beta-galactosidase and green fluorescent protein. Selection markers include drug and toxin resistance genes, and the like. Because of limitations in studying primary immune responses in vitro, in vivo studies are particularly useful screening methods.
  • the antigens are first introduced to test animals, and the immune responses are subsequently studied by analyzing protective immune responses or by studying the quality or strength of the induced immune response using lymphoid cells derived from the immunized animal.
  • spontaneous selection can and does occur in the course of natural evolution, in the present methods selection is performed by man.
  • a "selectable polynucleotide” is comprised of a 5' terminal region (or end region), an intermediate region (i.e. an internal or central region), and a 3' terminal region (or end region).
  • a 5' terminal region is a region that is located towards a 5' polynucleotide terminus (or a 5' polynucleotide end); thus it is either partially or entirely in a 5' half of a polynucleotide.
  • a 3' terminal region is a region that is located towards a 3' polynucleotide terminus (or a 3' polynucleotide end); thus it is either partially or entirely in a 3' half of a polynucleotide.
  • sequence identity means that two polynucleotide sequences are identical (i.e., on a nucleotide-by-nucleotide basis) over the window of comparison.
  • percentage of sequence identity is calculated by comparing two optimally aligned sequences over the window of comparison, determining the number of positions at which the identical nucleic acid base (e.g., A, T, C, G, U, or I) occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison (i.e., the window size), and multiplying the result by 100 to yield the percentage of sequence identity.
  • substantially identical denotes a characteristic of a polynucleotide sequence, wherein the polynucleotide comprises a sequence having at least 80 percent sequence identity, or at least 85 percent identity, often 90 to 95 percent sequence identity, and most commonly at least 99 percent sequence identity as compared to a reference sequence of a comparison window of at least 25-50 nucleotides, wherein the percentage of sequence identity is calculated by comparing the reference sequence to the polynucleotide sequence which may include deletions or additions which total 20 percent or less of the reference sequence over the window of comparison.
  • similarity between two enzymes is determined by comparing the amino acid sequence and its conserved amino acid substitutes of one enzyme to the sequence of a second enzyme. Similarity may be determined by procedures which are well- known in the art, for example, a BLAST program (Basic Local Alignment Search Tool at the National Center for Biological Information).
  • single-chain antibody refers to a polypeptide comprising a V H domain and a V domain in polypeptide linkage, generally liked via a spacer peptide (e.g., [Gly-Gly-Gly-Gly-Ser] x ), and which may comprise additional amino acid sequences at the amino- and/or carboxy- termini.
  • a single-chain antibody may comprise a tether segment for linking to the encoding polynucleotide.
  • a scFv is a single- chain antibody.
  • Single-chain antibodies are generally proteins consisting of one or more polypeptide segments of at least 10 contiguous amino substantially encoded by genes of the immunoglobulin superfamily (e.g., see Williams and Barclay, 1989, pp. 361-368, which is incorporated herein by reference), most frequently encoded by a rodent, non-human primate, avian, porcine bovine, ovine, goat, or human heavy chain or light chain gene sequence.
  • a functional single-chain antibody generally contains a sufficient portion of an immunoglobulin superfamily gene product so as to retain the property of binding to a specific target molecule, typically a receptor or antigen (epitope).
  • the specified antibodies bind to a particular protein and do not bind in a significant amount to other proteins present in the sample.
  • the antibodies raised against a multivalent antigenic polypeptide will generally bind to the proteins from which one or more of the epitopes were obtained. Specific binding to an antibody under such conditions may require an antibody that is selected for its specificity for a particular protein.
  • immunoassay formats may be used to select antibodies specifically immunoreactive with a particular protein.
  • solid-phase ELISA immunoassays, Western blots, or immunohistochemistry are routinely used to select monoclonal antibodies specifically immunoreactive with a protein. See Harlow and Lane (1988) Antibodies, A Laboratory Manual, Cold Spring Harbor Publications, New York “Harlow and Lane”), for a description of immunoassay formats and conditions that can be used to determine specific immunoreactivity.
  • a specific or selective reaction will be at least twice background signal or noise and more typically more than 10 to 100 times background.
  • the members of a pair of molecules are said to "specifically bind" to each other if they bind to each other with greater affinity than to other, non-specific molecules.
  • an antibody raised against an antigen to which it binds more efficiently than to a non-specific protein can be described as specifically binding to the antigen.
  • a nucleic acid probe can be described as specifically binding to a nucleic acid target if it forms a specific duplex with the target by base pairing interactions (see above).)
  • a "specific binding affinity" between two molecules means a preferential binding of one molecule for another in a mixture of molecules.
  • the binding of the molecules can be considered specific if the binding affinity is about 1 X
  • Specific hybridization is defined herein as the formation of hybrids between a first polynucleotide and a second polynucleotide (e.g., a polynucleotide having a distinct but substantially identical sequence to the first polynucleotide), wherein substantially unrelated polynucleotide sequences do not form hybrids in the mixture.
  • a second polynucleotide e.g., a polynucleotide having a distinct but substantially identical sequence to the first polynucleotide
  • the term "specific polynucleotide” means a polynucleotide having certain end points and having a certain nucleic acid sequence.
  • Two polynucleotides wherein one polynucleotide has the identical sequence as a portion of the second polynucleotide but different ends comprises two different specific polynucleotides.
  • the T m is the temperature (under defined ionic strength and pH) at which 50% of the target sequence hybridizes to a perfectly matched probe.
  • Very stringent conditions are selected to be equal to the T, relieve for a particular probe.
  • An example of stringent hybridization conditions for hybridization of complementary nucleic acids which have more than 100 complementary residues on a filter in a Southern or northern blot is 50% formamide with I mg of heparin at 42'C, with the hybridization being carried out overnight.
  • Stringent hybridization conditions means hybridization will occur only if there is at least 90% identity, or at least 95% identity, or, at least 97% identity between the sequences. See, e.g., Sambrook et al, 1989.
  • An example of highly “stringent” wash conditions is 0. 15M NaCl at 72'C for about 15 minutes.
  • An example of stringent wash conditions is a 0.2x SSC wash at 65'C for 15 minutes (see, Sambrook, infra., for a description of SSC buffer). Often, a high stringency wash is preceded by a low stringency wash to remove background probe signal.
  • An example medium stringency wash for a duplex of, e.g., more than 100 nucleotides, is lx SSC at 45°C for 15 minutes.
  • An example low stringency wash for a duplex of, e.g., more than 100 nucleotides, is 4-6x SSC at 40°C for 15 minutes.
  • stringent conditions typically involve salt concentrations of less than about 1.0 M Na + ion, typically about 0.01 to 1.0 M Na+ ion concentration (or other salts) at pH 7.0 to 8.3, and the temperature is typically at least about 30°C. Stringent conditions can also be achieved with the addition of destabilizing agents such as formamide.
  • a signal to noise ratio of 2x (or higher) than that observed for an unrelated probe in the particular hybridization assay indicates detection of a specific hybridization.
  • Nucleic acids which do not hybridize to each other under stringent conditions are still substantially identical if the polypeptides which they encode T cell receptor polypeptides and major histocompatibility molecules are substantially identical. This occurs, e.g., when a copy of a nucleic acid is created using the maximum codon degeneracy permitted by the genetic code.
  • Stringent hybridization conditions and “stringent hybridization wash conditions” in the context of nucleic acid hybridization experiments such as Southern and northern hybridizations are sequence dependent, and are different under different environmental parameters. Longer sequences hybridize specifically at higher temperatures. An extensive guide to the hybridization of nucleic acids is found in Tijssen (1993) Laboratory Techniques in Biochemistry and Molecular Biology— Hybridization with Nucleic Acid Probes part I chapter 2 “Overview of principles of hybridization and the strategy of nucleic acid probe assays", Elsevier, New York. Generally, highly stringent hybridization and wash conditions are selected to be about 5°C lower than the thermal melting point (T m ) for the specific sequence at a defined ionic strength and pH. Typically, under “stringent conditions” a probe will hybridize to its target subsequence, but to no other sequences.
  • T m thermal melting point
  • a "substantially identical" amino acid sequence is a sequence that differs from a reference sequence only by conservative amino acid substitutions, for example, substitutions of one amino acid for another of the same class (e.g., substitution of one hydrophobic amino acid, such as isoleucine, valine, leucine, or methionine, for another, or substitution of one polar amino acid for another, such as substitution of arginine for lysine, glutamic acid for aspartic acid, or glutamine for asparagine).
  • substantially identical in the context of two nucleic acids or polypeptides, refers to two or more sequences or subsequences that have at least 60%, or 80%, or 90-95% nucleotide or amino acid residue identity, when compared and aligned for maximum conespondence, as measured using one of the following sequence comparison algorithms or by visual inspection.
  • the substantial identity exists over a region of the sequences that is at least about 50 residues in length or about 100 residues, or, the sequences are substantially identical over at least about 150 residues. In some embodiments, the sequences are substantially identical over the entire length of the coding regions.
  • a “subsequence” refers to a sequence of nucleic acids or amino acids that comprise a part of a longer sequence of nucleic acids or amino acids (e. g., polypeptide) respectively.
  • a “substantially identical" amino acid sequence is a sequence that differs from a reference sequence or by one or more non-conservative substitutions, deletions, or insertions, particularly when such a substitution occurs at a site that is not the active site the molecule, and provided that the polypeptide essentially retains its behavioural properties.
  • one or more amino acids can be deleted from a phytase polypeptide, resulting in modification of the structure of the polypeptide, without significantly altering its biological activity.
  • amino- or carboxyl-terminal amino acids that are not required for phytase biological activity can be removed. Such modifications can result in the development of smaller active phytase polypeptides.
  • the present invention provides a "substantially pure enzyme".
  • substantially pure enzyme is used herein to describe a molecule, such as a polypeptide (e.g., a phytase polypeptide, or a fragment thereof) that is substantially free of other proteins, lipids, carbohydrates, nucleic acids, and other biological materials with which it is naturally associated.
  • a substantially pure molecule such as a polypeptide
  • a substantially pure molecule can be at least 60%, by dry weight, the molecule of interest.
  • the purity of the polypeptides can be determined using standard methods including, e.g., polyacrylamide gel electrophoresis (e.g., SDS-PAGE), column chromatography (e.g., high performance liquid chromatography (HPLC)), and amino-terminal amino acid sequence analysis.
  • substantially pure means an object species is the predominant species present (i.e., on a molar basis it is more abundant than any other individual macromolecular species in the composition); alternatively, a substantially purified fraction is a composition wherein the object species comprises at least about 50 percent (on a molar basis) of all macromolecular species present. Generally, a substantially pure composition will comprise more than about 80 to 90 percent of all macromolecular species present in the composition.
  • the object species is purified to essential homogeneity (contaminant species cannot be detected in the composition by conventional detection methods) wherein the composition consists essentially of a single macromolecular species.
  • variable segment refers to a portion of a nascent peptide which comprises a random, pseudorandom, or defined kernal sequence.
  • a variable segment refers to a portion of a nascent peptide which comprises a random pseudorandom, or defined kernal sequence.
  • a variable segment can comprise both variant and invariant residue positions, and the degree of residue variation at a variant residue position may be limited: both options are selected at the discretion of the practitioner.
  • variable segments are about 5 to 20 amino acid residues in length (e.g., 8 to 10), although variable segments may be longer and may comprise antibody portions or receptor proteins, such as an antibody fragment, a nucleic acid binding protein, a receptor protein, and the like.
  • wild-type means that the polynucleotide does not comprise any mutations.
  • a wild type protein means that the protein will be active at a level of activity found in nature and will comprise the amino acid sequence found in nature.
  • working as in “working sample”, for example, is simply a sample with which one is working.
  • a “working molecule” for example is a molecule with which one is working.
  • the invention provides methods for generating variant antigen binding sites, antibodies and specific domains or fragments of antibodies (e.g., Fab or Fc domains) by manipulating a template nucleic acid, as described herein.
  • the invention can be practiced in conjunction with any method or protocol or device known in the art, which are well described in the scientific and patent literature.
  • DNA, vectors, viruses or hybrids thereof may be isolated from a variety of sources, genetically engineered, amplified, and/or expressed/ generated recombinantiy (recombinant polypeptides can be modified or immobilized to anays in accordance with the invention).
  • Any recombinant expression system can be used, including bacterial, mammalian, yeast, insect or plant cell expression systems.
  • these nucleic acids can be synthesized in vitro by well-known chemical synthesis techniques, as described in, e.g., Carruthers (1982) Cold Spring Harbor Symp. Quant. Biol. 47:411-418; Adams (1983) J. Am. Chem. Soc. 105:661; Belousov (1997) Nucleic Acids Res. 25:3440-3444; Frenkel (1995) Free Radic. Biol. Med. 19:373-380; Blommers (1994) Biochemistry 33:7886-7896; Narang (1979) Meth. Enzymol. 68:90; Brown (1979) Meth. Enzymol. 68:109; Beaucage (1981) Tetra. Lett. 22:1859; U.S. Patent No. 4,458,066. Double stranded DNA fragments may then be obtained either by synthesizing the complementary strand and annealing the strands together under appropriate conditions, or by adding the complementary strand using DNA polymerase with a primer sequence.
  • nucleic acids such as, e.g., subcloning, labeling probes (e.g., random-primer labeling using Klenow polymerase, nick translation, amplification), sequencing, hybridization and the like are well described in the scientific and patent literature, see, e.g., Sambrook, ed., MOLECULAR CLONING: A LABORATORY MANUAL (2ND ED.), Vols. 1-3, Cold Spring Harbor Laboratory, (1989); CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, Ausubel, ed.
  • genomic nucleic acid used in the methods and compositions of the invention include genomic or cDNA libraries contained in, or comprised entirely of, e.g., mammalian artificial chromosomes (see, e.g., Ascenzioni (1997) Cancer Lett. 118:135-142; U.S. Patent Nos.
  • a template nucleic acid is amplified by an amplification reaction, such as a polymerase-based amplification, e.g., polymerase chain reaction (PCR).
  • amplification reaction is carried out using a 64-fold degenerate oligonucleotide for each codon to be mutagenized.
  • PCR polymerase chain reaction
  • Amplification methods are also well known in the art, and include, e.g., polymerase chain reaction, PCR (see, e.g., PCR PROTOCOLS, A GUIDE TO METHODS AND
  • RNA polymerase mediated techniques e.g., NASBA,
  • the invention provides methods for generating variant antigen binding sites, antibodies and specific domains or fragments of antibodies, e.g., Fab or Fc domains (defined above) by altering a template nucleic acid by saturation mutagenesis, an optimized directed evolution system, synthetic ligation reassembly, or a combination thereof.
  • Antigen binding sites, antibodies or fragments thereof generated by these methods can be analyzed, e.g., screened for antigen binding activity (e.g., affinity, avidity) using a novel capillary anay platform of the invention. All of an antibody sequence can be altered using one or more of these methods alone or in any order, or, subsequences or domains can be altered individually, and then can be reassembled in any order or orientation.
  • an Fc domain can be altered and screened for its ability to bind an Fc-cell surface receptor independently; the Fc segment can be religated to/ reassembled with an antigen binding domain afterwards.
  • the invention provides methods for generating variant nucleic acids from template sequences, such as antibody encoding sequences (e.g., genomic DNA or message) isolated from an organism, a cell or synthetically constructed.
  • template sequences such as antibody encoding sequences (e.g., genomic DNA or message) isolated from an organism, a cell or synthetically constructed.
  • These nucleic acid sequences encoding for specific antigens, e.g., the template nucleic acids of the invention can be generated by immunization followed by screening and isolation of the sequences encoding all or fragments of antibodies that can specifically bind to that antigen.
  • Antibodies also can be generated in vitro, e.g., using recombinant antibody binding site expressing phage display libraries, in addition to the traditional in vivo methods using animals. See, e.g., Huse (1989) Science 246:1275; Ward (1989) Nature 341:544; Hoogenboom (1997) Trends Biotechnol. 15:62-70; Katz (1997) Annu. Rev. Biophys. Biomol. Struct. 26:27-45. Human antibodies can be generated in mice engineered to produce only human antibodies, as described by, e.g., U.S. Patent Nos. 5,877,397; 5,874,299; 5,789,650; and 5,939,598.
  • B-cells from these mice can be immortalized using standard techniques (e.g., by fusing with an immortalizing cell line such as a myeloma or by manipulating such B-cells by other techniques to perpetuate a cell line) to produce a monoclonal human antibody-producing cell. See, e.g., U.S. Patent No. 5,916,771; 5,985,615.
  • human lymphocytes can be inserted into an immunocompromised animal model, such as a SCED mouse.
  • the animal is challenged with antigen one or more times and lymphocytes expressing an antibody specific for the antigen is isolated cloned.
  • mice comprising human antibody genes that only express human antibodies can be used (discussed above).
  • Nucleic acid sequences (e.g., from cDNA libraries, isolated from human antibody producing mice, etc.) encoding desired antibodies can be cloned and further manipulated (e.g., to be used as templates in the methods of the invention). For example, if the antibody is of non-human origin, it can be "humanized” for eventual administration to patients. Methods for making chimeric, e.g., "humanized,” antibodies are well known in the art, see e.g., U.S. Patent Nos. 5,811,522; 5,789,554; 5,861,155. Alternatively, recombinant antibodies can also be expressed by transient or stable expression vectors in mammalian, including human, cells and cell lines, as in Norderhaug (1997) J. Immunol. Methods
  • the methods of the invention provide for "affinity enrichment" of an antibody or an antigen binding site.
  • Antibody constant regions e.g., Fc domains
  • Fc domains can also be “affinity enriched” for their ability to specifically bind to an Fc receptor or a complement polypeptide.
  • Very large sets, or libraries, of variant antibodies, including, e.g., CDRs, Fabs, Fes, and single-chain antibodies, can be generated and screened for binding to ligand (e.g., antigen, complement, receptor, and the like).
  • the variant polynucleotide is isolated and further manipulated by a method described herein, e.g., shuffled to recombine combinatorially the amino acid sequence of the selected polypeptides, peptide(s) or predetermined portions thereof.
  • a method described herein e.g., shuffled to recombine combinatorially the amino acid sequence of the selected polypeptides, peptide(s) or predetermined portions thereof.
  • antibodies, antigen binding sites, Fc domains, and the like can be generated having a desired binding affinity for a molecule.
  • the peptide or antibody can then be synthesized in bulk by conventional means for any suitable use (e.g., as a therapeutic pharmaceutical, a diagnostic agent, or as an in vitro reagent). Saturation mutagenesis
  • This invention provides methods for generating variant antigen binding sites, antibodies and specific domains or fragments of antibodies (e.g., Fab or Fc domains), T cell receptor polypeptides and major histocompatibility molecules by altering template nucleic acids by saturation mutagenesis.
  • codon primers containing a degenerate N,N,G/T sequence are used to introduce point mutations into a polynucleotide, so as to generate a set of progeny polypeptides in which a full range of single amino acid substitutions is represented at each amino acid position.
  • oligonucleotides can comprise a contiguous first homologous sequence, a degenerate N,N,G/T sequence, and, optionally, a second homologous sequence.
  • the downstream progeny translational products from the use of such oligonucleotides include all possible amino acid changes at each amino acid site along the polypeptide, because the degeneracy of the N,N,G/T sequence includes codons for all 20 amino acids.
  • one such degenerate oligonucleotide (comprised of one degenerate N,N,G/T cassette) is used for subjecting each original codon in a parental polynucleotide template to a full range of codon substitutions.
  • at least two degenerate N,N,G/T cassettes are used - either in the same oligonucleotide or not, for subjecting at least two original codons in a parental polynucleotide template to a full range of codon substitutions.
  • more than one N,N,G/T sequence can be contained in one oligonucleotide to introduce amino acid mutations at more than one site.
  • This plurality of N,N,G/T sequences can be directly contiguous, or separated by one or more additional nucleotide sequence(s).
  • oligonucleotides serviceable for introducing additions and deletions can be used either alone or in combination with the codons containing an N,N,G/T sequence, to introduce any combination or permutation of amino acid additions, deletions, and/or substitutions.
  • simultaneous mutagenesis of two or more contiguous amino acid positions is done using an oligonucleotide that contains contiguous N,N,G/T triplets, i.e. a degenerate (N,N,G/T)n sequence.
  • degenerate cassettes having less degeneracy than the N,N,G/T sequence are used.
  • use of degenerate N,N,G/T triplets allows for systematic and easy generation of a full range of possible natural amino acids (for a total of 20 amino acids) into each and every amino acid position in a polypeptide (in alternative aspects, the methods also include generation of less than all possible substitutions per amino acid residue, or codon, position). For example, for a 100 amino acid polypeptide, 2000 distinct species (i.e. 20 possible amino acids per position X 100 amino acid positions) can be generated.
  • an oligonucleotide or set of oligonucleotides containing a degenerate N,N,G/T triplet 32 individual sequences can code for all 20 possible natural amino acids.
  • Nondegenerate oligos can optionally be used in combination with degenerate primers disclosed; for example, nondegenerate oligonucleotides can be used to generate specific point mutations in a working polynucleotide. This provides one means to generate specific silent point mutations, point mutations leading to conesponding amino acid changes, and point mutations that cause the generation of stop codons and the conesponding expression of polypeptide fragments.
  • each saturation mutagenesis reaction vessel contains polynucleotides encoding at least 20 progeny polypeptide molecules such that all 20 natural amino acids are represented at the one specific amino acid position conesponding to the codon position mutagenized in the parental polynucleotide (other aspects use less than all 20 natural combinations).
  • the 32-fold degenerate progeny polypeptides generated from each saturation mutagenesis reaction vessel can be subjected to clonal amplification (e.g. cloned into a suitable host, e.g., E. coli host, using, e.g., an expression vector) and subjected to expression screening.
  • an individual progeny polypeptide When an individual progeny polypeptide is identified by screening to display a favorable change in property (when compared to the parental polypeptide, such as increased affinity or avidity to an antigen), it can be sequenced to identify the conespondingly favorable amino acid substitution contained therein.
  • a favorable change in property when compared to the parental polypeptide, such as increased affinity or avidity to an antigen
  • it can be sequenced to identify the conespondingly favorable amino acid substitution contained therein.
  • favorable amino acid changes may be identified at more than one amino acid position.
  • One or more new progeny molecules can be generated that contain a combination of all or part of these favorable amino acid substitutions.
  • the permutations include 3 possibilities at each position (no change from the original amino acid, and each of two favorable changes) and 3 positions.
  • site-saturation mutagenesis can be used together with shuffling, chimerization, recombination and other mutagenizing processes, along with screening. This invention provides for the use of any mutagenizing process(es), including saturation mutagenesis, in an iterative manner.
  • the iterative use of any mutagenizing process(es) is used in combination with screening.
  • this invention provides for the use of saturation mutagenesis in combination with additional mutagenization processes, such as process where two or more related polynucleotides are introduced into a suitable host cell such that a hybrid polynucleotide is generated by recombination and reductive reassortment.
  • This invention provides methods for generating variant antigen binding sites, antibodies and specific domains or fragments of antibodies (e.g., Fab or Fc domains), T cell receptor polypeptides and major histocompatibility molecules by manipulating a nucleic acid by an optimized directed evolution system.
  • the invention further comprises mutagenizing a template nucleic acid, e.g., a nucleic acid encoding an antigen binding site, an antibody or fragment thereof, altered by saturation mutagenesis, by a method comprising an optimized directed evolution system.
  • Optimized directed evolution is directed to the use of repeated cycles of reductive reassortment, recombination and selection that allow for the directed molecular evolution of nucleic acids through recombination.
  • Optimized directed evolution allows generation of a large population of evolved chimeric sequences, wherein the generated population is significantly enriched for sequences that have a predetermined number of crossover events .
  • a crossover event is a point in a chimeric sequence where a shift in sequence occurs from one parental variant to another parental variant. Such a point is normally at the juncture of where oligonucleotides from two parents are ligated together to form a single sequence.
  • This method allows calculation of the conect concentrations of oligonucleotide sequences so that the final chimeric population of sequences is enriched for the chosen number of crossover events. This provides more control over choosing chimeric variants having a predetermined number of crossover events.
  • this method provides a convenient means for exploring a tremendous amount of the possible protein variant space in comparison to other systems.
  • Previously if one generated, for example, 10 13 chimeric molecules during a reaction, it would be extremely difficult to test such a high number of chimeric variants for a particular activity.
  • a significant portion of the progeny population would have a very high number of crossover events which resulted in proteins that were less likely to have increased levels of a particular activity.
  • the population of chimerics molecules can be enriched for those variants that have a particular number of crossover events.
  • each of the molecules chosen for further analysis most likely has, for example, only three crossover events.
  • the boundaries on the functional variety between the chimeric molecules is reduced. This provides a more manageable number of variables when calculating which oligonucleotide from the original parental polynucleotides might be responsible for affecting a particular trait, such as antigen binding.
  • One method for creating a chimeric progeny polynucleotide sequence is to create oligonucleotides conesponding to fragments or portions of each parental sequence.
  • Each oligonucleotide can include a unique region of overlap so that mixing the oligonucleotides together results in a new variant that has each oligonucleotide fragment assembled in the conect order. Additional information can also be found in U.S. Patent Application Number 09/332,835 entitled "Synthetic Ligation Reassembly in Directed Evolution" and filed on June 14, 1999, the disclosure of which has been incorporated by reference in its entirety.
  • the number of oligonucleotides generated for each parental variant bears a relationship to the total number of resulting crossovers in the chimeric molecule that is ultimately created.
  • three parental nucleotide sequence variants might be provided to undergo a ligation reaction in order to find a chimeric variant having, for example, greater activity at high temperature.
  • a set of 50 oligonucleotide sequences can be generated conesponding to each portions of each parental variant. Accordingly, during the ligation reassembly process there could be up to 50 crossover events within each of the chimeric sequences.
  • each of the generated chimeric polynucleotides will contain oligonucleotides from each parental variant in alternating order is very low. If each oligonucleotide fragment is present in the ligation reaction in the same molar quantity it is likely that in some positions oligonucleotides from the same parental polynucleotide will ligate next to one another and thus not result in a crossover event. If the concentration of each oligonucleotide from each parent is kept constant during any ligation step in this example, there is a 1/3 chance (assuming 3 parents) that an oligonucleotide from the same parental variant will ligate within the chimeric sequence and produce no crossover.
  • a probability density function can be determined to predict the population of crossover events that are likely to occur during each step in a ligation reaction given a set number of parental variants, a number of oligonucleotides conesponding to each variant, and the concentrations of each variant during each step in the ligation reaction.
  • PDF probability density function
  • a target number of crossover events can be predetermined, and the system then programmed to calculate the starting quantities of each parental oligonucleotide during each step in the ligation reaction to result in a probability density function that centers on the predetermined number of crossover events.
  • a crossover event is a point in a chimeric sequence where a shift in sequence occurs from one parental variant to another parental variant. Such a point is normally at the juncture of where oligonucleotides from two parents are ligated together to form a single sequence.
  • the method allows calculation of the conect concentrations of oligonucleotide sequences so that the final chimeric population of sequences is enriched for the chosen number of crossover events. This provides more control over choosing chimeric variants having a predetermined number of crossover events. In addition, these methods provide a convenient means for exploring a tremendous amount of the possible protein variant space in comparison to other systems.
  • the population of chimerics molecules can be enriched for those variants that have a particular number of crossover events. Thus, although one can still generate 10 13 chimeric molecules during a reaction, each of the molecules chosen for further analysis most likely has, for example, only three crossover events.
  • the method creates a chimeric progeny polynucleotide sequence by creating oligonucleotides conesponding to fragments or portions of each parental sequence
  • Each oligonucleotide can include a unique region of overlap so that mixing the oligonucleotides together results in a new variant that has each oligonucleotide fragment assembled in the conect order. Additional information can also be found in U.S. Patent Application No. 09/332,835 entitled “Synthetic Ligation Reassembly in Directed Evolution” and filed on June 14, 1999. The number of oligonucleotides generated for each parental variant bears a relationship to the total number of resulting crossovers in the chimeric molecule that is ultimately created.
  • three parental nucleotide sequence variants might be provided to undergo a ligation reaction in order to find a chimeric variant having, for example, greater activity at high temperature.
  • a set of 50 oligonucleotide sequences can be generated conesponding to each portions of each parental variant. Accordingly, during the ligation reassembly process there could be up to 50 crossover events within each of the chimeric sequences. The probability that each of the generated chimeric polynucleotides will contain oligonucleotides from each parental variant in alternating order is very low.
  • each oligonucleotide fragment is present in the ligation reaction in the same molar quantity it is likely that in some positions oligonucleotides from the same parental polynucleotide will ligate next to one another and thus not result in a crossover event. If the concentration of each oligonucleotide from each parent is kept constant during any ligation step in this example, there is a 1/3 chance (assuming 3 parents) that a oligonucleotide from the same parental variant will ligate within the chimeric sequence and produce no crossover.
  • a probability density function can be determined to predict the population of crossover events that are likely to occur during each step in a ligation reaction given a set number of parental variants, a number of oligonucleotides conesponding to each variant, and the concentrations of each variant during each step in the ligation reaction.
  • PDF probability density function
  • a target number of crossover events can be predetermined, and the system then programmed to calculate the starting quantities of each parental oligonucleotide during each step in the ligation reaction to result in a probability density function that centers on the predetermined number of crossover events.
  • Embodiments of the invention include a system and software that receive a desired crossover probability density function (PDF), the number of parent genes to be reassembled, and the number of fragments in the reassembly as inputs.
  • PDF crossover probability density function
  • the output of this program is a "fragment PDF" that can be used to determine a recipe for producing reassembled genes, and the estimated crossover PDF of those genes.
  • the processing described herein can be performed in MATLAB ® (The Mathworks, Natick, Massachusetts) a programming language and development environment for technical computing.
  • the computer system that carries out the methods described herein.
  • the computer system is a conventional personal computer such as those based on an Intel microprocessor and running a Windows operating system.
  • the output of the computer system is a fragment PDF that can be used as a recipe for producing reassembled progeny genes, and the estimated crossover PDF of those genes.
  • the processing described herein can be performed by a personal computer using the MATLAB ® programming language and development environment.
  • the invention is not limited to any particular hardware or software configuration. For example, computers based on other well- known microprocessors and running operating system software such as UNTX, Linux, MacOS and others are contemplated.
  • the methods generate sets of chimeric nucleic acid and protein molecules and then screen those molecules for a particular activity, such as the ability to bind to a desired antigen.
  • the invention is not limited to only a single round of screening. For example, a second round of screening can take place if nucleotide sequencing indicates that all of the chimeric progeny antibody polynucleotides having an increased affinity or specificity have a particular parental oligonucleotide in common. Based on this determination, a second round of reassembly can take place that enriches for progeny having that oligonucleotide.
  • oligonucleotide sequences from the other parental polynucleotides into the ligation reassembly reactions.
  • the only oligonucleotide that can be ligated into each gene will be the desired oligonucleotide.
  • a particular oligonucleotide has no affect at all on the desired trait (e.g., affinity for antigen)
  • it can be removed as a variable by synthesizing larger parental oligonucleotides that include the sequence to be removed.
  • Automated Control of Reactions The process of generating any of the reactions of the methods of the invention can be automated with the assistance of robotic instruments.
  • a TECAN GENESISTM programmable robot made by Tecan Corporation (Hombrechtikon, Switzerland) can be interfaced with a computer that determines the quantities of each oligonucleotide fragment to yield a resulting PDF.
  • a computer system that determines the proper quantities of each oligonucleotide By linking a computer system that determines the proper quantities of each oligonucleotide to an automated robot, a complete ligation reassembly system is produced.
  • one aspect of the invention is an automated system for generating nucleic acid sequences that encode variant antigen binding sites, such as variant antibodies having increased affinity to desired antigen.
  • the automated system includes a plurality of oligonucleotide fragments derived from a series of nucleic acid sequence variants, wherein said fragments are configured to join one another at unique overhangs.
  • the system also has a data input field configured to store a target number of crossover events in for each of the variant sequences.
  • a prediction module configured to determine the quantity of each of the fragments to admix together so that mixing the fragments results in a population of progeny molecules that are enriched for crossover events conesponding to the target number.
  • the system also provides a robotic arm linked to the prediction module through a communication interface for automatically mixing the fragments in the determined quantities.
  • the optimized directed evolution method can use oligonucleotides that have a 100% fidelity to their parent polynucleotide sequence, this level of fidelity is not required.
  • a set of three related parental polynucleotides are chosen to undergo ligation reassembly in order to create, e.g., an antibody having increase affinity to a desired antigen
  • a set of oligonucleotides having unique overlapping regions can be synthesized by conventional methods.
  • a set of mutagenized oligonucleotides could also be synthesized. These mutagenized oligonucleotides can be designed to encode silent, conservative, or non-conservative amino acids.
  • the choice to enter a silent mutation might be made to, for example, add a region of nucleotide homology two fragments, but not affect the final translated protein.
  • a non- conservative or conservative substitution is made to determine how such a change alters the function of the resultant polypeptide. This can be done if, for example, it is determined that mutations in one particular oligonucleotide fragment were responsible for increasing the activity of a peptide.
  • mutagenized oligonucleotides e.g.: those having a different nucleotide sequence than their parent
  • Another method for creating variants of a nucleic acid sequence using mutagenized fragments includes first aligning a plurality of nucleic acid sequences to determine demarcation sites within the variants that are conserved in a majority of said variants, but not conserved in all of said variants. A set of first sequence fragments of the conserved nucleic acid sequences are then generated, wherein the fragments bind to one another at the demarcation sites. A second set of fragments of the not conserved nucleic acid sequences are then generated by, for example, a nucleic acid synthesizer. However, the not conserved, sequences are generated to have mutations at their demarcation site so that the second fragments have the same nucleotide sequence at the demarcation sites as said first fragments.
  • This invention provides methods for generating variant antigen binding sites, antibodies and specific domains or fragments of antibodies (e.g., Fab or Fc domains) by altering template nucleic acids by synthetic ligation reassembly.
  • SLR is a method of ligating oligonucleotide fragments together non-stochastically. This method differs from stochastic oligonucleotide shuffling in that the nucleic acid building blocks are not shuffled, concatenated or chimerized randomly, but rather are assembled non-stochastically.
  • the SLRs used in the methods of the invention do not depend on the presence of high levels of homology between polynucleotides to be reananged.
  • this method can be used to non- stochastically generate libraries (or sets) of progeny molecules comprised of over 10 100 different chimeras.
  • SLR can be used to generate libraries comprised of over lo 1000 different progeny chimeras.
  • aspects of the present invention include non-stochastic methods of producing a set of finalized chimeric nucleic acid molecules (e.g., nucleic acids encoding antibodies or fragments thereof) having an overall assembly order that is chosen by design.
  • This method includes the steps of generating by design a plurality of specific nucleic acid building blocks having serviceable mutually compatible ligatable ends, and assembling these nucleic acid building blocks, such that a designed overall assembly order is achieved.
  • the mutually compatible ligatable ends of the nucleic acid building blocks to be assembled are considered to be "serviceable" for this type of ordered assembly if they enable the building blocks to be coupled in predetermined orders.
  • the overall assembly order in which the nucleic acid building blocks can be coupled is specified by the design of the ligatable ends. If more than one assembly step is to be used, then the overall assembly order in which the nucleic acid building blocks can be coupled is also specified by the sequential order of the assembly step(s).
  • the annealed building pieces are treated with an enzyme, such as a ligase (e.g. T4 DNA ligase), to achieve covalent bonding of the building pieces.
  • a ligase e.g. T4 DNA ligase
  • the design of the oligonucleotide building blocks is obtained by analyzing a set of progenitor nucleic acid sequence templates (parents, such as antibody coding sequences) that serve as a basis for producing a progeny set of finalized chimeric polynucleotide molecules (e.g., variant antibodies).
  • progenitor nucleic acid sequence templates parents, such as antibody coding sequences
  • progeny set of finalized chimeric polynucleotide molecules e.g., variant antibodies.
  • a chimerization of a set, or family, of related genes and their encoded set, or family, of polypeptides is provided.
  • the encoded products can be antibodies or fragments or subsequences thereof, such as Fc or Fab domains, antigen binding sites, CDRs, and the like.
  • the sequences of a plurality of parental nucleic acid templates are aligned in order to select one or more demarcation points.
  • the demarcation points can be located at an area of homology, and are comprised of one or more nucleotides. These demarcation points can be shared by at least two of the progenitor templates. The demarcation points can thereby be used to delineate the boundaries of oligonucleotide building blocks to be generated in order to rearrange the parental polynucleotides.
  • the demarcation points identified and selected in the progenitor molecules serve as potential chimerization points in the assembly of the final chimeric progeny molecules.
  • a demarcation point can be an area of homology (comprised of at least one homologous nucleotide base) shared by at least two parental polynucleotide sequences.
  • a demarcation point can be an area of homology that is shared by at least half of the parental polynucleotide sequences, or, it can be an area of homology that is shared by at least two thirds of the parental polynucleotide sequences.
  • a serviceable demarcation points is an area of homology that is shared by at least three fourths of the parental polynucleotide sequences, or, it can be shared by at almost all of the parental polynucleotide sequences.
  • a demarcation point is an area of homology that is shared by all of the parental polynucleotide sequences.
  • a ligation reassembly process is performed exhaustively in order to generate an exhaustive library of progeny chimeric polynucleotides.
  • all possible ordered combinations of the nucleic acid building blocks are represented in the set of finalized chimeric nucleic acid molecules.
  • the assembly order i.e. the order of assembly of each building block in the 5' to 3 sequence of each finalized chimeric nucleic acid
  • the assembly order is by design (or non-stochastic) as described above.
  • the ligation reassembly method is performed systematically.
  • the method is performed in order to generate a systematically compartmentalized library of progeny molecules, with compartments that can be screened systematically, e.g. one by one.
  • this invention provides that, through the selective and judicious use of specific nucleic acid building blocks, coupled with the selective and judicious use of sequentially stepped assembly reactions, a design can be achieved where specific sets of progeny products are made in each of several reaction vessels. This allows a systematic examination and screening procedure to be performed. Thus, these methods allow a potentially very large number of progeny molecules to be examined systematically in smaller groups.
  • the progeny molecules generated can comprise a library of finalized chimeric nucleic acid molecules having an overall assembly order that is chosen by design.
  • sets, or a library, of generated progeny molecules comprises greater than 10 3 different progeny molecular species, greater than 10 5 different progeny molecular species, greater than 10 10 different progeny molecular species, greater than 10 15 different progeny molecular species, greater than 10 20 different progeny molecular species, greater than 10 30 different progeny molecular species, greater than 10 40 different progeny molecular species, greater than 10 50 different progeny molecular species, greater than 10 60 different progeny molecular species, greater than 10 70 different progeny molecular species, greater than 10 80 different progeny molecular species, or greater than 10 100 different progeny molecular species, greater than 10 110 different progeny molecular species, greater than 10 120 different progeny molecular species, greater than 10 130 different progeny molecular species, greater than 10 140 different progeny molecular species, greater than 10 different progeny molecular species, greater than 10 1
  • the saturation mutagenesis and optimized directed evolution methods also can be used to generate these amounts of different progeny molecular species.
  • a set of finalized chimeric nucleic acid molecules, produced as described herein comprises a polynucleotide encoding a polypeptide.
  • this polynucleotide is a gene, which may be a man-made gene.
  • this polynucleotide is an antibody or a fragment thereof.
  • the invention provides freedom of choice and control regarding the selection of demarcation points, the size and number of the nucleic acid building blocks, and the size and design of the couplings. It is appreciated, furthermore, that the requirement for intermolecular homology is highly relaxed for the operability of this invention. In fact, demarcation points can even be chosen in areas of little or no intermolecular homology. For example, because of codon wobble, i.e. the degeneracy of codons, nucleotide substitutions can be introduced into nucleic acid building blocks without altering the amino acid originally encoded in the conesponding progenitor template. Alternatively, a codon can be altered such that the coding for an originally amino acid is altered.
  • nucleic acid building block in order to increase the incidence of intermolecularly homologous demarcation points and thus to allow an increased number of couplings to be achieved among the building blocks, which in turn allows a greater number of progeny chimeric molecules to be generated.
  • the synthetic nature of the step in which the building blocks are generated allows the design and introduction of nucleotides (e.g., one or more nucleotides, which may be, for example, codons or introns or regulatory sequences) that can later be optionally removed in an in vitro process (e.g. by mutageneis) or in an in vivo process (e.g. by utilizing the gene splicing ability of a host organism). It is appreciated that in many instances the introduction of these nucleotides may also be desirable for many other reasons in addition to the potential benefit of creating a serviceable demarcation point.
  • a nucleic acid building block can be used to introduce an intron.
  • functional introns may be introduced into a man-made gene manufactured according to the methods described herein.
  • functional introns may be introduced into a man-made antibody gene of this invention. Accordingly, these methods provide for the generation of a chimeric polynucleotide that is a man-made gene containing one (or more) artificially introduced intron(s).
  • the artificially introduced intron(s) are functional in one or more host cells for gene splicing much in the way that naturally- occurring introns serve functionally in gene splicing.
  • a process of producing man-made intron-containing polynucleotides to be introduced into host organisms for recombination and/or splicing is also contemplated. Screening methodologies
  • the set of progeny nucleic acids e.g., antibody-, Fc-, antigen binding site- encoding polynucleotides, T cell receptor polypeptides and major histocompatibility molecules are expressed.
  • These polypeptides can be expressed to screen for their ability to bind a ligand, e.g., an antigen (if, for example, affinity maturation of an antibody is desired), or, a receptor or a complement molecule (e.g., for Fc domains). Any method of expression or screening known in the art can be used.
  • the displayed peptide or polypeptide sequences can be of varying lengths, e.g., from 3-5000 amino acids long or longer, from 5-100 amino acids long, or from about 8-15 amino acids long.
  • a set, or library can comprise library members having varying lengths of displayed peptide sequence, or may comprise library members having a fixed length of displayed peptide sequence.
  • Exemplary display methods include methods for in vitro and in vivo display of single-chain antibodies, such as nascent scFv on polysomes or scfv displayed on phage, which enable large-scale screening of scfv libraries having broad diversity of variable region sequences and binding specificities.
  • the present invention also provides random, pseudorandom, and defined sequence framework nucleic acid and polypeptide libraries and methods for generating and screening those libraries to identify useful compounds (e.g., antibodies, including single-chain antibodies, Fc, and the like) that bind to receptor molecules or antigens or epitopes of interest.
  • useful compounds e.g., antibodies, including single-chain antibodies, Fc, and the like
  • the random, pseudorandom, and defined sequence framework peptides can be produced from libraries of peptide library members that comprise displayed peptides or displayed single-chain antibodies attached to a polynucleotide template from which the displayed peptide was synthesized.
  • the mode of attachment may vary according to the specific embodiment of the invention selected, and can include encapsulation in a phage particle or incorporation in a cell.
  • the variant nucleic acids are expressed and the generated polypeptides, e.g., antibodies, including antigen binding sites, CDRs, or Fab or Fc, are screened for their ability to specifically bind a molecule, e.g., an antigen, a complement molecule, an Fc receptor, by a method comprising a capillary anay, such as GIGAMATREXTM, Diversa Corporation, San Diego, CA.
  • a capillary anay such as GIGAMATREXTM, Diversa Corporation, San Diego, CA.
  • a sample screening apparatus includes a plurality of capillaries formed into an anay of adjacent capillaries, wherein each capillary comprises at least one wall defining a lumen for retaining a sample.
  • the apparatus further includes interstitial material disposed between adjacent capillaries in the array, and one or more reference indicia formed within of the interstitial material.
  • a capillary for screening a sample wherein the capillary is adapted for being bound in an array of capillaries, includes a first wall defining a lumen for retaining the sample, and a second wall formed of a filtering material, for filtering excitation energy provided to the lumen to excite the sample.
  • the invention provides a method for incubating a biomolecule of interest (e.g., the antibody or fragment thereof, or, a ligand, such as an epitope or antigen, to be screened) includes the steps of introducing a first component into at least a portion of a capillary of a capillary anay, wherein each capillary of the capillary anay comprises at least one wall defining a lumen for retaining the first component, and introducing an air bubble into the capillary behind the first component.
  • the method further includes the step of introducing a second component into the capillary, wherein the second component is separated from the first component by the air bubble.
  • a sample of interest is introduced as a first liquid labeled with a detectable particle into a capillary of a capillary anay, wherein each capillary of the capillary array comprises at least one wall defining a lumen for retaining the first liquid and the detectable particle, and wherein the at least one wall is coated with a binding material for binding the detectable particle to the at least one wall.
  • the method can further include removing the first liquid from the capillary tube, wherein the bound detectable particle is maintained within the capillary, and introducing a second liquid into the capillary tube.
  • variant polypeptide e.g., the antibody or fragment thereof
  • the capillary array or other device if another screening method is used
  • the ligand such as an epitope or antigen
  • the device e.g., the capillary array
  • the ligand such as an antigen
  • the capillary array includes a plurality of individual capillaries comprising at least one outer wall defining a lumen.
  • the outer wall of the capillary can be one or more walls fused together.
  • the wall can define a lumen that is cylindrical, square, hexagonal or any other geometric shape so long as the walls form a lumen for retention of a liquid or sample.
  • the capillaries of the capillary anay can be held together in close proximity to form a planar structure.
  • the capillaries can be bound together, by being fused (e.g., where the capillaries are made of glass), glued, bonded, or clamped side-by-side.
  • the capillary array can be formed of any number of individual capillaries, for example, a range from 100 to 4,000,000 capillaries.
  • a capillary anay can form a microtiter plate having about 100,000 or more individual capillaries bound together.
  • the capillaries can be formed with an aspect ratio of 50:1. Ln one aspect, each capillary has a length of approximately 10mm, and an internal diameter of the lumen of approximately 200 ⁇ m. However, other aspect ratios are possible, and range from 10:1 to well over 1000:1. Accordingly, individual capillaries have an inner diameter that ranges from 10-500 ⁇ m. A capillary having an internal diameter of 200 ⁇ m and a length of 1 cm has a volume of about 0.3 ⁇ l. The length and width of each capillary can be based on a desired volume and other characteristics, such as evaporation rate, etc.
  • the capillary anay can have a density of 500 to more than 1,000 capillaries per cm 2 , or about 5 capillaries per mm 2 .
  • the capillary anay can be formed to a width or diameter of about 0.5-20 mm and a height or thickness of 0.05 to about 10 cm. The capillary anay can have a thickness of about 0.1 to about 5 cm.
  • one or more particles are introduced into each capillary for screening.
  • Suitable particles include cells, cell clones, and other biological matter, chemical beads, or any other particulate matter.
  • the capillaries containing particles of interest can be exposed with various types of substances for screening for an activity of interest, e.g., antibody binding to antigen, Fc binding to complement, and the like.
  • a chemical solution containing new particles can be introduced to cause a combining event with other chemical beads already introduced into one or more capillaries.
  • the particles and resulting activity of interest are screened and analyzed using the capillary anay. Ln one aspect, the activity produces optical energy within the capillary, which can act as a waveguide for guiding the light energy to an analyzer.
  • the capillaries can be made according to various manufacturing techniques.
  • the capillaries are manufactured using a hollow-drawn technique.
  • a cylindrical, or other hollow shape, portion of glass is drawn out to continually longer lengths according to known techniques.
  • the glass is drawn to a desired diameter and then cut into portions of a specific length to form a capillary according to the invention.
  • a number of individual capillaries are bound together in an anay.
  • a glass etching process is used.
  • a solid tube of glass can be drawn out to a particular width, and cut into portions of a specific length. Then, each solid tube portion is center-etched with acid to form a capillary.
  • the tubes can be bound before or after the etch process.
  • a large number of materials can be suitably used to form a capillary anay according to the invention and depending on the manufacturing technique used, including without limitation, glass, metal, semiconductors such as silicon, quartz, ceramics, or various polymers and plastics including, among others, polyethylene, polystyrene, and polypropylene.
  • the internal walls of the capillary anay, or portions thereof, may be coated or silanized to modify their surface properties. For example, the hydrophilicity or hydrophobicity may be altered to promote or reduce wicking or capillary action, respectively.
  • the coating material includes, for example, ligands such as avidin, streptavidin, antibodies, antigens, and other molecules having specific binding affinity or which can withstand thermal or chemical sterilization.
  • a capillary anay may optionally include reference indicia for providing a positional or alignment reference.
  • the reference indicia may be formed of a pad of glass extending from the surface of the capillary array, or embedded in the interstitial material. Ln one aspect, the reference indicia are provided at one or more corners of a microtiter plate formed by the capillary anay. A corner of the plate or set of capillaries may be removed, and replaced with the reference indicia.
  • the reference indicia may also be formed at spaced intervals along a capillary array, to provide an indication of a subset of capillaries.
  • the capillary can include a first wall defining a lumen and a second wall sunounding the first wall.
  • the second wall has a lower index of refraction than the first wall.
  • the first wall is a sleeve glass having a high index of refraction, forming a waveguide in which light from excited fluorophores travels.
  • the second wall can be black
  • EMA glass having a low index of refraction, forming a cladding around the first wall against which light is refracted and directed along the first wall for total internal reflection within the capillary.
  • the second wall can thus be made with any material that reduces the "cross-talk" or diffusion of light between adjacent capillaries.
  • the inside surface of the first wall can be coated with a reflective substance to form a mirror, or minor-like structure, for specular reflection within the lumen.
  • Many different materials can be used in forming the first and second walls, creating different indices of refraction for desired purposes.
  • a filtering material can be formed around the lumen to filter energy to and from the lumen.
  • the inner wall of the first wall of each capillary of the array, or portion of the anay is coated with the filtering material.
  • the second wall includes the filtering material.
  • the second wall can be formed of the filtering material, such as filter glass for example, or in one aspect, the second wall is EMA glass that is doped with an appropriate amount of filtering material.
  • the filtering material can be formed of a color other than black and tuned for a desired excitation/ emission filtering characteristic.
  • the filtering material can allow transmission of excitation energy into the lumen, and blocks emission energy from the lumen except through one or more openings at either end of the capillary. Excitation energy is illustrated as a solid line, while emission energy is indicated by a broken line.
  • the second wall is formed with a filtering material
  • certain wavelengths of light representing excitation energy are allowed through to the lumen, and other wavelengths of light representing emission energy are blocked from exiting, except as directed within and along the first wall.
  • the entire capillary anay, or a portion thereof can be tuned to a specific individual wavelength or group of wavelengths, for filtering different bands of light in an excitation and detection process.
  • an excitation light is directed into the lumen contacting a particle (discussed above) and exciting a reporter fluorescent material causing emission of light.
  • the emitted light travels the length of the capillary until it reaches a detector.
  • the second wall is black EMA glass, emitted light cannot cross contaminate adjacent capillary tubes in a capillary anay. Ln addition, the black EMA glass refracts and directs the emitted light towards either end of the capillary tube thus increasing the signal detected by an optical detector (e.g., a CCD camera and the like).
  • an optical detection system is aligned with the array, which is then scanned for one or more bright spots, representing either a fluorescence or luminescence associated with a "positive."
  • the term "positive” refers to the presence of an activity of interest. Again, the activity can be a chemical event, or a biological event.
  • a capillary anay is immersed or contacted with a container containing particles or molecules of interest.
  • the particles can be cells, clones, molecules or compounds (e.g., antibodies or fragments thereof, antigens, and the like) suspended in a liquid. The liquid is wicked into the capillary tubes by capillary action.
  • a substrate for measuring biological activity can be contacted with the particles either before or after introduction of the particles into the capillaries in the capillary anay.
  • the substrate can include clones of a cell of interest, for example.
  • the substrate can be introduced simultaneously into the capillaries by placing an open end of the capillaries in the container containing a mixture of the particle-bearing liquid and the substrate.
  • the particle-bearing liquid may be wicked a portion of the way into the capillaries, and then the substrate is wicked into a remaining portion of the capillaries.
  • the mixture in the capillaries can then be incubated for producing a desired activity, e.g., a binding event, such as antibody binding to antigen, Fc to complement, and the like.
  • a binding event such as antibody binding to antigen, Fc to complement, and the like.
  • the incubation can be for a specific period of time and at an appropriate temperature or to allow the substrate to permeabilize the cell membrane to produce an optically detectable signal, or for a period of time and at a temperature for optimum binding activity.
  • the incubation can be performed, for example, by placing the capillary array in a humidified incubator or at ambient temperature in an apparatus containing a water source to ensure reduced evaporation within the capillary tubes.
  • the evaporative flow rate may be reduced by increasing the humidity (e.g., by placing the capillary anay in a humidified chamber).
  • the evaporation rate can also be reduced by capping the capillaries with an oil, wax, membrane or the like.
  • a high molecular weight fluid such as various alcohols, or molecules capable of forming a molecular monolayer, bilayers or other thin films (e.g., fatty acids), or various oils (e.g., mineral oil) can be used to reduce evaporation.
  • a first fluid is wicked into the capillary according to methods described above.
  • the capillary containing the substrate solution is then introduced to a fluid bath containing a second liquid.
  • the second liquid may or may not be the same as the first.
  • the first liquid may contain particles from which an activity is screened. The particles are suspended in liquid within the lumen, and gradually migrate toward the top of the lumen in the direction of the flow of liquid through the capillary.
  • the width of the lumen at the open end of the capillary can be sized to provide a particular surface area of liquid at the top of the lumen, for controlling the amount and rate of evaporation of the liquid mixture.
  • the first liquid from within the capillary will evaporate, and will be replenished by the second liquid from the fluid bath.
  • the amount of evaporation is balanced against possible diffusion of the contents of the capillary into the liquid, and against possible mechanical mixing of the capillary contents with the liquid due to vibration and pressure changes.
  • the greater the length of the capillary the less the capillary contents will mix with the liquid and be subject to diffusion.
  • the greater the width of the lumen the larger the amount of mechanical mixing. Therefore, the temperature and humidity level in the sunounding environment may be adjusted to produce the desired evaporative cycle, and the lumen width is sized to minimize mechanical mixing, in addition to produce a desired evaporation rate.
  • the non-submersed open end of the capillary may also be capped to create a vacuum force for holding the capillary contents within the capillary, and minimizing mechanical mixing and diffusion of the contents within the liquid.
  • the capillary will not experience evaporation.
  • a relatively high humidity level of the environment will slow the rate of evaporation and keep more liquid within the capillary.
  • a heat differential between the environment and capillary anay exceeds a certain level, however, evaporating or other liquid can condense on a top surface of tightly-packed capillaries of the capillary array.
  • the outer edge surface of the capillary walls can be a planar surface.
  • the wall of the capillary can be glass, the outer edge surface of the capillary wall can be polished glass.
  • a hydrophobic coating can be provided over the outer edge surface of the capillary walls.
  • the coating can reduce the tendency for water or other liquid to accumulate near the outer edge surface of the capillary wall.
  • the hydrophobic coating is TEFLONTM. Ln one configuration, the coating covers only the outer edge surfaces of the capillary walls. In another configuration, the coating can be formed over both the interstitial material and the outer edge surfaces of the capillary walls. Another advantage of a hydrophobic coating over the outer edge surface of the capillary tubes is during the initial wicking process, some fluidic material in the form of droplets will tend to stick to the surface in which the fluid is introduced. Therefore, the coating minimizes extraneous fluid from forming on the surface of a capillary array, dispensing with a need to shake or knock the extraneous fluid from the surface.
  • a process of dilution may be used to achieve a particular concentration, or series of dilutions, of particles.
  • a bolus of a first component is wicked into a capillary by capillary action until only a portion of the capillary is filled.
  • pressure is applied at one end of the capillary to prevent the first component from wicking into the entire capillary.
  • the end of the capillary may be completely or partially capped to provide the pressure.
  • An amount of air is then introduced into the capillary adjacent the first component. The air can be introduced by any number of processes.
  • One such process includes moving the first component in one direction within the capillary until a suitable amount of the air (84) is introduced behind the first component. Further movement of the first component by a pulling and/or pushing pressure causes a piston-like action by the first component on the air. The capillary or capillary anay is then contacted to a second component. The second component can be pulled into the capillary by the piston-like action created by movement of the first component until a suitable amount of the second component is provided in the capillary, separated from the first component by the air.
  • One of the first or second components may contain one or more particles of interest, and the other of the components may be a developer of the particles for causing an activity of interest.
  • the capillary or capillary anay can then be incubated for a period of time to allow the first and second components to reach an optimal temperature, or for a sufficient time to allow cell growth for example.
  • the air-bubble separating the two components can be disrupted in order to allow mix the two components together and initialize the desired activity. Ln one aspect, pressure is applied to either one of the components or to the entire capillary to collapse the bubble.
  • One of the components may contain paramagnetic beads or particles.
  • the paramagnetic beads can be used to disrupt the air bubble and/or mix the contents of the capillary tube or capillary anay. For example, paramagnetic beads can be magnetically attracted from one location in each to another location.
  • the paramagnetic beads are attracted by magnetic fields formed in proximity to the capillary or capillary anay. By alternating or adjusting the location of the magnetic field with respect to each capillary, the paramagnetic beads will move within each capillary to mix the liquid within the capillary in which the beads are suspended. Mixing the liquid can improve cell growth by increasing aeration of the cells. This aspect also improves consistency and detectability of the liquid sample among the capillaries.
  • a method of forming a multi-component assay includes providing one or more capsules of a second component within a first component.
  • the second component capsules can have an outer layer of a substance that melts or dissolves at a predetermined temperature, thereby releasing the second component into the first component and combining particles among the components.
  • One such substance is a thermally activated enzyme.
  • a "release on command" mechanism that is configured to release the second component upon a predetermined event or condition may also be used.
  • recombinant clones containing a reporter construct or a substrate are wicked into the capillary tubes of the capillary anay.
  • a substrate as the reporter construct or substrate contained in the clone can be readily detected using techniques known in the art.
  • a clone containing a reporter construct such as green fluorescent protein can be detected by exposing the clone or substrate within the clone to a wavelength of light that induces fluorescence.
  • Such reporter constructs can be implemented to respond to various conditions or upon exposure to various physical stimuli (including light and heat).
  • various compounds can be screened in a sample using similar techniques.
  • an antibody or antigen detectably labeled with a florescent molecule can be readily detected within a capillary tube of a capillary array.
  • a fluorescence-activated cell sorter (FACS) is used to separate and isolate particles or clones for delivery into the capillary anay; thus, one or more clones per capillary tube can be precisely achieved.
  • FACS fluorescence-activated cell sorter
  • Some assays may require an exchange of media within the capillary.
  • a media exchange process a first liquid containing the particles is wicked into a capillary. The first liquid is removed, and replaced with a second liquid while the particles remain suspended within the capillary. Addition of the second liquid to the capillary and contact with the particles can initialize an activity, such as an assay, for example.
  • the media exchange process may include a mechanism by which the particles in the capillary are physically maintained in the capillary while the first liquid is removed. Ln one aspect, the inner walls of the capillary anay are coated with antibodies to which an antigen, e.g., a cell, can bind.
  • the first liquid is removed, while the antigen remains bound to the antibodies, and the second liquid is wicked into the capillary.
  • the second liquid could be adapted to cause the antigens to unbind if desirable.
  • one or more walls of the capillary can be magnetized.
  • the particles are also magnetized and attracted to the walls.
  • magnetized particles are attracted and held against one side of the capillary upon application of a magnetic field near that side.
  • the capillary anay can be analyzed for identification of capillaries having a detectable signal, such as an optical signal (e.g., fluorescence), by a detector capable of detecting a change in light production or light transmission, for example.
  • a detectable signal such as an optical signal (e.g., fluorescence)
  • Detection may be performed using an illumination source that provides fluorescence excitation to each of the capillaries in the anay, and a photodetector that detects resulting emission from the fluorescence excitation.
  • Suitable illumination sources include, without limitation, a laser, incandescent bulb, light emitting diode (LED), and arch discharge.
  • Suitable photodetectors include, without limitation, a photodiode array, a charge-coupled device (CCD), or charge injection device (CED).
  • a detection system includes a laser source that produces a laser beam. The laser beam can be directed into a beam expander configured to produce a wider or less divergent beam for exciting the array of capillaries.
  • Suitable laser sources include argon or ion lasers. A cooled CCD can be used.
  • the light detector may be, for example, film, a photomultiplier tube, photodiode, avalanche photo diode, CCD or other light detector or camera.
  • the light detector may be a single detector to detect sequential emissions, such as a scanning laser.
  • the light detector may include a plurality of separate detectors to detect and spatially resolve simultaneous emissions at single or multiple wavelengths of emitted light.
  • the light emitted and detected may be visible light or may be emitted as non- visible radiation such as infrared or ultraviolet radiation.
  • a thermal detector may be used to detect an infrared emission.
  • the detector or detectors may be stationary or movable.
  • the emitted light or other radiation, such as illumination may be channeled to the detector or detectors by means of lenses, mirrors and fiber optic light guides or light conduits (single, multiple, fixed, or moveable) positioned on or adjacent to at least one surface of the capillary array.
  • the photodetector can comprise a CCD, CED or an anay of photodiode elements.
  • Detection of a position of one or more capillaries having an optical signal can then be determined from the optical input from each element.
  • the array may be scanned by a scanning confocal or phase-contrast fluorescence microscope or the like, where the anay is, for example, carried on a movable stage for movement in a X-Y plane as the capillaries in the anay are successively aligned with the beam to determine the capillary array positions at which an optical signal is detected.
  • a CCD camera or the like can be used in conjunction with the microscope.
  • the detection system can be computer-automated for rapid screening and recovery.
  • a telecentric lens can be used for detection. Magnification of the telecentric lens is adjusted to match the camera to the plane of view of the capillary array.
  • the change in the absorbance spectrum can be measured, such as by using a spectrophotometer or the like.
  • a spectrophotometer or the like.
  • Such measurements are usually difficult when dealing with a low- volume liquid because the optical path length is short.
  • the capillary approach of the present invention permits small volumes of liquid to have long optical path lengths (e.g., longitudinally along the capillary tube), thereby providing the ability to measure absorbance changes using conventional techniques.
  • binding or other activity is detected by using various electromagnetic detection devices, including, for example, optical, magnetic and thermal detection.
  • radioactivity can be detected within a capillary tube using detection methods known in the art. The radiation can be detected at either end of the capillary tube.
  • Other detection modes include, without limitation, luminescence, fluorescence polarization, time- resolved fluorescence.
  • Luminescence detection includes detecting emitted light that is produced by a chemical or physiological process associated with a sample molecule or cell.
  • Fluorescence polarization detection includes excitation of the contents of the lumen with polarized light. Under such environment, a fluorophore emits polarized light for a particular molecule.
  • Time-resolved fluorescence includes reading the fluorescence at a predetermined time after excitation.
  • the molecule is flashed with excitation energy, which produces emissions from the fluorophore as well as from other particles within the substrate. Emissions from the other particles causes background fluorescence.
  • the background fluorescence normally has a short lifetime relative to the long-life emission from the fluorophore.
  • the emission can be read after excitation is complete, at a time when all background fluorescence usually has short lifetime, and during a time in which the long-life fluorophores continues to fluoresce.
  • Time-resolved fluorescence can be a technique for suppressing background fluorescent activity.
  • a fluid within a capillary will usually form a meniscus at each end. Any light entering the capillary will be deflected toward the wall, except for paraxial rays, which enter the meniscus curvature at its center. The paraxial rays create a small bright spot in middle of capillary, representing the small amount of light that makes it through. Measurement of the bright spot provides an opportunity to measure how much light is being absorbed on its way through.
  • a detection system includes the use of two different wavelengths. A ratio between a first and a second wavelength indicates how much light is absorbed in the capillary. Alternatively, two images of the capillary can be taken, and a difference between them can be used to ascertain a differential absorbance of a chemical within the capillary.
  • the fluid bath can be contained in a clear, light-passing container, and the light source can be directed through the fluid bath into the capillary.
  • Recovery of putative hits (e.g., antigen binding to antibody) producing a detectable or optical signal can be facilitated by using position feedback from the detection system to automate positioning of a recovery device (e.g., a needle pipette tip or capillary tube).
  • a recovery device e.g., a needle pipette tip or capillary tube.
  • a support table supports a microtiter plate capillary anay and a light source. The light source is used with a camera assembly to find a location in the Z-axis of a needle connected to the recovery mechanism.
  • the support table moves in the axis of X and Y, to place the capillary anay underneath the needle, where the capillary array contains a "hit.”
  • the recovery mechanism guides the needle to a capillary containing a "hit” by overlapping the tip of the needle with the capillary containing the "hit,” in the Z direction, until the tip of the needle engages the capillary opening.
  • the needle may be attached to a spring or be of a material that flexes. Once in contact with the opening of the capillary the sample can be aspirated or expelled from the capillary.
  • a single camera is used for determining a location of a recovery tool, such as the tip of a needle, in the Z-plane.
  • the Z-plane determination can be accomplished using an auto-focus algorithm, or proximity sensor used in conjunction with the camera.
  • an image processing function can be executed to determine a precise location of the recovery tool in X and Y.
  • the recovery tool is back-lit to aid the image processing.
  • the capillary anay can be moved in X and Y relative to the precise location of the recovery tool, which can be moved along the Z axis for coupling with a target capillary.
  • two or more cameras are used for determining a location of the recovery tool. For instance, a first camera can determine X and Z coordinate locations of the recovery tool, such as the X, Z location of a needle tip. A second camera can determine Y and Z coordinate locations of the recovery tool. The two sets of coordinates can then be multiplexed for a complete X,Y,Z coordinate location. Next, the movement of the capillary array relative to the recovery tool can be executed.
  • the sample can be expelled by, for example, injecting a blast of inert gas into the capillary and collecting the ejected sample in a collection device at the opposite end of the capillary.
  • the diameter of the collection device can be larger than or equal to the diameter of the capillary.
  • the collected sample can then be further processed by, for example, extracting polynucleotides, proteins or by growing the clone in culture.
  • the sample is aspirated by use of a vacuum.
  • the needle contacts, or nearly contacts, the capillary opening and the sample is "vacuumed" or aspirated from the capillary tube onto or into a collection device.
  • the collection device may be a microfuge tube or a filter located proximal to the opening of the needle.
  • Suitable collection devices include a microfuge tube, a capillary tube, microtiter plate, cell culture plate, and the like.
  • the delivery of the sample can be accomplished by forcing another media, air or other fluid through the filter in the reverse direction.
  • the sample can also be expelled from a capillary by a sample ejector.
  • the ejector is a jet system where sample fluid at one end of the capillary tube is subjected to a high temperature, causing fluid at the other end of the capillary tube to eject out.
  • the heating of fluid can be accomplished mechanically, by applying a heated probe directly into one end of a capillary tube.
  • the heated probe can seal the one end, heats fluid in contact with the probe, and expels fluid out the other end of the capillary tube .
  • the heating and expulsion may also be accomplished electronically.
  • at least one wall of a capillary tube is metalized.
  • a heating element is placed in direct contact with one end of the wall.
  • the heating element may completely close off the one end, or partially close the one end.
  • the heating element charges up the metalized wall, which generates heat within the fluid.
  • the heating element can be an electricity source, such as a voltage source, or a cunent source.
  • a laser applies heat pulses to the fluid at one end of the capillary tube.
  • An electric field may be created in or near the fluid to create an electrophoretic reaction, which causes the fluid to move according to electromotive force created by the electric field.
  • An electric field may also assist in guiding a heated probe or electrically charged element to a target location near the fluid.
  • An electromagnetic field may also be used.
  • the capillary tube contains, in addition to the fluid, magnetically charged particles to help move the fluid out of the capillary anay.
  • Component modules provides genetic vaccine with the acquisition of or improvement in a useful property or characteristic.
  • the present invention provides multicomponent genetic vaccines that include one or more component modules, each of which provides the genetic vaccine with the acquisition of or an improvement in a property or characteristic useful in genetic vaccination.
  • a multicomponent genetic vaccine can, for example, contain a component that is optimized for optimal antigen expression, as well as a component that confers improved activation of cytotoxic T lymphocytes (CTLs) by enhancing the presentation of the antigen on dendritic cell MHC Class I molecules. Additional examples are described herein.
  • CTLs cytotoxic T lymphocytes
  • the invention provides a new approach to vaccine development, which is termed "antigen library immunization.”
  • antigen library immunization No other technologies are available for generating libraries of related antigens or optimizing known protective antigens.
  • the immunization protocols of the invention which use experimentally evolved (e.g.
  • antigen libraries provide a means to identify novel antigen sequences. Those antigens that are most protective can be selected from these pools by in vivo challenge models. Antigen library immunization dramatically expands the diversity of available immunogen sequences, and therefore, these antigen chimera libraries can also provide means to defend against newly emerging pathogen variants of the future.
  • the methods of the invention enable the identification of individual chimeric antigens that provide efficient protection against a variety of existing pathogens, providing improved vaccines for troops and civilian populations.
  • the methods of the invention provide an evolution-based approach, such as stochastic (e.g. polynucleotide shuffling & interrupted synthesis) and non-stochastic polynucleotide reassembly in particular, that is an optimal approach to improve the immunogenicity of many types of antigens.
  • the methods provide means of obtaining optimized cancer antigens useful for preventing and treating malignant diseases.
  • an increasing number of self-antigens, causing autoimmune diseases, and allergens, causing atopy, allergy and asthma have been characterized.
  • the immunogenicity and manufacturing of these antigens can likewise be improved with the methods of this invention.
  • the antigen library immunization methods of the invention provide a means by which one can obtain a recombinant antigen that has improved ability to induce an immune response to a pathogenic agent.
  • a "pathogenic agent” refers to an organism or virus that is capable of infecting a host cell. Pathogenic agents typically include and/or encode a molecule, usually a polypeptide, that is immunogenic in that an immune response is raised against the immunogenic polypeptide. Often, the immune response raised against an immunogenic polypeptide from one serotype of the pathogenic agent is not capable of recognizing, and thus protecting against, a different serotype of the pathogenic agent, or other related pathogenic agents. In other situations, the polypeptide produced by a pathogenic agent is not produced in sufficient amounts, or is not sufficiently immunogenic, for the infected host to raise an effective immune response against the pathogenic agent.
  • the methods of the invention typically involve reassembling (&/or subjecting to one or more directed evolution methods described herein) two or more forms of a nucleic acid that encode a polypeptide of the pathogenic agent, or antigen involved in another disease or condition.
  • reassembly methods including stochastic (e.g. polynucleotide shuffling & interrupted synthesis) and non- stochastic polynucleotide reassembly, use as substrates forms of the nucleic acid that differ from each other in two or more nucleotides, so a library of recombinant nucleic acids results.
  • the library is then screened to identify at least one optimized recombinant nucleic acid that encodes an optimized recombinant antigen that has improved ability to induce an immune response to the pathogenic agent or other condition.
  • the resulting recombinant antigens often are chimeric in that they are recognized by antibodies (Abs) reacting against multiple pathogen strains, and generally can also elicit broad spectrum immune responses. Specific neutralizing antibodies are known to mediate protection against several pathogens of interest, although additional mechanisms, such as cytotoxic T lymphocytes, are likely to be involved. The concept of chimeric, multivalent antigens inducing broadly reacting antibody responses is further illustrated herein.
  • the different forms of the nucleic acids that encode antigenic polypeptides are obtained from members of a family of related pathogenic agents.
  • This scheme of performing stochastic (e.g. polynucleotide shuffling & interrupted synthesis) and non-stochastic polynucleotide reassembly using nucleic acids from different organisms is shown schematically herein. Therefore, these stochastic (e.g. polynucleotide shuffling & interrupted synthesis) and non-stochastic polynucleotide reassembly methods provide an effective approach to generate multivalent, crossprotective antigens. The methods are useful for obtaining individual chimeras that effectively protect against most or all pathogen variants.
  • immunizations using entire libraries or pools of experimentally evolved (e.g. by polynucleotide reassembly &/or polynucleotide site-saturation mutagenesis) antigen chimeras can also result in identification of chimeric antigens that protect against pathogen variants that were not included in the starting population of antigens (for example, protection against strain C by experimentally evolved (e.g. by polynucleotide reassembly &/or polynucleotide site-saturation mutagenesis) library of chimeras/mutants of strains A and B).
  • sequence reassembly (&/or one or more additional directed evolution methods described herein) can be achieved in many different formats and permutations of formats, as described in further detail below. These formats share some common principles.
  • the targets for modification vary in different applications, as does the property sought to be acquired or improved.
  • candidate targets for acquisition of a property or improvement in a property include genes that encode proteins which have immunogenic and/or toxigenic activity when introduced into a host organism.
  • the methods use at least two variant forms of a starting target.
  • the variant forms of candidate substrates can show substantial sequence or secondary structural similarity with each other, but they should also differ in at least one, or, alternatively, in at least two positions.
  • the initial diversity between forms can be the result of natural variation, e.g., the different variant forms (homologs) are obtained from different individuals or strains of an organism, or constitute related sequences from the same organism (e.g. , allelic variations), or constitute homologs from different organisms (interspecific variants).
  • initial diversity can be induced, e.g., the variant forms can be generated by enor-prone transcription, such as an enor-prone PCR or use of a polymerase which lacks proof-reading activity (see, Liao (1990) Gene 88:107-111), of the first variant form, or, by replication of the first form in a mutator strain (mutator host cells are discussed in further detail below, and are generally well known).
  • a mutator strain can include any mutants in any organism impaired in the functions of mismatch repair. These include mutant gene products of mutS, mutT, mutH, mutL, ovrD, dcm, vsr, umuC, umuD, sbcB, recJ, etc.
  • Impairment can be of the genes noted, or of homologous genes in any organism.
  • Other methods of generating initial diversity include methods well known to those of skill in the art, including, for example, treatment of a nucleic acid with a chemical or other mutagen, through spontaneous mutation, and by inducing an enor-prone repair system (e.g., SOS) in a cell that contains the nucleic acid.
  • SOS enor-prone repair system
  • Polynucleotide sequences that can positively or negatively affect the immunogenicity of an antigen encoded by the polynucleotide are often scattered throughout the entire antigen gene. Several of these factors are shown diagrammatically herein.
  • stochastic e.g. polynucleotide shuffling & interrupted synthesis
  • non-stochastic polynucleotide reassembly followed by selection for those chimeric polynucleotides that encode an antigen that can induce an improved immune response, one can obtain primarily sequences that have a positive influence on antigen immunogenicity. Those sequences that negatively affect antigen immunogenicity are eliminated. One need not know the particular sequences involved.
  • the present invention provides methods for obtaining polynucleotide sequences that, either directly or indirectly (i.e., through encoding a polypeptide), can modulate an immune response when present on a genetic vaccine vector.
  • the invention provides methods for optimizing the transport and presentation of antigens.
  • the optimized immunomodulatory polynucleotides obtained using the methods of the invention are particularly suited for use in conjunction with vaccines, including genetic vaccines.
  • One of the advantages of genetic vaccines is that one can incorporate genes encoding immunomodulatory molecules, such as cytokines, costimulatory molecules, and molecules that improve antigen transport and presentation into the genetic vaccine vectors. This provides opportunities to modulate immune responses that are induced against the antigens expressed by the genetic vaccines.
  • the present invention provides methods of obtaining components for use in genetic vaccines, including the multicomponent vaccines, that are more effective in conferring a desired immune response property upon a genetic vaccine.
  • the methods involve creating a library of recombinant nucleic acids and screening the library to identify those library members that exhibits an enhanced capacity to confer a desired property upon a genetic vaccine.
  • Those recombinant nucleic acids that exhibit improved properties can be used as components in a genetic vaccine, either directly as a polynucleotide or as a protein that is obtained by expression of the component nucleic acid. Improvement goals
  • the properties or characteristics that are acquired or improved by the methods of the invention vary widely, and, of course depend on the choice of substrate.
  • antibodies include "affinity maturation," or, the generation of antibodies with a higher affinity for an antigen.
  • affinity maturation or, the generation of antibodies with a higher affinity for an antigen.
  • T cell receptors this can include an increased or decreased affinity for antigen, as presented by a major histocompatibility complex molecule.
  • improvement goals include higher titer, more stable expression, improved stability, higher specificity targeting, higher or lower frequency of integration, reduced immunogenicity of the vector or an expression product thereof, increased immunogenicity of the antigen, higher expression of gene products, and the like.
  • Other properties for which optimization is desired include the tailoring of an immune response to be most effective for a particular application. Examples of genetic vaccine components are shown, described &/or referenced herein (including inco ⁇ orated by reference). Two or more components can be included in a single vector molecule, or each component can be present in a genetic vaccine formulation as a separate molecule.
  • Sequence reassembly (&/or one or more additional directed evolution methods described herein) can be achieved through different formats which share some common principles
  • At least two variant forms of a nucleic acid are reassembled (&/or subjected to one or more directed evolution methods described herein) to produce a library of recombinant nucleic acids, which is then screened to identify at least one recombinant component that is optimized for the particular vaccine property.
  • improvements are achieved after one round of reassembly (&/or one or more additional directed evolution methods described herein) and selection.
  • Sequence reassembly (&/or one or more additional directed evolution methods described herein) can be achieved in many different formats and permutations of formats, as described in further detail below. These formats share some common principles.
  • reassembly &/or one or more additional directed evolution methods described herein
  • diversity resulting from reassembly can be augmented in any cycle by applying prior methods of mutagenesis (e.g., enor-prone PCR or cassette mutagenesis) to either the substrates or products of reassembly (&/or one or more additional directed evolution methods described herein).
  • a new or improved property or characteristic can be achieved after only a single cycle of in vivo or in vitro reassembly (&/or one or more additional directed evolution methods described herein), as when using different, variant forms of the sequence, as homologs from different individuals or strains of an organism, or related sequences from the same organism, as allelic variations.
  • recursive sequence reassembly &/or one or more additional directed evolution methods described herein
  • which entails successive cycles of reassembly &/or one or more additional directed evolution methods described herein
  • polynucleotides that encode optimized recombinant antigens are subjected to molecular backcrossing, which provides a means to breed the experimentally evolved (e.g. by polynucleotide reassembly &/or polynucleotide site-saturation mutagenesis) chimeras/mutants back to a parental or wild-type sequence, while retaining the mutations that are critical to the phenotype that provides the optimized immune responses.
  • molecular backcrossing can also be used to characterize which of the many mutations in an improved variant contribute most to the improved phenotype. This cannot be accomplished in an efficient library fashion by any other method.
  • Backcrossing is performed by reassembling (optionally in combination with other directed evolution methods described herein) the improved sequence with a large molar excess of the parental sequences.
  • Stochastic e.g. polynucleotide shuffling & interrupted synthesis
  • non-stochastic polynucleotide reassembly is used to obtain the library of recombinant nucleic acids, using a variety of substrates to acquire or improve various properties for different applications. Creation of Recombinant Libraries
  • the invention involves creating recombinant libraries of polynucleotides that are then screened to identify those library members that exhibit a desired property.
  • the recombinant libraries can be created using any of various methods.
  • the substrate nucleic acids used for the reassembly can vary depending upon the particular application. For example, where a polynucleotide that encodes a nucleic acid binding domain or a ligand for a cell-specific receptor is to be optimized, different forms of nucleic acids that encode all or part of the nucleic acid binding domain or a ligand for a cell-specific receptor are subjected to reassembly (&/or one or more additional directed evolution methods described herein).
  • stochastic e.g. polynucleotide shuffling & interrupted synthesis
  • non-stochastic polynucleotide reassembly is used to obtain the library of recombinant nucleic acids
  • stochastic e.g. polynucleotide shuffling & interrupted synthesis
  • non-stochastic polynucleotide reassembly which is described herein, can result in optimization of a desired property even in the absence of a detailed understanding of the mechanism by which the particular property is mediated.
  • the substrates for this modification, or evolution vary in different applications, as does the property sought to be acquired or improved.
  • candidate substrates for acquisition of a property or improvement in a property include viral and nonviral vectors used in genetic vaccination, as well as nucleic acids that are involved in mediating a particular aspect of an immune response.
  • the methods require at least two variant forms of a starting substrate.
  • the variant forms of candidate components can have substantial sequence or secondary structural similarity with each other, but they should also differ in at least two positions.
  • the initial diversity between forms can be the result of natural variation, e.g., the different variant forms (homologs) are obtained from different individuals or strains of an organism (including geographic variants) or constitute related sequences from the same organism (e.g., allelic variations).
  • the initial diversity can be induced, e.g., the second variant form can be generated by enor- prone transcription, such as an enor- prone PCR or use of a polymerase which lacks proofreading activity (see, Liao (1990) Gene 88:107-111), of the first variant form, or, by replication of the first form in a mutator strain (mutator host cells are discussed in further detail below).
  • the initial diversity between substrates is greatly augmented in subsequent steps of recursive sequence reassembly (&/or one or more additional directed evolution methods described herein).
  • the library is subjected to selection and/or screening to identify those library members that encode antigenic peptides that have improved ability to induce an immune response to the pathogenic agent.
  • Selection and screening of experimentally generated polynucleotides that encode polypeptides having an improved ability to induce an immune response can involve either in vivo and in vitro methods, but most often involves a combination of these methods.
  • the members of a library of recombinant nucleic acids are picked, either individually or as pools.
  • the clones can be subjected to analysis directly, or can be expressed to produce the conesponding polypeptides.
  • an in vitro screen is performed to identify the best candidate sequences for the in vivo studies.
  • the library can be subjected to in vivo challenge studies directly.
  • the analyses can employ either the nucleic acids themselves (e.g., as genetic vaccines), or the polypeptides encoded by the nucleic acids.
  • a cycle of reassembly (&/or one or more additional directed evolution methods described herein) is usually followed by at least one cycle of screening or selection for molecules having a desired property or characteristic. If a cycle of reassembly (&/or one or more additional directed evolution methods described herein) is performed in vitro, the products of reassembly (&/or one or more additional directed evolution methods described herein), i.e., recombinant segments, are sometimes introduced into cells before the screening step. Recombinant segments can also be linked to an appropriate vector or other regulatory sequences before screening.
  • products of reassembly (&/or one or more additional directed evolution methods described herein) generated in vitro are sometimes packaged as viruses (in viruses- e.g., bacteriophage) before screening.
  • viruses viruses- e.g., bacteriophage
  • product of reassembly (&/or one or more additional directed evolution methods described herein) can sometimes be screened in the cells in which reassembly (&/or one or more additional directed evolution methods described herein) occuned.
  • recombinant segments are extracted from the cells, and optionally packaged as viruses, before screening.
  • a genetic vaccine vector can have many component sequences each having a different intended role (e.g., coding sequence, regulatory sequences, targeting sequences, stability-conferring sequences, immunomodulatory sequences, sequences affecting antigen presentation, and sequences affecting integration). Each of these component sequences can be varied and reassembled
  • Screening/selection can then be performed, for example, for recombinant segments that have increased episomal maintenance in a target cell without the need to attribute such improvement to any of the individual component sequences of the vector.
  • initial round(s) of screening can sometimes be performed in bacterial cells due to high transfection efficiencies and ease of culture.
  • test animals are used for library expression and screening.
  • Later rounds, and other types of screening which are not amenable to screening in bacterial cells are generally performed (in cells selected for use in an environment close to that of their intended use) in mammalian cells to optimize recombinant segments for use in an environment close to that of their intended use.
  • Final rounds of screening can be performed in the cell type of intended use (e.g., a human antigen-presenting cell).
  • this cell can be obtained from a patient to be treated with a view, for example, to minimizing problems of immunogenicity in this patient.
  • use of a genetic vaccine vector in treatment can itself be used as a round of screening. That is, genetic vaccine vectors that are successively taken up and/or expressed by the intended target cells in one patient are recovered from those target cells and used to treat another patient.
  • the genetic vaccine vectors that are recovered from the intended target cells in one patient are enriched for vectors that have evolved, i.e., have been modified by recursive reassembly (&/or one or more additional directed evolution methods described herein), toward improved or new properties or characteristics for specific uptake, immunogenicity, stability, and the like.
  • the screening or selection step identifies a subpopulation of recombinant segments that have evolved toward acquisition of a new or improved desired property or properties useful in genetic vaccination.
  • the recombinant segments can be screened as components of cells, components of viruses or other vectors, or in free form. More than one round of screening or selection can be performed after each round of reassembly (&/or one or more additional directed evolution methods described herein). The second round of reassemblv (&/or one or more additional directed evolution methods described herein)
  • At least one and usually a collection of recombinant segments surviving a first round of screening/selection are subject to a further round of reassembly (&/or one or more additional directed evolution methods described herein).
  • These recombinant segments can be reassembled (&/or subjected to one or more directed evolution methods described herein) with each other or with exogenous segments representing the original substrates or further variants thereof.
  • reassembly (&/or one or more additional directed evolution methods described herein) can proceed in vitro or in vivo.
  • the components can be subjected to further reassembly (&/or one or more additional directed evolution methods described herein) in vivo, or can be subjected to further reassembly (&/or one or more additional directed evolution methods described herein) in vitro, or can be isolated before performing a round of in vitro reassembly (&/or one or more additional directed evolution methods described herein).
  • the previous screening step identifies desired recombinant segments in naked form or as components of viruses or other vectors, these segments can be introduced into cells to perform a round of in vivo reassembly (&/or one or more additional directed evolution methods described herein).
  • the second round of reassembly (&/or one or more additional directed evolution methods described herein), inespective how performed, generates further recombinant segments which encompass additional diversity compared to recombinant segments resulting from previous rounds. Additional rounds of reassembly (&/or one or more additional directed evolution methods described hereinVscreenin to sufficiently evolve the recombinant segments
  • the second round of reassembly (&/or one or more additional directed evolution methods described herein) can be followed by a further round of screening/selection according to the principles discussed above for the first round.
  • the stringency of screening/selection can be increased between rounds.
  • the nature of the screen and the property being screened for can vary between rounds if improvement in more than one property is desired or if acquiring more than one new property is desired.
  • Additional rounds of reassembly (&/or one or more additional directed evolution methods described herein) and screening can then be performed until the recombinant segments have sufficiently evolved to acquire the desired new or improved property or function.
  • the practice of this invention involves the construction of recombinant nucleic acids and the expression of genes in transfected host cells.
  • Molecular cloning techniques to achieve these ends are known in the art.
  • a wide variety of cloning and in vitro amplification methods suitable for the construction of recombinant nucleic acids such as expression vectors are well-known to persons of skill.
  • General texts which describe molecular biological techniques useful herein, including mutagenesis include Berger and Kimmel, Guide to Molecular Cloning Techniques, Methods in Enzymology volume 152 Academic Press, Inc., San Diego, CA (Berger); Sambrook et al., Molecular Cloning - A Laboratory Manual (2nd Ed.), Vol.
  • Oligonucleotides for use as probes e.g., in in vitro amplification methods, for use as gene probes, or as reassembly targets (e.g., synthetic genes or gene segments) are typically synthesized chemically according to the solid phase phosphoramidite triester method described by Beaucage and Caruthers (1981) Tetrahedron Letts., 22(20): 1859-1862, e.g., using an automated synthesizer, as described in Needham- VanDevanter et al. (1984) Nucleic Acids Res., 12:6159-6168. Oligonucleotides can also be custom made and ordered from a variety of commercial sources known to persons of skill.
  • nucleic acid with a known sequence can be custom ordered from any of a variety of commercial sources, such as The Midland Certified Reagent Company (mcrc@oligos.com), The Great American Gene Company, ExpressGen Inc., Operon Technologies Inc. (Alameda, CA) and many others.
  • peptides and antibodies can be custom ordered from any of a variety of sources, such as PeptidoGenic (pkim@ccnet.com), HTI Bio-products, Inc., BMA Biomedicals Ltd (U.K.), Bio-Synthesis, Inc., and many others.
  • Different formats are available for performing reassembly (&/or additional directed evolution methods described herein) and screening/selection which allow for large numbers of mutations in a minimum number of selection cycles and does not require the extensive analysis and computation required by conventional methods.
  • a number of different formats are available by which one can create a library of recombinant nucleic acids for screening.
  • the methods of the invention entail performing reassembly (&/or one or more additional directed evolution methods described herein) and screening or selection to "evolve" individual genes, whole plasmids or viruses, multigene clusters, or even whole genomes (Stemmer (1995) Bio/Technology 13:549-553).
  • Reiterative cycles of reassembly (&/or one or more additional directed evolution methods described herein) and screening/selection can be performed to further evolve the nucleic acids of interest.
  • Such techniques do not require the extensive analysis and computation required by conventional methods for polypeptide engineering.
  • Reassembly allows the combination of large numbers of mutations in a minimum number of selection cycles, in contrast to traditional, pair wise recombiantion events (e.g., as occur during sexual replication).
  • the directed evolution techniques described herein provide particular advantages in that they provide reassembly (optionally in combination with one or more additional directed evolution methods described herein) between any or all of the mutations, thereby providing a very fast way of exploring the manner in which different combinations of mutations can affect a desired result. Ln some instances, however, structural and/or functional information is available which, although not required for sequence reassembly (&/or one or more additional directed evolution methods described herein), provides opportunities for modification of the technique.
  • the stochastic (e.g. polynucleotide shuffling & interrupted synthesis) and non- stochastic polynucleotide reassembly methods can involve one or more of at least four different approaches to improve immunogenic activity as well as to broaden specificity.
  • stochastic (e.g. polynucleotide shuffling & interrupted synthesis) and non-stochastic polynucleotide reassembly can be performed on a single gene.
  • stochastic (e.g. polynucleotide shuffling & interrupted synthesis) and non-stochastic polynucleotide reassembly can be performed on a single gene.
  • several highly homologous genes can be identified by sequence comparison with known homologous genes. These genes can be synthesized and experimentally evolved (e.g.
  • polynucleotide reassembly &/or polynucleotide site-saturation mutagenesis as a family of homologs, to select recombinants with the desired activity.
  • the experimentally evolved (e.g. by polynucleotide reassembly &/or polynucleotide site-saturation mutagenesis) genes can be introduced into appropriate host cells, which can include E. coli, yeast, plants, fungi, animal cells, and the like, and those having the desired properties can be identified by the methods described herein.
  • whole genome reassembly can be performed to shuffle genes that can confer a desired property upon a genetic vaccine (along with other genomic nucleic acids).
  • polypeptide- encoding genes can be codon modified to access mutational diversity not present in any naturally occurring gene.
  • Kits for mutagenesis are commercially available (e.g., Bio-Rad, Amersham International, Yalen Biotechnology).
  • the starting materials must differ from each other in at least two nucleotide positions.
  • the reassembly procedure starts with at least two substrates that generally show substantial sequence identity to each other (i.e., at least about 30%, 50%, 70%, 80% or 90% sequence identity), but differ from each other at certain positions.
  • the difference can be any type of mutation, for example, substitutions, insertions and deletions.
  • different segments differ from each other in about 5-20 positions.
  • the starting materials must differ from each other in at least two nucleotide positions. That is, if there are only two substrates, there should be at least two divergent positions.
  • the starting DNA segments can be natural variants of each other, for example, allelic or species variants.
  • the segments can also be from nonallelic genes showing some degree of structural and usually functional relatedness (e.g., different genes within a superfamily, such as the family of Yersinia V- antigens, for example).
  • the starting DNA segments can also be induced variants of each other.
  • one DNA segment can be produced by enor-prone PCR replication of the other, the nucleic acid can be treated with a chemical or other mutagen, or by substitution of a mutagenic cassette.
  • Induced mutants can also be prepared by propagating one (or both) of the segments in a mutagenic strain, or by inducing an enor-prone repair system in the cells.
  • the different segments forming the starting materials are related, and might or might not be of similar length
  • the second DNA segment is not a single segment but a large family of related segments.
  • the different segments forming the starting materials are often the same length or substantially the same length. However, this need not be the case; for example; one segment can be a subsequence of another.
  • the segments can be present as part of larger molecules, such as vectors, or can be in isolated form.
  • the starting DNA segments are reassembled (&/or subjected to one or more directed evolution methods described herein) to generate a library of recombinant DNA segments varying in size which will include full length coding sequences and any essential regulatory
  • the starting DNA segments are reassembled (&/or subjected to one or more directed evolution methods described herein) by any of the sequence reassembly (&/or one or more additional directed evolution methods described herein) formats provided herein to generate a diverse library of recombinant DNA segments.
  • Such a library can vary widely in size from having fewer than 10 to more than 10 5 , 10 9 , 10 12 or more members.
  • the starting segments and the recombinant libraries generated will include full-length coding sequences and any essential regulatory sequences, such as a promoter and polyadenylation sequence, required for expression.
  • the recombinant DNA segments in the library can be inserted into a common vector providing sequences necessary for expression before performing screening/selection. Using reassemblv PCR to assemble multiple segments that have been separately evolved into a full length nucleic acid template such as a gene
  • a further technique for recombining mutations in a nucleic acid sequence utilizes "reassembly PCR".
  • This method can be used to assemble multiple segments that have been separately evolved into a full length nucleic acid template such as a gene.
  • This technique is performed when a pool of advantageous mutants is known from previous work or has been identified by screening mutants that may have been created by any mutagenesis technique known in the art, such as PCR mutagenesis, cassette mutagenesis, doped oligo mutagenesis, chemical mutagenesis, or propagation of the DNA template in vivo in mutator strains.
  • Boundaries defining segments of a nucleic acid sequence of interest can lie in intergenic regions, introns, or areas of a gene not likely to have mutations of interest.
  • Oligos are synthesized for PCR amplification of segments of the nucleic acid sequence of interest so that the oligos overlap the junctions of two segments by, typically, about 10 to 100 nucleotides
  • oligonucleotide primers are synthesized for PCR amplification of segments of the nucleic acid sequence of interest, such that the sequences of the oligonucleotides overlap the junctions of two segments.
  • the overlap region is typically about 10 to 100 nucleotides in length.
  • Each of the segments is amplified with a set of such primers.
  • the PCR products are then "reassembled” according to assembly protocols such as those discussed herein to assemble non-stochastically generated nucleic acid building blocks &/or randomly fragmented genes. In brief, in an assembly protocol the PCR products are first purified away from the primers, by, for example, gel electrophoresis or size exclusion chromatography.
  • Purified products are mixed together and subjected to about 1-10 cycles of denaturing, reannealing, and extension in the presence of polymerase and deoxynucleoside triphosphates (dNTP's) and appropriate buffer salts in the absence of additional primers ("self-priming").
  • dNTP's polymerase and deoxynucleoside triphosphates
  • self-priming additional primers
  • Subsequent PCR with primers flanking the gene are used to amplify the yield of the fully reassembled and experimentally evolved (e.g. by polynucleotide reassembly &/or polynucleotide site-saturation mutagenesis) genes.
  • PCR primers are used to introduce variation into the gene of interest and the mutations at sites of interest are screened or selected by sequencing homologues of the nucleic acid sequence
  • PCR primers for amplification of segments of the nucleic acid sequence of interest are used to introduce variation into the gene of interest as follows. Mutations at sites of interest in a nucleic acid sequence are identified by screening or selection, by sequencing homologues of the nucleic acid sequence, and so on. Using oligonucleotide PCR primers (encoding wild type or mutant information) in PCR to generate libraries of full length genes encoding permutations of said info, where the alternative screening or selection process is expensive, cumbersome, or impractical
  • Oligonucleotide PCR primers are then synthesized which encode wild type or mutant information at sites of interest. These primers are then used in PCR mutagenesis to generate libraries of full length genes encoding permutations of wild type and mutant information at the designated positions. This technique is typically advantageous in cases where the screening or selection process is expensive, cumbersome, or impractical relative to the cost of sequencing the genes of mutants of interest and synthesizing mutagenic oligonucleotides.
  • the invention provides multicomponent genetic vaccines, and methods of obtaining genetic vaccine components that improve the capability of the genetic vaccine for use in nucleic acid-mediated immunomodulation.
  • a general approach for evolution of genetic vaccines and components by stochastic (e.g. polynucleotide shuffling & interrupted synthesis) and non-stochastic polynucleotide reassembly is shown schematically herein. Including an origin of replication is useful to obtain sufficient quantities of the vector prior to administration to a patient, but might be undesirable if the vector is designed to integrate into host chromosomal DNA or bind to host mRNA or DNA.
  • a genetic vaccine vector is an exogenous polynucleotide which produces a medically useful phenotypic effect upon the mammalian cell(s) and organisms into which it is transferred.
  • a vector may or may not have an origin of replication.
  • the origin of replication can be removed before administration, or an origin can be used that functions in the cells used for vector production but not in the target cells.
  • Vectors used in genetic vaccination can be viral or nonviral.
  • Viral vectors are usually introduced into a patient as components of a virus.
  • Dlustrative viral vectors into which one can incorporate nucleic acids that are modified by the stochastic (e.g. polynucleotide shuffling & interrupted synthesis) and non-stochastic polynucleotide reassembly methods of the invention include, for example, adenovirus-based vectors (Cantwell (1996) Blood 88:4676-4683; Ohashi (1997) Proc. Nat'l. Acad. Sci USA 94:1287-1292), Epstein-Ban virus- based vectors (Mazda (1997) J. Immunol.
  • Techniques for transferring DNA into a cell useful in vivo naked DNA delivered using liposomes fusing to cellular membrane or entering through endocytosis; permeabilize the cells and use DNA binding protein to transport into cell; and bombardment of skin with particles coated with DNA delivered mechanically
  • Nonviral vectors can be transfened as naked DNA or associated with a transfer-enhancing vehicle, such as a receptor- recognition protein, liposome, lipoamine, or cationic lipid.
  • This DNA can be transfened into a cell using a variety of techniques well known in the art.
  • naked DNA can be delivered by the use of liposomes which fuse with the cellular membrane or are endocytosed, i.e., by employing ligands attached to the liposome, or attached directly to the DNA, that bind to surface membrane protein receptors of the cell resulting in endocytosis.
  • the cells may be permeabilized to enhance transport of the DNA into the cell, without injuring the host cells.
  • DNA binding protein e.g., HBGF-1
  • DNA can be delivered by bombardment of the skin by gold or other particles coated with DNA which are delivered by mechanical means, e.g., pressure.
  • liposomes particularly where the liposome surface carries ligands specific for target cells, or are otherwise preferentially directed to a specific organ, one may provide for the introduction of the DNA into the target cells/organs in vivo.
  • Viral Vectors Structure of viral vectors often consist of a modified viral genome and a coat structure sunounding it.
  • viral vectors such as retroviruses, adenoviruses, adenoassociated viruses and herpes viruses, are commonly used in genetic vaccination. They are often made up of two components, a modified viral genome and a coat structure sunounding it (see generally Smith (1995) Annu. Rev. Microbiol. 49, 807-83 8), although sometimes viral vectors are introduced in naked form or coated with proteins other than viral proteins. Most current viral vectors have coat structures similar to a wild type virus. This structure packages and protects the viral nucleic acid and provides the means to bind and enter target cells.
  • the viral nucleic acid in a vector designed for genetic vaccination can be changed in many ways.
  • the goals of these changes can be, for example, to enhance or reduce replication of the virus in target cells while maintaining its ability to grow in vector form in available packaging or helper cells, to incorporate new sequences that encode and enable appropriate expression of a gene of interest (e.g., an antigen-encoding gene), and to alter the immunogenicity of the viral vector itself
  • Viral vector nucleic acids generally comprise two components: essential cis- acting viral sequences for replication and packaging in a helper line and a transcription unit for the exogenous gene. Other viral functions can be expressed in trans in a specific packaging or helper cell line.
  • Adenoviruses adenoviruses
  • Adenoviruses comprise a large class of nonenveloped viruses that contain linear double- stranded DNA.
  • the normal life cycle of the virus does not require dividing cells and involves productive infection in permissive cells during which large amounts of virus accumulate.
  • the productive infection cycle takes about 32-36 hours in cell culture and comprises two phases, the early phase, prior to viral DNA synthesis, and the late phase, during which structural proteins and viral DNA are synthesized and assembled into virions.
  • adenovirus infections are associated with mild disease in humans.
  • E3-deletion vectors studied replication in cultured cells does not require E3 region, allowing insertion of exogenous DNA sequences to yield vectors capable of productive infection and the transient synthesis of relatively large amounts of encoded protein.
  • Adenovirus vectors are somewhat larger and more complex than retrovirus or AAV vectors, partly because only a small fraction of the viral genome is removed from most cunent vectors. If additional genes are removed, they are provided in trans to produce the vector, which so far has proved difficult. Instead, two general types of adenovirus-based vectors have been studied, E3-deletion and El-deletion vectors. Some viruses in laboratory stocks of wild-type lack the E3 region and can grow in the absence of helper.
  • E3 gene products are not necessary in the wild, only that replication in cultured cells does not require them.
  • Deletion of the E3 region allows insertion of exogenous DNA sequences to yield vectors capable of productive infection and the transient synthesis of relatively large amounts of encoded protein.
  • El replacement vectors grown in 293 cells utilized in most gene therapy applications involving adenoviruses. Deletion of the El region disables the adenovirus, but such vectors can still be grown because there exists an established human cell line (called "293") that contains the El region of Ad5 and that constitutively expresses the El proteins. Most recent gene-therapy applications involving adenovirus have utilized El replacement vectors grown in 293 cells.
  • Adenovirus vectors capable of efficient episomal gene transfer, easy to grow, can be topically applied to skin for antigen delivery, induction of antigen specific immune responses can be observed, but host response limits duration of expression and ability to repeat dosing in cases with high doses of first generation vectors
  • adenovirus vectors are capable of efficient episomal gene transfer in a wide range of cells and tissues and that they are easy to grow in large amounts.
  • Adenovirus-based vectors can also be used to deliver antigens after topical application onto the skin, and induction of antigen-specific immune responses can be observed following delivery to the skin (Tang et al. (1997) Nature 388: 729-730).
  • the main disadvantage is that the host response to the virus appears to limit the duration of expression and the ability to repeat dosing, at least with high doses of first- generation vectors.
  • This invention provides for the first time a phagemid system capable of cloning large DNA inserts of over 10 kilobases and generating ssDNA in vitro and in vivo conesponding to those large inserts.
  • the directed evolution methods of the invention are used to construct a novel adenovirus-phagemid capable of packaging DNA inserts over 10 kilobases in size.
  • Incorporation of a phage origin in a plasmid using the methods of the invention also generates a novel in vivo reassembly or shuffling format capable of evolving whole genomes of viruses, such as the 36 kb family of human adenoviruses.
  • the widely used human adenovirus type 5 (Ad5) has a genome size of 36 kb. It is difficult to shuffle this large genome in vitro without creating an excessive number of changes which may cause a high percentage of nonviable recombinant variants.
  • Ad5 adenovirus-phagemid
  • the Ad- phagemid has been demonstrated to accept inserts as large as 15 and 24 kilobases and to effectively generate ssDNA of that size.
  • larger DNA inserts as large as 50 to 100 kb are inserted into the Ad-phagemid of the invention; with generation of full length ssDNA conesponding to those large inserts.
  • Generation of such large ssDNA non- stochastically generated nucleic acid building blocks &/or fragments provides a means to evolve, i.e.
  • this invention provides for the first time a unique phagemid system capable of cloning large DNA inserts (>10 KB) and generating ssDNA in vitro and in vivo conesponding to those large inserts.
  • a unique phagemid system capable of cloning large DNA inserts (>10 KB) and generating ssDNA in vitro and in vivo conesponding to those large inserts.
  • In vivo reassemblv or shuffling of the genomes of related serotypes of human adenoviruses using system is useful for creation of recombinant adenovirus variants with changes in multiple genes.
  • the genomes of related serotypes of human adenovirus are experimentally evolved (e.g. by polynucleotide reassembly &/or polynucleotide site-saturation mutagenesis) in vivo using this unique phagemid system, as described in International Application No. PCT/US97/17302 (Publ. No. W098/13485).
  • the genomic DNA is first cloned into a phagemid vector, and the resulting plasmid, designated an "Admid,” can be used to produce single-stranded (ss) Admid phage by using a helper M13 phage.
  • ssAdmid phages containing the genome of homologous human adenoviruses are used to perform high multiplicity of infection (MOI) on F + MutS E. coli cells.
  • MOI multiplicity of infection
  • the ssDNA is a better substrate for reassembly (&/or one or more additional directed evolution methods described herein) enzymes such as RecA.
  • the high MOI ensures that the probability of having multiple cross-overs between copies of the infecting ssAdmid DNA is high.
  • the experimentally evolved e.g.
  • adenovirus genome is generated by purification of the double stranded Admid DNA from the infected cells and is introduction into a permissive human cell line to produce the adenovirus library.
  • This genomic reassembly strategy is useful for creation of recombinant adenovirus variants with changes in multiple genes. This allows screening or selection of recombinant variant phenotypes resulting from combinations of variations in multiple genes.
  • Adeno-Associated Virus Adeno-Associated Virus
  • AAV is a small, simple, nonautonomous virus containing linear single-stranded DNA. See, Muzycka, Cunent Topics Microbiol. Immunol. 158, 97- 129 (1992). The virus requires co-infection with adenovirus or certain other viruses in order to replicate. AAV is widespread in the human population, as evidenced by antibodies to the virus, but it is not associated with any known disease. AAV genome organization is straightforward, comprising only two genes: rep and cap. The termini of the genome comprises terminal repeats (ITR) sequences of about 145 nucleotides.
  • ITR terminal repeats
  • AAV-based vectors typically contain only the ITR sequences flanking the transcription unit of interest.
  • the length of the vector DNA cannot greatly exceed the viral genome length of 4680 nucleotides.
  • growth of AAV vectors is cumbersome and involves introducing into the host cell not only the vector itself but also a plasmid encoding rep and cap to provide helper functions.
  • the helper plasmid lacks ITRs and consequently cannot replicate and package.
  • helper virus such as adenovirus is often required.
  • Advantage long-term expression in nondividing cells.
  • AAV vectors appear capable of long-term expression in nondividing cells, possibly, though not necessarily, because the viral DNA integrates.
  • the vectors are structurally simple, and they may therefore provoke less of a host- cell response than adenovirus.
  • Papillomaviruses are small, nonenveloped, icosahedral DNA viruses that replicate in the nucleus of squamous epithelial cells.
  • Papillomaviruses consist of a single molecule of double-stranded circular DNA about 8,000 bp in size within a spherical protein coat of 72 capsomeres.
  • Such papillomaviruses are classified by the species they infect (e.g., bovine, human, rabbit) and by type within species.
  • HPV human papillomaviruses
  • Papillomaviruses display a marked degree of cellular tropism for epithelial cells. Specific viral types have a preference for either cutaneous or mucosal epithelial cells. Benign, low-risk, intermediate-risk, and high-risk HPVs.
  • All papillomaviruses have the capacity to induce cellular proliferation. The most common clinical manifestation of proliferation is the production of benign warts. However, many papillomaviruses have capacity to be oncogenic in some individuals and some papillomaviruses are highly oncogenic. Based on the pathology of the associated lesions, most human papillomaviruses (HPVs) can be classified in one of four major groups, benign, low-risk, intermediate-risk and high-risk (Fields Virology, (Fields et al., eds., Lippincott- Raven, Philadelphia, 3d ed. 1996); DNA Tumor Viruses: Papilloma in (Encyclopedia of Cancer, Academic Press) Vol.
  • viruses HPV-1, HPV-2, HPV-3, HPV-4, and HPV-27 are associated with benign cutaneous lesions.
  • viruses HPV-6 and HPV- 11 are associated with vulval, penile, and laryngeal warts and are considered low-risk viruses as they are rarely associated with invasive carcinomas.
  • Viruses HPV-16, HPV-18, HPV-31, and HPV-45 are considered high risk virus as they are associated with a high frequency with adeno- and squamous carcinoma of the cervix.
  • Viruses HPV- 5 and HPV-8 are associated with benign cutaneous lesion in a multifactorial disease Epidermodysplasia Venuciformis (EV). Such lesions, however, can progress into squamous cell carcinomas.
  • HPVs classified for risk based on frequency of cancerous lesions relative to previously classified HPVs.
  • HPVs can classified for risk based on the frequency of cancerous lesions relative to that of HPVs that have already been classified for risk.
  • HPV vectors can be subjected to iterative cycles of reassembly (&/or one or more additional directed evolution methods described herein) and screening with a view to obtaining vectors with improved properties. Improved properties include increased tissue specificity, altered tissue specificity, increased expression level, prolonged expression, increased episomal copy number, increased or decreased capacity for chromosomal integration, increased uptake capacity, and other properties as discussed herein.
  • the starting materials for reassembling are typically vectors of the kind described above constructed from different strains of human papillomaviruses, or segments or variants of such generated by e.g., enor- prone PCR or cassette mutagenesis.
  • the human papillomaviruses, or at least the El and E2 coding regions thereof can be human cutaneous papillomaviruses.
  • Retroviruses comprise a large class of enveloped viruses that contain single- stranded
  • RNA as the viral genome.
  • viral RNA is reverse-transcribed to yield double-stranded DNA that integrates into the host genome and is expressed over extended periods.
  • infected cells shed virus continuously without apparent harm to the host cell.
  • the viral genome is small (approximately 10 kb), and its prototypical organization is extremely simple, comprising three genes encoding gag, the group specific antigens or core proteins; pol, the reverse transcriptase; and env, the viral envelope protein.
  • the termini of the RNA genome are called long terminal repeats (LTRs) and include promoter and enhancer activities and sequences involved in integration.
  • LTRs long terminal repeats
  • the genome also includes a sequence required for packaging viral RNA and splice acceptor and donor sites for generation of the separate envelope mRNA.
  • Most retroviruses can integrate only into replicating cells, although human immunodeficiency virus (HTV) appears to be an exception.
  • HTV human immunodeficiency virus
  • Retrovirus vectors are relatively simple, containing the 5' and 3' LTRs, a packaging sequence, and a transcription unit composed of the gene or genes of interest, which is typically an expression cassette.
  • a packaging cell line Such a cell is engineered to contain integrated copies of gag, pol, and env but to lack a packaging signal so that no helper virus sequences become encapsidated. Additional features added to or removed from the vector and packaging cell line reflect attempts to render the vectors more efficacious or reduce the possibility of contamination by helper virus. Potentially capable of long-term expression, can be grown in large amounts, but must ensure the absence of helper virus.
  • retroviral vectors have the advantage of being able integrate in the chromosome and therefore potentially capable of long-term expression. They can be grown in relatively large amounts, but care is needed to ensure the absence of helper virus.
  • Nonviral nucleic acid vectors used in genetic vaccination include plasmids, RNAs, polyamide nucleic acids, and yeast artificial chromosomes (YACs), and the like. Vector organization; insertion of enhancer sequence increases transcription.
  • Such vectors typically include an expression cassette for expressing a polypeptide against which an immune response is induced.
  • the promoter in such an expression cassette can be constitutive, cell type-specific, stage-specific, and/or modulatable (e. g., by tetracycline ingestion; tetracycline-responsive promoter). Transcription can be increased by inserting an enhancer sequence into the vector.
  • Enhancers are cis-acting sequences, typically between 10 to 300 base pairs in length, that increase transcription by a promoter. Enhancers can effectively increase transcription when either 5' or 3' to the transcription unit. They are also effective if located within an intron or within the coding sequence itself.
  • viral enhancers are used, including SV40 enhancers, cytomegalovirus enhancers, polyoma enhancers, and adenovirus enhancers. Enhancer sequences from mammalian systems are also commonly used, such as the mouse immunoglobulin heavy chain enhancer.
  • Methods for introduction of nonviral vectors into an animal Nonviral vectors encoding products useful in gene therapy can be introduced into an animal by means such as lipofection, biolistics, virosomes, liposomes, immunoliposomes, polycatiomnucleic acid conjugates, naked DNA injection, artificial virions, agent-enhanced uptake of DNA, ex vivo transduction.
  • Lipofection is described in e.g., US Patent Nos. 5,049,386, 4,946,787; and 4,897,355) and lipofection reagents are sold commercially (e.g., TRANSFECTAMTM and LIPOFECTINTM).
  • Cationic and neutral lipids that are suitable for efficient receptor-recognition lipofection of polynucleotides include those of Feigner, WO 91/17424, WO 91/16024.
  • Naked DNA genetic vaccines are described in, for example, US Patent No. 5,589,486.
  • Multicomponent Genetic Vaccines Use of two or more separate genetic vaccine components for immunization, providing a means for eliciting differentiated responses in different cell types.
  • the invention provides multicomponent genetic vaccines that are designed to obtain an optimal immune response upon administration to a mammal.
  • two or more separate genetic vaccine components are used for immunization.
  • they are in the same formulation.
  • Each component can be optimized for particular functions that will occur in some cells and not in others, thus providing a means for eliciting differentiated responses in different cell types.
  • mutually incompatible consequences are derived from use of one plasmid, those activities are separated into different vectors that will have different fates and effects in vivo.
  • Genetic vaccines are ideal for the formulation of several biologically active entities into one preparation.
  • the vectors can be all of the same chemical type so there is no incompatibility of this nature, and can all be manufactured by the same chemical and/or biological processes.
  • the vaccine preparation can consist of a defined molar ratio of the separate vector components that can be formulated exactly and repeatedly.
  • AR Genetic vaccine vector component "AR” is designed to provide optimal release of antigen in a form that will be recognized by antigen presenting cells (APC) and taken up by those cells for efficient intracellular processing and presentation to T helper (T H ) cells.
  • APC antigen presenting cells
  • T H T helper cells
  • AR plasmids typically have one or more of the following properties, each of which can be optimized using the stochastic (e.g. polynucleotide shuffling & interrupted synthesis) and non-stochastic polynucleotide reassembly methods of the invention.
  • stochastic e.g. polynucleotide shuffling & interrupted synthesis
  • non-stochastic polynucleotide reassembly methods of the invention Optimal plasmid binding to and uptake by the chosen antigen expressing cells (e.g., myocytes for intramuscular immunization or epithelial cells for mucosal immunization)
  • Optimal vector binding to the target cell includes not only the concept of very avid binding and subsequent intemalization into target cells, but relative inability to bind to and enter other cells. Optimization of this ratio of desired binding to undesired binding will significantly increase the number of target cells transfected.
  • This property can be optimized using stochastic (e.g. polynucleotide shuffling & interrupted synthesis) and non-stochastic polynucleotide reassembly according to the present invention as described herein. For example, variant vector component sequences obtained by stochastic (e.g.
  • polynucleotide shuffling & interrupted synthesis and non-stochastic polynucleotide reassembly, combinatorial assembly of vector components, insertion of random oligonucleotide sequences, and the like, can first be selected for those that bind to target cells, after which this population of cells is depleted for those that bind to other cells.
  • Vector components for targeting genetic vaccine vectors to particular cell types, and methods of obtaining improved targeting, are described in
  • the present invention provides methods by which one can obtain genetic vaccine components that are optimal for such properties.
  • cell-specific promoters are used that only allow transcription of the genes when the vector is within the nucleus of the target cell type. In this case, specificity is derived not only from selective vector entry into target cells.
  • the methods of the invention are used to obtain optimal 3' and 5' non-translated regions of the mRNA. (d) optimal translation of the mRNA.
  • the stochastic (e.g. polynucleotide shuffling & interrupted synthesis) and non- stochastic polynucleotide reassembly methods are used to obtain optimized recombinant sequences which exhibit optimal ribosome binding and assembly of translational machinery, plus optimal codon preference.
  • optimal antigen structure for efficient uptake by APC Extracellular antigen is taken up by APC by at least five non-exclusive mechanisms. One mechanism is sampling of the external fluid phase by micropinocytosis and internalization of a vesicle.
  • the first mechanism has, as far as is presently known, no structural requirements for an antigen in the fluid phase and is therefore not relevant to considerations of designing antigen structure.
  • a second mechanism involves binding of antigen to receptors on the APC surface; such binding occurs according to rules that are only now being studied (these receptors are not immunoglobulin family members and appear to represent several families of proteins and glycoproteins capable of binding different classes of extracellular proteins/glycoproteins). This type of binding is followed by receptor- mediated internalization, also in a vesicle. Because this mechanism is poorly understood at present, elements of antigen design cannot be incorporated in a rational design process. However, application of stochastic (e.g.
  • polynucleotide shuffling & interrupted synthesis and non- stochastic polynucleotide reassembly methods, an empirical approach of selection of variant DNA molecules most successful at entry into APC, can select for variants that are improved throughout this mechanism.
  • the other three mechanisms all relate to specific antibody recognition of the extracellular antigen.
  • the first mechanism involves immunoglobulin- mediated recognition of the specific antigen via IgG that is bound to Fc receptors on the cell surface.
  • APC such as monocytes, macrophages and dendritic cells can be decorated with surface membrane IgG of diverse specificities. In a primary response, this mechanism will not be operative.
  • IgG on the surface of APC can specifically bind extracellular antigen and mediate uptake of the bound antigen into an intracellular endosomal compartment.
  • Another mechanism involves binding to clonally-derived surface membrane immunoglobulin which is present on each B cells (IgM in the case of primary B cells and
  • B cells are efficient APC.
  • Extracellular antigen can bind specifically to surface lg and be internalized and processed in a membrane compartment for presentation on the B cell surface. Finally, extracellular antigen can be recognized by specific soluble immunoglobulin (IgM in the case of a primary immunization and IgG in the previously immunized animals). Complexing with lg will elicit binding to the surface of APC (via Fc receptor recognition in the case of IgG) and internalization.
  • IgM specific soluble immunoglobulin
  • APC via Fc receptor recognition in the case of IgG
  • Antibodies can recognize linear protein epitopes as well as conformational epitopes determined by the three dimensional structure of the protein antigen.
  • Protective antibodies that will recognize an extracellular virus or bacterial pathogen and by binding to its surface prevent infection or mediate its immune destruction (complement mediated lysis, immune complex formation and phagocytosis) are almost exclusively generated against conformational determinants on the proteins with native structure displayed on the surface of the pathogen.
  • T helper cells after intracellular processing of the antigen and presentation of degradation peptides in the context of MHC Class U.
  • This T help will allow selective proliferation of the relevant B cells with consequent mutation of antibody and antigen driven selection for antibodies with increased specificity, as well as antibody class switching.
  • Native protein conformation includes appropriate protein folding, glycosylation and any other post- translational modifications necessary for optimal reactivity with the receptors (immunoglobulin and possibly non-immunoglobulin) on APC.
  • the structure (and sequence) can be optimized for increased protein stability outside the expressing cell, until the time when it is recognized by immune cells, including APCs.
  • the reassembly (&/or one or more additional directed evolution methods described herein) and screening methods of the invention can be used to optimize the antigen structure (and sequence) for subsequent processing after uptake by APC so that intracellular processing results in derivation of the required peptide fragments for presentation on Class I or Class II on APC and desired immune responses. ( ) optimal partitioning of the nascent antigen into the desired subcellular compartment or compartments.
  • a variation on items (f) and (g) is to design the expression of the antigen within the cytoplasm of the factory cell followed by lysis of that cell to release soluble antigen.
  • Cell death can be engineered by expression on the same genetic vaccine vector of an intracellular protein that will elicit apoptosis. In this case, the timing of cell death is balanced with the need for the cell to produce antigen, as well as the potential deleterious effect of killing some cells in a designed process.
  • items (a) -(h) lead to a variety of scenarios for the optimizing the longevity and extent of antigen expression. It is not always desirable that the antigen be expressed for the longest time at the highest level. In certain clinical applications, it will be important to have antigen expression that is short time-low expression, short time-high expression, long time-low expression, long time-high expression or somewhere in between.
  • Plasmid AR can be designed to express one or more variants of a single antigen gene or several quite different targets for immunization. Methods for obtaining optimized antigens for use in genetic vaccines are described herein. Multiple antigens can be expressed from a monocistronic or multicistronic form of the vector.
  • CTL-DC Vector Components
  • CTL-LC Vector Components
  • CTL-MM Vector Components
  • CTL-DC cytotoxic CD8 + lymphocytes
  • CTL-LC dendritic cells
  • CTL-LC Langerhan's cells
  • CTL-MM monocytes and macrophages
  • Cells transfected with CTL vector components can be considered as the direct activators of this arm of specific immunity that is usually critically important for protection against viral diseases.
  • CTL vector components are typically designed to have one or more of the following properties, each of which can be optimized using the stochastic (e.g. polynucleotide shuffling & interrupted synthesis) and non-stochastic polynucleotide reassembly methods of the invention:
  • the chosen antigen presenting cells e.g., dendritic cells, monocytes/macrophages, Langerhan's cells.
  • CTL series vectors do not bind to or enter cells that are chosen to be the extracellular antigen expression host via AR vectors.
  • This separation of functions is critical, as the intracellular fate and trafficking of antigen destined for stimulation of immune cells after release from an antigen expressing cell is quite different than the fate of antigen destined to be presented on the cell surface in association with MHC Class I. Ln the former case, antigen is directed via a signal secretion sequence to be delivered intact to the lumen of the rough endoplasmic reticulum (RER) and then secreted.
  • RER rough endoplasmic reticulum
  • antigen is directed to remain in the cytoplasm and there be degraded into peptide fragments by the proteasomal system followed by delivery to the lumen of the RER for association with MHC Class I. These complexes of peptide and MHC Class I are then delivered to the cell surface for specific interaction with CD8 + cytotoxic T cells.
  • Vector components, and methods for obtaining optimized vector components, that are optimized for targeting to desired cell types are described in Optimizing transcription of the antigen gene(s")
  • Optimal 3' and 5' non-translated regions of the mRNA can be obtained using the methods of the invention. (c) optimal translation of the mRNA.
  • stochastic e.g. polynucleotide shuffling & interrupted synthesis
  • non- stochastic polynucleotide reassembly and selection methods of the invention can be used to obtain polynucleotide sequences for optimal ribosome binding and assembly of translational machinery, as well as optimal codon preference.
  • the optimal protein conformation yields appropriate cytoplasmic proteolysis and production of the conect peptides for presentation on MHC Class I and elicitation of the desired specific CTL responses, rather than a conformation that will interact with specific antibody or other receptors on the surface of APC.
  • proteolytic cleavages will depend on the nature of protein folding and the nature of proteases either in the cytoplasm or in the proteasome.
  • Vector CTL can be designed to express one or more variants of a single antigen gene or several different targets for immunization. Multiple optimized antigens can be expressed from a monocistronic or multicistronic form of the vector.
  • Target cells can be either the predominant cell type in the immunized tissue or immune cells such dendritic cells (M-DC),
  • M-LC Langerhan's cells
  • M-MM monocytes & macrophages
  • These vectors direct simultaneous expression of optimal levels of several immune cell “modulators” (cytokines, growth factors, and the like) such that the immune response is of the desired type, or combination of types, and of the desired level.
  • Cells transfected with M vectors can be considered as the directors of the nature of the vaccine immune response (CTL versus THI versus T H 2 versus NK cell, etc.) and its magnitude.
  • CTL THI versus T H 2 versus NK cell, etc.
  • the properties of these vectors reflect the nature of the cell in which the vectors are designed to operate. For example, the vectors are designed to bind to and enter the desired cell type, and/or can have cell-specific regulated promoters that drive transcription in the desired cell type.
  • the vectors can also be engineered to direct maximal synthesis and release of the cell modulator proteins from the target cells in the desired ratio.
  • "M" genetic vaccine vectors are typically designed to have one or more of the following properties, each of which can be optimized using the stochastic (e.g. polynucleotide shuffling & interrupted synthesis) and non-stochastic polynucleotide reassembly methods of the invention:
  • Suitable expressing cells include, for example, muscle cells, epithelial cells or other dominant (by number) cell types in the target tissue, antigen presenting cells (e.g. dendritic cells, monocytes/macrophages, Langerhans cells). This is a critical property which differentiates M series vectors from those designed to bind to and enter other cells.
  • Optimal 3' and 5' non-translated regions of the mRNA can be obtained using the methods of the invention.
  • stochastic e.g. polynucleotide shuffling & interrupted synthesis
  • non- stochastic polynucleotide reassembly and selection methods of the invention can be used to obtain polynucleotide sequences for optimal ribosome binding and assembly of translational machinery, as well as optimal codon preference.
  • Anchored modulator can be retained on the surface of the synthesizing cell by, for example, a hydrophobic tail and phosphoinositol glycan linkage.
  • the optimal protein conformation is that which allows extracellular modulator and/or cell membrane anchored modulator to interact with the relevant receptor.
  • T H response e.g., production of EL-2 and/or EFN ⁇
  • T H 2 response e.g., EL-4, EL-5, EL-13
  • Vector M can be designed to express one or more modulators.
  • Optimized immunomodulators, and methods for obtaining optimized immunomodulators, are described herein. These optimized immunomodulatory sequences are particularly suitable for use as components of the multicomponent genetic vaccines of the invention.
  • Multiple modulators can be expressed from a monocistronic or multicistronic form of the vector.
  • Vectors "CK" Designed To Direct Release Of Chemokines
  • CK Genetic vaccine vectors designated "CK” are designed to direct optimal release of chemokines from target cells.
  • Target cells can be either the predominant cell type in the immunized tissue, or can be immune cells such as dendritic cells (CK-DC), Langerhan's cells (CK-LC), or monocytes and macrophages (CK-MM).
  • CK-DC dendritic cells
  • CK-LC Langerhan's cells
  • CK-MM monocytes and macrophages
  • the vectors are designed to bind to and enter the desired cell type, and/or can have cell-specific regulated promoters that drive transcription in the desired cell type.
  • the vectors are also engineered to direct maximal synthesis and release of the chemokines from the target cells in the desired ratio. Genetic vaccine components, and methods for obtaining components, that provide optimal release of chemokines are described herein.
  • CK vectors are typically designed to have one or more of the following properties, each of which can be optimized using the stochastic (e.g. polynucleotide shuffling & interrupted synthesis) and non-stochastic polynucleotide reassembly methods of the invention:
  • Suitable cells include, for example, muscle cells, epithelial cells, or cell types that are dominant (by number) in the particular tissue of interest. Also suitable are antigen presenting cells (e.g. dendritic cells, monocytes and macrophages, Langerhans cells). This is a critical property which differentiates CK series vectors from those designed to bind to and enter other cells.
  • promoters, enhancers, introns, and the like can be optimized according to the methods of the invention.
  • Optimal 3' and 5' non-translated regions of the mRNA can be obtained using the methods of the invention.
  • stochastic e.g. polynucleotide shuffling & interrupted synthesis
  • non- stochastic polynucleotide reassembly and selection methods of the invention can be used to obtain polynucleotide sequences for optimal ribosome binding and assembly of translational machinery, as well as optimal codon preference.
  • the optimal protein conformation is that which allows extracellular chemokine/cell membrane anchored chemokine to interact with the relevant receptor.
  • CTL T H cells
  • B cells monocytes/macrophages
  • eosinophils eosinophils
  • neutrophils as appropriate.
  • Vector CK can be designed to express one or more chemokines. Multiple chemokines can be expressed from a monocistronic or multicistronic form of the vector. Other Vectors
  • genetic vaccines which contain one or more additional component vector moieties are also provided by the invention.
  • the genetic vaccine can include a vector that is designed to specifically enter dendritic cells and Langerhans cells, and will migrate to the draining lymph nodes.
  • This vector is designed to provide for expression of the target antigen(s). as well as a cocktail of cytokines and chemokines relevant to elicitation of the desired immune response in the node
  • the vector can be optimized for relatively long lived expression of the target antigen so that stimulation of the immune system is prolonged at the node.
  • Another example is a vector that specifically modulates MHC expression in B cells.
  • Such vectors are designed to specifically bind to and enter B cells, cells either resident in the injection site or attracted into the site. Within the B cell, this vector directs the association of antigen peptides derived from specific uptake of antigen into the endocytic compartment of the cell to either association with Class I or Class EC, hence directing the elicitation of specific immunity via CD4 + T helper cells or CD8 + cytotoxic lymphocytes. Numerous means exist for this intracellular direction of the fate of processed peptide that are discussed herein.
  • Examples of molecules that direct Class I presentation include tapasin, TAP-1 and TAP-2 (Koopman et al. (1997) Cun. Opin. Immunol. 9: 80-88), and those affecting Class El presentation include, for example, endosomal/lysosomal proteases (Peters (1997) Cun. Opin. Immunol. 9: 89-96). Genetic vaccine components, and methods for obtaining components, that provide optimized Class I presentation are described herein.
  • An optimal DNA vaccine could, for example, combine an AR vector (antigen release), a CTL-DC vector (CTL activation via dendritic cell presentation of antigen peptide on MHC Class I), an M-MM vector for release of EL- 12 and EFNg from resident tissue macrophages, and a CK vector for recruitment of T H cells into the immunization site.
  • Directed evolution aid the following DNA vaccination goals
  • DNA vaccination can be used for diverse goals that can include the following, among others: • stimulation of a CTL response and/or humoral response ready to react rapidly and aggressively against an invading bacterial or viral pathogen at some time in the distant future
  • a multicomponent format allows the generation of a portfolio of DNA vaccine vectors, some of which will be reconstructed on each occasion (e.g., those vectors containing antigen) while others will be used as well characterized and understood reagents for numerous different clinical applications (e.g., the same chemokine-expressing vector can be used in different situations).
  • Recombinant nucleic acid libraries that are obtained by the methods described herein are screened to identify those DNA segments that have a property which is desirable for genetic vaccination.
  • the particular screening assay employed will vary, as described below, depending on the particular property for which improvement is sought.
  • the experimentally evolved (e.g. by polynucleotide reassembly &/or polynucleotide site- saturation mutagenesis) nucleic acid library is introduced into cells prior to screening. If the stochastic (e.g.
  • polynucleotide shuffling & interrupted synthesis and non-stochastic polynucleotide reassembly format employed is an in vivo format, the library of recombinant DNA segments generated already exists in a cell. If the sequence reassembly (&/or one or more additional directed evolution methods described herein) is performed in vitro, the recombinant library can be introduced into the desired cell type before screening/selection. The members of the recombinant library can be linked to an episome or virus before introduction or can be introduced directly. Cell types
  • Cells of particular interest include many bacterial cell types that are used to deliver vaccines or vaccine antigens (Courvalin et al.(1995) C. R. Acad. Sci. 11118: 1207- 12), both gram- negative and gram-positive, such as salmonella (Attridge et al. (1997) Vaccine 15: 155-62), clostridium. (Fox et al. (1996) Gene Ther. 3: 173-8), lactobacillus, shigella (Sizemore et al. (1995) Science 270: 299-302), E.
  • the library is amplified in a first host, and is then recovered from that host and introduced to a second host more amenable to expression, selection, or screening, or any other desirable parameter.
  • the manner in which the library is introduced into the cell type depends on the DNA-uptake characteristics of the cell type, e.g., having viral receptors, being capable of conjugation, or being naturally competent. If the cell type is unsusceptible to natural and chemical-induced competence, but susceptible to electroporation, one would usually employ electroporation.
  • biolistics If the cell type is unsusceptible to electroporation as well, one can employ biolistics.
  • the biolistic PDS-1000 Gene Gun (Biorad, Hercules, CA) uses helium pressure to accelerate DNA-coated gold or tungsten microcarriers toward target cells. Competent or Potentially Competent Tissue
  • the process is applicable to a wide range of tissues, including plants, bacteria, fungi, algae, intact animal tissues, tissue culture cells, and animal embryos.
  • Novel methods for making cells competent are described in International Patent Application PCT/US97/04494 (Publ. No. W097/35957).
  • the cells are optionally propagated to allow expression of genes to occur. Identifying cells that contain a vector through inclusion of a selectable marker gene In many assays, a means for identifying cells that contain a particular vector is necessary.
  • Genetic vaccine vectors of all kinds can include a selectable marker gene. Under selective conditions, only those cells that express the selectable marker will survive.
  • Selectable Marker Genes Examples of suitable markers include, the dihydrofolate reductase gene (DHFR), the thymidine kinase gene (TK), or prokaryotic genes conferring drug resistance, gpt (xanthine- guanine phosphoribosyltransferase, which can be selected for with mycophenolic acid; neo (neomycin phosphotransferase), which can be selected for with G418, hygromycin, or puromycin; and DHFR (dihydrofolate reductase), which can be selected for with methotrexate (Mulligan � Southern & Berg (1982) J Mol. Appl. Genet. 1: 327). Identifying cells that contain a vector through inclusion of a screenable marker gene
  • a genetic vaccine vector can include a screenable marker which, when expressed, confers upon a cell containing the vector a readily identifiable phenotype.
  • gene that encodes a cell surface antigen that is not normally present on the host cell is suitable.
  • the detection means can be, for example, an antibody or other ligand which specifically binds to the cell surface antigen.
  • suitable cell surface antigens include any CD (cluster of differentiation) antigen (GDI to CD163) from a species other than that of the host cell which is not recognized by host-specific antibodies.
  • Other examples include green fluorescent protein (GFP, see, e.g., Chalfie et al.
  • the recovered vector molecules can be amplified in, for example, E. coli and/ or by PCR in vitro.
  • the PCR amplification can involve further polynucleotide (e.g.
  • the invention provides methods for identifying those vectors in a genetic vaccine population that exhibit not only the desired tissue localization and longevity of DNA integrity in vivo, but retention of maximal antigen expression (or expression of other genes such as cytokines, chemokines, cell surface accessory molecules, MHC, and the like).
  • the methods involve in vitro identification of cells which express the desired molecule using cells purified from the tissue of choice, under conditions that allow recovery of very small numbers of cells and quantitative selection of those with different levels of antigen expression as desired.
  • the recombinant library represents a population of vectors that differ in known ways (e.g., a combinatorial vector library of different functional modules), or has randomly generated diversity generated either by insertion of random nucleotide stretches, or has been experimentally evolved (e.g. by polynucleotide reassembly &/or polynucleotide site-saturation mutagenesis) in vitro to introduce low level mutations across all or part of the vector.
  • the invention method involves selection for expression of cell surface-localized antigen.
  • the antigen gene is engineered in the vaccine vector library such that it has a region of amino acids which is targeted to the cell membrane.
  • the region can encode a hydrophobic stretch of C-terminal amino acids which signals the attachment of a phosphoinositol-glycan (PIG) terminus on the expressed protein and directs the protein to be expressed on the surface of the transfected cell.
  • PAG phosphoinositol-glycan
  • an antigen that is naturally a transmembrane protein e.g., a surface membrane protein on pathogenic viruses, bacteria, protozoa or tumor cells
  • the extracellular domain can be engineered to be in fusion with the C- terminal sequence for signaling PIG-linkage.
  • the protein can be expressed in toto relying on the signaling of the host cell to direct it efficiently to the cell surface.
  • the antigen for expression will have an endogenous PIG terminal linkage (e.g., some antigens of pathogenic protozoa). Collection, purification, identification and separation of target cells
  • the vector library is delivered in vivo and, after a suitable interval of time tissue and/or cells from diverse target sites in the animal are collected.
  • Cells can be purified from the tissue using standard cell biological procedures, including the use of cell specific surface reactive monoclonal antibodies as affinity reagents. It is relatively facile to purify isolated epithelial cells from mucosal sites where epithelium may have been inoculated or myoblasts from muscle. In some embodiments, minimal physical purification is performed prior to analysis. It is sometimes desirable to identify and separate specific cell populations from various tissues, such as spleen, liver, bone manow, lymph node, and blood.
  • Blood cells can be fractionated readily by FACS to separate B cells, CD4 + or CD8 + T cells, dendritic cells, Langerhans cells, monocytes, and the like, using diverse fluorescent monoclonal antibody reagents. Identification and purification of cells expressing the antigen Those cells expressing the antigen can be identified with a fluorescent monoclonal antibody specific for the C-terminal sequence on PIG-linked forms of the surface antigen. FACS analysis allows quantitative assessment of the level of expression of the conect form of the antigen on the cell population. Cells expressing the maximal level of antigen are sorted and standard molecular biology methods used to recover the plasmid DNA vaccine vector that confened this reactivity.
  • one monoclonal antibody cannot define all aspects of conect folding of the target antigen, one can minimize the possibility of an antigen which reacts with high affinity to the diagnostic antibody but does not yield the conect conformation as defined by that in which the antigen is found on the surface of the target pathogen or as secreted from the target pathogen.
  • One way to minimize this possibility is to use several monoclonal antibodies, each known to react with different conformational epitopes in the conectly folded protein, in the selection process. This can be achieved by secondary FACS sorting for example.
  • the enriched plasmid population that successfully expressed sufficient of the antigen in the conect body site for the desired time is then used as the starting population for another round of selection, incorporating gene reassembling (optionally in combination with other directed evolution methods described herein) to expand the diversity.
  • gene reassembling optionally in combination with other directed evolution methods described herein to expand the diversity.
  • This method can also provide the best in vivo selected vectors that express immune accessory molecules that one may wish to incorporate into DNA vaccine constructs.
  • the accessory protein B7.1 or B7.2 in antigen- presenting- cells APC
  • APC antigen-presenting- cells
  • the invention also provides methods to identify plasmids in a genetic vaccine vector population that are optimal in secretion of soluble proteins that can affect the qualitative and quantitative nature of an elicited immune response.
  • the methods are useful for selecting vectors that are optimal for secretion of particular cytokines, growth factors and chemokines.
  • the goal of the selection is to determine which particular combinations of cytokines, chemokines and growth factors, in combination with different promoters, enhancers, polyA tracts, introns, and the like, elicits the required immune response in vivo.
  • Genes encoding the polypeptides are typically present in the vaccine vector library in combination with optimal signal secretion sequences (proteins are secreted from the cells.)
  • Combinations of the genes for the soluble proteins of interest can be present in the vectors; transcription can be either from a single promoter, or the genes can be placed in multicistronic anangements.
  • transcription can be either from a single promoter, or the genes can be placed in multicistronic anangements.
  • the genes encoding the polypeptides are present in the vaccine vector library in combination with optimal signal secretion sequences, such that the expressed proteins are secreted from the cells.
  • the first step in these methods is to generate vectors that are capable of secreting high (or in some case low) levels of different combinations of soluble factors in vitro and that will express those factors for a short or long time as desired.
  • This method allows one to select for and retain an inventory of plasmids which can be characterized by known patterns of soluble protein expression in known tissues for a known time. These vectors can then be tested individually for in vivo efficacy, after being placed in combination with the genetic vaccine antigen in an appropriate expression construct. Delivery of vector library and subsequent collection, testing, and purification using FACS sorting, affinity panning, rosetting, or magnetic bead separation to separate cell populations prior to identification
  • the vector library is delivered to a test animal and, after a chosen interval of time, tissue and/or cells from diverse sites on the animal are collected.
  • Cells are purified from the tissue using standard cell biological procedures, which often include the use of cell specific surface reactive monoclonal antibodies as affinity reagents.
  • cell specific surface reactive monoclonal antibodies as affinity reagents.
  • physical purification of separate cell populations can be performed prior to identification of cells which express the desired protein.
  • the target cells for expression of cytokines will most usually be APC or B cells or T cells rather than muscle cells or epithelial cells. In such cases FACS sorting by established methods can be used to separate the different cell types.
  • the different cell types described above may also be separated into relatively pure fractions using affinity panning, rosetting or magnetic bead separation with panels of existing monoclonal antibodies known to define the surface membrane phenotype of murine immune cells. Identifying and selecting purified cells through visual inspection or flow cytometry for use in another round of selection incorporating gene reassembling (optionally in combination with other directed evolution methods described herein) to expand the diversity.
  • Purified cells are plated onto agar plates under conditions that maintain cell viability.
  • Cells expressing the required conformational structure of the target antigen are identified using conformationally-dependent monoclonal antibodies that are known to react specifically with the same structure as expressed on the target pathogen. Release of the relevant soluble protein from the cells is detected by incubation with monoclonal antibody, followed by a secondary reagent that gives a macroscopic signal (gold deposition, color development, fluorescence, luminescence).
  • Cells expressing the maximal level of antigen can be identified by visual inspection, the cell or cell colony picked and standard molecular biology methods used to recover the plasmid DNA vaccine vector that confened this reactivity.
  • flow cytometry can be used to identify and select cells harboring plasmids that induce high levels of gene expression.
  • the enriched plasmid population that successfully expressed sufficient of the soluble factor in the conect body site for the desired time is then used as the starting population for another round of selection, incorporating gene reassembling (optionally in combination with other directed evolution methods described herein) to expand the diversity, if further improvement is desired.
  • gene reassembling optionally in combination with other directed evolution methods described herein
  • Flow cytometry provides a means to efficiently analyze the functional properties of millions of individual cells.
  • the cells are passed through an illumination zone, where they are hit by a laser beam; the scattered light and fluorescence is analyzed by computer-linked detectors.
  • Flow cytometry provides several advantages over other methods of analyzing cell populations. Thousands of cells can be analyzed per second, with a high degree of accuracy and sensitivity. Gating of cell populations allows multiparameter analysis of each sample. Cell size, viability, and morphology can be analyzed without the need for staining. When dyes and labeled antibodies are used, one can analyze DNA content, cell surface and intracytoplasmic proteins, and identify cell type, activation state, cell cycle stage, and detect apoptosis.
  • Screening for improved vaccination properties using various in vitro testing methods such as screening for improved adjuvant activity and immunostimulatory properties.
  • Genetic vaccine vectors and vector modules can be screened for improved vaccination properties using various in vitro testing methods that are known to those of skill in the art.
  • the optimized genetic vaccines can be tested for their effect on induction of proliferation of the particular lymphocyte type of interest, e.g., B cells, T cells, T cell lines, and T cell clones.
  • This type of screening for improved adjuvant activity and immunostimulatory properties can be performed using, for example, human or mouse cells. Screening for improved vaccination properties using various in vitro testing methods such as
  • a library of genetic vaccine vectors e.g. obtained either from polynucleotide reassembly (optionally in combination with other directed evolution methods described herein), or of vectors harboring genes encoding cytokines, costimulatory molecules etc.
  • cytokine production e.g., EL-2, EL-4, EL-5, EL-6, EL- 10, EL- 12, EL- 13, EL- 15, IFN- ⁇ , TNF- ⁇
  • B cells T cells, monocytes/macrophages, total human PBMC, or
  • Cytokines can be measured by ELISA or and cytoplasmic cytokine staining and flow cytometry (single-cell analysis). Based on the cytokine production profile, one can screen for alterations in the capacity of the vectors to direct T H 1/ T H 2 differentiation
  • Induction of APC activation can be detected based on changes in surface expression levels of activation antigens, such as B7-1 (CD80), 137-2 (CD86), MHC class I and R, CD 14, CD23, and Fc receptors, and the like. Analyzing genetic vaccine vectors for their capacity to induce T cell activation through
  • genetic vaccine vectors are analyzed for their capacity to induce T cell activation. More specifically, spleen cells from injected mice can be isolated and the capacity of cytotoxic T lymphocytes to lyse infected, autologous target cells is
  • T helper cell differentiation is analyzed by measuring proliferation or production of T H I (EL-2 and EFN- ⁇ ) and T H 2 (EL-4 and EL-5) cytokines by ELISA and directly in CD4 + T cells by cytoplasmic cytokine staining and flow cytometry.
  • T H I EL-2 and EFN- ⁇
  • T H 2 EL-4 and EL-5
  • cytokines cytokines by ELISA and directly in CD4 + T cells by cytoplasmic cytokine staining and flow cytometry.
  • Testing for ability to induce humoral immune responses with assays using, for example, so peripheral B lymphocytes from immunized individuals or other assays involving detection of antigen expression by the target cells
  • Genetic vaccines and vaccine components can also be tested for ability to induce humoral immune responses, as evidenced, for example, by induction of B cell production of antibodies specific for an antigen of interest.
  • assays can be conducted using, for example, peripheral B lymphocytes from immunized individuals. Such assay methods are known to those of skill in the art. Other assays involve detection of antigen expression by the target cells. For example, FACS selection provides the most efficient method of identifying cells which produce a desired antigen on the cell surface. Another advantage of FACS selection is that one can sort for different levels of expression; sometimes lower expression may be desired. Another method involves panning using monoclonal antibodies on a plate. This method allows large numbers of cells to be handled in a short time, but the method only selects for highest expression levels. Capture by magnetic beads coated with monoclonal antibodies provides another method of identifying cells which express a particular antigen. Screening for ability to inhibit proliferation of tumor cell lines in vitro
  • Genetic vaccines and vaccine components that are directed against cancer cells can be screened for their ability to inhibit proliferation of tumor cell lines in vitro. Such assays are known in the art.
  • An indication of the efficacy of a genetic vaccine against, for example, cancer or an autoimmune disorder is the degree of skin inflammation when the vector is injected into the skin of a patient or test animal. Strong inflammation is conelated with strong activation of antigen-specific T cells. Improved activation of tumor- specific T cells may lead to enhanced killing of the tumors.
  • autoantigens one can add immunomodulators that skew the responses towards T H 2.
  • Skin biopsies can be taken, enabling detailed studies of the type of immune response that occurs at the sites of each injection (in mice large numbers of injections/vectors can be analyzed)
  • Other suitable screening methods can involve detection of changes in expression of cytokines, chemokines, accessory molecules, and the like, by cells upon challenge by a library of genetic vaccine vectors.
  • screening involves expressing the recombinant peptides or polypeptides encoded by the experimentally generated polynucleotides of the library as fusions with a protein that is displayed on the surface of a replicable genetic package.
  • phage display can be used. See, e.g., Cwirla et al., Proc. Natl. Acad. Sci. USA 87: 6378-6382 (1990); Devlin et al., Science 249: 404-406 (1990), Scott � Ladner et al., US 5,571,698.
  • Other replicable genetic packages include, for example, bacteria, eukaryotic viruses, yeast, and spores. Purification and in vitro analysis of recombinant nucleic acids and polypeptides
  • the resulting library of experimentally generated polynucleotides can be subjected to purification and preliminary analysis in vitro, in order to identify the most promising candidate recombinant nucleic acids.
  • the assays can be practiced in a high-throughput format. For example, to purify individual experimentally evolved (e.g.
  • clones can robotically picked into 96- well formats, grown, and, if desired, frozen for storage.
  • the experimentally evolved (e.g. by polynucleotide reassembly &/or polynucleotide site-saturation mutagenesis) antigen-encoding polynucleotides are assayed as genetic vaccines.
  • Genetic vaccine vectors containing the experimentally evolved (e.g. by polynucleotide reassembly &/or polynucleotide site-saturation mutagenesis) antigen sequences can be prepared using robotic colony picking and subsequent robotic plasmid purification. Robotic plasmid purification protocols are available that allow purification of 600-800 plasmids per day. The quantity and purity of the DNA can also be analyzed in 96- well plates, for example.
  • the amount of DNA in each sample is robotically normalized, which can significantly reduce the variation between different batches of vectors.
  • proteins and/or nucleic acids are picked and purified as desired, they can be subjected to any of a number of in vitro analysis methods.
  • screenings include, for example, phage display, flow cytometry, and ELISA assays to identify antigens that are efficiently expressed and have multiple epitopes and a proper folding pattern.
  • the libraries may also be screened for reduced toxicity in mammalian cells.
  • monoclonal antibodies can be raised against various regions of immunogenic proteins (Alving et al. (1995) Immunol. Rev. 145: 5).
  • monoclonal antibodies that only recognize one strain of a given pathogen, and by definition, different serotypes of pathogens are recognized by different sets of antibodies.
  • a panel of monoclonal antibodies have been raised against VEE envelope proteins, thus providing a means to recognize different subtypes of the virus (Roehrig and Bolin (1997) J Clin. Microbiol.
  • Such antibodies can be used to enrich recombinant antigens that have epitopes from multiple pathogen strains. Flow cytometry based cell sorting will further allow for the selection of variants that are most efficiently expressed.
  • Phage display provides a powerful method for selecting proteins of interest from large libraries (Bass et al. (1990) Proteins: Struct. Funct. Genet. 8: 309; Lowman and Wells (1991) Methods: A Companion to Methods Enz. 3(3);205-216. Lowman and Wells (1993) J Mol. Biol. 234;564-578).
  • Some recent reviews on the phage display technique include, for example, McGregor (1996) Mol Biotechnol. 6(2): 15 5 -62; Dunn (1996) Cun. Opin. Biotechnol. 7(5):547-53; Hill et al. (1996) Mol Microbiol 20(4):685-92; Phage Display of Peptides and Proteins: A Laboratory Manual. BK.
  • Each phage particle displays a unique variant protein on its surface and packages the gene encoding that particular variant.
  • the experimentally evolved e.g. by polynucleotide reassembly &/or polynucleotide site-saturation mutagenesis
  • genes for the antigens are fused to a protein that is expressed on the phage surface, e.g., gene HI of phage M 13, and cloned into phagemid vectors.
  • a suppressible stop codon separates the genes so that in a suppressing strain of E. coli, the antigen-gulp fusion is produced and becomes incorporated into phage particles upon infection with M 13 helper phage.
  • the same vector can direct production of the unfused antigen alone in a nonsuppressing E. coli for protein purification.
  • the genetic packages most frequently used for display libraries are bacteriophage, particularly filamentous phage, and especially phage M13, Fd and FI. Most work has involved inserting libraries encoding polypeptides to be displayed into either glu or gVIII of these phage forming a fusion protein. See, e.g., Dower, WO 91/19818; Devlin, WO 91/18989; MacCafferty, WO 92/01047 (gene HI); Huse, WO 92/06204; Kang, WO 92/18619 (gene VuT).
  • Such a fusion protein comprises a signal sequence, usually but not necessarily, from the phage coat protein, a polypeptide to be displayed and either the gene HI or gene VEH protein or a fragment thereof.
  • Exogenous coding sequences are often inserted at or near the N-terminus of gene UI or gene VHI although other insertion sites are possible.
  • Eukaryotic viruses can be used to display polypeptides in an analogous manner. For example, display of human heregulin fused to gp70 of Moloney murine leukemia virus has been reported by Han et al., Proc. Natl. Acad. Sci. USA 92: 9747-9751 (1995). Spores can also be used as replicable genetic packages. In this case, polypeptides are displayed from the outer surface of the spore. For example, spores from B. subtilis have been reported to be suitable. Sequences of coat proteins of these spores are provided by Donovan et al., J. Mol. Biol. 196, 1-10 (1987). Cells can also be used as replicable genetic packages.
  • Polypeptides to be displayed are inserted into a gene encoding a cell protein that is expressed on the cells surface.
  • Bacterial cells can include Salmonella typhimurium, Bacillus subtilis, Pseudomonas aeruginosa, Vibrio cholerae, Klebsiella pneumonia, Neisseria gonorrhoeae, Neisseria meningitidis, Bacteroides nodosus, Moraxella bovis, and especially Escherichia coli. Details of outer surface proteins are discussed by Ladner et al., US 5,571,698 and references cited therein. For example, the lam , protein of E. coli is suitable.
  • a basic concept of display methods that use phage or other replicable genetic package is the establishment of a physical association between DNA encoding a polypeptide to be screened and the polypeptide. This physical association is provided by the replicable genetic package, which displays a polypeptide as part of a capsid enclosing the genome of the phage or other package, wherein the polypeptide is encoded by the genome.
  • the establishment of a physical association between polypeptides and their genetic material allows simultaneous mass screening of very large numbers of phage bearing different polypeptides.
  • Phage displaying a polypeptide with affinity to a target bind to the target and these phage are enriched by affinity screening to the target.
  • the identity of polypeptides displayed from these phage can be determined from their respective genomes.
  • polypeptide identified as having a binding affinity for a desired target can then be synthesized in bulk by conventional means, or the polynucleotide that encodes the peptide or polypeptide can be used as part of a genetic vaccine.
  • Variants with specific binding properties are easily enriched by panning with immobilized antibodies.
  • Antibodies specific for a single family are used in each round of panning to rapidly select variants that have multiple epitopes from the antigen families.
  • A-family specific antibodies can be used to select those experimentally evolved (e.g. by polynucleotide reassembly &/or polynucleotide site-saturation mutagenesis) clones that display A-specific epitopes in the first round of panning.
  • a second round of panning with B-specific antibodies will select from the "A" clones those that display both A- and B-specific epitopes.
  • a third round of panning with C- specific antibodies will select for variants with A, B, and C epitopes.
  • a continual selection exists during this process for clones that express well in E. coli and that are stable throughout the selection. Improvements in factors such as transcription, translation, secretion, folding and stability are often observed and will enhance the utility of selected clones for use in vaccine production.
  • Phage ELISA methods can be used to rapidly characterize individual variants. These assays provide a rapid method for quantitation of variants without requiring purification of each protein. Individual clones are anayed into 96-well plates, gown, and frozen for storage. Cells in duplicate plates are infected with helper phage, grown overnight and pelleted by centrifugation. The supematants containing phage displaying particular variants are incubated with immobilized antibodies and bound clones are detected by anti- M13 antibody conjugates. Titration series of phage particles, immobilized antigen, and/or soluble antigen competition binding studies are all highly effective means to quantitate protein binding.
  • Variant antigens displaying multiple epitopes will be further studied in appropriate animal challenge models.
  • Several groups have reported an in vitro ribosome display system for the screening and selection of mutant proteins with desired properties from large libraries. This technique can be used similarly to phage display to select or enrich for variant antigens with improved properties such as broad cross reactivity to antibodies and improved folding (see, e.g., Hanes et al. (1997) Proc. Nat'l. A cad. Sci. USA 94(10):493 7-42; Mattheakis et al. (1994) Proc. Nat 7. Acad. Sci. USA 91(19):9022-6; He et al. (1997) Nucl. Acids Res. (24):5132-4; Nemoto et al. (1997) FEBS Lett. 414(2):405-8).
  • the various display methods and ELISA assays can be used to screen for experimentally evolved (e.g. by polynucleotide reassembly &/or polynucleotide site-saturation mutagenesis) antigens with improved properties such as presentation of multiple epitopes, improved immunogenicity, increased expression levels, increased folding rates and efficiency, increased stability to factors such as temperature, buffers, solvents, improved purification properties, etc. Selection of experimentally evolved (e.g.
  • antigens with improved expression, folding, stability and purification profile under a variety of chromatographic conditions can be very important improvements to incorporate for the vaccine manufacturing process.
  • flow cytometry is a useful technique.
  • Flow cytometry provides a method to efficiently analyze the functional properties of millions of individual cells.
  • Very large numbers (> 10 7 ) of cells can be evaluated in a single vial experiment, and the pool of the best individual sequences can be recovered from the sorted cells.
  • These methods are particularly useful in the case of, for example, Hantaan virus glycoproteins, which are generally very poorly expressed in mammalian cells.
  • This approach provides a general solution to improve expression levels of pathogen antigens in mammalian cells, a phenomenon that is critical for the function of genetic vaccines.
  • polypeptides that are not expressed on the cell surface can be produced by using flow cytometry to analyze polypeptides that are not expressed on the cell surface.
  • the polynucleotide is expressed as a fusion protein that has a region of amino acids which is targeted to the cell membrane.
  • the region can encode a hydrophobic stretch of C-terminal amino acids which signals the attachment of a phosphoinositol- glycan (PIG) terminus on the expressed protein and directs the protein to be expressed on the surface of the transfected cell (Whitehorn et al. (1995) Biotechnology (N Y) 13:1215-9).
  • PAG phosphoinositol- glycan
  • an antigen that is naturally a soluble protein this method will likely not affect the three dimensional folding of the protein in this engineered fusion with a new C-terminus.
  • an antigen that is naturally a transmembrane protein e.g., a surface membrane protein on pathogenic viruses, bacteria, protozoa or tumor cells
  • the extracellular domain can be engineered to be in fusion with the C-terminal sequence for signaling PIG-linkage.
  • the protein can be expressed in toto relying on the signaling of the host cell to direct it efficiently to the cell surface.
  • the antigen for expression will have an endogenous PIG terminal linkage (e.g., some antigens of pathogenic protozoa).
  • Those cells expressing the antigen can be identified with a fluorescent monoclonal antibody specific for the C-terminal sequence on PIG-linked forms of the surface antigen. FACS analysis allows quantitative assessment of the level of expression of the conect form of the antigen on the cell population. Cells expressing the maximal level of antigen are sorted and standard molecular biology methods are used to recover the plasmid DNA vaccine vector that confened this reactivity.
  • An alternative procedure that allows purification of all those cells expressing the antigen is to rosette or pan-purify the cells expressing surface antigen.
  • Rosettes can be formed between antigen expressing cells and erythrocytes bearing covalently coupled antibody to the relevant antigen. These are readily purified by unit gravity sedimentation. Panning of the cell population over petri dishes bearing immobilized monoclonal antibody specific for the relevant antigen can also be used to remove unwanted cells.
  • each well of a microtiter plate can be used to run a separate assay, or, if concentration or incubation time effects are to be observed, every 5 -10 wells can test a single variant.
  • a single standard microtiter plate can assay about 100 (e.g., 96) reactions. Lf 1536 well plates are used, then a single plate can easily assay from about 100 to about 1500 different reactions.
  • library members e.g., cells, viral plaques, or the like
  • solid media e.g., cells, viral plaques, or the like
  • colonies or plaques are identified, picked, and up to 10,000 different mutants inoculated into 96 well microtiter dishes, optionally containing glass balls in the wells to prevent aggregation.
  • the Q-bot does not pick an entire colony but rather inserts a pin through the center of the colony and exits with a small sampling of cells (or viruses in plaque applications).
  • the uniform process of the Q-bot decreases human handling enor and increases the rate of establishing cultures (roughly 10,000/4 hours). These cultures are then shaken in a temperature and humidity controlled incubator.
  • the glass balls in the microtiter plates act to promote uniform aeration of cells dispersal of cells, or the like, similar to the blades of a fermentor.
  • Clones from cultures of interest can be cloned by limiting dilution. Plaques or cells constituting libraries can also be screened directly for production of proteins, either by detecting hybridization, protein activity, protein binding to antibodies, or the like.
  • High throughput screening systems are commercially available (see, e.g., Zymark Corp., Hopkinton, MA; Air Technical Industries, Mentor, OH; Beckman Instruments, Inc. Fullerton, CA; Precision Systems, Inc., Natick, MA, etc.). These systems typically automate 5 entire procedures including all sample and reagent pipetting, liquid dispensing, timed incubations, and final readings of the microplate in detector(s) appropriate for the assay. These configurable systems provide high throughput and rapid start up as well as a high degree of flexibility and customization.
  • Zymark Corp. provides technical bulletins describing screening systems for detecting the modulation of gene transcription, ligand binding, and the like.
  • Microfluidic approaches to reagent manipulation have also been developed, e.g., by Caliper Technologies (Palo Alto, CA).
  • Optical images viewed (and, optionally, recorded) by a camera or other recording device are optionally further processed in any of the embodiments herein, e.g., by digitizing the image and/or storing and analyzing the image on a computer.
  • the signals resulting from assays are florescent, making optical detection approaches appropriate in these instances.

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