EP1572345A2 - Automatisation de conception de proteines pour la conception de bibliotheques de proteines a antigenicite modifiee - Google Patents

Automatisation de conception de proteines pour la conception de bibliotheques de proteines a antigenicite modifiee

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Publication number
EP1572345A2
EP1572345A2 EP02784886A EP02784886A EP1572345A2 EP 1572345 A2 EP1572345 A2 EP 1572345A2 EP 02784886 A EP02784886 A EP 02784886A EP 02784886 A EP02784886 A EP 02784886A EP 1572345 A2 EP1572345 A2 EP 1572345A2
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EP
European Patent Office
Prior art keywords
protein
sequences
sequence
proteins
computational
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
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Application number
EP02784886A
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German (de)
English (en)
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EP1572345A3 (fr
Inventor
Arthur Chirino
Bassil I. Dahiyat
John Desjarlais
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Xencor Inc
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Xencor Inc
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Publication date
Priority claimed from US09/903,378 external-priority patent/US20020119492A1/en
Application filed by Xencor Inc filed Critical Xencor Inc
Priority claimed from PCT/US2002/000165 external-priority patent/WO2003006154A2/fr
Publication of EP1572345A2 publication Critical patent/EP1572345A2/fr
Publication of EP1572345A3 publication Critical patent/EP1572345A3/fr
Withdrawn legal-status Critical Current

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/04—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length on carriers
    • C07K1/047—Simultaneous synthesis of different peptide species; Peptide libraries
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/473—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used alpha-Glycoproteins

Definitions

  • the present invention relates to the use of a variety of computational methods for modulating the immunogenicity of proteins by identifying and then altering potential amino acid sequences that elicit an immune response in a host organism.
  • proteins will be screened for MHC binding motifs, T cell receptor, and B cell receptor binding sequences.
  • Immunoglobulin is the crux of the humoral immune response. As a cell surface receptor on B lymphocytes, immunoglobulin is responsible for instigating cellular responses as diverse as activation, differentiation, and programmed cell death. As secreted in antibody, immunoglobulin can bind a foreign antigen, neutralizing it directly or initiating steps necessary to arm and recruit effector systems such as complement or antibody dependent cell cytolysis by monocytic phagocytes (Fundamental Immunology, 4th edition, W. E. Paul, ed., Lippincott-Raven Publishers, 1999, Chapter 3, pp 37-74).
  • T cells are responsible for cellular immunity. T cells are known to directly kill target cells, to provide help for such killers, to activate other immune system cells (i.e., macrophages), to help B cells make an antibody response, to down modulate the activities of various immune system cells, and to secrete cytokines, chemokines, and other mediators. These activities are often mediated by distinct types of T cells, such as ⁇ : ⁇ T cells, type 1 and type 2 helper cells. Activation of a T cell occurs when it recognizes a particular antigen via receptors displayed on its surface (i.e. T cell receptors or TCRs).
  • T cell receptors i.e. T cell receptors or TCRs
  • ⁇ : ⁇ T cells i.e., CD8+ and CD4+ T cells
  • MHC major histocompatibility complex
  • MHC Major Histocompatibility Complex
  • MHC I or the proteins ingested from the immediate extracellular environment (MHC II) (Fundamental Immunology. 4th edition, W. E. Paul, ed., Lippincott-Raven Publishers, 1999, Chapter 8, pp 263-285).
  • TCR-peptide-MHC recognition regulates immune responses including graft and tumor rejection, anti-viral cytolysis, and the recruitment and control of other immune cells such as antibody producing B cells (Madden, D.R., (1995) Annu. Rev. Immunol., 13:587-622).
  • MHC molecules are highly polymorphic and display allelic variation among different human populations (Buus, supra). Hundreds of MHC class I and II alleles are known, each exhibiting different binding affinities for specific antigenic peptide sequences. The structural basis for this allelic dependent peptide preference has been localized to differences in amino acid residues within the MHC peptide binding pocket (Buus, supra). X-ray crystal structure of MHC class I and II molecules bound to specific antigenic peptides reveal that peptide residues at the N and C termini, i.e., the anchor positions, are in close physical contact with the MHC class I binding pocket, while peptides bound to class II are more extended with additional peptide residues making contact with the MHC class II pocket (Buus, supra).
  • B lymphocytes An important component of humoral immunity is the diverse repertoire of antibodies (i.e., immunoglobulins) produced by B lymphocytes. Antigen contact with a specific B cell triggers the transmembrane signaling function of the B cell antigen receptor (BCR). This, in turn, induces early events in B cell activation, including increased expression of MHC class II molecules and formation of antibody secreting cells.
  • BCR B cell antigen receptor
  • One way to overcome these problems is to use computational methods to design sequences that are more or less immunogenic relative to a target protein, but retain the structural properties to ensure proper folding and activity.
  • the present invention provides methods for generating polypeptides exhibiting enhanced immunogenicity comprising the steps of inputting a target protein backbone structure with variable residue positions into a computer, computationally generating a set of primary variant amino acid sequences by applying at least one protein design algorithm, and computationally analyzing said set of primary variant amino acid sequences by applying a computational immunogenicity filter.
  • the candidate protein is then made and tested to determine if the immunogenicity of the candidate protein is enhanced relative to the target protein. This same method may be used to generate polypeptides exhibiting reduced immunogenicity.
  • the present invention provides methods for generating polypeptides exhibiting enhanced immunogenicity comprising the steps of inputting a target protein backbone structure with variable residue positions into a computer, applying at least one computational immunogenicity filter to generate a set of primary variant amino acid sequences, computationally analyzing said set of primary variant amino acid sequences using at least one protein design algorithm and identifying at least one variant protein with enhanced immunogenicity. This same method may be used to generate polypeptides exhibiting reduced immunogenicity.
  • the present invention provides methods for generating polypeptides exhibiting enhanced immunogenicity comprising the steps of inputting a target protein backbone structure with variable residue positions into a computer, computationally generating a set of primary amino acid sequences by applying at least one protein design algorithm comprising at least one scoring function comprising at least one computational immunogenicity filter and identifying at least one variant protein with enhanced immunogenicity. This same method may be used to generate polypeptides exhibiting reduced immunogenicity.
  • the present invention provides methods for generating a polypeptide exhibiting enhanced immunogenicity comprising the steps of inputting a target protein backbone structure with variable residue positions into a computer, applying in any order at least one computational protein design algorithm and at least one computational immunogenicity filter and identifying at least one variant protein with enhanced immunogenicity. This same method may be used to generate polypeptides exhibiting reduced immunogenicity.
  • the present invention provides methods for eliciting an enhanced immune response in a patient comprising the steps of inputting a target protein backbone structure with variable residue positions into a computer, applying in any order at least one computational protein design algorithm and at least one computational immunogenicity filter, identifying at least one variant protein with enhanced immunogenicity, and administering said variant protein to a patient.
  • the computational design algorithm may be applied prior to or after the application of the computational immunogenicity filter.
  • the computational protein design algorithm comprises the computational filter as a scoring function.
  • the computationally generating step may include applying a computational immunogenicity filter comprising a scoring function for MHC class I motifs, MHC class II motifs, B cell epitopes or T cell epitopes.
  • Other computational steps include a Dead-End Elimination (DEE) computation, a Monte Carlo search, or use of a genetic algorithm.
  • Additional scoring functions include Van der Waals potential scoring function, a hydrogen bond potential scoring function, an atomic solvation scoring function, a secondary structure propensity scoring function and electrostatic scoring function.
  • the polypeptide may comprise one or more immunogenic sequences.
  • the immunogenic sequences may be identical or different.
  • the immunogenic sequences may be selected from the group consisting of MHC Class I motifs, MHC class II motifs, B cell epitopes and T cell epitopes.
  • the target protein is selected from the group comprising Zn-alpha2- glycoprotein, human serum albumin, immunoglobulin G, and other non-immunogenic proteins.
  • Figure 1 depicts the synthesis of a full-length gene and all possible mutations by PCR.
  • Overlapping oligonucleotides corresponding to the full-length gene black bar, Step 1 are synthesized, heated and annealed. Addition of Pfu DNA polymerase to the annealed oligonucleotides results in the 5' -3' synthesis of DNA (Step 2) to produce longer DNA fragments (Step 3). Repeated cycles of heating, annealing (Step 4) results in the production of longer DNA, including some full-length molecules. These can be selected by a second round of PCR using primers (arrows) corresponding to the end of the full-length gene (Step 5).
  • Figure 2 depicts a preferred scheme for synthesizing a library of the invention.
  • the wild-type gene, or any starting gene, such as the gene for the global minima gene, can be used.
  • Oligonucleotides comprising different amino acids at the different variant positions can be used during PCR using standard primers. This generally requires fewer oligonucleotides and can result in fewer errors.
  • Figure 3 depicts an overlapping extension method.
  • the template DNA showing the locations of the regions to be mutated (black boxes) and the binding sites of the relevant primers (arrows).
  • the primers R1 and R2 represent a pool of primers, each containing a different mutation; as described herein, this may be done using different ratios of primers if desired.
  • the variant position is flanked by regions of homology sufficient to get hybridization.
  • three separate PCR reactions are done for step 1.
  • the first reaction contains the template plus oligos F1 and R1.
  • the second reaction contains template plus F2 and R2, and the third contains the template and F3 and R3.
  • the reaction products are shown.
  • Step 2 the products from Step 1 tube 1 and Step 1 tube 2 are taken.
  • Step 3 the purified product from Step 2 is used in a third PCR reaction, together with the product of Step 1 , tube 3 and the primers F1 and R3.
  • the final product corresponds to the full length gene and contains the required mutations.
  • Figure 4 depicts a ligation of PCR reaction products to synthesize the libraries of the invention.
  • the primers also contain an endonuclease restriction site (RE), either blunt, 5' overhanging or 3' overhanging.
  • RE endonuclease restriction site
  • the first reaction contains the template plus oligos F1 and R1.
  • the second reaction contains template plus F2 and R2, and the third contains the template and F3 and R3.
  • the reaction products are shown.
  • the products of step 1 are purified and then digested with the appropriate restriction endonuclease.
  • the digestion products from Step 2, tube 1 and Step 2 tube 2 are ligated togther with DNA ligase (step 3).
  • the products are then amplified in Step 4 using primer F1 and R4.
  • the whole process is then repeated by digesting the amplified products, ligating them to the digested products of Step 2, tube 3, and amplifying the final product by primers F1 and R3. It would also be possible to ligate all three PCR products from Step 1 together in one reaction, providing the two restriction sites (RET and RE2) were different.
  • Figure 5 depicts blunt end ligation of PCR products.
  • the primers such as F1 and R1 do not overlap, but they abut. Again three separate PCR reactions are performed. The products from tube 1 and tube 2 are ligated, and then amplified with outside primers F1 and R4. This product is then
  • the present invention is directed to methods of using computational screening of protein sequence libraries (that can comprise up to 10 80 or more members) to select smaller libraries of protein sequences (that can comprise up to 10 13 members) with altered immunogenicity.
  • a computational filter can be use to identify and replace residues known to elicit a immune response with compensatory residues that maintain the native fold and stability of the protein resulting in a protein that is non-immunogenic or less immunogenic than the starting protein.
  • the computational filter can be applied to modify residues to introduce an antigenic motif to ensure proper folding and stability of the resultant protein.
  • this can be done in one of two general ways. In a first embodiment, computational processing is used to generate a list of variant proteins that have an altered property such as stability. Then a computational filter is applied to select those variants with a high propensity for altered immunogenicity.
  • the computational filter is first applied to generate a list of variants with a propensity for altered immunogenicity, and then computational processing is done to select those variant that are likely to fold or to be stable.
  • a computational filter is used to screen for peptide fragments or amino acid residues that have the potential to bind to MHC class I and class II molecules, T cells and B cells.
  • databases for MHC ligands and peptide motifs can be searched and used to identify potential MHC class I or class II binding sequences (Rammensee, H., et al. (1999) Immunogenetics, 50:213-219).
  • Computational methods are then used to structurally and chemically compensate for amino acid residues involved in binding to MHC molecules.
  • computational methods can be used identify peptide sequences or amino acid residues predicted to elicit an immune response, replace these residues with residues predicted to be non immunogenic and then screen the resulting sequences for sequences that fold properly and are stable.
  • T cell epitopes there are also situations where it is desirable to increase the immunogenicity of a target protein. For example, activating populations of T cells toward a specific epitope has implications for controlling or eliminating viral pathogens or neoplasia.
  • computational methods can be used to introduce T cell epitopes anywhere within the target protein.
  • T cell epitopes also can be introduced into less rigid, less structurally restricted regions of a target protein, such as a loop region. Computational methods can then be used to modify the residues adjacent to the epitope insertion, ensuring energetic compatibility between the native protein and the grafted epitope. Accordingly, the present invention provides methods for modulating the immunogenicity of a target protein.
  • modulating herein is meant that the immune response to a target protein is altered. That is, if a target protein elicits an immune response in a given species, the amino acid sequence of the target protein is changed such that the immune response is either reduced or enhanced.
  • reduced herein is meant that at least one immunological response is decreased relative to the wild-type protein.
  • enhanced herein is meant that at least one immunological response is increased relative to the wild-type protein.
  • immune responses are generally not mounted against autologous circulating proteins, such as immunoglobulins and other serum proteins. Therefore, at least 5% of the sequences that are capable of eliciting a response are altered.
  • At least 10% of the sequences are altered, more preferred is where at least 15% of the sequence are altered, even more preferred is when at least 20% of the sequences are altered, even more preferred is when at least 30% of the sequences are altered, even more preferred is when at least 40% of the sequences are altered, more preferred are where at least 50% of the sequences are altered, and most preferred is when 100% of the sequences are altered.
  • altered immunogenicity is defined within a particular host organism. That is, in a preferred embodiment, target proteins (as defined below) are altered to exhibit altered immunogenicity within a human.
  • Alternate host organisms include, bur are not limited to, rodents, (rats, mice, hamster, guinea pigs, etc.), primates, farm animals (including sheep, goats, pigs, cows, horses, etc.), and domestic animals, (including cats, dogs, rabbits, etc).
  • immunogenicity refers to the ability of a protein to elicit an immune response.
  • the ability of a protein to elicit an immune response depends on the amino acid sequence or sequences within the protein. Immunogenicity includes both the humoral and the cellular component of the immune response as outlined below. Amino acid sequences capable of eliciting an immune response are referred to herein as “immunogenic sequences".
  • immunogenic sequences comprise "MHC binding sites (i.e., MHC binding motifs)", “T cell epitopes” and “B cell epitopes” as outlined below.
  • immunogenicity is sufficiently broad to include the term
  • Antigenicity refers to the ability of a protein by itself to elicit an antibody response when recognized as a non-self molecule.
  • immunogenic response in the context of the invention includes any component of the humoral or cellular immune response.
  • immunogenic response in the context of the invention includes any component of the humoral or cellular immune response.
  • a protein with immunogenic sequences when administered to a human, that protein is subjected to surveillance by both the humoral and cellular arms of the immune system. The immune system will respond to the protein if it is recognized as foreign and if the immune system is not already tolerant to the immunogenic sequence within the protein.
  • immature B cells displaying surface immunoglobulins can bind to one or more sequences within the protein (B cell epitopes) if there is an affinity fit with the individual immunoglobulin and if the B cell epitope is exposed such that the Igs can access the B cell epitope.
  • the process of Ig binding to the protein can, in the presence of suitable cytokines, stimulate the B cell to differentiate and divide to provide soluble forms of the original Ig, which can complex with the protein to facilitate its clearance from an individual.
  • An effective B cell response also includes a parallel T cell response in order to provide the cytokines and other signals necessary to give rise to soluble antibodies.
  • An effective T cell response requires the uptake of a protein fragment by antigen presenting cells (APCs); APCs include B cells or other cells such as macrophages, dendritic cells and other monocytes. The APCs then present the protein complexed with an MHC class II molecule at the cell surface. Such peptide-MHC II complexes can be recognized by helper T cells via the T cell receptor (TCR) and this results in stimulation of the T cells and secretion of cytokines that provide help for B cells in their differentiation to antibody producing cells.
  • TCR T cell receptor
  • an effective primary immune response to an immunogenic protein generally requires a combination of B and T cell responses to B and T cell specific sequences or epitopes.
  • MHC class I molecules gather fragments of proteins derived from infecting pathogens or "self " molecules and then display these fragments at the surface of an APC.
  • the bound peptides are recognized by the TCRs of cytotoxic T lymphocytes and are the primary antigenic determinants of the cellular immune response.
  • modulation of immunogenicity includes identifying peptides that stimulate T cell responses, termed T cell epitopes, changing the sequence of these peptides such that the cellular response to the protein is either reduced or enhanced.
  • modulation of immunogenicity also includes identifying peptides that stimulate B cell responses, termed “B cell epitopes” or “BCRs", changing the sequence of these peptides such that the humoral response to the protein is altered.
  • B cell epitopes identifying peptides that stimulate B cell responses
  • BCRs B cell epitopes
  • a single protein may contain both T and B cell epitopes, such that modification of both may alter both the humoral and cellular arms of the immune system.
  • the target protein is altered such that its MHC I response is altered.
  • MHC class I molecules gather fragments of proteins derived from infecting viruses, intracellular parasites, or self molecules, either normally expressed or deregulated by tumorigenesis, and then displays these molecular fragments at the_cell surface.
  • the cell-bound MHC l-peptide complex exposed on the APC is displayed to T cells.
  • the second characteristic of the MHC I molecule is the ability to interact with TCR which allows the APC bearing a particular MHC-peptide complex to engage an appropriate TCR. This is the first step in the activation of a cellular program leading to cytolysis of the APC as a target and/or the secretion of lymphokines by the T cell.
  • MHC class I molecules show preferential restriction to CD8+ cells.
  • An additional function of MHC class I molecules is to serve as elements for signal transduction to natural killer cells (Fundamental Immunology, 4th edition, W. E. Paul, ed., Lippincott-Raven Publishers, 1999, Chapter 8, pp 263-285).
  • the target protein is altered such that its MHC II response is altered.
  • MHC class II molecules bind peptides derived from the degradation of proteins ingested by MHC II expressing APCs, and displays them at the cell surface for recognition by specific T cells.
  • the MHC II antigen presentation pathway is based on the initial assembly of the MHC II ⁇ heterodimer with a dual function molecule, the invariant chain (Ii) that serves as a chaperone to direct the ⁇ heterodimer to an endosomal, acidic protein processing location where it encounters antigenic peptides.
  • MHC II recognizing T cells then secrete lymphokines and may be induced to proliferate.
  • MHC class II molecules show preferential restriction to CD4+ cells (Fundamental Immunology, 4th edition, W. E.
  • the target protein is altered such that its TCR response is altered.
  • TCRs occur as either of two distinct heterodimers, ⁇ or ⁇ , both of which are expressed with the non- polymorphic CD3 polypeptides y, ⁇ , ⁇ , ⁇ .
  • the CD3 polypeptides, especially ⁇ and its variants, are critical for intracellular signaling.
  • the ⁇ TCR heterodimer expressing cells predominate in most lymphoid compartments and are responsible for the classical helper or cytotoxic T cell responses.
  • the ⁇ TCR ligand is a peptide antigen bound to a class I or a class II MHC molecule (Fundamental Immunology, 4th edition, W. E. Paul, ed., Lippincott-Raven Publishers, 1999, Chapter 10, pp 341-367).
  • the target protein is altered such that its BCR response is altered.
  • Antigen contact with a specific B cell triggers the transmembrane signaling function of the B cell antigen receptor (BCR).
  • BCR molecules are rapidly internalized after antigen binding, leading to antigen uptake and degradation in endosomes or lysosomes.
  • antigen-derived peptides bind in the groove of class II MHC molecules. Upon binding, this complex is sent to the cell surface, where it serves as a stimulus for specific helper T cells. Antigen recognition by the helper T cell induces it to form a tight and long lasting interaction with the B cell and to synthesize B cell growth and differentiation factors.
  • B cells activated in this way may proliferate and terminally differentiate to antibody secreting cells (also called plasma cells) (Fundamental Immunology, 4th edition, W. E. Paul, ed., Lippincott-Raven Publishers, 1999, Chapters 6-7, pp 183-261)
  • target protein herein is meant at least two covalently attached amino acids, which includes proteins, polypeptides, oligopeptides and peptides.
  • the protein may be made up of naturally occurring amino acids and peptide bonds, or synthetic peptidomimetic structures, i.e., “analogs” such as peptoids [see Simon et al., Proc. Natl. Acad. Sci. U.S.A. 89(20:9367-71 (1992)], generally depending on the method of synthesis.
  • amino acid or “peptide residue”, as used herein means both naturally occurring and synthetic amino acids.
  • amino acids for the purposes of the invention.
  • amino acid also includes imino acid residues such as proline and hydroxyproline.
  • any amino acid representing a component of the variant proteins of the present invention can be replaced by the same amino acid but of the opposite chirality.
  • any amino acid naturally occurring in the L-configuration (which may also be referred to as the R or S, depending upon the structure of the chemical entity) may be replaced with an amino acid of the same chemical structural type, but of the opposite chirality, generally referred to as the D- amino acid but which can additionally be referred to as the R- or the S-, depending upon its composition and chemical configuration.
  • Such derivatives generally have the property of greatly increased stability, and therefore are advantageous in the formulation of compounds which may have longer in vivo half lives, when administered by oral, intravenous, intramuscular, intraperitoneal, topical, rectal, intraocular, or other routes.
  • the amino acids are in the (S) or L-configuration. If non-naturally occurring side chains are used, non-amino acid substituents may be used, for example to prevent or retard in vivo degradations. Proteins including non-naturally occurring amino acids may be synthesized or in some cases, made recombinantly; see van Hest et al., FEBS Lett 428:(1-2) 68-70 May 22 1998 and Tang et al., Abstr. Pap Am. Chem. S218: U138 Part 2 August 22, 1999, both of which are expressly incorporated by reference herein.
  • Aromatic amino acids may be replaced with D- or L-naphylalanine, D- or L-phenylglycine, D- or L-2- thieneylalanine, D- or L-1-, 2-, 3- or 4-pyreneylalanine, D- or L-3-thieneylalanine, D- or L-(2-pyridinyl)- alanine, D- or L-(3-pyridinyl)-alanine, D- or L-(2-pyrazinyl)-alanine, D- or L-(4-isopropyl)-phenylglycine, D-(trifluoromethyl)-phenylglycine, D-(trifluoromethyl)-phenylalanine, D-p-fluorophenylalanine, D- or L- p-biphenylphenylalanine, D- or L-p-methoxybiphenylphenylalanine, D- or L-2-indole(al
  • Acidic amino acids can be substituted with non-carboxylate amino acids while maintaining a negative charge, and derivatives or analogs thereof, such as the non-limiting examples of (phosphono)alanine, glycine, leucine, isoleucine, threonine, or serine; or sulfated (e.g., -S0 3 H) threonine, serine, or tyrosine.
  • (phosphono)alanine glycine, leucine, isoleucine, threonine, or serine
  • sulfated e.g., -S0 3 H
  • alkyl refers to a branched or unbranched saturated hydrocarbon group of 1 to 24 carbon atoms, such as methyl, ethyl, n-propyl, isoptopyl, n- butyl, isobutyl, t-butyl, octyl, decyl, tetradecyl, hexadecyl, eicosyl, tetracisyl and the like.
  • Alkyl includes heteroalkyl, with atoms of nitrogen, oxygen and sulfur.
  • alkyl groups herein contain 1 to 12 carbon atoms.
  • Basic amino acids may be substituted with alkyl groups at any position of the naturally occurring amino acids lysine, arginine, ornithine, citrulline, or (guanidino)-acetic acid, or other (guanidino)alkyl-acetic acids, where "alkyl" is define as above.
  • Nitrile derivatives e.g., containing the
  • CN-moiety in place of COOH may also be substituted for asparagine or glutamine, and methionine sulfoxide may be substituted for methionine.
  • Methods of preparation of such peptide derivatives are well known to one skilled in the art.
  • any amide linkage in any of the variant polypeptides can be replaced by a ketomethylene moiety.
  • Such derivatives are expected to have the property of increased stability to degradation by enzymes, and therefore possess advantages for the formulation of compounds which may have increased in vivo half lives, as administered by oral, intravenous, intramuscular, intraperitoneal, topical, rectal, intraocular, or other routes.
  • Additional amino acid modifications of amino acids of variant polypeptides of to the present invention may include the following: Cysteinyl residues may be reacted with alpha-haloacetates (and corresponding amines), such as 2-chloroacetic acid or chloroacetamide, to give carboxymethyl or carboxyamidomethyl derivatives.
  • Cysteinyl residues may also be derivatized by reaction with compounds such as bromotrifluoroacetone, alpha-bromo-beta-(5-imidozoyl)propionic acid, chloroacetyl phosphate, N-alkylmaleimides, 3-nitro-2-pyridyl disulfide, methyl 2-pyridyl disulfide, p- chloromercuribenzoate, 2-chloromercuri-4-nitrophenol, or chloro-7-nitrobenzo-2-oxa-1 ,3-diazole.
  • compounds such as bromotrifluoroacetone, alpha-bromo-beta-(5-imidozoyl)propionic acid, chloroacetyl phosphate, N-alkylmaleimides, 3-nitro-2-pyridyl disulfide, methyl 2-pyridyl disulfide, p- chloromercuribenzoate, 2-chloromercuri-4-nitrophenol, or
  • Histidyl residues may be derivatized by reaction with compounds such as diethylprocarbonate e.g., at pH 5.5-7.0 because this agent is relatively specific for the histidyl side chain, and para-bromophenacyl bromide may also be used; e.g., where the reaction is preferably performed in 0.1 M sodium cacodylate at pH 6.0. Lysinyl and amino terminal residues may be reacted with compounds such as succinic or other carboxylic acid anhydrides. Derivatization with these agents is expected to have the effect of reversing the charge of the lysinyl residues.
  • compounds such as diethylprocarbonate e.g., at pH 5.5-7.0 because this agent is relatively specific for the histidyl side chain, and para-bromophenacyl bromide may also be used; e.g., where the reaction is preferably performed in 0.1 M sodium cacodylate at pH 6.0.
  • Suitable reagents for derivatizing alpha-amino-containing residues include compounds such as imidoesters, e.g., as methyl picolinimidate; pyridoxal phosphate; pyridoxal; chloroborohydride; tnnitrobenzenesulfonic acid; O-methylisourea; 2,4 pentanedione; and transaminase-catalyzed reaction with glyoxylate.
  • Arginyl residues may be modified by reaction with one or several conventional reagents, among them phenylglyoxal, 2,3-butanedione, 1 ,2-cyclohexanedione, and ninhydrin according to known method steps.
  • arginine residues requires that the reaction be performed in alkaline conditions because of the high pKa of the guanidine functional group. Furthermore, these reagents may react with the groups of lysine as well as the arginine epsilon-amino group.
  • the specific modification of tyrosyl residues per se is well known, such as for introducing spectral labels into tyrosyl residues by reaction with aromatic diazonium compounds or tetranitromethane.
  • N-acetylimidizol and tetranitromethane may be used to form O-acetyl tyrosyl species and 3-nitro derivatives, respectively.
  • Carboxyl side groups (aspartyl or glutamyl) may be selectively modified by reaction with carbodiimides (R'-N-C-N-R') such as 1-cyclohexyl-3-(2-morpholinyl- (4-ethyl) carbodiimide or 1-ethyl-3-(4-azonia-4,4- dimethylpentyl) carbodiimide.
  • aspartyl and glutamyl residues may be converted to asparaginyl and glutaminyl residues by reaction with ammonium ions.
  • Glutaminyl and asparaginyl residues may be frequently deamidated to the corresponding glutamyl and aspartyl residues. Alternatively, these residues may be deamidated under mildly acidic conditions. Either form of these residues falls within the scope of the present invention.
  • the target protein may be any protein for which a three dimensional structure is known or can be generated; that is, for which there are three dimensional coordinates for each atom of the protein.
  • this can be determined using X-ray crystallographic techniques, NMR techniques, de novo modeling, homology modeling, etc. In general, if X-ray structures are used, structures at 2 ⁇ resolution or better are preferred, but not required.
  • the target proteins of the present invention may be from prokaryotes and eukaryotes, such as bacteria (including extremeophiles such as the archebacteria), fungi, insects, fish, and mammals.
  • Suitable mammals include, but are not limited to, rodents (rats, mice, hamsters, guinea pigs, etc.), primates, farm animals (including sheep, goats, pigs, cows, horses, etc) and in the most preferred embodiment, from humans.
  • target protein herein is meant a protein for which a library of variants, preferably with altered immunogenicity is desired.
  • protein any number of target proteins will find use in the present invention.
  • fragments and domains of known proteins including functional domains such as enzymatic domains, binding domains, etc., and smaller fragments, such as turns, loops, etc. That is, portions of proteins may be used as well.
  • protein as used herein includes proteins, oligopeptides and peptides.
  • protein variants i.e. non-naturally occurring protein analog structures, may be used.
  • Suitable proteins include, but are not limited to, industrial, pharmaceutical, and agricultural proteins, including ligands, cell surface receptors, antigens, antibodies, cytokines, hormones, transcription factors, signaling modules, cytoskeletal proteins and enzymes.
  • Suitable classes of enzymes include, but are not limited to, hydrolases such as proteases, carbohydrases, lipases; isomerases such as racemases, epimerases, tautomerases, or mutases; transferases, kinases, oxidoreductases, and phophatases. Suitable enzymes are listed in the Swiss-Prot enzyme database.
  • Suitable protein backbones include, but are not limited to, all of those found in the protein data base compiled and serviced by the Research Collaboratory for Structural Bioinformatics (RCSB, formerly the Brookhaven
  • preferred pharmaceutical target proteins include, but are not limited to, those with known structures (including variants) including cytokines (IL-1ra (+receptor complex), IL-1 (receptor alone), IL-1a, IL-1b (including variants and or receptor complex), lL-2, IL-3, IL-4, IL-5, IL-6, IL-8, IL-10, IFN- ⁇ ,
  • INF-Y IFN- ⁇ -2a
  • IFN- ⁇ -2B TNF- ⁇
  • CD40 ligand (chk) Human Obesity Protein Leptin, Granulocyte Colony-Stimulating Factor, Bone Morphogenetic Protein-7, Ciliary Neurotrophic Factor, Granulocyte- Macrophage Colony-Stimulating Factor, Monocyte Chemoattractant Protein 1, Macrophage Migration Inhibitory Factor, Human Glycosylation-lnhibiting Factor, Human Rantes, Human Macrophage Inflammatory Protein 1 Beta, human growth hormone, Leukemia Inhibitory Factor, Human Melanoma
  • VEGF Vascular Endothelial growth factor
  • acidic Fibroblast growth factor basic Fibroblast growth factor
  • Endothelial growth factor Nerve growth factor
  • Brain Derived Neurotrophic Factor Ciliary Neurotrophic Factor
  • Platelet Derived Growth Factor Human Hepatocyte Growth Factor, Glial Cell-Derived Neurotrophic Factor, (as well as the 55 cytokines in PDB 1/12/99)
  • urokinase Erythropoietin
  • other extracellular signaling moieties including, but
  • soluble proteins that can serve as vehicles for the delivery of immunogenic sequences.
  • soluble proteins include, but are not limited to, albumins, globulins, other proteins present in the blood and other body fluids, and any other substantially non-immunogenic proteins.
  • substantially non-immunogenic proteins herein is meant any protein that does not elicit an immune response in a subject.
  • substantially non-immunogenic proteins may be naturally occurring, synthetic, or modified using recombinant techniques known to one of skill in the art.
  • soluble proteins used as delivery vehicles include, but are not limited to, Zn-alpha2-glycoprotein (Sanchez, L.M., (1997) Proc. Natl.
  • HSA human serum albumin
  • IgG immunoglobulin G
  • preferred industrial target proteins include, but are not limited to, those with known structures (including variants) including proteases, (including, but not limited to papains, subtilisins), cellulases (including , but not limited to, endoglucanases I, II, and III, exoglucanases, xylanases, ligninases, cellobiohydrolases I, II, and III, carbohydrases (including, but not limited to glucoamylases, ⁇ -amylases, glucose isomerases) and lipases.
  • proteases including, but not limited to papains, subtilisins
  • cellulases including , but not limited to, endoglucanases I, II, and III, exoglucanases, xylanases, ligninases, cellobiohydrolases I, II, and III
  • carbohydrases including, but not limited to glucoamylases, ⁇ -amylases, glucose isome
  • preferred agricultural target proteins include, but are not limited to, those with known structures (including variants) including xylose isomerase, pectinases, cellulases, peroxidases, rubisco, ADP glucose pyrophosphorylase, as well as enzymes involved in oil biosynthesis, sterol biosynthesis, carbohydrate biosynthesis, and the synthesis of secondary metabolites.
  • the methods of the invention involve starting with a target protein and using computational analysis to generate a set of primary sequences.
  • computational methods There are a wide variety of computational methods that can be used including sequence alignments of related proteins, structural alignments, structural prediction models, databases, or (preferably) protein design automation computational analysis. Collectively, these computational methods are referred to herein as
  • libraries of primary variant sequences can be generated via sequence screening using a set of scaffold structures that are created by perturbing the starting structure (using any number of techniques such as molecular dynamics, Monte Carlo analysis) to make changes to the protein (including backbone and side-chain torsion angle changes).
  • Optimal sequences can be selected for each starting structures (or, some set of the top sequences) to make libraries of primary variant sequences.
  • lists of sequences that are generated without ranking can then be ranked or filtered using techniques as outlined below.
  • sequence based methods are used.
  • structure based methods such as PDATM, described in detail below, are used.
  • Other models for assessing the relative energies of sequences with high precision include Warshel, Computer Modeling of Chemical Reactions in Enzymes and Solutions. Wiley & Sons, New York, (1991), hereby expressly incorporated by reference.
  • molecular dynamics calculations can be used to computationally screen sequences by individually calculating mutant sequence scores and compiling a rank ordered list.
  • residue pair potentials can be used to score sequences (Miyazawa et al., Macromolecules 18(3):534-552 (1985), expressly incorporated by reference) during computational screening.
  • sequence profile scores (Bowie et al., Science 253(5016): 164-70 (1991), incorporated by reference) and/or potentials of mean force (Herium et al., J. Mol. Biol. 216(1):167- 180 (1990), also incorporated by reference) can also be calculated to score sequences.
  • These methods assess the match between a sequence and a 3D protein structure and hence can act to screen for fidelity to the protein structure.
  • scoring functions can be used to rank sequences, different regions of sequence space can be sampled in the computational screen.
  • scoring functions can be used to screen for sequences that would create metal or co- factor binding sites in the protein (Hellinga, Fold Des. 3(1):R1-8 (1998), hereby expressly incorporated by reference).
  • scoring functions can be used to screen for sequences that would create disulfide bonds in the protein.
  • sequence and/or structural alignment programs can be used to generate primary libraries.
  • sequence-based alignment programs including for example, Smith-Waterman searches, Needleman-Wunsch, Double Affine Smith- Waterman, frame search, Gribskov/GCG profile search, Gribskov/GCG profile scan, profile frame search, Bucher generalized profiles, Hidden Markov models, Hframe, Double Frame, Blast, Psi-Blast, Clustal, and GeneWise.
  • the source of the sequences can vary widely, and include taking sequences from one or more of the known databases, including, but not limited to, SCOP (Hubbard, et al., Nucleic Acids Res 27(1):254-
  • sequences from these databases can be subjected to continguous analysis or gene prediction; see Wheeler, et al., Nucleic Acids Res 28(1 ):10-14. (2000) and Burge and Karlin, J Mol Biol 268(1 ):78-94. (1997), both of which are expressly incorporated herein by reference.
  • sequence alignment methodologies can be used. For example, sequence homology based alignment methods can be used to create sequence alignments of proteins related to the target structure (Altschul et al., J. Mol. Biol. 215(3):403 (1990), incorporated by reference). These sequence alignments are then examined to determine the observed sequence variations. These sequence variations are tabulated to define a primary library. In addition, as is further outlined below, these methods can also be used to generate secondary libraries. Sequence based alignments can be used in a variety of ways.
  • a number of related proteins can be aligned, as is known in the art, and the "variable” and “conserved” residues defined; that is, the residues that vary or remain identical between the family members can be defined.
  • These results can be used to generate a probability table, as outlined below.
  • these sequence variations can be tabulated and a secondary library defined from them as defined below.
  • the allowed sequence variations can be used to define the amino acids considered at each position during the computational screening.
  • Another variation is to bias the score for amino acids that occur in the sequence alignment, thereby increasing the likelihood that they are found during computational screening but still allowing consideration of other amino acids. This bias would result in a focused primary library but would not eliminate from consideration amino acids not found in the alignment.
  • bias may be introduced.
  • diversity may be forced; that is, a "conserved" residue is chosen and altered to force diversity on the protein and thus sample a greater portion of the sequence space.
  • positions of high variability between family members i.e. low conservation
  • outlier residues either positional outliers or side chain outliers, may be eliminated.
  • structural alignment of structurally related proteins can be done to generate sequence alignments.
  • structural alignment programs known. See for example VAST from the NCBI (http://www.ncbi.nlm.nih.gov:80/Structure/VAST/vast.shtml); SSAP (Orengo and , Taylor, Methods Enzymol 266(617-635 (1996)) SARF2 (Alexandrov, Protein Eng 9(9):727-732.
  • Libraries of primary variant sequences can be generated by predicting secondary structure from sequence, and then selecting sequences that are compatible with the predicted secondary structure.
  • secondary structure prediction methods including, but not limited to, threading (Bryant and Altschul, Curr Opin Struct Biol 5(2):236-244. (1995)), Profile 3D (Bowie, et al., Methods Enzymol 266(598-616 (1996); MONSSTER (Skolnick, et al., J Mol Biol 265(2):217-241. (1997);
  • DISCOVER cvff and cff91
  • AMBER AMBER force fields
  • INSIGHT molecular modeling package Biosym/MSI, San Diego California
  • HARMM is used in the QUANTA molecular modeling package (Biosym/MSI, San Diego California), all of which are expressly incorporated by reference.
  • these force field methods may be used to generate the secondary library directly; that is, no primary library is generated; rather, these methods can be used to generate a probability table from which the secondary library is directly generated, for example by using these forcefields during an SCMF calculation.
  • the computational method used to generate the primary library is Protein
  • the PDATM protein design technology can be described as follows: A known protein structure is used as the starting point. The residues to be optimized are then identified, which may be the entire sequence or subset(s) thereof. The side chains of any positions to be varied are then removed. The resulting structure consisting of the protein backbone and the remaining sidechains is called the template. Each variable residue position is then preferably classified as a core residue, a surface residue, or a boundary residue; each classification defines a subset of possible amino acid residues for the position (for example, core residues generally will be selected from the set of hydrophobic residues, surface residues generally will be selected from the hydrophilic residues, and boundary residues may be either).
  • Each amino acid can be represented by a discrete set of all allowed conformers of each side-chain, called rotamers.
  • rotamers To arrive at an optimal sequence for a backbone, all possible sequences of rotamers must be screened, where each backbone position can be occupied either by each amino acid in all its possible rotameric states, or a subset of amino acids, and thus a subset of rotamers.
  • Two sets of interactions are then calculated for each rotamer at every position: the interaction of the rotamer side chain with all or part of the backbone (the “singles” energy, also called the rotamer/template or rotamer/backbone energy), and the interaction of the rotamer side chain with all other possible rotamers at every other position or a subset of the other positions (the “doubles” energy, also called the rotamer/rotamer energy).
  • the energy of each of these interactions is calculated through the use of a variety of scoring functions, which include the energy of van der Waal's forces, the energy of hydrogen bonding, the energy of secondary structure propensity, the energy of surface area solvation and the electrostatics.
  • the total energy of each rotamer interaction, both with the backbone and other rotamers is calculated, and stored in a matrix form.
  • rotamer sets allow a simple calculation of the number of rotamer sequences to be tested.
  • a backbone of length n with m possible rotamers per position will have m n possible rotamer sequences, a number which grows exponentially with sequence length and renders the calculations either unwieldy or impossible in real time.
  • a "Dead End Elimination" (DEE) calculation is performed.
  • the DEE calculation is based on the fact that if the worst total interaction of a first rotamer is still better than the best total interaction of a second rotamer, then the second rotamer cannot be part of the global optimum solution.
  • a Monte Carlo search may be done to generate a rank- ordered list of sequences in the neighborhood of the DEE solution.
  • Starting at the DEE solution random positions are changed to other rotamers, and the new sequence energy is calculated. If the new sequence meets the criteria for acceptance, it is used as a starting point for another jump. After a predetermined number of jumps, a rank-ordered list of sequences is generated.
  • Monte Carlo searching is a sampling technique to explore sequence space around the global minimum or to find new local minima distant in sequence space.
  • sampling techniques including Boltzman sampling, genetic algorithm techniques and simulated annealing.
  • the kinds of jumps allowed can be altered (e.g. random jumps to random residues, biased jumps (to or away from wild- type, for example), jumps to biased residues (to or away from similar residues, for example), etc.).
  • the acceptance criteria of whether a sampling jump is accepted can be altered.
  • the protein backbone (comprising (for a naturally occurring protein) the nitrogen, the carbonyl carbon, the ⁇ -carbon, and the carbonyl oxygen, along with the direction of the vector from the ⁇ -carbon to the ⁇ -carbon) may be altered prior to the computational analysis, by varying a set of parameters called supersecondary structure parameters.
  • the protein backbone structure contains at least one variable residue position.
  • the residues, or amino acids, of proteins are generally sequentially numbered starting with the N- terminus of the protein.
  • a protein having a methionine at it's N-terminus is said to have a methionine at residue or amino acid position 1 , with the next residues as 2, 3, 4, etc.
  • the wild type (i.e. naturally occurring) protein may have one of at least 20 amino acids, in any number of rotamers.
  • variant residue position herein is meant an amino acid position of the protein to be designed that is not fixed in the design method as a specific residue or rotamer, generally the wild-type residue or rotamer.
  • all of the residue positions of the protein are variable. That is, every amino acid side chain may be altered in the methods of the present invention. This is particularly desirable for smaller proteins, although the present methods allow the design of larger proteins as well. While there is no theoretical limit to the length of the protein that may be designed this way, there is a practical computational limit.
  • residue positions of the protein are variable, and the remainder are "fixed", that is, they are identified in the three dimensional structure as being in a set conformation.
  • a fixed position is left in its original conformation (which may or may not correlate to a specific rotamer of the rotamer library being used).
  • residues may be fixed as a non-wild type residue; for example, when known site-directed mutagenesis techniques have shown that a particular residue is desirable (for example, to eliminate a proteolytic site or alter the substrate specificity of an enzyme), the residue may be fixed as a particular amino acid.
  • the methods of the present invention may be used to evaluate mutations de novo, as is discussed below.
  • a fixed position may be "floated"; the amino acid at that position is fixed, but different rotamers of that amino acid are tested.
  • the variable residues may be at least one, or anywhere from 0.1 % to 99.9% of the total number of residues. Thus, for example, it may be possible to change only a few (or one) residues, or most of the residues, with all possibilities in between.
  • residues that can be fixed include, but are not limited to, structurally or biologically functional residues; alternatively, biologically functional residues may specifically not be fixed.
  • residues which are known to be important for biological activity such as the residues which form the active site of an enzyme, the substrate binding site of an enzyme, the binding site for a binding partner (ligand/receptor, antigen/antibody, etc.), phosphorylation or glycosylation sites which are crucial to biological function, or structurally important residues, such as disulfide bridges, metal binding sites, critical hydrogen bonding residues, residues critical for backbone conformation such as praline or glycine, residues critical for packing interactions, etc. may all be fixed in a conformation or as a single rotamer, or "floated".
  • residues which may be chosen as variable residues may be those that confer undesirable biological attributes, such as susceptibility to proteolytic degradation, dimerization or aggregation sites, glycosylation sites which may lead to immune responses, unwanted binding activity, unwanted allostery, undesirable enzyme activity but with a preservation of binding, etc.
  • each variable position is classified as either a core, surface or boundary residue position, although in some cases, as explained below, the variable position may be set to glycine to minimize backbone strain.
  • residues need not be classified, they can be chosen as variable and any set of amino acids may be used. Any combination of core, surface and boundary positions can be utilized: core, surface and boundary residues; core and surface residues; core and boundary residues, and surface and boundary residues, as well as core residues alone, surface residues alone, or boundary residues alone.
  • the classification of residue positions as core, surface or boundary may be done in several ways, as will be appreciated by those in the art.
  • the classification is done via a visual scan of the original protein backbone structure, including the side chains, and assigning a classification based on a subjective evaluation of one skilled in the art of protein modeling.
  • a preferred embodiment utilizes an assessment of the orientation of the C ⁇ -C ⁇ vectors relative to a solvent accessible surface computed using only the template C ⁇ atoms, as outlined in U.S.S.N.s 60/061 ,097, 60/043,464, 60/054,678, 09/127,926 and PCT US98/07254.
  • a surface area calculation can be done.
  • a set of amino acid side chains is assigned to each position. That is, the set of possible amino acid side chains that the program will allow to be considered at any particular position is chosen. Subsequently, once the possible amino acid side chains are chosen, the set of rotamers that will be evaluated at a particular position can be determined.
  • a core residue will generally be selected from the group of hydrophobic residues consisting of alanine, valine, isoleucine, leucine, phenylalanine, tyrosine, tryptophan, and methionine (in some embodiments, when the ⁇ scaling factor of the van der Waals scoring function, described below, is low, methionine is removed from the set), and the rotamer set for each core position potentially includes rotamers for these eight amino acid side chains (all the rotamers if a backbone independent library is used, and subsets if a rotamer dependent backbone is used).
  • surface positions are generally selected from the group of hydrophilic residues consisting of alanine, serine, threonine, aspartic acid, asparagine, glutamine, glutamic acid, arginine, lysine and histidine.
  • the rotamer set for each surface position thus includes rotamers for these ten residues.
  • boundary positions are generally chosen from alanine, serine, threonine, aspartic acid, asparagine, glutamine, glutamic acid, arginine, lysine histidine, valine, isoleucine, leucine, phenylalanine, tyrosine, tryptophan, and methionine.
  • the rotamer set for each boundary position thus potentially includes every rotamer for these seventeen residues (assuming cysteine, glycine and praline are not used, although they can be). Additionally, in some preferred embodiments, a set of 18 naturally occurring amino acids (all except cysteine and praline, which are known to be particularly disruptive) are used.
  • proline, cysteine and glycine are not included in the list of possible amino acid side chains, and thus the rotamers for these side chains are not used.
  • the variable residue position has a ⁇ angle (that is, the dihedral angle defined by 1) the carbonyl carbon of the preceding amino acid; 2) the nitrogen atom of the current residue; 3) the ⁇ - carbon of the current residue; and 4) the carbonyl carbon of the current residue) greater than 0°
  • the position is set to glycine to minimize backbone strain.
  • processing proceeds as outlined in U.S.S.N. 09/127,926 and PCT US98/07254.
  • This processing step entails analyzing interactions of the rotamers with each other and with the protein backbone to generate optimized protein sequences.
  • the processing initially comprises the use of a number of scoring functions to calculate energies of interactions of the rotamers, either to the backbone itself or other rotamers.
  • Preferred PDATM technology scoring functions include, but are not limited to, a Van der Waals potential scoring function, a hydrogen bond potential scoring function, an atomic solvation scoring function, a secondary structure propensity scoring function and an electrostatic scoring function.
  • At least one scoring function is used to score each position, although the scoring functions may differ depending on the position classification or other considerations, like favorable interaction with an ⁇ -helix dipole.
  • the total energy which is used in the calculations is the sum of the energy of each scoring function used at a particular position, as is generally shown in Equation 1:
  • Equation 1 the total energy is the sum of the energy of the van der Waals potential (E vdw ), the energy of atomic solvation (E as ), the energy of hydrogen bonding (E h . bond ⁇ ng ), the energy of secondary structure (E ss ) and the energy of electrostatic interaction (E ete0 ).
  • the term n is either 0 or 1 , depending on whether the term is to be considered for the particular residue position.
  • the preferred first step in the computational analysis comprises the determination of the interaction of each possible rotamer with all or part of the remainder of the protein. That is, the energy of interaction, as measured by one or more of the scoring functions, of each possible rotamer at each variable residue position with either the backbone or other rotamers, is calculated. In a preferred embodiment, the interaction of each rotamer with the entire remainder of the protein, i.e. both the entire template and all other rotamers, is done.
  • portion refers to a fragment of that protein. This fragment may range in size from 10 amino acid residues to the entire amino acid sequence minus one amino acid.
  • portion refers to a fragment of that nucleic acid. This fragment may range in size from 10 nucleotides to the entire nucleic acid sequence minus one nucleotide.
  • the first step of the computational processing is done by calculating two sets of interactions for each rotamer at every position: the interaction of the rotamer side chain with the template or backbone (the “singles” energy), and the interaction of the rotamer side chain with all other possible rotamers at every other position (the “doubles” energy), whether that position is varied or floated.
  • the backbone in this case includes both the atoms of the protein structure backbone, as well as the atoms of any fixed residues, wherein the fixed residues are defined as a particular conformation of an amino acid.
  • “singles” (rotamer/template) energies are calculated for the interaction of every possible rotamer at every variable residue position with the backbone, using some or all of the scoring functions.
  • the hydrogen bonding scoring function every hydrogen bonding atom of the rotamer and every hydrogen bonding atom of the backbone is evaluated, and the E HB is calculated for each possible rotamer at every variable position.
  • the van der Waals scoring function every atom of the rotamer is compared to every atom of the template (generally excluding the backbone atoms of its own residue), and the E vdW is calculated for each possible rotamer at every variable residue position.
  • every atom of the first rotamer is compared to every atom of every possible second rotamer, and the E vdW is calculated for each possible rotamer pair at every two variable residue positions.
  • the surface of the first rotamer is measured against the surface of every possible second rotamer, and the E as for each possible rotamer pair at every two variable residue positions is calculated.
  • the secondary structure propensity scoring function need not be run as a "doubles" energy, as it is considered as a component of the "singles" energy.
  • AMBER 3.0 force field U.C. Singh et al., Proc. Natl. Acad. Sci. USA. 82:755-759
  • CHARMM and CHARMM22 Brooks, et al., J. Comp. Chem. v4, pp 187-217)
  • cvff3.0 Dauber-Osguthorpe, et al.,(1988) Proteins: Structure, Function and Genetics, v4,pp31-47
  • cff91 Maple, et al., J. Comp. Chem.
  • DISCOVER cvff and cff91
  • AMBER forcefields are used in the INSIGHT molecular modeling package (Biosym/MSI, San Diego California) and HARMM is used in the
  • DEE Dead End Elimination
  • PDATM technology viewed broadly, has three components that may be varied to alter the output (e.g. the primary library): the scoring functions used in the process; the filtering technique, and the sampling technique. These functions may be used sequentially or substantially simultaneously. For example, a scoring function may be used in parallel with a filtering technique.
  • the scoring functions may be altered.
  • the scoring functions outlined above may be biased or weighted in a variety of ways. For example, a bias towards or away from a reference sequence or family of sequences can be done; for example, a bias towards wild-type or homolog residues may be used.
  • the entire protein or a fragment of it may be biased; for example, the active site may be biased towards wild-type residues, or domain residues towards a particular desired physical property can be done.
  • a bias towards or against increased energy can be generated.
  • Additional scoring function biases include, but are not limited to applying electrostatic potential gradients or hydrophobicity gradients, adding a substrate or binding partner to the calculation, or biasing towards a desired charge or hydrophobicity.
  • Additional scoring functions include, but are not limited to torsional potentials, or residue pair potentials, or residue entropy potentials. Such additional scoring functions can be used alone, or as functions for processing the library after it is scored initially.
  • a variety of process filtering techniques can be done, including, but not limited to, DEE and its related counterparts. Additional filtering techniques include, but are not limited to branch-and-bound techniques for finding optimal sequences (Gordon and Mayo, Structure Fold. Des. 7:1089-98, 1999), and exhaustive enumeration of sequences. It should be noted however, that some techniques may also be done without any filtering techniques; for example, sampling techniques can be used to find good sequences, in the absence of filtering.
  • sampling methods can include the use of amino acid substitutions, insertions or deletions, or recombinations of one or more sequences.
  • a preferred embodiment utilizes a
  • Monte Carlo search which is a series of biased, systematic, or random jumps.
  • sampling techniques including Boltzman sampling, genetic algorithm techniques and simulated annealing.
  • the.kinds of jumps allowed can be altered (e.g. random jumps to random residues, biased jumps (to or away from wild- type, for example), jumps to biased residues (to or away from similar residues, for example), etc.).
  • the preferred methods of the invention result in a rank ordered list of sequences; that is, the sequences are ranked or filtered on the basis of some objective criteria.
  • it is possible to create a set of non-ordered sequences for example by generating a probability table directly (for example using SCMF analysis or sequence alignment techniques) that lists sequences without ranking them.
  • the sampling techniques outlined herein can be used in either situation.
  • Boltzman sampling is done.
  • the temperature criteria for Boltzman sampling can be altered to allow broad searches at high temperature and narrow searches close to local optima at low temperatures (see e.g., Metropolis et al., J. Chem.
  • the sampling technique utilizes genetic algorithms, e.g., such as those described by Holland (Adaptation in Natural and Artificial Systems, 1975, Ann Arbor, U. Michigan Press). Genetic algorithm analysis generally takes generated sequences and recombines them computationally, similar to a nucleic acid recombination event, in a manner similar to "gene shuffling". Thus the "jumps" of genetic algorithm analysis generally are multiple position jumps. In addition, as outlined below, correlated multiple jumps may also be done. Such jumps can occur with different crossover positions and more than one recombination at a time, and can involve recombination of two or more sequences. Furthermore, deletions or insertions (random or biased) can be done. In addition, as outlined below, genetic algorithm analysis may also be used after the secondary library has been generated.
  • Genetic algorithm analysis may also be used after the secondary library has been generated.
  • the sampling technique utilizes simulated annealing, e.g., such as described by Kirkpatrick et al. (Science, 220:671-680, 1983). Simulated annealing alters the cutoff for accepting good or bad jumps by altering the temperature. That is, the stringency of the cutoff is altered by altering the temperature. This allows broad searches at high temperature to new areas of sequence space, altering with narrow searches at low temperature to explore regions in detail.
  • these sampling methods can be used to further process a secondary library to generate additional secondary libraries (sometimes referred to herein as tertiary libraries).
  • the primary library can be generated in a variety of computational ways, including structure based methods such as PDATM, or sequence based methods, or combinations as outlined herein.
  • Optimized variant candidate protein sequences are generally different from the target protein sequence in regions critical for MHC, TCR or BCR binding.
  • each optimized variant candidate sequence comprises at least about 1 variant amino acid from the starting or target sequence, with 3-5 being preferred.
  • the variant residues are located in noncontiguous regions.
  • the present invention is directed to methods of computationally processing a target protein, or fragment thereof, to produce a variant candidates protein or a set of variant candidates protein sequences.
  • the variant candidate proteins of the invention have an amino acid sequence that differs from the target protein in at least one MHC, TCR, or BCR binding site.
  • the candidate variant protein differs from the target protein by the elimination of at least one MHC, TCR, or BCR binding site.
  • the candidate variant protein differs from the target protein via the addition of at least one MHC, TCR, or BCR binding site.
  • each optimized protein sequence preferably comprises at least about 5-10% variant amino acids from the starting target or wild-type sequence, with at least about 15-20% changes being preferred and at least about 30% changes being particularly preferred.
  • a computational immunogenicity filter is applied to the set of primary library sequences.
  • computational immunogenicity filter herein is meant any one of a number of scoring functions derived from data on binding of peptides to MHC molecules, or T cell epitopes or B cell epitopes.
  • the computational immunogenecity filter can be applied as part of the original computation (e. g., substantially simultaneously; for example as one of the computational steps or as a scoring fucntion in the original computation), prior to the computation (e.g. as a pre-filter), or after the original computation (e.g., as a post-filter).
  • the computational immunogenicity filter is used as a post-filter: that is, the scoring functions are used to rescore the set of primary library sequences to eliminate potentially immunogenic sequences, or to introduce non- immunogenic sequences.
  • the computational immunogenicity filter is applied during the same time, i.e., substantially simultaneously, when the primary library sequences are generated. In other preferred embodiments, the computational immunogenicity filter is applied before the computational generation of a set of primary sequences. Using this approach, a set of primary sequences is generated that potentially either lack or include immunogenic sequences depending on the desired result. The PDATM technology is then run on these sequences to identify those sequences that retain the native fold and are at least as stable as the starting target protein.
  • the PDATM technology is used to structurally and chemically compensate for either the removal or addition of amino acid residues encoding linear epitopes displayed by MHC class I and II molecules that are recognized by TCRs.
  • the PDATM technology is used to structurally and chemically compensate for either the removal or addition of amino acid residues encoding conformational epitopes, that are sensed by membrane bound antibodies on naive B cells.
  • MHC-peptide complexes The current understanding of the rules for peptide selection by MHC molecules is derived from sequencing of peptides and natural peptide libraries extracted from MHC proteins, from analyses of the effects of mutations in sequences of unknown CTL epitopes on peptide binding to MHC molecules and on T cell responses, as well as from crystal structure analyses and molecular dynamic studies of defined MHC-peptide complexes (Meister, G.E., et al. (1995) Vaccine, 13:581-591 ; Malios, R.R., (1999) Bioinformatics Savoie, C.J. etal. (1999) Pac Symp Biocomput., 182-9; Brusic, V., etal., (1998)
  • primary variant sequences are screened for peptide fragments potentially capable of binding to MHC class I molecules.
  • the MHC I ligands are mostly octa-or nonapeptides and show MHC allele specific sequence motifs as determined by pool sequencing of natural isolates. Crystal structure analysis has identified a peptide binding cleft, i.e., groove, framed by two ⁇ helices and a ⁇ pleated sheet. The cleft is stabilized from beneath by the noncovalently associated ⁇ 2 microglobulin. Specific pockets in the binding groove accommodate the anchor residues of the peptide. The orientation of the peptides is determined by conserved side chains of the MHC I protein that compensate the NH 2 . and COOH- terminal charges.
  • a given MHC class I peptide binding groove can bind hundreds or thousands of different peptides, identical or homologous at only a few side chain positions. Comparisons of the structures of numerous class I peptide-MHC complexes reveals that this flexibility is achieved by the structurally equivalent binding of a small subset of each peptide's residues. Among these, the binding of charged and polar atoms of the peptide main chain provides essential side-chain-independent peptide MHC interactions. This collection of hydrogen bonds and van der Waals contacts helps to stabilize the binding of any peptide capable of adopting the required backbone conformation.
  • MCH binding peptide such as SYPEITHI and MHCPEP
  • SYPEITHI and MHCPEP are also available and may be used to identify potential MHC I binding sites.
  • Other methods for identifying MHC binding motifs include allele-specific polynomial algorithms described by Fikes, J., et al., WO 01/41788.
  • potential MHC class I binding sites will be replaced with amino acid residues that structurally and chemically compensate for the anchor residues removed to reduce or eliminate peptide binding to MHC class I molecules.
  • Potential MHC I binding motifs will be identified either by matching a database of published motifs, such as SYFPEITHI (Rammensee, H., et al., (1999) Immunogenetics, 50:213-219; http://134.2.96.221/scripts/MHCServer.dll/home.html)); http://wehih.wehi.edu.au/mhcpep/, MHCEP (Brusic, B., et al., supra) or by either established methods such as neural net (Gulukota, K, supra), polynomial (Gulukota, K., supra) rank ordering (Parker, K.C., supra), and allele-specific allele-specific polynomial algorithms (Fikes, J., et al., WO 01/41788
  • non-anchoring residues will be replaced.
  • specific cleavage motifs for antigen processing and presentation are removed.
  • specific cleavage motif herein is meant a motif specifically recognized as a proteolytic cleavage site by proteases implicated in the processing of antigenic determinants present in a given protein (see Schneider, S.C., et al., (2000) J. Immunol., 165:20-23; incorporated by reference in its entirety).
  • specific cleavage motifs are motifs that when present can render antigenic determinants more available for binding to MHC molecules and subsequent presentation on the surface of APCs.
  • proteasomal cleavage sites are removed to reduce the availability of antigenic determinants for binding to MHC class I molecules. Potential proteasomal cleavage sites will be identified by using a prediction algorithm, such as the one described by Kutter, C, et al., (2000) J.
  • potential MHC class I binding sites are added to a target protein as a means of inducing cellular immunity.
  • Suitable target proteins include, but are not limited to, soluble proteins, such as Zn- alpha2-glycoprotein (Sanchez, L.M., et al., (1999) Science 283:1914-9) or primary sequence libraries generated using other target proteins of interest.
  • MHC I binding motifs will be identified either by matching a database of published motifs, such as SYFPEITHI (Rammensee, H., et al., (1999) Immunogenetics, 50:213-219; http://134-2.96.221/scripts/MHCServer.dll/home.html)); http://wehih.wehi.edu.au/mhcpep/, MHCEP (Brusic, B., et al., supra) or by established methods such as neural net (Gulukota, K, supra), polynomial (Gulukota, K., supra), rank ordering (Parker, K.C., supra), and allele-specific polynomial algorithms described (Fikes, J., et al., WO 01/41788).
  • proteasomal cleavage sites are added to enhance the availability of antigenic determinants for binding to MHC class I molecules.
  • Potential proteasomal cleavage sites will be identified by using a prediction algorithm, such as the one described by Kutter, C, et al., (2000) J. Mol. Biol., 298:417-429 and Nussbaum, A. K., et al., (2001) Immunogenetics, 53:87-94; both of which are incorporated by reference in their entirety.
  • primary variant sequences will be screened for peptide fragments predicted to bind to MHC class II molecules.
  • Class II ligands consist of 12 to 25 amino acids, nine of which occupy the binding groove; between two and four are anchored in the pockets. As in the class I ligands, the nonanchoring amino acids play a secondary, but still significant role (Rammensee, H., et al., (1999) Immunogenetics, 50:213-219). Rules for identifying MHC II binding sites have been described in Hammer, J. et al., (1994) Behring. Inst. Mitt., 94: 124-132; Hammer, J. et al., (1994) J.
  • potential MHC class II binding sites will be replaced with amino acid residues which structurally and chemically compensate for anchor residues removed to eliminate MHC II binding sites.
  • potential MHC II binding sites will be identified by matching a database of published motifs, such as SYFPEITHI (Rammensee, H., et al., (1999) Immunogenetics, 50:213-219; http://134.2.96.221/scripts/MHCServer.dll/home.html) or http://wehih.wehi.edu.au/mhcpep/), or MHCEP (Brusic, B., et al., supra).
  • prediction of binding to class II molecules will use the method of virtual matrices (see Sturniolo, T, et al. (1999) Nature Biotechnology, 17:555-561) and Raddrizzani, L. and Hammer, J., (2000) Brief Bioinform., 1 :179-89; hereby incorporated by reference in their entirety) or allele-specific polynomial algorithms described by Fikes, J., et al., WO 01/41788.
  • non-anchoring residues will be replaced.
  • proteolytic cleavage sites are removed to reduce the availability of antigenic determinants for binding to MHC class II molecules. Potential proteolytic cleavage sites will be identified as described by Schneider, S.C., et al., (2000) J.
  • potential MHC class II binding sites are added to a target protein as a means of inducing cellular immunity.
  • at least one MHC class II binding site is added per target protein. More preferably at least 2 MHC class II binding sites are added per target protein. More preferably between 3 to 5 MHC class II binding sites are added per target protein. In other embodiments, up to 16 MHC class II binding sites may be added per target protein (see
  • Suitable target proteins include, but are not limited to, soluble proteins, such as Zn-alpha2-glycoprotein (Sanchez, L.M., et al., (1999) Science 283:1914-9) or primary sequence libraries generated using other target proteins of interest.
  • MHC II binding motifs will be identified either by matching a database of published motifs, such as SYFPEITHI (Rammensee, H., et al., (1999) Immunogenetics, 50:213-219; http://134.2.96.221/scripts/MHCServer.dll/home.html)); http://wehih.wehi.edu.au/mhcpep/, MHCEP (Brusic, B., et al., supra) or by established methods such as virtual matrices (Sturniolo, T, et al. (1999) Nature Biotechnology, 17:555-561; Raddrizzani, L. and Hammer, J., (2000) Brief Bioinform., 1:179-89) and allele-specific polynomial algorithms (Fikes, J., et al., WO 01/41788).
  • proteolytic cleavage sites for cathepsins B, D, E, L and asparaginyl endopeptidase are added to enhance the availability of antigenic determinants for binding to MHC class II molecules.
  • Potential proteolytic cleavage sites will be identified as described by Schneider, S.C., et al., (2000) J. Immunol., 165:20-23; and, Medd and Chain, (2000) Cell & Developmental Biology, 11 :203-210; both of which are incorporated by reference in their entirety.
  • potential MHC class I and class II binding sites are added to a target protein or primary sequence libraries generated using other target proteins of interest as a means of inducing cellular immunity as described above.
  • peptide sequences present in autologous proteins i.e., circulating human proteins such as immunoglobulins, albumin, etc. are ignored.
  • primary variant sequences will be screened for peptide fragments predicted to function as T cell epitopes.
  • potential T cell epitopes will be replaced with amino acid residues that structurally and chemically compensate for the residues removed to eliminate the T cell epitope.
  • potential T cell epitopes will be identified by matching a database of published motifs (Walden, P., (1996) Curr. Op. Immunol., 8:68-74). Other methods of identifying T cell epitopes which are useful in the present invention include those described by Hemmer, B., et al. (1998) J. Immunol., 160:3631-3636; Walden, P., et al. (1995) Biochemical Society Transactions, 23; Anderton, S.M., ef al., (1999) Eur. J. Immunol., 29:1850-1857; Correia-
  • proteolytic cleavage sites may removed to reduce the availability of antigenic determinants for binding to MHC class II molecules.
  • proteasomal cleavage sites may be removed to reduce the availability of antigenic determinants for binding to MHC class I molecules.
  • non-peptide backbone elements are incorporated into T cell epitopes to generate MHC class I or class II ligands with antagonistic properties.
  • non-peptide backbone elements herein is meant non-naturally occurring or synthetic amino acids as described above.
  • antagonistic herein is meant epitopes that are recognized by T cells, but block their activation even in the presence of the activating epitope, i.e., the cognate epitope.
  • antagonistics are derived from known epitopes by amino acid replacements that introduce charge or bulky size modification of peptide side chains.
  • N-hydroxylated peptide derivatives, or ⁇ -amino acids are introduced into T cell epitopes to generate antagonists (see for example, Hin, S., et al., (1999) J. Immunology, 163:2363-2367; Reinelt, S., et al., (2001) J. Biol. Chemistry, 276:24525-24530; both incorporated by reference in their entirety).
  • T cell epitopes will be introduced into primary sequence libraries in regions that will not affect the native folding and stability of the target protein.
  • T cell epitopes will be selected from databases of known MHC I binding peptides, MHC II binding peptides, and T cell epitopes.as described above.
  • T cell epitopes may be added per target protein (see Stienekemeier, M., et al., (2001) Proc Natl Acad Sci USA, 98:13872-13877; hereby incorporated by reference in its entirety).
  • the PDATM technology will be used to ensure proper folding and stability of the modified target protein.
  • specific cleavage motifs as defined above for antigen processing and presentation are added. Cleavage sites implicated in the processing of antigenic determinants present for MHC class I and/or class II molecules are added as described above. Thus, proteolytic cleavage sites may added to enhance the availability of antigenic determinants for binding to MHC class II molecules. In addition, proteasomal cleavage sites may be added to enhance the availability of antigenic determinants for binding to MHC class I molecules.
  • non-peptide backbone elements are incorporated into T cell epitopes to generate MHC class I or class II ligands with agonist properties.
  • agonist herein is meant epitopes that are recognized and activate T cells.
  • primary variant sequences will be screened for peptide fragments predicted to bind to antibodies.
  • potential B cell epitopes will be replaced with smaller neutral residues to reduce the immunogenicity of the sequence as described by Meyer ef al. (Meyer, D.L., et al. (2001), Protein Sci., 10:491-503; see also Schwartz, HL, ef al. (1999) J. Mol.
  • B cell epitopes will be introduced into primary sequence libraries or soluble target proteins in regions that will not affect the native folding and stability of the target protein.
  • charged, aromatic, or large hydrophobic residues on the surface of the target protein are added.
  • any combination of T cell epitopes, B cell epitopes, MHC class I and/or MHC class II binding motifs will be introduced into primary sequence libraries or into a soluble target protein, such as Zn-alpha2-glycoprotein, as described above.
  • At least one candidate variant protein is identified in which at least one sequence capable of interacting with an MHC class I or class II molecule, a TCR or BCR has been altered.
  • Any method of identifying potential or actual MHC, TCR or BCR sequences can be used in the invention.
  • Acceptable methods include computational or physical methods.
  • Acceptable computational methods include the use of algorithms such as OptiMer and EpiMer (Meister, GE., et al.
  • Acceptable physical methods include high affinity binding assays (Hammer, J., et al. (1993) Proc. Natl. Acad. Sci. USA, 91 :4456-4460; Sarobe, P. et al. (1998) J. Clin. Invest., 102:1239-1248), T cell proliferation and CTL assays (Hemmer, B., et al., (1998) J. Immunol., 160:3631-3636); stabilization assays, competitive inhibition assays to purified MHC molecules or cells bearing MHC, or elution followed by sequencing (Brusic, V., et al., (1998) Nucleic Acids Res., 26:368-371). All references cited in this paragraph are hereby incorporated in their entirety.
  • these sequences are then modified by the replacement of one or more amino acids as described below.
  • the protein is then tested to determine if its activity is similar to the target protein.
  • the variant may retain full activity, or retain a sufficient proportion of its activity to be useful.
  • variant proteins and nucleic acids of the invention are distinguishable from the naturally occurring target protein.
  • naturally occurring or “wild type” or grammatical equivalents, herein is meant an amino acid sequence or a nucleotide sequence that is found in nature and includes allelic variations; that is, an amino acid sequence or a nucleotide sequence that usually has not been intentionally modified.
  • non-naturally occurring or “synthetic” or “recombinant” or grammatical equivalents thereof, herein is meant an amino acid sequence or a nucleotide sequence that is not found in nature; that is, an amino acid sequence or a nucleotide sequence that usually has been intentionally modified.
  • nucleic acid once a recombinant nucleic acid is made and reintroduced into a host cell or organism, it will replicate non-recombinantly, i.e., using the in vivo cellular machinery of the host cell rather than in vitro manipulations, however, such nucleic acids, once produced recombinantly, although subsequently replicated non-recombinantly, are still considered recombinant for the purpose of the invention.
  • the variant proteins and nucleic acids of the invention are non- naturally occurring; that is, they do not exist in nature.
  • the variant protein has an amino acid sequence that differs from a target sequence by at least 1-5% of the residues. That is, the variant proteins of the invention are less than about 97-99% identical to a target amino acid sequence. Accordingly, a protein is a "candidate variant protein" if the overall homology of the protein sequence to the target sequence is preferably less than about 99%, more preferably less than about 98%, even more preferably less than about 97% and more preferably less than about 95%. In some embodiments, the homology will be as low as about 75-80%.
  • sequence similarity means sequence similarity or identity, with identity being preferred.
  • a number of different programs can be used to identify whether a protein (or nucleic acid as discussed below) has sequence identity or similarity to a known sequence. Sequence identity and/or similarity is determined using standard techniques known in the art, including, but not limited to, the local sequence identity algorithm of Smith & Waterman, Adv. Appl. Math., 2:482 (1981), by the sequence identity alignment algorithm of Needleman & Wunsch, J. Mol. Biol., 48:443 (1970), by the search for similarity method of Pearson & Lipman, Proc. Natl. Acad. Sci.
  • PILEUP creates a multiple sequence alignment from a group of related sequences using progressive, pairwise alignments. It can also plot a tree showing the clustering relationships used to create the alignment. PILEUP uses a simplification of the progressive alignment method of Feng & Doolittle, J. Mol. Evol. 35:351-360 (1987); the method is similar to that described by Higgins & Sharp CABIOS 5:151-153 (1989).
  • Useful PILEUP parameters including a default gap weight of 3.00, a default gap length weight of 0.10, and weighted end gaps.
  • BLAST algorithm Another example of a useful algorithm is the BLAST algorithm, described in: Altschul et al., J. Mol. Biol. 215, 403-410, (1990); Altschul et al., Nucleic Acids Res. 25:3389-3402 (1997); and Karlin et al., Proc. Natl. Acad. Sci. U.S.A. 90:5873-5787 (1993).
  • a particularly useful BLAST program is the WU- BLAST-2 program which was obtained from Altschul et al., Methods in Enzymology, 266:460-480
  • WU-BLAST-2 uses several search parameters, most of which are set to the default values.
  • the HSP S and HSP S2 parameters are dynamic values and are established by the program itself depending upon the composition of the particular sequence and composition of the particular database against which the sequence of interest is being searched; however, the values may be adjusted to increase sensitivity.
  • An additional useful algorithm is gapped BLAST as reported by Altschul et al., Nucl. Acids Res., 25:3389-3402.
  • Gapped BLAST uses BLOSUM-62 substitution scores; threshold T parameter set to 9; the two-hit method to trigger ungapped extensions; charges gap lengths of k a cost of 10+/c; X u set to 16, and X g set to 40 for database search stage and to 67 for the output stage of the algorithms. Gapped alignments are triggered by a score corresponding to ⁇ 22 bits.
  • a % amino acid sequence identity value is determined by the number of matching identical residues divided by the total number of residues of the "longer" sequence in the aligned region.
  • the "longer” sequence is the one having the most actual residues in the aligned region (gaps introduced by WU- Blast-2 to maximize the alignment score are ignored).
  • percent (%) nucleic acid sequence identity with respect to the coding sequence of the polypeptides identified herein is defined as the percentage of nucleotide residues in a candidate sequence that are identical with the nucleotide residues in the coding sequence of the target protein.
  • a preferred method utilizes the BLASTN module of WU-BLAST-2 set to the default parameters, with overlap span and overlap fraction set to 1 and 0.125, respectively.
  • the alignment may include the introduction of gaps in the sequences to be aligned.
  • the percentage of sequence identity will be determined based on the number of identical amino acids in relation to the total number of amino acids. In percent identity calculations relative weight is not assigned to various manifestations of sequence variation, such as, insertions, deletions, substitutions, etc.
  • identities are scored positively (+1) and all forms of sequence variation including gaps are assigned a value of "0", which obviates the need for a weighted scale or parameters as described below for sequence similarity calculations.
  • Percent sequence identity can be calculated, for example, by dividing the number of matching identical residues by the total number of residues of the "shorter" sequence in the aligned region and multiplying by 100. The "longer" sequence is the one having the most actual residues in the aligned region.
  • the variant proteins of the present invention may be shorter or longer than the target protein. Included within the definition of variant proteins are portions or fragments of the target sequence. Fragments of variant proteins are considered variant ⁇ proteins if they share a) at least one antigenic epitope; b) have at least the indicated homology; c) and preferably exhibit the biological activity of the target protein.
  • the candidate variant proteins include further amino acid variations, as compared to a target protein, than those outlined herein.
  • any of the variations depicted herein may be combined in any way to form additional novel variant proteins.
  • candidate variant proteins can be made that are longer than the target protein, for example, by the addition of other sequences, such as purification tags, fusion sequences, etc, as described in U.S.S.N. 09/798,789, incorporated herein by reference in its entirety.
  • the variant proteins of the invention may be fused to other therapeutic proteins or to other proteins such as Fc or serum albumin for pharmacokinetic purposes. See for example U.S. Patent No. 5,766,883 and
  • variant proteins comprising variable residues in core, surface, and boundary residues.
  • the variant proteins of the invention are human conformers.
  • conformer herein is meant a protein that has a protein backbone 3D structure that is virtually the same but has significant differences in the amino acid side chains. That is, the variant proteins of the invention define a conformer set, wherein all of the proteins of the set share a backbone structure and yet have sequences that differ by at least 1-3-5%.
  • the three-dimensional backbone structure of a variant protein thus substantially corresponds to the three dimensional backbone structure of human target protein.
  • Backbone in this context means the non-side chain atoms: the nitrogen, carbonyl carbon and oxygen, and the ⁇ -carbon, and the hydrogens attached to the nitrogen and ⁇ -carbon.
  • a protein must have backbone atoms that are no more than 2 A from the human target protein structure, with no more than 1.5 A being preferred, and no more than 1 A being particularly preferred. In general, these distances may be determined in two ways. In one embodiment, each potential conformer is crystallized and its three dimensional structure determined. Alternatively, as the former is technically challenging, the sequence of each potential conformer is run in the PDATM program to determine whether it is a conformer.
  • Candidate variant proteins may also be identified as being encoded by candidate variant nucleic acids.
  • the overall homology of the nucleic acid sequence is commensurate with amino acid homology but takes into account the degeneracy in the genetic code and codon bias of different organisms. Accordingly, the nucleic acid sequence homology may be either lower or higher than that of the protein sequence, with lower homology being preferred.
  • a candidate variant nucleic acid encodes a candidate variant protein.
  • an extremely large number of nucleic acids may be made, all of which encode the variant proteins of the present invention.
  • those skilled in the art could make any number of different nucleic acids, by simply modifying the sequence of one or more codons in a way that does not change the amino acid sequence of the variant protein.
  • the nucleic acid homology is determined through hybridization studies.
  • stringent conditions are selected to be about 5-10'C lower than the thermal melting point (TJ for the specific sequence at a defined ionic strength and pH.
  • T m is the temperature (under defined ionic strength, pH and nucleic acid concentration) at which 50% of the probes complementary to the target hybridize to the target sequence at equilibrium (as the target sequences are present in excess, at T m , 50% of the probes are occupied at equilibrium).
  • Stringent conditions will be those in which the salt concentration is less than about 1.0 M sodium ion, typically about 0.01 to 1.0 M sodium ion concentration (or other salts) at pH 7.0 to 8.3 and the temperature is at least about 30 * C for short probes (e.g. 10 to 50 nucleotides) and at least about 60°C for long probes (e.g. greater than 50 nucleotides).
  • Stringent conditions may also be achieved with the addition of destabilizing agents such as formamide.
  • less stringent hybridization conditions are used; for example, moderate or low stringency conditions may be used, as are known in the art; see Maniatis and Ausubel, supra, and Tijssen, supra.
  • nucleic acid may refer to either DNA or RNA, or molecules that contain both deoxy- and ribonucleotides.
  • the nucleic acids include genomic DNA, cDNA and oligonucleotides including sense and anti-sense nucleic acids.
  • Such nucleic acids may also contain modifications in the ribose- phosphate backbone to increase stability and half-life of such molecules in physiological environments.
  • the nucleic acid may be double stranded, single stranded, or contain portions of both double stranded or single stranded sequence.
  • the depiction of a single strand also defines the sequence of the other strand (“Crick”); thus the sequence depicted in Figure 6 also includes the complement of the sequence.
  • recombinant nucleic acid herein is meant nucleic acid, originally formed in vitro, in general, by the manipulation of nucleic acid by endonucleases, in a form not normally found in nature.
  • an isolated candidate variant nucleic acid, in a linear form, or an expression vector formed in vitro by ligating DNA molecules that are not normally joined are both considered recombinant for the purposes of this invention.
  • nucleic acid once a recombinant nucleic acid is made and reintroduced into a host cell or organism, it will replicate non-recombinantly, i.e. using the in vivo cellular machinery of the host cell rather than in vitro manipulations; however, such nucleic acids, once produced recombinantly, although subsequently replicated non-recombinantly, are still considered recombinant for the purposes of the invention.
  • a "recombinant protein” is a protein made using recombinant techniques, i.e. through the expression of a recombinant nucleic acid as depicted above.
  • a recombinant protein is distinguished from naturally occurring protein by at least one or more characteristics.
  • the protein may be isolated or purified away from some or all of the proteins and compounds with which it is normally associated in its wild type host, and thus may be substantially pure.
  • an isolated protein is unaccompanied by at least some of the material with which it is normally associated in its natural state, preferably constituting at least about 0.5%, more preferably at least about 5% by weight of the total protein in a given sample.
  • a substantially pure protein comprises at least about 75% by weight of the total protein, with at least about 80% being preferred, and at least about 90% being particularly preferred.
  • the definition includes the production of a candidate variant protein from one organism in a different organism or host cell. Alternatively, the protein may be made at a significantly higher concentration than is normally seen, through the use of a inducible promoter or high expression promoter, such that the protein is made at increased concentration levels.
  • all of the variant proteins outlined herein are in a form not normally found in nature, as they contain amino acid substitutions, insertions and deletions, with substitutions being preferred, as discussed below.
  • candidate variant proteins of the present invention are amino acid sequence variants of the candidate variant sequences outlined herein. That is, the candidate variant proteins may contain additional variable positions as compared to the target protein. These variants fall into one or more of three classes: substitutional, insertional or deletional variants. These variants ordinarily are prepared by site specific mutagenesis of nucleotides in the DNA encoding a candidate variant protein, using cassette or PCR mutagenesis or other techniques well known in the art, to produce DNA encoding the variant, and thereafter expressing the DNA in recombinant cell culture as outlined above. However, candidate variant protein fragments having up to about 100-150 residues may be prepared by in vitro synthesis using established techniques.
  • Amino acid sequence variants are characterized by the predetermined nature of the variation, a feature that sets them apart from naturally occurring allelic or interspecies variation of the candidate variant protein amino acid sequence.
  • the variants typically exhibit the same qualitative biological activity as the naturally occurring analogue, although variants can also be selected which have modified characteristics as will be more fully outlined below.
  • the mutation per se need not be predetermined.
  • random mutagenesis may be conducted at the target codon or region and the expressed variant proteins screened for the optimal combination of desired activity.
  • Techniques for making substitution mutations at predetermined sites in DNA having a known sequence are well known, for example, M13 primer mutagenesis and PCR mutagenesis.
  • Amino acid substitutions are typically of single residues; insertions usually will be on the order of from about 1 to 20 amino acids, although considerably larger insertions may be tolerated. Deletions range from about 1 to about 20 residues, although in some cases deletions may be much larger.
  • substitutions may be used to arrive at a final derivative. Generally these changes are done on a few amino acids to minimize the alteration of the molecule. However, larger changes may be tolerated in certain circumstances. When small alterations in the characteristics of the variant protein are desired, substitutions are generally made in accordance with the chart 1 :
  • substitutions that are less conservative than those shown in Chart I.
  • substitutions may be made which more significantly affect: the structure of the polypeptide backbone in the area of the alteration, for example the alpha-helical or beta-sheet structure; the charge or hydrophobicity of the molecule at the target site; or the bulk of the side chain.
  • the substitutions which in general are expected to produce the greatest changes in the polypeptide's properties are those in which (a) a hydrophilic residue, e.g. seryl or threonyl, is substituted for (or by) a hydrophobic residue, e.g.
  • leucyl isoleucyl, phenylalanyl, valyl or alanyl
  • a cysteine or proline is substituted for (or by) any other residue
  • a residue having an electropositive side chain e.g. lysyl, arginyl, or histidyl
  • an electronegative residue e.g. glutamyl or aspartyl
  • a residue having a bulky side chain e.g. phenylalanine, is substituted for (or by) one not having a side chain, e.g. glycine.
  • the variants typically exhibit the same qualitative biological activity, however the immune response may be altered from that of the original candidate variant protein, as needed.
  • the variant may be designed such that the biological activity of the candidate variant protein is altered. For example, glycosylation sites may be altered or removed. Similarly, the biological function may be altered.
  • a change in oxidative stability is evidenced by at least about 20%, more preferably at least about 50% increase of activity of a variant protein when exposed to various oxidizing conditions as compared to that of wild-type protein. Oxidative stability is measured by known procedures.
  • a change in alkaline stability is evidenced by at least about a 5% or greater increase or decrease
  • alkaline stability is measured by known procedures.
  • a change in thermal stability is evidenced by at least about a 5% or greater increase or decrease
  • thermal stability is measured by known procedures.
  • candidate variant proteins and nucleic acids of the invention can be made in a number of ways.
  • nucleic acids and proteins can be made as known in the art and outlined below. Alternatively, libraries of candidate variant proteins can be made for testing.
  • the library of candidate variant proteins is generated from a probability distribution table.
  • a probability distribution table As outlined herein, there are a variety of methods of generating a probability distribution table, including using PDATM technology, sequence alignments, forcefield calculations such as self-consistent meant field (SCMF) calculations, etc.
  • the probability distribution can be used to generate information entropy scores for each position, as a measure of the mutational frequency observed in the library.
  • the frequency of each amino acid residue at each variable position in the list is identified.
  • Frequencies can be thresholded, wherein any variant frequency lower than a cutoff is set to zero. This cutoff is preferably about 1%, 2%, 5%, 10% or 20%, with about 10% being particularly preferred.
  • These frequencies are then built into the library of candidate variant proteins. That is, as above, these variable positions are collected and all possible combinations are generated, but the amino acid residues that "fill" the library of candidate variant proteins are utilized on a frequency basis.
  • a variable position that has 5 possible residues will have about 20% of the proteins comprising that variable position with the first possible residue, 20% with the second, etc.
  • the actual frequency may depend on the method used to actually generate the proteins; for example, exact frequencies may be possible when the proteins are synthesized. However, when the frequency-based primer system outlined below is used, the actual frequencies at each position will vary, as outlined below.
  • SCMF self-consistent mean field
  • HARMM is used in the QUANTA molecular modeling package (Biosym/MSI, San Diego California); all references hereby expressly incorporated by reference in their entirety.
  • a preferred method of generating a probability distribution table is through the use of sequence alignment programs.
  • the probability table can be obtained by a combination of sequence alignments and computational approaches. For example, one can add amino acids found in the alignment of homologous sequences to the result of the computation. Preferable one can add the wild type amino acid identity to the probability table if it is not found in the computation.
  • a library of candidate variant proteins created by recombining variable positions and/or residues at the variable position may not be in a rank-ordered list. In some embodiments, the entire list may just be made and tested.
  • the secondary library is also in the form of a rank ordered list. This may be done for several reasons, including the size of the secondary library is still too big to generate experimentally, or for predictive purposes. This may be done in several ways. In one embodiment, the secondary library is ranked or filtered using the scoring functions of PDATM to rank or filter the library members. Alternatively, statistical methods could be used.
  • the secondary library may be ranked or filtered by frequency score; that is, proteins containing the most of high frequency residues could be ranked higher, etc. This may be done by adding or multiplying the frequency at each variable position to generate a numerical score.
  • the secondary library different positions could be weighted and then the proteins scored; for example, those containing certain residues could be arbitrarily ranked or filtered.
  • the different protein members of the candidate variant library may be chemically synthesized. This is particularly useful when the designed proteins are short, preferably less than 150 amino acids in length, with less than 100 amino acids being preferred, and less than 50 amino acids being particularly preferred, although as is known in the art, longer proteins can be made chemically or enzymatically. See for example Wilken et al, Curr. Opin. Biotechnol. 9:412-26 (1998), hereby expressly incorporated by reference.
  • the candidate variant sequences are used to create nucleic acids such as DNA which encode the member sequences and which can then be cloned into host cells, expressed and assayed, if desired.
  • nucleic acids, and particularly DNA can be made which encodes each member protein sequence. This is done using well known procedures. The choice of codons, suitable expression vectors and suitable host cells will vary depending on a number of factors, and can be easily optimized as needed.
  • multiple PCR reactions with pooled oligonucleotides is done, as is generally depicted in Figure 1.
  • overlapping oligonucleotides are synthesized which correspond to the full length gene. Again, these oligonucleotides may represent all of the different amino acids at each variant position or subsets.
  • these oligonucleotides are pooled in equal proportions and multiple PCR reactions are performed to create full length sequences containing the combinations of mutations defined by the secondary library. In addition, this may be done using error-prone PCR methods.
  • the different oligonucleotides are added in relative amounts corresponding to the probability distribution table.
  • the multiple PCR reactions thus result in full length sequences with the desired combinations of mutation in the desired proportions.
  • the total number of oligonucleotides needed is a function of the number of positions being mutated and the number of mutations being considered at these positions:
  • Mn total number of oligos required), where Mn is the number of mutations considered at position n in the sequence.
  • each overlapping oligonucleotide comprises only one position to be varied; in alternate embodiments, the variant positions are too close together to allow this and multiple variants per oligonucleotide are used to allow complete recombination of all the possibilities. That is, each oligo can contain the codon for a single position being mutated, or for more than one position being mutated. The multiple positions being mutated must be close in sequence to prevent the oligo length from being impractical. For multiple mutating positions on an oligonucleotide, particular combinations of mutations can be included or excluded in the library by including or excluding the oligonucleotide encoding that combination.
  • clusters there may be correlations between variable regions; that is, when position X is a certain residue, position Y must (or must not) be a particular residue.
  • These sets of variable positions are sometimes referred to herein as a "cluster".
  • the clusters When the clusters are comprised of residues close together, and thus can reside on one oligonucleotide primer, the clusters can be set to the "good” correlations, and eliminate the bad combinations that may decrease the effectiveness of the library. However, if the residues of the cluster are far apart in sequence, and thus will reside on different oligonucleotides for synthesis, it may be desirable to either set the residues to the "good” correlation, or eliminate them as variable residues entirely.
  • the library may be generated in several steps, so that the cluster mutations only appear together.
  • This procedure i.e., the procedure of identifying mutation clusters and either placing them on the same oligonucleotides or eliminating them from the library or library generation in several steps preserving clusters, can considerably enrich the experimental library with properly folded protein.
  • Identification of clusters can be carried out by a number of ways, e.g. by using known pattern recognition methods, comparisons of frequencies of occurrence of mutations or by using energy analysis of the sequences to be experimentally generated (for example, if the energy of interaction is high, the positions are correlated). These correlations may be positional correlations (e.g. variable positions 1 and 2 always change together or never change together) or sequence correlations (e.g. if there is a residue A at position 1 , there is always residue B at position 2). See: Pattern discovery in Biomolecular Data: Tools, Techniques, and Applications; edited by Jason
  • correlations and shuffling can be fixed or optimized by altering the design of the oligonucleotides; that is, by deciding where the oligonucleotides (primers) start and stop (e.g. where the sequences are "cut").
  • the start and stop sites of oligos can be set to maximize the number of clusters that appear in single oligonucleotides, thereby enriching the library with higher scoring sequences.
  • Different oligonucleotides start and stop site options can be computationally modeled and ranked or filtered according to number of clusters that are represented on single oligos, or the percentage of the resulting sequences consistent with the predicted library of sequences.
  • the total number of oligonucleotides required increases when multiple mutable positions are encoded by a single oligonucleotide.
  • the annealed regions are the ones that remain constant, i.e. have the sequence of the reference sequence.
  • Oligonucleotides with insertions or deletions of codons can be used to create a library expressing different length proteins.
  • computational sequence screening for insertions or deletions can result in secondary libraries defining different length proteins, which can be expressed by a library of pooled oligonucleotide of different lengths.
  • the secondary library is done by shuffling the family (e.g. a set of variants); that is, some set of the top sequences (if a rank-ordered list is used) can be shuffled, either with or without error-prone PCR.
  • shuffling in this context means a recombination of related sequences, generally in a random way. It can include “shuffling” as defined and exemplified in U.S. Patent Nos. 5,830,721; 5,811 ,238; 5,605,793; 5,837,458 and PCT US/19256, all of which are expressly incorporated by reference in their entirety.
  • This set of sequences can also be an artificial set; for example, from a probability table (for example generated using SCMF) or a Monte Carlo set.
  • the "family" can be the top 10 and the bottom 10 sequences, the top 100 sequences, etc.
  • in silico shuffling is done using the computational methods described therein. That is, starting with either two libraries or two sequences, random recombinations of the sequences can be generated and evaluated.
  • error-prone PCR is done to generate the secondary library. See U.S. Patent Nos. 5,605,793, 5,811 ,238, and 5,830,721, all of which are hereby incorporated by reference. This can be done on the optimal sequence or on top members of the library, or some other artificial set or family.
  • the gene for the optimal sequence found in the computational screen of the primary library can be synthesized.
  • Error prone PCR is then performed on the optimal sequence gene in the presence of oligonucleotides that code for the mutations at the variant positions of the secondary library (bias oligonucleotides). The addition of the oligonucleotides will create a bias favoring the incorporation of the mutations in the secondary library. Alternatively, only oligonucleotides for certain mutations may be used to bias the library.
  • gene shuffling with error prone PCR can be performed on the gene for the optimal sequence, in the presence of bias oligonucleotides, to create a DNA sequence library that reflects the proportion of the mutations found in the secondary library.
  • bias oligonucleotides can be done in a variety of ways; they can chosen on the basis of their frequency, i.e.
  • oligonucleotides encoding high mutational frequency positions can be used; alternatively, oligonucleotides containing the most variable positions can be used, such that the diversity is increased; if the secondary library is ranked or filtered, some number of top scoring positions can be used to generate bias oligonucleotides; random positions may be chosen; a few top scoring and a few low scoring ones may be chosen; etc. What is important is to generate new sequences based on preferred variable positions and sequences.
  • PCR using a wild type gene or target gene can be used, as is schematically depicted in Figure 1.
  • a starting gene is used; generally, although this is not required, the gene is the wild type gene. In some cases it may be the gene encoding the global optimized sequence, or any other sequence of the list.
  • oligonucleotides are used that correspond to the variant positions and contain the different amino acids of the secondary library. PCR is done using PCR primers at the termini, as is known in the art. This provides two benefits; the first is that this generally requires fewer oligonucleotides and can result in fewer errors. In addition, it has experimental advantages in that if the wild type gene is used, it need not be synthesized.
  • a variety of additional steps may be done to one or more candidate variant secondary libraries; for example, further computational processing can occur, candidate variant secondary libraries can be recombined, or cutoffs from different candidate variant secondary libraries can be combined.
  • a candidate variant secondary library may be computationally remanipulated to form an additional secondary library (sometimes referred to herein as "tertiary libraries").
  • additional secondary library sometimes referred to herein as "tertiary libraries"
  • any of the candidate variant secondary library sequences may be chosen for a second round of PDATM, by freezing or fixing some or all of the changed positions in the first secondary library. Alternatively, only changes seen in the last probability distribution table are allowed. Alternatively, the stringency of the probability table may be altered, either by increasing or decreasing the cutoff for inclusion.
  • the candidate variant secondary library may be recombined experimentally after the first round; for example, the best gene/genes from the first screen may be taken and gene assembly redone (using techniques outlined below, multiple PCR, error prone
  • the fragments from one or more good gene(s) to change probabilities at some positions. This biases the search to an area of sequence space found in the first round of computational and experimental screening.
  • a tertiary library can be generated from combining candidate variant secondary libraries.
  • a probability distribution table from a candidate variant secondary library can be generated and recombined, whether computationally or experimentally, as outlined herein.
  • a PDATM technology candidate variant secondary library may be combined with a sequence alignment secondary library, and either recombined (again, computationally or experimentally) or just the cutoffs from each joined to make a new tertiary library.
  • the top sequences from several libraries can be recombined.
  • Primary and secondary libraries can similarly be combined. Sequences from the top of a library can be combined with sequences from the bottom of the library to more broadly sample sequence space, or only sequences distant from the top of the library can be combined.
  • Candidate variant secondary libraries that analyzed different parts of the protein can be combined to a tertiary library that treats the combined parts of the protein.
  • a tertiary library can be generated using correlations in the candidate variant secondary library. That is, a residue at a first variable position may be correlated to a residue at second variable position (or correlated to residues at additional positions as well). For example, two variable positions may sterically or electrostatically interact, such that if the first residue is X, the second residue must be Y. This may be either a positive or negative correlation.
  • the expression vectors may be either self-replicating extrachromosomal vectors or vectors which integrate into a host genome. Generally, these expression vectors include transcriptional and translational regulatory nucleic acid operably linked to the nucleic acid encoding the library protein.
  • control sequences refers to DNA sequences necessary for the expression of an operably linked coding sequence in a particular host organism.
  • the control sequences that are suitable for prokaryotes include a promoter, optionally an operator sequence, and a ribosome binding site. Eukaryotic cells are known to utilize promoters, polyadenylation signals, and enhancers.
  • Nucleic acid is "operably linked" when it is placed into a functional relationship with another nucleic acid sequence.
  • DNA for a presequence or secretory leader is operably linked to DNA for a polypeptide if it is expressed as a preprotein that participates in the secretion of the polypeptide;
  • a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the sequence; or
  • a ribosome binding site is operably linked to a coding sequence if it is positioned so as to facilitate translation.
  • "operably linked” means that the DNA sequences being linked are contiguous, and, in the case of a secretory leader, contiguous and in reading phase.
  • transcriptional and translational regulatory nucleic acid will generally be appropriate to the host cell used to express the library protein, as will be appreciated by those in the art; for example, transcriptional and translational regulatory nucleic acid sequences from Bacillus are preferably used to express the library protein in Bacillus. Numerous types of appropriate expression vectors, and suitable regulatory sequences are known in the art for a variety of host cells.
  • the transcriptional and translational regulatory sequences may include, but are not limited to, promoter sequences, ribosomal binding sites, transcriptional start and stop sequences, translational start and stop sequences, and enhancer or activator sequences.
  • the regulatory sequences include a promoter and transcriptional start and stop sequences.
  • Promoter sequences include constitutive and inducible promoter sequences.
  • the promoters may be naturally occurring promoters, hybrid or synthetic promoters. Hybrid promoters, which combine elements of more than one promoter, are also known in the art, and are useful in the present invention.
  • the expression vector may comprise additional elements.
  • the expression vector may have two replication systems, thus allowing it to be maintained in two organisms, for example in mammalian or insect cells for expression and in a prokaryotic host for cloning and amplification.
  • the expression vector contains at least one sequence homologous to the host cell genome, and preferably two homologous sequences that flank the expression construct.
  • the integrating vector may be directed to a specific locus in the host cell by selecting the appropriate homologous sequence for inclusion in the vector.
  • the expression vector contains a selection gene to allow the selection of transformed host cells containing the expression vector, and particularly in the case of mammalian cells, ensures the stability of the vector, since cells that do not contain the vector will generally die. Selection genes are well known in the art and will vary with the host cell used.
  • selection gene herein is meant any gene that encodes a gene product that confers resistance to a selection agent. Suitable selection agents include, but are not limited to, neomycin (or its analog G418), blasticidin S, histinidol D, bleomycin, puromycin, hygromycin B, and other drugs.
  • the expression vector contains a RNA splicing sequence upstream or downstream of the gene to be expressed in order to increase the level of gene expression. See Barret et al., Nucleic Acids Res. 1991; Groos et al., Mol. Cell. Biol. 1987; and Budiman et al., Mol. Cell. Biol. 1988.
  • a preferred expression vector system is a retroviral vector system such as is generally described in
  • the candidate variant library proteins of the present invention are produced by culturing a host cell transformed with nucleic acid, preferably an expression vector, containing nucleic acid encoding an library protein, under the appropriate conditions to induce or cause expression of the library protein.
  • nucleic acid preferably an expression vector, containing nucleic acid encoding an library protein
  • the conditions appropriate for candidate variant library protein expression will vary with the choice of the expression vector and the host cell, and will be easily ascertained by one skilled in the art through routine experimentation.
  • constitutive promoters in the expression vector will require optimizing the growth and proliferation of the host cell, while the use of an inducible promoter requires the appropriate growth conditions for induction.
  • the timing of the harvest is important.
  • the baculoviral systems used in insect cell expression are lytic viruses, and thus harvest time selection can be crucial for product yield.
  • the type of cells used in the present invention can vary widely. Basically, a wide variety of appropriate host cells can be used, including yeast, bacteria, archaebacteria, fungi, and insect and animal cells, including mammalian cells. Of particular interest are Drosophila melanogaster cells, Saccharomyces cerevisiae and other yeasts, E. coli, Bacillus subtilis, SF9 cells, C129 cells, 293 cells, Neurospora, BHK, CHO, COS, and HeLa cells, fibroblasts, Schwanoma cell lines, immortalized mammalian myeloid and lymphoid cell lines, Jurkat cells, mast cells and other endocrine and exocrine cells, and neuronal cells.
  • the cells may be genetically engineered, that is, contain exogenous nucleic acid, for example, to contain target molecules.
  • the candidate variant library proteins are expressed in mammalian cells. Any mammalian cells may be used, with mouse, rat, primate and human cells being particularly preferred, although as will be appreciated by those in the art, modifications of the system by pseudotyping allows all eukaryotic cells to be used, preferably higher eukaryotes.
  • a screen will be set up such that the cells exhibit a selectable phenotype in the presence of a random library member.
  • cell types implicated in a wide variety of disease conditions are particularly useful, so long as a suitable screen may be designed to allow the selection of cells that exhibit an altered phenotype as a consequence of the presence of a library member within the cell.
  • suitable mammalian cell types include, but are not limited to, tumor cells of all types (particularly melanoma, myeloid leukemia, carcinomas of the lung, breast, ovaries, colon, kidney, prostate, pancreas and testes), cardiomyocytes, endothelial cells, epithelial cells, lymphocytes (T-cell and B cell) , mast cells, eosinophils, vascular intimal cells, hepatocytes, leukocytes including mononuclear leukocytes, stem cells such as haemopoetic, neural, skin, lung, kidney, liver and myocyte stem cells (for use in screening for differentiation and de-differentiation factors), osteoclasts, chondrocytes and other connective tissue cells, keratinocytes, melanocytes, liver cells, kidney cells, and adipocytes.
  • Suitable cells also include known research cells, including, but not limited to, Jurkat T cells, NIH3T3 cells, CHO, Cos, etc
  • a mammalian promoter is any DNA sequence capable of binding mammalian RNA polymerase and initiating the downstream (3') transcription of a coding sequence for library protein into mRNA.
  • a promoter will have a transcription initiating region, which is usually placed proximal to the 5' end of the coding sequence, and a TATA box, using a located 25-30 base pairs upstream of the transcription initiation site. The TATA box is thought to direct RNA polymerase II to begin RNA synthesis at the correct site.
  • a mammalian promoter will also contain an upstream promoter element (enhancer element), typically located within 100 to 200 base pairs upstream of the TATA box.
  • An upstream promoter element determines the rate at which transcription is initiated and can act in either orientation.
  • mammalian promoters are the promoters from mammalian viral genes, since the viral genes are often highly expressed and have a broad host range. Examples include the SV40 early promoter, mouse mammary tumor virus LTR promoter, adenovirus major late promoter, herpes simplex virus promoter, and the CMV promoter.
  • transcription termination and polyadenylation sequences recognized by mammalian cells are regulatory regions located 3' to the translation stop codon and thus, together with the promoter elements, flank the coding sequence.
  • the 3' terminus of the mature mRNA is formed by site-specific post-transactional cleavage and polyadenylation.
  • transcription terminator and polyadenylation signals include those derived from SV40.
  • candidate variant library proteins are expressed in bacterial systems.
  • Bacterial expression systems are well known in the art.
  • a suitable bacterial promoter is any nucleic acid sequence capable of binding bacterial RNA polymerase and initiating the downstream (3') transcription of the coding sequence of library protein into mRNA.
  • a bacterial promoter has a transcription initiation region that is usually placed proximal to the 5' end of the coding sequence. This transcription initiation region typically includes an RNA polymerase binding site and a transcription initiation site. Sequences encoding metabolic pathway enzymes provide particularly useful promoter sequences.
  • promoter sequences derived from sugar metabolizing enzymes such as galactose, lactose and maltose
  • sequences derived from biosynthetic enzymes such as tryptophan.
  • Promoters from bacteriophage may also be used and are known in the art.
  • synthetic promoters and hybrid promoters are also useful; for example, the tac promoter is a hybrid of the frp and lac promoter sequences.
  • a bacterial promoter can include naturally occurring promoters of non-bacterial origin that have the ability to bind bacterial RNA polymerase and initiate transcription.
  • the ribosome binding site is called the Shine-Delgarno (SD) sequence and includes an initiation codon and a sequence 3-9 nucleotides in length located 3 - 11 nucleotides upstream of the initiation codon.
  • SD Shine-Delgarno
  • the expression vector may also include a signal peptide sequence that provides for secretion of the library protein in bacteria.
  • the signal sequence typically encodes a signal peptide comprised of hydrophobic amino acids ' which direct the secretion of the protein from the cell, as is well known in the art.
  • the protein is either secreted into the growth media (gram-positive bacteria) or into the periplasmic space, located between the inner and outer membrane of the cell (gram-negative bacteria).
  • the bacterial expression vector may also include a selectable marker gene to allow for the selection of bacterial strains that have been transformed.
  • Suitable selection genes include genes which render the bacteria resistant to drugs such as ampicillin, chloramphenicol, erythromycin, kanamycin, neomycin and tetracycline.
  • Selectable markers also include biosynthetic genes, such as those in the histidine, tryptophan and leucine biosynthetic pathways.
  • Expression vectors for bacteria are well known in the art, and include vectors for Bacillus subtilis, E. coli, Streptococcus cremoris, and Streptococcus lividans, among others.
  • the bacterial expression vectors are transformed into bacterial host cells using techniques well known in the art, such as calcium chloride treatment, electroporation, and others.
  • candidate variant library proteins are produced in insect cells.
  • Expression vectors for the transformation of insect cells, and in particular, baculovirus-based expression vectors, are well known in the art and are described e.g., in O'Reilly et al., Baculovirus Expression Vectors: A Laboratory Manual (New York: Oxford University Press, 1994).
  • candidate variant library protein is produced in yeast cells.
  • Yeast expression systems are well known in the art, and include expression vectors for Saccharomyces cerevisiae, Candida albicans and C. maltosa, Hansenula polymorpha, Kluyveromyces fragilis and K. lactis, Pichia guillerimondii and P. pastoris, Schizosaccharomyces pombe, and Yarrowia lipolytica.
  • Preferred promoter sequences for expression in yeast include the inducible GAL1 , 10 promoter, the promoters from alcohol dehydrogenase, enolase, glucokinase, glucose-6-phosphate isomerase, glyceraldehyde-3-phosphate-dehydrogenase, hexokinase, phosphofructokinase, 3-phosphoglycerate mutase, pyruvate kinase, and the acid phosphatase gene.
  • Yeast selectable markers include ADE2, H1S4, LEU2, TRP1, and ALG7, which confers resistance to tunicamycin; the neomycin phosphotransferase gene, which confers resistance to G418; and the CUP1 gene, which allows yeast to grow in the presence of copper ions.
  • the candidate variant library protein may also be made as a fusion protein, using techniques well known in the art.
  • the library protein may be fused to a carrier protein to form an immunogen.
  • the library protein may be made as a fusion protein to increase expression, or for other reasons.
  • the nucleic acid encoding the peptide may be linked to other nucleic acid for expression purposes.
  • fusion partners may be used, such as targeting sequences which allow the localization of the library members into a subcellular or extracellular compartment of the cell, rescue sequences or purification tags which allow the purification or isolation of either the library protein or the nucleic acids encoding them; stability sequences, which confer stability or protection from degradation to the library protein or the nucleic acid encoding it, for example resistance to proteolytic degradation, or combinations of these, as well as linker sequences as needed.
  • suitable targeting sequences include, but are not limited to, binding sequences capable of causing binding of the expression product to a predetermined molecule or class of molecules while retaining bioactivity of the expression product, (for example by using enzyme inhibitor or substrate sequences to target a class of relevant enzymes); sequences signaling selective degradation, of itself or co-bound proteins; and signal sequences capable of constitutively localizing the candidate expression products to a predetermined cellular locale, including a) subcellular locations such as the Golgi, endoplasmic reticulum, nucleus, nucleoli, nuclear membrane, mitochondria, chloroplast, secretory vesicles, lysosome, and cellular membrane; and b) extracellular locations via a secretory signal. Particularly preferred is localization to either subcellular locations or to the outside of the cell via secretion.
  • the candidate variant library member comprises a rescue sequence.
  • a rescue sequence is a sequence that may be used to purify or isolate either the candidate agent or the nucleic acid encoding it.
  • peptide rescue sequences include purification sequences such as the His 6 tag for use with Ni affinity columns and epitope tags for detection, immunoprecipitation or FACS (fluoroscence-activated cell sorting).
  • Suitable epitope tags include myc (for use with the commercially available 9E10 antibody), the BSP biotinylation target sequence of the bacterial enzyme BirA, flag tags, lacZ, and GST.
  • the rescue sequence may be a unique oligonucleotide sequence that serves as a probe target site to allow the quick and easy isolation of the retroviral construct, via PCR, related techniques, or hybridization.
  • the fusion partner is a stability sequence to confer stability to the library member or the nucleic acid encoding it.
  • peptides may be stabilized by the incorporation of glycines after the initiation methionine (MG or MGGO), for protection of the peptide to ubiquitination as per Varshavsky's N-End Rule, thus conferring long half-life in the cytoplasm.
  • MG(X) n GGPP where X is any amino acid and n is an integer of at least four.
  • the candidate variant library nucleic acids, proteins and antibodies of the invention are labeled.
  • labeled herein is meant that nucleic acids, proteins and antibodies of the invention have at least one element, isotope or chemical compound attached to enable the detection of nucleic acids, proteins and antibodies of the invention.
  • labels fall into three classes: a) isotopic labels, which may be radioactive or heavy isotopes; b) immune labels, which may be antibodies or antigens; and c) colored or fluorescent dyes. The labels may be incorporated into the compound at any position.
  • the candidate variant library protein is purified or isolated after expression.
  • Library proteins may be isolated or purified in a variety of ways known to those skilled in the art depending on what other components are present in the sample.
  • Standard purification methods include electrophoretic, molecular, immunological and chromatographic techniques, including ion exchange, hydrophobic, affinity, and reverse-phase HPLC chromatography, and chromatofocusing.
  • the library protein may be purified using a standard anti-library antibody column. Ultrafiltration and diafiltration techniques, in conjunction with protein concentration, are also useful. For general guidance in suitable purification techniques, see Scopes, R., Protein Purification,
  • the candidate variant protein is purified or isolated after expression.
  • Variant proteins may be isolated or purified in a variety of ways known to those skilled in the art depending on what other components are present in the sample. Standard purification methods include electrophoretic, molecular, immunological and chromatographic techniques, including ion exchange, hydrophobic, affinity, and reverse-phase HPLC chromatography, and chromatofocusing.
  • the variant protein may be purified using a standard anti-library antibody column. Ultrafiltration and diafiltration techniques, in conjunction with protein concentration, are also useful.
  • the candidate variant library proteins and nucleic acids can be tested for altered immunogenicity.
  • Suitable methods include measuring of the binding of MHC peptide complexes to TCRs, measurement of MHC/peptide interactions(Sidney, J., et al.,
  • candidate variant proteins and nucleic acids of the invention find use in a number of applications.
  • candidate variant proteins that are less immunogenic than the target protein are used as therapeutic proteins.
  • exogenous proteins can be effective in vivo as artificial receptors for the capture of radionuclides, as toxins, or as catalysts for the activation of pro-drugs (Meyer, DL., ef al.
  • candidate variant proteins that are more immunogenetic than the target protein are used in the development of vaccines and immunotherapeutics for autoimmune disease and cancer.
  • vaccines can be made that are more effective at inducing an immune response by inserting a linear amino acid sequence epitope that has increased affinity for MHC class I or class II molecules (see for example, Sarobe, P., et al. (1998) J. Clin. Invest, 102:1239-1248; Thimme, R., et al. (2001) J.
  • vaccines are made that are more effective at inducing an immune response by inserting at least one T cell epitope (de Lalla, C, et al., (1999) J. Immunology, 163:1725-
  • vaccines are made that are more effective at inducing an immune response by inserting a sequence encoding at least one conformational three dimensional epitope that interacts with membrane bound antibodies on naive B cells (see Criag, L, et al., (1998) J. Mol. Biol., 281:183- 201 ; Buttinelli, G., et al., (2001) Virology, 281 :265-271 ; Saphire, E.O., et al., (2001) Science, 293:1155; Mascola and Nabel, (2001) Curr. Op. Immunology, 13:489-495; all references hereby incorporated by reference in their entirety).
  • vaccines are made that are more effective at inducing an immune response by inserting any combination of B cell epitopes, MHC class I binding motifs, MHC class II binding motifs, and T cell epitopes (see for example WO 01/41788 and U.S. Patent No. 6,037,135).
  • Vaccines may be designed that are effective against allergens, bacterial pathogens, viral pathogens and tumors. See for example, WO/41788; U.S. Patent No. 6,322,789; U.S. Patent No. 6,329,505; WO 01/41799; WO 01/42267; WO 01/42270; and, WO 01/45728
  • vaccines may be designed against one or more allergens, including but not limited to, chemical allergens, food allergens, pollen allergens, fungal allergens, pet dander, mites, etc (see
  • vaccines are made against viral pathogens, including but not limited to, Hepatitis A, Hepatitis B, Hepatitis C, poliovirus, HIV, herpes simplex I and II, small pox, human papillomavirus, cytomeglovirus, hantavirus, rabies, Ebola virus, yellow fever virus, rotavirus, rubella, measles virus, mumps virus, Varicella (i.e., chicken pox), influenza, encephalitis, Lassa Fever virus, etc.
  • viral pathogens including but not limited to, Hepatitis A, Hepatitis B, Hepatitis C, poliovirus, HIV, herpes simplex I and II, small pox, human papillomavirus, cytomeglovirus, hantavirus, rabies, Ebola virus, yellow fever virus, rotavirus, rubella, measles virus, mumps virus, Varicella (i.e., chicken pox), influenza
  • vaccines are made against bacterial pathogens, including but not limited to, the causative agent of Lyme disease, diphtheria, anthrax, botulism, pertussis, whooping cough*, tetanus, cholera, typhoid, typhus, plague, Hansen's disease, tuberculosis (including multidrug resistant forms), staphylococcal infections, streptococcal infections, Listeria, meningococcal meningitis, pneumococcal infections, legionnaires' disease, ulcers, conjunctivitis, etc.
  • the causative agent of Lyme disease diphtheria, anthrax, botulism, pertussis, whooping cough*, tetanus, cholera, typhoid, typhus, plague, Hansen's disease, tuberculosis (including multidrug resistant forms), staphylococcal infections, streptococcal infections, Listeria, meningococcal
  • Vaccines also may be made against other infectious agents, including but not limited to the causative agent of dengue fever, malaria, African Sleeping Sickness, dysentery, Rocky Mountain Spotted Fever, Schistosomiasis, Diarrhea, West Nile Fever, Leishmaniasis, Giardiasis, etc.
  • the candidate variant proteins are more immunogenic toward different cancers including solid tumors such as skin, breast, brain, cervical carcinomas, testicular carcinomas, etc.
  • cancers that may be treated by the compositions and methods of the invention include, but are not limited to: Cardiac: sarcoma (angiosarcoma, fibrosarcoma, rhabdomyosarcoma, liposarcoma), myxoma, rhabdomyoma, fibroma, lipoma and teratoma; Lung: bronchogenic carcinoma
  • squamous cell undifferentiated small cell, undifferentiated large cell, adenocarcinoma
  • alveolar bronchiolar carcinoma
  • bronchial adenoma sarcoma
  • lymphoma chondromatous hamartoma
  • mesothelioma Gastrointestinal: esophagus (squamous cell carcinoma, adenocarcinoma, leiomyosarcoma, lymphoma), stomach (carcinoma, lymphoma, leiomyosarcoma), pancreas (ductal adenocarcinoma, insulinoma, glucagonoma, gastrinoma, carcinoid tumors, vipoma), small bowel
  • vaccines are directed to p53 bearing tumors, melanoma antigen genes (MAGE; see WO 01/42267); carcinoembryonic antigen (CEA; see WO 01/42270), prostate cancer antigens (see WO 01/45728 and U.S. Patent No. 6,329,505), such as prostate specific antigen (PSA), prostate specific membrane antigen (PSM), prostatic acid phosphatase (PAP), and human kallikrein2
  • PSA prostate specific antigen
  • PSM prostate specific membrane antigen
  • PAP prostatic acid phosphatase
  • human kallikrein2 human kallikrein2
  • hK2 or HuK2 breast cancer antigens(i.e., her2/neu; see AU 2087401).
  • a therapeutically effective dose of a candidate variant protein is administered to a patient in need of treatment.
  • therapeutically effective dose herein is meant a dose that produces the effects for which it is administered. The exact dose will depend on the purpose of the treatment, and will be ascertainable by one skilled in the art using known techniques.
  • dosages of about 5 ⁇ g/kg are used, administered intraveneously, peritoneally, or subcutaneously.
  • a “patient” for the purposes of the present invention includes both humans and other animals, particularly mammals, and organisms. Thus the methods are applicable to both human therapy and veterinary applications.
  • the patient is a mammal, and in the most preferred embodiment the patient is human.
  • treatment in the instant invention is meant to include therapeutic treatment, as well as prophylactic, or suppressive measures for the disease or disorder.
  • successful administration of a candidate variant protein prior to onset of the disease results in "treatment” of the disease.
  • successful administration of a variant protein after clinical manifestation of the disease to combat the symptoms of the disease comprises “treatment” of the disease.
  • Treatment also encompasses administration of a variant protein after the appearance of the disease in order to eradicate the disease.
  • Successful administration of an agent after onset and after clinical symptoms have developed, with possible abatement of clinical symptoms and perhaps amelioration of the disease, comprises "treatment" of the disease.
  • Those "in need of treatment” include mammals already having the disease or disorder, as well as those prone to having the disease or disorder, including those in which the disease or disorder is to be prevented.
  • the administration of the candidate variant proteins of the present invention can be done in a variety of ways, including, but not limited to, orally, subcutaneously, intravenously, intranasally, transdermally, intraperitoneally, intramuscularly, intrapulmonary, vaginally, rectally, or intraocularly.
  • the candidate variant protein may be directly applied as a solution or spray.
  • the pharmaceutical composition may be formulated in a variety of ways.
  • the concentration of the therapeutically active candidate variant protein in the formulation may vary from about 0.1 to 100 weight %. In another preferred embodiment, the concentration of the candidate variant protein is in the range of 0.003 to 1.0 molar, with dosages from 0.03, 0.05, 0.1, 0.2, and 0.3 millimoles per kilogram of body weight being preferred.
  • compositions of the present invention comprise a candidate variant protein in a form suitable for administration to a patient.
  • the pharmaceutical compositions are in a water soluble form, such as being present as pharmaceutically acceptable salts, which is meant to include both acid and base addition salts.
  • “Pharmaceutically acceptable acid addition salt” refers to those salts that retain the biological effectiveness of the free bases and that are not biologically or otherwise undesirable, formed with inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid and the like, and organic acids such as acetic acid, propionic acid, glycolic acid, pyruvic acid, oxalic acid, maleic acid, malonic acid, succinic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, salicylic acid and the like.
  • inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid and the like
  • organic acids such as acetic acid, propionic acid, glycolic acid, pyruvic acid,
  • “Pharmaceutically acceptable base addition salts” include those derived from inorganic bases such as sodium, potassium, lithium, ammonium, calcium, magnesium, iron, zinc, copper, manganese, aluminum salts and the like. Particularly preferred are the ammonium, potassium, sodium, calcium, and magnesium salts.
  • Salts derived from pharmaceutically acceptable organic non-toxic bases include salts of primary, secondary, and tertiary amines, substituted amines including naturally occurring substituted amines, cyclic amines and basic ion exchange resins, such as isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine, and ethanolamine.
  • compositions may also include one or more of the following: carrier proteins such as serum albumin; buffers such as NaOAc; fillers such as microcrystalline cellulose, lactose, corn and other starches; binding agents; sweeteners and other flavoring agents; coloring agents; and polyethylene glycol.
  • carrier proteins such as serum albumin
  • buffers such as NaOAc
  • fillers such as microcrystalline cellulose, lactose, corn and other starches
  • binding agents such as microcrystalline cellulose, lactose, corn and other starches
  • sweeteners and other flavoring agents such as a variety of formulations. See for example, Goodman and Gilman, incorporated herein by reference in its entirety.
  • the candidate variant proteins are added in a micellular formulation; see U.S. Patent No.5, 833, 948, hereby expressly incorporated by reference in its entirety.
  • compositions comprising mixtures of variant proteins exhibiting enhanced immunogenicity selected from the group consisting of variants of soluble proteins such as, zinc-alpha2-glycoprotein, human serum albumin, immunoglobulin G (IgG) and other modified non-immunogenic proteins may be administered to a patient.
  • pharmaceutical compositions comprising mixtures of variant proteins exhibiting enhanced immunogenicity selected from the group consisting of variants of soluble proteins such as, zinc-alpha2-glycoprotein, human serum albumin, immunoglobulin G (IgG) and other modified non-immunogenic proteins may be administered to a patient.
  • the compositions may be administered in combination with other therapeutics.
  • antibodies including but not limited to monoclonal and polyclonal antibodies, are raised against variant proteins using methods known in the art (see Soren, M., et al., EP 0 752 886; incorporated herein by reference in its entirety).
  • these anti- variant antibodies are used for immunotherapy.
  • methods of immunotherapy are provided.
  • immunotherapy is meant treatment of an autoimmune disease associated with the production of self- proteins.
  • self-proteins are conjugated to a T cell epitope to make an autovaccine (see for example, WO 95/05849 and WO 00/20027; both of which are incorporated by reference in their entirety).
  • Self proteins of use in the present invention include TNF ⁇ , and ⁇ -interferon for the treatment of cancer, IgE for the treatment of allergy, and TNF ⁇ , TNF ⁇ , and or interleukin 1 for the treatment of chronic inflammatory diseases.
  • immunotherapy can be passive or active.
  • Passive immunotherapy is the passive transfer of antibody to a recipient (patient).
  • Active immunization is the induction of antibody and/or T-cell responses in a recipient (patient).
  • Induction of an immune response can be the consequence of providing the recipient with a variant protein antigen comprising a T cell epitope and a self-protein to which antibodies are raised.
  • the variant protein antigen may be provided by injecting a variant polypeptide against which antibodies are desired to be raised into a recipient, or contacting the recipient with a variant protein encoding nucleic acid, capable of expressing the variant protein antigen, under conditions for expression of the variant TNF- ⁇ protein antigen.
  • candidate variant proteins are administered as therapeutic agents, and can be formulated as outlined above.
  • candidate variant genes (including both the full-length sequence, partial sequences, or regulatory sequences of the variant coding regions) can be administered in gene therapy applications, as is known in the art.
  • These variant genes can include antisense applications, either as gene therapy (i.e. for incorporation into the genome) or as antisense compositions, as will be appreciated by those in the art.
  • the nucleic acid encoding the candidate variant proteins may also be used in gene therapy.
  • genes are introduced into cells in order to achieve in vivo synthesis of a therapeutically effective genetic product, for example for replacement of a defective gene.
  • Gene therapy includes both conventional gene therapy where a lasting effect is achieved by a single treatment, and the administration of gene therapeutic agents, which involves the one time or repeated administration of a therapeutically effective DNA or mRNA.
  • Antisense RNAs and DNAs can be used as therapeutic agents for blocking the expression of certain genes in vivo. It has already been shown that short antisense oligonucleotides can be imported into cells where they act as inhibitors, despite their low intracellular concentrations caused by their restricted uptake by the cell membrane.
  • oligonucleotides can be modified to enhance their uptake, e.g. by substituting their negatively charged phosphodiester groups by uncharged groups.
  • nucleic acids there are a variety of techniques available for introducing nucleic acids into viable cells.
  • the techniques vary depending upon whether the nucleic acid is transferred into cultured cells in vitro, or in vivo in the cells of the intended host.
  • Techniques suitable for the transfer of nucleic acid into mammalian cells in vitro include the use of liposomes, electroporation, microinjection, cell fusion, DEAE-dextran, the calcium phosphate precipitation method, etc.
  • the currently preferred in vivo gene transfer techniques include transfection with viral (typically retroviral) vectors and viral coat protein- liposome mediated transfection [Dzau et al., Trends in Biotechnology 11:205-210 (1993)].
  • the nucleic acid source with an agent that targets the target cells, such as an antibody specific for a cell surface membrane protein or the target cell, a ligand for a receptor on the target cell, etc.
  • an agent that targets the target cells such as an antibody specific for a cell surface membrane protein or the target cell, a ligand for a receptor on the target cell, etc.
  • proteins which bind to a cell surface membrane protein associated with endocytosis may be used for targeting and/or to facilitate uptake, e.g. capsid proteins or fragments thereof tropic for a particular cell type, antibodies for proteins which undergo internalization in cycling, proteins that target intracellular localization and enhance intracellular half-life.
  • the technique of receptor-mediated endocytosis is described, for example, by Wu et al., J. Biol. Chem. 262:4429-4432 (1987); and Wagner et al., Proc. Natl. Acad. Sci. U.S.A.
  • candidate variant genes are administered as DNA vaccines, either single genes or combinations of candidate variant genes.
  • Naked DNA vaccines are generally known in the art. Brower, Nature Biotechnology, 16:1304-1305 (1998). Methods for the use of genes as DNA vaccines are well known to one of ordinary skill in the art, and include placing a candidate variant gene or portion of a variant gene under the control of a promoter for expression in a patient in need of treatment.
  • the variant gene used for DNA vaccines can encode full-length variant proteins, but more preferably encodes portions of the variant proteins including peptides derived from the variant protein.
  • a patient is immunized with a DNA vaccine comprising a plurality of nucleotide sequences derived from a variant gene.
  • a DNA vaccine comprising a plurality of nucleotide sequences derived from a variant gene.
  • expression of the polypeptide encoded by the DNA vaccine, cytotoxic T-cells, helper T-cells and antibodies are induced which recognize and destroy or eliminate cells expressing TNF- ⁇ proteins.
  • the DNA vaccines include a gene encoding an adjuvant molecule with the DNA vaccine.
  • adjuvant molecules include cytokines that increase the immunogenic response to the variant polypeptide encoded by the DNA vaccine. Additional or alternative adjuvants are known to those of ordinary skill in the art and find use in the invention.

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Abstract

L'invention concerne l'utilisation d'un éventail de procédés de calcul pour la modulation de l'antigénicité de protéines, à travers l'identification puis la modification de séquences d'acides aminés potentielles qui induisent une réponse immunitaire dans un organisme hôte. En particulier, on effectue un criblage des protéines visant à déterminer les séquences de liaison du CMH, les épitopes de lymphocytes T et les épitopes de lymphocytes B.
EP02784886A 2001-07-10 2002-01-04 Automatisation de conception de proteines pour la conception de bibliotheques de proteines a antigenicite modifiee Withdrawn EP1572345A3 (fr)

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US09/903,378 US20020119492A1 (en) 2000-07-10 2001-07-10 Protein design automation for designing protein libraries with altered immunogenicity
WOPCT/US01/21823 2001-07-10
US903378 2001-07-10
PCT/US2001/021823 WO2002005146A2 (fr) 2000-07-10 2001-07-10 Automatisation de conception de proteines pour la conception de bibliotheques de proteines a antigenicite modifiee
PCT/US2002/000165 WO2003006154A2 (fr) 2001-07-10 2002-01-04 Automatisation de conception de proteines pour la conception de bibliotheques de proteines a antigenicite modifiee

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