Novel methods of generating antibodies

Abstract

To provide novel methods of generating antibodies.SOLUTION: The invention describes a method of generating antibodies to a mixture of peptidogenic proteins, where the peptidogenic proteins have altered conformational dynamics as compared to a starting protein and where the peptidogenic proteins have similar conformations to the starting peptide. The peptidogenic proteins can be used to induce an immune response, which can lead to the generation of antibodies and / or can be used to vaccinate a mammal.SELECTED DRAWING: None

Description

Technical Field

[0001] Cross - Reference to Related Applications This application claims priority based on U.S. Provisional Application No. 62 / 207,022, filed on August 19, 2015, the entire disclosure of which is incorporated herein by reference.

[0002] Sequence Listing This application contains a sequence listing submitted in ASCII format via EFS - Web, the entire disclosure of which is incorporated herein by reference. The ASCII copy was created on August 16, 2016, named Combined_SA_01_PCT_ST25_V2.txt, and is 10,883,263 bytes in size.

Background Art

[0003] Introduction Methods for making antibodies have existed for approximately over 100 years and have been routinely used by those skilled in the art. See, for example, Morrison et al., Science 229:1202 (1985); Oi et al., BioTechniques 4:214 (1986); Cabilly et al., U.S. Patent No. 4,816,567; Taniguchi et al., EP171496; Morrison et al., EP 173494; Neuberger et al., WO8601533; Robinson et al., WO8702671; Boulianne et al., Nature 312:643 (1984); Neuberger et al., Nature 314:268 (1985). Improved methods for producing antibodies have extended these initial methods and have been used to produce many of the currently marketed therapeutic antibodies. For example, techniques such as phage display and transgenic mice, i.e., mice containing human immunoglobulin genes, have been used to produce fully human antibodies. However, certain antigens continue to challenge researchers' ability to elicit antibodies even when using the latest techniques.

[0004] To induce a cell-mediated immune response in the human body, foreign proteins are typically degraded into smaller peptides between 8 and 24 amino acids in length and bound to MHC molecules for display on the surface of antigen-presenting cells. MHC-bound peptides are presented to T cells to elicit a cell-mediated immune response.

[0005] The three-dimensional (3D) structure of proteins has been associated as a factor in proteolytic processing and epitope presentation (see Carmicle et al., Molecular Immunology (2007) 44:1159-1168). Further, Ohkuri et al. (see Ohkuri et al., J. Immunol., (2010), 185:4199-4205) have recognized that the conformational stability of proteins is an immunologically dominant factor. However, there is no consensus on exactly how the 3D structure affects the immune response.

[0006] Delamarre et al. (see Delamarre et al., JEM, (2006), 203:2049-2055) found that proteins in a less digestible form, which are less susceptible to lysosomal proteolysis, are more immunogenic, and thus concluded that increasing protein stability improves the immune response. For example, Delamarre et al. showed that the immunogenicity of a protein antigen can be improved by decreasing its susceptibility to proteolysis. Similarly, Mirano-Bascos et al. (see Mirano-Bascos et al., J. of Virology, (2010), 84:3303-3311) mutated cysteine residues to prevent the formation of each of three disulfide bonds and determined that the CD4+ T cell response was broadly decreased for all three mutants. Mirano-Bascos et al. also concluded that globally destabilizing the 3-D structure of a protein decreases antigen presentation and suppresses the immune response. For example, in other studies such as Nguyen et al., Vaccine, (2015), 33:2887-2896, external domain disulfide bonds were deleted and such deletions were expected to improve antigen presentation. Instead, a typical pattern of epitope dominance was observed and the authors concluded that it may not be possible to generate a substantially stronger immune response.

[0007] Other groups have similarly concluded that protein stabilization is required for an immune response. For example, Deressa et al. (see Deressa et al., PLOS, (2014), 9:1-12) concluded that even minor modifications to the amino acid sequence of an antigen can cause fundamental quantitative and qualitative changes in the immune response. Similarly, Porta et al. (see Porta et al., PLOS, (2013), 9:1-8) reported that stability is required to induce an immune response. Other groups such as Thomas (see Thomas et al., Human Vaccines & Immunotherapeutics, (2013), 9:744-752) have similarly concluded that increasing the thermal stability of a peptide induces a better immune response.

[0008] In contrast, other groups such as So (see So et al., Immunology, (2001), 104:259-268) have reported contrary results. So et al. investigated the effect of cross-linking on the magnitude of the in vivo T cell response (e.g., removing and adding cross-links), and found that removing such cross-links led to better antigen processing and an improved immune response. Similarly, Thai et al. (J. Biol. Chem. (2004) 279:50257-50266) reported that mutating residues that can be exposed on the surface decreased stability and increased conformational dynamics, thereby increasing the immunogenicity of the protein antigen. Thai et al. also targeted the administration of a single antigen. There is no consensus regarding whether removing or adding cross-links improves or inhibits antigen processing. Thus, it is not clear in the art whether increasing or decreasing protein stability improves the immune response, which includes a broad and diverse array of antibodies. Therefore, there continues to be a need to develop new and improved methods for producing antibodies that can provide a broader repertoire of antibodies than previously obtained.

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Summary of the Invention

Means for Solving the Problems

[0011] Gist of the Invention This gist is provided to introduce in a simplified form a selection of concepts further described in the following detailed description. This gist is not intended to identify important or essential features of the claimed subject matter, nor is it intended to be used as an aid in determining the scope of the claimed subject matter.

[0012] As described herein, the present invention is a method of eliciting an immune response, comprising the step of designing a mixture of peptidegenic proteins derived from a starting protein, wherein the peptidegenic proteins have conformational dynamics that are altered compared to the starting protein and the peptidegenic proteins are structurally similar to the starting protein, the step of introducing the peptidegenic protein into an animal, and the step of generating an immune response. The peptidegenic protein can be introduced directly into the animal (e.g., by inoculation or immunization) or can be expressed in vivo by a polynucleotide that is introduced into the animal and encodes the peptidegenic protein. When these peptidegenic proteins are expressed, an immune response is elicited, producing antibodies against both the peptidegenic protein and the original starting protein.

[0013] In preferred embodiments, the conformational dynamics of the starting protein preferably change by altering the thermodynamic stability of the starting protein. In further preferred embodiments, the conformational dynamics of the starting protein change by replacing at least one non-surface amino acid residue of the starting protein to modify the peptidogenicity of the protein. Methods of altering conformational dynamics include, but are not limited to, examining a model of the 3-D structure of the starting protein (predicted experimentally or based on homology), identifying non-surface amino acid residues of the starting protein, replacing at least one non-surface amino acid residue in the starting protein to create a peptidogenic protein, and / or comparing patterns of amino acid homology conserved across orthologous proteins from different species to tentatively identify non-surface amino acid residues (e.g., conserved hydrophobic residues) of the starting protein and replacing at least one non-surface amino acid residue in the starting protein to create a peptidogenic protein. Other methods of predicting or empirically discovering non-surface (i.e., buried) amino acid residues can also be used. These methods include using bioinformatics tools that predict secondary structure and / or identify disordered regions of the starting protein to identify at least one non-surface amino acid residue within these structured or ordered regions for substitution, as well as replacing at least one non-surface amino acid residue to create a peptidogenic protein (see, e.g., Cheng et al., Nucleic Acids Res (2005) 33:W72-6; Huang et al. (2014), DisMeta: A Meta Server for Construct Design and Optimization In Chen editor, Structural Genomics, Humana Press 3-16). In some embodiments, substitutions in disordered regions are avoided. For example, disorder predictors can be used to identify ordered / structured regions for selection of regions in which to make mutations [ibid].Still other methods involve using biochemical experiments to identify core residues, such as through alanine scanning of hydrophobic residues or comparable methods, to identify at least one non-surface amino acid residue within these structures or regions for substitution, and creating a peptidogenic protein by replacing at least one non-surface amino acid residue. Thus, in some embodiments, residues for substitution can be identified based on known structures, and in other embodiments, residues for substitution can be identified based on conserved hydrophobic residues.

[0014] In a preferred embodiment, the non-surface amino acid residues are replaced with smaller amino acid residues. In a more preferred embodiment, the smaller amino acid is alanine or glycine. In another preferred embodiment, at least 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids are replaced in the starting protein. In yet another preferred embodiment, at least 10 amino acids, at least 20 amino acids, at least 30 amino acids, at least 40 amino acids or at least 50 amino acids are replaced in the starting protein. In still another preferred embodiment, the multiple amino acid substitutions are distributed across a mixture of proteins. For example, in one embodiment, to mutate 10 different residues, the starting protein is mutated 10 different times to create 10 different peptidic proteins, each having a single amino acid substitution. Each of the 10 proteins is then mixed together and inoculated into an animal. In some cases, the wild-type starting protein, i.e., the protein without mutations, is part of the mixture. In a further preferred embodiment, at least one disulfide bond is removed from the starting protein, such as by replacing cysteine with alanine, serine, and / or glycine, etc. In a further preferred embodiment, both cysteines involved in the formation of at least one disulfide bond in the starting protein are replaced with alanine, serine, and / or glycine, or preferably alanine or glycine, etc.In a more preferred embodiment, the conformational dynamics of the starting protein are altered by replacing (a) at least one threonine with valine, alanine, glycine, or serine; or (b) at least one cysteine with alanine, valine, glycine, serine, or threonine; or (c) at least one valine with alanine, glycine, leucine, or isoleucine; or (d) at least one leucine with alanine, valine, glycine, or isoleucine; or (e) at least one isoleucine with alanine, valine, leucine, or glycine; or (f) at least one proline, methionine, phenylalanine, tyrosine, or tryptophan with alanine, valine, leucine, isoleucine, or glycine; or (g) at least one aspartic acid or asparagine with glycine, serine, threonine, alanine, valine, leucine, or isoleucine; or (h) at least one glutamic acid or glutamine with aspartic acid, asparagine, glycine, serine, threonine, alanine, valine, leucine, or isoleucine; or (i) at least one lysine with arginine, histidine, glycine, serine, threonine, alanine, valine, methionine, leucine, or isoleucine; or (j) at least one arginine with lysine, histidine, glycine, serine, threonine, alanine, valine, methionine, leucine, or isoleucine; or (k) at least one histidine with lysine, arginine, glycine, serine, threonine, alanine, valine, glutamine, asparagine, leucine, or isoleucine; or (l) at least one alanine with glycine; or (m) at least one residue with a non-natural amino acid; and / or (n) any combination of the above.

[0015] In an even more preferred embodiment, the conformational dynamics of the starting protein are such that (a) at least one tryptophan is replaced by tyrosine, phenylalanine, methionine, histidine, isoleucine, leucine, valine, alanine or glycine; or (b) at least one tyrosine is replaced by phenylalanine, methionine, histidine, isoleucine, leucine, valine, alanine or glycine; or (c) at least one phenylalanine is replaced by tyrosine, methionine, histidine, isoleucine, leucine, valine, alanine or glycine; or (d) at least one proline is replaced by methionine, leucine, isoleucine, valine, alanine, or glycine; or (e) at least one histidine is replaced by phenylalanine, tyrosine, methionine, isoleucine, leucine, valine, alanine, glycine, lysine, arginine, serine, threonine, asparagine, or glutamine; or (f) at least one methionine is replaced by isoleucine, leucine, valine, alanine or glycine; or (g) at least one isoleucine is replaced by leucine, valine, alanine or glycine; or (h) at least one leucine is replaced by isoleucine, valine, alanine or glycine; or (i) at least one valine is replaced by alanine, glycine, leucine, or isoleucine; or (j) at least one cysteine is replaced by alanine, valine, glycine, serine or threonine; or (k) at least one aspartic acid is replaced by glutamic acid, glutamine, asparagine, glycine, serine, threonine, alanine, valine, leucine, or isoleucine; or (l) at least one glutamic acid is replaced by aspartic acid, glutamine, asparagine, glycine, serine, threonine, alanine, valine, leucine, or isoleucine; or (m) at least one alanine is replaced by glycine or proline; or (n) at least one serine is replaced by alanine or glycine; or (o) at least one glycine is replaced by alanine or proline; or (p) at least one lysine is replaced by arginine, histidine, glycine, serine, threonine, alanine, valine, methionine, leucine or isoleucine;or (q) at least one asparagine with glycine, alanine, serine, threonine, valine, leucine, isoleucine, glutamine, aspartic acid or glutamic acid; or (r) at least one glutamine with glycine, alanine, serine, threonine, valine, leucine, isoleucine, glutamine, aspartic acid, glutamic acid, or histidine; or (s) at least one arginine with lysine, histidine, glycine, serine, threonine, alanine valine, methionine, leucine, or isoleucine; or (t) at least one threonine with valine, alanine, glycine or serine; or (u) a hydrophobic residue with a smaller similar hydrophobic residue; or (v) at least one residue with a non-natural amino acid; or (w) by any combination of the above replacements. Optimal substitutions to increase peptidogenicity may be determined using a combinatorial approach.;

[0016] In a preferred embodiment, the change in the conformational dynamics of the peptidogenic protein is measured by the change in the melting temperature and / or by measuring the change in the Gibbs free energy of stabilization compared to the starting protein. Preferred methods for measuring the Gibbs free energy include, but are not limited to, denaturant-modulated equilibrium unfolding. Preferred denaturants are urea and / or guanidinium hydrochloride. Alternatively, the change in conformational dynamics can be assayed by detecting a change in a proteolysis susceptibility assay, such as by measuring digestion using cathepsin and / or other proteases and then analyzing the mixture by mass spectrometry (MS) or sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE).

[0017] In a preferred embodiment, the determination of whether the peptidic protein has a three-dimensional structure similar to that of the starting protein can be measured by cross-reactive antibodies that bind to three-dimensional conformational epitopes (which are often discontinuous epitopes) on both the peptidic protein and the starting protein. Methods for measuring antibody binding include, but are not limited to, immunoprecipitation assays, surface plasmon resonance, isothermal titration calorimetry, oblique incidence reflectivity difference (OI-RD), Western blot, radioimmunoassay, ELISA (enzyme-linked immunosorbent assay), "sandwich" immunoassays, gel diffusion precipitation reactions, immunodiffusion assays, agglutination assays, complement fixation assays, immunoradiometric assays, fluorescence immunoassays, and / or protein A immunoassays.

[0018] In a further preferred embodiment, the test for measuring cross-reactivity is by a binding assay. In a further preferred embodiment, the antibodies (including cross-reactive antibodies) that bind to the peptidic protein have a dissociation constant (KD) of less than or equal to 10 -9 M, less than or equal to 10 -8 M, less than or equal to 10 -7 M, and / or less than or equal to 10 -6 M.

[0019] In a preferred embodiment, the starting protein is selected from human immunodeficiency virus (HIV) envelope glycoproteins, HIV gp120, HIV gp41, HIV gp160, Ebola antigen, hepatitis C virus (HCV) antigen, hepatitis B virus (HBV) antigen, Middle East respiratory syndrome coronavirus (MERS-CoV) antigen, Zika virus antigen, influenza virus antigen, viral antigens, malaria antigens, bacterial antigens, parasitic antigens, allergens, venoms, toxins, or tumor-associated antigens, transmembrane domain proteins, ion channel proteins, and / or G protein-coupled receptors.

[0020] In a more preferred embodiment, the tumor-associated antigen is MAGE-A1, MAGE-A2, MAGE-A3, MAGE-A4, MAGE-A5, MAGE-A6, MAGE-A7, MAGE-A8, MAGE-A9, MAGE-A10, MAGE-A11, MAGE-A12, GAGE-I, GAGE-2, GAGE-3, GAGE-4, GAGE-5, GAGE-6, GAGE-7, GAGE-8, BAGE-I, RAGE-1, LB33 / MUM-1, PRAME, NAG, MAGE-Xp2 (MAGE-B2), MAGE-Xp3 (MAGE-B3), MAGE-Xp4 (MAGE-B4), MAGE-C1 / CT7, MAGE-C2, NY-ESO-I, LAGE-I, SSX-I, SSX-2 (HOM-MEL-40), SSX-3, SSX-4, SSX-5, SCP-I and XAGE, melanocyte differentiation antigen, p53, ras, CEA, MUC1, PMSA, PSA, tyrosinase, Melan A, MART-1, gp100, gp75, alpha-actinin-4, Bcr-Abl fusion protein, Casp-8, beta-catenin, cdc27, cdk4, cdkn2a, coa-1, dek-can fusion protein, EF2, ETV6-AML1 fusion protein, LDLR-fucosyltransferase AS fusion protein, HLA-A2, HLA-A11, hsp70-2, KIAAO205, Mart2, Mum-2, and 3, neo-PAP, myosin class I, OS-9, pml-RAR alpha fusion protein, PTPRK, K-ras, N-ras, triosephosphate isomerase, GnTV, Herv-K-mel, NA-88, SP17, and TRP2-Int2, (MART-I), E2A-PRL, H4-RET, IGH-IGK, MYL-RAR, Epstein-Barr virus antigen, EBNA, human papillomavirus (HPV) antigens E6 and E7, TSP-180, MAGE-4, MAGE-5, MAGE-6, p185erbB2, p180erbB-3, c-met, nm-23H1, PSA, TAG-72-4, CA 19-9, CA 72-4, CAM 17.1, NuMa, K-ras, alpha-fetoprotein, 13HCG, BCA225, BTAA, CA 125, CA It is selected from 15-3 (CA 27.29\BCAA), CA 195, CA 242, CA-50, CAM43, CD68\KP1, CO-029, FGF-5, G250, Ga733 (EpCAM), HTgp-175, M344, MA-50, MG7-Ag, MOV18, NB\170K, NY-CO-1, RCAS1, SDCCAG16, TA-90 (Mac-2 binding protein\cyclophilin C-related protein), TAAL6, TAG72, TLP, TPS, tyrosine-related protein, TRP-1, TRP-2, and cytomegalovirus phosphoprotein 65 (pp65).

[0021] In another preferred embodiment, the peptide - antigenic protein is a protein that is part of a complex. For example, the buried interface residues may be targeted for amino acid substitution in a protein such as gp120, which is an envelope glycoprotein that forms a trimeric complex and is involved in HIV infection.

[0022] In a preferred embodiment, the mixture of polynucleotides encoding the peptide - antigenic protein can be synthesized in vitro. The polynucleotide can preferably contain either DNA or mRNA. In a preferred embodiment, the polynucleotide is in vitro - transcribed (IVT) mRNA. The mRNA containing IVT mRNA can further contain a poly(A) tail and / or a 5' cap. In another preferred embodiment, the mRNA can be translated in vitro, including by the use of coupled in vitro transcription / translation (IVTT), to produce the peptide - antigenic protein.

[0023] The mixture of polynucleotides can comprise sequences encoding different peptidic proteins derived from the same starting protein or from multiple starting proteins. In a further preferred embodiment, the polynucleotide can associate with a targeting component that directs the polynucleotide to a cell or an organ. Alternatively, the polynucleotide can be unable to associate with a targeting component. The polynucleotide encoding the peptidic protein may comprise a vector sequence.

[0024] Also contemplated are these mixtures of polynucleotides and animals (genetically modified or non-genetically modified) expressing the mixtures of polynucleotides. In a preferred embodiment, the animal is a mammal, and in a further preferred embodiment, the mammal is a human, mouse, rabbit, llama, or cow.

[0025] In a further preferred embodiment, the method induces an immune response. The immune response can occur in vivo, ex vivo and / or in vitro.

[0026] The polynucleotide encoding the peptidic protein, including but not limited to the mixture of polynucleotides, can be delivered to an animal by injection. In a preferred embodiment, the injection is performed in the muscle of the animal. The delivery of the polynucleotide to the animal can be used for vaccination purposes, research, or antibody development.

[0027] In a further preferred embodiment, the antibodies produced by the described method are recovered and isolated. In a preferred embodiment, the antibodies are full human antibodies, chimeric antibodies, single-chain antibodies, camelid antibodies, humanized antibodies, polyclonal antibodies or monoclonal antibodies. In a preferred embodiment, the polyclonal antibodies are further fractionated into a single isolated antibody species. In other preferred embodiments, the antibodies produced are affinity matured by, for example, phage display, yeast display, ribosome display or panning techniques.

[0028] Also contemplated are polynucleotides encoding antibodies generated by the methods described herein. The polynucleotides encoding these antibodies can also include heterologous promoter and / or vector sequences.

[0029] It is also possible to vaccinate a mammal using a mixture of a peptidogenic protein and / or a polynucleotide encoding a peptidogenic protein. In a preferred embodiment, the vaccine is a cancer vaccine, an HIV vaccine, an HCV vaccine, an HBV vaccine, an influenza virus vaccine, a MERS-CoV vaccine, a Zika vaccine, a malaria vaccine, and / or an Ebola virus vaccine that includes a peptidogenic protein.

[0030] In a further preferred embodiment, the invention is a method of processing a peptidogenic protein, comprising introducing the peptidogenic protein into an antigen-presenting cell, wherein the peptidogenic protein has altered conformational dynamics compared to the starting protein and the peptidogenic protein has a conformational structure similar to the starting protein, and enabling the antigen-presenting cell to process and display a T cell epitope derived from the peptidogenic protein.

[0031] In a preferred embodiment, the antigen-presenting cell is a dendritic cell, a B cell, a monocyte or a macrophage. In a further preferred embodiment, the method is performed in vitro or ex vivo. In a further preferred embodiment, the antigen-presenting cell is transfected with and / or contacted with a polynucleotide encoding the peptidogenic protein(s). In a further preferred embodiment, the antigen-presenting cell undergoes phagocytosis or pinocytosis of the peptidogenic protein(s) or polynucleotide(s). In embodiments of the present invention, for example, the following items are provided. (Item 1) A method of generating an immune response, comprising a. Designing a mixture of peptide - antigenic proteins derived from a starting protein, wherein the peptide - antigenic proteins have altered conformational dynamics compared to the starting protein and are conformationally similar to the starting peptide; b. Introducing the mixture of peptide - antigenic proteins into an animal; c. Generating an immune response A method comprising the above steps. (Item 2) Altering the conformational dynamics by a. Examining the 3 - D structure of the starting protein, identifying non - surface amino acid residues of the starting protein, and creating the peptide - antigenic protein by replacing at least one non - surface amino acid residue in the starting protein; or b. Examining a model of the 3 - D structure of the starting protein, identifying non - surface amino acid residues of the starting protein, and creating the peptide - antigenic protein by replacing at least one non - surface amino acid residue in the starting protein; or c. Comparing patterns of amino acid homology conserved across orthologous proteins of the starting protein from different species to identify non - surface amino acid residues of the starting protein, and creating the peptide - antigenic protein by replacing at least one non - surface amino acid residue in the starting protein; or d. Creating the peptide - antigenic protein by replacing at least one non - surface amino acid residue of the starting protein; or e. Replacing at least one non - surface amino acid residue with a smaller amino acid residue; or f. Replacing at least one non - surface amino acid residue with alanine or glycine; or g. Removing at least one disulfide bond in the starting protein The method according to item 1. (Item 3) The method according to either item 1 or 2, wherein at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids are replaced within the starting protein. (Item 4) The conformational dynamics of the starting protein is a. at least one threonine with valine, alanine, glycine or serine; or b. at least one cysteine with alanine, valine, glycine, serine or threonine; or c. at least one valine with alanine, glycine, leucine or isoleucine; or d. at least one leucine with alanine, valine, glycine, or isoleucine; or e. at least one isoleucine with alanine, valine, leucine, or glycine; or f. at least one proline with methionine, alanine, valine, leucine, isoleucine, or glycine; or g. at least one methionine with alanine, valine, leucine, isoleucine or glycine; or h. at least one phenylalanine with tyrosine, methionine, histidine, alanine, valine, leucine, isoleucine or glycine; or i. at least one tyrosine with phenylalanine, methionine, histidine, alanine, valine, leucine, isoleucine or glycine; or j. at least one tryptophan with tyrosine, phenylalanine, methionine, histidine, alanine, valine, leucine, isoleucine or glycine; or k. at least one aspartic acid with glutamic acid, glutamine, asparagine, glycine, serine, threonine, alanine, valine, leucine, or isoleucine; or l. at least one asparagine with glycine, serine, threonine, alanine, valine, leucine, isoleucine, glutamine, glutamic acid or aspartic acid; or m. at least one glutamic acid with aspartic acid, asparagine, glutamine, glycine, serine, threonine, alanine, valine, leucine, or isoleucine; or n. at least one glutamine with glutamic acid, aspartic acid, asparagine, glutamine, glycine, serine, threonine, alanine, valine, leucine, or isoleucine; or o. at least one lysine with arginine, histidine, glycine, serine, threonine, alanine, valine, methionine, leucine, or isoleucine; or p. at least one arginine with lysine, histidine, glycine, serine, threonine, alanine, valine, methionine, leucine, or isoleucine; or q. at least one histidine with phenylalanine, tyrosine, lysine, arginine, glycine, serine, threonine, alanine, valine, glutamine, asparagine, leucine, methionine or isoleucine; or r. at least one alanine with glycine or proline; or s. at least one glycine with alanine or proline; or t. at least one serine with alanine or glycine; or u. at least one residue with a non-natural amino acid; or v. any of the above combinations The method according to any one of items 1 to 3, which is changed by replacing with. (Item 5) The change in the conformational dynamics of the peptidogenic protein is: a. The change in the melting temperature compared to the starting protein; or b. The change in the Gibbs free energy of stabilization or the proteolysis susceptibility assay; or c. The change in Gibbs free energy of stabilization, as measured by denaturant-adjusted equilibrium unfolding, such as urea or guanidinium hydrochloride unfolding, of the change in Gibbs free energy The method according to any one of items 1 to 4, as measured by (Item 6) A similar three-dimensional structure is: a. A cross-reactive antibody that binds to both the peptidic protein and the starting protein; or b. The cross-reactivity is measured by an immunoprecipitation assay, surface plasmon resonance, isothermal titration calorimetry, oblique incidence reflectivity difference (OI-RD), Western blot, radioimmunoassay, ELISA (enzyme-linked immunosorbent assay), "sandwich" immunoassay, gel diffusion precipitation reaction, immunodiffusion assay, agglutination assay, complement fixation assay, immunoradiometric assay, fluorescence immunoassay, and / or protein A immunoassay, the cross-reactive antibody of (a); or c. The cross-reactivity is measured by a binding assay, the cross-reactive antibody of (a); or d. 10 -9 The cross-reactive antibody of (a) having a dissociation constant (KD) of less than or equal to 10 M; or e. 10 -8 Less than or equal to 10 M, 10 -7 Less than or equal to 10 M, or 10 -6 The cross-reactive antibody of (a) having a dissociation constant (KD) of less than or equal to 10 M The method according to any one of items 1 to 5, as measured by (Item 7) The starting protein is: a. Human immunodeficiency virus (HIV) envelope glycoproteins, HIV gp120, HIV gp41, HIV gp160, Ebola antigen, viral antigen, bacterial antigen, parasitic antigen, allergen, venom, toxin, tumor-associated antigen, transmembrane domain protein, G protein-coupled receptor, ion channel, hepatitis C virus antigen, hepatitis B virus antigen, MERS-CoV antigen, Zika virus antigen, influenza virus antigen, malaria antigen; and / or b. Any one of the malaria antigens listed in Table 2; and / or c. Any one of the targets listed in Table 5; and / or d. MAGE-A1, MAGE-A2, MAGE-A3, MAGE-A4, MAGE-A5, MAGE-A6, MAGE-A7, MAGE-A8, MAGE-A9, MAGE-A10, MAGE-A11, MAGE-A12, GAGE-I, GAGE-2, GAGE-3, GAGE-4, GAGE-5, GAGE-6, GAGE-7, GAGE-8, BAGE-I, RAGE-1, LB33 / MUM-1, PRAME, NAG, MAGE-Xp2 (MAGE-B2), MAGE-Xp3 (MAGE-B3), MAGE-Xp4 (MAGE-B4), MAGE-C1 / CT7, MAGE-C2, NY-ESO-I, LAGE-I, SSX-I, SSX-2 (HOM-MEL-40), SSX-3, SSX-4, SSX-5, SCP-I and XAGE, melanocyte differentiation antigen, p53, ras, CEA, MUC1, PMSA, PSA, tyrosinase, Melan A, MART-1, gp100, gp75, alpha-actinin-4, Bcr-Abl fusion protein, Casp-8, beta-catenin, cdc27, cdk4, cdkn2a, coa-1, dek-can fusion protein, EF2, ETV6-AML1 fusion protein, LDLR-fucosyltransferase AS fusion protein, HLA-A2, HLA-A11, hsp70-2, KIAAO205, Mart2, Mum-2, and 3, neo-PAP, myosin class I, OS-9, pml-RAR alpha fusion protein, PTPRK, K-ras, N-ras, triosephosphate isomerase, GnTV, Herv-K-mel, NA-88, SP17, and TRP2-Int2, (MART-I), E2A-PRL, H4-RET, IGH-IGK, MYL-RAR, Epstein-Barr virus antigen, EBNA, human papillomavirus (HPV) antigens E6 and E7, TSP-180, MAGE-4, MAGE-5, MAGE-6, p185erbB2, p180erbB-3, c-met, nm-23H1, PSA, TAG-72-4, CA 19-9, CA 72-4, CAM 17.1, NuMa, K-ras, alpha-fetoprotein, 13HCG, BCA225, BTAA, CA 125, CA 15-3 (CA 27.Tumor-associated antigens selected from 29\BCAA), CA 195, CA 242, CA-50, CAM43, CD68\KP1, CO-029, FGF-5, G250, Ga733 (EpCAM), HTgp-175, M344, MA-50, MG7-Ag, MOV18, NB\170K, NY-CO-1, RCAS1, SDCCAG16, TA-90 (Mac-2 binding protein\cyclophilin C-related protein), TAAL6, TAG72, TLP, TPS, tyrosine-related protein, TRP-1, TRP-2, or cytomegalovirus phosphoprotein 65 (pp65). A method according to any one of items 1 to 8, selected from (Item 8) The method according to any one of items 1 to 7, wherein the peptide antigenic protein is directly administered to the animal. (Item 9) The peptide antigenic protein is: a. The same starting protein; or b. A plurality of starting proteins; or c. A plurality of related starting proteins A method according to any one of items 1 to 8, which is derived from (Item 10) a. Obtaining a mixture of polynucleotides encoding the peptide antigenic protein; and b. Introducing the mixture of polynucleotides into an animal, wherein the peptide antigenic protein is expressed from the polynucleotides The method according to any one of items 1 to 9, further comprising (Item 11) The polynucleotide is: a. Synthesized in vitro; or b. DNA; or c. In vitro transcribed (IVT) mRNA; or d. IVT mRNA containing a poly(A) tail; or e. IVT mRNA containing a 5' cap The method according to item 10, which is (Item 12) The polynucleotide is: a. not associated with any targeting component; or b. associated with a targeting component capable of directing the polynucleotide to a cell or organ; or c. associated with a targeting component capable of directing the polynucleotide to a cell or organ, wherein the targeting component is a vector. The method according to any one of Items 10 or 11. (Item 13) An animal comprising the peptidic protein according to any one of Items 1 to 9 or the polynucleotide according to any one of Items 10 to 12. (Item 14) The method according to any one of Items 1 to 12 or the animal according to Item 13, wherein the animal is a mammal, human, mouse, rabbit, llama, or cow. (Item 15) The method according to any one of Items 10 to 12 or 14 or the animal according to Item 13, wherein the animal has been injected with the polynucleotide. (Item 16) The polynucleotide is a. directly injected into the muscle of the animal; b. injected into the animal multiple times. The method according to Item 15. (Item 17) The method according to any one of Items 1 to 12 or 14 to 17, wherein the immune response produces an antibody. (Item 18) The method according to Item 17, further comprising the step of isolating the antibody. (Item 19) The method according to Item 18, wherein the antibody is a fully human antibody, chimeric antibody, humanized antibody, monoclonal antibody, and / or polyclonal antibody. (Item 20) An antibody produced by the method according to any one of Items 17 to 19. (Item 21) The method according to item 19, wherein the polyclonal antibody is further fractionated to obtain a single isolated antibody species. (Item 22) A single antibody species produced by the method according to item 21. (Item 23) The method according to any one of items 17 to 19 or 21, or the antibody produced in item 20 or the single antibody species produced in item 22, wherein the antibody is affinity matured. (Item 24) The affinity maturation is: a. phage display, yeast display, or ribosome display; or b. panning technique The method or antibody according to item 23, which occurs by. (Item 25) An antibody produced by the method according to any one of items 23 or 24. (Item 26) A polynucleotide encoding the antibody according to any one of items 20, 22, or 25. (Item 27) The polynucleotide according to item 26, further comprising a heterologous promoter. (Item 28) The polynucleotide according to any one of items 26 or 27, further comprising a vector sequence. (Item 29) A host cell comprising the polynucleotide according to any one of items 11 to 12 or 25 to 28. (Item 30) A mixture of polynucleotides encoding a mixture of peptidic proteins. (Item 31) The polynucleotide is: a. encoding a mixture of peptidic proteins derived from the same starting protein; or b. encoding a mixture of peptidic proteins derived from a plurality of starting proteins; or c. encoding a mixture of peptidic proteins derived from a plurality of related starting proteins; or d.synthesized in vitro; or e.DNA; or f.mRNA transcribed in vitro (IVT); or g.IVT mRNA containing a poly(A) tail; or h.IVT mRNA containing a 5’ cap, A mixture of polynucleotides according to item 30.

Mode for Carrying Out the Invention

[0032] Detailed Description of the Invention Overview This specification describes a novel method of generating an immune response, including enhancing antibody production by using the "petidogenic potency" of a protein by altering the conformational dynamics of the initiating protein while maintaining the 3-D conformation of the protein. These peptidogenic proteins can then be used as vaccines to initiate an immune response and / or to produce antibodies.

[0033] Accordingly, the present invention is directed to a method of eliciting an immune response, comprising designing a mixture of peptidogenic proteins derived from an initiating protein, wherein the peptidogenic proteins have altered conformational dynamics compared to the initiating protein and the peptidogenic proteins are conformationally similar to the initiating protein, and introducing the peptidogenic proteins into an animal to generate an immune response. The peptidogenic proteins can be introduced directly into the animal (e.g., by inoculation or immunization) or can be expressed in vivo by a polynucleotide introduced into the animal that encodes the peptidogenic protein. When these peptidogenic proteins are expressed, an immune response is elicited, preferably producing antibodies against both the peptidogenic protein and the original initiating protein.

[0034] The introduction of polynucleotides can be carried out, for example, directly or after first performing ex vivo transfection of dendritic cells. Further, polynucleotides encoding peptidic proteins can be created and introduced into an animal. The peptidic proteins can then be produced in the animal to generate antibodies against the peptidic proteins. The methods described herein have the potential to greatly influence the immunogenicity of proteins. Preferred biophysical and biochemical properties that vary in proteins include, but are not limited to, protein conformational dynamics, thermodynamic stability, MHC-II binding, and / or protease sensitivity of the initiating protein. The methods described herein can also be used to simultaneously generate cross-reactive antibodies against different peptidic proteins (derived from either the same or different initiating proteins), which has the potential to greatly change the way antibodies are currently produced as a repertoire of antibodies that can be obtained by single injection in an animal and has the potential to rationalize antibody development and vaccination efficacy.

[0035] The inventors recognized that the conformational dynamics of proteins are extremely important for the ability of the protein to initiate an immune response. The inventors named the tendency of an antigen to efficiently yield peptide fragments in vivo after immunization "peptidic". Having the ability to modify the conformational dynamics of the initiating protein to design mixtures of peptidic proteins that can be administered directly as a mixture of proteins or co-expressed in an animal by a mixture of polynucleotides has the potential to produce a broad repertoire of antibodies using single injection in a cost-effective manner.

[0036] Thus, as disclosed herein, immunizing an animal with a mixture of peptidic proteins can strongly stimulate the immune system and, when brought into contact with antigen-presenting cells, can generate a more powerful and / or superior immune system.

[0037] Immunization with a mixture of peptidic proteins (or combinatorial cocktails) is advantageous due to the complexity of proteolytic attack on the protein antigen(s) that generate the peptides. For example, providing a plurality of different peptidic proteins with different amino acid sequences creates an environment where the “tuning mutation(s)” optimal for the generation of a given peptide (T cell epitope) within an appropriate time frame may differ from the mutations optimal for the generation of another peptide. For example, some cells such as dendritic cells mediate a T cell response during the activation period. If these cells are presented with an antigen outside of this activation window (e.g., before or after activation), a T cell response may not occur. Thus, T cells need to be presented with an antigen at an appropriate time in order to mount an immune response, and this time is governed by the rate of proteolysis (e.g., proteolysis) in the antigen-presenting cell. By presenting the antigen as a mixture, a very large number of different peptidic proteins can be endocytosed by a single cell, thereby theoretically maximizing the diversity of peptides produced and displayed by that cell. Alternatively, by presenting the antigen as a mixture, a very large number of different peptidic proteins can be endocytosed by a plurality of cells, thereby theoretically maximizing the diversity of peptides produced and displayed by these cells. Furthermore, peptidic proteins with increased conformational dynamics may lead to improved MHC class II binding, which is expected to maximize the immune response. For example, proteins that are relatively non-immunogenic and / or not good vaccine components because they are too stable and thus protease degradation is inhibited, thereby reducing subsequent peptide presentation and attenuating the immune response in adaptive immunity, such proteins can be altered as described herein to create a mixture of peptidic proteins with altered conformational dynamics while maintaining a similar conformation compared to the starting protein.

[0038] In a preferred embodiment, an initiation protein, also referred to as a test initiation protein, is systematically mutated to vary the thermodynamic stability of the initiation protein without significantly changing the three-dimensional structure of the corresponding folded protein, thereby creating a peptide antigenic protein that displays substantially the same 3D (steric structure) surface epitope as the initiation protein while increasing peptide antigenicity.

[0039] Thus, by increasing the immunogenicity of the initiation protein by changing its conformational dynamics to generate a number of peptide antigenic proteins, which can then be simultaneously introduced into an animal, a strong immune response can be generated, producing a broader repertoire of polyclonal antibodies and having the potential to further fractionate into a single isolated species (e.g., by molecular cloning via their respective encoded mRNAs). Definitions

[0040] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art, such as in the technical fields of peptide chemistry, cell culture and phage display, nucleic acid chemistry, and biochemistry. Standard techniques incorporated herein by reference are used in molecular biology, genetic, and biochemical methods (see Sambrook et al., Molecular Cloning: A Laboratory Manual, 3rd ed., 2001, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY; Ausubel et al., Short Protocols in Molecular Biology (1999) 4th ed., John Wiley & Sons, Inc.).

[0041] As used herein, "peptidogenicity" refers to the tendency of a protein to efficiently produce a diverse set of robust peptides that can be used to elicit an immune response. There are various assays for measuring peptidogenicity (see, e.g., So et al., Figs. 2c-d; Thai et al., Fig. 7c-f; and Delamarre et al., Figs. 1b-c, 4b-c and 5a-b).

[0042] As used herein, "peptidogenic protein" refers to a mutated protein whose amino acid sequence has been modified to change its conformational dynamics compared to the starting protein sequence while maintaining a conformational structure similar to the starting protein.

[0043] As used herein, "non-surface residue" is a residue that cannot be exposed on the surface with respect to the 3D structure of a protein, e.g., a residue buried inside the 3D structure of an unfolded protein. In a preferred embodiment, "non-surface" residues are defined by the method of Lee and Richards (see, e.g., Lee B et al., J. Mol. Biol. (1971); 55(3):379-IN4.doi:http: / / dx.doi.org / 10.1016 / 0022-2836(71)90324-X), and the relative solvent exposure of a residue in an unfolded protein is less than 50%, less than 40%, less than 30%, less than 25%, less than 20%, less than 10%, less than 5%, or 0%, or by the same method, the difference between the absolute solvent-exposed surface area and the surface area of a fully extended Ala-X-Ala tripeptide (see, e.g., Gready JE et al., Protein Science. (1997); 6(5):983-98.doi:10.1002 / pro.5560060504.) is greater than 40 Å 2 greater than 50 Å 2 greater than 60 Å 2 greater than 70 Å 2 greater than 80 Å 2 greater than 90 Å 2Greater than, 100 Å 2 Greater than, 110 Å 2 Greater than, or 120 Å 2 Greater than. In a further preferred embodiment, a "non-surface" residue, when calculated by a structural analysis software package well known to those skilled in the art, is 10 Å 2 Less than, 5 Å 2 Less than, 2.5 Å 2 Less than, or 1 Å 2 Less than and is defined as a residue having a solvent-exposed surface area (e.g., see UCSF Chimera (e.g., Pettersen EF et al., J. Comput. Chem. (2004); 25(13):1605-12. Epub 2004 / 07 / 21), PyMol (e.g., Schrodinger, LLC. The PyMOL Molecular Graphics System, version 1.8. 2015), etc.).

[0044] As used herein, "starting protein" or "test starting protein" refers to the amino acid sequence of the "first" or "reference" protein used to derive a peptidogenic protein. In some examples, the "starting protein" can be a further modified peptidogenic protein.

[0045] As used herein, "immune response" refers to a humoral immune response and / or a cell-mediated immune response caused by antigen-presenting cells after processing a protein. In a humoral immune response, B lymphocytes produce antibodies that react with unmodified unprocessed antigens. These antigen-antibody reactions may in some cases include cell surface antigens that activate the complement cascade, which causes lysis of the cells carrying those antigens. In a cell-mediated immune response, T lymphocytes mobilize macrophages in the presence of processed peptide antigens recognized as foreign. Activated T lymphocytes can also directly attack cells carrying foreign antigens.

[0046] As used herein, "antigen-presenting cell" refers to a cell that can degrade ( "process") a protein into peptides and present the peptides on the cell surface in conjunction with MHC alleles, preferably major HLA complex class I or class II molecules. Examples of antigen-presenting cells include, but are not limited to, dendritic cells, macrophages, B cells, and monocytes.

[0047] As used herein, "structural dynamics" is defined as the conformational changes and flexibility of a protein structure in the spatial arrangement of atoms or groups of atoms relative to each other in a protein molecule. Structural dynamics includes "breathing" motions, are joined by covalent bonds in a protein molecule, and are governed by intrinsic restoring forces, but are regulated by non-covalent interactions such as hydrogen bonds, van der Waals forces, and electrostatic interactions, including vibrations, flexions, curvatures, rotations, and other allowed modes of motion of atoms. These motions can subtly change the geometry of the protein on a time scale of picoseconds or less, and can give rise to a vast diversity of conformational states on a time scale of microseconds to milliseconds. The structural molecular dynamics of proteins are often studied using computer simulations. See, for example, Shaw et al., (2010) Science 330, 341. Further, as used herein, the structural dynamics of an initial protein can be altered by chemical modification, amino acid substitution, and other mutations such as deletions, insertions, truncations, or any combination thereof. To state that the structural dynamics of a peptidic protein are diverse with respect to the wild-type protein means that one or more amino acid substitutions in the peptidic protein result in altered structural dynamics compared to the wild-type protein.

[0048] As used herein, "thermodynamic stability" is defined in terms of a chemical system that has no or minimal energy released or consumed, and thus has no or minimal thermal energy change and is in its lowest energy state under a given set of experimental conditions. Further as used herein, "decrease in thermodynamic stability" or "decreased thermodynamic stability" means that a parameter related to the thermodynamic stability of a peptidic protein is attenuated compared to the parameter of the starting protein measured under the same conditions, and this decrease can be achieved, but is not limited to, by chemical modifications, amino acid substitutions, and other genetic mutations that alter the molecular structure of the starting protein in the peptidic protein. Methods for measuring a decrease in thermodynamic stability are known in the art and are described herein, including protocols that incorporate measurements of parameters such as melting temperature and urea or guanidinium hydrochloride-induced equilibrium unfolding (denaturation). These parameters are typically reached by monitoring the protein unfolding reaction as a function of temperature or denaturant concentration under equilibrium or quasi-equilibrium conditions. Methods for monitoring the unfolding reaction by measuring the concentration of the unfolded state compared to the concentration of the folded state include, but are not limited to, UV absorption, fluorescence, and circular dichroism. This approach enables the calculation of the stabilization free energy (Gibbs free energy) of the mutant protein compared to that of the starting protein measured under the same conditions. The difference in free energy is typically represented by ΔΔG = ΔG 変異体 - ΔG 標準(例えば、wt) wherein ΔG 変異体 and ΔG 標準(例えば、wt) are the stabilization free energies of the mutant and "standard" (e.g., wt or wild-type) proteins, respectively, and ΔΔG is the difference. ΔΔG > 0 indicates a mutant protein that is less stable than the standard protein, and ΔΔG < 0 indicates a mutant protein that is more stable than the standard protein.

[0049] As used herein, a peptidic protein has a "similar three-dimensional structure" to a starting protein if, after mutating the non-surface residues of the protein (and consequently potentially modifying its overall conformational dynamics), the 3-D structure is sufficiently maintained such that an antibody cross-reacts with both the peptidic protein and the starting protein. "Cross-reactivity" can be measured by binding assays described herein or well-known in the art, and is measured as "binding affinity" based on the dissociation constant (K D ), off-rate (k off ), and / or on-rate (k on ). The peptidic protein need not have the same 3-D structure as the starting protein, but only a sufficiently similar structure that exhibits similar 3D conformational epitopes (including discontinuous epitopes) that enable the antibody to recognize both proteins, even if the binding affinities may not be identical.

[0050] In the present invention, the term "antibody" refers to an immunoglobulin molecule and immunologically active portions of immunoglobulin molecules, i.e., molecules containing antigen-binding sites that immunospecifically bind to a peptidic protein and / or a starting protein. Thus, the term antibody encompasses not only whole antibody molecules, but also antibody fragments and variants of antibodies and antibody fragments (including derivatives such as fusion proteins). Examples of molecules described by the term "antibody" in this application include, but are not limited to, fragments comprising or consisting of any of single-chain Fv (scFv), Fab fragment, Fab' fragment, F(ab') 2 , disulfide-linked Fv (sdFv), Fv, and fragments comprising or consisting of either the VL or VH domain. As used herein, the term "single-chain Fv" or "scFv" refers to a polypeptide comprising the VL domain of an antibody linked to the VH domain of the antibody. See Carter (2006) Nature Rev. Immunol. 6:243.

[0051] Furthermore, the antibodies of the present invention include, but are not limited to, monoclonal, multispecific, bispecific, human, humanized, mouse, or chimeric antibodies, single-chain antibodies, camelid antibodies, Fab fragments, F(ab’) fragments, anti-idiotype (anti-Id) antibodies (including, for example, anti-Id antibodies against the antibodies of the present invention), domain antibodies, and any epitope-binding fragments of the foregoing. The immunoglobulin molecules of the present invention can be of any class (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), subclass (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2) or type of immunoglobulin molecule.

[0052] Most preferably, the antibody is a human antibody. As used herein, a "human" antibody includes antibodies having the amino acid sequence of a human immunoglobulin, including antibodies isolated from a human immunoglobulin library and antibodies isolated from transgenic mice or other organisms genetically engineered to produce human antibodies. For a detailed discussion of a few of the techniques for generating human antibodies and human monoclonal antibodies and protocols for generating such antibodies, see, for example, PCT Publication WO98 / 24893; WO92 / 01047; WO96 / 34096; WO96 / 33735; European Patent No. 0598877; U.S. Patent Nos. 5,413,923; 5,625,126; 5,633,425; 5,569,825; 5,661,016; 5,545,806; 5,814,318; 5,885,793; 5,916,771; and 5,939,598; as well as Lonberg and Huszar, Int. Rev. Immunol. 13:65-93 (1995).

[0053] Human antibodies or "humanized" chimeric monoclonal antibodies can be produced using techniques described herein or otherwise known in the art. For example, methods for producing chimeric antibodies are known in the art. For reviews, see the following references: Morrison, Science 229:1202 (1985); Oi et al., BioTechniques 4:214 (1986); Cabilly et al., U.S. Patent No. 4,816,567; Taniguchi et al., EP 171496; Morrison et al., EP 173494; Neuberger et al., WO 8601533; Robinson et al., WO 8702671; Boulianne et al., Nature 312:643 (1984); Neuberger et al., Nature 314:268 (1985).

[0054] The antibodies of the present invention may be monovalent, bivalent, trivalent or multivalent. For example, monovalent scFv can be multimerized chemically or by association with another protein or substance. ScFv fused to a hexahistidine tag or Flag tag can be multimerized using Ni-NTA agarose (Qiagen) or using an anti-Flag antibody (Stratagene, Inc.).

[0055] The antibodies of the present invention may be monospecific, bispecific, trispecific or of greater multispecificity. Multispecific antibodies may be specific for a peptidic protein, for more than one peptidic protein, for an initiation protein, or multispecific antibodies may be specific for both a peptidic protein and / or an initiation protein as well as for heterologous epitopes such as heterologous polypeptides or solid support materials. See, for example, PCT Publications WO 93 / 17715; WO 92 / 08802; WO 91 / 00360; WO 92 / 05793; Tutt et al., J. Immunol. 147:60-69 (1991); U.S. Patent Nos. 4,474,893; 4,714,681; 4,925,648; 5,573,920; 5,601,819; Kostelny et al., J. Immunol. 148:1547-1553 (1992).

[0056] As used herein, the term "fragment" refers to a polypeptide comprising an amino acid sequence of at least 5 amino acid residues, at least 10 amino acid residues, at least 15 amino acid residues, at least 20 amino acid residues, at least 25 amino acid residues, at least 30 amino acid residues, at least 35 amino acid residues, at least 40 amino acid residues, at least 45 amino acid residues, at least 50 amino acid residues, at least 60 amino acid residues, at least 70 amino acid residues, at least 80 amino acid residues, at least 90 amino acid residues, at least 100 amino acid residues, at least 125 amino acid residues, at least 150 amino acid residues, at least 175 amino acid residues, at least 200 amino acid residues or at least 250 amino acid residues of an amino acid sequence of a peptidogenic protein or an initiating protein. In some embodiments, a fragment may also refer to a polypeptide comprising an amino acid sequence of about 8-24 amino acid residues or about 5-30 amino acid residues.

[0057] As used herein, the term "fusion protein" refers to a polypeptide comprising or consisting of an amino acid sequence of a peptidogenic protein, an initiating protein, and / or an antibody produced against a peptidogenic protein, as well as an amino acid sequence of one or more heterologous peptides and / or polypeptides. In vaccine applications, the heterologous polypeptide sequence fused to the peptidogenic protein is preferably derived from a viral protein.

[0058] As used herein, the term "host cell" refers to a particular target cell transfected with a nucleic acid molecule and the progeny or potential progeny of such a cell. The progeny may not be identical to the parental cell transfected with the nucleic acid molecule due to mutations that may occur later in time, environmental influences, or developmental steps or the incorporation of the nucleic acid molecule into the host cell genome.

[0059] "Conservative amino acid substitution" refers to a substitution in which an amino acid residue is replaced with an amino acid residue having a side chain with similar chemical properties (e.g., size, charge, steric characteristics [e.g., beta-branched vs. non-beta-branched], polarity [hydrophilic vs. hydrophobic], aromatic vs. non-aromatic, etc.). Whether a particular substitution is considered "conservative" can also depend on the structural context of the folded protein in which the substitution occurs. Amino acid side chains can be chemically similar in some respects but not in others, and the context can determine which of these properties is dominant in terms of how "conservative" (i.e., least disruptive) a particular substitution is. Families of amino acid residues with chemically similar side chains are defined in the art. These families include amino acids having basic side chains (e.g., lysine, arginine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., asparagine, glutamine, serine, threonine), nonpolar side chains (e.g., glycine, alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Some side chains have hybrid properties that are pH-dependent within a physiologically relevant pH range. For example, histidine (pKa ~6) becomes more positively charged (basic) below pH 6 and polar but substantially uncharged at pH 7.5 and above. Cysteine (pKa ~8.5) is substantially uncharged (and particularly nonpolar) below pH 8 but negatively charged (and acidic) at pH 9. The tyrosine phenol side chain is also partially ionized and negatively charged at higher pH. Furthermore, the remaining local electrostatic environment (context) of the protein can substantially shift these effective pH values. Additionally, the acidic protein cysteine thiolate side chain can react with another protein cysteine thiol via thiol-disulfide exchange involving intermediate disulfide-containing compounds such as oxidized glutathione to form an intramolecular disulfide bond, and such bonds are highly hydrophobic (nonpolar).Furthermore, both naturally occurring and / or non-naturally occurring amino acids can be used in the peptidogenic protein.

[0060] Mutations can be introduced in a site-specific manner or randomly along all or part of the coding sequence. A library of mutants can be designed to introduce up to 19 amino acid substitutions at a given residue site, such as single amino acid substitutions, 2 amino acid substitutions, 3 amino acid substitutions, 4 amino acid substitutions, etc. In yet other embodiments, a library of mutants can be designed to introduce more than 19 amino acid substitutions (including natural and non-natural amino acids) at a given residue site. Furthermore, the library can be combinatorially designed to simultaneously generate multiple mutations at two sites, three sites, four sites, etc. Following mutagenesis, the encoded protein may be routinely expressed, and the conformational dynamics and / or peptidogenicity of the encoded protein can be determined using the techniques described herein or by routinely modifying techniques known in the art. The resulting mutant proteins can be screened and evaluated for altered thermodynamic stability, peptidogenicity, or a conformational structure similar to the starting protein. Alternatively, the expressed protein “output” from the designed library can be used to immunize animals without pre-screening for protein properties.

[0061] As used herein, "patient" or "subject suitable for treatment" may be a mammal such as a rodent (e.g., guinea pig, hamster, rat, mouse), murine (e.g., mouse), canine (e.g., dog), feline (e.g., cat), equine (e.g., horse), primate, simian (e.g., monkey or ape), monkey (e.g., marmoset, baboon, rhesus monkey), ape (e.g., gorilla, chimpanzee, orangutan, langur), or human. In other embodiments, non-human mammals, particularly mammals customarily used as models for demonstrating therapeutic effects in humans (e.g., murine, primates, pigs, canine, camels, llamas, or rabbits) may be used.

[0062] Other aspects and embodiments of the present invention provide the term "comprising" as replaced by the term "consisting of" in the aspects and embodiments described herein, and the term "comprising" as replaced by the term "consisting essentially of" in the aspects and embodiments described above.

[0063] As used herein, "and / or" should be interpreted as specific disclosure of each of two or more specific features or components, including or excluding the others. For example, "A, B and / or C" should be interpreted as (i) A, (ii) B, (iii) C, (iv) A and B, (v) A and C, (vi) B and C, and (vii) A and B and C, as if each were presented individually. Method for changing the conformational dynamics of a protein

[0064] Peptide - antigenic proteins can be created using standard molecular biology mutagenesis techniques well - known in the art. For example, peptide - antigenic proteins can be created by random mutagenesis well - known in the art, such as error - prone PCR, random nucleotide insertions or deletions, or other methods prior to recombination.

[0065] To create peptide - antigenic proteins, protein engineering may be used. Recombinant DNA techniques known to those skilled in the art, including single or multiple amino acid substitutions, deletions, insertions, or fusion proteins, can be used to generate peptide - antigenic proteins. Such peptide - antigenic proteins may be screened against peptide - antigenic proteins having altered conformational dynamics while maintaining a conformational structure similar to the starting protein described herein.

[0066] For example, to increase the conformational dynamics of peptide - antigenic proteins, Table 1 below shows the average change in Gibbs free energy for exemplary amino acid substitutions in a range of proteins derived from Tables 1 and 2 of Loladze et al., J. Mol. Biol. 320, 343 - 357 (2002) [Note: This research paper uses a non - standard convention when representing the Gibbs free energy between mutants and wild - type proteins, i.e., using negative values (ΔΔG = ΔG(mutant)-ΔG(WT)) to indicate destabilization; the standard convention is that a positive change indicates destabilization (ΔΔG = ΔG(WT)-ΔG(mutant), see paragraph

[0040] above)]. For example, Val and Leu (and other larger non - polar amino acid residues) can be substituted with smaller amino acid residues such as Ala, Thr, Asn, and / or Gly. Further, buried sites of Glu in the native protein structure can be substituted with Leu, Val, Asn, Thr, Ser, Ala, and / or Gly. Single - site amino acid substitutions of the types shown generally have little effect on the overall conformational structure of the starting protein.

Table 1

[0067] Another research paper that is useful for another explanation describing destabilizing mutations that increase conformational dynamics in the protein core is Kim et al., (1993) Protein Sci. 2:588-596. In this study, the authors showed that the mutations Phe22->Ala (2.1 kcal / mol), Tyr23->Ala (7.0 kcal / mol), Tyr35->Gly (5.7 kcal / mol), Asn43->Gly (6.0 kcal / mol), and Phe45->Ala (7.2 kcal / mol) destabilize bovine pancreatic trypsin inhibitor (BPTI) by the respective amounts shown in parentheses at pH 3.5 without significantly disrupting the overall 3D structure of BPTI.

[0068] Furthermore, gene deletions, insertions, inversions, duplications, and type substitutions selected according to general rules known in the art should have little effect on activity. For example, guidance on how to make phenotypically silent amino acid substitutions is provided in Bowie, J. U. et al., "Deciphering the Message in Protein Sequences: Tolerance to Amino Acid Substitutions", Science 247:1306-1310 (1990), where the authors show that there are two main approaches for studying the tolerance of amino acid sequences to change. The first method relies on the process of evolution, where mutations are either accepted or rejected by natural selection. The second approach uses genetic manipulation to introduce amino acid changes at specific positions in the cloned gene and selection or screening to identify sequences that maintain functionality.

[0069] As the authors have stated, these studies have revealed that proteins are surprisingly tolerant of amino acid substitutions. The authors further show which amino acid changes are likely to be tolerated at a particular position in a protein. For example, most buried amino acid residues require nonpolar side chains and have few features of the generally conserved surface side chains. Other such phenotypically silent substitutions are described in Bowie, J.U. et al., supra, and the references cited therein. Substitutions that are typically seen as conservatively substituted are the replacements of one another among the aliphatic amino acids Ala, Val, Leu, and Ile; the interchange of the hydroxyl-bearing residues, Ser and Thr; the exchange of the acidic residues, Asp and Glu; the substitution between the side-chain amide-bearing residues, Asn and Gln; the exchange of the basic amino acids, Lys and Arg; and the replacement of one another between the aromatic residues, Phe and Tyr.

[0070] In a preferred embodiment, the conformational dynamics of the starting protein are such that (a) at least one threonine is replaced with valine, alanine, glycine or serine; or (b) at least one cysteine is replaced with alanine, valine, glycine, serine or threonine; or (c) at least one valine is replaced with alanine, glycine, leucine or isoleucine; or (d) at least one leucine is replaced with alanine, valine, glycine or isoleucine; or (e) at least one isoleucine is replaced with alanine, valine, isoleucine or glycine; or (f) at least one proline, methionine, phenylalanine, tyrosine or tryptophan is replaced with alanine, valine, leucine, isoleucine or glycine; or (g) at least one aspartic acid is replaced with glutamic acid, glutamine, asparagine, glycine, serine, threonine, alanine, valine, leucine, isoleucine; or (h) at least one glutamic acid is replaced with aspartic acid, glutamine, asparagine, glycine, serine, threonine, alanine, valine, leucine or isoleucine; or (i) at least one lysine is replaced with arginine, histidine, glycine, serine, threonine, alanine, valine, leucine or isoleucine; or (j) at least one arginine is replaced with lysine, histidine, glycine, serine, threonine, alanine, valine, leucine or isoleucine; or (k) at least one histidine is replaced with lysine, arginine, glycine, serine, threonine, alanine, valine, leucine, isoleucine or glutamine; or (l) at least one alanine is replaced with glycine or proline; or (m) at least one asparagine is replaced with glycine, alanine, serine, threonine, glutamine, aspartic acid or glutamic acid; or (n) at least one glutamine is replaced with glycine, alanine, serine, threonine, asparagine, aspartic acid, glutamic acid or histidine; or (o) at least one glycine is replaced with alanine or proline; or (p) at least one residue is replaced with a non-natural amino acid;Or it can be changed by replacing with any combination of (q)(a) to (p). In an even more preferred embodiment, the conformational dynamics of the starting protein is such that (a) at least one tryptophan is replaced with tyrosine, phenylalanine, methionine, histidine, isoleucine, leucine, valine, alanine or glycine; or (b) at least one tyrosine is replaced with phenylalanine, methionine, histidine, isoleucine, leucine, valine, alanine or glycine; or (c) at least one phenylalanine is replaced with tyrosine, methionine, histidine, isoleucine, leucine, valine, alanine or glycine; or (d) at least one proline is replaced with methionine, leucine, isoleucine, valine, alanine, or glycine; or (e) at least one histidine is replaced with phenylalanine, tyrosine, methionine, isoleucine, leucine, valine, alanine, glycine, lysine, arginine, serine, threonine, asparagine, or glutamine; or (f) at least one methionine is replaced with isoleucine, leucine, valine, alanine or glycine; or (g) at least one isoleucine is replaced with leucine, valine, alanine or glycine; or (h) at least one leucine is replaced with isoleucine, valine, alanine or glycine; or (i) at least one valine is replaced with alanine, glycine, leucine, or isoleucine; or (j) at least one cysteine is replaced with alanine, valine, glycine, serine or threonine; or (k) at least one aspartic acid is replaced with glutamic acid, glutamine, asparagine, glycine, serine, threonine, alanine, valine, leucine, or isoleucine; or (l) at least one glutamic acid is replaced with aspartic acid, glutamine, asparagine, glycine, serine, threonine, alanine, valine, leucine, or isoleucine; or (m) at least one alanine is replaced with glycine or proline; or (n) at least one serine is replaced with alanine or glycine; or (o) at least one glycine is replaced with alanine or proline;or (p) at least one lysine with arginine, histidine, glycine, serine, threonine, alanine, valine, methionine, leucine or isoleucine; or (q) at least one asparagine with glycine, alanine, serine, threonine, valine, leucine, isoleucine, glutamine, aspartic acid or glutamic acid; or (r) at least one glutamine with glycine, alanine, serine, threonine, valine, leucine, isoleucine, glutamine, aspartic acid, glutamic acid, or histidine; or (s) at least one arginine with lysine, histidine, glycine, serine, threonine, alanine valine, methionine, leucine, or isoleucine; or (t) at least one threonine with valine, alanine, glycine or serine; or (u) a hydrophobic residue with a smaller similar hydrophobic residue; or (v) at least one residue with a non-natural amino acid; or (w) is varied by replacement with any of the above combinations. In some embodiments, hydrophobic residues are targeted for substitution.;

[0071] Amino acids in the starting protein that are essential for function, conformation, and / or structure and are located on the protein surface versus the interior can be identified by methods known in the art such as site-directed mutagenesis or alanine-scanning mutagenesis (Cunningham and Wells, Science 244:1081-1085 (1989)). In the latter procedure, a single alanine mutation is introduced into every residue in the molecule. The resulting mutant molecules are then tested against molecules with altered conformational dynamics while maintaining a conformation similar to the starting protein.;

[0072] In additional embodiments, the amino acid sequence of the starting protein is replaced with one or more amino acids (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, or 50 amino acids) of the above-described substituted amino acids (either conservative or non-conservative substitutions) to generate a peptidic protein. For example, substitutions can be readily made at positions that do not contain the activity of the starting protein and / or within the interior of the protein structure. Sites that are extremely important for ligand-receptor binding can also be determined by structural analysis such as crystallization, nuclear magnetic resonance, or photoaffinity labeling (Smith et al., J. Mol. Biol. 224:899-904 (1992); and de Vos et al., Science 255:306-312 (1992)).

[0073] Using recombinant DNA techniques that employ combinatorial mutagenesis and synthetic DNA synthesis approaches known to those of skill in the art, peptidic proteins can also be created that include single or multiple amino acid substitutions, deletions, additions, or fusion proteins. Such modified polypeptides can then be screened against altered conformational dynamics while maintaining a conformation similar to the starting protein.

[0074] In this way, a peptidic protein with one or more amino acid residues deleted, added, or substituted can be created to produce a peptidic protein with altered conformational dynamics. For example, residues in the hydrophobic "core" of a protein can be substituted with nonpolar residues having smaller side chains (see above) to create a cavity in the core and disrupt packing, and cysteine residues can be deleted or substituted with other amino acid residues to remove disulfide bridges (which are often found in the protein core). In some embodiments, for example, at least one disulfide bond is removed in the starting protein, such as by replacing cysteine with alanine, serine, and / or glycine, etc. In a further preferred embodiment, both cysteines involved in the formation of at least one disulfide bond are replaced with alanine, serine, and / or glycine, or preferably alanine or glycine, etc.

[0075] The peptidic protein is preferably provided in an isolated form and is preferably substantially purified. Further, the peptidic protein exhibits a stable 3D conformational epitope for B cell activation, and synthetic peptides (such as by chemical synthesis) can be co-administered, thereby optimizing the epitope for MHC-II presentation. Alternatively, the peptidic protein and the peptide can be expressed by a mixture of polynucleotides. In yet other embodiments, the peptidic protein can be combined with wild-type starting protein and synthetic peptide(s) to induce an immune response.

[0076] In some embodiments, the rate of polypeptide degradation may be adjusted to produce an optimal mix of peptides and allow for the greatest diversity of peptides displayed on antigen-presenting cells within an appropriate time frame.

[0077] Due to the complexity of proteolytic attack on the protein antigen(s) that generate peptides for presentation, immunization with a mixture of antigens (such as a combinatorial cocktail) is advantageous. Thus, the “tuning mutation(s)” optimal for the generation of a given peptide (T cell epitope) within an appropriate time frame may differ from the mutations optimal for the generation of another peptide. By presenting the antigen as a mixture, a very large number of different mutant proteins can be endocytosed by a single cell or multiple cells, thereby maximizing the diversity of peptides generated and presented by that cell.

[0078] Combinatorial immunization, in which the subject is immunized with two or more distinct antigens that have the same overall surface characteristics (i.e., cross-reactive B cell epitopes) but different conformational dynamics, enriches the diversity of T cell epitopes. This combinatorial approach, with a single inoculation (both protein-based and nucleotide-based) containing hundreds or thousands of different immunogens, can greatly increase the B cell epitope repertoire. This is because all molecules in the mix can contribute to one or more unique T cell epitopes while maintaining a wild-type-like conformation. In some embodiments, the B cell epitope repertoire is biased towards the most stable (and presumably wild-type-like) molecules in the ensemble, in order to maintain the wild-type conformation. The peptidogenic protein has a conformation similar to the starting protein

[0079] An operational test of whether the peptidogenic protein has a “conformation similar to the starting protein” is whether cross-reactive antibodies, particularly antibodies that recognize conformational (3D) epitopes, specifically bind to both the peptidogenic protein and the starting protein. In the present invention, “cross-reactivity” or “cross-reactive antibody” is defined in terms of “binding affinity” that can be measured based on the dissociation constant (K D ), off-rate (k off ), and / or on-rate (k on ).

[0080] For example, the cross-reactive antibody binds to both the peptidic protein and the starting protein with a dissociation constant or K -6 M, 10 -6 M, 5×10 -7 M, 10 -7 M, 5×10 -8 M or 10 -8 M or less or equal dissociation constant. Further more preferably, the cross-reactive antibody binds to both the peptidic protein and the starting protein with a dissociation constant or K D M, 10 -9 M, 5×10 -9 M, 10 -10 M, 5×10 -10 M, 10 -11 M, 5×10 -11 M, 10 -12 M, 5×10 -12 M, 10 -13 M, 5×10 -13 M, 10 -14 M, or 10 -14 M or less or equal dissociation constant K D . The present invention encompasses dissociation constants or K D for peptidic proteins and / or starting proteins within any one of the ranges between each of the individually listed values. Further, it is specifically contemplated that the K D for an antibody binding to a peptidic protein may not be the same as its K D for the starting protein, and in a preferred embodiment, the K D for an antibody binding to a peptidic protein is lower than its K D for its binding to the starting protein. Operationally, it is understood that in this case K D refers to the functional affinity of the antibody for the antigen. The functional or "apparent" affinity may be enhanced with a multivalent antibody containing multiple interaction sites (e.g., Fab arms) capable of binding to the antigen ("avidity effect").

[0081] Furthermore, the cross-reactive antibody binds to both the peptidic protein and the starting protein with a dissociation constant of 5×10 -2 sec-1 and 10 -2 sec -1 or 5×10 -3 sec -1 or 10 -3 sec -1 bind at an off-rate (k off ) less than or equal to this. More preferably, the cross-reactive antibody binds to both the peptidic protein and the starting protein at 5×10 -4 sec -1 or 10 -4 sec -1 or 5×10 -5 sec -1 or 10 -5 sec -1 or 5×10 -6 sec -1 or 10 -6 sec -1 or 5×10 -7 sec -1 or 10 -7 sec -1 bind at an off-rate (k off ) less than or equal to this. The present invention encompasses off-rates (k off ) for peptidic proteins and / or starting proteins within any one of the ranges between each of the individually listed values. Furthermore, it is specifically contemplated that the k off of the antibody to the peptidic protein may not be the same as the k off of the starting protein, and in a preferred embodiment, the (k off ) for binding of the antibody to the peptidic protein is greater than the (k off ) for binding of the antibody to the starting protein.

[0082] Assays for testing cross-reactivity are described herein or are known in the art. For example, binding assays can be performed in solution (e.g., Houghten, Bio / Techniques 13:412-421 (1992)), on beads (e.g., Lam, Nature 354:82-84 (1991)), on chips (e.g., Fodor, Nature 364:555-556 (1993)), on bacteria (e.g., U.S. Patent No. 5,223,409), on spores (e.g., Patent Nos. 5,571,698; 5,403,484; and 5,223,409), on plasmids (e.g., Cull et al., Proc. Natl. Acad. Sci. USA 89:1865-1869 (1992)) or on phage (e.g., Scott and Smith, Science 249:386-390 (1990); Devlin, Science 249:404-406 (1990); Cwirla et al., Proc. Natl. Acad. Sci. USA 87:6378-6382 (1990); and Felici, J. Mol. Biol. 222:301-310 (1991)). Examples of such assays are further described in the examples below. Use of a Peptide-Provoking Protein for Producing Antibodies

[0083] Antibodies can be produced using a peptide-provoking protein by methods well known to those of skill in the art, such as, for example, the methods described in the art. See, for example, Sutcliffe et al., (supra); Wilson et al., (supra); Chow et al., Proc. Natl. Acad. Sci. USA 82:910-914 (1985); and Bittle et al., J. Gen. Virol. 66:2347-2354 (1985). When using in vivo immunization, the animals may be immunized with the peptide-provoking protein described herein and / or a polynucleotide encoding the peptide-provoking protein.

[0084] Animals such as rabbits, rats, mice, llamas, camels, and / or cows can be immunized with a peptidic protein and / or a polynucleotide encoding a peptidic protein. For example, intraperitoneal and / or intradermal injection of an emulsion containing about 100 micrograms of a peptidic protein or carrier protein and Freund's adjuvant or any other adjuvant known to stimulate an immune response may be used. For example, several booster immunizations may be required, for example, at intervals of about two weeks, to provide a useful titer of anti-peptidic protein antibodies that can be detected by an ELISA assay using a free peptidic protein adsorbed directly or indirectly (e.g., via a biotinylated AviTag) to a solid surface. The titer of anti-peptidic protein antibodies in the serum of immunized animals may be increased, for example, by selecting anti-peptidic protein antibodies by adsorption to a peptidic protein on a solid support and elution of the selected antibodies according to methods well known in the art. Such selection can also be performed using the starting protein.

[0085] Furthermore, the antibodies produced by the disclosed methods can be affinity matured using display technologies such as phage display, yeast display, or ribosome display. In one example, single-chain antibody molecules ("scFv") displayed on the surface of phage particles are screened to identify scFv that immunospecifically bind to a peptidic protein and / or a starting protein. The present invention encompasses both scFv that have been confirmed to immunospecifically bind to a peptidic protein and / or a starting protein and portions thereof. Such scFv can be routinely "converted" into immunoglobulin molecules, for example, by inserting a nucleotide sequence encoding the VH and / or VL domain of the scFv into an expression vector that has been engineered to contain a constant domain sequence and direct the expression of an immunoglobulin molecule.

[0086] For the recombinant expression of an antibody produced using a polynucleotide encoding a peptide - antigenic protein and / or a peptide - antigenic protein of the present invention (including other molecules (e.g., the heavy or light chain of an antibody of the present invention or a portion thereof or a single - chain antibody of the present invention) comprising or consisting of an scFv and its antibody fragments or variants), it is necessary to construct an expression vector(s) containing a polynucleotide encoding the antibody or its fragment or variant. After obtaining a polynucleotide encoding an antibody molecule of the present invention (e.g., a whole antibody, a heavy or light chain of an antibody, or a variant or portion thereof (preferably containing but not necessarily containing a heavy or light chain variable domain)), the vector(s) for the production of the antibody molecule may be produced by recombinant DNA techniques using techniques well - known in the art. Thus, methods for preparing an antibody by expressing a polynucleotide containing a nucleotide sequence encoding an antibody that encodes a coding nucleotide sequence (as well as a sequence encoding a peptide - antigenic protein, of course) are described herein. Expression vectors containing an antibody - coding sequence (as well as a sequence encoding a peptide - antigenic protein) and appropriate transcriptional and translational control signals can be constructed using methods well - known to those skilled in the art. These methods include, for example, in vitro recombinant DNA techniques, synthetic techniques, and in vivo genetic recombination. Thus, the present invention provides a replicable vector comprising a nucleotide sequence encoding either a peptide - antigenic protein operably linked to a promoter or an antibody produced against the peptide - antigenic protein (e.g., a whole antibody, a heavy or light chain of an antibody, a heavy or light chain variable domain of an antibody, or a portion thereof, or a heavy or light chain CDR, a single - chain Fv, or a fragment or variant thereof). Such a vector can include a nucleotide sequence encoding a constant region of the antibody molecule (see, for example, PCT Publication WO / 86 / 05807; PCT Publication WO / 89 / 01036, and U.S. Patent No. 5,122,464), and the variable domain of the antibody may be cloned into such a vector for expression of the whole heavy chain, whole light chain, or both the whole heavy and light chains.

[0087] The expression vector(s) can be introduced into the host cell by conventional techniques, and then the transfected cells are cultured by conventional techniques to generate either the peptidic protein or the antibody produced against the peptidic protein. Accordingly, the present invention includes host cells containing polynucleotide(s) encoding a peptidic protein or an antibody produced against the peptidic protein (e.g., a whole antibody, its heavy or light chain, or a portion thereof, or a single-chain antibody of the present invention, or a fragment or variant thereof) operably linked to a heterologous promoter. In a preferred embodiment, as detailed below, for the expression of whole antibody molecules, vectors encoding both the heavy and light chains may be co-expressed in a host cell for the expression of whole immunoglobulin molecules.

[0088] Using various host expression vector systems, it is possible to express a peptide - antigenic protein or an antibody produced against a peptide - antigenic protein. Such host expression systems represent the medium in which the coding sequence of interest is created and subsequently purified, but when transformed or transfected with an appropriate nucleotide coding sequence, they also represent cells that can express a peptide - antigenic protein or an antibody produced against a peptide - antigenic protein. These include microorganisms such as bacteria (e.g., E. coli, B. subtilis) transformed with a recombinant bacteriophage DNA, plasmid DNA or cosmid DNA expression vector containing the sequence; yeast (e.g., Saccharomyces, Pichia) transformed with a recombinant yeast expression vector containing the coding sequence; an insect cell line infected with a recombinant virus expression vector (e.g., baculovirus); a plant cell line infected with a recombinant virus expression vector (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) or transformed with a recombinant plasmid expression vector containing the coding sequence (e.g., Ti plasmid); or a mammalian cell line (e.g., COS, CHO, BHK, 293, 3T3 cells) harboring a recombinant expression construct containing a promoter derived from the genome of a mammalian cell (e.g., metallothionein promoter) or from a mammalian virus (e.g., adenovirus late promoter; vaccinia virus 7.5K promoter), but are not limited to these. Preferably, bacterial cells such as Escherichia coli, and more preferably, eukaryotic cells are used for the expression of either a peptide - antigenic protein or a recombinant antibody molecule. For example, mammalian cells such as Chinese hamster ovary cells (CHO), in combination with a vector such as the major intermediate early gene promoter element derived from human cytomegalovirus, are an effective expression system (Foecking et al., Gene 45:101 (1986); Cockett et al., Bio / Technology 8:2 (1990)).

[0089] In the bacterial system, several expression vectors can be advantageously selected according to the purpose of use. For example, when attempting to generate large amounts of protein (whether it is a peptidic protein or an antibody produced against a peptidic protein), a vector that directs the expression of a high level of fusion protein product that can be easily purified may be desirable. Such vectors may include, but are not limited to, E. coli expression vector pUR278 (Ruther et al., EMBO J. Vol. 2: 1791 (1983)), in which the coding sequences may be individually ligated in-frame with the lacZ coding region in the vector so that a fusion protein is generated; pIN vectors (Inouye and Inouye, Nucleic Acids Res. Vol. 13: 3101 - 3109 (1985); Van Heeke and Schuster, J. Biol. Chem. Vol. 24: 5503 - 5509 (1989)); and the like. The pGEX vectors can be used to express a foreign polypeptide as a fusion protein with glutathione S-transferase (GST). Generally, such fusion proteins are soluble and can be easily purified from lysed cells by adsorption and binding to matrix glutathione agarose beads followed by elution in the presence of free glutathione. The pGEX vectors are designed to contain thrombin or factor Xa protease cleavage sites so that the cloned target gene product can be released from the GST moiety.

[0090] In the insect system, the Autographa californica nuclear polyhedrosis virus (AcNPV) can be used as a vector to express a peptidic protein or an antibody produced against a peptidic protein. The virus grows in Spodoptera frugiperda cells. The coding sequences can be individually cloned into a non-essential region of the virus (e.g., the polyhedrin gene) and placed under the control of an AcNPV promoter (e.g., the polyhedrin promoter).

[0091] In mammalian host cells, several virus-based expression systems may be utilized. When adenovirus is used as an expression vector, the coding sequence of interest may be ligated to an adenovirus transcription / translation control complex, such as a late promoter and a tripartite leader sequence. This chimeric gene may then be inserted into the adenovirus genome by in vitro or in vivo recombination.

[0092] Insertion into a non-essential region of the viral genome (e.g., region E1 or E3) results in a recombinant virus that is viable and capable of expressing a peptidic protein or an antibody produced against a peptidic protein in an infected host (see, for example, Logan and Shenk, Proc. Natl. Acad. Sci. USA 81:355-359 (1984)).

[0093] Efficient translation of the inserted coding sequence may also require specific initiation signals, which include the ATG initiation codon and adjacent sequences. Furthermore, the initiation codon must be in phase with the reading frame of the desired coding sequence to ensure translation of the entire insert. These exogenous translation control signals and initiation codons may be of various origins, either natural or synthetic. The efficiency of expression may be enhanced by including appropriate transcriptional enhancer elements, transcriptional terminator factors, etc. (see, for example, Bittner et al., Methods in Enzymol. 153:51-544 (1987)).

[0094] Furthermore, a host cell line that regulates the expression of the inserted array or modifies and processes the gene product in a desired specific form may be selected. Such modifications (e.g., glycosylation) and processing (e.g., cleavage) of the protein product can be important for the function of the protein. Different host cells have characteristic and specific mechanisms for the post-translational processing and modification of proteins and gene products. An appropriate cell line or host system can be selected to ensure the correct modification and processing of the expressed foreign protein, and for this purpose, eukaryotic host cells having cell machinery for the appropriate processing of the primary transcript, glycosylation, and phosphorylation of the gene product may be used. Such mammalian host cells include, but are not limited to, CHO, VERY, BHK, Hela, COS, NSO, MDCK, 293, 3T3, W138, and, in particular, breast cancer cell lines such as, for example, BT483, Hs578T, HTB2, BT2O, and T47D, and normal mammary gland cell lines such as, for example, CRL7O3O and HsS78Bst.

[0095] For the long-term high-yield production of recombinant proteins, stable expression is preferred. For example, a cell line that stably expresses a peptidic protein or an antibody produced against a peptidic protein may be engineered. Rather than using an expression vector containing a viral origin of replication, the host cells can be transformed with a polynucleotide and a selectable marker controlled by appropriate expression control elements (e.g., promoter, enhancer, sequence, transcription terminator, polyadenylation site, etc.). Following the introduction of the foreign polynucleotide, the engineered cells may be grown in enriched medium for 1-2 days and then switched to selective medium. The selectable marker in the recombinant plasmid confers resistance to the selection, enabling the cells to stably integrate the plasmid into their chromosomes and grow to form cell growth colonies, which can then be cloned and expanded into cell lines. This method may be advantageously used to engineer a cell line that expresses a peptidic protein or an antibody produced against a peptidic protein.

[0096] Some selection systems may be used, including but not limited to the genes for herpes simplex virus thymidine kinase (Wigler et al., Cell 11:223 (1977)), hypoxanthine-guanine phosphoribosyl transferase (Szybalska and Szybalski, Proc. Natl. Acad. Sci. USA 48:202 (1992)), and adenine phosphoribosyl transferase (Lowy et al., Cell 22:17 (1980)), and these genes can be used in tk-, hgprt-, or aprt-cells, respectively. Further, the following genes: dhfr that confers methotrexate resistance (Wigler et al., Natl. Acad. Sci. USA 77:357 (1980); O’Hare et al., Proc. Natl. Acad. Sci. USA 78:1527 (1981)); gpt that confers mycophenolic acid resistance (Mulligan and Berg, Proc. Natl. Acad. Sci. USA 78:2072 (1981)); neo that confers aminoglycoside G-418 resistance (Goldspiel et al., Clinical Pharmacy, 12:488-505 (1993); Wu and Wu, Biotherapy 3:87-95(1991); Tolstoshev, Ann.Rev.Pharmacol.Toxicol. 32:573-596(1993); Mulligan, Science 260:926-932(1993); and Morgan and Anderson, Ann.Rev.Biochem. 62:191-217(1993); TIB TECH 11(5):155-215(May;1993)) and hygro which confers hygromycin resistance (Santerre et al., Gene 30:147(1984)) can be used as a criterion for selection of antimetabolite resistance. Methods generally known in the art of recombinant DNA technology can be routinely applied to select the desired recombinant clone, such methods being described, for example, in Ausubel et al., (eds.) Current Protocols in Molecular Biology, John Wiley & Sons, NY(1993); Kriegler, Gene Transfer and Expression, A Laboratory Manual, Stockton Press, NY(1990); and Chapters 12 and 13, Dracopoli et al., (eds) Current Protocols in Human Genetics, John Wiley & Sons, NY(1994); Colberre-Garapin et al., J.Mol.Biol. 150:1(1981).

[0097] The expression level of a peptidic protein or an antibody produced against a peptidic protein can be increased by vector amplification (for a review, see Bebbington and Hentschel, The use of vectors based See, e.g., DNA Cloning, Vol. 3 (Academic Press, New York, 1987), for gene amplification for the expression of cloned genes in mammalian cells. If a marker in a vector system expressing a peptide - antigenic protein or an antibody produced against the peptide - antigenic protein is amplifiable, an increase in the level of an inhibitor present in the host cell culture will increase the copy number of the marker gene. Since the amplified region is associated with the coding sequence, production of the peptide - antigenic protein or the antibody produced against the peptide - antigenic protein also increases (Crouse et al., Mol. Cell. Biol. 3:257 (1983)).

[0098] Other elements that can be included in the vector sequence are heterologous signal peptides (secretion signals), membrane anchor sequences, introns, alternative splicing sites, translation start and stop signals, inteins, biotinylation sites and other sites that facilitate post - translational modification, purification tags, sequences encoding fusions to other proteins or peptides, separate coding regions separated by an internal ribosome entry site, e.g., sequences encoding "marker" proteins that confer selectability (e.g., antibiotic resistance) or sortability (e.g., fluorescence), modified nucleotides, and other known polynucleotide cis - acting features, but are not limited to these examples.

[0099] In the case of antibodies, the host cells may be co-transfected with two expression vectors of the present invention, a first vector encoding a heavy-chain-derived polypeptide and a second vector encoding a light-chain-derived polypeptide. The two vectors may contain the same selectable marker that allows for equivalent expression of the heavy and light chain polypeptides. Alternatively, a single vector capable of encoding and expressing both the heavy-chain polypeptide and the light-chain polypeptide may be used. In such a situation, the light chain is preferably placed before the heavy chain to avoid excessive toxic free heavy chain (Proudfoot, Nature 322:52 (1986); Kohler, Proc. Natl. Acad. Sci. USA 77:2 197 (1980)). The coding sequences for the heavy and light chains may include cDNA or genomic DNA or synthetic DNA sequences.

[0100] After a peptide antigenic protein or an antibody produced against a peptide antigenic protein has been produced by recombinant expression, the antibody produced against the peptide antigenic protein or the peptide antigenic protein may be purified by any method known in the art for protein purification, for example, chromatography (e.g., ion exchange, affinity (in particular, by protein A affinity and immunological affinity for a specific antigen), and sizing column chromatography), centrifugation, differential solubility, or any other standard technique for protein purification. Further, the antibody produced against the peptide antigenic protein or the peptide antigenic protein may be fused to a heterologous polypeptide sequence described herein or otherwise known in the art to facilitate purification.

[0101] In one example, an antibody produced against a peptidic protein or a peptidic protein described herein may be fused with a constant domain of an immunoglobulin (IgA, IgE, IgG, IgM) or a portion thereof (CH1, CH2, CH3, or any combination and portions thereof), or albumin (including but not limited to recombinant human albumin or fragments or variants thereof (see, e.g., U.S. Patent No. 5,876,969 issued March 2, 1999, European Patent No. 0413622, and U.S. Patent No. 5,766,883 issued June 16, 1998)) to obtain a chimeric polypeptide. Such fusion proteins may facilitate purification and may increase the half-life in vivo. This has been shown for chimeric proteins consisting of the first two domains of the human CD4 polypeptide and various domains of the constant regions of the heavy or light chains of mammalian immunoglobulins. See, e.g., EP394,827; Traunecker et al., Nature, 331:84-86 (1988). Enhanced delivery of antigens through epithelial barriers to the immune system has been demonstrated for antigens (e.g., insulin) conjugated to an FcRn binding partner such as IgG or an Fe fragment (see, e.g., PCT Publication WO96 / 22024 and WO99 / 04813). IgG fusion proteins having a disulfide-linked dimer structure due to IgG partial disulfide bonds have also been found to be more efficient in binding and neutralizing other molecules than monomeric polypeptides or fragments thereof alone. See, e.g., Fountoulakis et al., J. Biochem., 270:3958-3964 (1995). Nucleic acids encoding the peptidic proteins or antibodies described herein can also be recombined with the gene of interest as an epitope tag (e.g., hemagglutinin ("HA") tag or flag tag) to aid in the detection and purification of the expressed polypeptide. For example, the system described by Janknecht et al. allows for the easy purification of non-denatured fusion proteins expressed in human cell lines (Janknecht et al., 1991, Proc. Natl. Acad. Sci. USA 88:8972-897).In this system, the gene of interest is subcloned into a vaccinia recombinant plasmid such that the open reading frame of the gene is translationally fused to an amino-terminal tag consisting of six histidine residues. The tag serves as a matrix binding domain for the fusion protein. Extracts from cells infected with recombinant vaccinia virus are loaded onto a Ni2+-nitrilotriacetic acid agarose column, and the histidine-tagged protein can be selectively eluted with an imidazole-containing buffer. Vaccination

[0102] Animals can be vaccinated using a mixture of a peptidogenic protein and / or a polynucleotide encoding a peptidogenic protein. This vaccination may produce antibodies against the peptidogenic protein. Suitable subjects for the above treatment may be mammals such as rodents (e.g., guinea pigs, hamsters, rats, mice), murine (e.g., mice), canine (e.g., dogs), feline (e.g., cats), equine (e.g., horses), primates, simian (e.g., monkeys or apes), monkeys (e.g., marmosets, baboons, rhesus monkeys), apes (e.g., gorillas, chimpanzees, orangutans, langurs), or humans. In some preferred embodiments, the subject is human. In other embodiments, non-human mammals, particularly mammals customarily used as models for demonstrating therapeutic effects in humans (e.g., murine, primates, pigs, canine, or rabbit animals) may be used.

[0103] In some embodiments, the peptidogenic protein is a chimeric fusion protein used for the vaccine, e.g., a viral protein fused to another protein.

[0104] The vaccination strategy can be based on the repeated administration of a peptidic protein and / or a polynucleotide encoding a peptidic protein to a subject as described herein, in order to enable the generation of memory B cells and memory T cells against the peptidic protein. Vaccination can be performed either prophylactically or therapeutically. The peptidic protein can be derived from the same starting protein or from multiple starting proteins. A prophylactic vaccination strategy aims to stimulate the subject's immune system to generate prophylactic adaptive immunity against a pathogen, while a therapeutic vaccination strategy is performed to improve the clinical situation that exists in the subject after the disease has already been established.

[0105] Proteolytic processing involves the processing of an antigen, such as a peptidic protein, in an antigen-presenting cell after endocytosis and fusion of the endosome with lysosomes of the endosome. The endosome then merges with exocytic vesicles derived from the Golgi apparatus containing class II MHC molecules, to which the resulting peptides bind. The MHC-peptide complex then travels to the cell membrane, where the antigen is available for presentation to CD4 + T cells. Any limitation in the proteolytic processing of the peptidic protein may promote narrowing of the diversity of peptide products, thus reducing the options for selecting a stable binding partner provided by class II MHC molecules, and for this reason, the phenomenon of dominant epitopes may deteriorate. Enhanced immunodominance, in turn, can increase the proportion of non-responders in the population. This is because immune responsiveness is governed by the genetic characteristics of class II MHC alleles. Thus, a vaccine using a mixture of a peptidic protein and / or a polynucleotide encoding a peptidic protein as described herein should increase the diversity of antigenic peptides resulting from endosomal proteolytic processing, and thus is expected to increase the effectiveness of the vaccine. Introduction of a polynucleotide encoding a peptidic protein into an animal

[0106] Polynucleotides encoding peptide - antigenic proteins can also be introduced directly into animals. For example, U.S. Patent Nos. 5,676,954; 6,875,748; 5,661,133; Sahin et al., Nat Rev Drug Discov, 2014 Oct;13(10):759 - 80; Kariko et al., Mol Ther, 2008 Nov;16(11):1833 - 40; Kariko et al., Nucleic Acid Res, 2011, Nov;39(21):e142; see U.S. Patent No. 6,511,832. In one example, a polynucleotide such as a DNA sequence encoding a mixture of peptide - antigenic proteins is directly injected into a host animal, the polynucleotide enters the nucleus and is transcribed into mRNA, generating a peptide - antigenic protein.

[0107] Similarly, the polynucleotide can also be an mRNA sequence such as in vitro transcribed mRNA (IVT mRNA). Essentially, synthetic mRNA can be engineered to express peptide - antigenic proteins and, ideally, the mRNA is translated in the cytoplasm of the cell without entering the nucleus. In the cytoplasm, the mRNA is decoded by ribosomes and translated into peptide - antigenic proteins.

[0108] In either method, next, the peptide - antigenic protein is processed and used to produce antibodies, similar to immunization with a protein. Polynucleotides encoding peptide - antigenic proteins can be synthesized using gene - codon degeneracy and standard DNA synthesis techniques. Mixtures of different polynucleotides encoding the same peptide - antigenic protein, different peptide - antigenic proteins derived from the same starting protein, and / or different peptide - antigenic proteins derived from different starting proteins can be used.

[0109] Animals that can be used to produce antibodies include, but are not limited to, rabbits, rats, mice, llamas, and / or cows. The polynucleotides disclosed herein can be injected into animals via intramuscular, intradermal, intranasal, subcutaneous, intravenous, intratracheal, and intrathecal delivery. This method of producing antibodies allows for the simultaneous generation of antibodies of many species compared to conventional methodologies and substantially increases the repertoire of antibodies produced. Formulation

[0110] The pharmaceutical composition may comprise a peptidic protein described herein, a polynucleotide encoding the peptidic protein, or an antibody produced against the peptidic protein, together with one or more pharmaceutically acceptable carriers, adjuvants, excipients, diluents, fillers, buffers, stabilizers, preservatives, lubricants, or other materials well known to those skilled in the art. Suitable materials are sterile and pyrogen-free and have appropriate isotonicity and stability. Examples include sterile saline (e.g., 0.9% NaCl), water, glucose, glycerol, ethanol or the like or combinations thereof. Such materials should be non-toxic and should not interfere with the effectiveness of the active compound. The exact nature of the carrier or other material depends on the route of administration, which may be by bolus, infusion, injection or any other suitable route considered below. The composition may further contain auxiliary agents such as wetting agents, emulsifying agents, pH buffering agents or the like. Suitable carriers, excipients, etc. can be found in standard pharmaceutical texts, e.g., Remington’s Pharmaceutical Sciences, 18th Edition, Mack Publishing Company, Easton, Pa., 1990.

[0111] As used herein, the term "pharmaceutically acceptable" relates to compounds, materials, compositions, and / or dosage forms that are commensurate with a reasonable benefit / risk ratio and are suitable for use in contact with the tissues of a subject (e.g., a human) within the scope of sound medical judgment, without undue toxicity, irritation, allergic response, or other problems or complications. Each carrier, excipient, etc. must also be "acceptable" in the sense of being compatible with the other ingredients in the formulation.

[0112] In some embodiments, a peptidogenic protein, a polynucleotide encoding a peptidogenic protein, or an antibody produced against a peptidogenic protein may be provided in lyophilized form for reconstitution prior to administration. For example, a lyophilized reagent may be reconstituted with sterile water and mixed with physiological saline prior to administration to a subject.

[0113] Furthermore, "cocktails" of peptidogenic proteins, polynucleotides encoding peptidogenic proteins, or antibodies produced against peptidogenic proteins are specifically contemplated. For example, a mixture of different peptidogenic proteins or polynucleotides encoding different peptidogenic proteins from the same starting protein can be used to initiate an immune response. Alternatively, a mixture of different peptidogenic proteins or polynucleotides encoding different peptidogenic proteins from different starting materials can also be used to initiate an immune response.

[0114] The formulations may conveniently be presented in unit dosage form and may be prepared by any methods well known in the pharmaceutical art. Such methods include the step of bringing the active compound into association with a carrier which constitutes one or more accessory ingredients. In general, the formulations are prepared by uniformly and intimately bringing the active compound into association with a liquid carrier or a finely divided solid carrier or both, and then, if necessary, shaping the product.

[0115] The formulation may be in the form of a liquid, solution, suspension, emulsion, elixir, syrup, tablet, lozenge, granule, powder, capsule, cachet, pill, ampoule, suppository, vaginal suppository, ointment, gel, paste, cream, spray, mist, foam, lotion, oil, bolus, liniment, or aerosol.

[0116] Optionally, other therapeutic or prophylactic agents may be included in the pharmaceutical composition or formulation.

[0117] The treatment may be any treatment and therapy, whether in humans or animals (e.g., veterinary uses), and to some extent a desired therapeutic effect, such as inhibition or delay of the progression of a condition, is achieved. The therapeutic effect includes a decrease in the rate of progression, suppression of the rate of progression, remission of the condition, cure or alleviation (partial or total) of the condition, prevention, delay, reduction or cessation of one or more symptoms and / or signs of the condition, or prolongation of the survival of the subject or patient longer than expected if no treatment were given.

[0118] Treatment as a prophylactic measure (i.e., prophylaxis) is also included. For example, a subject who is susceptible to or at risk of developing or recurring a disease may be treated as described herein. Such treatment may prevent or delay the development or recurrence of the disease in the subject.

[0119] As used herein, the term "therapeutically effective amount" relates to the amount of an antibody produced against a peptidogenic protein or peptidogenic protein that is effective to produce a desired therapeutic effect, balanced with a reasonable benefit / risk ratio.

[0120] It is recognized that the appropriate dosage of a peptidic protein or an antibody produced against a peptidic protein can vary from patient to patient. Determining the optimal dosage generally involves balancing the level of therapeutic benefit against any risks or adverse side effects of administration. The dosage level selected depends on a variety of factors including the route of administration, the time of administration, the active compound, other drugs, compounds, and / or materials used in combination, and the rate of excretion, as well as the age, sex, weight, condition, general health, and past medical history of the patient, but is not limited thereto. The amount and route of administration of a peptidic protein, a polynucleotide encoding a peptidic protein or an antibody produced against a peptidic protein are ultimately at the discretion of the physician, but generally the dosage should achieve the concentration of the active compound at the site of treatment without substantially harming or causing adverse side effects.

[0121] Generally, the appropriate dosage of a peptidic protein or an antibody produced against a peptidic protein is in the range of about 100 μg to about 250 mg per kilogram body weight of the subject per day. If the peptidic protein or an antibody produced against a peptidic protein is a salt, ester, prodrug, or the like, the amount administered is calculated based on the parent compound, so the actual weight used increases proportionally.

[0122] Administration in vivo can be carried out in a single dose, continuously or intermittently (e.g., in divided doses at appropriate intervals). The most effective means of administration and methods for determining dosage are well known to those skilled in the art and vary depending on the formulation used for treatment, the purpose of treatment, the target cells being treated, and the subject being treated. Single or multiple administrations can be carried out using the dosage levels and patterns selected by the physician.

[0123] "Concurrent" administration means that the peptidic protein, the polynucleotide encoding the peptidic protein, or the antibody produced against the peptidic protein is administered to the subject in a single dose by the same route of administration.

[0124] "Separate" administration means that the peptidic protein, the polynucleotide encoding the peptidic protein, or the antibody produced against the peptidic protein is administered to the subject by two different routes of administration occurring simultaneously. This may occur, for example, when one agent is administered by injection or parenterally and the other agent is given orally during the course of the injection or parenteral administration.

[0125] "Sequential" means that the peptidic protein, the polynucleotide encoding the peptidic protein, or the antibody produced against the peptidic protein is administered at different times under the condition that the activity of the first administered agent is present and ongoing in the subject at the time the second agent is administered. Preferably, sequential dosing occurs such that the second of the two agents is administered within 48 hours, preferably within 24 hours, such as within 12, 6, 4, 2 or 1 hour of the first agent.

[0126] Multiple doses of a peptidic antigenic protein, a polynucleotide encoding the peptidic antigenic protein and / or an antibody produced against the peptidic antigenic protein may be administered. For example, 2, 3, 4, 5 or more than 5 doses may be administered after administration of the peptidic antigenic protein, the polynucleotide encoding the peptidic antigenic protein and / or the antibody produced against the peptidic antigenic protein. Administration of the peptidic antigenic protein, the polynucleotide encoding the peptidic antigenic protein and / or the antibody produced against the peptidic antigenic protein may continue for a long time after the first administration. For example, treatment with the peptidic antigenic protein, the polynucleotide encoding the peptidic antigenic protein, or the antibody produced against the peptidic antigenic protein may continue for at least 1 week, at least 2 weeks, at least 3 weeks, at least 1 month or at least 2 months. Treatment with the peptidic antigenic protein, the polynucleotide encoding the peptidic antigenic protein, or the antibody produced against the peptidic antigenic protein may continue for as long as necessary to achieve a therapeutic response.

[0127] The peptidic antigenic protein, the polynucleotide encoding the peptidic antigenic protein, or the antibody produced against the peptidic antigenic protein and compositions containing these molecules may be administered to a subject by any convenient route of administration, systemically / peripherally or at the desired site of action, including, but not limited to, orally (e.g., by oral ingestion); and by injection, including, for example, subcutaneously, intradermally, intramuscularly, intravenously, intraarterially, intracardially, intrathecal, intraspinal, intracapsular, subcapsular, intraorbital, intraperitoneal, intratracheal, subepidermal, intraarticular, intrathecal, and intrasternal; for example, parenterally by implantation of a depot subcutaneously or intramuscularly. Usually, administration is by the intravenous route, but other routes such as intraperitoneal, subcutaneous, transdermal, oral, nasal, intramuscular or other convenient routes are not excluded.

[0128] A pharmaceutical composition comprising a peptidic antigenic protein, a polynucleotide encoding the peptidic antigenic protein, or an antibody produced against the peptidic antigenic protein may be formulated into suitable dosage unit formulations appropriate for the intended route of administration.

[0129] Formulations suitable for oral administration (e.g., by oral ingestion) may be presented as discrete units such as capsules, cachets, or tablets, each containing a predetermined amount of the active compound as a powder or granules; as a solution or suspension in aqueous or non-aqueous liquids; or as an oil-in-water liquid emulsion or a water-in-oil liquid emulsion; as a bolus; as a lozenge; or as a paste.

[0130] Tablets may be made by conventional means, for example, by compression or molding, optionally with one or more accessory ingredients. Compressed tablets may be prepared by compressing, in a suitable machine, the active compound, optionally with one or more binders (e.g., povidone, gelatin, acacia, sorbitol, tragacanth, hydroxypropylmethylcellulose); fillers or diluents (e.g., lactose, microcrystalline cellulose, calcium hydrogen phosphate); lubricants (e.g., magnesium stearate, talc, silica); disintegrants (e.g., sodium starch glycolate, cross-linked povidone, cross-linked sodium carboxymethylcellulose); surface-active or dispersing or wetting agents (e.g., sodium lauryl sulfate); and preservatives (e.g., methyl p-hydroxybenzoate, propyl p-hydroxybenzoate, sorbic acid) in a fluid form such as a powder or granules. Moulded tablets may be made by molding, in a suitable machine, a mixture of the powdered compound moistened with an inert liquid diluent. Tablets may be optionally coated or scored and may be formulated so as to provide for slow or controlled release of the active compound therein using, for example, hydroxypropylmethylcellulose in varying proportions to provide the desired release profile. Tablets may optionally be enteric-coated to provide release in parts of the intestine other than the stomach.

[0131] Formulations suitable for parenteral administration (e.g., by injection including intradermal, subcutaneous, intramuscular, intravenous, and intradermal) may contain antioxidants, buffers, preservatives, stabilizers, bacteriostatic agents, and solutes that render the formulation isotonic with the blood of the intended recipient, and may be aqueous and non-aqueous isotonic sterile injectable solutions that are pyrogen-free; and aqueous and non-aqueous sterile suspensions that may contain suspending and thickening agents, and liposomes or other microparticle systems designed to direct the compound to the target blood component or one or more organs. Examples of suitable isotonic solvents for use in such formulations include sodium chloride injection, Ringer's solution, or lactated Ringer's injection. Typically, the concentration of the active compound in the solution is from about 1 ng / ml to about 10 μg / ml, for example, about 10 ng / ml to about 1 μg / ml, about 1 μg / ml to about 10 mg / ml, about 10 μg / ml to about 1 mg / ml, about 1 mg / ml to about 20 mg / ml, about 10 mg / ml to about 120 mg / ml, or any other concentration suitable for the administration of a biological drug (e.g., protein, antibody, etc.). The formulation may be presented in a sealed container, e.g., ampoules and vials, for single or multiple doses, and may be stored in a freeze-dried (lyophilized) state that requires only the addition of a sterile liquid carrier, e.g., water for injection, immediately prior to use. Immediate injectable solutions and suspensions may be prepared from sterile powders, granules, and tablets. The formulation may also be in the form of liposomes or other microparticle systems designed to direct the active compound to the target blood component or one or more organs.

[0132] Compositions containing a propeptide protein, a polynucleotide encoding the propeptide protein, and / or an antibody produced against the propeptide protein may be prepared in the form of a concentrate for subsequent dilution, or in the form of a divided dose ready for administration. Alternatively, the reagents may be provided separately in a kit for mixing prior to administration to a human or animal subject.

[0133] The peptidic antigenic protein, the polynucleotide encoding the peptidic antigenic protein, and / or the antibody produced against the peptidic antigenic protein may be administered alone or in combination with other treatments, simultaneously or sequentially, depending on the individual circumstances. For example, the peptidic antigenic protein, the polynucleotide encoding the peptidic antigenic protein, or the antibody produced against the peptidic antigenic protein described herein may be administered in combination with one or more additional active compounds.

[0134] Various further aspects and embodiments of the present invention will be apparent to those skilled in the art in light of the present disclosure.

[0135] It should be understood that this application discloses all possible combinations with each other of the above aspects and any of the embodiments described above, unless the context requires otherwise. Similarly, this application discloses all combinations of preferred and / or optional features, either singly or together with any of the other aspects, unless the context requires otherwise.

[0136] Modifications, further embodiments and modifications of the above embodiments will be apparent to those skilled in the art upon reading the present disclosure, and accordingly, these modifications are within the scope of the present invention. All documents and sequence database entries referred to herein are hereby incorporated by reference in their entirety for all purposes. The present invention is further described below with reference to the following examples.

Examples

[0137] (Example 1) Production of peptidic antigen To create peptide - antigenic proteins, the starting protein can be modified at its core residues (e.g., one or more mutations) to alter its conformational dynamics. Multiple different peptide - antigenic proteins can be designed and expressed to immunize an animal (e.g., rabbit) to generate a polyclonal antibody response. Alternatively, a polynucleotide encoding a peptide - antigenic protein can be directly administered to an animal to create the peptide - antigenic protein in vivo. The response is monitored by the following two complementary and mutually reinforcing methods (Georgiou et al., 2014; Figure 2): (a) Purifying B cells from the blood, spleen, and bone marrow of the immunized animal, isolating cDNA from the mRNA encoding the variable regions of the heavy and light chains, and analyzing this repertoire via deep DNA sequencing; and (b) Immunoaflfinity - purifying polyclonal Fab or (Fab’)2 fragments from immune serum using antigen attached to a solid support, digesting the eluted Fab / (Fab’)2 with protease, and sequencing the resulting peptides using LC / MS / MS.

[0138] Specifically, conformational studies can be performed in test animals (rabbits) with various peptide - antigenic proteins (or polynucleotides encoding peptide - antigenic proteins) whose conformation is similar to the starting protein but whose conformational dynamics vary. Next - generation DNA sequencing techniques can be used to comprehensively characterize the humoral response in the animal. Immunoglobulin V regions can be cloned from B lymphocytes and subjected to deep sequencing in large - scale parallel (5 - 8). Along with this, polyclonal antibodies from the same test animals can be purified by immunoaffinity chromatography, then digested with protease, and subjected to LC - MS / MS to determine the peptide sequences (9, 10). Comparison of these two datasets reveals the repertoire of individual antibodies, including the polyclonal response (9).

[0139] For example, small mammalian proteins that have been biophysically well-characterized can be used as test antigens. Preferred examples include, but are not limited to, bovine pancreatic trypsin inhibitor and / or the Alzheimer's amyloid precursor protein Kunitz domain. Alternatively, antigens relevant to unmet vaccine needs, such as, for example, P. falciparum sporozoite antigen, can also be produced and tested in this method. In addition, optimization (or re-optimization) of synthetic vaccines with respect to the conformational dynamics of the component proteins (perhaps by replacing a single component with a combinatorial cocktail of several versions of the same antigen with different core destabilizing mutations) can also be generated. Testing these new vaccines in clinical trials may involve monitoring vaccinated individuals using similar DNA sequence analysis of the blood-derived B cell V region repertoire and proteomic characterization of immunoaffinity-purified polyclonal antibody peptides, similar to the procedures described above.

[0140] Other preferred examples of antigens that can be used in accordance with embodiments of the invention described herein include malaria polypeptides such as thrombospondin-related adhesion protein (TRAP) and / or apical membrane antigen 1 (AMA1), human immunodeficiency virus (HIV) gp120 and gp41, hepatitis C (HCV) envelope glycoproteins E1 and E2, Middle East respiratory syndrome coronavirus (MERS-CoV) spike glycoprotein, human influenza virus hemagglutinin (HA) and neuraminidase, hepatitis B virus (HBV) capsid core, and antigens or antigens derived from antigens from related viruses that infect apes, monkeys, chickens, pigs, camels, and other animals, but are not limited thereto.

[0141] In a preferred embodiment, any one of the P. falciparum protein antigens listed in Table 2 below can be used as a starting protein for inducing a peptidic protein. In addition, a plurality of antigens listed in Table 2 can be used as starting proteins for inducing a plurality of different peptidic proteins that generate an immune response, including generating antibodies.

Table 2-1

Table 2-2

Table 2-3

[0142] In order to modify the conformational dynamics of the starting protein, the changes in Gibbs free energy shown in Table 3 below can be considered:

Table 3

[0143] As discussed by Loladze et al. (J. Mol. Biol. 320, 343-357 (2002)), the following amino acid substitutions can reduce the thermodynamic stability (e.g., as reflected in Gibbs free energy) and change the conformational dynamics of the starting protein. For example, Val and Leu (and other larger nonpolar amino acid residues) can be substituted with smaller ones, such as Ala, Thr, Asn, and / or Gly. In addition, the buried sites of Glu in the starting protein can be substituted with Leu, Val, Asn, Thr, Ser, Ala, and / or Gly. These single-site amino acid substitutions are expected to create peptidic proteins with lower stability than the starting protein but with similar conformations.

[0144] Alternatively, the conformational dynamics of the starting protein can be altered by replacing (a) at least one threonine with valine, alanine, glycine, or serine; or (b) at least one cysteine with alanine, valine, glycine, serine, or threonine; or (c) at least one valine with alanine, glycine, leucine, or isoleucine; or (d) at least one leucine with alanine, valine, glycine, or isoleucine; or (e) at least one isoleucine with alanine, valine, leucine, or glycine; or (f) at least one proline, methionine, phenylalanine, tyrosine, or tryptophan with alanine, valine, leucine, isoleucine, or glycine; or (g) at least one aspartic acid or asparagine with glycine, serine, threonine, alanine, valine, leucine, or isoleucine; or (h) at least one glutamic acid or glutamine with aspartic acid, asparagine, glycine, serine, threonine, alanine, valine, leucine, or isoleucine; or (i) at least one lysine with arginine, histidine, glycine, serine, threonine, alanine, valine, methionine, leucine, or isoleucine; or (j) at least one arginine with lysine, histidine, glycine, serine, threonine, alanine, valine, methionine, leucine, or isoleucine; or (k) at least one histidine with lysine, arginine, glycine, serine, threonine, alanine, valine, glutamine, asparagine, leucine, or isoleucine; or (l) at least one alanine with glycine; or (m) at least one residue with a non-natural amino acid; and / or (n) any combination of the above.

[0145] In an even more preferred embodiment, the conformational dynamics of the starting protein are such that (a) at least one tryptophan is replaced by tyrosine, phenylalanine, methionine, histidine, isoleucine, leucine, valine, alanine or glycine; or (b) at least one tyrosine is replaced by phenylalanine, methionine, histidine, isoleucine, leucine, valine, alanine or glycine; or (c) at least one phenylalanine is replaced by tyrosine, methionine, histidine, isoleucine, leucine, valine, alanine or glycine; or (d) at least one proline is replaced by methionine, leucine, isoleucine, valine, alanine, or glycine; or (e) at least one histidine is replaced by phenylalanine, tyrosine, methionine, isoleucine, leucine, valine, alanine, glycine, lysine, arginine, serine, threonine, asparagine, or glutamine; or (f) at least one methionine is replaced by isoleucine, leucine, valine, alanine or glycine; or (g) at least one isoleucine is replaced by leucine, valine, alanine or glycine; or (h) at least one leucine is replaced by isoleucine, valine, alanine or glycine; or (i) at least one valine is replaced by alanine, glycine, leucine, or isoleucine; or (j) at least one cysteine is replaced by alanine, valine, glycine, serine or threonine; or (k) at least one aspartic acid is replaced by glutamic acid, glutamine, asparagine, glycine, serine, threonine, alanine, valine, leucine, or isoleucine; or (l) at least one glutamic acid is replaced by aspartic acid, glutamine, asparagine, glycine, serine, threonine, alanine, valine, leucine, or isoleucine; or (m) at least one alanine is replaced by glycine or proline; or (n) at least one serine is replaced by alanine or glycine; or (o) at least one glycine is replaced by alanine or proline; or (p) at least one lysine is replaced by arginine, histidine, glycine, serine, threonine, alanine, valine, methionine, leucine or isoleucine;or (q) at least one asparagine with glycine, alanine, serine, threonine, valine, leucine, isoleucine, glutamine, aspartic acid or glutamic acid; or (r) at least one glutamine with glycine, alanine, serine, threonine, valine, leucine, isoleucine, glutamine, aspartic acid, glutamic acid, or histidine; or (s) at least one arginine with lysine, histidine, glycine, serine, threonine, alanine valine, methionine, leucine, or isoleucine; or (t) at least one threonine with valine, alanine, glycine or serine; or (u) a hydrophobic residue with a smaller similar hydrophobic residue; or (v) at least one residue with a non-natural amino acid; or (w) by any combination of the above. An optimal substitution to increase immunogenicity can be determined using a combinatorial approach.; (Example 2) The propeptide protein of bovine pancreatic trypsin inhibitor.

[0146] Bovine pancreatic trypsin inhibitor (BPTI) is a small protein that has been extremely well characterized, and there is a substantial body of literature describing its folding, structure, activity, thermodynamic properties, expression characteristics, and protease specificity (15). Our own laboratory was the first to express recombinant BPTI and manipulate its properties using site-directed mutagenesis (16, 17). Wild-type BPTI has three disulfide bonds, with disulfides 14–38, 30–51, and 5–55 cross-linked; the 14–38 disulfide is on the surface, and the other two disulfides are deeply buried in the hydrophobic core of the protein. Mutations that replace any one of these disulfides with two cysteine-to-alanine residues destabilize the BPTI molecule (18). All possible combinations of BPTI disulfide bond mutants have been made by ourselves and others, and all are significantly destabilized. Importantly, when all combinations of mutants of BPTI in which one or two native disulfide bonds have been knocked out were examined, the proteins nevertheless maintain a similar three-dimensional structure and trypsin inhibitor activity comparable to wild-type BPTI (19–22). Thus, despite the stability differences manifested by Tm values in the range of >100°C to <40°C, all of the wild-type molecule and its disulfide mutants show similarity, if not substantial identity of the 3D conformational epitope. For example, at 40°C, almost 50% of the mutant proteins are unfolded at body temperature, and at >100°C (higher than the boiling point of water), the wild-type protein is one of the most heat-resistant proteins known.

Table 4

[0147] Since the inventors were interested in protease digestion and peptide production in vivo from antigens, for the immunological experiments with BPTI by the inventors, the inventors generate the BPTI variants described in Table 4. The mutated residues are underlined and the cysteine residues are shown in bold. These consist of wild-type BPTI in which the surface disulfide 14 - 38 is mutated to alanine, which has been shown to confer increased stability against reducing agents. BPTI variants are also generated in the [Lys15→Ala] background. The Lys15 - Ala mutation excises the P1 residue side chain and reduces the affinity of BPTI for trypsin and other proteases by about 107 - fold, rendering it essentially inactive as a protease inhibitor (23). Additional mutations within the core of the protein (e.g., F22A, Y23A, N43G, F45A) contribute to destabilizing the protein, still maintaining a 3D structure comparable to wild-type BPTI while changing the conformational dynamics to various degrees. This table is intended to be non-limiting with respect to embodiments of the present invention.

[0148] Similar mutations can be made in the Alzheimer's amyloid precursor protein Kunitz inhibitor (APP-KI), a human protease inhibitor homologous to BPTI. Since APP-KI has a relatively low isoelectric point (pI), unlike BPTI, it should be electrostatically almost neutral in charge at lysosomal pH. Similar to BPTI, APP-KI has been previously expressed, characterized in terms of folding, activity, and 3D structure, and has exactly three disulfide bonds homologous to those found in BPTI.

[0149] The inventors express variants both with and without a flanking tag array. Tags used by the inventors to vary solubility include the calmodulin-binding peptide (CBP) tag, which is highly soluble, and the TrpLE tag, which is highly insoluble (24). However, in particularly preferred constructs, the inventors use the three-part tag, AviTag-hexaHis-TEV protease cleavage site. This tag confers moderate solubility, can be biotinylated using BirA biotin ligase (25), enables binding to a HisTrap column for purification and / or on-column refolding (Campbell and Anderson, in preparation), and can cleave the antigen, if desired. The antigen can be used to immunize animals either without the tag (i.e., after TEV cleavage) or with the tag intact, followed by depletion of anti-tag antibodies (S. Blackshaw and D. Eichinger, personal communication). (Example 3) Preferred target of the present invention.

[0150] As used herein, a "target" is a protein that is disclosed in Table 5 and specifically selected, and can be modified to have improved peptide antigenicity as described herein. The first column lists the sequence numbers corresponding to the sequences provided in the sequence listing. The second column lists the "protein name" of each target, and the third column provides the "UniProt reference number", which is a unique "catalog" number used in the art that provides both known and established explanations of the function, expression, and sequence information for each target listed in the second column (the UniProt reference number provides a mapping of the proteome to a reference genome assembly, such as that created by the Genome Reference Consortium (GRC)). This public information (searched from the UniProt database (http: / / www.uniprot.org) on August 10, 2016), including the sequence information corresponding to each target, is hereby incorporated by reference in its entirety. The sequence listing and Table 5 describe the positions of specific residues in each target protein that can be mutated to create corresponding peptide antigenic proteins along specific amino acids that can be substituted at each position. In a preferred embodiment, multiple substitutions can be made in each target protein as shown in the reference positions in the sequence listing and in Table 5. In a further preferred embodiment, at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more residues listed in the sequence listing and / or Table 5 for each target protein can be changed in any combination in each starting target protein listed in the second column using the amino acids listed in the sequence listing and / or as described in the last two paragraphs of Example 1. By spreading mutations across multiple positions and / or target proteins and by mixing these mutated molecules together, an immune cocktail can be created.

Table 5-1

Table 5-2

Table 5-3

Table 5-4

Table 5-5

Table 5-6

Table 5-7

Table 5-8

Table 5-9

Table 5-10

Table 5-11

Table 5-12

Table 5-13

Table 5-14

Table 5-15

Table 5-16

Table 5-17

Table 5-18

Table 5-19

Table 5-20

Table 5-21

Table 5-22

Table 5-23

Table 5-24

Table 5-25

Table 5-26

Table 5-27

Table 5-28

Table 5-29

Table 5-30

Table 5-31

Table 5-32

Table 5-33

Table 5-34

Table 5-35

Table 5-36

Table 5-37

Table 5-38

Table 5-39

Table 5-40

Table 5-41

Table 5-42

Table 5-43

Table 5-44

Table 5-45

Table 5-46

Table 5-47

Table 5-48

Table 5-49

Table 5-50

Table 5-51

Table 5-52

Table 5-53

Table 5-54

Table 5-55

Table 5-56

Table 5-57

Table 5-58

Table 5-59

Table 5-60

Table 5-61

Table 5-62

Table 5-63

Table 5-64

Table 5-65

Table 5-66

Table 5-67

Table 5-68

Table 5-69

Table 5-70

Table 5-71

Table 5-72

Table 5-73

Table 5-74

Table 5-75

Table 5-76

Table 5-77

Table 5-78

Table 5-79

Table 5-80

Table 5-81

Table 5-82

Table 5-83

Table 5-84

Table 5-85

Table 5-86

Table 5-87

Table 5-88

Table 5-89

Table 5-90

Table 5-91

Table 5-92

Table 5-93

Table 5-94

Table 5-95

Table 5-96

Table 5-97

Table 5-98

Table 5-99

Table 5-100

Table 5-101

[0151] Changes in the conformational dynamics can be measured by standard methods known in the art. In a preferred embodiment, changes in the conformational dynamics can be demonstrated by measuring the change in melting temperature in urea-induced equilibrium unfolding studies and / or in the Gibbs free energy compared to the starting protein.

[0152] The change in melting temperature can be demonstrated by the following protocol. For example, the peptidic protein (0.20 mg / ml) and the starting protein (as a control) are heated from 10 °C to 72 °C in a 0.1 cm quartz cuvette at a heating rate of 1 °C x min-1, controlled by a Jasco programmable Peltier element. The dichroic activity at 209 nm and the photomultiplier tube voltage (PMTV) are continuously monitored in parallel every 0.5 °C. All thermal scans are corrected for solvent contributions at different temperatures. The melting temperature (Tm) value is calculated by taking the first derivative of the ellipticity at 209 nm with respect to temperature. All denaturation experiments are performed in triplicate (see Lori et al., PLoS One, 5;8(6):e64824 (2013)).

[0153] Changes in urea-induced equilibrium unfolding can be demonstrated by the following protocol. Peptidic proteins (final concentration 40 μg / ml) and starting proteins (as controls) are incubated at 10 °C in increasing concentrations of urea (0 - 8 M) in 25 mM Tris / HCl, pH 7.5 in the presence of 0.2 M NaCl and 2 mM DTT (for non-disulfide-containing proteins). After 10 minutes, equilibrium is reached and the intrinsic fluorescence emission, absorbance at 287 nm, and / or far-UV CD spectrum (0.5 cm cuvette) are recorded in parallel at 10 °C. To test the reversibility of unfolding, peptidic proteins are unfolded at 10 °C in 7.0 M urea at a protein concentration of 0.4 mg / ml in 25 mM Tris / HCl, pH 7.5 in the presence of 2 mM DTT and 0.2 M NaCl. After 10 minutes, refolding is initiated at 10 °C by 10-fold dilution of the unfolded mixture into a solution of the same buffer containing decreasing urea concentration used for unfolding. The final protein concentration is 40 μg / ml. After an incubation period of 15 minutes to 24 hours, the intrinsic fluorescence emission, absorbance at 287 nm, and / or CD spectrum are recorded as a function of urea concentration at 10 °C (see Lori et al., PLoS One, 5;8(6):e64824 (2013)).

[0154] The change in Gibbs free energy can be shown by the following protocol. To measure the Gibbs free energy, a differential scanning calorimetry (DSC) experiment is performed on a VP-DSC (Microcal Inc., Northampton, MA) instrument at a scan rate of 1.5 deg / min. If possible, the temperature-induced unfolding of the peptidic protein is confirmed for reversibility by comparing the first and second DSC scans. It is understood that reversibility of folding and unfolding is not a requirement for the peptidic proteins described herein. The partial molar heat capacity at constant pressure Cp,pr(T) of the protein is obtained from the experimental measurement of the apparent heat capacity difference ΔC_p^app(T) between the sample (containing the protein solution) and reference (containing the corresponding buffer) cells. The protein concentration is measured spectrophotometrically using a known molar extinction coefficient. Analysis of the heat capacity profile according to the two-state model is performed using the non-linear regression routine NLREG and a script written in-house. The standard thermodynamic functions under reference conditions are calculated as follows: [Number]

[0155] Where ΔH(T), ΔS(T), and ΔG(T) in the formula are the enthalpy, entropy, and Gibbs energy functions of the peptidic protein, respectively, ΔHcal is the enthalpy of unfolding at the transition temperature Tm, and ΔCp is the heat capacity of unfolding (see Loladze et al., J. Mol. Biol. 320, 343-357 (2002)). (Example 5) Assay for measuring peptidogenicity

[0156] As described herein, one intracellular state that can be involved in the processing of peptidogenic proteins is proteolysis. The effect of differential stability of peptidogenic proteins in proteolysis can be determined using one of several in vitro or ex vivo assays.

[0157] (a) Cathepsin L proteolysis In one embodiment, the examination of the behavior of peptidogenic proteins towards proteolysis is measured by subjecting them to the action of cathepsin L, one of the enzymes known to be critically important for protein antigen processing (Hsieh, C. S., deRoos, P., Honey, K., Beers, C., and Rudensky, A. Y. (2002) J. Immunol. 168, 2618 - 2625). The sensitivity of peptidogenic proteins to proteolysis is assessed using lysosomal cathepsin L. Peptidogenic proteins (0.5 μg / μl) are incubated at 37°C for various lengths of time in the presence of various amounts (e.g., 1.5 m units) of the enzyme in 50 mM sodium acetate buffer pH 4.5. Digestion is stopped using 0.1% TFA, and proteolysis is monitored by reverse-phase HPLC on a C18 reverse-phase column (Vydac, Hesperia, CA). Elution of proteolytic products is performed with a linear gradient of acetonitrile / water containing 0.1% TFA.

[0158] (b) Proteolysis using alpha-chymotrypsin and carboxypeptidase Y In another embodiment, the examination of the behavior of peptidic proteins towards proteolysis is measured by subjecting them to the action of alpha-chymotrypsin and carboxypeptidase Y. Alpha-chymotrypsin is an endopeptidase that cleaves at the carboxyl terminus of aromatic amino acids; carboxypeptidase Y is an exopeptidase that sequentially removes amino acids from the carboxyl terminus. Proteolytic digestion with these enzymes is specific for unstable conformations, and thus the conformational stability of peptidic proteins determines their resistance / sensitivity to proteolytic digestion. Peptidic proteins at 1 mg / ml in 0.5 ml of 20 mM HEPES buffered saline pH 7.5 are incubated at 37 °C with 100 μg of alpha-chymotrypsin from bovine pancreas and carboxypeptidase Y from yeast. Each incubation is terminated at various time points and the digested samples are stored at -20 °C until analyzed. Samples are analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) under reducing conditions through a 15% acrylamide gel and are then stained with Coomassie Brilliant Blue for visualization.

[0159] (c) Proteolysis using lysosomal extracts In another embodiment, the examination of the behavior of peptidic proteins towards proteolysis is measured by subjecting them to the action of a lysosomal extract of bone marrow-derived dendritic cells. The peptidic proteins are incubated at various concentrations in the presence of an equal amount of protein from a crude lysosomal extract from bone marrow-derived dendritic cells. The mixtures are incubated at pH 4.5 in 0.1 M sodium citrate buffer, 0.5% Triton X-100, and 2 mM dithiothreitol. Each incubation is terminated at various time points and the digested samples are stored at -20 °C until analyzed. The samples are analyzed by SDS-PAGE. The experiment is repeated with and without pre-adsorption of the peptidic proteins in an adjuvant such as aluminum hydroxide. Bone marrow-derived dendritic cells are purified from bone marrow cultured in granulocyte macrophage colony-stimulating factor by using anti-CD11c microbeads. See, for example, FIG. 4 of Delamarre et al. (d) Proteolysis after internalization by bone marrow-derived dendritic cells

[0160] In another embodiment, the examination of the behavior of peptidic proteins towards proteolysis is measured by labeling them with FITC according to the manufacturer's protocol, where bone marrow-derived dendritic cells are incubated with FITC protein and the percentage of FITC+, CD11c+ cells is measured over time. Bone marrow-derived dendritic cells are loaded with 0.5 mg / ml of FITC-labeled peptidic protein for 1 hour, washed, and then cultured at 37 °C for various amounts of time. FACS is then used to determine the percentage of FITC+, CD11c+ cells at each time point minus the percentage of FITC+, CD11c+ cells at 0 hours. This represents the percentage of proteolysis of the peptidic protein. The experiment is repeated with and without pre-adsorption of the FITC-labeled peptidic protein in an adjuvant such as aluminum hydroxide. (Example 6) Antibody production and sequencing

[0161] Ig-seq of the antibody repertoire may be performed with minor modifications to the previously described protocols (10, 29). B cells can be isolated from the serum, spleen, or other tissues of hyperimmunized rabbits. To reduce the complexity of the sequencing library, this population is sorted to enrich for CD19 + CD3 - CD27 + CD38 int memory B cells or B cells that recognize the target antigen (5, 30, 31). These cells are then lysed, mRNA is isolated using standard methods, and reverse transcribed into cDNA using 5’ RACE with a 3’ primer specific for the IgH or IgL constant region (9, 32). The cDNA library is then amplified with primers containing the required pair of end adapter sequences and optional barcodes, and error correction is enabled by quantifying the template and averaging multiple reads (8, 9).

[0162] Complete determination of the antibody sequence requires identification of the native VH-VL pair. Since each VH and VL sequence is encoded by a separate mRNA, cloning and sequencing can be performed by isolating single B cells in wells with volumes below nanoliter capacity (5) or microemulsions (9) prior to mRNA isolation, reverse transcription, and overlap extension or ligation PCR. Alternatively, the endogenous VH-VL pair can be identified by partially cross-linking the purified Fab prior to LC-MS / MS. This results in a portion of the Fab heavy and light chains forming an inter-chain cross-link under appropriate conditions, and the resulting peptide masses are used to determine native pairing.

[0163] To identify antibodies raised in response to a mixture of peptide - antigenic proteins, sequence information can be combined with data from high - resolution mass spectrometry. Protein A - purified IgG can be digested with papain to release two Fabs from the Fc domain. These can then be immunoaffinity purified on a custom column prepared using the peptide - antigenic proteins immobilized on a solid support, the eluted Fabs are proteolytically digested, and the peptide products are subjected to mass spectrometry. By comparing the resulting peptide masses with complete antibody - sequencing data, the CDR sequences that recognize the antigen can be identified. Pairing of IgG VH and VL sequences can be achieved by chemical cross - linking of immunoaffinity - purified Fabs prior to proteolytic digestion; Young et al. demonstrated the potential of this approach (33). (Example 7) Immunization using a mixture of peptide - antigenic proteins

[0164] Methods for generating antibodies in mammals are well known in the art. In one example, polyclonal antisera against a peptidogenic protein are generated by immunizing pathogen-free rabbits with a total of 500 μg of a mixture of peptidogenic proteins over a period of two months. For example, the peptidogenic protein can be dissolved in PBS and emulsified with an equal volume of Freund's adjuvant. After the final booster, the rabbit serum can be separated to determine the titer of the polyclonal antiserum. To obtain monoclonal antibodies, 4- to 6-week-old Balb / c mice can be immunized with a peptidogenic protein (e.g., 10-100 μg / injection at two-week intervals four times in Freund's complete adjuvant for the first injection and Freund's incomplete adjuvant for subsequent immunizations). Spleen cells are isolated, fused with a fusion cell line, such as Sp2 / 0 myeloma cells, and subsequently limiting diluted. Growing clones are screened using, for example, an enzyme-linked immunosorbent assay (ELISA). A 96-well plate is coated with the peptidogenic protein or a control protein. Culture supernatant is added, followed by washing, and a labeled anti-mouse antibody is added for detection. After limiting dilution cloning of peptidogenic protein-specific antibody-producing hybridomas, stable hybridomas are obtained. From each cell, the supernatant is collected, and the monoclonal antibody can be purified by affinity chromatography using a protein A Sepharose column. (Example 8) Another example of immunization using a mixture of peptidogenic proteins

[0165] In additional animal models, groups of five mice (C57BL / 6J; Jackson Labs) can be subcutaneously immunized with 5 μg of endotoxin-free peptide antigenic protein emulsified in alum, the adjuvant most commonly used in human vaccines. Three weeks later, the mice are bled, and the presence of peptide antigenic protein-specific antibodies can be determined by titrating the sera by ELISA (direct or indirect (via biotinylated tags and streptavidin) binding of antibodies in the sera to wild-type BPTI or APP-KI coating in wells). To confirm that the peptide antigenic protein has a similar conformation to the starting protein, a competitive inhibition assay is performed, in which the titrated amounts of the starting protein and peptide antigenic protein are pre-incubated with the sera before being added to the starting protein-coated plates. This provides additional evidence that the 3D structure of the peptide antigenic protein has not been impaired by the engineered mutations, with the immunological probe.

[0166] To determine whether the peptide antigenic protein elicits a better secondary antibody response, groups of mice can be immunized as described above, and six weeks after the primary immunization, they can be given a second immunization. One week after the secondary immunization, the mice are bled, and the antigen-specific antibody response is determined by ELISA as described above. Mouse dendritic cells are pulsed in vitro with the peptide antigenic protein capable of generating a strong antibody response, and 24 hours later, peptides derived from the peptide antigenic protein presented by MHCII are isolated and their masses analyzed by liquid chromatography and mass spectrometry (LC / MS). For these studies, a large number (>10 7) dendritic cells are required. To enable MHCII-peptide quantification by peak identification and mass spectrometry, the peptide antigenic protein can be biosynthetically labeled with stable isotopes, such as 13C and 15N (see above during the preparation of recombinant proteins), prior to feeding to the DCs (Hoedt et al. 2014). (Example 9) Immunity using an array encoding a mixture of peptide antigenic proteins

[0167] Methods of directly injecting polynucleotides into animals are well described in the art. See, for example, U.S. Patent Nos. 5,676,954; 6,875,748; 5,661,133. Briefly, using the known degeneracy of the genetic code, polynucleotides encoding the mixtures of peptide antigenic proteins described herein can be synthesized using standard DNA synthesis techniques. The polynucleotides can then be directly injected into animals, such as mice. Specifically, a mixture of polynucleotides encoding a mixture of peptide antigenic proteins can be injected into the quadriceps muscle of restrained, awake mice (female 6-12 weeks old BALB / c or Nude (nu / nu), Harlan Sprague Dawley, Indianapolis, Ind.). In one embodiment, a 50 μg polynucleotide in 50 μl solution is used and injected into the mice as described in Hartikka, J. et al., Hum. Gene Ther. 7:1205-1217 (1996), using a disposable sterile plastic insulin syringe and a 28G 1 / 2 needle (Becton-Dickinson, Franklin Lakes, N.J., catalog number 329430) fitted with a plastic collar cut from a micropipette tip.

[0168] Alternatively, 6-week-old Sprague Dawley female mice (weighing 20-25 grams) may be given 5000 ppm ZnOSO4 in their drinking water during the first 24 hours prior to injection. This amount of zinc has been shown to be able to activate the metallothionein promoter. Each mouse is then injected intravenously by tail vein puncture with a 25-gauge needle with a mixture of 30 μg of a polynucleotide encoding a peptidogenic protein complexed with 150 μg of liposome™ in a total volume of 30 μl. In one embodiment, the polynucleotide mixture injected into the mouse encodes different peptidogenic proteins related to the same initiating protein. Alternatively, a library of peptidogenic proteins may be encoded by a mixture of polynucleotides where the library is related to different initiating proteins. Animal care is maintained throughout the study and should be conducted in accordance with "Guide for the Use and Care of Laboratory Animals" (Institute of Laboratory Animal Resources, Commission on Life Sciences, National Research Council, National Academy Press).

[0169] After a polynucleotide encoding an injected, peptide - antigenic protein has been delivered to a cell of an animal, the peptide - antigenic protein is then expressed in vivo. The peptide - antigenic protein can then stimulate the production of antibodies specific to the peptide - antigenic protein. These antibodies can be isolated and used as a polyclonal mixture or further isolated into a single species or monoclonal. The process of the immune response and antibody production against a foreign antigen in vivo is well - known in the art. Different from the traditional protocols of antibody production, the platform invention described herein can simultaneously generate a group of antibodies against multiple peptide - antigenic proteins (regardless of whether they depend on the same starting protein). The simultaneous production of antibodies against multiple proteins using this mixture of polynucleotides has the potential to change the way antibody production is currently carried out. (Example 10) Immunization using mRNA encoding a peptide - antigenic protein

[0170] Methods of directly injecting in vitro - transcribed (IVT) mRNA into animals are well - known in the art. See Sahin et al., Nat Rev Drug Discov. October 2014; 13(10):759 - 80; Kariko et al., Mol Ther, November 2008; 16(11):1833 - 40; Kariko et al., Nucleic Acid Res, November 2011; 39(21):e142; U.S. Patent No. 6,511,832. For example, a linearized DNA plasmid template encoding a mixture of peptide - antigenic proteins can be used. All mRNAs can be designed to contain 5' and 3' untranslated regions, open reading frames, and long poly(A) tails that can assist in determining the translational activity and stability of the mRNA molecules after transfer to cells.

[0171] For example, mRNA (including a poly(A) tail) encoding a peptide antigenic protein can be synthesized using pseudouridine triphosphate derivatives and 5-methylcytidine (m5C) (TriLink) to generate modified nucleoside-containing RNA. A 5’ cap can be added to the mRNA by supplementing the transcription reaction with 6 mmol / l of 3’-O-Me-m7GpppG, an irreversible cap analog (New England Biolabs, Beverly, MA), and reducing the concentration of guanosine triphosphate (3.75 mmol / l). Purification of the transcript can be carried out by Turbo DNase (Ambion, Austin, TX) digestion, followed by LiCl precipitation and 75% ethanol washing. The concentration of RNA reconstituted in water can be determined by measuring the optical density at 260 nm. Efficient incorporation of modified nucleotides into the transcript can be determined by HPLC analysis. All RNA samples can be analyzed by denaturing agarose gel electrophoresis for quality assurance. Next, lipofectin (Invitrogen, Carlsbad, CA) and mRNA are complexed in phosphate buffer to enhance transfection. To assemble 50 μl of the RNA-lipofectin complex, first 0.4 μl of potassium phosphate buffer (0.4 mol / l, pH 6.2) containing 10 μg / μl bovine serum albumin (Sigma, St. Louis, MO) is added to 6.7 μl of DMEM, then 0.8 μl of lipofectin is mixed in, and the sample is incubated for 10 minutes. In a separate tube, 0.25 - 3 μg of RNA is added to DMEM to a final volume of 3.3 μl. The diluted RNA is added to the lipofectin mix and incubated for 10 minutes. Finally, the RNA-lipofectin complex is further diluted by adding 38.8 μl of DMEM.

[0172] RNA encoding a peptidic antigenic protein can then be injected into the mouse model described herein. Generally, a composition containing 1 μl of lipofectin and polynucleotides encoding different amounts of peptidic antigenic protein in a final volume of 60 μl is injected into the lateral vein using a 1 ml syringe with a 27G1 / 2 needle (Becton Dickinson, San Diego, CA). Alternatively, the polynucleotides can be injected via intramuscular, intradermal, intranasal, subcutaneous, intravenous, intratracheal, and intrathecal delivery. After the polynucleotides are delivered to the cells, the peptidic antigenic protein is synthesized in vivo. The immune response elicited by the peptidic antigenic protein and the subsequent production of antibodies in the animals are described herein. (Example 11) Affinity matured antibodies against peptidic antigenic proteins using phage display

[0173] Once an antibody has been generated against a peptidic antigenic protein by presenting the peptidic antigenic protein and subjecting it to processing by antigen-presenting cells as described, for example, in the examples herein, the generated antibody can be matured using a display approach. For example, a library of phages displaying scFv or Fab derived from B cell mRNA encoding a target-specific antibody can be screened in an assay to identify phages displaying scFv or Fab that immunospecifically bind to the peptidic antigenic protein or to the initiating protein. Phages displaying scFv or Fab that bind to the immobilized peptidic antigenic protein or initiating protein can be identified after panning on the immobilized peptidic antigenic protein or initiating protein and ELISA assessment for binding to the immobilized peptidic antigenic protein or initiating protein. The peptidic antigenic protein or initiating protein immobilized on plates for these assays can be purified from the supernatant of Sf9 cells infected with a baculovirus expression construct or from the supernatant of HEK293 cells as described by Moore et al., Science 285:260-263. Each of the identified scFv or Fab can then be sequenced.

[0174] To determine the specificity of each unique scFv or Fab, phage ELISA can be performed for each scFv or Fab against a peptide antigenic protein or starting protein and control wells. Individual E. coli colonies containing a phagemid presenting one of the unique scFvs or Fabs can be inoculated into a 96-well plate containing 100 μl of 2TYAG medium per well. The plate is incubated with shaking at 37 °C for 4 h. M13K07 helper phage is then added to each well up to an MOI of 10, and the plate is incubated for a further 1 h at 37 °C. The plate is centrifuged in a tabletop centrifuge at 2000 rpm for 10 min. The supernatant is removed, and the cell pellet is resuspended in 100 μl of 2TYAK and incubated with shaking overnight at 30 °C.

[0175] The next day, the plate is centrifuged at 2000 rpm for 10 min, and 100 μl of the phage-containing supernatant from each well is carefully transferred to a fresh 96-well plate. 20 μl of 6xMPBS is added to each well and incubated for 1 h at room temperature to pre-block the phage before ELISA.

[0176] The flexible 96-well plate (Falcon) is coated overnight at 4°C with peptidic protein in PBS (either directly or indirectly at 1 mg / ml), BSA in PBS (1 g / ml), or PBS. After coating, the solution is removed from the wells and the plate is blocked in MPBS for 1 hour at room temperature. The plate is washed three times with PBS and then 50 μl of pre-blocked phage is added to each well. The plate is incubated for 1 hour at room temperature and then washed by replacing PBST three times followed by replacing PBS three times. To each well, 50 μl of anti-gene VIII-HRP conjugate (Pharmacia) diluted 1 to 5000 in MPBS is added and the plate is incubated for 1 hour at room temperature. Each plate is washed three times with PBST followed by three times with PBS. Next, 50 μl of HRP-labeled anti-mouse antibody (DAKO EnVision) diluted 1 / 50 in 3% MPBS is added and incubated for 1 hour at room temperature. Each plate is then washed three times with PBST followed by three times with PBS. 50 μl of TMB substrate is then added to each well and incubated at room temperature for 30 minutes or until color develops. The reaction is stopped by the addition of 25 μl of 0.5 M H2SO4. The generated signal is measured by reading the absorbance at 450 nm (A450) using a microtiter plate reader (Bio-Rad 3550).

[0177] The conversion of scFv or Fab to the IgG1 form can be carried out as follows. The VH and VL domains of the scFv or Fab that the inventors desire to convert to an IgG molecule can be cloned into a vector containing the nucleotide sequence of an appropriate heavy (human IgG1, IgG2, etc.) or light chain (human kappa or human lambda) constant region such that the complete heavy or light chain molecule can be expressed from these vectors when transfected into an appropriate host cell. Further, when the cloned heavy and light chains are expressed in one cell line (from either one or two vectors), they can be assembled into a complete functional antibody molecule that is secreted into the cell culture medium. Methods for converting scFv or Fab to conventional antibody molecules are well known in the art.

[0178] The purification of IgG from the fermentation broth is carried out using a combination of conventional techniques commonly used for antibody production. Typically, the culture harvest is clarified to remove cells and cell debris prior to initiating the purification scheme. This is usually accomplished using either centrifugation or filtration of the harvest. After clarification, the antibody is typically captured and significantly purified using affinity chromatography on protein A sepharose. The antibody is bound to the protein A sepharose at basic pH and eluted by reduction of the pH after washing of the matrix. Further purification of the antibody is then achieved by gel filtration. This step can also be used to buffer exchange into the desired final formulation buffer, as well as to remove components having different molecular weights than the antibody. (Example 12) Assays used to measure the characterization and cross-reactivity of the antibody

[0179] Antibodies (whether cross - reactive with or generating antibodies against the peptidic protein) (including scFv or Fab and other molecules comprising antibody fragments or variants thereof, or other molecules consisting of scFv or Fab and antibody fragments or variants thereof) can be screened in various assays, some of which are described below for identifying antibodies that bind to the peptidic protein and / or the starting protein.

[0180] In one particular assay, antibodies (which cross-react with or give rise to antibodies against a peptidic protein) that bind to a biotinylated protein (peptidic protein and / or initiating protein) can be captured on streptavidin-coated magnetic beads. This assay can be applied to identify antibodies (which cross-react with or give rise to antibodies against a peptidic protein) that neutralize and / or bind to a peptidic protein and / or initiating protein. Additionally, the antibodies can be assayed in a neutralization assay as described herein or otherwise known in the art. For example, if the target protein is BlyS, the antibodies can be tested for their ability to inhibit the peptidic protein and / or initiating protein from binding to IM9 cells. In this assay, a labeled peptidic protein and / or initiating protein (e.g., biotinylated) is incubated with the antibody to form a protein-antibody complex. After incubation, an aliquot of the protein-antibody sample is added to IM9 cells. Binding can be determined using techniques known in the art. For example, the binding of the biotinylated protein (peptidic protein and / or initiating protein) to IM9 cells can be detected using a fluorometer after the addition of streptavidin-delphia. If the protein is not bound by an antibody that neutralizes the protein, the biotinylated protein can bind to the cells and be detected. Thus, an antibody that reduces the amount of biotinylated protein that binds to IM9 cells (compared to a control sample in which the protein is pre-incubated with an irrelevant antibody or no antibody at all) is identified as one that binds to and neutralizes the protein.

[0181] Other immunoassays that can be used to analyze cross-reactivity and / or characterize antibodies raised against a peptidic antigen include, but are not limited to, competitive and non-competitive assay systems that utilize techniques such as Western blot, radioimmunoassay, ELISA (enzyme-linked immunosorbent assay), "sandwich" immunoassay, immunoprecipitation assay, precipitation reaction, gel diffusion precipitation reaction, immunodiffusion assay, agglutination assay, complement fixation assay, immunoradiometric assay, fluorescence immunoassay, and protein A immunoassay. Such assays are routine and well known in the art (see, e.g., Ausubel et al., eds., 1994, Current Protocols in Molecular Biology, Vol. 1, John Wiley & Sons, Inc., New York).

[0182] Exemplary immunoassays are briefly described below (however, these are not intended for purposes of limitation). Immunoprecipitation protocols generally involve lysing cells in a lysis buffer such as RIPA buffer (1% NP-40 or Triton X-100, 1% sodium deoxycholate, 0.1% SDS, 0.15 M NaCl, 0.01 M sodium phosphate, pH 7.2, 1% Trasylol) supplemented with a protein phosphatase and / or protease inhibitor (e.g., EDTA, PMSF, aprotinin, sodium vanadate), adding an antibody of interest that cross-reacts to the cell lysate, incubating for a period (e.g., 1 to 4 hours) at 4° C., adding protein A and / or protein G sepharose beads to the cell lysate, incubating for about 1 hour or more at 4° C., washing the beads in lysis buffer and resuspending the beads in SDS / sample buffer. The ability of an antibody to immunoprecipitate a peptidogenic protein and / or an initiating protein can be assessed, for example, by Western blot analysis or mass spectrometry. One of ordinary skill in the art will be familiar with parameters that can be modified to increase antibody binding to a peptidogenic protein and / or an initiating protein and to decrease background (e.g., preclearing the cell lysate with sepharose beads). For further considerations regarding immunoprecipitation protocols, see, for example, Ausubel et al., eds., 1994, Current Protocols in Molecular Biology, Volume 1, John Wiley & Sons, Inc., New York at 10.16.1.

[0183] Western blot analysis generally involves preparing a protein sample, electrophoresing the protein sample in a polyacrylamide gel (e.g., 8%-20% SDS-PAGE depending on the molecular weight of the antigen), transferring the protein sample from the polyacrylamide gel to a membrane such as nitrocellulose, PVDF, or nylon, blocking the membrane in a blocking solution (e.g., PBS with 3% BSA or fat-free milk), washing the membrane in a wash buffer (e.g., PBS-Tween20), blocking the membrane with a primary antibody (the antibody of interest) diluted in the blocking buffer, washing the membrane in the wash buffer, blocking the membrane with a secondary antibody (which recognizes the primary antibody, e.g., an anti-human antibody) conjugated to an enzyme substrate (e.g., horseradish peroxidase or alkaline phosphatase) or a radioactive molecule (e.g., 32P or 1211) diluted in the blocking buffer, washing the membrane in the wash buffer, and detecting the presence of the antigen. One skilled in the art will be familiar with the parameters that can be modified to increase the detected signal and reduce background noise. For further considerations regarding Western blot protocols, see, for example, Ausubel et al., eds., 1994, Current Protocols in Molecular Biology, Volume 1, John Wiley & Sons, Inc., New York at 10.8.1.

[0184] In a further example, ELISA comprises preparing a peptidic protein and / or an initiating protein, coating the wells of a 96-well microtiter plate (either directly or indirectly) with the peptidic protein and / or the initiating protein, washing away peptidic protein and / or initiating protein that does not bind to the wells, adding to the wells an antibody of interest conjugated to a detectable compound such as an enzyme substrate (e.g., horseradish peroxidase or alkaline phosphatase), incubating for a period of time, washing away unbound or nonspecifically bound antibody, and detecting the presence of antibody-specific binding to the peptidic protein and / or initiating protein coating the wells. In ELISA, the antibody of interest is not necessarily conjugated to a detectable compound; instead, a secondary antibody (which recognizes the antibody of interest) conjugated to a detectable compound can be added to the wells.

[0185] Furthermore, instead of coating the wells with an antigen, the wells can be coated with an antibody. In this case, the detectable molecule can be a peptidic protein and / or an initiating protein conjugated to a detectable compound, such as an enzyme substrate (e.g., horseradish peroxidase or alkaline phosphatase). One of ordinary skill in the art will be familiar with the parameters that can be modified to increase the detected signal, as well as other variations of ELISA known in the art. For further considerations regarding ELISA, see, for example, Ausubel et al., eds., 1994, Current Protocols in Molecular Biology, Volume 1, John Wiley & See Sons, Inc., New York at 11.2.1. The binding affinity of an antibody for a peptidic protein and / or an initiating protein, as well as the off-rate of the antibody-protein interaction, can be determined by a competitive binding assay. One example of a competitive binding assay is a radioimmunoassay that involves incubating a labeled peptidic protein and / or an initiating protein (e.g., 3H or 125I) with the antibody of interest in the presence of increasing amounts of unlabeled peptidic protein and / or an initiating protein, and detecting the binding of the antibody to the labeled peptidic protein and / or an initiating protein. The affinity and binding off-rate of the antibodies of the invention for a peptidic protein and / or an initiating protein can be determined from data by Scatchard plot analysis. Competition with a secondary antibody can also be determined using a radioimmunoassay. In this case, the peptidic protein and / or an initiating protein is incubated with the antibody of interest conjugated to a labeled compound (e.g., 3H or 125I) in the presence of increasing amounts of unlabeled secondary anti-peptidic protein antibody.

[0186] In a preferred embodiment, BIAcore kinetic analysis can be used to determine the binding and off-rates in an antibody for a peptidic protein and / or an initiating protein. BIAcore kinetic analysis involves analyzing the binding and dissociation of a peptidic protein and / or an initiating protein from a chip having an antibody immobilized on its surface. (Example 13) Vaccination

[0187] Furthermore, as described herein, the mixture of peptide - antigenic proteins can be used as a vaccine. For example, a concentration of 320 μg / mL of the peptide - antigenic protein in phosphate - buffered saline (PBS, 155 mM NaCl, 1 mM KH2PO4, 3 mM Na2HPO3) is aseptically emulsified with an equal volume of Montanide ISA 51 to obtain a final vaccine concentration of 160 μg / mL. The emulsion is achieved by homogenizing a 200 - mL volume mixture in a 400 - mL container at 6000 rpm for 6 minutes at room temperature using an Omni Mixer - ES homogenizer (Omni International, Warrenton, VA). Each vaccine undergoes comprehensive quality control analysis to ensure general safety, purity, identity, integrity, and a uniform water - in - oil droplet size. The stability of the vaccine stored at 2 - 8 °C is periodically evaluated using mouse immunogenicity tests as well as physical and biochemical assays to verify vaccine safety, potency, uniformity, purity, and integrity up to 4 - 10 months after the end of human immunization. The 160 μg / mL peptide - antigenic protein vaccine is diluted with PBS / ISA51 (adjuvant control vaccine) to 10 μg / mL or 40 μg / mL in the final dosage form before immunization. As a result of different degrees of dilution, these vaccines contain two different ratios of vaccine - containing to vaccine - free water droplets, namely 10 μg / mL and 40 μg / mL formulations with ratios of 1:15 and 1:3 respectively. The test and control vaccines are highly viscous and may require vortexing before and after manipulation to ensure uniformity. The vaccine can be administered intramuscularly by needle and syringe. The vaccine - induced T - cell response is further evaluated by means of a qualified intracellular cytokine staining assay. Peripheral blood mononuclear cells are quantified to determine the ratios of total and memory CD4 and CD8 T cells that produce interleukin - 2, interferon - γ, or tumor necrosis factor (TNF).

[0188] A polynucleotide encoding a mixture of peptide antigenic proteins can also be used as a vaccine by administering the polynucleotide described herein to a patient. [Cited Document]

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Claims

[Claim 1] The method or antibody described in the specification.

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