WO2019074934A1 - Immunogènes pour réponse immunitaire dirigée et anticorps issus de ceux-ci - Google Patents

Immunogènes pour réponse immunitaire dirigée et anticorps issus de ceux-ci Download PDF

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Publication number
WO2019074934A1
WO2019074934A1 PCT/US2018/055027 US2018055027W WO2019074934A1 WO 2019074934 A1 WO2019074934 A1 WO 2019074934A1 US 2018055027 W US2018055027 W US 2018055027W WO 2019074934 A1 WO2019074934 A1 WO 2019074934A1
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Prior art keywords
target protein
antibody
immunogen
bound
existing antibodies
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PCT/US2018/055027
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English (en)
Inventor
Mohan Srinivasan
Jon MELNICK
Matthew Greving
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Healthtell Inc.
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Publication of WO2019074934A1 publication Critical patent/WO2019074934A1/fr

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6854—Immunoglobulins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/0005—Vertebrate antigens
    • A61K39/0011—Cancer antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/0005—Vertebrate antigens
    • A61K39/0011—Cancer antigens
    • A61K39/001102—Receptors, cell surface antigens or cell surface determinants
    • A61K39/001116—Receptors for cytokines
    • A61K39/001119—Receptors for interleukins [IL]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/0005—Vertebrate antigens
    • A61K39/0011—Cancer antigens
    • A61K39/001102—Receptors, cell surface antigens or cell surface determinants
    • A61K39/001116—Receptors for cytokines
    • A61K39/001121—Receptors for chemokines
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/0005—Vertebrate antigens
    • A61K39/0011—Cancer antigens
    • A61K39/001102—Receptors, cell surface antigens or cell surface determinants
    • A61K39/001129—Molecules with a "CD" designation not provided for elsewhere
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/39—Medicinal preparations containing antigens or antibodies characterised by the immunostimulating additives, e.g. chemical adjuvants
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/24—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against cytokines, lymphokines or interferons
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C07K16/2818—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against CD28 or CD152
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2866—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against receptors for cytokines, lymphokines, interferons
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6803—General methods of protein analysis not limited to specific proteins or families of proteins
    • G01N33/6845—Methods of identifying protein-protein interactions in protein mixtures
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6878—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids in eptitope analysis
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/33—Crossreactivity, e.g. for species or epitope, or lack of said crossreactivity
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/34—Identification of a linear epitope shorter than 20 amino acid residues or of a conformational epitope defined by amino acid residues
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding

Definitions

  • Antibodies and monoclonal antibodies for therapeutic and research use are generally created by immunizing a mammal, such as a mouse, with an immunogen which comprises a target protein or a portion of a target protein. Often, the resulting antibodies bind only to a few regions of the target leaving other regions where no antibodies are observed to bind. Needed are methods and compositions that provide novel immunogens that target portions of the target where no antibodies are observed to bind.
  • the disclosure provides a method of designing an immunogen to a target protein, the method comprising a) obtaining polypeptide sequence(s) on the target protein bound by existing antibodies; b) identifying at least one region of the target protein not bound by the existing antibodies; and c) selecting an immunogen polypeptide comprising an amino acid sequence of a region of the target protein not bound by the existing antibodies, thereby designing an immunogen to the target protein.
  • the method further comprises mapping regions of the target protein bound by the existing antibodies, wherein mapping the region of the target protein bound by the existing antibodies comprises detecting binding of the existing antibodies to a library of peptides.
  • the library of peptides comprises peptides having amino acid sequences identical or similar to a wildtype protein from one or more mammals, and the sequences can be about 75% identical or about 90% similar to the sequences of a wildtype protein from the one or more mammals.
  • the one or more mammals can be a human, a mouse, a hamster, or a monkey.
  • the monkey can be selected from a cynomolgus monkey, a macaque monkey, and a rhesus macaque monkey.
  • mapping the region of the target protein bound by the existing antibodies comprises detecting binding of the existing antibodies to a peptide array.
  • the peptide array can comprise a diverse set of peptides or a related set of peptides.
  • the related set of peptides can be 75 % identical or 90 % similar.
  • obtaining a polypeptide sequence on the target protein bound by the existing antibodies comprises identifying peptide sequences bound by the existing antibodies from a database of peptide sequences known to be bound by the existing antibodies.
  • the method further comprises (i) identifying amino acids that reside on the surface of the target protein when natively folded; and (ii) selecting the immunogen polypeptide from the amino acids that reside on the surface of the target protein when natively folded.
  • (i) can comprise obtaining a structure of the target protein when natively folded or (i) can comprise obtaining a predicted structure of the target protein when natively folded from an algorithm, where the algorithm may predict the folded structure of the target protein.
  • the immunogen polypeptide comprises at least one linker.
  • the linker can comprise an amino acid sequence having one or more glycine residues and one or more serine residues, and the linker may have an amino acid sequence selected from at least one of GSG, (GGGGS)n, (GSG)n , GGGSGGGGS, GGGGSGGGS, (PGSG) n , PGSGSG where n is an integer between 1 and 10.
  • the method further comprises determining cross-species homology of the region not bound by the existing antibodies.
  • the immunogen comprises an amino acid sequence having at least 75% identity to a wildtype protein from one or more mammals.
  • the immunogen polypeptide comprises at least two amino acid sequences from two distinct regions of the target protein not bound by the existing antibodies.
  • the amino acid sequence of the region of the target protein comprises at least a first domain and a second domain.
  • the first domain and the second domain are operatively connected in a non-native order.
  • the target protein is a therapeutic target protein.
  • the disclosure provides an immunogen comprising a region of a target protein not bound by existing antibodies, comprising: a) obtaining polypeptide sequence(s) on the target protein bound by the existing antibodies; b) identifying at least one region of the target protein not bound by the existing antibodies; and c) selecting at least one immunogen comprising an amino acid sequence of the region of the target protein not bound by the existing antibodies.
  • it further comprises mapping a region of the target protein bound by the existing antibodies. Mapping the region of the target protein bound by the existing antibodies can comprise detecting binding of the existing antibodies to a library of peptides.
  • the library of peptides can comprise peptides having amino acid sequences identical or similar to a wildtype protein from one or more mammals.
  • the immunogen comprises at least one region having an amino acid sequence common to one or more mammals are selected from human, mouse, hamster, and monkey.
  • the monkey can be selected from a cynomolgus monkey, a macaque monkey, and a rhesus macaque monkey.
  • Mapping the region of the target protein bound by the existing antibodies can comprise detecting binding of the existing antibodies to a peptide array.
  • the peptide array can comprise a diverse set of peptides or a related set of peptides.
  • the related set of peptides can be at least 75%, at least 80%, at least 85%, at least 95%, or at least 99% identical.
  • Obtaining a polypeptide sequence on the target protein bound by the existing antibodies can comprise identifying peptide sequences bound by the existing antibodies from a database of peptide sequences known to be bound by the existing antibodies.
  • the method further comprises (i) identifying amino acids that reside on the surface of the target protein when natively folded; and (ii) selecting the immunogen polypeptide amino acids that reside on the surface of the target protein when natively folded.
  • (i) comprises obtaining a structure of the target protein when natively folded.
  • the structure of the natively- folded target protein or folded structure of the target protein can be predicted from an algorithm.
  • the immunogen polypeptide comprises at least 2 amino acids or at least 30 amino acids.
  • the immunogen polypeptide comprises at least one linker.
  • the linker can comprise an amino acid sequence having one or more glycine residues, one or more serine residues, or one or more proline residues.
  • the linker can have an amino acid sequence selected from at least one of GSG, (GGGGS)n, (GSG)n, GGGSGGGGS, GGGGSGGGS, (PGSG) n , PGSGSG where n is an integer between 1 and 10.
  • the method further comprises determining cross-species homology of the region not bound by the existing antibodies.
  • the immunogen comprises an amino acid sequence having at least 95% identity to a wildtype protein from one or more mammals.
  • the immunogen polypeptide can comprise at least two amino acid sequences from two distinct regions of the target protein not bound by the existing antibodies.
  • the at least two amino acid sequences do not correspond to the sequential positions in the native protein.
  • the amino acid sequence of the region of the target protein comprises at least a first domain and a second domain.
  • the first domain and the second domain are operatively connected in a non-native order.
  • the target protein is a therapeutic target protein.
  • the disclosure provides a method of creating an antibody to a region of a target protein not bound by existing antibodies, the method comprising: a) obtaining polypeptide sequence(s) on the target protein bound by existing antibodies; b) identifying a region on the target protein not bound by the existing antibodies; c) preparing at least one immunogen polypeptide comprising an amino acid sequence of the region of the target protein not bound by the existing antibodies; d) immunizing a mammal with the immunogen polypeptide; and e) obtaining from the mammal an antibody that specifically binds to the region identified in step b), thereby creating the antibody.
  • the method further comprises mapping of the target protein region(s) bound by the existing antibodies.
  • mapping the region of the target protein bound by the existing antibodies comprises detecting binding of the existing antibodies to a library of peptides.
  • the library of peptides can comprise peptides having amino acid sequences identical or similar to a wildtype protein from one or more mammals.
  • the one or more mammals can be selected from human, mouse, hamster, and monkey.
  • the monkey can be selected from a cynomolgus monkey, a macaque monkey, and a rhesus macaque monkey.
  • Mapping the region of the target protein bound by the existing antibodies can comprise detecting binding of the existing antibodies to a peptide array.
  • Obtaining a sequence on the target protein bound by the existing antibodies can comprise identifying peptide sequences bound by the existing antibodies from a database of peptide sequences known to be bound by the existing antibodies.
  • the method further comprises: (i) identifying amino acids that reside on the surface of the target protein when natively folded; and (ii) selecting the immunogen polypeptide from the amino acids that reside on the surface of the target protein when natively folded, (i) may comprise obtaining a structure of the target protein when natively folded. Alternatively, (i) may comprise obtaining a predicted structure of the target protein when natively folded from an algorithm.
  • Immunizing the mammal with the immunogen polypeptide can comprise administering at least one dose of a vaccine composition comprising the immunogen polypeptide and an adjuvant.
  • at least one dose comprises the target protein.
  • at least one dose does not comprise the target protein.
  • Creating the antibody from the mammal may comprise isolating a B cell which expresses the antibody.
  • Some embodiments further comprise fusing the B cell with a myeloma cell to create a hybridoma which expresses the antibody.
  • the method further comprises determining an epitope for the antibody. The method may comprise binding the antibody to a focused array.
  • the focused array can comprise a library of peptides having an amino acid sequence at least 100% identical to at least a portion of the immunogen polypeptide.
  • determining the epitope for the antibody can comprise measuring binding of the Ab to a library of peptides.
  • determining the epitope for the antibody can comprise identifying the peptides bound by the Ab measuring binding of the antibody to a peptide array.
  • antibodies binding to two or more distinct epitopes are discarded.
  • the method further comprises determining a biological effect for the antibody.
  • the biological effect for the antibody can comprise at least one of inhibiting an activity of the target protein, increasing an activity of the target protein, inhibiting binding of the target protein to a binding partner, stabilizing binding of the target protein to a binding partner, increasing half-life of the target protein, or decreasing half-life of the target protein.
  • the target protein can be at least one of PD-1, PD-L1, CD25, IL2, MIF or CXCR4.
  • the immunogen polypeptide can comprise at least 2 amino acids, or at least 30 amino acids.
  • the immunogen polypeptide comprises at least one linker.
  • the linker comprises an amino acid sequence having one or more glycine residues, one or more serine residues, or one or more proline residues.
  • the linker can have an amino acid sequence selected from at least one of GSG, (GGGGS)n, (GSG)n, GGGSGGGGS, GGGGSGGGS, (PGSG) n , PGSGSG where n is an integer between 1 and 10. Some instances further comprise determining cross-species homology of the target protein region not bound by the existing antibodies.
  • the immunogen polypeptide can comprise at least two amino acid sequences from two distinct regions of the target protein not bound by the existing antibodies. An antibody can also be created with the aforementioned methods.
  • the amino acid sequence of the region of the target protein comprises at least a first domain and a second domain. In some cases, the first domain and the second domain are operatively connected in a non-native order.
  • the target protein is a therapeutic target protein.
  • the disclosure provides, a method of treating an individual in need thereof, comprising: a) obtaining polypeptide sequence(s) on a target protein bound by existing antibodies; b) identifying at least one region of the target protein not bound by the existing antibodies; c) preparing at least one immunogen polypeptide comprising an amino acid sequence of the region of the target protein not bound by the existing antibodies; d) immunizing a mammal with the immunogen polypeptide; e) obtaining from the mammal an antibody that specifically binds to the region identified in step b); and f) administering the antibody to the individual, thereby treating the individual in need thereof.
  • the process may further comprise mapping a region of the target protein bound by the existing antibodies.
  • Mapping the region of the target protein bound by the existing antibodies may comprise detecting binding of the existing antibodies to a library of peptides.
  • the library of peptides can comprise peptides having amino acid sequences identical or similar to a wildtype protein from one or more mammals.
  • the one or more mammals can be selected from human, mouse, hamster, and monkey.
  • the monkey can be selected from a cynomolgus monkey, a macaque monkey, and a rhesus macaque monkey.
  • Mapping the region of the target protein bound by the existing antibodies can comprise detecting binding of the existing antibodies to a peptide array.
  • Obtaining polypeptide sequence(s) on the target protein bound by the existing antibodies may comprise identifying peptide sequences bound by the existing antibodies from a database of peptide sequences known to be bound by the existing antibodies.
  • the process further comprises: (i) identifying amino acids that reside on the surface of the target protein when natively folded; and (ii) selecting the immunogen polypeptide from the amino acids that reside on the surface of the target protein when natively folded.
  • (i) comprises obtaining a structure of the target protein when natively folded.
  • (i) comprises obtaining a predicted structure of the target protein when natively folded from an algorithm. In some instances the algorithm predicts the folded structure of the target protein.
  • immunizing the mammal with the immunogen polypeptide comprises administering at least one dose of a vaccine composition comprising the immunogen polypeptide and an adjuvant.
  • at least one dose comprises the target protein.
  • at least one dose does not comprise the target protein.
  • Creating the antibody from the mammal can comprise isolating a B cell which expresses the antibody.
  • the method further comprises fusing the B cell with a myeloma cell to create a hybridoma which expresses the antibody.
  • the method further comprises determining an epitope for the antibody. Determining the epitope for the antibody can comprise measuring binding of the antibody to a library of peptides.
  • Determining the epitope for the antibody can comprise measuring binding of the antibody to a peptide array. In some instances, antibodies binding to two or more distinct epitopes are discarded. In other instances, the method further comprises determining a biological effect for the antibody.
  • the biological effect for the antibody can comprise at least one of inhibiting an activity the target protein, increasing an activity of the target protein, inhibiting binding of the target protein to a binding partner, stabilizing binding of the target protein to a binding partner, increasing half-life of the target protein, and decreasing half-life of the target protein.
  • the target protein can be at least one of PD-1 , PD-L1, CD25, IL2, MIF or CXCR4.
  • the immunogen polypeptide can comprise at least 2 amino acids or at least 30 amino acids.
  • the immunogen polypeptide can comprise at least one linker.
  • the linker can comprise an amino acid sequence having one or more glycine residues, one or more serine residues, or one or more proline residues.
  • the linker can have an amino acid sequence selected from at least one of GSG, (GGGGS)n, (GSG)n, GGGSGGGGS, and GGGGSGGGS, where n is an integer between 1 and 10.
  • the method can further comprise determining cross- species homology of the region not bound by the existing antibodies.
  • the immunogen polypeptide comprises at least two amino acid sequences from two distinct regions of the target protein not bound by the existing antibodies.
  • the amino acid sequence of the region of the target protein comprises at least a first domain and a second domain. In some cases, the first domain and the second domain are operatively connected in a non-native order.
  • the target protein is a therapeutic target protein.
  • the disclosure provides an antibody for use as a medicament, wherein the antibody is produced by: a) obtaining polypeptide sequence(s) on a target protein bound by existing antibodies; b) identifying at least one region of the target protein not bound by the existing antibodies; c) preparing at least one immunogen polypeptide comprising an amino acid sequence of the region of the target protein not bound by the existing antibodies; d) immunizing a mammal with the immunogen polypeptide; and e) obtaining from the mammal an antibody that specifically binds to the region identified in step b).
  • the process may further comprise mapping a region of the target protein bound by the existing antibodies.
  • Mapping the region of the target protein bound by the existing antibodies may comprise detecting binding of the existing antibodies to a library of peptides.
  • the library of peptides can comprise peptides having amino acid sequences identical or similar to a wildtype protein from one or more mammals.
  • the one or more mammals can be selected from human, mouse, hamster, and monkey.
  • the monkey can be selected from a cynomolgus monkey, a macaque monkey, and a rhesus macaque monkey.
  • Mapping the region of the target protein bound by the existing antibodies can comprise detecting binding of the existing antibodies to a peptide array.
  • Obtaining polypeptide sequence(s) on the target protein bound by the existing antibodies may comprise identifying peptide sequences bound by the existing antibodies from a database of peptide sequences known to be bound by the existing antibodies.
  • the process further comprises: (i) identifying amino acids that reside on the surface of the target protein when natively folded; and (ii) selecting the immunogen polypeptide from the amino acids that reside on the surface of the target protein when natively folded.
  • (i) comprises obtaining a structure of the target protein when natively folded.
  • (i) comprises obtaining a predicted structure of the target protein when natively folded from an algorithm. In some instances the algorithm predicts the folded structure of the target protein.
  • immunizing the mammal with the immunogen polypeptide comprises administering at least one dose of a vaccine composition comprising the immunogen polypeptide and an adjuvant.
  • at least one dose comprises the target protein.
  • at least one dose does not comprise the target protein.
  • Creating the antibody from the mammal can comprise isolating a B cell which expresses the antibody.
  • the method further comprises fusing the B cell with a myeloma cell to create a hybridoma which expresses the antibody.
  • the method further comprises determining an epitope for the antibody. Determining the epitope for the antibody can comprise measuring binding of the antibody to a library of peptides.
  • Determining the epitope for the antibody can comprise measuring binding of the antibody to a peptide array. In some instances, antibodies binding to two or more distinct epitopes are discarded. In other instances, the method further comprises determining a biological effect for the antibody.
  • the biological effect for the antibody can comprise at least one of inhibiting an activity the target protein, increasing an activity of the target protein, inhibiting binding of the target protein to a binding partner, stabilizing binding of the target protein to a binding partner, increasing half-life of the target protein, and decreasing half-life of the target protein.
  • the target protein can be at least one of PD-1, PD-L1, CD25, IL2, MIF or CXCR4.
  • the immunogen polypeptide can comprise at least 2 amino acids or at least 30 amino acids.
  • the immunogen polypeptide can comprise at least one linker.
  • the linker can comprise an amino acid sequence having one or more glycine residues, one or more serine residues, or one or more proline residues.
  • the linker can have an amino acid sequence selected from at least one of GSG, (GGGGS)n, (GSG)n, GGGSGGGGS, and GGGGSGGGS, where n is an integer between 1 and 10.
  • the method can further comprise determining cross- species homology of the region not bound by the existing antibodies.
  • the immunogen polypeptide comprises at least two amino acid sequences from two distinct regions of the target protein not bound by the existing antibodies.
  • the amino acid sequence of the region of the target protein comprises at least a first domain and a second domain.
  • the first domain and the second domain are operatively connected in a non-native order.
  • the target protein is a therapeutic target protein.
  • the disclosure provides the use of an antibody in the manufacture of a medicament, wherein the antibody is produced by: a) obtaining polypeptide sequence(s) on a target protein bound by existing antibodies; b) identifying at least one region of the target protein not bound by the existing antibodies; c) preparing at least one immunogen polypeptide comprising an amino acid sequence of the region of the target protein not bound by the existing antibodies; d) immunizing a mammal with the immunogen polypeptide; and e) obtaining from the mammal an antibody that specifically binds to the region identified in step b).
  • the process may further comprise mapping a region of the target protein bound by the existing antibodies.
  • Mapping the region of the target protein bound by the existing antibodies may comprise detecting binding of the existing antibodies to a library of peptides.
  • the library of peptides can comprise peptides having amino acid sequences identical or similar to a wildtype protein from one or more mammals.
  • the one or more mammals can be selected from human, mouse, hamster, and monkey.
  • the monkey can be selected from a cynomolgus monkey, a macaque monkey, and a rhesus macaque monkey.
  • Mapping the region of the target protein bound by the existing antibodies can comprise detecting binding of the existing antibodies to a peptide array.
  • Obtaining polypeptide sequence(s) on the target protein bound by the existing antibodies may comprise identifying peptide sequences bound by the existing antibodies from a database of peptide sequences known to be bound by the existing antibodies.
  • the process further comprises: (i) identifying amino acids that reside on the surface of the target protein when natively folded; and (ii) selecting the immunogen polypeptide from the amino acids that reside on the surface of the target protein when natively folded.
  • (i) comprises obtaining a structure of the target protein when natively folded.
  • (i) comprises obtaining a predicted structure of the target protein when natively folded from an algorithm. In some instances the algorithm predicts the folded structure of the target protein.
  • immunizing the mammal with the immunogen polypeptide comprises administering at least one dose of a vaccine composition comprising the immunogen polypeptide and an adjuvant.
  • at least one dose comprises the target protein.
  • at least one dose does not comprise the target protein.
  • Creating the antibody from the mammal can comprise isolating a B cell which expresses the antibody.
  • the method further comprises fusing the B cell with a myeloma cell to create a hybridoma which expresses the antibody.
  • the method further comprises determining an epitope for the antibody. Determining the epitope for the antibody can comprise measuring binding of the antibody to a library of peptides.
  • Determining the epitope for the antibody can comprise measuring binding of the antibody to a peptide array. In some instances, antibodies binding to two or more distinct epitopes are discarded. In other instances, the method further comprises determining a biological effect for the antibody.
  • the biological effect for the antibody can comprise at least one of inhibiting an activity the target protein, increasing an activity of the target protein, inhibiting binding of the target protein to a binding partner, stabilizing binding of the target protein to a binding partner, increasing half-life of the target protein, and decreasing half-life of the target protein.
  • the target protein can be at least one of PD-1, PD-L1, CD25, IL2, MIF or CXCR4.
  • the immunogen polypeptide can comprise at least 2 amino acids or at least 30 amino acids.
  • the immunogen polypeptide can comprise at least one linker.
  • the linker can comprise an amino acid sequence having one or more glycine residues, one or more serine residues, or one or more proline residues.
  • the linker can have an amino acid sequence selected from at least one of GSG, (GGGGS)n, (GSG)n, GGGSGGGGS, and GGGGSGGGS, where n is an integer between 1 and 10.
  • the method can further comprise determining cross- species homology of the region not bound by the existing antibodies.
  • the immunogen polypeptide comprises at least two amino acid sequences from two distinct regions of the target protein not bound by the existing antibodies.
  • the amino acid sequence of the region of the target protein comprises at least a first domain and a second domain. In some cases, the first domain and the second domain are operatively connected in a non-native order.
  • the target protein is a therapeutic target protein.
  • the immunogens are also designed to include residues (for example, in some embodiments charged amino acids such as histidine) that assume different protonated states when in an acidic environment, such as a tumor micro environment.
  • residues for example, in some embodiments charged amino acids such as histidine
  • the CD25 immunogens disclosed herein are designed to represent conformational and configurational domains of CD25 that will undergo protonation under acidic environment. Antibodies derived using such immunogen constructs are likely to recognize the native form of the immunogen differentially in a neutral and acidic environment.
  • T regulatory cells By mediating the pH of the designed immunogens presented herein and thereby the environment to which antibodies recognize the immunogen, T regulatory cells (Tregs), can be selectively targeted in the periphery (for example, normal tissue), or selectively targeted in a tumor micro environment, for example CD25 antigens that are found on the surface of cells in the periphery or in tumor cells.
  • these immunogens could be used to make antibodies that can recognize the antigen target selectively in a neutral pH environment, such as in the periphery, or in an acidic pH environment, such as a tumor micro environment.
  • Other amino acid properties may also be exploited to direct selective targeting by an antibody as disclosed herein, including structural conformation differences, charge, hydrophobicity or reactive oxygen species status.
  • selective targeting of regulatory T cells (Tregs) will help alleviate some of the severe toxicities associated with checkpoint immunotherapy.
  • the disclosure provides an immunogen comprising at least two domains of a target protein, the immunogen comprising a polypeptide sequence of a first domain of the target protein operatively connected to a polypeptide sequence of a second domain of the target protein in a non-native order.
  • the first domain of the target protein is operatively connected to the second domain of the target protein by a linker molecule.
  • the target protein is a therapeutic target protein.
  • FIG. 1 shows an alignment of mouse, human, and cynomolgus monkey sequences of PD-1.
  • the darker regions are the residues of the protein that interact with PD-L1.
  • the darker regions are residues of PD-1 that are conserved across human, cynomolgus monkey, and mouse.
  • the lighter highlighted regions are the epitope for the Opdivo® PD-1 therapeutic antibody.
  • FIG. 2 shows epitope mapping of Nivolumab on the V13 peptide array.
  • FIG. 3 shows a PD-1 immunogen sequence and the position of the residues on a three dimensional structure of PD-1.
  • FIG. 4 shows additional exemplary immunogens for PD-1.
  • FIG. 5 shows exemplary immunization protocols for creating an antibody using the novel immunogen.
  • FIG. 6 shows an exemplary immunogen sequence for CD25 comprising two histidines, a disulfide bond, and point mutations designed to minimize the likelihood of cross- reactivity with mouse and monkey antibodies.
  • FIG.7 shows an exemplary immunogen sequence
  • FIG. 8 shows the 3D structural model for an exemplary CD-25 immunogen sequence.
  • FIG. 9 shows an additional exemplary model for a CD-25 immunogen sequence.
  • amino acids 144-160 of CD-25 are targeted.
  • FIG. 10 shows an exemplary embodiment for an IL2 binding domain.
  • FIG. 11 illustrates the application of competition-free epitope binning assays on peptide arrays to classify anti-Her2 antibodies according to their consensus binding characteristics.
  • Use of peptide arrays enables the identification of high-resolution immunogen positioning and amino acid binding contributions by applying the methods and devices disclosed herein. This includes: 1) providing a quantitative metric for epitope coverage of the target protein; 2) providing putative epitope identification for each classification of "bin"; 3) indicating regions of the target that are not covered by selected clones, which allows for immunization with specific target regions to improve epitope coverage; and 4) providing position-level amino acid binding contribution, which is important for screening and identification of pan-species selectivity of clones.
  • FIG. 12 shows an alignment MIF-1 and MIF-2 (DDT) sequences, and an MIF-HT (hybrid) sequence of MIF-1 and MIF-2.
  • the bolded residues are surface exposed, and not part of the trimer interface.
  • the residues outlined in a solid box are the same between MIF-1 and MIF-2 (DDT), and residues that are gray indicate that the residues are different between MIF-1 and MIF-2 (DDT).
  • Residues highlighted and outlined in a dashed box are mutated to cysteine to lock the trimer mutated residue.
  • the single white stars in dark boxes above the sequences indicates a CD74 binding region; the double white stars in grey boxes indicates a CXCR4 binding region.
  • the large starred region towards the top of the figure indicates residue(s) of interest for monoclonal antibody development.
  • FIG. 13 A and FIG. 13B show an interspecies alignment of MIF-1 (FIG. 13A) and MIF2 (DDT; FIG. 13B).
  • the alignment includes mouse, human, cynomolgus monkey and rabbit sequences of MIF-1 and MIF-2 (DDT).
  • FIG. 14A and FIG. 14B shows an exemplary model of MIF-1 (FIG. 14A) and MIF-2 (FIG. 14B); with location of residues in common in red.
  • FIG. 15 depicts the structure of PD-1, denoting which surface residues interact with known antibodies Nivolumab (green) and Pembrolizumab (blue) and which surface residues were used to engineer a different immunogen (red). Some surface residues interact with both Nivolumab and Pembrolizumab (orange).
  • the engineered immunogen of Example 1 was designed based on the area that is known not to be bound by Nivolumab.
  • FIG. 16 is a chart summarizing the immunization schedule of five different cohorts of mice, as described in Example 14.
  • FIG. 17 is a graph depicting ELIS A data detecting binding of sera from selected mice in Cohorts 1, 2, and 4, and non-immunized mouse sera, to Peptide Immunogen 1, as described in Example 14.
  • FIG. 18 is a graph depicting ELISA data detecting binding of sera from the same mice to full-length human PD-1, as described in Example 14.
  • FIG. 19 is a graph of the flow cytometry results, evaluating the binding of PD-1 expressing CHO cells to sera from selected mice in Cohort 3 and Cohort 5, and sera from a mouse that was not immunized, as described in Example 14.
  • FIG. 20 is flow cytometry data evaluating antibody (serum) binding of each mouse cohort to PD-1 expressing CHO cells (graphs second from left) and control cells that did not express PD-1 (right-most graphs). The histograms with a solid outline are hybridoma libraries at 1/2 dilution, while the ones without solid outline indicate cells + secondary only.
  • FIG. 21A is a graph of the binding of sera from Cohorts 1, 2, and 4 to human PD-1, as evaluated by ELISA. The x-axis is the dilution factor of the sample. Cohort 1 is the top line. Cohort 2 and Cohort 4 are overlapping, with Cohort 2 the darker line above Cohort 4 after 1000 factor dilution.
  • FIG. 2 IB is a graph of the binding of the same cohorts with mouse PD-1, as evaluated by ELISA.
  • Cohort 1 is the top line.
  • Cohorts 2 and 4 are overlapping with Cohort 2 being the darker line at the bottom between 10 and 100 dilution.
  • FIG. 21C is a graph of the binding of the same cohorts with cyno PD-1, as evaluated by ELISA.
  • Cohort 1 is the top line.
  • Cohorts 2 and 4 are overlapping with Cohort 2 being the darker bottom line between 100 and 1000 dilution.
  • FIG. 21D is a summary of the fraction of serum antibodies binding to mouse vs. cyno PD-1, relative to the binding to human PD-1, in the immunized mice described in Example 14.
  • FIG. 22A is a bar graph of ELISA results evaluating the binding of supernatant from Cohort 2 hybridomas with human, mouse, and cyno PD-1. For each x-axis entry in each figure, human PD-1 binding is the left bar, cyno PD-1 binding is the middle bar, and mouse PD-1 binding is the right bar.
  • FIG. 22B is a bar graph of ELISA results evaluating the binding of supernatant from Cohort 3 hybridomas with human, mouse, and cyno PD-1. For each x-axis entry in each figure, human PD-1 binding is the left bar, cyno PD-1 binding is the middle bar, and mouse PD-1 binding is the right bar.
  • FIG. 22C is a bar graph of ELISA results evaluating the binding of supernatant from Cohort 4 hybridomas with human, mouse, and cyno PD-1. For each x-axis entry in each figure, human PD-1 binding is the left bar, cyno PD-1 binding is the middle bar, and mouse PD-1 binding is the right bar.
  • FIG. 22D is a bar graph of ELISA results evaluating the binding of supernatant from Cohort 5 hybridomas with human, mouse, and cyno PD-1. For each x-axis entry in each figure, human PD-1 binding is the left bar, cyno PD-1 binding is the middle bar, and mouse PD-1 binding is the right bar.
  • FIG. 23 is a bar graph of the binding of monoclonal antibodies (and polyclonal C3- Hl) derived from different mouse cohorts, to human, mouse, and cyno PD-1 at two different concentrations (10 nm and 0.1 nm) as evaluated by ELISA.
  • human ⁇ binding is the bar on the left, human 0.1 nm the middle bar, and cyno 10 nM binding the bar on the right.
  • the top line is cyno 0.1 nM binding, mouse 10 nM binding is the darkest line, and mouse 0.1 nM binding the bottom line.
  • FIG. 24 is a bar graph of a reporter assay evaluating blocking of the PD-1/PD-L1 interaction by monoclonal antibodies (and polyclonal C3-H1) derived from different mouse cohorts immunized with engineered immunogens, according to Example 14.
  • FIG. 25 is a graph demonstrating the blocking of PD-1/PD-L1 binding by the supernatant of monoclonal (M) and polyclonal (H) hybridoma derived from mice immunized with engineered immunogens, according to Example 14.
  • FIGS. 26A-26C are functional epitope maps of three different antibodies indicated by the arrows in FIG. 23 and FIG. 24.
  • FIG. 26A is the map for antibody C3H1 ;
  • FIG. 26B is for C4M3;
  • FIG. 26C is for C2M1.
  • the structures are heat-mapped to indicate epitope confidence, which is (true peptide array binding hit alignments )/(random alignments).
  • FIGS. 27A-27C are Spearman's rank correlations that were performed with the data evaluating cohort sera from different blood draw days (average across each cohort for each separate date), as described in Example 14.
  • FIG. 28 is a table summarizing the number of target binding hits produced in each mouse cohort immunized according to Example 14.
  • FIGS. 29A-29E are epitope maps of PD-1 that were produced using day 35 blood draw serum from the five different mice cohorts immunized according to Example 14.
  • the dark structure is PD-1
  • the ribbon is PD-L1
  • the mesh is the engineered immunogen (in structures on the right). In these maps, red is highest confidence, blue is lowest, and white/pink is in the middle.
  • FIG. 29 A is Cohort 1.
  • FIG. 29B is Cohort 2.
  • FIG. 29C is Cohort 3.
  • FIG. 29D is Cohort 4.
  • FIG. 29E is Cohort 5.
  • FIG. 30 is a sequence alignment of human/cyno PD-1 , and human/mouse PD-1. DETAILED DESCRIPTION
  • novel immunogens are novel immunogens, methods and compositions for designing such immunogens, and use of such immunogens to generate novel antibodies.
  • the immunogens can be from a known target protein related to, or involved in a disease process, a therapeutic target.
  • the applications include generating antibodies binding to the unexplored region of the target protein.
  • Other methods and compositions disclosed herein relate to the use of novel peptide array formats that characterize or enable targeting of the identified unexplored regions of a target protein to create custom immunogens for the purposes disclosed herein.
  • an immunogen such as an immunogen comprising a region of a target polypeptide (e.g. a therapeutic target) not bound by existing antibodies.
  • the method comprises: a) obtaining a sequence of the target polypeptide (e.g. therapeutic target polypeptide) bound by each of the existing antibodies; b) identifying a region of the target polypeptide not bound by each of the existing antibodies; and c) selecting an immunogen polypeptide comprising an amino acid sequence of the region of the target polypeptide not bound by the existing antibodies, thereby designing the immunogen.
  • the method comprises targeting and mutating select amino acids, for example histidines, in a designated immunogen sequence in order to select antibodies that could potentially recognize the antigen under various pH conditions, thereby targeting the antibody to recognize the target sequence only when the immunogen is expressed in the targeted pH environment.
  • select amino acids for example histidines
  • the CD25 immunogens disclosed herein are designed to represent conformational and configurational domains of CD25 that can undergo protonation under acidic environment.
  • Antibodies derived using such immunogen constructs are likely to differentially recognize the native form of the immunogen in a neutral or acidic environment. These immunogens could thus be used to make antibodies that can recognize the antigen target selectively in a neutral pH environment, such as in the periphery, or in an acidic pH environment, such as a tumor micro environment.
  • the methods and compositions described herein for generating immunogens for target polypeptides may include, for example, wherein the target polypeptide is a therapeutic target, or a target relevant for an assay, or a target relevant for a reagent, or another type of target.
  • the target polypeptide is a therapeutic target.
  • the target polypeptide is an assay target.
  • the target polypeptide is a reagent target.
  • the existing antibody(ies) is an antibody that is commercially available or an antibody that has been disclosed in a patent or non-patent publication.
  • the methods comprise mapping regions of a target protein bound by existing antibodies by detecting binding of the existing antibodies to a library of peptides.
  • the library of peptides may comprise peptides having amino acid sequences identical or similar to a wildtype protein from one or more mammals.
  • the library of peptides comprise sequences that are at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, or at least 99% identical to each other. In other cases the library of peptides comprise sequences that are at least 75% similar, at least 80% similar, at least 85% similar, at least 90% similar, at least 95% similar, or at least 99% similar to each other.
  • Wildtype mammalian proteins can be protein sequences that prevail among individuals in natural conditions, as distinct from an atypical mutant type.
  • mapping the region of the target protein bound by the existing antibodies comprises detecting binding of the existing antibodies to a library of peptides.
  • the library of peptides can comprise peptides having amino acid sequences identical or similar to a wildtype protein from one or more mammals.
  • the library of peptides comprise peptides having amino acid sequences that are at least 60% identical, at least 65% identical, at least 70% identical, at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, or at least 99% identical to the wildtype protein of from one or more mammals.
  • the library of peptides comprise peptides having amino acid sequences that are at most 60% identical, at most 65% identical, at most 70% identical, at most 75% identical, at most 80% identical, at most 85% identical, at most 90% identical, at most 95% identical, or at most 99% identical to the wildtype protein of from one or more mammals.
  • the library of peptides comprise peptides having amino acid sequences that are between 50% to 99% similar to one another, between 50% to 95% similar to one another, between 50% to 90% similar to one another, between 50% to 85% similar to one another, between 50% to 80% similar to one another, between 50% to 75% similar to one another, between 50% to 70% similar to one another, or between 50% to 65% similar to one another.
  • Mammals include humans, mice, rabbits, rats, dogs, hamsters, monkeys.
  • Monkeys include monkeys from the Catarrhine family (Cercopithecoidea superfamily) such as cynomolgus monkey, macaque monkey, and rhesus macaque monkey; monkeys from the Callitrichidae family; the Cebidae family; the Aotidae family; the Pitheciidae family; or the Atelidae family.
  • An apparent binding affinity to the at least one region of the target protein can be used to inform the binding, or a lack of binding, of the existing antibodies to a region of the target polypeptide.
  • a binding can be any interaction with an affinity ranging from the micromolar (10 ⁇ 6 M) range to the nanomolar (10 ⁇ 9 M) range or lower.
  • Orthogonal affinity measurements can also be used to further characterize a binding interaction, such as an enzyme-linked immunosorbent assay (ELISA), nuclear magnetic resonance (NMR), isothermal titration calorimetry (ITC), dynamic light scattering (DLS), surface plasmon resonance (SPR), dual polarisation interferometry (DPI), bi-layer interferometry (BLI), microscale thermophoresis (MST), or the like which can be used to assess whether the antibody is not bound effectively to at least one region of the target protein.
  • ELISA enzyme-linked immunosorbent assay
  • NMR nuclear magnetic resonance
  • ITC isothermal titration calorimetry
  • DLS dynamic light scattering
  • SPR surface plasmon resonance
  • DPI dual polarisation interferometry
  • BLI bi-layer interferometry
  • MST microscale thermophoresis
  • a computer database or computer model can be used to identify or predict a polypeptide sequence that is not bound by the existing antibodies, a sequence that is known to be bound, or a sequence that is predicted to be bound by the existing antibodies.
  • the computer database may contain data related to antibody epitopes for a variety of species, such as antibody epitopes for humans, non-human primates, rodents, and other animal species.
  • obtaining a polypeptide sequence on the target protein bound by the existing antibodies comprises identifying peptide sequences bound by the existing antibodies from a database of peptide sequences known to be bound by the existing antibodies.
  • the method can further comprise (i) identifying amino acids that reside on the surface of the target protein when natively folded; and (ii) selecting the immunogen polypeptide from the amino acids that reside on the surface of the target protein when natively folded.
  • (i) comprises obtaining a structure of the target protein when natively folded and in others
  • (i) comprises obtaining a predicted structure of the target protein when natively folded from an algorithm.
  • antigenic epitopes are classified as either continuous or discontinuous.
  • a continuous (also called linear) epitope is a consecutive fragment from a protein sequence, and a discontinuous epitope is composed of several fragments scattered along the protein sequence, which form the antigen-binding interface.
  • linear epitope is a consecutive fragment from a protein sequence
  • discontinuous epitope is composed of several fragments scattered along the protein sequence, which form the antigen-binding interface.
  • the majority of available epitope prediction methods focus on continuous epitopes due to the convenience of the investigation in which the amino acid sequence of a protein is taken as the input. Such prediction methods are based upon the amino acid properties including hydrophilicity, solvent accessibility, secondary structure, flexibility, and antigenicity.
  • databases exist that provide the identity of known linear epitopes for existing antibodies, including databases such as Bcipep, FIMM, and SVMTrip.
  • HMM Hidden Markov Model
  • ANN Artificial Neural Network
  • SVM Support Vector Machine
  • one or more of the algorithms described herein can be used to predict the folded structure of the target protein.
  • immunogens such as immunogens comprising a region of a target polypeptide (e.g. therapeutic target polypeptide) not bound by existing antibodies.
  • Immunogens herein are designed using one or more methods provided herein, such as a process comprising: a) obtaining a sequence of the target polypeptide bound by each of the existing antibodies; b) identifying a region of the target polypeptide not bound by each of the existing antibodies; and c) preparing at least one immunogen polypeptide comprising an amino acid sequence of the region of the target polypeptide not bound by the existing antibodies.
  • the target polypeptide is a therapeutic target polypeptide.
  • the immunogen can be designed to contain one or more segments or domains of the native target or protein antigen, wherein each segment could be of at least one amino acid in length.
  • the different segments that comprise the custom immunogen may be connected by linkers in an order that may or may not represent their corresponding order of sequence in the native target or protein antigen. Linking such different domains of the protein antigen provides uniqueness of the immunogen that could capture the 'unexplored' epitopes or some unique features of the protein antigen, such as pH sensitive domains or conformations.
  • Discrete domains of protein antigens are connected with linkers to be unique conformational and configurational representations of the native protein antigen.
  • a custom immunogen can comprise a re-ordered domain sequence of a protein.
  • a native protein that has four domains may be said to have a sequence with four native domains: "domain 1", "domain 2", “domain 3", and "domain 4.”
  • a custom immunogen derived from such protein can comprise one or more, two or more, three or more, or all of the
  • the non-native order of the aforementioned custom immunogen can be, for example: a) “domain 1", “domain 3", “domain 4", “domain 2"; b) “domain 4", “domain 3", “domain 2", “domain 1”; c) "domain 3", “domain 4", “domain 2", “domain 1”; d) "domain 4", "domain 3", “domain 2", “domain 1”; e) "domain 1", “domain 3", “domain 2", “domain 1”; or any other non-native suitable order.
  • a custom immunogen comprising an amino acid sequence of the region of the target protein comprising at least a first domain and a second domain in a non-native order. In some instances, these domains are linked in a custom immunogen by a linker.
  • a custom immunogen can comprise a re-ordered domain sequence of a protein comprising at least two domains of the native target protein. In some cases, the at least two domains (such as the first domain and the second domain) are operatively connected in a non-native order.
  • Operatively connected domains of an immunogen can include, for example, domains that are connected such that the immunogen can elicit an immune response in a mammal (for example, the connection itself does not prevent immune response).
  • the disclosure provides a method of creating an antibody to a region of a target protein not bound by existing antibodies, in some cases that target protein is PD-1.
  • PD-1 is an inhibitory receptor belonging to the CD28/CTLA4 family and is expressed on the surface of activated T lymphocytes, B cells, monocytes, DCs, Natural Killer (NK) cells, MDSCs (myeloid derived suppressor cells) and Tregs.
  • CTLA4 the major role of PD-1 is limitation of activity of T cells in peripheral tissues at the time of an inflammatory response to infection and to limit autoimmunity. Chronic antigen exposure can lead to persistently -high levels of PD-1 expression, which can induce a state of exhaustion or anergy of antigen-specific T-cells, which can be at least partially reversed by PD-1 blockade.
  • Two ligands for PD-1, PD-L1 and PD-L2 are expressed on T cells, APCs, and malignant cells, and function to suppress self-reactive lymphocytes and to inhibit the effector function of TAA-specific cytotoxic T lymphocytes (CTLs). Accordingly, a therapy that targets PD-1 and ligands thereof has the potential to restore the cytotoxic activity of TAA- specific T cells.
  • PD-1 can inhibit kinases involved in T-cell activation through the phosphatase, SHP2.
  • PD-1 can limit the activity of T-cells in peripheral tissues at the time of an inflammatory response to infection and limit autoimmunity. The decrease in the proliferation of T-cells can lead to a decrease in IL-2 secretion.
  • PD-1 can also be highly expressed on regulatory T-cells, which can have an immunosuppressive function, and further increase the proliferation of regulatory T-cells. Tumors can be highly infiltrated with regulatory T-cells; thus, blockade of PD-1 can diminish the immunosuppressive function of the intratumoral regulatory T-cells.
  • PD-1 can also enhance NK activity in tumors or tissues.
  • PD-1 can increase antibody production through PD-1 + B-cells.
  • Chronic antigen exposure observed in viral infection and cancer can lead to persistent PD-1 activation, and can induce a state of anergy among the cognate antigen-specific T-cells. This anergic state can be reversed through a blockade of PD-1.
  • PD-1 can also be expressed on tumor infiltrating lymphocytes (TILs) in many tumor types.
  • TILs tumor infiltrating lymphocytes
  • the enhanced PD-1 expression of CD4 + cells can reflect the high expression of PD-1 on regulatory T-cells within tumors.
  • PD-1 can also be highly expressed on CD8 + cells and can reflect an anergic state. Consistent with the increased expression of PD-1 on lymphocytes from many tumors, the ligands of PD-1 can also be highly expressed on the tumor cell surface.
  • PD-L1 can be highly expressed on, for example, melanoma, ovarian cancer, lung cancer, and renal cancer cells.
  • PD-L2 can be highly expressed on, for example, primary mediastinal B-cell lymphoma, follicular cell B-cell lymphoma, and Hodgkin's lymphoma.
  • Anti-PD-1 antibodies can induce regression of several tumor types including colon, renal, lung, and melanoma.
  • Therapies targeting PD-1 directly or the interaction between PD-1 and a ligand include MDX-1106, BMS-936558, MK3475, CT-01 1, and AMP-224.
  • the disclosure provides a method of creating an antibody to a region of a target protein not bound by existing antibodies, in some cases that target protein is PD-1.
  • the disclosure provides a method of creating an antibody to a region of a target protein not bound by existing antibodies, in some cases that target protein is CD-25.
  • CD-25 is a type I transmembrane protein present on activated T cells, activated B cells, some thymocytes, myeloid precursors, and oligodendrocytes. Reductions in the amount of CD-25 expressed on the surface of immune cells can be associated with infections by the protozoan Trypanosoma cruzi, and such reductions can lead to chronic immune suppression.
  • the disclosure provides a method for obtaining a polypeptide sequence on the target protein bound by one or more existing antibodies, in some instances the antibody is daclizumab and/or bacilixamab.
  • the disclosure provides a method of creating an antibody to a region of a target protein not bound by existing antibodies, in some cases that target protein is CXCR4.
  • CXCR-4 is an alpha-chemokine receptor specific for stromal-derived-factor-1 (SDF-1 also called CXCL12), a molecule with potent chemotactic activity for lymphocytes.
  • SDF-1 stromal-derived-factor-1
  • Various molecules can function as ligands for CXCR4, including, for example, the macrophage migration inhibitory factor (MIF or MMIF).
  • MIF macrophage migration inhibitory factor
  • the disclosure provides a method of creating an antibody to a region of a target protein, including for example, MIF.
  • the disclosure provides a method of creating an antibody to a region of a target protein not bound by existing antibodies, in some cases that target protein is IL2.
  • IL2 is a cytokine molecule that regulates the activities of leukocytes (including lymphocytes) responsible for immune regulation, including discrimination of self and foreign antigens. IL2 mediates its effects by binding to IL-2 receptors in the body, which are expressed by lymphocytes.
  • the immunogen polypeptide comprise the immunogen polypeptide comprises at least 2 amino acids, at least 3 amino acids, at least 4 amino acids, at least 5 amino acids, at least 6 amino acids, at least 7 amino acids, at least 8 amino acids, at least 9 amino acids, at least 10 amino acids, at least 11 amino acids, at least 12 amino acids, at least 13 amino acids, at least 14 amino acids, at least 15 amino acids, at least 16 amino acids, at least 17 amino acids, at least 18 amino acids, at least 19 amino acids, at least 20 amino acids, at least 21 amino acids, at least 22 amino acids, at least 23 amino acids, at least 24 amino acids, at least 25 amino acids, at least 26 amino acids, at least 27 amino acids, at least 28 amino acids, at least 29 amino acids, at least 30 amino acids, or more.
  • the immunogen polypeptide comprises at most 2 amino acids, at most 3 amino acids, at least 4 amino acids, at most 5 amino acids, at most 6 amino acids, at most 7 amino acids, at most 8 amino acids, at most 9 amino acids, at most 10 amino acids, at most 11 amino acids, at most 12 amino acids, at most 13 amino acids, at most 14 amino acids, at most 15 amino acids, at most 16 amino acids, at most 17 amino acids, at most 18 amino acids, at most 19 amino acids, at most 20 amino acids, at most 21 amino acids, at most 22 amino acids, at most 23 amino acids, at most 24 amino acids, at most 25 amino acids, at most 26 amino acids, at most 27 amino acids, at most 28 amino acids, at most 29 amino acids, at most 30 amino acids, at most 40 amino acids, at most 50 amino acids, at most 60 amino acids, at most 70 amino acids, at most 80 amino acids, at most 90 amino acids, at most 100 amino acids, at most 150 amino acids, at most 200 amino acids, at most 250 amino acids, or at most 300 amino acids.
  • the methods and compositions comprises targeting and mutating select amino acids, for example histidines, in a designated immunogen sequence in order to select antibodies that have the ability to recognize the target sequence only when the immunogen is expressed in the targeted pH environment.
  • the methods and compositions comprises targeting and mutating select amino acids in a designated immunogen sequence in order to mediate the structural confirmation of the immunogen, thereby targeting the antibody to the mutated immunogen to recognize the target sequence only when the immunogen is expressed in a particular structural confirmation present only in, for example, tumor cells.
  • the methods and composition comprises targeting and mutating select amino acids in a designated immunogen sequence in order to improve tumor access, including for example, manipulating charge (anionic or neutral, for example) of the immunogen, thereby targeting the antibody to the mutated immunogen to recognize the target sequence only when the immunogen is expressed as a charged molecule.
  • the methods and compositions comprises targeting and mutating select amino acids in a designated immunogen sequence in order to mediate the hydrophobicity of the immunogen, thereby targeting the antibody to the mutated immunogen to recognize the target sequence only when the immunogen is expressed with a particular hydrophobicity level present only in, for example, tumor cells.
  • the methods and compositions comprises targeting and mutating select amino acids in a designated immunogen sequence in order to represent the target protein that can mediate production of the reactive oxygen species, thereby targeting the antibody to the mutated immunogen to recognize the target sequence on the immunogen to increase reactive oxygen species in the host tumor cell.
  • the tumor cell is also present in the hypoxic environment.
  • CD25 immunogens are disclosed herein, which are designed to represent conformational and configurational domains of CD25 that will undergo protonation under acidic environment.
  • Antibodies derived using such immunogen constructs are likely to differentially recognize the native form of the immunogen in a neutral or acidic environment. These immunogens could thus be used to make antibodies that can recognize the antigen target selectively in a neutral pH environment, such as in the periphery, or in an acidic pH environment, such as a tumor micro environment.
  • Amino acid residues targeted for modifying in an immunogen include, for example, charged amino acids such as histidine that could be modified to, for example, arginine or lysine such that modifications of the protonated state of the immunogen would allow selection of antibodies that recognize the target only when the immunogen is expressed in the targeted pH, e.g., the acidic
  • Antigen recognition may also be mediated by modifying amino acid residues such that the resulting immunogen becomes neutral or lacks hydrogen bonding in its properties. For example, amino acid residue 68 in PD-1 , which is tyrosine in human and cynomolgus monkey and arginine in mouse (see FIG.
  • alanine or glycine which are smaller amino acids that lack or have a diminished capacity to form side-chain hydrogen bonding. This may assist in allowing selection of antibodies that recognize the target across different species (i. e. , pan-species antibody).
  • the immunogen sequence may be modified to exploit reactive oxygen species status of the antibody-antigen targeting.
  • increased reactive oxygen species concentrations have been shown to lead to cell death in non-apoptotic pathways.
  • Increased binding of antibodies to certain epitopes of CXCR4 levels have been correlated with high reactive oxygen species concentrations, wherein increased CXCR4 levels are also correlated with poor prognosis and metastasis.
  • modifying amino acid residues such that the resulting immunogen promotes reactive oxygen species generation upon binding of the antibody to the target could be beneficial in selecting antibodies that recognize the target when presented in an increased hypoxic microenvironment, e.g. , in a tumor
  • antigen recognition may also be mediated by modifying amino acid residues such that the resulting immunogen changes its charge or hydrophobic/hydrophilic state, allowing selection of antibodies that recognize the target when the immunogen is expressed in an alternate charge, hydrophobic or hydrophilic state.
  • This includes, for example, targeting amino acids with hydrophobic side chains (e.g. , alanine, isoleucine, leucine, methionine or valine) and substituting with amino acids with hydrophobic side chains (e.g. , phenylalanine, tryptophan or tyrosine), or vice versa.
  • amino acids with polar neutral side chains in the identified immunogen sequence e.g.
  • amino acids may be substituted with amino acids that have charged amino acids, such as basic (e.g. , arginine, histidine or lysine) or acidic (e.g. , aspartic acid or glutamic acid) amino acids.
  • basic e.g. , arginine, histidine or lysine
  • acidic e.g. , aspartic acid or glutamic acid
  • mapping regions of a target protein not bound by existing antibodies comprise detecting binding, or lack of binding, of the existing antibodies to a peptide array.
  • the array platforms may comprise a plurality of individual features on the surface of the array. Each feature typically comprises a plurality of individual molecules synthesized in situ on the surface of the array, wherein the molecules are identical within a feature, but the sequence or identity of the molecules differ between features.
  • the array molecules include, but are not limited to nucleic acids (including DNA, RNA, nucleosides, nucleotides, structure analogs or combinations thereof), peptides, peptide- mimetics, and combinations thereof and the like, wherein the array molecules may comprise natural or non-natural monomers within the molecules.
  • Such array molecules include the synthesis of large synthetic peptide arrays.
  • the peptides on the array are cyclized on the surface of the array.
  • a molecule in an array is a mimotope, a molecule that mimics the structure of an epitope and is able to bind an epitope-elicited antibody.
  • a molecule in the array is a paratope or a paratope mimetic, comprising a site in the variable region of an antibody (or T cell receptor) that binds to an epitope of an antigen.
  • an array of the invention is a peptide array comprising random, semi-random or diverse peptide sequences.
  • the diverse peptide sequences may be derived from a proteome library, for example, from a specific organism (see, e.g., Mycobacterium tuberculosis (Mtb) proteome library (Schubert et al., Cell Host Microbe (2013) 13(5):602-12), or organelle (see, e.g., Mitochondrial (Mtd) proteome library (Calvo and Mootha, Annu. Rev. Genomics (2010) 11 :25-44), and the like.
  • Mtb Mycobacterium tuberculosis
  • organelle see, e.g., Mitochondrial (Mtd) proteome library (Calvo and Mootha, Annu. Rev. Genomics (2010) 11 :25-44
  • the peptide array may comprise a diverse set of peptides, a related set of peptides or both. In some cases the related set of peptides are 75% identical. In other cases, a related set of peptides can be 90% similar to one another. In some embodiments, the peptide array comprises at least 1000 unique peptides. In other embodiments, the peptide array comprises at least 10,000 unique peptides. In still other embodiments, the peptide array comprises at least 100,000 unique peptides. In yet other embodiments, the peptide array comprises at least 1,000,000 unique peptides.
  • the peptide array comprises at least 5000, at least 10,000, at least 50,000, at least 100,000, at least 250,000, at least 500,000, at least 750,000, at least 1,000,000, at least 2,000,000, at least 3,000,000 or more unique peptides.
  • the peptide array is in situ synthesized.
  • the diverse peptide sequences may be derived from a set of all known combinations of amino acids, for example at least 100% of all possible tetramers, at least 90% of all possible tetramers, at least 85% of all possible tetramers, at least 80% of all possible tetramers, at least 75% of all possible tetramers, at least 70% of all possible tetramers, at least 65% of all possible tetramers, at least 60% of all possible tetramers, at least 55% of all possible tetramers, at least 50% of all possible tetramers, at least 45% of all possible tetramers, at least 40% of all possible tetramers, at least 35% of all possible tetramers, at least 30% of all possible tetramers, or at least 25% of all possible tetramers.
  • the diverse peptide sequences may be derived from a set of all possible pentamers, for example, at least 100% of all possible pentamers, at least 95% of all possible pentamers, at least 90% of all possible pentamers, at least 85% of all possible pentamers, at least 80% of all possible pentamers, at least 75% of all possible pentamers, at least 70% of all possible pentamers, at least 65% of all possible pentamers, at least 60% of all possible pentamers, at least 55% of all possible pentamers, at least 50% of all possible pentamers, at least 45% of all possible pentamers, at least 40% of all possible pentamers, at least 35% of all possible pentamers, at least 30% of all possible pentamers or at least 25% of all possible pentamers.
  • the diverse peptide sequences of an array may be derived from a set of amino acid combinations, for example from 25%-100% of all possible hexamers, from 25%-100% of all possible septamers, from 25%-100% of all possible octamers, from 25%- 100% of all possible nonamers or from 25%- 100% of all possible decamers, or combinations thereof. Representation of the diverse peptide sequences is only limited by the size of the array.
  • large arrays for example, at least 1 million, at least 2 million, at least 3 million, at least 4 million, at least 5 million, at least 6 million, at least 7 million, at least 8 million, at least 9 million, at least 10 million or more peptides can be used with the methods, systems and assays disclosed herein.
  • multiple substantially non-overlapping peptide libraries/arrays may be synthesized to cover the sequence space needed for resolution of the peptide sequences or motif(s) that could be recognized by the antibody.
  • the individual sequences can share a % homology to an amino acid sequence of a polypeptide from a related species.
  • a polypeptide sequence can share at most 10% homology, at most 20% homology, at most 30% homology, at most 40% homology, at most 50% homology, at most 60% homology, at most 70% homology, at most 80% homology, at most 90% homology, or at most 99% homology with an amino acid sequence of a related peptide.
  • Various methods and software programs can be used to determine the homology between two or more peptides, such as NCBI BLAST, Clustal W, MAFFT, Clustal Omega, AlignMe, Praline, or another suitable method or algorithm.
  • the individual peptides on the array are of variable and/or different lengths.
  • the peptides are between about 6-20 amino acids in length, or between about 7-18 amino acids in length, or between about 8-15 amino acids in length, or between about 9-14 amino acids in length.
  • the peptides are at least 6 amino acids, at least 7 amino acids, at least 8 amino acids, at least 9 amino acids, at least 10 amino acids, at least 1 1 amino acids, at least 12 amino acids, at least 13 amino acids, at least 14 amino acids, at least 15 amino acids in length.
  • the peptides are not more than 15 amino acids, not more than 14 amino acids, not more than 13 amino acids, not more than 12 amino acids, not more than 11 amino acids, not more than 10 amino acids, not more than 9 amino acids or not more than 8 amino acids in length.
  • the peptides on the array have an average length of about 6 amino acids, about 7 amino acids, about 8 amino acids, about 9 amino acids, about 10 amino acids, about 11 amino acids, about 12 amino acids, about 13 amino acids, about 14 amino acids, or about 15 amino acids.
  • the amino acid building blocks for the peptides on the array comprises all natural amino acids.
  • the amino acid building blocks for the peptides on the array are comprised of non-natural or synthetic amino acids.
  • only 19 amino acids are used as the building blocks for synthesizing the peptides on the array.
  • only 18 amino acids, only 17 amino acids, only 16 amino acids, only 15 amino acids or only 14 amino acids are used as the building blocks for synthesizing the peptides on the array.
  • cysteine is omitted during peptide synthesis.
  • methionine is omitted during peptide synthesis.
  • an array of the invention is a peptide array comprising a focused or limited set of peptide sequences, all derived from an input amino acid or peptide sequence, or an input amino acid or peptide motif.
  • One or more peptide arrays may be used with the methods, systems and assays disclosed herein, including a diverse or semi-random peptide array and/or a focused or limited set of peptide sequences.
  • the methods, systems and assays disclosed herein may utilize both a diverse set of peptides and a focused or limited set of peptides are chosen.
  • the peptide arrays may be used either in parallel or sequentially with a biological sample as disclosed herein.
  • a diverse peptide array may be used initially, and at least one motif (either sequence or structure-based) or sequence is obtained for a monoclonal antibody, for example, with an unknown binding profile.
  • the identified motif or sequence may be then used as the input sequence for the creation of at least one focused or limited set of peptide sequences, and assays performed as described herein. Using the methods and arrays described herein, multiple focused or limited set of peptide arrays may be used to characterize antibody binding for the antibody.
  • the technologies disclosed herein include a photolithographic array synthesis platform that merges semiconductor manufacturing processes and combinatorial chemical synthesis to produce array-based libraries on silicon wafers. Further, by sequentially applying another mask with UV light exposure, various array features can be established. By utilizing the tremendous advancements in photolithographic feature patterning, the array synthesis platform is highly-scalable and capable of producing combinatorial chemical libraries with 40 million features on an 8-inch wafer. Photolithographic array synthesis is performed using semiconductor wafer production equipment in a class 10,000 cleanroom to achieve high reproducibility. When the wafer is diced into standard microscope slide dimensions, each slide contains more than 3 million distinct chemical entities. Exemplary embodiments of focused and targeted arrays are described in detail in PCT/US2017/025546, entitled "Array- Based Peptide Libraries for Therapeutic Antibody Characterization", which is incorporated by reference herein for such disclosure.
  • arrays with chemical libraries produced by the technologies disclosed herein are used for immune-based diagnostic assays, for example called
  • immunosignature assays are being developed for clinical application to diagnose/monitor autoimmune diseases and to assess response to autoimmune treatments. Exemplary embodiments of immunosignature assays is described in detail in US Pre-Grant Publication No. 2012/0190574, entitled “Compound Arrays for Sample Profiling” and US Pre-Grant Publication No. 2014/0087963, entitled “Immunosignaturing: A Path to Early Diagnosis and Health Monitoring", both of which are incorporated by reference herein for such disclosure.
  • the arrays developed herein incorporate analytical measurement capability within each synthesized array using orthogonal analytical methods including ellipsometry, mass spectrometry and fluorescence. These measurements enable longitudinal qualitative and quantitative assessment of array synthesis performance.
  • the peptide arrays are high density peptide arrays.
  • the arrays comprise individual peptides within a feature on the array spaced less than 0.5 nm, less than 1 nm, less than 2 nm, less than 3 nm, less than 4 nm, less than 5 nm, less than 6 nm, less than 7 nm, less than 8 nm, less than 9 nm, less than 10 nm apart, less than 1 1 nm apart, less than 12 nm apart, less than 13 nm apart, less than 14 nm part or less than 15 nm apart.
  • Focused and diverse peptide arrays can comprise a number of different peptides.
  • the size of the peptide array is dependent on the desired coverage of a proteome or of a target protein.
  • the methods of the invention can effectively provide for: the selection of a monoclonal antibody with specific binding; the elimination of dead-end monoclonal antibodies from a library of binding domains; the selection of a monoclonal antibody that binds to multiple epitopes in a target protein; the selection of a monoclonal antibody that binds to at least two homologs of a target protein; the selection of a monoclonal antibody that binds to an active domain, a functional domain, or to a target epitope of a target protein; or the selection of a multi-specific monoclonal antibody by screening the antibody against a peptide array that comprises no more than 2,000 peptides; no more than 5,000 peptides; no more than 10,000 peptides; no more than
  • a peptide array comprises at least 2,000 peptides; at least 3,000 peptides; at least 4,000 peptides; at least 5,000 peptides; at least 6,000 peptides; at least 7,000 peptides; at least 8,000 peptides; at least 9,000 peptides; at least 10,000 peptides; at least 11 ,000 peptides; at least 12,000 peptides; at least 13,000 peptides; at least 14,000 peptides; at least 15,000 peptides; at least 16,000 peptides; at least 17,000 peptides; at least 18,000 peptides; at least 19,000 peptides; at least 20,000 peptides; at least 21,000 peptides; at least 22,000 peptides; at least 23,000 peptides; at least 24,000 peptides; at least 25,000 peptides; at least 30,000 peptides; at least 40,000 peptides; at least 50,000
  • a peptide can be physically tethered to a peptide array by a linker molecule.
  • the N- or the C-terminus of the peptide can be attached to a linker molecule.
  • a linker molecule can be, for example, a functional plurality or molecule present on the surface of an array, such as an imide functional group, an amine functional group, a hydroxyl functional group, a carboxyl functional group, an aldehyde functional group, and/or a sulfhydryl functional group.
  • a linker molecule can be, for example, a polymer. In some embodiments the linker is maleimide.
  • the linker is a glycine-serine-cysteine (GSC) or glycine- glycine-cysteine (GGC) linker.
  • the linker consists of a polypeptide of various lengths or compositions.
  • the linker is polyethylene glycol of different lengths.
  • the linker is hydroxymethyl benzoic acid, 4-hydroxy-2-methoxy benzaldehyde, 4-sulfamoyl benzoic acid, or other suitable for attaching a peptide to the solid substrate.
  • the linker molecule is cyclized. Exemplary embodiments of cyclized linkers include, but are not limited to, embodiments disclosed in PCT Application No. PCT/US2017/025546 entitled "Array -Based Peptide Libraries for Therapeutic Antibody Characterization" (filed March 31, 2017), whose disclosure is incorporated herein in its entirety.
  • a surface of a peptide array can comprise a plurality of different materials.
  • a surface of a peptide array can be, for example, glass.
  • materials that can comprise a surface of a peptide array include glass, functionalized glass, silicon, germanium, gallium arsenide, gallium phosphide, silicon dioxide, sodium oxide, silicon nitride, nitrocellulose, nylon, polytetrafluoroethylene, polyvinylidendifluoride, polystyrene, polycarbonate, methacrylates, or combinations thereof.
  • a surface of a peptide array can also comprise semi-conductor wafers, for example, silicon wafers, derivatized with, for example, aminosilane molecules, which allows spotting or in situ synthesis on the surface of the array.
  • a surface of a peptide array can be flat, concave, or convex.
  • a surface of a peptide array can be homogeneous and a surface of an array can be heterogeneous.
  • the surface of a peptide array is flat.
  • the surface of a peptide array is round, such as the surface of a bead.
  • a surface of a peptide array can be coated with a coating.
  • a coating can, for example, improve the adhesion capacity of an array of the invention.
  • a coating can, for example, reduce background adhesion of a biological sample to an array of the invention.
  • a peptide array of the invention comprises a glass slide or silicon wafer with an aminosilane-coating.
  • the immunogen polypeptide comprises at least one linker.
  • the linker can comprises an amino acid sequence having one or more glycine residues, one or more serine residues, or one or more proline residues.
  • the linker can have an amino acid sequence selected from at least one of GSG, (GGGGS)n, (GSG)n, GGGSGGGGS, GGGGSGGGS, (PGSG) n , PGSGSG where n is an integer between 1 and 10.
  • an immunogen can be designed to contain two or more sequences of residues present on the surface of a target protein that are known not to be bound by an antibody.
  • Example 1 describes the design of a novel immunogen for PD-1 designed to contain two sequences of residues present on the surface of PD-1 that are known not to be bound by Nivolumab.
  • Example 1 describes the design of a novel immunogen for PD-1. In that example, the N-terminus to C-terminus directionality of each sequence was maintained, but the order of the sequences was reversed relative to their positions/order in the native PD-1 protein to provide a PD-1 immunogen having the second sequence linked to the first sequence.
  • the present disclosure provides competition-free, high throughput binning assays to characterize and/or binding of antibodies based on the methods and devices disclosed herein.
  • binning or “bin”, as used herein, is meant the classification of antibodies in distinct categories based on target sequence analysis. For example, as seen in FIG.
  • the peptide array platforms are able to identify two distinct epitope bins ("Epitope Bin 1" (left panel) and “Epitope Bin 2" (right panel)) based on identification and comparison of the peptide sequences bound to several Her2 antibodies (Thermo MA5-13675, Santa Cruz SC-33684 and Cell Signaling 2165). Accordingly, the epitope binning data obtained provided putative epitope
  • the epitope binning data also provided position-level amino acid binding contribution ⁇ compare amino acid distribution and coverage for each binding peptide analyzed), which may be important, for example, for pan-species selectivity of clones.
  • the method further comprises determining cross-species homology of the region not bound by the existing antibodies.
  • the method comprises: a) obtaining a sequence of a target bound by each of the existing antibodies; b) identifying a region of the target polypeptide not bound by each of the existing antibodies; c) preparing at least one immunogen comprising an amino acid sequence of the region of the target polypeptide not bound by the existing antibodies; d) immunizing a mammal with the immunogen polypeptide; and e) obtaining from the mammal a monoclonal antibody that specifically binds to the region identified in step b), thereby creating the monoclonal antibody.
  • the target polypeptide is a therapeutic target polypeptide.
  • a target polypeptide e.g. therapeutic target polypeptide
  • the method comprising: a) obtaining a sequence of a target bound by existing antibodies; b) identifying a region of the target polypeptide not comprising the binding region for each of the existing antibodies; c) preparing at least one immunogen comprising an amino acid sequence of the region of the target polypeptide not comprising the binding region for each of the existing antibodies; d) immunizing a mammal with the immunogen polypeptide; and e) obtaining from the mammal a monoclonal antibody that specifically binds to the region identified in step b), thereby creating the monoclonal antibody.
  • the target polypeptide is a therapeutic target polypeptide.
  • Some such methods comprise creating a monoclonal antibody in a region of a target polypeptide (e.g., a therapeutic target polypeptide) not bound by existing antibodies, the method comprising: a) obtaining a sequence of a target bound by each of the existing antibodies; b) identifying a region of the target polypeptide not bound by each of the existing antibodies; c) preparing at least one immunogen comprising an amino acid sequence of the region of the target polypeptide targeted to the region not bound by the existing antibodies; d) immunizing a mammal with the immunogen polypeptide; e) obtaining from the mammal a monoclonal antibody that specifically binds to the region identified in step b); and f) determining binding of the monoclonal antibody to a focused array comprising a library of peptides having an amino acid sequence identical to a portion of the target polypeptide,
  • Immunizing the mammal with the immunogen polypeptide comprises
  • the mammal can be immunized with a full length protein, a cell, a DNA, or one of the selected immunogens.
  • a booster round of immunization can be performed with the full length protein, the cell, the DNA, or one of the selected immunogens.
  • a mammal is immunized with one of the selected immunogens, and then a booster round of immunization can be performed with the full length target protein, a target cell, or the selected immunogen, or a combination thereof (for example the full length target protein in combination with the selected immunogen).
  • FIG. 5 shows exemplary immunization protocols for creating an antibody. Other methods for immunization are contemplated herein, and would be understood by a skilled artisan.
  • Functional assays comprise at least one of inhibiting an activity the arget polypeptide, increasing an activity of the target polypeptide, inhibiting binding of the target polypeptide to a binding partner, stabilizing binding of the target polypeptide to a binding partner, increasing half-life of the target polypeptide, and decreasing half-life of the target polypeptide.
  • the target polypeptide is a therapeutic target polypeptide.
  • an assay can inform whether an antibody designed with the disclosed methods inhibits PD-1, PD-Ll, CD25, IL2, MIF or CXCR4, or another biological target.
  • Non-limiting examples of functional assays can include: a) assays that inform the interactions of proteins with DNA, such as DNase footprinting assay and gel shift assays; b) assays that inform the integrity of an RNA molecule, such as nuclear run-on assays; c) end point assays, which can measure quantitatively or qualitatively the end result of an assay; d) kinetic assays, which can evaluate readings of data points at multiple time intervals and compare the kinetics of a biological process; e) semi-quantitative assays, which provide a read-out that can be quantitated within a context, such as western-blots, clotting and agglutination assays; f) immunoassays, which evaluate the response of an antigen antibody binding type of reaction, for example, the human anti-D immunoglobulin assay measures levels of a specific immunoglobulin, and immunization assays such as the assay for pertussis vaccine, tetanus
  • monoclonal antibodies created by methods disclosed herein. Some such monoclonal antibodies bind a region of a target polypeptide (e.g. a therapeutic target polypeptide) not bound by existing antibodies and are created by a process comprising: a) obtaining a sequence of a target bound by the existing antibodies; b) identifying a region of the target polypeptide not bound by each of the existing antibodies; c) preparing at least one immunogen comprising an amino acid sequence of the region of the target polypeptide not bound by the existing antibodies; d) immunizing a mammal with the immunogen polypeptide; and e) obtaining from the mammal a monoclonal antibody that specifically binds to the region identified in step b), thereby creating the monoclonal antibody.
  • the target polypeptide is a therapeutic target polypeptide.
  • Monoclonal antibodies obtained as provided herein may be evaluated using one or more methods known to the skilled artisan.
  • the antibody is characterized using two peptide arrays.
  • this application cross-references to the following patent application: Attorney Docket No. 43638-727.101 , previously filed on October 9, 2017, U. S. Provisional Application No. 62/569,926, which is incorporated herein by reference in its entirety; and the international application entitled "INTEGRATED PLATFORM FOR TARGET AND SPECIFICITY INFORMATION-DERIVED BINDING PARTNER SELECTION", Attorney Docket No. RBYC-019/01WO 334002-2071 , filed concurrently herewith, which is incorporated herein by reference in its entirety. Methods from these incorporated applications may be used, in certain embodiments, to evaluate antibodies obtained as described herein.
  • methods of treatment comprising administration of monoclonal antibodies created by methods provided herein. Some such methods comprise administering an effective amount of a monoclonal antibody binding a region of a target polypeptide (e.g., a therapeutic target polypeptide) not bound by existing antibodies, wherein the monoclonal antibody is created by a process comprising: a) obtaining a sequence of a target polypeptide bound by each of the existing antibodies; b) identifying a region of the target polypeptide not bound by each of the existing antibodies; c) preparing at least one immunogen comprising an amino acid sequence of the region of the target polypeptide not bound by the existing antibodies; d) immunizing a mammal with the immunogen
  • the target polypeptide is a therapeutic target polypeptide.
  • monoclonal antibodies created by methods provided herein for use as a medicament. Some such monoclonal antibodies bind a region of a target polypeptide (e.g., a therapeutic target polypeptide) not bound by existing antibodies.
  • a target polypeptide e.g., a therapeutic target polypeptide
  • monoclonal antibodies for use as a medicament created by a process comprising: a) obtaining a sequence of a target bound by each of the existing antibodies; b) identifying a region of the target polypeptide not bound by each of the existing antibodies; c) preparing at least one immunogen comprising an amino acid sequence of the region of the target polypeptide not bound by the existing antibodies; d) immunizing a mammal with the immunogen polypeptide; and e) obtaining from the mammal a monoclonal antibody that specifically binds to the region identified in step b), thereby creating the monoclonal antibody.
  • the target polypeptide is a therapeutic target polypeptide.
  • Non-limiting examples of target proteins include PD-1, PD-L1, CD-25, CXCR4, and MIF.
  • the method of treating an individual may comprise treating a subject (e.g. a patient with a disease and/or a lab animal with a condition) with an antibody of the disclosure.
  • the method may comprise a) obtaining polypeptide sequence(s) on a target protein bound by existing antibodies; b) identifying at least one region of the target protein not bound by the existing antibodies; c) preparing at least one immunogen polypeptide comprising an amino acid sequence of the region of the target protein not bound by the existing antibodies; d) immunizing a mammal with the immunogen polypeptide; e) obtaining from the mammal an antibody that specifically binds to the region identified in step b); and f) administering the antibody to the individual, thereby treating the individual in need thereof.
  • the treatment may treat an autoimmune disease or a cancer.
  • the subject may be a human.
  • Treatment may be provided to the subject before clinical onset of disease.
  • Treatment may be provided to the subject after clinical onset of disease.
  • Treatment may be provided to the subject after 1 day, 1 week, 6 months, 12 months, or 2 years after clinical onset of the disease.
  • Treatment may be provided to the subject for more than 1 day, 1 week, 1 month, 6 months, 12 months, 2 years or more after clinical onset of disease.
  • Treatment may be provided to the subject for less than 1 day, 1 week, 1 month, 6 months, 12 months, or 2 years after clinical onset of the disease.
  • Treatment may also include treating a human in a clinical trial.
  • a treatment can comprise administering to a subject a pharmaceutical composition, such as a pharmaceutical composition comprising the disclosed antibody.
  • Multiple antibodies can be administered in any order or simultaneously. If simultaneously, the antibodies can be provided in a single, unified form, such as an intravenous injection, or in multiple forms, for example, as multiple intravenous injections or pills.
  • the antibodies can be packed together or separately, in a single package or in a plurality of packages.
  • One or all of the antibodies can be given in multiple doses. If not simultaneous, the timing between the multiple doses may vary to as much as about a month.
  • compositions comprising the antibodies described herein can be in unit dosage forms suitable for single administration of precise dosages.
  • the formulation is divided into unit doses containing appropriate quantities of one or more compounds.
  • the unit dosage can be in the form of a package containing discrete quantities of the formulation.
  • Non-limiting examples are packaged tablets or capsules, and powders in vials or ampoules.
  • Aqueous suspension compositions can be packaged in single-dose non- reclosable containers. Multiple-dose reclosable containers can be used, for example, in combination with a preservative or without a preservative.
  • the pharmaceutical composition does not comprise a preservative.
  • Formulations for parenteral injection can be presented in unit dosage form, for example, in ampoules, or in multi-dose containers with a preservative.
  • An antibody described herein can be present in a composition in a range of from about 1 ng/kg to about 10 ng/kg, from about 1 ng/kg to about 100 ng/kg, from about 1 ng/kg to about 1 mg/kg, from about 1 ng/kg to about 10 mg/kg, from about 1 ng/kg to about 100 mg/kg, from about 1 ng/kg to about 250 mg/kg, from about 1 ng/kg to about 500 mg/kg, from about 1 ng/kg to about 750 mg/kg, from about 1 ng/kg to about 1,000 mg/kg, from about 1 ng/kg to about 1,250 mg/kg, from about 1 ng/kg to about 1,500 mg/kg, from about 1 ng/kg to about 1,750 mg/kg, from about 1 ng/kg to about 2,000 mg/kg, from about 10 ng/kg to about 100 ng/kg, from about 10 ng/kg to about 1 mg/kg, from about 10 ng/kg to about 10 mg/kg, from about
  • an immune disorder is susceptible to treatment with an antibody as disclosed herein.
  • immune diseases or conditions that can be treated with an antibody disclosed herein include rheumatoid arthritis, multiple sclerosis, experimental autoimmune encephalomyelitis, psoriasis, uveitis, diabetes mellitus type 1, systemic lupus erythematosus (SLE), eczema, scleroderma, ulcerative proctitis, severe combined immunodeficiency (SCID), DiGeorge syndrome, ataxia-telangiectasia, seasonal allergies, perennial allergies, food allergies, anaphylaxis, mastocytosis, allergic rhinitis, atopic dermatitis, Parkinson's, Alzheimer's, hypersplenism, leukocyte adhesion deficiency, X-linked lymphoproliferative disease, X-linked agammaglobulinemia, selective
  • immunoglobulin A deficiency hyper IgM syndrome, HIV, autoimmune lymphoproliferative syndrome, Wiskott-Aldrich syndrome, chronic granulomatous disease, common variable immunodeficiency (CVID), hyperimmunoglobulin E syndrome, Hashimoto's thyroiditis, acute inflammatory conditions, chronic inflammatory conditions, and cancer.
  • CVID common variable immunodeficiency
  • a cancer is susceptible to treatment with an antibody disclosed herein.
  • cancers include: acute lymphoblastic leukemia, acute myeloid leukemia, adrenocortical carcinoma, AIDS-related cancers, AIDS-related lymphoma, anal cancer, appendix cancer, astrocytomas, neuroblastoma, basal cell carcinoma, bile duct cancer, bladder cancer, bone cancers, brain tumors, such as cerebellar astrocytoma, cerebral astrocytoma/malignant glioma, ependymoma, medulloblastoma, supratentorial primitive neuroectodermal tumors, visual pathway and hypothalamic glioma, breast cancer, bronchial adenomas, Burkitt lymphoma, carcinoma of unknown primary origin, central nervous system lymphoma, cerebellar astrocytoma, cervical cancer, childhood cancers, chronic lymphocytic leukemia, chronic lymphocytic leukemia, chronic lymph
  • bone/osteosarcoma medulloblastoma, melanomas, mesothelioma, metastatic squamous neck cancer with occult primary, mouth cancer, multiple endocrine neoplasia syndrome, myelodysplasia syndromes, myeloid leukemia, nasal cavity and paranasal sinus cancer, nasopharyngeal carcinoma, neuroblastoma, non-Hodgkin lymphoma, non-small cell lung cancer, oral cancer, oropharyngeal cancer, osteosarcoma/malignant fibrous histiocytoma of bone, ovarian cancer, ovarian epithelial cancer, ovarian germ cell tumor, pancreatic cancer, pancreatic cancer islet cell, paranasal sinus and nasal cavity cancer, parathyroid cancer, penile cancer, pharyngeal cancer, pheochromocytoma, pineal astrocytoma, pineal germinoma, pituitary a
  • An antibody of the disclosure can be combined with other chemical components, such as carriers, stabilizers, diluents, dispersing agents, suspending agents, thickening agents, and/or excipients.
  • the pharmaceutical composition facilitates administration of the antibody to a subject.
  • Pharmaceutical compositions can be administered in therapeutically-effective amounts as pharmaceutical compositions by various forms and routes including, for example, intratumor, intravenous, subcutaneous, intramuscular, rectal, aerosol, parenteral, ophthalmic, pulmonary, transdermal, vaginal, optic, nasal, and topical administration.
  • An antibody can be administered in a local or systemic manner, for example, via injection of the antibody directly into an organ, optionally in a depot.
  • compositions can be formulated using one or more physiologically-acceptable carriers comprising excipients and auxiliaries, which facilitate processing of the active compounds into preparations that can be used pharmaceutically. Formulation can be modified depending upon the route of administration chosen.
  • compositions comprising an antibody or immunogen described herein can be manufactured, for example, by mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping, or compression processes.
  • the pharmaceutical compositions can include at least one pharmaceutically acceptable carrier, diluent, or excipient and compounds described herein as free-base or pharmaceutically-acceptable salt form.
  • Non-limiting examples of pharmaceutically-acceptable excipients can be found, for example, in Remington: The Science and Practice of Pharmacy, Nineteenth Ed (Easton, Pa. : Mack Publishing Company, 1995); Hoover, John E., Remington 's Pharmaceutical Sciences, Mack Publishing Co., Easton, Pennsylvania 1975; Liberman, H.A. and Lachman, L., Eds., Pharmaceutical Dosage Forms, Marcel Decker, New York, N.Y., 1980; and Pharmaceutical Dosage Forms and Drug Delivery Systems, Seventh Ed. (Lippincott Williams &
  • Parenteral injections can be formulated for bolus injection or continuous infusion.
  • the pharmaceutical compositions can be in a form suitable for parenteral injection as a sterile suspension, solution or emulsion in oily or aqueous vehicles, and can contain formulatory agents such as suspending, stabilizing and/or dispersing agents.
  • Pharmaceutical formulations for parenteral administration include aqueous solutions of the antibody(ies) in water-soluble form. Suspensions of the PD-1, PD-L1 , CD25, IL2, MIF or CXCR4 and other antibodies disclosed herein can be prepared as oily injection suspensions.
  • Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acid esters, such as ethyl oleate or triglycerides, or liposomes.
  • Aqueous injection suspensions can contain substances which increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, or dextran.
  • the suspension can also contain suitable stabilizers or agents which increase the solubility and/or reduces the aggregation of the antibodies to allow for the preparation of highly concentrated solutions.
  • the antibodies can be lyophilized or in powder form for re-constitution with a suitable vehicle, e.g. , sterile pyrogen-free water, before use.
  • a suitable vehicle e.g. , sterile pyrogen-free water, before use.
  • an antibody of the invention is administered
  • the antibodies disclosed herein can be administered topically and can be formulated into a variety of topically administrable compositions, such as solutions, suspensions, lotions, gels, pastes, medicated sticks, balms, creams, and ointments.
  • Such pharmaceutical compositions can contain solubilizers, stabilizers, tonicity enhancing agents, buffers and preservatives.
  • methods described herein are used in conjunction with computer systems, platforms, software, and networks, that facilitate the detection of the binding domains to one or more peptides of a peptide array by the at least one of
  • the aforementioned computer systems, platforms, software, and networks may include a digital processing device, or use of the same.
  • the digital processing device includes one or more hardware central processing units (CPUs), i.e., processors that carry out the device's functions.
  • the digital processing device further comprises an operating system configured to perform executable instructions.
  • the digital processing device is optionally connected a computer network.
  • the digital processing device is optionally connected to the Internet such that it accesses the World Wide Web.
  • the digital processing device is optionally connected to a cloud computing infrastructure.
  • the digital processing device is optionally connected to an intranet.
  • the digital processing device is optionally connected to a data storage device.
  • suitable digital processing devices include, by way of non-limiting examples, server computers, desktop computers, laptop computers, notebook computers, sub-notebook computers, netbook computers, netpad computers, set-top computers, handheld computers, Internet appliances, mobile smartphones, tablet computers, personal digital assistants, video game consoles, and vehicles.
  • server computers desktop computers, laptop computers, notebook computers, sub-notebook computers, netbook computers, netpad computers, set-top computers, handheld computers, Internet appliances, mobile smartphones, tablet computers, personal digital assistants, video game consoles, and vehicles.
  • smartphones are suitable for use in the system described herein.
  • Suitable tablet computers include those with booklet, slate, and convertible configurations, known to those of skill in the art.
  • a digital processing device includes an operating system configured to perform executable instructions.
  • the operating system is, for example, software, including programs and data, which manages the device's hardware and provides services for execution of applications.
  • suitable server operating systems include, by way of non-limiting examples, FreeBSD, OpenBSD, NetBSD ® , Linux, Apple ® Mac OS X Server ® , Oracle ® Solaris ® , Windows Server ® , and Novell ® NetWare ® .
  • suitable personal computer operating systems include, by way of non-limiting examples, Microsoft ® Windows ® , Apple ® Mac OS X ® , UNIX ® , and UNIX-like operating systems such as GNU/Linux ® .
  • the operating system is provided by cloud computing.
  • suitable mobile smart phone operating systems include, by way of non-limiting examples, Nokia ® Symbian ® OS, Apple ® iOS ® , Research In Motion ®
  • BlackBerry OS ® , Google ® Android ® , Microsoft ® Windows Phone ® OS, Microsoft ® Windows Mobile ® OS, Linux ® , and Palm ® WebOS ® .
  • a digital processing device includes a storage and/or memory device.
  • the storage and/or memory device is one or more physical apparatuses used to store data or programs on a temporary or permanent basis.
  • the device is volatile memory and requires power to maintain stored information.
  • the device is non-volatile memory and retains stored information when the digital processing device is not powered.
  • the non-volatile memory comprises flash memory.
  • the non-volatile memory comprises dynamic random-access memory (DRAM).
  • the non-volatile memory comprises ferroelectric random access memory (FRAM).
  • the non-volatile memory comprises phase-change random access memory (PRAM).
  • the device is a storage device including, by way of non-limiting examples, CD-ROMs, DVDs, flash memory devices, magnetic disk drives, magnetic tapes drives, optical disk drives, and cloud computing based storage.
  • the storage and/or memory device is a combination of devices such as those disclosed herein.
  • a digital processing device includes a display to send visual information to a user.
  • the display is a cathode ray tube (CRT).
  • the display is a liquid crystal display (LCD).
  • the display is a thin film transistor liquid crystal display (TFT-LCD).
  • the display is an organic light emitting diode (OLED) display.
  • OLED organic light emitting diode
  • on OLED display is a passive-matrix OLED (PMOLED) or active-matrix OLED (AMOLED) display.
  • the display is a plasma display.
  • the display is a video projector.
  • a digital processing device includes an input device to receive information from a user.
  • the input device is a keyboard.
  • the input device is a pointing device including, by way of non-limiting examples, a mouse, trackball, track pad, joystick, game controller, or stylus.
  • the input device is a touch screen or a multi-touch screen. In other words,
  • the input device is a microphone to capture voice or other sound input.
  • the input device is a video camera to capture motion or visual input.
  • the input device is a combination of devices such as those disclosed herein.
  • a digital processing device includes a digital camera.
  • a digital camera captures digital images.
  • the digital camera is an autofocus camera.
  • a digital camera is a charge- coupled device (CCD) camera.
  • a digital camera is a CCD video camera.
  • a digital camera is a complementary metal-oxide- semiconductor (CMOS) camera.
  • CMOS complementary metal-oxide- semiconductor
  • a digital camera captures still images.
  • a digital camera captures video images.
  • suitable digital cameras include 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, and higher megapixel cameras, including increments therein.
  • a digital camera is a standard definition camera. In other embodiments, a digital camera is an HD video camera. In further embodiments, an HD video camera captures images with at least about 1280 x about 720 pixels or at least about 1920 x about 1080 pixels. In some embodiments, a digital camera captures color digital images. In other embodiments, a digital camera captures grayscale digital images. In various embodiments, digital images are stored in any suitable digital image format.
  • Suitable digital image formats include, by way of non-limiting examples, Joint Photographic Experts Group (JPEG), JPEG 2000, Exchangeable image file format (Exif), Tagged Image File Format (TIFF), RAW, Portable Network Graphics (PNG), Graphics Interchange Format (GIF), Windows ® bitmap (BMP), portable pixmap (PPM), portable graymap (PGM), portable bitmap file format (PBM), and WebP.
  • digital images are stored in any suitable digital video format.
  • Suitable digital video formats include, by way of non-limiting examples, AVI, MPEG, Apple ® QuickTime ® , MP4, AVCHD ® , Windows Media ® , DivXTM, Flash Video, Ogg Theora, WebM, and RealMedia.
  • methods described herein are used in conjunction with computer systems, platforms, software, and networks, that facilitate the detection of the binding domains to one or more peptides of a peptide array.
  • the systems, platforms, software, networks, and methods include one or more non-transitory computer readable storage media encoded with a program including instructions executable by the operating system of an optionally networked digital processing device.
  • a computer readable storage medium is a tangible component of a digital processing device.
  • a computer readable storage medium is optionally removable from a digital processing device.
  • a computer readable storage medium includes, by way of non-limiting examples, CD-ROMs, DVDs, flash memory devices, solid state memory, magnetic disk drives, magnetic tape drives, optical disk drives, cloud computing systems and services, and the like.
  • the program and instructions are permanently, substantially permanently, semi-permanently, or non-transitorily encoded on the media.
  • the systems, platforms, software, networks, and methods disclosed herein include at least one computer program.
  • a computer program includes a sequence of instructions, executable in the digital processing device's CPU, written to perform a specified task.
  • a computer program may be written in various versions of various languages.
  • a computer program comprises one sequence of instructions.
  • a computer program comprises a plurality of sequences of instructions.
  • a computer program is provided from one location. In other embodiments, a computer program is provided from a plurality of locations.
  • a computer program includes one or more software modules.
  • a computer program includes, in part or in whole, one or more web applications, one or more mobile applications, one or more standalone applications, one or more web browser plug-ins, extensions, add-ins, or add-ons, or combinations thereof.
  • Non- limiting examples of computer programs that can be used with the present disclosure include databases that provide the identity of known linear epitopes for existing antibodies, including databases such as Bcipep, FIMM, and SVMTrip.
  • machine learning algorithms such as Hidden Markov Model (HMM) , Artificial Neural Network (ANN) , and Support Vector Machine (SVM) can be used to characterize binding domains that bind to one or more peptides specifically or off-target.
  • HMM Hidden Markov Model
  • ANN Artificial Neural Network
  • SVM Support Vector Machine
  • a computer program includes a web application.
  • a web application in various embodiments, utilizes one or more software frameworks and one or more database systems.
  • a web application is created upon a software framework such as Microsoft ® .NET or Ruby on Rails (RoR).
  • a web application utilizes one or more database systems including, by way of non-limiting examples, relational, non-relational, object oriented, associative, and XML database systems.
  • suitable relational database systems include, by way of non-limiting examples, Microsoft ® SQL Server, mySQLTM, and Oracle ® .
  • a web application in various embodiments, is written in one or more versions of one or more languages.
  • a web application may be written in one or more markup languages, presentation definition languages, client-side scripting languages, server-side coding languages, database query languages, or combinations thereof.
  • a web application is written to some extent in a markup language such as Hypertext Markup Language (HTML), Extensible Hypertext Markup Language (XHTML), or extensible Markup Language (XML).
  • a web application is written to some extent in a presentation definition language such as Cascading Style Sheets (CSS).
  • CSS Cascading Style Sheets
  • a web application is written to some extent in a client-side scripting language such as Asynchronous Javascript and XML (AJAX), Flash ® Actionscript, Javascript, or Silverlight ® .
  • AJAX Asynchronous Javascript and XML
  • Flash ® Actionscript Javascript
  • Javascript or Silverlight ®
  • a web application is written to some extent in a server- side coding language such as Active Server Pages (ASP), ColdFusion ® , Perl, JavaTM, JavaServer Pages (JSP), Hypertext Preprocessor (PHP), PythonTM, Ruby, Tel, Smalltalk, WebDNA ® , or Groovy.
  • a web application is written to some extent in a database query language such as Structured Query Language (SQL).
  • SQL Structured Query Language
  • a web application integrates enterprise server products such as IBM ® Lotus Domino ® .
  • a web application for providing a career development network for artists that allows artists to upload information and media files includes a media player element.
  • a media player element utilizes one or more of many suitable multimedia technologies including, by way of non-limiting examples, Adobe ® Flash ® , HTML 5, Apple ® QuickTime ® , Microsoft ® Silverlight ® , JavaTM, and Unity ® .
  • a computer program includes a mobile application provided to a mobile digital processing device.
  • the mobile application is provided to a mobile digital processing device at the time it is manufactured.
  • the mobile application is provided to a mobile digital processing device via the computer network described herein.
  • a mobile application is created by techniques known to those of skill in the art using hardware, languages, and development environments known to the art. Those of skill in the art will recognize that mobile applications are written in several languages. Suitable programming languages include, by way of non-limiting examples, C, C++, C#, Objective-C, JavaTM, Javascript, Pascal, Object Pascal, PythonTM, Ruby, VB.NET, WML, and XHTML/HTML with or without CSS, or combinations thereof.
  • Suitable mobile application development environments are available from several sources. Commercially available development environments include, by way of non-limiting examples, AirplaySDK, alcheMo, Appcelerator ® , Celsius, Bedrock, Flash Lite, .NET Compact Framework, Rhomobile, and WorkLight Mobile Platform. Other development environments are available without cost including, by way of non-limiting examples, Lazarus, MobiFlex, MoSync, and Phonegap. Also, mobile device manufacturers distribute software developer kits including, by way of non-limiting examples, iPhone and iPad (iOS) SDK, AndroidTM SDK, BlackBerry® SDK, BREW SDK, Palm® OS SDK, Symbian SDK, webOS SDK, and Windows® Mobile SDK.
  • iOS iPhone and iPad
  • a computer program includes a standalone application, which is a program that is run as an independent computer process, not an add-on to an existing process, e.g., not a plug-in.
  • a compiler is a computer program(s) that transforms source code written in a programming language into binary object code such as assembly language or machine code. Suitable compiled programming languages include, by way of non-limiting examples, C, C++, Objective-C, COBOL, Delphi, Eiffel, JavaTM, Lisp, PythonTM, Visual Basic, and VB .NET, or combinations thereof. Compilation is often performed, at least in part, to create an executable program.
  • a computer program includes one or more executable complied applications.
  • a software module comprises a file, a section of code, a programming object, a programming structure, or combinations thereof.
  • a software module comprises a plurality of files, a plurality of sections of code, a plurality of programming objects, a plurality of programming structures, or combinations thereof.
  • the one or more software modules comprise, by way of non-limiting examples, a web application, a mobile application, and a standalone application.
  • software modules are in one computer program or application. In other embodiments, software modules are in more than one computer program or application. In some embodiments, software modules are hosted on one machine. In other embodiments, software modules are hosted on more than one machine. In further embodiments, software modules are hosted on cloud computing platforms. In some embodiments, software modules are hosted on one or more machines in one location. In other embodiments, software modules are hosted on one or more machines in more than one location.
  • Embodiment 1-1 A method of designing an immunogen polypeptide to a target protein, the method comprising:
  • Embodiment 1-2 The method of Embodiment 1-1, further comprising mapping regions of the target protein bound by the existing antibodies.
  • Embodiment 1-3 The method of Embodiment 1-2, wherein mapping the region of the target protein bound by the existing antibodies comprises detecting binding of the existing antibodies to a library of peptides.
  • Embodiment 1-4 The method of Embodiment 1-3, wherein the library of peptides comprises peptides having amino acid sequences identical or a similar to a wildtype protein from one or more mammals.
  • Embodiment 1-5 The method of Embodiment 1-4, wherein the one or more mammals are selected from human, mouse, hamster, and monkey.
  • Embodiment 1-6 The method of Embodiment 1-5, wherein the monkey is selected from a cynomolgus monkey, a macaque monkey, and a rhesus macaque monkey.
  • Embodiment 1-7 The method of any one of Embodiments 1-2 to 1-6, wherein mapping the region of the target protein bound by the existing antibodies comprises detecting binding of the existing antibodies to a peptide array.
  • Embodiment 1-8 The method of Embodiment 1-7, wherein the peptide array comprises a diverse set of peptides.
  • Embodiment 1-9 The method of Embodiment 1-7, wherein the peptide array comprises a related set of peptides.
  • Embodiment I- 10 The method of Embodiment 1-9, wherein the related set of peptides are 75 % identical.
  • Embodiment 1-11 The method of any one of Embodiments 1-1 to I- 10, wherein obtaining a polypeptide sequence on the target protein bound by the existing antibodies comprises identifying peptide sequences bound by the existing antibodies from a database of peptide sequences known to be bound by the existing antibodies.
  • Embodiment 1-12 The method of any one of Embodiments 1-1 to 1-11 , wherein the method further comprises:
  • Embodiment 1-13 The method of Embodiment 1-12, wherein (i) comprises obtaining a structure of the target protein when natively folded.
  • Embodiment 1-14 The method of Embodiment 1-12, wherein (i) comprises obtaining a predicted structure of the target protein when natively folded from an algorithm.
  • Embodiment 1-15 The method of Embodiment 1-14, wherein the algorithm predicts the folded structure of the target protein.
  • Embodiment 1-16 The method of any one of Embodiments 1-1 to 1-15, wherein the immunogen polypeptide comprises at least 2 amino acids.
  • Embodiment 1-17 The method of any one of Embodiments 1-1 to 1-16, wherein the immunogen polypeptide comprises at least 30 amino acids.
  • Embodiment 1-18 The method of any one of Embodiments 1-1 to 1-17, wherein the immunogen polypeptide comprises at least one linker.
  • the linker comprises an amino acid sequence having one or more glycine residues, one or more serine residues, or one or more proline residues.
  • Embodiment 1-20 The method of Embodiment 1-18 or 1-19, wherein the linker has an amino acid sequence selected from at least one of GSG, (GGGGS)n, (GSG)n ,
  • Embodiment 1-21 The method of any one of Embodiments 1-1 to 1-20, wherein the method further comprises determining cross-species homology of the region not bound by the existing antibodies.
  • Embodiment 1-22 The method of any one of Embodiments 1-1 to 1-21, wherein the immunogen polypeptide comprises an amino acid sequence having at least 75% identity to a wildtype protein from one or more mammals.
  • Embodiment 1-2 The method of any one of Embodiments 1-1 to 1-22, wherein the immunogen polypeptide comprises at least two amino acid sequences from two distinct regions of the target protein not bound by the existing antibodies.
  • Embodiment 1-24 The method of any one of Embodiments 1-1 to 1-23, wherein the immunogen polypeptide is mutated such that the immunogen exhibits a different physical characteristic in a tumor microenvironment as compared to the normal tissue periphery.
  • Embodiment 1-25 The method of Embodiment 1-24, wherein the different physical characteristic include different protonated state in a tumor microenvironment as compared to the normal tissue periphery.
  • Embodiment 1-26 The method of Embodiment 1-25, wherein histi dines in the immunogen polypeptide are substituted with arginine or lysine.
  • Embodiment 1-27 The method of Embodiment 1-24, wherein the different physical characteristic includes protonated state, charge, structural conformation, hydrophobicity, hydrophilicity, reactive oxygen species status, or combinations thereof.
  • Embodiment 1-28 The method of any one of Embodiments 1-1 to 1-27, wherein the amino acid sequence of the region of the target protein comprises at least a first domain and a second domain.
  • Embodiment 1-29. The method of Embodiment 1-28, wherein the first domain and the second domain are operatively connected in a non-native order.
  • Embodiment 1-30 An immunogen comprising a region of a target protein not bound by existing antibodies, comprising:
  • Embodiment 1-3 The immunogen of Embodiment 1-30, further comprising mapping a region of the target protein bound by the existing antibodies.
  • Embodiment 1-32 The immunogen of Embodiment 1-31, wherein mapping the region of the target protein bound by the existing antibodies comprises detecting binding of the existing antibodies to a library of peptides.
  • Embodiment 1-33 The immunogen of Embodiment 1-32, wherein the library of peptides comprises peptides having amino acid sequences identical or similar to a wildtype protein from one or more mammals.
  • Embodiment 1-34 The immunogen of any one of Embodiments 1-30 to 1-33, wherein the immunogen comprises at least one region having an amino acid sequence common to one or more mammals are selected from human, mouse, hamster, and monkey.
  • Embodiment 1-35 The immunogen of Embodiment 1-34, wherein the monkey is selected from a cynomolgus monkey, a macaque monkey, and a rhesus macaque monkey.
  • Embodiment 1-36 The immunogen of any one of Embodiments 1-31 to 1-36, wherein mapping the region of the target protein bound by the existing antibodies comprises detecting binding of the existing antibodies to a peptide array.
  • Embodiment 1-37 The immunogen of Embodiment 1-36, wherein the peptide array comprises a diverse set of peptides.
  • Embodiment 1-38 The immunogen of Embodiment 1-36, wherein the peptide array comprises a related set of peptides.
  • Embodiment 1-39 The immunogen of Embodiment 1-38, wherein the related set of peptides are 75% identical.
  • Embodiment 1-40 The immunogen of any one of Embodiments 1-30 to 1-39, wherein obtaining a polypeptide sequence on the target protein bound by the existing antibodies comprises identifying peptide sequences bound by the existing antibodies from a database of peptide sequences known to be bound by the existing antibodies.
  • Embodiment 1-4 The immunogen of any one of Embodiments 1-30 1-40, further comprising:
  • Embodiment 1-42 The immunogen of Embodiment 1-41, wherein (i) comprises obtaining a structure of the target protein when natively folded.
  • Embodiment 1-43 The immunogen of Embodiment 1-41, wherein the structure of the natively -folded target protein is predicted from an algorithm.
  • Embodiment 1-44 The immunogen of Embodiment 1-43, wherein the algorithm predicts the folded structure of the target protein.
  • Embodiment 1-45 The immunogen of any one of Embodiments 1-30 to 1-44, wherein the immunogen polypeptide comprises at least 2 amino acids.
  • Embodiment 1-46 The immunogen of any one of Embodiments 1-30 to 1-45, wherein the immunogen polypeptide comprises at least 30 amino acids.
  • Embodiment 1-47 The immunogen of any one of Embodiments 1-30 to 1-46, wherein the immunogen polypeptide comprises at least one linker.
  • Embodiment 1-48 The immunogen of Embodiment 1-47, wherein the linker comprises an amino acid sequence having one or more glycine residues, one or more serine residues, or one or more proline residues.
  • Embodiment 1-49 The immunogen of Embodiment 1-47 or 1-48, wherein the linker has an amino acid sequence selected from at least one of GSG, (GGGGS)n, (GSG)n , GGGSGGGGS, GGGGSGGGS, (PGSG) n , PGSGSG where n is an integer between 1 and 10.
  • Embodiment 1-50 The immunogen of any one of Embodiments 1-30 to 1-49, further comprising determining cross-species homology of the region not bound by the existing antibodies.
  • Embodiment 1-51 The immunogen of any one of Embodiments 1-30 to 1-50, wherein the immunogen comprises an amino acid sequence having at least 75% identity to a wildtype protein from one or more mammals.
  • Embodiment 1-52 The immunogen of any one of Embodiments 1-30 to 1-51, wherein the immunogen polypeptide comprises at least two amino acid sequences from two distinct regions of the target protein not bound by the existing antibodies.
  • Embodiment 1-53 The immunogen of Embodiment 1-52, wherein the at least two amino acid sequences do not correspond to the sequential positions in the native protein.
  • Embodiment 1-54 The immunogen of any one of Embodiments 1-30 to 1-53, wherein the immunogen is mutated such that the immunogen exhibits a different physical characteristic in a tumor microenvironment as compared to the normal tissue periphery.
  • Embodiment 1-55 The immunogen of Embodiment 1-54, wherein the different physical characteristic include different protonated state in a tumor microenvironment as compared to the normal tissue periphery.
  • Embodiment 1-56 The immunogen of Embodiment 1-54, wherein histidines in the immunogen polypeptide are substituted with arginine or lysine.
  • Embodiment 1-57 The immunogen of Embodiment 1-54, wherein the different physical characteristic includes protonated state, charge, structural conformation, hydrophobicity, hydrophilicity, reactive oxygen species status, or combinations thereof.
  • Embodiment 1-58 The method of any one of Embodiments 1-30 to 1-57, wherein the amino acid sequence of the region of the target protein comprises at least a first domain and a second domain.
  • Embodiment 1-59 The method of Embodiment 1-58, wherein the first domain and the second domain are operatively connected in a non-native order.
  • Embodiment 1-60 A method of creating an antibody to a region of a target protein not bound by existing antibodies, the method comprising:
  • step e obtaining from the mammal an antibody that specifically binds to the region identified in step b), thereby creating the antibody.
  • Embodiment 1-61 The method of Embodiment 1-60, further comprising mapping of the target protein region(s) bound by the existing antibodies.
  • Embodiment 1-62 The method of Embodiment 1-61, wherein mapping the region of the target protein bound by the existing antibodies comprises detecting binding of the existing antibodies to a library of peptides.
  • Embodiment 1-63 The method of Embodiment 1-62, wherein the library of peptides comprises peptides having amino acid sequences identical or similar to a wildtype protein from one or more mammals.
  • Embodiment 1-64 The method of Embodiment 1-63, wherein the one or more mammals are selected from human, mouse, hamster, and monkey.
  • Embodiment 1-65 The method of Embodiment 1-64, wherein the monkey is selected from a cynomolgus monkey, a macaque monkey, and a rhesus macaque monkey.
  • Embodiment 1-66 The method of any one of Embodiments 1-61 to 1-65, wherein mapping the region of the target protein bound by the existing antibodies comprises detecting binding of the existing antibodies to a peptide array.
  • Embodiment 1-67 The method of any one of Embodiments 1-60 to 1-66, wherein obtaining a sequence on the target protein bound by the existing antibodies comprises identifying peptide sequences bound by the existing antibodies from a database of peptide sequences known to be bound by the existing antibodies.
  • Embodiment 1-68 The method of Embodiment 1-60 to 1-67, wherein the method further comprises:
  • Embodiment 1-69 The method of Embodiment 1-68, wherein (i) comprises obtaining a structure of the target protein when natively folded.
  • Embodiment 1-70 The method of Embodiment 1-68, wherein (i) comprises obtaining a predicted structure of the target protein when natively folded from an algorithm.
  • Embodiment 1-71 The method of any one of Embodiments 1-60 to 1-70, wherein immunizing the mammal with the immunogen polypeptide comprises administering at least one dose of a vaccine composition comprising the immunogen polypeptide and an adjuvant.
  • Embodiment 1-72 The method of Embodiment 1-70, wherein at least one dose comprises the target protein.
  • Embodiment 1-73 The method of Embodiment 1-70, wherein at least one dose does not comprise the target protein.
  • Embodiment 1-75 The method of claim 74, further comprising fusing the B cell with a myeloma cell to create a hybridoma which expresses the antibody.
  • Embodiment 1-76 The method of any one of Embodiments 1-60 to 1-75, wherein the method further comprises determining an epitope for the antibody.
  • Embodiment 1-77 The method of Embodiment 1-76, comprising binding the antibody to a focused array.
  • Embodiment 1-78 The method of Embodiment 1-77, wherein the focused array comprises a library of peptides having an amino acid sequence at least 100% identical to at least a portion of the immunogen polypeptide.
  • Embodiment 1-79 The method of any one of Embodiments 1-76 to 1-78, wherein determining the epitope for the antibody comprises measuring binding of the antibody to a library of peptides.
  • Embodiment 1-80 The method of any one of Embodiments 1-76 to 1-79, wherein determining the epitope for the antibody comprises measuring binding of the Ab measuring binding of the antibody to a peptide array.
  • Embodiment 1-81 The method of any one of Embodiments 1-76 to 1-80, wherein antibodies binding to two or more distinct epitopes are discarded.
  • Embodiment 1-82 The method of any one of Embodiments 1-60 to 1-81, wherein the method further comprises determining a biological effect for the antibody.
  • Embodiment 1-83 The method of Embodiment 1-82, wherein the biological effect for the antibody comprises at least one of inhibiting an activity of the target protein, increasing an activity of the target protein, inhibiting binding of the target protein to a binding partner, stabilizing binding of the target protein to a binding partner, increasing half-life of the target protein, and decreasing half-life of the target protein.
  • Embodiment 1-84 The method of any one of Embodiments 1-60 to 1-83, wherein the target protein is at least one of PD-1, PD-L1, CD25, IL2, MIF or CXCR4.
  • Embodiment 1-85 The method of any one of Embodiments 1-60 to 1-84, wherein the immunogen polypeptide comprises at least 2 amino acids.
  • Embodiment 1-86 The method of any one of Embodiments 1-60 to 1-85, wherein the immunogen polypeptide comprises at least 30 amino acids.
  • Embodiment 1-87 The method of any one of Embodiments 1-60 to 1-86, wherein the immunogen polypeptide comprises at least one linker.
  • Embodiment 1-88 The method of Embodiment 1-87, wherein the linker comprises an amino acid sequence having one or more glycine residues, one or more serine residues, or one or more proline residues.
  • Embodiment 1-89 The method of Embodiment 1-87 or 1-88, wherein the linker has an amino acid sequence selected from at least one of GSG, (GGGGS)n , (GSG)n ,
  • GGGSGGGGS and GGGGSGGGS, where n is an integer between 1 and 10.
  • Embodiment 1-90 The method of any one of Embodiments 1-60 to 1-89, further comprising determining cross-species homology of the target protein region not bound by the existing antibodies.
  • Embodiment 1-91 The method of any one of Embodiments 1-60 to 1-90, wherein the immunogen polypeptide comprises at least two amino acid sequences from two distinct regions of the target protein not bound by the existing antibodies.
  • Embodiment 1-92 The method of any one of Embodiments 1-60 to 1-91, wherein the immunogen polypeptide is mutated such that the immunogen exhibits a different physical characteristic in a tumor microenvironment as compared to the normal tissue periphery.
  • Embodiment 1-93 The method of Embodiment 1-92, wherein the different physical characteristic include different protonated state in a tumor microenvironment as compared to the normal tissue periphery.
  • Embodiment 1-94 The method of Embodiment 1-93, wherein histi dines in the immunogen polypeptide are substituted with arginine or lysine.
  • Embodiment 1-95 The method of Embodiment 1-94, wherein the different physical characteristic includes protonated state, charge, structural conformation, hydrophobicity, hydrophilicity, reactive oxygen species status, or combinations thereof.
  • Embodiment 1-96 The method of any one of Embodiments 1-60 to 1-95, wherein the amino acid sequence of the region of the target protein comprises at least a first domain and a second domain.
  • Embodiment 1-97 The method of Embodiment 1-96, wherein the first domain and the second domain are operatively connected in a non-native order.
  • Embodiment 1-98 An antibody created by the method of any one of Embodiments 1-60 to 1-97.
  • Embodiment 1-99 A method of treating an individual in need thereof, comprising: a) obtaining polypeptide sequence(s) on a target protein bound by existing antibodies; b) identifying at least one region of the target protein not bound by the existing antibodies;
  • step b obtaining from the mammal an antibody that specifically binds to the region identified in step b);
  • Embodiment I- 100 The method of Embodiment 1-99, wherein the process further comprises mapping a region of the target protein bound by the existing antibodies.
  • Embodiment I- 101 The method of Embodiment I- 100, wherein mapping the region of the target protein bound by the existing antibodies comprises detecting binding of the existing antibodies to a library of peptides.
  • Embodiment I- 102 The method of Embodiment I- 101 , wherein the library of peptides comprises peptides having amino acid sequences identical or similar to a wildtype protein from one or more mammals.
  • Embodiment 1-103 The method of Embodiment 1-102, wherein the one or more mammals are selected from human, mouse, hamster, and monkey.
  • Embodiment 1-104 The method of Embodiment 1-103, wherein the monkey is selected from a cynomolgus monkey, a macaque monkey, and a rhesus macaque monkey.
  • Embodiment 1-105 The method of any one of Embodiments I- 100 to 1-104, wherein mapping the region of the target protein bound by the existing antibodies comprises detecting binding of the existing antibodies to a peptide array.
  • Embodiment 1-106 The method of any one of Embodiments 1-99 to 1-105, wherein obtaining polypeptide sequence(s) on the target protein bound by the existing antibodies comprises identifying peptide sequences bound by the existing antibodies from a database of peptide sequences known to be bound by the existing antibodies.
  • Embodiment 1-107 The method of any one of Embodiments 1-99 to 1-106, wherein the process further comprises:
  • Embodiment 1-108 The method of Embodiment 1-107, wherein (i) comprises obtaining a structure of the target protein when natively folded.
  • Embodiment 1-109 The method of Embodiment 1-107, wherein (i) comprises obtaining a predicted structure of the target protein when natively folded from an algorithm.
  • Embodiment I- 110 The method of Embodiment 1-109, wherein the algorithm predicts the folded structure of the target protein.
  • Embodiment 1-11 1. The method of any one of Embodiments 1-99 to 1-109, wherein immunizing the mammal with the immunogen polypeptide comprises administering at least one dose of a vaccine composition comprising the immunogen polypeptide and an adjuvant.
  • Embodiment 1-112. The method of Embodiment I- 11 1 , wherein at least one dose comprises the target protein.
  • Embodiment 1-113 The method of Embodiment 1-11 1, wherein at least one dose does not comprise the target protein.
  • Embodiment 1-114 The method of any one of Embodiments 1-99 to 1-113, wherein creating the antibody from the mammal comprises isolating a B cell which expresses the antibody.
  • Embodiment 1-115 The method of Embodiment 1-114, further comprising fusing the B cell with a myeloma cell to create a hybridoma which expresses the antibody.
  • Embodiment 1-116 The method of any one of Embodiments 1-99 to I- 115, wherein the method further comprises determining an epitope for the antibody.
  • Embodiment 1-117 The method of Embodiment 1-116, wherein determining the epitope for the antibody comprises measuring binding of the antibody to a library of peptides.
  • Embodiment 1-118 The method of Embodiment 1-116, wherein determining the epitope for the antibody comprises measuring binding of the antibody to a peptide array.
  • Embodiment 1-119 The method of any one of Embodiments 1-1 16 to 1-120, wherein antibodies binding to two or more distinct epitopes are discarded.
  • Embodiment 1-120 The method of any one of Embodiments 1-99 to 1-119, wherein the method further comprises determining a biological effect for the antibody.
  • Embodiment 1-121 The method of Embodiment 1-120, wherein the biological effect for the antibody comprises at least one of inhibiting an activity the target protein, increasing an activity of the target protein, inhibiting binding of the target protein to a binding partner, stabilizing binding of the target protein to a binding partner, increasing half-life of the target protein, and decreasing half-life of the target protein.
  • Embodiment 1-122 The method of any one of Embodiments 1-99 to 1-121, wherein the target protein is at least one of PD-1, PD-L1, CD25, IL2, MIF or CXCR4.
  • Embodiment 1-123 The method of any one of Embodiments 1-99 to 1-122, wherein the immunogen polypeptide comprises at least 2 amino acids.
  • Embodiment 1-124 The method of any one of Embodiments 1-99 to 1-123, wherein the immunogen polypeptide comprises at least 30 amino acids.
  • Embodiment 1-125 The method of any one of Embodiments 1-99 to 1-124, wherein the immunogen polypeptide comprises at least one linker.
  • Embodiment 1-126 The method of Embodiment 1-125, wherein the linker comprises an amino acid sequence having one or more glycine residues, one or more serine residues, or one or more proline residues.
  • Embodiment 1-127 The method of Embodiment 1-125 or 1-126, wherein the linker has an amino acid sequence selected from at least one of GSG, (GGGGS)n, (GSG)n , GGGSGGGGS, GGGGSGGGS, (PGSG) n , PGSGSG where n is an integer between 1 and 10.
  • Embodiment 1-128 The method of any one of Embodiments 1-99 to 1-127, wherein the method further comprises determining cross-species homology of the region not bound by the existing antibodies.
  • Embodiment 1-129 The method of any one of Embodiments 1-99 to 1-128, wherein the immunogen polypeptide comprises at least two amino acid sequences from two distinct regions of the target protein not bound by the existing antibodies.
  • Embodiment 1-130 The method of any one of Embodiments 1-99 to 1-129, wherein the immunogen polypeptide is mutated such that the immunogen exhibits a different physical characteristic in a tumor microenvironment as compared to the normal tissue periphery.
  • Embodiment 1-131 The method of Embodiment 1-130, wherein the different physical characteristic include different protonated state in a tumor microenvironment as compared to the normal tissue periphery.
  • Embodiment 1-132 The method of Embodiment 1-130, wherein histidines in the immunogen polypeptide are substituted with arginine or lysine.
  • Embodiment 1-133 The method of Embodiment 1-130, wherein the different physical characteristic includes protonated state, charge, structural conformation, hydrophobicity, hydrophilicity, reactive oxygen species status, or combinations thereof.
  • Embodiment 1-134 An antibody for use as a medicament, wherein the antibody is produced by:
  • step b obtaining from the mammal an antibody that specifically binds to the region identified in step b).
  • Embodiment 1-135. The antibody for use of Embodiment 1-134, wherein the process further comprises mapping a region of the target protein bound by the existing antibodies.
  • Embodiment 1-136 The antibody for use of Embodiment 1-135, wherein mapping the region of the target protein bound by the existing antibodies comprises detecting binding of the existing antibodies to a library of peptides.
  • Embodiment 1-137 The antibody for use of Embodiment 1-136, wherein the library of peptides comprises peptides having amino acid sequences identical or similar to a wildtype protein from one or more mammals.
  • Embodiment 1-138 The antibody for use of Embodiment 1-137, wherein the one or more mammals are selected from human, mouse, hamster, and monkey.
  • Embodiment 1-139 The antibody for use of Embodiment 1-138, wherein the monkey is selected from a cynomolgus monkey, a macaque monkey, and a rhesus macaque monkey.
  • Embodiment 1-140 The antibody for use of any one of Embodiments 1-135 to I-
  • mapping the region of the target protein bound by the existing antibodies comprises detecting binding of the existing antibodies to a peptide array.
  • Embodiment 1-141 The antibody for use of any one of Embodiments 1-34 to I-
  • obtaining polypeptide sequence(s) on the target protein bound by the existing antibodies comprises identifying peptide sequences bound by the existing antibodies from a database of peptide sequences known to be bound by the existing antibodies.
  • Embodiment 1-142 The antibody for use of any one of Embodiments 1-134 to I-
  • Embodiment 1-143 The antibody for use of Embodiment 1-142, wherein (i) comprises obtaining a structure of the target protein when natively folded.
  • Embodiment 1-144 The antibody for use of Embodiment 1-142, wherein (i) comprises obtaining a predicted structure of the target protein when natively folded from an algorithm.
  • Embodiment 1-145 The antibody for use of Embodiment 1-144, wherein the algorithm predicts the folded structure of the target protein.
  • Embodiment 1-146 The antibody for use of any one of Embodiments 1-134 to I- 144, wherein immunizing the mammal with the immunogen polypeptide comprises administering at least one dose of a vaccine composition comprising the immunogen polypeptide and an adjuvant.
  • Embodiment 1-147 The antibody for use of Embodiment 1-146, wherein at least one dose comprises the target protein.
  • Embodiment 1-148 The antibody for use of Embodiment 1-146, wherein at least one dose does not comprise the target protein.
  • Embodiment 1-149 The antibody for use of any one of Embodiments 1-134 to I- 148, wherein creating the antibody from the mammal comprises isolating a B cell which expresses the antibody.
  • Embodiment 1-150 The antibody for use of Embodiment 1-149, further comprising fusing the B cell with a myeloma cell to create a hybridoma which expresses the antibody.
  • Embodiment 1-151 The antibody for use of any one of Embodiments 1-134 to I- 150, wherein the method further comprises determining an epitope for the antibody.
  • Embodiment 1-152 The antibody for use of Embodiment 1-151, wherein determining the epitope for the antibody comprises measuring binding of the antibody to a library of peptides.
  • Embodiment 1-153 The antibody for use of Embodiment 1-151 or 1-152, wherein determining the epitope for the antibody comprises measuring binding of the antibody to a peptide array.
  • Embodiment 1-154 The antibody for use of any one of Embodiments 1-151 to I-
  • Embodiment 1-155 The antibody for use of any one of Embodiments 1-134 to I-
  • the method further comprises determining a biological effect for the antibody.
  • Embodiment 1-156 The antibody for use of Embodiment 1-155, wherein the biological effect for the antibody comprises at least one of inhibiting an activity the target protein, increasing an activity of the target protein, inhibiting binding of the target protein to a binding partner, stabilizing binding of the target protein to a binding partner, increasing half-life of the target protein, and decreasing half-life of the target protein.
  • Embodiment 1-157 The antibody for use of any one of Embodiments 1-134 to I-
  • target protein is at least one of PD-1, PD-L1, CD25, IL2, MIF or CXCR4.
  • Embodiment 1-158 The antibody for use of any one of Embodiments 1-134 to I-
  • immunogen polypeptide comprises at least 2 amino acids.
  • Embodiment 1-159 The antibody for use of any one of Embodiments 1-134 to I-
  • Embodiment 1-160 The antibody for use of any one of Embodiments 1-134 to I- 159, wherein the immunogen polypeptide comprises at least one linker.
  • Embodiment 1-161 The antibody for use of Embodiment 1-160, wherein the linker comprises an amino acid sequence having one or more glycine residues, one or more serine residues, or one or more proline residues.
  • Embodiment 1-162 The antibody for use of Embodiment 1-160 or 1-161, wherein the linker has an amino acid sequence selected from at least one of GSG, (GGGGS)n, (GSG)n , GGGSGGGGS, GGGGSGGGS, (PGSG) n , PGSGSG where n is an integer between 1 and 10.
  • Embodiment 1-163 The antibody for use of any one of Embodiments 1-134 to I-
  • the method further comprises determining cross-species homology of the region not bound by the existing antibodies.
  • Embodiment 1-164 The antibody for use of any one of Embodiments 1-134 to I-
  • immunogen polypeptide comprises at least two amino acid sequences from two distinct regions of the target protein not bound by the existing antibodies.
  • Embodiment 1-165 The antibody for use of any one of Embodiments 1-134 to I-
  • the immunogen polypeptide is mutated such that the immunogen exhibits a different physical characteristic in a tumor microenvironment as compared to the normal tissue periphery.
  • Embodiment 1-166 The antibody for use of Embodiment 1-165, wherein the different physical characteristic include different protonated state in a tumor
  • Embodiment 1-167 The antibody for use of Embodiment 1-166, wherein histi dines in the immunogen polypeptide are substituted with arginine or lysine.
  • Embodiment 1-168 The antibody for use of Embodiment 1-165, wherein the different physical characteristic includes protonated state, charge, structural conformation, hydrophobicity, hydrophilicity, reactive oxygen species status, or combinations thereof.
  • Embodiment 1-169 An antibody for use in the manufacture of a medicament, wherein the antibody is produced by:
  • step b obtaining from the mammal an antibody that specifically binds to the region identified in step b).
  • Embodiment 1-170 The antibody for use of Embodiment 1-169, wherein the process further comprises mapping a region of the target protein bound by the existing antibodies.
  • Embodiment 1-171 The antibody for use of Embodiment 1-170, wherein mapping the region of the target protein bound by the existing antibodies comprises detecting binding of the existing antibodies to a library of peptides.
  • Embodiment 1-172 The antibody for use of Embodiment 1-171, wherein the library of peptides comprises peptides having amino acid sequences identical or similar to a wildtype protein from one or more mammals.
  • Embodiment 1-173 The antibody for use of Embodiment 1-172, wherein the one or more mammals are selected from human, mouse, hamster, and monkey.
  • Embodiment 1-174 The antibody for use of Embodiment 1-173, wherein the monkey is selected from a cynomolgus monkey, a macaque monkey, and a rhesus macaque monkey.
  • Embodiment 1-175. The antibody for use of any one of Embodiments 1-170 to I- 174, wherein mapping the region of the target protein bound by the existing antibodies comprises detecting binding of the existing antibodies to a peptide array.
  • Embodiment 1-176 The antibody for use of Embodiment 1-175, wherein obtaining polypeptide sequence(s) on the target protein bound by the existing antibodies comprises identifying peptide sequences bound by the existing antibodies from a database of peptide sequences known to be bound by the existing antibodies.
  • Embodiment 1-177 The antibody for use of Embodiment 1-169, wherein the process further comprises: (i) identifying amino acids that reside on the surface of the target protein when natively folded; and
  • Embodiment 1-178 The antibody for use of Embodiment 1-177, wherein (i) comprises obtaining a structure of the target protein when natively folded.
  • Embodiment 1-179 The antibody for use of Embodiment 1-177, wherein (i) comprises obtaining a predicted structure of the target protein when natively folded from an algorithm.
  • Embodiment 1-180 The antibody for use of Embodiment 1-179, wherein the algorithm predicts the folded structure of the target protein.
  • Embodiment 1-181 The antibody for use of any one of Embodiments 1-169 to I- 180, wherein immunizing the mammal with the immunogen polypeptide comprises administering at least one dose of a vaccine composition comprising the immunogen polypeptide and an adjuvant.
  • Embodiment 1-182 The antibody for use of Embodiment 1-181, wherein at least one dose comprises the target protein.
  • Embodiment 1-183 The antibody for use of Embodiment 1-181, wherein at least one dose does not comprise the target protein.
  • Embodiment 1-184 The antibody for use of any one of Embodiments 1-169 to I-
  • creating the antibody from the mammal comprises isolating a B cell which expresses the antibody.
  • Embodiment 1-185 The antibody for use of Embodiment 1-184, further comprising fusing the B cell with a myeloma cell to create a hybridoma which expresses the antibody.
  • Embodiment 1-186 The antibody for use of any one of Embodiments 1-169 to I-
  • the method further comprises determining an epitope for the antibody.
  • Embodiment 1-187 The antibody for use of Embodiment 1-186, wherein determining the epitope for the antibody comprises measuring binding of the antibody to a library of peptides.
  • Embodiment 1-188 The antibody for use of Embodiment 1-186, wherein determining the epitope for the antibody comprises measuring binding of the antibody to a peptide array.
  • Embodiment 1-189 The antibody for use of any one of Embodiments 1-186 to I-
  • Embodiment 1-190 The antibody for use of any one of Embodiments 1-169 to I-
  • the method further comprises determining a biological effect for the antibody.
  • Embodiment 1-191 The antibody for use of Embodiment 1-190, wherein the biological effect for the antibody comprises at least one of inhibiting an activity the target protein, increasing an activity of the target protein, inhibiting binding of the target protein to a binding partner, stabilizing binding of the target protein to a binding partner, increasing half-life of the target protein, and decreasing half-life of the target protein.
  • Embodiment 1-192 The antibody for use of any one of Embodiments 1-169 to I-
  • target protein is at least one of PD-1, PD-L1, CD25, IL2, MIF or CXCR4.
  • Embodiment 1-193 The antibody for use of any one of Embodiments 1-169 to I-
  • the immunogen polypeptide comprises at least 2 amino acids.
  • Embodiment 1-194 The antibody for use of any one of Embodiments 1-169 to I-
  • immunogen polypeptide comprises at least 30 amino acids.
  • Embodiment 1-195 The antibody for use of any one of Embodiments 1-169 to I-
  • immunogen polypeptide comprises at least one linker.
  • Embodiment 1-196 The antibody for use of Embodiment 1-195, wherein the linker comprises an amino acid sequence having one or more glycine residues, one or more serine residues, or one or more proline residues.
  • Embodiment 1-197 The antibody for use of Embodiment 1-195 or 1-196, wherein the linker has an amino acid sequence selected from at least one of GSG, (GGGGS)n, (GSG)n , GGGSGGGGS, GGGGSGGGS, (PGSG) n , PGSGSG where n is an integer between 1 and 10.
  • Embodiment 1-198 The antibody for use of any one of Embodiments 1-169 to I- 197, wherein the method further comprises determining cross-species homology of the region not bound by the existing antibodies.
  • Embodiment 1-199 The antibody for use of any one of Embodiments 1-169 to I-
  • the immunogen polypeptide comprises at least two amino acid sequences from two distinct regions of the target protein not bound by the existing antibodies.
  • Embodiment 1-200 The antibody for use of any one of Embodiments 1-169 to I-
  • the immunogen polypeptide is mutated such that the immunogen exhibits a different physical characteristic in a tumor microenvironment as compared to the normal tissue periphery.
  • Embodiment 1-201. The antibody for use of Embodiment 1-200, wherein the different physical characteristic include different protonated state in a tumor
  • Embodiment 1-202. The antibody for use of Embodiment 1-201, wherein histi dines in the immunogen polypeptide are substituted with arginine or lysine.
  • Embodiment 1-203 The antibody for use of Embodiment 1-200, wherein the different physical characteristic includes protonated state, charge, structural conformation, hydrophobicity, hydrophilicity, reactive oxygen species status, or combinations thereof.
  • Embodiment 1-204. An immunogen comprising at least two domains of a target protein, the immunogen comprising a polypeptide sequence of a first domain of the target protein operatively connected to a polypeptide sequence of a second domain of the target protein in a non-native order.
  • Embodiment 1-205 The immunogen of Embodiment 1-204, wherein the first domain of the target protein is operatively connected to the second domain of the target protein by a linker molecule.
  • Embodiment 1-206 The immunogen of Embodiment 1-204 or 1-205, wherein the target protein is a therapeutic target protein.
  • Embodiment 1-207 The method of any one of Embodiments 1-1 to 1-29, wherein the target protein is a therapeutic target protein.
  • Embodiment 1-208 The immunogen of any one of Embodiments 1-30 to 1-59, wherein the target protein is a therapeutic target protein.
  • Embodiment 1-209 The method of any one of Embodiments 1-60 to 1-97, wherein the target protein is a therapeutic target protein.
  • Embodiment 1-210 The method of any one of Embodiments 1-99 to 1-133, wherein the target protein is a therapeutic target protein.
  • Embodiment 1-211 The antibody for use of any one of Embodiments 1-134 to I- 168, wherein the target protein is a therapeutic target protein.
  • Embodiment 1-212 The antibody for use of any one of Embodiments 1-169 to I- 203, wherein the target protein is a therapeutic target protein.
  • Example 1 Designing a Novel Immunogen for PD-1
  • the VI 3 library was a diverse combinatorial library of 126,009 peptides with a median length of 9 residues, ranging from 5 to 13 amino acids, and designed to include 99.9% of all possible 4-mers and 48.3% of all possible 5-mers of 16 amino acids (methionine, M;
  • cysteine, C; isoleucine, I; and threonine, T were excluded).
  • the peptides were synthesized on an 200 mm silicon oxide wafer using standard semiconductor photolithography tools adapted for /er/-butyloxycarbonyl (BOC) protecting group peptide chemistry. Regions of the protein not bound by Nivolumab were determined and a three dimensional structure of PD-1 was obtained in order to determine which of these regions were found on the surface of the folded protein. Results of the mapping and three dimensional structure analysis are shown in FIG. 2.
  • RRNDSGAYLSGAISL (SEQ ID NO: 2) (amino acids 114-128) by a GSG linker.
  • the N- terminus to C-terminus directionality of each sequence was maintained, but the order of the sequences was reversed relative to their positions/order in the native PD-1 protein to provide a PD-1 immunogen having the second sequence linked to the first sequence.
  • LNWARMSPSNQTDK (SEQ ID NO: 3) and its location on the 3-D structure of PD-1 are shown in FIG. 3.
  • FIG. 3 Various other exemplary PD-1 immunogens were designed using different parameters and the sequences are shown in FIG. 4. The immunogens shown in FIG. 4 were designed to reverse the order of two identified sequences as given for the immunogen of FIG. 1.
  • Amino acids at position 68 N (mouse) and Y (human and cynomolgus monkey) were substituted by A to reduce the size of the side chain of N and Y.
  • amino acids at position 120 I (mouse) and T (human and cynomolgus monkey), were substituted by A.
  • Substitution of 2 of 32 amino acids provided an immunogen having 93.75% identity (2/32aa) to each of the native protein sequences.
  • a mouse or a rabbit is immunized with a full length protein or one of the selected immunogens.
  • the mouse or rabbit is then boosted with either the full length protein or one of the selected immunogens.
  • B cells are isolated from the immunized mouse or rabbit and cells are fused to make a hybridoma.
  • ELISA is used to test monoclonal antibodies for binding to the full length protein or one of the selected immunogens. Further fusions and refining of the monoclonal antibody are conducted as needed.
  • Example 3 Designing a Novel Immunogen for CD-25: Illustrative First Design
  • CD-25 In order to design a novel immunogen for CD-25, the human, mouse, and cynomolgus monkey sequences of CD-25 were aligned and regions having high similarity were identified. Next the regions of CD-25 bound by an existing antibody, Daclizumab or Baciliximab were determined using an epitope mapping on a V13 peptide array. Regions of the protein not bound by Daclizumab and by Baciliximab were determined and a three dimensional structure of CD-25 was obtained in order to determine which of these regions were found on the surface of the folded protein. A resulting immunogen sequence and its location on the 3-D structure of CD-25 are determined.
  • results of the mapping and three dimensional structure analysis of an illustrative design for CD-25 are shown in FIG. 6.
  • the immunogen illustrated on FIG. 6 comprises two histidine's and it illustrates the surrounding residues that may have pH-dependent conformational changes.
  • FIG. 6 also shows that the immunogen overlaps with a portion of the IL2 ligand binding domain that is not covered by existing therapeutics in later-stage development.
  • the immunogen illustrated on FIG. 6 comprises a disulfide bond for enhanced stability and conformational retention.
  • the immunogen illustrated on FIG. 6 comprises point mutations designed to minimize a likelihood of cross-reactivity with mouse and a cynomolgus monkey.
  • the CD-25 immunogen can comprise the entirety or a portion of the sequence GHCREGS GRIYHF VGGQGSGSGAYKEGTMLNGS GYMACTGNS S AS SW (SEQ ID NO: 4), illustrated in a three-dimensional model on FIG. 6.
  • CD-25 In order to design a novel immunogen for CD-25, the human, mouse, and cynomolgus monkey sequences of CD-25 were aligned and regions having high similarity were identified. Next the regions of CD-25 bound by an existing antibody, Daclizumab or Baciliximab were determined using an epitope mapping on a V 13 peptide array. Regions of the protein not bound by Daclizumab and by Baciliximab were determined and a three dimensional structure of CD-25 was obtained in order to determine which of these regions were found on the surface of the folded protein. A resulting immunogen sequence and its location on the 3-D structure of CD-25 are determined.
  • results of the mapping and three dimensional structure analysis of an illustrative design are shown in FIG. 7.
  • the immunogen illustrated on FIG. 7 covers the entire IL2 interface and comprises two histidines.
  • the CD-25 immunogen can comprise the entirety or a portion of the sequence
  • CD-25 In order to design a novel immunogen for CD-25, the human, mouse, and cynomolgus monkey sequences of CD-25 were aligned and regions having high similarity were identified. Next the regions of CD-25 bound by an existing antibody, Daclizumab or Baciliximab were determined using an epitope mapping on a V 13 peptide array. Regions of the protein not bound by Daclizumab and by Baciliximab were determined and a three dimensional structure of CD-25 was obtained in order to determine which of these regions were found on the surface of the folded protein. A resulting immunogen sequence and its location on the 3-D structure of CD-25 are determined.
  • results of the mapping and three dimensional structure analysis of an illustrative design are shown in FIG. 8.
  • the immunogen illustrated on FIG. 8 divides CD25 into two domains retaining all histidines.
  • the CD-25 immunogen described herein can comprise the entirety or a portion of the sequence
  • CD-25 In order to design a novel immunogen for CD-25, the human, mouse, and cynomolgus monkey sequences of CD-25 were aligned and regions having high similarity were identified. Next the regions of CD-25 bound by an existing antibody, Daclizumab or Baciliximab were determined using an epitope mapping on a V 13 peptide array. Regions of the protein not bound by Daclizumab and by Baciliximab were determined and a three dimensional structure of CD-25 was obtained in order to determine which of these regions were found on the surface of the folded protein. A resulting immunogen sequence and its location on the 3-D structure of CD-25 are determined.
  • results of the mapping and three dimensional structure analysis of an illustrative design are shown in FIG. 8.
  • the immunogen illustrated on FIG. 8 divides CD25 into two domains retaining all histidines.
  • the CD-25 immunogen described herein can comprise the entirety or a portion of the sequence
  • ELCDDDPPEIPHATFKAMAYFVVGQMVYYQCVQGYRALHRGPAESVCKMTHGKTR WTQPQLICT (SEQ ID NO: 7), illustrated in the colors white and purple within a three- dimensional model on FIG. 8.
  • Example 7 Designing a Novel Immunogen for CD-25: Illustrative Fifth Design
  • CD-25 immunogen for CD-25, the human, mouse, and cynomolgus monkey sequences of CD-25 were aligned and regions having high similarity were identified. Next the regions of CD-25 bound by an existing antibody, Daclizumab or Baciliximab were determined using an epitope mapping on a V 13 peptide array. Regions of the protein not bound by Daclizumab and by Baciliximab were determined and a three dimensional structure of CD-25 was obtained in order to determine which of these regions were found on the surface of the folded protein. A resulting immunogen sequence and its location on the 3-D structure of CD-25 are determined. The immunogen described herein divides CD25 into two domains, mutating all histidines to arginine's to mimic low tumor pH. The CD-25 immunogen described herein can comprise the entirety or a portion of the sequence
  • Example 8 Designing a Novel Immunogen for CD-25: Illustrative Sixth Design
  • CD-25 immunogen for CD-25, the human, mouse, and cynomolgus monkey sequences of CD-25 were aligned and regions having high similarity were identified. Next the regions of CD-25 bound by an existing antibody, Daclizumab or Baciliximab were determined using an epitope mapping on a V 13 peptide array. Regions of the protein not bound by Daclizumab and by Baciliximab were determined and a three dimensional structure of CD-25 was obtained in order to determine which of these regions were found on the surface of the folded protein. A resulting immunogen sequence and its location on the 3-D structure of CD-25 are determined. The immunogen described herein divides CD25 into two domains, mutating all histi dines to arginine's to mimic low tumor pH. The CD-25 immunogen described herein can comprise the entirety or a portion of the sequence
  • ELCDDDPPEIPRATFKAMAYFVVGQMVYYQCVQGYRALRRGPAESVCKMTRGKTR WTQPQLICT (SEQ ID NO: 9).
  • ESVSKMTHGKTRWTQPQ 160 (SEQ ID NO: 10).
  • Example 10 Designing a Novel Immunogen for IL2 binding domain of CD25: Illustrative Eight Design
  • an additional novel immunogen was designed for IL2 binding domain of CD25.
  • the immunogen described herein as seen in FIG. 10, divides IL2 binding domain of CD25_into two separate domains, and includes the sequence 8 PEIPH 12 -GP- 134 GYRALHRGPAE 144 -GP- 159 PQLIST 164 (SEQ ID NO: 11).
  • CXC chemokine receptor 4 (CXCR4) is overexpressed in many cancers including non-small cell lung cancer (NSCLC) and triple negative breast cancer (TNBC).
  • NSCLC non-small cell lung cancer
  • TNBC triple negative breast cancer
  • the human, mouse, and cynomolgus monkey sequences of CXCR4 can be aligned and regions having high similarity were identified.
  • the regions of CXCR4 bound by an existing antibody can be determined using an epitope mapping on a VI 3 or V14 OR V15 OR V16 peptide array.
  • the VI 3, VI 4, and VI 5 peptide arrays are described in greater detail in U.S. Provisional Application No.
  • the VI 3 peptide array was a diverse combinatorial library of 126,009 peptides with a median length of 9 residues, ranging from 5 to 13 amino acids, and designed to include 99.9% of all possible 4-mers and 48.3% of all possible 5-mers of 16 amino acids (methionine, M;
  • the V14 peptide array was prepared as a library of 16,920 peptides using a series of 24 overlapping masks, which resulted in synthesized peptides with a median length of 0 to 17 amino acid residues.
  • the V15 peptide array comprised 3.3M peptides, and was designed to include 100% of all possible 4-mers to 6-mers.
  • the V16 array is a 3.3M feature array of 3.2M unique peptides.
  • the V 16 array comprises a library of peptides synthesized from 18 of the 20 naturally occurring amino acids by excluding cysteine (C) and methionine (M).
  • Peptides range from 5 to 16 amino acids in length, having a median length of 8 amino acids.
  • the array comprises a low-bias library of peptides, which is a high sequence-diversity library of unique peptides designed to cover sequence space evenly based on the 18 amino acids; and a library of peptides designed to map particular sequences including epitope sequences.
  • Regions of the protein not bound by the CXCR4 antibody can be determined and a three dimensional structure of CXCR4 can be obtained in order to determine which of these regions were found on the surface of the folded protein.
  • an immunogen can be designed to contain two sequences of residues present on the surface of CXCR4 that are known not to be bound by the CXCR4 antibody.
  • Various other exemplary CXCR4 immunogens can be designed using different parameters. Cyclized versions of the above mentioned peptide arrays could be utilized to design immunogens.
  • Macrophage migration inhibitory factor also known as glycosylation-inhibiting factor (GIF), L-dopachrome isomerase, or phenylpyruvate tautomerase is a protein that is an important regulator of innate immunity.
  • MIF may function as a ligand for CD74 and/or CXCR4 receptors. MIF is involved in catalyzing the tautormerization of p-hydroxyphenylpyruvate to D-dopachrome, the catalytic domain possibly localized near the N-terminus of the protein.
  • MIF contributes to both autoimmune disorders (by, for example, blocking the effects of steroids) and cancer (by, for example, possessing proliferative, anti-apoptotic, migration and/or other tumor stroma activity).
  • MIF-1 shares approximately 30% homology with MIF-2 (also known as DDT), which has a similar function to MIF-1.
  • MIF-2 also known as DDT
  • MIF-1 shares approximately 30% homology with MIF-2 (also known as DDT), which has a similar function to MIF-1.
  • Regions of the protein not bound by the MIF antibody can be determined and a three dimensional structure of MIF can be obtained in order to determine which of these regions are present on the surface of the folded protein.
  • an immunogen can be designed to contain two sequences of residues present on the surface of MIF that are known not to be bound by the MIF antibody.
  • Various other exemplary MIF immunogens can be designed using different parameters.
  • Example 13 Designing a Hybrid MIF-l/MIF-2 (MIF-HD Immunogen
  • hybrid MIF-l/MIF-2 hybrid MIF-HT
  • Antibody attributes may include, for example, the inhibition of both MIF-1 and MIF-2 (DDT) activity, the blocking or inhibition of catalytic activity, the blocking or inhibition of CD74 binding and/or the blocking or inhibition of CXCR4 binding.
  • the design may include a monoclonal antibody centered or approximately around lysine 32 (K32), which could potentially block all activities.
  • the immunogen described herein is a hybrid (MIF-HT) between MIF-1 and MIF-2 (DDT) sequences, in which residues in common are preserved and residues that differ are mutated to less-biasing residues (e.g., mutating charged amino acid residues to alanine). Additionally, asparagine at position 111 (Nl 11) of MIF-1, which corresponds to position 112 in the alignment in FIG. 12 has been mutated to cysteine to force the expressed protein into its physiologically -relevant trimeric form. This should allow for the identification of an antibody that blocks both MIF-1 and MIF-2 binding to their receptors in vivo.
  • An exemplary sequence of the hybrid immunogen MIF-HT as shown in FIG. 12 includes the sequence:
  • FIG. 12 depicts an alignment of mouse, human, cynomolgus monkey and rabbit MIF-1 (A) and MIF-2 (DDT; B).
  • FIG. 14 depicts an exemplary model of MIF-1 (A) and MIF-2 (B); the locations of residues common to MIF-1 and MIF-2, which correspond to the residues shown in red in the alignment of FIG. 12.
  • mice are immunized with the hybrid MIF-l/MIF-2 sequence, MIF-HT, and antibodies screened and isolated as detailed above.
  • An alternative immunization strategy may include first immunizing with MIF-1, then boosting with an immunogen directed against MIF-2, as see in FIG. 12. The procedure is repeated and the antibodies screened for cross-reactivity with MIF-1 and MIF-2 (DDT) immunogens.
  • immunogens can be expressed in E. coli, wherein the immunogens are preferably tagged at the C-terminus, although no tags may be used for immunogen expression. Tagging at the N-terminus will likely perturb immunogen structure, and should be avoided.
  • the phage display mimotope MSTPLGQYTGTK SEQ ID NO: 13
  • K32 lysine 32 residue
  • Cohort 1 was immunized with the full-length PD-1 protein.
  • Cohorts 2 and 3 were immunized with Peptide Immunogen 1, which is immunogen 1 in FIG. 4, and has the sequence RRNDSGAYLSGAISLHPDLMLNWARMSPSNQTDK (SEQ ID NO: 14).
  • Cohort 2 also received boosters comprising the full-length PD-1 protein in combination with the immunogen 1, as shown on the summary chart.
  • Cohorts 4 and 5 were immunized with Peptide Immunogen 2, which is immunogen 4 in FIG.
  • FIG. 17 ' is a graph depicting ELIS A data detecting binding of sera from selected mice in Cohorts 1, 2, and 4, and non-immunized mouse sera, to Peptide Immunogen 1. As can be seen in this graph, sera from mice that were not immunized or that were immunized with the full-length PD-1 did not bind to Peptide Immunogen 1.
  • FIG. 18 is a graph depicting ELIS A data detecting binding of sera from the same mice to full-length human PD-1. As seen in this figure, sera from all of the immunized mice tested bind to PD-1.
  • FIG. 19 is a graph of the flow cytometry results, evaluating sera from selected mice in Cohort 3 and Cohort 5, and sera from a mouse that was not immunized. All of the evaluated sera from immunized mice exhibited binding to the PD-1 expressing CHO cells, while the sera from a non-immunized mouse exhibited no binding. Additional flow cytometry data is presented in FIG. 20, evaluating antibody (serum) binding of each mouse cohort to PD-1 expressing CHO cells (graphs second from left) and control cells that did not express PD-1 (right-most graphs). The histograms with a solid outline are hybridoma libraries at 1/2 dilution, while the ones without solid outline indicate cells + secondary only.
  • FIG. 21A is a graph of the binding of sera from Cohorts 1, 2, and 4 to human PD-1, as evaluated by ELIS A. The x-axis is the dilution factor of the sample. Cohort 1 is the top line. Cohort 2 and Cohort 4 are overlapping, with Cohort 2 the darker line above Cohort 4 after 1000 factor dilution.
  • FIG. 21B is a graph of the binding of the same cohorts with mouse PD-1, as evaluated by ELISA. Cohort 1 is the top line. Cohorts 2 and 4 are overlapping with Cohort 2 being the darker line at the bottom between 10 and 100 dilution. FIG.
  • 21C is a graph of the binding of the same cohorts with cyno PD-1, as evaluated by ELISA.
  • Cohort 1 is the top line.
  • Cohorts 2 and 4 are overlapping with Cohort 2 being the darker bottom line between 10 and 1000 dilution.
  • Shown in FIG. 2 ID is a summary of the fraction of serum antibodies binding to mouse vs. cyno PD-1, relative to the binding to human PD-1. A higher percentage indicates a greater number of the antibodies are reactive across the three different species.
  • B cells were isolated from the immunized mice cohorts 2-5 and fused to make hybridomas, and the hybridoma supernatants were evaluated for binding to human, cyno, and mouse PD-1 by ELISA. The results are shown in FIGS. 22A-22D, for cohorts 2-5, respectively. For each x-axis entry in each figure, human PD-1 binding is the left bar, cyno PD-1 binding is the middle bar, and mouse PD-1 binding is the right bar.
  • the monoclonal antibodies were also evaluated for blocking the PD-l/PD-Ll interaction by a reporter assay.
  • a Jurkat T cell line was engineered to stably express human PD-1 and a luciferase reporter that was triggered by IL-2, NFAT, or NF-kB response elements in the promoter region. Interaction of the Jurkat T cells with PD-Ll stably expressing cell line inhibited the intracellular mechanism by which the
  • promoter/lucif erase constructs were activated, which prevented luciferase expression.
  • FIG. 25 is a graph demonstrating the blocking of PD-l/PD-Ll binding by the supernatant of monoclonal (M) and polyclonal (H) hybridoma.
  • FIGS. 26A-26C ior antibodies C3H1 , C4M3, and C2M1.
  • the methods for developing the epitope mapping are described in greater detail in U.S. Provisional
  • the structures are heat-mapped to indicate epitope confidence, which is (true peptide array binding hit alignments )/(random alignments).
  • FIG. 28 is a table summarizing the number of target binding hits produced in each cohort. Using the engineered immunogen (Cohorts 2-5) produced significantly more target binding hits than the traditional immunization with full- length PD-1 (Cohort 1).
  • FIGS. 29A-29E Epitope maps of PD-1 were produced using day 35 blood draw serum from the five different cohorts. Shown in FIGS. 29A-29E are these maps, in which the dark structure is PD-1, the ribbon is PD-L1, and the mesh is the engineered immunogen (in structures on the right). The V14 peptide array was used to develop these maps. In these maps, red is highest confidence, blue is lowest, and white/pink is in the middle. Red, pink, and purple areas are epitopes that elicit immune response. These maps demonstrate that the methods described herein result in the development of immunogens that effectively elicit immune response, but avoid the antigenic sink caused by immunodominant epitopes.

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Abstract

L'invention concerne des compositions et des procédés pour créer des immunogènes représentant des régions d'une protéine cible non ciblée par des anticorps disponibles, et leur utilisation pour générer des anticorps spécifiques de régions.
PCT/US2018/055027 2017-10-09 2018-10-09 Immunogènes pour réponse immunitaire dirigée et anticorps issus de ceux-ci WO2019074934A1 (fr)

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