WO2016164308A1 - De novo binding domain containing polypeptides and uses thereof - Google Patents

De novo binding domain containing polypeptides and uses thereof Download PDF

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Publication number
WO2016164308A1
WO2016164308A1 PCT/US2016/025880 US2016025880W WO2016164308A1 WO 2016164308 A1 WO2016164308 A1 WO 2016164308A1 US 2016025880 W US2016025880 W US 2016025880W WO 2016164308 A1 WO2016164308 A1 WO 2016164308A1
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WIPO (PCT)
Prior art keywords
seq
dbdpp
amino acid
target
polypeptide
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
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PCT/US2016/025880
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English (en)
French (fr)
Inventor
David William LAFLEUR
David M. Hilbert
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Arcellx Inc
Subdomain LLC
Original Assignee
Arcellx Inc
Subdomain LLC
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Priority to EP16777109.6A priority Critical patent/EP3280433B1/en
Priority to HK18109015.2A priority patent/HK1249421B/en
Priority to CN201680032016.8A priority patent/CN107708720A/zh
Priority to JP2018503726A priority patent/JP6873101B2/ja
Priority to US15/564,319 priority patent/US10647775B2/en
Priority to CA2981711A priority patent/CA2981711A1/en
Application filed by Arcellx Inc, Subdomain LLC filed Critical Arcellx Inc
Priority to ES16777109T priority patent/ES2901273T3/es
Priority to DK16777109.6T priority patent/DK3280433T3/da
Publication of WO2016164308A1 publication Critical patent/WO2016164308A1/en
Anticipated expiration legal-status Critical
Priority to US16/824,809 priority patent/US20210002381A1/en
Ceased legal-status Critical Current

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    • 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
    • C07K16/2878—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 NGF-receptor/TNF-receptor superfamily, e.g. CD27, CD30, CD40, CD95
    • 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
    • C07K16/2827—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 B7 molecules, e.g. CD80, CD86
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
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    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/177—Receptors; Cell surface antigens; Cell surface determinants
    • A61K38/1774—Immunoglobulin superfamily (e.g. CD2, CD4, CD8, ICAM molecules, B7 molecules, Fc-receptors, MHC-molecules)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/10—Cellular immunotherapy characterised by the cell type used
    • A61K40/11—T-cells, e.g. tumour infiltrating lymphocytes [TIL] or regulatory T [Treg] cells; Lymphokine-activated killer [LAK] cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/30—Cellular immunotherapy characterised by the recombinant expression of specific molecules in the cells of the immune system
    • A61K40/31—Chimeric antigen receptors [CAR]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
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    • A61K40/4202—Receptors, cell surface antigens or cell surface determinants
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    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0019—Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
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    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C—CHEMISTRY; METALLURGY
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    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
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    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70503—Immunoglobulin superfamily
    • C07K14/7051—T-cell receptor (TcR)-CD3 complex
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70575—NGF/TNF-superfamily, e.g. CD70, CD95L, CD153, CD154
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/08—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses
    • C07K16/10—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses from RNA viruses
    • C07K16/1027—Paramyxoviridae, e.g. respiratory syncytial virus
    • 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
    • C07K16/30—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells
    • C07K16/3007—Carcino-embryonic Antigens
    • 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
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57484—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites
    • A—HUMAN NECESSITIES
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    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K40/00
    • A61K2239/27—Indexing codes associated with cellular immunotherapy of group A61K40/00 characterized by targeting or presenting multiple antigens
    • A61K2239/29—Multispecific CARs
    • A—HUMAN NECESSITIES
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    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
    • 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
    • C07K2317/31—Immunoglobulins specific features characterized by aspects of specificity or valency multispecific
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/565—Complementarity determining region [CDR]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/567—Framework region [FR]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
    • C07K2317/62—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising only variable region components
    • C07K2317/622—Single chain antibody (scFv)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2318/00—Antibody mimetics or scaffolds
    • C07K2318/20—Antigen-binding scaffold molecules wherein the scaffold is not an immunoglobulin variable region or antibody mimetics
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/02—Fusion polypeptide containing a localisation/targetting motif containing a signal sequence
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/03—Fusion polypeptide containing a localisation/targetting motif containing a transmembrane segment
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/035—Fusion polypeptide containing a localisation/targetting motif containing a signal for targeting to the external surface of a cell, e.g. to the outer membrane of Gram negative bacteria, GPI- anchored eukaryote proteins
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/20—Fusion polypeptide containing a tag with affinity for a non-protein ligand
    • C07K2319/21—Fusion polypeptide containing a tag with affinity for a non-protein ligand containing a His-tag
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/40—Fusion polypeptide containing a tag for immunodetection, or an epitope for immunisation
    • C07K2319/41—Fusion polypeptide containing a tag for immunodetection, or an epitope for immunisation containing a Myc-tag
    • C—CHEMISTRY; METALLURGY
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    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/40—Fusion polypeptide containing a tag for immunodetection, or an epitope for immunisation
    • C07K2319/43—Fusion polypeptide containing a tag for immunodetection, or an epitope for immunisation containing a FLAG-tag
    • C—CHEMISTRY; METALLURGY
    • C40—COMBINATORIAL TECHNOLOGY
    • C40B—COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES
    • C40B40/00—Libraries per se, e.g. arrays, mixtures
    • C40B40/04—Libraries containing only organic compounds
    • C40B40/10—Libraries containing peptides or polypeptides, or derivatives thereof
    • G—PHYSICS
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    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/705—Assays involving receptors, cell surface antigens or cell surface determinants
    • G01N2333/70596—Molecules with a "CD"-designation not provided for elsewhere in G01N2333/705

Definitions

  • Antibody-based reagents have accelerated the pace of biological research and development. Antibody compositions represent one of the most important and successful classes of therapeutic and diagnostic agents utilized in the pharmaceutical industry. However, cost, time and efficacy have motivated the development of alternative affinity reagents.
  • the polypeptide specifically binds a target of interest (such as a cancer marker or other distinctive marker related to a target of interest), and the specific binding of the polypeptide to the target of interest is greater than binding of a polypeptide according to SEQ ID NO: l to the target of interest.
  • the polypeptide does not contain the sequence of SEQ ID NO: 50.
  • a DBDpp having an amino acid sequence MGSWXjEFXsXsRLXizAIXisXieRLXigALGGSEAELAAFEKEIAAFESELQAYKGKGNPEVEXs 5 LRX 58 X 59 AAX 62 IRX 65 X 66 LQAYRHN (SEQ ID NO:4), and wherein X n is a natural or non-natural amino acid.
  • a DBDpp having an amino acid sequence at least 85% identical to the amino acid sequence of any one of SEQ ID NO:60 - SEQ ID NO: 136.
  • Still further embodiments provide for a fusion protein that binds to CD 123 (or other target of interest disclosed herein) and further comprises one or more additional DBDpp exhibiting binding specificity for a tumor target.
  • the target-binding agent e.g., a polypeptide
  • a therapeutic or cytotoxic agent e.g., chemotherapeutic agent, radiotherapeutic agent, etc.
  • the target-binding agent may optionally comprise a pharmaceutically acceptable carrier.
  • methods of treating a subject having cancer comprising administering to the subject an immune cell comprising a chimeric antigen receptor (CAR), wherein the CAR comprises a target binding domain, wherein the target binding domain comprises a polypeptide having an amino acid sequence comprising: MGSWX 5 EFX 8 X 9 RLX 12 AIX 15 X 16 RLX 19 ALGGSEAELAAFEKEIAAFESELQAYKGKGNPEVEX 5 5LRX58 59AAX 62 IRX 65 X66LQAYRHN (SEQ ID NO:4), a transmembrane domain, and an intracellular domain (comprising a signaling domain).
  • CAR chimeric antigen receptor
  • methods of treating a subject having cancer comprising, administering to the subject an immune cell comprising a chimeric antigen receptor (CAR), wherein the CAR comprises a target binding domain, wherein the target binding domain comprises a polypeptide having an amino acid sequence selected from of SEQ ID N0 2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6, wherein no cysteine or proline residues are substituted into any of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO: 5, and SEQ ID NO: 6, wherein the polypeptide specifically binds a target of interest expressed by a cancer cell, and wherein the polypeptide's specific binding to the target of interest is greater than binding of a polypeptide according to SEQ ID NO: l to the target of interest, a transmembrane domain, and an intracellular domain, wherein the intracellular domain comprises a signaling domain, wherein, upon administration
  • CAR chimeric antigen receptor
  • a method for purifying a target of interest comprising contacting a sample comprising a target of interest with a composition comprising a virus-like particle coupled to a solid support, wherein the virus-like particle expresses a polypeptide as a membrane protein, the polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO: 6, wherein the polypeptide has an amino acid sequence that differs from SEQ ID NO: l, wherein the polypeptide specifically binds the target of interest, wherein the polypeptide's specific binding to the target of interest is greater than binding of a polypeptide according to SEQ ID NO: 1 to the target of interest; and the contacting performed under conditions that permit binding of the composition to the target of interest; and removing a portion of the sample that is not bound to the composition.
  • no cysteine or proline residues are substitute
  • compositions for use in protein purification comprising a polypeptide agent having an amino acid sequence comprising a sequence selected from the group consisting of: MGSWX 5 X 6 FKX 9 XioLA 13 lKXi 6 Xi 7 LEALGGSEAELA 3 oFEX 33 X 34 lA 37 FEX 4 oX 4 iLQX 44 YKG KGNPEVEALRKEAAAIRDELQAYRHN (SEQ ID NO:2),
  • 1 to 25, 1 to 30, 1 to 35, 5 to 40, 5 to 45, 5 to 50, 5 to 55, or 5 to 60 of the substitutions are at amino acid residues of SEQ ID NO: l selected from the group consisting of: Ml, G2, S3, W4, A5, E6, K8, Q9, RIO, AI2, AI3, KI5, TI6, R17, E19, A20, L21, G22, G23, S24, E25, A26, E27, A29, A30, E32, K33, E34, A36, A37, E39, S40, E41, Q43, A44, Y45, K46, G47, K48, G49, N50, P51, E52, E54, A55, R57, K58, E59, A61, A62, R64, D65, E66, Q68, A69, Y70, R71, H72, and N73.
  • SEQ ID NO: l selected from the group consisting of: Ml, G2, S3, W4,
  • the substitution of both accessible and inaccessible residues confers a greater degree of target specificity as compared to substitution of only accessible or only inaccessible residues.
  • the substituted residues corresponding to a solvent inaccessible residue of SEQ ID NO: l are selected from the group consisting of: F7, Ll l, 114, L18, L28, F31, 135, F38, L42, V53, L56, A60, 163, and L67, and Y70.
  • L21 and Y45 are also included in the group of substituted, solvent inaccessible residues.
  • the host cell is a prokaryote or a eukaryote that display the DBDpp on its surface.
  • the host cell is a phage that displays the DBDpp on its surface.
  • the host cell is a human immune cell that expresses a DBDpp fusion protein on its surface. Libraries comprising a plurality of DBDpp are also provided.
  • the vector library comprises: (a) nucleic acids encoding 3 DBDpp that specifically bind to different targets; (b) nucleic acids encoding 3 DBDpp having different sequences that specifically bind to the same target; (c) nucleic acids encoding 3 DBDpp having different sequences that specifically bind to the same epitope of a target; (d) nucleic acids encoding 3 DBDpp having different sequences that specifically bind to different epitopes of a target; (e) nucleic acids encoding 3 DBDpp having different sequences that compete for binding to the same target; or (f) 3 different nucleic acid sequences encoding the same DBDpp sequence. Host cells containing the vectors are also provided.
  • 1 to 5, 5 to 25, 5 to 30, 5 to 35, 5 to 40, 5 to 45, 5 to 50, 5 to 55, or 5 to 60 of the encoded substitutions are at amino acid residues of SEQ ID NO: l selected from the group consisting of one or more of: Ml, G2, S3, W4, A5, E6, K8, Q9, R10, A12, A13, K15, T16, R17, E19, A20, L21, G22, G23, S24, E25, A26, E27, A29, A30, E32, K33, E34, A36, A37, E39, S40, E41, Q43, A44, Y45, K46, G47, K48, G49, N50, P51, E52, E54, A55, R57, K58, E59, A61, A62, R64, D65, E66, Q68, A69, Y70, R71, H72, and N73.
  • SEQ ID NO: l selected from the group consisting
  • the library comprises nucleic acids encoding at least 2, 3, 4, 5, 10, 25, 50, 75, 100, 250, 500, or 1000 different DBDpp that specifically bind a target disclosed herein.
  • a plurality of the vectors of the vector library are contained in host cells.
  • the host cells e.g., viral particles
  • the host cells are phage that display DBDpp on their surface.
  • the host cells are mammalian cells.
  • One embodiment provides a method of treating a disease or disorder comprising administering a therapeutically effective amount of a DBDpp (e.g., a DBDpp fusion protein) that specifically binds a therapeutic target of interest to a subject in need thereof.
  • a DBDpp e.g., a DBDpp fusion protein
  • the disease or disorder is cancer, a disease or disorder of the immune system, or an infection.
  • Methods of treating a disease or disorder that comprises co-administering an additional therapeutic agent along with a DBDpp are also provided.
  • ELISA-based binding assessment demonstrated that purified FLAG-pb04 (targets PD-L1) binds in a dose dependent manner to PD-L1- Fc coated microtiter wells, whereas FLAG-a3D (the reference sequence of SEQ ID 49, with an N- terminal FLAG tag) exhibits no change in binding.
  • FIGS. 4A-D DBDpp have novel binding specificities and impart these novel binding specificities to another molecule (e.g., an antibody) as part of a fusion protein (e.g., an antibody-DBDpp fusion protein).
  • a fusion protein e.g., an antibody-DBDpp fusion protein
  • FIGS. 4A-D Schematic depicting the recombinant fusion of DBDpp (shown as circle) to the C-terminus (FIG. 4A) and N-terminus (FIG. 4B) of an antibody heavy chain.
  • DBDpp - antibody fusions were created using an RSV-specific antibody (SYN) and either the targetless peptide of SEQ ID NO: 1 (DBD) or the CD137-specific DBDpp (bblO).
  • FIGS. 6A-C Multi-specific DBDpp fusions recognize cell surface targets.
  • FACS analysis indicates that bblO-SYN and SYNbblO bispecific antibodies bind (shaded histogram) to activated CEM cell at levels greater than SYN alone (black outline).
  • the weaker binding observed with the bblO N-terminal fusion (FIG. 6A) as compared to the C-terminal fusion (FIG. 6B) is consistent with the above ELISA data.
  • U E1 is a recombinant antibody constructed formed from variable domains of the CD137-targeting, urelumab fused to IgG scaffold.
  • FIG. 8 In vivo stability is critical to the clinical efficacy of most biotherapeutics. Pharmacokinetic measurements of bblO fusions were performed to assess the relative stability of DBDpp as compared to the antibody fusion partner. The in vivo stability was determined by analysis of both the RSV and CD137 binding of the bi-specific antibody present in serum from CDl mice that received a single intravenous injection (1 mg/kg) of the fusion. Serum samples were collected at 15 minutes and 48 hours, and were assayed by ELISA. Both N-terminal and C-terminal DBDpp fusion proteins demonstrate sustained stability in vivo.
  • FIG. 11 depicts a deconvoluted electrospray ionisation mass spectrometry (ESI-MS) spectrum of SEQ ID NO. 54.
  • 12B, 12D, 12F, 12H, 12J, 12L, 12N, and 12P depict the corresponding steady state binding data for DBDpps of SEQ ID 51 (12B), 52 (12D), 53 (12F), 54 (12H), 55 (12J), 56 (12L), 57 (12N) and 58 (12P).
  • FIGS. 14A-14B Analysis of proteins purified using DBDpp.
  • FIG. 14A depicts a Coomassie stained gel loaded with purified fractions from DBDpp purification columns. Lane 1 is a molecular weight marker. Lane 2 is IMAC-purified CD 137 protein, and Lanes 3-8 are eluates from columns with various DBDpp according to several embodiments herein.
  • FIG. 14B is a western blot analysis with corresponding samples to those shown in FIG. 14A.
  • FIG. 18 DBDpp mediate signal transduction.
  • FIG 18 depicts data related to the expression and ability of DBDpp-CAR Jurkat cells to function through an intracellular signaling pathway.
  • FIGS. 19A-19B CD 123 -DBDpp-CAR T cells produce cytokines in response to target binding.
  • FIG. 19A shows data related to the production of interferon gamma (IFNy) by T cells expressing DBDpp-CARs that target CD 123.
  • FIG. 19B depicts similar data measuring the production of interleukin 2 (IL2) by CD 123 -targeting DBDpp-CAR T cells.
  • IFNy interferon gamma
  • IL2 interleukin 2
  • an immunoglobulin Fc region or simply “Fc” is understood to mean the carboxyl-terminal portion of an immunoglobulin chain constant region, preferably an immunoglobulin heavy chain constant region, or a portion thereof
  • an immunoglobulin Fc region may comprise (1) a CHI domain, a CH2 domain, and a CH3 domain, (2) a CHI domain and a CH2 domain, (3) a CHI domain and a CH3 domain, 4) a CH2 domain and a CH3 domain, or (5) a combination of two or more domains and an immunoglobulin hinge region.
  • the immunoglobulin Fc region comprises at least an immunoglobulin hinge region a CH2 domain and a CH3 domain, and preferably lacks the CHI domain.
  • the class of immunoglobulin from which the heavy chain constant region is derived is IgG (Igy) ( ⁇ subclasses 1, 2, 3, or 4).
  • Other classes of immunoglobulin, IgA (Ig ), IgD (lg6), IgE (Igs) and IgM (3 ⁇ 4 ⁇ ) may be used.
  • IgG immunoglobulin
  • IgA Ig
  • IgD IgD
  • IgE IgE
  • IgM 3 ⁇ 4 ⁇
  • ADCC antibody-dependent cell-mediated cytotoxicity
  • FcRs Fc receptors
  • NK Natural Killer
  • macrophages a cell-mediated reaction in which nonspecific cytotoxic cells that express Fc receptors (FcRs) (e.g., Natural Killer (NK) cells, neutrophils, and macrophages) recognize bound antibody on a target cell and subsequently cause lysis (or other cytotoxic effects) of the target cell.
  • FcRs Fc receptors
  • NK Natural Killer
  • PBMC peripheral blood mononuclear cells
  • NK Natural Killer
  • ADCC activity of the molecule of interest can be assessed in vivo, e.g., in an animal model such as that disclosed in Clynes et al. PNAS 95:652-656 (1998).
  • single chain variable fragment(s),” or “scFv” antibodies refer to forms of antibodies (e.g., antibody fragments) comprising the variable regions of only the heavy and light chains, connected by a linker peptide.
  • a DBDpp fusion protein comprises a DBDpp and a scFv.
  • Target refers to any molecule or combination of molecules that can be bound by a DBDpp such as a DBDpp fusion protein, or other component of the DBDpp fusion protein such as an antibody or antibody variable domain fragment.
  • a "peptide tag” as used herein refers to a peptide sequence that is part of or attached (for instance through genetic engineering) to another protein, to provide a function to the resultant fusion.
  • Peptide tags are usually relatively short in comparison to a protein to which they are fused; by way of example, peptide tags are, in several embodiments, four or more amino acids in length, such as, 5, 6, 7, 8, 9, 10, 15, 20, or 25 or more amino acids.
  • the DBDpp is a fusion protein that contains a peptide tag. In other embodiments, the DBDpp specifically binds a peptide tag. Numerous peptide tags that have uses as provided herein are known in the art.
  • non-conservative amino acid substitution is one in which one amino acid residue is replaced with another amino acid residue having a dissimilar side chain.
  • non-conservative substitutions in the sequences of the DBDpp result in the specific binding of the DBDpp containing the substitution to the target of interest to which it binds.
  • non-conservative substitutions in the sequences of the DBDpp do not abrogate the binding of the DBDpp containing the substitution to the target of interest to which it binds
  • non-natural amino acids that can be substituted in a DBDpp include, but are not limited to molecules such as: D- isomers of the common amino acids, 2,4-diaminobutyric acid, alpha-amino isobutyric acid, A- aminobutyric acid, Abu, 2-amino butyric acid, gamma- Abu, epsilon-Ahx, 6-amino hexanoic acid, Aib, 2-amino isobutyric acid, 3 -amino propionic acid, ornithine, norleucine, norvaline, hydroxyproline, sarcosine, citrulline, homocitrulline, cysteic acid, t-butylglycine, t-butylalanine, phenylglycine, cyclohexylalanine, beta-alanine, lanthionine, dehydroalanine, ⁇ -aminobutyric acid, selenoc
  • polynucleotide and “nucleic acid,” used interchangeably herein, refer to a polymeric form of nucleotides of any length, either ribonucleotides or deoxyribonucleotides. These terms include, but are not limited to, DNA, RNA, cDNA (complementary DNA), mRNA (messenger RNA), rRNA (ribosomal RNA), shRNA (small hairpin RNA), snRNA (small nuclear RNA), snoRNA (short nucleolar RNA), miRNA (microRNA), genomic DNA, synthetic DNA, synthetic RNA, and/or tRNA.
  • vector refers to the vehicle by which a nucleic acid sequence (e.g., a DBDpp coding sequence) can be maintained or amplified in a host cell (e.g., cloning vector) or introduced into a host cell, so as to transform the host and promote expression (e.g., transcription and translation) of the introduced sequence.
  • vectors include plasmids, phages, viruses, etc.
  • a "host cell” includes an individual cell or cell culture which can be or has been a recipient of nucleic acids encoding a DBDpp.
  • a "library" of DBDpp refers to a plurality of unique DBDpp, and optionally including multiple DBDpp that bind to the same target, but with varied binding sites and/or specificities.
  • an "effective amount" of a DBDpp such as a DBDpp fusion protein as provided herein is an amount sufficient to carry out a specifically stated purpose such as to bring about an observable change in the level of one or more biological activities related to the target to which the DBDpp (e.g., a DBDpp fusion protein) binds.
  • the change increases the level of target activity.
  • the change decreases the level of target activity.
  • An "effective amount" can be determined empirically and in a routine manner, in relation to the stated purpose.
  • terapéuticaally effective amount refers to an amount of a DBDpp such as a DBDpp fusion protein, or other therapeutic agent effective to "treat” (e.g., reduce symptoms of) a disease or disorder in a subject (mammal).
  • a “prophylactically effective amount” refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired prophylactic result.
  • "Patient,” “subject,” “animal” and “mammal” are used interchangeably and refer to mammals such as human patients and non-human primates, as well as experimental animals such as rabbits, rats, and mice, and other animals. Animals include all vertebrates, e.g., mammals and non-mammals, such as chickens, amphibians, and reptiles.
  • DBDpp fusion proteins include without limitation, breast, lung, brain, bone, liver, kidney, colon, head and neck, ovarian, hematopoietic (e.g., leukemia), and prostate cancer.
  • Other types of cancer and tumors that may be treated using DBDpp-containing antibodies are described herein or otherwise known in the art.
  • TAAs and TSAs that may be specifically bound by a DBDpp include but are not limited to: CD 19, CD20, CD22, ROR 1, mesothelin, CD33/lL3Ra, cMet, PSMA, Glycolipid F77, EGFRvIII, GD2, NY-ESO- ITCR, MAGE A3 TCR MARTI, gplOO (Pmel 17), tyrosinase, TRP1, TRP2, MAGE1, MAGE3, BAGE, GAGE1, GAGE2, pi5, CEA; p53, Ras, HER-2/neu; BCR-ABL, E2A-PRL, H4-RET, 1GH-IGK, MYL-RAR; EBVA, HPV antigens E6 and E7, TSP-180, MAGE4, MAGE5, MAGE6, RAGE, NY-ESO, pl85erbB2, P 180erbB3, nm-23Hl, PSA, CA 19-9, CA72-4, CAM 17.1,
  • the DBDpp does not contain CDR2. In another embodiment, the DBDpp in not derived from protein A. In another embodiment the DBDpp is not derived from a natural bacterial receptor. In another embodiment the DBDpp is not derived from fibronectin. In another embodiment the DBDpp is not derived from fibronectin type III domain. In yet another embodiment, the DBDpp is not derived from a knottin protein. In yet another embodiment, the DBDpp is not derived from a lipocalin. In yet another embodiment, the DBDpp is not derived from an affibody.
  • longer DBDpp are employed, for example DBDpp ranging in length from about 150 to about 160 amino acids, about 160 to about 170 amino acids, about 170 to about 180 amino acids, about 180 to about 190 amino acids, about 190 to about 200 amino acids, or any length between those listed (including endpoints).
  • the relative accessibility of a residue within the domain (area D) as compared to the isolated state (area I) is represented as a percent value (%A).
  • Amino residues of SEQ ID NO: l that have %A values less than about 10% to 11% e.g., residues corresponding to F7, Ll l, 114, L18, L21, S24, L28, F31, 135, F38, L42, Y45, G49, V53, L56, A60, 163, and L67, of SEQ ID NO: l
  • amino acid residues of SEQ ID NO: l with %A values that are greater than about 10% to 11% are believed to occupy positions that have greater potential for interaction a target of interest.
  • Binding surfaces of proteins are typically composed of several amino residues that are either adjacent, or in close proximity, to each other in three-dimensional space. Therefore, a secondary consideration in the construction of libraries, according to several embodiments herein, was the relative proximity of these selected residues within the predicted secondary and tertiary structure of the DBDpp.
  • At least 3, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, or 35 of the solvent accessible acid residues are substituted. In some embodiments, at least 3, 5, 6, 7, 8, 9, 10, 15, 20, 25, or 30 of the solvent accessible acid residues of SEQ ID NO: l are substituted with conservative amino acid residue substitutions. In some embodiments, at least 3, 5, 6, 7, 8, 9, 10, 15, 20, 25, or 30 of the solvent accessible acid residues of SEQ ID NO: 1 are substituted with non-conservative amino acid residue substitutions. In some embodiments, the amino acid substitutions do not contain proline. In some embodiments, the amino acid substitutions do not contain cysteine or proline. In some embodiments, the amino acid residue substitutions include no more than one cysteine.
  • the DBDpp is a fusion protein.
  • at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 of the substituted amino acid residues of SEQ ID NO: l are substituted with conservative amino acid residue substitutions.
  • at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 of the above amino acid residues of SEQ ID NO: l are substituted with non-conservative amino acid residue substitutions.
  • the amino acid substitutions do not contain proline.
  • the amino acid substitutions do not contain cysteine.
  • neither proline nor cysteine is included in the amino acid substitutions.
  • the amino acid residue substitutions include no more than one cysteine.
  • X trap is a natural amino acid residue.
  • X n is a natural amino acid residue other than cysteine or proline.
  • the DBDpp does not contain the amino acid sequence LAAIKTRLQ (SEQ ID NO:50).
  • the DBDpp is a fusion protein.
  • at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 of the substituted amino acid residues of SEQ ID NO: l are substituted with conservative amino acid residue substitutions.
  • at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 of the above amino acid residues of SEQ ID NO: l are substituted with non-conservative amino acid residue substitutions.
  • the DBDpp specifically binds a target of interest selected from the group consisting of: a nucleic acid, an oligosaccharide, a peptide, a protein, a cell surface antigen, and a small organic molecule.
  • the DBDpp specifically binds a protein selected from the group consisting of: an immunoglobulin, an enzyme, a hormone, a serum protein, a cell surface protein, a therapeutic protein, a TSA, a CSA, and a protein containing a peptide tag.
  • the DBDpp specifically binds a target disclosed herein.
  • a library containing a plurality of DBDpp is provided.
  • the DBDpp specifically binds a target of interest selected from the group consisting of: a nucleic acid, an oligosaccharide, a peptide, a protein, a cell surface antigen, and a small organic molecule.
  • the DBDpp specifically binds a protein selected from the group consisting of: an immunoglobulin, an enzyme, a hormone, a serum protein, a cell surface protein, a therapeutic protein, a TSA, a CSA, and a protein containing a peptide tag.
  • the DBDpp specifically binds a target disclosed herein.
  • a library containing a plurality of DBDpp is provided.
  • Nucleic acids encoding the DBDpp and vectors containing the nucleic acids are also provided.
  • Host cells (including viral particles) containing the nucleic acids and vectors are also provided.
  • the host cell is a prokaryote or a eukaryote that display the DBDpp on its surface.
  • the host cell displays the DBDpp on its surface.
  • the host cell is a phage that displays the DBDpp on its surface.
  • the host cell is a human immune cell that expresses a DBDpp fusion protein on its surface.
  • the DBDpp is attached to a solid support.
  • the solid support is selected from the group consisting of: a bead, a glass slide, a chip, a gelatin, and an agarose.
  • the host cell is a prokaryote or a eukaryote that display the DBDpp on its surface. In some embodiments, the host cell displays the DBDpp on its surface. In a further embodiment, the host cell is a phage that displays the DBDpp on its surface. In a further embodiment, the host cell is a human immune cell that expresses a DBDpp fusion protein on its surface. In one embodiment, the DBDpp is attached to a solid support. In a further embodiment, the solid support is selected from the group consisting of: a bead, a glass slide, a chip, a gelatin, and an agarose.
  • X n is a natural amino acid residue. In a further embodiment, X n is a natural amino acid residue other than cysteine or proline. In a particular embodiment, the DBDpp does not contain the amino acid sequence LAAIKTRLQ (SEQ ID NO:50). In an additional embodiment, the DBDpp is a fusion protein. In some embodiments, at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 of the substituted amino acid residues of SEQ ID NO: l are substituted with conservative amino acid residue substitutions. In some embodiments, at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 of the above amino acid residues of SEQ ID NO: l are substituted with non-conservative amino acid residue substitutions.
  • the DBDpp specifically binds a target of interest selected from the group consisting of: a nucleic acid, an oligosaccharide, a peptide, a protein, a cell surface antigen, and a small organic molecule.
  • the DBDpp specifically binds a protein selected from the group consisting of: an immunoglobulin, an enzyme, a hormone, a serum protein, a cell surface protein, a therapeutic protein, a TSA, a CSA, and a protein containing a peptide tag.
  • the DBDpp specifically binds a target disclosed herein.
  • a library containing a plurality of DBDpp is provided.
  • the amino acid substitutions do not contain proline. In some embodiments, the amino acid substitutions do not contain cysteine. In some embodiments, neither proline nor cysteine is included in the amino acid substitutions. In some embodiments, the amino acid residue substitutions include no more than one cysteine. In some embodiments, 1 to 12 of the solvent accessible acid residues are substituted with conservative substitutions and 1 to 12 of the solvent accessible acid residues are substituted with non-conservative substitutions, or 5 to 12 of the solvent accessible acid residues are substituted with conservative substitutions and 5 to 12 of the solvent accessible amino acid residues are substituted with non-conservative substitutions.
  • the DBDpp specifically binds a target of interest selected from the group consisting of: a nucleic acid, an oligosaccharide, a peptide, a protein, a cell surface antigen, and a small organic molecule.
  • the DBDpp specifically binds a protein selected from the group consisting of: an immunoglobulin, an enzyme, a hormone, a serum protein, a cell surface protein, a therapeutic protein, a TSA, a CSA, and a protein containing a peptide tag.
  • the DBDpp specifically binds a target disclosed herein.
  • a library containing a plurality of DBDpp is provided.
  • the DBDpp comprises a substitution at a corresponding position in the sequence of SEQ ID NO: l selected from the group consisting of: G2, S3, W4, A5, E6, K8, Q9, RIO, A12, A13, K15, T16, R17, E19, A20, A29, A30, E32, K33, E34, A36, A37, E39, S40, E41, Q43, A44, E52, E54, A55, R57, K58, E59, A61, A62, R64, D65, E66, Q68, A69, and Y70.
  • the DBDpp comprises substitutions of at least 1, 5, 10, 15, 20, or 30 of the above positions in the sequence of SEQ ID NO: l . These substitutions can be conservative, non-conservative, or a mix of conservative and non-conservative substitutions. In some embodiments, the substitutions do not include the addition of a proline or cysteine. In some embodiments, the substitutions include no more than a single cysteine. In some DBDpp, these residues may be greater than 90% identical to SEQ ID NO: 1. In other DBDpp, these residues may be greater than 80% identical to SEQ ID NO: l . In other DBDpp, these residues may be greater than 70%) identical to SEQ ID NO: l .
  • these residues may be greater than 60% identical to SEQ ID NO: l . In other DBDpp, these residues may be greater than 50% identical to SEQ ID NO: l . In other DBDpp, these residues may be greater than 40% identical to SEQ ID NO: l . In other DBDpp, these residues may be greater than 30% identical to SEQ ID NO: l . In other DBDpp, these residues are greater than 20% identical to SEQ ID NO: 1. In other DBDpp, these residues are greater than 10% identical to SEQ ID NO: 1.
  • the DBDpp contains a sequence with 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 amino acid residues deleted from the amino terminus of the DBDpp sequence corresponding to the sequence of SEQ ID NO: l . In some embodiments the DBDpp contains a sequence with 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 amino acid residues deleted from the carboxy terminus of the DBDpp sequence corresponding to the sequence of SEQ ID NO: l .
  • the DBDpp contains a sequence with 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 11, or 12 amino acid residues deleted from the amino terminus of the corresponding sequence of SEQ ID NO: 1 and a sequence with 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 amino acid residues deleted from the carboxy terminus corresponding to the sequence of SEQ ID NO: l .
  • the DBDpp contains a sequence with 1-5, 1-10, or 1 to 15 amino acid residues deleted from the carboxy terminus of the sequence corresponding to SEQ ID NO: l .
  • the DBDpp contains a sequence that differs from the corresponding sequence in reference SEQ ID NO: l in 2 or more categories of sequence modifications (i.e., substitutions, deletions, insertions, and additions).
  • DBDpp may include combinations of amino acid deletions, insertions and substitutions compared to the corresponding sequence in the reference polypeptide sequence.
  • the DBDpp contains 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10 amino acid deletions within the sequence reference sequence shown in SEQ ID NO: l.
  • the DBDpp contains 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10 amino acid insertions within the reference sequence shown in SEQ ID NO: I.
  • DBDpp can bind to a target of interest, and in several embodiments, have no discernable impact on the function of the target.
  • DBDpp can bind to a target of interest and completely or partially inhibit, antagonize, agonize, block, increase, stimulate or interfere with the biological activity of that target. Binding can be identified as agonistic or antagonistic and determined using or routinely modifying assays, bioassays, and/or animal models known in the art for evaluating such activity.
  • a DBDpp binds a target of interest with a KD of less than or equal to 5 l(T 6 M, 1(T 6 M, 5 x l(T 7 M, 1(T 7 M, 5 l(T 8 M, or 1(T 8 M.
  • the DBDpp generated by the methods disclosed herein have a dissociation constant selected from the group consisting of between 10 "4 M and 10 "5 M, between 10 "5 M and 10 "6 M, between 10 “6 M and 10 “7 M, between 10 “7 M and 10 “8 M, between 10 “8 M and 10 “9 M, between 10 "9 M and 10 “10 M, between 10 "10 M and 10 "11 M and between 10 "11 M and 10 ⁇ 12 M.
  • a DBDpp binds a target of interest in active form.
  • a DBDpp reversibly binds a target of interest in active form and also releases the bound target in active form.
  • a DBDpp binds a target of interest in the native form.
  • DBDpp bind targets of interest with off-rates or K 0ff of greater than or equal to 10 -10 sec -1 , 5x l(T 9 sec -1 , 10 -9 sec -1 , 5x l0 -8 sec -1 , 10 -8 sec -1 , 5 x l0 -7 sec -1 , 10 -7 sec -1 , 5x l0 -6 sec -1 , 10 -6 sec -1 , 5 ⁇ 10 -5 sec -1 , 10 -5 sec -1 , 5 ⁇ 10 -4 sec -1 , 10 -4 sec -1 , 5 ⁇ 10 -3 sec -1 , 10 -3 sec -1 , 5 l0 -2 sec -1 , 10 -2 sec -1 , 5 x l0 -1 sec -1 , or 10 -1 sec -1 .
  • Binding experiments to determine KD and off-rates can be performed in a number of conditions including, but not limited to, [pH 6.0, 0.01% Tween 20], [pH 6.0, 0.1% gelatin], [pH5.0, 0.01% Tween 20], [pH9.0, 0.1% Tween 20], [pH6.0, 15% ethylene glycol, 0.01% Tween 20], [pH5.0, 15% ethylene glycol, 0.01% Tween 20], and [pH9.0, 15% ethylene glycol, 0.01% Tween 20],
  • the buffers in which to make these solutions can readily be determined by one of skill in the art, and depend largely on the desired pH of the final solution.
  • a DBDpp specifically binds a target of interest with a K 0ff
  • a DBDpp e.g., a DBDpp fusion protein
  • a DBDpp binds a target of interest with an off rate (K 0 g) of less than 5 X 10 "4 sec “1 , 10 “4 sec “1 , 5 X 10 “5 sec “1 , or 10 "5 sec “1 , 5 XI 0 "6 sec “1 , 10 “6 sec “1 , 5 X 10 “7 sec “1 , or 10 "7 sec “1 .
  • a DBDpp specifically binds a target of interest with a Ko n ranging from 10 3 to 10 7 M-l sec-1, lO 3 to 10 6 M-l sec-1, or lO 3 to lO 5 M-lsec-1.
  • a DBDpp e.g., a DBDpp fusion protein
  • a DBDpp binds a target of interest with a K 0n of greater than 10 5 Nf 1 , 5 X 10 5 Nf 1 , 10 6 M “1 sec “1 , or 5 X 10 6 NfW 1 , or 10 7 M “1 sec “1 .
  • DBDpp Targets of Interest binds a target of interest with a K 0n of greater than 10 5 Nf 1 , 5 X 10 5 Nf 1 , 10 6 M “1 sec “1 , or 5 X 10 6 NfW 1 , or 10 7 M “1 sec “1 .
  • the target of interest specifically bound by a DBDpp can be any molecule for which it is desirable for a DBDpp to bind.
  • the targets specifically bound by DBDpp can be any target of purification, manufacturing, formulation, therapeutic, diagnostic, or prognostic relevance or value.
  • a number of exemplary targets are provided herein, by way of example, and are intended to be illustrative and not limiting.
  • the target of interest can be naturally occurring or synthetic.
  • the target of interest can be an extracellular component or an intracellular component, a soluble factor (e.g., an enzyme, hormone, cytokine, and growth factor, toxin, venom, pollutant, etc.), or a transmembrane protein (e.g., a cell surface receptor).
  • the target of interest specifically bound by a DBDpp is itself a DBDpp having a different sequence.
  • a DBDpp fusion protein specifically binds a target of interest on the surface of a target cell. In a further embodiment, the DBDpp fusion protein specifically binds a cell surface receptor. In one embodiment, a DBDpp fusion protein specifically binds a target of interest that is a member of a family selected from: a growth factor receptor, a tyrosine kinase receptor, a TNF family receptor, a G-protein-coupled receptor, and a chemokine receptor. In some embodiments, the DBDpp fusion protein binds multiple members of the same family (e.g., the TNF receptors TRAILRl and TRAILR2).
  • a DBDpp specifically binds a target of interest that is a serum protein.
  • a DBDpp specifically binds a serum protein selected from: serum albumin (e.g., human serum albumin (HSA)), thyroxin-binding protein, transferrin, fibrinogen, and an immunoglobulin (e.g., IgG, IgE and IgM).
  • serum albumin e.g., human serum albumin (HSA)
  • thyroxin-binding protein thyroxin-binding protein
  • transferrin thyroxin-binding protein
  • fibrinogen e.g., transferrin, fibrinogen
  • an immunoglobulin e.g., IgG, IgE and IgM
  • a DBDpp specifically binds CD19 (B-CLL, B-ALL, leukemia, lymphoma, BNHL/CLL, ALL post-HCST, B lymphoid malignancies, B lineage malignancies), CD20 (mantle cell lymphoma/indolent B-NHL), PMSA (prostate cancer), CEA (breast cancer, colorectal cancer), Her2/neu (lung cancer, osteosarcoma, glioblastoma), kappa light chain (B-NHL and B-CLL).
  • a target of interest specifically bound by a DBDpp is an antigen associated with an autoimmune disorder, inflammatory or other disorder of the immune system or is associated with regulating an immune response.
  • a DBDpp specifically binds CD 137.
  • a DBDpp specifically binds CD 137 and comprises an amino acid sequence selected from: (a) MGSWVEFGHRLWAIDQRLYALGGSEAELAAFEKEIAAFESELQAYKGKGNPEVEK LRQRAAFIRFRLQAYRHN (SEQ ID NO: 12), (b) MGSWVEFANRLWAIDQRLFALGGS EAELAAFEKEIAAFESELQAYKGKGNPEVEHLRDQAAFIRHKLQAYRHN (SEQ ID NO: 13), (c) MGSWYEFRHRLWAIDQRLYALGGSEAELAAFEKEIAAFESELQAYKGK GNPEVEGLREAAAFIRAKLQAYRHN (SEQ ID NO: 14), (d) MGS WYEF SMRLWAIDQ RLYALGGSEAELAAFEKEIAAFESELQAYKGKGNPEVEALRAKAAYIRWKLQAY
  • DBDpp and polypeptides that completely or partially bind to the same epitope of KIR as an above DBDpp are provided. Additionally, DBDpp and polypeptides that completely or partially compete with an above DBDpp for binding to KIR are also provided. Nucleic acids encoding the DBDpp are also provided, as are vectors containing the nucleic acids and host cells containing the nucleic acids and vectors.
  • a DBDpp specifically binds PD-Ll .
  • a DBDpp specifically binds PD-Ll and comprises an amino acid sequence selected from (a) MGSWTEFQSRLDAIHSRLRALGGSEAELAAFEKEIAAFESELQAYKGKGNPEVELLR DDAAFIRHFLQAYRHN (SEQ ID NO: 38), (b) MGSWQEFDDRLNAIKARLQALGGSEA ELAAFEKEIAAFESELQAYKGKGNPEVEDLRDDAAFIRRFLQAYRHN (SEQ ID NO:39), (c) MGSWYEFQNRLHAIHERLNALGGSEAELAAFEKEIAAFESELQAYKGK GNPEVELLRDDAAFIRHFLQAYRHN (SEQ ID NO:40), (d) MGSWFEFQDRLTATNER LSALGGSEAELAAFEKEIAAFESELQAYKGKGNPEVETLRSDAAFIRRFLQAYRHN
  • DBDpp and polypeptides that completely or partially bind to the same epitope of PD-Ll as an above DBDpp are provided. Additionally, DBDpp and polypeptides that completely or partially compete with a DBDpp for binding to PD-Ll are also provided. Nucleic acids encoding the DBDpp are also provided, as are vectors containing the nucleic acids and host cells containing the nucleic acids and vectors.
  • a DBDpp specifically binds PD1.
  • a DBDpp specifically binds PD1 and comprises an amino acid sequence selected from (a) MGSWTIFKEWLAFIKTDLEALGGSEAELAFFEGWIASFEMELQKYKGKGNPEVEAL RKEAAAIRDELQAYRHN (SEQ ID NO:46), (b) MGSWVMFKWLLADIKSHLEALGG SEAELAFFEGFIAAFETHLQVYKGKGNPEVEALRKEAAAIRDELQAYRHN (SEQ ID NO:47), and (c) MGSWYAFKDYLADIKGWLEALGGSEAELAFFEIFIARFELELQAY
  • a DBDpp specifically binds TIM3.
  • a DBDpp specifically binds TIM3 and comprises an amino acid sequence of MGSWHEFHD RLQAIHERLYALGGSEAELAAFEKEIAAFESELQAYKGKGNPEVESLRIAAAHI QV LQAYRHN (SEQ ID NO:45).
  • Other DBDpp and polypeptides that completely or partially bind to the same epitope of TIM3 as the above DBDpp are provided.
  • DBDpp and polypeptides that completely or partially compete with the DBDpp for binding to TIM3 are also provided.
  • Nucleic acids encoding the DBDpp are also provided, as are vectors containing the nucleic acids and host cells containing the nucleic acids and vectors.
  • the DBDpp fusion protein comprises a DBDpp and a member selected from: an antibody, an antibody fragment (e.g., an antigen binding domain or portion thereof (e.g., an ScFv), an effector domain or portion thereof, an FcRn binding domain or portion thereof, and an Fc or a portion thereof), a serum protein (e.g., albumin or a portion thereof), a cytokine, a growth factor, a hormone, an imaging agent, a labeling agent, and a peptide tag.
  • the DBDpp fusion protein comprises an Fc domain of an immunoglobulin (e.g., a human Fc domain) or a portion thereof.
  • the Fc domain is a variant human Fc domain.
  • the DBDpp provided herein include DBDpp fusion proteins.
  • a DBDpp and any polypeptide of interest can be operably linked to form a DBDpp fusion protein.
  • the DBDpp is incorporated into a larger, multi-domain molecular complex (e.g., a monomeric or multimeric DBDpp fusion protein) and in so doing, imparts the functional attributes of the incorporated DBDpp to the resultant fusion protein.
  • the DBDpp fusion protein contains one DBDpp. In some embodiments, the DBDpp fusion protein comprises at least 2, 3, 4, or 5, or more than 5 DBDpp. In some embodiments, the DBDpp fusion protein contains 1-3, 1-4, 1-5, or more than 5 different DBDpp. In some embodiments, the DBDpp fusion protein contains at least 2, 3, 4, or 5, or more than 5 different DBDpp.
  • a DBDpp fusion protein can be a monomeric DBDpp (i.e., containing one DBDpp) or multimeric DBDpp (i.e., containing more than one DBDpp in tandem optionally operably connected by a linker). Non-limiting embodiments of such multimeric DBDpp are shown in FIG.
  • the fusion proteins may comprises multimers of about 5 to about 10 DBDpp subunits, about 10 to about 15 subunits, about 15 to about 20 subunits, about 20 to about 25 subunits, or about 25 to about 30 subunits (including numbers in between those listed as well as endpoints).
  • multiple tandem components of a DBDpp fusion protein can contain the same or different DBDpp.
  • the DBDpp are present as a monomer, or in homomultimers or heteromers such as, homodimers or heterodimers, homotrimers or heterotrimers, homotetramers or heterotetramers.
  • Examples of fusion partners of a DBDpp includes but is not limited to, antibodies, antibody subdomains (e.g., scFv or Fc domains), serum albumin, serum albumin subdomains, cell surface receptors, an alpha chain of a T cell receptor (TCR), a beta chain of a T cell receptor, cell surface receptor subdomains, peptides, peptide tags (e.g., FLAG or myc), fibronectin type III repeats, z-domains, elastin-like polypeptides.
  • TCR T cell receptor
  • TCR alpha chain of a T cell receptor
  • peptides peptide tags (e.g., FLAG or myc)
  • fibronectin type III repeats e.g., FLAG or myc
  • the number and location of DBDpp and their respective positions within the fusion protein can vary.
  • DBDpp(s) can be located at one or all termini of a fusion partner and/or interspersed within heterologous
  • the antibody component of the antibody-DBDpp fusion enhances half-life, and increase or decrease antibody dependent cellular cytotoxicity (ADCC), and/or complement dependent cytotoxicity (CDC) activity.
  • ADCC antibody dependent cellular cytotoxicity
  • CDC complement dependent cytotoxicity
  • the antibodies are human, murine, donkey, rabbit, goat, guinea pig, camel, llama, horse, or chicken antibodies. In specific embodiments, the antibodies are human.
  • the DBDpp fusion contains a whole antibody or an antibody fragment that is an antigen-binding fragment.
  • the antibody or antibody fragment binds a disease-related antigen.
  • the DBDpp fusion protein comprises an antibody or an antibody fragment that specifically binds a cancer antigen.
  • the DBDpp fusion protein comprises an antibody or an antibody fragment that specifically binds a particular pathogen (e.g., a bacterial cell (e.g., tuberculosis, smallpox, anthrax)), a virus (e.g., HIV), a parasite (e.g., malaria, leishmaniosis), a fungal infection, a mold, a mycoplasm, a prion antigen,
  • the DBDpp fusion protein comprises an antibody or an antibody fragment that specifically binds a particular pathogen (e.g., a bacterial cell (e.g., tuberculosis, smallpox, anthrax)), a virus (e.g., HIV), a parasite (e.g., malaria, leishmaniosis), a fungal infection, a mold, a mycoplasm, or a prion antigen.
  • the DBDpp fusion protein comprises an antibody or an antibody fragment that specifically binds a particular path
  • the DBDpp fusion protein comprises an antibody fragment that confers upon the DBDpp fusion protein a biological or biochemical characteristic of an immunoglobulin.
  • the antibody fragment confers a characteristic selected from: the ability to non-covalently dimerize, the ability to localize at the site of a tumor, and an increased serum half-life when compared to the DBDpp fusion protein in which said one or more DBDpp have been deleted.
  • the DBDpp fusion protein is at least as stable as the corresponding antibody without the attached DBDpp. In certain embodiments, the DBDpp fusion protein is more stable than the corresponding antibody without the attached DBDpp.
  • a DBDpp fusion contains an immunoglobulin effector domain or half-life influencing domain that corresponds to an immunoglobulin domain or fragment in which at least a fraction of one or more of the constant region domains has been altered so as to provide desired biochemical characteristics such as reduced or increased effector functions, the ability to non-covalently dimerize, increased ability to localize at the site of a tumor, reduced serum half-life, or increased serum half-life when compared with an immunoglobulin fragment having the corresponding unaltered immunoglobulin sequence.
  • These alterations of the constant region domains can be amino acid substitutions, insertions, or deletions.
  • a DBDpp fusion protein comprises a sequence of an immunoglobulin effector domain that has been modified to decrease ADCC (see, e.g., Idusogie et al., J. Immunol. 166:2571-2575 (2001); Sazinsky et al., PNAS 105:20167-20172 (2008); Davis et al., J. Rheumatol. 34:2204-2210 (2007); Bolt et al., Eur. J. Immunol. 23 :403-411 (1993); Alegre et al., Transplantation 57: 1537-1543 (1994); Xu et al., Cell Immunol.
  • a DBDpp fusion protein comprises an amino acid sequence of an immunoglobulin effector domain, or a derivative of an immunoglobulin effector domain, that confers the ability to bind neonatal receptor FcRn to the to the DBDpp fusion.
  • a DBDpp fusion protein comprises a sequence of an immunoglobulin FcRn binding domain that has been modified to enhance binding to FcRn (see, e.g., Petkova et al., Int. Immunol. 18: 1759-1769 (2006); Dall'Acqua et al, J.
  • the DBDpp-receptor fusion protein comprises a chimeric antigen receptor (CAR), or DBDpp-CAR, composed of the following elements: an extracellular targeting domain, a transmembrane domain and a cytoplasmic domain wherein the cytoplasmic domain comprises the signaling domain.
  • the DBDpp-CAR is composed of an extracellular targeting domain and a transmembrane domain.
  • the DBDpp-CAR is comprised of an extracellular domain composed of one or more DBDpp, in which each DBDpp constitutes a target-specific binding domain with the same or different specificities.
  • the DBDpp- CAR may also comprise an epitope tag.
  • the cytoplasmic domain of the DBDpp-CAR can include but is not limited to CD3-zeta, 4-1BB and CD28 signaling modules and combinations thereof.
  • the transmembrane domain can be derived either from a natural or from a synthetic source. Where the source is natural, the domain can be derived from any membrane-bound or transmembrane protein.
  • Transmembrane regions of particular use for the purposes herein may be derived from (i.e., comprise at least the transmembrane region(s) of) a member selected from the group: the alpha, beta or zeta chain of the T-cell receptor; CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, and CD154.
  • the transmembrane domain can be synthetic, in which case the DBDpp-CAR transmembrane domain will comprise predominantly hydrophobic residues such as leucine and valine.
  • the transmembrane domain comprises the triplet of phenylalanine, tryptophan and valine at each end of a synthetic transmembrane domain.
  • Further embodiments employ a fragment of the CD8 hinge region that has been further modified, through amino acid substitution, to improve expression function or immunogenicity. Further embodiments employ a fragment of the extracellular region derived from the human CD28 Further embodiments employ a fragment of the CD28 extracellular region that has been further modified, through amino acid substitution, to improve expression function or immunogenicity.
  • intracellular signaling domain refers to the portion of a DBDpp-CAR protein which transduces the effector function signal and directs the cell to perform a specialized function. While typically the entire intracellular signaling domain corresponding to a naturally occurring receptor can be employed, in many cases it is not necessary to use the entire chain. To the extent that a truncated portion of the intracellular signaling domain is used, such truncated portion can be used in place of the intact chain as long as it transduces the effector function signal.
  • intracellular signaling domain is thus meant to include any truncated portion of the intracellular signaling domain sufficient to transduce the effector function signal.
  • Primary cytoplasmic signaling sequences regulate primary activation of the TCR complex either in a stimulatory way, or in an inhibitory way.
  • Primary cytoplasmic signaling sequences that act in a stimulatory manner may contain signaling motifs which are known as immunoreceptor tyro sine-based activation motifs (ITAMs).
  • ITAMs immunoreceptor tyro sine-based activation motifs
  • Examples of such molecules include CD27, CD28, 4-1BB (CD 137), OX40, CD30, CD40, PD1, ICOS, lymphocyte function-associated antigen-1 (LFAl), CD2, CD7, LIGHT, NKG2C, B7H3, T1M1, and LAG-3.
  • Suitable linkers for operably linking a DBDpp and an additional component of a DBDpp fusion protein in a single-chain amino acid sequence include but are not limited to, polypeptide linkers such as glycine linkers, serine linkers, mixed glycine/serine linkers, glycine- and serine-rich linkers or linkers composed of largely polar polypeptide fragments.
  • the DBDpp fusion protein linker is made up of one or more of the amino acids selected from glycine, alanine, proline, asparagine, aspartic acid, threonine, glutamine, and lysine.
  • the DBDpp fusion protein linker is made up of a majority of amino acids that are sterically unhindered.
  • a linker in which the majority of amino acids are glycine, serine, and/or alanine.
  • the peptide linker is selected from polyglycines (such as (Gly)5, and (Gly)8, poly(Gly-Ala), and polyalanines.
  • the peptide linker contains the sequence of Gly-Gly-Gly-Gly-Thr-Gly-Gly-Gly-Gly- Ser. In some embodiments, the peptide linker contains the sequence of Gly-Gly-Gly-Gly-Asp-Gly- Gly-Gly-Gly-Ser.
  • a DBDpp fusion comprises a DBDpp directly attached (i.e., without a linker) to another component of the DBDpp fusion protein. In one embodiment, a DBDpp fusion comprises at least 2, at least 3, at least 4, DBDpp directly attached to another component of the DBDpp fusion.
  • linker(s) may have some influence on the properties of the final polypeptide of the invention, including but not limited to the affinity, specificity or avidity for a target of interest, or for one or more other target proteins of interest.
  • these linkers may be the same or different.
  • a person skilled in the art will be able to routinely determine the optimal linker composition and length for the purpose of operably linking a DBDpp and other components of a DBDpp fusion protein.
  • the linker can also be a non-peptide linker such as an alkyl linker, or a PEG linker.
  • These alkyl linkers may further be substituted by any non-sterically hindering group such as lower alkyl e.g., CI C6) lower acyl, halogen (e.g., CI, Br), CN, NH2, phenyl, etc.
  • An exemplary non- peptide linker is a PEG linker.
  • the PEG linker has a molecular weight of about 100 to 5000 kDa, or about 100 to 500 kDa.
  • Suitable linkers for coupling DBDpp and DBDpp fusion protein components by chemical cross-linking include, but are not limited to, homo-bifunctional chemical cross-linking compounds such as glutaraldehyde, imidoesters such as dimethyl adipimidate (DMA), dimethyl suberimidate (DMS) and dimethyl pimelimidate (DMP) or N-hydroxysuccinimide (NHS) esters such as dithiobis(succinimidylpropionate) (DSP) and dithiobis (sulfosuccinimidylpropionate) (DTSSP).
  • homo-bifunctional chemical cross-linking compounds such as glutaraldehyde, imidoesters such as dimethyl adipimidate (DMA), dimethyl suberimidate (DMS) and dimethyl pimelimidate (DMP) or N-hydroxysuccinimide (NHS) esters such as dithiobis(succinimidylpropionate) (DSP)
  • linkers for coupling DBDpp and DBDpp fusion protein components of hetero- bifunctional reagents for cross-linking include, but are not limited to, cross-linkers with one amine- reactive end and a sulfhydryl-reactive moiety at the other end, or with a NHS ester at one end and an SH-reactive group (e.g., a maleimide or pyridyl).
  • one or more of the linkers in the DBDpp fusion protein is cleavable.
  • cleavable linkers include, without limitation, a peptide sequence recognized by proteases (in vitro or in vivo) of varying type, such as Tev, thrombin, factor Xa, plasmin (blood proteases), metalloproteases, cathepsins (e.g., GFLG, etc.), and proteases found in other corporeal compartments.
  • the linker is a "cleavable linker" that facilitates the release of a DBDpp or cytotoxic agent in a cell.
  • a DBDpp or cytotoxic agent e.g., a DBDpp or cytotoxic agent in a cell.
  • an acid-labile linker e.g., hydrazone
  • protease- sensitive linker e.g., peptidase-sensitive linker
  • photolabile linker e.g., dimethyl linker or disulfide-containing linker
  • Linker optimization can be evaluated using techniques described herein and/or otherwise known in the art.
  • linkers do not disrupt the ability of a DBDpp to bind a target molecule and/or another DBDpp fusion protein component such as an antibody domain or fragment to bind an antigen.
  • the production of the DBDpp may be carried out using a variety of standard techniques for chemical synthesis, semi- synthetic methods, and recombinant DNA methodologies known in the art. Also provided is a method for producing a DBDpp, individually or as part of multi-domain fusion protein, as soluble agents and cell associated proteins.
  • a natural amino acid e.g., one of alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, or valine
  • the modifications do not include substituting in either a cysteine or a proline.
  • the reference sequence and/or the modified polypeptides can be de-immunized
  • residues or motifs that are potentially immunogenic can be identified and modified in order to reduce or eliminate potential immune responses to the DBDpp. Additional details regarding various embodiments of the production, selection, and isolation of DBDpp are provided in more detail below.
  • a vector comprising a DBDpp encoding nucleic acid is introduced into a host cell (e.g., phagemid) for expression of a DBDpp.
  • the vector can remain episomal or become chromosomally integrated, as long as the insert encoding therapeutic agent can be transcribed.
  • Vectors can be constructed by standard recombinant DNA technology. Vectors can be plasmids, phages, cosmids, phagemids, viruses, or any other types known in the art, which are used for replication and expression in prokaryotic or eukaryotic cells.
  • Prokaryotes useful as host cells in producing a DBDpp such as DBDpp fusion protein include gram negative or gram positive organisms such as, E. coli and B. subtilis.
  • Expression vectors for use in prokaryotic host cells generally contain one or more phenotypic selectable marker genes (e.g., genes encoding proteins that confer antibiotic resistance or that supply an autotrophic requirement).
  • prokaryotic host expression vectors examples include the pKK223-3 (Pharmacia, Uppsala, Sweden), pGEMl (Promega, Wis., USA), pET (Novagen, Wis., USA) and pRSET (Invitrogen, Calif, USA) series of vectors (see, e.g., Studier, J. Mol. Biol. 219:37 (1991) and Schoepfer, Gene 124:83 (1993)).
  • a eukaryotic host cell systems including yeast cells transformed with recombinant yeast expression vectors containing the coding sequence of a DBDpp, such as, the expression systems taught in U.S. Appl. No. 60/344, 169 and WO03/056914 (methods for producing humanlike glycoprotein in a non-human eukaryotic host cell) (the contents of each of which are incorporated by reference in their entirety).
  • yeast that can be used to produce compositions of the invention, such as, DBD include yeast from the genus Saccharomyces, Pichia, Actinomycetes and Kluyveromyces.
  • Yeast vectors typically contain an origin of replication sequence from a 2mu yeast plasmid, an autonomously replicating sequence (ARS), a promoter region, sequences for polyadenylation, sequences for transcription termination, and a selectable marker gene.
  • ARS autonomously replicating sequence
  • promoter sequences in yeast expression constructs include, promoters from metallothionein, 3-phosphoglycerate kinase (Hitzeman, J. Biol. Chem.
  • glycolytic enzymes such as, enolase, glyceraldehyde-3 -phosphate dehydrogenase, hexokinase, pyruvate decarboxylase, phosphofructokinase, glucose-6-phosphate isomerase, 3- phospho glycerate mutase, pyruvate kinase, triosephosphate isomerase, phosphoglucose isomerase, and glucokinase.
  • Additional suitable vectors and promoters for use in yeast expression as well as yeast transformation protocols are known in the art. See, e.g., Fleer, Gene 107:285-195 (1991) and Hinnen, PNAS 75: 1929 (1978).
  • Insect and plant host cell culture systems are also useful for producing the compositions of the invention.
  • host cell systems include for example, insect cell systems infected with recombinant virus expression vectors (e.g., baculovirus) containing the coding sequence of a DBD; plant cell systems infected with recombinant virus expression vectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) or transformed with recombinant plasmid expression vectors (e.g., Ti plasmid) containing the coding sequence of a DBD, including, but not limited to, the expression systems taught in U. S. Pat. No. 6,815, 184; U.S. Publ. Nos. 60/365,769, and 60/368,047; and WO2004/057002, WO2004/024927, and WO2003/078614, the contents of each of which is herein incorporated by reference in its entirety.
  • the host cell systems may be used, including animal cell systems infected with recombinant virus expression vectors (e.g., adenoviruses, retroviruses, adeno-associated viruses, herpes viruses, lentiviruses) including cell lines engineered to contain multiple copies of the DNA encoding a DBDpp either stably amplified (CHO/dhfr) or unstably amplified in double-minute chromosomes (e.g., murine cell lines).
  • the vector comprising the polynucleotide(s) encoding the DBDpp is polycistronic.
  • Exemplary mammalian cells useful for producing these compositions include 293 cells (e.g., 293T and 293F), CHO cells, BHK cells, NS0 cells, SP2/0 cells, YO myeloma cells, P3X63 mouse myeloma cells, PER cells, PER.C6 (Crucell, Netherlands) cells VERY, Hela cells, COS cells, MDCK cells, 3T3 cells, W138 cells, BT483 cells, Hs578T cells, HTB2 cells, BT20 cells, T47D cells, CRL7O30 cells, HsS78Bst cells, hybridoma cells, and other mammalian cells.
  • 293 cells e.g., 293T and 293F
  • CHO cells e.g., 293T and 293F
  • BHK cells e.g., NS0 cells, SP2/0 cells
  • YO myeloma cells e.g., P3X63 mouse myel
  • Transcriptional and translational control sequences for mammalian host cell expression vectors are frequently derived from viral genomes.
  • Commonly used promoter sequences and enhancer sequences in mammalian expression vectors include, sequences derived from Polyoma virus, Adenovirus 2, Simian Virus 40 (SV40), and human cytomegalovirus (CMV).
  • Exemplary commercially available expression vectors for use in mammalian host cells include pCEP4 (Invitrogen) and pcD A3 (Invitrogen).
  • a nucleic acid into a host cell e.g., a mammalian host cell
  • a host cell e.g., a mammalian host cell
  • Methods for producing cells comprising vectors and/or exogenous nucleic acids are well-known in the art. See, for example, Sambrook et al. (2001, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York).
  • Chemical means for introducing a polynucleotide into a host cell include colloidal dispersion systems, such as macromolecule complexes, nanocapsules, microspheres, beads, and lipid-based systems including oil-in-water emulsions, micelles, mixed micelles, and liposomes.
  • An exemplary colloidal system for use as a delivery vehicle in vitro and in vivo is a liposome (e.g., an artificial membrane vesicle).
  • an exemplary delivery vehicle is a liposome.
  • the use of lipid formulations is contemplated for the introduction of the nucleic acids into a host cell (in vitro, ex vivo or in vivo).
  • Lipid, lipid DNA or lipid/expression vector associated compositions are not limited to any particular structure in solution. For example, they can be present in a bilayer structure, as micelles, or with a "collapsed" structure. They can also simply be interspersed in a solution, possibly forming aggregates that are not uniform in size or shape.
  • Lipids are fatty substances which can be naturally occurring or synthetic lipids.
  • lipids include the fatty droplets that naturally occur in the cytoplasm as well as the class of compounds which contain long-chain aliphatic hydrocarbons and their derivatives, such as fatty acids, alcohols, amines, amino alcohols, and aldehydes.
  • Liposome is a generic term encompassing a variety of single and multilamellar lipid vehicles formed by the generation of enclosed lipid bilayers or aggregates. Liposomes can be characterized as having vesicular structures with a phospholipid bilayer membrane and an inner aqueous medium. Multilamellar liposomes have multiple lipid layers separated by aqueous medium. They form spontaneously when phospholipids are suspended in an excess of aqueous solution.
  • Reporter genes are used for identifying potentially transfected cells and for evaluating the functionality of regulatory sequences.
  • a reporter gene is a gene that is not present in or expressed by the recipient organism, tissue, or cell and that encodes a polypeptide whose expression is manifested by some easily detectable property, e.g., enzymatic activity. Expression of the reporter gene is assayed at a suitable time after the DNA has been introduced into the recipient cells.
  • reporter genes can include genes encoding luciferase, beta-galactosidase, chloramphenicol acetyl transferase, secreted alkaline phosphatase, or the green fluorescent protein gene (e.g., Ui-Tei et al., FEBS Lett. 479:79-82 (2000)).
  • Suitable expression systems are known in the art and can be prepared using known techniques or obtained commercially.
  • the construct with the minimal 5' flanking region showing the highest level of expression of reporter gene is identified as the promoter.
  • Such promoter regions can routinely be linked to a reporter gene and used to evaluate agents for the ability to modulate promoter-driven transcription.
  • a number of selection systems can be used in mammalian host-vector expression systems, including, but not limited to, the herpes simplex virus thymidine kinase, hypoxanthine- guanine phosphoribosyltransferase and adenine phosphoribosyltransferase (Lowy et al., Cell 22:817 (1980)) genes, which can be employed in tk " , hgprf or aprt " cells, respectively.
  • a DBDpp such as a DBDpp fusion protein
  • it can be purified by any method known in the art for purification of a recombinant protein, for example, by chromatography (e.g., ion exchange, affinity, and sizing column chromatography), centrifugation, differential solubility, or by any other standard technique for the purification of proteins.
  • the DBDpp are optionally fused to heterologous polypeptide sequences described herein or otherwise known in the art to facilitate purification.
  • ligands e.g., antibodies and other affinity matrices
  • DBDpp affinity columns for affinity purification
  • DBDpp or other components of the DBDpp fusion composition that are bound by these ligands are removed from the composition prior to final preparation of the DBDpp using techniques known in the art.
  • DBDpp production of DBDpp result in cell associated DBDpp compositions.
  • the expression of recombinant vectors that encode DBDpp operably linked to a cell membrane anchor or transmembrane domain have the potential to remain cell associated.
  • DBDpp comprising chimeric antigen receptors are intentionally cell associated and used in the context of the cell in which they are expressed.
  • One particular embodiment relates to a strategy of adoptive cell transfer of T cells which have been transduced to express a DBDpp chimeric antigen receptor (CAR).
  • the cell can be genetically modified to stably express a DBDpp on its surface, conferring novel target specificity that is MHC independent.
  • This promoter sequence is a strong constitutive promoter sequence capable of driving high levels of expression of any polynucleotide sequence operatively linked thereto.
  • a suitable promoter is EF- la.
  • other constitutive promoter sequences can also be used, including, but not limited to the simian virus 40 (SV40) early promoter, mouse mammary tumor virus (MMTV), human immunodeficiency virus (HIV) long terminal repeat (LTR) promoter, MoMuLV promoter, an avian leukemia virus promoter, an Epstein-Barr virus immediate early promoter, a Rous sarcoma virus promoter, as well as human gene promoters such as, but not limited to, the actin promoter, the myosin promoter, the hemoglobin promoter, and the creatine kinase promoter.
  • Inducible promoters include, but are not limited to a metallothionein promoter, a glucocorticoid promoter, a progesterone promote
  • DBDpp production may also be carried out using organic chemical synthesis of the desired polypeptide using a variety of liquid and solid phase chemical processes known in the art.
  • Various automatic synthesizers are commercially available and can be used in accordance with known protocols. See, for example, Tarn et al., J. Am. Chem. Soc, 105:6442 (1983); Merrifield, Science 232:341-347 (1986); Barany and Merrifield, The Peptides, Gross and Meienhofer, eds, Academic Press, New York, 1- 284; Barany et al., Int. J. Pep. Protein Res., 30:705 739 (1987); Kelley et al.
  • the peptides are acetylated at the N-terminus and/or amidated at the C-terminus.
  • any of numerous chemical modifications may be carried out by known techniques, including, but not limited to acetylation, formylation, etc. Additionally, the derivative may contain one or more non-classical amino acids.
  • a "library” of DBDpp refers to a plurality of unique DBDpp.
  • a “vector library” of DBDpp refers to a plurality of unique nucleic acids encoding DBDpp.
  • DBDpp are represented by a mixed population, or library, of different DBDpp molecules.
  • a library of DBDpp does not imply any particular size limitation to the number unique polypeptide molecules.
  • a library can contain as few as 3, 5, 6, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 75, or 100 unique DBDpp, and can range to greater than 1020 different DBDpp.
  • the library has up to about 10 4 , 10 5 , 10 6 , 10 7 , or 10 8 unique DBDpp. In further embodiments the library has up to about 10 12 different DBDpp.
  • a population of polypeptide variants is based on a sequence of core residues and variant residues.
  • SEQ ID NO: 3 variant residues are denoted by an X, where X can be any amino acid residue independent of the identity of any other residue denoted X in the sequence.
  • X can comprise a null position (e.g., no amino acid at that site).
  • the different varied amino acids X may be chosen from all 20 naturally occurring amino acid residues in such a way that any of these 20 naturally occurring amino acid residues may be present at the corresponding X position in any given variant. The selection of amino acid residue in each position is more or less randomized, depending on the embodiment.
  • the variant residues are not replaced by cysteine and/or proline.
  • the variability in different positions can be adjusted individually, between one, meaning no randomization, up to all 20 amino acids. Random introduction of a smaller subset of amino acids may be obtained by careful selection of the deoxyribonucleotide bases introduced, for example the codons T(A/C)C may be introduced to obtain a random introduction of either serine or tyrosine at a given position in the polypeptide chain.
  • the codons (T/C/A/G)CC can be introduced to obtain a random introduction of phenylalanine, leucine, alanine and valine at a given position in the polypeptide chain.
  • deoxyribonucleotide base combinations can be used to obtain different combinations of amino acids at a given position in the polypeptide chain.
  • the set of amino acids that can appear at a given position in the polypeptide chain can also be determined by the introduction of trinucleotides during the oligonucleotide synthesis, instead of one deoxyribonucleotide base at a time.
  • 5 to 25, 5 to 30, 5 to 35, 5 to 40, 5 to 45, or 5 to 50 of the modified amino acid residues are conservative substitutions. In some embodiments, 5 to 25, 5 to 30, 5 to 35, 5 to 40, 5 to 45, or 5 to 50 of the modified amino acid residues are non-conservative substitutions. In a further embodiment, 5 to 15, 5 to 20, 5 to 25, 5 to 30, 5 to 35, 5 to 40, or 5 to 45 of the amino acid residue modifications are conservative substitutions and 5 to 15, 5 to 20, 5 to 25, 5 to 30, 5 to 35, 5 to 40, or 5 to 45 of the amino acid residue modifications are non-conservative substitutions.
  • 5 to 25, 5 to 30, 5 to 35, 5 to 40, 5 to 45, 5 to 50, 5 to 55, or 5 to 60 of the substitutions are at amino acid residues of SEQ ID NO: l selected from the group consisting of: Ml, G2, S3, W4, A5, E6, K8, Q9, R10, A12, A13, K15, T16, R17, E19, A20, L21, G22, G23, S24, E25, A26, E27, A29, A30, E32, K33, E34, A36, A37, E39, S40, E41, Q43, A44, Y45, K46, G47, K48, G49, N50, P51, E52, E54, A55, R57, K58, E59, A61, A62, R64, D65, E66, Q68, A69, Y70, R71, H72, and N73.
  • SEQ ID NO: l selected from the group consisting of: Ml, G2, S3,
  • 1 to 20, 1 to 30, or 1 to 40 of the substitutions are at amino acid residues of SEQ ID NO: 1 selected from the group consisting of: G2, S3, W4, A5, E6, K8, Q9, R10, A12, A13, K15, T16, R17, E19, A20, A29, A30, E32, K33, E34, A36, A37, E39, S40, E41, Q43, A44, E52, E54, A55, R57, K58, E59, A61, A62, R64, D65, E66, Q68, A69, and Y70.
  • SEQ ID NO: 1 selected from the group consisting of: G2, S3, W4, A5, E6, K8, Q9, R10, A12, A13, K15, T16, R17, E19, A20, A29, A30, E32, K33, E34, A36, A37, E39, S40, E41, Q43, A44, E52, E54, A55,
  • the library comprises at least 2, 3, 4, 5, 10, 25, 50, 75, 100, 250, 500, or 1000 different DBDpp that specifically bind a protein target selected from the group consisting of: an immunoglobulin, an enzyme, a hormone, a serum protein, a cell surface protein, a therapeutic protein, a TSA, a CSA, and a protein containing a sequence tag.
  • the library comprises at least 2, 3, 4, 5, 10, 25, 50, 75, 100, 250, 500, or 1000 different DBDpp that specifically bind a target disclosed herein.
  • the library is a vector library or a host cell library.
  • the vector library is a library of host cells.
  • the host cell library comprises a plurality of host cells that display the DBDpp on their surface.
  • the host cells are phage that display the DBDpp on their surface.
  • the DBDpp library comprises: (a) 3 DBDpp that specifically bind to different targets; (b) 3, 4, 5, 6, 7, 8, 9, 10, or more than 10 DBDpp having different sequences that specifically bind to the same target; (c) 3, 4, 5, 6, 7, 8, 9, 10, or more than 10 DBDpp having different sequences that specifically bind to the same epitope of a target of interest; (d) 3, 4, 5, 6, 7, 8, 9, 10, or more than 10 DBDpp having different sequences that specifically bind to different epitopes of a target; or (e) 3, 4, 5, 6, 7, 8, 9, 10, or more than 10 DBDpp having different sequences that compete for binding to the same target.
  • the DBDpp library comprises a plurality of different DBDpp that comprise the amino acid sequence of SEQ ID NO: l wherein 5 to 25, 5 to 30, 5 to 35, 5 to 40, 5 to 45, 5 to 50, 5 to 55, or 5 to 60 amino acid residues have been modified; and wherein the DBDpp specifically binds a target of interest.
  • 5 to 25, 5 to 30, 5 to 35, 5 to 40, 5 to 45, 5 to 50, 5 to 55, or 5 to 60 of the modified amino acid residues are substitutions.
  • 5 to 25, 5 to 30, 5 to 35, 5 to 40, 5 to 45, or 5 to 50 of the modified amino acid residues are conservative substitutions. In some embodiments, 5 to 25, 5 to 30, 5 to 35, 5 to 40, 5 to 45, or 5 to 50 of the modified amino acid residues are non-conservative substitutions. In a further embodiment, 5 to 15, 5 to 20, 5 to 25, 5 to 30, 5 to 35, 5 to 40, or 5 to 45 of the amino acid residue modifications are conservative substitutions and 5 to 15, 5 to 20, 5 to 25, 5 to 30, 5 to 35, 5 to 40, or 5 to 45 of the amino acid residue modifications are non-conservative substitutions.
  • 5 to 25, 5 to 30, 5 to 35, 5 to 40, 5 to 45, 5 to 50, 5 to 55, or 5 to 60 of the substitutions are at amino acid residues of SEQ ID NO: l selected from the group consisting of: Ml, G2, S3, W4, A5, E6, K8, Q9, R10, A12, A13, K15, T16, R17, E19, A20, L21, G22, G23, S24, E25, A26, E27, A29, A30, E32, K33, E34, A36, A37, E39, S40, E41, Q43, A44, Y45, K46, G47, K48, G49, N50, P51, E52, E54, A55, R57, K58, E59, A61, A62, R64, D65, E66, Q68, A69, Y70, R71, H72, and N73.
  • SEQ ID NO: l selected from the group consisting of: Ml, G2, S3,
  • 1 to 20, 1 to 30, or 1 to 40 of the substitutions are at amino acid residues of SEQ ID NO: 1 selected from the group consisting of: G2, S3, W4, A5, E6, K8, Q9, R10, A12, A13, K15, T16, R17, E19, A20, A29, A30, E32, K33, E34, A36, A37, E39, S40, E41, Q43, A44, E52, E54, A55, R57, K58, E59, A61, A62, R64, D65, E66, Q68, A69, and Y70.
  • SEQ ID NO: 1 selected from the group consisting of: G2, S3, W4, A5, E6, K8, Q9, R10, A12, A13, K15, T16, R17, E19, A20, A29, A30, E32, K33, E34, A36, A37, E39, S40, E41, Q43, A44, E52, E54, A55,
  • the library comprises at least 2, 3, 4, 5, 10, 25, 50, 75, 100, 250, 500, or 1000 different DBDpp that specifically binding different targets.
  • the different targets bound by DBDpp in the library are selected from the group consisting of: a nucleic acid, an oligosaccharide, a peptide, a protein, a cell surface antigen, and a small organic molecule.
  • the DBDpp library contains a plurality of different nucleic acid sequences encoding DBDpp, that comprise the amino acid sequence of SEQ ID NO: l wherein a total of 5 to 25, 5 to 30, 5 to 35, 5 to 40, 5 to 45, 5 to 50, 5 to 55, or 5 to 60 amino acid residues have been modified; and wherein the DBDpp specifically binds a target of interest.
  • 5 to 25, 5 to 30, 5 to 35, 5 to 40, 5 to 45, 5 to 50, 5 to 55, or 5 to 60 of the modified amino acid residues encoded by the nucleic acids sequences are substitutions.
  • 5 to 25, 5 to 30, 5 to 35, 5 to 40, 5 to 45, or 5 to 50 of the modified amino acid residues are conservative substitutions.
  • 5 to 25, 5 to 30, 5 to 35, 5 to 40, 5 to 45, or 5 to 50 of the encoded modified amino acid residues are non-conservative substitutions.
  • 5 to 15, 5 to 20, 5 to 25, 5 to 30, 5 to 35, 5 to 40, or 5 to 45 of the encoded amino acid residue modifications are conservative substitutions and 5 to 15, 5 to 20, 5 to 25, 5 to 30, 5 to 35, 5 to 40, or 5 to 45 of the encoded amino acid residue modifications are non-conservative substitutions.
  • 5 to 25, 5 to 30, 5 to 35, 5 to 40, 5 to 45, 5 to 50, 5 to 55, or 5 to 60 of the encoded substitutions are at amino acid residues of SEQ ID NO: l selected from the group consisting of: Ml, G2, S3, W4, A5, E6, K8, Q9, R10, A12, A13, K15, T16, R17, E19, A20, L21, G22, G23, S24, E25, A26, E27, A29, A30, E32, K33, E34, A36, A37, E39, S40, E41, Q43, A44, Y45, K46, G47, K48, G49, N50, P51, E52, E54, A55, R57, K58, E59, A61, A62, R64, D65, E66, Q68, A69, Y70, R71, H72, and N73.
  • SEQ ID NO: l selected from the group consisting of: Ml, G2,
  • the nucleic acids optionally encode a DBDpp that further comprises an amino acid sequence wherein 1 to 5, 5 to 15, 5 to 20, 5 to 25, 5 to 30, 5 to 35, 5 to 40, or 5 to 45 of the residues corresponding to the solvent inaccessible residues of the amino acid sequence of SEQ ID NO: 1 are substituted and wherein the DBDpp specifically binds a target of interest.
  • the library comprises nucleic acids encoding at least 2, 3, 4, 5, 10, 25, 50, 75, 100, 250, 500, or 1000 different DBDpp that specifically bind different targets.
  • the different targets bound by DBDpp in the library are selected from the group consisting of: a nucleic acid, an oligosaccharide, a peptide, a protein, a cell surface antigen, and a small organic molecule.
  • the library comprises nucleic acids encoding at least 2, 3, 4, 5, 10, 25, 50, 75, 100, 250, 500, or 1000 different DBDpp that specifically bind a protein target selected from the group consisting of: an immunoglobulin, an enzyme, a hormone, a serum protein, a cell surface protein, a therapeutic protein, a TSA, a CSA, and a protein containing a peptide tag.
  • the library comprises nucleic acids encoding at least 2, 3, 4, 5, 10, 25, 50, 75, 100, 250, 500, or 1000 different DBDpp that specifically bind a target disclosed herein.
  • the vector library is contained in host cells (e.g., viral particles).
  • the library comprises a plurality of host cells that display the DBDpp on their surface.
  • the host cells are phage that display the DBDpp on their surface.
  • the DBDpp library comprises: (a) 3, 4, 5, 6, 7, 8, 9, 10, or more than 10 DBDpp that specifically bind to different targets; (b) 3, 4, 5, 6, 7, 8, 9, 10, or more than 10 DBDpp having different sequences that specifically bind to the same target; (c) 3, 4, 5, 6, 7, 8, 9, 10, or more than 10 DBDpp having different sequences that specifically bind to the same epitope of a target; (d) 3, 4, 5, 6, 7, 8, 9, 10, or more than 10 DBDpp having different sequences that specifically bind to different epitopes of a target; or (e) 3, 4, 5, 6, 7, 8, 9, 10, or more than 10 DBDpp having different sequences that compete for binding to the same target.
  • Nucleic acids encoding DBDpp such as DBDpp fusion proteins are also provided.
  • the host cell containing the nucleic acids is a bacteria, yeast, fungal or mammalian cell.
  • the host cell is an immune cell.
  • the host cell is a human immune cell.
  • the human immune cell expresses the DBDpp on its cell surface.
  • the nucleic acid encode a DBDpp fusion protein.
  • the host cell expresses the DBDpp as a fusion protein on the cell surface.
  • vector libraries comprising a plurality of nucleic acids encoding the DBDpp.
  • a vector library comprises a plurality of different nucleic acids encoding DBDpp, wherein the encoded DBDpp comprises an amino acid sequence selected from the group consisting of: (a) MGSWX 5 EFX 8 X 9 RLXi 2 AIXi 5 Xi 6 RL Xi 9 ALGGSEAELAAFEKEIAAFESELQAYKGKGNPEVEX 55 LRX 58 X 59 AAX 62 IRX 65 X 66 L
  • QAYRHN (SEQ ID NO:4), wherein 3 ⁇ 4, X 8 , X 9 , Xn, Xis, Xie, 3 ⁇ 4, X55, X 58 , X59, X 62 , Xes, and X 66 , is a natural and/or non-natural amino acid residue; (b) MGSWX 5 X 6 FKX 9 X 10 LA Xi 3 lKXi 6 Xi 7 LEALGGSEAELAX 3 oFEX 33 X 34 lAX 37 FEX 4 oX 4 iLQX 44 YKGKGNPEVEALRK
  • EAAAIRDELQAY HN (SEQ ID NO:2), wherein X 5 , X 6 , X 9 , X 10 , 3 ⁇ 4, Xie, Xn, X30, X 33 , X 34 , X37, X 4 o, X 4 i, and X 4 4, is a natural and/or non-natural amino acid residue; (c) MGSWAEFKQRLAAIKTRLEALGGSEAELAAFX32X 3 3EIX36AFX39X oELX 43 AYKGKGKG
  • X n is a natural amino acid residue other than cysteine or proline.
  • a plurality of the vectors in the library encode a DBDpp fusion protein.
  • the library comprises nucleic acids encoding at least 2, 3, 4, 5, 10, 25, 50, 75, 100, 250, 500, or 1000 different DBDpp that specifically bind different targets.
  • the different targets bound by DBDpp encoded by the nucleic acids in the library are selected from the group consisting of: a nucleic acid, an oligosaccharide, a peptide, a protein, a cell surface antigen, and a small organic molecule.
  • the library comprises nucleic acids encoding at least 2, 3, 4, 5, 10, 25, 50, 75, 100, 250, 500, or 1000 different DBDpp that specifically bind a protein target selected from the group consisting of: an immunoglobulin, an enzyme, a hormone, a serum protein, a cell surface protein, a therapeutic protein, a TSA, a CSA, and a protein containing a peptide tag.
  • the library comprises nucleic acids encoding at least 2, 3, 4, 5, 10, 25, 50, 75, 100, 250, 500, or 1000 different DBDpp that specifically bind a target disclosed herein.
  • a plurality of the vectors of the vector library are contained in host cells (e.g., viral particles such as phage), E. coli, yeast, and mammalian cells.
  • the host cells display DBDpp on their surface.
  • the host cells are phage that display DBDpp on their surface.
  • the vector library comprises: (a) nucleic acids encoding 3, 4, 5, 6, 7, 8, 9, 10, or more than 10 DBDpp that specifically bind to different targets; (b) nucleic acids encoding 3, 4, 5, 6, 7, 8, 9, 10, or more than 10 DBDpp 3, 4, 5, 6, 7, 8, 9, 10, or more than 10 DBDpp having different sequences that specifically bind to the same target; (c) nucleic acids encoding 3, 4, 5, 6, 7, 8, 9, 10, or more than 10 DBDpp having different sequences that specifically bind to the same epitope of a target; (d) nucleic acids encoding 3, 4, 5, 6, 7, 8, 9, 10, or more than 10 DBDpp having different sequences that specifically bind to different epitopes of a target; (e) nucleic acids encoding 3, 4, 5, 6, 7, 8, 9, 10, or more than 10 DBDpp having different sequences that compete for binding to the same target; or (f) 3, 4, 5, 6, 7, 8, 9, 10, or more than 10 different nucleic acids encoding the same DBDpp. Host
  • a vector library comprises a plurality of nucleic acids encoding DBDpp comprising an amino acid sequence selected from the group consisting of: (a) MGS WX 5 EFX 8 X 9 RLXi 2 AIXi 5 Xi 6 RLXi 9 ALZiEAELAAFEKEIAAFESELQAYZ 2 NPEVEX 5 oLR X53X54AAX57IRX60X61LQAYRH (SEQ ID NO:9), wherein X 5 , X 8 , X 9 , X12, X15, Xie, X19, X50, X53, X54, X57, ⁇ ⁇ ⁇ , and Xei, is a natural and/or non-natural amino acid residue, and Zi and Z 2 is 2 to 30 natural and/or non-natural amino acid residues; (b) MGSWX 5 X 6 FKX 9 X 10 LA Xi 3 lKXi 6 X
  • IRDELQAYRHN (SEQ ID N0 7), wherein X 5 , X 6 , X 9 , X10, X 13 , 3 ⁇ 4, X17, X 28 , X31, X32, X35, X 38 , X39, and X 42 , is a natural and/or non-natural amino acid residue, and Zi and Z2 is 2 to 30 natural and/or non-natural amino acid residues; (c) MGSWAEFKQRLAAIKTRLEALZiEAEL AAFX 3 oX 3 iEIX 34 AFX 37 X 38 ELX 4 iAYZ 2 , EVEALX 52 X 53 EAX 56 AIX 59 X 6 oELX 63 AYRHN (SEQ ID NO:8), wherein X 30 , X31, X34, X37, 3 8 , X41, Xs 2 , X53, X56, X59, X
  • X n is a natural amino acid residue. In a further embodiment, X n is a natural amino acid residue other than cysteine or proline.
  • a plurality of the vectors in the library encode a DBDpp fusion protein.
  • the library comprises nucleic acids encoding at least 2, 3, 4, 5, 10, 25, 50, 75, 100, 250, 500, or 1000 different DBDpp that specifically bind different targets.
  • the different targets bound by DBDpp encoded by the nucleic acids in the library are selected from the group consisting of: a nucleic acid, an oligosaccharide, a peptide, a protein, a cell surface antigen, and a small organic molecule.
  • 4, 5, 10 or more DBDpp encoded by the nucleic acids in the library specifically bind different targets.
  • a vector library comprises comprising a plurality of different nucleic acid sequences encoding DBDpp, that comprise the amino acid sequence of SEQ ID NO: l wherein a total of 5 to 25, 5 to 30, 5 to 35, 5 to 40, 5 to 45, 5 to 50, 5 to 55, or 5 to 60 amino acid residues have been modified; and wherein the DBDpp specifically binds a target of interest.
  • 5 to 25, 5 to 30, 5 to 35, 5 to 40, 5 to 45, 5 to 50, 5 to 55, or 5 to 60 of the modified amino acid residues encoded by the nucleic acids sequences are substitutions.
  • 5 to 25, 5 to 30, 5 to 35, 5 to 40, 5 to 45, 5 to 50, 5 to 55, or 5 to 60 of the encoded substitutions are at amino acid residues of SEQ ID NO: l selected from the group consisting of: Ml, G2, S3, W4, A5, E6, K8, Q9, R10, A12, A13, K15, T16, R17, E19, A20, L21, G22, G23, S24, E25, A26, E27, A29, A30, E32, K33, E34, A36, A37, E39, S40, E41, Q43, A44, Y45, K46, G47, K48, G49, N50, P51, E52, E54, A55, R57, K58, E59, A61, A62, R64, D65, E66, Q68, A69, Y70, R71, H72, and N73.
  • SEQ ID NO: l selected from the group consisting of: Ml, G2,
  • 1 to 20, 1 to 30, or 1 to 40 of the encoded substitutions are at amino acid residues of SEQ ID NO: 1 selected from the group consisting of: G2, S3, W4, A5, E6, K8, Q9, R10, A12, A13, K15, T16, R17, E19, A20, A29, A30, E32, K33, E34, A36, A37, E39, S40, E41, Q43, A44, E52, E54, A55, R57, K58, E59, A61, A62, R64, D65, E66, Q68, A69, and Y70.
  • the nucleic acids optionally encode a DBDpp that further comprises an amino acid sequence wherein 1 to 5, 5 to 15, 5 to 20, 5 to 25, 5 to 30, 5 to 35, 5 to 40, or 5 to 45 of the residues corresponding to the solvent inaccessible residues of the amino acid sequence of SEQ ID NO: 1 are substituted and wherein the DBDpp specifically binds a target of interest.
  • the library comprises nucleic acids encoding at least 2, 3, 4, 5, 10, 25, 50, 75, 100, 250, 500, or 1000 different DBDpp that specifically bind different targets.
  • the different targets bound by DBDpp in the library are selected from the group consisting of: a nucleic acid, an oligosaccharide, a peptide, a protein, a cell surface antigen, and a small organic molecule.
  • the library comprises nucleic acids encoding at least 2, 3, 4, 5, 10, 25, 50, 75, 100, 250, 500, or 1000 different DBDpp that specifically bind a protein target selected from the group consisting of: an immunoglobulin, an enzyme, a hormone, a serum protein, a cell surface protein, a therapeutic protein, a TSA, a CSA, and a protein containing a peptide tag.
  • the library comprises nucleic acids encoding at least 2, 3, 4, 5, 10, 25, 50, 75, 100, 250, 500, or 1000 different DBDpp that specifically bind a target disclosed herein.
  • the vector library is contained in host cells (e.g., viral particles).
  • the library comprises a plurality of host cells that display the DBDpp on their surface.
  • the host cells are phage that display the DBDpp on their surface.
  • the vector library comprises: (a) nucleic acids encoding 3, 4, 5, 6, 7, 8, 9, 10, or more than 10 DBDpp that specifically bind to different targets; (b) nucleic acids encoding 3, 4, 5, 6, 7, 8, 9, 10, or more than 10 DBDpp having different sequences that specifically bind to the same target; (c) nucleic acids encoding 3, 4, 5, 6, 7, 8, 9, 10, or more than 10 DBDpp having different sequences that specifically bind to the same epitope of a target; (d) nucleic acids encoding 3, 4, 5, 6, 7, 8, 9, 10, or more than 10 DBDpp having different sequences that specifically bind to different epitopes of a target; or (e) nucleic acids encoding 3, 4, 5, 6, 7, 8, 9, 10, or more than 10 DBDpp having different sequences that compete for binding to the same target; or (f) 3, 4, 5, 6, 7, 8, 9, 10, or more than 10 different nucleic acids encoding the same DBDpp. Host cells containing the vectors are also provided.
  • the vector library comprises: (a) nucleic acids encoding 3 DBDpp that specifically bind to different targets of interest; (b) nucleic acids encoding 3, 4, 5, 6, 7, 8, 9, 10, or more than 10 DBDpp having different sequences that specifically bind to the same target of interest; (c) nucleic acids encoding 3, 4, 5, 6, 7, 8, 9, 10, or more than 10 DBDpp having different sequences that specifically bind to the same epitope of a target of interest; (d) nucleic acids encoding 3, 4, 5, 6, 7, 8, 9, 10, or more than 10 DBDpp having different sequences that specifically bind to different epitopes of a target of interest; (e) nucleic acids encoding 3, 4, 5, 6, 7, 8, 9, 10, or more than 10 DBDpp having different sequences that compete for binding to the same target of interest; or (f) 3 different nucleic acid sequences encoding the same DBDpp sequence.
  • the vector library comprises a plurality of nucleic acids encoding DBDpp comprising an amino acid selected from the group consisting of: (a) MGSWX 5 EFX 8
  • X 57 IRX 6 oX 6 iLQAYRHN (SEQ ID NO:9), wherein X 5 , X 8 , X 9 , X12, X15, Xie, 3 ⁇ 4 3 ⁇ 4 X50, X53, X 54 , X57, X60, and X 6 i, is a natural and/or non-natural amino acid residue, and Zi and Z 2 is 2 to 30 natural and/or non-natural amino acid residues; (b) MGSWX5X6FKX9X10LAX13IK Xi 6 Xi 7 LEALZiEAELAX2 8 FEX 3 iX 3 2lAX 3 5FEX 38 X 39 LQX 4 2YZ 2 NPEVEALRKEAAAIRDE
  • LQAYRHN (SEQ ID NO:7), wherein X 5 , X 6 , X 9 , X 10 , X 13 , Xie, X17, 28, X31, X 32 , X35, X 38 , X39, and X42, is a natural and/or non-natural amino acid residue, and Zi and Z 2 is 2 to 30 natural and/or non- natural amino acid residues; (c) MGS WAEFKQRLAAIKTRLEALZ iE AELAA X3oX 3 iElX34AFX3 7 X38ELX41AYZ 2 PEVEALX5 2 X5 3 EAX56AlX59X6oELX63AYRHN (SEQ ID NO: 8), wherein X 30 , X31, X34, X37, X38, X41, X52, X53, X56, X59, Xeo, and X 63 is a natural and
  • X n is a natural amino acid residue. In a further embodiment, X n is a natural amino acid residue other than cysteine or proline.
  • a plurality of the vectors in the library encode a DBDpp fusion protein.
  • the library comprises nucleic acids encoding at least 2, 3, 4, 5, 10, 25, 50, 75, 100, 250, 500, or 1000 different DBDpp that specifically bind different targets.
  • the different targets bound by DBDpp encoded by the nucleic acids in the library are selected from the group consisting of: a nucleic acid, an oligosaccharide, a peptide, a protein, a cell surface antigen, and a small organic molecule.
  • the host cells e.g., viral particles
  • the host cells are phage that display DBDpp on their surface.
  • at least two, three, four, five, or ten of the DBDpp encoded in the vector library specifically bind different targets.
  • the DBDpp binds a target of interest selected from the group consisting of: a nucleic acid, an oligosaccharide, a peptide, a protein, a cell surface antigen, and a small organic molecule.
  • the DBDpp target of interest is a protein selected from the group consisting of: an immunoglobulin, an enzyme, a hormone, a serum protein, a cell surface protein, a therapeutic protein, a TSA, a CSA, and a protein containing a peptide tag.
  • the DBDpp specifically binds a target disclosed herein.
  • a library of host cells e.g., viral particles
  • the library contains a plurality of host cells (e.g., viral particles) that display the DBDpp on their surface.
  • the host cells are phage that display the DBDpp on their surface.
  • a method of screening a library of DBDpp for binding with a binding partner comprises: (a) obtaining a population displaying a library of DBDpp; (b) contacting the population with the target of interest under conditions suitable for binding; and (c) identifying those DBDpp that bind to the target.
  • Two exemplary DBDpp display selection processes include panning and cell- based screening selection.
  • DBDpp phage display libraries are prepared and screened for DBDpp having desired properties, including the ability to specifically bind numerous validated therapeutic and diagnostic targets.
  • Representative DBDpp identified in these screens are further characterized and demonstrated to display desirable properties useful in for example, purification, diagnostic, and therapeutic applications.
  • Nucleic acids encoding DBDpp may be obtained using standard methods in the art, such as chemical synthesis, recombinant methods and/or obtained from biological sources. Nucleic acid of interest may be placed under the control of one or more elements necessary for their expression in any particular host cell.
  • a variety of host cells are available to propagate nucleic acids encoding DBDpp, and display methods are known in the art and described herein that may be used in display DBDpp on their surface. Display methods include without limitation phage display, bacterial display, yeast display, ribosome display, and mRNA display.
  • the generation of a (partially) randomized DBDpp library requires the (partial) randomization of specific positions within the reference scaffold sequence of SEQ ID NO: l .
  • other reference sequences may be used and modified according to the methods disclosed herein.
  • a DBDpp library for use in the methods provided herein are generated by recombinant DNA techniques.
  • libraries of nucleic acid sequences encoding DBDpp each differing in sequence at particular amino acid positions can be obtained by site-directed or random mutagenesis of a template sequence.
  • the number of substituted amino acid residue positions in DBDpp libraries provided herein range from 5 to 20 amino acid residue positions.
  • a defined set of substituted amino acid residue positions in a DBDpp library provided herein comprise 5 to 20 defined substituted amino acid residue positions, such as 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20, defined substituted amino acid residue positions.
  • the substituted amino acid residues are natural or non-natural amino acids.
  • any of the 20 natural amino acids can be used.
  • the substitutions do not result in the replacement of any amino acids with a cysteine and/or a proline.
  • a library of DBDpp can contain any suitable number of different DBDpp sequences.
  • the library of DBDpp contain least 2, at least 5, at least 10, at least 50, at least 100, at least 1000, at least 10,000, at least 105, at least 106, at least 107, at least 108, at least 109 or more different DBDpp sequences (e.g., DBDpp fusion proteins).
  • substituted amino acid residue position when referring to a library of different-DBDpp sequences, refers to an amino acid residue position at which at least two different amino acid residue types are located when at least two of the amino acid sequences of the different DBDpp from a library of DBDpp are compared to each other.
  • the disclosure encompasses methods of producing a library (i.e., a collection or plurality) of DBDpp which differ from each other in at least one of a defined set of 5 to 20 substituted amino acid residue positions. Therefore, the sequences within a library of DBDpp differ from each other at any one or more particular amino acid positions that are comprised in a selected, defined, or random set. Accordingly, the term “different sequences” or “different DBDpp sequences” refers to the occurrence of sequence variation or sequence differences in a defined set of amino acid residue positions between two or more DBDpp in a library.
  • the population or library of molecules is displayed on a typical display vehicle (e.g., bacteriophage, E.coli, ribosome) that affords the coupling of phenotype to genotype.
  • a typical display vehicle e.g., bacteriophage, E.coli, ribosome
  • the DBDpp of the library are displayed on the surface of a phage particle, a ribosome, a bacterium, a yeast cell, a mammalian cell or any other suitable (micro)organism, so as to facilitate screening or selection to isolate the desired DBDpp sequences having detectable binding affinity for, or detectable in vitro activity on the target of interest.
  • a major advantage of this technology is the coupling of genotype (i.e., the encapsulated DNA encoding the displayed protein) and phenotype (i.e., the displayed protein such as a DBDpp provided herein) which allows affinity-based selection from libraries with millions to trillions of polypeptide variants in a relatively simple in vitro assay.
  • Suitable methods, techniques and host organisms for displaying and selecting or screening a library of substituted DBDpp sequences or nucleotide sequences encoding such substituted DBDpp sequences, and which are applicable to DBDpp having desired features, are known to the person skilled in the art. Such methods are described, for example, in Georgiou, Nat. Biotechnol. 15:29-34 (1997); Wittrup, Curr. Opin. Biotechnol.
  • phage library display and the selection by means of a phage display technique may be chosen as a method for high-throughput identification of protein- specific binders, because it is one of the most robust and versatile selection techniques available (Scott, Science 249:386-390 (1990); Bratkovic, Cell. Mol. Life Sci. 67:749-767 (2010)).
  • a typical phage display protocol involves the use of a filamentous phage (phagemid) surface expression system, production of phage particles in a bacterial host with each particle displaying the gene product of one member of the gene library as a fusion with one type of its coat proteins (gill or gVIII proteins).
  • a library of phage particles is taken through a selection process for binding to an immobilized target molecule ('biopanning') involving binding of the phage library to the target, washing steps to remove non-bound phage DBDpp, and elution of bound particles.
  • 'biopanning' immobilized target molecule
  • washing steps to remove non-bound phage DBDpp washing steps to remove non-bound phage DBDpp
  • elution of bound particles usually several rounds of panning are necessary to select molecules with the desired characteristics involving reamplification of eluted phage in the bacterial host and selection on the immobilized target.
  • a given DBDpp library may be represented by a collection of phagemids each of which encodes for a fusion protein comprising a member of the DBDpp library fused to the minor coat protein pill.
  • These phagemids can be introduced into suitable E. coli cells (e.g. TGI) by electroporation or other means.
  • E. coli cells e.g. TGI
  • helper phage phage are produced (packaging also the phagemid genome) that display the DBDpp -fusion protein.
  • These phage can be used to select binders against a given target and the selected phage can be propagated by infecting E. coli TGI (Stratagene).
  • the DBDpp libraries are provided as a phage library and binding DBDpp are identified by contacting the phage with the labeled target of interest, after which binding phages are retrieved by detection or selective collection of the labeled, bound target.
  • a biotinylated target is used, whereby phage which generate a DBDpp that specifically binds to the target are captured with a streptavidin-coated support (e.g., magnetic beads).
  • the selection steps of the methods for producing one or more DBDpp having detectable binding affinity for a target of interest may comprise the (further) enrichment of the DBDpp library or the mixture of DBDpp libraries for DBDpp having detectable binding affinity for the target of interest by iterative execution of the steps of contacting a target of interest with a DBDpp library or with a mixture of DBDpp libraries (including a plurality of DBDpp) of the invention and subsequently identifying from the DBDpp library or mixture of DBDpp libraries being contacted with the protein, one or more DBDpp having detectable binding affinity for the target of interest.
  • phage display methods are used to display and screen DBDpp for the ability to specifically bind a target of interest
  • DBD domain can be displayed and selected on the surface of phage.
  • Different libraries of DBDpp based on the scaffold of SEQ ID NO: l, and described herein in the examples, were prepared and subjected to phage display methods to demonstrate that DBDpp can be produced that specifically bind to different targets of interest including CD137, CD47, CTLA4, DR5, KIR, PD-L1, PD1 and TIM3.
  • the library screening techniques include a cell surface display system.
  • the cell surface display system may comprise prokaryotic cells, such as Gram+ cells, or eukaryotic cells, such as yeast cells.
  • prokaryotic cells such as Gram+ cells
  • eukaryotic cells such as yeast cells.
  • Numerous cell surface display systems are known in the art and can routinely be adapted for screening DBDpp libraries.
  • Prokaryotic systems are, for example, described in Francisco et al., PNAS 90: 10444-10448 (1993) and Lee et al, Trends Biotechnol 21 :45-52 (2003).
  • Eukaryotic systems are described for example in Boder et al, Nat. Biotechnol. 15:553-557 (1997) and Gai et al., Curr. Opin. Struct. Biol.
  • E. coli display methods such as peptidoglycan-associated lipoprotein (PAL) fusion are also encompassed herein.
  • PAL peptidoglycan-associated lipoprotein
  • a DBDpp peptide can be fused to the carboxyl terminus of the lac repressor and expressed in E. coli.
  • the bacterial display and yeast display technologies known in the art allow expression of recombinant proteins on the surface of yeast cells S. cerevisiae (Boder, Nat. Biotechnol. 15:553-557 (1997) or bacteria (E. coli, Staphylococcus carnosus) (Daugherty., 1998, Wernerus, Appl. Environ. Microbiol. 69(9):5328-5335 (2003)) as a fusion with the a-agglutinin yeast adhesion receptor or a bacterial outer membrane protein (OMP) respectively.
  • OMP bacterial outer membrane protein
  • the expressed fusion proteins also contain a peptide tag allowing quantification of the library surface expression by flow cytometry. Combined with indirect fluorescent labeling of the ligand, anti-tag labeling allows cell sorting by FACS (fluorescence activated cell sorting) and the determination of the binding affinities of the interactions (Feldhaus et al., Nat. Biotechnol. 21 : 163-70 (2003); Wernerus et al. Appl. Environ. Microbiol. 69(9):5328-35 (2003)).
  • FACS fluorescence activated cell sorting
  • In vitro (also known as cell-free or acellular) display methods may also be employed to select for, isolate and identify DBDpp that bind a target of interest.
  • translation of random RNA is halted prior to ribosome release, resulting in a library of polypeptides with their associated RNA still attached. This and related methods are collectively referred to as "ribosome display.”
  • Other known methods employ chemical linkage of peptides to RNA. See, for example, Roberts et al., PNAS 94: 12297-303 (1997).
  • RNA-peptide screening RNA display and mRNA display
  • in vitro display methods may employ DNA as the genetic component to which the expressed polypeptide is coupled.
  • a method known as cis-display affords the in vitro selection of peptides from libraries of protein-DNA complexes and is described in US7842476 B2, the contents of which are herein incorporated by reference in its entirety.
  • Chemically derived peptide libraries have been developed in which peptides are immobilized on stable, non-biological materials, such as polyethylene rods or solvent-permeable resins.
  • Another chemically derived peptide library uses photolithography to scan peptides immobilized on glass slides.
  • Chemical-peptide screening may be advantageous in that it allows use of D-amino acids and other unnatural analogues, as well as non-peptide elements. Both biological and chemical methods are reviewed in Wells, Curr. Opin. Biotechnol. 3 :355-362 (1992).
  • Biopanning is a known iterative selection and screening method to enrich an initial population of different molecules (such as a DBDpp library) for molecules having an affinity for a target of choice.
  • Library members that have affinity for the target are allowed to bind.
  • Non- specifically or weakly bound members are washed from the support.
  • the bound library members are recovered (e.g., by elution) from the support.
  • Recovered library members are collected for further analysis (e.g., screening) or pooled for an additional round of selection.
  • the target is captured on the solid support after incubation with the phage library.
  • the immobilization of the target can be performed by many different methods known in the art.
  • solid support are microtiter plates or tubes (e.g. Maxisorp plates, Maxisorp tubes, Nunc) or magnetic beads (Dynabead®, Invitrogen).
  • the target can either be directly coated on the plastic or the beads (surface activated Dynabeads, e.g. Dynabeads M270 Epoxy, Invitrogen) or via streptavidin when the target is biotinylated (e.g. Dynabeads MyOne Streptavidin Tl, Invitrogen).
  • the target may be bound non-covalently to the bead via an intermediate affinity molecule such as an antibody or protein A directed against the target or a target-associated peptide tag.
  • Peptide tags such as His-tags or alternatively, an antibody directed against the target can also be used to capture the target on the support.
  • These alternative peptide tags are also compatible with the Dynabeads (Dynabeads His-tag isolation and pull down, Invitrogen) and Protein A or Protein G coupled Dynabeads (Dynabeads-Protein A/G, Invitrogen).
  • Dynabeads Dynabeads
  • Protein A or Protein G coupled Dynabeads Dynabeads-Protein A/G, Invitrogen
  • the capturing step may then consist of trapping the target to coated magnetic beads, thereby capturing indirectly phage bound to the target.
  • the target-phage interaction is performed in solution.
  • the target needs to be immobilized on a solid support.
  • the immobilization of the target in the soluble biopanning method is identical to the immobilization possibilities in the direct biopanning protocols.
  • step (4) amplification of the eluted binders from step (4) or from step (3) (in case step (4)) was omitted in consecutive screening rounds).
  • the steps 1 to 5 will be repeated two, three, four or more times to isolate from the initial library target-specific binders. After the biopanning, the target- specificity of the binders isolated from the different selection rounds is typically analyzed in ELISA assays or similar assays.
  • a method of screening for DBDpp that specifically bind a target of interest comprises the steps of: (a) contacting a target of interest with a plurality of DBDpp; and (b) identifying a DBDpp that specifically binds the target of interest.
  • the step of contacting the target of interest with the plurality of DBDpp may be affected in any way known in the art.
  • the target of interest is immobilized on a solid support and contacting a solution containing the plurality of DBDpp molecules with the immobilized target of interest.
  • Such a procedure is akin to an affinity chromatographic process, with the affinity matrix being comprised of the immobilized target of interest.
  • the DBDpp having a selective affinity for the target of interest can then be purified using techniques known in the art, such as affinity selection.
  • affinity selection The composition of the solid support, process for attaching the target of interest to the solid support, and the reagents, conditions and methods of screening for and isolating DBDpp having a selective affinity for a target of interest are largely conventional and known to those of ordinary skill in the art.
  • Such a wash step may be particularly desirable when the target of interest is bound to a solid support.
  • the selection step of the methods described herein can be performed by way of a method commonly known as a selection method or a by way of a method commonly known as a screening method. Both methods envisage the identification and subsequent isolation (e.g., the selection step) of desirable components (e.g., DBDpp library members) from an original ensemble comprising both desirable and non-desirable components (e.g., a DBDpp library).
  • desirable components e.g., DBDpp library members
  • non-desirable components e.g., a DBDpp library
  • library members will typically be isolated by a step wherein the desired property is applied to obtain the desired goal; in such case, the desired property is usually restricted to the property of a high affinity for a given target interest.
  • library members will typically be isolated by a step wherein all library members, or at least a substantial collection of library members, are individually examined with respect to a given desired property, and wherein members having such desired property are retained whereas members not having such desired property are discarded; in such case, and in the context provided herein, desired properties may relate to either a high affinity for a target of interest, or a functional activity such as, the inhibition, reduction and/or prevention of the activity of a target of interest.
  • the selection step of the methods may be accomplished by either an (affinity) selection technique or by an affinity-based or activity-based functional screening technique, both techniques resulting in the selection of one or more DBDpp having beneficial (favorable, desirable, superior) affinity or activity properties compared to the non- selected DBDpp of the DBDpp.
  • the identified members of the library can be individually isolated and screened.
  • a screening differs from a selection in that a screen is characterized by the analysis of library members individually (or in pools) whereas a selection is characterized by analysis of library members that are separated from other members during the process (e.g., retained, eluted, or washed off).
  • a collection of library members is directly screened, without being subjected to a selection step. This approach, for example, can be used during affinity maturation protocols that are known and can be routinely applied.
  • DBDpp-target interaction can be assayed as described in the Examples below or alternatively, using in vitro or in vivo binding assays such as western blots, radioimmunoassays, ELISA (enzyme linked immunosorbent assay), "sandwich” immunoassays, immunoprecipitation assays, fluorescent immunoassays, protein A immunoassays, immunohistochemistry (IHC) and BIAcore analysis.
  • in vitro or in vivo binding assays such as western blots, radioimmunoassays, ELISA (enzyme linked immunosorbent assay), "sandwich” immunoassays, immunoprecipitation assays, fluorescent immunoassays, protein A immunoassays, immunohistochemistry (IHC) and BIAcore analysis.
  • the ability of a DBDpp to specifically bind a target of interest and to alter the biological activity of the target can be determined using or routinely modifying assays and other methodologies described herein or otherwise known in the art.
  • Assays evaluating the ability of a DBDpp to functionally affect its target e.g., assays to measure signaling, proliferation, migration etc.
  • DBDpp can be identified based on their effects in assays that measure particular pathways or activities.
  • assays that measure signaling pathways e.g., phosphorylation studies or multimerization
  • ion channel fluxes e.g., intracellular cAMP levels
  • cellular activities such as migration, adherence, proliferation, or apoptosis
  • viral entry, replication, budding, or integration can be used to identify, characterize, and improve the desired properties of the DBDpp.
  • the ability of a DBDpp to competitively inhibit another DBDpp-containing sequence can be determined using techniques known in the art, including ELISA and BIAcore analysis.
  • the nucleic acid coding sequence can be isolated and routinely determined. It is possible to isolate from said cell or phage or particle, the nucleotide sequence that encodes that DBDpp sequence. In this way, the nucleotide sequence of the selected DBDpp library member(s) can be determined by routine sequencing methods.
  • DBDpp library members that are specific for the target can be characterized by nucleic acid sequencing. Sequence information is used to classify the members and to remove redundant members (i.e., members that encode that same DBDpp).
  • DBDpp libraries and library members include, but are not limited to those identified in below in Table 1. Additionally included are those DBDpp that correspond to any of SEQ ID NOS: 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11, wherein one or more of the X n positions are substituted with a natural or non-natural amino acid. In some embodiments, the DBDpp that correspond to any of SEQ TD NOS: 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 do not include cysteine and/or proline residues that are substituted into an X n position.
  • the article of manufacture may comprise a container and a label or package insert on or associated with the container.
  • Suitable containers include, for example, bottles, vials or syringes.
  • the containers may be formed from a variety of materials such as glass or plastic.
  • the container holds one or more DBDpp, nucleic acids encoding DBDpp and/or vectors or host cells of the present disclosure.
  • the label or package insert may include directions for performing affinity based screening, detection, and/or purification.
  • the detectable marker or label can be any which is capable of producing, either directly or indirectly, a measurable signal, such as a radioactive, chromogenic, luminescence, or fluorescent signal, which can be used to quantitate the amount of bound detectable moiety or label in a sample.
  • a measurable signal such as a radioactive, chromogenic, luminescence, or fluorescent signal
  • a DBDpp such as a DBDpp fusion protein (e.g., a DBDpp-Fc, DBDpp-CAR, a DBDpp-scFv), or other molecule is said to "competitively inhibit" binding of a reference molecule to a given epitope if it binds to that epitope to the extent that it blocks, to some degree, binding of the reference molecule to the epitope.
  • a DBDpp fusion protein e.g., a DBDpp-Fc, DBDpp-CAR, a DBDpp-scFv
  • DBDpp such as a DBDpp fusion protein
  • a DBDpp fusion protein e.g., a DBDpp-Fc, DBDpp CAR, a DBDpp-scFv, and an antibody-comprising a DBDpp
  • a standard competition assay as described herein or otherwise known in the art, including, but not limited to, competitive assay systems using techniques such as radioimmunoassays (RIA), enzyme immunoassays (EIA), preferably the enzyme linked immunosorbent assay (ELISA), "sandwich” immunoassays, immunoradiometric assays, fluorescent immunoassays, luminescent, electrochemical luminescent, and Immunoelectrophoresis assays.
  • DBD described herein are useful in a variety of applications including, but not limited to, therapeutic treatment methods, which may be in vitro, ex vivo, or in vivo methods.
  • the DBDpp is a soluble fusion protein (schematically shown in FIG. 5C and made up of an optional epitope tag 10 and a targeting domain 20) that binds to a target that is associated with a disease or disorder of the metabolic, cardiovascular, musculoskeletal, neurological, or skeletal system.
  • the DBDpp is a soluble fusion protein that binds to a target that is associated with yeast, fungal, viral or bacterial infection or disease.
  • the DBDpp is a soluble fusion protein that binds to a target that is associated with a disease or disorder of the immune system.

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