WO2024145687A1 - Protéines bioréactives contenant un acide aminé non naturel et de l'arginine - Google Patents

Protéines bioréactives contenant un acide aminé non naturel et de l'arginine Download PDF

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WO2024145687A1
WO2024145687A1 PCT/US2024/010060 US2024010060W WO2024145687A1 WO 2024145687 A1 WO2024145687 A1 WO 2024145687A1 US 2024010060 W US2024010060 W US 2024010060W WO 2024145687 A1 WO2024145687 A1 WO 2024145687A1
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protein
receptor
substituted
amino acid
unsubstituted
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PCT/US2024/010060
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Lei Wang
Li Cao
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The Regents Of The University Of California
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    • 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
    • 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
    • C07K16/2863—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against receptors for growth factors, growth regulators
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • 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
    • C07K16/283—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 Fc-receptors, e.g. CD16, CD32, CD64
    • 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
    • C07K2317/569—Single domain, e.g. dAb, sdAb, VHH, VNAR or nanobody®

Definitions

  • proteins comprising: (i) an unnatural amino acid, and (ii) a non- naturally occurring arginine; wherein: (a) the non-naturally occurring arginine is proximal to the –S(O 2 )F group in the unnatural amino acid side chain; and (b) the unnatural amino comprises a side chain of the substituents are defined herein.
  • a single-chain variable fragment such as a single-domain antibody, an affibody, or an antigen-binding fragment.
  • FIG.6 is an SDS-PAGE analysis of nanobody 2Rs15d(D54FFY) at left and nanobody 2Rs15d(D54FFY/D56R) at right crosslinking with HER2 ECD protein.
  • FIG.7 is an SDS-PAGE analysis of nanobody 7D12(Y109FFY) at left and nanobody 7D12(Y109FFY/E44R) at right crosslinking with EGFR ECD protein.
  • FIG.8 is an SDS-PAGE analysis of WT BiKE 7D12(Y109FSY)-C21 at left and Arg- BiKE 7D12(Y109FSY/E44R)-C21 at right crosslinking with EGFR ECD protein.
  • proximal means up to about 15 angstroms. In embodiments, “proximal” means up to about 10 angstroms. In embodiments, “proximal” means from about 1 angstrom to about 25 angstroms. In embodiments, “proximal” means from about 1 angstrom to about 20 angstroms. In embodiments, “proximal” means from about 1 angstrom to about 15 angstroms. In embodiments, “proximal” means from about 1 angstrom to about 12 angstroms. In embodiments, “proximal” means from about 1 angstrom to about 10 angstroms. In embodiments, “proximal” means from about 1 angstrom to about 8 angstroms.
  • the naturally or non-naturally occurring arginine is within 1 to about 2 amino acid residues of the unnatural amino acid. In embodiments, the naturally or non-naturally occurring arginine is within 2 to about 6 amino acid residues of the unnatural amino acid. In embodiments, the naturally or non-naturally occurring arginine is within 2 to about 5 amino acid residues of the unnatural amino acid. In embodiments, the naturally or non-naturally occurring arginine is within 2 to about 4 amino acid residues of the unnatural amino acid. In embodiments, the naturally or non-naturally occurring arginine is within 2 to about 3 amino acid residues of the unnatural amino acid.
  • polyclonal antibodies can be selected to obtain only a subset of antibodies that are specifically immunoreactive with the selected antigen and not with other proteins. This selection may be achieved by subtracting out antibodies that cross-react with other molecules.
  • a variety of immunoassay formats may be used to select antibodies specifically immunoreactive with a particular protein.
  • solid-phase ELISA immunoassays are routinely used to select antibodies specifically immunoreactive with a protein (e.g., Harlow & Lane, Using Antibodies, A Laboratory Manual (1998) for a description of immunoassay formats and conditions that can be used to determine specific immunoreactivity).
  • target protein refers to a targeting molecule having, e.g., a regulatory role in a cell.
  • Examples of such analogs include, without limitation, phosphodiester derivatives including, e.g., phosphoramidate, phosphorodiamidate, phosphorothioate (also known as phosphorothioate having double bonded sulfur replacing oxygen in the phosphate), phosphorodithioate, phosphonocarboxylic acids, phosphonocarboxylates, phosphonoacetic acid, phosphonoformic acid, methyl phosphonate, boron phosphonate, or O-methylphosphoroamidite linkages (see Eckstein, Oligonucleotides and Analogues: A Practical Approach, Oxford University Press) as well as modifications to the nucleotide bases such as in 5-methyl cytidine or pseudouridine and peptide nucleic acid backbones and linkages.
  • phosphodiester derivatives including, e.g., phosphoramidate, phosphorodiamidate, phosphorothioate (also known as phosphorothioate having double bonded sulfur
  • Amino acid analogs refers to compounds that have the same basic chemical structure as a naturally occurring amino acid, i.e., an ⁇ -carbon that is bound to a hydrogen, a carboxyl group, an amino group, and an R group, e.g., homoserine, norleucine, methionine sulfoxide, methionine methyl sulfonium. Such analogs have modified R groups (e.g., norleucine) or modified peptide backbones, but retain the same basic chemical structure as a naturally occurring amino acid.
  • Amino acid mimetics refers to chemical compounds that have a structure that is different from the general chemical structure of an amino acid, but that functions in a manner similar to a naturally occurring amino acid.
  • unnatural e functional substituent of compounds that have the same basic chemical structure as a naturally occurring amino acid i.e., an ⁇ carbon that is bound to a hydrogen, a carboxyl group, an amino group, and an R group, e.g., homoserine, norleucine, methionine sulfoxide, methionine methyl sulfonium, allylalanine, 2- aminoisobutryric acid.
  • Unnatural amino acids are non-proteinogenic amino acids that either occur naturally or are chemically synthesized. Such analogs have modified R groups (e.g., norleucine) or modified peptide backbones, but retain the same basic chemical structure as a naturally occurring amino acid.
  • a protein that is the predominant species present in a preparation is substantially purified.
  • Contacting is used in accordance with its plain ordinary meaning and refers to the process of allowing at least two distinct species (e.g., protein and a target protein) to become sufficiently proximal to react, interact, or physically touch. It should be appreciated that the resulting reaction product can be produced directly from a reaction between the added reagents or from an intermediate from one or more of the added reagents that can be produced in the reaction mixture.
  • the term “contacting” may include allowing two species to react, interact, or physically touch, wherein the two species may be proteins as described herein.
  • intermolecular linker refers to a linking group between two proteins.
  • a protein described in WO 2019/173760 is enhanced green fluorescent protein (EGFP), Z SPA affibody, maltose binding protein, protein Z, thioredoxin (TRX1), and human growth hormone.
  • EGFP enhanced green fluorescent protein
  • TRX1 thioredoxin
  • human growth hormone The amino acid sequence of each of these proteins is set forth in WO 2020/072674 or is known in the art.
  • the term “a protein described in WO 2020/072674” refers to any protein, including, without limitation, an antibody or an antibody variant, described in WO 2020/072674.
  • a protein described in WO 2020/072674 is bovine serum albumin, peptide 7KR, glutathione S-transferase, 14-3-3 protein, and single-strand DNA binding protein (SSB).
  • a protein described in WO 2022/232377 is a CRISPR protein, Hfq, ACE2 receptor protein, SARS-CoV-2 spike (S) protein, SR4 nanobody, MR17K99Y nanobody, nanobody H11D4, mNb6 nanobody, nanobody 2rs15d (NbHER2), nanobody C21, nanobody NB13, nanobody NB17B05, MS211, ZHER2:2891, ZHER2:342, F57 (or 5F7), nanobody 7D12 (NbEGFR), trastuzumab, trastuzumab Fab, neuregulin 1 ⁇ (NRG1b), thioredoxin (TRX), NK035, A1 nanobody, and C6 nanobody.
  • the alkyl may include a designated number of carbons (e.g., C 1 -C 10 means one to ten carbons).
  • Alkyl is an uncyclized chain.
  • saturated hydrocarbon radicals include, but are not limited to, groups such as methyl, ethyl, n-propyl, isopropyl, n-butyl, t-butyl, isobutyl, sec-butyl, methyl, homologs and isomers of, for example, n-pentyl, n-hexyl, n-heptyl, n-octyl, and the like.
  • An unsaturated alkyl group is one having one or more double bonds or triple bonds.
  • alkenyl may include more than one double bond and/or one or more triple bonds in addition to the one or more double bonds.
  • An alkynyl may include more than one triple bond and/or one or more double bonds in addition to the one or more triple bonds.
  • alkylene by itself or as part of another substituent, means, unless otherwise stated, a divalent radical derived from an alkyl, as exemplified by, e.g., -CH2CH2CH2CH2-.
  • an alkyl (or alkylene) group will have from 1 to 24 carbon atoms, with those groups having 10 or fewer carbon atoms being preferred herein.
  • a “lower alkyl” or “lower alkylene” is a shorter chain alkyl or alkylene group, generally having eight or fewer carbon atoms.
  • alkenylene by itself or as part of another substituent, means, unless otherwise stated, a divalent radical derived from an alkene.
  • heteroalkyl by itself or in combination with another term, means, unless otherwise stated, a stable straight or branched chain, or combinations thereof, including at least one carbon atom and at least one heteroatom (e.g., O, N, P, Si, and S), and wherein the nitrogen and sulfur atoms may optionally be oxidized, and the nitrogen heteroatom may optionally be quaternized.
  • heteroatom(s) may be placed at any interior position of the heteroalkyl group or at the position at which the alkyl group is attached to the remainder of the molecule.
  • a heteroalkyl moiety may include one heteroatom.
  • a heteroalkyl moiety may include two optionally different heteroatoms.
  • a heteroalkyl moiety may include three optionally different heteroatoms.
  • a heteroalkyl moiety may include four optionally different heteroatoms.
  • a heteroalkyl moiety may include five optionally different heteroatoms.
  • a heteroalkyl moiety may include up to 8 optionally different heteroatoms.
  • the term “heteroalkenyl,” by itself or in combination with another term, means, unless otherwise stated, a heteroalkyl including at least one double bond.
  • heteroalkyl groups include those groups that are attached to the remainder of the molecule through a heteroatom, such as - C(O)R', -C(O)NR', -NR'R'', -OR', -SR', and/or -SO2R'.
  • heteroalkyl is recited, followed by recitations of specific heteroalkyl groups, such as -NR'R'' or the like, it will be understood that the terms heteroalkyl and -NR'R'' are not redundant or mutually exclusive.
  • cycloalkyl examples include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, 1-cyclohexenyl, 3-cyclohexenyl, cycloheptyl, and the like.
  • bicyclic ring systems include, but are not limited to, bicyclo[3.1.1]heptane, bicyclo[2.2.1]heptane, bicyclo[2.2.2]octane, bicyclo[3.2.2]nonane, bicyclo[3.3.1]nonane, and bicyclo[4.2.1]nonane.
  • fused bicyclic cycloalkyl ring systems contain a monocyclic cycloalkyl ring fused to either a phenyl, a monocyclic cycloalkyl, a monocyclic cycloalkenyl, a monocyclic heterocyclyl, or a monocyclic heteroaryl.
  • the bridged or fused bicyclic cycloalkyl is attached to the parent molecular moiety through any carbon atom contained within the monocyclic cycloalkyl ring.
  • cycloalkyl groups are optionally substituted with one or two groups which are independently oxo or thia.
  • cycloalkyl groups include, but are not limited to tetradecahydrophenanthrenyl, perhydrophenothiazin-1-yl, and perhydrophenoxazin-1-yl.
  • ring A is a 5-membered monocyclic cycloalkyl, a 5-membered monocyclic heterocycloalkyl, or a 5-membered monocyclic heteroaryl.
  • a cycloalkyl is a cycloalkenyl.
  • cycloalkenyl is used in accordance with its plain ordinary meaning.
  • a cycloalkenyl is a monocyclic, bicyclic, or a multicyclic cycloalkenyl ring system.
  • monocyclic cycloalkenyl ring systems are cyclic hydrocarbon groups containing from 3 to 8 carbon atoms, where such groups are unsaturated (i.e., containing at least one annular carbon carbon double bond), but not aromatic. Examples of monocyclic cycloalkenyl ring systems include cyclopentenyl and cyclohexenyl.
  • bicyclic cycloalkenyl rings are bridged monocyclic rings or a fused bicyclic rings.
  • bridged monocyclic rings contain a monocyclic cycloalkenyl ring where two non adjacent carbon atoms of the monocyclic ring are linked by an alkylene bridge of between one and three additional carbon atoms (i.e., a bridging group of the form (CH2)w, where w is 1, 2, or 3).
  • alkylene bridge of between one and three additional carbon atoms
  • bicyclic cycloalkenyls include, but are not limited to, norbornenyl and bicyclo[2.2.2]oct 2 enyl.
  • multicyclic cycloalkenyl is attached to the parent molecular moiety through any carbon atom contained within the base ring.
  • multicyclic cycloalkenyl rings contain a monocyclic cycloalkenyl ring (base ring) fused to either (i) one ring system selected from the group consisting of a bicyclic aryl, a bicyclic heteroaryl, a bicyclic cycloalkyl, a bicyclic cycloalkenyl, and a bicyclic heterocyclyl; or (ii) two ring systems independently selected from the group consisting of a phenyl, a monocyclic heteroaryl, a monocyclic cycloalkyl, a monocyclic cycloalkenyl, and a monocyclic heterocyclyl.
  • heterocyclyl monocyclic heterocycle is connected to the parent molecular moiety through any carbon atom or any nitrogen atom contained within the heterocyclyl monocyclic heterocycle.
  • Representative examples of heterocyclyl monocyclic heterocycles include, but are not limited to, azetidinyl, azepanyl, aziridinyl, diazepanyl, 1,3-dioxanyl, 1,3-dioxolanyl, 1,3-dithiolanyl, 1,3-dithianyl, imidazolinyl, imidazolidinyl, isothiazolinyl, isothiazolidinyl, isoxazolinyl, isoxazolidinyl, morpholinyl, oxadiazolinyl, oxadiazolidinyl, oxazolinyl, oxazolidinyl, piperazinyl, piperidinyl, pyranyl, pyrazoliny
  • bicyclic heterocyclyls include, but are not limited to, 2,3-dihydrobenzofuran-2-yl, 2,3-dihydrobenzofuran-3-yl, indolin-1-yl, indolin-2-yl, indolin-3-yl, 2,3-dihydrobenzothien-2-yl, decahydroquinolinyl, decahydroisoquinolinyl, octahydro-1H-indolyl, and octahydrobenzofuranyl.
  • heterocyclyl groups are optionally substituted with one or two groups which are independently oxo or thia.
  • the bicyclic heterocyclyl is a 5 or 6 membered monocyclic heterocyclyl ring fused to a phenyl ring, a 5 or 6 membered monocyclic cycloalkyl, a 5 or 6 membered monocyclic cycloalkenyl, a 5 or 6 membered monocyclic heterocyclyl, or a 5 or 6 membered monocyclic heteroaryl, wherein the bicyclic heterocyclyl is optionally substituted by one or two groups which are independently oxo or thia.
  • Multicyclic heterocyclyl ring systems are a monocyclic heterocyclyl ring (base ring) fused to either (i) one ring system selected from the group consisting of a bicyclic aryl, a bicyclic heteroaryl, a bicyclic cycloalkyl, a bicyclic cycloalkenyl, and a bicyclic heterocyclyl; or (ii) two other ring systems independently selected from the group consisting of a phenyl, a bicyclic aryl, a monocyclic or bicyclic heteroaryl, a monocyclic or bicyclic cycloalkyl, a monocyclic or bicyclic cycloalkenyl, and a monocyclic or bicyclic heterocyclyl.
  • multicyclic heterocyclyl is attached to the parent molecular moiety through any carbon atom or nitrogen atom contained within the base ring.
  • multicyclic heterocyclyl ring systems are a monocyclic heterocyclyl ring (base ring) fused to either (i) one ring system selected from the group consisting of a bicyclic aryl, a bicyclic heteroaryl, a bicyclic cycloalkyl, a bicyclic cycloalkenyl, and a bicyclic heterocyclyl; or (ii) two other ring systems independently selected from the group consisting of a phenyl, a monocyclic heteroaryl, a monocyclic cycloalkyl, a monocyclic cycloalkenyl, and a monocyclic heterocyclyl.
  • multicyclic heterocyclyl groups include, but are not limited to 10H-phenothiazin-10-yl, 9,10- dihydroacridin-9-yl, 9,10-dihydroacridin-10-yl, 10H-phenoxazin-10-yl, 10,11-dihydro-5H- dibenzo[b,f]azepin-5-yl, 1,2,3,4-tetrahydropyrido[4,3-g]isoquinolin-2-yl, 12H- benzo[b]phenoxazin-12-yl, and dodecahydro-1H-carbazol-9-yl.
  • halo(C 1 -C 4 )alkyl includes, but is not limited to, fluoromethyl, difluoromethyl, trifluoromethyl, 2,2,2-trifluoroethyl, 4-chlorobutyl, 3-bromopropyl, and the like.
  • aryl means, unless otherwise stated, a polyunsaturated, aromatic, hydrocarbon substituent, which can be a single ring or multiple rings (preferably from 1 to 3 rings) that are fused together (i.e., a fused ring aryl) or linked covalently.
  • a fused ring aryl refers to multiple rings fused together wherein at least one of the fused rings is an aryl ring.
  • heteroaryl refers to aryl groups (or rings) that contain at least one heteroatom such as N, O, or S, wherein the nitrogen and sulfur atoms are optionally oxidized, and the nitrogen atom(s) are optionally quaternized.
  • alkyl is meant to include groups including carbon atoms bound to groups other than hydrogen groups, such as haloalkyl (e.g., -CF3 and -CH2CF3) and acyl (e.g., -C(O)CH3, -C(O)CF3, -C(O)CH2OCH3, and the like).
  • haloalkyl e.g., -CF3 and -CH2CF3
  • acyl e.g., -C(O)CH3, -C(O)CF3, -C(O)CH2OCH3, and the like.
  • the ring-forming substituents are attached to non-adjacent members of the base structure.
  • Two of the substituents on adjacent atoms of the aryl or heteroaryl ring may optionally form a ring of the formula -T-C(O)-(CRR') q -U-, wherein T and U are independently -NR-, -O-, -CRR'-, or a single bond, and q is an integer of from 0 to 3.
  • a “lower substituent” or “ lower substituent group,” as used herein, means a group selected from all of the substituents described above for a “substituent group,” wherein each substituted or unsubstituted alkyl is a substituted or unsubstituted C1-C8 alkyl, each substituted or unsubstituted heteroalkyl is a substituted or unsubstituted 2 to 8 membered heteroalkyl, each substituted or unsubstituted cycloalkyl is a substituted or unsubstituted C3-C7 cycloalkyl, each substituted or unsubstituted heterocycloalkyl is a substituted or unsubstituted 3 to 7 membered heterocycloalkyl, each substituted or unsubstituted aryl is a substituted or unsubstituted C6-C10 aryl, and each substituted or unsubstituted heteroaryl is a substituted
  • each substituted or unsubstituted alkyl is a substituted or unsubstituted C 1 -C 8 alkyl
  • each substituted or unsubstituted heteroalkyl is a substituted or unsubstituted 2 to 8 membered heteroalkyl
  • each substituted or unsubstituted cycloalkyl is a substituted or unsubstituted C 3 -C 7 cycloalkyl
  • each substituted or unsubstituted heterocycloalkyl is a substituted or unsubstituted 3 to 7 membered heterocycloalkyl
  • each substituted or unsubstituted aryl is a substituted or unsubstituted C 6 -C 10 aryl
  • each substituted or unsubstituted heteroaryl is a substituted or unsubstituted 5 to 9 membered heteroaryl.
  • each size-limited substituent group is different.
  • a substituted moiety e.g., substituted alkyl, substituted heteroalkyl, substituted cycloalkyl, substituted heterocycloalkyl, substituted aryl, substituted heteroaryl, substituted alkylene, substituted heteroalkylene, substituted cycloalkylene, substituted heterocycloalkylene, substituted arylene, and/or substituted heteroarylene
  • each lower substituent group is different.
  • a substituted moiety e.g., substituted alkyl, substituted heteroalkyl, substituted cycloalkyl, substituted heterocycloalkyl, substituted aryl, substituted heteroaryl, substituted alkylene, substituted heteroalkylene, substituted cycloalkylene, substituted heterocycloalkylene, substituted arylene, and/or substituted heteroarylene
  • the term “isomers” refers to compounds having the same number and kind of atoms, and hence the same molecular weight, but differing in respect to the structural arrangement or configuration of the atoms.
  • the term “tautomer,” as used herein, refers to one of two or more structural isomers which exist in equilibrium and which are readily converted from one isomeric form to another. It will be apparent to one skilled in the art that certain compounds of this disclosure may exist in tautomeric forms, all such tautomeric forms of the compounds being within the scope of the disclosure. Unless otherwise stated, structures depicted herein are also meant to include all stereochemical forms of the structure; i.e., the R and S configurations for each asymmetric center.
  • variable e.g., moiety or linker
  • a compound or of a compound genus e.g., a genus described herein
  • the unfilled valence(s) of the variable will be dictated by the context in which the variable is used.
  • variable is the divalent form of a standalone compound (e.g., if the variable is assigned to “PEG” or “polyethylene glycol” in an embodiment but the variable is connected by two separate bonds to the remainder of the compound, a person of ordinary skill in the art would understand that the variable is a divalent (i.e., capable of forming two bonds through two unfilled valences) form of PEG instead of the standalone compound PEG).
  • bond refers to direct bonds, such as covalent bonds (e.g., direct or a linking group), or indirect bonds, such as non-covalent bond (e.g., electrostatic interactions (e.g., ionic bond, hydrogen bond, halogen bond), van der Waals interactions (e.g., dipole-dipole, dipole-induced dipole, London dispersion), ring stacking (pi effects), hydrophobic interactions, and the like).
  • bioconjugate and “bioconjugate linker” refers to the resulting association between atoms or molecules of “bioconjugate reactive groups” or “bioconjugate reactive moieties”. The association can be direct or indirect.
  • bioconjugates or bioconjugate linkers are formed using bioconjugate chemistry (i.e. the association of two bioconjugate reactive groups) including, but are not limited to nucleophilic substitutions (e.g., reactions of amines and alcohols with acyl halides, active esters), electrophilic substitutions (e.g., enamine reactions) and additions to carbon-carbon and carbon-heteroatom multiple bonds (e.g., Michael reaction, Diels-Alder addition).
  • bioconjugate chemistry i.e. the association of two bioconjugate reactive groups
  • nucleophilic substitutions e.g., reactions of amines and alcohols with acyl halides, active esters
  • electrophilic substitutions e.g., enamine reactions
  • additions to carbon-carbon and carbon-heteroatom multiple bonds e.g., Michael reaction, Diels-Alder addition.
  • the first bioconjugate reactive group e.g., unnatural amino acid side chain
  • the second bioconjugate reactive group e.g., a hydroxyl group
  • the unnatural amino acid comprises a side chain of Formula (II) as described herein.
  • the unnatural amino acid comprises a side chain of Formula (III) as described herein.
  • the unnatural amino acid comprises a side chain of Formula (IV) as described herein.
  • the unnatural amino acid comprises a side chain of Formula (V) as described herein.
  • the unnatural amino acid comprises a side chain of Formula (VI) as described herein.
  • the unnatural amino acid comprises a side chain of Formula (VII) as described herein.
  • R 1 is hydrogen, halogen, -CX 1 3, -CHX 1 2, -CH2X 1 , -OCX 1 3, -OCH2X 1 , -OCHX 1 2, -CN, -SO n1 R 1A , -SO v1 NR 1A R 1B , -NHC(O)NR 1A R 1B , -N(O) m1 , -NR 1A R 1B , -C(O)R 1A , -C(O)-OR 1A , -C(O)NR 1A R 1B , -OR 1A , -NR 1A SO2R 1B , -NR 1A C(O)R 1B , -NR 1A C(O)OR 1B , -NR 1A OR 1B , unsubstituted C 1-8 alkyl, or unsubstituted 2 to 8 membered heteroalkyl; R 1A is hydrogen, unsubstituted
  • R 1 is hydrogen or halogen.
  • proteins comprising: (i) an unnatural amino acid, and (ii) a naturally-occurring arginine; wherein: (a) the naturally occurring arginine is proximal to the - S(O2)F group in the unnatural amino acid side chain; and (b) the unnatural amino comprises a side chain of Formula (II): ; wherein: ring A is heterocycloalkyl, or a 5- membered heteroaryl; x is an integer from 0 to 8; L 1 is a bond, substituted or unsubstituted alkylene, or substituted or unsubstituted heteroalkylene; R 1 is hydrogen, halogen, -CX 1 3 , -CHX 1 2, -CH2X 1 , -OCX 1 3, -OCH2X 1 , -OCHX 1 2, -CN, -SOn1R 1A , -SOv1NR
  • the protein when A is a 5-membered cycloalkyl, a 5- membered heterocycloalkyl, or a 5-membered heteroaryl, then the protein is not enhanced green fluorescent protein, a maltose binding protein, or protein Z. In embodiments, the protein does not comprise a non-naturally occurring arginine.
  • proteins comprising: (i) an unnatural amino acid, and (ii) a non- naturally occurring arginine; wherein: (a) the non-naturally occurring arginine is proximal to the -S(O 2 )F group in the unnatural amino acid side chain; and (b) the unnatural amino comprises a side chain of Formula (FSY): .
  • proteins comprising: (i) an unnatural amino acid, and (ii) a non- naturally occurring arginine; wherein: (a) the non-naturally occurring arginine is proximal to the -S(O 2 )F group in the unnatural amino acid side chain; and (b) the unnatural amino comprises a side chain of Formula (mFSY): .
  • the protein is a single-chain variable fragment, a single-domain antibody, an affibody, or an antigen-binding fragment.
  • the protein is a single-chain variable fragment.
  • the protein is a single-domain antibody.
  • the protein is an affibody.
  • proteins comprising: (i) an unnatural amino acid, and (ii) a non- naturally occurring arginine; wherein: (a) the non-naturally occurring arginine is proximal to the -S(O 2 )F group in the unnatural amino acid side chain; and (b) the unnatural amino comprises a side chain of Formula (FFY): .
  • the protein protein is a single-chain variable fragment, a single-domain antibody, an affibody, or an antigen-binding fragment.
  • the protein is a single-chain variable fragment.
  • the protein is a single-domain antibody.
  • the protein is an affibody.
  • the protein is not epidermal growth factor receptor, protein tyrosine phosphatase 1B, P-selectin glycoprotein ligand 1, complement component 5a receptor, chemokine receptor D6, CXCR4, thymopentin, oxytocin, arginine vasopressin, indolicidin, or a protein described in any one of WO 2017/161183, WO 2019/173760, WO 2020/072674, WO 2020/206341, and WO 2022/232377.
  • proteins comprising: (i) an unnatural amino acid, and (ii) a non- naturally occurring arginine; wherein: (a) the non-naturally occurring arginine is proximal to the -S(O 2 )F group in the unnatural amino acid side chain; and (b) the unnatural amino comprises a side chain of Formula (mFSK): .
  • the protein is a single-chain variable fragment.
  • the protein is a single-domain antibody.
  • the protein is an affibody.
  • the protein is a single-chain variable fragment. In embodiments, the protein is a single-domain antibody. In embodiments, the protein is an affibody. In embodiments, the protein is an antigen-binding fragment. In embodiments, the protein does not comprise a non-naturally occurring arginine.
  • the protein is not epidermal growth factor receptor, protein tyrosine phosphatase 1B, P-selectin glycoprotein ligand 1, complement component 5a receptor, chemokine receptor D6, CXCR4, thymopentin, oxytocin, arginine vasopressin, indolicidin, or a protein described in any one of WO 2017/161183, WO 2019/173760, WO 2020/072674, WO 2020/206341, and WO 2022/232377.
  • proteins comprising: (i) an unnatural amino acid, and (ii) a non- naturally occurring arginine; wherein: (a) the non-naturally occurring arginine is proximal to the -S(O2)F group in the unnatural amino acid side chain; and (b) the unnatural amino comprises a side chain of Formula (IV): .
  • the is a single-chain variable fragment, a single-domain antibody, an affibody, or an antigen-binding fragment.
  • the protein is a single-chain variable fragment.
  • the protein is a single-domain antibody.
  • the protein is an affibody.
  • the protein is an antigen-binding fragment.
  • the protein further comprises a naturally occurring arginine. In embodiments, the protein further comprises a naturally occurring arginine that is not proximal to the –S(O2)F group in the unnatural amino acid side chain.
  • proteins comprising: (i) an unnatural amino acid, and (ii) a naturally occurring arginine; wherein: (a) the naturally occurring arginine is proximal to the – S(O2)F group in the unnatural amino acid side chain; and (b) the unnatural amino comprises a side chain of Formula (IV): .
  • the is a single-chain variable fragment, a single-domain antibody, an affibody, or an antigen-binding fragment.
  • the protein is a single-chain variable fragment. In embodiments, the protein is a single-domain antibody. In embodiments, the protein is an affibody. In embodiments, the protein is an antigen-binding fragment. In embodiments, the protein does not comprise a non-naturally occurring arginine.
  • proteins comprising: (i) an unnatural amino acid, and (ii) a non- naturally occurring arginine; wherein: (a) the non-naturally occurring arginine is proximal to the -S(O 2 )F group in the unnatural amino acid side chain; and (b) the unnatural amino comprises a side chain of Formula (V): ; wherein: ring A is phenyl, heterocycloalkyl, or a 5- membered heteroaryl; x is an integer from 0 to 8; L 1 is a bond, substituted or unsubstituted alkylene, or substituted or unsubstituted heteroalkylene; R 1 is hydrogen, halogen, -CX 1 3 , -CHX 1 2, -CH2X 1 , -OCX 1 3, -OCH2X 1 , -OCHX 1 2, -CN, -SOn1R 1A , -SOv1NR 1A R 1B
  • R 1 is hydrogen, halogen, -CX 1 3, -CHX 1 2, -CH2X 1 , -OCX 1 3, -OCH2X 1 , -OCHX 1 2, -CN, -SO n1 R 1A , -SO v1 NR 1A R 1B , -NHC(O)NR 1A R 1B , -N(O) m1 , -NR 1A R 1B , -C(O)R 1A , -C(O)-OR 1A , -C(O)NR 1A R 1B , -OR 1A , -NR 1A SO2R 1B , -NR 1A C(O)R 1B , -NR 1A C(O)OR 1B , -NR 1A OR 1B , unsubstituted C 1-8 alkyl, or unsubstituted 2 to 8 membered heteroalkyl; R 1A is hydrogen, unsubstituted
  • R 1 is hydrogen or halogen.
  • proteins comprising: (i) an unnatural amino acid, and (ii) a naturally occurring arginine; wherein: (a) the naturally occurring arginine is proximal to the – S(O2)F group in the unnatural amino acid side chain; and (b) the unnatural amino comprises a side chain of Formula (V): ; wherein: ring A is phenyl, heterocycloalkyl, or a 5- membered heteroaryl; x is an integer from 0 to 8; L 1 is a bond, substituted or unsubstituted alkylene, or substituted or unsubstituted heteroalkylene; R 1 is hydrogen, halogen, -CX 1 3 , -CHX 1 2, -CH2X 1 , -OCX 1 3, -OCH2X 1 , -OCHX 1 2, -CN, -SOn1R 1A , -SOv1
  • R 1 is hydrogen, halogen, -CX 1 3, -CHX 1 2, -CH2X 1 , -OCX 1 3, -OCH2X 1 , -OCHX 1 2, -CN, -SO n1 R 1A , -SO v1 NR 1A R 1B , -NHC(O)NR 1A R 1B , -N(O) m1 , -NR 1A R 1B , -C(O)R 1A , -C(O)-OR 1A , -C(O)NR 1A R 1B , -OR 1A , -NR 1A SO2R 1B , -NR 1A C(O)R 1B , -NR 1A C(O)OR 1B , -NR 1A OR 1B , unsubstituted C 1-8 alkyl, or unsubstituted 2 to 8 membered heteroalkyl; R 1A is hydrogen, unsubstituted
  • R 1 is hydrogen or halogen.
  • proteins comprising: (i) an unnatural amino acid, and (ii) a non- naturally occurring arginine; wherein: (a) the non-naturally occurring arginine is proximal to the -S(O2)F group in the unnatural amino acid side chain; and (b) the unnatural amino comprises a side chain of Formula (VI): ; wherein ring A is phenyl, heterocycloalkyl, or a 5- membered heteroaryl; x is an integer from 0 to 8; L 1 is a bond, substituted or unsubstituted alkylene, or substituted or unsubstituted heteroalkylene.
  • the protein further comprises a naturally occurring arginine. In embodiments, the protein further comprises a naturally occurring arginine that is not proximal to the –S(O2)F group in the unnatural amino acid side chain.
  • proteins comprising: (i) an unnatural amino acid, and (ii) a naturally occurring arginine; wherein: (a) the naturally occurring arginine is proximal to the – S(O2)F group in the unnatural amino acid side chain; and (b) the unnatural amino comprises a side chain of Formula (VI): I); wherein ring A is phenyl, a d heterocycloalkyl, or a 5- membered heteroaryl; x is an integer from 0 to 8; L is a bond, substituted or unsubstituted alkylene, or substituted or unsubstituted heteroalkylene provided that when A is phenyl, then the protein is not epidermal growth factor receptor, protein t
  • the protein when A is a 5-membered cycloalkyl, a 5- membered heterocycloalkyl, or a 5-membered heteroaryl, then the protein is not enhanced green fluorescent protein, a maltose binding protein, or protein Z. In embodiments, the protein does not comprise a non-naturally occurring arginine.
  • ring A is phenyl, a 5-membered cycloalkyl, a 5-membered heterocycloalkyl, or a 5-membered heteroaryl. In embodiments, ring A is phenyl.
  • ring A is a 5-membered cycloalkyl, a 5-membered heterocycloalkyl, or a 5-membered heteroaryl.
  • ring A is a 5-membered cycloalkyl.
  • ring A is a 5-membered heterocycloalkyl.
  • ring A is a 5- membered heterocycloalkyl having no double bonds. In embodiments, ring A is a 5-membered heterocycloalkyl having one double bond. [0146] In embodiments, ring A is a 5-membered heteroaryl. In embodiments, ring A is a 5- membered heteroaryl containing 1 to 4 heteroatoms selected from the group consisting of oxygen, nitrogen, and sulfur. In embodiments, ring A is a 5-membered heteroaryl containing 1 to 3 heteroatoms selected from the group consisting of oxygen, nitrogen, and sulfur. In embodiments, ring A is a 5-membered heteroaryl containing 1 or 2 heteroatoms selected from the group consisting of oxygen, nitrogen, and sulfur.
  • ring A is a 5-membered heteroaryl containing 1 heteroatom selected from the group consisting of oxygen, nitrogen, and sulfur. In embodiments, ring A is a 5-membered heteroaryl containing 2 heteroatoms selected from the group consisting of oxygen, nitrogen, and sulfur. In embodiments, ring A is a 5- membered heteroaryl containing 3 heteroatoms selected from the group consisting of oxygen, nitrogen, and sulfur. In embodiments, ring A is pyrrole, pyrazole, imidazole, triazole, furan, thiophene, phosphole, oxazole, isoxazole, thiazole, or isothiazole. In embodiments, ring A is pyrrole.
  • ring A is pyrazole. In embodiments, ring A is imidazole. In embodiments, ring A is triazole. In embodiments, ring A is furan. In embodiments, ring A is thiophene. In embodiments, ring A is phosphole. In embodiments, ring A is oxazole. In embodiments, ring A is isoxazole. In embodiments, ring A is thiazole. In embodiments, ring A is isothiazole. In embodiments, L 1 is attached to a heteroatom in the 5-membered heteroaryl. In embodiments, L 1 is attached to a carbon atom in the 5-membered heteroaryl.
  • the –S(O2)F moiety is attached to a heteroatom in the 5-membered heteroaryl. In embodiments, the –S(O 2 )F moiety is attached to a carbon atom in the 5-membered heteroaryl. In embodiments, L 1 is attached to a carbon atom in the 5-membered heteroaryl and the –S(O2)F moiety is attached to a carbon atom in the 5-membered heteroaryl. In embodiments, L 1 is attached to a heteroatom in the 5-membered heteroaryl and the –S(O2)F moiety is attached to a carbon atom in the 5- membered heteroaryl.
  • L 1 is attached to a carbon atom in the 5-membered heteroaryl and the —S(O2)F moiety is attached to a heteroatom in the 5-membered heteroaryl. In embodiments, L 1 is attached to a heteroatom in the 5-membered heteroaryl, and the —S(O 2 )F moiety is attached to a heteroatom in the 5-membered heteroaryl.
  • proteins comprising: (i) an unnatural amino acid, and (ii) a non- naturally occurring arginine; wherein: (a) the non-naturally occurring arginine is proximal to the -S(O2)F group in the unnatural amino acid side chain; and (b) the unnatural amino comprises a side chain of Formula (NHSF): .
  • the protein is a single-chain variable fragment, a single-domain antibody, an affibody, or an antigen-binding fragment.
  • the protein is a single-chain variable fragment.
  • the protein is a single-domain antibody.
  • the protein is an affibody.
  • the protein is an antigen-binding fragment.
  • the protein further comprises a naturally occurring arginine.
  • the protein further comprises a naturally occurring arginine that is not proximal to the –S(O2)F group in the unnatural amino acid side chain.
  • proteins comprising: (i) an unnatural amino acid, and (ii) a naturally occurring arginine; wherein: (a) the naturally occurring arginine is proximal to the – S(O2)F group in the unnatural amino acid side chain; and (b) the unnatural amino comprises a side chain of Formula (NHSF): .
  • the protein is not epidermal growth factor receptor, protein tyrosine phosphatase 1B, P-selectin glycoprotein ligand 1, complement component 5a receptor, chemokine receptor D6, CXCR4, thymopentin, oxytocin, arginine vasopressin, indolicidin, or a protein described in any one of WO 2017/161183, WO 2019/173760, WO 2020/072674, WO 2020/206341, and WO 2022/232377.
  • the protein is an antigen-binding fragment.
  • the protein further comprises a naturally occurring arginine.
  • the protein further comprises a naturally occurring arginine that is not proximal to the –S(O 2 )F group in the unnatural amino acid side chain.
  • proteins comprising: (i) an unnatural amino acid, and (ii) a naturally occurring arginine; wherein: (a) the naturally occurring arginine is proximal to the – S(O2)F group in the unnatural amino acid side chain; and (b) the unnatural amino comprises a side chain of Formula (VII): .
  • the protein is an antigen-binding fragment.
  • the protein further comprises a naturally occurring arginine.
  • the protein further comprises a naturally occurring arginine that is not proximal to the –S(O 2 )F group in the unnatural amino acid side chain.
  • proteins comprising: (i) an unnatural amino acid, and (ii) a naturally occurring arginine; wherein: (a) the naturally occurring arginine is proximal to the – S(O 2 )F group in the unnatural amino acid side chain; and (b) the unnatural amino comprises a side chain of Formula (VIII): .
  • the is a single-chain variable fragment, a binding fragment.
  • the protein is a single-chain variable fragment. In embodiments, the protein is a single-domain antibody. In embodiments, the protein is an affibody. In embodiments, the protein is an antigen-binding fragment. In embodiments, the protein does not comprise a non-naturally occurring arginine. In embodiments, the protein is not enhanced green fluorescent protein, a maltose binding protein, or protein Z.
  • proteins comprising: (i) an unnatural amino acid, and (ii) a non- naturally occurring arginine; wherein: (a) the non-naturally occurring arginine is proximal to the -S(O 2 )F group in the unnatural amino acid side chain; and (b) the unnatural amino comprises a side chain of Formula (X): .
  • the is a single-chain variable fragment, a single-domain antibody, an affibody, or an antigen-binding fragment.
  • the protein is a single-chain variable fragment.
  • the protein is a single-domain antibody.
  • the protein is an affibody.
  • the unnatural amino acid and the arginine are within a CDR region of the antibody.
  • the unnatural amino acid is within a framework region of the antibody and the arginine (non-naturally occurring or naturally occurring) are within a CDR region of the antibody.
  • the unnatural amino acid and the arginine (non-naturally occurring or naturally occurring) are within the same CDR region of the antibody.
  • the unnatural amino acid and the arginine (non-naturally occurring or naturally occurring) are within different CDR regions of the antibody.
  • a method of covalently binding an affibody to protein Z comprising contacting the affibody with the protein Z; wherein the affibody comprises SEQ ID NO:14 (or any embodiment thereof), thereby covalently binding the affibody to the protein Z.
  • a biomolecule conjugate comprising an affibody covalently bonded to protein Z, wherein the affibody comprises SEQ ID NO:14 (or any embodiment thereof).
  • the affibody has at least 90% sequence identity to SEQ ID NO:15, provided that the position corresponding to position 36 in SEQ ID NO:15 is FSY and the position corresponding to position 32 in SEQ ID NO:15 is arginine.
  • a method of covalently binding an affibody to protein Z comprising contacting the affibody with the protein Z; wherein the affibody comprises SEQ ID NO:15 (or any embodiment thereof), thereby covalently binding the affibody to the protein Z.
  • a biomolecule conjugate comprising an affibody covalently bonded to protein Z, wherein the affibody comprises SEQ ID NO:15 (or any embodiment thereof).
  • the protein is a receptor protein.
  • the receptor protein is a PD-L1 receptor. In embodiments, the receptor protein is a PD-1 receptor. [0172] In embodiments, the receptor protein is a receptor expressed on a cancer cell. In embodiments, the receptor protein is a receptor overexpressed on a cancer cell relative to a control. [0173] In embodiments, the receptor protein is a G protein-coupled receptor. In embodiments, the receptor protein is a receptor tyrosine kinase. In embodiments, the receptor protein is a an ErbB receptor. In embodiments, the receptor protein is an epidermal growth factor receptor (EGFR). In embodiments, the receptor protein is epidermal growth factor receptor 1 (HER1).
  • EGFR epidermal growth factor receptor
  • HER1 epidermal growth factor receptor 1
  • a “detectable agent” or “detectable moiety” is a composition detectable by appropriate means such as spectroscopic, photochemical, biochemical, immunochemical, chemical, magnetic resonance imaging, or other physical means.
  • the proteins described herein are bonded to a detectable agent.
  • the fusion proteins described herein are bonded to a detectable agent.
  • an antibody or antibody variant is bonded to a detectable agent.
  • a nanobody is bonded to a detectable agent.
  • the bond is noncovalent or covalent.
  • the bond is covalent.
  • the protein is covalently bonded to a detectable agent.
  • the fusion protein is covalently bonded to a detectable agent.
  • the antibody or antibody variant is covalently bonded to a detectable agent.
  • a nanobody is covalently bonded to a detectable agent.
  • the covalent bond is between the detectable agent and a naturally-occurring amino acid in the protein or fusion protein.
  • the nanobody is covalently bonded to a detectable agent, the covalent bond is between the detectable agent and a naturally- occurring amino acid in the nanobody.
  • Detectable agents include 18 F, 32 P, 33 P, 45 Ti, 47 Sc, 52 Fe, 59 Fe, 62 Cu, 64 Cu, 67 Cu, 67 Ga, 68 Ga, 77 As, 86 Y, 90 Y. 89 Sr, 89 Zr, 94 Tc, 94 Tc, 99m Tc, 99 Mo, 105 Pd, 105 Rh, 111 Ag, 111 In, 123 I, 124 I, 125 I, 131 I, 142 Pr, 143 Pr, 149 Pm, 153 Sm, 154-1581 Gd, 161 Tb, 166 Dy, 166 Ho, 169 Er, 175 Lu, 177 Lu, 186 Re, 188 Re, 189 Re, 194 Ir, 198 Au, 199 Au, 211 At, 211 Pb, 212 Bi, 212 Pb, 213 Bi, 223 Ra, 225 Ac, Cr, V, Mn, Fe, Co, Ni, Cu, La, Ce, Pr, Nd, Pm, Sm
  • a detectable moiety is a monovalent detectable agent or a detectable agent capable of forming a bond with another composition.
  • paramagnetic ions that may be used as imaging agents in accordance with the embodiments of the disclosure include, e.g., ions of transition and lanthanide metals (e.g., metals having atomic numbers of 21-29, 42, 43, 44, or 57- 71). These metals include ions of Cr, V, Mn, Fe, Co, Ni, Cu, La, Ce, Pr, Nd, Pm, Sm, Eu, Gd, Tb, Dy, Ho, Er, Tm, Yb and Lu.
  • an or to a a nanobody is bonded to a radioisotope.
  • the bond is noncovalent or covalent.
  • the bond is covalent.
  • the protein is covalently bonded to a radioisotope.
  • the antibody or antibody variant is covalently bonded to a radioisotope.
  • a nanobody is covalently bonded to a radioisotope.
  • the covalent bond is between the radioisotope and an unnatural amino acid in the nanobody.
  • Methods for covalently bonding radioisotopes to proteins are well-known in the art.
  • the radioisotope is 123 I, 124 I, 125 I, or 131 I.
  • the radioisotope is 123 I.
  • the radioisotope is 124 I.
  • the radioisotope is 125 I.
  • the radioisotope is 131 I.
  • the radioisotope is a positron-emitting radioisotope.
  • the positron-emitting radioisotope is 11 C, 13 N, 15 O, 18 F, 64 Cu, 68 Ga, 78 Br, 82 Rb, 86 Y, 89 Zr, 90 Y, 22 Na, 26 Al, 40 K, 83 Sr, or 124 I.
  • the positron-emitting radioisotope is 11 C.
  • the positron-emitting radioisotope is 13 N.
  • the positron-emitting radioisotope is 15 O.
  • the positron-emitting radioisotope is 18 F.
  • the positron-emitting radioisotope is 64 Cu. In embodiments, the positron-emitting radioisotope is 168 Ga. In embodiments, the positron-emitting radioisotope is 78 Br. In embodiments, the positron- emitting radioisotope is 82 Rb. In embodiments, the positron-emitting radioisotope is 86 Y. In embodiments, the positron-emitting radioisotope is 89 Zr. In embodiments, the positron-emitting radioisotope is 90 Y. In embodiments, the positron-emitting radioisotope is 22 Na.
  • the positron-emitting radioisotope is 26 Al. In embodiments, the positron-emitting radioisotope is 40 K. In embodiments, the positron-emitting radioisotope is 83 Sr. In embodiments, the positron- emitting radioisotope is 124 I. In embodiments, the radioisotope is an alpha-emitting radioisotope. In embodiments, the alpha-emitting radioisotope is 211 At, 227 Th, 225 Ac, 223 Ra, 213 Bi, or 212 Bi. In embodiments, the alpha-emitting radioisotope is 211 At.
  • “Small-molecule drugs” refers to chemical compounds with low molecular weight that are capable of treating and/or preventing diseases.
  • the proteins described herein are bonded to a therapeutic agent.
  • an antibody or antibody variant is bonded to a therapeutic agent.
  • a nanobody is bonded to a therapeutic agent.
  • the bond is noncovalent or covalent.
  • the bond is covalent.
  • the protein is covalently bonded to a therapeutic agent.
  • the antibody or antibody variant is covalently bonded to a therapeutic agent.
  • a nanobody is covalently bonded to a therapeutic agent.
  • the covalent bond is between the therapeutic agent and a naturally-occurring amino acid in the protein.
  • the covalent bond is between the therapeutic agent and a naturally-occurring amino acid in the nanobody.
  • R 1 is hydrogen, halogen, -CX 1 3, -CHX 1 2 , -CH 2 X 1 , -OCX 1 3 , -OCH 2 X 1 , -OCHX 1 2 , -CN, -SO n1 R 1A , -SO v1 NR 1A R 1B , -NHC(O)NR 1A R 1B , -N(O)m1, -NR 1A R 1B , -C(O)R 1A , -C(O)-OR 1A , -C(O)NR 1A R 1B , -OR 1A , -NR 1A SO 2 R 1B , -NR 1A C(O)R 1B , -NR 1A C(O)OR 1B , -NR 1A OR 1B , -NR 3 + , substituted or unsubstit
  • R 1 is hydrogen, halogen, -CX 1 3 , -CHX 1 2, -CH2X 1 , -OCX 1 3, -OCH2X 1 , -OCHX 1 2, -CN, -SOn1R 1A , -SOv1NR 1A R 1B , -NHC(O)NR 1A R 1B , -N(O) m1 , -NR 1A R 1B , -C(O)R 1A , -C(O)-OR 1A , -C(O)NR 1A R 1B , -OR 1A , -NR 1A SO2R 1B , -NR 1A C(O)R 1B , -NR 1A C(O)OR 1B , -NR 1A OR 1B , -NR3 + , substituted or unsubstituted alkyl, or substituted or unsubstituted heteroalkyl.
  • R 1A and R 1B are hydrogen.
  • R 1 is an electron-donating group.
  • the electron-donating group is –Cl, -Br, -I, -CX 2 3, -CHX 2 2, -OCX 1 3, -OCH2X 1 , -OCHX 1 2, -OCOR 1A , -OC(O)R 1A , -OC(O)NR 1A R 1B , -SR 1A , -PR 1A R 1B -NHC(O)NR 1A R 1B , -NR 1A R 1B , -OR 1A , -NR 1A SO2R 1B , -NR 1A C(O)R 1B , -NR 1A C(O)OR 1B , substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted aryl, or substituted or un
  • the substituted or unsubstituted alkyl is substituted or unsubstituted alkene.
  • the electron-donating group is unsubstituted alkene.
  • the substituted or unsubstituted alkyl is substituted or unsubstituted alkyne.
  • R 1A and R 1B are hydrogen.
  • the electron-donating group is unsubstituted alkyne.
  • R 1 is substituted or unsubstituted heteroalkyl.
  • R 1 is unsubstituted heteroalkyl.
  • R 1 is unsubstituted 2 to 8 membered heteroalkyl.
  • R 1 is chlorine. In embodiments, R 1 is bromine. In embodiments, R 1 is iodine. [0187] In embodiments, R 1 is -CX 1 3, -CHX 1 2, or -CH2X 1 , wherein X 1 is halogen. In embodiments, R 1 is -CH2X 1 . In embodiments, R 1 is -CHX 1 2. In embodiments, R 1 is -CX 1 3. In embodiments, R 1 is -CF3. In embodiments, R 1 is -CHF2. In embodiments, R 1 is -CH2F. In embodiments, R 1 is -CCl3. In embodiments, R 1 is -CHCl2. In embodiments, R 1 is -CH2Cl.
  • R 1 is an alkyl group substituted with an electron-withdrawing group.
  • R 1 is a halogen-substituted alkyl group.
  • w is 1.
  • w is 2.
  • w is 3.
  • w is 4.
  • w is 5.
  • R 1A is hydrogen, substituted or unsubstituted alkyl, or substituted or unsubstituted heteroalkyl. In embodiments, R 1A is hydrogen, unsubstituted alkyl, or unsubstituted heteroalkyl. In embodiments, R 1A is hydrogen, substituted or unsubstituted C 1-4 alkyl, or substituted or unsubstituted 2 to 4 membered heteroalkyl. In embodiments, R 1A is hydrogen, unsubstituted C1-4 alkyl, or unsubstituted 2 to 4 membered heteroalkyl. In embodiments, R 1A is hydrogen.
  • R 1A is unsubstituted C1-4 alkyl. In embodiments, R 1A is unsubstituted 2 to 4 membered heteroalkyl. In embodiments, R 1A is hydrogen and R 1B is hydrogen. [0190] In embodiments of the proteins described herein, R 1B is hydrogen, substituted or unsubstituted alkyl, or substituted or unsubstituted heteroalkyl. In embodiments, R 1B is hydrogen, unsubstituted alkyl, or unsubstituted heteroalkyl. In embodiments, R 1B is hydrogen, substituted or unsubstituted C1-4 alkyl, or substituted or unsubstituted 2 to 4 membered heteroalkyl.
  • R 1B is hydrogen, unsubstituted C 1-4 alkyl, or unsubstituted 2 to 4 membered heteroalkyl. In embodiments, R 1B is hydrogen. In embodiments, R 1B is unsubstituted C 1-4 alkyl. In embodiments, R 1B is unsubstituted 2 to 4 membered heteroalkyl. In embodiments, R 1A is hydrogen and R 1B is hydrogen. [0191] In embodiments of the proteins described herein, X 1 is independently –F, -Cl, -Br, or – I. In embodiments, X 1 is independently –F, -Cl, or -Br.
  • m1 is 2. [0194] In embodiments of the proteins described herein, v1 is 1 or 2. In embodiments, v1 is 1. In embodiments, v1 is 2. [0195] In embodiments of the proteins described herein, x is an integer from 0 to 8. In embodiments, x is an integer from 1 to 8. In embodiments, x is an integer from 1 to 7. In embodiments, x is an integer from 1 to 6. In embodiments, x is an integer from 1 to 5. In embodiments, x is an integer from 1 to 4. In embodiments, x is an integer from 1 to 3. In embodiments, x is an integer of 1 or 2. In embodiments, x is 1. In embodiments, x is 2. In embodiments, x is 3.
  • L 1 is a bond, substituted or unsubstituted alkylene, or substituted or unsubstituted heteroalkylene. In embodiments, L 1 is a bond. In embodiments, L 1 is substituted or unsubstituted alkylene. In embodiments, L 1 is substituted or unsubstituted C1-6 alkylene. In embodiments, L 1 is substituted or unsubstituted C1- 4 alkylene. In embodiments, L 1 is unsubstituted alkylene.
  • L 1 is unsubstituted C1-6 alkylene. In embodiments, L 1 is unsubstituted C1-4 alkylene. In embodiments, L 1 is methylene. In embodiments, L 1 is ethylene. In embodiments, L 1 is propylene. In embodiments, L 1 is substituted or unsubstituted heteroalkylene. In embodiments, L 1 is substituted or unsubstituted 2 to 8 membered heteroalkylene. In embodiments, L 1 is substituted or unsubstituted 2 to 6 membered heteroalkylene.
  • L 1 is –NH-C(O)-(CH2)y- or – NH-C(O)-O-(CH 2 ) y -, and y is an integer from 0 to 6. In embodiments, L 1 is –NH-C(O)-(CH 2 ) y - or –NH-C(O)-O-(CH2)y-, and y is an integer from 0 to 5. In embodiments, L 1 is –NH-C(O)- (CH 2 ) y - or –NH-C(O)-O-(CH 2 ) y -, and y is an integer from 0 to 4.
  • L 1 is —NH-C(O)-(CH 2 )- In embodiments, L 1 is -NH-C(O)-(CH 2 ) 2 -. In embodiments, L 1 is –NH-C(O)-(CH2)3-. In embodiments, L 1 is –NH-C(O)-O-(CH2)y-, and y is an integer from 0 to 3. In embodiments, L 1 is –NH-C(O)-O-. In embodiments, L 1 is –NH-C(O)- O-(CH2)-. In embodiments, L 1 is –NH-C(O)-O-(CH2)2-.
  • L 1 is –NH-C(O)-O- (CH 2 ) 3 -.
  • –(CH 2 ) x -L 1 - is –(CH 2 ) x NHC(O)- or –(CH2)xNHC(O)O-, where x is as defined herein.
  • –(CH2)x-L 1 - is –(CH 2 ) x NHC(O)-, where x is as defined herein.
  • –(CH 2 ) x -L 1 - is – (CH2)NHC(O)-.
  • Adjuvants can be administered as a component of a therapeutic composition with an active agent or can be administered separately, before, concurrently with, or after administration of the therapeutic agent.
  • An adjuvant can be administered with an immunogen as a single composition, or can be administered before, concurrent with or after administration of the immunogen.
  • Immunogen and adjuvant can be packaged and supplied in the same vial or can be packaged in separate vials and mixed before use. Immunogen and adjuvant are typically packaged with a label indicating the intended therapeutic application. If immunogen and adjuvant are packaged separately, the packaging typically includes instructions for mixing before use.
  • an adjuvant and/or carrier depends on the stability of the immunogenic formulation containing the adjuvant, the route of administration, the dosing schedule, the efficacy of the adjuvant for the species being vaccinated, and, in humans, a pharmaceutically acceptable adjuvant is one that has been approved or is approvable for human administration by pertinent regulatory bodies. For example, Complete Freund's adjuvant is not suitable for human administration.
  • Alum, MPL and QS-21 are preferred.
  • two or more different adjuvants can be used simultaneously. Preferred combinations include alum with MPL, alum with QS-21, MPL with QS-21, MPL or RC-529 with GM-CSF, and alum, QS-21 and MPL together.
  • amino comprises a side chain of Formula (IV): V).
  • Embodiment 43 ein the unnatural amino comprises a side chain of Formula (NHSF): .
  • Embodiment 44. the unnatural amino comprises a side chain of Formula (VII): .
  • unnatural amino comprises a side chain of Formula (VIII): .
  • unnatural amino comprises a side chain of Formula (IX): .
  • Embodiment unnatural amino comprises a side chain of Formula (X): X).
  • Embodiment 47, wherein the protein is an antibody.
  • Embodiment 49. The protein of any one of embodiments 1 to 47, wherein the protein is an antibody variant.
  • Embodiment 84 The protein of embodiment 83, wherein the detectable agent is a radioisotope.
  • Embodiment 85 The protein of any one of embodiments 1 to 84, further comprising a therapeutic agent.
  • Embodiment 86 A nucleic acid encoding the protein of any one of embodiments 1 to 85.
  • Embodiment 87 A vector comprising a nucleic acid of embodiment 86.
  • Embodiment 88. A cell comprising: (i) the protein of any one of embodiments 1 to 85; (ii) the nucleic acid of embodiment 86, or (iii) the vector of embodiment 87. [0314] Embodiment 89.
  • the target protein is a programmed death-ligand 1 receptor, a programmed cell death protein 1 receptor, a 5- hydroxytryptamine receptor, an acetylcholine receptor, an adenosine receptor, an adenosine A2A receptor, an adenosine A2B receptor, an angiotensin receptor, an apelin receptor, a bile acid receptor, a bombesin receptor, a bradykinin receptor, a cannabinoid receptor, a chemerin receptor, a chemokine receptor, a cholecystokinin receptor, a Class A Orphan receptor, a dopamine receptor, an endothelin receptor, an epidermal growth factor receptor, a formyl peptide receptor, a free fatty acid receptor, a galanin receptor, a ghrelin receptor, a glycoprotein hormone receptor, a gonadotrophin-releasing hormone receptor, a G protein-coup
  • a method of covalently binding a single domain antibody to c-2 comprising contacting the single domain antibody with the human epidermal growth factor receptor-2; wherein the single domain antibody is the protein of any one of embodiments 64 to 70, thereby covalently binding the single-domain antibody to the human epidermal growth factor receptor-2.
  • Embodiment 116 The method of embodiment 115, wherein the first amino acid is Arg, Ala, Ile, Leu, Met, Val, Phe, Trp, Tyr, Asn, Cys, Gln, Ser, Thr, Asp, Glu, His, Lys, Gly, or Pro; and wherein the second amino acid is Ala, Ile, Leu, Met, Val, Phe, Trp, Tyr, Asn, Cys, Gln, Ser, Thr, Asp, Glu, His, Lys, Gly, or Pro.
  • Embodiment 117 Embodiment 117.
  • Nanobody 7D12(Y109FSY/E44R) and control nanobody 7D12(Y109FSY) were expressed and purified and the from E. coli, and 5 ⁇ M of the nanobody protein were individually incubated with 0.4 ⁇ M EGFR extracellular domain (ECD) in PBS buffer for crosslinking. At different time points, an aliquot of the reaction mixture was taken and the reaction was stopped by adding SDS-loading buffer.
  • Example 2 The inventors also mutated residues T107 and L108 to Arg separately, as they are close to Y109 site on 1D amino acid sequence. However, introducing Arg at these two sites did not significantly increase the crosslinking rate (FIGS.1C-1D). These results indicate that the introduced Arg needs to be in an appropriate orientation with FSY in the 3D structure to accelerate the reaction.
  • Example 2 The inventor used nanobody 2Rs15d, which binds to HER2. On the basis of the crystal structure of 2Rs15d in complex with HER2, FSY was incorporated at D54 in the nanobody to target Lys150 of HER2, and Asp56 in 2Rs15d was mutated into Arg (FIG.2A).
  • FSY was incorporated at site D36 and F32 was mutated into Arg in the affibody (FIG.3A).
  • the Z protein was fused with MBP with residue Asn6 mutated into Lys, His or Tyr for reaction with FSY.
  • These mutant affibody and Z proteins were expressed and purified from E. coli, and then incubated in PBS buffer for crosslinking (concentration 10 ⁇ M for each). The reaction mixture was analyzed with SDS-PAGE to determine crosslinking extent.
  • FIGS.3B-3D in comparison with the control affibody, introduction of Arg close to FSY accelerated its crosslinking reaction with His, Lys, and Tyr, respectively.
  • Example 5 The 7D12 nanobody specific for EGFR was used here.
  • mFSY has the fluorosulfonate warhead at the meta position, in contrast to the para-positioned fluorosulfonate in Uaa FSY. See Klauser et al, Chemical communications 2022, 58 (48), 6861–6864.
  • mFSY was incorporated at site Y109, and mutated E44 to Arg.3 ⁇ M of 7D12 nanobody protein was incubated with 0.3 ⁇ M of EGFR ECD protein for different time followed with SDS-PAGE analysis under denatured conditions. For 7D12(Y109mFSY), crosslinking was detectable after 4 h incubation.
  • FFY was incorporated at site 54 in nanobody 2Rs15d and mutated residue D56 to Arg.3 mM of 2Rs15d nanobody protein was incubated with 0.3 mM of HER2 ECD protein for different time followed with SDS- PAGE analysis under denatured conditions. While 2Rs15d(54FFY) could rapidly crosslink in 10 minutes, the Arg mutant 2Rs15d(54FFY/D56R) showed even faster crosslinking within 5 minutes.
  • FIG.6 [0362]
  • Example 7 [0363] Acceleration of FFY-mediated crosslinking was also verified in nanobody 7D12 specific for the EGFR receptor.
  • BiKE bispecific NK-cell engager
  • Arg-BiKE and WT-BiKE in activating NK toward EGFR-expressing cells
  • the EGFR+ A431 cells (10K) were incubated with different concentrations of BiKE (50 nM, 100 nM, 200 nM, 400 nM) for 1 h, followed with washing to remove the BiKE (10 min each for 3 times).
  • NK cells (100K) were then added and incubated with the treated A431 cells for 4 h. The supernatant of the cells was assayed with the human perforin ELISA kit to measure perforin, a marker for NK cell activity.
  • TMG has been frequently used as a catalyst to accelerate the SuFEx reaction between small molecules (Homer et al, Nat Rev Methods Primers 2023, 3 (1), 58. doi.org/10.1038/s43586-023- 00241-y).
  • the inventors found that TMG had no effect on SuFEx crosslinking in the protein context.20 mM of TMG was added into all protein pairs described in the examples above, followed with SDS-PAGE analysis of the crosslinking efficiency. There was no detection of any acceleration of the reaction rate or increase in the crosslinking yield by TMG (data not shown).
  • Example 10 [0371] Methods of incorporating unnatural amino acid side chains, such as those described herein, into proteins are known in the art and described, for example, in US Publication No. 2020/010411, US Publication No.2021/002325, US Publication No.2022/107327, US Publication No.2022/371986, and WO 2022/232377, the disclosures of each of which are incorporated by reference herein in their entirety. [0372] References: (1) Li et al, Cell 2020, 182 (1), 85–97.e16; (2) Yu et al, Chem 2022, 8 (10), 2766–2783; (3) Wang et al, J. Am. Chem.

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Abstract

La présente invention concerne, entre autres, des protéines contenant des acides aminés non naturels et de l'arginine, des compositions pharmaceutiques comprenant les protéines, et des méthodes d'amélioration de la bioréactivité de protéines. Dans des modes de réalisation, l'acide aminé non naturel présente une chaîne latérale de formule (I), les substituants étant tels que définis dans la description.
PCT/US2024/010060 2022-12-30 2024-01-02 Protéines bioréactives contenant un acide aminé non naturel et de l'arginine WO2024145687A1 (fr)

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