Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
  • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
  • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
  • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
  • A61K47/6801—Drug-antibody or immunoglobulin conjugates defined by the pharmacologically or therapeutically active agent
  • A61K47/6803—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates
  • A61K47/6811—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates the drug being a protein or peptide, e.g. transferrin or bleomycin
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
  • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
  • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
  • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
  • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
  • A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
  • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
  • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
  • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
  • A61K47/65—Peptidic linkers, binders or spacers, e.g. peptidic enzyme-labile linkers
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
  • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
  • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
  • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
  • A61K47/6801—Drug-antibody or immunoglobulin conjugates defined by the pharmacologically or therapeutically active agent
  • A61K47/6803—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
  • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
  • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
  • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
  • A61K47/6835—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site
  • A61K47/6843—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site the antibody targeting a material from animals or humans
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
  • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
  • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
  • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
  • A61K47/6889—Conjugates wherein the antibody being the modifying agent and wherein the linker, binder or spacer confers particular properties to the conjugates, e.g. peptidic enzyme-labile linkers or acid-labile linkers, providing for an acid-labile immuno conjugate wherein the drug may be released from its antibody conjugated part in an acidic, e.g. tumoural or environment
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
  • C07K7/04—Linear peptides containing only normal peptide links
  • C07K7/06—Linear peptides containing only normal peptide links having 5 to 11 amino acids
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
  • C07K16/18—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans

Definitions

• the present disclosure relates to conjugated therapy enhancers that are useful for preventing and/or treating various conditions, disorders, or diseases. Specifically, the present disclosure relates to protein conjugates such as antibody-drug conjugates that are capable of acting as therapy enhancers.
• Conjugated therapy enhancers have been extensively used for preventing and/or treating various conditions, disorders, and diseases.
• Such enhancers typically include a therapeutically active molecule, such as an antibody, linked to a moiety having affinity to a particular target implicated in the condition, disorder, or disease.
• a therapeutically active molecule such as an antibody
• conjugation techniques are not directed to a specific site of the therapeutically active molecule, and usually result in a mixture of conjugates. There remains a need in the development of site-specific conjugation techniques that provide reaction products with high degree of homogeneity.
• compositions that include therapy enhancer agents containing moieties of interest conjugated to target agent moieties at specific locations.
• composition including; a first compound having the structure of formula (P-ll):
• P-N is a protein agent moiety including a lysine residue
• L PM is a linker
• MOI is a moiety of interest; and a second compound having the structure: LG-OH (LG-I) wherein LG is a group including a target binding moiety that binds to a target agent.
• composition further includes:
• LG is a group including a target binding moiety that binds to a target agent, which is identical to LG in formula (LG-I);
• RG is a reactive group
• L RM is a linker, which is identical to in formula (P-ll); and MOI is a moiety of interest.
• FIGURE 1 A representative UPLC/UV280 nm trace is provided for compound 1-36 (MATE reagent 1-20 conjugated to IVIG). The UPLC conditions are given in Example 2. uABT indicates universal Antibody Binding Terminal, DAR is Drug Antibody Ratio.
• FIGURE 2 UPLC traces of unconjugated IVIG (FIG. 2A), MATE impurities pooled standard (FIG. 2B), and 1-36 Conjugate (FIG. 2C).
• FIGURE 3 Calibration curves for uABT, MATE-Linker, and MATE-Reagent pooled and individual impurities. Samples are prepared and analyzed by the methods given in Example 2.
• the embodiments are merely described below, by referring to structures and schemes, to explain aspects of the present description.
• the term “and/or” includes any and all combinations of one or more of the associated listed items.
• the term “or” means “and/or.” Expressions such as "at least one of,” when preceding a list of elements, modify the entire list of elements and do not modify the individual elements of the list.
• first, second, third etc. may be used herein to describe various elements, components, regions, layers, and/or sections, these elements, components, regions, layers, and/or sections should not be limited by these terms. These terms are only used to distinguish one element, component, region, layer, or section from another element, component, region, layer, or section. Thus, a first element, component, region, layer, or section discussed below could be termed a second element, component, region, layer, or section without departing from the teachings of the present embodiments.
• substituted refers to a group substituted with deuterium, a halogen (-F, -Cl, -Br, -I), a hydroxy group (- OH), an amino group (-NH2), a carboxyl group (-CO2H), a substituted or unsubstituted C1-C10 amine group, a nitro group (-N0 2 ), a C1-C10 alkyl group, a C3-C10 cycloalkyl group, a C6-C12 aryl group, a Cl- C10 alkoxy group, a Cl to CIO trifluoroalkyl group such as a trifluoromethyl group (-CF 3 ) and the like, or a cyano group (-CN) instead of at least one hydrogen of a substituting group or compound.
• compositions that include therapy enhancer agents containing moieties of interest conjugated to target agent moieties at specific locations.
• composition including;
• P-N is a protein agent moiety including a lysine residue
• L PM is a linker
• MOI is a moiety of interest
• the composition further includes: a third compound having the formula (R-l):
• LG is a group including a target binding moiety that binds to a target agent, which is identical to LG in formula (LG-I);
• RG is a reactive group
• L RM is a linker, which is identical to in formula (P-ll).
• MOI is a moiety of interest.
• target agents are or include a protein agent, a nucleic acid, or a combination thereof.
• a target agent is or includes a protein agent.
• a target agent is a protein agent.
• a target agent is a natural protein in a cell, tissue, organ or organism.
• a target agent is an endogenous protein.
• a target agent is an exogenous protein.
• a target agent is a manufactured protein, e.g., a protein produced using various biotechnologies.
• a target agent is an antibody agent.
• a target agent is an antibody useful as therapeutics. Various such antibodies are known in the art and can be utilized as target agents.
• an antibody is a monoclonal antibody.
• an antibody is a polyclonal antibody. In some embodiments, an antibody is an IgG antibody. In some embodiments, an antibody is IVIG (in some embodiments, pooled from healthy donors). In some embodiments, a protein includes a Fc region. In some embodiments, an antibody includes a Fc region.
• a Fc region includes a single heavy chain or a fragment thereof. In some embodiments, a Fc region includes two heavy chains or fragments thereof.
• an antibody is a human antibody. In some embodiments, an antibody is a chimeric antibody. In some embodiments, an antibody is a humanized antibody. In some embodiments, an antibody is a mouse antibody.
• digestions are performed, e.g., enzyme digestions using IdeZ, IdeS, etc., so that certain regions of antibodies (e.g., Fab) are removed to provide compositions with improved homogeneity for characterization (e.g., by MS).
• an antibody is a therapeutic antibody, e.g., a FDA-approved antibody for therapeutic uses.
• a therapeutic antibody is useful for treating cancer.
• an antibody is adalimumab, alemtuzumab, atezolizumab, avelumab, ipilimumab, cetuximab, daratumumab, dinutuximab, elotuzumab, ibritumomab tiuxetan, imgatuzumab, infliximab, ipilimumab, necitumumab, obinutuzumab, ofatumumab, pertuzumab, reslizumab, rituximab, trastuzumab, mogamulizumab, AMP-224, FS-102, GSK-2857916, ARGX-111, ARGX-110, A
• an antibody is rituximab, basiliximab, infliximab, cetuximab, siltuximab, dinutuximab, altertoxaximab, daclizumab, palivizumab, trastuzumab, alemtuzumab, omalizumab, efalizumab, bevacizumab, natalizumab, tocilizumab, eculizumab, mogamulizumab, pertuzumab, obinutuzumab, vedolizumab, pembrolizumab, mepolizumab, elotuzumab, daratumumab, ixekizumab, reslizumab, and atezolizumab, adalimumab, panitumumab, golimumab, ustekinumab, canakinumab, ofatumumab,
• an antibody is daratumumab. In some embodiments, an antibody is cetuximab. In some embodiments, a provided compound or agent including an antibody agent moiety is useful for treating a condition, disorder or disease that may be treated by the antibody agent.
• Antibodies may be prepared in a number of technologies in accordance with the present disclosure.
• antibodies may have engineered structures compared to natural immunoglobulins.
• antibodies may include certain tags for purification, identification, assessment, etc.
• antibodies may contain fragments (e.g., CDR and/or Fc, etc.) and not full immunoglobulins.
• an amino acid residue may not be at the exact numbered site but may be at a site that corresponds to that numbered site per, e.g., EU numbering and/or sequence homology (e.g., homologues of the same or different species).
• target agents are or include native antibody agents.
• target agents are or include engineered antibody agents.
• target agents, e.g., antibodies include no engineered unnatural amino acid residues.
• LG is R LG -L lg ;
• R LG is ⁇ , R c -(Xaa)z-, a nucleic acid moiety, or a small molecule moiety; each Xaa is independently a residue of an amino acid or an amino acid analog; t is 0-50; z is 1-50; each R c is independently -L a -R'; each L a is independently a covalent bond, or an optionally substituted bivalent group selected from C 1 -C 2 o aliphatic or C1-C20 heteroaliphatic having 1-5 heteroatoms, wherein one or more methylene units of the group are optionally and independently replaced with -C(R')2-, -Cy-, -O-, -S-, -S-S-, -N(R')-, -C(O)-, -C(S)-, -C(NR')-, -C(O)N(R')-, -N(R')C(O)N(R')-
• RG is -L RG1 -L RG2 -, — L LG4 — L RG1 — L RG2 — , -L LG3 -L LG4 -L RG1 -L RG2 -, or -L LG2 -L LG3 -L LG4 -L RG1 -L RG2 -; each of L lg1 , L LG2 , L lgs , L LG4 , L rg1 , L RG2 , and L RM is independently L; each L is independently a covalent bond, or a bivalent optionally substituted, linear or branched C 1-100 group including one or more aliphatic moieties, aryl moieties, heteroaliphatic moieties each independently having 1-20 heteroatoms, heteroaromatic moieties each independently having 1-20 heteroatoms, or any combinations of any one or more of such moieties, wherein one or more methylene units of the group are optionally and independently replaced
• LG is or includes a target binding moiety that binds to a target agent, wherein the target agent is an antibody agent.
• LG is or includes a target binding moiety that binds to a Fc region
• R LG is or includes DCAWXLGELVWCT (SEQ ID NO:l), wherein the two cysteine residues optionally form a disulfide bond, and X is an amino acid residue.
• LG is or includes a target binding moiety having the structure of A-l to A-50:
• moieties of interest are or include detectable moieties. Among other things, such moieties can be useful for detection, quantification, diagnosis, treatment, etc.
• a moiety of interest is or includes a radioactive label.
• a moiety of interest is or includes a label that can be detected through spectroscopy.
• a moiety of interest is or includes a fluorophore such as FITC moiety.
• a moiety of interest may be a moiety having affinity to a particular target implicated in a medical condition, disorder, or disease.
• moieties of interest are or include therapeutic agent moieties.
• a moiety of interest is or includes a drug moiety, e.g., a drug moiety in an antibody-drug conjugate.
• a moiety of interest is or includes a toxic agent.
• a moiety of interest is or includes a cytotoxic agent.
• a moiety of interest is or includes an anti-cancer agent.
• an anti-cancer agent is a chemotherapeutic agent.
• moieties of interest are or include moieties that can interact and/or recruit other agents, such as proteins, nucleic acids, cells, etc.
• moieties of interest interact with proteins expressed by certain cell types, e.g., immune cells, disease cells, etc.
• moieties of interest are immune cell binders.
• moieties of interest recruit immune cells.
• moieties of interest trigger, promote and/or enhance one or more immune activities, e.g., for removing, killing, and/or inhibiting desired targets (e.g., cancer cells, antigens, etc.).
• moieties of interest interact, recruit and/or bind to disease cells, and trigger, promote and/or enhance removing, killing, and/or inhibiting disease cells.
• a moiety of interest is or includes a small molecule agent (e.g., one can bind specifically to its protein targets, cells targets, etc.).
• a moiety of interest is or includes a peptide or protein agent (e.g., scFv, a peptide binder to specific target, etc.).
• a moiety of interest is or includes a nucleic acid agent (e.g., an oligonucleotide, mRNA, etc.).
• a moiety of interest is or includes a carbohydrate agent.
• a moiety of interest is or includes a lipid agent.
• a moiety of interest is or includes a protein complex (e.g., Fab).
• Fab protein complex
• a moiety of interest is or includes a fluorophore. In some embodiments, a moiety of interest is or includes a cytotoxic small molecule agent. In some embodiments, a moiety of interest is or includes a cytotoxic peptide agent.
• a moiety of interest is an adjuvant.
• adjuvants can be utilized as moieties of interest in accordance with the present disclosure.
• an adjuvant is one described in US 2019/0015516, which is incorporated herein in its entirety by reference.
• a moiety of interest stimulates an immune system.
• a moiety of interest is or includes a particle.
• a particle is or includes a nanoparticle.
• a moiety of interest is or includes a nucleic acid moiety. In some embodiments, a moiety of interest is or includes an oligonucleotide. In some embodiments, a moiety of interest is or includes an aptamer.
• a moiety of interest is an antibody agent. In some embodiments, a moiety of interest is or includes an antibody fragment. In some embodiments, a moiety of interest is an antibody agent moiety that does not contain a region to which a target binding moiety binds. In some embodiments, a moiety of interest is an antibody agent that contains no Fc region. In some embodiments, a moiety of interest is or includes a scFv. In some embodiments, a scFv is for a different antigen than an antibody target agent.
• moieties of interest are or include reactive moieties, particularly those reaction partners for bio-orthogonal reactions. Suitable reactive moieties, including those for bio- orthogonal reactions, are widely known in the art and can be utilized herein.
• a bio-orthogonal reaction is a cycloaddition reaction, e.g., click chemistry.
• a moiety of interest is or includes -N3.
• a moiety of interest is or includes an alkyne.
• a moiety of interest may be a moiety that binds to a SARS-CoV-2 virus that is implicated in the COVID-19 disease.
• the moiety that binds to a SARS-CoV-2 virus may be a polypeptide disclosed in L. Cao et al., "De novo design of picomolar SARS-CoV-2 mini- protein inhibitors” Science 370, 426-431 (2020), which is incorporated herein in its entirety by reference.
• Such a polypeptide moiety may result in binding to SARS-CoV-2 spike proteins, inhibition, reduction and prevention of binding and/or infection of cells, inhibition, killing, and removal of SARS- CoV-2 viruses and/or cells infected thereby, etc.
• a moiety of interest improves one or more properties and/or activities of a target agent.
• a moiety of interest is or includes a stability enhancer.
• a moiety of interest improves one or more pharmacodynamic and/or pharmacokinetic properties of a target agent.
• At least one of the following conditions is met:
• the moiety of interest is or includes a therapeutic agent
• the moiety of interest is or includes a moiety that can bind to a protein, nucleic acid or a cell;
• the moiety of interest is or includes a reactive moiety suitable for a bio-orthogonal reaction.
• MOI is or includes a therapeutic agent moiety; and/or MOI is or includes an antibody agent.
• moieties are optionally connected to each other through linker moieties.
• a reactive group e.g., RG
• a moiety of interest e.g., MOI
• a linker e.g., L RM
• a moiety e.g., LG
• L LG may also include one or more linkers, e.g., L lg1 , I 1 - 22 , L lgs , L LG4 , etc., to link various portions.
• L LG is a linker moiety described herein.
• L lg1 is a linker moiety described herein.
• L LG2 is a linker moiety described herein.
• L LG3 is a linker moiety described herein.
• L LG4 is a linker moiety described herein.
• L RM is a linker moiety described herein.
• L PM is L as described herein. In some embodiments, L PM is a linker moiety described herein. In some embodiments, L PM is L as described herein.
• Linker moieties of various types and/or for various purposes e.g., those utilized in antibody-drug conjugates, etc., may be utilized in accordance with the present disclosure.
• Linker moieties can be either bivalent or polyvalent depending on how they are used. In some embodiments, a linker moiety is bivalent. In some embodiments, a linker is polyvalent and connecting more than two moieties.
• L LM includes one or more -[(CFhJn-Ojm-, wherein each n is independently 1-20, and m is 1-100.
• L RM the linker includes one or more -[(CFbJn-Ojm-, wherein each n is independently 1-20, and m is 1-100.
• provided compounds include reactive groups (e.g., RG).
• reactive groups e.g., RG
• first groups e.g., LG
• moieties of interest e.g., MOI
• RG is a reaction group as described herein.
• reactive groups when utilized in compounds that include no target binding moieties react slowly and provide low level of, in some embodiments, substantially no conjugation of moieties of interest with target agents.
• combination of reactive groups with target binding moieties in the same compounds e.g., as in compounds of formula R-l or salts thereof, can, among other things, promote reactions between reactive groups and target agents, enhance reaction efficiency, reduce side reactions, and/or improve reaction selectivity (e.g., in terms of target sites wherein conjugation of moieties of interest with target agents occurs).
• Reactive groups in provided compounds can react with various types of groups in target agents.
• reactive groups in provided compounds selectively react with amino groups of target agents, e.g., -IMH2 groups on side chains of lysine residues of proteins.
• reactive groups when utilized in provided compounds e.g., those of formula R-l or salts thereof, selectively react with particular sites of target agents, e.g., as shown in examples herein, one or more of K246, K248, K288, K290, K317, etc. of IgGl, K251, K 253, etc. for lgG2, K239, K241 for lgG4, etc.
• a site is K246 or K248 of an antibody heavy chain.
• sites are K246 and/or K248 of an antibody heavy chain.
• a site is K246 of an antibody heavy chain.
• a site is K248 of an antibody heavy chain.
• a site is K288 or K290 of an antibody heavy chain.
• a site is K288 of an antibody heavy chain.
• a site is K290 of an antibody heavy chain.
• a site is K317.
• a site is K414 of an antibody heavy chain.
• a site is K185 of an antibody light chain.
• a site is K187 of an antibody light chain.
• sites are K251 and/or K253 of an lgG2 heavy chain. In some embodiments, a site is K251 of an lgG2 heavy chain. In some embodiments, a site is K253 of an lgG2 heavy chain. In some embodiments, sites are K239 and/or K241 of an lgG4 heavy chain. In some embodiments, a site is K239 of an lgG4 heavy chain. In some embodiments, a site is K241 of an lgG4 heavy chain. In some embodiments, conjugation selectively occurs at one or more heavy chain sites over light chain sites. In some embodiments, for technologies without target binding moieties, conjugation occurs at light chain sites more than heavy chain sites (e.g., see Figure 15).
• a reactive group e.g., RG
• a reactive group is or includes an ester group.
• a reactive group e.g., RG
• an electrophilic group e.g., a Michael acceptor.
• RG is a group of the formula -L LG2 , — L LG2 — L LG3 — L LG4 — L rg1 — or - _I_RGI_
• L LG2 is -NH-, -NHC(O)-,-(CH 2 )n-NHC(O)-, -(CH 2 )n-OC(O)-, -(CH 2 )n-OC(O)NH-, -C(O)-NHCH 2 -, -C(O)-NHCH 2 CH 2 -, -C(O)O-CH 2 -, or NH-C(0)0-CH 2 -;
• L LGS is an optionally substituted aryl ring
• I 1 - 24" is a bond, -NH- or -O-;
• L RG1 is -O-C(O)-, -C(O) -, -S(O)-, -OS(O) 2 - or -OP(O(OR)-;
• L RG2 is -CH 2 -C(O)-, -C(O)-, or-CH 2 -;
• L LG is -(O)C-[(CH 2 )nO] m (CH 2 )nNH- -(O)C-[(CH 2 ) n O] m (CH 2 ) n NH- -[(CH 2 )nO] m NHC(O)[(CH 2 )nO] m NH- -[(CH 2 )nO] m ⁇ NHC(O)[(CH 2 )nO] m ⁇ p NH- -[(CH 2 )nO] m Cy[(CH 2 ) n O] m NH- -[(CH 2 ) n O] m Cy[(CH 2 ) n O] m NHC(O)[(CH 2 ) n O] m NH- or -[(CH 2 ) n O] m Cy
• RG is a group of the formula -
• n is 2 at each occurrence.
• the reactive group is or includes -C(O)-O- or -O-C(O)-.
• the reactive group includes an aryl group, optionally bonded to -C(O)-O- or -O-C(O)- and substituted with one or more electron-withdrawing groups.
• the aryl group has the structure of or , wherein R s is independently chosen at each occurrence from halogen, -NO 2 , -F, -L-R',
• R' is H or C 1- C 6 alkyl.
• the compositions may include the first and second compounds in equimolar amount.
• the amount of the second compound may be 50 mole percent (mole%) or less based on the total number of moles of the first and second compounds in the composition.
• the amount of the second compound may be 50 mole% or less, 45 mole% or less, 40 mole% or less, 35 mole% or less, 30 mole% or less, 25 mole% or less, 20 mole% or less, 15 mole% or less, 10 mole% or less, or 5 mole% or less based on the total number of moles of the first and second compounds in the composition.
• the amount of the second compound may be 5% or less, 4% or less, 3% or less, 2% or less, or 1% or less based on the total number of moles of the first and second compounds in the composition. In some embodiments, the amount of the second compound may be 1.0% or less, 0.9% or less, 0.8% or less, 0.7% or less, 0.6% or less, 0.5% or less, 0.4% or less, 0.3% or less, 0.2% or less, 0.1% or less based on the total number of moles of the first and second compounds in the composition.
• the amount of the second compound may be 0.10% or less, 0.09% or less, 0.08% or less, 0.07% or less, 0.06% or less, 0.05% or less, 0.04% or less, 0.03% or less, 0.02% or less, 0.01% or less based on the total number of moles of the first and second compounds in the composition. In some embodiments, the amount of the second compound may be 0.010% or less, 0.009% or less, 0.008% or less, 0.007% or less, 0.006% or less, 0.005% or less, 0.004% or less, 0.003% or less, 0.002% or less, 0.001% or less based on the total number of moles of the first and second compounds in the composition.
• the amount of the second compound may be 0.0010% or less, 0.0009% or less, 0.0008% or less, 0.0007% or less, 0.0006% or less, 0.0005% or less, 0.0004% or less, 0.0003% or less, 0.0002% or less, 0.0001% or less based on the total number of moles of the first and second compounds in the composition.
• the amount of the second compound may be 0.00010% or less, 0.00009% or less, 0.00008% or less, 0.00007% or less, 0.00006% or less, 0.00005% or less, 0.00004% or less, 0.00003% or less, 0.00002% or less, 0.00001% or less based on the total number of moles of the first and second compounds in the composition.
• the amount of the second compound may be 0.000010% or less, 0.000009% or less, 0.000008% or less, 0.000007% or less, 0.000006% or less, 0.000005% or less, 0.000004% or less, 0.000003% or less, 0.000002% or less, 0.000001% or less based on the total number of moles of the first and second compounds in the composition.
• the compositions may further include a third compound, a fourth compound, or a combination thereof.
• the amount of the third compound, the fourth compound, or the combination thereof may be 5% or less, 4% or less, 3% or less, 2% or less, or 1% or less based on the number of moles of the first compound in the composition.
• the amount of the third compound, the fourth compound, or the combination thereof may be 1.0% or less, 0.9% or less, 0.8% or less, 0.7% or less, 0.6% or less, 0.5% or less, 0.4% or less, 0.3% or less, 0.2% or less, 0.1% or less based on the number of moles of the first compound in the composition.
• the amount of the third compound, the fourth compound, or the combination thereof may be 0.10% or less, 0.09% or less, 0.08% or less, 0.07% or less, 0.06% or less, 0.05% or less, 0.04% or less, 0.03% or less, 0.02% or less, 0.01% or less based on the number of moles of the first compound in the composition.
• the amount of the third compound, the fourth compound, or the combination thereof may be 0.010% or less, 0.009% or less, 0.008% or less, 0.007% or less, 0.006% or less, 0.005% or less, 0.004% or less, 0.003% or less, 0.002% or less, 0.001% or less based on the number of moles of the first compound in the composition.
• the amount of the third compound, the fourth compound, or the combination thereof may be 0.0010% or less, 0.0009% or less, 0.0008% or less, 0.0007% or less, 0.0006% or less, 0.0005% or less, 0.0004% or less, 0.0003% or less, 0.0002% or less, 0.0001% or less based on the number of moles of the first compound in the composition.
• the amount of the third compound, the fourth compound, or the combination thereof may be 0.00010% or less, 0.00009% or less, 0.00008% or less, 0.00007% or less, 0.00006% or less, 0.00005% or less, 0.00004% or less, 0.00003% or less, 0.00002% or less, 0.00001% or less based on the number of moles of the first compound in the composition.
• the amount of the third compound, the fourth compound, or the combination thereof may be 0.000010% or less, 0.000009% or less, 0.000008% or less, 0.000007% or less, 0.000006% or less, 0.000005% or less, 0.000004% or less, 0.000003% or less, 0.000002% or less, 0.000001% or less based on the number of moles of the first compound in the composition.
• a composition including: a first compound having the structure of formula (P-ll):
• P-N is a protein agent moiety including a lysine residue
• L PM is a linker
• MOI is a moiety of interest; and at least one of a second compound having the structure:
• LG-OH wherein LG is a group including a target binding moiety that binds to a target agent; and a third compound having the formula (R-l):
• LG is a group including a target binding moiety that binds to a target agent, which is identical to LG in formula (LG-I);
• RG is a reactive group
• L RM is a linker, which is identical to in formula (P-ll); and MOI is a moiety of interest.
• composition may further include: a fourth compound having the formula (R-lll):
• Example 1 Certain technologies for preparing agents: 1-29, 1-30, 1-31, 1-32, 1-33, 1-34, 1-35, 1-36.
• the present disclosure provides technologies for preparing MATE agents and compositions thereof.
• provided technologies include reacting a composition including a plurality of antibody agents (e.g., IVIG compositions such as Gamunex-C) with a composition including a plurality of agents each including a target binding moiety, an antibody binding moiety, and a reactive group in between (and optional linker moieties linking such moieties) (e.g., 1-3, I- 7, 1-8, 1-15, 1-19, 1-20, 1-21, 1-22, 1-23, etc.). Described below as examples are preparations of certain MATE agents and compositions thereof.
• DMSO volume (mL) (Solid weight (mg)/Molecular weight) x purity (%) / 5 mM x 10 s .
• reaction mixtures included the following product, which was formed in equimolar amount to the agent 1-29, 1-30, 1-31, 1-32, 1-33, 1-34, 1-35, and 1-36:
• the concentration of protein conjugates in supernatant was determined by UV-Vis Nanodrop instrument using extinction coefficient 1.41 mL * mg-lcm-l.
• concentration, volume, and yield of the final conjugates from certain preparations are summarized in the table below.
• MS Waters Xevo G2-QTOF Processing Software: MassLynx Mobile A: water + 0.1% formic acid Mobile B: acetonitrile + 0.1% formic acid
• a total ion chromatogram (TIC) is obtained.
• a TIC region is selected for spectral analysis. The region is selected to encompass both conjugated and unconjugated Fc with no bias.
• a MS profile is obtained from the TIC.
• a charge state envelope is selected for deconvolution. The raw spectrum is deconvoluted using MaxEntl deconvolution tool to obtain the zero charge spectrum.
• BAR Binder distribution ratio in Fc x Fc per antibody
• UPLC analytical method allows simultaneous determination of species with different DARs and MATE-reagent related impurities.
• Drug substance/ Drug Product (DR/DP) and DAR/DAR distribution is calculated based on individual DAR component HPLC/UV280 nm signal area.
• MATE-reagent related DS/DP impurity content (MATE-reagent, MATE-linker and universal antibody binding terminal (uABT)) is determined as a ratio of sample impurity concentration to DS/DP protein concentration (reported as % wt. impurity/wt. protein).
• the identity of UPLC signals was confirmed by protein and individual impurity RS and LCMS analysis.
• a representative UPLC/UV280 Trace is shown for compound 1-36 in FIG. 1.
• DS is the desired product (MATE), and the impurity is expressed as a weight % of that product (DS/DP).
• the UPLC method is performed with a WATERS ACQUITY UPLC system, UV 280 nm detection.
• the MS detector is a WATERS XEVO G2-XS QTof detector. This detector is used for species molecular weight determination.
• the autosampler functions at ambient conditions, 22-23 °C.
• a HALO 1000 A Diphenyl column, 2.7 pm, 2.1 x 50 mm with a column temperature of 80 °C is used.
• the flow rate is 0.5 mL/ min.
• Mobile phase A is 0.5% trifluoroacetic acid; mobile phase B is acetonitrile with 0.05% trifluoroacetic acid.
• the DS/DP sample is diluted to a protein concentration of approximately 1.5 mg/mL, corresponding to a 50-fold DS/DP dilution in a DMSO: water dilution vehicle, 6:94 v/v.
• diluted sample can be stored before analysis at 2-8°C for 24 hours.
• a pooled standard stock solution (PL STOCK) is prepared as follows. Individual uABT, MATE-Reagent (MR) and MATE-Linker standard (STD) stock solutions are prepared in DMSO at STD stock solution concentrations of 2.00 mg/mL. Equal volumes of individual 1 mg/mL DMSO stock solutions are combined. Resulting individual pooled stock solution concentration is 0.667 mg/mL (PL Stock). Store DMSO standard stock solutions at -70oC for up to one month.
• a calibration curve having an individual impurity concentration range 0.004 mg/mL to 0.04 mg/mL (or 0.02 ug/injection to 0.2 ug/injection for 5-uL injection volume) is needed to quantitate most impurities formed during the MATE reaction.
• DMSO solutions are thawed at ambient temperature, and then vortex thoroughly.
• IVIG working stock (82.5 mg/ mL) is prepared by 2-fold dilution of 165 mg/ mL IVIG reference material with water.
• Calibration curve standards are according to Table 5.
• 6% DMSO concentration and constant IVIG Pooled Impurity ratio (w/w) is maintained for all calibrators.
• Calibrator solutions are stored at ambient temperature. Calibration solutions have limited stability; therefore, analysis should be performed within 4 hours after calibrator preparation.
• the calibration curve was found to be reproducible for individual and pooled impurities.
• Calibration curve data for uABT, MATE-Linker, and MATE-Reagent samples are shown in FIG. 3.
• the calibration curve calculators for uABT, MATE-Linker, and MATE-Reagent for 1-36/ 1-20 were extracted from the calibration curves and are presented in Table 7.

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