EP4637835A1 - Procédés de préparation de conjugués anticorps-polynucléotide - Google Patents

Procédés de préparation de conjugués anticorps-polynucléotide

Info

Publication number
EP4637835A1
EP4637835A1 EP23906194.8A EP23906194A EP4637835A1 EP 4637835 A1 EP4637835 A1 EP 4637835A1 EP 23906194 A EP23906194 A EP 23906194A EP 4637835 A1 EP4637835 A1 EP 4637835A1
Authority
EP
European Patent Office
Prior art keywords
antibody
polynucleotide
peg
mal
bis
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
EP23906194.8A
Other languages
German (de)
English (en)
Inventor
Shalom Goldberg
Rebecca WISSNER
JR. Michael MCCAULEY
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Janssen Pharmaceutica NV
Original Assignee
Janssen Pharmaceutica NV
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Janssen Pharmaceutica NV filed Critical Janssen Pharmaceutica NV
Publication of EP4637835A1 publication Critical patent/EP4637835A1/fr
Pending legal-status Critical Current

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    • 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
    • A61K47/6807—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates the drug or compound being a sugar, nucleoside, nucleotide, nucleic acid, e.g. RNA antisense
    • 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
    • A61K47/6835—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
    • A61K47/6851—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 determinant of a tumour cell
    • A61K47/6855—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 determinant of a tumour cell the tumour determinant being from breast cancer cell
    • 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
    • A61K47/6851—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 determinant of a tumour cell
    • A61K47/6869—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 determinant of a tumour cell the tumour determinant being from a cell of the reproductive system: ovaria, uterus, testes, prostate
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
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    • 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
    • A61K47/6875—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 being a hybrid immunoglobulin
    • A61K47/6879—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 being a hybrid immunoglobulin the immunoglobulin having two or more different antigen-binding sites, e.g. bispecific or multispecific immunoglobulin
    • 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
    • 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
    • C07K16/28—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/30—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells
    • C07K16/3069—Reproductive system, e.g. ovaria, uterus, testes, prostate
    • 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
    • C07K16/32—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against translation products of oncogenes
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/111—General methods applicable to biologically active non-coding nucleic acids
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/52—Constant or Fc region; Isotype
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
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    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/94—Stability, e.g. half-life, pH, temperature or enzyme-resistance
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/14—Type of nucleic acid interfering nucleic acids [NA]
    • C—CHEMISTRY; METALLURGY
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    • C12N2310/32—Chemical structure of the sugar
    • C12N2310/323—Chemical structure of the sugar modified ring structure
    • C12N2310/3233—Morpholino-type ring
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2310/00—Structure or type of the nucleic acid
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    • C12N2310/35—Nature of the modification
    • C12N2310/351—Conjugate
    • C12N2310/3513—Protein; Peptide

Definitions

  • Exogenous polynucleotides can exert diverse effects on cellular physiology which can be utilized for a variety of purposes.
  • delivery of polynucleotides has emerged as a promising therapeutic strategy for the treatment of various diseases.
  • polynucleotides are typically too large, hydrophilic, and negatively charged to spontaneously cross the cell membrane.
  • polynucleotides may be susceptible to degradation by extracellular and intracellular nucleases.
  • Short interfering RNAs are a class of double-stranded nucleotides that can drive the sequence-specific degradation of target mRNAs by engaging the RNA-induced silencing complex (RISC). These molecules typically suffer from being too large, hydrophilic, and negatively charged to spontaneously cross the cell membrane. Further, unmodified siRNAs are also susceptible to degradation.
  • RISC RNA-induced silencing complex
  • an antibody-polynucleotide conjugate comprising contacting an antibody comprising two reactive bioorthogonal groups, and a polynucleotide comprising a bifurcated linker that comprises two reactive groups, wherein the reactive groups on the bifurcated linker selectively interact with the biorthogonal groups to form a conjugate having an antibody-polynucleotide ratio that is about 1 : 1.
  • an antibody-polynucleotide conjugate comprising contacting an antibody and a polynucleotide, wherein each antibody heavy chain constant region comprises an azide group, and the polynucleotide comprises a bifurcated linker that comprises two alkyne groups, wherein each azide group reacts with an alkyne group to form a conjugate having an antibody-polynucleotide ratio that is about 1 : 1.
  • compositions comprising a Mal-PEG(X)-bis-PEG(X)-BCN or a Mal-PEG(X)-bis-PEG(X)-DBCO conjugated to a polynucleotide.
  • polynucleotide-conjugated antibodies comprising a polynucleotide attached to a bifurcated linker, and an antibody comprising two heavy chain constant regions that each comprise a bioorthogonal group, wherein the bifurcated linker is attached to the bioorthogonal groups of each heavy chain constant region.
  • an antibody-polynucleotide conjugate comprising contacting an antibody comprising a F405L substitution and a R409K substitution on one heavy chain constant region and an azide group on the other heavy chain constant region, and a polynucleotide to form a conjugate having an antibody-polynucleotide ratio that is about 1 : 1.
  • Disclosed herein are methods of promoting Fab arm exchange comprising contacting a first antibody comprising a F405L substitution and a R409K substitution, and a second antibody that does not have the F405L substitution and the R409K substitution and that comprises an azide group on one or both of the antibody heavy chain constant regions to form an antibody comprising the F405L substitution and the R409K substitution on one heavy chain constant region and the azide group on the other heavy chain constant region.
  • FIG. 1 illustrates an exemplary scheme depicting the generation of 1 : 1 mAb- polynucleotide conjugates using an siRNA as an exemplary polynucleotide.
  • FIG. 2 illustrates an exemplary synthesis reaction for generating a Mal-bis-BCN- polynucleotide.
  • a Mal-bis-BCN-polynucleotideCTNNBl siRNA was generated.
  • FIG. 3 illustrates an SDS-PAGE analysis of reduced PSMB127-siRNA conjugates.
  • FIG. 4 illustrates an SDS-PAGE analysis of reduced Trastuzumab-siRNA conjugates.
  • FIG. 5A and FIG. 5B illustrate analytical SEC chromatograms (280 nm absorbance trace) corresponding to 1 : 1 PSMB127-siRNA conjugates generated by controlled Fab arm exchange (cFAE) (FIG. 5A) and by the stitching method using azido-modified antibodies and Mal-bis-BCN siRNAs (FIG. 5B).
  • cFAE controlled Fab arm exchange
  • FIG. 6A, FIG. 6B, FIG. 6C, and FIG. 6D illustrate analytical SEC chromatograms (280 nm absorbance trace) corresponding to 1 : 1 Trastuzumab-siRNA conjugates generated by chromatographic isolation (FIG. 6A and FIG. 6C) and by the stitching method using azidomodified antibodies and Mal-bis-BCN siRNAs (FIG. 6B and FIG. 6D).
  • FIG. 7 illustrates CTNNB1 mRNA knockdown following treatment of PSMA- expressing HEK-293T cells with PSMB127 antibody :CTNNB1 -siRNA conjugates.
  • FIG. 8 illustrates CTNNB1 mRNA knockdown following treatment of HCC1954 cells with Trastuzumab:CTNNBl-siRNA conjugates.
  • FIG. 9 illustrates the pharmacokinetics of Trastuzumab-siRNA conjugates in C57BL/6 mice over time.
  • conjugated polynucleotide molecules e.g., siRNA conjugated molecules
  • polynucleotide-conjugated antibodies may be understood more readily by reference to the following detailed description taken in connection with the accompanying figures, which form a part of this disclosure.
  • the disclosed methods and compositions are not limited to the specific methods and compositions described and/or shown herein, and the terminology used herein is for the purpose of describing particular embodiments by way of example only and is not intended to be limiting of the claimed methods and compositions.
  • any description as to a possible mechanism or mode of action or reason for improvement is meant to be illustrative only, and the disclosed methods and compositions are not to be constrained by the correctness or incorrectness of any such suggested mechanism or mode of action or reason for improvement.
  • compositions and methods of using said compositions refer to compositions and methods of using said compositions.
  • a feature or embodiment associated with a composition such a feature or embodiment is equally applicable to the methods of using said composition.
  • a feature or embodiment associated with a method of using a composition such a feature or embodiment is equally applicable to the composition.
  • administering and similar terms indicate a procedure by which an antibody -polynucleotide conjugate is exposed to target cells in vitro, or is injected into a subject such that target cells, tissues, or segments of the body of the subject are contacted with the antib ody-poly nucl eoti de conj ugate .
  • subject as used herein is intended to mean any animal, in particular, mammals. Although treatment of in mice with an antibody-polynucleotide conjugate is exemplified herein, any type of mammal can be treated using the disclosed methods. Any type of cell or mammal can be treated using the disclosed methods. Thus, the methods are applicable to human and nonhuman animals, although preferably used with mice and humans, and most preferably with humans. “Subject” and “patient” are used interchangeably herein. The subjects herein can be a subject that is in need thereof.
  • antibody and like terms is meant in a broad sense and includes immunoglobulin molecules including, monoclonal antibodies, antibody fragments, bispecific or multispecific antibodies, dimeric, tetrameric or multimeric antibodies, and single chain antibodies.
  • Immunoglobulins can be assigned to five major classes, namely IgA, IgD, IgE, IgG, and IgM, depending on the heavy chain constant domain amino acid sequence.
  • IgA and IgG are further sub-classified as the isotypes IgAl, IgA2, IgGl, IgG2, IgG3, and IgG4.
  • Antibody light chains of any vertebrate species can be assigned to one of two clearly distinct types, namely kappa (K) and lambda (X), based on the amino acid sequences of their constant domains.
  • Stitched refers to antibody (Ab):polynucleotide conjugates, such as an Ab: siRNA conjugate, wherein the Ab has attached thereto a bifurcated linker, such that the bifurcated linker is attached to two different protein chains of the Ab via a biorthogonal group (such as one end of the bifurcated linker being attached to a biorthogonal group on one heavy chain constant region and the other end of the bifurcated linker being attached to a biorthogonal group on the other heavy chain constant region) and whereby the bifurcated linker is attached to a polynucleotide or payload, such as an siRNA.
  • a biorthogonal group such as one end of the bifurcated linker being attached to a biorthogonal group on one heavy chain constant region and the other end of the bifurcated linker being attached to a biorthogonal group on the other heavy chain constant region
  • mAbs a monoclonal antibody
  • mAbs exhibit long serum half-lives and selectively bind their target antigens with high affinity, rendering cell-type specific targeting possible.
  • mAbs that target internalizing cell-surface receptors can be further exploited to deliver conjugated payloads, including polynucleotides such as siRNAs, through the endo/lysosomal pathway.
  • siRNAs for example, to exert its gene silencing effect, it must ultimately be delivered to the cytosol to engage the RISC complex.
  • the methods can comprise contacting an antibody comprising two reactive bioorthogonal groups and an polynucleotide comprising a bifurcated linker that comprises two reactive groups wherein the reactive groups on the bifurcated linker selectively interact with the biorthogonal groups to form a conjugate having an antibody-polynucleotide ratio that is about 1 : 1.
  • Bioorthogonal groups can be chemical moieties that take part in chemical reactions that are selective enough to proceed in complex biological environments without being impacted by the presence of amino acids, nucleic acids, etc.
  • the methods can comprise contacting an antibody and polynucleotide, wherein each antibody heavy chain constant region comprises an azide group, and the polynucleotide comprises a bifurcated linker that comprises two alkyne groups, wherein each azide groups reacts with an alkyne group to form a conjugate having an antibody-polynucleotide ratio that is about 1 : 1.
  • the polynucleotide conjugated to the antibody can be DNA or RNA, or analogs thereof.
  • the DNA or the RNA is a phosphorodiamidate morpholino oligomer (PMO).
  • PMO phosphorodiamidate morpholino oligomer
  • Suitable RNAs include siRNA, miRNA, gRNA, shRNA, or an antisense oligonucleotide.
  • the polynucleotide may be coding or noncoding.
  • the polynucleotide may comprise DNA or RNA nucleotide modifications, such as methylation.
  • the polynucleotide may be single stranded or double stranded.
  • the polynucleotide may be linear or circular.
  • the polynucleotide may be in various secondary or tertiary conformations due to internal base pairing.
  • the reactive biorthogonal groups can be located within the antibody’s heavy chain constant region.
  • one antibody heavy chain constant region comprises one reactive biorthogonal group and the other antibody heavy chain constant region comprises the other reactive biorthogonal group.
  • Each of the two reactive biorthogonal groups can comprise an azide group.
  • Suitable azide groups include, but are not limited to, 3 -azidopropylamine, N- azidoacetylgalactosamine, 6-azido GalNAc, azido-butylamine, azidepentylamine, and azide- PEG3-amine.
  • at least one of the azide groups can be 3 -azidopropylamine.
  • At least one of the azide groups can be N-azidoacetylgalactosamine. In some embodiments, at least one of the azide groups can be 6-azido GalNAc. In some embodiments, at least one of the azide groups can be azido-butylamine. In some embodiments, at least one of the azide groups can be azidepentylamine. In some embodiments, at least one of the azide groups can be azide-PEG3 -amine.
  • Each antibody heavy chain can comprise the same bioorthogonal group. In some embodiments, each antibody heavy chain can comprise 3- azidopropylamine. In some embodiments, each antibody heavy chain can comprise N- azidoacetylgalactosamine.
  • each antibody heavy chain can comprise 6- azido GalNAc. In some embodiments, each antibody heavy chain can comprise azido- butylamine. In some embodiments, each antibody heavy chain can comprise azidepentylamine. In some embodiments, each antibody heavy chain can comprise azide-PEG3 -amine. Alternatively, each antibody heavy chain can comprise a different bioorthogonal group. For example, one antibody heavy chain can comprise 3 -azidopropylamine and one antibody heavy chain can comprise N-azidoacetylgalactosamine.
  • the CH2 region of the antibody can bear the reactive bioorthogonal group.
  • Suitable antibody heavy chain positions for the bioorthogonal group include Q295 and N297.
  • Suitable azides at position Q295 include 3 -azidopropylamine, azido-butylamine, azidepentylamine, and azide-PEG3 -amine.
  • the antibody has 3 -azidopropylamine at amino acid position Q295.
  • One or both Q295 positions can bear 3 -azidopropylamine.
  • Suitable azides at position N297 include N-azidoacetylgalactosamine and 6-azido GalNAc.
  • the antibody has N-azidoacetylgalactosamine at amino acid position N297.
  • One or both N297 positions can bear N-azidoacetylgalactosamine.
  • Suitable bifurcated linkers include those capable of reacting with the bioorthogonal group present on the antibody heavy chains.
  • the bifurcated linker can comprise alkyne reactive groups.
  • the alkyne group can comprise a cyclooctyne.
  • the bifurcated linker can include two reactive groups, wherein each group reacts with a bioorthogonal group on the antibody heavy chain.
  • the bifurcated linker can be a Mal-PEG(X)-bis-PEG(X)-BCN linker or a Mal-PEG(X)- bis-PEG(X)-DBCO linker, wherein (X) represents the number of PEG units.
  • the number of PEG units in the general formulas Mal-PEG(X)-bis-PEG(X)-BCN linker and Mal-PEG(X)-bis- PEG(X)-DBCO can be about 1 to 10.
  • the bifurcated linker is a Mal- PEG2-bis-PEG3-BCN.
  • compositions comprising a Mal-PEG(X)-bis-PEG(X)-BCN or a Mal-PEG(X)-bis-PEG(X)-DBCO conjugated to a polynucleotide, wherein (X) represents the number of PEG units.
  • the number of PEG units in the general formulas Mal-PEG(X)-bis- PEG(X)-BCN linker and Mal-PEG(X)-bis-PEG(X)-DBCO can be about 1 to 10.
  • Mal-PEG(X)-bis-PEG(X)-BCN is conjugated to the polynucleotide.
  • Mal-PEG(X)-bis-PEG(X)-DBCO is conjugated to the polynucleotide.
  • the bifurcated linker is a Mal-PEG2-bis-PEG3-BCN.
  • the Mal-PEG(X)-bis- PEG(X)-BCN or Mal-PEG(X)-bis-PEG(X)-DBCO can be attached to the polynucleotide by a dihexyldisulfide linker.
  • the Mal-PEG(X)-bis-PEG(X)-BCN or Mal-PEG(X)-bis-PEG(X)-DBCO can be attached to the 5’ end of the polynucleotide.
  • the Mal-PEG(X)-bis- PEG(X)-BCN or Mal-PEG(X)-bis-PEG(X)-DBCO is attached to the 5’ end of the sense strand of the polynucleotide.
  • the Mal-PEG2-bis-PEG3-BCN conjugated polynucleotide molecule can be reacted with an antibody that comprises bioorthogonal groups to form a polynucleotide- conjugated antibody as described herein.
  • the polynucleotide can be DNA or RNA, or analogs thereof.
  • the DNA or the RNA is a PMO.
  • Suitable RNAs include siRNA, miRNA, gRNA, shRNA, or an antisense oligonucleotide.
  • the polynucleotide may be coding or noncoding.
  • the polynucleotide may comprise DNA or RNA nucleotide modifications, such as methylation.
  • the polynucleotide may be single stranded or double stranded.
  • the polynucleotide may be linear or circular.
  • the polynucleotide may be in various secondary or tertiary conformations due to internal base pairing.
  • polynucleotide-conjugated antibodies comprising a polynucleotide attached to a bifurcated linker and an antibody comprising two heavy chain constant regions that each comprise a bioorthogonal group, wherein the bifurcated linker is attached with the bioorthogonal group of each heavy chain constant region.
  • the bifurcated linker can be a Mal- PEG(X)-bis-PEG(X)-BCN or a Mal-PEG(X)-bis-PEG(X)-DBCO, wherein (X) represents the number of PEG units.
  • the number of PEG units in the general formulas Mal-PEG(X)-bis- PEG(X)-BCN linker and Mal-PEG(X)-bis-PEG(X)-DBCO can be about 1 to 10.
  • the bifurcated linker can be Mal-PEG2-bis-PEG3-BCN.
  • the polynucleotide can be conjugated to the antibody by a triazole.
  • the polynucleotide-conjugated antibody can be generated by the methods described herein.
  • the polynucleotide conjugated to the antibody can be DNA or RNA, or analogs thereof.
  • the DNA or the RNA is a PMO.
  • Suitable RNAs include siRNA, miRNA, gRNA, shRNA, or an antisense oligonucleotide.
  • the polynucleotide may be coding or noncoding.
  • the polynucleotide may comprise DNA or RNA nucleotide modifications, such as methylation.
  • the polynucleotide may be single stranded or double stranded.
  • the polynucleotide may be linear or circular.
  • the polynucleotide may be in various secondary or tertiary conformations due to internal base pairing.
  • Any of the herein described polynucleotides can be delivered to a target via the described polynucleotide- conjugated antibodies.
  • Polynucleotides can be delivered for a wide variety of reasons such as genomic modification, introduction of exogenous polynucleotides for use by the cellular machinery, or induction of cellular responses that are sensitive to the delivered polynucleotide.
  • Suitable polynucleotides delivered for genomic modification include, but are not limited to, gRNAs and insertion cassettes.
  • Suitable polynucleotides delivered for use by the cellular machinery include but are not limited to, mRNA, tRNA, miRNA, or coding DNA.
  • Suitable polynucleotides delivered for induction of cellular responses, such as the immune response include single stranded RNA, double stranded RNA, and CpG containing DNA.
  • RNA silencing or reducing gene expression comprising administering any of the herein disclosed polynucleotide-conjugated antibodies.
  • Polynucleotides capable of promoting gene silencing or reducing gene expression include those that reduce the transcription of translation of mRNAs. Coding or noncoding RNAs can be targeting for gene silencing or a reduction in gene expression.
  • the RNA to be silenced is an mRNA.
  • Suitable polynucleotides administered for gene silencing or reducing gene expression include, but are not limited to, siRNA, shRNA, and PMO polynucleotides.
  • the polynucleotide-conjugated antibody can be administered to a subject in need thereof.
  • the subject can be a human subject or a non-human subject.
  • the subject can be a subject in need thereof.
  • the polynucleotide-conjugated antibody can be administered to cells in vitro.
  • an antibody-polynucleotide conjugate comprising contacting an antibody comprising a F405L substitution and a R409K substitution, according to EU numbering index, on one heavy chain constant region and an azide group on the other heavy chain constant region and a polynucleotide to form a conjugate having an antibody-polynucleotide ratio that is about 1 : 1.
  • the methods comprise, prior to contacting the antibody and the polynucleotide, contacting an antibody comprising a F405L substitution and an R409K substitution, according to EU numbering index, and an antibody that does not have the F405L substitution and the R409K substitution and that comprises an azide group on one or both of the antibody heavy chain constant regions to promote Fab arm exchange and the formation of an antibody comprising the F405L substitution and the R409K substitution on one heavy chain constant region and the azide group on the other heavy chain constant region.
  • Disclosed herein are methods of promoting Fab arm exchange comprising contacting a first antibody comprising a F405L substitution and a R409K substitution, according to EU numbering index, and a second antibody that does not have the F405L substitution and the R409K substitution and that comprises an azide group on one or both of the antibody heavy chain constant regions, to form an antibody comprising the F405L substitution and the R409K substitution on one heavy chain constant region and the azide group on the other heavy chain constant region.
  • Fab arm exchange refers to the process by which half antibodies (a light chain and heavy chain pair) associate to form intact antibodies (two light chain and heavy chain pairs).
  • the Fab arm exchange reaction is the result of a disulfide-bond isomerization reaction and dissociation-association of CH3 domains.
  • the heavychain disulfide bonds in the hinge regions of the antibodies are reduced, and the resulting free cysteines of the antibody can form an inter heavy-chain disulfide bond with cysteine residues of another antibody.
  • CH3 domains of the antibodies simultaneously release and reform by dissociation-association.
  • Fab arm exchange can be used to generate heterodimers (such as bispecific antibodies) by introducing amino acid substitutions (such as a F405L substitution and an R409K substitution) into the heavy chain CH3 domains of mono-specific bivalent antibodies to thereby favor formation of the heterodimer.
  • amino acid substitutions such as a F405L substitution and an R409K substitution
  • antibody halves containing the amino acid substitutions will preferably associate with antibody halves not having the amino acid substitutions to thereby drive formation of the heterodimer.
  • Fab arm exchange is described in, for example, U.S. Pub. No. US20220267438.
  • the antibody that does not have the F405L substitution and the R409K substitution comprises an azide group on one of the heavy chain constant regions.
  • the antibody that does not have the F405L substitution and the R409K substitution comprises azide groups on both of the heavy chain constant regions.
  • the CH2 region of the antibody can bear the azide group.
  • Suitable azide groups on one or both of the antibody heavy chain constant regions include, but are not limited to, 3 -azidopropylamine, N- azidoacetylgalactosamine, 6-azido GalNAc, azido-butylamine, azidepentylamine, and azide- PEG3-amine.
  • Suitable antibody positions for the azide group include Q295 and N297.
  • Suitable azides at position Q295 include 3 -azidopropylamine, azido-butylamine, azidepentylamine, and azide-PEG3 -amine.
  • the antibody has 3 -azidopropylamine at amino acid position Q295.
  • One or both Q295 positions can bear 3 -azidopropylamine.
  • Suitable azides at position N297 include N-azidoacetylgalactosamine and 6-azido GalNAc.
  • the antibody has N-azidoacetylgalactosamine at amino acid position N297.
  • One or both N297 positions can bear N-azidoacetylgalactosamine.
  • Polynucleotides suitable for contact with the antibody comprising the F405L substitution and the R409K substitution on one heavy chain constant region and the azide group on the other heavy chain constant region include those capable of reacting with the azide group.
  • the polynucleotide can comprise one or more reactive groups capable of reacting with the azide group.
  • the polynucleotide can comprise one or more alkyne reactive groups capable of reacting with the azide.
  • the alkyne can comprise a cyclooctyne.
  • the alkene reactive group is dibenzocyclooctyne (DBCO).
  • the antibody comprising the F405L substitution and the R409K substitution can be an IgG4 antibody.
  • the antibody that does not have the F405L substitution and the R409K substitution comprising the azide group(s) can be an IgG4.
  • both the antibody comprising the F405L substitution and the R409K substitution and the antibody that does not have the F405L substitution and the R409K substitution comprising the azide group(s) can be an IgG4.
  • the antibody comprising the F405L substitution and the R409K substitution can be monospecific, bispecific, or multispecific.
  • the antibody that does not have the F405L substitution and the R409K substitution comprising the azide group(s) can be monospecific, bispecific, or multispecific. In some embodiments the antibody comprising the F405L substitution and the R409K substitution binds to the same antigen as the antibody that does not have the F405L substitution and the R409K substitution comprising the azide group(s). In some embodiments, the antibody comprising the F405L substitution and the R409K substitution binds to a different antigen as the antibody that does not have the F405L substitution and the R409K substitution comprising the azide group(s).
  • the antibody comprising the F405L substitution and the R409K substitution on one heavy chain constant region and the azide group on the other heavy chain constant region can be monospecific, bispecific, or multispecific.
  • the antibody-polynucleotide conjugate described throughout has similar or improved biophysical stability profiles compared to non-conjugated antibodies and/or polynucleotides. For example, thermal stability of the antibody-polynucleotide conjugate is retained compared to the parental antibody and synthetic intermediates such as apa and GlaNAz. Differential scanning fluorometry can be used to analyze biophysical stability of the conjugates. Further analysis of the antibody-polynucleotide conjugates (by e.g., analytical size exclusion chromatography) demonstrates that the conjugates are predominantly monomeric and intact.
  • the antibody-polynucleotide conjugates also induce efficient gene silencing and is at least equivalent or better than an antibody-polynucleotide conjugates generated by other methods.
  • other methods of generating antibody-polynucleotide conjugates include chromatographic isolation or Fab-arm exchange.
  • the antibody-polynucleotide conjugate is produced by the methods described throughout can lead to an overall yield of greater than other known methods such as chromatographic isolation.
  • chromatographic isolation typically produces antibody- polynucleotide conjugate yields at around 30% at best.
  • the methods described throughout produce antibody-polynucleotide conjugates at an overall yield of greater than about 50%.
  • the yield can be between about 50% to about 100%.
  • the yield can be between about 55% to about 85%.
  • the yield can be between about 55% to about 75%.
  • the yield can be between about 55% to about 70%.
  • the antibody-polynucleotide conjugates produced by the methods described throughout have an improved PK profile (e.g., plasma concentration) compared to an antibody- polynucleotide conjugate that has a 1 :2 ratio (even when produced by the same methods described throughout).
  • PK profile e.g., plasma concentration
  • the method described throughout ultimately leads to an easier and more efficient method of producing antibody-polynucleotide conjugates.
  • Rapid PNGase F was added to each mAb (5 pL/mg mAb) and the reaction was incubated overnight at 37 °C.
  • the deglycosylated mAbs (1 mg/mL) were reacted with 100 equivalents of 3-azidopropylamine (3- apa) and MTG (Activa TI, 20% w/v) in Dulbecco’s phosphate buffered saline (DPBS) for 4-6 hours at 37 °C. Reaction progress was monitored by mass spectrometry.
  • the apa-modified mAbs were purified by protein A affinity chromatography and analyzed by mass spectrometry and analytical size-exclusion chromatography. 3-azidopropylamine was conjugated to amino acid position Q295 using this procedure.
  • mAb Fc glycans Site-specific azido-modification of mAb Fc glycans.
  • mAbs PSMB127 and Trastuzumab
  • EndoS2 A0-GL8-020, GlycINATOR LE, Genovis
  • GlycNAc N-Acetylglucosamine residues of the Fc glycans, leaving the innermost GlcNAc resides intact (one per heavy chain).
  • GlycINATOR LE was added to each mAb (1-5 pL/mg mAb) and incubated overnight at 37 °C.
  • the core GlcNAc residues were subsequently conjugated to N-azidoacetylgalactosamine (GalNAz) using components from the SiteClickTM antibody azido modification system kit (SI 0901, Thermo Fisher Scientific). Briefly, the deglycosylated mAbs were exchanged into tris buffered saline (TBS, pH 7.4) and concentrated to 6 mgs/mL. The mAbs were then reacted with the provided GalT enzyme (60 pL/mg mAb) in the presence of UDP-GalNAz (2.5 mM) overnight at 30 °C. Reaction progress was monitored by mass spectrometry.
  • GalNAz- modified mAbs were purified by diafiltration and analyzed by mass spectrometry and analytical size exclusion chromatography. N-azidoacetylgalactosamine (GalNAz) was conjugated to amino acid position N297 using this procedure.
  • the deprotected siRNA was exchanged into 20 mM Tris, pH 8.0 by diafiltration and subsequently reacted with 10 equivalents of Mal-PEG2-bis-PEG3-BCN (“Mal-bis-BCN”) (CP- 2097, Conju-Probe) to yield the Mal-bis-BCN-CTNNBl siRNA. If the succinimide moiety of a maleimide-thiol conjugate is hydrolyzed, the ring-opened product is stabilized toward cleavage in vivo as it is no longer susceptible to thiol exchange reactions.
  • Mal-bis-BCN Mal-bis-BCN
  • the reaction pH was adjusted to 9.0 by adding sodium bicarbonate to a final concentration of 100 mM to drive the formation of the ring-opened product (FIG. 2).
  • the reaction was incubated overnight at 4 °C and the extent of maleimide ring-opening was monitored by mass spectrometry.
  • the Mal-bis-BCN-CTNNBl siRNA was purified by anion exchange chromatography, dialyzed into DPBS, and stored at -20 °C until further use.
  • the bifurcated linker (Mal-bis-BCN) was conjugated to the siRNA at the 5’ end at an inverted abasic residue (i.e. at the 5’ OH of the abasic group). The modification occurred on the sense strand.
  • the mAb-siRNA conjugates were purified by size exclusion chromatography or anion-exchange chromatography over a TSK-gelTM BioAssistTM Q column.
  • 1 mAb-siRNA conjugates PSMB127-GalNAz-siRNA stitched DOL 1, Trastuzumab-GalNAz-siRNA stitched DOL 1, and Trastuzumab-apa-siRNA stitched DOL 1 were analyzed by reduced SDS-PAGE and analytical size exclusion chromatography.
  • mAb-CTNNBl siRNA conjugates Preparation of 1:2 mAb-CTNNBl siRNA conjugates. 1 :2 mAb-siRNA conjugates were prepared using strain-promoted azide-alkyne cycloaddition chemistry (SPAAC). Azidomodified mAbs (apa or GalNAz as described above) were diluted to 1 mg/mL in DPBS and reacted with 5 equivalents of a chemically stabilized dibenzocyclooctyne (DBCO)-modified siRNA targeting CTNNB1 (DBCO-CTNNB1 siRNA) overnight at 37 °C.
  • DBCO dibenzocyclooctyne
  • the mAb-siRNA conjugates (PSMB127-apa-siRNA degree of labeling (DOL) of 2, Trastuzumab-apa-siRNA DOL 2, and Trastuzumab -GalNAz-siRNA DOL 2) were purified by size exclusion chromatography and analyzed by reduced SDS-PAGE and analytical size exclusion chromatography.
  • PSMB127-apa (1 mg/mL) was combined with PSMB127 F405L/R409K (1 mg/mL) in the presence of 50 mM cysteamine. The reaction was incubated overnight at 37 °C. After the fab-arm exchange reaction was complete, the resultant bispecific mAb (PSMB127-apa DOL 1) was exchanged into DPBS using a desalting column and allowed to re-oxidize at 4 °C for 2 days. Next, PSMB127-apa DOL 1 was reacted with 5 equivalents of a DBCO-CTNNBl siRNA overnight at 37 °C. The 1 : 1 mAb-siRNA conjugate (PSMB127-apa-siRNA cFAE DOL 1) was purified by size exclusion chromatography and analyzed by reduced SDS-PAGE and analytical size exclusion chromatography.
  • Trastuzumab-siRNA conjugate (Trastuzumab- GalNAz-siRNA DOL 1) were combined and exchanged into DPBS using a desalting column.
  • Trastuzumab-GalNAz-siRNA DOL 1 was analyzed by reduced SDS-PAGE and analytical size exclusion chromatography.
  • Trastuzumab-apa-siRNA DOL 1 was prepared in a similar manner as described above, except only 1 equivalent of DBC0-CTNNB1 siRNA was used to generate the crude reaction mixture.
  • the obtained yields of the 1 : 1 mAb-siRNA conjugates (from each respective azido-mAb intermediate) were approximately 30% in each case.
  • PSMB127-siRNA conjugates were characterized more fully to assess the impact of the siRNA conjugation strategy on the thermal stability of the mAb.
  • Melting temperature (T m ) was determined by differential scanning fluorimetry using a nanoDSF instrument from Nanotemper. T m was determined by monitoring changes in fluorescence intensity at 330 and 350 nm upon thermal scanning from 20 to 95°C. While a lower T O nset (start of unfolding) was observed for the modified PSMB127 variants, the T m was not significantly impacted by conjugation to siRNA.
  • T m of both 1 : 1 mAb-siRNA conjugates generated by the herein disclosed methods or by cFAE were similar, demonstrating that the disclosed methods do not grossly impact the thermal stability the parental mAb.
  • PSMB127 and Trastuzumab-siRNA conjugates were assessed for their ability to mediate knockdown of CTNNB1 mRNA.
  • PSMB127-siRNA conjugates were evaluated in stably transfected prostate-specific membrane antigen (PSMA)-expressing HEK-293T cells and Trastuzumab-siRNA conjugates were evaluated in HCC1954 cells. Cells were treated with siRNA conjugates for 72 hours in the absence of transfection reagent. The extent of mRNA knockdown was determined using real-time quantitative PCR (RT-qPCR) (FIG. 7 and FIG. 8).
  • the 1 : 1 Trastuzumab-siRNA conjugates exhibited significantly higher plasma concentrations at each time point compared to the 1 :2 Trastuzumab-siRNA conjugates (Table 2).
  • the total in vivo exposures observed for the 1 : 1 GalNAz-siRNA conjugates were significantly higher ( ⁇ 2-4 fold) than those observed for the 1 : 1 apa-siRNA conjugates.
  • 1 : 1 mAb-siRNA conjugates generated by the disclosed methods retained favorable biophysical stability profiles and induced efficient gene silencing equivalent to 1 : 1 mAb-siRNA conjugates generated by other methods, such as chromatographic isolation. Furthermore, pharmacokinetic (PK) studies in mice revealed that the in vivo exposures of the mAb-siRNA conjugates generated by the disclosed methods are comparable to those 1 : 1 mAb-siRNA conjugates generated by other methods and are substantially improved relative to analogous 1:2 mAb-siRNA controls.
  • PK pharmacokinetic
  • the disclosed methods of generating 1 : 1 mAb-siRNA conjugates provides significant improvements in synthetic strategy and yield while maintaining the potency of 1 : 1 mAb-siRNA conjugates prepared by other methods, and dramatically improves circulation pharmacokinetics compared to 1 :2 mAb-siRNA conjugates.

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Abstract

L'invention concerne des procédés de formation d'un conjugué anticorps-polynucléotide ayant un rapport anticorps-polynucléotide qui est d'environ 1:1 et des compositions de celui-ci. Ces procédés permettent la génération efficace de conjugués anticorps-polynucléotides avec des stœchiométries précises. En outre, ces conjugués ont des propriétés PK améliorées et sont plus faciles à fabriquer par rapport à d'autres procédés.
EP23906194.8A 2022-12-22 2023-12-14 Procédés de préparation de conjugués anticorps-polynucléotide Pending EP4637835A1 (fr)

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