Method for preparing antibody-polynucleotide conjugates

Abstract

Provided herein are methods of forming antibody-polynucleotide conjugates having an antibody-polynucleotide ratio of about 1: 1 and compositions thereof. These methods allow for efficient production of antibody-polynucleotide conjugates with precise stoichiometry. In addition, these conjugates have improved PK properties and are easier to manufacture compared to other methods.

Description

[0001] Cross-references

[0002] This patent application claims priority to U.S. Provisional Application No. 63 / 434,517, filed on December 22, 2023, and U.S. Provisional Application No. 63 / 434,532, filed on December 22, 2023, the contents of which are incorporated by reference in their entirety.

[0003] Incorporated by reference

[0004] All publications, patents, and patent applications are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. In the event of a conflict between a term in this document and that in an incorporated reference, the term in this document controls. Background Art

[0005] Exogenous polynucleotides can play a variety of roles in cell physiology, and they can be used for multiple purposes. Specifically, the delivery of polynucleotides (such as interfering RNA) has become a promising therapeutic strategy for treating various diseases. However, polynucleotides are usually too large, hydrophilic and negatively charged and cannot spontaneously pass through the cell membrane. In addition, polynucleotides may be susceptible to the degradation of extracellular and intracellular nucleases.

[0006] Short interfering RNA (siRNA) is a type of double-stranded nucleotide that can drive sequence-specific degradation of target mRNA by engaging the RNA-induced silencing complex (RISC). These molecules are typically too large, hydrophilic, and negatively charged to spontaneously cross the cell membrane. In addition, unmodified siRNA is also prone to degradation. Summary of the Invention

[0007] Disclosed herein are methods for forming antibody-polynucleotide conjugates, the method comprising contacting an antibody comprising two reactive bioorthogonal groups with a polynucleotide comprising a bifurcated junction comprising two reactive groups, wherein the reactive groups on the bifurcated junction selectively interact with the bioorthogonal groups to form a conjugate having an antibody-to-polynucleotide ratio of about 1:1.

[0008] Disclosed herein are methods of forming 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 comprising two alkyne groups, wherein each azide group reacts with the alkyne group to form a conjugate at a ratio of about 1:1 antibody to polynucleotide.

[0009] Disclosed herein are compositions comprising Mal-PEG(X)-bis-PEG(X)-BCN or Mal-PEG(X)-bis-PEG(X)-DBCO conjugated to a polynucleotide.

[0010] Disclosed herein are polynucleotide-conjugated antibodies comprising a polynucleotide attached to a bifurcated linker, and an antibody comprising two heavy chain constant regions, each comprising a bioorthogonal group, wherein a bifurcated linker is attached to the bioorthogonal group of each heavy chain constant region.

[0011] Disclosed herein are methods of forming 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 with a polynucleotide to form a conjugate at an antibody-to-polynucleotide ratio of about 1:1.

[0012] Disclosed herein are methods of promoting Fab arm exchange, the method comprising contacting a first antibody comprising a F405L substitution and a R409K substitution with 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 a F405L substitution and a R409K substitution on one heavy chain constant region and an azide group on the other heavy chain constant region. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] The Summary of the Invention and the following Detailed Description will be further understood when read in conjunction with the accompanying drawings. The accompanying drawings show exemplary embodiments of the methods and compositions for purposes of illustrating the disclosed methods, conjugated polynucleotide molecules (such as siRNA molecules), and polynucleotide-conjugated antibodies; however, the methods and compositions are not limited to the specific embodiments disclosed. In the drawings:

[0014] Figure 1 Shown is an exemplary scheme depicting the generation of a 1:1 mAb-polynucleotide conjugate using siRNA as an exemplary polynucleotide.

[0015] Figure 2 An exemplary synthesis reaction for producing Mal-bis-BCN-polynucleotide is shown. In this case, Mal-bis-BCN-polynucleotide CTNNB1 siRNA is produced.

[0016] Figure 3 Shown is SDS-PAGE analysis of reduced PSMB127-siRNA conjugate.

[0017] Figure 4 Shown is SDS-PAGE analysis of reduced trastuzumab-siRNA conjugates.

[0018] Figure 5A and Figure 5B The corresponding Fab arm exchange (cFAE) is shown. Figure 5A ) and by a stitching method using an azide-modified antibody and Mal-bis-BCN siRNA ( Figure 5B ) Analytical SEC chromatogram of the 1:1 PSMB127-siRNA conjugate produced (280 nm absorbance trace).

[0019] Figure 6A 、 Figure 6B 、 Figure 6C and Figure 6D Analytical SEC chromatograms (280 nm absorbance trace) are shown, corresponding to the chromatographic separation ( Figure 6A and Figure 6C ) and a 1:1 trastuzumab-siRNA conjugate produced by a stitching method using an azide-modified antibody and Mal-bis-BCN siRNA ( Figure 6B and Figure 6D ).

[0020] Figure 7 Shown is CTNNB1 mRNA knockdown following treatment of PSMA-expressing HEK-293T cells with PSMB127 antibody:CTNNB1-siRNA conjugate.

[0021] Figure 8 Shown is CTNNB1 mRNA knockdown following treatment of HCC1954 cells with trastuzumab:CTNNB1-siRNA conjugate.

[0022] Figure 9 Shown are the pharmacokinetics of trastuzumab-siRNA conjugates in C57BL / 6 mice over time. DETAILED DESCRIPTION

[0023] The disclosed methods, conjugated polynucleotide molecules (e.g., siRNA-conjugated molecules), and polynucleotide-conjugated antibodies may be more readily understood by reference to the following detailed description taken in conjunction with the accompanying drawings, 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 merely for the purpose of describing particular embodiments by way of example and is not intended to limit the claimed methods and compositions.

[0024] Unless specifically stated otherwise, any description of possible mechanisms or modes of action or causes of improvement is intended for illustrative purposes only, and the disclosed compositions and methods are not bound by the correctness or incorrectness of any such suggested mechanisms or modes of action or causes of improvement.

[0025] In this document, descriptions refer to compositions and methods of using the compositions. When the disclosure describes or claims features or embodiments related to compositions, such features or embodiments also apply to methods of using the compositions. Similarly, when the disclosure describes or claims features or embodiments related to methods of using compositions, such features or embodiments also apply to the compositions.

[0026] For the sake of clarity, certain features of the disclosed methods and compositions that are described in the context of separate embodiments may also be provided in combination in a single embodiment. Conversely, different features of the disclosed methods and compositions that are described in the context of a single embodiment for the sake of brevity may also be provided separately or in any subcombination.

[0027] As used herein, the singular forms "a," "an," and "the" include plural forms.

[0028] Various terms related to various aspects of the specification are used throughout the specification and claims. Unless otherwise indicated, such terms are given their ordinary meaning in the art. Other specifically defined terms should be understood in a manner consistent with the definitions provided herein.

[0029] The term “comprising” is intended to include examples encompassed by the terms “consisting essentially of and “consisting of;” similarly, the term “consisting essentially of is intended to include examples encompassed by the term “consisting of.

[0030] When used in reference to a numerical range, a cutoff value, or a specific value, the term "about" is used to indicate that the recited value may vary from the listed value by up to 10%. Thus, the term "about" is used to encompass variations of ±10% or less, ±5% or less, ±1% or less, ±0.5% or less, or ±0.1% or less from the specified value.

[0031] As used herein, "administering" and like terms refer to the procedure of exposing the antibody-polynucleotide conjugate to target cells in vitro or injecting it into a subject to contact target cells, tissues or fragments of the subject's body with the antibody-polynucleotide conjugate.

[0032] As used herein, the term "subject" is intended to refer to any animal, particularly a mammal. Although the treatment of mice with antibody-polynucleotide conjugates 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. Therefore, the methods are applicable to humans and non-human animals, but are preferably used in mice and humans, and most preferably in humans. "Subject" and "patient" are used interchangeably herein. The subject herein can be a subject in need thereof.

[0033] The term "antibody" and similar terms are broad and include immunoglobulin molecules, including monoclonal antibodies, antibody fragments, bispecific or multispecific antibodies, dimers, tetramers or multimeric antibodies and single-chain antibodies. According to the heavy chain constant domain amino acid sequence, immunoglobulins can be designated as five major types, i.e., IgA, IgD, IgE, IgG and IgM. IgA and IgG are further subdivided into isotypes IgA1, IgA2, IgG1, IgG2, IgG3 and IgG4. Based on the amino acid sequence of its constant domain, the antibody light chain of any vertebrate species can be designated as one of two completely different types, i.e., kappa and lambda.

[0034] As used herein, "stitching" refers to an antibody (Ab):polynucleotide conjugate, such as an Ab:siRNA conjugate, in which the Ab has been attached to a bifurcation junction such that the bifurcation junction is attached to two different protein chains of the Ab via biorthogonal groups (such as one end of the bifurcation junction is attached to a biorthogonal group on one heavy chain constant region and the other end of the bifurcation junction is attached to a biorthogonal group on the other heavy chain constant region), and thereby the bifurcation junction is attached to a polynucleotide or payload, such as an siRNA.

[0035] A kind of method for polynucleotide delivery is to conjugate nucleic acid chemistry with biologically active targeting moieties, such as monoclonal antibodies (mAb). mAb shows long serum half-life, and selectively binds their target antigen with high affinity, making cell type specific targeting possible. The mAb of target internalization cell surface receptor can be further used for delivering the useful load of conjugation by endosome / lysosome pathway, including polynucleotide, such as siRNA. For example, for siRNA, in order to play its gene silencing effect, it must eventually be delivered to cytosol to engage RISC complex. It is necessary to overcome multiple obstacles to improve the efficacy in vivo of polynucleotide, such as inefficient endosome / lysosome escape, polynucleotide may be on the impact of the serum half-life of mAb and polynucleotide with appropriate ratio and mAb conjugated to realize effective delivery and functional challenge.

[0036] Disclosed herein are methods for forming antibody-polynucleotide conjugates. The methods may include contacting an antibody comprising two reactive bioorthogonal groups with a polynucleotide comprising a bifurcated linker comprising two reactive groups, wherein the reactive groups on the bifurcated linker selectively interact with the bioorthogonal groups to form a conjugate having an antibody-polynucleotide ratio of approximately 1:1. The bioorthogonal groups may be chemical groups that participate in a chemical reaction that is selective enough to proceed in a complex biological environment without being affected by the presence of amino acids, nucleic acids, etc.

[0037] The method may comprise contacting an antibody with a polynucleotide, wherein each antibody heavy chain constant region comprises an azide group and the polynucleotide comprises a bifurcated linker comprising two alkyne groups, wherein each azide group reacts with the alkyne group to form a conjugate at an antibody-to-polynucleotide ratio of about 1:1.

[0038] The polynucleotide conjugated to the antibody can be DNA or RNA or its analogs. For example, in some embodiments, the DNA or RNA is a phosphorodiamidate morpholino oligomer (PMO). Suitable RNA includes siRNA, miRNA, gRNA, shRNA or antisense oligonucleotides. The polynucleotide can be coded or non-coding. The polynucleotide can include DNA or RNA nucleotide modifications, such as methylation. The polynucleotide can be single-stranded or double-stranded. The polynucleotide can be linear or circular. Due to internal base pairing, the polynucleotide can be in various secondary conformations or tertiary conformations.

[0039] Reactive biorthogonal groups may be located within the heavy chain constant region of an antibody. In some embodiments, one antibody heavy chain constant region comprises one reactive biorthogonal group, and the other antibody heavy chain constant region comprises another reactive biorthogonal group. Each of the two reactive biorthogonal groups may comprise an azide group. Suitable azide groups include, but are not limited to, 3-azidopropylamine, N-azidoacetylgalactosamine, 6-azidoGalNAc, azido-butylamine, azidopentylamine, and azido-PEG3-amine. In some embodiments, at least one of the azide groups may be 3-azidopropylamine. In some embodiments, at least one of the azide groups may be N-azidoacetylgalactosamine. In some embodiments, at least one of the azide groups may be 6-azidoGalNAc. In some embodiments, at least one of the azide groups may be azido-butylamine. In some embodiments, at least one of the azide groups may be azidopentylamine. In some embodiments, at least one of the azide groups may be azido-PEG3-amine. Each antibody heavy chain may comprise the same bioorthogonal group. In some embodiments, each antibody heavy chain may comprise 3-azidopropylamine. In some embodiments, each antibody heavy chain may comprise N-azidoacetylgalactosamine. In some embodiments, each antibody heavy chain may comprise 6-azidoGalNAc. In some embodiments, each antibody heavy chain may comprise azido-butylamine. In some embodiments, each antibody heavy chain may comprise azidopentylamine. In some embodiments, each antibody heavy chain may comprise azido-PEG3-amine. Alternatively, each antibody heavy chain may comprise different bioorthogonal groups. For example, one antibody heavy chain may comprise 3-azidopropylamine and one antibody heavy chain may comprise N-azidoacetylgalactosamine.

[0040] The CH2 region of an antibody may carry a reactive bioorthogonal group. Suitable antibody heavy chain positions for bioorthogonal groups include Q295 and N297. Suitable azides at position Q295 include 3-azidopropylamine, azido-butylamine, azidopentylamine, and azido-PEG3-amine. In some embodiments, the antibody has 3-azidopropylamine at amino acid position Q295. One or both Q295 positions may carry 3-azidopropylamine. Suitable azides at position N297 include N-azidoacetylgalactosamine and 6-azidoGalNAc. In some embodiments, the antibody has N-azidoacetylgalactosamine at amino acid position N297. One or both N297 positions may carry N-azidoacetylgalactosamine.

[0041] Suitable bifurcated joints include those that can react with bioorthogonal groups present on the antibody heavy chain. For example, in the case of an antibody comprising a bioorthogonal group having an azide group, the bifurcated joint may comprise an alkyne reactive group. The alkyne group may comprise a cyclooctyne. The bifurcated joint may comprise two reactive groups, each of which reacts with a bioorthogonal group on the antibody heavy chain. The bifurcated joint may be a Mal-PEG(X)-bis-PEG(X)-BCN joint or a Mal-PEG(X)-bis-PEG(X)-DBCO joint, where (X) represents the number of PEG units. The number of PEG units in the general formula Mal-PEG(X)-bis-PEG(X)-BCN joint and Mal-PEG(X)-bis-PEG(X)-DBCO may be about 1 to 10. In some embodiments, the bifurcated joint is Mal-PEG2-bis-PEG3-BCN.

[0042] Disclosed herein are compositions comprising Mal-PEG(X)-bis-PEG(X)-BCN or 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 from about 1 to 10. In some embodiments, Mal-PEG(X)-bis-PEG(X)-BCN is conjugated to a polynucleotide. In some embodiments, Mal-PEG(X)-bis-PEG(X)-DBCO is conjugated to a polynucleotide. In some embodiments, the bifurcated linker is Mal-PEG2-bis-PEG3-BCN. Mal-PEG(X)-bis-PEG(X)-BCN or Mal-PEG(X)-bis-PEG(X)-DBCO can be attached to the polynucleotide via a dihexyl disulfide linker. 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. In some embodiments, 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. Mal-PEG2-bis-PEG3-BCN conjugated polynucleotide molecules can be reacted with antibodies comprising bioorthogonal groups to form polynucleotide-conjugated antibodies as described herein.

[0043] In the compositions described herein comprising Mal-PEG(X)-bis-PEG(X)-BCN or Mal-PEG(X)-bis-PEG(X)-DBCO conjugated to a polynucleotide, the polynucleotide can be DNA or RNA or an analog thereof. For example, in some embodiments, the DNA or RNA is a PMO. Suitable RNAs include siRNA, miRNA, gRNA, shRNA, or antisense oligonucleotides. The polynucleotide can be coding or non-coding. The polynucleotide can contain DNA or RNA nucleotide modifications, such as methylation. The polynucleotide can be single-stranded or double-stranded. The polynucleotide can be linear or circular. Due to internal base pairing, the polynucleotide can be in various secondary or tertiary conformations.

[0044] Disclosed herein are polynucleotide-conjugated antibodies comprising a polynucleotide attached to a bifurcated junction, and an antibody comprising two heavy chain constant regions, each of which comprises a bioorthogonal group, wherein the bifurcated junction is attached to the bioorthogonal group of each heavy chain constant region. The bifurcated junction can be Mal-PEG(X)-bis-PEG(X)-BCN or Mal-PEG(X)-bis-PEG(X)-DBCO, where (X) represents the number of PEG units. The number of PEG units in the general formula Mal-PEG(X)-bis-PEG(X)-BCN junction and Mal-PEG(X)-bis-PEG(X)-DBCO can be about 1 to 10. The bifurcated junction can be Mal-PEG2-bis-PEG3-BCN. The polynucleotide can be conjugated to the antibody through triazole. The polynucleotide-conjugated antibody can be produced by the methods described herein.

[0045] In the polynucleotide-conjugated antibodies described herein, the polynucleotide conjugated to the antibody can be DNA or RNA or an analog thereof. For example, in some embodiments, the DNA or RNA is a PMO. Suitable RNAs include siRNA, miRNA, gRNA, shRNA, or antisense oligonucleotides. The polynucleotide can be coding or non-coding. The polynucleotide can contain DNA or RNA nucleotide modifications, such as methylation. The polynucleotide can be single-stranded or double-stranded. The polynucleotide can be linear or circular. Due to internal base pairing, the polynucleotide can be in various secondary or tertiary conformations.

[0046] Described herein is a method for delivering polynucleotides to a target, the method including administering any one of the antibodies conjugated with polynucleotides disclosed herein. Any one of the polynucleotides described herein can be delivered to a target via the antibodies conjugated with polynucleotides. Polynucleotides can be delivered for a variety of reasons, such as genome modification, introduction of exogenous polynucleotides for use by cell mechanisms, or induction of cellular responses sensitive to the polynucleotides delivered. Suitable polynucleotides for genome modification are delivered, including but not limited to gRNA and insertion cassettes. Suitable polynucleotides for use by cell mechanisms include but are not limited to mRNA, tRNA, miRNA, or encoding DNA. Suitable polynucleotides for inducing cell responses (such as immune responses) include single-stranded RNA, double-stranded RNA, and DNA containing CpG.

[0047] Described herein are methods for gene silencing or reducing gene expression comprising administering any of the polynucleotide-conjugated antibodies disclosed herein. Polynucleotides capable of promoting gene silencing or reducing gene expression include those that reduce the translation and transcription of mRNA. Coding RNA or non-coding RNA can target gene silencing or reduction of gene expression. In some embodiments, the RNA to be silenced is mRNA. Suitable polynucleotides for administering gene silencing or reducing gene expression include, but are not limited to, siRNA, shRNA, and PMO polynucleotides.

[0048] 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.

[0049] Disclosed herein are methods of forming an antibody-polynucleotide conjugate, the method comprising contacting an antibody comprising a F405L substitution and a R409K substitution according to the EU numbering index on one heavy chain constant region and an azide group on the other heavy chain constant region with a polynucleotide to form a conjugate at an antibody-to-polynucleotide ratio of about 1:1.

[0050] In some embodiments, the method comprises contacting an antibody comprising a F405L substitution and a R409K substitution according to the EU numbering index with an antibody that does not have the F405L substitution and the R409K substitution and comprises an azide group on one or both of the antibody's heavy chain constant regions, prior to contacting the antibody with the polynucleotide, to promote Fab arm exchange and formation of the 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.

[0051] Disclosed herein are methods of promoting Fab arm exchange, the method comprising contacting a first antibody comprising a F405L substitution and a R409K substitution according to the EU numbering index with 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 a F405L substitution and a R409K substitution on one heavy chain constant region and an azide group on the other heavy chain constant region.

[0052] As used herein, "Fab arm exchange" (or half molecule exchange) refers to the process in which half antibodies (light chain and heavy chain pairs) associate to form complete antibodies (two light chain and heavy chain pairs). The Fab arm exchange reaction is the result of a disulfide bond isomerization reaction and CH3 domain dissociation-association. The heavy chain disulfide bonds in the antibody hinge region are reduced, and the resulting free cysteines of the antibody can form inter-heavy chain disulfide bonds with cysteine residues of another antibody. The CH3 domains of the antibody are simultaneously released and reformed by dissociation-association. Fab arm exchange can be used to facilitate the formation of heterodimers by introducing amino acid substitutions (such as F405L substitutions and R409K substitutions) into the heavy chain CH3 domains of monospecific bivalent antibodies to produce heterodimers (such as bispecific antibodies). After the monospecific bivalent antibody dissociates, for example, the half antibody containing the amino acid substitution will preferably associate with the half antibody without the amino acid substitution, thereby driving the formation of heterodimers. Fab arm exchange is described, for example, in U.S. Pub. No. US20220267438.

[0053] In some embodiments, antibodies without the F405L substitution and the R409K substitution comprise an azide group on one of the heavy chain constant regions. In some embodiments, antibodies without the F405L substitution and the R409K substitution comprise an azide group on both heavy chain constant regions. The CH2 region of the antibody may carry an 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-azidoGalNAc, azido-butylamine, azidopentylamine, and azido-PEG3-amine. Suitable antibody positions for azide groups include Q295 and N297. Suitable azides at position Q295 include 3-azidopropylamine, azido-butylamine, azidopentylamine, and azido-PEG3-amine. In some embodiments, the antibody has 3-azidopropylamine at amino acid position Q295. One or both Q295 positions may carry 3-azidopropylamine. Suitable azides at position N297 include N-azidoacetylgalactosamine and 6-azidoGalNAc. In some embodiments, the antibody has N-azidoacetylgalactosamine at amino acid position N297. One or both N297 positions may carry N-azidoacetylgalactosamine.

[0054] Suitable for contacting polynucleotides of antibodies comprising a F405L substitution and a R409K substitution on one heavy chain constant region and an azide group on another heavy chain constant region, including those capable of reacting with an azide group. The polynucleotide may comprise one or more reactive groups capable of reacting with an azide group. For example, the polynucleotide may comprise one or more alkyne reactive groups capable of reacting with an azide. In some embodiments, the alkyne may comprise cyclooctyne. In some embodiments, the olefin reactive group is dibenzocyclooctyne (DBCO).

[0055] The antibody comprising F405L substitution and R409K substitution can be an IgG4 antibody. The antibody not having F405L substitution and R409K substitution comprising an azido group can be an IgG4. In some embodiments, the antibody comprising F405L substitution and R409K substitution and the antibody comprising an azido group without F405L substitution and R409K substitution can both be IgG4. The antibody comprising F405L substitution and R409K substitution can be monospecific, bispecific or multispecific. The antibody not having F405L substitution and R409K substitution comprising an azido group can be monospecific, bispecific or multispecific. In some embodiments, the antibody comprising F405L substitution and R409K substitution binds to the same antigen as the antibody comprising an azido group without F405L substitution and R409K substitution. In some embodiments, an antibody comprising a F405L substitution and a R409K substitution binds to a different antigen than an antibody comprising an azide group that does not have the F405L substitution and the R409K substitution. 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 can be monospecific, bispecific, or multispecific.

[0056] The antibody-polynucleotide conjugates described throughout the text have similar or improved biophysical stability characteristics compared to non-conjugated antibodies and / or polynucleotides. For example, the thermal stability of the antibody-polynucleotide conjugates is retained compared to the parent antibody and synthetic intermediates (such as apa and GlaNAz). Differential scanning fluorimetry can be used to analyze the biophysical stability of the conjugates. Further analysis of the antibody-polynucleotide conjugates (by, for example, analytical size exclusion chromatography) demonstrated that the conjugates were primarily monomeric and intact.

[0057] The antibody-polynucleotide conjugates also induce efficient gene silencing and are at least equivalent to or better than antibody-polynucleotide conjugates produced by other methods. For example, other methods for producing antibody-polynucleotide conjugates include chromatography or Fab arm exchange.

[0058] Importantly, the antibody-polynucleotide conjugates produced by the methods described herein can result in a higher overall yield than other known methods (such as chromatographic separation). For example, chromatographic separation typically produces an antibody-polynucleotide conjugate yield of up to about 30%. The methods described herein produce antibody-polynucleotide conjugates in an overall yield greater than about 50%. For example, in some cases, the yield may be between about 50% and about 100%. In other cases, the yield may be between about 55% and about 85%. In other cases, the yield may be between about 55% and about 75%. In other cases, the yield may be between about 55% and about 70%.

[0059] Antibody-polynucleotide conjugates produced by the methods described throughout have improved PK characteristics (e.g., plasma concentrations) compared to antibody-polynucleotide conjugates having a 1:2 ratio (even when produced by the same methods described throughout).

[0060] The methods described throughout ultimately lead to easier and more efficient methods of producing antibody-polynucleotide conjugates.

[0061] Example

[0062] The following examples are provided to further describe some of the embodiments disclosed herein.These examples are intended to illustrate, but not to limit, the embodiments disclosed herein.

[0063] Example 1

[0064] Preparation of azide-modified mAbs.

[0065] Conjugation of 3-azidopropylamine to mAb position Q295 catalyzed by microbial transglutaminase (MTG). All mAbs were expressed in CHO cells and purified by protein A chromatography using standard methods. mAbs (PSMB127 and trastuzumab) were completely deglycosylated using Rapid PNGase F (P0710, New England Biolabs). Briefly, Rapid PNGase F was added to each mAb (5 μL / mg mAb) and the reaction was incubated at 37°C overnight. 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) at 37°C for 4-6 hours. The 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. This procedure was used to conjugate 3-azidopropylamine to amino acid position Q295.

[0066] Site-specific azido modification of mAb Fc glycans. mAbs (PSMB127 and trastuzumab) were deglycosylated using a hydrolase, EndoS2 (A0-GL8-020, GlycINATOR LE, Genovis), which hydrolyzes the β1,4 linkages between the core N-acetylglucosamine (GlcNAc) residues of the Fc glycan, leaving the innermost GlcNAc residues intact (one per heavy chain). GlycINATOR LE was added to each mAb (1-5 μL / mg mAb) and incubated overnight at 37°C. TM The components of the Antibody Azide Modification System Kit (S10901, Thermo Fisher Scientific) conjugate the core GlcNAc residue to N-azidoacetylgalactosamine (GaInAz). Briefly, the deglycosylated mAb was exchanged into tris-buffered saline (TBS, pH 7.4) and concentrated to 6 mg / mL. The mAb was then reacted with the provided GalT enzyme (60 μL / mg mAb) in the presence of UDP-GalNAz (2.5 mM) at 30°C overnight. The reaction progress was monitored by mass spectrometry. The GalNAz-modified mAb was purified by diafiltration and analyzed by mass spectrometry and analytical size exclusion chromatography. This procedure was used to conjugate N-azidoacetylgalactosamine (GaInAz) to amino acid position N297.

[0067] Preparation of siRNA with forked junctions

[0068] Preparation of Mal-bis-BCN-CTNNB1 siRNA. A variant of a chemically stabilized siRNA targeting CTNNB1 containing a 5'-dihexyl disulfide linker on the sense strand was prepared by solid phase synthesis. The disulfide-protected siRNA was dissolved in water and deprotected overnight at 37°C using 15 equivalents of tris(2-carboxyethyl)phosphine (TCEP). 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 produce Mal-bis-BCN-CTNNB1 siRNA. If the succinimide portion of the maleimide-thiol conjugate is hydrolyzed, the ring-opened product is stable to cleavage in vivo because it is no longer sensitive to the thiol exchange reaction. After Mal-PEG2-bis-PEG3-BCN conjugation, 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-opening product ( Figure 2). The reaction was incubated at 4 ° C overnight, and the extent of maleimide ring opening was monitored by mass spectrometry. Upon complete conversion to the maleimide ring-opening product, Mal-bis-BCN-CTNNB1 siRNA was purified by anion exchange chromatography, dialyzed into DPBS, and stored at -20 ° C until further use. Using this method, a forked linker (Mal-bis-BCN) was conjugated to the siRNA at the 5' end, at the reverse abasic residue (i.e., at the 5'OH of the abasic group). Modification occurred on the sense strand.

[0069] Preparation of mAb-siRNA conjugates

[0070] Preparation of "stitched" 1:1 mAb-siRNA conjugates using azide-modified mAbs and Mal-bis-BCN siRNA. Heavy chain "stitched" 1:1 mAb-siRNA conjugates were prepared by strain-promoted azide-alkyne cycloaddition chemistry (copper-free click chemistry) by reacting azide-modified mAbs (PSMB127-GalNAz, trastuzumab-apa, and trastuzumab-GaInAz) with 2-5 equivalents of Mal-bis-BCN-CTNNB1 siRNA at 37°C overnight. The reaction progress was monitored by SDS-PAGE analysis. The siRNAs were separated by size exclusion chromatography or anion exchange chromatography on TSK-gel. TM BioAssist TM mAb-siRNA conjugates were purified on a Q column. 1:1 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 reducing SDS-PAGE and analytical size exclusion chromatography. SDS-PAGE analysis of the reduced mAbs confirmed the formation of covalently linked heavy chain dimers in each case, as evidenced by the presence of a protein band extending between the 100 kDa and 150 kDa molecular weight markers ( Figure 3 and Figure 4 Analytical size exclusion chromatography confirmed that the mAb was monomeric and intact ( Figure 5A 、 Figure 5B 、 Figure 6A 、 Figure 6B 、 Figure 6C and Figure 6DThe retention times of the 1:1 mAb-siRNA conjugates were comparable to those observed for 1:1 mAb-siRNA conjugates prepared by other methods (cFAE or chromatography). The yields of the 1:1 conjugates (from each corresponding azido-mAb intermediate) ranged from 55-70%, nearly double the yields obtained by chromatography.

[0071] Preparation of 1:2 mAb-CTNNB1 siRNA conjugates. 1:2 mAb-siRNA conjugates were prepared using strain-promoted azide-alkyne cycloaddition chemistry (SPAAC). Azide-modified mAb (apa or GaInAz as described above) was diluted to 1 mg / mL in DPBS and reacted with 5 equivalents of chemically stabilized dibenzocyclooctyne (DBCO)-modified siRNA targeting CTNNB1 (DBCO-CTNNB1 siRNA) at 37°C overnight. mAb-siRNA conjugates (PSMB127-apa-siRNA degree of labeling (DOL) 2, trastuzumab-apa-siRNA DOL 2, and trastuzumab-GaInAz-siRNA DOL 2) were purified by size exclusion chromatography and analyzed by reducing SDS-PAGE and analytical size exclusion chromatography.

[0072] A 1:1 PSMB127-CTNNB1 siRNA conjugate was prepared by a controlled Fab arm exchange method. IgG4 mAbs, due to their flexible hinge regions and slightly labile CH3 domains, can exchange half-molecules with other IgG4 mAbs. This process can be facilitated by combining a wild-type IgG4 mAb with a mutant mAb containing two point mutations (F405L / R409K); the resulting heterodimer formation is energetically favored over either homodimer combination. This controlled Fab arm exchange method was used to generate PSMB127 containing a single azide moiety for subsequent SPAAC conjugation of siRNA to produce a 1:1 mAb-siRNA conjugate. PSMB127-apa (1 mg / mL) was mixed 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 completed, the resulting bispecific mAb (PSMB127-apa DOL 1) was exchanged into DPBS using a desalting column and allowed to oxidize for another 2 days at 4°C. Next, PSMB127-apa DOL 1 was reacted with 5 equivalents of DBCO-CTNNB1 siRNA overnight at 37°C. The 1:1 mAb-siRNA conjugate (PSMB127-apa-siRNA cFPE DOL 1) was purified by size exclusion chromatography and analyzed by reducing SDS-PAGE and analytical size exclusion chromatography.

[0073] A 1:1 trastuzumab-CTNNB1 siRNA conjugate was prepared by chromatography. Trastuzumab-GaInAz (1 mg / mL) was reacted with 1.5 equivalents of DBCO-CTNNB1 siRNA at 37°C overnight. The resulting crude reaction mixture contained 1:0, 1:1, and 1:2 trastuzumab-siRNA conjugates. To separate the 1:1 trastuzumab-siRNA conjugate, the reaction mixture was filtered through a TSK-gel. TM BioAssist TMQ column (Tosoh) purification. The chromatographic fractions containing 1: 1 trastuzumab-siRNA conjugate (trastuzumab-GaInAz-siRNA DOL 1) were merged and exchanged into DPBS using a desalting column. Trastuzumab-GaInAz-siRNA DOL 1 was analyzed by reducing SDS-PAGE and analytical size exclusion chromatography. Trastuzumab-apa-siRNA DOL 1 was prepared in a manner similar to that described above, except that only 1 equivalent of DBCO-CTNNB1 siRNA was used to produce the crude reaction mixture. In each case, the yield of the 1: 1 mAb-siRNA conjugate obtained (from each corresponding azido-mAb intermediate) was approximately 30%.

[0074] Example 2

[0075] Thermal stability analysis of PSMB127-siRNA conjugates.

[0076] The PSMB127-siRNA conjugate was more fully characterized to evaluate the effect of the siRNA conjugation strategy on the thermal stability of the mAb. m ) was determined by differential scanning fluorimetry using a nanoDSF instrument from Nanotemper. T was determined by monitoring the change in fluorescence intensity at 330 nm and 350 nm after a thermal scan from 20°C to 95°C. m Although lower T was observed for the modified PSMB127 variants 开始 (beginning of unfolding), but T m Notably, the T values of two 1:1 mAb-siRNA conjugates (PSMB127-GalNAz-siRNA DOL 1 and PSMB127-apa-siRNA cFAE DOL 1) produced by the methods disclosed herein or by cFAE were significantly higher than those of the two 1:1 mAb-siRNA conjugates (PSMB127-GalNAz-siRNA DOL 1 and PSMB127-apa-siRNA cFAE DOL 1). m were similar, demonstrating that the disclosed method does not severely affect the thermal stability of the parent mAb.

[0077] Construct <![CDATA[T 开始 (℃)]]> <![CDATA[T m1 (℃)]]> PSMB127 59.9 65.3 PSMB127-apa 48.7 63.5 PSMB127-GalNAz 52.3 63.3 PSMB127-GalNAz-siRNA stitched DOL 1 56.0 64.3 PSMB127-apa-siRNA cFAE DOL 1 53.5 63.8 PSMB127-apa siRNA DOL 2 47.1 63.4

[0078] Table 1. Thermal stability analysis of PSMB127-siRNA conjugates .

[0079] Target mRNA knockdown following mAb-mediated siRNA uptake .

[0080] The ability of PSMB127 and trastuzumab-siRNA conjugates to mediate CTNNB1 mRNA knockdown was evaluated. PSMB127-siRNA conjugates were evaluated in stably transfected HEK-293T cells expressing prostate-specific membrane antigen (PSMA), 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). Figure 7 and Figure 8 ). PSMB127 and trastuzumab-siRNA conjugates had equivalent potency in their respective cell lines. Taken together, these data indicate that the disclosed method has no distinguishable effect on mAb uptake and siRNA delivery when directly compared to conventionally prepared 1:1 or 1:2 mAb-siRNA conjugates.

[0081] Pharmacokinetic evaluation of trastuzumab-siRNA conjugates in mice .

[0082] To evaluate the pharmacokinetic properties of trastuzumab-siRNA conjugates, 1 mg / kg (siRNA) of each conjugate was administered intravenously to C57BL.6 mice (n=3 per group). Blood samples were collected 1, 24, 72, and 168 hours after a single dose. Blood samples were immediately centrifuged after collection, and the resulting plasma was frozen at -80°C until further analysis. The plasma concentration of CTNNB1 siRNA at each time point was determined using a stem-loop RT-qPCR method ( Figure 9 ). Overall, the 1:1 trastuzumab-siRNA conjugate exhibited significantly higher plasma concentrations at each time point compared to the 1:2 trastuzumab-siRNA conjugate (Table 2). Unexpectedly, the total in vivo exposure observed for the 1:1 GaInAz-siRNA conjugate was significantly higher than that observed for the 1:1 apa-siRNA conjugate (approximately 2-4 times).

[0083]

[0084] Table 2. Mean % of each trastuzumab-siRNA conjugate treated with a single dose (1 mg / kg siRNA) siRNA plasma concentration (nM) and total siRNA exposure (nmol siRNA*day / L) .

[0085] The 1:1 mAb-siRNA conjugates produced by the disclosed method retain favorable biophysical stability characteristics and induce effective gene silencing equivalent to 1:1 mAb-siRNA conjugates produced by other methods such as chromatographic separation. In addition, pharmacokinetic (PK) studies in mice revealed that the in vivo exposure of the mAb-siRNA conjugates produced by the disclosed method was comparable to those 1:1 mAb-siRNA conjugates produced by other methods and significantly improved relative to similar 1:2 mAb-siRNA controls. In summary, the disclosed method for producing 1:1 mAb-siRNA conjugates provides a significant improvement in synthesis strategy and yield while maintaining the efficacy of 1:1 mAb-siRNA conjugates prepared by other methods and significantly improving circulating pharmacokinetics compared to 1:2 mAb-siRNA conjugates.

[0086] Those skilled in the art will appreciate that many changes and modifications may be made to the preferred embodiments disclosed herein, and that such changes and modifications may be made without departing from the spirit of the invention. Therefore, the appended claims are intended to cover all such equivalent variations that fall within the true spirit and scope of the invention.

Claims

1. A method for forming an antibody-polynucleotide conjugate, the method comprising contacting: (a) an antibody comprising two reactive bioorthogonal groups; and (b) a polynucleotide comprising a forked linker comprising two reactive groups, wherein the reactive group on the bifurcated linker selectively interacts with the biorthogonal group to form a conjugate having an antibody-to-polynucleotide ratio of about 1:

1.

2. The method of claim 1, wherein the polynucleotide is selected from DNA or RNA.

3. The method of claim 2, wherein the DNA or the RNA is a phosphorodiamidate morpholino oligomer.

4. The method of claim 2, wherein the RNA is siRNA, miRNA, gRNA, shRNA, or antisense oligonucleotide.

5. The method of any one of the preceding claims, wherein one antibody heavy chain constant region comprises one reactive biorthogonal group and the other antibody heavy chain constant region comprises another reactive biorthogonal group.

6. The method of any one of the preceding claims, wherein each of the two reactive biorthogonal groups comprises an azide group.

7. The method of claim 6, wherein at least one of the azide groups is selected from 3-azidopropylamine, N-azidoacetylgalactosamine, 6-azidoGalNAc, azido-butylamine, azidopentylamine, or azido-PEG3-amine.

8. The method of claim 7, wherein the antibody has 3-azidopropylamine at amino acid position Q295.

9. The method of claim 7, wherein the antibody has N-azidoacetylgalactosamine at amino acid position N297.

10. The method of any preceding claim, wherein the bifurcated linker comprises two alkyne groups. The method of claim 10 , wherein the alkyne group comprises cyclooctyne.

12. The method of any one of the preceding claims, wherein the bifurcated linker is a Mal-PEG(X)-bis-PEG(X)-BCN linker or a Mal-PEG(X)-bis-PEG(X)-DBCO linker.

13. The method of claim 12, wherein the bifurcated linker is Mal-PEG2-bis-PEG3-BCN.

14. The method of any one of the preceding claims, wherein the antibody-polynucleotide conjugate has (a) improved biophysical stability characteristics, such as thermal stability; and (b) Inducing efficient gene silencing equivalent antibody-polynucleotide conjugates produced by other methods such as chromatographic separation or Fab arm exchange.

15. The method of claim 14, wherein the biophysical stability profile is analyzed by differential scanning fluorimetry.

16. The method of any one of the preceding claims, wherein the antibody-polynucleotide conjugate is produced in an overall yield greater than other methods such as chromatographic separation.

17. The method of any of the preceding claims, wherein the antibody-polynucleotide conjugate is produced with an overall yield greater than about 50%, e.g., an overall yield between about 50% to about 100%; about 55% to about 85%; about 55% to about 75%; or about 55% to about 70%.

18. The method of any one of the preceding claims, wherein the antibody-polynucleotide conjugate has improved PK characteristics (eg, plasma concentration) compared to an antibody-polynucleotide conjugate having a 1:2 ratio.

19. A method of forming an antibody-polynucleotide conjugate, the method comprising contacting an antibody and a polynucleotide, in (a) each antibody heavy chain constant region comprises an azide group; and (b) the polynucleotide comprises a forked linker comprising two alkyne groups, wherein each azide group reacts with an alkyne group to form a conjugate having an antibody-to-polynucleotide ratio of approximately 1:

1.

20. The method of claim 18, wherein the polynucleotide is selected from DNA or RNA.

21. The method of claim 19, wherein the DNA or the RNA is a phosphorodiamidate morpholino oligomer.

22. The method of claim 19, wherein the RNA is siRNA, miRNA, gRNA, shRNA, or an antisense oligonucleotide.

23. The method according to any one of claims 18 to 21, wherein at least one of the azide groups is selected from 3-azidopropylamine, N-azidoacetylgalactosamine, 6-azidoGalNAc, azido-butylamine, azidopentylamine, azido-PEG3-amine.

24. The method of claim 22, wherein the antibody has 3-azidopropylamine at amino acid position Q295.

25. The method of claim 22, wherein the antibody has N-azidoacetylgalactosamine at amino acid position N297.

26. The method of any one of claims 18 to 24, wherein the alkyne group comprises cyclooctyne.

27. The method of any one of claims 18 to 25, wherein the bifurcated linker is Mal-PEG(X)-bis-PEG(X)-BCN or Mal-PEG(X)-bis-PEG(X)-DBCO.

28. The method of claim 26, wherein the bifurcated linker is Mal-PEG2-bis-PEG3-BCN.

29. A composition comprising Mal-PEG(X)-bis-PEG(X)-BCN or Mal-PEG(X)-bis-PEG(X)-DBCO conjugated to a polynucleotide.

30. The composition of claim 28, wherein the Mal-PEG(X)-bis-PEG(X)-BCN is Mal-PEG2-bis-PEG3-BCN.

31. The composition of claim 28 or 29, wherein the polynucleotide is selected from DNA or RNA.

32. The composition of claim 30, wherein the DNA or the RNA is a phosphorodiamidate morpholino oligomer.

33. The composition of claim 30, wherein the RNA is siRNA, miRNA, gRNA, shRNA, or an antisense oligonucleotide.

34. The composition of any one of claims 28 to 32, wherein the Mal-PEG(X)-bis-PEG(X)-BCN or the Mal-PEG(X)-bis-PEG(X)-DBCO is attached to the polynucleotide via a dihexyl disulfide linker.

35. The composition of any one of claims 28 to 33, wherein the Mal-PEG(X)-bis-PEG(X)-BCN or the Mal-PEG(X)-bis-PEG(X)-DBCO is attached to the 5' end of the polynucleotide.

36. A polynucleotide-conjugated antibody, comprising: a polynucleotide attached to the fork junction; and an antibody comprising two heavy chain constant regions, each of the two heavy chain constant regions comprising a bioorthogonal group; wherein the bifurcated linker is attached to the bioorthogonal group of each heavy chain constant region.

37. The polynucleotide-conjugated antibody of claim 35, wherein the polynucleotide is selected from DNA or RNA.

38. The polynucleotide-conjugated antibody of claim 36, wherein the DNA or the RNA is a phosphorodiamidate morpholino oligomer.

39. The polynucleotide-conjugated antibody of claim 36, wherein the RNA is siRNA, miRNA, gRNA, shRNA, or an antisense oligonucleotide.

40. The polynucleotide-conjugated antibody according to any one of claims 35 to 38, wherein the polynucleotide is conjugated to the antibody via a triazole.

41. The polynucleotide-conjugated antibody according to any one of claims 35 to 39, wherein the bifurcated linker is Mal-PEG(X)-bis-PEG(X)-BCN or Mal-PEG(X)-bis-PEG(X)-DBCO.

42. The polynucleotide-conjugated antibody of claim 40, wherein the bifurcated linker is Mal-PEG2-bis-PEG3-BCN.

43. A method of delivering a polynucleotide to a target, the method comprising administering the conjugated antibody according to any one of claims 35 to 41.

44. A method of reducing gene expression comprising administering an RNA-conjugated antibody according to any one of claims 38 to 42.

45. The method of claim 42 or 43, wherein the polynucleotide-conjugated antibody is administered to a subject in need thereof.

46. The method of claim 42 or 43, wherein the polynucleotide-conjugated antibody is administered to cells in vitro.

47. A method of forming an antibody-polynucleotide conjugate, the method comprising contacting: (a) 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 (b) polynucleotides; to form a conjugate with an antibody-to-polynucleotide ratio of approximately 1:

1.

48. A method for promoting Fab arm exchange, the method comprising contacting: (a) a first antibody comprising an F405L substitution and an R409K substitution; and (b) a second antibody that does not have said F405L substitution and said R409K substitution and comprises an azide group on one or both of said 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.

49. The method of claim 46 or 47, wherein the azide group on one or both of the antibody heavy chain constant regions is selected from 3-azidopropylamine, N-azidoacetylgalactosamine, 6-azidoGalNAc, azido-butylamine, azidopentylamine, or azido-PEG3-amine.

50. The method of claim 48, wherein the azide group at amino acid position Q295 is 3-azidopropylamine.

51. The method of claim 48, wherein the azide group at amino acid position N297 is N-azidoacetylgalactosamine.

52. The method of any one of claims 46 to 50, wherein 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 is a bispecific antibody.

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