An antibody-oligonucleotide conjugate and uses thereof

Abstract

The application discloses an antibody-oligonucleotide conjugate and application thereof, relates to targeted protein degradation, and particularly relates to an antibody-oligonucleotide conjugate. The antibody-oligonucleotide conjugate with a guanylate residue (G) constructed by the application can guide cell surface proteins into the macroautophagy-lysosome pathway for selective proteolysis. After coupling of a G4 structure and cetuximab (Ctx-G4), a concentration of only 1 nM is sufficient to strongly degrade epidermal growth factor receptor (EGFR) in HeLa and A549 cells. The modular nature of the platform is further verified by successfully degrading HER2 and PD-L1 by simply replacing the antibody, highlighting its wide applicability. In general, the Ab-G4 platform provides a synthetic feasible and multifunctional cell surface protein directed degradation (TPD) strategy, and extends the autophagy-driven degradation tool to extracellular proteins.

Description

Technical Field

[0001] This invention relates to targeted protein degradation, and more particularly to antibody-oligonucleotide conjugates. Background Technology

[0002] Targeted protein degradation (TPD) has emerged as a versatile therapeutic paradigm, utilizing the body's own protein clearance mechanisms to eliminate disease-associated targets. To date, TPD primarily utilizes two endogenous pathways: the ubiquitin-proteasome system (UPS), mainly responsible for the renewal of cytoplasmic and nuclear proteins, and the lysosomal degradation pathway, including autophagy, endocytosis, and microautophagy, which clear extracellular ligands, membrane-associated receptors, and even entire organelles. By modulating these differentially differentiated but complementary protein degradation pathways, TPD can efficiently and selectively remove pathogenic proteins, thus demonstrating considerable potential for treating various human diseases.

[0003] Autophagy, particularly macroautophagy (hereinafter referred to as autophagy), is a major degradation pathway in higher eukaryotic cells. This process involves the encapsulation of cytoplasmic components into autophagosomes, which then fuse with lysosomes, where resident hydrolases mediate the breakdown of the encapsulated material. In 2019, Arimoto et al. demonstrated that protein S-guanylation alone is sufficient to be an independent signal triggering selective autophagic clearance. Building on this, they reported a targeted clearance platform, named AUTAC (Autophagy-Targeted Chimera), which consists of a guanine-based degradation tag and a targeting recognition portion. Consistent with the broad substrate spectrum of autophagy, this chimera not only promotes protein degradation but also facilitates the clearance of mitochondrial debris. Subsequently, autophagy-targeted chimeras (AUTOTACs) and autophagy-anchored compounds (ATTECs) have both belonged to this class of degradative agents. Compared to ubiquitin-proteasome system (UPS)-based methods, these autophagy-based degradative agents have significant advantages in clearing large substrates, including protein aggregates and organelles. To date, autophagy-targeted degradative agents (AUTACs) have been successfully used to clear damaged mitochondria, while ATTECs and AUTOTACs have shown promise in eliminating pathological protein aggregates within neurons. However, these autophagy-targeting chimeras are currently limited to cytoplasmic proteins and organelles because their design relies on small molecules or peptides that bind to intracellular targets.

[0004] In a recent study, Yang et al. discovered an autophagy-lysosomal pathway mediated by MAP1LC3B–SNX18, responsible for the degradation of extracellular vesicles (EVs) and mechanistically linked to autophagy through an LC3 interaction region (LIR)-dependent sorting process. Building on this discovery, they further constructed LIR-modified EVs and conjugated them with target-specific antibodies, forming a system called AETAC for protein-targeted degradation (TPD) of EGFR, PD-L1, and VEGF. This platform uniquely utilizes the natural degradation mechanism of EVs and combines it with the multivalent binding effect on the EV surface, thus providing a conceptually innovative and efficient TPD approach.

[0005] However, despite its innovative design, the AETAC system faces significant engineering and translational challenges in practical applications. Its preparation process is inherently complex, requiring the engineering of EVs to simultaneously express LIR motifs and streptavidin, followed by the introduction of biotinylated monoclonal antibodies to anchor the target molecule to the EV surface. This multi-step process significantly increases production complexity and introduces substantial batch-to-batch variability. Furthermore, current processes heavily rely on flow cytometry sorting for separation and characterization, lacking standardized affinity tags to simplify purification and achieve stable quality control—crucial conditions for large-scale production and clinical translation.

[0006] In contrast, traditional antibody-drug conjugate systems (such as antibody-drug conjugates (ADCs) or direct antibody-effect molecule fusion proteins) have significant advantages in terms of production scalability, precise stoichiometric control, and mature purification systems. For example, methods such as protein A / G affinity chromatography are widely used in GMP-level production processes and have a mature and reliable industrial foundation.

[0007] Therefore, although AETAC demonstrates the biological potential of using EV transport mechanisms to achieve protein degradation, the complex engineering burden and quality control uncertainties introduced during EV surface modification, as well as the additional steps required for antibody-biotin conjugation, may make it less advantageous than direct antibody conjugation strategies in terms of industrialization.

[0008] In 2025, Li et al. engineered antibodies with polyethyleneimine (PEI) using chemical methods to activate the endogenous autophagy pathway, thereby guiding cell membrane proteins into lysosomes for degradation. Although these autophagy-inducible antibodies (AUTABs) exhibit strong activity, their positive charge-dependent mechanism also presents significant limitations. Their cationic nature is associated with accelerated in vivo clearance and increased immunogenicity risk, factors that collectively weaken their pharmacokinetic performance and translational potential. Furthermore, compared to unmodified parent antibodies, AUTABs show very limited improvement in antitumor efficacy, leaving their therapeutic advantage controversial. More importantly, their reported membrane damage effects further raise safety concerns, thus limiting their clinical application prospects.

[0009] In summary, these limitations highlight the urgent need to develop novel autophagy-guided antibody-conjugate systems that can achieve efficient targeted degradation of membrane proteins while avoiding the inherent safety risks of the current AUTAB platform. Summary of the Invention

[0010] (a) Technical problems to be solved Therefore, one of the main objectives of this invention is to provide an antibody-oligonucleotide conjugate (AOC) and its applications. The antibody-oligonucleotide conjugate constructed in this invention, containing a guanylic acid residue (G), can guide cell surface proteins into the macroautophagy-lysosome pathway for selective proteolysis. After conjugation of the G4 structure with cetuximab (Ctx-G4), a concentration of only 1 nM is sufficient to potently degrade epidermal growth factor receptor (EGFR) in HeLa and A549 cells. Mechanistic analysis shows that Ctx-G4 is primarily taken up by cells via clathrin-mediated endocytosis and then enters the macroautophagy-lysosome pathway, a process independent of the ubiquitin-proteasome system. The modular nature of this platform is further validated by the successful degradation of HER2 and PD-L1 through simple antibody replacement, highlighting its broad applicability. In summary, the Ab-G4 platform provides a synthetically feasible and multifunctional strategy for directed degradation (TPD) of cell surface proteins, extending autophagy-driven degradation tools to extracellular proteins.

[0011] (II) Technical Solution To achieve the above objectives, the present invention provides an antibody-oligonucleotide conjugate, wherein the oligonucleotide comprises A, T, C and / or G.

[0012] In one embodiment, the antibody includes an EGFR antibody.

[0013] In one embodiment, the EGFR antibody includes cetuximab, panitumumab, nimotuzumab, and / or nexituzumab.

[0014] In one embodiment, the EGFR antibody is cetuximab.

[0015] In one embodiment, the number of oligonucleotides is 1 to 25.

[0016] In one embodiment, the oligonucleotide is G.

[0017] In one embodiment, the number of G is 1 to 14.

[0018] In one embodiment, the number of G is 4, 7, or 14.

[0019] In one embodiment, the number of G is 14.

[0020] In another aspect, the present invention provides a pharmaceutical composition comprising: (1) A therapeutically effective amount of the above-mentioned antibody-oligonucleotide conjugate; (2) Pharmaceutically or immunologically acceptable carriers or excipients.

[0021] In another aspect, the present invention provides a pharmaceutical preparation comprising the above-described pharmaceutical composition.

[0022] In another aspect, the present invention also provides a pharmaceutical product comprising the above-described pharmaceutical preparation.

[0023] In one embodiment, the pharmaceutical product includes a vial or box.

[0024] In another aspect, the present invention also provides the above-mentioned antibody-oligonucleotide conjugates, pharmaceutical compositions, pharmaceutical formulations and / or pharmaceutical products for use in the preparation of drugs for the prevention and / or treatment of tumors.

[0025] In one embodiment, the tumor includes EGFR, HER2, and / or PD-L1 mediated tumors.

[0026] In one embodiment, the tumor includes non-small cell lung cancer, breast cancer, gastric cancer, gastroesophageal junction adenocarcinoma, colorectal cancer, bile duct cancer, cervical cancer, urothelial carcinoma, esophageal cancer, head and neck squamous cell carcinoma, and / or lung adenocarcinoma.

[0027] (III) Beneficial Effects This invention provides an antibody-oligonucleotide conjugate and its applications. Compared with the prior art, it has the following advantages: 1. When the G4 structure is conjugated with cetuximab, only a concentration of 1 nM is required in HeLa and A549 cells to potently degrade the epidermal growth factor receptor (EGFR).

[0028] 2. It can achieve concentration-dependent degradation of HER2 and PD-L1, and has universal applicability. Attached Figure Description

[0029] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0030] Figure 1 This is a schematic diagram of the structure of an antibody-oligonucleotide conjugate; Figure 2 This is a graph showing the degradation efficiency of different oligonucleotides; Figure 3 This is a graph showing the degradation efficiency of Ctx-G4; Figure 4 It's CTX-G 14 Degradation efficiency results graph; Figure 5 This is a graph showing the results of the universality verification of antibody-oligonucleotide conjugates. Detailed Implementation

[0031] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0032] Terms and Definitions As used herein, the term "pharmaceutical composition" refers to a composition comprising an antibody-oligonucleotide conjugate formulated with one or more pharmaceutically acceptable carriers.

[0033] The formulation of a pharmaceutical composition can be tailored to the application. In particular, pharmaceutical compositions can be formulated using methods known in the art to provide rapid, continuous, or delayed release of the active ingredient upon administration to mammals. For example, the formulation can be selected from any of the following: plasters, granules, lotions, liniments, lemonade, aromatic water, powders, syrups, eye ointments, liquids and solutions, aerosols, sprays, extracts, elixirs, ointments, fluid extracts, emulsions, suspensions, decoctions, infusions, eye drops, tablets, suppositories, injections, alcoholic preparations, capsules, creams, lozenges, tinctures, pastes, pills, and soft or hard gelatin capsules.

[0034] As used herein, the term "pharmaceuticalally acceptable" refers to a substance that is suitable for use in humans and / or animals without excessive adverse effects (such as toxicity, irritation, and allergic reactions), i.e., a reasonable benefit / risk ratio.

[0035] As used herein, the term "pharmaceutically acceptable carrier" or "pharmaceutically acceptable excipient" refers to a carrier used for the administration of therapeutic agents, encompassing a variety of excipients and diluents. This term refers to pharmaceutical carriers that are not essential active ingredients themselves and do not cause excessive toxicity upon administration. Suitable carriers are well known to those skilled in the art, and a thorough discussion of pharmaceutically acceptable excipients can be found in Remington's Pharmaceutical Sciences (Mack Pub. Co., NJ 1991).

[0036] Pharmaceutically acceptable carriers in a composition include any and all solvents, dispersion media, preservatives, antioxidants, coatings, isotonic and absorption-delaying agents, surfactants, fillers, disintegrants, binders, diluents, lubricants, flow aids, pH adjusters, buffers, enhancers, wetting agents, solubilizers, surfactants, antioxidants, etc., compatible with drug administration. The use of such media and agents for pharmaceutically active substances is well known in the art. The composition may contain other active compounds that provide complementary, additional, or enhanced therapeutic functions. Solid carriers or excipients, such as lactose, starch, or talc, or liquid carriers, such as water, fatty oils, or liquid paraffin, are possible. Other examples of carriers include culture media, such as DMEM or RPMI; and cryogenic storage media containing components that scavenge free radicals, provide pH buffering, osmotic / osmotic support, energy substrates, and ion concentrations to balance intracellular states at low temperatures; and mixtures of organic solvents with water.

[0037] The dosage forms of the pharmaceutical compositions disclosed in this invention can be granules, tablets, lyophilized powders, suppositories, capsules, sublingual tablets, liquid solutions, nasal drops, sprays, and metered-dose sprays.

[0038] The pharmaceutical compositions of the present invention can be administered using any known method. One of a variety of methods known to those skilled in the art can be used to administer the substance, compound, or agent to a subject using the terms "give" or "apply".

[0039] For example, compounds or agents can be administered intranasally (e.g., by inhalation), intrathecally (into the spinal canal or subarachnoid space), intraarterially, intradermally, intramuscularly, intraperitoneally, intravenously, subcutaneously, ocularly, sublingually, orally (by ingestion), intracerebrally, and transdermally (by absorption, e.g., through a skin catheter). Compounds or agents can also be suitably introduced via rechargeable or biodegradable polymeric devices or other devices (e.g., patches and pumps or formulations) that provide prolonged, slowed, or controlled release of the compound or agent. Administration can also be performed, for example, once, multiple times, and / or over one or more prolonged periods.

[0040] As used herein, the term “therapeutic effective dose” refers to a dose sufficient to treat a disease with a reasonable benefit / risk ratio suitable for medical treatment, and the effective dose level includes subject type and severity, age, sex, drug activity, drug sensitivity, time of administration, route of administration and excretion rate, duration of treatment, factors including concomitant drugs, and other factors known in the medical field.

[0041] As used herein, the term “treatment” for a symptom or patient refers to steps taken to achieve a beneficial or desired outcome, including clinical outcomes. Beneficial or desired clinical outcomes include, but are not limited to, eliminating, substantially inhibiting, slowing, or reversing the progression of a disease, symptom, or condition; substantially improving or alleviating the clinical or aesthetic symptoms of a symptom; substantially preventing the clinical or aesthetic symptoms of a disease, symptom, or condition; and avoiding harmful or unpleasant symptoms. Treatment also refers to accomplishing one or more of the following: (a) reducing the severity of the symptom; (b) limiting the development of characteristic symptoms of the symptom being treated; (c) limiting the exacerbation of characteristic symptoms of the symptom being treated; (d) limiting the recurrence of the symptom in patients who previously had the symptom; and / or (e) limiting the recurrence of symptoms in patients who previously did not have symptoms of the symptom.

[0042] As used in this article, the term "prevention" refers to reducing the likelihood of the onset (or recurrence) of a disease, disorder, condition, or associated symptoms.

[0043] As used in this article, “containing,” “having,” or “including” includes “containing,” “mainly composed of,” “substantially composed of,” and “composed of”; “mainly composed of,” “substantially composed of,” and “composed of” are subordinate concepts of “containing,” “having,” or “including.”

[0044] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, and the reagents, methods and equipment used are conventional reagents, methods and equipment in this technical field.

[0045] Example 1: Preparation of antibody-oligonucleotide conjugates: 1. Antibody labeling: Cetuximab (Ctx) was dissolved in 50mM phosphate buffer (pH=7.4) to prepare an antibody solution (5~100μM, preferably 30μM). A chemical linker (preferably DBCO-PEG4-NHS ester) was added to the solution, and the mixture was reacted at 25°C for 24h. The solution was then dialyzed using a 100kDa MWCO dialysis bag, and the retentate was the labeled antibody. The molar ratio of antibody to chemical linker in the antibody solution was 1:25. 2. The obtained antibody marker was prepared to a preferred concentration of 10 μM, and 25 equivalents of different azide-modified oligonucleotides (A, T, C and / or G) were added. The mixture was shaken at 25 °C for 48 h. The reaction solution was dialyzed using a 100 kDa MWCO dialysis bag. The retentate was collected to obtain the antibody-oligonucleotide conjugate. The protein labeling status could be detected by gel imaging and the sample concentration could be detected by BCA method.

[0046] A schematic diagram of the antibody-oligonucleotide conjugate structure is shown below. Figure 1 As shown.

[0047] Example 2: Screening of oligonucleotide species: Human cervical cancer cells (HeLa) were seeded into 12-well plates, and 1000 μL of DMEM medium containing 10 vol% FBS and 1 wt% penicillin and streptomycin (final concentrations of 100 units / ml penicillin and 100 μg / ml streptomycin) was added. The plates were incubated at 37°C until the cell density reached 70-85%. The medium was removed, the cells were washed once with PBS, and then Ctx-A was added. 25 / T 25 / C 25 / G 14 Alternatively, cells were incubated in DMEM medium containing Ctx (10 nM) at 37°C for 24 h, and then collected for immunoblotting experiments.

[0048] The results are as follows Figure 2 As shown in A, only Ctx-G 14 It can significantly reduce the content of EGFR protein.

[0049] Example 3 Screening for oligonucleotide numbers: Human cervical cancer cells (HeLa) were seeded into 12-well plates, and 1000 μL of DMEM medium containing 10 vol% FBS and 1 wt% penicillin and streptomycin (final concentrations of 100 units / ml penicillin and 100 μg / ml streptomycin) was added. The plates were incubated at 37°C until the cell density reached 70-85%. The medium was removed, the cells were washed once with PBS, and then filled with a solution containing Ctx-G4 / G7 / G... 14 Alternatively, cells were incubated in DMEM medium (10 nM Ctx) at 37°C for 24 hours, and then collected for Western blotting. Results are as follows: Figure 2As shown in B, Ctx-G4 / G7 / G 14 All of these significantly reduced the content of EGFR protein. Furthermore, different concentrations of Ctx-G4 / G... 14 Co-incubation with cells revealed a positive correlation between degradation efficiency and concentration; Ctx-G4 at a concentration of 1 nM was sufficient to potently degrade EGFR. Figure 3 , 4 ).

[0050] Example 4: Validation of the universality of antibody-oligonucleotide conjugates: Following the method in Example 1, a degradative agent capable of targeting and degrading HER2 and PD-L1 was prepared by replacing cetuximab with pertuzumab / durvalumab, and its ability to degrade these targets was verified at the live cell level. The results are as follows: Figure 5 As shown, it can achieve concentration-dependent degradation of HER2 and PD-L1.

[0051] It should be noted that, in this document, relational terms such as "first" and "second" are used only to distinguish one entity or operation from another, and do not necessarily require or imply any such actual relationship or order between these entities or operations. Furthermore, the terms "comprising," "including," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such a process, method, article, or apparatus. Without further limitations, an element defined by the phrase "comprising one..." does not exclude the presence of other identical elements in the process, method, article, or apparatus that includes said element.

[0052] The above embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit it. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. An antibody-oligonucleotide conjugate, characterized in that, The oligonucleotides include A, T, C and / or G.

2. The antibody-oligonucleotide conjugate according to claim 1, characterized in that, The antibodies include EGFR antibodies, HER2 antibodies, and / or PD-L1 antibodies.

3. The antibody-oligonucleotide conjugate according to claim 2, characterized in that, The EGFR antibodies include cetuximab, panitumumab, nimotuzumab, and / or nexituzumab.

4. The antibody-oligonucleotide conjugate according to claim 1, characterized in that, The number of oligonucleotides is 1 to 25.

5. The antibody-oligonucleotide conjugate according to claim 1, characterized in that, The oligonucleotide is G.

6. The antibody-oligonucleotide conjugate according to claim 5, characterized in that, The number of G is 1 to 14.

7. A pharmaceutical composition, characterized in that, include: (1) A therapeutically effective amount of the antibody-oligonucleotide conjugate according to any one of claims 1 to 6; (2) Pharmaceutically or immunologically acceptable carriers or excipients.

8. A pharmaceutical preparation, characterized in that, Includes the pharmaceutical composition of claim 7.

9. A pharmaceutical product, characterized in that, Includes the pharmaceutical preparation described in claim 8.

10. The use of the antibody-oligonucleotide conjugate of any one of claims 1 to 6, the pharmaceutical composition of claim 7, the pharmaceutical formulation of claim 8, and / or the pharmaceutical product of claim 9 as a preparation of a drug for the prevention and / or treatment of tumors.

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