Bispecific antibody for cell surface receptor-based endocytosis-mediated extracellular antigen degradation

Abstract

The present invention relates to the technical field of biomedicine, and specifically to a bispecific antibody for cell surface receptor-based endocytosis-mediated extracellular antigen degradation. Provided is a bispecific antibody binding to a cell surface receptor and a target protein, the bispecific antibody comprising a first antigen binding portion binding to the target protein and a second antigen binding portion binding to the cell surface receptor. The cell surface receptor is a cell surface receptor capable of mediating endocytosis and recycling. The provided bispecific antibody can bind to both a target protein such as an extracellular soluble protein or a pathological protein aggregate and a cell surface receptor to form a ternary complex. By means of the endocytosis mediated by the cell surface receptor, the antigen is separated from the antibody in an acidic environment and degraded, and the antibody is recycled by means of the cell surface receptor, thereby exhibiting a significant clearance effect on the extracellular soluble protein or the pathological protein aggregate, providing a new therapeutic solution for the treatment of related diseases.

Description

Bispecific antibodies based on cell surface receptor endocytosis-mediated degradation of extracellular antigens Technical Field

[0001] This application relates to the field of biomedical technology, specifically to a bispecific antibody based on cell surface receptor endocytosis-mediated degradation of extracellular antigens. Background Technology

[0002] Neutralizing antibodies are an effective treatment for diseases caused by abnormally high levels of cytokines or extracellular proteins, such as cytokines like TNF-α, IL-4, IL-6, IL-12, IL-17, and VEGF; extracellular pathogenic factors like IgE and C5; and other rapidly degradable soluble antigens, including PCSK9, MCP1, β-amyloid, hepcidin, α-synuclein, TREM2, and Tau. However, traditional neutralizing antibodies have limitations: they cannot degrade antigens, leading to antigen accumulation in the blood, and each antibody can only neutralize and clear one antigen at a time.

[0003] Taking IgE as an example, clinically common IgE elevations are mainly seen in allergic diseases such as allergic rhinitis, bronchial asthma, and atopic conjunctivitis, as well as parasitic infections, inflammatory diseases, primary immunodeficiency, malignant tumors, cardiovascular diseases, and psoriasis. IgE binds to its high-affinity receptor FcεRI and is mainly involved in type I hypersensitivity reactions. IgE-related allergic diseases can be treated with antibody neutralization, such as omalizumab. Omalizumab is a recombinant humanized anti-IgE monoclonal antibody and is the first targeted drug for the treatment of asthma. It inhibits the binding of IgE to FcεRI by neutralizing free IgE, thereby inhibiting the type I hypersensitivity reaction caused by the activation of corresponding effector cells. However, the complex formed by omalizumab and IgE, once inside the cell via endocytosis, cannot be completely degraded. Instead, it returns to the extracellular space through FcRn-mediated recycling, leading to the continuous accumulation of the IgE-omalizumab complex in the patient's blood. This necessitates high doses of omalizumab, increasing the risk of adverse events and the patient's treatment burden. Furthermore, discontinuation of the drug can easily cause a rebound in the patient's condition.

[0004] Taking PCSK9 as an example, PCSK9, short for proprotein convertase subtilisin / kexin type 9, is mainly produced by the liver and regulates cholesterol homeostasis. Normally, low-density lipoprotein cholesterol (LDL-C) in the blood binds to LDL-R receptors on the cell membrane surface. After being endocytosed into the cell, it separates in the endoplasmic reticulum. LDL-C is then digested and degraded by lysosomes, while LDL-R circulates back to the cell. However, PCSK9 competitively binds to LDL-R on the cell surface, forming a PCSK9-LDL-R complex, which is then endocytosed and degraded, thus inhibiting LDL-R circulation. This reduces the number of LDL-R on the cell membrane, preventing LDL-C from entering the liver for metabolism, leading to elevated plasma LDL-C levels and further causing hypercholesterolemia, a leading risk factor for atherosclerotic cardiovascular disease. PCSK9 monoclonal antibodies, such as evolocumab and alilicumab, can bind to free PCSK9 in plasma, inhibit PCSK9-mediated LDL-R degradation, and reduce plasma LDL-C levels, thereby achieving a lipid-lowering effect. However, PCSK9 monoclonal antibodies also have the problem that antibody-antigen complexes, after entering cells via endocytosis, cannot be completely degraded, but instead recycle back into the extracellular space through FcRn binding.

[0005] As can be seen, when traditional neutralizing antibodies bind to soluble antigens, they form antigen-antibody immune complexes. Unlike free antigens, which are taken up by cells and transported to lysosomes for complete degradation, a portion of the antigen-antibody complex enters the lysosome for degradation, while the remainder returns to the plasma through FcRn-mediated recycling due to the interaction between the antibody's Fc terminus and the neonatal Fc receptor (FcRn). This leads to the accumulation of antigen-antibody complexes in the plasma. Furthermore, the patient's body continuously produces and secretes antigens, resulting in high antigen concentrations in the blood or extracellular fluid. Therefore, high doses of neutralizing antibodies are required to ensure effective treatment, sometimes exceeding the clinically achievable range. The above process also shows that traditional neutralizing antibodies can only bind to an antigen once and cannot clear total antigens from the plasma. Traditional blocking antibodies, such as adalimumab, can specifically bind to soluble targets to slow disease progression. However, traditional antibodies have some limitations. Traditional therapeutic antibodies bind to only two target antigen molecules during their lifespan. Therefore, when antigens are abundant, large doses of conventional antibodies are needed to completely neutralize their function. For example, the recommended dose of eculizumab for treating paroxysmal nocturnal hemoglobinuria is approximately 1000 mg every two weeks. Furthermore, because antibody-antigen complexes are recycled by FcRn in endosomes, antibody binding to antigens can significantly prolong the half-life of the antigen. It has been observed that total IgE accumulates to 5-10 times the basal IgE level after administration.

[0006] To address these issues, various extracellular targeted protein degradation (eTPD) platforms have been developed, degrading extracellular antigens by transporting them to lysosomes for degradation. Igawa et al. developed "sweeping technology," where antibodies exhibit pH-responsive binding to antigens, releasing the bound antigens in acidic endosomes while the antibody remains attached to the FcRn and recycles back to the plasma membrane. Further enhancement of antibody internalization rates can be achieved by modifying the Fc region to have higher affinity for FcRn or FcγR2B receptors. Their work led to the approval of two drugs, satralizumab and SKY59. Bertozzi and colleagues pioneered the use of glycan-targeted recycling receptors, such as the cation-independent mannose-6-phosphate receptor (CI-M6PR) or ASGPR, to facilitate lysosomal transport and degradation of target proteins, constructing LYTAC. The LYTAC molecule forms a ternary complex with the target protein and the lysosomal receptor, inducing the internalization of this complex. Upon internalization, the LYTAC molecule dissociates from the receptor in an acidic environment, leading to the degradation of the target protein and the LYTAC molecule in the lysosome, and the recycling of the lysosomal receptor. Around the same time, some research groups created bifunctional molecules by linking ASGPR-targeting small molecules or aptamers to another molecule that binds to the protein of interest.

[0007] Compared to traditional neutralizing antibodies, eTPD antibodies offer significant advantages. First, an eTPD antibody can catalyze antigen degradation via lysosomal-mediated protein degradation, thus avoiding the accumulation of antigen-antibody immune complexes. Second, eTPDs can eliminate some antigens resistant to conventional blocking antibodies, such as β-amyloid (Aβ). However, current eTPD systems still have room for improvement. Sweep-through antibodies can compete with the large amounts of naturally occurring immunoglobulins present in plasma and tissues, and antibody internalization via Fc region binding to cell surface Fc receptors is inefficient. Glycan-based degraders, such as LYTAC, are difficult to internalize, requiring extensive glycan modification for effective internalization. Glycan-based degradation products separate from internalized receptors and cannot be effectively recovered, leading to target-mediated drug clearance. Furthermore, the heterogeneous bioconjugation of sugars and antibodies increases the difficulty of development and manufacturing. Whether degradation products interfere with the endogenous function of certain receptors remains to be investigated. Furthermore, LYTACs molecules are also degraded within lysosomes, still exhibiting the limitation that an antibody molecule can only degrade one antigen. LYTACs are limited to the types of receptors targeted by lysosomes, and the development of glycopeptides targeting receptors and their connection to antibodies via in vitro conjugation makes the production process complex. Summary of the Invention

[0008] After conventional neutralizing antibodies bind to antigens, the antigen-antibody complex enters the endosome with low efficiency through phagocytosis or macrophage. Under acidic pH conditions, the antibody cannot separate from the antigen, and the antigen-antibody complex is recycled back to the extracellular space via FcRn, accumulating continuously in the blood. Based on the shortcomings of the prior art, this application proposes a design strategy for bispecific antibodies that can bind to two antigen sites, based on cell membrane receptor endocytosis-mediated degradation of extracellular antigens. One end of the bispecific antibody targets the cell membrane receptor that mediates endocytosis; by screening for antibodies with strong endocytosis, the degradation and clearance of the antigen-antibody complex are accelerated. The other end targets the extracellular target protein, and through antibody engineering, it is modified to have pH-dependent antigen-binding properties. The novel bispecific antibody designed in this application can simultaneously bind to extracellular antigens and a recyclable cell surface receptor that mediates endocytosis, forming a ternary complex. Through endocytosis mediated by this cell surface receptor, efficient endocytosis of the complex is promoted. The extracellular antigen separates from the antibody in the acidic environment of the endocytic vesicles, and the antigen enters the lysosomal degradation pathway, while the antibody is recycled through the cell surface receptor. Therefore, in this application, a single bispecific antibody can efficiently degrade multiple antigens, thereby improving the antibody's efficiency in inhibiting antigens and reducing the effective concentration of the antibody. Based on the research in this application, it is anticipated that antibodies binding to different circulating cell surface receptors that mediate endocytosis can be arbitrarily combined with antibodies binding to different target proteins at the other end, achieving the same effect as described in this application.

[0009] The first aspect of this application provides a bispecific antibody that binds to a cell surface receptor and a target protein, comprising a first antigen-binding portion that binds to the target protein and a second antigen-binding portion that binds to the cell surface receptor, wherein the cell surface receptor is a cyclic cell surface receptor capable of mediating endocytosis, and the cell surface receptor is selected from FcγR2B receptor, ASGPR receptor, EGFR receptor, TfR receptor, HSPG receptor, IGF2R receptor, and CI-M6PR receptor.

[0010] The target protein is selected from extracellular soluble proteins or pathological protein aggregates.

[0011] The first antigen-binding portion binds to the target protein in a pH-dependent or pH-independent manner, preferably in a pH-dependent manner.

[0012] A second aspect of this application provides a polynucleotide that encodes the bispecific antibody described in the first aspect above.

[0013] A third aspect of this application provides a construct containing the polynucleotide described in the second aspect above.

[0014] A fourth aspect of this application provides a host cell containing the construct described in the third aspect above, or the host cell genome integrating the polynucleotides described in the second aspect above.

[0015] The fifth aspect of this application provides a drug or kit comprising the bispecific antibody described in the first aspect above.

[0016] The sixth aspect of this application provides the use of the bispecific antibody described in the first aspect above, or the polynucleotide described in the second aspect above, or the construct described in the third aspect above, or the host cell described in the fourth aspect above, or the drug or kit described in the fifth aspect above, in the preparation of a drug for the prevention and / or treatment of diseases related to target protein overexpression.

[0017] The seventh aspect of this application provides a method for preventing and / or treating diseases related to target protein overexpression.

[0018] The target protein overexpression-related diseases are selected from diseases associated with IgE overexpression, PCSK9 overexpression, TNF-α overexpression, IL-4 overexpression, IL-6 overexpression, IL-12 overexpression, IL-17 overexpression, VEGF overexpression, C5 overexpression, MCP1 overexpression, β-amyloid overexpression, hepcidin overexpression, α-synuclein overexpression, TREM2 overexpression, and Tau overexpression.

[0019] The beneficial effects of this application are as follows:

[0020] Traditional monoclonal antibodies (MCIs) form complexes with antigens that are endocytosed into the endosome. In the acidic environment of the endosome, the antigen and antibody remain bound. Subsequently, the antibody is circulated out of the cell by FcRn, and the antigen circulates along with it, leading to serum antigen accumulation. In contrast, the bispecific antibody provided in this application binds to the antigen and enters the endosome through highly efficient endocytosis via a circulating cell surface receptor that mediates endocytosis. In the acidic environment, the antigen and antibody dissociate, the antigen enters the lysosome for degradation, and the antibody circulates out of the cell via the cell surface receptor, continuing to exert its effect.

[0021] The bispecific antibody of this application targets membrane receptors that mediate endocytosis at one end, accelerating the degradation and clearance of antigen-antibody complexes by screening for antibodies with strong endocytosis activity. The other end of the antibody targets extracellular target proteins and is engineered to possess pH-dependent antigen-binding properties. The bispecific antibody can simultaneously bind to extracellular antigens and recyclable cell surface receptors that mediate endocytosis, forming a ternary complex. Through endocytosis mediated by these cell surface receptors, efficient endocytosis of the complex is promoted. The extracellular antigen separates from the antibody in the acidic environment of the endocytic vesicles, and the antigen is degraded via lysosomes, while the antibody is recycled through the cell surface receptors. Furthermore, the one-end antibody, which binds to different recyclable cell surface receptors that mediate endocytosis, can be arbitrarily combined with the other-end antibody, which binds to different extracellular soluble proteins or pathological protein aggregates, achieving the desired effect. It can be used to degrade different extracellular soluble proteins or pathological protein aggregates, and has therapeutic effects on diseases with overexpression of these target proteins.

[0022] The bispecific antibody of this application can efficiently bind to cyclic cell surface receptors that mediate endocytosis, such as FcγR2B receptor, ASGPR receptor, EGFR receptor, TfR receptor, HSPG receptor, IGF2R receptor, and CI-M6PR receptor, thereby promoting the endocytosis of the bispecific antibody. Simultaneously, the bispecific antibody can efficiently bind to target proteins such as extracellular soluble proteins or pathological protein aggregates. After antibody endocytosis, these target proteins are effectively degraded, and under acidic conditions, the antigen and antibody separate, allowing the antibody to be reused. Therefore, this application allows a single bispecific antibody to efficiently degrade multiple antigens, thereby improving the antibody's efficiency in inhibiting antigens. The effective concentration of the antibody is reduced, and the bispecific antibody of this application has high safety. It has a significant effect on clearing exogenous and endogenous target proteins in cells and mice that overexpress target proteins. Therefore, it is expected to provide a new solution for the prevention and treatment of diseases with overexpression of target proteins such as IgE, PCSK9, TNF-α, IL-4, IL-6, IL-12, IL-17, VEGF, C5, MCP1, β-amyloid, hepcidin, α-synuclein, TREM2, and Tau, such as allergic diseases, parasitic infections, inflammatory diseases, primary immunodeficiency, malignant tumors, cardiovascular diseases, psoriasis, and atherosclerotic cardiovascular diseases induced by hyperlipidemia. Attached Figure Description

[0023] Figure 1 shows the screening and characterization results of hFcγR2B-binding single-chain antibodies. Figure 1a is a schematic diagram of phage display screening of single-chain antibodies targeting FcγR2B. Figure 1b shows the ELISA experiment evaluating the binding ability of single-chain antibody-Fc fusion protein (SKX-Fcs) to human FcγR2B; SKX-Fcs were serially diluted and added to a solid-phase carrier coated with hFcγR2B. Figure 1c shows the flow cytometry analysis of Huvec-2b cells; Huvec or Huvec-2b cells were co-incubated with NK-2-12-SK3, and flow cytometry was performed using Alexa Fluor. TM Detection of NK-2-12-SK3 binding using 488-labeled goat anti-human IgG (H+L) antibody.

[0024] Figure 2 shows the construction and characterization of FcRTAC for IgE degradation; Figure 2a is a schematic diagram of the mechanism of action of FcRTAC. FcRTAC binds to the target antigen at one end and FcγR2B at the other, effectively triggering FcγR2B receptor-mediated internalization. After internalization, the FcRTAC-antigen complex is transported to acidic endosomes, where the antigen dissociates from FcRTAC based on pH-dependent binding characteristics and then targets lysosomes for proteolytic degradation. The FcRTAC-FcγR2B complex returns to the cell surface for subsequent antigen capture cycles. Figure 2b shows the flow cytometry analysis of the binding of single-chain antibody-Fc fusion protein (SKX-Fcs) to hFcγR2B on the surface of Huvec-2b cells. Huvec-2b cells or control Huvec cells were co-incubated with different SKX-Fcs, and the binding was detected using AF488-labeled secondary antibodies. Fluorescence intensity was measured by flow cytometry to assess the binding ability of SKX-Fcs to FcγR2B; Figure 2c shows the internalization assay of SKX-Fcs. SKX-Fcs were premixed with an anti-human Fc fragment labeled with a pH-sensitive dye and co-incubated with Huvec-2b cells. When internalization occurred, the pH-sensitive dye emitted red fluorescence in acidic endosomes. Fluorescence changes were monitored hourly over 24 hours; Figure 2d shows the SPR analysis of antibody binding to hFcγR2B at pH 7.4 and pH 6.0. SK3-Fc or SK4-Fc was immobilized on a protein A biosensor chip, allowing hFcγR2B to flow through the detection channel. Solid lines represent raw data, and dashed lines represent fitted curves; Figure 2e compares the internalization efficiency of different FcRTAC conformations; Figures 2f and 2g show the SPR analysis of FcRTAC binding to FcγR2B and IgE. FcRTAC was immobilized on a protein A biosensor chip, and hFcγR2B and IgE were injected sequentially; Figure 2h shows a live-cell imaging experiment mediated by FcRTAC-mediated IgE internalization. Cy5-labeled IgE (red) was co-incubated with Huvec-2b cells for 1 hour in the presence or absence of FcRTAC, and the cell nuclei were counterstained with Hoechst (blue). Scale bar: 20 μm.

[0025] Figure 3 shows the effect of IgE targeting FcRTAC in clearing human IgE in a mouse model. Figure 3a is a schematic diagram of the IgE clearance experiment. Figures 3b-3g: FcγR2B humanized mice (be; n=5) or FcγR2B knockout mice (f, g; n=4) were injected with 5 mg / kg FcRTACs, followed by 2.5 mg / kg IgE 30 minutes later. Blood samples were collected at specified time points (red arrows) to detect serum total IgE (b, d, f, g) and free IgE (c, e) levels. Data are expressed as mean ± standard error. Statistical significance was determined using two-way ANOVA (P < 0.05, P < 0.01, P < 0.001, ****P < 0.0001). The curves for the "IgE only," "omalizumab," and "NK-2-12" groups share data between figures b and d, and between figures c and e.

[0026] Figure 4 shows the toxicity assessment of FcRTAC; Figures 4a-4c show the blood samples collected from FcγR2B humanized mice after a single intravenous injection of the antibody (10 mg / kg) 24 hours later for biochemical analysis (n=3); Figure 4d shows the hematoxylin-eosin staining results of tissues from mice treated with FcRTACs. Scale bar: 200 μm.

[0027] Figure 5 shows the PCSK9 clearance activity of PCSK9-targeted FcRTACs in a mouse model. Figure 5a shows the binding affinity of pH-insensitive and pH-sensitive PCSK9-targeting antibodies (300N, L300H) or FcRTACs (300N-SK3, L300H-SK3) to PCSK9 at pH 6.0 and pH 7.4. Human PCSK9 was immobilized on a CM5 biosensor chip, and the antibody solution flowed through the detection channel. Solid lines represent raw data, and dashed lines represent fitted curves. Figure 5b shows the quantitative detection of serum PCSK9 concentration (n=5) at multiple time points after intraperitoneal injection of 5 mg / kg PCSK9 neutralizing antibodies (300N, L300H) or PCSK9-targeted FcRTACs (300N-SK3, L300H-SK3) into humanized PCSK9 mice. Data are expressed as mean ± standard error, and statistical comparisons were performed using two-way ANOVA (P<0.05, P<0.01, P<0.001, ****P<0.0001).

[0028] Figure 6 shows the antigen clearance efficiency of FcRTAC constructed using affinity-matured FcγR2B-binding antibody; Figure 6a shows that FcRTAC constructed using the affinity-matured FcγR2B-binding antibody mutant SK3hi exhibits enhanced and sustained antigen clearance efficiency. Affinity assays of SK3 and SK3hi binding to FcγR2B were performed based on spr at pH 7.4 and pH 6.0. SK3 or SK3hi was immobilized on a protein A biosensor chip, and then FcγR2B was injected into the mobile phase. Solid lines represent raw data, and dashed lines represent fitted curves; Figure 6b shows live-cell imaging of NK-2-12-sk3hi-mediated IgE endocytosis. Cy3-labeled IgE (red) was incubated with Huvec-2b cells for 2 hours with or without Cy5-labeled NK-2-12-SK3hi (white). Cells were confocally imaged using LysoTracker (green) and Hoechst (blue) staining. Images were acquired using super-resolution confocal microscopy. Scale bar: 5 μm; Figures 6c-6d show the antibody rechallenge experiment. One hour after injection of NK-2-12-SK3 or NK-2-12-SK3hi, FcγR2B humanized mice were first injected with 2.5 mg / kg IgE, and one day later, the mice were injected again with 2.5 mg / kg IgE. Blood samples were collected to measure serum IgE levels. Data are expressed as mean ± SEM. Two-way ANOVA was used for statistical comparison (*P<0.05, **P<0.01, ***P<0.001, ****P<0.0001).

[0029] Figure 7 shows the efficacy evaluation of NK-2-12-SK3ph; Figure 7a compares the binding of NK-2-12-SK3 and NK-2-12-SK3ph to FcγR2B using SPIR-based affinity measurements under the same experimental conditions at pH 7.4 and pH 6.0. Antibodies were immobilized on a protein a biosensor chip, and then FcγR2B was injected into the mobile phase. Solid curves represent the original sensor plot, and dashed curves represent the fitted binding model. The binding affinity of NK-2-12-SK3, NK-2-12-SK4, NK-2-12-SK3hi, and NK-2-12-SK3ph to FcγR2B was determined in the same experiment; Figure 7b shows the IgE clearance assay in humanized FcγR2B mice. Humanized FcγR2B mice were intraperitoneally injected with 5 mg / kg fcrtac. 30 minutes later, IgE 2.5 mg / kg was injected. Serum total IgE levels were quantitatively measured at specified time points (n=5); Figure 7c shows a pharmacokinetic comparison of NK-2-12-SK3 and NK-2-12-SK3ph (n=5); Figure 7d proposes the mechanism of action of NK-2-12-SK3ph. NK-2-12-SK3ph binds to IgE at one end and FcγR2B at the other. This dual binding triggers FcγR2B-mediated complex endocytosis. After internalization, the FcγR2B / NK-2-12-SK3ph / IgE ternary complex dissociates in an acidic endosome environment. The released IgE is then guided to lysosomes for proteolytic degradation, while NK-2-12-SK3ph binds to FcRn in the endosome. The FcRn / NK-2-12-SK3ph complex then cycles to the cell surface, and NK-2-12-SK3ph is released back into circulation upon exposure to physiological pH (7.4), thus achieving multiple rounds of target binding. Data are expressed as mean ± SEM. Two-way ANOVA was used to determine statistical significance (*P<0.05, **P<0.01, ***P<0.001, ****P<0.0001).

[0030] Figure 8 shows the internalization detection of the mutant antibody in the HUVEC-2B cell line.

[0031] Figure 9 shows the experimental results of NK-2-12-SK3 NGR mice; Figure 9A shows the results of the IgE clearance experiment in 2B mice; Figure 9B shows the half-life detection.

[0032] Figure 10 shows a comparison between FcRTAC and cross-scan antibody technology; Figure 10a is a schematic diagram of the NK-2-12-V12 structure; Figure 10b shows the results of the cell internalization experiment; Figure 10c assesses the contribution of different endocytic pathways of NK-2-12-V12; Figure 10d is an immunofluorescence microscopy observation; Figure 10e is a high-resolution microscopy observation; Figure 10f shows the results of the IgE clearance experiment; Figure 10g is a pharmacokinetic comparison of NK-2-12-SK3 and NK-2-12-V12.

[0033] Figure 11 shows the screening and characterization results of ASGPR-binding single-chain antibodies; Figure 11a shows the identification of ASGPR-binding single-chain antibodies. After ASGPR-targeting biopanning of a human single-chain antibody phage display library, ASGPR-specific single-chain antibodies were screened using phage ELISA technology; Figure 11b shows the internalization experiment of the single-chain antibody-Fc fusion protein. AT-Fc (single-chain antibody-Fc fusion protein) was pre-incubated with pH-sensitive dye-labeled anti-human Fc antibody and then added to HepG2 cells expressing ASGPR. Fluorescence intensity changes were monitored hourly over 24 hours; Figure 11c shows the amino acid sequences of AT5 and AT10; Figure 11d is a schematic diagram of the design of an ASGPR-targeting IgE degrading agent. An NK-2-12-AT degrading agent was constructed by fusing an ASGPR-binding, internalization-triggering single-chain antibody with the IgE-targeting antibody NK-2-12; Figure 11e shows the internalization of the ASGPR-targeting degrading agent. The efficiency of NK-2-12-AT-mediated internalization was assessed; Figure 11f shows live-cell imaging of IgE and IgE degrading agents internalized. HepG2 cells were co-incubated for 12 hours with Cy3-labeled IgE (red) and Cy5-labeled NK-2-12-AT5 and NK-2-12-AT10 (white). Lysosomes and nuclei were labeled using LysoTracker (green) and Hoechst (blue), respectively. Images were acquired using a 40x objective lens on a rotating confocal microscope (scale bar: 40 μm); Figure 11g shows the IgE clearance effect of ASGPR-targeted IgE degrading agents. Wild-type mice were intraperitoneally injected with 5 mg / kg NK-2-12, NK-2-12-AT5, NK-2-12-AT10, or NK-2-12-SK3, followed by 2.5 mg / kg IgE 30 minutes later. Serum IgE levels were quantitatively measured at specified time points (n=5). Data are expressed as mean ± standard error, and two-way ANOVA is used in conjunction with... Statistical analysis was performed using multiple comparison tests (P<0.05, P<0.01, P<0.001, ****P<0.0001). Detailed Implementation

[0034] The following specific examples illustrate the implementation of the present invention. Those skilled in the art can easily understand other advantages and effects of the present invention from the content disclosed in this specification. The present invention can also be implemented or applied through other different specific embodiments, and various details in this specification can also be modified or changed based on different viewpoints and applications without departing from the spirit of the present invention.

[0035] Before further describing specific embodiments of the present invention, it should be understood that the scope of protection of the present invention is not limited to the specific embodiments described below; it should also be understood that the terminology used in the embodiments of the present invention is for describing specific embodiments and not for limiting the scope of protection of the present invention; in the specification and claims of the present invention, unless otherwise expressly stated in the text, the singular forms "a", "an" and "this" include the plural forms.

[0036] When numerical ranges are given in the embodiments, it should be understood that, unless otherwise stated in the present invention, both endpoints of each numerical range and any value between the two endpoints may be selected. Unless otherwise defined, all technical and scientific terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art. In addition to the specific methods, apparatus, and materials used in the embodiments, based on the knowledge of the prior art possessed by one of ordinary skill in the art and the description of this invention, any prior art methods, apparatus, and materials similar to or equivalent to those described, apparatus, and materials in the embodiments of this invention may be used to implement the present invention.

[0037] This application utilizes the characteristic that bispecific antibodies can bind to two antigen sites to propose a design strategy for bispecific antibodies based on cell membrane receptor endocytosis-mediated degradation of extracellular antigens. One end of the bispecific antibody targets the membrane receptor that mediates endocytosis, and the degradation and clearance of antigen-antibody complexes are accelerated by screening for antibodies with strong endocytosis. The other end targets extracellular target proteins, and the antibody is engineered to have pH-dependent antigen-binding properties. The novel bispecific antibody designed in this application can simultaneously bind to extracellular antigens and a recyclable cell surface receptor that mediates endocytosis, forming a ternary complex. Through endocytosis mediated by this cell surface receptor, the complex is promoted to achieve efficient endocytosis. The extracellular antigen separates from the antibody in the acidic environment of the endocytic vesicles and enters the lysosomal degradation pathway, while the antibody is recycled through the cell surface receptor. Thus, a single bispecific antibody can efficiently degrade multiple antigens, thereby improving the antibody's efficiency in inhibiting antigens and reducing the effective concentration of the antibody. Based on the research in this application, it is expected that antibodies binding to different recyclable cell surface receptors that mediate endocytosis can be arbitrarily combined with antibodies binding to different target proteins to achieve the same effect as described in this application.

[0038] This application first provides a bispecific antibody that binds to a cell surface receptor and a target protein, comprising a first antigen-binding portion that binds to the target protein and a second antigen-binding portion that binds to the cell surface receptor, wherein the cell surface receptor is a cyclic cell surface receptor capable of mediating endocytosis.

[0039] Bispecific antibodies are antibodies capable of simultaneously recognizing and binding to two different antigens or epitopes, possessing two distinct antigen-binding sites (i.e., two different Fab fragments). Therefore, in a specific embodiment of this application, the bispecific antibody includes a first antigen-binding portion and a second antigen-binding portion that respectively recognize and bind to two antigens. The antigen-binding portion refers to the region in the antibody molecule that binds to a specific antigenic epitope. The first antigen-binding portion specifically recognizes and binds to extracellular target proteins, while the second antigen-binding portion specifically recognizes and binds to cyclic cell surface receptors capable of mediating endocytosis.

[0040] Endocytosis refers to the process by which cells invaginate their cell membranes to form vesicles, bringing extracellular substances into the cell. Receptor-mediated endocytosis (RME) typically involves specific cell surface receptors that recognize and bind to specific ligands, triggering the endocytosis process. Cyclicity refers to the process by which cell surface receptors, after binding to ligands, are transported into the cell via endocytosis and subsequently recycled back to the cell surface. Cyclic cell surface receptors that mediate endocytosis are those that, after binding to ligands, enter the cell via endocytosis and can be reclaimed from the ligand within the endosome and returned to the cell surface.

[0041] The cell surface receptor is selected from any one of the following: FcγR2B receptor, ASGPR receptor, EGFR receptor, TfR receptor, HSPG receptor, IGF2R receptor, and CI-M6PR receptor. This application only provides examples.

[0042] In a specific embodiment of this application, the second antigen-binding portion includes a single-chain antibody or a fragment thereof. The single-chain antibody may be scFv.

[0043] scFv is composed of single-chain variable region fragments, which are formed by linking heavy chain variable regions and light chain variable regions through a flexible short peptide using genetic engineering technology. The scFv includes heavy chain variable regions and light chain variable regions, and the variable regions include complementarity-determining regions (CDRs).

[0044] FcγRII is a single-chain glycoprotein molecule with a molecular weight of approximately 40 kDa. Normally, FcγR2B receptors recognize and bind to the Fc fragment of IgG antibodies; this binding is typically low-affinity. The antibody-antigen complex binds to multiple FcγR2B receptors via the Fc fragment, leading to receptor aggregation. This aggregation triggers intracellular signaling pathways, activating a series of signaling events, including the activation of protein tyrosine kinases, which further activate other signaling molecules. Signaling results in the formation of an endocytic vesicle containing the immune complex around the receptor-antibody-antigen complex on the cell membrane, isolating it from the extracellular environment. After endocytosis, the vesicle undergoes maturation, fusing with early endosomes and then moving into the late endosome and lysosomal systems within the cell. Inside the endocytic vesicle, the immune complex is broken down, and the antigen is processed into small peptide fragments.

[0045] ASGPR is a cell surface receptor primarily expressed on the surface of hepatocytes in the liver. In the blood, it recognizes and binds to desialyl glycoprotein. ASGPR binds to its ligand, forming a receptor-ligand complex. The receptor-ligand complex triggers invagination of the cell membrane, forming endosomes containing these complexes. The endosomes then fuse with lysosomes within the cell, where enzymes break down the ligands. After the ligands are broken down by enzymes within the lysosomes, their metabolites can be further processed by the cell or excreted from the body.

[0046] In some embodiments of this application, when the cell surface receptor is an FcγR2B receptor, i.e., the second antigen-binding region specifically recognizes and binds to the FcγR2B receptor, the sequence of CDR1 of the heavy chain variable region is as shown in SEQ ID NO.1, the sequence of CDR2 of the heavy chain variable region is as shown in SEQ ID NO.2, the sequence of CDR3 of the heavy chain variable region is as shown in SEQ ID NO.3, the sequence of CDR1 of the light chain variable region is as shown in SEQ ID NO.4, the sequence of CDR2 of the light chain variable region is as shown in SEQ ID NO.5, and the sequence of CDR3 of the light chain variable region is as shown in SEQ ID NO.6, as specifically shown in Table 1.

[0047] Table 1. CDR region sequence of FcγR2B-scFv3(SK3)

[0048] In some other embodiments of this application, when the cell surface receptor is an FcγR2B receptor, i.e., the second antigen-binding region specifically recognizes and binds to the FcγR2B receptor, the sequence of CDR1 of the heavy chain variable region is as shown in SEQ ID NO.7, the sequence of CDR2 of the heavy chain variable region is as shown in SEQ ID NO.8, the sequence of CDR3 of the heavy chain variable region is as shown in SEQ ID NO.9, the sequence of CDR1 of the light chain variable region is as shown in SEQ ID NO.10, the sequence of CDR2 of the light chain variable region is as shown in SEQ ID NO.11, and the sequence of CDR3 of the light chain variable region is as shown in SEQ ID NO.12, as specifically shown in Table 2.

[0049] Table 2. CDR region sequence of FcγR2B-scFv4(SK4)

[0050] In some other embodiments of this application, when the cell surface receptor is an AsGPR receptor, i.e., the second antigen-binding portion specifically recognizes and binds to the AsGPR receptor, the sequence of CDR1 of the heavy chain variable region is shown in SEQ ID NO.13, the sequence of CDR2 of the heavy chain variable region is shown in SEQ ID NO.14, the sequence of CDR3 of the heavy chain variable region is shown in SEQ ID NO.15, the sequence of CDR1 of the light chain variable region is shown in SEQ ID NO.16, the sequence of CDR2 of the light chain variable region is shown in SEQ ID NO.17, and the sequence of CDR3 of the light chain variable region is shown in SEQ ID NO.18, as specifically shown in Table 3.

[0051] Table 3. CDR region sequence of AsGPR-scFv5(AT5)

[0052] In some other embodiments of this application, when the cell surface receptor is an AsGPR receptor, i.e., the second antigen-binding part specifically recognizes and binds to the AsGPR receptor, the sequence of CDR1 of the heavy chain variable region is shown in SEQ ID NO.19, the sequence of CDR2 of the heavy chain variable region is shown in SEQ ID NO.20, the sequence of CDR3 of the heavy chain variable region is shown in SEQ ID NO.21, the sequence of CDR1 of the light chain variable region is shown in SEQ ID NO.22, the sequence of CDR2 of the light chain variable region is shown in SEQ ID NO.23, and the sequence of CDR3 of the light chain variable region is shown in SEQ ID NO.24, as specifically shown in Table 4.

[0053] Table 4. CDR region sequence of AsGPR-scFv10(AT10)

[0054] To further improve the therapeutic effect, this application involves mutation of the heavy chain variable region of scFv3.

[0055] In some other embodiments of this application, when the cell surface receptor is an FcγR2B receptor, i.e., the second antigen-binding region specifically recognizes and binds to the FcγR2B receptor, the sequence of CDR1 of the heavy chain variable region is as shown in SEQ ID NO. 59, the sequence of CDR2 of the heavy chain variable region is as shown in SEQ ID NO. 2, the sequence of CDR3 of the heavy chain variable region is as shown in SEQ ID NO. 60, the sequence of CDR1 of the light chain variable region is as shown in SEQ ID NO. 4, the sequence of CDR2 of the light chain variable region is as shown in SEQ ID NO. 5, and the sequence of CDR3 of the light chain variable region is as shown in SEQ ID NO. 6, as specifically shown in Table 5.

[0056] Table 5. CDR region sequence of FcγR2B-scFv3hi (SK3hi)

[0057] In some other embodiments of this application, when the cell surface receptor is an FcγR2B receptor, i.e., the second antigen-binding region specifically recognizes and binds to the FcγR2B receptor, the sequence of CDR1 of the heavy chain variable region is as shown in SEQ ID NO. 61, the sequence of CDR2 of the heavy chain variable region is as shown in SEQ ID NO. 62, the sequence of CDR3 of the heavy chain variable region is as shown in SEQ ID NO. 63, the sequence of CDR1 of the light chain variable region is as shown in SEQ ID NO. 4, the sequence of CDR2 of the light chain variable region is as shown in SEQ ID NO. 5, and the sequence of CDR3 of the light chain variable region is as shown in SEQ ID NO. 6, as specifically shown in Table 6.

[0058] Table 6. CDR region sequence of FcγR2B-scFv3ph (SK3ph)

[0059] In some other embodiments of this application, when the cell surface receptor is an FcγR2B receptor, i.e., the second antigen-binding region specifically recognizes and binds to the FcγR2B receptor, the sequence of CDR1 of the heavy chain variable region is as shown in SEQ ID NO. 59, the sequence of CDR2 of the heavy chain variable region is as shown in SEQ ID NO. 64, the sequence of CDR3 of the heavy chain variable region is as shown in SEQ ID NO. 3, the sequence of CDR1 of the light chain variable region is as shown in SEQ ID NO. 4, the sequence of CDR2 of the light chain variable region is as shown in SEQ ID NO. 5, and the sequence of CDR3 of the light chain variable region is as shown in SEQ ID NO. 6, as specifically shown in Table 7.

[0060] Table 7. CDR region sequences of FcγR2B-scFv3NGR (SK3 NGR)

[0061] In some embodiments of this application, when the cell surface receptor is an FcγR2B receptor, i.e., the second antigen-binding part specifically recognizes and binds to the FcγR2B receptor, the sequence of the heavy chain variable region of the scFv is as shown in SEQ ID NO.25, and the sequence of the light chain variable region of the scFv is as shown in SEQ ID NO.26, as specifically shown in Table 8.

[0062] Table 8 Variable region sequences of FcγR2B-scFv3(SK3)

[0063] In some other embodiments of this application, when the cell surface receptor is an FcγR2B receptor, i.e., the second antigen-binding part specifically recognizes and binds to the FcγR2B receptor, the sequence of the heavy chain variable region of the svFv is as shown in SEQ ID NO.27, and the sequence of the light chain variable region of the scFv is as shown in SEQ ID NO.28, as specifically shown in Table 9.

[0064] Table 9. Variable region sequences of FcγR2B-scFv4(SK4)

[0065] In some other embodiments of this application, when the cell surface receptor is an AsGPR receptor, i.e., the second antigen-binding part specifically recognizes and binds to the AsGPR receptor, the sequence of the heavy chain variable region of the scFv is as shown in SEQ ID NO.29, and the sequence of the light chain variable region of the scFv is as shown in SEQ ID NO.30, as specifically shown in Table 10.

[0066] Table 10 Variable region sequence of AsGPR-scFv5(AT5)

[0067] In some other embodiments of this application, when the cell surface receptor is an AsGPR receptor, i.e., the second antigen-binding part specifically recognizes and binds to the AsGPR receptor, the sequence of the heavy chain variable region of the scFv is as shown in SEQ ID NO.31, and the sequence of the light chain variable region of the scFv is as shown in SEQ ID NO.32, as specifically shown in Table 11.

[0068] Table 11 Variable region sequence of AsGPR-scFv10 (AT10)

[0069] In some embodiments of this application, when the cell surface receptor is an FcγR2B receptor, i.e., the second antigen-binding part specifically recognizes and binds to the FcγR2B receptor, the sequence of the heavy chain variable region of the scFv is as shown in SEQ ID NO.68, and the sequence of the light chain variable region of the scFv is as shown in SEQ ID NO.26, as specifically shown in Table 12.

[0070] Table 12 Variable region sequences of FcγR2B-scFv3hi (SK3hi)

[0071] In some embodiments of this application, when the cell surface receptor is an FcγR2B receptor, i.e., the second antigen-binding part specifically recognizes and binds to the FcγR2B receptor, the sequence of the heavy chain variable region of the scFv is as shown in SEQ ID NO.69, and the sequence of the light chain variable region of the scFv is as shown in SEQ ID NO.26, as specifically shown in Table 13.

[0072] Table 13 Variable region sequences of FcγR2B-scFv3ph (SK3ph)

[0073] In some embodiments of this application, when the cell surface receptor is an FcγR2B receptor, i.e., the second antigen-binding part specifically recognizes and binds to the FcγR2B receptor, the sequence of the heavy chain variable region of the scFv is as shown in SEQ ID NO.70, and the sequence of the light chain variable region of the scFv is as shown in SEQ ID NO.26, as specifically shown in Table 14.

[0074] Table 14 Variable region sequences of FcγR2B-scFv3NGR (SK3 NGR)

[0075] In a specific embodiment of this application, the scFv further includes a short peptide connecting the heavy chain variable region and the light chain variable region. The short peptide connects the heavy chain variable region and the light chain variable region in a single-chain antibody or a fragment thereof. The sequence of the short peptide is shown in SEQ ID NO.33.

[0076] SEQ ID NO.33: GGGGSGGGGSGGGGS

[0077] In a specific embodiment of this application, the full-length amino acid sequence of FcγR2B-scFv3(SK3) is shown in SEQ ID NO.34.

[0078] SEQ ID NO.34: QVLVETGGGVVQPGRSLRLSCAASGFTFSSYGMHWVRQAPGKGLEWVAVISYDGDDKDYADSVKGRFTISRDNADSSVFLQMNSLRAEDTALYYCAREGQWVGGALDIWGQGTMVTVS SGGGGSGGGGSGGGGSDIVMTQSPSTLSASLGDTVTITCRASQSINGWLAWYQQKPGKAPNLLIYQAPNLESGVPSRFSGSGSGTEFTLTINGLQPDDFATYYCQKYDNAPHTFGQGTKVEIKR

[0079] In a specific embodiment of this application, the full-length amino acid sequence of FcγR2B-scFv4(SK4) is shown in SEQ ID NO.35.

[0080] SEQ ID NO.35: QVQLVETGGGVVQVGRSLRLSCAASGFTFSHYGMHWVRQAPGKGLEWVSGISWNSDRRGYADSVKGRFTISRDNTKNSLYLQMNSLRAEDTAVYYCVREFYDAFDIWGQGTMVTVSS GGGGSGGGGSGGGGSDIQMTQSPSSLPAYVGDRVTISCRASQSIRTYLNWYRQKVGKAPELLVYDASSLHKGVPSRFSASGSGTDFSLTISSLQPEDFATYYCQQSFSSPRTFGQGTKVEIKR

[0081] In a specific embodiment of this application, the full-length amino acid sequence of AsGPR-scFv5(AT5) is shown in SEQ ID NO.36.

[0082] SEQ ID NO.36: QVQLQESGPGLVKPSETLSLNCSVSGGSFNNYYWNWIRQAPGGGLEWIGSVYYSGSASYNPSLKSRVTISDTSKNQFSLKLTSVTAADTAVYFCAREWGAGSFDIWGQGTLVTVSS GGGGSGGGGSGGGGSDIQMTQSPSSVSASVGDSVTITCRASEDIVSRLAWYQQRPGKAPKLLINAASSLQRGVPSRFSGSGSGTEYILTISSLQPDDIATYYCQQYIAFPLTFGGGTKVEIKR

[0083] In a specific embodiment of this application, the full-length amino acid sequence of AsGPR-scFv10(AT10) is shown in SEQ ID NO.37.

[0084] SEQ ID NO.37: QVQLVQSGGGLVQPGRSLRIPCAASGFTFDDSAMHWVRQAPGKGLEWVSAISGSGGRTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAREPMDVWGKGTLVTVSSGGGGS GGGGSGGGGSQAVLTQPSSLSASPGASVSLTCTLRSGINVGAYRIYWYQQKPGSPPQFLLRYKSDSDKQQGSGVPSRFSGSRDASANAGILLISGLRSEDEANYYCMIWHSSAWVFGGGTQLTVLG

[0085] In a specific embodiment of this application, the full-length amino acid sequence of FcγR2B-scFv3hi (SK3hi) is shown in SEQ ID NO.72.

[0086] SEQ ID NO.72: QVLVETGGGVVQPGRSLRLSCAASGFTFSSNGMHWVRQAPGKGLEWVAVISYDGDDKDYADSVKGRFTISRDNADSSVFLQMNSLRAEDTALYYCASEGQGVGGALDIWGQGTMVTVS SGGGGSGGGGSGGGGSDIVMTQSPSTLSASLGDTVTITCRASQSINGWLAWYQQKPGKAPNLLIYQAPNLESGVPSRFSGSGSGTEFTLTINGLQPDDFATYYCQKYDNAPHTFGQGTKVEIKR

[0087] In a specific embodiment of this application, the full-length amino acid sequence of FcγR2B-scFv3ph (SK3ph) is shown in SEQ ID NO.73.

[0088] SEQ ID NO.73: QVLVETGGGVVQPGRSLRLSCAASGFTFSSHGMHWVRQAPGKGLEWVAVISYDGHDKDYADSVKGRFTISRDNADSSVFLQMNSLRAEDTALYYCAREGQWHGGALDIWGQGTMVTVS SGGGGSGGGGSGGGGSDIVMTQSPSTLSASLGDTVTITCRASQSINGWLAWYQQKPGKAPNLLIYQAPNLESGVPSRFSGSGSGTEFTLTINGLQPDDFATYYCQKYDNAPHTFGQGTKVEIKR

[0089] In a specific embodiment of this application, the full-length amino acid sequence of FcγR2B-scFv3 NGR (SK3 NGR) is shown in SEQ ID NO.74.

[0090] SEQ ID NO.74: QVLVETGGGVVQPGRSLRLSCAASGFTFSSNGMHWVRQAPGKGLEWVAVISYNGRDKDYADSVKGRFTISRDNADSSVFLQMNSLRAEDTALYYCASEGQGVGGALDIWGQGTMVTVS SGGGGSGGGGSGGGGSDIVMTQSPSTLSASLGDTVTITCRASQSINGWLAWYQQKPGKAPNLLIYQAPNLESGVPSRFSGSGSGTEFTLTINGLQPDDFATYYCQKYDNAPHTFGQGTKVEIKR

[0091] Based on the principles of this application, the target protein is not limited. The target protein only needs to be able to bind to the antibody binding to the first antigen-binding portion of the target protein and be degraded by the lysosomal degradation pathway. For example, the target protein can be an extracellular soluble protein or a pathological protein aggregate. The target protein binds to the antibody binding to the first antigen-binding portion, and then enters the cell through the binding of the second antigen-binding portion to a circulating cell surface receptor capable of mediating endocytosis. It is then degraded via the lysosomal degradation pathway.

[0092] In specific embodiments of this application, the target proteins include, but are not limited to, IgE, PCSK9, TNF-α, IL-4, IL-6, IL-12, IL-17, VEGF, C5, MCP1, β-amyloid, hepcidin, α-synuclein, TREM2, and Tau. This application is merely illustrative and not intended to limit the target proteins.

[0093] In a specific embodiment of this application, the first antigen-binding portion can be a full-length antibody that binds to the target protein. The full-length antibody can be an existing antibody targeting the target protein, as long as it can specifically recognize and bind to the target protein. It can be obtained by purchasing or synthesizing based on a known sequence. For example, when the target protein is IgE, the first antigen-binding portion can be an existing IgE antibody, including but not limited to Omalizumab and Ligelizumab; when the target protein is PCSK9, the first antigen-binding portion can be an existing PCSK9 antibody, for example, as referenced in patent CN104540852A. The first antigen-binding portion can bind to the target protein in a pH-dependent manner or in a pH-independent manner.

[0094] The pH-dependent binding of target proteins refers to the ability of a protein or antibody to bind to a target protein in a way that changes with pH value; under specific pH conditions, this binding ability may be enhanced or weakened.

[0095] The pH-independent binding of target proteins means that the binding of proteins or antibodies to target proteins does not change with pH value; that is, the binding capacity remains relatively constant under different pH conditions.

[0096] In a preferred embodiment of this application, the first antigen-binding portion binds to the target protein in a pH-dependent manner.

[0097] In a specific embodiment of this application, when the first antigen-binding portion of the target protein binds to the target protein in a pH-dependent manner, the target protein separates from the antibody in the acidic environment of the endocytic vesicle, the target protein enters the lysosomal degradation pathway, and the antibody can be recycled.

[0098] Those skilled in the art will recognize that antibodies can be modified to make their binding to target proteins pH-dependent. In this application, it is sufficient to make the antibody binding to the target protein of the first antigen binding part pH-dependent. Therefore, there are no restrictions on the modification method of the antibody of the first antigen part. For example, it can be amino acid mutation, introduction of pH-sensitive amino acids, fusion of pH-sensitive peptides, introduction of ionizable functional groups on the antibody by chemical methods, or use of pH-sensitive binding ligands.

[0099] Those skilled in the art will recognize that the N297A mutation is a type of mutation targeting the Fc region of an antibody. In human IgG1 antibodies, amino acid residue N297 is located in the CH2 region of the antibody, near the end of the Fc fragment. The asparagine (Asn, N) residue at this position can undergo glycosylation in wild-type (unmutated) antibodies. Glycosylation regulates the affinity for the Fc receptor. The N297A mutation reduces the antibody's affinity for FcγR by removing the glycosylation site. The mutation site that performs the same function as the N297A mutation varies among different antibodies; therefore, the N297A mutation does not refer to the amino acid site of the antibody in this application, but only to this type of mutation.

[0100] Therefore, this application introduces specific amino acid mutations into the heavy chain variable region and / or light chain variable region of the basic antibody to make the first antigen binding moiety pH dependent. On this basis, it also introduces a conventional N297A amino acid mutation into the heavy chain constant region to reduce the affinity of the first antigen binding moiety for FcγR.

[0101] For example, in a specific embodiment of this application, when the first antigen-binding part is an IgE antibody, the first antigen-binding part can be either pH-independent binding target protein Omalizumab or pH-dependent binding target protein Omalizumab (NK-2-12-N297A). The pH-dependent binding target protein Omalizumab (NK-2-12-N297A) is obtained by modifying Omalizumab. The modification is based on the amino acid sequence of Omalizumab, and amino acid substitutions are performed. Specifically, specific amino acid substitutions are performed on the heavy chain variable region and the light chain variable region of Omalizumab, and conventional N297A amino acid substitutions are performed on the heavy chain constant region of Omalizumab. The light chain constant region of Omalizumab remains unchanged, so that NK-2-12-N297A can bind IgE in a pH-dependent manner.

[0102] The heavy chain variable region of Omalizumab includes the amino acid sequence shown in SEQ ID NO.38, the light chain variable region of Omalizumab is shown in SEQ ID NO.39, the heavy chain constant region of Omalizumab is shown in SEQ ID NO.40, and the light chain constant region of Omalizumab is shown in SEQ ID NO.41, as detailed in Table 15.

[0103] Table 15 Variable and constant region sequences of Omalizumab

[0104] The sequence of the heavy chain variable region of the modified pH-dependent binding target protein Omalizumab (NK-2-12-N297A) is shown in SEQ ID NO.42; the sequence of the light chain variable region of the modified Omalizumab (NK-2-12-N297A) is shown in SEQ ID NO.43; the sequence of the heavy chain constant region of the modified Omalizumab (NK-2-12-N297A) is shown in SEQ ID NO.44; the sequence of the light chain constant region of the modified Omalizumab (NK-2-12-N297A) remains unchanged relative to Omalizumab, as shown in SEQ ID NO.41, as detailed in Table 16.

[0105] Table 16. Sequences of the variable and constant regions of the modified Omalizumab (NK-2-12-N297A)

[0106] For example, in a specific embodiment of this application, when the first antigen-binding part is a PCSK9 antibody, the first antigen-binding part can be a pH-independent 300N antibody that binds to the target protein, or a pH-dependent 300N antibody (L30H antibody) that binds to the target protein. The pH-dependent 300N antibody (L30H antibody) is obtained by modifying 300N. The modification refers to replacing amino acids based on the amino acid sequence of the 300N antibody. Specifically, it involves making specific amino acid replacements in the light chain variable region of the 300N antibody and making conventional N297A amino acid replacements in the heavy chain constant region of the 300N antibody, while the heavy chain variable region and the light chain constant region of the 300N antibody remain unchanged, so that the L30H antibody can bind to PCSK9 in a pH-dependent manner.

[0107] The 300N antibody is referenced in CN104540852A. The sequence of the heavy chain variable region of the 300N antibody is shown in SEQ ID NO.45, the sequence of the light chain variable region of the 300N antibody is shown in SEQ ID NO.46, the sequence of the heavy chain constant region of the 300N antibody is shown in SEQ ID NO.47, and the sequence of the light chain constant region of the 300N antibody is shown in SEQ ID NO.48, as detailed in Table 17.

[0108] Table 17. Variable and constant region sequences of the 300N antibody.

[0109] The modified pH-dependent target protein binding 300N antibody (L30H-N297A) retains the same heavy chain variable region sequence as the 300N antibody, as shown in SEQ ID NO. 45; the modified light chain variable region sequence is shown in SEQ ID NO. 49; the modified heavy chain constant region sequence is shown in SEQ ID NO. 50; and the modified light chain constant region sequence remains unchanged compared to the 300N antibody, as shown in SEQ ID NO. 48, as detailed in Table 18.

[0110] Table 18. Variable and constant region sequences of the modified 300N antibody (L30H-N297A)

[0111] The first antigen-binding portion and the second antigen-binding portion can be directly fused, or the first antigen-binding portion can be fused to the second antigen-binding portion through a linker peptide. The linker peptide refers to a short peptide or polypeptide sequence used to connect two molecular structures in biochemistry and molecular biology, including but not limited to flexible linker peptides composed of GGGGS sequences or other small side chain amino acids, or rigid linker peptides composed of proline.

[0112] In a specific embodiment of this application, the first antigen-binding portion is fused to the second antigen-binding portion via a linker peptide, the amino acid sequence of which is shown in SEQ ID NO. 51. SEQ ID NO. 51: GGGGSGGGGSGGGGS

[0113] In a specific embodiment of this application, the heavy chain variable region of the second antigen-binding portion is fused with the C-terminus of the heavy chain or light chain of the first antigen-binding portion. In a preferred embodiment, the heavy chain variable region of the second antigen-binding portion is fused with the C-terminus of the heavy chain of the first antigen-binding portion.

[0114] In the specific embodiments of this application, the second antigen-binding portion of different cell surface receptors capable of mediating endocytosis can be arbitrarily combined with the first antigen-binding portion binding to different target proteins. All combinations allow the bispecific antibody to target the cell surface receptor mediating endocytosis at one end and the extracellular target protein at the other, forming a ternary complex. Through cell surface receptor-mediated endocytosis, the complex undergoes efficient endocytosis. The extracellular antigen separates from the antibody in the acidic environment of the endocytic vesicles, and the antigen enters the lysosomal degradation pathway. The antibody is then recycled through the cell surface receptor. Thus, a single bispecific antibody can efficiently degrade multiple antigens, thereby improving the antibody's efficiency in inhibiting antigens and reducing the effective concentration of the antibody. Based on the prior art and the research of this application, it is anticipated that the second antigen-binding portion of different cell surface receptors capable of mediating endocytosis can be arbitrarily combined with the first antigen-binding portion binding to different target proteins, achieving the effects of this application and falling within the scope of protection of this application.

[0115] In some embodiments of this application, the second antigen-binding portion is scFv3(SK3) which binds to the FcγR2B cell surface receptor, and the first antigen-binding portion is an amino acid-mutated, pH-dependent IgE antibody NK-2-12-N297A. The C-terminus of the heavy chain of NK-2-12-N297A is fused to the variable region of the heavy chain of scFv3(SK3) to form the heavy chain of the bispecific antibody NK-2-12-SK3, and the light chain of NK-2-12-N297A forms the light chain of the bispecific antibody NK-2-12-SK3. The full-length amino acid sequence of the heavy chain of the bispecific antibody NK-2-12-SK3 is shown in SEQ ID NO. 52, and the full-length amino acid sequence of the light chain of the bispecific antibody NK-2-12-SK3 is shown in SEQ ID NO. 53.

[0116] SEQ ID NO.52:EVQLVESGGGLVQPGGSLRLSCAVSGYSITSGYSWNWIRQAPGKGLEWVASITHDGSTNYNPSVKGRITISRDDSKNTFYLQMNSLRAEDTAVYYCARGSHYFGHWHFAVWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGGGGSGGGGSGGGGSMAQVLVETGGGVVQPGRSLRLSCAASGFTFSSYGMHWVRQAPGKGLEWVAVISYDGDDKDYADSVKGRFTISRDNADSSVFLQMNSLRAEDTALYYCAREGQWVGGALDIWGQGTMVTVSSGGGGSGGGGSGGGGSDIVMTQSPSTLSASLGDTVTITCRASQSINGWLAWYQQKPGKAPNLLIYQAPNLESGVPSRFSGSGSGTEFTLTINGLQPDDFATYYCQKYDNAPHTFGQGTKVEIKR

[0117] SEQ ID NO.53:DIQLTQSPSSLSASVGDRVTITCRASQSVHHHGDHHMNWYQQKPGKAPKLLIYAAHHHLESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSHEDPYTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0118] In some embodiments of this application, the second antigen-binding portion is scFv4(SK4) which binds to the FcγR2B cell surface receptor, and the first antigen-binding portion is an amino acid-mutated, pH-dependent IgE antibody NK-2-12-N297A. The C-terminus of the heavy chain of NK-2-12-N297A is fused to the variable region of the heavy chain of scFv4(SK4) to form the heavy chain of the bispecific antibody NK-2-12-SK4, and the light chain of NK-2-12-N297A forms the light chain of the bispecific antibody NK-2-12-SK4. The full-length amino acid sequence of the heavy chain of the bispecific antibody NK-2-12-SK4 is shown in SEQ ID NO. 54, and the full-length amino acid sequence of the light chain of the bispecific antibody NK-2-12-SK4 is shown in SEQ ID NO. 53.

[0119] SEQ ID NO.54:EVQLVESGGGLVQPGGSLRLSCAVSGYSITSGYSWNWIRQAPGKGLEWVASITHDGSTNYNPSVKGRITISRDDSKNTFYLQMNSLRAEDTAVYYCARGSHYFGHWHFAVWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGGGGSGGGGSGGGGSMAQVQLVETGGGVVQVGRSLRLSCAASGFTFSHYGMHWVRQAPGKGLEWVSGISWNSDRRGYADSVKGRFTISRDNTKNSLYLQMNSLRAEDTAVYYCVREFYDAFDIWGQGTMVTVSSGGGGSGGGGSGGGGSDIQMTQSPSSLPAYVGDRVTISCRASQSIRTYLNWYRQKVGKAPELLVYDASSLHKGVPSRFSASGSGTDFSLTISSLQPEDFATYYCQQSFSSPRTFGQGTKVEIKR

[0120] In some embodiments of this application, the second antigen-binding portion is scFv5(AT5) which binds to the AsGPR cell surface receptor, and the first antigen-binding portion is an amino acid-mutated, pH-dependent IgE antibody NK-2-12-N297A. The C-terminus of the heavy chain of NK-2-12-N297A is fused to the variable region of the heavy chain of scFv5(AT5) to form the heavy chain of the bispecific antibody NK-2-12-AT5, and the light chain of NK-2-12-N297A is formed by the heavy chain of the bispecific antibody NK-2-12-AT5. The full-length amino acid sequence of the heavy chain of the bispecific antibody NK-2-12-AT5 is shown in SEQ ID NO. 55, and the full-length amino acid sequence of the light chain of the bispecific antibody NK-2-12-AT5 is shown in SEQ ID NO. 53.

[0121] SEQ ID NO.55:EVQLVESGGGLVQPGGSLRLSCAVSGYSITSGYSWNWIRQAPGKGLEWVASITHDGSTNYNPSVKGRITISRDDSKNTFYLQMNSLRAEDTAVYYCARGSHYFGHWHFAVWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGGGGSGGGSGGGSGGGSMAQVQLQESGPGLVKPSETLSLNCSVSGGSFNNYYWNWIRQAPGGGLEWIGSVYYSGSASYNPSLKSRVTISVDTSKNQFSLKLTSVTAADTAVYFCAREWGAGSFDIWGQGTLVTVSSGGGGSGGGGSGGGGSDIQMTQSPSSVSASVGDSVTITCRASEDIVSRLAWYQQRPGKAPKLLINAASSLQRGVPSRFSGSGSGTEYILTISSLQPDDIATYYCQQYIAFPLTFGGGTKVEIKR

[0122] In some embodiments of this application, the second antigen-binding portion is scFv10 (AT10) that binds to the AsGPR cell surface receptor, and the first antigen-binding portion is an amino acid-mutated, pH-dependent IgE antibody NK-2-12-N297A. The C-terminus of the heavy chain of NK-2-12-N297A is fused to the variable region of the heavy chain of scFv10 (AT10) to form the heavy chain of the bispecific antibody NK-2-12-AT10, and the light chain of NK-2-12-N297A forms the light chain of the bispecific antibody NK-2-12-AT10. The full-length amino acid sequence of the heavy chain of the bispecific antibody NK-2-12-AT10 is shown in SEQ ID NO. 56, and the full-length amino acid sequence of the light chain of the bispecific antibody is shown in SEQ ID NO. 53.

[0123] SEQ ID NO.56:EVQLVESGGGLVQPGGSLRLSCAVSGYSITSGYSWNWIRQAPGKGLEWVASITHDGSTNYNPSVKGRITISRDDSKNTFYLQMNSLRAEDTAVYYCARGSHYFGHWHFAVWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGGGGSGGGSGGGSGGGSMAQVQLVQSGGGLVQPGRSLRIPCAASGFTFDDSAMHWVRQAPGKGLEWVSAISGSGGRTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAREPMDVWGKGTLVTVSSGGGGSGGGGSGGGGSQAVLTQPSSLSASPGASVSLTCTLRSGINVGAYRIYWYQQKPGSPPQFLLRYKSDSDKQQGSGVPSRFSGSRDASANAGILLISGLRSEDEANYYCMIWHSSAWVFGGGTQLTVLG

[0124] In some embodiments of this application, the second antigen-binding portion is scFv3 (SK3) that binds to the FcγR2B cell surface receptor, and the first antigen-binding portion is a pH-dependent PCSK9 antibody L30H-N297A that has undergone amino acid mutation. The C-terminus of the heavy chain of L30H-N297A is fused to the variable region of the heavy chain of scFv3 (SK3) to form the heavy chain of the bispecific antibody L30H-SK3, and the light chain of L30H-N297A forms the light chain of the bispecific antibody L30H-SK3. The full-length amino acid sequence of the heavy chain of the bispecific antibody L30H-SK3 is shown in SEQ ID NO. 57, and the full-length amino acid sequence of the light chain of the bispecific antibody is shown in SEQ ID NO. 58.

[0125] SEQ ID NO.57:EMQLVESGGGLVQPGGSLRLSCAASGFTFSSHWMKWVRQAPGKGLEWVANINQDGSEKYYVDSVKGRFTISRDNAKNSLFLQMNSLRAEDTAVYYCARDIVLMVYDMDYYYYGMDVWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGGGGSGGGSGGGSGGGSMAQVLVETGGGVVQPGRSLRLSCAASGFTFSSYGMHWVRQAPGKGLEWVAVISYDGDDKDYADSVKGRFTISRDNADSSVFLQMNSLRAEDTALYYCAREGQWVGGALDIWGQGTMVTVSSGGGGSGGGGSGGGGSDIVMTQSPSTLSASLGDTVTITCRASQSINGWLAWYQQKPGKAPNLLIYQAPNLESGVPSRFSGSGSGTEFTLTINGLQPDDFATYYCQKYDNAPHTFGQGTKVEIKR

[0126] SEQ ID NO.58:DIVMTQSPLSLPVTPGEPASISCRSSQSLHHSNGNNYLDWYLQKPGQSPQLLIYLGSNRASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQTLQTPLTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0127] In some embodiments of this application, the second antigen-binding portion is scFv3hi (SK3hi) which binds to the FcγR2B cell surface receptor, and the first antigen-binding portion is an amino acid-mutated, pH-dependent IgE antibody NK-2-12-N297A. The C-terminus of the heavy chain of NK-2-12-N297A is fused to the variable region of the heavy chain of scFv3hi (SK3hi) to form the heavy chain of the bispecific antibody NK-2-12-SK3hi. The light chain of NK-2-12-N297A forms the light chain of the bispecific antibody NK-2-12-SK3hi. The full-length amino acid sequence of the heavy chain of the bispecific antibody NK-2-12-SK3hi is shown in SEQ ID NO. 76, and the full-length amino acid sequence of the light chain of the bispecific antibody NK-2-12-SK3hi is shown in SEQ ID NO. 53.

[0128] SEQ ID NO.76:EVQLVESGGGLVQPGGSLRLSCAVSGYSITSGYSWNWIRQAPGKGLEWVASITHDGSTNYNPSVKGRITISRDDSKNTFYLQMNSLRAEDTAVYYCARGSHYFGHWHFAVWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGGGGSGGGSGGGSGGGSMAQVLVETGGGVVQPGRSLRLSCAASGFTFSSNGMHWVRQAPGKGLEWVAVISYDGDDKDYADSVKGRFTISRDNADSSVFLQMNSLRAEDTALYYCASEGQGVGGALDIWGQGTMVTVSSGGGGSGGGGSGGGGSDIVMTQSPSTLSASLGDTVTITCRASQSINGWLAWYQQKPGKAPNLLIYQAPNLESGVPSRFSGSGSGTEFTLTINGLQPDDFATYYCQKYDNAPHTFGQGTKVEIKR

[0129] In some embodiments of this application, the second antigen-binding portion is scFv3ph(SK3ph) which binds to the FcγR2B cell surface receptor, and the first antigen-binding portion is an amino acid-mutated, pH-dependent IgE antibody NK-2-12-N297A. The C-terminus of the heavy chain of NK-2-12-N297A is fused to the variable region of the heavy chain of scFv3ph(SK3ph) to form the heavy chain of the bispecific antibody NK-2-12-SK3ph. The light chain of NK-2-12-N297A forms the light chain of the bispecific antibody NK-2-12-SK3ph. The full-length amino acid sequence of the heavy chain of the bispecific antibody NK-2-12-SK3ph is shown in SEQ ID NO. 77, and the full-length amino acid sequence of the light chain of the bispecific antibody NK-2-12-SK3ph is shown in SEQ ID NO. 53.

[0130] SEQ ID NO.77:EVQLVESGGGLVQPGGSLRLSCAVSGYSITSGYSWNWIRQAPGKGLEWVASITHDGSTNYNPSVKGRITISRDDSKNTFYLQMNSLRAEDTAVYYCARGSHYFGHWHFAVWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGGGGSGGGSGGGSGGGSMAQVLVETGGGVVQPGRSLRLSCAASGFTFSSHGMHWVRQAPGKGLEWVAVISYDGHDKDYADSVKGRFTISRDNADSSVFLQMNSLRAEDTALYYCAREGQWHGGALDIWGQGTMVTVSSGGGGSGGGGSGGGGSDIVMTQSPSTLSASLGDTVTITCRASQSINGWLAWYQQKPGKAPNLLIYQAPNLESGVPSRFSGSGSGTEFTLTINGLQPDDFATYYCQKYDNAPHTFGQGTKVEIKR

[0131] In some embodiments of this application, the second antigen-binding portion is scFv3 NGR (SK3 NGR) that binds to the FcγR2B cell surface receptor, and the first antigen-binding portion is an amino acid-mutated, pH-dependent IgE antibody NK-2-12-N297A. The C-terminus of the heavy chain of NK-2-12-N297A is fused to the variable region of the heavy chain of scFv3 NGR (SK3 NGR) to form the heavy chain of the bispecific antibody NK-2-12-SK3 NRG. The light chain of NK-2-12-N297A forms the light chain of the bispecific antibody NK-2-12-SK3 NRG. The full-length amino acid sequence of the heavy chain of the bispecific antibody NK-2-12-SK3 NRG is shown in SEQ ID NO. 78, and the full-length amino acid sequence of the light chain of the bispecific antibody NK-2-12-SK3 NRG is shown in SEQ ID NO. 53.

[0132] SEQ ID NO.78: EVQLVESGGGLVQPGGSLRLSCAVSGYSITSGYSWNWIRQAPGKGLEWVASITHDGSTNYNPSVKGRITISRDDSKNTFYLQMNSLRAEDTAVYYCARGSHYFGHWHFAVWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHT FPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVY TLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGGGGSGGGSGGGSGGGSMAQVLVETGGGVVQPGRSLRLSCAASGFTFSSYGMHWVRQAPGKGLEWVAVISYNGRDKDYADS VKGRFTISRDNADSSVFLQMNSLRAEDTALYYCAREGQWVGGALDIWGQGTMVTVSSGGGGSGGGGSGGGGSDIVMTQSPSTLSASLGDTVTITCRASQSINGWLAWYQQKPGKAPNLLIYQAPNLESGVPSRFSGSGSGTEFTLTINGLQPDDFATYYCQKYDNAPHTFGQGTKVEIKR

[0133] This application also provides a polynucleotide encoding the aforementioned bispecific antibody. The polynucleotide is obtained based on the amino acid sequence of the bispecific antibody of this application according to conventional genetic codon rules in the art.

[0134] This application may also provide a construct comprising the aforementioned polynucleotides. The construct may be selected from DNA vectors, RNA vectors, plasmids, transposon vectors, CRISPR / Cas9 vectors, or viral vectors, etc.

[0135] This application may also provide a host cell containing the above-described construct or whose genome integrates the above-described polynucleotides.

[0136] The host cells can be selected from bacterial cells, fungal cells, insect cells, plant cells, mammalian cells, etc. Specifically, they can be selected from Escherichia coli, Streptomyces, Salmonella typhimurium, yeast, filamentous fungi, Drosophila S2 or Sf9 cells, CHO cells, COS cells, HEK293F cells, Bowes melanoma cells, NS0 cells, BHK cells, PER.C6 cells, etc.

[0137] The bispecific antibody provided in this application can effectively bind to target proteins including IgE, PCSK9, TNF-α, IL-4, IL-6, IL-12, IL-17, VEGF, C5, MCP1, β-amyloid, hepcidin, α-synuclein, TREM2, and Tau. This allows for the treatment of diseases related to the overexpression of these target proteins through neutralizing antibodies targeting the first antigen-binding portion. For example, when treating IgE overexpression-related diseases with neutralizing IgE antibodies, the type I hypersensitivity reaction caused by the binding of IgE to its high-affinity receptor FcεRI can be blocked, thereby treating allergic diseases. Similarly, when treating PCSK9 overexpression-related diseases with neutralizing PCSK9 antibodies, the binding of PCSK9 to LDL-R can be blocked, thereby inhibiting LDL-R degradation and reducing plasma LDL-C levels, thus achieving a lipid-lowering effect.

[0138] This application also provides a pharmaceutical composition or kit comprising an effective amount of the above-described bispecific antibody.

[0139] This application also provides the use of the above-described bispecific antibody, or the above-described polynucleotide, or the above-described construct, or the above-described host cell, or the above-described preparation method, or the above-described drug or kit in the preparation of products for the prevention and / or treatment of diseases related to target protein overexpression.

[0140] This application also provides a method for preventing and / or treating diseases related to target protein overexpression, comprising administering an effective amount of the above-described bispecific antibody, or the above-described drug or kit, to a subject, and adjusting the actual dose according to the subject's age, weight, and response.

[0141] In this application, the target protein overexpression-related diseases mentioned above are selected from IgE overexpression-related diseases, PCSK9 overexpression-related diseases, TNF-α overexpression-related diseases, IL-4 overexpression-related diseases, IL-6 overexpression-related diseases, IL-12 overexpression-related diseases, IL-17 overexpression-related diseases, VEGF overexpression-related diseases, C5 overexpression-related diseases, MCP1 overexpression-related diseases, β-amyloid overexpression-related diseases, hepcidin overexpression-related diseases, α-synuclein overexpression-related diseases, TREM2 overexpression-related diseases, and Tau overexpression-related diseases.

[0142] The diseases associated with IgE overexpression include, but are not limited to, allergic diseases, parasitic infections, inflammatory diseases, primary immunodeficiency, tumors, cardiovascular diseases, and psoriasis with IgE overexpression. In these diseases, patients have higher IgE levels than healthy individuals.

[0143] Specifically, allergic diseases with IgE overexpression include, but are not limited to, allergic rhinitis, bronchial asthma, and atopic conjunctivitis. Tumors with IgE overexpression include, but are not limited to, lymphoma, ovarian epithelial carcinoma, multiple myeloma, and skin cancer.

[0144] The diseases associated with PCSK9 overexpression include, but are not limited to, diseases induced by hyperlipidemia and tumors caused by PCSK9 overexpression, in which patients have elevated PCSK9 levels compared to healthy individuals. Specifically, diseases induced by hyperlipidemia and PCSK9 overexpression include, but are not limited to, atherosclerotic cardiovascular disease. Tumors caused by PCSK9 overexpression include gastric cancer, lung cancer, breast cancer, cervical squamous cell carcinoma, colonic adenocarcinoma, esophageal cancer, head and neck squamous cell carcinoma, hepatocellular carcinoma, rectal adenocarcinoma, gastric adenocarcinoma, thyroid carcinoma, and endometrial cancer.

[0145] The diseases associated with TNF-α overexpression include, but are not limited to, autoimmune diseases, inflammatory bowel disease, diabetes, atherosclerosis, asthma, psoriatic arthritis, and tumors, in which patients have elevated TNF-α levels compared to healthy individuals. The diseases associated with IL-4 overexpression include, but are not limited to, tumors, asthma, allergies and atopic dermatitis, and pulmonary fibrosis, in which patients have elevated IL-4 levels compared to healthy individuals. The diseases associated with IL-6 overexpression include, but are not limited to, non-small cell lung cancer, pulmonary hypertension, autoimmune diseases, tumors, and neurological diseases, in which patients have elevated IL-6 levels compared to healthy individuals. The diseases associated with IL-12 overexpression include, but are not limited to, psoriasis, systemic lupus erythematosus, Crohn's disease, and malignant melanoma, in which IL-12 overexpression is present. The diseases associated with IL-17 overexpression include, but are not limited to, autoimmune diseases, inflammatory skin diseases, hidradenitis suppurativa, viral infections, and parasitic infections, in which patients have elevated IL-17 levels compared to healthy individuals. The VEGF overexpression-related diseases include, but are not limited to, neurological diseases, eye diseases, respiratory diseases, liver diseases, tumors, and inflammatory diseases caused by VEGF overexpression. In these diseases, patients have higher VEGF levels than healthy individuals. The C5 overexpression-related diseases include, but are not limited to, colorectal cancer, mesangial proliferative glomerulonephritis, and non-small cell lung cancer caused by C5 overexpression. In these diseases, patients have higher C5 levels than healthy individuals. The MCP1 overexpression-related diseases include, but are not limited to, diabetic complications, inflammatory diseases, brain diseases, cardiovascular diseases, rheumatoid arthritis, neuroinflammatory diseases, tumors, pulmonary fibrosis, and autoimmune thyroid diseases caused by MCP1 overexpression. In these diseases, patients have higher MCP1 levels than healthy individuals. The β-amyloid protein overexpression-related diseases include, but are not limited to, Alzheimer's disease and tumors caused by β-amyloid protein overexpression. In these diseases, patients have higher β-amyloid protein levels than healthy individuals. The diseases associated with hepcidin overexpression include, but are not limited to, chronic anemia due to hepcidin overexpression, anemia due to chronic kidney disease, Castleman's disease anemia, iron overload anemia, primary myelofibrosis, and tumors. In these diseases, patients have elevated hepcidin levels compared to healthy individuals. The diseases associated with α-synuclein overexpression include, but are not limited to, Parkinson's disease and tumors. In these diseases, patients have elevated α-synuclein levels compared to healthy individuals. The diseases associated with TREM2 overexpression include, but are not limited to, tumors, Alzheimer's disease, and obesity. In these diseases, patients have elevated TREM2 levels compared to healthy individuals.The diseases associated with Tau overexpression include, but are not limited to, Alzheimer's disease, frontotemporal dementia, corticobasal degeneration, progressive supranuclear palsy, Pick's disease, prion disease, and tumors, in which patients have higher Tau levels than healthy individuals.

[0146] The present application is further illustrated below by way of examples, but these examples do not limit the scope of the application. Unless otherwise stated, the experimental methods, detection methods, and preparation methods disclosed in this invention all employ conventional techniques in this technical field. Unless otherwise specified, the instruments, materials, and reagents used in the examples can all be obtained through conventional means. The antibodies used in the examples of this application are shown in Table 19 below.

[0147] Table 19 Antibodies in the Examples

[0148] Example 1: Screening, internalization assay, and construction of FcγR2B-targeting chimeras of human FcγR2B-binding monoclonal antibodies.

[0149] Experimental methods:

[0150] 1. Construction of phage antibody library and screening of FcγR2B antibody scFvs

[0151] Humanized single-chain antibody (scFv) phage display library: The humanized single-chain antibody (scFv) phage display library previously constructed in our laboratory was used as the starting library. This library contains approximately 10... 10 A unique human scFv clone provides diversity assurance for subsequent screening of FcγR2B-specific antibodies.

[0152] FcγR2B protein: ACROBiosystems, CDB-H82E0.

[0153] Phage library screening: Biotinylated human FcγR2B extracellular protein was co-incubated with the phage library. Specific phages binding the protein were adsorbed using streptavidin-conjugated magnetic beads, followed by acid elution. During panning, the screening pressure was increased by gradually reducing the amount of antigen and introducing competitive antigens to enrich high-affinity specific phage clones. After 3-4 rounds of panning, approximately 200 enriched specific binding phages were obtained.

[0154] Antibody screening: The phage library was incubated with biotinylated human FcγR2B outdomain protein (ACROBiosystems, CDB-H82E0) at room temperature for 2 hours, followed by capture of the phage-antigen complex using Dynabeads M280 magnetic beads (Invitrogen, 11206D). Next, the bound phages were eluted with glycine-hydrochloric acid solution (pH 2.2) at room temperature for 10 minutes, and neutralized to a final pH of 7.5 with Tris-HCl solution (pH 8). The eluted phages were amplified and used for the next round of screening. Three rounds of screening were performed to enrich phages binding to FcγRIIB. Subsequently, single phage clones were amplified and detected by phage ELISA. For ELISA-positive clones, phage DNA was extracted using a plasmid miniprep kit (TIANGEN, DP118-02) and sequenced using the Sanger DNA method.

[0155] 2. Construction of FcγR2B antibody scFvs-Fc

[0156] The ScFv-Fc (SKX-Fc) protein was constructed by fusing an FcγR2B-binding scFv to the N-terminus of a human IgG1 Fc fragment via a glycine-serine (G4S)3 linker. FcRTACs, on the other hand, involved fusing an FcγR2B-binding scFv to the C-terminus of a full-length antibody (including Omalizumab, NK-2-12, 300N, L30H, and lecanemab) heavy chain, with an N297A mutation introduced into the Fc region of all FcRTACs. The SKX-Fc and FcRTAC constructs were cloned into the mammalian expression vector pTT5, and the plasmids were transfected into HEK293F cells, where they were expressed for 5 days in Freestyle medium (Thermo, cat 12338018). After expression, cells were centrifuged at 200×g for 10 minutes at 4°C, and the supernatant was collected and filtered through a 0.45 μm filter membrane (Cobetter, OBT-1051). The supernatant was then processed using a HiTrap Protein A column (GE, 17040301). Protein purification was performed on the purification system. The antibody was further purified by gel filtration chromatography in PBS buffer using a Superose 6Increase 10 / 300 column (Cytiva, 29091596). Finally, the protein was concentrated using ultrafiltration centrifuge tubes (Merck / Millipore, UFC903024).

[0157] 3. Flow cytometry (FACS) detection of antibody and cell surface antigen binding.

[0158] First, HUVEC or HUVEC-2b cells were co-incubated with 2 μg / mL anti-FcγR2B SKX-Fc protein at 4°C for 30 minutes. After washing with PBS containing 2% FBS, the cells were stained with AF488-labeled goat anti-human IgG (H+L) (Invitrogen, A-11013) at a 1:500 dilution for 30 minutes. After two washes, the fluorescence signal was detected by flow cytometry, and the data were analyzed using FlowJo X software.

[0159] 4. Detection of FcγR2B antibody scFv-Fc internalization efficiency

[0160] Antibody internalization screening for functional antibodies: utilizing pH-sensitive... The purified FcγR2B antibody scFv-Fc was labeled with FabFluor-pH Red reagent and added to a 96-well plate containing a stable HUVEC-2b cell line. The live cell analysis system captures images every 60 minutes (20x magnification) and monitors changes in red fluorescence intensity for 48 hours. Analysis is performed using integrated software.

[0161] Experimental results:

[0162] In this study, we designed a novel bispecific antibody-based FcγR2B-targeting chimeric (FcRTAC) platform comprising two distinct functional arms: one specifically targeting soluble antigens, and the other involved in receptor reactivation to facilitate efficient endocytosis. We strategically selected FcγR2B, a physiologically relevant receptor responsible for immune complex clearance, as the optimal receptor for FcRTAC construction (Figure 2a).

[0163] To obtain a monoclonal antibody against human FcγR2B, we screened for human FcγR2B extracellular domain conjugates using a human single-chain variable fragment (scFv) phage display library (Figure 1a), excluding redundant or poorly developed sequences. Eight scFvs were converted into Fc fusions for further characterization. The binding of these scFv-Fcs (SKX-Fcs) to FcγR2B was detected by enzyme-linked immunosorbent assay (ELISA) (Figure 1b). Subsequently, we successfully generated a stable Huvec cell line expressing FcγR2B (named Huvec-2b) (Figure 1c).

[0164] Flow cytometry analysis showed that these SKX-Fcs bound to the native conformation of FcγR2B on Huvec-2b (Fig. 2b). Next, we evaluated the internalization efficiency of the eight SKX-Fcs in the Huvec-2b cell line. The results showed that all eight SKX-Fcs could be internalized, with SK3-Fc and SK4-Fc exhibiting the highest antibody endocytosis rates (Fig. 2c). Therefore, we selected SK3 and SK4 for further studies. The binding affinity of SK3-Fc and SK4-Fc to FcγR2B was determined by surface plasmon resonance (SPR) at pH 6 and pH 7. The affinity of SK3 for FcγR2B was approximately one order of magnitude higher than that of SK4 (Fig. 2d, Table A).

[0165] Table A | Equilibrium dissociation constant and kinetic binding constant of FcγR2B specific antibodies

[0166] To ensure data comparability, the binding affinity of all analytes in the table was determined in the same SPR run.

[0167] This invention further designs FcRTACs based on bispecific antibodies, with one arm binding FcγR2B and the other arm binding the target antigen to promote the clearance of the target antigen. To determine the effect of the spatial arrangement between the two FcγR2B scFv domains and the distance between the binding antibodies, we designed two different structures: in configuration FcRTAC-1, the FcγR2B conjugate is fused to the C-terminus (C-terminus) of the heavy chain of the pH-sensitive IgE-targeting NK-2-12; while in configuration FcRTAC-2, the binding antibody is positioned at the C-terminus of the light chain to maximize the distance between the binding antibodies. We also designed FcRTAC-3 (Figure 2e) containing a single FcγR2B binding antibody and two IgE binding antibodies using an in-pore knob (KIH) platform.

[0168] Internalization experiments in the Huvec-2b cell line revealed that FcRTAC-3 failed to initiate endocytosis, indicating that receptor-mediated endocytosis requires molecular aggregation promoted by two FcγR2B conjugates. Compared to FcRTAC-1, FcRTAC-2 exhibited significantly reduced internalization efficiency. The shorter interbindus distance in FcRTAC-1, compared to FcRTAC-2, may contribute to more efficient receptor aggregation, thereby enhancing its internalization capacity (Figure 1e). Based on these results, we constructed NK-2-12-SK3 and NK-2-12-SK4 to deplete IgE by fusing SK3 or SK4 scFv to the C-terminus of the IgE-targeting NK-2-12 heavy chain.

[0169] SPR results showed that both FcRTACs could interact with IgE and FcγR2B simultaneously (Fig. 2f, g). NK-2-12-SK3 and NK-2-12-SK4 significantly increased IgE uptake in Huvec-2b cells (Fig. 2h).

[0170] Example 2: FcRTAC can rapidly clear plasma targets and improve clearance efficiency by binding to the target in a pH-sensitive manner.

[0171] Experimental Methods: Six-week-old humanized hFCGR2B mice (purchased from Southern Model Biotechnology Co., Ltd.), weighing 18-20g and raised under SPF conditions, were intraperitoneally injected with bispecific antibodies binding FcγR2B and IgE, NK-2-12-SK3, NK-2-12-SK4, NK-2-12-SK9, Omalizumab, and NK-2-12, at a dose of 5 mg / kg. Thirty minutes later, 2.5 mg / kg IgE was injected intraperitoneally. Blood samples were collected at 1h, 2h, 3h, 8h, and 22h, and serum human IgE levels were measured using ELISA. Mice injected with PBS and those with anti-IgE antibodies alone served as negative controls to compare the efficacy of different bispecific antibodies in clearing endogenous IgE.

[0172] Experimental Results: To evaluate the effect of FcRTAC in enhancing antigen clearance and to explore whether its performance can be optimized by pH-sensitive antigen binding, we conducted in vivo experiments using FcγR2B humanized mice.

[0173] The experimental group included: (1) pH-sensitive IgE-targeting FcRTACs (NK-2-12-SK3 and NK-2-12-SK4); (2) pH-insensitive IgE antibody FcRTACs (Omalizumab-SK3 and Omalizumab-SK4); (3) pH-sensitive NK-2-12; and (4) pH-insensitive IgE-targeting antibody Omalizumab. Except for the commercially available Omalizumab, NK-2-12 and all FcRTACs contained the N297A mutation, which eliminates the binding of the Fc domain to the Fc receptor. These drugs were administered to FcγR2B humanized mice via intraperitoneal injection, followed by IgE administration. Total IgE and free IgE levels were then quantified using ELISA (Figure 3a). Compared to the IgE-only group, omalizumab treatment rapidly reduced free IgE while significantly increasing plasma total IgE levels (Figures 3b-e), summarizing the clinical phenomenon of total IgE accumulating to 5-10 times the baseline IgE level after omalizumab treatment. Omalizumab-SK3 treatment reduced total IgE levels compared to omalizumab (Figure 3b), while omalizumab-SK4 did not increase total IgE levels compared to the IgE-only group (Figure 3d). Both NK-2-12-SK3 and NK-2-12-SK4 treatments significantly accelerated the clearance of total IgE compared to omalizumab-SK3 or omalizumab-SK4 treatments, reducing both total IgE and free IgE levels below detectable levels (Figures 3b-e).

[0174] In summary, our results indicate that IgE-targeted FcRTACs, especially engineered proteins with pH-dependent binding properties, exhibit strong IgE scavenging capabilities.

[0175] To investigate whether FcRTAC antigen clearance depends on the FcγR2B receptor, we analyzed the antigen clearance activity of FcRTAC in FcγR2B knockout mice. In FcγR2B knockout mice, the IgE clearance capacity mediated by different FcRTACs was significantly reduced (Fig. 3f, g), confirming the antigen clearance capacity of FcRTAC mediated by the FcγR2B receptor.

[0176] To assess the toxicity of FcRTAC, FcγR2B humanized mice were intravenously injected with NK-2-12-SK3, NK-2-12-SK4, or Omalizumab at a dose of 10 mg / kg. Blood samples were collected after administration, and different organs were dissected for analysis. The levels of AST, ALT, and LDH in the blood samples did not increase significantly (Figures 4a-c), and H&E staining showed that FcRTAC did not cause inflammation or damage to organs such as the spleen and liver (Figure 4d).

[0177] Example 3: Construction and Evaluation of FcRTAC for Clearing PCSK9 Antigen

[0178] 1. Construction of antibodies targeting PCSK9

[0179] Following the method described in the patent "Anti-PCSK9 Antibody with pH-dependent Binding Properties" (Publication No. CN 104540852A), the leucine at position 30 of the variable region of the light chain of PCSK9 antibody 300N was replaced with histidine to obtain the pH-dependent PCSK9 antibody L30H; then, an N297A mutation was introduced into the constant region of the heavy chain of PCSK9 antibody L30H to obtain L30H-N297A. The amino acid sequences of PCSK9 antibody 300N and L30H-N297A are shown in Table 20. PCSK9 antibody 300N and L30H-N297A are used as the anti-PCSK9 ends of the bispecific antibodies, respectively.

[0180] Table 20 Amino acid sequences of PCSK9 antibodies 300N and L30H-N297A

[0181] 2. Design and expression of bispecific antibodies combining FcγR2B and PCSK9

[0182] PCSK9 antibody 300N and a pH-dependent mutant antibody L30H-N297A that binds to 300N were used as the anti-PCSK9 ends of bispecific antibodies, respectively. The heavy chain variable region of the anti-FcγR2B scFv3 (SK3) screened in Example 1 was linked to the C-terminus of the heavy chain of the anti-PCSK9 antibody (PCSK9 antibody 300N or L30H-N297A) via a linker peptide to assemble bispecific antibodies. The linker peptide sequence was GGGGSGGGGSGGGGS (SEQ ID NO. 51), and they were named 300N-SK3 and L30H-SK3.

[0183] Specifically, the genes of the bispecific antibodies 300N-SK3 and L30H-SK3 were synthesized according to the rules of the genetic code based on their amino acid sequences. The amino acid sequence of L30H-3H is shown in Table 21. The heavy chain genes of the bispecific antibodies 300N-SK3 and L30H-SK3, which bind to FcγR2B and PCSK9, were cloned into the ptt5 expression vector, and the light chain genes of the same antibodies were cloned into the ptt5 expression vector. Sequencing confirmed the correct plasmid construction. The cloned ptt5 expression vectors were transiently transfected into HEK 293F cells to express and secrete the bispecific antibodies. The culture supernatant was purified by Protein A affinity to obtain high-purity bispecific antibodies (see the expression and purification methods in Example 1 for details). SDS-PAGE was used to identify the purity, and flow cytometry was used to detect the bispecific binding activity. Both bispecific antibodies were able to interact with both FcγR2B and PCSK9 simultaneously.

[0184] Table 21 Amino acid sequence of L30H-SK3

[0185] Six-week-old, 18-20g humanized hFCGR2B mice (purchased from Southern Model Biotechnology Co., Ltd.) raised under SPF conditions were intraperitoneally injected with the bispecific antibody L30H-SK3, which binds to FcγR2B and PCSK9, as well as 300N-SK3, L30H, and 300N, at a dose of 100 μg / kg. Serum was collected at 0, 4h, 16h, 24h, 38h, and 72h, and PCSK9 concentration was detected using a PCSK9 ELISA kit. Mice injected with PBS were used as negative controls to compare the efficacy of different bispecific antibodies in clearing endogenous PCSK9.

[0186] To assess whether FcRTACs could be used to deplete multiple pathological antigens other than IgE, we generated two different PCSK9-targeting antibodies: 300N (pH insensitive) and L30H (pH sensitive), along with their corresponding FcRTACs (300N-SK3 and L30H-SK3) (Figure 5a and Table B). In a PCSK9 humanized mouse model, neither 300N nor L30H showed a significant reduction in circulating PCSK9 levels, while the derived FcRTACs demonstrated PCSK9 clearance capabilities. Among them, L30H-SK3 showed the most significant reduction in plasma PCSK9 concentration (Figure 5b).

[0187] Table B | Equilibrium dissociation constant of anti-PCSK9 antibody

[0188] Example 4: FcRTAC constructed using an affinity-matured FcγR2B binding antibody exhibited a more durable antigen clearance efficiency.

[0189] To further improve the efficacy of antibody therapy, we optimized the SK3 binding of FcγR2B. A mutant library was constructed by introducing mutations in all three CDRs of the SK3 heavy chain. After repeated phage display screening of FcγR2B under acidic conditions, a variant called SK3hi showed an affinity one order of magnitude higher than the parental SK3 molecule under both physiological (pH 7.4) and endosome (pH 6.0) conditions (Fig. 6a). Confocal microscopy analysis showed that NK2-12-SK3hi exhibited preferential plasma membrane localization with no detectable accumulation in the lysosomal compartment (Fig. 6b). In the re-excitation experiment, mice receiving sk3hi-based FcRTAC maintained significantly better IgE clearance capacity after IgE re-injection on day 2 than mice receiving sk3-based FcRTAC, without additional FcRTAC administration (Fig. 6c-d).

[0190] Subsequently, we selected pH-dependent binders by adding histidine substitutions to the interacting residues of SK3 and FcγR2B, constructed a phage display library, and screened for SK3ph. A variant containing three histidine substitutions was the clone with the highest enrichment after screening. SPR analysis showed that SK3ph binds to FcγR2B at pH 7.4 with a Kd of 2.65 × 10⁻⁶. -9 M decreases to 9.37 × 10 at pH 6.0. -9 M (Fig. 7a, Table C). The antigen clearance rate of FcRTAC-SK3ph was comparable to that of NK-2-12-SK3 (Fig. 7b). This pH-sensitive FcγR2B dissociation and conversion prolonged the serum half-life of FcRTAC-SK3ph in vivo (Fig. 7c). Data indicate that pH-dependent FcγR2B dissociation promotes the recycling of FcRTAC via the FcRn pathway, ultimately leading to its release back into circulation and prolonging its in vivo half-life (Fig. 7d).

[0191] Table C | Equilibrium dissociation constant and kinetic binding constant of anti-FcγR2B bispecific antibody

[0192] To ensure data comparability, the binding affinity of all analytes in the table was determined in the same SPR run.

[0193] We also designed a point mutation of SK3, SK3 NGR, through structural analysis. It can be internalized in the Huvec-2b cell line (Figure 8) and can prolong the serum half-life of FcRTAC in vivo (Figure 9).

[0194] Example 5: Comparison of FcRTAC and cross-scan antibody technology

[0195] To evaluate the performance of FcRTAC relative to sweep antibody technology, we designed NK-2-12-V12, a pH-dependent hIgG1 variant with selectively enhanced affinity for human FcγR2B binding (Figure 10a). The full-length amino acid sequence of the heavy chain of NK-2-12-V12 is shown in SEQ ID NO. 80, and the full-length amino acid sequence of the light chain is shown in SEQ ID NO. 53.

[0196] Internalization assays showed that NK-2-12-V12 uptake efficiency was significantly reduced compared to NK-2-12-SK3 (Fig. 10b). To characterize the mechanism of NK-2-12-V12 internalization, we systematically evaluated the contributions of different endocytic pathways using pathway-specific drug inhibitors. Chlorpromazine (clathrin inhibition), cytochalasin D (actin polymerization blockade), and amiloride (macrophage inhibition) significantly impaired NK-2-12-V12 uptake (Fig. 10c), indicating that clathrin-mediated endocytosis, phagocytosis, and macrophage activity are simultaneously involved. In contrast, nystatin-mediated disruption of pit-dependent endocytosis had no significant effect on internalization efficiency. After incubating Huvec-2b cells with labeled IgE and NK-2-12-V12, the subcellular localization of endocytic IgE and NK-2-12-V12 was assessed using immunofluorescence microscopy. Most of NK-2-12-V12 is localized to the cell membrane, with only a small fraction detected in the cytoplasm. In contrast, a large amount of NK-2-12-SK3 is internalized into the cytoplasm in addition to its presence on the cell surface (Fig. 10d). High-resolution microscopy revealed different IgE transport patterns: after NK-2-12-V12 treatment, IgE was mainly localized to the cell surface with minimal lysosomal co-localization, while NK-2-12-SK3 treatment induced significant lysosomal IgE recruitment (Fig. 10e). Although cell biology studies revealed different mechanisms of action between FcRTAC and the sweep antibody, NK-2-12-V12 at 5 mg / kg exhibited IgE-depleting efficacy comparable to FcRTAC (Fig. 10f). Notably, the sweep antibody mechanism conferred prolonged serum persistence (Fig. 10g).

[0197] Example 6: Screening, detection, and construction of bispecific antibodies targeting AsGPR

[0198] 1. Screening AsGPR antibodies according to the method in Example 1.

[0199] Eight scFv sequences were finally obtained, named AT4, AT5, AT10, AT13, AT16, AT17 and AT18, and further expressed as the corresponding AsGPR antibody ATs-Fc.

[0200] 2. Internalization assay of AsGPR antibody scFvs

[0201] The internalization experiment was performed according to "4. Internalization Efficiency Detection" in Example 1: scFv-Fc was first mixed with a pH-sensitive dye Fab fragment containing anti-human Fc, and then co-incubated with HepG2 cells. After internalization, the pH-sensitive dye showed red fluorescence in acidic environments (such as endosomes and lysosomes). The internalization efficiency of the AsGPR antibody scFv-Fc was assessed by capturing changes in the intensity of the red fluorescence over 24 consecutive hours. AT5-Fc and AT10-Fc were selected as the best candidates due to their high internalization efficiency in HepG2 cells (Figures 11b and 11c).

[0202] 3. Construction of bispecific antibodies combining AsGPR and IgE

[0203] Referring to Example 1, the pH-dependent IgE-binding mutant antibody NK-2-12-N297A was used as the anti-IgE end of the bispecific antibody. The heavy chain variable region of the anti-AsGPR antibody scFvs (AT5, AT10) screened in Example 5 was linked to the C-terminus of the heavy chain of NK-2-12-N297A via a linker peptide to assemble bispecific antibodies. The linker peptide sequence was GGGGSGGGGSGGGGS (SEQ ID NO. 51). Two bispecific antibodies were constructed and named NK-2-12-AT5 and NK-2-12-AT10, respectively.

[0204] Table 22 Sequences of AT5 and AT10

[0205] Table 23. Amino acid sequences of the bispecific antibodies NK-2-12-AT5 and NK-2-12-AT10 that bind AsGPR and IgE.

[0206] Experimental results:

[0207] Phage display screening yielded several AsGPR-specific scFvs (Fig. 11a), with AT5-Fc and AT10-Fc emerging as the top candidates due to their high internalization efficiency in HepG2 cells (Fig. 11b, c). These lead conjugates were subsequently engineered into bispecific antibodies with both an IgE-targeting arm and an AsGPR-binding arm (Fig. 11d). Both NK-2-12-AT5 and NK-2-12-AT10 showed reduced internalization efficiency in HepG2 cells compared to NK-2-12-SK3 in HUVEC-2b cells (Fig. 11e, f). While NK-2-12-AT5 effectively reduced IgE levels in mice, its effect was slightly less than that of FcRTAC NK-2-12-SK3 (Fig. 11g). Overall, these data suggest that any receptor can serve as a platform for developing eTPDs capable of mediating robust antigen clearance in circulation.

[0208] The above embodiments are merely illustrative of the principles and effects of this application and are not intended to limit this application. Any person skilled in the art can modify or alter the above embodiments without departing from the spirit and scope of this application. Therefore, all equivalent modifications or alterations made by those skilled in the art without departing from the spirit and technical concept disclosed in this application should still be covered by the claims of this application.

Claims

1. A bispecific antibody that binds to a cell surface receptor and a target protein, comprising a first antigen-binding portion that binds to the target protein and a second antigen-binding portion that binds to a cell surface receptor, wherein the cell surface receptor is a circulating cell surface receptor capable of mediating endocytosis, and the cell surface receptor is selected from FcγR2B receptor, ASGPR receptor, EGFR receptor, TfR receptor, HSPG receptor, IGF2R receptor, and CI-M6PR receptor.

2. The bispecific antibody according to claim 1, characterized in that, The second antigen-binding portion includes a single-chain antibody or a fragment thereof.

3. The bispecific antibody according to claim 2, characterized in that, The single-chain antibody or its fragment is scFv.

4. The bispecific antibody according to any one of claims 2 to 3, wherein the single-chain antibody or a fragment thereof comprises a heavy chain variable region and a light chain variable region, and the single-chain antibody or a fragment thereof has any one of the following characteristics: 1) The heavy chain variable region includes CDR1 as shown in SEQ ID NO.1, CDR2 as shown in SEQ ID NO.2, and CDR3 as shown in SEQ ID NO.3, and the light chain variable region includes CDR1 as shown in SEQ ID NO.4, CDR2 as shown in SEQ ID NO.5, and CDR3 as shown in SEQ ID NO.6; 2) The heavy chain variable region includes CDR1 as shown in SEQ ID NO.7, CDR2 as shown in SEQ ID NO.8, and CDR3 as shown in SEQ ID NO.9; the light chain variable region includes CDR1 as shown in SEQ ID NO.10, CDR2 as shown in SEQ ID NO.11, and CDR3 as shown in SEQ ID NO.

12. 3) The heavy chain variable region includes CDR1 as shown in SEQ ID NO.13, CDR2 as shown in SEQ ID NO.14, and CDR3 as shown in SEQ ID NO.15, and the light chain variable region includes CDR1 as shown in SEQ ID NO.16, CDR2 as shown in SEQ ID NO.17, and CDR3 as shown in SEQ ID NO.18; 4) The heavy chain variable region includes CDR1 as shown in SEQ ID NO.19, CDR2 as shown in SEQ ID NO.20, and CDR3 as shown in SEQ ID NO.21; the light chain variable region includes CDR1 as shown in SEQ ID NO.22, CDR2 as shown in SEQ ID NO.23, and CDR3 as shown in SEQ ID NO.

24. 5) The heavy chain variable region includes CDR1 as shown in SEQ ID NO.59, CDR2 as shown in SEQ ID NO.2, and CDR3 as shown in SEQ ID NO.60; the light chain variable region includes CDR1 as shown in SEQ ID NO.4, CDR2 as shown in SEQ ID NO.5, and CDR3 as shown in SEQ ID NO.

6. 6) The heavy chain variable region includes CDR1 as shown in SEQ ID NO.61, CDR2 as shown in SEQ ID NO.62, and CDR3 as shown in SEQ ID NO.63; the light chain variable region includes CDR1 as shown in SEQ ID NO.4, CDR2 as shown in SEQ ID NO.5, and CDR3 as shown in SEQ ID NO.

6. 7) The heavy chain variable region includes CDR1 as shown in SEQ ID NO.59, CDR2 as shown in SEQ ID NO.64, and CDR3 as shown in SEQ ID NO.3, and the light chain variable region includes CDR1 as shown in SEQ ID NO.4, CDR2 as shown in SEQ ID NO.5, and CDR3 as shown in SEQ ID NO.

6.

5. The bispecific antibody according to claim 4, characterized in that, The single-chain antibody or its fragment also has any of the following characteristics: 1) The sequence of the heavy chain variable region of the single-chain antibody or its fragment is shown in SEQ ID NO.25, and the sequence of the light chain variable region of the single-chain antibody or its fragment is shown in SEQ ID NO.26; 2) The sequence of the heavy chain variable region of the single-chain antibody or its fragment is shown in SEQ ID NO.27, and the sequence of the light chain variable region of the single-chain antibody or its fragment is shown in SEQ ID NO.28; 3) The sequence of the heavy chain variable region of the single-chain antibody or its fragment is shown in SEQ ID NO.29, and the sequence of the light chain variable region of the single-chain antibody or its fragment is shown in SEQ ID NO.30; 4) The sequence of the heavy chain variable region of the single-chain antibody or its fragment is shown in SEQ ID NO.31, and the sequence of the light chain variable region of the single-chain antibody or its fragment is shown in SEQ ID NO.32; 5) The sequence of the heavy chain variable region of the single-chain antibody or its fragment is shown in SEQ ID NO. 68, and the sequence of the light chain variable region of the single-chain antibody or its fragment is shown in SEQ ID NO. 26; 6) The sequence of the heavy chain variable region of the single-chain antibody or its fragment is shown in SEQ ID NO. 69, and the sequence of the light chain variable region of the single-chain antibody or its fragment is shown in SEQ ID NO. 26; 7) The sequence of the heavy chain variable region of the single-chain antibody or its fragment is shown in SEQ ID NO.70, and the sequence of the light chain variable region of the single-chain antibody or its fragment is shown in SEQ ID NO.

26.

6. The bispecific antibody according to any one of claims 4 to 5, characterized in that, The single-chain antibody or its fragments further include a short peptide connecting the heavy chain variable region and the light chain variable region.

7. The bispecific antibody according to claim 6, characterized in that, The sequence of the short peptide is shown in SEQ ID NO.

33.

8. The bispecific antibody according to any one of claims 1 to 7, characterized in that, The target protein is selected from extracellular soluble proteins or pathological protein aggregates.

9. The bispecific antibody according to claim 8, characterized in that, The target proteins are selected from IgE, PCSK9, TNF-α, IL-4, IL-6, IL-12, IL-17, VEGF, C5, MCP1, β-amyloid, hepcidin, α-synuclein, TREM2, and Tau.

10. The bispecific antibody according to any one of claims 1 to 9, wherein the first antigen-binding portion is a full-length antibody that binds to the target protein.

11. The bispecific antibody according to any one of claims 1 to 10, characterized in that, The first antigen-binding site binds to the target protein in a pH-dependent or pH-independent manner.

12. The bispecific antibody according to claim 11, characterized in that, The first antigen-binding portion binds to the target protein in a pH-dependent manner.

13. The bispecific antibody according to any one of claims 1 to 12, characterized in that, The first antigen-binding portion has any of the following characteristics: 1) The sequence of the heavy chain variable region of the first antigen-binding region is shown in SEQ ID NO.42; the sequence of the light chain variable region of the first antigen-binding region is shown in SEQ ID NO.43; the sequence of the heavy chain constant region of the first antigen-binding region is shown in SEQ ID NO.44; and the sequence of the light chain constant region of the first antigen-binding region is shown in SEQ ID NO.

41. 2) The sequence of the heavy chain variable region of the first antigen-binding region is shown in SEQ ID NO.45; the sequence of the light chain variable region of the first antigen-binding region is shown in SEQ ID NO.49; the sequence of the heavy chain constant region of the first antigen-binding region is shown in SEQ ID NO.50; and the sequence of the light chain constant region of the first antigen-binding region is shown in SEQ ID NO.

48.

14. The bispecific antibody according to any one of claims 1 to 13, characterized in that, The first antigen-binding portion is fused to the second antigen-binding portion via a linker peptide; And / or, the heavy chain variable region of the second antigen-binding portion fuses with the C-terminus of the heavy chain and / or light chain of the first antigen-binding portion.

15. The bispecific antibody according to claim 14, characterized in that, The amino acid sequence of the linker peptide is shown in SEQ ID NO. 60; And / or, the heavy chain variable region of the second antigen-binding portion fuses with the C-terminus of the heavy chain of the first antigen-binding portion.

16. A polynucleotide encoding a bispecific antibody as described in any one of claims 1-15.

17. A construct comprising the polynucleotide of claim 16.

18. A host cell containing the construct of claim 17, or the host cell genome having the polynucleotide of claim 16 integrated therein.

19. A pharmaceutical composition or kit comprising the bispecific antibody as described in any one of claims 1-15.

20. Use of the bispecific antibody of any one of claims 1-15, or the polynucleotide of claim 16, or the construct of claim 17, or the host cell of claim 18, or the pharmaceutical composition or kit of claim 19 in the preparation of products for the prevention and / or treatment of diseases related to target protein overexpression.

21. A method for preventing and / or treating diseases related to target protein overexpression, comprising administering to a subject a bispecific antibody as described in any one of claims 1-15, or a pharmaceutical composition or kit as described in claim 19.

22. The use according to claim 20, or the method according to claim 21, characterized in that, The target protein overexpression-related diseases are selected from diseases associated with IgE overexpression, PCSK9 overexpression, TNF-α overexpression, IL-4 overexpression, IL-6 overexpression, IL-12 overexpression, IL-17 overexpression, VEGF overexpression, C5 overexpression, MCP1 overexpression, β-amyloid overexpression, hepcidin overexpression, α-synuclein overexpression, TREM2 overexpression, and Tau overexpression.

23. The use according to claim 20, or the method according to claim 21, characterized in that, The diseases associated with IgE overexpression are selected from allergic diseases, parasitic infections, inflammatory diseases, primary immunodeficiency, tumors, cardiovascular diseases, and psoriasis caused by IgE overexpression. The diseases associated with PCSK9 overexpression are selected from diseases induced by hyperlipidemia and tumors caused by PCSK9 overexpression. The diseases associated with TNF-α overexpression are selected from autoimmune diseases, inflammatory bowel disease, diabetes, atherosclerosis, asthma, psoriatic arthritis, and tumors that overexpress TNF-α. The IL-4 overexpression-related diseases are selected from tumors, asthma, allergies and atopic dermatitis, and pulmonary fibrosis that overexpress IL-4. The IL-6 overexpression-related diseases are selected from non-small cell lung cancer, pulmonary hypertension, autoimmune diseases, tumors, and nervous system diseases that overexpress IL-6. The IL-12 overexpression-related diseases are selected from psoriasis, systemic lupus erythematosus, Crohn's disease, and malignant melanoma, all of which involve IL-12 overexpression. The IL-17 overexpression-related diseases are selected from autoimmune diseases, inflammatory skin diseases, hidradenitis suppurativa, viral infections, and parasitic infections caused by IL-17 overexpression. The VEGF overexpression-related diseases are selected from neurological diseases, eye diseases, respiratory diseases, liver diseases, tumors, and inflammatory diseases that involve VEGF overexpression. The C5 overexpression-related diseases are selected from C5-overexpressing colorectal cancer, mesangial proliferative glomerulonephritis, and non-small cell lung cancer; The diseases associated with MCP1 overexpression are selected from diabetic complications, inflammatory diseases, brain diseases, cardiovascular diseases, rheumatoid arthritis, neuroinflammatory diseases, tumors, pulmonary fibrosis, and autoimmune thyroid diseases. The diseases associated with β-amyloid overexpression are selected from Alzheimer's disease and tumors caused by β-amyloid overexpression. The diseases associated with hepcidin overexpression are selected from chronic anemia caused by hepcidin overexpression, anemia caused by chronic kidney disease, Castleman's disease anemia, iron overload anemia, primary myelofibrosis, and tumors. The diseases associated with α-synuclein overexpression are selected from Parkinson's disease and tumors caused by α-synuclein overexpression. The Tau overexpression-related diseases are selected from Tau-overexpressing Alzheimer's disease, frontotemporal dementia, corticobasal degeneration, progressive supranuclear palsy, Pick's disease, prion diseases, and tumors.

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