Bispecific nano antibody targeting FcRn and Fc gamma Rs as well as preparation method and application of bispecific nano antibody

Abstract

The invention provides a separated nucleic acid molecule. The nucleotide sequence of the separated nucleic acid molecule is shown as SEQ ID NO. 1. The invention also provides an antibody coded by the nucleic acid molecule. The invention also provides a carrier which contains the nucleic acid molecule. The invention also provides a method for producing the antibody. The method comprises the following steps: transfecting antibody expression plasmids in competent cells, culturing, collecting cell supernatant, centrifuging to remove precipitate, purifying antibody protein, collecting eluent, and concentrating to obtain the antibody. The invention also provides an application of the antibody, the carrier or the pharmaceutical composition in preparation of drugs for treating autoimmune diseases. The antibody disclosed by the invention can be specifically combined with FcRn and Fc gamma Rs, is a potent inhibitor for FcRn-mediated IgG recirculation and Fc gamma R-dependent immune complex activation, is a therapeutic drug for autoimmune diseases, and has huge potential.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine and relates to an antibody and its uses, specifically a bispecific nanobody targeting FcRn and FcγRs, its preparation method, and its uses. Background Technology

[0002] Autoimmune diseases are caused by the immune system's erroneous attack on the body's own tissues, ultimately leading to widespread tissue damage and systemic lesions. Immunoglobulin G (IgG) antibodies that target autoantigens are a core driver of this destructive process, contributing to the pathogenesis of many diseases, including rheumatoid arthritis (RA) and immune thrombocytopenic purpura (ITP).

[0003] In these diseases, IgG-autoantigen immune complexes (ICs) trigger a strong inflammatory cascade by binding to Fcγ receptors (FcγRs), which continuously exacerbates tissue damage. For example, antiplatelet antibodies lead to excessive platelet destruction in ITP, and immune complexes induce synovial inflammation and erode joints in RA.

[0004] Current treatment strategies primarily aim to suppress pathogenic IgG levels or block their destructive effector pathways. While plasma exchange, intravenous immunoglobulin (IVIg), and B-cell depletion therapies (such as rituximab) have demonstrated clinical efficacy, significant limitations remain: antibody drugs are expensive and require frequent injections; IVIg, as a blood product, carries the potential risk of pathogen contamination; and widespread B-cell depletion leads to excessive immunosuppression, with significant individual variability in efficacy. Therefore, the urgent need to develop precise targeting strategies against IgG-driven pathological processes is increasingly evident.

[0005] FcRn-mediated IgG recycling effectively protects IgG from lysosomal degradation through a pH-dependent binding-dissociation cycle, significantly reducing the need for biosynthesis while maintaining antibody homeostasis. Based on this mechanism, FcRn-targeting therapies such as Fc-modified monoclonal antibodies (mAbs) developed in recent years can significantly accelerate the clearance of endogenous IgG (including pathogenic antibodies) by competitively inhibiting IgG recycling, with efficacy comparable to plasma exchange. However, single-target FcRn strategies still face three key challenges in complex autoimmune diseases: limited tissue clearance, the immune risk of non-selective clearance, and compensatory antibody rebound. Therefore, developing bifunctional antibodies that simultaneously target FcRn and FcγR holds promise for overcoming the clinical limitations of current therapies by synergistically blocking IgG recycling and complement activation, and selectively clearing pathogenic antibodies.

[0006] FcγR is a core hub in the pathogenic effect of autoantibodies. Its activation depends on the cross-linking of immune complexes (ICs) with activating receptors (FcγR1, FcγR1A, FcγRIIIa) via the Fc segment, triggering phosphorylation of the immune receptor tyrosine activation motif (ITAM) and activating the spleen tyrosine kinase (SYK) signaling pathway. This drives effector functions such as antibody-dependent cell-mediated cytotoxicity (ADCC) and antibody-dependent cell-mediated phagocytosis (ADCP), ultimately leading to tissue damage. Current therapeutic strategies targeting FcγR mainly include SYK inhibitors (such as fotatinib), which exert broad-spectrum effects by blocking downstream signal transduction. Inhibition; Fc engineered antibodies (such as LALA mutants) eliminate FcγR binding ability; and Fc multimers (such as stradomers, M045, multi-Fc variant GL-2045, hexamer IgG1-Fc and trimer Fc) regulate immune responses through competitive binding. However, existing therapies still face three major challenges: (1) broad-spectrum inhibition leads to infection risk (such as SYK inhibitors); (2) impaired effector function (such as Fc engineered antibodies); and (3) inflammation risk caused by multimer cross-linking (such as Fc multimers).

[0007] Therefore, next-generation treatment strategies need to focus on the precise regulation of monomeric Fc and the synergistic design of dual-targeting drugs. Monomeric Fc can circumvent the non-specific activation of multimers; while dual-targeting drugs (such as FcγR / FcRn) can simultaneously block the generation and effector function of pathogenic antibodies, achieving safer and more durable immune regulation. This direction is expected to drive the treatment of autoimmune diseases from "broad-spectrum inhibition" to "precision intervention," providing more optimized solutions for clinical practice. Summary of the Invention

[0008] To address the aforementioned technical problems in the prior art, this invention provides a bispecific nanobody targeting FcRn and FcγRs, its preparation method, and its uses. This bispecific nanobody targeting FcRn and FcγRs, its preparation method, and its uses aim to solve the technical problem of poor therapeutic effects of existing drugs on autoimmune diseases.

[0009] The present invention provides an isolated nucleic acid molecule, the nucleotide sequence of which is shown in SEQ ID NO.1.

[0010] The present invention also provides an antibody encoded by the above-mentioned nucleic acid molecule, the amino acid sequence of which is shown in SEQ ID NO.4.

[0011] The present invention also provides a carrier comprising the above-described nucleic acid molecules.

[0012] The present invention also provides a host cell comprising the above-described nucleic acid molecules or the above-described carriers.

[0013] The present invention also provides a pharmaceutical composition comprising the antibody described above and a pharmaceutically acceptable carrier.

[0014] The present invention also provides a method for generating the above-mentioned antibody, wherein a plasmid containing the above-mentioned antibody nucleotide sequence is transfected into competent cells, the cell supernatant is collected after culture, the precipitate is removed by centrifugation, the antibody protein is purified, the eluent is collected and concentrated to obtain the antibody.

[0015] The present invention also provides the use of the above-described antibody, the above-described carrier, or the above-described pharmaceutical composition in the preparation of a medicament for treating autoimmune diseases.

[0016] Furthermore, in the above applications, the antibody is administered in combination with chemotherapeutic agents, radiotherapy, and / or other agents used for cancer immunotherapy.

[0017] Furthermore, the autoimmune disease mentioned refers to rheumatoid arthritis or immune thrombocytopenic purpura.

[0018] The present invention also provides a recombinant protein containing the nucleic acid molecule shown in SEQ ID NO.1.

[0019] To overcome these technical problems in existing technologies, this invention designs Fc-3-18—a 15 kDa bispecific nanobody that can simultaneously block FcRn and FcγRs. Utilizing the ASyNaL phage display platform, this invention designs a high-affinity, dual-targeting nanomonoclonal antibody (Fc-3-18, 15 kDa) that specifically binds to FcRn and FcγRs. Through iterative screening of Fc mutant libraries and selection guided by biolayer interference (BLI), this invention determines that Fc-3-18 is a potent inhibitor of FcRn-mediated IgG recycling and FcγR-dependent immune complex activation, without inducing IVIg-like immune responses. This invention rigorously validates the efficacy of Fc-3-18 in mouse ITP and CIA models, demonstrating its significant potential as a next-generation therapeutic agent for antibody-driven autoimmune diseases.

[0020] Compared with existing technologies, the technical effects of this invention are positive and obvious.

[0021] 1) From the perspective of mechanism of action, Fc-3-18's pH-dependent Fc receptor binding characteristic is particularly prominent. This molecule maintains high affinity (KD = 5.7 nM) even at the endosomal pH of 6.0, while appropriately dissociating under physiological pH conditions. This allows it to effectively clear IgG antibodies without disrupting normal immune homeostasis. This contrasts sharply with traditional Fc receptor inhibitors—which often lead to excessive IgG consumption and may weaken the body's defense capabilities.

[0022] 2) From a transformational perspective, the 15 kDa molecular weight of Fc-3-18 offers the following advantages: enhanced tissue permeability, simplified manufacturing process, and potentially lower immunogenicity risk. Since there is no risk of complement activation or cytokine release syndrome, long-term use is generally safe, and the dual mechanism of action can reduce the probability of drug resistance. Attached Figure Description

[0023] Figure 1 The expression efficiency and high affinity of Fc-3-18, as well as its binding ability to FcRn and FcγRs, were demonstrated.

[0024] Figure 2 This demonstrates that Fc-3-18 can effectively inhibit IgG-mediated immune effector function.

[0025] Figure 3 The in vitro safety of Fc-3-18 was demonstrated.

[0026] Figure 4 The therapeutic effect of Fc-3-18 in a mouse model of autoimmune disease (rheumatoid arthritis) was demonstrated.

[0027] Figure 5 The changes in B cells in the bone marrow, lymph nodes, and peripheral blood of rheumatoid arthritis mice treated with Fc-3-18 are shown.

[0028] Figure 6 The therapeutic effect of Fc-3-18 in a mouse model of autoimmune disease (immune thrombocytopenic purpura) was demonstrated.

[0029] Figure 7 This study identifies several candidate molecules with neutralizing potential using the ASyNaL platform. Detailed Implementation Example 1: Construction, design, and expression preparation of dual-targeting nanoclonal antibodies

[0030] (1) Construction Design This invention designs a dual-targeting Fc fragment using the ASyNaL platform through a multi-stage process. The specific steps are as follows: First, a large library of nanobody molecules was screened based on multiple components of FcRn. Several candidate molecules with neutralizing potential (Fc mutants with high affinity for the Fc receptor (FcRn)) were identified (using competitive binding assays, biolayer interferometry (BLI), and other biomolecular interaction analysis techniques). After screening with FcγR, 10 potential neutralizing antibodies were obtained (e.g., Figure 7 (As shown).

[0031] Then, using the FcγR antibody as the parent, an affinity-matured library targeting FcγR was constructed using phage display technology.

[0032] To rule out non-specific binding, the binding affinity of different nanobodies to various antigens was evaluated (using competitive binding assays, biolayer interferometry (BLI), and other biomolecular interaction analysis techniques), and the Fc-3-18 nanobodies (C5G2) were ultimately selected. The partial CDR sequence of the Fc-3-18 nanobody (C5G2) is shown below: CDR H1(27 - 33) CDR H2(51 - 58) CDR H3(99 - 112) KFSHLVF GLGAYESG LVVLSRDNTEFIAH SDS-PAGE analysis showed that Fc-3-18 (C5G2) expression was highly efficient and showed no significant degradation, indicating excellent protein expression levels suitable for subsequent functional validation. Figure 1 A).

[0033] The binding dynamics of engineered Fc fragments to FcRn and Fcγ receptors were systematically characterized using a biolayer interferometer (BLI) system (Sartorius). Two different capture strategies were employed for parallel receptor interaction analysis.

[0034] To address FcRn interactions, when using a CAP sensor chip, biotinylated anti-FcRn antibodies were captured as an intermediate layer using a biotin capture kit (GE Healthcare). Subsequently, biotinylated human FcRn (α-chain / β2-microglobulin heterodimer) was immobilized at a density of 50-100 reaction units (RU) to ensure optimal receptor orientation and reduce steric hindrance.

[0035] When studying Fcγ receptors, recombinant human Fcγ receptors (FcγRI / CD64, FcγRIIa / CD32a, and FcγRIIIa / CD16a) with histidine tags were immobilized at a density of 100-150 RU to ensure consistent presentation of the extracellular domains of the functional receptors.

[0036] BLI analysis showed that Fc-3-18 exhibited high affinity for FcγR and slow dissociation, indicating potential for further development. Figure 1B). Furthermore, experimental results at different concentrations and pH conditions showed that, at pH 6, the Fc-3-18 mutant of this invention exhibited excellent binding ability to both FcRn and FcγRs. Figure 1 C).

[0037] (2) Expression preparation The antibody expression plasmid (containing the nucleotide sequence shown in SEQ ID NO.1) was transfected into competent cells. After culturing, the cell supernatant was collected and added to PBS stock solution to achieve a concentration of 1×PBS. The precipitate was removed by high-speed centrifugation, and the antibody protein was purified. ①. Equilibration: Use 1×PBS to equilibrate the chromatography column, 5 column volumes; ②. Sample loading: The entire cell supernatant is loaded through the chromatography column, then equilibrated by top washing with PBS until the UV280 drops to near 0 and stops decreasing; ③. Washing: Use wash buffer to wash away impurities. An impurity peak may appear in this step. Wait until the UV280 drops to a stable level. ④. Elution: Elute the target antibody using elution buffer. This elution step is extremely fast, and you must remain on the equipment at all times, ready to inoculate the sample. Immediately after inoculation, add 1 / 10 volume of 1 M Tris-base Buffer pH 8.0 (1 MTB pH 8.0) to neutralize the acidity. Then, replace the PBS with an ultrafiltration tube, and finally determine the concentration and store the sample.

[0038] ⑤. Cleaning and regeneration of the chromatography column: Clean the column with 0.1M NaOH for 10-15 min, and rinse with buffer for 5 column volumes; ⑥. Preservation of chromatography column: Store in 20% ethanol.

[0039] All purified proteins were centrifuged using ultrafiltration tubes to replace the PBS buffer, and the protein concentration was determined. High-concentration proteins were diluted with PBS to a concentration of 5 mg / ml, aliquoted into 1.5 ml EP tubes, and stored at -20°C for later use.

[0040] The synthesized nanobody Fc-3-18 exhibits high efficiency, broad-spectrum activity, and unique binding properties: it can simultaneously bind to two different epitopes (FcRn and FcγRs) of the same antigen. Unlike other IgG antibodies and bispecific antibodies, this antibody exists in the form of a nanobody (15 kDa).

[0041] To compare with existing Fc variants, we synthesized Fc-YTE (a validated serum-long half-life Fc mutation) using the existing ASyNaL platform. Using the wild-type Fc sequence (Fc-Tag) from the ASyNaL platform, we introduced the YTE mutation through site-directed mutagenesis: changing the amino acid sequences at positions 32, 34, and 36 from those encoding MST to those encoding YTE. The PCR amplification product was then digested with enzymes to remove the template plasmid. The digested PCR product was transformed into competent cells, plated on antibiotic resistance plates to select single clones, and the plasmids from these clones were extracted for DNA sequencing to verify the correct introduction of the three mutation sites. Subsequently, the Fc-YTE protein was expressed and purified following the same expression preparation steps as Fc-3-18 described above.

[0042] The DNA nucleotide sequences of the dual-targeting antibody Fc-3-18, its variant Fc-YTE, and the wild-type Fc (Fc-Tag) involved in this invention are shown in the table below: Name Sequence Fc-3-18 ATGAATCTCCTGCTAATTCTTACGTTCGTCGCGGCCGCAGTCGCCTCTTCTGATAAGACCCATACTTGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTTCCGGGCGCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCATGCACCAGGACTGGGTCAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTAGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATACACGAGGCTCTGCACCAACACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAATGA (SEQ ID NO.1). Fc-YTE ATGAATCTCCTGCTAATTCTTACGTTCGTCGCGGCCGCAGTCGCCTCTTCTGATAAGACCCATACTTGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCTATATCACCCGGGAACCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTAGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCACGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAATGA(SEQID NO.2)。 Fc-Tag ATGAATCTCCTGCTAATTCTTACGTTCGTCGCGGCCGCAGTCGCCTCTTCTGATAAGACCCATACTTGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCACGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAATGA (SEQ ID NO.3) The amino acid sequences of the dual-targeting antibody Fc-3-18, its variant Fc-YTE, and the wild-type Fc (Fc-Tag) involved in this invention are shown in the table below: Name Sequence Fc-3-18 MNLLLILTFVAAAVASSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTHTCPPCPAPELLGGPSVFLFPPKPKDTLMIFRAPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVMHQDWVNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVIHEALHQHYTQKSLSLSPGK* (SEQ ID NO.4) Fc-YTE MNLLLILTFVAAAVASSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLYITREPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK* (SEQ ID NO.5) Fc-Tag MNLLLILTFVAAAVASSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK* (SEQ ID NO.6) The mutant amino acid sequences of the dual-targeting antibody Fc-3-18, variant Fc-YTE, and wild-type Fc (Fc-Tag) involved in this invention are shown in the table below: Example 2: Fc-3-18 can effectively inhibit IgG-mediated immune effector function.

[0043] (1) Anti-D antibody-mediated inhibition of RhD+ erythrocyte cytotoxicity (ADCC) assay Take 2 mL of fresh blood from a healthy person and dilute it 1:1 with PBS. Prepare 2 mL of lymphocyte separation medium in a sterile centrifuge tube. Pipette the diluted blood and slowly add it along the tube wall, 1 cm above the separation medium, ensuring the diluted blood overlaps the separation medium and forms a clear interface. Centrifuge at 2000 rpm for 20 minutes at room temperature. At this point, five layers will form in the centrifuge tube: the top layer is plasma, and between the plasma layer and the lymphocyte separation medium is a white membrane-like layer of lymphocytes. Carefully aspirate the middle layer of mononuclear cells, ensuring all mononuclear cells are removed. Wash once with at least 5 times the volume of PBS and centrifuge at 1500 rpm for 5 minutes.

[0044] Discard the supernatant, add 300 μL of PBS, mix well, and take 10 μL for cell counting. Adjust the cell concentration for later use. Use RhD+ red blood cells (purified from whole blood after three washes with PBS) as target cells, and add PBMCs (1 × 10⁻⁶) to the cell line. 6 1 cell / well; 25 µL, 4 × 10 7 (cells / mL) and 50 μL of anti-D antibody (IgG) (Solarbio) and RhD-positive red blood cells (0.5 × 10⁻⁶ cells / mL) 6 1 cell / well; 25 µL, 2×10 7 Cells / mL were co-incubated at 37°C for 16 hours in the presence of Fc-YTE, Fc-3-18 and IVIg (effect-to-target ratio 2:1).

[0045] The amount of hemoglobin released from the supernatant generated by anti-D antibody (IgG) induced lysis was quantitatively detected using visible light spectrophotometry (BioTek Synergy HTX). Cytotoxic activity was then determined using a colorimetric method based on hemoglobin release, thereby quantifying the inhibitory effect of Fc-3-18 on antibody-mediated cell lysis.

[0046] The results showed that Fc-3-18 exhibited superior protective efficacy compared to therapeutic intravenous immunoglobulin (IVIg) and the control Fc variant (Fc-YTE). Figure 2 A).

[0047] (2) Inhibition experiment of interleukin (IL)-2 secretion Raji cells expressing CD20 (2.5 × 10⁻⁶) 5 Cells / well) and anti-CD20 monoclonal antibody (rituximab, 10 mg / mL) (Roche), Jurkat cells (0.5 × 10⁻⁶ cells / well) 5 Cells / well), 10 ng / mL phorbol ester (PMA) (MedChemExpress), Fc-YTE, Fc-3-18 and IVIg were co-cultured at 37°C for 16 hours.

[0048] IL-2 secretion levels were quantitatively detected using an ELISA assay (Human IL-2 ELISA kit #E-EL-H0099; Elabscience). The results showed that a 10 μL dose of Fc-3-18 inhibited IL-2 secretion by 75.9 ± 37.9%, significantly exceeding the inhibitory effect of IVIg, indicating that this drug can more effectively block the FcγR-mediated immune cell activation pathway. This finding has important implications for autoimmune diseases—in which immune complex-driven cytokine production is a major driver of pathological processes. Figure 2 B).

[0049] (3) Antibody-mediated platelet phagocytosis assay Platelets were isolated from human peripheral blood labeled with CFSE (BD Horizon™) and co-cultured with human THP1 monocytes. IVIg, Fc-3-18, and Fc-YTE were then added, and the cells were incubated at 37°C for 30 minutes. Antibody-mediated platelet phagocytosis was subsequently detected using flow cytometry (BD AriaIII).

[0050] Flow cytometry analysis showed equally significant inhibitory effects: Fc-3-18 reduced THP-1-mediated opsonized platelet clearance by 6.70 ± 1.34%, almost reaching the inhibitory effect of IVIg (8.03 ± 0.79%) by 0.83 ± 0.19 times (p < 0.01). This potent blockade of Fcγ receptor-dependent phagocytosis highlights the molecule's potential to alleviate antibody-mediated cytopenia by directly interfering with myeloid effector cell function (Figures 2C and 2D).

[0051] In these diverse experimental systems, Fc-3-18 consistently exhibits multimodal FcγR blocking activity, making it a highly promising therapeutic candidate for antibody-mediated diseases. Its broad-spectrum inhibitory properties target multiple pathological mechanisms simultaneously—from direct cytotoxicity to phagocytic clearance and pro-inflammatory signaling—while maintaining superior specificity for pathogenic immune complexes compared to physiological immune complexes. These properties promise to translate into enhanced clinical efficacy in complex autoimmune diseases, where current Fc-targeted therapies have limited effectiveness.

[0052] Example 3: In vitro safety assessment of Fc-3-18 (1) Complement activation experiment Whole blood (100 μL) from healthy individuals was incubated overnight at 37°C with CaCl2 (8 μL, 75 mM) and different volumes of Fc-3-18, IVIg (1.5 μL, 15 μL, 150 μL). The reaction was terminated by adding 5 μL of 100 mM EDTA. After centrifugation at 1700 rpm for 2 minutes, the supernatant was collected, and the levels of C3 and C4 were quantitatively analyzed using immunoturbidimetry (Roche 8000). Quantitative detection of complement components C3 and C4 in whole blood revealed that Fc-3-18 did not activate the classical complement pathway in the concentration range of 1.5–150 μL. Figure 3 A). Notably, at the highest tested concentration (150 μL), both C3 (0.38 ± 0.12%, p < 0.05) and C4 (0.10 ± 0.03%, p < 0.05) levels showed a statistically significant, albeit insignificant, decrease compared to baseline, suggesting a possible complement depletion inhibition phenomenon.

[0053] (2) Whole blood cytokine release test Whole blood from healthy individuals was incubated with different concentrations of Fc-3-18 (1.5 μL, 15 μL, 150 μL) and IVIg (1.5 μL, 15 μL, 150 μL) at 37°C for 16 hours. The cytokine levels in the supernatant were then measured using a human cytokine assay kit (magnetic bead flow cytometry) (Saige Biotechnology).

[0054] The levels of interleukin-1β (IL-1β), interleukin-2 (IL-2), interleukin-5 (IL-5), interleukin-6 (IL-6), interleukin-8 (IL-8), interleukin-10 (IL-10), interleukin-12 / p70 (IL-12 / p70), interleukin-17A (IL-17A), tumor necrosis factor-α (TNF-α), interferon-α (IFN-α), and interferon-γ (IFN-γ) were quantitatively detected using CBA technology via flow cytometry.

[0055] The results showed that the levels of pro-inflammatory cytokines (IL-1β, IL-2, IL-6, IL-12 / p70, TNF-α, IFN-α / γ, IL-17A) and anti-inflammatory cytokines (IL-5, IL-10) were not significantly increased. Figure 3 B). However, the pro-inflammatory cytokine IL-8 was slightly elevated, which may be related to environmental or exogenous factors.

[0056] (3) Induction and activation of platelet aggregation Whole blood from healthy individuals was centrifuged at 1500 rpm for 12 minutes, and the supernatant was platelet-rich plasma (PRP). The PRP was then washed three times with PBS at 3500 rpm for 5 minutes each time.

[0057] Fc-3-18 and IVIg were added to 25 µL of PRP and incubated at 37 °C for 20 min. TRAP-6 (AbMole BioScience) was used as a positive control at a final concentration of 50 μM. Platelet activation was quantified by detecting the expression levels of CD42b and CD62P (BioLegend) using flow cytometry (BD Aria III).

[0058] Flow cytometry analysis showed that neither Fc-3-18 nor IVIg induced CD62P or CD42b expression. Figure 3 (C and 3D). In contrast, TRAP-6 was significantly elevated in the positive control group. This indicates that although Fc-3-18 optimizes its binding affinity to FcγRIIa, it does not promote platelet activation or subsequent prothrombotic responses.

[0059] These findings demonstrate that Fc-3-18 exhibits a superior safety profile compared to existing Fc-targeted therapies, making it a highly promising therapeutic candidate. This drug overcomes a key deficiency of current immunomodulatory biologics by not activating the complement system, stimulating platelets, or inducing cytokines. Comprehensive safety assessments provide ample evidence for advancing in vivo toxicology trials.

[0060] Example 4: Therapeutic effect of Fc-3-18 in a mouse model of autoimmune disease (1) Collagen-induced arthritis (CIA) mouse model On day 0, arthritis was induced in 8-12 week old male DBA / 1 mice (GemPharmatech) by subcutaneous injection of bovine type II collagen (Chondrex) emulsified with complete Freund's adjuvant (containing 5 mg / ml Mycobacterium tuberculosis) at the base of the tail. Figure 4 A). On day 21, mice were boosted with a complete Freund's adjuvant emulsified bovine type II collagen containing 1 mg / ml Mycobacterium tuberculosis (Sigma). Arthritis symptoms were monitored every three days after the booster. Three weeks after the second immunization, mice were randomly assigned to a control group and a treatment group (PBS, Fc-3-18) (n=3). Each group was treated every other day with an intraperitoneal injection of Fc-3-18 (100 μL / mouse) or PBS for two weeks. After sacrifice, limbs were collected for hematoxylin-eosin staining, and bone marrow, lymph nodes, and peripheral blood samples were collected for flow cytometry (BD Aria III) analysis of immune cells. Anti-CD19 (BioLegend), anti-CD38 (BioLegend), anti-GL7 (BioLegend), and anti-CD138 antibodies (BD Horizon) were used for staining.

[0061] The clinical parameter assessment criteria are as follows: Arthritis is graded on a scale of 0-4 for each paw, where 0 indicates no swelling; 1 indicates mild swelling with redness and a paw size increase of ≥0.1 mm; 2 indicates swelling with redness and a paw size increase of ≥0.2 mm; 3 indicates extensive swelling ≥0.3 mm with severe joint deformity or ankylosis; and 4 indicates significant swelling ≥0.45 mm with joint deformity or ankylosis. Each mouse's limbs were scored separately, with a maximum total score of 16.

[0062] In the untreated group, the symptoms gradually worsened, manifested as persistent redness and swelling of the paws and a continuous increase in clinical scores. Notably, compared with baseline before drug administration, the paw swelling in the Fc-3-18 treated group was significantly reduced. Figure 4 B), and the clinical score also gradually decreased ( Figure 4C). No significant change in mouse body weight was observed before and after treatment. Figure 4 D).

[0063] Histopathological analysis showed that, compared with the blank control group and the untreated group, Fc-3-18 effectively inhibited joint inflammation, synovial hyperplasia, and bone erosion. Figure 4 E). Post-treatment analysis of B cells in bone marrow, lymph nodes, and peripheral blood showed that, compared with the untreated group, mice treated with Fc-3-18 had a significantly reduced number of CD19+ B cells in bone marrow, but no significant differences were observed in lymph nodes and peripheral blood. Conversely, the number of plasma cells in the lymph nodes of mice treated with Fc-3-18 was significantly reduced compared with the untreated group, but no significant changes were observed in bone marrow and peripheral blood. No significant changes in activated B cells were observed after treatment. Figure 5 A, 5B, and 5C).

[0064] (2) Passive immune thrombocytopenic purpura (ITP) mouse model This invention established a passive antibody-mediated immune thrombocytopenic purpura (ITP) model using 7-8 week old BALB / c mice (GemPharmatech) and 8-week-old male guinea pigs (JSJ). All experimental animals were housed in a specific pathogen-free environment, with a male-to-female ratio of 50%.

[0065] The platelet separation procedure is as follows: First, anticoagulated whole blood is collected from anesthetized BALB / c mice. After gradient centrifugation, the blood is washed three times with PBS buffer, resuspended, counted, and adjusted to a final concentration of 2.5 × 10⁻⁶. 6Platelets were then uniformly emulsified with equal volumes of complete Freund's adjuvant (Sigma) and incomplete Freund's adjuvant (Sigma). At week 0, 1 ml of platelet antigen emulsified with complete Freund's adjuvant was injected subcutaneously into guinea pigs at at least four injection sites, including the paws, back, and abdomen. During weeks 1, 2, and 3, booster immunizations were administered with 1 ml of platelet antigen emulsified with incomplete Freund's adjuvant, using the same method. At week 4, unanticoagulated whole blood was collected from the guinea pig heart, centrifuged at 3500 rpm for 10 minutes after standing, and the supernatant was used as guinea pig anti-mouse platelet serum (GP-APS). This serum was neutralized by inactivating complement activity at 56°C for 30 minutes, followed by two adsorption treatments with an equal volume of 5% BALB / c mouse erythrocyte suspension. The treated serum was then diluted 1:4 for use. Thrombocytopenia was induced by administration of GP-APS (5 μl / g body weight). Baseline platelet counts were measured 24 hours before GP-APS stimulation (day 0) via retroorbital blood sampling (50 μL) and analyzed using a fully automated veterinary blood analyzer (Mindray BC-5000 Vet). Two hours before GP-APS administration (day 1), mice received prophylactic treatment: Fc-3-18 (5 μl / g PBS, intraperitoneal injection), IVIg (5 μl / g, positive control), or PBS (solvent control). Platelet counts were reassessed 24 hours after GP-APS administration (day 2) to confirm the development of thrombocytopenia. Continuous monitoring was conducted from 24 to 96 hours post-induction to assess the duration of the protective effect. After monitoring, bone marrow smears were prepared from mouse femurs, stained with Gymza stain, and examined under an optical microscope. The number of megakaryocytes within a diameter of 1.5cm × 3.0cm in bone marrow smears was counted, and the megakaryocyte classification (primitive, immature, granular, naked nucleus, and platelet-producing megakaryocytes) was assessed simultaneously.

[0066] Intraperitoneal injection of Fc-3-18 (5µl / g) two hours prior to GP-APS induction provided significant protection against thrombocytopenia. Figure 6 A). Twenty-four hours after serum injection (day 2), platelet counts were significantly decreased in all groups of mice. However, in mice that received an intraperitoneal injection of Fc-3-18 two hours before serum injection, platelet counts recovered to 315.7 ± 73.5 × 10⁻⁶ by day 3. 9 / L (P<0.05), compared to the ITP model group (179.3±14.0×10 9 The platelet count in the IVIg group was 1.76 ± 0.43 times higher than that in the normal range. In contrast, the platelet count in the IVIg group was 233.3 ± 45.8 × 10⁹ / L. 9 / L, no statistically significant difference was observed ( Figure 6B). Megakaryocyte maturation arrest is a typical pathological feature of ITP. It is mainly manifested as an increase in the total number of megakaryocytes, but a decrease in the number of platelet-producing megakaryocytes. In these cases, immature megakaryocytes predominate. Bone marrow smear observation and statistical analysis revealed that the number of megakaryocytes in the ITP model group (166.7±11.51) was significantly higher than that in the control group (71.33±14.57) (P<0.001). Furthermore, most of these megakaryocytes were in a primitive or immature stage, which is highly consistent with the pathological characteristics of ITP.

[0067] In contrast, the number of megakaryocytes in mice receiving prophylactic Fc-3-18 treatment was 96.7±22.2, significantly lower than that in the ITP model group (P < 0.01). The megakaryocyte count in mice receiving IVIg treatment was 116.0±16.4, also significantly lower than that in the ITP model group (P < 0.05). Notably, both treatment groups showed the presence of granular megakaryocytes (precursors of platelet-producing megakaryocytes) and platelet-producing megakaryocytes, indicating that drug treatment significantly improves thrombocytopenia symptoms. Figure 6 C and 6D).

[0068] Preclinical studies have confirmed that Fc-3-18 is significantly more effective than intravenous immunoglobulin (IVIg), thanks to its unique dual regulatory mechanism—maintaining antibody persistence while simultaneously activating effector cells. This drug not only improves clinical symptoms but also effectively regulates B cell populations and plasma cell function. These advantages provide strong clinical research evidence for the treatment of antibody-mediated autoimmune diseases.

Claims

1. An isolated nucleic acid molecule, characterized in that, Its nucleotide sequence is shown in SEQ ID NO.

1.

2. An antibody encoded by the nucleic acid molecule of claim 1.

3. A carrier, characterized in that, It comprises the nucleic acid molecule as described in claim 1.

4. A host cell, characterized in that, It comprises the nucleic acid molecule of claim 1 or the vector of claim 3.

5. A pharmaceutical composition, characterized in that, It comprises the antibody as described in claim 2 and a pharmaceutically acceptable carrier.

6. A method for producing the antibody of claim 2, characterized in that, The competent cells were transfected with a plasmid containing the nucleic acid molecule shown in SEQ ID NO.

1. After culturing, the cell supernatant was collected, centrifuged to remove the precipitate, and the antibody protein was purified. The eluent was collected and concentrated to obtain the antibody.

7. The use of the antibody of claim 2, the carrier of claim 3, or the pharmaceutical composition of claim 5 in the preparation of a medicament for treating autoimmune diseases.

8. The use as described in claim 7, characterized in that, The antibody is administered in combination with chemotherapy agents, radiotherapy, and / or other agents used for cancer immunotherapy.

9. The application according to claim 7, characterized in that, The autoimmune diseases mentioned are rheumatoid arthritis or immune thrombocytopenic purpura.

10. A recombinant protein, characterized in that, The recombinant protein contains the nucleic acid molecule shown in SEQ ID NO.1.

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