Anti-Dsg1 antibody and mouse model and application thereof

Abstract

The invention relates to an anti-Dsg1 antibody as well as a mouse model and application thereof. The antibody is a P22344 antibody. The antibody disclosed by the invention can be quickly synthesized in quantity, is used for constructing pemphigus mouse models, and has the advantages of high accessibility and standardization; a disease phenotype and disease severity evaluation method of the Dsg1 antibody induced adult pemphigus mouse model is comprehensively observed and developed, and an animal model which is high in accessibility, standardized and capable of simulating skin lesion changes of pemphigus patients is constructed; the evaluation method is used for evaluating the pemphigus disease system and the effect of local intervention means, and has the advantage of standardization.

Description

Technical Field

[0001] This invention belongs to the field of pemphigus treatment technology, and specifically relates to an anti-Dsg1 antibody, its mouse model, and its application. Background Technology

[0002] Pemphigus is an organ-specific autoimmune disease induced by autoantibodies, characterized by vesicular erosions of the skin and mucous membranes. It can be divided into pemphigus vulgaris and pemphigus foliaceus. The target antigens of pemphigus autoantibodies are desmoglein 1 and 3 (Dsg), which are located between desmosomes in keratinocytes. Their distribution differs between the skin and mucous membranes. Pemphigus foliaceus is caused by anti-Dsg1 antibodies and only presents with skin lesions; while pemphigus vulgaris is caused by both anti-Dsg1 and anti-Dsg3 antibodies, resulting in both skin and mucous membrane lesions. Pemphigus is a relatively rare disease, but it severely impacts patients' quality of life and has a high mortality rate. Although the use of hormones and biologics has significantly improved the prognosis of pemphigus patients, some patients still cannot tolerate the side effects of hormone and biologic therapy or resist existing treatments.

[0003] Animal models are helpful for in-depth research into the pathogenesis of skin diseases and the development of new treatments. Among other animal models of skin diseases, the imiquimod-induced psoriasis-like skin inflammation mouse model is characterized by its simplicity, rapid model establishment, and low cost. It is the most widely used psoriasis mouse model to date, used to simulate acute and chronic relapsing skin lesions of psoriasis, and is widely used in research related to skin T-cell activation, microbiota, systemic and topical drug treatments. Other commonly used mouse models, such as the 2,4-dinitrochlorobenzene sensitization-induced atopic dermatitis mouse model and the UVA and UVB exposure-induced skin photoaging mouse model, are characterized by their simplicity, ability to simulate changes in the skin lesion microenvironment, and the availability of disease severity assessment systems. However, current basic and clinical translational research on pemphigus foliaceus is limited by the lack of highly accessible, standardized, and long-term chronic animal models.

[0004] The most classic animal model of pemphigus is the neonatal mouse model, which involves transferring serum from pemphigus patients (containing anti-Dsg1 and / or anti-Dsg3 antibodies) into newborn BALB / c mice, resulting in observed skin blisters. However, this model can only be used for short-term studies (a few hours), and the thin skin and underdeveloped immune system of newborn mice cannot simulate the chronic skin changes of pemphigus patients. Mouse models of pemphigus vulgaris are relatively mature, and existing adult mouse models include: ① Dsg3- / - mice: Significantly reduced weight and hair loss 8-10 days after birth; developmental delays may be related to restricted feeding. ② Lymphocyte transfer-induced model of actively producing anti-Dsg3 antibodies: Dsg3- / - mice are immunized with recombinant mouse Dsg3, and spleen cells from the immunized Dsg3- / - mice are transferred to Dsg3-expressing Rag2- / - immunodeficient mice. The recipient mice produce stable anti-Dsg3 antibodies and exhibit the disease phenotype. The distribution of Dsg1 and Dsg3 proteins in mouse skin is similar to, but slightly different from, that in humans. Dsg3 protein is distributed in mucous membranes and hair follicles, while Dsg1a / b / c is mainly distributed in the epidermis. Therefore, these existing mouse models of pemphigus vulgaris do not exhibit obvious skin vesicles, erosions, or rashes; they can only partially mimic the mucosal damage of pemphigus and cannot simulate acantholysis and inflammatory changes in the skin. Furthermore, their high construction cost, requirement for special diets, and high mortality rate limit their application.

[0005] Knocking out the Dsg1 gene or using anti-Dsg1 antibodies may induce skin lesions in mice similar to those seen in patients with pemphigus foliaceus. However, Dsg1- / - mice suffer from significant physiological structural defects and die during the embryonic stage; Dsg1- / - mice cannot survive, making it impossible to establish a mouse model of lymphocyte transfer induced by active anti-Dsg1 antibody production. Developing synthetic pathogenic anti-Dsg1 antibodies for constructing a passive transfer model of pemphigus in adult mice would be beneficial for regulating antibody titers, standardizing research, and promoting widespread application. Currently available pathogenic anti-Dsg1 antibodies are derived from phage display technology (PF1-8-15, PF24-9, patent name: Isolation of anti-desmoglein 1 antibodies by phage display of pemphigusfoliaceus autoantibodies, patent number: US8846867B2). Modifications have been made to PF1-8-15 and PF24-9 ​​antibodies to weaken or enhance the Fc-Fcγr interaction (patent name: Anti-Dsg1 antibody and its application, patent number: CN114591428A). While the development of single-cell immune repertoire sequencing technology has facilitated antibody development and application in recent years, research and translation in autoimmune diseases are still lacking.

[0006] Clinical practice has revealed that pemphigus foliaceus is characterized by localized inflammatory responses and chronic, refractory nature. However, a suitable, standardized model is currently lacking for in-depth research and drug efficacy testing. In 2022, Bi et al. injected pathogenic anti-Dsg1 antibodies into adult mice with a wild-type C57BL / 6 background, observing exposed skin damage, ear skin thickening, and leukocyte infiltration, preliminarily confirming the feasibility, advantages, and potential of an anti-Dsg1 antibody-mediated passive transfer model of pemphigus in adult mice. However, this study has limitations in the comprehensive evaluation and testing of mouse models; and it failed to establish a long-term chronic pemphigus foliaceus mouse model. Summary of the Invention

[0007] The technical problem to be solved by this invention is to provide an anti-Dsg1 antibody and its mouse model and application. This invention has developed a novel anti-Dsg1 antibody, comprehensively observed and developed a method for evaluating the disease phenotype and disease severity of an adult pemphigus mouse model induced by this anti-Dsg1 antibody, and constructed an animal model that is highly accessible, standardized, and simulates the skin lesion changes of pemphigus patients.

[0008] This invention provides an anti-Dsg1 antibody or its antigen-binding fragment, wherein the antibody comprises a heavy chain variable region and a light chain variable region; the sequence of the heavy chain variable region is shown in SEQ ID NO:1; the sequence of the light chain variable region is shown in SEQ ID NO:3, and is designated as the P2_234_4 antibody.

[0009] Furthermore, the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:2, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:4.

[0010] The present invention also provides a nucleic acid molecule or a vector containing the nucleic acid molecule, said nucleic acid molecule having: a polynucleotide sequence encoding the anti-Dsg1 antibody variant or its antigen-binding fragment; and / or a complementary sequence of said polynucleotide sequence.

[0011] The present invention also provides a host cell that expresses the aforementioned anti-Dsg1 antibody variant or its antigen-binding fragment; and / or contains the aforementioned nucleic acid molecule or a vector containing the nucleic acid molecule.

[0012] The present invention also provides a mouse model constructed with the aforementioned anti-Dsg1 antibody or its antigen-binding fragment.

[0013] Furthermore, the construction method includes tail vein injection or intradermal injection in the back of the neck.

[0014] Furthermore, the severity of the disease in the mouse model was scored by calculating the affected skin area (%BSA) of each site and multiplying it by the skin lesion severity weighting coefficient (1 = vesicles, erosions, ulcers, crusts; 0.5 = erythema, desquamation, hair loss; 0.2 = reepithelialization; 0 = no skin lesions). This was used to evaluate the effectiveness of systemic and local interventions for pemphigus disease.

[0015] The present invention also provides the use of the aforementioned anti-Dsg1 antibody variant or its antigen-binding fragment in the preparation of a medicament for treating pemphigus.

[0016] Furthermore, the pemphigus is Dsg1-mediated pemphigus.

[0017] Furthermore, the pemphigus described is pemphigus foliaceus.

[0018] Beneficial effects

[0019] The antibody of this invention can be synthesized rapidly and in large quantities to construct a pemphigus mouse model, which has the advantages of high accessibility and standardization. The invention also comprehensively observes and develops a method for evaluating the disease phenotype and severity of the adult pemphigus mouse model induced by the anti-Dsg1 antibody, thus constructing an animal model that is highly accessible, standardized, and simulates the skin lesion changes of pemphigus patients. This evaluation method has the advantage of standardization in evaluating the effects of systemic and local interventions for pemphigus disease. Attached Figure Description

[0020] Figure 1 This invention provides the antibody synthesis and detection process.

[0021] Figure 2 AE represents the antigen specificity and pathogenicity test results of the antibodies of this invention.

[0022] Figure 3 The procedure for constructing a pemphigus mouse model by tail vein injection of the antibody of the present invention is shown in (A); skin lesions were observed around the mouth, eyes, and mandible of the control mice and the pemphigus mouse model (B); images of the mandible and neck of the control mice and the pemphigus mouse model after hair removal (C); HE-stained images (20×) of skin lesions of the control mice and the pemphigus mouse model and the results of direct immunofluorescence detection of the skin next to the lesions (D).

[0023] Figure 4 Antibody was constructed for intradermal injection into the neck and back of a pemphigus mouse model.

[0024] Figure 5 This is an example of the observation and scoring method for a mouse model of pemphigus induced by tail vein injection of the antibody of the present invention. Detailed Implementation

[0025] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Furthermore, it should be understood that after reading the teachings of this invention, those skilled in the art can make various alterations or modifications to the invention, and these equivalent forms also fall within the scope defined by the appended claims.

[0026] Example 1

[0027] 1. P2_234_4 antibody synthesis method and sequence information

[0028] Ectopic lymphoid structures at different levels exist in the skin lesions of pemphigus patients, which are considered key sites for autoantibody production and autoimmune responses; antibody-secreting cells in the skin lesions may directly secrete anti-autoantibodies. To obtain anti-Dsg antibody sequences, CD45 was enriched and sorted from pemphigus patient skin lesion samples. + Immune cells, or CD19+ / CD138 + B cells and antibody-secreting cells were subjected to joint sequencing of single-cell transcriptomes and TCR / BCR immune repertoires. Figure 1 In the study and sequencing analysis, it was found that over 45% of antibody-secreting cells in pemphigus lesions exhibited characteristics of IgG4 antibody subtype switching and BCR clonal amplification, with a significantly increased proportion of Dsg-reactive cells. The variable regions of the BCR heavy and light chains from the clonal amplification of antibody-secreting cells obtained by single-cell BCR sequencing were extracted, and the constant region sequences were completed. These sequences were then expressed in vitro using the pcDNA3.1 plasmid and a Chinese hamster ovary cell expression system to synthesize monoclonal antibodies. Figure 1 The antibody comprises a heavy chain variable region and a light chain variable region; the sequence of the heavy chain variable region is shown in SEQ ID NO:1; the sequence of the light chain variable region is shown in SEQ ID NO:3, and it is designated as antibody P2_234_4. The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:2, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:4. The synthesized antibody was tested for antigen binding capacity by enzyme-linked immunosorbent assay (ELISA) and indirect immunofluorescence assay. Antibody P2_234_4 showed Dsg1 antigen specificity (Table 1). Figure 2 AB). Simultaneously, the pathogenicity of the P2_234_4 antibody was demonstrated by co-incubating the P2_234_4 antibody with immortalized human keratinocytes (HaCaT) cells that were connected in sheets, and then mechanically blowing the cells. In the isotype control group (Iso), the intercellular connections of HaCaT cells were difficult to disrupt, and they remained connected in sheets; in the experimental group incubated with the P2_234_4 antibody, the intercellular connections were easily disrupted, forming multiple cell fragments. Figure 2C). The in vivo pathogenicity of the P2_234_4 antibody was also evaluated. Recombinant mAb was subcutaneously injected into the back of newborn Balb / c mice. Six hours after injection, abdominal skin was harvested for direct immunofluorescence assay. The assay showed that the P2_234_4 antibody formed a network of fluorescent IgG deposits between mouse epidermal cells. Figure 2 D). Observations were conducted 24 hours after injection. Newborn mice injected with the P2_234_4 antibody showed grossly visible severe epidermal cell detachment, and intradermal vesicle formation was observed in skin pathological sections. Figure 2 E).

[0029] Table 1. Antibody source information for P2_234_4

[0030]

[0031] 2. Methods for establishing an adult mouse model of pemphigus

[0032] The P2_234_4 anti-Dsg1 antibody-mediated adult mouse model has the advantages of being simple and able to simulate the skin acantholysis and inflammatory changes in patients with pemphigus.

[0033] (1) Establishment of a mouse model of tail vein injection of P2_234_4 antibody

[0034] The in vivo pathogenicity of the recombinant mAb was tested using an adult mouse model. Adult C57 / BL6 mice were injected intravenously with 0.5 mg of the synthetic mAb on Day 0 and Day 2, respectively. Observation and sampling were performed on Day 4 and 5. Figure 3 A). Mice injected with P2_234_4 antibody showed desquamative skin lesions around the eyes and mouth. Figure 3 B). After hair removal treatment of the perioral and neck / chest areas of mice, pemphigus mouse models injected with P2_234_4 antibody showed obvious erythematous, erosive, and crust-like skin lesions on the neck and chest. Figure 3 C). Skin samples were taken from the neck and chest lesions of mice injected with P2_234_4 antibody for pathological sectioning and HE staining. Acantholysis and intraepidermal vesicle formation were observed. Immunofluorescence staining of dorsal skin samples revealed IgG fluorescent deposition between acanthocytes. Figure 3 D). This mouse model showed extensive skin involvement. In addition to obvious erythematous, erosive, and crust-like lesions on the neck and chest, varying degrees of skin lesions may also appear around the eyes, mouth, ears, upper limbs, and tail.

[0035] (2) Establishment of a mouse model of intradermal injection of P2_234_4 antibody in the neck and back

[0036] In a mouse model of scleroderma, repeated local intradermal injections of bleomycin induced collagen deposition and skin thickening at the injection site, mimicking the pathogenesis of scleroderma. To further verify the local pathogenicity of the P2_234_4 antibody and expand the methods for constructing mouse models, adult C57 / BL6 mice were further intradermally injected with the P2_234_4 antibody to induce pemphigus. After hair removal on the back of the mice, 0.05 mg ( Figure 4 The recombinant mAb showed significant erosion and crusting at the injection site from Day 2 to 5, similar to the tail vein injection model. In the later stages, Days 8 to 11, desquamation and skin thickening were observed, consistent with the clinical characteristics of some patients with chronic pemphigus foliaceus. This further demonstrates that the P2_234_4 antibody has direct pathogenicity in the local skin, inducing skin inflammation, and that inducing a chronic pemphigus model via intradermal injection is feasible.

[0037] 3. Observation, evaluation and analysis methods of pemphigus mouse model

[0038] To assess the phenotypic severity of pemphigus mouse models, investigate whether these models can mimic the clinical presentation of patients, and quantify the extent to which drugs improve pemphigus disease during preclinical animal experiments, it is crucial to establish a comprehensive and universally accepted observation and evaluation system for mouse models. Previous studies have used a scoring system that incorporates lesion location (perioral, perioral, periauricular, back, chest, abdomen, limbs, tail) and lesion type (erosion, alopecia) to evaluate the severity of lymphocyte metastasis-induced pemphigus in mice. However, due to the inherent phenotypic limitations of this model, this evaluation system does not include other lesion types, such as vesicles, crusts, desquamation, and erythema, and cannot reflect the lesion area.

[0039] Based on the phenotypic characteristics of the established anti-Dsg1 antibody-mediated pemphigus mouse model, and referring to the commonly used clinical scoring systems for pemphigus patients, PDAI (Pemphigus Disease Area Index) and ABSIS (Autoimmune Bullous Skin Disorder Intensity-Score), a scoring system for evaluating the severity of pemphigus in mice was developed (Table 2). The affected skin area (%BSA) at each site (mandible and neck, chest and abdomen, back, perioral region, periorbital region, ears and periorbital region, limbs, and tail) was calculated, multiplied by a skin lesion severity weighting coefficient (1 = vesicles, erosions, ulceration, crusting; 0.5 = erythema, desquamation, alopecia; 0.2 = reepithelialization; 0 = no lesions), and then summed.

[0040] The specific methods for observation and evaluation were as follows: Two days before the tail vein injection of 0.5 mg anti-Dsg1 antibody (Day 2), hair removal cream was used to remove hair from the neck, chest, and upper abdomen areas of mice most prone to skin lesions. After 3 minutes of application, the hair was wiped away with sterile saline to prevent skin irritation. Following antibody injection, the skin characteristics of the above-mentioned areas were regularly observed, recorded, and scored until the mice were euthanized as specified in the experiment. Changes in skin lesions in the mouse model established by tail vein injection of P2_234_4 antibody were recorded and scored. Figure 5 The disease manifests most severely on Day 4-5, with the highest score; afterwards, the scabs fall off, the erosions heal, and the skin lesions tend to re-epithelialize, resulting in a decrease in the score. Figure 5 ).

[0041] Table 2. Disease Severity Scoring Table for Antibody-Injected Pemphigus Mouse Model

[0042]

[0043] Pemphigus mouse model disease severity = Σ%BSA × skin lesion severity, maximum 100 points; *BSA = Body Surface Area.

Claims

1. An anti-Dsg1 antibody or its antigen-binding fragment, characterized in that: The antibody comprises a heavy chain variable region and a light chain variable region; the sequence of the heavy chain variable region is shown in SEQ ID NO:1; the sequence of the light chain variable region is shown in SEQ ID NO:

3.

2. The anti-Dsg1 antibody or its antigen-binding fragment according to claim 1, characterized in that: The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:2, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:

4.

3. A nucleic acid molecule or a carrier containing the nucleic acid molecule, said nucleic acid molecule having: a polynucleotide sequence encoding the anti-Dsg1 antibody variant of claim 1 or an antigen-binding fragment thereof; and / or a complementary sequence of said polynucleotide sequence.

4. A host cell that expresses the anti-Dsg1 antibody variant of claim 1 or its antigen-binding fragment; and / or contains the nucleic acid molecule of claim 3 or a vector containing the nucleic acid molecule.

5. A mouse model constructed with the anti-Dsg1 antibody as described in claim 1 or its antigen-binding fragment.

6. The mouse model according to claim 5, characterized in that: The construction method includes tail vein injection or intradermal injection in the back of the neck.

7. The mouse model according to claim 5, characterized in that: The severity of the disease in the mouse model was scored by calculating its severity.

8. Use of the anti-Dsg1 antibody variant or its antigen-binding fragment as described in claim 1 in the preparation of a medicament for treating pemphigus.

9. The application according to claim 8, characterized in that, The pemphigus described is Dsg1-mediated pemphigus.

10. The application according to claim 8, characterized in that, The pemphigus mentioned is the leaf-type pemphigus.

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