CN117467006A - anti-TSLP antibody, preparation and application thereof - Google Patents

Abstract

To an isolated anti-TSLP antibody for use in the treatment of TSLP-mediated diseases and conditions. The antibody preparation has strong stability under the conditions of freeze thawing, oscillation, acceleration and long term, can ensure that the preparation has good stability in the preparation, transportation and storage processes, and ensures the safety and quality controllability of clinical medication.

Description

An anti-TSLP antibody and its preparation and application

Technical field

The invention belongs to the field of biomedicine technology, and specifically relates to an anti-TSLP antibody and its preparation and application.

Background technique

Thymic stromal lymphopoietin (TSLP) is a cytokine that signals through a heterodimeric receptor composed of IL-7Rα subunit and TSLP-R. TSLP-R is a unique component that is homologous to the common cytokine receptor γ chain (γc) (Pandey et al., Nat. Immunol. 2000, 1(1): 59-64 ). TSLP is produced by epithelial cells on the barrier surface and activates DCs expressing the TSLP receptor (TSLPR) to induce functional Th2 cells (Roan F et al., J Clin Invest 2019;129:1441-51). Additionally, TSLP is known to be associated with other diseases, including autoimmune diseases and cancer (Varricchi G et al., Front Immunol 2018;9:1595). The TSLP receptor (TSLPR) complex consists of TSLPR and IL-7 receptor alpha (IL-7Rα) (Park LS, et al., J Exp Med 2000; 192:659-670). The combination of TSLPR and IL-7R chains results in high-affinity binding and activation of signal transducer and activator of transcription 3 (STAT3) and STAT5 (Pandey A, et al., Nat Immunol 2000; 1:59-64). Such broad pathophysiological features have prompted TSLP- and TSLPR-mediated signaling as therapeutic targets in type 2 cytokine-mediated allergic diseases. AMG157 is a fully human antibody against TSLP (used as a control in the examples) developed by Amgen for the treatment of asthma and atopic dermatitis. This antibody is described in U.S. Patent No. 7,982,016. The disclosures in all publications, patents, patent applications, and published patent applications mentioned herein are hereby incorporated by reference in their entirety.

The present invention provides an isolated anti-TSLP antibody and an anti-TSLP antibody preparation with strong stability for treating TSLP-mediated diseases and disorders.

Contents of the invention

The invention discloses an isolated anti-TSLP antibody. The antibody includes _VH . The _VH includes: HC-CDR1, which includes the amino acid sequence SEQ ID NO: 1, and HC-CDR2, which includes the amino acid sequence SEQ ID NO: 2, and HC-CDR3, which comprises the amino acid sequence SEQ ID NO: 3; and _VL , which V _L comprises: LC-CDR1, which comprises the amino acid sequence SEQ ID NO: 7, LC-CDR2, which comprises the amino acid sequence SEQ ID NO: 8, and LC-CDR3 comprising the amino acid sequence SEQ ID NO: 9; or

The antibody comprises a V _H comprising: HC- _CDR1 , which comprises the amino acid sequence SEQ ID NO: 4, HC-CDR2, which comprises the amino acid sequence SEQ ID NO: 5, and HC-CDR3, which comprises the amino acid sequence SEQ ID NO: 6; and V _L comprising: LC-CDR1 comprising the amino acid sequence SEQ ID NO: 10, LC-CDR2 comprising the amino acid sequence SEQ ID NO: 11 _, and LC-CDR3 comprising Amino acid sequence SEQ ID NO:12.

In some embodiments of the invention, the antibody comprises _VH , comprising the amino acid sequence SEQ ID NO: 13, and _VL , comprising the amino acid sequence SEQ ID NO: 14; or

The antibody comprises _VH , comprising the amino acid sequence SEQ ID NO:15, and _VL , comprising the amino acid sequence SEQ ID NO:16.

In some embodiments of the invention, the antibody further comprises a heavy chain constant region comprising the amino acid sequence SEQ ID NO: 17 and a light chain constant region comprising the amino acid sequence SEQ ID NO. NO:18.

On the other hand, the present invention also provides an anti-TSLP antibody preparation, the antibody preparation comprising any of the isolated anti-TSLP antibodies as described above.

In some embodiments of the present invention, the antibody concentration is 1 mg/ml-300 mg/ml; preferably, the antibody concentration is 10 mg/ml-200 mg/ml; further preferably 100 mg/ml-200 mg/ml; more Preferably, it is 135mg/ml-165mg/ml; in specific embodiments of the invention, the antibody concentration is 1mg/ml, 10mg/ml, 30mg/ml, 50mg/ml, 80mg/ml, 100mg/ml, 120mg/ml. ml, 135mg/ml, 140mg/ml, 145mg/ml, 150mg/ml, 155mg/ml, 160mg/ml, 165mg/ml, 180mg/ml, 190mg/ml, 200mg/ml, 230mg/ml, 250mg/ml, 280mg/ml or 300mg/ml.

In some embodiments of the invention, the antibody preparation further includes a surfactant, and the surfactant is polysorbate and/or poloxamer; preferably, the polysorbate is polysorbate 20 and / or polysorbate 80; preferably, the poloxamer is poloxamer 188.

In some embodiments of the invention, the concentration of the surfactant is 0.02mg/ml-2.0mg/ml, preferably 0.52mg/ml-1.65mg/ml, more preferably 0.65mg/ml-1.52mg/ ml; in specific embodiments of the invention, the concentration of the surfactant is 0.02mg/ml, 0.03mg/ml, 0.05mg/ml, 0.06mg/ml, 0.08mg/ml, 0.10mg/ml, 0.12 mg/ml, 0.15mg/ml, 0.20mg/ml, 0.3mg/ml, 0.4mg/ml, 0.5mg/ml, 0.6mg/ml, 0.8mg/ml, 1.0mg/ml, 1.2mg/ml, 1.5 mg/ml or 2.0mg/ml.

In some embodiments of the present invention, the surfactant is polysorbate and poloxamer; preferably, the concentration of the polysorbate is 0.02 mg/ml-0.15 mg/ml, preferably 0.1 mg/ml ; In specific embodiments of the present invention, the concentration of polysorbate is 0.02mg/ml, 0.03mg/ml, 0.05mg/ml, 0.06mg/ml, 0.08mg/ml, 0.10mg/ml, 0.12mg /ml or 0.15mg/ml; the concentration of the poloxamer is 0.5-1.5mg/ml, preferably 1.0mg/ml; in specific embodiments of the invention, the concentration of the poloxamer is 0.5 mg/ml, 0.6mg/ml, 0.8mg/ml, 1.0mg/ml, 1.2mg/ml or 1.5mg/ml.

In some embodiments of the invention, the antibody preparation further includes a stabilizer. Preferably, the stabilizer is sucrose, trehalose, maltose, sorbitol, mannitol, sodium chloride, arginine, glycine, proline. Any one or more of amino acid and lysine; more preferably, the stabilizer is any one or more of sucrose, trehalose, sodium chloride or arginine.

In some embodiments of the present invention, the stabilizer is: sucrose or trehalose with a concentration of 40 mg/ml-80 mg/ml, preferably 50 mg/ml-80 mg/ml, and further preferably 50 mg/ml-70 mg/ml. , maltose, sorbitol and/or mannitol; or sodium chloride, arginine, glycine, proline or lysine at a concentration of 100mM-180mM, preferably 130mM-150mM; in specific embodiments of the invention, The stabilizer is sucrose, trehalose, maltose, sorbitol and/or mannitol at a concentration of 40mg/ml, 50mg/ml, 60mg/ml, 70mg/ml or 80mg/ml, or the stabilizer is 100mM, 110mM , 120mM, 130mM, 140mM, 150mM, 160mM, 170mM, or 180mM sodium chloride, arginine, glycine, proline or lysine.

In some embodiments of the invention, the antibody preparation further includes a buffer. Preferably, the buffer is a citrate buffer, an acetate buffer and/or a histidine hydrochloride buffer.

In some embodiments of the invention, the concentration of the buffer is 3mM-50mM; preferably, the concentration of the buffer is 10mM-30mM; in specific embodiments of the invention, the concentration of the buffer is 3mM, 5mM, 8mM, 10mM, 15mM, 20mM, 25mM or 30mM.

In some embodiments of the invention, the pH value of the preparation is 4.8-5.5, preferably 5.0-5.4; in specific embodiments of the invention, the pH value of the preparation is 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4 or 5.5.

In some embodiments of the invention, the antibody concentration is 100mg/ml-200mg/ml; the stabilizer is 40mg/ml-80mg/ml sucrose and/or trehalose, or the stabilizer is 100mM- 180mM sodium chloride or arginine; the buffer is 3mM-50mM acetate buffer, citrate buffer and/or histidine hydrochloride buffer; the pH value of the preparation is 4.8- 5.5; The surfactant is 0.02mg/ml-0.15mg/ml polysorbate and 0.5mg/ml-1.5mg/ml poloxamer.

In some embodiments of the invention, the antibody concentration is 135 mg/ml-165 mg/ml; the stabilizer is 50 mg/ml-80 mg/ml sucrose and/or trehalose, or the stabilizer is 130 mM- 150mM sodium chloride or arginine; the buffer is 10mM-30mM acetate buffer, citrate buffer and/or histidine hydrochloride buffer; the pH value of the preparation is 4.8-30mM 5.5; The surfactant is 0.02mg/ml-0.15mg/ml polysorbate and 0.5mg/ml-1.5mg/ml poloxamer.

In some embodiments of the invention, the antibody concentration is 135 mg/ml-165 mg/ml; the stabilizer is 50 mg/ml-70 mg/ml sucrose and/or trehalose, or the stabilizer is 130 mM- 150mM sodium chloride or arginine; the buffer is 10mM-30mM acetate buffer, citrate buffer and/or histidine hydrochloride buffer; the pH value of the preparation is 5.0-30mM 5.4; The surfactant is 0.02 mg/ml-0.15 mg/ml polysorbate and 0.5 mg/ml-1.5 mg/ml poloxamer.

In some embodiments of the invention, the formulation is any one of the following antibody formulations:

(1) The antibody concentration is 135mg/ml, 140mg/ml, 145mg/ml, 150mg/ml, 155mg/ml, 160mg/ml or 165mg/ml; the stabilizer is 50mg/ml-70mg/ml sucrose and/or trehalose, or the stabilizer is 130mM-150mM sodium chloride or arginine; the buffer is 10mM-30mM acetate buffer, citrate buffer and/or histidine salt Acid buffer; the pH value of the preparation is 5.0-5.4; the surfactant is 0.02mg/ml-0.15mg/ml polysorbate and 0.5mg/ml-1.5mg/ml poloxamer ;

(2) The antibody concentration is 135mg/ml-165mg/ml; the stabilizer is 50mg/ml, 60mg/ml or 70mg/ml sucrose and/or trehalose, or the stabilizer is 130mM, 140mM or 150mM sodium chloride or arginine; the buffer is 10mM-30mM acetate buffer, citrate buffer and/or histidine hydrochloride buffer; the pH value of the preparation is 5.0-5.4 ; The surfactant is 0.02mg/ml-0.15mg/ml polysorbate and 0.5mg/ml-1.5mg/ml poloxamer;

(3) The antibody concentration is 135mg/ml-165mg/ml; the stabilizer is 50mg/ml-70mg/ml sucrose or trehalose, or the stabilizer is 130mM-150mM sodium chloride or arginine ; The buffer is 10mM, 20mM or 30mM acetate buffer, citrate buffer and/or histidine hydrochloride buffer; the pH value of the preparation is 5.0-5.4; the surfactant It is 0.02mg/ml-0.15mg/ml polysorbate and 0.5mg/ml-1.5mg/ml poloxamer;

(4) The antibody concentration is 135mg/ml-165mg/ml; the stabilizer is 50mg/ml-70mg/ml sucrose or trehalose, or the stabilizer is 130mM-150mM sodium chloride or arginine ; The buffer is 10mM-30mM acetate buffer, citrate buffer and/or histidine hydrochloride buffer; the pH value of the preparation is 5.0, 5.1, 5.2, 5.3 or 5.4; so The surfactant is 0.02mg/ml-0.15mg/ml polysorbate and 0.5mg/ml-1.5mg/ml poloxamer;

(5) The antibody concentration is 135mg/ml-165mg/ml; the stabilizer is 50mg/ml-70mg/ml sucrose or trehalose, or the stabilizer is 130mM-150mM sodium chloride or arginine ; The buffer is 10mM-30mM acetate buffer, citric acid buffer and/or histidine hydrochloride buffer; the pH value of the preparation is 5.0-5.4; the surfactant is 0.02 mg/ml, 0.03mg/ml, 0.05mg/ml, 0.06mg/ml, 0.08mg/ml, 0.10mg/ml, 0.12mg/ml or 0.15mg/ml polysorbate and 0.5mg/ml-1.5mg /ml of poloxamer;

(6) The antibody concentration is 135mg/ml-165mg/ml; the stabilizer is 50mg/ml-70mg/ml sucrose or trehalose, or the stabilizer is 130mM-150mM sodium chloride or arginine ; The buffer is 10mM-30mM acetate buffer, citric acid buffer and/or histidine hydrochloride buffer; the pH value of the buffer is 5.0-5.4; the surfactant is 0.02mg/ml-0.15mg/ml polysorbate and 0.5mg/ml, 0.6mg/ml, 0.8mg/ml, 1.0mg/ml, 1.2mg/ml or 1.5mg/ml poloxamer;

The polysorbate is polysorbate 80 or polysorbate 20.

In some embodiments of the invention, the antibody concentration is 135mg/ml-165mg/ml; the stabilizer is 50mg/ml-70mg/ml sucrose; the buffer is 10mM-30mM acetate buffer liquid; the pH value of the preparation is 5.0-5.4; the surfactant is 0.02mg/ml-0.15mg/ml polysorbate and 0.5mg/ml-1.5mg/ml poloxamer.

In some embodiments of the invention, the antibody formulation is any one of the following formulations:

(1) The antibody concentration is 150mg/ml; the stabilizer is 130mM sodium chloride; the buffer is 20mM acetate buffer; the pH value of the buffer is 5.4; the surfactant is 0.1 mg/ml polysorbate and 1.0 mg/ml poloxamer;

(2) The antibody concentration is 150mg/ml; the stabilizer is 60mg/ml sucrose; the buffer is 20mM citrate buffer; the pH value of the buffer is 5.0; the surface activity The agents are 0.1 mg/ml polysorbate and 1.0 mg/ml poloxamer;

(3) The antibody concentration is 135 mg/ml; the stabilizer is 50 mg/ml sucrose; the buffer is 10 mM acetate buffer; the pH value of the buffer is 5.4; the surface activity The dosage is 0.02 mg/ml polysorbate and 1.5 mg/ml poloxamer;

(4) The antibody concentration is 150mg/ml; the stabilizer is 60mg/ml sucrose; the buffer is 20mM acetate buffer; the pH value of the buffer is 5.2; the surface activity The agents are 0.1 mg/ml polysorbate and 1.0 mg/ml poloxamer;

(5) The antibody concentration is 165mg/ml; the stabilizer is 70mg/ml sucrose; the buffer is 30mM acetate buffer; the pH value of the buffer is 5.0; the surface activity The dosage is 0.15 mg/ml polysorbate and 0.5 mg/ml poloxamer;

(6) The antibody concentration is 150mg/ml; the stabilizer is 80mg/ml sucrose; the buffer is 30mM acetate buffer; the pH value of the buffer is 5.4; the surface activity The agents are 0.1 mg/ml polysorbate and 1.0 mg/ml poloxamer;

(7) The antibody concentration is 150mg/ml; the stabilizer is 150mM arginine; the buffer is 20mM acetate buffer; the pH value of the buffer is 5.2; the surface activity The agents are 0.1 mg/ml polysorbate and 1.0 mg/ml poloxamer;

(8) The antibody concentration is 160mg/ml; the stabilizer is 60mg/ml sucrose; the buffer is 20mM citrate buffer; the pH value of the buffer is 5.0; the surface activity The agents are 0.1 mg/ml polysorbate and 1.0 mg/ml poloxamer;

(9) The antibody concentration is 150mg/ml; the stabilizer is 60mg/ml sucrose; the buffer is 20mM citrate buffer; the pH value of the preparation is 4.8; the surfactant It is 0.1 mg/ml polysorbate and 1.0 mg/ml poloxamer;

(10) The antibody concentration is 150 mg/ml; the stabilizer is 60 mg/ml sucrose; the buffer is 20 mM histidine hydrochloride buffer; the pH value of the preparation is 5.5; The surfactants are 0.1 mg/ml polysorbate and 1.0 mg/ml poloxamer;

Preferably, the polysorbate is polysorbate 80 or polysorbate 20;

Preferably, the poloxamer is poloxamer 188.

In some embodiments of the invention, the antibody formulation further comprises antioxidants and/or preservatives. The preservative or antioxidant is commonly used in antibody preparations. The concentration of the oxidant and/or preservative is 0-20mM. In some embodiments, the concentration of the antioxidant is 0, 5mM, 8mM, 10mM, 12mM, 15mM, 18mM or 20mM. In some embodiments, the preservative is ethylenediaminetetraacetic acid (EDTA), diethylenetriaminepentaacetic acid (DTPA) or a combination thereof; the antioxidant is methionine.

In some embodiments of the invention, the antibody preparation is a liquid preparation or an injection powder.

In another aspect, the present invention also provides the use of the above-mentioned antibody or antibody preparation in the preparation of medicaments for treating diseases; preferably, the diseases include inflammatory or autoimmune diseases.

In some embodiments of the invention, the disease is selected from the group consisting of asthma, idiopathic pulmonary fibrosis, atopic dermatitis, allergic conjunctivitis, allergic rhinitis, Netherton syndrome, eosinophilic esophagitis (EoE), food Allergy, allergic diarrhea, eosinophilic gastroenteritis, allergic bronchopulmonary aspergillosis (ABPA), allergic fungal sinusitis, cancer, rheumatoid arthritis, chronic obstructive pulmonary disease (COPD), systemic sclerosis , keloids, ulcerative colitis, chronic rhinosinusitis (CRS), nasal polyposis, chronic eosinophilic pneumonia, eosinophilic bronchitis, celiac disease, Churg-Strauss syndrome, eosinophilic myalgia syndrome, eosinophilic Granulocytosis syndrome, eosinophilic granulomatosis with polyangiitis, inflammatory bowel disease.

In another aspect, the invention provides a method of treating a disease or condition in a desired individual, comprising administering to said individual an effective amount of any of the antibody formulations described above. The administration methods include: intravenous injection, intraarterial administration, intraperitoneal injection, intrapulmonary administration, intravascular administration, intramuscular injection, intratracheal administration, subcutaneous injection, intraocular administration, intrathecal administration, and mucosal administration. drug or transdermal administration. In some embodiments, the formulation is administered subcutaneously. In some embodiments, the formulation is administered intramuscularly.

"Antibodies" as used herein include full-length antibodies and antigen-binding fragments thereof. Full-length antibodies include two heavy chains and two light chains. The variable regions of the light and heavy chains are responsible for antigen binding. The variable regions in the two chains usually include three hypervariable loops, called complementarity determining regions (CDRs) (light chain (LC) CDRs include LC-CDR1, LC-CDR2 and LC-CDR3, heavy chain (HC) )CDRs include HC-CDR1, HC-CDR2 and HC-CDR3). The CDR boundaries of the antibodies or antigen-binding fragments disclosed herein may be defined or identified by the Kabat, Chothia or Al-Lazikani conventions (Al-Lazikani 1997; Chothia 1985; Chothia 1987; Chothia 1989; Kabat 1987; Kabat 1991). The three CDR regions of the heavy or light chain are inserted between flanking segments called framework regions (FRs), which are more conserved than the CDR regions and form a scaffold supporting the hypervariable loops. The constant regions of the heavy and light chains are not involved in antigen binding but exhibit a variety of effector functions. Antibodies are classified based on the amino acid sequence of their heavy chain constant region. The five major classes or isotypes of full-length antibodies of the present invention are IgA, IgD, IgE, IgG and IgM, characterized by alpha, delta, epsilon, gamma and mu heavy chains respectively. Furthermore, several major antibody categories described in the present invention can be further divided into subcategories, including: IgG1 (γ1 heavy chain), IgG2 (γ2 heavy chain), IgG3 (γ3 heavy chain), IgG4 (γ4 heavy chain). chain), IgA1 (α1 heavy chain) or IgA2 (α2 heavy chain).

As used herein, the term "antigen-binding fragment" includes antibody fragments, e.g., diabodies, Fab, Fab', F(ab')2, Fv fragments, disulfide-stabilized Fv fragments (dsFv), (dsFv) 2. Bispecific dsFv (dsFv-dsFv'), disulfide bond stabilized diabody (ds diabody), single chain antibody (scFv), scFv dimer (bivalent diabody), consisting of a Or multispecific antibodies, single domain antibodies, nanobodies, domain antibodies, bivalent domain antibodies composed of antibody fragments of multiple CDRs, or any other antibody fragments that can bind to antigens but do not contain a complete antibody structure.

The antibodies and antibody preparations provided by the invention have the following excellent effects:

(1) The anti-TSLP antibody provided by the present invention has good effects in inhibiting Stat5 activation induced by human TSLP, inhibiting TSLP-induced Ba/F3 cell proliferation, and can also inhibit the release of TARC induced by recombinant human TSLP from human PBMC, Having better ability to inhibit human TSLP to promote TARC release, the anti-TSLP antibody of the present invention has a good effect in treating TSLP-mediated diseases.

(2) The anti-TSLP antibody preparation of the present invention uses a combination of dual surfactants (polysorbate and poloxamer), which has strong stability under freeze-thaw, shaking, acceleration and long-term conditions, which can ensure the preparation and transportation of the preparation. and maintain good stability during storage to ensure clinical drug safety and quality controllability.

Detailed ways

In order to make the technical problems to be solved, the technical solutions adopted and the advantages of the present invention clearer, the present invention will be described in detail below with reference to specific embodiments. The following examples are used to illustrate the invention but are not intended to limit the scope of the invention.

The reagents used in the following examples, unless otherwise specified, are all prepared using conventional methods or obtained from commercial sources; the experimental methods used, unless otherwise specified, are conventional methods; the materials, instruments, etc. used, Unless otherwise stated, all materials were obtained from commercial sources.

Example 1. Antibody preparation and screening

Preparation of scFv antibody yeast display library: Extract RNA from 2000 human blood samples, obtain cDNA through reverse transcription, use V _H and V _K specific primers to amplify V _H and V _K fragments, and after gel recovery and purification, connect V _H and V _K , construct scFv and clone it into the yeast display plasmid PYD1. Subsequently, the plasmid is electroporated into yeast to obtain a scFv antibody yeast display library.

Magnetic activated cell sorting (MACS) was used to enrich cells expressing anti-TSLP scFv antibodies. In order to prepare a phage display library, the scFv fragment was cloned into the phage display vector pDAN5 using SfiI. After ligation, the vector was transformed into TG1 phage display. Electroporate competent cells to obtain scFv antibody phage display libraries. Continue the repeated screening to isolate TSLP-specific scFv antibodies from the phage display library. Then select monoclones for combined ELISA identification and in vitro biological activity evaluation to obtain lead antibodies TSLP-1 and TSLP-2.

To reduce the potential immunogenicity risk of the candidate molecule, two framework residues of the lead antibody TSLP-1V _H were individually or collectively replaced with human germline residues according to their most homologous germline. One framework residue of 1V _L was replaced with a human germline residue. One scFv (TSLP-01) is assembled by pairing different heavy chain and light chain variable regions. For the scFv of TSLP-2, the two framework residues of the TSLP-2V _H were replaced individually or collectively with human germline residues. One scFv (TSLP-02) is assembled by pairing different heavy and light chain variable domains. Full-length anti-TSLP-01-IgG1 and TSLP-02-IgG1 antibodies were constructed and screened.

The above antibodies were expressed and purified for subsequent experiments. The amino acid sequences of the antibodies are shown in Tables 1 and 2.

Table 1 Anti-TSLP antibody CDR sequences

Table 2. Amino acid sequences of anti-TSLP antibodies

Example 2. Activity detection of anti-TSLP antibodies

1. TSLP-induced Stat5 activation inhibition experiment

The antibodies TSLP-01-IgG1 and TSLP-02-IgG1 shown in Table 1 were evaluated for their effectiveness in inhibiting human TSLP-stimulated signal transducer and transcription activator 5 activation in Ba/F3 stable cells using cell analysis. The results are shown in Table 3 As shown, the TSLP-01-IgG1 antibody and the TSLP-02-IgG1 antibody showed better effects than the reference antibody AMG157 in inhibiting human TSLP-induced Stat5 activation.

2. TSLP-induced Ba/F3 cell proliferation inhibition experiment

Anti-TSLP antibodies were evaluated for their ability to inhibit TSLP-induced proliferation of Ba/F3 cells expressing human TSLP receptor (hTSLPR) and human IL-7Ra (hIL7Ra). The results are shown in Table 3. Compared with the reference antibody AMG157, the anti-TSLP antibody showed a better effect in inhibiting TSLP-induced Ba/F3 cell proliferation.

3. TARC release inhibition experiment in human PBMCs

To determine whether anti-TSLP antibodies are able to neutralize TSLP in primary cell-driven responses, human TSLP-induced TARC secretion from human PBMCs was tested in the presence or absence of anti-TSLP antibodies. The results are shown in Table 3. The TSLP-01-IgG1 antibody and the TSLP-02-IgG1 antibody inhibited the ability of human TSLP (hTSLP) to promote the release of TARC from human PBMC and showed better inhibition compared with the reference antibody AMG157. The ability of human TSLP to promote TARC release.

Table 3. Activity detection results of anti-TSLP antibodies

Example 3. Prescription screening of anti-TSLP antibody preparations

In this experiment, the prescriptions of antibody preparations were screened under shaking conditions. The screening prescription design is shown in Table 4. Shake conditions (2-8°C, 300rpm, two months) to examine the stability of different prescriptions. Test items include visible foreign matter, concentration, pH, osmotic pressure, biological activity (relative to the labeled amount, %), insoluble particles, Aggregation properties. Among them, insoluble particles were detected using the Flowcam method, and aggregate properties were detected using the SEC method and NR-CE-SDS method. The test results are shown in Tables 5 and 6.

Table 4. Screening prescription design plan

Table 5. Visible foreign matter and insoluble particulate results from prescription screening

Table 6. Polymer test results for formulation screening

After shaking for 2 months, there were no visible foreign matter in the antibody preparations of each group. The pH, osmotic pressure, protein concentration, and biological activity of the protein samples (relative to the labeled amount) of the antibody preparations were basically stable.

Flowcam test results showed that at 2 months compared with day 0, the number of particles >25 μm in each group of antibody preparations increased by 14-85, and the number of particles >10 μm increased by 179-264, all meeting the standards related to antibody preparations. Relatively speaking, the number of >25 μm particles in the antibody preparations of prescription groups 3-5 only increased by 14-17, and the number of >10 μm particles only increased by 179-196, showing better stability.

SEC-HPLC test results showed that at 2 months compared with day 0, the main peak of the antibody preparations in each group decreased by 0.18%-4.0%, all of which met the standards related to antibody preparations. Relatively speaking, the main peak of the antibody preparation in prescription groups 3-5 was only reduced by 0.18-0.21%, showing better stability. The conclusions obtained from the NR-CE test results are also consistent with this.

In summary, each group maintained good antibody preparation stability under shaking conditions. Relatively speaking, the stability of antibody preparations with 3-5 groups of prescriptions is better.

Example 4. Confirmation of prescription of high-concentration antibody preparation

Refer to the prescription in Table 7 to prepare antibody preparations, and test the stability of TSLP antibody preparations of different sequences under repeated freezing and thawing, shaking, acceleration, and long-term conditions.

Table 7. Confirmation of high concentration antibody prescription

(1) Repeated freezing and thawing stability test

The stability of the antibody preparation was tested under freeze-thaw conditions (-80°C freeze/room temperature thaw, 5 cycles). Table 8 shows the test results of each antibody preparation: no visible foreign matter was found within 5 freeze-thaw times. Compared with 0 freeze-thaw times, the concentration, pH, osmotic pressure, biological activity, and insoluble particles of the antibody preparation basically did not change; SEC test The change of the main peak is very small, within 0.05%; the main peak detected by NR-CE and the change amplitude are within 0.4%. The above results demonstrate the excellent stability of the antibody formulation of the present invention under freeze-thaw conditions.

Table 8. Freeze-thaw test results of antibody preparations

(2) Oscillation stability test:

The stability of the antibody preparation was tested under shaking conditions (2-8°C, 300 rpm, upright). Table 9 shows the test results of each antibody preparation: each preparation recipe also showed excellent stability under shaking conditions: within 2 months of shaking, no visible foreign matter was found. Compared with day 0, the concentration, pH, and There is basically no change in osmotic pressure, biological activity, and insoluble particles; the main peak detected by SEC changes very little, all within 0.29%; the main peak detected by NR-CE and the change range are within 0.7%. The above results demonstrate the excellent stability of the formulation of the antibody of the present invention under shaking conditions.

Table 9. Oscillation test results of antibody preparations

(3) Acceleration stability:

The stability of the antibody preparation was tested under accelerated conditions (25°C, 2 months). Table 10 shows the test results of each antibody preparation: within 2 months of acceleration, no visible foreign matter was found. Compared with day 0, the concentration, pH, osmotic pressure, biological activity, and insoluble particles of the antibody preparation basically did not change; the main peak of SEC detection The changes are small, all within 1.09%; the main peak and change range of NR-CE detection are within 1.3%. The above results indicate that the formulation of the antibody of the present invention can maintain excellent stability under accelerated conditions.

Table 10. Accelerated test results of antibody preparations

(4) Long-term stability:

The stability of the antibody preparation was tested under long-term conditions (2-8°C, upright, 6 months). Table 11 shows the test results of each antibody preparation: under long-term conditions for 6 months, no visible foreign matter was found. Compared with day 0, the concentration, pH, osmotic pressure, biological activity, and insoluble particles of the antibody preparation basically did not change; SEC test The changes in the main peaks are small, all within 0.18%; the main peaks and changes in NR-CE detection are within 0.3%. The above results indicate that the formulation of the antibody of the present invention can maintain excellent stability under long-term conditions.

Table 11. Long-term test results of antibody preparations

In summary, the antibody preparation of the present invention has strong stability under freeze-thaw, shaking, acceleration and long-term conditions, which can ensure that the preparation maintains good stability during preparation, transportation and storage, and ensures the safety and quality of clinical medication. Controllability.

Claims (20)

1. An isolated anti-TSLP antibody, comprising V _H The V is _H Comprising: HC-CDR1 comprising the amino acid sequence SEQ ID NO:1, HC-CDR2 comprising the amino acid sequence SEQ ID NO:2, and HC-CDR3 comprising the amino acid sequence SEQ ID NO:3; v (V) _L The V is _L Comprising: LC-CDR1 comprising the amino acid sequence SEQ ID NO.7, LC-CDR2 comprising the amino acid sequence SEQ ID NO. 8, and LC-CDR3 comprising the amino acid sequence SEQ ID NO. 9; or (b)

The antibody comprises V _H The V is _H Comprising: HC-CDR1 comprising the amino acid sequence SEQ ID NO. 4, HC-CDR2 comprising the amino acid sequence SEQ ID NO. 5, and HC-CDR3 comprising the amino acid sequence SEQ ID NO. 6; v (V) _L The V is _L Comprising: LC-CDR1 comprising the amino acid sequence SEQ ID NO 10, LC-CDR2 comprising the amino acid sequence SEQ ID NO 11, and LC-CDR3 comprising the amino acid sequence SEQ ID NO 12.

2. The antibody of claim 1, wherein the antibody comprises V _H Comprising the amino acid sequence SEQ ID NO. 13, and V _L Comprising the amino acid sequence SEQ ID NO. 14; or (b)

The antibody comprises V _H Comprising the amino acid sequence SEQ ID NO. 15, and V _L Which comprises the amino acid sequence SEQ ID NO. 16.

3. The antibody of claim 2, further comprising a heavy chain constant region comprising the amino acid sequence of SEQ ID No. 17 and a light chain constant region comprising the amino acid sequence of SEQ ID No. 18.

4. An anti-TSLP antibody preparation, comprising the isolated anti-TSLP antibody of any one of claims 1-3.

5. The antibody preparation according to claim 4, wherein the antibody concentration is 1mg/ml to 300mg/ml; preferably, the antibody concentration is 10mg/ml to 200mg/ml; further preferably 100mg/ml to 200mg/ml; more preferably 135mg/ml to 165mg/ml.

6. The antibody formulation of claim 4 or 5, further comprising a surfactant, the surfactant being polysorbate and/or poloxamer; preferably, the polysorbate is polysorbate 20 and/or polysorbate 80; preferably, the poloxamer is poloxamer 188.

7. The antibody preparation according to claim 6, wherein the concentration of the surfactant is 0.02mg/ml-2.0mg/ml, preferably 0.52mg/ml-1.65mg/ml, more preferably 0.65mg/ml-1.52mg/ml.

8. The antibody formulation of any one of claims 5-7, wherein the surfactant is polysorbate and poloxamer; preferably, the concentration of said polysorbate is between 0.02mg/ml and 0.15mg/ml, preferably 0.1mg/ml; the concentration of poloxamer is 0.5-1.5mg/ml, preferably 1.0mg/ml.

9. The antibody preparation according to any one of claims 4-8, further comprising a stabilizer, preferably any one or more of sucrose, trehalose, maltose, sorbitol, mannitol, sodium chloride, arginine, glycine, proline, lysine; more preferably, the stabilizer is any one or more of sucrose, trehalose, sodium chloride or arginine.

10. The formulation of claim 9, wherein the stabilizer is: sodium chloride, arginine, glycine, proline or lysine at a concentration of 100mM-180mM, preferably 130mM-150 mM; or sucrose, trehalose, maltose, sorbitol and/or mannitol at a concentration of 40mg/ml to 80mg/ml, preferably 50mg/ml to 80mg/ml, further preferably 50mg/ml to 70 mg/ml.

11. Antibody preparation according to any one of claims 4-10, characterized in that it further comprises a buffer, preferably the buffer is a citrate buffer, an acetate buffer and/or a histidine hydrochloride buffer; preferably, the pH of the formulation is in the range of 4.8 to 5.5, preferably 5.0 to 5.4.

12. The antibody preparation of claim 11, wherein the concentration of the buffer is 3mM-50mM; preferably, the concentration of the buffer is 10mM-30mM.

13. Antibody preparation according to any one of claims 4 to 12, characterized in that the pH of the preparation is 4.8 to 5.5, preferably 5.0 to 5.4.

14. The antibody preparation according to claim 4, wherein the antibody concentration is 100mg/ml-200mg/ml; the stabilizer is sucrose and/or trehalose of 40mg/ml-80mg/ml, or the stabilizer is 100mM-180mM sodium chloride or arginine; the buffer solution is 3mM-50mM acetate buffer solution, citrate buffer solution and/or histidine hydrochloride buffer solution; the pH value of the preparation is 4.8-5.5; the surfactant is polysorbate 0.02mg/ml-0.15mg/ml and poloxamer 0.5mg/ml-1.5 mg/ml.

15. The antibody preparation of claim 4, wherein the antibody concentration is 135mg/ml-165mg/ml; the stabilizer is sucrose and/or trehalose of 50mg/ml-80mg/ml, or 130mM-150mM sodium chloride or arginine; the buffer solution is 10mM-30mM acetate buffer solution, citrate buffer solution and/or histidine hydrochloride buffer solution; the pH value of the preparation is 4.8-5.5; the surfactant is polysorbate 0.02mg/ml-0.15mg/ml and poloxamer 0.5mg/ml-1.5 mg/ml.

16. The antibody preparation of claim 4, wherein the antibody concentration is 135mg/ml-165mg/ml; the stabilizer is sucrose and/or trehalose of 50mg/ml-70mg/ml, or 130mM-150mM sodium chloride or arginine; the buffer solution is 10mM-30mM acetate buffer solution, citrate buffer solution and/or histidine hydrochloride buffer solution; the pH value of the preparation is 5.0-5.4; the surfactant is polysorbate 0.02mg/ml-0.15mg/ml and poloxamer 0.5mg/ml-1.5 mg/ml.

17. The antibody preparation of claim 4, wherein the preparation is any one of the following antibody preparations:

(1) The concentration of the antibody is 135mg/ml, 140mg/ml, 145mg/ml, 150mg/ml, 155mg/ml, 160mg/ml or 165mg/ml; the stabilizer is sucrose and/or trehalose of 50mg/ml-70mg/ml, or 130mM-150mM sodium chloride or arginine; the buffer solution is 10mM-30mM acetate buffer solution, citrate buffer solution and/or histidine hydrochloride buffer solution; the pH value of the preparation is 5.0-5.4; the surfactant is polysorbate 0.02mg/ml-0.15mg/ml and poloxamer 0.5mg/ml-1.5 mg/ml;

(2) The concentration of the antibody is 135mg/ml-165mg/ml; the stabilizer is sucrose and/or trehalose of 50mg/ml, 60mg/ml or 70mg/ml, or 130mM, 140mM or 150mM sodium chloride or arginine; the buffer solution is 10mM-30mM acetate buffer solution, citric acid buffer solution and/or histidine hydrochloride buffer solution; the pH value of the preparation is 5.0-5.4; the surfactant is polysorbate 0.02mg/ml-0.15mg/ml and poloxamer 0.5mg/ml-1.5 mg/ml;

(3) The concentration of the antibody is 135mg/ml-165mg/ml; the stabilizer is sucrose or trehalose of 50mg/ml-70mg/ml, or 130mM-150mM sodium chloride or arginine; the buffer is 10mM, 20mM or 30mM acetate buffer, citric acid buffer and/or histidine hydrochloride buffer; the pH value of the preparation is 5.0-5.4; the surfactant is polysorbate 0.02mg/ml-0.15mg/ml and poloxamer 0.5mg/ml-1.5 mg/ml;

(4) The concentration of the antibody is 135mg/ml-165mg/ml; the stabilizer is sucrose or trehalose of 50mg/ml-70mg/ml, or 130mM-150mM sodium chloride or arginine; the buffer solution is 10mM-30mM acetate buffer solution, citric acid buffer solution and/or histidine hydrochloride buffer solution; the pH of the formulation is 5.0, 5.1, 5.2, 5.3 or 5.4; the surfactant is polysorbate 0.02mg/ml-0.15mg/ml and poloxamer 0.5mg/ml-1.5 mg/ml;

(5) The concentration of the antibody is 135mg/ml-165mg/ml; the stabilizer is sucrose or trehalose of 50mg/ml-70mg/ml, or 130mM-150mM sodium chloride or arginine; the buffer solution is 10mM-30mM acetate buffer solution, citric acid buffer solution and/or histidine hydrochloride buffer solution; the pH value of the preparation is 5.0-5.4; the surfactant is polysorbate 0.02mg/ml, 0.03mg/ml, 0.05mg/ml, 0.06mg/ml, 0.08mg/ml, 0.10mg/ml, 0.12mg/ml or 0.15mg/ml and poloxamer 0.5mg/ml-1.5 mg/ml;

(6) The concentration of the antibody is 135mg/ml-165mg/ml; the stabilizer is sucrose or trehalose of 50mg/ml-70mg/ml, or 130mM-150mM sodium chloride or arginine; the buffer solution is 10mM-30mM acetate buffer solution, citric acid buffer solution and/or histidine hydrochloride buffer solution; the pH value of the buffer solution is 5.0-5.4; the surfactant is polysorbate 0.02mg/ml-0.15mg/ml and poloxamer 0.5mg/ml, poloxamer 0.6mg/ml, poloxamer 0.8mg/ml, poloxamer 1.0mg/ml, poloxamer 1.2mg/ml or poloxamer 1.5 mg/ml;

preferably, the polysorbate is polysorbate 80 or polysorbate 20;

preferably, the poloxamer is poloxamer 188.

18. The antibody formulation of any one of claims 4-17, further comprising an antioxidant and/or preservative.

19. The antibody preparation according to any one of claims 4-18, wherein the antibody preparation is a liquid preparation or a powder for injection.

20. Use of an antibody according to any one of claims 1-3 or an antibody preparation according to any one of claims 4-19 in the manufacture of a medicament for the treatment of a disease;

preferably, the disease comprises an inflammatory or autoimmune disease;

more preferably, the disease is selected from asthma, idiopathic pulmonary fibrosis, atopic dermatitis, allergic conjunctivitis, allergic rhinitis, netherton's syndrome, eosinophilic esophagitis (EoE), food allergy, allergic diarrhea, eosinophilic gastroenteritis, allergic bronchopulmonary aspergillosis (ABPA), allergic mycotic sinusitis, cancer, rheumatoid arthritis, chronic Obstructive Pulmonary Disease (COPD), systemic sclerosis, keloids, ulcerative colitis, chronic Rhinosinusitis (CRS), nasal polyposis, chronic eosinophilic pneumonia, eosinophilic bronchitis, celiac disease, churg-Strauss syndrome, eosinophilic myalgia syndrome, eosinophilic granulomatosis with polyangiitis, inflammatory bowel disease.