CN113069543B - Liquid composition comprising monoclonal antibodies against thymic stromal lymphopoietin - Google Patents

Abstract

The present invention provides a liquid composition comprising a monoclonal antibody against thymic stromal lymphopoietin. The liquid composition contains the monoclonal antibody at a high concentration, the antibody can be kept stable for a long period of time, and the preparation has appropriate viscosity and osmotic pressure, and can be used as a subcutaneous injection preparation. In addition, the invention also provides the use of the liquid composition in the preparation of a medicament for treating a disease or condition associated with TSLP signaling and/or TSLP overexpression; a container containing the liquid composition; and kits comprising the same.

Description

Liquid composition comprising monoclonal antibodies against thymic stromal lymphopoietin

Technical Field

The present invention relates to the field of biopharmaceutical formulations, and in particular, to a liquid pharmaceutical formulation comprising a stable anti-Thymic Stromal Lymphopoietin (TSLP) antibody.

Background

Thymic Stromal Lymphopoietin (TSLP), a cytokine produced by epithelial cells upon pro-inflammatory stimulation, acts on Dendritic Cells (DCs) and mast cells to drive the body's allergic inflammatory response. TSLP-activated DCs express the chemical factors TARC and MDC that aggregate Th 2. In addition, TSLP-activated DCs (TSLP-DCs) also initiate the transformation of CD 4T cell precursors to Th2, whereas Th2 cells produce the allergic factors IL-4, IL-5, IL-13 and TNF- α, while reducing IL-10 and IFN- γ production.

TSLP plays an important role in the development of allergic asthma, and targeting TSLP inhibits multiple asthma-associated biological pathways, including IL-4, IL-5, and IL-13. TSLP is active in the early upstream phase of the inflammatory cascade, making it a clear target for the treatment of asthma. In the clinical study of the IIb stage, the annual incidence rate of asthma of a drug group is reduced by 60-70% compared with that of a control group by an anti-TSLP fully humanized antibody Tezepelumab jointly developed by an Angin and Alikang.

However, the best mode of administration of antibody drugs is subcutaneous injection, as is antibody drugs directed against the TSLP target. Because of the high antibody doses required to exert an effect, it is often necessary to prepare antibody preparations in high concentrations. However, the increase in antibody concentration is accompanied by various drawbacks, for example, excessive viscosity of the antibody preparation, resulting in difficulty in injection, pain at the injection site, remaining of the antibody in the container, difficulty in production, and the like. In addition, high concentrations of antibody tend to aggregate, form microparticles, cause formulation instability, increased immunogenicity, etc., and are difficult to store and store for extended periods of time.

Therefore, there is still a need in the art to develop a novel antibody targeting thymic stromal lymphopoietin and a formulation containing the same, which satisfy the need for a long-term stable, aggregation-free, low viscosity drug capable of treating thymic stromal lymphopoietin-related diseases.

Disclosure of Invention

The technical problem to be solved by the invention is to obtain a new antibody with high affinity and high functional activity to human TSLP by hybridoma screening and humanization technology; and the new antibody is used as a medicinal active ingredient to obtain a high-concentration and stable liquid medicinal preparation.

In view of the above technical problems, it is an object of the present invention to provide a liquid composition comprising anti-Thymic Stromal Lymphopoietin (TSLP).

The invention provides the following technical scheme:

in one aspect, the present invention provides a liquid composition comprising an antibody or fragment thereof against thymic stromal lymphopoietin, said antibody or fragment thereof having a heavy chain variable region and a light chain variable region and comprising any one selected from the group consisting of:

(1) the heavy chain CDR1 (GYTFTNY), the heavy chain CDR2 (DAFSGG) and the heavy chain CDR3 (ESEVGEGFAY) shown in sequence in SEQ ID NO 7, 17 and 9; and, the light chain CDR1 (RASQDISNYLN), light chain CDR2 (YTSTLHS), light chain CDR3 (QQGNTLPYT) shown sequentially in SEQ ID NOs 10, 11, 18;

(2) 13, 19, 9, respectively, heavy chain CDR1 (NYFID), heavy chain CDR2 (VIDAFSGGSNFNEKFKG), heavy chain CDR3 (ESEVGEGFAY); and, the light chain CDR1 (RASQDISNYLN), light chain CDR2 (YTSTLHS), light chain CDR3 (QQGNTLPYT) shown sequentially in SEQ ID NOs 10, 11, 18;

(3) 15, 20, 9, respectively, heavy chain CDR1 (GYTFTNYFID), heavy chain CDR2 (VIDAFSGGSN), heavy chain CDR3 (ESEVGEGFAY); and, the light chain CDR1 (RASQDISNYLN), light chain CDR2 (YTSTLHS), light chain CDR3 (QQGNTLPYT) shown sequentially in SEQ ID NOs 10, 11, 18; and

(4) 15, 19, 9, respectively, heavy chain CDR1 (GYTFTNYFID), heavy chain CDR2 (VIDAFSGGSNFNEKFKG), heavy chain CDR3 (ESEVGEGFAY); and, the light chain CDR1 (RASQDISNYLN), light chain CDR2 (YTSTLHS), light chain CDR3 (QQGNTLPYT) shown sequentially in SEQ ID NOs 10, 11, 18.

The above-mentioned heavy chain and light chain CDR combinations of groups (1) to (4) are obtained by CDR-partitioning of SEQ ID NO: 5 (humanized antibody heavy chain variable region) and SEQ ID NO: 6 (humanized antibody light chain variable region) provided below using different antibody numbering schemes: (1) chothia; (2) kabat; (3) abm, respectively; (4) CCG. See in particular the examples section below.

Preferably, in the liquid composition provided by the present invention, the antibody is preferably a monoclonal antibody, preferably a humanized monoclonal antibody. Preferably, in the liquid composition provided by the present invention, the heavy chain variable region of the monoclonal antibody or fragment thereof comprises the amino acid sequence shown in SEQ ID NO. 5 and the light chain variable region or fragment thereof comprises the amino acid sequence shown in SEQ ID NO. 6. As for the fragment of the monoclonal antibody, the fragment is a functional fragment of the monoclonal antibody capable of binding thymic stromal lymphopoietin, and can be scFv, dsFv, (dsFv)₂、Fab、Fab'、F(ab')₂Or a Fv fragment. The monoclonal antibody or fragment thereof can be obtained using methods known in the art.

The antibody or fragment thereof in the liquid composition of the present invention has a heavy chain variable region and a light chain variable region comprising the above domain components in the order of FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, wherein FR is a framework region, according to domain composition of a monoclonal antibody heavy chain variable region and light chain variable region known in the art.

In the context of the present invention, the thymic stromal lymphopoietin is a mammalian TSLP, e.g., a human TSLP.

More preferably, the liquid composition provided by the present invention comprises said monoclonal antibody. Wherein the monoclonal antibody comprises a heavy chain constant region of an IgG, IgA, IgM, IgD or IgE and/or a light chain constant region of a kappa or lambda type. More preferably, the heavy chain constant region of the monoclonal antibody is of the IgG1 or IgG2 type and the light chain constant region is of the kappa type. According to a specific embodiment of the present invention, the heavy chain constant region of the monoclonal antibody comprises the amino acid sequence shown in SEQ ID NO. 25 and the light chain constant region comprises the amino acid sequence shown in SEQ ID NO. 26.

Preferably, the liquid compositions provided herein are in the form of a solution, emulsion or suspension in water. More preferably, the liquid composition is a liquid formulation for parenteral administration. Further preferably, the liquid composition is a formulation for administration by injection. According to a particular embodiment of the invention, the liquid composition is a subcutaneous injection formulation.

Preferably, the liquid composition provided by the present invention has a pH of 5.0 to 6.0; more preferably 5.5 to 6.0.

Preferably, the liquid composition comprises the monoclonal antibody at a concentration of 120-150 mg/mL.

Preferably, the liquid composition provided by the invention further comprises histidine at a concentration of 10-20 mmol/L.

Preferably, the liquid composition provided by the invention also comprises arginine hydrochloride with the concentration of 20-50 mmol/L.

Preferably, the liquid composition provided by the invention further comprises sucrose at a concentration of 180-200 mmol/L.

Preferably, the liquid composition provided by the present invention further comprises tween 80 at a concentration of 0.01% to 0.05% (said "%" is in weight (mg)/volume (L)).

According to a particular embodiment of the invention, there is provided a liquid composition comprising: the monoclonal antibody at a concentration of 120-; 10mmol/L histidine; arginine hydrochloride of 30 mmol/L; 185-190mmol/L sucrose; 0.01-0.03% tween 80. More preferably, the present invention provides a liquid composition having the composition of the aforementioned ingredients and the contents thereof.

According to a particular embodiment of the invention, the liquid composition provided herein has a pH of 5.5.

In another aspect, the invention provides the use of the liquid composition in the manufacture of a medicament for the treatment of a disease or condition associated with TSLP signaling and/or TSLP overexpression, e.g., an inflammatory disease or tumor. Preferably, the inflammatory disease is selected from asthma, allergic dermatitis, chronic obstructive pulmonary disease, and allergic rhinitis. More preferably, the asthma comprises Th 2-type and non-Th 2-type asthma. Preferably, the tumor is selected from hodgkin's lymphoma, breast cancer, pancreatic cancer, melanoma and lung cancer.

In yet another aspect, the present invention provides a container comprising a liquid composition as described herein. Preferably, the container is a syringe.

In a further aspect, the present invention provides a kit comprising a container according to the invention.

The invention provides a novel humanized monoclonal antibody against TSLP. Experiments prove that the monoclonal antibody can be combined with TSLP with high affinity; capable of blocking the binding of TSLP to its receptor (TSLPR); can inhibit BAF/3-TSLPR cell reporter gene expression; can inhibit dendritic cells from secreting CCL17 and OPG. Also, the monoclonal antibody does not recognize IL 7.

Based on the provision of such monoclonal antibodies, the present invention further provides liquid compositions comprising antibodies or fragments thereof against TSLP, which can be used as antibody pharmaceutical formulations for the treatment of TSLP-related diseases. Through screening of antibody pharmaceutical preparation components such as buffer system composition, pH, protective agent, surfactant and the like, experiments prove that the invention provides a stable liquid preparation containing a high-concentration anti-TSLP humanized monoclonal antibody, the antibody can stably exist under long-term storage, and simultaneously, the low viscosity and the osmotic pressure close to isotonic of the preparation are realized; in addition, the administration volume of such formulations can be reduced and subcutaneous administration achieved.

Drawings

Embodiments of the invention are described in detail below with reference to the attached drawing figures, wherein:

FIG. 1 shows the TSLP antibody titers in the sera of 10 mice.

FIG. 2 shows the TSLPR blocking capacity of culture supernatants of hybridoma monoclonal cells, wherein 2A to 2D are the results of the monoclonal cells of batches 1 to 4, respectively.

FIG. 3 shows the results of the detection of the inhibitory effect of hybridoma monoclonal cell culture supernatant on the expression of BAF/3-TSLPR cell reporter gene.

FIG. 4 shows the results of the detection of the inhibitory effect of the chimeric antibody on the expression of BAF/3-TSLPR cell reporter gene.

FIG. 5 shows the results of the antibody blocking the binding of TSLP to TSLPR.

Figure 6 shows the results of binding kinetic measurements of humanized antibodies to TSLP, where 6A: hzD01Lm 12; 6B: hzD01Hm5Lm 4.

FIG. 7 shows the results of the measurement of the inhibitory effect of the humanized antibody on the expression of BAF/3-TSLPR cell reporter gene.

Fig. 8 shows the results of the detection of the inhibitory effect of the humanized antibody on the secretion of CCL17 and OPG by dendritic cells, wherein 8A: CCL 17; 8B: OPG.

Figure 9 shows the species cross-binding assay results for humanized antibodies to human TSLP, mouse TSLP and monkey TSLP proteins and human IL7 protein, where 9A: hzD01Hm5Lm 4; 9B: hzD01m 12; 9C: AMG 157; BLK: blank.

FIG. 10 shows the first round of prescribed SEC-HPLC results of humanized antibody hzD01Hm5Lm4 (70 mg/ml) at accelerated conditions of 40 ℃.

FIG. 11 shows the first round of prescribed CEX-HPLC results of humanized antibody hzD01Hm5Lm4 (70 mg/ml) under accelerated conditions at 40 ℃.

FIG. 12 shows the first round of prescribed SEC-HPLC results of humanized antibody hzD01Hm5Lm4 (150 mg/ml) at accelerated conditions of 40 ℃.

FIG. 13 shows the first round of prescribed CEX-HPLC results for humanized antibody hzD01Hm5Lm4 (150 mg/ml) at accelerated conditions of 40 ℃.

FIG. 14 shows humanized antibody hzD01Hm5Lm4 (150 mg/ml) particle size as determined by DLS first round prescription.

Figure 15 shows the second round of prescribed SEC-HPLC results of humanized antibody hzD01Hm5Lm4 under accelerated conditions at 40 ℃.

FIG. 16 shows the second round of prescribed CEX-HPLC results of humanized antibody hzD01Hm5Lm4 under accelerated conditions at 40 ℃.

FIG. 17 shows the SEC-HPLC results of a second round of the prescription of humanized antibody hzD01Hm5Lm4 at 2-8 ℃.

FIG. 18 shows the second round of prescribed CEX-HPLC results for humanized antibody hzD01Hm5Lm4 at 2-8 ℃.

FIG. 19 shows the third round of SEC-HPLC results for humanized antibody hzD01Hm5Lm4 at 40 ℃.

FIG. 20 shows a SEC-HPLC profile corresponding to the third round of the prescription of humanized antibody hzD01Hm5Lm4 at 40 ℃.

FIG. 21 shows the third round of prescription CEX-HPLC results for humanized antibody hzD01Hm5Lm4 at 40 ℃.

FIG. 22 shows a third round of prescribed CEX-HPLC profiles for humanized antibody hzD01Hm5Lm4 at 40 ℃.

Detailed Description

The invention is illustrated below with reference to specific examples. It will be understood by those skilled in the art that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention in any way.

The experimental procedures in the following examples are conventional unless otherwise specified. The raw materials, reagents and the like used in the following examples are all commercially available products unless otherwise specified. Wherein:

human TSLP: near shore company, catalog # CK 16.

Mouse (mouse) TSLP: casalog # 51005-M08H.

Monkey (rhesus) TSLP: near shore company, catalog # CR 62.

Human TSLPR: casalog #29749-H08H, Chinesis.

Human IL 7: near shore company, catalog # C086.

Human TSLPR (CRLF 2) CDS: NM _ 022148.3.

Human IL7R CDS: NM _ 002185.4.

The following known antibodies were used as control antibodies:

AMG 157: the heavy chain is shown in SEQ ID NO. 1 and the light chain is shown in SEQ ID NO. 2. Respectively cloning the fully-synthesized AMG157 antibody light chain and heavy chain genes into eukaryotic expression vectors to obtain AMG157 light and heavy chain expression plasmids, transferring the AMG157 light and heavy chain expression plasmids into escherichia coli for amplification, separating to obtain a large amount of plasmids containing AMG157 antibody light chains and heavy chains, mixing the AMG157 light chains and heavy chains with Polyethyleneimine (PEI), and then co-transfecting into HEK293 cells. And (3) transfecting the cells for 5-6 days, taking culture supernatant, and purifying the expression supernatant by using a ProA affinity chromatography column to obtain the AMG157 antibody recombinant protein.

Heavy chain constant region of IgG2 subclass: 25 in SEQ ID NO; light chain constant region of kappa subclass: 26 SEQ ID NO

The following detection method is adopted in the screening of the composition of the antibody preparation:

SEC-HPLC detection:

high performance liquid chromatograph: waters column: waters Xbridge BEH200A

Mobile phase: 0.1mol/L PB +0.1mol/L Na₂SO₄（pH 6.80）

Sample preparation: diluting the sample to about 1.0mg/ml with mobile phase, centrifuging at 12000rpm for 10min, collecting supernatant, and analyzing

Analysis conditions were as follows: flow rate: 0.6 ml/min; column temperature: 30 ℃; detection wavelength: 280 nm.

And (3) testing the applicability of the system: taking 20 mu L of working reference sample for HPLC analysis according to analysis conditions, wherein the theoretical plate number of the monomer peak is as follows: not less than 5000.

CEX-HPLC detection:

high performance liquid chromatograph: waters column: ProPacTMElite WCX

Mobile phase: phase A: 4 mmol/L Tris + 4 mmol/L piperazine + 4 mmol/L imidazole (pH 5.0) phase B: 4 mmol/L Tris + 4 mmol/L piperazine + 4 mmol/L imidazole

And C phase: 500 mmol/L NaCl

Sample preparation: after a sample was diluted to about 1mg/mL with ultrapure water, 5. mu.L of CpB enzyme (2.0 mg/mL) was added to 200. mu.L of the dilution, and after 30min of water bath at 37 ℃, the mixture was centrifuged at 12000rpm for 10min, and the supernatant was sampled and analyzed.

Analysis conditions were as follows: flow rate: 0.5 mL/min; column temperature: 40 ℃; analyzing the wavelength: 280 nm; sample introduction amount: 50 mu L of the solution; temperature of the sample pan: 6 ℃ is adopted.

And (3) viscosity detection:

a detection instrument: m-VROC microfluidics rheology measurement viscometer

Minimum sample size: 20 mu l detection temperature: 25 deg.C

Sample preparation: final sample after ultrafiltration

Sample detection: and opening the software for connection, after the connection is successful, firstly, cleaning and testing by using a special cleaning solution for a viscometer, and then, rinsing the inner pipe wall by using the protein to be detected. And sucking 500 mul of sample into each tube, adjusting the temperature of the detection pool to 25 ℃ for temperature balance, and performing viscosity test according to different shear rates after the temperature is constant to 25 ℃.

And (3) DLS detection:

a detection instrument: DynaPro Plate Reader III type high flux dynamic light scattering instrument

Detection of the well plate: detection volume of 384-well plate: detection temperature of 25 μ l: 25 deg.C

Sample preparation: the sample is firstly placed in a 1.5ml EP tube for centrifugation at 10000rpm for 10min, then the supernatant is taken and transferred to a 384-well plate, the 384-well plate is centrifuged at 4000rpm for 20min, and the reading is carried out when no air bubbles exist in the 384-well plate.

Sample detection: and opening software to connect the devices, taking 25 mu l of the processed sample per tube after the connection is successful, adding the sample into a porous plate, and centrifuging by using a plate centrifuge. Removing bubbles in the porous plate, putting the porous plate into an equipment sample chamber, closing the sample chamber, editing an experimental method, and selecting micropores corresponding to a fixed temperature (25 ℃) and a sample to perform particle size test.

Example 1Screening of murine monoclonal antibodies

1.1 preparation of monoclonal cells

10 Balb/c mice were immunized with human TSLP protein according to the conventional methods described in the literature (Lonberg, N., et al., Nature 368(1994) 856-859; Fishwild, D.M., et al., Nat. Biotechnol. 14(1996) 845-851 and WO 98/24884), and the anti-TSLP titers in the sera of the immunized mice were determined by antigen-specific ELISA.

TSLP solution (in pH9.6, 0.1M NaHCO 3) at a concentration of 1ug/ml was coated in 96-well plates at 100ul per well and incubated overnight at 4 ℃. The coating solution was then washed off, added to each well using blocking solution, and incubated at room temperature for 2 hours. Mouse sera were pre-diluted 400-fold in PBSA (PBS with 1% BSA) and serially diluted 10-fold to 204800-fold in 1: 2. After washing off the blocking solution, diluted serum was added to the plate wells and incubated at room temperature for 1 hour. After washing the 96-well plate with PBST (0.05% Tween 20-PBS), a secondary antibody (HRP-labeled goat anti-mouse IgG (Fc. gamma.)) diluted at 1/30000 ul/well was added, incubated for 45 minutes at room temperature, washed three times with PBST, 20 ul/well of TMB equilibrated at room temperature was added, incubated for 10 minutes at room temperature, and the absorbance was measured at 450 nm. Serum TSLP antibody titers from 10 mice are shown in figure 1.

Mice with serum titers against TSLP greater than 1:100000 were boosted once with another intraperitoneal injection of TSLP 4 days prior to fusion. Splenocytes separated from the spleen of the mouse are fused with myeloma cells P3X63Ag8.653 by an electrofusion method.

The fused hybridoma cells are inoculated in a 384-well plate, after 14 days of culture, an enzyme label plate is coated with human TSLP protein (1 ug/ml, pH9.6, 0.1M NaHCO 3), HRP-goat anti-mouse IgG (Fc gamma) is added, and an A490 value is read by an enzyme label instrument, so that the antibody in the culture supernatant of the hybridoma clone is detected. The positive result is judged if the value of the A450 value of the detection hole is more than 3 times larger than the value of the A490 value of the negative control hole.

A total of 4 batches of immune fusions and screens were performed. Detecting and identifying to obtain multiple antibody secretion positive hybridoma clones, screening the hybridoma clones secreting anti-human TSLP antibody by combining experiments, unicellularizing by a limiting dilution method, and only secreting one antibody for each hybridoma clone obtained after two rounds of subcloning.

1.2 detection of monoclonal cell supernatants

And performing amplification culture on 4 batches of monoclonal cells, and taking supernatant to perform TSLPR blocking capacity detection.

Coating the enzyme label plate with 1ug/ml human TSLPR protein and NaHCO3 buffer solution (pH9.6, 0.1M), and standing at 4 deg.C overnight; blocking with 4% skimmed milk powder-PBS for 2 hours at 37 ℃; the plates were washed three times with PBST (0.05% Tween 20-PBS), and TSLP-biotin (Acro, cat # TSP-H82E0, 0.5 ug/ml) solution (in PBSA (1% BSA in PBS)) was added, with the negative clone supernatant as a control. Adding the monoclonal cell culture supernatant in the same volume, mixing and standing for 1 hour at normal temperature. Washing with PBST (0.05% Tween 20-PBS) for three times, adding 1:40000 diluted Streptavidin-HRP, and incubating at 37 deg.C for 1 hr; washing with PBST (0.05% Tween 20-PBS), adding TMB color developing solution, reacting for 10min in dark, adding 2M H2SO4 to terminate the reaction, and reading the A450 value with an enzyme-labeling instrument.

Blocking inhibition (%) = ((negative control well-monoclonal supernatant well)/negative control well) × 100%. Through detection and identification, a plurality of hybridoma monoclonals with blocking capacity are obtained, and are shown in 2A to 2D in figure 2.

In addition, the supernatants were tested for inhibition of reporter gene expression.

Human TSLPR and human IL7R molecules were overexpressed on BAF/3 cells and stably transfected with STAT 5-luciferase reporter gene, and stably transfected cells 1E6 were starved for 4 hours with 50ul per well of white-bottom plates. The medium was used as a negative control (N.C.), and the negative control and the clone supernatants were 100ul/well incubated with an equal volume of human TSLP protein solution (200 ng/ml, PBSA (1% BSA in PBS)) for 30 minutes in a U-plate. 50ul of the TSLP-test sample mixed solution was added to 50ul/well cells, incubated in a 37 ℃ 5% CO2 cell incubator for 16 hours, 100ul of Bio-Glo Luciferase Assay Reagent was added and shaken for 5 minutes, and the chemiluminescence results were read. See fig. 3.

And selecting the antibody clone with strong capability according to the results of the blocking experiment and the reporter gene expression inhibition experiment. After the hybridoma cells are subjected to expanded culture, extracting total RNA of the cells according to the steps of an RNAfast200 kit (Shanghai Feijie Biotechnology Co., Ltd.); reverse transcribing hybridoma cell total RNA to cDNA using 5 XPrimeScript RT Master Mix (Takara); amplifying antibody light chain variable region IgVL (κ) and heavy chain variable region VH sequences using degenerate primers (ake krebber.1997) and Extaq PCR reagents (Takara); purifying the PCR amplification product by using a PCR clean-up Gel extraction kit (Macherey-Nagel Co.); connecting the amplified PCR product to a T Vector according to the specification of a pClone007 Simple Vector Kit (Scopheraceae Biotechnology limited), converting escherichia coli competent cells, amplifying the strain, extracting plasmids, and performing DNA sequencing to obtain the variable region sequence of the monoclonal antibody.

Clone A20

>A20-VH

QIQLVQSGPELKKPGETVKISCKASGYSFATYGVSWLKQAPGKGLKWMGWMNTKSGVPTYADDFKGRFAFSWETSASTAYLLITNLKNEDTASYFCARNGQSGYDDYFDYWGQGTTLTVSS

>A20-VL

DIQLTQTTSSLSASLGDRVTISCSASQVISNYLNWYQQKPDGTLKLLIYYTSSLQSGVPSRFSGSGSGTDYSLTISNLESEDIATYYCQQYRKLPLTFGAGTKLELK

Clone B01

>B01-VH

EVHLQQSGAELVRPGASVKLSCIVSGFNIKDDFLHWVKQRPEQGLEWVGWIDPENGDTEYASKFQGKATVTADTSSNTAYLHLSSLTSEDTAVYYCVHQDYDEAFAYWGQGTLVTVSA

>B01-VL

DVVMTQTPLTLSITIGQPASISCKSSQSLLDSDEKTYLNWLLQRPGQSPKRLIYLVSKLDSGVPDRFTGSGSGTDFTLKISRVEAEDLGVYYCWQGTHFPQTFGGGTKLEIK

Clone C13

>C13-VH

EVQLQQSGPELVKPGTSMKISCKASGFSFTDYTMNWVKQSHGKSLEWIGLINPYNRGPTYNPKFKGKATLAVDKSSTTAYMELHSLTSEDSAVYYCAIEGGYRYDGAWFAYWGQGTLVTVSA

>C13-VL

DVLMTQTPLSLPVSLGDQASISCRSSQSIVHSNGNTYLEWYLQKPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYYCFQGSHVPLTFGAGTKLELK

Clone D01

>D01-VH（SEQ ID NO: 21）

QVQLQQSGAELVRPGTSVKVSCKASGYAFTNYFIDWVKQRPGQGLEWIGVINPGSGGTNFNEKFKGKATLTADKSSSTAYMQLSSLTSEDSAVYFCARESEVGEGFAYWGQGTLVTVSA

>D01-VL（SEQ ID NO: 22）

DIQMTQTSSSLSASLGDRVTISCRASQDISNYLNWYQQKPDGTVKLLIYYTSTLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTKLEIK

Clone D02

>D02-VH

QVQLKQSGPGLVQPSQSLSITCTVSGFSLTNYGLHWVRLSPGKGLEWLGVMWSGGGTDYNAAFISRLTISKDNSKSQVFFKMKSLQAADTAIYYCARRNSENEGYALDYWGQGTSVTVSS

>D02-VL

QIVLTQSPVIMSASLGEEITLTCSASSSVTYMHWYQQKSGTSPKLLIYSTSNLASGVPSRFSGSGSGTFYSLTISSVEAEDAADYYCHQWSTYMNTFGGGTKLEIK

Clone D04

>D04-VH

QVQLKQSGPGLVQPSQSLSITCTVSGFSLTNYGLHWVRLSPGKGLEWLGVMWSGGGTDYNAALISRLTISKDNSKSQVFFKMKSLQAADTAIYYCARRSSENEGYALDYWGQGTSVTVSS

>D04-VL

QIVLTQSPVIMSASLGEEITLTCSASSSVTYMHWYQQKSGTSPKLLIYSTSNLASGVPSRFSGSGSGTFYSLTISSVEAEDAADYYCHQWSTYMNTFGGGTKLEIK

Example 2Kinetic study of in vitro affinity of murine anti-human TSLP antibodies

Binding kinetics parameters of murine antibody and human TSLP protein were analyzed using a Fortebio (BLITZ pro1.1.0.28) instrument.

Before measurement, AMC biological probe is soaked in kinetic buffer (PBS containing 0.01% BSA, 0.002% Tween20, pH7.4) for 10 min; then, the probe was placed in a kinetic buffer (PBS containing 0.01% BSA, 0.002% Tween20, pH 7.4) containing 100nM of a murine antibody for 1000 seconds to capture the murine antibody; further performing binding reaction on the probe and an antigen solution (PBS, containing 0.01% BSA, 0.002% Tween20, pH7.4) containing 100nM for 600 seconds; the probe was then transferred to a kinetic buffer (PBS containing 0.01% BSA, 0.002% Tween20, pH7.4) for a dissociation reaction time of 600 seconds. After the experiment, blank control response values were deducted, and the software was run for 1:1 Langmuir binding pattern was fitted and kinetic constants for antigen-antibody binding were calculated and the results are shown in Table 1.

Example 3Preparation of chimeric anti-human TSLP antibodies

Selecting high affinity, high blocking ability and cell activity molecule D01, and the heavy and light chain variable region and CDR domain sequences of the antibody are shown in Table 2.

Splicing the heavy chain variable region sequence of a murine anti-human TSLP monoclonal antibody and a publicly published heavy chain constant region sequence of an IgG2 subclass of a human monoclonal antibody to construct a mammalian cell expression vector; the variable region sequence of the light chain of the murine anti-human TSLP monoclonal antibody D01 is spliced with the publicly published constant region sequence of the light chain of the kappa subclass of the human monoclonal antibody to construct a mammalian cell expression vector. The constructed heavy chain vector and light chain vector of the anti-human TSLP chimeric antibody are mixed in a matched mode, a HEK293 cell is transfected by Polyethyleneimine (PEI), cell supernatant is collected after about 7 days, and the anti-human TSLP chimeric antibody protein xiD01 is obtained through purification by ProteinA.

The binding kinetics of the chimeric antibody to the human TSLP protein was analyzed using a Fortebio instrument according to the detection method described in example 2, and the results are shown in Table 3.

The chimeric antibody is tested for cellular activity, i.e., inhibition of reporter gene expression.

The ability of the antibody to inhibit the activity of the reporter gene was examined as above using a stably transfected BAF/3 cell line overexpressing human TSLPR, human IL7R and STAT 5-luciferase. Human TSLPR and human IL7R molecules were overexpressed on BAF/3 cells and stably transfected with STAT 5-luciferase reporter gene, and stably transfected cells 1E6 were starved for 4 hours with 50ul per well of white-bottom plates. 1mg/ml of antibody was serially diluted 1:2 to 8 gradients using PBSA (1% BSA in PBS), 100ul/well in U-bottom plate and 200ng/ml human TSLP PBSA (1% BSA in PBS), incubated for 30 minutes. 50ul of the TSLP-antibody mixture was added to 50ul/well cells, incubated in a 37 ℃ 5% CO2 cell incubator for 16 hours, shaken for 5 minutes with 100ul of Bio-Glo Luciferase Assay Reagent and detected for chemiluminescence. The results are shown in FIG. 4.

Example 4Humanized anti-human TSLP murine antibodyTransformation of chemo-and affinity maturation

Combining the antibody coding schemes of Kabat, Chothia, Abm, CCG, the amino acid sequence regions of the 6 antigen Complementarity Determining Regions (CDRs) of the heavy and light chains of murine antibodies and the framework regions (framework regions) that support the conserved three-dimensional conformation of the antibodies were determined. And then, analyzing and searching the known human antibody sequence, selecting the human antibody heavy chain variable region sequence which is most similar and close to the murine antibody, selecting the antibody framework region sequence as a template, combining the murine antibody heavy chain CDR with the human antibody framework region, and finally generating the humanized antibody heavy chain variable region sequence. The same procedure produces the humanized antibody light chain variable region sequence.

The binding activity of antibody molecules of which the CDR of the murine antibody is directly transplanted to a human framework region is reduced, then the affinity maturation modification is carried out on the molecules by adopting a yeast display technology, random mutation library construction is carried out on the amino acids in the CDR regions of the heavy chain variable region and the light chain variable region of the template molecules, and candidate antibody molecules with higher affinity are screened out from the library. And carrying out back mutation on individual amino acids in the framework region, and changing the amino acids from human sources to mouse sources. Determining the back mutation site, namely checking the difference of amino acids by contrasting the designed humanized antibody sequence and the original murine antibody sequence; the second is to check whether these amino acids play an important role in supporting antibody structure or in binding to antigen, and whether there are some potential post-translational modification sites, such as N (asparagine) glycosylation site, N deamidation site, D (aspartic acid) isomerization site, etc. The humanized sequences are shown below.

Heavy chain variable region

>hzD01 H（SEQ ID NO: 3）

EVQLVQSGAEVKKPGASVKVSCKASGYTFTNYFIDWVRQAPGQGLEWIGVINPGSGGTNFNEKFKGRVTMTRDTSTSTAYMELSSLRSEDTAVYFCARESEVGEGFAYWGQGTLVTVSS

>hzD01 Hm5（SEQ ID NO: 5）

EVQLVQSGAEVKKPGASVKVSCKASGYTFTNYFIDWVRQAPGQGLEWIGVIDAFSGGSNFNEKFKGRVTMTRDTSTSTAYMELSSLRSEDTAVYFCARESEVGEGFAYWGQGTLVTVSS

>hzD01 Hm6

EVQLVQSGAEVKKPGASVKVSCKASGYTFTNYFIDWVRQAPGQGLEWIGVIDSFSGGINFNEKFKGRVTMTRDTSTSTAYMELSSLRSEDTAVYFCARESEVGEGFAYWGQGTLVTVSS

Light chain variable region

>hzD01 L

DIQMTQSPSSLSASVGDRVTISCRASQDISNYLNWYQQKPGKAPKLLIYYTSTLHSGVPSRFSGSGSGTDFTLTISSLQPEDIATYFCQQGNTLPYTFGQGTKLEIK

>hzD01 Lm4（SEQ ID NO: 6）

DIQMTQSPSSLSASVGDRVTISCRASQDISNYLNWYQQKPGKAPKLLIYYTSTLHSGVPSRFSGSGSGTDYTLTISSLQPEDIATYFCQQGNTLPYTFGQGTKLEIK

>hzD01 Lm12（SEQ ID NO: 4）

DIQMTQSPSSLSASVGDRVTISCRASQDISNYLNWYQQKPGKAPKLLIYYTSTLHSGVPSRFSGSGSGTDYTLTISSLQPEDIATYFCQQFDTLPYPFGQGTKLEIK

Constructing the engineered humanized antibody variable region heavy chain gene into a mammalian cell expression vector containing a heavy chain constant region gene of a subclass of human monoclonal antibody IgG 2; the light chain gene is constructed into a mammalian cell expression vector containing the light chain constant region gene of the kappa subclass of the human monoclonal antibody. And (3) pairing and mixing a heavy chain vector and a light chain vector of the constructed anti-human TSLP humanized antibody, transfecting HEK293 cells by using Polyethyleneimine (PEI), collecting cell supernatants after about 7 days, and purifying by using ProteinA to obtain the anti-human TSLP humanized antibody protein.

Example 5In vitro blocking assay for humanized antibodies

APC were conjugated to human TSLP protein using Dojindo kit (Dojindo # LK 21). In addition, using human TSLPR CDS and CHO cells to construct TSLPR over-expression cells, antibiotic screening for 4 weeks to obtain stable cells.

The test antibody was diluted 3-fold in gradient from an initial concentration of 100ug/mL (PBSA (1% BSA in PBS)) for a total of 11 concentration points, and 100ul of the antibody at each concentration point was added to a 96-well plate and incubated with 100ul of APC-conjugated TSLP antigen (1.6 ug/mL) in PBSA (1% BSA in PBS) for 1 hour. 100ul of TSLPR overexpressing CHO cells (1E 5/well) were added and incubated for 1 hour, the cells were washed once with 0.5% BSA in PBS, and the cell population was analyzed for mean fluorescence readings by flow cytometry. The results are shown in FIG. 5.

As a result of the assay, both humanized antibody molecules achieved substantially equivalent blocking effects to control antibody AMG157 with respect to blocking binding of TSLP to TSLPR. The variable regions of the heavy and light chains of the antibody and the CDR domain sequences thereof are shown in tables 4-1 and 4-2.

Example 6In vitro binding assay for anti-human TSLP humanized antibodies

With reference to the procedure of example 2, kinetic studies of in vitro affinity of the humanized antibody against human TSLP were performed. The results are shown in Table 5 and 6A and 6B in FIG. 6.

Example 7In vitro cytology assays for anti-human TSLP humanized antibodies

7.1 inhibition of BAF/3-TSLPR cell reporter Gene expression

Humanized TSLP antibodies were graded for dilution and evaluated for their ability to inhibit luciferase from BAF/3-TSLPR cells according to the assay described for chimeric antibodies in example 3. The results are shown in FIG. 7.

7.2 inhibition of dendritic cell secretion of CCL17 and OPG

Dendritic cells were sorted using human peripheral blood lymphocytes according to America gentle reagent instructions (catalog # 130-090-506), plated in 96-well plates at 100ul per well (5E 4 cells/well), and observed for cell spreading after 24 hours. Human TSLP protein was prepared to be detected at a concentration of 40ng/ml in PBSA (1% BSA in PBS), antibody concentration 4000ng/ml was diluted at the start of a 1:2 gradient (in PBSA (1% BSA in PBS), antibody and TSLP solution were mixed in equal volumes and allowed to stand for 30 minutes, 100ul of the mixture was added to the cell culture wells and incubation was continued for 48 hours, and the supernatants were tested for concentrations of both cytokines using CCL17 and OPG ELISA kits, see 8A and 8B in FIG. 8.

Example 8Species Cross-binding of anti-human TSLP humanized antibodies and Cross-binding study of the structurally similar molecule IL7

Coating 96-well plate with human TSLP, mouse TSLP and monkey TSLP protein, and human IL7 protein (1 ug/ml, pH9.6, 0.1M NaHCO 3), respectively, at 4 deg.C overnight; blocking with 4% skimmed milk powder-PBS, and sealing at 37 deg.C for 2 hr; PBST (0.05% Tween 20-PBS) was washed three times, anti-human TSLP humanized antibody was added, and incubated at 37 ℃ for 1 hour. Washing for three times by PBST, adding HRP-goat anti-human IgG (Jackson # 109-; washing with PBST for three times, adding TMB color developing solution, developing for 10min in dark place, and adding 2M H2SO4 to terminate the reaction; the microplate reader reads the A490 values.

The results show that the antibody molecules hzD01Hm5Lm4 and hzD01Lm12 of the invention both recognize human TSLP and monkey TSLP molecules, while IL7 does not recognize non-specifically. See 9A, 9B and 9C in fig. 9.

Example 9Preparation of protein stock solution of humanized antibody hzD01Hm5Lm4 against human TSLP

A fermentation broth of the humanized antibody hzD01Hm5Lm4 prepared as described in example 4 was cultured in Dynamis broth with cell boost 7a/7b fed-in, maintaining pH 6.95. + -. 0.2, initial culture temperature 36.8 ℃ and fifth day down to 33 ℃ for 12-14 days. And (3) performing affinity chromatography, low pH incubation, anion chromatography, cation chromatography and ultrafiltration on the fermentation liquor, and storing the final sample in 10mmol/L histidine, pH5.5 and protein concentration of 70mg/ml, wherein the protein stock solution is used for the subsequent prescription screening experiment.

1) Affinity chromatography

Buffer solution: AC-A1 buffer (20 mmol/L PB, 0.15 mol/L NaCl, pH 6.0. + -. 0.1)

AC-A2 buffer (20 mmol/L acetic acid-sodium acetate, pH5.5 + -0.1)

AC-B buffer (9.6 mmol/L disodium hydrogen phosphate-13.8 mmol/L citric acid, pH3.5 + -0.1)

Filler loading: less than or equal to 50 g/L

Filling: mabselect SureLX

Sample preparation: cell fermentation broth

The experimental process comprises the following steps: the buffer solution of AC-A1 is balanced for 1-3CV (column volume), the fermentation liquid is loaded, the buffer solution of AC-A1 is balanced for 1-2CV, the buffer solution of AC-A2 is balanced for 2-3CV, the buffer solution of AC-B is eluted for 3-5CV, the elution peak starts to collect when reaching the absorption of 280mAU, and the collection is stopped when the elution peak reaches 250mAU, and the buffer solution of AC-A1 is balanced for 1 CV. The sample collected at this step was designated as MF 2.

2) Low pH incubation

Sample preparation: affinity chromatography elution sample MF2

The experimental process comprises the following steps: the pH of the affinity chromatography elution sample MF2 is adjusted to 3.5 +/-0.1 by using 1mol/L citric acid, virus inactivation is carried out after the sample MF2 is placed for more than 30min at room temperature, and then the pH is adjusted to 5.5 +/-0.1 by using 2mol/L Tris. The sample collected at this step was designated LPH.

3) Anion chromatography

Buffer solution: AEC-A buffer (25.7 mmol/L disodium hydrogen phosphate-7.2 mmol/L citric acid, pH6.5 + -0.1)

AEC-B buffer (15.4 mmol/L disodium hydrogen phosphate-2.3 mmol/L citric acid, 0.5mol/L NaCl, pH6.5 + -0.1)

Filler loading: less than or equal to 125 g/L

Filling: q Sepharose Fastflow

Sample preparation: low pH incubated sample LPH

The experimental process comprises the following steps: AEC-B buffer solution pre-equilibrium 1-2 CV-AEC-A buffer solution equilibrium 7-10 CV-after incubation at low pH the sample is adjusted to pH6.5 +0.1 with 2mol/L Tris, sample loading is carried out after the conductivity is adjusted to be lower than 5 mS/cm with water for injection, and flow-through liquid is collected, after sample collection is completed, AEC-A buffer solution is used for re-equilibrium 2-3CV, peak collection is started when the elution peak reaches 280mAU absorption, and collection is stopped when the elution peak reaches 250mAU, namely 1-2CV of an AEC-B buffer solution equilibrium system. The flow-through collected at this step was designated as QF 1.

4) Cation chromatography

Buffer solution: CEC-A equilibration buffer (20 mmol/L PB, pH 6.0. + -. 0.1)

CEC-B elution buffer (20 mmol/L PB, pH 7.0. + -. 0.1)

Filler loading: less than or equal to 55g/L

Filling: capto S ImpAct

Sample preparation: anion chromatography was run through liquid QF 1.

The experimental process comprises the following steps: CEC-B buffer solution treatment 1-2 CV-CEC-A buffer solution balance 2-3 CV-QF 1 sample pH is adjusted to 6.0 +/-0.1, conductivity is adjusted to be lower than 5 mS/cm by using water for injection, sample loading is carried out, CEC-A buffer solution balance 1-2 CV-0% -100% CEC-B buffer solution is carried out for linear elution, peak collection is started when elution peak reaches 200mAU absorption, and collection is stopped after 3-5CV, CEC-A buffer solution balance 2-3 CV. The eluate collected at this step was designated as SF 2.

5) Ultrafiltration

Buffer solution: UF buffer (10 mmol/L histidine-histidine hydrochloride, 30mmol/L arginine hydrochloride, 185mmol/L sucrose, pH 5.5. + -. 0.1)

Sample preparation: sample SF2 after cation chromatography

The experimental process comprises the following steps: washing the membrane package with 20 kg/m2 of water for injection, using 10kg/m2 of ultrafiltration buffer balance membrane package to concentrate samples, using UF buffer to flow and add the replacement buffer when the concentration is 20-30g/L, stopping replacement after 8-10 times of volume of the replacement, collecting samples, keeping the concentration of the samples at 70mg/ml, and finally obtaining the protein stock solution after the samples are sterilized and filtered.

Example 10Liquid formulation prescription screening (first round experiment) of anti-human TSLP humanized antibody hzD01Hm5Lm4

The detection shows that the isoelectric point of the humanized antibody hzD01Hm5Lm4 is 7.2, the pH of the preparation prescription is easy to cause protein precipitation near the isoelectric point, the pH of the preparation prescription is higher than the isoelectric point, and the protein is in an alkaline environment and is not beneficial to the storage of the protein, so the pH of the preparation prescription is selected to be less than 7.2. Therefore, the pH values of the initial experiments of this round were 4.5, 5.5 and 6.5, and the protein molecules were examined at two different concentrations, 70mg/ml and 150 mg/ml.

Sample information: the protein stock obtained in example 9.

10.170 mg/ml protein concentration screening buffer system and pH

Buffer salt species: buffer1 (10 mmol/L citric acid, pH 4.5);

buffer2 (10 mmol/L histidine, pH 5.5);

Buffer3（10 mmol/L PB，pH 6.5）。

sample preparation method: taking a proper amount of the protein stock solution obtained in the example 9, desalting and replacing the protein stock solution to Buffer1-3 through a G25 desalting column respectively, and finely adjusting the sample by using an ultrafiltration concentration tube to reach corresponding buffering pH conditions, so that the concentration of the antibody in the sample reaches 70mg/ml finally.

The experimental process comprises the following steps: the three groups of samples are placed in a stability inspection box at 40 ℃ for 7 days of accelerated experiments, and SEC-HPLC and CEX-HPLC are detected. The results are shown in FIGS. 10 and 11.

Accelerated experiments SEC-HPLC results show that in the pH 4.5-6.5 range examined, the SEC-HPLC mer content does not change much when the pH4.5 citric acid, pH5.5 histidine group is accelerated to 7 days, while the mer content of the pH6.5 PB buffer group increases significantly.

The result of an accelerated test CEX-HPLC shows that under the conditions of pH4.5 citric acid and pH5.5 histidine, the content of an acidic peak is lower, and a histidine buffer system is slightly superior to a citric acid buffer system; whereas the acidic peak increases faster at pH6.5 PB, but within an acceptable range.

Since there is a risk of increased aggregation and decreased protein activity in the antibody stock solution at low pH, accelerated comparisons of both pH5.5 and pH6.5 were made at the next higher concentration sample investigation.

10.2150 mg/ml protein high concentration screening buffer system and pH

Further examine the optimal pH and buffer system of the protein at high concentrations. Based on previous knowledge of high concentration protein production: the greater the ultrafiltration resistance, the greater the viscosity as the protein concentration increases. Therefore, 30mM arginine was added to each group of samples during the preparation process to reduce the viscosity of the samples, so as to obtain samples with a concentration of 150 mg/ml.

Buffer salt species: buffer1 (20 mmol/L histidine +30mM arginine, pH 5.5);

buffer2 (20 mmol/L PB +30mM arginine, pH 6.5).

Sample preparation method: and (3) taking a proper amount of the protein stock solution obtained in the example 9, carrying out equal-volume ultrafiltration and replacement for 7-8 times to a corresponding buffer system, and concentrating to finally enable the concentration of the antibody in the sample to reach 150 mg/ml.

The experimental process comprises the following steps: and (3) placing the two groups of samples in a stability inspection box at 40 ℃ to carry out an accelerated experiment, and detecting SEC-HPLC and CEX-HPLC. The results are shown in fig. 12 and 13.

As can be seen from the SEC-HPLC and CEX-HPLC results, after 14 days at 40 ℃ acceleration, both the pH5.5 histidine-dimer content and the acid content were less than those of the pH6.5 PB group; the subsequent optimal buffer condition was therefore pH5.5 histidine.

The viscosity and DLS of the set were determined and the results are shown in table 6, table 7 and figure 14.

The detection result shows that under the condition of the concentration, the humanized antibody hzD01Hm5Lm4 has fewer macromolecular particles, and the protein viscosity is 6.0cp, thereby meeting the use requirement.

Example 11Liquid formulation prescription screening of anti-human TSLP humanized antibody hzD01Hm5Lm4 (second round experiment)

The experiment is examined from many aspects such as protein concentration, arginine hydrochloride content, buffer solution system and pH value, etc., so as to select the optimal buffer condition.

Buffer system: buffer A: 30mmol/L arginine hydrochloride +10mmol/L histidine pH5.0

Buffer B: 40mmol/L arginine hydrochloride +10mmol/L histidine pH5.0

Buffer C: 50mmol/L arginine hydrochloride +10mmol/L histidine pH5.0

Buffer D: 30mmol/L arginine hydrochloride +10mmol/L histidine pH5.0

Buffer E: 30mmol/L arginine hydrochloride +10mmol/L histidine pH5.0

Buffer F: 20mmol/L arginine hydrochloride +10mmol/L histidine at pH5.0

Buffer G: 20mmol/L arginine hydrochloride +10mmol/L histidine at pH5.5

Buffer H: 20mmol/L arginine hydrochloride +10mmol/L histidine pH6.0

Buffer I: 20mmol/L arginine hydrochloride +10mmol/L citric acid pH4.5

Buffer J: 20mmol/L arginine hydrochloride +10mmol/L citric acid pH5.0

Buffer K: 20mmol/L arginine hydrochloride +10mmol/L citric acid pH5.5

Buffer L: 20mmol/L arginine hydrochloride +10mmol/L citric acid pH6.0

Buffer M: 20mmol/L arginine hydrochloride +10mmol/L acetic acid pH4.5

Buffer N: 20mmol/L arginine hydrochloride +10mmol/L acetic acid pH5.0

Buffer O: 20mmol/L arginine hydrochloride +10mmol/L acetic acid pH5.5

Sample preparation method: 50ml of the protein stock solution obtained in example 9 were introduced into an ultrafiltration system and subjected to a corresponding buffer exchange by feeding at a flow rate of 120LMH (liter/square meter/hour, L/m)²H), replacing 7-8CV, and determining buffer replacement completion through pH measurement and conductivity of the permeation end. After the displacement is completed, the sample concentration is performed while keeping the flow rate constant, and finally the concentrations of all component samples are adjusted to the corresponding concentrations.

The experimental process comprises the following steps: and (3) placing each group of samples at 40 ℃ for an accelerated experiment and a long-term stability investigation experiment at 2-8 ℃, and detecting SEC-HPLC and CEX-HPLC. The results are shown in fig. 15 to 18.

The results of the accelerated experiment SEC-HPLC show that: the increase of the aggregate of the group I (10 mmol/L citric acid, pH4.5, 20mmol/L arginine hydrochloride, protein concentration of 150 mg/ml) is the highest, and the increase of the aggregate of the group M (10 mmol/L acetic acid, pH4.5, 20mmol/L arginine hydrochloride, protein concentration of 150 mg/ml) is the second, mainly due to the influence of pH, and the antibody is easy to form the aggregate at high concentration and low pH; under the same conditions, the concentration of arginine hydrochloride has little influence on the polymer. In summary, the content of the polymers and monomers in each group (pH 5.0-6.0) of the histidine buffer system is not changed greatly, while the acetic acid system and the citric acid system are influenced greatly by pH, so the histidine system is selected in the following experiment.

The result of accelerated experiment CEX-HPLC shows that: the acidic peak content of the sample in the histidine buffer system is lower than that of an acetic acid system and a citric acid system (except when the pH value is 6.0), so that a 10mmol/L histidine buffer system is selected. The temperature is accelerated for 30 days at 40 ℃, the acid peaks of the histidine system pH5.0 and pH5.5 groups are not increased greatly, the acid peak is obviously increased when the pH value is 6.0, and the acid peaks are obviously lower than the pH value of 5.0 when the pH value is 5.5 and pH6.0 when the result is examined for 30 days at 2-8 ℃, so that the histidine system with the pH value of 5.5 is selected in the experiment.

The viscosity of each group was measured and the results are shown in Table 8.

TABLE 8 humanized antibody hzD01Hm5Lm4 initial sample viscosity test results

The result shows that when the concentration of the arginine hydrochloride is 20-50mmol/L, the corresponding viscosity is the lowest when the concentration of the arginine hydrochloride is 30mmol/L and the pH value is the same, and the concentration of the protein has a significant positive correlation effect on the viscosity, so that the concentration of the protein needs to be less than 150mg/ml to meet the follow-up clinical use.

Example 12Liquid formulation prescription screening of anti-human TSLP humanized antibody hzD01Hm5Lm4 (third round of experiment)

On the basis of the second round of experiment, a buffer system of 30mmol/L arginine hydrochloride, 10mmol/L histidine and pH5.5 was selected. According to the previous experimental data, under the same conditions, the viscosity is 6.30cp at a protein concentration of 120mg/ml and 9.47cp at a protein concentration of 150mg/ml, so that the protein concentration of 120mg/ml and 150mg/ml is considered to be selected in this round. Since humanized antibody hzD01Hm5Lm4 was administered by subcutaneous injection, the volume of administration was limited, and the concentration required was as high as possible, and since the dilution ability of the subcutaneous body fluid was limited, it was necessary to ensure that the injection was maintained under isotonic conditions, and at the same time, the viscosity was required to be 20cp or less to reduce the pain of injection. The range of the osmolality of normal human blood is 285-310 mosm, and various preparations are required to be isotonic or nearly isotonic with blood in principle in pharmacopoeia. Therefore, the influence of different concentrations of sucrose on osmotic pressure is investigated in the experiment, and the most appropriate sucrose concentration is selected. Meanwhile, in order to reduce the aggregation of the high polymer, different amounts of Tween 80 (0.01% -0.03%) are considered to be added in the experiment of the round.

Buffer solution:

buffer A: 10mmol/L histidine +30mmol/L arginine +190mmol/L sucrose +0.01% Tween 80, pH5.5

Buffer B: 10mmol/L histidine +30mmol/L arginine +185mmol/L sucrose +0.03% Tween 80, pH5.5

Buffer C: 10mmol/L histidine +30mmol/L arginine +185mmol/L sucrose +0.01% Tween 80, pH5.5

Preparation of a sample: 30ml of the protein stock solution obtained in example 9 was taken into a sample cell of an ultrafiltration system, and substitution was performed by a fed-batch method (the substitution solution was 10mmol/L histidine +30mmol/L arginine, pH 5.5), the flow rate was controlled at 120LMH, and the completion of buffer substitution was determined by measuring the pH at the permeation end and the conductance at the permeation end after 7-8CV substitution. After the replacement is finished, the flow rate is kept unchanged to concentrate the sample, finally, the concentration of all component samples is over 130mg/ml, sucrose and Tween are added according to requirements, the corresponding concentration of Buffer A-B is 130mg/ml, and the corresponding concentration of Buffer C is 120 mg/ml.

The experimental process comprises the following steps: all samples were placed in a stability box at 40 ℃ for 30 days to speed up the experiment and tested for SEC-HPLC, CEX-HPLC, osmolality, viscosity. The results are shown in fig. 19 to 22 and table 9.

As can be seen from the accelerated SEC-HPLC and CEX-HPLC results, when the protein concentration is 120-130mg/mL, the SEC and CEX purity changes insignificantly; both osmotic pressure and viscosity meet the use requirements, so the final prescription conditions are: 10mmol/L histidine, 30mmol/L arginine hydrochloride, 185-190mmol/L sucrose, 0.01-0.03% Tween 80, pH 5.5.

The above description of the specific embodiments of the present invention is not intended to limit the present invention, and those skilled in the art may make various changes and modifications according to the present invention without departing from the spirit of the present invention, which is defined by the scope of the appended claims.

Sequence listing

<110> Miwei (Shanghai) Biotech Co., Ltd

Jiangsu Maiweikang New drug research and development Limited company

<120> liquid composition comprising monoclonal antibody against thymic stromal lymphopoietin

<130> LC21110016

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<170> PatentIn version 3.3

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Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asn Tyr

20 25 30

Phe Ile Asp Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile

35 40 45

Gly Val Ile Asp Ala Phe Ser Gly Gly Ser Asn Phe Asn Glu Lys Phe

50 55 60

Lys Gly Arg Val Thr Met Thr Arg Asp Thr Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Phe Cys

85 90 95

Ala Arg Glu Ser Glu Val Gly Glu Gly Phe Ala Tyr Trp Gly Gln Gly

100 105 110

Thr Leu Val Thr Val Ser Ser

115

<210> 6

<211> 107

<212> PRT

<213> Artificial sequence (artificial sequence)

<220>

<223> VL

<400> 6

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Ser Cys Arg Ala Ser Gln Asp Ile Ser Asn Tyr

20 25 30

Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Tyr Thr Ser Thr Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Ile Ala Thr Tyr Phe Cys Gln Gln Gly Asn Thr Leu Pro Tyr

85 90 95

Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 7

<211> 7

<212> PRT

<213> Artificial sequence (artificial sequence)

<220>

<223> H-CDR1

<400> 7

Gly Tyr Thr Phe Thr Asn Tyr

1 5

<210> 8

<211> 6

<212> PRT

<213> Artificial sequence (artificial sequence)

<220>

<223> H-CDR2

<400> 8

Asn Pro Gly Ser Gly Gly

1 5

<210> 9

<211> 10

<212> PRT

<213> Artificial sequence (artificial sequence)

<220>

<223> H-CDR3

<400> 9

Glu Ser Glu Val Gly Glu Gly Phe Ala Tyr

1 5 10

<210> 10

<211> 11

<212> PRT

<213> Artificial sequence (artificial sequence)

<220>

<223> L-CDR1

<400> 10

Arg Ala Ser Gln Asp Ile Ser Asn Tyr Leu Asn

1 5 10

<210> 11

<211> 7

<212> PRT

<213> Artificial sequence (artificial sequence)

<220>

<223> L-CDR2

<400> 11

Tyr Thr Ser Thr Leu His Ser

1 5

<210> 12

<211> 9

<212> PRT

<213> Artificial sequence (artificial sequence)

<220>

<223> L-CDR3

<400> 12

Gln Gln Phe Asp Thr Leu Pro Tyr Pro

1 5

<210> 13

<211> 5

<212> PRT

<213> Artificial sequence (artificial sequence)

<220>

<223> H-CDR1

<400> 13

Asn Tyr Phe Ile Asp

1 5

<210> 14

<211> 17

<212> PRT

<213> Artificial sequence (artificial sequence)

<220>

<223> H-CDR2

<400> 14

Val Ile Asn Pro Gly Ser Gly Gly Thr Asn Phe Asn Glu Lys Phe Lys

1 5 10 15

Gly

<210> 15

<211> 10

<212> PRT

<213> Artificial sequence (artificial sequence)

<220>

<223> H-CDR1

<400> 15

Gly Tyr Thr Phe Thr Asn Tyr Phe Ile Asp

1 5 10

<210> 16

<211> 10

<212> PRT

<213> Artificial sequence (artificial sequence)

<220>

<223> H-CDR2

<400> 16

Val Ile Asn Pro Gly Ser Gly Gly Thr Asn

1 5 10

<210> 17

<211> 6

<212> PRT

<213> Artificial sequence (artificial sequence)

<220>

<223> H-CDR2

<400> 17

Asp Ala Phe Ser Gly Gly

1 5

<210> 18

<211> 9

<212> PRT

<213> Artificial sequence (artificial sequence)

<220>

<223> L-CDR3

<400> 18

Gln Gln Gly Asn Thr Leu Pro Tyr Thr

1 5

<210> 19

<211> 17

<212> PRT

<213> Artificial sequence (artificial sequence)

<220>

<223> H-CDR2

<400> 19

Val Ile Asp Ala Phe Ser Gly Gly Ser Asn Phe Asn Glu Lys Phe Lys

1 5 10 15

Gly

<210> 20

<211> 10

<212> PRT

<213> Artificial sequence (artificial sequence)

<220>

<223> H-CDR2

<400> 20

Val Ile Asp Ala Phe Ser Gly Gly Ser Asn

1 5 10

<210> 21

<211> 119

<212> PRT

<213> Artificial sequence (artificial sequence)

<220>

<223> VH

<400> 21

Gln Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Val Arg Pro Gly Thr

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Ala Phe Thr Asn Tyr

20 25 30

Phe Ile Asp Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile

35 40 45

Gly Val Ile Asn Pro Gly Ser Gly Gly Thr Asn Phe Asn Glu Lys Phe

50 55 60

Lys Gly Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Ser Thr Ala Tyr

65 70 75 80

Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Phe Cys

85 90 95

Ala Arg Glu Ser Glu Val Gly Glu Gly Phe Ala Tyr Trp Gly Gln Gly

100 105 110

Thr Leu Val Thr Val Ser Ala

115

<210> 22

<211> 107

<212> PRT

<213> Artificial sequence (artificial sequence)

<220>

<223> VL

<400> 22

Asp Ile Gln Met Thr Gln Thr Ser Ser Ser Leu Ser Ala Ser Leu Gly

1 5 10 15

Asp Arg Val Thr Ile Ser Cys Arg Ala Ser Gln Asp Ile Ser Asn Tyr

20 25 30

Leu Asn Trp Tyr Gln Gln Lys Pro Asp Gly Thr Val Lys Leu Leu Ile

35 40 45

Tyr Tyr Thr Ser Thr Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Tyr Ser Leu Thr Ile Ser Asn Leu Glu Gln

65 70 75 80

Glu Asp Ile Ala Thr Tyr Phe Cys Gln Gln Gly Asn Thr Leu Pro Tyr

85 90 95

Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 23

<211> 7

<212> PRT

<213> Artificial sequence (artificial sequence)

<220>

<223> H-CDR1

<400> 23

Gly Tyr Ala Phe Thr Asn Tyr

1 5

<210> 24

<211> 10

<212> PRT

<213> Artificial sequence (artificial sequence)

<220>

<223> H-CDR1

<400> 24

Gly Tyr Ala Phe Thr Asn Tyr Phe Ile Asp

1 5 10

<210> 25

<211> 326

<212> PRT

<213> Artificial sequence (artificial sequence)

<220>

<223> CH

<400> 25

Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg

1 5 10 15

Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr

20 25 30

Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser

35 40 45

Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser

50 55 60

Leu Ser Ser Val Val Thr Val Pro Ser Ser Asn Phe Gly Thr Gln Thr

65 70 75 80

Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys

85 90 95

Thr Val Glu Arg Lys Cys Cys Val Glu Cys Pro Pro Cys Pro Ala Pro

100 105 110

Pro Val Ala Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp

115 120 125

Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp

130 135 140

Val Ser His Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly

145 150 155 160

Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn

165 170 175

Ser Thr Phe Arg Val Val Ser Val Leu Thr Val Val His Gln Asp Trp

180 185 190

Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro

195 200 205

Ala Pro Ile Glu Lys Thr Ile Ser Lys Thr Lys Gly Gln Pro Arg Glu

210 215 220

Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn

225 230 235 240

Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile

245 250 255

Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr

260 265 270

Thr Pro Pro Met Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys

275 280 285

Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys

290 295 300

Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu

305 310 315 320

Ser Leu Ser Pro Gly Lys

325

<210> 26

<211> 107

<212> PRT

<213> Artificial sequence (artificial sequence)

<220>

<223> CL

<400> 26

Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu

1 5 10 15

Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe

20 25 30

Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln

35 40 45

Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser

50 55 60

Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu

65 70 75 80

Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser

85 90 95

Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

100 105

Claims (28)

1. A liquid composition comprising an antibody or fragment thereof against thymic stromal lymphopoietin, said antibody or fragment thereof having and comprising in the heavy chain variable region and the light chain variable region any one selected from the group consisting of:

(1) the heavy chain CDR1, the heavy chain CDR2 and the heavy chain CDR3 shown in sequence in SEQ ID NO 7, 17 and 9; and, the light chain CDR1, light chain CDR2, light chain CDR3 shown in sequence in SEQ ID NOs 10, 11, 18;

(2) 13, 19, 9, respectively, heavy chain CDR1, heavy chain CDR2, heavy chain CDR 3; and, the light chain CDR1, light chain CDR2, light chain CDR3 shown in sequence in SEQ ID NOs 10, 11, 18;

(3) heavy chain CDR1, heavy chain CDR2, heavy chain CDR3 shown in SEQ ID NO. 15, 20, 9 in sequence; and, the light chain CDR1, light chain CDR2, light chain CDR3 shown in sequence in SEQ ID NOs 10, 11, 18; and

(4) heavy chain CDR1, heavy chain CDR2, heavy chain CDR3 shown in SEQ ID NO. 15, 19, 9 in sequence; and, the light chain CDR1, light chain CDR2, light chain CDR3 shown in sequence in SEQ ID NOs 10, 11, 18.

2. The liquid composition of claim 1, wherein the antibody is a monoclonal antibody.

3. The liquid composition of claim 2, wherein the antibody is a humanized monoclonal antibody.

4. The liquid composition of claim 3, wherein the heavy chain variable region of the antibody or fragment thereof comprises the amino acid sequence set forth in SEQ ID NO. 5 and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO. 6.

5. The liquid composition of claim 4, wherein the fragment is an scFv, a dsFv, (dsFv) of the antibody₂、Fab、Fab'、F(ab')₂Or Fv fragment form.

6. The liquid composition of claim 4, wherein the liquid composition comprises a humanized monoclonal antibody and the monoclonal antibody comprises a heavy chain constant region of an IgG, IgA, IgM, IgD or IgE and/or a light chain constant region of a kappa or lambda type.

7. The liquid composition of claim 6, wherein the monoclonal antibody has a heavy chain constant region of the IgG1 or IgG2 type and a light chain constant region of the kappa type.

8. The liquid composition of claim 7, wherein the heavy chain constant region of the monoclonal antibody comprises the amino acid sequence set forth in SEQ ID NO. 25 and the light chain constant region comprises the amino acid sequence set forth in SEQ ID NO. 26.

9. The liquid composition according to claim 8, characterized in that it is in the form of a solution, emulsion or suspension in water.

10. The liquid composition of claim 9, wherein the liquid composition is a parenterally administered formulation.

11. The liquid composition of claim 10, wherein the liquid composition is a formulation for administration by injection.

12. The liquid composition of claim 11, wherein the liquid composition is a subcutaneous injection formulation.

13. The liquid composition of claim 12, wherein the liquid composition has a pH of 5.0 to 6.0.

14. The liquid composition of claim 13, wherein the liquid composition has a pH of 5.5 to 6.0.

15. The liquid composition of claim 14, wherein the liquid composition comprises the monoclonal antibody at a concentration of 100-150 mg/mL.

16. The liquid composition of claim 15, further comprising histidine at a concentration of 10-20 mmol/L.

17. The liquid composition of claim 16, further comprising arginine hydrochloride at a concentration of 20 to 50 mmol/L.

18. The liquid composition as claimed in claim 17, further comprising sucrose at a concentration of 180-200 mmol/L.

19. The liquid composition of claim 18, further comprising tween 80 at a concentration of 0.01% to 0.05%.

20. The liquid composition according to any one of claims 1 to 19, characterized in that it comprises: the antibody at a concentration of 120-130 mg/mL; 10mmol/L histidine; arginine hydrochloride of 30 mmol/L; 185-190mmol/L sucrose; 0.01-0.03% tween; and has a pH of 5.5.

21. Use of the liquid composition of any one of claims 1 to 20 in the manufacture of a medicament for treating a disease or disorder associated with TSLP signaling and/or TSLP overexpression.

22. The use according to claim 21, wherein the disease or condition is an inflammatory disease or a tumor.

23. Use according to claim 22, characterized in that said inflammatory disease is selected from asthma, allergic dermatitis, chronic obstructive pulmonary disease and allergic rhinitis.

24. The use according to claim 23, wherein the asthma comprises Th 2-type and non-Th 2-type asthma.

25. Use according to claim 22, characterized in that said tumors are selected from hodgkin's lymphoma, breast cancer, pancreatic cancer, melanoma and lung cancer.

26. A container comprising the liquid composition of any one of claims 1 to 20.

27. The container of claim 26, wherein the container is a syringe.

28. A kit comprising the container of claim 26 or 27.

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