WO2023001257A1 - 抗肿瘤抑制素2的抗体以及包含其的液体组合物 - Google Patents
抗肿瘤抑制素2的抗体以及包含其的液体组合物 Download PDFInfo
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Definitions
- the invention relates to the field of biopharmaceutical preparations, in particular, the invention relates to an anti-tumor inhibin 2 (suppression of tumorigenicity 2, ST2) antibody and a stable liquid pharmaceutical preparation containing it.
- an anti-tumor inhibin 2 suppression of tumorigenicity 2, ST2
- Interleukin 33 (IL-33, also known as IL-1F11), as an alarm molecule, is mainly produced by Th2 cells, mast cells, endothelial cells and innate lymphocytes. Interleukin-33 acts on its receptors (IL-33R, ST2, ST2L, T1, Fit-1, DER-4, IL-1R4), and exerts its functions in vivo and in vitro by interacting with its receptors.
- IL-33 binds to ST2 on the cell membrane and recombinant human interleukin-1 receptor accessory protein (Recombinant Human Interleukin-1 Receptor Accessory Protein, IL-1RAcP) to form a heterodimer, transduces the signal into the cell, and recruits downstream Signaling molecules myeloid differentiation factor 88, IL-1 receptor-associated kinase, and tumor necrosis factor receptor-associated factor and activate downstream mitogen-activated protein kinase (MAPKK), which in turn activates protein-1 through c-jun N-terminal kinase ; Tumor necrosis factor receptor-associated factor 6 also activates the nuclear factor- ⁇ B (NF- ⁇ B) kinase inhibitor complex, leading to the release of NF- ⁇ B from the complex, which ultimately leads to the Th2 cytokines IL-4, IL-5 and IL-13 produces and exerts biological functions.
- This pathway is closely related to allergic inflammation, chronic obstructive pulmonary disease, asthma and other diseases.
- inhibitors targeting the above-mentioned pathways mainly include: IL33 antibodies (such as MEDI3506, ANB020, REGN3500, MT-2990, LY-3375880, PF-06817024) and ST2 antibodies (such as CNTO7160, AMG-282), all of which are in clinical 1
- the targeted indications include allergic rhinitis, atopic dermatitis, chronic obstructive pulmonary disease, asthma, etc.
- antibody molecules are biomacromolecules with complex structures.
- physical changes such as aggregation, denaturation, and precipitation, and chemical changes such as isomerization, deamidation, and oxidation will occur, especially under high concentration conditions. The changes are more pronounced. These changes will affect the safety and effectiveness of the product.
- the antibody as an inhibitor of the above-mentioned pathway is made into a preparation. It is necessary to ensure that the preparation is stable so that the antibody still has the biological activity required for treatment before it is used in a patient.
- high-concentration antibody preparations in addition to the solubility of the antibody protein itself, it is also necessary to pay attention to the viscosity of the antibody preparation, the storage stability of the finished preparation, and the stability of the injection site.
- the technical problem to be solved by the present invention is to obtain a new antibody with high affinity and high functional activity to human ST2 through hybridoma screening and humanization technology; to use the new antibody as an active ingredient of a drug to obtain a high-concentration, stable liquid pharmaceutical preparations.
- the object of the present invention is to provide an anti-tumor inhibin 2 (ST2) antibody.
- Another object of the present invention is to provide a liquid composition comprising an anti-tumor inhibin 2 (ST2) antibody.
- the present invention provides an anti-tumor inhibin 2 (ST2) antibody or a fragment thereof comprising a heavy chain variable region and a light chain variable region and in the heavy chain variable region and the light chain variable region Contain heavy chain CDR1 (H-CDR1), CDR2 (H-CDR2), CDR3 (H-CDR3) and light chain CDR1 (L-CDR1), CDR2 (L-CDR2), CDR3 (L-CDR3), respectively, wherein:
- H-CDR1 comprises the amino acid sequence shown in SEQ ID NO.8
- H-CDR2 comprises the amino acid sequence shown in SEQ ID NO.9
- H-CDR3 comprises the amino acid sequence shown in SEQ ID NO.10
- L-CDR1 Contains the amino acid sequence shown in SEQ ID NO.11
- L-CDR2 contains the amino acid sequence shown in SEQ ID NO.12
- L-CDR3 contains the amino acid sequence shown in SEQ ID NO.13.
- the anti-ST2 antibody provided by the present invention can be in the form of anti-ST2 monoclonal antibody, scFv, BsFv, dsFv, (dsFv)2, Fab, Fab', F(ab')2 or Fv and other antibody forms.
- the anti-ST2 antibody may be an anti-ST2 murine antibody, chimeric antibody, fully or partially humanized antibody.
- the ST2 is mammalian ST2, preferably primate ST2, more preferably human ST2.
- both the heavy chain variable region (VH) and the light chain variable region (VL) of the anti-ST2 antibody of the present invention or a fragment thereof include the above-mentioned CDRs and a spaced framework region (framework region, FR), each structure
- the domains are arranged in the following manner: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4.
- the heavy chain variable region (VH) of the anti-ST2 antibody or fragment thereof comprises the amino acid sequence shown in SEQ ID NO.7 or a variant thereof
- the light chain variable region (VL) comprises SEQ ID NO.7 The amino acid sequence shown in ID NO.6 or its variant.
- a "variant of an amino acid sequence” means having at least 75% sequence identity (e.g. at least 80%, preferably at least 85%, more preferably at least 90%, further preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or even 99% identity, etc. >75% identity of any percentage of amino acid sequences).
- the heavy chain variable region and the light chain variable region of an anti-ST2 antibody or fragment thereof of the present invention at most 25% of the difference in amino acid sequence resulting from said "at least 75% sequence identity" may exist in the heavy chain In any framework region in the variable region or light chain variable region.
- the differences may result from amino acid deletions, additions or substitutions at any position, where the substitutions may be conservative or non-conservative.
- the ST2 antibody or fragment thereof provided by the present invention further comprises a constant region.
- the anti-ST2 antibody further comprises a human or murine heavy chain constant region (CH) and/or light chain constant region (CL), more preferably a heavy chain selected from IgG, IgA, IgM, IgD or IgE Constant region and/or kappa or lambda type light chain constant region.
- CH human or murine heavy chain constant region
- CL light chain constant region
- the antibody is a monoclonal antibody, preferably a murine, chimeric or humanized monoclonal antibody; more preferably, the heavy chain constant region of the monoclonal antibody is IgG1 or IgG4 subtype, and the light chain The constant region is of the kappa type.
- the antibody is an immunoglobulin, specifically IgA, IgD, IgE, IgG or IgM, such as a human subtype of IgA, IgD, IgE, IgG or IgM, more preferably a human IgG1, IgG2, IgG3 or IgG4 subtype type.
- the anti-ST2 antibody or fragment thereof comprises a heavy chain constant region, and the heavy chain constant region comprises the amino acid sequence shown in SEQ ID NO.14 or a variant thereof.
- the anti-ST2 antibody or fragment thereof comprises a light chain constant region, and the light chain constant region comprises an amino acid sequence as shown in SEQ ID NO.16 or a variant thereof.
- a "variant of an amino acid sequence" means having at least 75% sequence identity to said amino acid sequence.
- the anti-ST2 antibody provided by the present invention comprises a heavy chain and/or a light chain, preferably two heavy chains and/or two light chains.
- the anti-ST2 antibody is an isolated monoclonal antibody of human IgG4/ ⁇ subtype, which contains two identical light chains and two identical heavy chains, and the two can be separated by a disulfide bond. connect.
- the heavy chain includes the amino acid sequence shown in SEQ ID NO.17
- the light chain includes the amino acid sequence shown in SEQ ID NO.18.
- the present invention also provides a nucleic acid molecule, which comprises encoding the light chain variable region, heavy chain variable region, heavy chain variable region contained in the antibody of the present invention or its fragment. Nucleotide sequence of chain or light chain.
- the invention provides a vector comprising a nucleic acid molecule of the invention.
- the vector can be a eukaryotic expression vector, a prokaryotic expression vector, an artificial chromosome, a phage vector, and the like.
- the vector or nucleic acid molecule of the present invention can be used to transform or transfect host cells or enter host cells in any way for the purpose of storing or expressing antibodies and the like.
- the present invention provides a host cell comprising, or transformed or transfected with, a nucleic acid molecule and/or vector of the present invention.
- the host cell can be any prokaryotic or eukaryotic cell, such as a bacterial or insect, fungal, plant or animal cell.
- the antibodies or fragments thereof, nucleic acid molecules, vectors and/or host cells provided by the present invention can be obtained by using any conventional techniques known in the art.
- the antibodies or fragments thereof, nucleic acid molecules, vectors and/or host cells can be contained in compositions (such as pharmaceutical compositions), more particularly in pharmaceutical preparations, so as to be used for various purposes according to actual needs.
- the present invention also provides a composition comprising the antibody or fragment thereof, nucleic acid molecule, vector and/or host cell of the present invention.
- the composition is a pharmaceutical composition, which optionally further comprises pharmaceutically acceptable excipients.
- the invention also provides a liquid antibody preparation comprising an anti-tumor inhibin 2 (ST2) antibody or a fragment thereof, the antibody as defined above.
- ST2 anti-tumor inhibin 2
- the liquid antibody preparation provided by the present invention comprises the antibody, a buffer system, a protective agent and a surfactant, and may optionally contain an antioxidant and/or a functional adjuvant.
- the liquid antibody preparations provided by the invention are in the form of solutions, emulsions or suspensions in water. More preferably, the liquid antibody formulation is a liquid formulation for parenteral administration. Further preferably, the liquid antibody preparation is an injection, preferably for subcutaneous injection or intravenous injection; or an infusion, such as for intravenous infusion. According to a specific embodiment of the present invention, the liquid antibody preparation is a subcutaneous injection preparation.
- the buffer system is a pharmaceutical buffer system as a solvent for the antibody.
- the pharmaceutical buffer system is citrate buffer, histidine buffer, phosphate buffer or acetate buffer; preferably, the citrate buffer contains citric acid/sodium citrate mixture; the composition
- the acid buffer contains a histidine/histidine hydrochloride mixture;
- the phosphate buffer contains a sodium dihydrogen phosphate/disodium hydrogen phosphate mixture; and the acetate buffer contains an acetic acid/sodium acetate mixture.
- the protective agent is sugar or alcohol, preferably one or more selected from sucrose, trehalose, sorbitol and mannitol.
- the surfactant is, for example, polysorbate.
- the antioxidant is an amino acid, such as methionine.
- the functional adjuvant such as hyaluronidase.
- the pH of the liquid antibody preparation is 5.5-6.5, preferably 5.5-6.1, more preferably 5.5-6.0.
- said liquid antibody formulation comprises said anti-ST2 antibody or fragment thereof at a concentration of 30-180 mg/mL, preferably 100-150 mg/mL, more preferably 140-150 mg/mL.
- the liquid antibody formulation comprises a histidine buffer as a buffer system.
- the liquid antibody preparation comprises a histidine buffer at a concentration of 5-25 mM, preferably 20-25 mM, and a pH buffer range of 5.5-6.5, preferably 5.5-6.1, more preferably 5.5-6.0.
- the liquid antibody formulation comprises sucrose as a protective agent.
- said liquid antibody formulation comprises sucrose at a concentration of 3%-9% (w/v), preferably 5-7% (w/v).
- the liquid antibody formulation comprises methionine as an antioxidant.
- said liquid antibody formulation comprises methionine at a concentration of 0-20 mM, preferably 5-10 mM.
- the liquid antibody formulation comprises polysorbate 20 or polysorbate 80 as a surfactant, eg, 0.005%-0.1% (w/v) polysorbate 20 or polysorbate 80.
- said liquid antibody preparation comprises polysorbate 20 at a concentration of 0.02%-0.1% (w/v), preferably 0.04%-0.06% (w/v); or, preferably, said liquid antibody preparation comprises Polysorbate 80 at a concentration of 0.005-0.1% (w/v), preferably 0.03%-0.05% (w/v).
- w/v means weight (mg)/volume (L).
- the liquid antibody formulation comprises hyaluronidase as a functional excipient.
- the liquid antibody preparation comprises hyaluronidase with an enzyme activity of 0-12800 units/mL, preferably 0-6400 units/mL.
- the hyaluronidase is a commercially available recombinant hyaluronidase.
- the liquid antibody preparation provided by the invention preferably comprises:
- the pH is 5.5-7.5.
- liquid antibody formulation comprises:
- the pH is 5.5-7.5.
- liquid antibody preparation provided by the present invention comprises:
- liquid antibody preparation provided by the present invention comprises:
- the pH is 5.5-6.0.
- the liquid antibody preparation comprises:
- the liquid antibody preparation comprises:
- the liquid antibody preparation comprises:
- the pH is 5.5-6.0.
- the present invention provides the use of the anti-ST2 antibody or its fragment, nucleic acid molecule, vector, host cell, composition, liquid antibody preparation and/or solid antibody preparation in the preparation of a drug, the drug For preventing, treating and/or improving inflammatory, allergic or autoimmune diseases.
- the present invention provides a method for preventing, treating or improving diseases, the method comprising administering the anti-ST2 antibody or fragment thereof, nucleic acid molecule, vector, host cell, composition to a subject in need thereof , a liquid antibody preparation and/or a solid antibody preparation, the disease is an inflammatory, allergic or autoimmune disease.
- the present invention provides a new anti-tumor inhibin 2 (ST2) antibody
- experiments prove that the antibody can bind to human ST2 with high affinity, block the binding of human IL-33 and human ST2, This effectively prevents the activation of the IL-33/ST2 signaling pathway; in addition, the antibody has no obvious ADCC and CDC effects.
- the present invention further provides a liquid composition containing an anti-ST2 antibody, which can be used as an antibody pharmaceutical preparation for the prevention of diseases related to the activation of the IL-33/ST2 signaling pathway, treatment or improvement. After screening the composition of the buffer system and its pH, protective agent, surfactant and other antibody pharmaceutical preparation components, experiments have proved that the present invention provides a stable liquid preparation containing high-concentration antibody.
- Figure 2 shows the results of the antibody inhibiting the activity of recombinant human IL-33 to promote the production of IL-6 in HUVEC cells.
- Figure 3 shows the results of the antibody inhibiting the activity of recombinant human IL-33 to promote the production of IL-8 in HMC-1 cells.
- Fig. 4 shows the change ratio (Ratio) of skin swelling area of cynomolgus monkeys detected in Example 13, wherein 4A: physiological saline challenge; 4B: histamine challenge; 4C: challenge with Ascaris suum egg extract. *p ⁇ 0.05, **p ⁇ 0.01, ***p ⁇ 0.001, G2-G4 vs G1 (One-way ANOVA/Dunnett's).
- Figure 6 shows the thermal stability results of the samples, where 6A: Tm; 6B: Tagg.
- Figure 7 shows the viscosity of the samples at different concentrations.
- Figure 8 shows the change trend of particles in samples containing polysorbate 20, where 8A: ⁇ 5 ⁇ m; 8B: ⁇ 10 ⁇ m; 8C: ⁇ 25 ⁇ m; 8D: ⁇ 50 ⁇ m.
- Figure 9 shows the change trend of particles in samples containing polysorbate 80, wherein 9A: ⁇ 5 ⁇ m; 9B: ⁇ 10 ⁇ m; 9C: ⁇ 25 ⁇ m; 9D: ⁇ 50 ⁇ m.
- Figure 10 shows the comparison of particles ⁇ 10 ⁇ m and ⁇ 25 ⁇ m in samples containing different proportions of polysorbate 20 and polysorbate 80, where 10A: after 28 days of high temperature storage; 10B: after shaking for 5 days; 10C: after 10 freeze-thaws .
- Figure 11 shows the change trend of particles in samples containing different proportions of polysorbate 80 in prefilled needles, where 11A: ⁇ 5 ⁇ m; 11B: ⁇ 10 ⁇ m; 11C: ⁇ 25 ⁇ m; 11D: ⁇ 50 ⁇ m.
- Figure 12 shows the trend of SEC aggregates in the samples containing different proportions of polysorbate 80 in the prefilled needles.
- Human ST2-his Human ST2 with 6 histidine tags fused at the C-terminus
- Human IL33 NP_254274.1 (Ser112-Thr270)
- Human IL33-his Human IL33 with 6 histidine tags fused to the C-terminus
- Control antibody CNTO7160 the heavy chain is shown in SEQ ID NO.19, and the light chain is shown in SEQ ID NO.20.
- mice were immunized by conventional immunization procedures, and then the serum of the immunized mice was tested by ELISA with the antigen, and the spleens of mice with positive serum reactions were obtained.
- the antigen was used for panning and culturing of B cells.
- the culture supernatant of B cells was taken, and ELISA screening was performed again against the antigen Human ST2-his; in addition, KU812 NF- ⁇ B reporter gene screening was performed.
- the inhibition rate of each clone was calculated according to the values of the negative control and the positive control, and the results of some B cell clones are shown in Figure 1.
- the mRNA of B cells is extracted, reverse-transcribed into cDNA, and the antibody sequence is amplified by PCR.
- the light and heavy chains from the same B cell clone were transfected into CHO-K1 cells, and the mouse antibody was obtained from the supernatant after the expression was completed. Take the obtained mouse antibody and carry out the screening of binding human ST2 activity, the screening of inhibiting IL33 binding human ST2 activity, the screening of inhibiting IL33 activation of KU812 NF- ⁇ B reporter gene activity, affinity determination and in vitro pharmacological research, ST2 binding experiments of different species, etc., and Compared with the control antibody CNTO7160.
- the heavy and light chain variable region sequences of the 5886-156H+L murine antibody were compared to the human germline sequence using a blast search of the IMGT database. Redundant genes as well as those with unpaired cysteines were removed from the set of human germline genes. The remaining closest matching human germline genes were selected as recipient human frameworks in both framework and CDR regions. FR-4 was selected based on sequence similarity to the IGHJ/IGJK germline genes.
- Table 1 is the mouse antibody of 5886-156H+L and its humanized sequence form, in which the HZ0 version represents only CDR transplantation, and the HZ1 version introduces a back mutation. The specific sequence after humanization is shown in Table 1, wherein the corresponding CDRs are shown underlined (according to the definition of enhanced Chothia/AbM CDR).
- KU812 cells in the logarithmic growth phase were centrifuged, 100,000 cells/well, 50 ⁇ L/well were added to the above-mentioned 96-well plate, and incubated for 48 hours.
- the concentration of IL5 in the cell culture supernatant was determined according to the instructions of the Human IL-5 DuoSet ELISA kit.
- Table 4 The results of humanized antibodies inhibiting the activity of IL33 to promote KU812-IL5 production and the relative activity compared with CNTO7160
- Antibody IC50 ( ⁇ g/mL) relative activity maximum inhibition rate 5886-156-H1L0 0.03539 181.12% 98.07% 5886-156-H1L1 0.02893 221.57% 94.95% CNTO7160 the the 97.55%
- the anti-human ST2 antibody was diluted to 2 ⁇ g/mL and captured on the surface of the Protein A chip for 60 s. After antibody capture, human ST2-his in the form of a solution (initial concentration 20 nM, 2-fold serial dilution of 6 concentrations) was injected. Association was monitored for 180 s, dissociation was monitored for 700 s, and regeneration of the sensor surface was achieved by injection of a glycine solution at pH 2.0. Kinetic data were analyzed using a 1:1 binding model.
- Dissociation kinetic affinity experiment between Human ST2 and antibody at pH 7.4 All experiments were performed in HBS-EP (1 ⁇ ) buffer (pH 7.4) at 25°C.
- the anti-human Fc antibody was coupled to the CM5 chip, and the 5886-156-H2L1 antibody diluted to 5.0 ⁇ g/ml was captured in the channel of the CM5 chip with the anti-human Fc antibody for 30 seconds.
- different concentrations of Human ST2 antigen were injected (initial concentration 16nM, 2-fold ratio dilution to 7 concentrations).
- the monitoring binding time was 180s and the dissociation time was 700s.
- the chip was regenerated using glycine at pH 1.5. Kinetic data were analyzed using a 1:1 binding model. The results are shown in Table 6.
- the range of 5886-156-H2L1 blocking IL-33 and ST2 binding ability (IC50) was determined to be 869.6-994.9 ng/mL.
- HUVECs expressing ST2 in the membrane were used as target cells, and the blocking activity of 5886-156-H2L1 antibody was evaluated indirectly by detecting the production of IL-6. Specifically, 10,000 HUVEC cells/well were incubated in 100 ⁇ L of the system at 37° C. and 5% CO 2 for 18-24 hours. Dilute human IL33-his to 10ng/mL with medium, 5886-156-H2L1 to 400 ⁇ g/mL with medium, 4-fold dilution, a total of 11 concentrations, mix the two 1:1 and add 100 ⁇ L/well to Incubate in the above 96-well plate at 37° C. and 5% CO 2 for 18-24 hours.
- Antibody preparation composition screening was performed using the antibody 5886-156-H2L1 in section (1) above. Among them, the following detection methods are used:
- the main steps are as follows: dilute the reference product and the test product to 2mg/ml with the sample diluent, and add the diluted reference product and the test product to the CpB enzyme working solution respectively, and the mass ratio of the protein to the CpB enzyme is 40: 1. After mixing by vortexing, place at 37°C and incubate for 30 minutes. Vortex and mix all processed samples, centrifuge at 10000-14000g for 3-5min, take the supernatant and fill it into a sample bottle.
- Test items include UNcle, SEC, CEX.
- Table 15 and Table 16 show that among different protective agents, there is no significant difference in the decrease rate of SEC purity and the increase rate of CEX main peak, so sucrose commonly used in biopharmaceuticals was selected as the additive.
- sucrose was selected as the protective agent
- the effects of different concentrations of sucrose on the 5886-156-H2L1 protein were investigated with reference to the above experimental operation. The results are shown in Table 17 and Table 18.
- the 5886-156-H2L1 sample was replaced with 20mM histidine buffer containing different concentrations of methionine, the concentration was 140mg/mL (pH5.8 ⁇ 0.3), and the oxidation analysis was carried out after 30 days at high temperature or 14 days under light. The results are shown in Table 21.
- the 5886-156-H2L1 antibody is relatively stable.
- the stability of the 5886-156-H2L1 sample containing hyaluronidase was investigated by high temperature stability test, freeze-thaw stability test, vibration stability test and light test.
- the 5886-156-H2L1 protein used in the experiment was a three-step purified sample, which was formulated into a prescription: antibody 140mg/ml, 25mM histidine buffer, 7% sucrose, 10mM methionine, 0.03% polysorbate 80, and added to the sample Add hyaluronidase in the sample so that the hyaluronidase enzyme activity in the sample is about 5500 units/ml (recombinant hyaluronidase, purchased from Shanghai Baoji Biology), after sterilizing and filtering, it is divided into vials, and The detection of monomer content (SEC), charge variant main peak content (CEX) and insoluble particles (photoresistance method) is performed, and the results are shown in Table 28.
- SEC monomer content
- CEX
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Abstract
一种抗肿瘤抑制素2(suppression of tumorigenicity 2,ST2)抗体或其片段。该抗ST2抗体或其片段可阻断IL-33与ST2蛋白的结合,抑制其下游信号通路,从而在与IL-33/ST2信号通路激活相关的疾病中发挥治疗作用。另外还提供包含所述抗ST2抗体或其片段的稳定的液体抗体制剂。
Description
相关申请的交叉引用
本专利申请要求于2021年7月21日提交的申请号为CN202110825833.1的中国发明专利申请的优先权权益,在此将其全部内容引入作为参考。
本发明涉及生物药物制剂领域,具体而言,本发明涉及一种抗肿瘤抑制素2(suppression of tumorigenicity 2,ST2)抗体以及包含其的稳定的液体药物制剂。
白介素33(IL-33,又名IL-1F11)作为一种警报素分子,主要由Th2细胞、肥大细胞、内皮细胞和先天性的淋巴细胞产生。白介素33作用于其受体(IL-33R、ST2、ST2L、T1、Fit-1、DER-4、IL-1R4),通过与其受体相互作用在体内外发挥功能。例如,IL-33通过结合到胞膜上ST2和重组人白介素1受体辅助蛋白(Recombinant Human Interleukin-1Receptor Accessory Protein,IL-1RAcP)组成异二聚体,将信号转导至胞内,募集下游信号分子髓样分化因子88、IL-1受体相关激酶和肿瘤坏死因子受体相关因子并激活下游丝裂原活化蛋白激酶(MAPKK),MAPKK反过来通过c-jun N端激酶激活蛋白-1;肿瘤坏死因子受体相关因子6还能激活核因子-κB(NF-κB)激酶抑制剂复合物,导致NF-κB从复合物中释放,最终导致Th2细胞因子IL-4、IL-5和IL-13产生并发挥生物学功能。该通路与过敏性炎症、慢性阻塞性肺疾病、哮喘等疾病息息相关。
目前针对上述通路的抑制剂主要包括:IL33抗体(如MEDI3506、ANB020、REGN3500、MT-2990、LY-3375880、PF-06817024)和ST2抗体(如CNTO7160、AMG-282),这些抗体均处于临床1期和2期,针对的适应症包括过敏性鼻炎、特应性皮炎、慢性阻塞性肺病、哮喘等。
但是,抗体分子为生物大分子,结构复杂,在生产和贮存过程中会发生聚集、变性、沉淀等物理变化及异构化、脱酰胺和氧化等化学变化,尤其在 高浓度条件下,此类变化更明显。这些改变会影响产品的安全性及有效性,作为上述通路的抑制剂的抗体在被制成制剂时也是如此,需要确保制剂稳定从而保证抗体用于患者体内前仍具有治疗所需的生物活性。并且,在开发高浓度的抗体制剂时,除了抗体蛋白本身的溶解度,同时还需要注意抗体制剂的粘度、制剂成品的储存稳定性及注射部位稳定性等方面的问题。
因此,就上述通路抑制剂而言,本领域尚需研发一种以肿瘤抑制素2为靶点的新型抗体以及包含其的稳定的制剂,满足对有效治疗相关疾病且长期稳定的高浓度抗体药物制剂、尤其是皮下注射制剂的需求。
发明内容
本发明要解决的技术问题是,通过杂交瘤筛选和人源化技术,获得对人ST2具有高亲和力和高功能活性的新抗体;将该新抗体作为药物活性成分,获得一种高浓度、稳定的液体药物制剂。
针对上述技术问题,本发明的目的是提供一种抗肿瘤抑制素2(ST2)的抗体。本发明的另一个目的是提供一种包含抗肿瘤抑制素2(ST2)抗体的液体组合物。
本发明提供以下技术方案:
本发明提供一种抗肿瘤抑制素2(ST2)抗体或其片段,所述抗体或其片段包含重链可变区和轻链可变区并且在重链可变区和轻链可变区中分别包含重链CDR1(H-CDR1)、CDR2(H-CDR2)、CDR3(H-CDR3)和轻链CDR1(L-CDR1)、CDR2(L-CDR2)、CDR3(L-CDR3),其中:
H-CDR1包含SEQ ID NO.8所示的氨基酸序列,H-CDR2包含SEQ ID NO.9所示的氨基酸序列,H-CDR3包含SEQ ID NO.10所示的氨基酸序列;和,L-CDR1包含SEQ ID NO.11所示的氨基酸序列,L-CDR2包含SEQ ID NO.12所示的氨基酸序列,L-CDR3包含SEQ ID NO.13所示的氨基酸序列。
本发明提供的所述抗ST2抗体可以为抗ST2的单克隆抗体、scFv、BsFv、dsFv、(dsFv)2、Fab、Fab'、F(ab')2或Fv等抗体形式。或者,所述抗ST2抗体可以为抗ST2的鼠抗体、嵌合抗体、完全或部分人源化抗体。所述ST2为哺乳动物ST2,优选灵长类动物ST2,更优选人ST2。
特别地,本发明的抗ST2抗体或其片段的重链可变区(VH)和轻链可变区(VL)二者均包括上述CDR以及间隔的框架区(framework region,FR), 各个结构域的排列方式为:FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4。
优选地,所述抗ST2抗体或其片段的所述重链可变区(VH)包含SEQ ID NO.7所示的氨基酸序列或其变体,所述轻链可变区(VL)包含SEQ ID NO.6所示的氨基酸序列或其变体。
本发明的上下文中,“氨基酸序列的变体”是指与所述氨基酸序列具有至少75%序列同一性(例如至少80%、优选至少85%、更优选至少90%、进一步优选至少91%、92%、93%、94%、95%、96%、97%、98%或甚至99%同一性等≥75%的任何百分比的同一性)的氨基酸序列。
因此,就本发明抗ST2抗体或其片段的重链可变区和轻链可变区而言,所述“至少75%序列同一性”导致的氨基酸序列的至多25%差异可存在于重链可变区或轻链可变区中的任意框架区中。所述差异可以由任何位置的氨基酸缺失、添加或置换产生,其中置换可以是保守置换或非保守置换。
优选地,本发明提供的所述ST2抗体或其片段还包含恒定区。优选地,所述抗ST2抗体还包含人或鼠的重链恒定区(CH)和/或轻链恒定区(CL),更优选地包含选自IgG、IgA、IgM、IgD或IgE的重链恒定区和/或κ或λ型轻链恒定区。
优选地,所述抗体为单克隆抗体,优选为鼠源、嵌合或人源化的单克隆抗体;更优选地,所述单克隆抗体的重链恒定区为IgG1或IgG4亚型,轻链恒定区为κ型。或者,例如,所述抗体为免疫球蛋白,具体为IgA、IgD、IgE、IgG或IgM,例如IgA、IgD、IgE、IgG或IgM的人亚型,更优选为人IgG1、IgG2、IgG3或IgG4亚型。
根据本发明的具体实施方式,所述抗ST2抗体或其片段包含重链恒定区,所述重链恒定区包含如SEQ ID NO.14所示的氨基酸序列或其变体。和/或,所述抗ST2抗体或其片段包含轻链恒定区,所述轻链恒定区包含如SEQ ID NO.16所示的氨基酸序列或其变体。如上文所限定,“氨基酸序列的变体”是指与所述氨基酸序列具有至少75%序列同一性。
优选地,本发明提供的抗ST2抗体包含重链和/或轻链,优选地包含两条重链和/或两条轻链。根据本发明的具体实施方式,所述抗ST2抗体为分离的人IgG4/κ亚型的单克隆抗体,其包含两条相同的轻链和两条相同的重链,二者可通过二硫键连接。其中,重链包含SEQ ID NO.17所示的氨基酸序列,轻链包含SEQ ID NO.18所示的氨基酸序列。
另一方面,基于本发明的抗ST2抗体或其片段,本发明还提供一种核酸分子,其包含编码本发明的抗体或其片段中包含的轻链可变区、重链可变区、重链或轻链的核苷酸序列。
还一方面,本发明提供一种载体,其包含本发明的核酸分子。所述载体可以为真核表达载体、原核表达载体、人工染色体及噬菌体载体等。
本发明的载体或核酸分子可以用于转化或转染宿主细胞或以任何方式进入宿主细胞内,用于保存或表达抗体等目的。因此,另一方面,本发明提供一种宿主细胞,所述宿主细胞包含本发明的核酸分子和/或载体,或者所述宿主细胞被本发明的核酸分子和/或载体转化或转染。宿主细胞可以是任何原核或真核细胞,例如细菌或昆虫、真菌、植物或动物细胞。
基于本发明的公开内容,本发明提供的抗体或其片段、核酸分子、载体和/或宿主细胞可以通过使用本领域已知的任何常规技术方法获得。所述抗体或其片段、核酸分子、载体和/或宿主细胞可以被包含在组合物(例如药物组合物)中,更特别地被包含在药物制剂中,从而根据实际需要用于各种目的。
因此,在又一方面,本发明还提供一种组合物,所述组合物包含本发明所述的抗体或其片段、核酸分子、载体和/或宿主细胞。优选地,所述组合物为药物组合物,其任选地还包含药学上可接受的辅料。
为了利于本发明提供的抗体作为药物的应用,本发明还提供一种包含抗肿瘤抑制素2(ST2)的抗体或其片段的液体抗体制剂,所述抗体如上文所定义。
本发明提供的液体抗体制剂包含所述抗体、缓冲体系、保护剂和表面活性剂,并可选地包含抗氧化剂和/或功能性辅料。
优选地,本发明提供的液体抗体制剂为在水中的溶液、乳液或悬浮液的形式。更优选地,所述液体抗体制剂为经肠胃外施用的液体制剂。进一步优选地,所述液体抗体制剂为注射剂,优选用于皮下注射或静脉注射;或者为输注剂,例如用于静脉内输注。根据本发明的具体实施方式,所述液体抗体制剂为皮下注射制剂。
其中,所述缓冲体系为药用缓冲体系,作为该抗体的溶剂。优选地,所述药用缓冲体系为柠檬酸缓冲液、组氨酸缓冲液、磷酸缓冲液或醋酸缓冲液;优选地,所述柠檬酸缓冲液包含柠檬酸/柠檬酸钠混合物;所述组氨酸缓冲液包含组氨酸/组氨酸盐酸盐混合物;所述磷酸缓冲液包含磷酸二氢钠/磷酸氢 二钠混合物;所述醋酸缓冲液包含醋酸/醋酸钠混合物。
任选地,所述保护剂为糖或醇,优选为选自蔗糖、海藻糖、山梨醇和甘露醇中的一种或多种。
任选地,所述表面活性剂例如为聚山梨酯。
任选地,所述抗氧化剂为氨基酸,例如蛋氨酸。
任选地,所述功能性辅料例如透明质酸酶。
任选地,所述液体抗体制剂的pH为5.5-6.5,优选5.5-6.1,更优选5.5-6.0。
任选地,所述液体抗体制剂包含浓度为30-180mg/mL、优选100-150mg/mL、更优选140-150mg/mL的所述抗ST2抗体或其片段。
任选地,所述液体抗体制剂包含组氨酸缓冲液作为缓冲体系。优选地,所述液体抗体制剂包含浓度为5-25mM、优选20-25mM的组氨酸缓冲液,pH缓冲范围为5.5-6.5,优选5.5-6.1,更优选5.5-6.0。
任选地,所述液体抗体制剂包含蔗糖作为保护剂。优选地,所述液体抗体制剂包含浓度为3%-9%(w/v)、优选5-7%(w/v)的蔗糖。
任选地,所述液体抗体制剂包含蛋氨酸作为抗氧化剂。优选地,所述液体抗体制剂包含浓度为0-20mM、优选5-10mM的蛋氨酸。
任选地,所述液体抗体制剂包含聚山梨酯20或聚山梨酯80作为表面活性剂,例如0.005%-0.1%(w/v)的聚山梨酯20或聚山梨酯80。优选地,所述液体抗体制剂包含浓度为0.02%-0.1%(w/v)、优选0.04%-0.06%(w/v)的聚山梨酯20;或者,优选地,所述液体抗体制剂包含浓度为0.005-0.1%(w/v)、优选0.03%-0.05%(w/v)的聚山梨酯80。
本发明中,“w/v”是指以重量(mg)/体积(L)计。
任选地,所述液体抗体制剂包含透明质酸酶作为功能性辅料。优选地,所述液体抗体制剂包含酶活为0-12800单位/mL、优选0-6400单位/mL的透明质酸酶。根据本发明的具体实施方式,所述透明质酸酶为可商购得到的重组透明质酸酶。
本发明提供的液体抗体制剂优选地包含:
30-180mg/mL抗ST2抗体或其片段;
5-25mM缓冲体系;
3%-9%(w/v)保护剂;以及
0.005%-0.1%(w/v)表面活性剂,
可选地,0-20mM抗氧化剂和/或0-12800单位/mL功能性辅料,
并且,pH为5.5-7.5。
例如,所述液体抗体制剂包含:
30-180mg/mL抗ST2抗体或其片段;
5-25mM柠檬酸缓冲液、磷酸缓冲液、醋酸缓冲液或组氨酸缓冲液;3%-9%(w/v)蔗糖、海藻糖、山梨醇或甘露醇;以及
0.005%-0.1%(w/v)聚山梨酯20或聚山梨酯80;
可选地,0-20mM蛋氨酸和/或0-12800单位/mL透明质酸酶,
并且,pH为5.5-7.5。
又例如,本发明提供的液体抗体制剂包含:
30-180mg/mL抗ST2抗体或其片段;
5-25mM组氨酸缓冲液;
3%-9%(w/v)蔗糖;以及
0.005%-0.1%(w/v)聚山梨酯20或聚山梨酯80;
可选地,0-20mM蛋氨酸和/或0-12800单位/mL透明质酸酶,
并且pH为5.5-6.0。
又例如,本发明提供的液体抗体制剂包含:
140-150mg/mL抗ST2抗体或其片段;
20-25mM组氨酸缓冲液;
3%-9%(w/v)蔗糖;以及
0.03-0.05%(w/v)聚山梨酯80;
可选地,5-10mM蛋氨酸和/或0-6400单位/mL透明质酸酶,
并且,pH为5.5-6.0。
又例如,所述液体抗体制剂包含:
140-150mg/mL抗ST2抗体或其片段;
20-25mM组氨酸缓冲液;
3%-9%(w/v)蔗糖;以及
0.03%-0.05%(w/v)聚山梨酯80;
可选地,5-10mM蛋氨酸和/或0-6400单位/mL透明质酸酶,
并且,pH为5.5-6.0。
又例如,所述液体抗体制剂包含:
140-150mg/ml抗ST2抗体或其片段;
25mM组氨酸缓冲液;
7%(w/v)蔗糖;以及
0.03%(w/v)聚山梨酯80;
可选地,10mM蛋氨酸和/或5500单位/mL透明质酸酶,
并且,pH为5.5-6.0。
又例如,所述液体抗体制剂包含:
150mg/ml抗ST2抗体或其片段;
20mM组氨酸缓冲液;
6%(w/v)蔗糖;以及
0.04%(w/v)聚山梨酯80;
可选地,10mM蛋氨酸和/或6400单位/mL透明质酸酶,
并且,pH为5.5-6.0。
还一方面,本发明提供一种固体抗体制剂,其通过固化本发明提供的液体抗体制剂而获得,所述固体抗体制剂例如是冻干粉针剂。
本发明还提供上述主题的相关应用。具体而言,再一方面,本发明提供所述抗ST2抗体或其片段、核酸分子、载体、宿主细胞、组合物、液体抗体制剂和/或固体抗体制剂在制备药物中的用途,所述药物用于预防、治疗和/或改善炎性、过敏性或自身免疫性疾病。优选地,所述疾病与IL-33/ST2信号传导通路的激活相关;更优选地,所述疾病为子宫内膜异位、心力衰竭、过敏性皮炎、过敏性或变应性鼻炎、鼻息肉、特应性皮炎、慢性阻塞性肺病、哮喘、肺纤维化、败血症、炎性肠病、系统性红斑狼疮、类风湿性关节炎、全身性硬化症、韦格纳氏肉芽肿或化疗相关性腹泻。
或者,本发明提供一种用于预防、治疗或改善疾病的方法,所述方法包括给有此需要的受试者施用所述抗ST2抗体或其片段、核酸分子、载体、宿主细胞、组合物、液体抗体制剂和/或固体抗体制剂,所述疾病为炎性、过敏性或自身免疫性疾病。优选地,所述疾病与IL-33/ST2信号传导通路的激活相关;更优选地,所述疾病为子宫内膜异位、心力衰竭、过敏性鼻炎、鼻息肉、特应性皮炎、慢性阻塞性肺病、哮喘、肺纤维化、败血症、炎性肠病、系统性红斑狼疮、类风湿性关节炎、全身性硬化症、韦格纳氏肉芽肿或化疗相关性腹泻。任选地,所述受试者为哺乳类动物,优选地,所述受试者为人。
与现有技术相比,本发明提供了一种新的抗肿瘤抑制素2(ST2)抗体,实验证明该抗体能够以高亲和力与人ST2结合,阻断人IL-33与人ST2的结合,由此有效阻止IL-33/ST2信号传导通路的激活;此外,该抗体无明显的ADCC和CDC效应。在提供所述抗体的基础上,本发明进一步提供了一种包含抗ST2抗体的液体组合物,其可以作为抗体药物制剂,用于与IL-33/ST2信号传导通路的激活相关的疾病预防、治疗或改善。经过就缓冲体系组成及其pH、保护剂以及表面活性剂等抗体药物制剂成分的筛选,实验证明,本发明提供了一种含高浓度抗体的稳定的液体制剂。
以下,结合附图来详细说明本发明的实施方案,其中:
图1示出了来自不同编号的小鼠的B细胞培养上清液的NF-κB报告基因活性抑制率。
图2示出了抗体抑制重组人IL-33促HUVEC细胞IL-6生成活性结果。
图3示出了抗体抑制重组人IL-33促HMC-1细胞IL-8生成活性结果。
图4示出了实施例13中检测的食蟹猴皮肤肿胀面积变化比值(Ratio),其中4A:生理盐水激发;4B:组胺激发;4C:猪蛔虫卵提取物激发。*p<0.05,**p<0.01,***p<0.001,G2-G4vs G1(One-way ANOVA/Dunnett’s)。
图5示出了实施例14中检测的食蟹猴疾病相关症状和严重程度,其中X+2A:体重变化;X+2B:腹泻评分;X+2C:活动评分;X+2D:食欲评分;X+2E:严重程度的AUC。*p<0.05,**p<0.01,***p<0.001,G1、G3vs G2(Two-way ANOVA/Dunnett’s)。
图6示出了样品的热稳定性结果,其中6A:Tm;6B:Tagg。
图7示出了样品在不同浓度下的粘度。
图8示出了含聚山梨酯20的样品中微粒变化趋势,其中8A:≥5μm;8B:≥10μm;8C:≥25μm;8D:≥50μm。
图9示出了含聚山梨酯80的样品中微粒变化趋势,其中9A:≥5μm;9B:≥10μm;9C:≥25μm;9D:≥50μm。
图10示出了含不同比例聚山梨酯20和聚山梨酯80的样品中≥10μm和≥25μm的微粒对比,其中10A:高温放置28天后;10B:震荡5天后;10C:冻融10次后。
图11示出了预充针中含不同比例聚山梨酯80的样品中微粒变化趋势,其中11A:≥5μm;11B:≥10μm;11C:≥25μm;11D:≥50μm。
图12示出了预充针中含不同比例聚山梨酯80的样品中SEC聚体变化趋势。
实施发明的最佳方式
以下参照具体的实施例来说明本发明。本领域技术人员能够理解,这些实施例仅用于说明本发明,其不以任何方式限制本发明的范围。
下述实施例中的实验方法,如无特殊说明,均为常规方法。下述实施例中所用的原料、试剂等,如无特殊说明,均为市售购买产品。
(一)人源化抗体的获得
采用了以下试剂:
Human ST2:NP_003847.2(Met1-Phe328)
Human ST2-his:C末端融合了6个组氨酸标签的Human ST2
Human IL33:NP_254274.1(Ser112-Thr270)
Human IL33-his:C末端融合了6个组氨酸标签的Human IL33
human ST2-fc:C末端融合了human IgG1 Fc标签的Human ST2
对照抗体CNTO7160:重链示于SEQ ID NO.19,轻链示于SEQ ID NO.20。
实施例1抗ST2鼠抗的筛选
以Human ST2-his为抗原,通过常规免疫程序免疫小鼠,然后以该抗原对经免疫小鼠的血清进行ELISA检测,获得血清为阳性反应的小鼠的脾脏。用该抗原进行B细胞淘选、培养。
取B细胞的培养上清,再次针对抗原Human ST2-his进行ELISA筛选;此外,进行KU812 NF-κB报告基因法筛选。根据阴性对照和阳性对照的值计算各克隆的抑制率,部分B细胞克隆的结果见图1。
提取B细胞的mRNA,逆转录成cDNA,PCR扩增抗体序列。将来自相同B细胞克隆的轻重链搭配转染CHO-K1细胞,从表达完成后的上清中获取鼠抗。取获得的鼠抗,进行结合human ST2活性筛选、抑制IL33结合human ST2活性筛选、抑制IL33激活KU812 NF-κB报告基因活性筛选、亲 和力测定和体外药理研究、不同种属的ST2结合实验等,并且与对照抗体CNTO7160相比较。
最终确定了选择鼠抗“5886-156H+L”进行人源化。鼠抗“5886-156H+L”来自5886-156细胞克隆,其重链为5886-156H,轻链为5886-156L。
实施例2人源化单克隆抗体的制备
将5886-156H+L鼠抗的重链和轻链可变区序列与人种系序列进行比较,该比较是对IMGT数据库进行的blast搜索。从该组人种系基因中去除冗余基因以及那些具有未配对半胱氨酸的基因。在框架和CDR区两者中选择其余最接近匹配人种系基因作为受体人框架。基于IGHJ/IGJK种系基因的序列相似性选择FR-4。表1是5886-156H+L的鼠抗及其人源化序列形式,其中HZ0版本代表仅CDR移植的,HZ1版本引入了回复突变。人源化后的具体序列见表1,其中以下划线示出了相应的CDR(根据增强Chothia/AbM CDR的定义)。
表1 5886-156重轻链可变区的人源化序列形式
以SEQ ID NO.15所示重链恒定区和SEQ ID NO.16所示轻链恒定区,构建源自于相同鼠抗的人源化轻重链,进行组合并转染CHO-K1细胞,转染24h后加入10μg/mL MSX进行加压筛选,待细胞密度和活力恢复后接种进行Feed-batch表达,表达完成后,从上清获得蛋白并经protein A纯化,定量。
人源化抗体及其重轻链可变区的搭配方式见表2,后缀“ix”表示该抗体为相应的嵌合抗体。
表2 5886-156人源化抗体的轻重链可变区序列搭配
实施例3人源化单克隆抗体的活性表征
(1)人源化抗体抑制IL33结合human ST2活性筛选
将human ST2-fc蛋白包被过夜,将稀释好的human IL33-his与稀释好的抗体1:1混合,按照50μL/孔加入到96孔板中,室温孵育1h;加入组氨酸标签二抗(1:2500),50μL/孔,室温孵育1h;TMB显色10min,2M硫酸100μL/孔终止。结果见表3。
表3人源化抗体抑制IL33结合human ST2的活性以及相较于CNTO7160的相对活性
抗体 | IC 50(μg/mL) | 相对活性 | 最大抑制率 |
5886-156-H1L0 | 1.217 | 103.12% | 89.24% |
5886-156-H1L1 | 1.004 | 125.00% | 88.05% |
CNTO7160 | 92.55% |
(2)人源化抗体抑制IL33促KU812-IL5生成活性筛选
将human IL33-his用培养基稀释到80ng/mL,抗体用培养基稀释到640μg/mL,4倍稀释,共11个浓度,将二者1:1混合后50μL/孔加到96孔板中。取对数生长期的KU812细胞离心,按照100000个/孔,50μL/孔加到上述96孔板中,孵育48h。按照Human IL-5 DuoSet ELISA试剂盒说明书测定细胞培养上清中IL5的浓度。
结果见表4。
表4人源化抗体抑制IL33促KU812-IL5生成活性结果以及相较于CNTO7160的相对活性
抗体 | IC 50(μg/mL) | 相对活性 | 最大抑制率 |
5886-156-H1L0 | 0.03539 | 181.12% | 98.07% |
5886-156-H1L1 | 0.02893 | 221.57% | 94.95% |
CNTO7160 | 97.55% |
(3)人源化抗体亲和力测定
抗体与human ST2-his相互作用实验使用Biacore X100进行。
(3-1)human ST2与抗体在pH7.4条件下解离动力学亲和力实验:实验均在25℃下在HBS-EP(1×)缓冲液(pH7.4)中操作。
抗human ST2抗体稀释至2μg/mL,捕获于Protein A芯片表面,捕获时间60s。抗体捕获之后,注射溶液形式的human ST2-his(起始浓度20nM,2倍梯度稀释6个浓度)。监测缔合180s,监测解离700s,通过注射pH2.0的甘氨酸溶液获得传感器表面的再生。动力学数据使用1:1结合模型分析。
(3-2)human ST2与抗体在pH5.5条件下解离动力学亲和力实验:
参照(6-1)中描述的实验过程进行,区别在于使解离在pH5.5的HBS-EP(1×)缓冲液条件下进行,监测解离600s。
结果见表5。
表5人源化抗体pH7.4和pH5.5条件下的亲和力
*:CN104334582B中的抗体Ab2
(6)5886-156-H1L1人源化抗体的进化
为了进一步减少5886-156-H1L1人源化抗体翻译后修饰和提高人源化程度,对原5886-156-H1L1人源化抗体的重链可变区序列按照氨基酸顺序进行Q1E和R72V突变形成5886-156H-VH-HZ2。以该突变后的重链可变区和5886-156L-VL-HZ1(SEQ ID NO.6)构建人IgG4/κ亚型的单克隆抗体(等电点为7.3,完整分子量为147kDa(含糖链修饰)),抗体命名为5886-156-H2L1,重链和轻链序列如下:
重链(SEQ ID NO.17)
(可变区(粗体):SEQ ID NO.7;H-CDR1/2/3(粗体带下划线):SEQ ID NO.8/9/10;恒定区(斜体带下划线):SEQ ID NO.14)
轻链(SEQ ID NO.18)
(可变区(粗体):SEQ ID NO.6;L-CDR1/2/3(粗体带下划线):SEQ ID NO.11/12/13;恒定区(斜体带下划线):SEQ ID NO.16)
实施例4 5886-156-H2L1与重组人ST2蛋白的结合活性(Biacore法)
该抗体与human ST2相互作用的实验使用Biacore T200进行。
Human ST2与抗体在pH7.4条件下解离动力学亲和力实验:实验均在25℃下在HBS-EP(1×)缓冲液(pH7.4)中操作。将anti-human Fc抗体偶联至CM5芯片中,将稀释至5.0μg/ml的5886-156-H2L1抗体用anti-human Fc抗体捕获于CM5芯片通道中,捕获时间30s。抗体捕获后,注射不同浓度的Human ST2抗原(起始浓度16nM,2倍比稀释7个浓度)。监测结合时间180s,解离时间700s。最后使用pH1.5的甘氨酸对芯片再生。动力学数据使用1:1结合模型分析。结果见表6。
表6 5886-156-H2L1在pH7.4条件下的亲和力
实施例5 5886-156-H2L1抑制IL33促KU812-IL5生成活性筛选
将human IL33-his用培养基稀释到80ng/mL,抗体用培养基稀释到640μg/mL,4倍稀释,共11个浓度,将二者1:1混合后50μL/孔加到96孔板中。取对数生长期的KU812细胞离心,按照100000个/孔,50μL/孔加到上述96孔板中,孵育48h。按照Human IL-5 DuoSet ELISA试剂盒说明书测定细胞培养上清中IL5的浓度。结果见表7。
表7人源化抗体抑制IL33促KU812-IL5生成活性结果以及相较于CNTO7160的相对活性
抗体 | IC 50(μg/mL) | 相对活性 | 最大抑制率 |
5886-156-H2L1 | 0.0343 | 403.79% | 94.16% |
CNTO7160 | 0.1385 | 95.21% |
实施例6 5886-156-H2L1与重组人ST2蛋白的结合活性(ELISA法)
Human ST2-his用包被缓冲液稀释到1μg/mL,按照50μL/孔加入96孔板内,4℃包被过夜;第二天将包被板取出用PBST洗涤3次;再用封闭液室温孵育1h,PBST再洗涤3次;5886-156-H2L1从100ng/mL起始3倍稀释,8个点,按照50μL/孔加入96孔板内;室温孵育1h,PBST洗涤三次,加入羊抗人二抗(1:10000),按照50μL/孔加入96孔板内;室温孵育1h,PBST洗涤三次,按照50μL/孔加TMB到96孔板内,避光显色10min,2M 硫酸100μL/孔终止;在酶标仪上读取OD450值。
检测5886-156-H2L1与人ST2重组蛋白的相对结合活性(EC50)范围为16.34~17.32ng/mL。
实施例7 5886-156-H2L1与ST2结合的种属特异性(ELISA法)
将5886-156-H2L1原液置换入PBS缓冲液内,加入Sulfo-NHS-LC-LC-Biotin试剂,Biotin与5886-156-H2L1分子的摩尔比为10:1。混匀后,室温反应30分钟。再将标记后的溶液超滤至PBS缓冲,去除多余的标记试剂。分装,冻存于超低温冰箱。
将人、食蟹猴、大鼠和小鼠的ST2稀释至1μg/mL,包被至ELISA板上。封闭洗涤后,将Biotin标记后的5886-156-H2L1原液稀释至1μg/mL,后续2倍梯度稀释至0.977ng/mL,共11个梯度。加入封闭好的ELISA板内,室温孵育约2hr,洗涤3次。加入10000倍稀释的Streptavidin-HRP,室温孵育30分钟,洗涤3次。加入已平衡至室温的TMB溶液,避光反应8分钟,终止反应,于450/650nm读数。使用仪器自带软件进行拟合。结果表明,5886-156-H2L1能够与人和食蟹猴的ST2特异性结合,亲和力相似,EC50分别为7.044ng/mL(人)、7.588ng/mL(食蟹猴)。5886-156-H2L1不结合大鼠和小鼠的ST2。
实施例8 5886-156-H2L1对重组人ST2蛋白和重组人IL-33结合的相对阻断活性(ELISA法)
将human ST2-fc蛋白包被过夜,将稀释好的human IL33-his与稀释好的5886-156-H2L1 1:1混合,按照50μL/孔加入到96孔板中,室温孵育1h;加入组氨酸标签二抗(1:2500),50μL/孔,室温孵育1h;TMB显色10min,2M硫酸100μL/孔终止。
测定5886-156-H2L1阻断IL-33与ST2结合能力(IC50)范围869.6~994.9ng/mL。
实施例9 5886-156-H2L1抑制重组人IL-33促HUVEC细胞IL-6生成活性(ELISA法)
采用膜表达ST2的HUVEC作为靶细胞,通过检测IL-6的产生量来间 接评价5886-156-H2L1抗体阻断活性功能。具体将HUVEC细胞按照10000个/孔,100μL体系,37℃、5%CO2条件下孵育18-24h。将human IL33-his用培养基稀释到10ng/mL,5886-156-H2L1用培养基稀释到400μg/mL,4倍稀释,共11个浓度,将二者1:1混合后100μL/孔加到上述96孔板中,37℃、5%CO2条件下孵育18-24h。按照Human IL-6 DuoSet ELISA试剂盒说明书测定细胞培养上清中IL6浓度。实验结果表明,5886-156-H2L1有显著的量效曲线,具有良好的阻断IL-33与ST2结合的生物学活性。结果见图2。
实施例10 5886-156-H2L1抑制重组人IL-33促HMC-1细胞IL-8生成活性(ELISA法)
采用膜表达ST2的HMC-1作为靶细胞,通过检测IL-8的产生量来间接评价5886-156-H2L1抗体阻断活性功能。具体将human IL33-his稀释到终浓度1000ng/mL,5886-156-H2L1按照起始640μg/mL,4倍稀释,共11个浓度,将二者1:1混合后按照50μL/孔加入到96孔板中。取对数生长期的HMC-1细胞按照50000个/孔,50μL/孔加入到上述96孔板中,37℃、5%CO2条件下孵育18-24h。按照Human IL-8 DuoSet ELISA试剂盒说明书测定细胞培养上清中IL8浓度。实验结果表明,5886-156-H2L1有显著的量效曲线,具有良好的阻断IL-33与ST2结合的生物学活性。结果见图3。
实施例11 5886-156-H2L1的ADCC效应实验
(1)PBMC法
将SK-BR-3细胞消化处理后重悬在ADCC分析培养基中,取样计数后将其细胞密度稀释调整到5×10
4个细胞/mL,在96孔细胞培养板中每孔加入100μl细胞稀释液,在ESR(Effector Spontaneous Release,效应细胞自发释放)孔、CMB(Culture Medium Background,空白培养基)孔和VCC(Volume Correction Control,体积校正)孔中加入100μl ADCC分析培养基,将加好样的96孔细胞板置于37℃,CO
2培养箱孵育18±2h。取对数生长期的KU812细胞,取样计数,1000rpm离心处理5min,分析培养基重悬稀释细胞密度为2×10
5cells/ml。在与加入SK-BR-3细胞平行的孔内分别加入25μl的KU812细胞稀释液。将阳性对照(Herceptin)用分析培养基稀释到浓度为500ng/ml、50ng/ml、5ng/ml、1.25ng/ml、0.313ng/ml、0.0781ng/ml、0.00781ng/ml 和0.000781ng/ml,之后取50μl阳性对照加入到已加入SK-BR-3细胞的孔内。将5886-156-H2L1单抗用分析培养基稀释到浓度为1000000ng/ml、100000ng/ml、10000ng/ml、1000ng/ml、100ng/ml、10ng/ml、1ng/ml和0.1ng/ml,之后取50μl加到已加入KU812细胞的孔内,TSR(Target Spontaneous Release,靶细胞自发释放)孔、TMR(Target Maximum Release,靶细胞最大释放)孔每孔加入100μl分析培养基,(E+T)SR孔和ESR孔每孔加入50μl分析培养基。将加完样品的96孔细胞板置于微孔板恒温振荡器中,100rpm混匀30min。将PBMC细胞400×g离心10min,弃上清后用分析培养基重悬计数,取适量该细胞悬液调整细胞密度至2.5×10
6cells/ml和5.0×10
6cells/ml,在阳性对照样品孔内加入50μl密度为2.5×10
6cells/ml的PBMC细胞稀释液,在待测抗体样品孔内加入25μl密度为5×10
6cells/ml的PBMC细胞稀释液,在(E+T)SR孔和ESR孔每孔加入50μl各自对应浓度的PBMC稀释液。加样完成后的96孔细胞板置于,CO
2培养箱37℃孵育4h。根据Cytotoxicity LDH Assay Kit-WST试剂盒,在孵育完成的TMR孔和VCC孔中加入10μl Lysis Buffer,在CO
2培养箱37℃孵育30min,之后在每孔中加入100μl Working Solution,避光室温反应30min,最后在每孔中加入50μl Stop Solution,混合后读取吸光度值(OD490nm)。
计算公式:
%Target cell Lysis=[(OD
ER–OD
(T+E)SR)/(OD
TMR-OD
TSR)]×100
12.2报告基因法
将SK-BR-3细胞消化处理后重悬在ADCC分析培养基中,取样计数后将其细胞密度稀释调整到2×10
5cells/mL,在96孔细胞培养板中每孔加入100μl细胞稀释液,将加好样的96孔细胞板置于37℃,CO
2培养箱孵育22±2h。取对数生长期的KU812细胞,取样计数,1000rpm离心处理5min,分析培养基重悬稀释细胞密度为1.33×10
6cells/ml。在与加入SK-BR-3细胞平行的孔内分别加入15μl的KU812细胞稀释液。将阳性对照(Herceptin)用分析培养基稀释到浓度为1500ng/ml、750ng/ml、375ng/ml、187.5ng/ml、93.75ng/ml、46.88ng/ml、23.44ng/ml、11.72ng/ml、5.86ng/ml和2.93ng/ml,将孵育好的SK-BR-3细胞组中的培养液吸弃,之后在每孔中加入30μl稀释好的阳性对照。将5886-156-H2L1单抗用分析培养基稀释到浓度为1000000ng/ml、100000ng/ml、10000ng/ml、1000ng/ml、100ng/ml、10ng/ml、1ng/ml、 0.1ng/ml、0.01ng/ml和0.001ng/ml,之后取30μl加到已加入KU812细胞的孔内。将加完样的96孔细胞板置于CO
2培养箱37℃孵育30min。期间制备效应细胞,将效应细胞Jurkat-ADCC12细胞400×g离心5min后用分析培养基重悬计数,之后调整细胞密度至4×10
6cells/ml和8×10
6cells/ml,在阳性对照样品孔内加入30μl密度为4×10
6cells/ml的效应细胞稀释液,在5886-156-H2L1单抗样品孔内加入15μl密度为8×10
6cells/ml的效应细胞稀释液。期间设置阴性对照(30μl SK-BR-3细胞+30μl分析培养基+30μl密度为4×10
6cells/ml的效应细胞和15μl KU812细胞+30μl分析培养基+15μl密度为8×10
6cells/ml的效应细胞),空白对照(60μl分析培养基)。将加完样的96孔细胞板置于CO
2培养箱37℃孵育16-24h。孵育结束后加入60μl/孔Bio-Glo Luciferase Assay System试剂,室温900rpm避光震荡2分钟后迅速用酶标仪检测光信号,选择化学发光模式,在30分钟内完成检测。
计算公式:
背景均值:空白对照孔RLU的均值;阴性对照均值:阴性对照孔RLU的均值。
实验结果表明,在两种不同检测体系内,5886-156-H2L1无明显的ADCC作用。
实施例12 5886-156-H2L1的CDC效应实验
将Raji细胞300×g离心5min,去上清后加入CDC分析培养基重悬,取样计数后将其细胞密度稀释调整到1×10
6cells/mL。取对数生长期的KU812细胞,取样计数,300g离心5min,分析培养基重悬稀释细胞密度为1×10
6cells/ml。分别在不同96孔细胞培养板中每孔加入50μl制好两种细胞稀释液。将阳性对照(MabThera)用分析培养基稀释到浓度为10000ng/ml、5000ng/ml、2500ng/ml、1250ng/ml、625ng/ml、312.5ng/ml、156.3ng/ml、78.1ng/ml、39.1ng/ml和19.5ng/ml,取50μl稀释好的阳性对照加入有Raji细胞的孔内。将5886-156-H2L1单抗用分析培养基稀释到浓度为1000000ng/ml、100000ng/ml、10000ng/ml、1000ng/ml、100ng/ml、10ng/ml、1ng/ml、 0.1ng/ml、0.01ng/ml和0.001ng/ml,之后取50μl加到已加入KU812细胞的孔内。将加好样的96孔细胞板置于CO
2培养箱37℃孵育30min。孵育结束后每孔加入50μl含有24%补体的分析培养基,再将细胞板置于CO
2培养箱37℃孵育2h。期间需设置无补体对照、无抗体对照和空白对照。之后将96孔板每孔加入10μl的Resazurin后,放置在恒温震荡器中室温震荡1-2min后,继续在培养箱内孵育16-20h后取出读板。
计算公式:CDC%=100×(E-S)/(M-S)
E:experimental well的信号;S(自发杀伤):无抗体对照的信号;M(最大杀伤):空白对照的信号。
实验结果表明,5886-156-H2L1不具有明显的激活补体系统介导产生CDC效应的活性。
实施例13 5886-156-H2L1在猪蛔虫卵提取物诱导的过敏性皮炎中的药效研究
本实施例中,采用猪蛔虫卵提取物(Ascaris.Suum,厂家:STALLERGENES GREER;批号:302145)诱导的食蟹猴过敏性皮炎模型,研究5886-156-H2L1单抗对过敏性皮炎与特应性皮炎的预防或治疗作用。
将24只对猪蛔虫卵提取物天然敏感的食蟹猴分成4组,每组6只,依次为溶媒对照组、阳性对照组(苯海拉明)、5886-156-H2L1低剂量组(10mg/kg)和高剂量组(30mg/kg)。其中,对于两个5886-156-H2L1组,提前(-4h)静脉注射给予由生理盐水稀释的5886-156-H2L1;对于阳性对照组,提前(-24h和-1h)苯海拉明2mg/kg灌胃;对于溶媒对照组,提前(-4h)静脉注射生理盐水(2mL/kg)。
给药后激发皮肤肿胀反应:各组中动物分别在腹部皮内注射生理盐水1个点,组胺(Histamine)1个点和A.suum 3个点。在生理盐水、组胺和A.suum激发后0min和20min测量激发点肿胀面积,以两者的比值(Ratio=20min面积/0min面积)来反映皮肤的肿胀反应。
结果表明与溶媒对照组相比,5886-156-H2L1高剂量组(30mg/kg)给药可以显著降低皮内注射A.suum引起的皮肤肿胀反应(p<0.01);与溶媒对照组相比,5886-156-H2L1低剂量组和高剂量组(10mg/kg和30mg/kg)预防给药可以显著降低皮内注射组胺引起的皮肤肿胀反应。结果如图4所示。
实施例14 5886-156-H2L1在伊利替康诱导的肠黏膜炎模型中的药效研究
本实施例中,采用伊立替康(CPT-11,盐酸伊立替康,湖北盛天恒创生物科技有限公司;Cas号:136572-09-3)诱导的食蟹猴粘膜炎模型,研究5886-156-H2L1对伊利替康诱导的肠黏膜炎的预防或治疗作用。
将9只食蟹猴分成3组(G1组、G2组、G3组),每组3只。在Day 0~2这三天,G1组静脉注射CPT-11溶媒(<3%DMSO),G2和G3组静脉注射CPT-11(30mg/kg),每天一次;在Day-1、2、5、8,G2组静脉注射生理盐水,G3组静脉注射5886-156-H2L1(用生理盐水稀释)(10mg/kg);Day12动物解剖取肠组织进行病理检查。
与正常对照组(G1)相比,伊利替康静脉注射引起了粘膜炎,表现为腹泻,食欲下降,体重降低,肠道炎症和粘液分泌减少。与生理盐水组(G2)相比,5886-156-H2L1显示出缓解疾病症状(腹泻、体重下降、食欲不振和缺乏活动)的功效。疾病相关症状变化和严重程度的AUC如图5所示。
(二)抗体制剂的组成筛选
采用上文第(一)部分中的抗体5886-156-H2L1进行抗体制剂组成筛选。其中采用以下检测方法:
UNcle:
采用Unchained Labs的UNcle多功能蛋白稳定分析系统测试样品的热稳定性;根据制造商的说明操作,其中每个样品孔加入9μl样品,然后打开控制软件,选择应用“Tm&Tagg with optional DLS”,设置运行模式为“Linear”,Acquisitions设定为4,Acquisition times设定为5s;起始温度设定为25℃,孵育时间180s,升温速率0.3℃/min,板层搁置时间0s,终止温度95℃。实验结束后,对原始数据进行二阶导积分计算Tm和Tagg值。
体积排阻色谱法(SEC):
检测样品中的纯度,比较各个样品中聚集体和单体的差异。主要步骤:取一定量样品于EP管中,10000~14000g离心3~5min,取上清液,用TOSOH/TSKgel G3000SWxl色谱柱进行分离,通过高效液相色谱(Agilent1260或者Waters/Aqcuity H Class Bio)进行分析和数据收集;运行 完成后,对结果进行积分,使用峰面积归一化法计算样品的单体峰面积百分比、聚合物峰面积百分比、其他峰面积百分比。
弱阳离子色谱法(CEX):
分析样品的电荷异质性,比较不同样品的酸性峰、碱性峰的差异。主要步骤如下:用样品稀释液将参比品和供试品稀释至2mg/ml,取稀释后的参比品和供试品分别加入CpB酶工作液,蛋白与CpB酶的质量比为40:1,旋涡振荡混匀后,置于37℃恒温孵育30min。将所有处理好的样品涡旋混匀,10000-14000g离心3-5min,取上清液填充至样品瓶中。用弱阳离子色谱柱进行分离,通过Waters H-Class高效液相色谱进行分离和数据采集。运行完成后,对结果进行积分,使用峰面积归一化法计算样品的酸性峰面积百分比、主峰面积百分比、碱性峰面积百分比。
粘度:
运用Rheosense的m-ROV粘度计对样品进行粘度测试。主要步骤如下:启动m-VROC控制软件,在Temp Ctrl/Measmt Adviscr中设置温度为25℃,用注射器吸取1ml左右样品,将注射器和芯片放到相应的夹套上,然后设置样品名称、预估粘度值和若干不同的剪切速率,设置好后点击START按钮开始测量,测量过程注意观察FULL SCALE,有效范围为5%-95%。
氧化分析:
对样品经盐酸胍变性,二硫苏糖醇(DTT)还原和碘乙酰胺(IAM)烷基化后,用Lys-C酶对样品酶解4h,通过反相色谱柱对肽段进行分离,通过Waters H-Class超高效液相色谱串联Waters Vion IMS Q-TOF高分辨质谱进行分离和数据采集,并使用UNIFI软件进行数据分析,获得各肽段的氧化位点和比例。
不溶性微粒:
应用Beckman公司HIAC 9703+型液体颗粒计数器检测样品中不溶性微粒。主要步骤如下:用水将容器外壁洗净,小心翻转20次,使溶液混合均匀,静置2分钟,小心开启容器,直接将供试品容器置于取样器上,以手缓缓转动使溶液混匀,由仪器直接抽取适量溶液,采集10μm和25μm通道的微粒数,依法测定4次,弃第一次测定数据,取后续3次测定数据的平均值作为测定结果。
实施例15 pH及缓冲体系选择
将5886-156-H2L1置换至不同缓冲液中,浓度为30mg/mL,无菌过滤、分装,分别放置于4℃和40℃进行稳定性考察,0天、7天、14天取样检测,检测项目包括UNcle、SEC、CEX。
缓冲液组成见表8。
表8.缓冲液组成
使用Uncle测定5886-156-H2L1在上述各缓冲液中0天样品的蛋白热稳定性,比较不同条件下Tm和Tagg的差异,结果如表9、图6所示。
表9.不同缓冲体系5886-156-H2L1的热稳定性结果
结果显示,Tm1随着pH增加而升高,磷酸缓冲液中Tm1显著高于其他缓冲液。在组氨酸缓冲液中pH=6.0时,达到最高值,随后降低,同时Tagg显著高于其他缓冲液中,表明组氨酸缓冲体系对5886-156-H2L1有更好的抑制聚集作用,从热稳定性角度考虑,组氨酸缓冲体系整体优于其他体系。
通过SEC方法考察5886-156-H2L1在40℃条件下聚体含量变化,从而确定适合该抗体的最佳缓冲体系及pH,结果如表10所示。
表10.不同缓冲液高温SEC聚体考察结果
从表10可以看出磷酸缓冲液中5886-156-H2L1的聚体含量增加明显多于其他体系,柠檬酸缓冲液中pH=5.0时,聚体增加量也高于其他pH,4种缓冲液中,组氨酸缓冲液中的聚体变化量最低。
通过CEX方法考察5886-156-H2L1在40℃条件下电荷异质性的变化,从而确定适合该抗体的最佳缓冲体系及pH,结果如表11所示。
表11.不同缓冲液高温稳定性CEX主峰考察结果
表11结果可以看出柠檬酸缓冲液pH 5.0、磷酸缓冲液pH 7.0-7.5中CEX主峰下降比例均超过20%,而组氨酸和醋酸缓冲液中主峰下降比例明显较低。
综合上述结果,选取缓冲范围为约pH 5.5-6.0的组氨酸缓冲液进行下一步研究。
实施例16蛋白浓度的选择
测定5886-156-H2L1抗体在组氨酸缓冲液(pH5.8±0.3)中不同浓度下的粘度,确定5886-156-H2L1可开发的浓度范围,结果如表12和图7所示。结果显示,当浓度达到180mg/ml以上时,粘度急剧升高,表明5886-156-H2L1的浓度应该控制在180mg/ml以下。
表12.不同浓度5886-156-H2L1样品的粘度结果
样品浓度(mg/ml) | 粘度(cP) |
50 | 1.666 |
120 | 4.136 |
150 | 8.305 |
180 | 19.616 |
200 | 36.872 |
250 | 89.653 |
备注:剪切速率为35000s-1,温度25℃
在最佳缓冲液组成确定以及粘度考察后,进一步考察不同蛋白浓度对于稳定性的影响。将5886-156-H2L1样品置换为组氨酸缓冲液中不同浓度,40℃放置进行加速稳定性实验,并在0天、7天、14天、28天时取样进行SEC和CEX检测。以实验起始0天、40℃放置7天、14天和28天的SEC和CEX主峰含量拟合直线,并计算主峰的下降速率(%/天),结果如表13和表14所示。
表13.不同蛋白浓度高温稳定性SEC主峰考察结果
表14.不同蛋白浓度高温稳定性CEX主峰考察结果
从表13和表14可以看出,在40℃条件放置28天后,高浓度样品的纯度下降速率与低浓度样品比较并没有显著增加,变化趋势在合理范围内。同时,在40℃条件放置28天后,高浓度样品的CEX主峰下降比例仅略高于低浓度样品。
结果显示,高浓度样品在高温放置28天后,纯度仅降低1.5%,样品中未出现大量聚体,表明5886-156-H2L1蛋白在可溶性方面具有开发成高浓度制剂的潜力。
实施例17保护剂选择
在上述条件确定后,进一步就添加保护剂进行研究比较,从而选出可使抗体最为稳定的辅料添加物。
将5886-156-H2L1样品置换至10mM浓度的组氨酸缓冲液中,浓度为50mg/mL,该缓冲液含不同保护剂,然后放置40℃进行高温稳定性实验,在0天、14天、28天时取样进行SEC和CEX检测,结果表15和表16所示。
表15.不同保护剂高温稳定性SEC主峰考察结果
表16.不同保护剂高温稳定性CEX主峰考察结果
表15和表16显示,不同保护剂中,SEC纯度的下降速率和CEX主峰的增加速率没有显著区别,因此选择生物药中常用的蔗糖作为添加剂。
确定选择蔗糖为保护剂后,参照上文实验操作,考察了不同浓度蔗糖对5886-156-H2L1蛋白的影响。结果如表17和表18所示。
表17.不同保护剂浓度高温稳定性SEC主峰考察结果
表18.不同保护剂高温稳定性CEX主峰考察结果
表17和表18显示,不同蔗糖浓度中抗体均相对稳定,不同蔗糖浓度之间没有显著区别。因此,蔗糖浓度可根据样品理化性质需求,在3%-9%之间进行调整。
实施例18缓冲液浓度选择
将5886-156-H2L1样品置换至不同浓度组氨酸缓冲液中,浓度为 150mg/mL,然后放置40℃进行加速稳定性实验,并在0天、14天、28天时取样进行pH和SEC检测,结果如表19和表20所示。
表19.不同缓冲液浓度高温稳定性SEC主峰考察结果
表20.不同缓冲液浓度pH考察结果
表19显示40℃条件放置28天后,5mM条件中的样品聚体略高于其他样品。表20显示缓冲液浓度为5mM时,置换后pH变化更大,说明低浓度的缓冲液缓冲能力不足,10mM和15mM时,高温放置过程中pH有升高趋势。因此,优选20-25mM组氨酸缓冲液作为5886-156-H2L1高浓度制剂的缓冲体系。
实施例19抗氧化剂选择
将5886-156-H2L1样品置换至含有不同浓度蛋氨酸的20mM组氨酸缓冲液,浓度为140mg/mL(pH5.8±0.3)中,高温放置30天或光照14天后进行氧化分析。结果如表21所示。
表21.不同蛋氨酸中5886-156-H2L1氧化结果
如上表所示,5886-156-H2L1高浓度制剂40℃放置30天或者光照14天后,检测到重链4个位点和轻链1个位点的甲硫氨酸发生氧化。样品中加入蛋氨酸后,重链4个位点的氧化比例均降低,表明蛋氨酸具有一定的抗氧化作用,因此考虑在配方中添加一定量的蛋氨酸作为抗氧化剂。当蛋氨酸浓度达到10mM时,高温和光照条件下其氧化比例均趋于平缓,能最大程度的减少M104或W107位点的氧化。因此,在5886-156-H2L1高浓度制剂中添加5-10mM蛋氨酸,降低其长期储存的氧化风险。
实施例20表面活性剂选择
将5886-156-H2L1样品置换至含有不同浓度聚山梨酯20和聚山梨酯80的20mM组氨酸缓冲液(pH5.8±0.3)中,浓度为150mg/mL。制备好的样品反复冻融10次、震荡5天及高温放置28天后测定样品中的不溶性微粒(MFI)。结果见表22、表23和图8至图10。
表22.不同比例聚山梨酯20样品不溶性微粒结果
表23.不同比例聚山梨酯80样品不溶性微粒结果
图8显示不含聚山梨酯20的样品不溶性微粒显著增加,≥0.04%的聚山梨酯20,抗聚集效果没有显著区别。图9显示≥0.02%的聚山梨酯80即能有效起到抗聚集作用。图10显示同等含量的聚山梨酯80和聚山梨酯20条件下,聚山梨酯80抑制微粒产生的效果略好于聚山梨酯20。因此,聚山梨酯20和聚山梨酯80均可作为5886-156-H2L1高浓度制剂的表面活性剂,但优先选择聚山梨酯80。
将含有不同浓度聚山梨酯80的样品分装至预充针中,反复冻融10次、震荡5天及高温放置28天后测定样品中的不溶性微粒和聚体变化。结果如表24、表25和图11、图12所示。
表24.预充针中不同比例聚山梨酯80样品不溶性微粒结果
表25.预充针中不同比例聚山梨酯80样品SEC结果
图11显示,不含聚山梨酯80的样品不溶性微粒显著增加,并且在震荡5天、冻融10次和高温放置28天后,出现浑浊现象。添加聚山梨酯80后,震荡、冻融对微粒产生的影响小于高温条件,说明高温对不溶性微粒产生的影响更显著。在高温放置28天后,添加0.04%聚山梨酯80的样品不溶性微粒显著下降。图12结果显示,震荡和高温对SEC聚体的产生有显著影响,但冻融对SEC聚体产生没有明显影响。不同比例聚山梨酯80的样品高温放置28天后,聚体产生的量没有显著差异,而在震荡5天后,聚山梨酯80含量低于0.03%的样品,SEC聚体高于其他样品;因此从抑制SEC聚体产生的角度看,聚山梨酯80含量应该≥0.03%。综合不溶性微粒及SEC聚体考虑,优选0.04%±0.01%聚山梨酯80作为5886-156-H2L1高浓度制剂的表面活性剂。
实施例21处方验证实验
通过高温稳定性试验、冻融稳定性试验、振荡稳定性试验和光照试验,考察5886-156-H2L1储存在最终处方的稳定性。实验所用5886-156-H2L1蛋白为经过三步纯化样品,将其配制成处方:抗体140mg/ml,25mM组氨酸缓冲液,7%蔗糖,10mM蛋氨酸,0.03%聚山梨酯80,分装至预充针中进行单体含量(SEC)、电荷异构体主峰含量(CEX)和不溶性微粒(MFI)检测,结果如表26所示。
表26. 5886-156-H2L1稳定性考察结果
在此处方下,5886-156-H2L1抗体较稳定。
实施例22处方验证实验
通过高温稳定性试验、冻融稳定性试验、振荡稳定性试验和光照试验,考察5886-156-H2L1储存在最终处方的稳定性。实验所用5886-156-H2L1蛋白为经过三步纯化样品,将其配制成处方:抗体150mg/ml,20mM组氨酸缓冲液,6%蔗糖,10mM蛋氨酸,0.04%聚山梨酯80,分装至预充针中进行单体含量(SEC)、电荷异构体主峰含量(CEX)和不溶性微粒(光阻法)检测,结果如表27所示。
表27. 5886-156-H2L1稳定性考察结果
在此处方下,5886-156-H2L1抗体较稳定。
实施例23添加透明质酸酶后的5886-156-H2L1样品稳定性考察
通过高温稳定性试验、冻融稳定性试验、振荡稳定性试验和光照试验,考察含有透明质酸酶的5886-156-H2L1样品的稳定性。实验所用5886-156-H2L1蛋白为经过三步纯化样品,将其配制成处方:抗体140mg/ml, 25mM组氨酸缓冲液,7%蔗糖,10mM蛋氨酸,0.03%聚山梨酯80,并向样品中添加透明质酸酶,使样品中的透明质酸酶酶活约为5500单位/ml(重组透明质酸酶,购自上海宝济生物),除菌过滤后分装至西林瓶中,并进行单体含量(SEC)、电荷异构体主峰含量(CEX)和不溶性微粒(光阻法)检测,结果如表28所示。
表28.含有透明质酸酶的5886-156-H2L1稳定性考察结果
*:抗体结合活性(%)是相对于参比品的结合活性的百分比,参比品为相同处方,但抗体浓度为50mg/ml;结合活性为针对人ST2抗原ELISA检测的结合活性。
表28结果显示添加透明质酸酶后未对抗体质量产生显著影响。高温和光照条件下,聚体少量增加、CEX主峰下降,透明质酸酶酶活显著下降、不溶性微粒和抗体结合活性没有显著变化;震荡5天、冻融5次对抗体及透明质酸酶均没有显著影响,因此该样品应该低温避光保存。
以上对本发明具体实施方式的描述并不限制本发明,本领域技术人员可 以根据本发明作出各种改变或变形,只要不脱离本发明的精神,均应属于本发明所附权利要求的范围。
Claims (16)
- 一种抗肿瘤抑制素2(ST2)抗体或其片段,所述抗体或其片段包含重链可变区和轻链可变区并且在重链可变区和轻链可变区中分别包含重链CDR1(H-CDR1)、CDR2(H-CDR2)、CDR3(H-CDR3)和轻链CDR1(L-CDR1)、CDR2(L-CDR2)、CDR3(L-CDR3),其中:H-CDR1包含SEQ ID NO.8所示的氨基酸序列,H-CDR2包含SEQ ID NO.9所示的氨基酸序列,H-CDR3包含SEQ ID NO.10所示的氨基酸序列;和,L-CDR1包含SEQ ID NO.11所示的氨基酸序列,L-CDR2包含SEQ ID NO.12所示的氨基酸序列,L-CDR3包含SEQ ID NO.13所示的氨基酸序列。
- 根据权利要求1所述的抗ST2抗体或其片段,其特征在于,所述抗ST2抗体为抗ST2的单克隆抗体、scFv、BsFv、dsFv、(dsFv)2、Fab、Fab'、F(ab')2或Fv等抗体形式;或者,所述抗ST2抗体为抗ST2的鼠抗体、嵌合抗体、完全或部分人源化抗体。
- 根据权利要求1或2所述的抗ST2抗体或其片段,其特征在于,所述抗ST2抗体的所述重链可变区(VH)包含SEQ ID NO.7所示的氨基酸序列或其变体,所述轻链可变区(VL)包含SEQ ID NO.6所示的氨基酸序列或其变体;优选地,所述抗ST2抗体为分离的人IgG4/κ亚型的单克隆抗体;更优选地,所述抗ST2抗体的重链包含SEQ ID NO.17所示的氨基酸序列,轻链SEQ ID NO.18所示的氨基酸序列。
- 一种核酸分子,其包含编码权利要求1至3中任一项所述的抗ST2抗体或其片段中包含的轻链可变区、重链可变区、重链或轻链的核苷酸序列。
- 一种载体,其包含权利要求4所述的核酸分子。
- 一种宿主细胞,其包含权利要求4所述的核酸分子和/或权利要求5所述的载体,或者所述宿主细胞被权利要求4所述的核酸分子和/或权利要求4所述的载体转化或转染。
- 一种组合物,所述组合物包含权利要求1至3中任一项所述的抗ST2抗体或其片段、权利要求4所述的核酸分子、权利要求5所述的载体和/或权利要求6所述的宿主细胞;优选地,所述组合物为药物组合物,其任选地还包含药学上可接受的辅料。
- 一种液体抗体制剂,其包含:权利要求1至3中任一项所述的抗ST2抗体或其片段;缓冲体系;保护剂;以及表面活性剂,可选地,抗氧化剂和/或功能性辅料。
- 根据权利要求8所述的液体抗体制剂,其特征在于,所述液体抗体制剂为注射剂,优选用于皮下注射或静脉注射,或者为输注剂,例如用于静脉内输注。
- 根据权利要求8或9所述的液体抗体制剂,其特征在于,所述缓冲体系为药用缓冲体系;优选地,所述药用缓冲体系为柠檬酸缓冲液、组氨酸缓冲液、磷酸缓冲液或醋酸缓冲液;任选地,所述保护剂为糖或醇,优选为选自蔗糖、海藻糖、山梨醇和甘露醇中的一种或多种;任选地,所述表面活性剂为聚山梨酯;任选地,所述抗氧化剂为氨基酸,例如蛋氨酸;任选地,所述功能性辅料为透明质酸酶。
- 根据权利要求8至10中任一项所述的液体抗体制剂,其特征在于,所述液体抗体制剂的pH为5.5-6.5,优选5.5-6.1,更优选5.5-6.0;任选地,所述液体抗体制剂包含浓度为30-180mg/mL、优选100-150mg/mL、更优选140-150mg/mL的所述抗ST2抗体或其片段;任选地,所述液体抗体制剂包含组氨酸缓冲液作为缓冲体系;优选地,所述液体抗体制剂包含浓度为5-25mM、优选20-25mM的组氨酸缓冲液,pH缓冲范围为5.5-6.5,优选5.5-6.1,更优选5.5-6.0。
- 根据权利要求7至11中任一项所述的液体抗体制剂,其特征在于,所述液体抗体制剂包含蔗糖作为保护剂;优选地,所述液体抗体制剂包含浓度为3%-9%(w/v)、优选5-7%(w/v)的蔗糖;任选地,所述液体抗体制剂包含蛋氨酸作为抗氧化剂;优选地,所述液体抗体制剂包含浓度为0-20mM、优选5-10mM的蛋氨酸;任选地,所述液体抗体制剂包含聚山梨酯20或聚山梨酯80作为表面活性剂,例如0.005%-0.1%(w/v)的聚山梨酯20或聚山梨酯80;优选地,所述液体抗体制剂包含浓度为0.02%-0.1%(w/v)、优选0.04%-0.06%(w/v)的聚山梨酯20;或者,优选地,所述液体抗体制剂包含浓度为0.005-0.1%(w/v)、优选0.03%-0.05%(w/v)的聚山梨酯80;任选地,所述液体抗体制剂包含透明质酸酶作为功能性辅料;优选地,所述液体抗体制剂包含酶活为0-12800单位/mL、优选0-6400单位/mL的透明质酸酶。
- 根据权利要求7至12中任一项所述的液体抗体制剂,其特征在于,所述液体抗体制剂包含:30-180mg/mL权利要求1至3中任一项所述的抗ST2抗体或其片段;5-25mM缓冲体系;3%-9%(w/v)保护剂;以及0.005%-0.1%(w/v)表面活性剂,可选地,0-20mM抗氧化剂和/或0-12800单位/mL功能性辅料,并且,pH为5.5-7.5;例如,所述液体抗体制剂包含:30-180mg/mL权利要求1至3中任一项所述的抗ST2抗体或其片段;5-25mM柠檬酸缓冲液、磷酸缓冲液、醋酸缓冲液或组氨酸缓冲液;3%-9%(w/v)蔗糖、海藻糖、山梨醇或甘露醇;以及0.005%-0.1%(w/v)聚山梨酯20或聚山梨酯80;可选地,0-20mM蛋氨酸和/或0-12800单位/mL透明质酸酶,并且,pH为5.5-7.5;或者,所述液体抗体制剂包含:30-180mg/mL权利要求1至3中任一项所述的抗ST2抗体或其片段;5-25mM组氨酸缓冲液;3%-9%(w/v)蔗糖;以及0.005%-0.1%(w/v)聚山梨酯20或聚山梨酯80;可选地,0-20mM蛋氨酸和/或0-12800单位/mL透明质酸酶,并且,pH为5.5-6.0;或者,所述液体抗体制剂包含:140-150mg/mL权利要求1至3中任一项所述的抗ST2抗体或其片段;20-25mM组氨酸缓冲液;3%-9%(w/v)蔗糖;以及0.03%-0.05%(w/v)聚山梨酯80;可选地,5-10mM蛋氨酸和/或0-6400单位/mL透明质酸酶,并且,pH为5.5-6.0;或者,所述液体抗体制剂包含:140-150mg/mL权利要求1至3中任一项所述的抗ST2抗体或其片段;20-25mM组氨酸缓冲液;3%-9%(w/v)蔗糖;以及0.03%-0.05%(w/v)聚山梨酯80;可选地,5-10mM蛋氨酸和/或0-6400单位/mL透明质酸酶,并且,pH为5.5-6.0;或者,所述液体抗体制剂包含:140-150mg/ml权利要求1至3中任一项所述的抗ST2抗体或其片段;25mM组氨酸缓冲液;7%(w/v)蔗糖;以及0.03%(w/v)聚山梨酯80;可选地,10mM蛋氨酸和/或5500单位/mL透明质酸酶,并且,pH为5.5-6.0;或者,所述液体抗体制剂包含:150mg/ml权利要求1至3中任一项所述的抗ST2抗体或其片段;20mM组氨酸缓冲液;6%(w/v)蔗糖;以及0.04%(w/v)聚山梨酯80;可选地,10mM蛋氨酸和/或6400单位/mL透明质酸酶,并且,pH为5.5-6.0。
- 一种固体抗体制剂,其通过固化权利要求8至13中任一项所述的液体抗体制剂而获得,所述固体抗体制剂例如是冻干粉针剂。
- 权利要求1至3中任一项所述的抗ST2抗体或其片段、权利要求4所述的核酸分子、权利要求5所述的载体、权利要求6所述的宿主细胞、权 利要求7所述的组合物、权利要求8至13中任一项所述的液体抗体制剂和/或权利要求14所述的固体抗体制剂在制备药物中的用途,所述药物用于预防、治疗和/或改善炎性、过敏性或自身免疫性疾病;优选地,所述疾病与IL-33/ST2信号传导通路的激活相关;更优选地,所述疾病包括子宫内膜异位、心力衰竭、过敏性皮炎、过敏性或变应性鼻炎、鼻息肉、特应性皮炎、慢性阻塞性肺病、哮喘、肺纤维化、败血症、炎性肠病、系统性红斑狼疮、类风湿性关节炎、全身性硬化症、韦格纳氏肉芽肿或化疗相关性腹泻。
- 一种用于预防、治疗或改善疾病的方法,所述方法包括给有此需要的受试者施用权利要求1至3中任一项所述的抗ST2抗体或其片段、权利要求4所述的核酸分子、权利要求5所述的载体、权利要求6所述的宿主细胞、权利要求7所述的组合物、权利要求8至13中任一项所述的液体抗体制剂和/或权利要求14所述的固体抗体制剂,所述疾病为炎性、过敏性或自身免疫性疾病;优选地,所述疾病与IL-33/ST2信号传导通路的激活相关;更优选地,所述疾病为子宫内膜异位、心力衰竭、过敏性鼻炎、鼻息肉、特应性皮炎、慢性阻塞性肺病、哮喘、肺纤维化、败血症、炎性肠病、系统性红斑狼疮、类风湿性关节炎、全身性硬化症、韦格纳氏肉芽肿或化疗相关性腹泻;任选地,所述受试者为哺乳类动物,优选地,所述受试者为人。
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