WO2022194311A2 - 一种IL-17RA抗体Fc融合蛋白及其用途 - Google Patents

一种IL-17RA抗体Fc融合蛋白及其用途 Download PDF

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WO2022194311A2
WO2022194311A2 PCT/CN2022/100748 CN2022100748W WO2022194311A2 WO 2022194311 A2 WO2022194311 A2 WO 2022194311A2 CN 2022100748 W CN2022100748 W CN 2022100748W WO 2022194311 A2 WO2022194311 A2 WO 2022194311A2
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fusion protein
nvs451
injection
pharmaceutical composition
buffer
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WO2022194311A4 (zh
WO2022194311A3 (zh
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张云涛
王健
刘建伟
闫甲丽
郭蓓蕾
刘明扬
李素贞
柳森
晁华
古琼
祁芳冰
雷永鹏
鲁慧
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国药中生生物技术研究院有限公司
Valin生物技术有限公司
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Publication of WO2022194311A2 publication Critical patent/WO2022194311A2/zh
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Definitions

  • the invention belongs to the field of medicine, and specifically relates to an IL-17RA fusion protein, a pharmaceutical composition, an injection and applications thereof.
  • Psoriasis commonly known as psoriasis, is a chronic autoimmune skin disease that is prone to recurrence. It usually causes great physical and psychological damage to patients. % to 3%, and there are currently 125 million psoriasis patients worldwide. The total prevalence rate in my country is 0.47%, and there are nearly 6.5 million clinically registered patients. Plaque psoriasis is one of the five most common forms of the disease, accounting for 80% to 90% of all cases, with moderate to severe patients. Accounting for 38%, the number of patients in the future will reach 9.5 million in 2030, and the proportion of moderate to severe patients will increase to 40%.
  • psoriasis The pathogenesis of psoriasis is associated with dysregulation of the innate and adaptive immune systems, including dendritic cell activation and pro-inflammatory cytokine secretion, leading to the development of skin inflammation characteristic of psoriasis.
  • monoclonal antibodies and antibody-based biological therapies have been approved for the treatment of psoriasis, and there are about 10 monoclonal antibody drugs in the clinical research stage, among which ustekinumab (IL- 12/IL-23 antibody, Johnson & Johnson) antibody, TNF-alpha drugs etanercept (Etanercept, Amgen) and adalimumab (Humira, AbbVie) were the top sellers.
  • the oral phosphodiesterase 4 (PDE4) selective inhibitor drug apremilast (Otezla, Celgene) was also approved in the United States in September 2014 for the treatment of refractory psoriasis.
  • PDE4 oral phosphodiesterase 4
  • apremilast Otezla, Celgene
  • the efficacy and safety of the treatment remain worrisome, and up to 40% of patients with moderate-to-severe psoriasis fail treatment with existing biologics, a phenomenon known as Fatigue phenomenon in biological products.
  • Th17 cells T17 helper cells
  • IL-17A proinflammatory cytokine IL-17A produced by Th17 cells and innate immune cells
  • the main cytokine in the mechanism, the immune system targets IL-23 in the IL-17-TH17 pathway, which indirectly contributes to the development of psoriasis through IL-17A.
  • inhibiting IL-17 may be a safer treatment option than other biologics.
  • the present invention provides IL-17RA fusion protein, pharmaceutical composition, injection and application thereof.
  • the present invention provides:
  • An IL-17RA fusion protein characterized by comprising an operably linked signal peptide, an IL-17RA extracellular domain and an IgG1 constant region in series in series.
  • IL-17RA fusion protein according to (1), wherein the amino acid sequence of the IL-17RA fusion protein is shown in SEQ ID NO.6, SEQ ID NO.7 or SEQ ID NO.8.
  • the IL-17RA fusion protein according to any one of (1)-(6) or the protein dimer according to claim 11 is prepared for the treatment of psoriasis, Crohn's disease, plaque Mass psoriasis, gastroenteritis, Behçet's syndrome, arthritis, uveitis, hidradenitis suppurativa, lichen planus, parapsoriasis, asthma, psoriatic arthritis, tendonitis, relapsing-remitting Multiple sclerosis, thyroid-related eye disease, juvenile rheumatoid arthritis, multiple sclerosis, lupus nephritis, spondyloarthritis, ankylosing spondylitis, rheumatoid arthritis, inflammatory bowel disease, nonalcoholic fatty liver disease, giant Cellular arteritis, nonradiographic axial spondyloarthritis, acne vulgaris, triple negative breast tumors, multiple myeloma, non-small cell
  • a pharmaceutical composition comprising a therapeutically effective amount of the IL-17RA fusion protein according to any one of (1)-(6) or the protein dimer according to claim 11 as an active ingredient and Medicinal excipients.
  • composition according to (14), wherein the buffer is selected from the group consisting of histidine-acetate buffer, Tris-acetate buffer, hydrochloric acid buffer, phosphate buffer, acetate buffer, histidine One or more of buffer, arginine buffer, succinate buffer, citrate buffer.
  • composition according to (14), wherein the protective agent is selected from one of trehalose, Tween-20, Tween-80, sucrose, amino acids, polyols, disaccharides, and polysaccharides or variety.
  • composition according to (14), wherein the surfactant is selected from one or more of Tween-20, Tween-80, and Poloxamers.
  • the intravenous preparation comprises 5 mg/ml-150 mg/ml of the IL-17RA fusion protein or the protein dimer, 2- 100 mM Tris-acetic acid, 10-250 mM arginine, 50-500 mM trehalose, 0.01-5% Tween-20.
  • the present invention has the following advantages and positive effects:
  • the IL-17RA fusion protein provided by the present invention is a fully human antibody Fc fusion protein drug.
  • the half-life of the drug molecule is prolonged, the longer-acting drug activity is obtained, and the immunogenicity is reduced compared with the antibody drug.
  • the present invention finds that selecting the Fc segment of IgG1 and further mutating the Fc sequence appropriately can greatly eliminate antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP) and complement Dependent cell-mediated cytotoxicity (CDC), and retains the effect of neonatal Fc receptor (FcRn)-mediated in vivo recycling, with low side effects compared to marketed IL-17A and IL-17RA antibodies and more secure.
  • ADCC antibody-dependent cell-mediated cytotoxicity
  • ADCP antibody-dependent cellular phagocytosis
  • CDC complement Dependent cell-mediated cytotoxicity
  • the present invention selects and designs the sequence of IL-17RA, so that the IL-17RA fusion protein can target multiple targets such as IL-17A, IL-17C, IL-17F, IL-17A/F, etc., and has high affinity, Thus, it can selectively block the binding of IL-17A, IL-17C, IL-17F and IL-17A/F to their receptors, thereby effectively blocking the biological activity of various pro-inflammatory IL-17 cytokines, inhibiting Therefore, it can more effectively alleviate the symptoms of autoimmune diseases, obtain better therapeutic benefits than single IL-17A target antibody drugs, and obtain better safety than IL-17RA monoclonal antibodies. Its mechanism of action is different from the IL-17 target drugs currently on the market and under development, and it is the first drug of its kind in the world.
  • the present invention is based on the binding affinity with IL-17A, biological activity cell experiments, GRO- ⁇ factor inhibitory ability, in vitro activity titer, in vivo titer, ADCC/CDC functional activity, FcRn binding affinity, safety pharmacology, pharmacokinetics
  • the activity and safety of the above drugs were comprehensively evaluated from the aspects of science and toxicology.
  • Figure 1 shows the amino acid sequence of the recombinant human IL-17RA fusion protein.
  • Figure 2 shows a map of the expression vector pCHO1.1/NVS451 used in one embodiment.
  • FIG. 3 shows the affinity measurement curve and fitting curve of IL-17RA fusion protein (NVS451) and human IL-17A protein in Experimental Example 1.
  • FIG. 3 shows the affinity measurement curve and fitting curve of IL-17RA fusion protein (NVS451) and human IL-17A protein in Experimental Example 1.
  • FIG. 4 shows the affinity measurement curve and fitting curve of secukinumab and human IL-17A protein in Experimental Example 1.
  • FIG. 5 shows the affinity measurement curve and fitting curve of wild-type IL-17RA and human IL-17A protein in Experimental Example 1.
  • FIG. 6 shows the affinity measurement curve and fitting curve of IL-17RA fusion protein (NVS451) and human IL-17F protein in Experimental Example 1.
  • FIG. 6 shows the affinity measurement curve and fitting curve of IL-17RA fusion protein (NVS451) and human IL-17F protein in Experimental Example 1.
  • FIG. 7 shows the affinity measurement curve and fitting curve of IL-17RA fusion protein (NVS451) and human IL-17A/F protein in Experimental Example 1.
  • FIG. 7 shows the affinity measurement curve and fitting curve of IL-17RA fusion protein (NVS451) and human IL-17A/F protein in Experimental Example 1.
  • FIG. 8 shows the affinity measurement curve and fitting curve of IL-17RA fusion protein (NVS451) and human IL-17C protein in Experimental Example 1.
  • FIG. 8 shows the affinity measurement curve and fitting curve of IL-17RA fusion protein (NVS451) and human IL-17C protein in Experimental Example 1.
  • FIG. 9 shows the S-curve of the inhibitory effect of IL-17RA fusion protein (NVS451) and secukinumab on GRO- ⁇ factor induced by IL-17A in Experimental Example 1.
  • FIG. 10 shows the S-curve of the inhibitory effect of IL-17RA fusion protein (NVS451) on GRO- ⁇ factor induced by IL-17A/F in Experimental Example 1.
  • FIG. 11 shows the S curve of the inhibitory effect of GRO- ⁇ factor on the induction of IL-17F by IL-17RA fusion protein (NVS451) in Experimental Example 1.
  • FIG. 11 shows the S curve of the inhibitory effect of GRO- ⁇ factor on the induction of IL-17F by IL-17RA fusion protein (NVS451) in Experimental Example 1.
  • FIG. 12 shows the appearance of skin after xenografted SCID mice were treated with different drugs in Experimental Example 1.
  • FIG. 13 shows the appearance of skin after xenografted SCID mice were treated with different drugs in Experimental Example 1.
  • FIG. 14 shows the skin pathological sections after xenografted skin-grafted SCID mice were treated with different drugs in Experimental Example 1.
  • FIG. 14 shows the skin pathological sections after xenografted skin-grafted SCID mice were treated with different drugs in Experimental Example 1.
  • Fig. 15 shows a comparison of the ADCC effect of RitxV301 and rituximab in Experimental Example 1.
  • FIG. 16 shows the comparison of the CDC effects of RitxV301 and Rituximab in Experimental Example 1.
  • FIG. 17 shows the SPR analysis patterns of NVS451 and human FcRn at different concentrations under acidic conditions (pH 6.0) in Experimental Example 1.
  • FIG. 17 shows the SPR analysis patterns of NVS451 and human FcRn at different concentrations under acidic conditions (pH 6.0) in Experimental Example 1.
  • Figure 19 shows the affinity comparison of IL-17RA fusion proteins NVS451 (V301), V302 and V303 with IL-17A in Experimental Example 1; the concentrations of V300, V301, V302, V303, V301*, V302*, V303* in the figure Both are 100ug/ml.
  • the Chinese hamster ovary cells provided by the present invention capable of stably expressing the IL-17RA fusion protein of the present invention have been deposited in the General Microorganism Center (CGMCC) of the China Microorganism Culture Collection and Management Committee on November 23, 2020, and the deposit address: Chaoyang, Beijing No. 3, No. 1 Yard, Beichen West Road, District, Zip code: 100101, preservation number: CGMCC No.21011.
  • CGMCC General Microorganism Center
  • injectable preparation refers to sterile solutions (including true solutions, emulsions and suspensions) made of drugs for infusion into the body, as well as for preparation of such sterile solutions just prior to use (including true solutions, emulsions and suspensions), lyophilized powders or concentrated solutions.
  • intravenous drip refers to a method of infusing a large amount of fluid containing a drug into the body intravenously through an infusion tube. Also known as “infusion”, “infusion”, “intravenous drip”, “hanging water”.
  • IL-17A refers to interleukin 17A.
  • IL-17RA refers to the receptor for IL-17A.
  • active ingredient refers to a drug molecule that has a therapeutic effect on a disease, such as the IL-17RA fusion protein described herein.
  • the present invention provides an IL-17RA fusion protein, which is characterized by comprising an operably linked signal peptide, an IL-17RA extracellular domain and an IgG1 constant region in series.
  • the role of signal peptides is mainly to guide the secretion of target proteins from the cytoplasm to the outside of the cell. Since this fusion protein has an IgG1 constant region, when it is secreted out of the cell, the two-molecule fusion protein forms a double chain through the combination of cysteines in the constant region and exerts activity.
  • the present invention adopts the extracellular domain of human IL-17RA (Gene bank number: NP_055154), and carries out R108K, D122G and H155D mutations, and the amino acid sequence of the mutant is shown in SEQ ID NO.1. Also preferably, the present invention adopts the extracellular domain of human IL-17RA, and carries out L9P, R108K, D122G and H155D mutations, and the amino acid sequence of the mutant is shown in SEQ ID NO.2.
  • the present inventors found that IL-17RA fusion proteins comprising these two mutants have increased thermostability, and increased binding affinity for IL-17A, compared to those comprising the wild type.
  • the amino acid numbering starts from position 1 of the amino acid sequence of the extracellular domain of the human IL-17RA.
  • the present invention adopts the extracellular domain of human IL-17RA, and carries out L9P, R108K, D122G, H155D, G243W and A267V mutations, and the amino acid sequence of the mutant is shown in SEQ ID NO.3.
  • the present inventors found that the IL-17RA fusion protein comprising the mutant has improved thermal stability and improved binding affinity to IL-17A, IL-17C, IL-17F, and IL-17A/F compared to the wild type. ; higher binding affinity for IL-17A compared to mutants containing the three and four mutations described above.
  • the present invention selects the constant region (Fc) of human IgG1 (Gene bank number: 3500) to form a fusion protein with IL-17RA.
  • the fusion protein not only retains the biological activity of the functional protein molecule, but also because the Fc part has certain antibody characteristics and is stable, the fused protein obtains a longer circulation life and prolongs the half-life.
  • IgG isotypes include IgG1, IgG2, and IgG4, which cause distinct ADCC, ADCP, and CDC effects that can have a major impact on toxicity in target and non-target tissues.
  • the constant region of IgG1 has strong ADCC, ADCP and CDC effects.
  • the present invention finds that selecting the constant region of IgG1 and further mutating the Fc sequence appropriately can reduce the ADCC, ADCP and CDC effects of the IL-17RA fusion protein.
  • the mutation sites are summarized in Table 1 below.
  • An additional mutation was to replace cysteine residues in the hinge region of IgG1 with serine residues to avoid the presence of unpaired cysteines in the fusion protein sequence.
  • the amino acid sequence of the IgG1-Fc mutant is shown in SEQ ID NO.4.
  • Signal peptides are one of the main factors affecting yield optimization and product quality. It is important that the signal peptide cleavage site should be well-defined by a single residue with a high probability of cleavage.
  • the natural signal peptide of human IL17RA is used for the fusion protein, and the amino acid sequence of the signal peptide is shown in SEQ ID NO.5.
  • a linker is used between the IL-17RA extracellular domain and the IgG1 constant region.
  • Linkers can be those commonly used in the art, such as one or more contiguous GSGs, one or more contiguous GGGGSs, and GSAGSAAGSG.
  • the amino acid sequence of the IL-17RA fusion protein is shown in SEQ ID NO.6, SEQ ID NO.7 or SEQ ID NO.8 (the IL-17RA extracellular domain has three mutations and four mutations, respectively). , six mutations), which has 522 amino acid residues.
  • SEQ ID NO. 8 the sequence of SEQ ID NO. 8 is shown in Figure 1, where the first 32 amino acids (bold) are the IL-17RA signal peptide, and the letters on a light gray background are recombination with 6 mutations (bold underlined letters)
  • Asterisks (*) indicate potential glycosylation sites.
  • the mechanism of action of the IL-17RA fusion protein provided by the present invention is that the decoy receptor (IL-17RA-Fc) is used to compete with the natural receptor for binding to IL-17 molecules, and the IL-17A, IL-17C, IL-17F and IL-17A/F binds with high affinity and selectively blocks the binding of IL-17A, IL-17C, IL-17F and IL-17A/F to their receptors, but not IL-17B, IL-17D, IL- Combined with 17E, it can effectively block the biological activity of a variety of pro-inflammatory IL-17 cytokines and inhibit the inflammatory signaling pathway, thereby more effectively alleviating the symptoms of autoimmune diseases. Good therapeutic benefit and a better safety profile than IL-17RA mAb.
  • the present invention also provides an isolated nucleic acid encoding the IL-17RA fusion protein according to the present invention.
  • the present invention utilizes molecular biology techniques to design the cDNA sequence of the fusion protein, and optimizes the codons to express it in CHO cells.
  • the optimized DNA sequences encoding the amino acid sequences SEQ ID NO.1-8 are shown in SEQ ID NO.9-16, respectively.
  • the present invention also provides an isolated mRNA transcribed from the DNA encoding the fusion protein of the present invention.
  • the present invention also provides an expression vector containing the nucleic acid according to the present invention operably linked to a promoter.
  • the present invention also provides a host cell containing the expression vector according to the present invention.
  • CHO cells suitable for growth in suspension and serum-free media are used as host cells.
  • the expression vector contains two selectable markers, puromycin and methotrexate, which facilitate the creation of high-yielding and stable cell lines.
  • the host cell has a deposit number of CGMCC 21011.
  • the construction of the host cell uses CHO-S TM cells of Thermo Scientific Company, which can stably express the IL-17RA fusion protein of the present invention after construction.
  • the host cell also has many advantages: (1) It has accurate post-transcriptional modification function, and the expressed protein is closest to the natural protein molecule in terms of molecular structure, physicochemical properties and biological functions; (2) It can grow on the wall.
  • the host cell has been deposited in the General Microbiology Center (CGMCC) of the China Microbial Culture Collection Management Committee (CGMCC) on November 23, 2020.
  • CGMCC General Microbiology Center
  • CGMCC China Microbial Culture Collection Management Committee
  • the deposit address is: No. 3, No. 1, Beichen West Road, Chaoyang District, Beijing, Zip Code: 100101, and the deposit number is: : CGMCC No.21011.
  • the present invention also provides a protein dimer formed by the IL-17RA fusion protein according to the present invention, the dimer is composed of two molecules of the IL-17RA fusion protein passing through the cysteine of the IgG1 constant region Acids combine to form double strands.
  • the two-molecule fusion protein of the present invention has an IgG1 constant region, when it is secreted to the outside of the cell, the two-molecule fusion protein forms a double chain through the combination of cysteines in the constant region and exerts activity.
  • the present invention also provides the IL-17RA fusion protein according to the present invention or the protein dimer formed by the IL-17RA fusion protein prepared for the treatment of psoriasis, Crohn's disease, plaque psoriasis, Gastroenteritis, Behcet's syndrome, arthritis, uveitis, hidradenitis suppurativa, lichen planus, parapsoriasis, asthma, psoriatic arthritis, tendinitis, relapsing-remitting multiple sclerosis, thyroid related eye disease, juvenile rheumatoid arthritis, multiple sclerosis, lupus nephritis, spondyloarthritis, ankylosing spondylitis, rheumatoid arthritis, inflammatory bowel disease, nonalcoholic fatty liver, giant cell arteritis, Non-radiological axial spondyloarthritis, acne vulgaris, triple negative breast tumor, multiple myeloma, non-small
  • the present invention is based on the binding affinity with IL-17A, biological activity cell experiments, GRO- ⁇ factor inhibitory ability, in vitro activity titer, in vivo titer, ADCC/CDC functional activity, FcRn binding affinity, safety pharmacology, pharmacokinetics
  • the pharmacological activity and safety of IL-17AR fusion protein were comprehensively evaluated in terms of science and toxicology.
  • the IL-17RA fusion protein can target multiple targets such as IL-17A, IL-17C, IL-17F, IL-17A/F, etc., and has high affinity, so that it can selectively block IL-17A , IL-17C, IL-17F and IL-17A/F are combined with their receptors, thereby effectively blocking the biological activity of a variety of pro-inflammatory IL-17 cytokines, inhibiting inflammatory signaling pathways, so it can be more effectively alleviated
  • better treatment benefits can be obtained than single IL-17A target antibody drugs, and better safety than IL-17RA monoclonal antibodies.
  • the present invention also provides a pharmaceutical composition, comprising a therapeutically effective amount of the IL-17RA fusion protein according to the present invention or a protein dimer formed by the IL-17RA fusion protein as an active ingredient and pharmaceutically acceptable excipients .
  • compositions can be selected according to the dosage form used and actual needs.
  • the present invention thoroughly studies and designs the preparation and preparation prescription of the IL-17RA fusion protein from the aspects of packaging material, buffer system, auxiliary materials, dosage, prescription composition, freeze-drying process and the like.
  • the pharmaceutically acceptable adjuvants of the pharmaceutical composition are selected from one or more of diluents, buffers, protective agents, surfactants, and antioxidants.
  • the buffer is selected from histidine-acetate buffer, Tris-acetate buffer, hydrochloric acid buffer, phosphate buffer, acetate buffer, histidine buffer, arginine buffer, succinate buffer, One or more of citrate buffers.
  • the protective agent is selected from one or more of trehalose, Tween-20, Tween-80, sucrose, amino acids (eg, arginine), polyols, disaccharides, and polysaccharides.
  • trehalose and the combination of trehalose and arginine are most preferred.
  • the trehalose is present as trehalose dihydrate.
  • the surfactant is selected from one or more of Tween-20, Tween-80, and poloxamer.
  • a single dose of the pharmaceutical composition comprises 5 mg/ml-150 mg/ml of the IL-17RA fusion protein or a protein dimer formed by the IL-17RA fusion protein.
  • the pharmaceutical composition of the present invention can be prepared into suitable dosage forms as required.
  • the IL-17RA fusion protein is preferably in the form of lyophilized preparation or injection.
  • the present invention also provides a method for the treatment of psoriasis, Crohn's disease, plaque psoriasis, gastroenteritis, Behcet's syndrome, arthritis, uveitis, hidradenitis suppurativa, flat lichen, parapsoriasis, asthma, psoriatic arthritis, tendonitis, relapsing-remitting multiple sclerosis, thyroid-related eye disease, juvenile rheumatoid arthritis, multiple sclerosis, lupus nephritis, spondyloarthritis, Ankylosing spondylitis, rheumatoid arthritis, inflammatory bowel disease, non-alcoholic fatty liver disease, giant cell arteritis, non-radiological axial spondyloarthritis, acne vulgaris, triple negative breast tumors, multiple myeloma, Injections for non-small cell lung cancer, adenocarcinoma, colorectal cancer,
  • the injection can be in the form of a lyophilized powder or a liquid preparation.
  • the injection is a subcutaneous injection or an intravenous drip. Most preferred are subcutaneous injections.
  • the injection preferably contains the IL-17RA fusion protein, Tris-acetic acid, arginine, trehalose, Tween-20 and a suitable solvent.
  • the injection comprises 5mg/ml-150mg/ml of the IL-17RA fusion protein or the protein dimer formed by the IL-17RA fusion protein, 2-100mM Tris-acetic acid, 10-250mM Arginine, 50-500 mM trehalose, 0.01-5% Tween-20 and a suitable solvent.
  • the solvent can be the solvent commonly used in the preparation of injections.
  • water for injection buffered saline solution, aqueous dextrose solution, aqueous sodium chloride solution or lactated Ringer's solution, etc.
  • the liquid preparation is prepared by using a solvent.
  • the liquid preparation can be prepared according to the formulation of the present invention by using methods commonly used in the pharmaceutical field.
  • Lyophilized powders can be prepared by freeze-drying the liquid formulation.
  • the lyophilization process includes pre-freezing, primary drying (sublimation) and secondary drying (decomposition).
  • Pre-freezing includes reducing the temperature from 5°C to -40°C and holding for a suitable time; primary drying includes raising the temperature to -5 to 0°C and holding for a suitable time; secondary drying includes raising the temperature to 25°C to 30°C for a suitable time.
  • the present invention has developed a pharmaceutical preparation with excellent drug stability and safe injection administration.
  • the percentage concentration (%) of each reagent refers to the volume percentage concentration (% (v/v)) of the reagent.
  • NVS451 (ie, IL-17RA fusion protein) consists of a signal peptide, rhIL17-RA ECD (ie, human IL17-RA extracellular domain), linker peptide and IgG1 Fc domain.
  • the signal peptide adopts the natural signal peptide of IL17RA.
  • the rhIL17-RA ECD contains six mutation sites, namely L9P, R108L, D122G, H155D, G243W and A267V (the amino acid numbering does not include the signal peptide sequence).
  • the linker peptide is GSG.
  • the IgG1 Fc domain employs a hybrid of IgG1, 2, 4 to remove potential ADCC, CDC and ADCP effects.
  • the amino acid sequence of NVS451 is shown in SEQ ID NO.8, and the nucleotide sequence is shown in SEQ ID NO.16.
  • Plasmids were constructed using the pCHO1.0 mammalian cell expression vector from Thermo Scientific.
  • the pCHO1.0 expression vector contains two expression cassettes. Since the expression of the NVS451 fusion protein requires only one expression cassette, the other expression cassette was removed using the SfiI restriction site. The removed expression cassette contains EcoRV and PacI clones site. The pCHO1.0 vector after removing one expression cassette was verified by sequence sequencing and named pCHO1.1.
  • the synthesized NVS451 fusion protein gene was inserted into the pCHO1.1 (Kan resistance) expression vector through the AvrII and Bstz17I cloning sites, and transformed into DH5 ⁇ competent cells, plated and screened, inoculated and amplified the clone containing the correct size plasmid, and the plasmid was extracted to obtain a large number of The constructed NVS451 fusion protein expression plasmid pCHO1.1-NVS4510. Gene sequencing verified that the pCHO1.1-NVS451 expression vector was constructed correctly.
  • NVS451 fusion protein cell line selected CHO-S TM (a cell bank (cGMP-banked) that complies with current drug production management standards), and the cell and the supporting expression vector pCHO1. Kit) cells as the primitive cell matrix.
  • the NVS451 fusion protein expression plasmid pCHO1.1-NVS451 contains selection markers for puromycin and MTX.
  • pCHO1.1-NVS4510 was transfected into CHO-S cells by transfection reagent.
  • the transfection procedure was as follows: 50 ⁇ g circular expression plasmid pCHO1.1-NVS451 was diluted to 1.5 ml volume by Opti PRO SFM, 50 ⁇ l Freestyle Max Transfection Reagent (GIBCO) was diluted to 1.5 ml by Opti PRO SFM, and then plasmid dilution and transfection Mix equal volumes of staining reagent diluents and add 30 ml of CHO-S cell suspension of 1E6 cells/ml. After 48 hours of culture of the transfected cells, a two-stage selection of pools of stably transfected clones (Pools) was performed using puromycin and MTX as the selection pressure.
  • CHO-S lacks pac (adenylate cyclase) activity and only has basal DHFR (dihydrofolate reductase) activity
  • pac adenylate cyclase
  • DHFR basal DHFR
  • CHO that was transfected with pCHO1.1-NVS4510 plasmid and integrated into the genome -S cells can survive and propagate in CD FortiCHO TM selection medium containing puromycin and MTX. Screening was performed in two phases, each using puromycin and MTX. Finally, four stable cloned cell pools (Pools) were formed, which were T3S1, T3S2, T3S3, and T3S4. Expression levels were assessed by simple fed-batch culture (SFB) for 14 days.
  • SFB simple fed-batch culture
  • T3S1 and T3S4 were selected for isolation of single clones (LDCs). Isolation of single clones Two rounds of limiting dilution were used to isolate single clones, and the cell plating concentration in each round was less than 1 cell/well (according to the statistical mathematical calculation method), and finally 5 candidate clones were obtained: T3S4-7E3-6G5; T3S4- 7E3-3C3; T3S4-17G2-6E2; T3S4-8F2-6E10; T3S1-18E7-6C5.
  • the stability of 5 candidate monoclones was evaluated at 70PDL (cell doubling time, equivalent to 70 passages). Based on the stability of expression stability, gene copy number, and mRNA transcription level, the final selection of monoclonal VAN301-T3S1- 18E7-6C5 was used as the main single clone (the deposit number was CGMCC 21011), and VAN301-T3S4-17G2-6E2 was selected as the backup clone.
  • VAN301-T3S1-18E7-6C5 was used to establish PCB (primary cell bank), and on this basis, MCB (master cell bank) and WCB (working cell bank) under GMP conditions were established.
  • the NVS451 fusion protein can be produced by a 14-day fed-Batch culture process.
  • Cell recovery Take a cell from the cell bank, recover it in a water bath at 37.0 ⁇ 0.5°C, transfer the seed solution to 10ml pre-warmed Dynamis (containing 8mM L-Gln, 1:100ACA and 1g/L P188) immediately after thawing for culture Centrifuge at 300g for 5 minutes in a 50ml centrifuge tube, discard the supernatant, add 10ml of pre-warmed Dynamis medium to resuspend the cells, and transfer the resuspended cell solution to a 125ml shake flask containing 28.0ml of Dynamis medium.
  • pre-warmed Dynamis containing 8mM L-Gln, 1:100ACA and 1g/L P188
  • the cell density was (0.15 ⁇ 0.35) ⁇ 10 6 cells/ml, and the cell viability was greater than 90%.
  • Seed passage and expansion the passage medium is Dynamis, the initial seeding density of cells in subculture should be (0.40 ⁇ 0.10) ⁇ 10 6 cells/mL, and the cell density reaches (2.0 ⁇ 5.0) ⁇ 10 6 cells after 3 days of culture /mL can be passaged, and the cell viability rate should be higher than 90.0% during the passage.
  • Reactor fed batch culture (Fed batch): the initial seeding density of cells was (0.40 ⁇ 0.10) ⁇ 10 6 cells/mL, fed with 2 ⁇ Feed C+ (Efficient FeedC+, Cat#: A2503101, Gibco), every day during the culture The contents of glucose and lactate were detected, and 450.0g/kg of glucose mother solution was added to make up to 5.0g/L on the 3rd/5/7/9/11/13 days respectively.
  • Cell culture harvest conditions Harvest when cultured to the 14th day or when the cell viability rate is lower than 80.0%, whichever comes first.
  • the cell culture harvest solution was first clarified by a two-stage depth filtration membrane package, and then 1% Tween 80, 0.3% tributyl phosphate was added for virus inactivation, and then the target protein was captured using GE's MabSelect SuRe affinity chromatography packing , and then use Millipore's Eshmuno CPX anion chromatography packing and GE's Capto Adhere cationic chromatography packing for two-step refinement, then use ASAHI KASEI's BioEX nanofiltration membrane for nanofiltration, use Millipore's Pellicon 2, interception
  • the PES material with a molecular weight of 50KDa and the C-channel ultrafiltration membrane are used for ultrafiltration diafiltration, and finally the auxiliary material polysorbate 20 is added, and the NVS451 stock solution is obtained after sterilization and filtration.
  • the inlet flow rate is less than or equal to 100LMH (based on A1HC)
  • the maximum load of DOHC is 60L/m 2
  • the maximum load of A1HC is 140L/m 2 .
  • the pressure is controlled, and the single-step recovery rate of deep filtration is generally about 90%.
  • Preliminary purification of the product was achieved by affinity chromatography using MabSelect SuRe media. Use 50 mM Tris-HAc, 150 mM NaCl, pH 7.4 as equilibration and post-loading rinse 1 buffer, then 20 mM Tris, 1 M NaCl, 0.5 M Arg, pH 8.6 for rinse 2, and finally 50 mM glycine, pH 3. 5 buffer to elute the target protein. The eluate was neutralized to pH 7.8-8.2 with 1 M Tris base.
  • Eshmuno CPX from Millipore was used as the cation chromatography packing.
  • the samples after neutralization by affinity chromatography were adjusted to pH 6.3-6.7 with 1M HAc, and the adjusted samples were used as samples for cation chromatography.
  • Product-related impurities such as HCP, protein A, DNA and some product analog impurities and fragments can be effectively removed after washing and gradient elution.
  • GE's Capto Adere packing was used as an anion chromatography packing to remove some product and process related impurities.
  • the cation chromatography eluent was first adjusted to pH 8.4-8.6 with 1M Tris base and water for injection, and the conductivity was 19.0-23.0.
  • the sample after pH adjustment was used as the sample for anion chromatography.
  • the nanofiltration process mainly utilizes the different molecular sizes of virus and protein products, the potential virus is intercepted by the nanofiltration membrane, and the target protein flows through, thereby realizing the separation of the two.
  • the pre-filtration membrane can adsorb impurities such as particles in the sample, and at the same time increase the processing capacity of the nanofiltration membrane.
  • Nanofiltration membranes can effectively retain potential viruses such as parvoviruses.
  • A1HC (Millipore Corporation) was used for prefiltration and BioEX (Asahi Kasei Corporation) to remove potential viruses.
  • the intermediate product was concentrated using a Pellicon 2 (Millipore Company) ultrafiltration membrane bag, and then the solution was changed to a buffer system of 20 mM Tris-HAc, 65 mM Arg, 120 mM trehalose, pH 7.5.
  • the ultrafiltration membrane package material is PES, the molecular weight cut off is 50kDa, and the C flow channel.
  • the loading capacity of ultrafiltration and diafiltration is ⁇ 300g/m 2 ;
  • the inlet flux is 150-300LMH, the transmembrane pressure (TMP) is ⁇ 1.5bar, and the concentration is 18.0-22.0g/L;
  • TMP transmembrane pressure
  • concentration is 18.0-22.0g/L;
  • the inlet flux is 150-300LMH, TMP ⁇ 2bar, the liquid exchange volume is ⁇ 5DV, the inlet flux is 150-300LMH, TMP ⁇ 2bar when overconcentrated, and the concentration is 80.0-90.0g/L.
  • the active molecule of NVS451 is a double-chain fusion protein composed of human IL-17RA extracellular segment mutant and human IgG1Fc mutant, which was obtained by recombinant expression in CHO cells. Therefore, the molecule can exhibit the biological functional properties of both IL-17RA and Fc molecules.
  • SPR surface plasmon resonance
  • in vitro target cell killing assay and other analytical techniques, aiming to clarify its in vitro pharmacodynamic properties.
  • NVS451 (at 75ug/ml) binds human IL-17A with high affinity.
  • Surface plasmon resonance was performed with 100nM-0.8nM human IL-17A (Acrobiosystems, Cat:ILA-H5118), and the experimental method was as follows:
  • Coating solution take 1.06g of Na 2 CO 3 and 0.84g of NaHCO 3 and fully dissolve them in ultrapure water, adjust the pH to 9.60 with concentrated hydrochloric acid, and then dilute to 200 ml, filter with 0.22 filter membrane, and store at 4°C;
  • 10 ⁇ TBS mother liquor Take 12.114g of Tris and 43.83g of NaCl into ultrapure water to fully dissolve, adjust the pH to 7.55 with hydrochloric acid, and then dilute to 500ml, filter with 0.22 membrane, and store at 4°C;
  • Substrate buffer mother solution Take 7.16g of NaHPO ⁇ 12HO and 2.1g of citric acid and fully dissolve in ultrapure water, adjust the pH value to 5.5 with NaOH, then dilute to 100ml, filter with 0.22 filter membrane, put Store at 4°C.
  • IL-17A Reconstitute IL-17A with ultrapure water according to COA, leave it for about 30 minutes to fully dissolve IL-17A, dilute IL-17A to 30 nM with a well-equilibrated coating solution at room temperature, and add 100 ⁇ l per well to the microtiter plate. , and the sealing film was placed at 4°C overnight (about 16h).
  • the lotion is prepared as it is. Take 100ml of 10 ⁇ TBS mother solution and add it to 900ml of ultrapure water to dilute to 1 ⁇ TBS, then add 2.5ml of 20% Tween 20, and wash the plate 4 times with the prepared lotion. Well 300 ⁇ l, pat dry.
  • the diluent is prepared now. Take 5ml of blocking solution and dilute it to 50ml with washing solution, prepare 0.5% BSA sample diluent, dilute the sample to 10 ⁇ g/ml by 2-fold gradient, and add the diluted sample to the ELISA plate. 100 ⁇ l/well, set background control (no coating + sample + secondary antibody, coating + no sample + secondary antibody, no coating + no sample + no secondary antibody). The plates were sealed with sealing film and incubated at 37°C for 1 h.
  • the diluent is prepared as it is, and 5 ml of the blocking solution is diluted to 50 ml with washing solution to prepare a 0.5% BSA antibody diluent.
  • the AffiniPure goat anti-human IgG and Fc ⁇ fragment-specific (min X Bov, Hrs, Ms Sr Prot) was diluted to 1:12000, 100 ⁇ l was added to each well, and the plate was sealed with a sealing film and incubated at 37°C for 1 h.
  • the absorbance (OD value) of each well was measured at a wavelength of 450 nm, and the average value was calculated.
  • the IL-17RA fusion protein (the amino acid sequence as SEQ ID NO.6 and shown in SEQ ID NO. 7).
  • the affinity of V302 and V303 to IL-17A was detected by affinity ELISA.
  • Two kits (R&D, Cat#: 317-ILB-050; Peprotech, Cat#: 900-K84) were used for detection according to the method provided by the manufacturer, and the results showed (Fig. 19) that NVS451 with six mutations and The percentage of IL-17A affinity (776% and 464% for the two kits above) was significantly higher than the affinity of V302 and V303 molecules for IL-17A.
  • the affinity of V302 and V303 molecules to IL-17A was comparable to that of wild-type human IL-17RA.
  • IL-17A molecules (KINGFISHER, Cat. Nos.: RP0921H-025, RP1031Y-025, RP0355M-025) were compared under the same conditions in 4 species (human, cynomolgus monkey, mouse, rat); The binding of rat Biolegend, 778704) and NVS451, the results show that NVS451 can bind to 4 different species of IL-17A, and the binding affinity is from high to low: human > cynomolgus > rat > mouse (KD See Table 3).
  • NVS451 In addition to binding to IL-17A molecules of the human IL-17 family, NVS451 also bound to human IL-17C, IL-17F, and IL-17AF (the results are shown in Figures 6-8, respectively). The KD results are summarized in Table 4.
  • the cell line CCD-1070Sk (human fibroepithelial cell line, ATCC CRL-2091) used for the in vitro activity assay of NVS451 belongs to the human fibroblast cell line, and on its surface there are many proteins including IL-17A, A/F and F. cytokine receptors.
  • IL-17A can bind to the IL-17A receptor IL-RA on the cell surface to stimulate the cells to produce the cytokine GRO- ⁇ , and its content can be accurately detected by ELISA double-antibody sandwich method.
  • NVS451 When IL-17A and NVS451 were co-incubated with CCD-1070Sk cells, NVS451 was able to inhibit the binding of IL-17A to the IL-17A receptor IL-17RA on the cell surface by competing with IL-17A binding, thereby reducing the cell supernatant The amount of GRO- ⁇ secreted in the liquid. And NVS451 can also reduce the secretion of GRO- ⁇ in the cell supernatant by combining with IL-17A/F and IL-17F based on the same competitive inhibitory mechanism. Therefore, the in vitro activity of NVS451 on multiple targets including IL-17A, IL-17A/F, and IL-17F can be determined by measuring the secretion of GRO- ⁇ in cells.
  • the relative potency of the NVS451 fusion protein in vitro was analyzed by measuring the inhibitory ability of IL-17A-induced GRO- ⁇ release from the human fibroblast cell line CCD-1070Sk, and the results were expressed as the median inhibitory concentration IC50 value (Table 6) .
  • NVS451 protein was diluted to an initial concentration of 4ug/ml (final concentration of 1ug/ml), and then a two-fold gradient was diluted to 9 points, and At the last 0ug/ml concentration point, 50ul per well was added to the cell plate, then IL-17A was diluted to a concentration of 40ng/ml (final concentration was 10ng/ml), and 50ul per well was added to each reaction well, and the cells were given The drug was incubated for 24 hours.
  • wash buffer needs to be left at room temperature one day in advance until fully dissolved.
  • sample diluent 50 ⁇ l of the pre-incubated sample (sup) was diluted in 200 ⁇ l of sample diluent (samples are now diluted 1:5) ready for loading.
  • the diluted detection antibody biotinylated goat anti-human GRO ⁇ antibody, R&D Cat# DY275-05-840256
  • diluent was 1.0 ⁇ g/ml (initial concentration 100 ⁇ g/ml).
  • P. Use a microplate reader to monitor the color development at 405nm, and the wavelength correction is set at 650nm.
  • the human fibroblast cell line CCD-1070Sk induced by IL-17A/F and IL-17F can also produce GRO- ⁇ cytokine, and the in vitro titer results of NVS451 fusion protein induced by different ligands Expressed as the median inhibitory concentration IC50 value (Table 7).
  • the S-curves of inhibition are shown in Figure 10 and Figure 11, respectively.
  • NVS451 inhibits the detection results of human skin fibroblasts CCD-1070Sk GRO-a secretion
  • Psoriasis is a chronic inflammatory skin disease characterized by excessive proliferation and abnormal differentiation of keratinocytes. It is caused by a variety of factors. The pathogenesis has not been fully elucidated. cells, T cells, endothelial cells, etc.), and cytokines are involved in the pathogenesis of psoriasis and the maintenance of the disease state [1].
  • the existing widely used animal models can be roughly divided into drug-induced acute inflammation models, genetic engineering models and xenograft models.
  • IMQ Imiquimod
  • TLR Toll-like receptor 7/8
  • Van der Fits[2] Van der Fits[2] and other studies found that IMQ-induced mouse psoriasis-like
  • TLR Toll-like receptor 7/8
  • IMQ-induced mouse psoriasis-like The changes in skin lesions are accompanied by changes in the IL-23/IL-17 axis, which have many similarities with the pathological changes of human psoriasis, and have the advantages of simple operation and low cost. Therefore, IMQ was applied to the skin of mice to establish psoriasis. It is a psoriasis model widely used at home and abroad.
  • SCID xenograft skin immunodeficiency mouse
  • NVS451 protein concentrations of NVS451 protein were formulated into a formulation of 20 mM Tris-acetic acid, 65 mM arginine, 120 mM trehalose, 0.02% Tween 20, pH 7.5.
  • mice Envigo, Jerusalem, Israel C.B-17/IcrHsd-scid-bg (beige-SCID) mice
  • mice After transplanting healthy human skin on the back of the mice, they were randomly divided into 6 groups of 10 in each group.
  • Mice negative control group (negative control group is using the above formulation to remove NVS451), hormone group (2mg/animal dexamethasone), secukinumab group (60mg/kg) and test article high dose (15mg /kg), middle dose (10mg/kg), low dose (5mg/kg) groups.
  • the negative control group, the test product group and the Sujin group were given subcutaneous injection every other day for a total of 28 days.
  • the hormone group was given topical dexamethasone twice a day for 28 days. At the end of the experiment, the appearance of the skin was observed (see Figure 12 for the results), and the thickness of the skin was measured. The results showed that NVS451 had a certain degree of improvement in redness, plaque and scale, and was positively correlated with the dose; there was no statistical difference in epidermal thickness between the 15 mg/kg dose group and the Sujin group (P>0.05). Similar healing effect to Sukin.
  • NVS451 the formulation described in 2.1
  • SCID mouse psoriasis model 15, 22.5, 30, 45, 60 mg/kg NVS451
  • the xenograft skin recovered completely with reduced epidermal thickness. However, in the 15 mg/kg twice-weekly group, the remaining 3/10 showed partial recovery compared with the other treatment groups, with a skin thickness of 334 ⁇ 174 ⁇ m and a histological score of 1.1 (according to the thickness of the skin lesions, thinning of the epidermis on the papilla, and keratinization). Hyperplasia, parakeratosis, agranulocytosis, Munro microabscess, normal or abnormal reticular crest elongation, vascular tortuosity, mononuclear cell infiltration in the papillary dermis and other indicators).
  • NVS451 exerting its efficacy is not dependent on ADCC/CDC activity, and the cytotoxic effect of Fc ADCC/CDC may bring unnecessary immune-related adverse events (irAEs), posing potential safety risks [9]. To avoid this effect, the relevant active site was mutated in the Fc portion of NVS451.
  • affinity analysis study seven CD molecules associated with ADCC and C1q molecules associated with CDC were evaluated. The results showed that the binding affinity of NVS451 (75ug/ml) to ADCC-related CD molecules was at non-binding or low levels (KD greater than the order of 10-5 M, see Table 12).
  • amino acid sequence of V301-wtFc is shown in SEQ ID NO.17.
  • the Fab regions of IgG1 and IgG3 subtype antibodies bind to target cells first, and then their Fc part binds to Fc receptors (such as Fc ⁇ RIII) on CTL cells such as NK, and then can induce ADCC.
  • Fc receptors such as Fc ⁇ RIII
  • Cell models lacking membrane-expressed IL-17 cannot be validated in vitro for ADCC production. Therefore, the Fab segment of Rituximab that can stimulate ADCC (Rituximab) and the Fc segment of NVS451 are fused to construct RitxV301 (i.e. RitxNVS451) to verify the chimeric molecule (the amino acid sequence of the light chain is shown in SEQ ID NO.18, The amino acid sequence of the heavy chain is shown in SEQ ID NO. 19).
  • ADCC/CDC effect is verified (analyze the experimental data with GraphPad Prism 5 analysis software, take the logarithm of the concentration of the antibody as the x-axis, and the corresponding calculated killing rate value is the y-axis, select a four-parameter equation regression simulation, and fit The measurement effect curve of the combined antibody.
  • Fc fusion proteins generally increase the half-life of target protein drugs and improve their pharmacokinetic characteristics through FcRn-mediated circulation of Fc [10].
  • the binding affinity to FcRn is related to the half-life of the drug in vivo [11].
  • NVS451 can bind to human, monkey and rat FcRn (acrobiosystems FCM-H5286, FCM-C5284, FCM-R5287). Under the same conditions, its affinity is at the same level as secukin antibody and wild-type IgG1Fc (ie V301-wtFc) (KD is in the order of 10 -8 M, see Table 14).
  • the experimental method is as follows:
  • Running reagent containing 2 mM KH 2 PO 4 , 10 mM Na 2 HPO 4 , 137 mM NaCl, 2.7 mM KCl, 0.05% Tween-20 (Tween-20), pH adjusted to 6.0;
  • His Capture Kit (Cat. No. 28-9950-56, GE), including: mouse anti-His antibody (1mg/mL), immobilization reagent (10mM sodium acetate, pH 4.5), regeneration reagent (glycine hydrochloride, pH 1.5);
  • Amino Coupling Kit (Cat. No. BR100050, GE), including: 115mg N-hydroxysuccinimide (NHS), 750mg 1-ethyl-(3-dimethylaminopropyl) carbodiimide salt acid (EDC) and 10.5 mL of 1 M ethanolamine (pH 8.5). Add 10 mL of deionized water to each tube of EDC and NHS, respectively, and store in aliquots at -18°C to a lower temperature. The shelf life is two months. (Refer to GE Amino Coupling Instruction Manual "22-0510-62AG").
  • the secukinumab and IL-17 RA-wtFc proteins were desalted using a desalting column and Running Buffer.
  • the concentration of the desalted protein was determined by UV-V.
  • the desalted protein should be aliquoted at a size greater than 10 ⁇ g per tube, and repeated freezing and thawing should be avoided.
  • the mouse anti-His antibody was diluted to 50 ⁇ g/mL with immobilization reagent (10 mM sodium acetate, pH 4.5). About 100 ⁇ L of mouse anti-His antibody was used for each channel of the chip, and about 190 ⁇ L of immobilization reagent was used to fix two channels and 10 ⁇ L of mouse anti-His antibody was added.
  • immobilization reagent 10 mM sodium acetate, pH 4.5.
  • mice anti-His antibody 50 ⁇ g/mL was injected into the experimental channel (FC4) at a flow rate of 10 ⁇ L/min for about 420 s, and the immobilized amount was about 9000 to 14000 RU. Finally, the chip was blocked with 1 M ethanolamine at 10 ⁇ L/min for 420 s.
  • the reference channel (FC3) performs the same operation as the test channel (FC4). (Refer to GE's His Capture Kit Instruction Manual "28-9974-71 AB").
  • FcRn stock solution was diluted to 0.5 ⁇ g/mL with running reagent and injected into the experimental channel (FC4) for approximately 40 RU at a flow rate of 10 ⁇ L/min.
  • FC3 The reference channel (FC3) does not require ligand capture.
  • KD values for each antibody were calculated using Biacore 8K analysis software.
  • the reference channel (FC3) is used for background subtraction.
  • NVS451 (concentration: 25nM-1.5625nM) can bind to FcRn of 3 different species of human, cynomolgus monkey and rat under acidic conditions of pH6.0 ( Figure 17); and at pH7.4 None of them bound under neutral conditions (NVS451 concentration 100 nM-1.5625 nM) ( Figure 18).
  • Cynomolgus monkeys purchased from Beijing Zhongke Lingrui Biotechnology Co., Ltd.
  • safety pharmacology test was carried out with long-term toxicity, subcutaneous injection of the prescription in Section 2.1 of Experimental Example 1, NVS451 doses of 15mg/kg, 50mg/kg and 150mg/ kg (administered twice a week for 4 consecutive weeks, a total of 9 doses, ie, administered on D1, D4, D8, D11, D15, D18, D22, D25 and D29, respectively).
  • an excipient control group was set up, and NVS451 was removed by using the prescription in Section 2.1 of Experimental Example 1.
  • the safety and pharmacological indicators were detected in combination with the observation of toxicity indicators.
  • Body temperature can be increased in 2 hours (excipient control group (that is, NVS451 is removed from the formulation) vs high dose group: 37.72 ⁇ 0.29°C vs 38.40 ⁇ 0.14°C); female animals in high, medium and low dose groups of the test product can see body temperature 24 hours after the drug increased, excipient control group vs high, medium and low dose groups: 37.60 ⁇ 0.28 °C vs 38.80 ⁇ 0.23, 38.72 ⁇ 0.44, 38.54 ⁇ 0.28 °C). There was no male and female consistency in the above changes in body temperature, and no regular changes were found, which was considered to be irrelevant to drug administration.
  • a total of 24 cynomolgus monkeys were used in the experiment, and they were divided into 4 groups (6 animals in each group, half male and half female).
  • Single subcutaneous and intravenous administration, group 1 to group 4 were given 5 (subcutaneous injection), 15 (subcutaneous injection), 50 (subcutaneous injection), 15 (intravenous injection) mg/kg of NVS451, administration volume 1mL/ kg.
  • Pharmacokinetic blood samples (about 1 mL) were collected from the non-administration site of the subcutaneous vein of the hind limbs of the animals to the tube without anticoagulant. 2h, 4h, 8h, 24h(D2), 32h(D2), 48h(D3), 56h(D3), 72h(D4), 96h(D5); the blood collection time points of the fourth group of animals were: before the drug (the day before ), 3 minutes, 1h, 2h, 6h, 24h(D2), 32h(D2), 48h(D3), 56h(D3), 72h(D4), 96h(D5) after the start of administration. Blood samples were used to prepare serum samples and for pharmacokinetic analysis (Table 15).
  • AUC ratio AUC last medium and high dose mean / AUC last low dose mean
  • F% (AUC last /dose (SC))/(AUC last /dose (IV))*100%.
  • test results showed that within the dose range of 5-50 mg/kg, after a single subcutaneous injection was administered to cynomolgus monkeys, the plasma concentration of NVS451 in cynomolgus monkeys increased with the dose; single subcutaneous injection and intravenous injection were administered to cynomolgus monkeys. After monkeys, no significant differences were observed between the sexes.
  • the mean Cmax and AUC last increased proportionally to NVS451 compared to dose proportionality.
  • the Cmax ratios of NVS451 in the single subcutaneous injection groups of 5, 15, and 50 mg/kg dose groups were 1:6.12:38.06 (male) and 1:5.46:34.29 (female), respectively; the AUC last ratios were 1:4.83:25.52, respectively (male) and 1:4.76:20.88 (female).
  • the independent samples t test showed that there was no statistical difference in the metabolic kinetic parameters between the subcutaneous injection and intravenous injection groups between the genders, and there was basically no significant gender difference in the metabolic characteristics of NVS451 in animals.
  • the T 1/2 of NVS451 in cynomolgus monkeys the average T 1/2 of male and female animals in each dose group was between 27.20 and 39.90; The drug concentration peaked in 4-8h.
  • Pharmacokinetic blood samples (about 0.4 mL) were collected from the animal's jugular vein in the experiment and placed in a tube without anticoagulant.
  • the blood collection time points of animals in groups 1-3 were: before administration (D-1), 1h, 2h, 4h, 8h, 24h, 32h, 48h, 56h, 72h, 96h; the blood collection time points of animals in group 4 were: before administration (D-1), 3min, 1h, 2h, 6h, 24h, 32h, 48h, 56h, 72h, 96h. Blood samples were used to prepare serum samples and for pharmacokinetic analysis (Table 16).
  • AUC ratio AUC last medium and high dose mean / AUC last low dose mean
  • F% (AUC last /dose (SC))/(AUC last /dose (IV))*100%.
  • test results showed that: within the dose range of 10-100 mg/kg, after a single subcutaneous injection was administered to SD rats, the plasma concentration of NVS451 in SD rats increased with the increase of the dose; single subcutaneous injection and intravenous injection of SD rats After mice, no significant differences were observed between the sexes.
  • the mean Cmax and AUClast of NVS451 increased proportionally less than the dose increased.
  • the ratios of Cmax of NVS451 in single subcutaneous injection of 10, 30 and 100 mg/kg groups were 1:2.03:6.56 (male) and 1:1.62:6.04 (female), respectively; the ratio of AUClast was 1:1.92:5.29 (male), respectively ) and 1:1.85:5.85 (female).
  • the T1/2 of NVS451 in SD rats and the T1/2 of male and female animals in each dose group were between 51.46 and 69.48; the Tmax of each dose group by subcutaneous injection was basically the same, and the drug concentration was between 8 ⁇ 24h peak.
  • the experiment consisted of 36 animals, divided into 6 groups, with 6 animals in each group, half male and half male.
  • the test article was administered as a single subcutaneous injection on the back of the neck at a dose of 30 mg/kg.
  • the radiochemical purity of the test product after 125I labeling was 99.03%, and the specific activity was 0.09KBq/ ⁇ g.
  • the feces and urine of the animals in the 5 groups were collected once a day for 5 days.
  • the 6 groups of animals collected bile every hour after injection for a total of 8 hours.
  • the drug After subcutaneous injection of 125I-NVS451 to rats, the drug is mainly distributed in intestinal contents, urine, bladder, back skin and other tissues/organs, but less in muscle, fat, and brain.
  • the peak time of the drug in most tissues is 4h to 24h, and then the concentration gradually decreases with the prolongation of time.
  • the drug has some distribution on the back skin (non-administration site).
  • the peak time is 24h, and the drug content of 4h and 120h is roughly equal.
  • the concentration of the drug decreases more slowly in the back skin.
  • NVS451 is a macromolecular protein drug that is expected to be degraded into peptides and amino acids in vivo and then excreted or re-used for in vivo protein or peptide synthesis , so the metabolism of NVS451 was not assessed.
  • Cynomolgus monkeys and SD rats were selected for toxicological studies, and NVS451 cross-reacted with cynomolgus monkeys and rat IL-17A, which were related species. All trials were conducted under GLP conditions, following the current Food and Drug Administration Good Laboratory Practice (21CFR Part 58), the State Drug Administration (formerly the State Food and Drug Administration) "Quality Management Practice for Nonclinical Drug Research” ” (Order No. 34 of the Bureau, September 2017).
  • the purpose is to evaluate the acute toxicity that may occur within 14 days after a single subcutaneous injection of NVS451 in cynomolgus monkeys, and set three dose groups of 45mg/kg, 150mg/kg and 450mg/kg. During the experiment, no animals in each group were found to be dead or dying. There was no abnormality in the clinical observation of animals in each administration group. Compared with its own pre-drug value and the excipient control group (with NVS451 removed from the formulation prescription), the body weight, body temperature, ECG parameters, blood cell count, coagulation index, blood biochemistry and urine examination of animals in each administration group showed no drug-related abnormalities. Change.
  • the purpose is to evaluate the acute toxicity that may occur within 14 days after single subcutaneous injection of NVS451 into SD rats, and set three dose groups of 90 mg/kg, 300 mg/kg and 900 mg/kg. During the experiment, no animals in each group were found to be dead or dying, and at the end of the observation period (D15), there was no abnormal change in the general observation of the animals in each group. Conclusion: Under the conditions of this experiment, all animals were not found dead or dying, and the maximum tolerated dose (MTD) of animals was greater than or equal to 900 mg/kg.
  • MTD maximum tolerated dose
  • mice 40 cynomolgus monkeys (20/sex), they were randomly divided into 4 groups (5/sex/group) according to gender, which were the excipient control group and the test product low-dose, medium-dose and high-dose groups.
  • Animals in excipient control group were given excipient control substance, 2mL/kg, and the low-dose, medium-dose and high-dose groups of test substance were given NVS451, the doses were 15, 50, and 150mg/kg, and the doses were 0.2, 0.67, and 2mL/kg, respectively. kg, and the administration concentration was 75 mg/mL.
  • mice All animals were subcutaneously injected into the hind limbs, twice a week for 4 consecutive weeks, 9 times in total, namely D1, D4, D8, D11, D15, D18, D22, D25 and D29.
  • the animals were subjected to clinical observation, body weight, body temperature, electrocardiogram, respiration (respiratory frequency and tidal volume), blood pressure, ophthalmological examination, blood cell count, coagulation function, blood biochemistry, urinalysis and immunological indicators (T lymphocyte subgroups).
  • RESULTS During the experiment, there was no death or dying in each group of animals. Clinical observation, body weight, body temperature, ophthalmological examination, urine examination, blood cell count, coagulation function (except FIB), blood biochemical indexes, lymphocyte subgroups of animals in each group were observed. Population, complement, cytokines (except IL-17A) and immunoglobulins showed no abnormal changes related to administration. There were no abnormal changes related to drug administration in the animals' ECG, respiratory and blood pressure parameters. There were no abnormal reactions such as erythema, edema, induration, and ulceration at the administration site.
  • the CRP increased (193.0%) in male animals in the high-dose group of the test product on the next day (D16) after 5 doses; the next day after the last dose (D30), Male animals in the low-dose, medium- and high-dose groups of the test article showed increased CRP (70.5%, 218.5%, 246.9%), and male animals in the high-dose group of the test article also increased FIB (56.4%), and the difference was statistically significant ( P ⁇ 0.05). After 4 weeks of drug withdrawal, the above changes were fully recovered.
  • the injection sites and inguinal lymph nodes of the animals in the 15, 50 and 150 mg/kg dose groups showed changes related to the test article, and the injection sites showed mild to moderate mononuclear cells in the dermis/subcutaneous/muscular layer Inflammation, which is an irritant reaction caused by the test product; mild to moderate increase in the number of lymphocytes in the paracortical area/medulla cord of the inguinal lymph nodes, which is related to the local mononuclear cell inflammation of the injection, and no obvious clinical pathological indicators. Changes are considered non-adverse reactions.
  • 120 rats in the main experimental group from groups 1 to 4 were used for toxicology studies (15 rats/sex/group), 5 Seventy-two rats in ⁇ 8 satellite groups were used for serum antibody and toxicokinetic testing (6-10 rats/sex/group).
  • the animals in the excipient control group were given NVS451 white excipient solution (4mL/kg); the low-dose, medium-dose and high-dose groups of the test product were given NVS451, respectively, at a dose of 30, 100, and 300 mg/kg, and the administration volume was 0.4, 1.33, 4 mL/kg.
  • the animals in the main experimental group were mainly subjected to clinical observation, and the body weight, food intake, body temperature, blood cell count, coagulation function, blood biochemistry, ophthalmological examination, urine examination, T lymphocyte subsets and cytokines were detected; the animals in the satellite group were examined.
  • Blood was collected before and after the first and eighth administrations for toxicokinetic testing, and at different time points (before the first administration (D-1), before the 15th administration (D14), before the last administration (D28) and the end of the recovery period (D56)) blood was collected for antibody determination.
  • the first 10 animals/sex/group in the main experimental group were euthanized on the next day after the last dose (D30), and the remaining animals were euthanized after the 4-week recovery period (D57). All animals in the main experimental group were subjected to gross anatomical observation, the main organs were weighed, and the relative organ weight was calculated; more than 40 kinds of tissues and organs were injected into the animals in the control group and high-dose group, and the animals in the low- and medium-dose groups were injected with local and tissue. Pathological examination.
  • mice in each group were not found dead or dying, and no abnormality was found in clinical observation. No erythema, hyperemia, swelling, ulcer and induration were found in the administration site. There were no abnormalities in the ophthalmological examination and urine examination of the animals in each group; compared with the control group of the same sex in the same period, the body weight, body temperature, food intake, blood cell count, coagulation function, blood biochemistry, and T lymphocyte subsets of the animals in each group were not found. Abnormal changes associated with administration.
  • the animals were euthanized.
  • the 30, 100 and 300 mg/kg dose groups showed local irritation related to the test article, manifested as mild to moderate mononuclear cell inflammation in the local subcutaneous and/or dermis injected.
  • the local inflammation of the injection was basically completely recovered.
  • the local tolerance toxicity test was carried out with the long-term toxicity. Both cynomolgus monkeys and SD rats were administered twice a week (30, 100 and 300 mg/kg in three dose groups for rats, 15, 50 mg/kg in three dose groups for cynomolgus monkeys). and 150 mg/kg) for 4 consecutive weeks, a total of 9 doses (ie D1, D4, D8, D11, D15, D18, D22, D25 and D29 doses). During the test, no abnormal reactions such as erythema, edema, induration, and ulceration were found in the local observation of animals in each group.
  • In vitro test tube method was used to observe the effect of 75mg/mL NVS451 on hemolysis and aggregation of human erythrocytes (experimental conditions were conventional). From 15 minutes after incubation in the incubator to the end of 3 hours of observation, the upper liquid of the test tube was colorless and clear, and the red blood cells at the bottom of the tube sank. Under the conditions of this test, 75mg/mL NVS451 has no hemolysis effect on human erythrocytes in vitro, and does not cause human erythrocyte aggregation.
  • NVS451 In vitro pharmacodynamics: Comparative analysis of binding affinity with IL-17A showed that NVS451 could bind to human IL-17A with high affinity, which was higher than that of secukinumab and wild-type human IL-17RA under the same conditions. In addition to binding to IL-17A molecules of the human IL-17 family, NVS451 also binds to IL-17C, IL-17F, and IL-17AF. The affinity of NVS451 to human IL-17C and IL-17F was higher than that of wild-type human IL-17RA.
  • NVS451 fusion protein showed inhibitory ability to IL-17A, IL-17A/F, IL-17F-induced GRO- ⁇ cytokines, and inhibited IL-17A-induced GRO- ⁇ cytokines.
  • the inhibitory ability is lower than the IC 50 value of secukinumab (Cosentyx).
  • NVS451 primarily acts by inhibiting the activities of IL-17A and IL-17C, IL-17F, and IL-17AF.
  • the interactions shown by the SPR experiments suggest that in vivo, the recommended dose of NVS451 exerts clinical activity mainly inhibiting IL-17A, supplemented by inhibition of IL-17C, IL-17F, and IL-17AF molecules in patients with psoriasis.
  • NVS451 binding affinity of NVS451 to ADCC-related CD molecules was at no binding or low level. Compared with native IgG1Fc, its affinity for ADCC/CDC-related Fc receptors was significantly reduced, proving that the ADCC binding site of NVS451 was removed by mutation . Binding affinity analysis to FcRn showed that NVS451 could bind to human, monkey and rat FcRn. Under the same conditions, its affinity is at the same level as secukinumab and wild-type IgG1Fc.
  • NVS451 can bind to FcRn of three different species of human, cynomolgus monkey and rat under the acidic condition of pH6.0, and it can bind to FcRn of three different species at pH7.4. Neither binds under neutral conditions.
  • NVS451 is a soluble antibody Fc fusion protein carrying the human IgG1 Fc domain, so it can theoretically interact with Fc receptors, but the mechanism of NVS451's drug efficacy does not depend on ADCC/CDC activity, and the cytotoxicity of Fc affects ADCC /CDC may bring unnecessary immune-related side effects and present potential safety risks. To avoid this effect, the relevant active site was mutated in the Fc portion of NVS451.
  • NVS451 Under GLP conditions, NVS451 was administered with a single subcutaneous injection of 30 mg/kg, 100 mg/kg and 300 mg/kg. The results showed that NVS451 had no effect on the central nervous system function of rats.
  • the safety pharmacology test of cynomolgus monkeys is carried out with long poison: subcutaneous injection at doses of 15, 30 and 150 mg/kg, the test substance has no obvious effect on the central nervous system, cardiovascular system and respiratory system of cynomolgus monkeys.
  • NVS451 cross-reacts with cynomolgus monkey and rodent IL-17A
  • cynomolgus monkey and rat were selected as relevant species for toxicity evaluation.
  • NVS451 was repeatedly subcutaneously injected into cynomolgus monkeys at doses of 15, 50 and 150 mg/kg, 2 times a week, for 4 consecutive weeks, for a total of 9 doses. There was no death or moribund in each group of animals. Obvious systemic toxicity, the no-adverse-effect dose (NOAEL) in this trial was considered to be 150 mg/kg.
  • NOAEL no-adverse-effect dose
  • the NVS451 fusion protein for injection was administered subcutaneously to SD rats at doses of 30, 100 and 300 mg/kg, 2 times a week, for 4 consecutive weeks, for a total of 9 times, and no obvious systemic toxicity was found in the animals. , the no-adverse reaction dose (NOAEL) was considered to be 300 mg/kg. Cynomolgus monkey and rat toxicokinetics showed no accumulation of NVS451 after repeated administration.
  • the T1/2 of NVS451 in cynomolgus monkeys within the dose range of 5-50 mg/kg, the T1/2 of NVS451 in cynomolgus monkeys, the male and female of each dose group The average T1/2 of animals was between 27.20 and 39.90h.
  • the T1/2 of NVS451 in SD rats ranged from 10 to 100 mg/kg, and the T1/2 of male and female animals in each dose group ranged from 51.46 to 69.48 h.
  • NVS451 is a high-affinity antibody Fc fusion protein that selectively targets IL-17A, IL-17C, IL-17F, and IL-17AF heterodimeric cytokines.
  • the target has a high affinity.
  • the present inventors conducted a summary of the non-clinical data of the project, which provided evidence of target specificity and mode of action, and treatment with NVS451 twice weekly or once weekly with high doses showed a The anti-positive control had a similar treatment effect.
  • NVS451 was well tolerated in comprehensive toxicology studies. All non-clinical evaluation studies provided strong evidence support for the efficacy and safety of NVS451 clinical treatment.

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Abstract

本发明提供了IL-17RA融合蛋白、药物组合物、注射剂及其应用。所述IL-17RA融合蛋白包括可操作地连接的、依次串联的信号肽、IL-17RA胞外结构域和IgG1恒定区。本发明的IL-17RA融合蛋白具有延长的半衰期，获得了更长效的药物活性，并且与抗体药物相比降低了免疫原性。本发明的设计使IL-17RA融合蛋白消除了ADCC、ADCP和CDC作用，并且保留新生儿Fc受体(FcRn)介导的体内循环利用的作用，与已上市的IL-17A和IL-17RA抗体相比，副反应低且更为安全。

Description

一种IL-17RA抗体Fc融合蛋白及其用途 技术领域
本发明属于医药领域，具体而言，涉及IL-17RA融合蛋白、药物组合物、注射剂及其应用。
背景技术
银屑病俗称牛皮癣，是一种慢性易于复发的自身免疫性皮肤病，通常会对患者生理及心理造成极大损害，WHO将其视为全球重大健康问题之一，发病率占世界人口的0.1％～3％，目前全球已有银屑病患者1.25亿。我国总患病率为0.47％，目前已有近650万临床登记患者，斑块银屑病是该疾病最常见的五种形式之一，占所有病例的80％～90％，其中中重度患者占38％，未来患者数量将在2030年达到950万人，中重度患者比例将提高到40％。银屑病发病机制和先天和适应性免疫***的失调，包括树突状细胞激活和促炎性细胞因子分泌，导致银屑病特征皮肤炎症的发生有关。近年来，近13种单克隆抗体和以抗体为基础的生物疗法已经被批准用于治疗银屑病，处于临床研究阶段的单抗药物约有10个左右，其中优特克(ustekinumab，IL-12/IL-23抗体，强生)抗体、TNF-α药物依那西普(Etanercept，安进)和阿达木单抗(Humira，艾伯维)是最畅销的药物。口服磷酸二酯酶4(PDE4)选择性抑制剂药物apremilast(Otezla，Celgene公司)也于2014年9月在美国获得批准，治疗难治性银屑病。然而，即便是这些生物药物作为二线药物来使用，治疗效果和安全性依然堪忧，而且多达40％的中度至重度银屑病患者使用现有的生物制剂治疗失败，这种现象被称为生物制品疲劳现象。
最新的研究表明：银屑病的发病机制和T17辅助性细胞(Th17细胞)相关，而由Th17细胞及先天性免疫细胞产生的促炎细胞因子IL-17A已被证实是在银屑病的发病机制中的主要细胞因子，免疫系 统靶向于IL-17-TH17通路中的IL-23，通过IL-17A间接导致银屑病的发生。从理论上讲，抑制IL-17相比于其他生物制剂可能是一种更安全的治疗选择，这在诺华已上市的针对IL-17A全人源IgG1单抗苏金单抗(secukinumab)、阿斯利康公司已上市的针对IL-17A受体全人源IgG2单抗(Brodalumab)、以及礼来公司的针对IL-17A全人源IgG4单抗(ixekizumab)与依那西普和优特克单抗(IL-12/IL-23抗体)头对头优效性比较研究中得到证实。研究表明，针对IL-17A靶标开发的抑制剂与目前的炎性疾病治疗药物(如TNF-α抑制剂)相比，可以提高疗效和安全性，且对TNF-α抑制剂及IL-12和IL-23抑制剂无效的银屑病患者有效，比IL-12和IL-23抑制剂具有更专一性。特别值得一提的是，于2015年1月上市的苏金单抗(商品名Cosentyx)，欧洲药品管理局(EMA)批准其可取代其他具有显著副作用的一线***疗法用于银屑病患者的一线治疗。
目前仍然需要更多的具有优良疗效的创新药物作为供患者选择的治疗途径。
发明内容
为解决上述现有技术中所存在的问题，本发明提供了IL-17RA融合蛋白、药物组合物、注射剂及其应用。
具体而言，本发明提供了：
(1)一种IL-17RA融合蛋白，其特征在于包括可操作地连接的、依次串联的信号肽、IL-17RA胞外结构域和IgG1恒定区。
(2)根据(1)所述的IL-17RA融合蛋白，其中所述IL-17RA胞外结构域的氨基酸序列如SEQ ID NO.1、SEQ ID NO.2或SEQ ID NO.3所示。
(3)根据(1)所述的IL-17RA融合蛋白，其中所述IgG1恒定区的氨基酸序列如SEQ ID NO.4所示。
(4)根据(1)所述的IL-17RA融合蛋白，其中所述信号肽为IL-17-RA天然信号肽，其氨基酸序列如SEQ ID NO.5所示。
(5)根据(1)所述的IL-17RA融合蛋白，其中所述IL-17RA 胞外结构域与所述IgG1恒定区之间采用接头连接。
(6)根据(1)所述的IL-17RA融合蛋白，其中所述IL-17RA融合蛋白的氨基酸序列如SEQ ID NO.6、SEQ ID NO.7或SEQ ID NO.8所示。
(7)一种分离的、编码根据(1)-(6)中任一项所述IL-17RA融合蛋白的核酸。
(8)一种表达载体，其含有与启动子有效连接的根据(7)所述的核酸。
(9)一种宿主细胞，其含有根据(8)所述的表达载体。
(10)根据(9)所述的宿主细胞，其中该宿主细胞的保藏编号为CGMCC 21011。
(11)一种由根据(1)-(6)中任一项所述的IL-17RA融合蛋白形成的蛋白二聚体，该二聚体由两分子所述IL-17RA融合蛋白通过所述IgG1恒定区的半胱氨酸结合形成双链。
(12)根据(1)-(6)中任一项所述的IL-17RA融合蛋白或根据权利要求11所述的蛋白二聚体在制备用于治疗银屑病,克罗恩病,斑块银屑病,胃肠炎,白塞病综合征,关节炎,葡萄膜炎,化脓性汗腺炎,扁平苔藓,副银屑病,哮喘,银屑病关节炎,肌腱炎,复发-缓解型多发性硬化,甲状腺相关性眼病,幼年型类风湿性关节炎,多发性硬化,狼疮肾炎,椎关节炎,强直性脊柱炎,类风湿关节炎,炎症性肠疾病,非酒精性脂肪肝,巨细胞性动脉炎,非放射学性轴性脊柱关节炎，寻常痤疮,三阴性乳腺肿瘤,多发性骨髓瘤，非小细胞肺癌,腺癌，结直肠癌，***癌，卡波济氏肉瘤,黑色素瘤,***和/或其他炎症性疾病的药物中的应用。
(13)一种药物组合物，包含治疗有效量的根据(1)-(6)中任意一项所述的IL-17RA融合蛋白或根据权利要求11所述的蛋白二聚体作为活性成分和可药用的辅料。
(14)根据(13)所述的药物组合物，其中所述可药用的辅料选自稀释剂、缓冲剂、保护剂、表面活性剂、抗氧化剂中的一种或多种。
(15)根据(14)所述的药物组合物，其中所述缓冲剂选自组氨酸-醋酸缓冲液、Tris-醋酸缓冲液、盐酸缓冲液、磷酸缓冲液、醋酸缓冲液、组氨酸缓冲液，精氨酸缓冲液，琥珀酸缓冲液，柠檬酸缓冲液中的一种或多种。
(16)根据(14)所述的药物组合物，其中所述保护剂选自海藻糖、吐温-20、吐温-80、蔗糖、氨基酸、多元醇、二糖、多糖中的一种或多种。
(17)根据(14)所述的药物组合物，其中所述表面活性剂选自吐温-20、吐温-80、泊洛沙姆中的一种或多种。
(18)根据(13)所述的药物组合物，其中单剂所述药物组合物包含5mg/ml-150mg/ml的所述IL-17RA融合蛋白或所述蛋白二聚体。
(19)根据(13)所述的药物组合物，其中所述药物组合物为冻干剂或注射液的形式。
(20)一种用于治疗银屑病,克罗恩病,斑块银屑病,胃肠炎,白塞病综合征,关节炎,葡萄膜炎,化脓性汗腺炎,扁平苔藓,副银屑病,哮喘,银屑病关节炎,肌腱炎,复发-缓解型多发性硬化,甲状腺相关性眼病,幼年型类风湿性关节炎,多发性硬化,狼疮肾炎,椎关节炎,强直性脊柱炎,类风湿关节炎,炎症性肠疾病,非酒精性脂肪肝,巨细胞性动脉炎,非放射学性轴性脊柱关节炎，寻常痤疮,三阴性乳腺肿瘤,多发性骨髓瘤，非小细胞肺癌,腺癌，结直肠癌，***癌，卡波济氏肉瘤,黑色素瘤,***和/或其他炎症性疾病的注射剂，包含(13)-(19)中任一项所述的药物组合物。
(21)根据(20)所述的注射剂，其中所述注射剂为冻干粉剂的形式或液体制剂的形式。
(22)根据(20)所述的注射剂，其中所述注射剂为皮下注射剂或静脉滴注剂。
(23)根据(21)所述的注射剂，其中在所述液体制剂中，所述静脉制剂包含5mg/ml-150mg/ml的所述IL-17RA融合蛋白或所述蛋白二聚体、2-100mM的Tris-醋酸、10-250mM的精氨酸、50-500mM 的海藻糖、0.01-5％的吐温-20。
(24)根据(21)所述的注射剂，其中所述液体制剂是采用注射用水、缓冲盐水溶液、葡萄糖水溶液、氯化钠水溶液或乳酸林格氏液配制得到的。
(25)根据(21)所述的注射剂，其中所述冻干粉剂是通过将所述液体制剂冷冻干燥制得的。
本发明与现有技术相比具有以下优点和积极效果：
本发明提供的IL-17RA融合蛋白是全人源抗体Fc融合蛋白药物。本发明通过将IL-17RA与抗体Fc段融合，延长了药物分子的半衰期，获得了更长效的药物活性，并且与抗体药物相比降低了免疫原性。
另外，本发明发现选择IgG1的Fc段，并且进一步对该Fc的序列进行合适的突变，能够大大消除抗体依赖的细胞介导的细胞毒作用(ADCC)、抗体依赖的细胞吞噬(ADCP)和补体依赖的细胞介导的细胞毒作用(CDC)，并且保留新生儿Fc受体(FcRn)介导的体内循环利用的作用，与已上市的IL-17A和IL-17RA抗体相比，副反应低且更为安全。
另外，本发明通过选择和设计IL-17RA的序列，使得IL-17RA融合蛋白能够靶向IL-17A、IL-17C、IL-17F、IL-17A/F等多个靶点，且亲和力高，从而能够选择性地阻断IL-17A、IL-17C、IL-17F和IL-17A/F与其受体的结合，进而有效阻断多种促炎性IL-17细胞因子的生物学活性，抑制炎症信号通路，因此可更加有效地缓解自身免疫性疾病的症状，可获得比单一IL-17A靶点抗体药物更好的治疗收益，获得比IL-17RA单抗更好的安全性。其作用机制与目前已上市和在研的IL-17靶点药物均不同，系全球同类首创的药物。
本发明从与IL-17A的结合亲和力、生物学活性细胞实验、GRO-α因子抑制能力、体外活性效价、体内效价、ADCC/CDC功能活性、FcRn结合亲和力、安全药理学、药代动力学、毒理学方面对上述药物活性和安全性进行了综合的评价。
附图说明
图1示出重组人IL-17RA融合蛋白的氨基酸序列。
图2示出在一个实施方案中所用表达载体pCHO1.1/NVS451的图谱。
图3示出在实验例1中IL-17RA融合蛋白(NVS451)与人IL-17A蛋白的亲和力测定曲线及拟合曲线。
图4示出在实验例1中苏金单抗与人IL-17A蛋白的亲和力测定曲线及拟合曲线。
图5示出在实验例1中野生型IL-17RA与人IL-17A蛋白的亲和力测定曲线及拟合曲线。
图6示出在实验例1中IL-17RA融合蛋白(NVS451)与人IL-17F蛋白的亲和力测定曲线及拟合曲线。
图7示出在实验例1中IL-17RA融合蛋白(NVS451)与人IL-17A/F蛋白的亲和力测定曲线及拟合曲线。
图8示出在实验例1中IL-17RA融合蛋白(NVS451)与人IL-17C蛋白的亲和力测定曲线及拟合曲线。
图9示出在实验例1中IL-17RA融合蛋白(NVS451)和苏金单抗对IL-17A诱导作用下GRO-α因子抑制作用的S曲线。
图10示出在实验例1中IL-17RA融合蛋白(NVS451)对IL-17A/F诱导作用下GRO-α因子抑制作用的S曲线。
图11示出在实验例1中IL-17RA融合蛋白(NVS451)对IL-17F诱导作用下GRO-α因子抑制作用的S曲线。
图12示出在实验例1中用不同药物处理异种皮肤移植SCID鼠之后的皮肤外观。
图13示出在实验例1中用不同药物处理异种皮肤移植SCID鼠之后的皮肤外观。
图14示出在实验例1中用不同药物处理异种皮肤移植SCID鼠之后的皮肤病理切片。
图15示出在实验例1中RitxV301与利妥昔单抗的ADCC效应比 较。
图16示出在实验例1中RitxV301与利妥昔单抗的CDC效应比较。
图17示出在实验例1中在酸性条件下(pH6.0)不同浓度NVS451与人FcRn的SPR分析图谱。
图18示出在实验例1中在中性条件下(pH7.4)不同浓度NVS451与人FcRn的SPR分析图谱。
图19示出在实验例1中IL-17RA融合蛋白NVS451(V301)、V302和V303与IL-17A的亲和力比较；图中V300、V301、V302、V303、V301*、V302*、V303*的浓度均为100ug/ml。
生物材料保藏信息
本发明提供的能够稳定表达本发明的IL-17RA融合蛋白的中华仓鼠卵巢细胞已于2020年11月23日保藏于中国微生物菌种保藏管理委员会普通微生物中心(CGMCC)，保藏地址：北京市朝阳区北辰西路1号院3号，邮编：100101，保藏编号为：CGMCC No.21011。
具体实施方式
以下通过具体实施方式的描述并参照附图对本发明作进一步说明，但这并非是对本发明的限制，本领域技术人员根据本发明的基本思想，可以做出各种修改或改进，但是只要不脱离本发明的基本思想，均在本发明的范围之内。
在本文中，术语“注射剂”是指：由药物制成的供注入体内的无菌溶液(包括真溶液、乳浊液和混悬液)，以及供临用前配制成所述无菌溶液(包括真溶液、乳浊液和混悬液)的冻干粉末或浓溶液。
在本文中，术语“静脉滴注”是指：通过输液管，将包含药物的大量液体由静脉输入体内的方法。又称“输液”、“点滴”、“静滴”、“挂水”。
在本文中，术语“IL-17A”是指白细胞介素17A。
在本文中，术语“IL-17RA”是指IL-17A的受体。
在本文中，术语“活性成分”是指对疾病具有治疗作用的药物分 子，例如本文所述的IL-17RA融合蛋白。
本发明提供了一种IL-17RA融合蛋白，其特征在于包含可操作地连接的、依次串联的信号肽、IL-17RA胞外结构域和IgG1恒定区。
信号肽的作用主要是引导目的蛋白从细胞胞质内分泌到细胞外。该融合蛋白由于存在IgG1恒定区，因此在分泌至细胞外时，两分子融合蛋白通过恒定区的半胱氨酸结合形成双链并发挥活性。
优选地，本发明采用人源IL-17RA的胞外结构域(Gene bank编号：NP_055154)，并且进行R108K、D122G和H155D突变，该突变体的氨基酸序列如SEQ ID NO.1所示。还优选地，本发明采用人源IL-17RA的胞外结构域，并且进行L9P、R108K、D122G和H155D突变，该突变体的氨基酸序列如SEQ ID NO.2所示。本发明发现，与包含野生型相比，包含这两个突变体的IL-17RA融合蛋白具有提高的热稳定性，和提高的对IL-17A的结合亲和力。所述氨基酸编号从所述人源IL-17RA的胞外结构域氨基酸序列的第1位起算。
还优选地，本发明采用人源IL-17RA的胞外结构域，并且进行L9P、R108K、D122G、H155D、G243W和A267V突变，该突变体的氨基酸序列如SEQ ID NO.3所示。本发明发现，IL-17RA融合蛋白包含该突变体与包含野生型相比具有提高的热稳定性，和提高的对IL-17A、IL-17C、IL-17F、IL-17A/F的结合亲和力；与包含上述带有三个和四个突变的突变体相比，具有更高的对IL-17A的结合亲和力。
本发明选择人源IgG1(Gene bank编号：3500)的恒定区(Fc)与IL-17RA形成融合蛋白。该融合蛋白不仅保留了功能蛋白分子的生物学活性，并且由于Fc部分具有一定抗体的特性且稳定，使得融合后的蛋白获得更长的循环寿命，半衰期延长。IgG同种型包括IgG1，IgG2和IgG4，其会引起不同的ADCC、ADCP和CDC效应，可对靶组织和非靶组织的毒性产生重大影响。IgG1的恒定区具有较强的ADCC、ADCP和CDC效应。本发明发现选择IgG1的恒定区，并且进一步对该Fc的序列进行合适的突变，能够降低IL-17RA融合蛋白的ADCC、ADCP和CDC效应。
在优选的实施方案中，突变位点总结于下表1。另外的突变是用 丝氨酸残基替换IgG1铰链区的半胱氨酸残基，以避免融合蛋白序列中存在未配对的半胱氨酸。
表1
Figure PCTCN2022100748-appb-000001
注：表1中所述的氨基酸编号是从人源IgG1氨基酸序列的第1位起算的。
该IgG1-Fc的突变体的氨基酸序列如SEQ ID NO.4所示。
信号肽是影响产量优化和产品质量的主要因素之一。重要的是信号肽切割位点应由切割清晰、切割概率高的单个残基明确界定。优选地，将人源IL17RA的天然信号肽用于融合蛋白，该信号肽的氨基酸序列如SEQ ID NO.5所示。
优选地，所述IL-17RA胞外结构域与所述IgG1恒定区之间采用接头连接。接头可以为本领域常用的那些，例如一个或多个连续的GSG、一个或多个连续的GGGGS、和GSAGSAAGSG。
优选地，所述IL-17RA融合蛋白的氨基酸序列如SEQ ID NO.6、SEQ ID NO.7或SEQ ID NO.8所示(IL-17RA胞外结构域分别带有三个突变、四个突变、六个突变)，其具有522个氨基酸残基。
例如，SEQ ID NO.8的序列如图1所示，其中前32个氨基酸(粗体)为IL-17RA信号肽，浅灰色背景的字母为经6个突变(粗体加下划线字母)的重组人源IL-17RA的氨基酸序列；黑色背景字母为接头氨基酸序列；深灰色背景字母为经表1所述3个修饰(粗体加下划线字母)的Fc部分的氨基酸序列，其中两个粗体半胱氨酸残基负 责Fc的二聚化。星号(*)示出潜在的糖基化位点。
本发明提供的IL-17RA融合蛋白的作用机理是，使用诱饵受体(IL-17RA-Fc)与天然受体竞争结合IL-17分子，通过与IL-17A、IL-17C、IL-17F及IL-17A/F以高亲和力结合并选择性地阻断IL-17A、IL-17C、IL-17F和IL-17A/F与其受体的结合，不与IL-17B、IL-17D、IL-17E结合，有效阻断多种促炎性IL-17细胞因子的生物学活性，抑制炎症信号通路，从而更加有效地缓解自身免疫性疾病的症状，可获得比单一IL-17A靶点抗体药物更好的治疗收益，并获得比IL-17RA单抗更好的安全性。
本发明还提供了一种分离的、编码根据本发明所述IL-17RA融合蛋白的核酸。
在一个优选的实施方案中，本发明利用分子生物学技术设计了融合蛋白的cDNA序列，并优化了密码子使其在CHO细胞中表达。优化后的编码所述氨基酸序列SEQ ID NO.1-8的DNA序列分别如SEQ ID NO.9-16所示。
本发明还提供了一种分离的、由本发明所述的编码所述融合蛋白的DNA转录得到的mRNA。
本发明还提供了一种表达载体，其含有与启动子有效连接的根据本发明所述的核酸。
本发明还提供了一种宿主细胞，其含有根据本发明所述的表达载体。
在一些优选的实施方案中，使用适于在悬浮和无血清培养基中生长的CHO细胞用作宿主细胞。还优选地，所述表达载体中包含两个选择性标记嘌呤霉素和甲氨蝶呤，其可促进高产和稳定细胞系的创建。
在更优选的实施方案中，宿主细胞的保藏编号为CGMCC 21011。该宿主细胞的构建使用Thermo Scientific公司的CHO-S TM细胞，构建后其可以稳定表达本发明的IL-17RA融合蛋白。此外，该宿主细胞还具有许多优点：(1)具有准确的转录后修饰功能，表达的蛋白在分子结构、理化特性和生物学功能方面最接近于天然蛋白分 子；(2)既可贴壁生长，又可以悬浮培养，且有较高的耐受剪切力和渗透压能力；(3)具有重组基因的高效扩增和表达能力，外源蛋白的整合稳定；(4)具有产物胞外分泌功能，并且很少分泌自身的内源蛋白，便于下游产物分离纯化；(5)能以悬浮培养方式或在无血清培养基中达到高密度培养。且培养体积能达到1000L以上，可以大规模生产。因此，该CHO细胞是重组糖基蛋白生产的首选体系。该宿主细胞已于2020年11月23日保藏于中国微生物菌种保藏管理委员会普通微生物中心(CGMCC)，保藏地址：北京市朝阳区北辰西路1号院3号，邮编：100101，保藏编号为：CGMCC No.21011。
本发明还提供了一种由根据本发明所述的IL-17RA融合蛋白形成的蛋白二聚体，该二聚体由两分子所述IL-17RA融合蛋白通过所述IgG1恒定区的半胱氨酸结合形成双链。
本发明所述的IL-17RA融合蛋白由于存在IgG1恒定区，因此在分泌至细胞外时，两分子融合蛋白通过恒定区的半胱氨酸结合形成双链并发挥活性。
本发明还提供了根据本发明所述的IL-17RA融合蛋白或由该IL-17RA融合蛋白形成的蛋白二聚体在制备用于治疗银屑病,克罗恩病,斑块银屑病,胃肠炎,白塞病综合征,关节炎,葡萄膜炎,化脓性汗腺炎,扁平苔藓,副银屑病,哮喘,银屑病关节炎,肌腱炎,复发-缓解型多发性硬化,甲状腺相关性眼病,幼年型类风湿性关节炎,多发性硬化,狼疮肾炎,椎关节炎,强直性脊柱炎,类风湿关节炎,炎症性肠疾病,非酒精性脂肪肝,巨细胞性动脉炎,非放射学性轴性脊柱关节炎，寻常痤疮,三阴性乳腺肿瘤,多发性骨髓瘤，非小细胞肺癌,腺癌，结直肠癌，***癌，卡波济氏肉瘤,黑色素瘤,***等和/或其他炎症性疾病的药物中的应用
本发明从与IL-17A的结合亲和力、生物学活性细胞实验、GRO-α因子抑制能力、体外活性效价、体内效价、ADCC/CDC功能活性、FcRn结合亲和力、安全药理学、药代动力学、毒理学方面对IL-17AR融合蛋白的药物活性和安全性进行了综合的评价。结果证明，所述IL-17RA融合蛋白能够靶向IL-17A、IL-17C、IL-17F、IL-17A/F等 多个靶点，且亲和力高，从而能够选择性地阻断IL-17A、IL-17C、IL-17F和IL-17A/F与其受体的结合，进而有效阻断多种促炎性IL-17细胞因子的生物学活性，抑制炎症信号通路，因此可更加有效地缓解自身免疫性疾病的症状，可获得比单一IL-17A靶点抗体药物更好的治疗收益，获得比IL-17RA单抗更好的安全性。
本发明还提供了一种药物组合物，包含治疗有效量的根据本发明所述的IL-17RA融合蛋白或由该IL-17RA融合蛋白形成的蛋白二聚体作为活性成分和可药用的辅料。
可药用的辅料可以根据所用的剂型和实际需要进行选择。
本发明从包材、缓冲体系、辅料、剂量、处方组成、冻干工艺等方面深入研究和设计了所述IL-17RA融合蛋白的制剂及制剂处方。
在一些优选的实施方案中，所述药物组合物的可药用辅料选自稀释剂、缓冲剂、保护剂、表面活性剂、抗氧化剂中的一种或多种。
优选地，缓冲剂选自组氨酸-醋酸缓冲液、Tris-醋酸缓冲液、盐酸缓冲液、磷酸缓冲液、醋酸缓冲液、组氨酸缓冲液，精氨酸缓冲液，琥珀酸缓冲液，柠檬酸缓冲液中的一种或多种。
优选地，保护剂选自海藻糖、吐温-20、吐温-80、蔗糖、氨基酸(例如精氨酸)、多元醇、二糖、多糖中的一种或多种。其中最优选为海藻糖、以及海藻糖+精氨酸的组合。
在一些实施方案中，海藻糖以二水海藻糖的形式存在。
优选地，表面活性剂选自吐温-20、吐温-80、泊洛沙姆(poloxamer)中的一种或多种。
在一些优选的实施方案中，单剂所述药物组合物包含5mg/ml-150mg/ml的所述IL-17RA融合蛋白或由该IL-17RA融合蛋白形成的蛋白二聚体。
可以根据需要将本发明所述的药物组合物制成合适的剂型。在本发明中，所述IL-17RA融合蛋白优选为冻干剂或注射液的形式。
因此，本发明还提供了一种用于治疗银屑病,克罗恩病,斑块银屑病,胃肠炎,白塞病综合征,关节炎,葡萄膜炎,化脓性汗腺炎,扁平苔藓,副银屑病,哮喘,银屑病关节炎,肌腱炎,复发-缓解型多发 性硬化,甲状腺相关性眼病,幼年型类风湿性关节炎,多发性硬化,狼疮肾炎,椎关节炎,强直性脊柱炎,类风湿关节炎,炎症性肠疾病,非酒精性脂肪肝,巨细胞性动脉炎,非放射学性轴性脊柱关节炎，寻常痤疮,三阴性乳腺肿瘤,多发性骨髓瘤，非小细胞肺癌,腺癌，结直肠癌，***癌，卡波济氏肉瘤,黑色素瘤,***等和/或其他炎症性疾病的注射剂，其包含本发明所述的药物组合物。
所述注射剂可以为冻干粉剂的形式或液体制剂的形式。
优选地，所述注射剂为皮下注射剂或静脉滴注剂。最优选为皮下注射剂。
在液体制剂中，所述注射剂优选包含所述IL-17RA融合蛋白、Tris-醋酸、精氨酸、海藻糖、吐温-20以及合适的溶剂。
更优选地，所述注射剂包含5mg/ml-150mg/ml的所述IL-17RA融合蛋白或由该IL-17RA融合蛋白形成的蛋白二聚体、2-100mM的Tris-醋酸、10-250mM的精氨酸、50-500mM的海藻糖、0.01-5％的吐温-20以及合适的溶剂。
所述溶剂可以采用配制注射剂常用的溶剂。例如注射用水、缓冲盐水溶液、葡萄糖水溶液、氯化钠水溶液或乳酸林格氏液等。
所述液体制剂是采用溶剂配制得到的。
可以采用制药领域常用的方法按照本发明所述的处方配制所述液体制剂。
冻干粉剂可以通过将所述液体制剂冷冻干燥而制得。
在优选的实施方案中，冻干工艺包括预冻、一次干燥(升华)和二次干燥(解析)。预冻包括将温度由5℃降至-40℃，并保持合适的时间；一次干燥包括将温度升至-5至0℃，并保持合适的时间；二次干燥包括将温度升至25℃至30℃，并保持合适的时间。
根据本发明对IL-17RA融合蛋白的制剂及制剂处方的研究，本发明开发了药品稳定性优良、可以安全注射给药的药物制剂。
以下通过例子的方式进一步解释或说明本发明的内容，但这些例子不应被理解为对本发明的保护范围的限制。
例子
以下除非特别说明，否则以下例子中所用实验方法均使用生物工程和制药领域的常规实验流程、操作、材料和条件进行。
以下除非特别说明，否则各试剂的百分浓度(％)均指该试剂的体积百分浓度(％(v/v))。
制备例1：质粒构建
NVS451(即，IL-17RA融合蛋白)由信号肽、rhIL17-RA ECD(即，人源IL17-RA胞外结构域)、接头肽段和IgG1 Fc结构域组成。其中信号肽采用IL17RA的天然信号肽。rhIL17-RA ECD中包含六个突变位点，分别为L9P、R108L、D122G、H155D、G243W和A267V(氨基酸编号不包含信号肽序列)。接头肽段为GSG。IgG1 Fc结构域采用IgG1、2、4的杂合体，以去除潜在的ADCC、CDC和ADCP效应。NVS451的氨基酸序列如SEQ ID NO.8所示，核苷酸序列如SEQ ID NO.16所示。
采用Thermo Scientific公司的pCHO1.0哺乳动物细胞表达载体构建质粒。pCHO1.0表达载体含有两个表达框，由于NVS451融合蛋白的表达只需要一个表达框，因此使用SfiI酶切位点移除了另外一个表达框，被移除的表达框包含有EcoRV和PacI克隆位点。移除一个表达框之后的pCHO1.0载体通过序列测序来验证，并命名为pCHO1.1。合成的NVS451融合蛋白基因通过AvrII和Bstz17I克隆位点***pCHO1.1(Kan抗性)表达载体，并转化DH5α感受态细胞，涂板筛选，接种扩增含有正确大小质粒的克隆，质粒提取获得大量构建好的NVS451融合蛋白表达质粒pCHO1.1-NVS4510。基因测序验证pCHO1.1-NVS451表达载体构建正确。
制备例2：细胞株筛选及稳定性评价
NVS451融合蛋白细胞株的构建及筛选选用CHO-S TM(符合现行药品生产管理规范的细胞库(cGMP-banked)，该细胞以及配套的表达载体pCHO1.0源于Thermo Scientific公司的
Figure PCTCN2022100748-appb-000002
Figure PCTCN2022100748-appb-000003
Kit)细胞作为原始细胞基质。NVS451融合蛋白表达质粒pCHO1.1-NVS451含有嘌呤霉素和MTX的筛选标记。pCHO1.1-NVS4510通过转染试剂转入CHO-S细胞。转染过程如下：50μg环状的表达质粒pCHO1.1-NVS451通过Opti PRO SFM稀释至1.5ml体积，50μl Freestyle Max转染试剂(GIBCO)通过Opti PRO SFM稀释至1.5ml，然后质粒稀释液和转染试剂稀释液等体积混合，加入30ml 1E6细胞/ml的CHO-S细胞悬液。转染细胞培养48小时后使用嘌呤霉素和MTX作为筛选压力进行两阶段的稳转克隆细胞池(Pools)筛选。由于未转染的CHO-S缺乏pac(腺苷酸环化酶)活性，仅具有基础DHFR(二氢叶酸还原酶)活性，因此只有被pCHO1.1-NVS4510质粒转染并整合入基因组的CHO-S细胞才能在含有嘌呤霉素和MTX的CD FortiCHO TM筛选培养基中存活和繁殖。筛选分两个阶段进行，每个阶段都使用嘌呤霉素和MTX进行筛选。最后形成4个稳转克隆细胞池(Pool)，分别为T3S1，T3S2，T3S3，T3S4。通过14天的简单流加批次培养(SFB)来评价表达量。结合基于细胞的GRO-α抑制活性实验、亲和ELISA和SPR分析方法(采用常规方法，故不赘述)，选出T3S1和T3S4两个稳转克隆池用于分离单克隆(LDC)。单克隆的分离采用两轮有限稀释分离单克隆，每轮细胞铺板浓度小于1个细胞/孔(按照统计学的数学计算方法)，最终获得5个候选单克隆：T3S4-7E3-6G5；T3S4-7E3-3C3；T3S4-17G2-6E2；T3S4-8F2-6E10；T3S1-18E7-6C5。
对5个候选单克隆进行70PDL(细胞倍增时间，相当于70个代次)的稳定性评价，基于表达稳定性、基因拷贝数、以及mRNA转录水平的稳定性，最终选择单克隆VAN301-T3S1-18E7-6C5作为主单克隆(保藏编号为CGMCC 21011)，选择VAN301-T3S4-17G2-6E2作为备份克隆。
使用该单克隆VAN301-T3S1-18E7-6C5建立PCB(原始细胞库)，并在此基础上建立GMP条件的MCB(主细胞库)和WCB(工作细胞库)。
制备例3：细胞培养
NVS451融合蛋白可通过14天fed-Batch培养工艺进行生产。细胞复苏：从细胞库中取一支细胞，37.0±0.5℃水浴复苏，解冻后立即将种子液转入装有10ml预热Dynamis(含8mM L-Gln、1:100ACA和1g/L P188)培养基的50ml离心管中，300g离心5分钟，弃上清后再加入10ml预热的Dynamis培养基重悬细胞，将重悬后的细胞液转移至装有28.0ml Dynamis培养基的125ml摇瓶中，细胞密度在(0.15～0.35)×10 6细胞/ml，细胞活率大于90％。种子传代与扩增：传代培养基为Dynamis，传代培养中细胞的初始接种密度应为(0.40±0.10)×10 6细胞/mL，培养3天后，细胞密度达到(2.0～5.0)×10 6细胞/mL即可传代，传代过程中应保证细胞活率高于90.0％。
反应器流加培养(Fed batch)：细胞初始接种密度为(0.40±0.10)×10 6细胞/mL，流加2×补料C+(Efficient FeedC+,Cat#:A2503101，Gibco)，培养过程中每天检测葡萄糖和乳酸含量，第3/5/7/9/11/13天分别流加450.0g/kg的葡萄糖母液补至5.0g/L。
细胞培养收获条件：当培养至第14天或细胞活率低于80.0％时收获，以先到达条件为准。
制备例4：NVS451融合蛋白纯化
细胞培养收获液首先通过两级深层过滤膜包进行澄清处理，然后加入1％吐温80，0.3％磷酸三丁酯进行病毒灭活，之后使用GE公司的MabSelect SuRe亲和层析填料捕获目的蛋白，接着使用Millipore公司的Eshmuno CPX阴离子层析填料和GE公司的Capto Adhere阳离子层析填料进行两步精细纯化，然后使用ASAHI KASEI公司的BioEX纳滤膜进行纳滤，使用Millipore公司的Pellicon 2、截留分子量50KDa的PES材质、C流道超滤膜进行超滤渗滤，最后加入辅料聚山梨酯20，经除菌过滤后得到NVS451原液。
1.深层过滤
使用串联的深层过滤膜(一级深层过滤膜D0HC和二级过滤器A1HC，Millipore公司)进行深层过滤，去除细胞等杂质，收获含目 的蛋白的澄清液。过滤样品时，进口流速≤100LMH(基于A1HC)，D0HC的最大载量为60L/m 2，A1HC最大载量为140L/m 2，控制压力，深层过滤的单步回收率一般在90％左右。
2.去垢剂病毒灭活
在澄清收获液中加入50mM Tris-HAc,150mM NaCl,25％PS80,pH7.4使得最终吐温80的浓度为1.0％。加入100％磷酸三丁酯使最终浓度为0.3％。16～26℃下孵育360–480分钟，然后进行亲和层析。
3.亲和层析
使用MabSelect SuRe填料进行亲和层析，实现产品的初步纯化。使用50mM Tris-HAc,150mM NaCl,pH 7.4作为平衡和上样后淋洗1缓冲液，再用20mM Tris,1M NaCl,0.5M Arg,pH8.6进行淋洗2，最后用50mM甘氨酸，pH3.5缓冲液洗脱目的蛋白。洗脱液使用1M Tris碱中和至pH7.8-8.2。
4.阳离子层析
使用Millipore公司的Eshmuno CPX作为阳离子层析填料。亲和层析中和后样品用1M HAc调节至pH6.3-6.7，调节后的样品作为阳离子层析上样样品。经过淋洗和梯度洗脱，能有效去除HCP、蛋白A、DNA以及一些产品类似物杂质及片段等与产品相关的杂质。
用20mM PB,pH6.5作为平衡和上样后淋洗缓冲液，25mM PB,110mM NaCl，pH6.5作为淋洗2，洗脱使用25mM PB,110mM NaCl,pH6.5拉梯度至20mM PB,1M NaCl,pH6.5，0-50％B(10CV)进行梯度洗脱。
5.阴离子层析
使用GE公司的Capto Adere填料作为阴离子层析填料，以去除部分与产品和工艺相关的杂质。阳离子层析洗脱液首先用1M Tris碱和注射用水调节pH至8.4-8.6，电导19.0-23.0，调节pH电导后的样品作为阴离子层析上样样品。
用20mM Tris,0.2M NaCl,pH8.5作为平衡和上样后淋洗缓冲液，20mM Tris,0.2M NaCl,60mM Arg,pH8.0作为淋洗2缓冲液，使用20mM Tris,0.2M NaCl,290mM Arg,pH7.1缓冲液洗脱目的蛋 白。
6.纳滤
阴离子层析洗脱样品使用旭化成公司纳滤器BioEX进行除病毒过滤。
纳滤工艺主要利用病毒和蛋白产品的分子大小不同，潜在病毒被纳滤膜截留，目的蛋白流穿，从而实现两者分离。预过滤膜能够吸附上样样品中的颗粒等杂质，同时增加纳滤膜处理量。纳滤膜可有效截留细小病毒等潜在病毒。采用A1HC(Millipore公司)进行预过滤和BioEX(旭化成公司)去除潜在的病毒。过滤样品时，控制预过滤膜上压力差≤2bar，纳滤膜上压力差≤3bar，纳滤膜的载量≤600g/m 2。
7.超滤渗滤
纳滤后中间品采用Pellicon 2(Millipore公司)超滤膜包进行浓缩，然后再换液至20mM Tris-HAc,65mM Arg,120mM海藻糖，pH7.5缓冲体系中。超滤膜包材质为PES，截留分子量50kDa，C流道。超滤渗滤的载量为≤300g/m 2；浓缩时进口通量150-300LMH,跨膜压(TMP)≤1.5bar，浓缩至浓度18.0-22.0g/L；然后进行换液，换液时进口通量150-300LMH,TMP≤2bar，换液体积≥5DV，过浓缩时进口通量150-300LMH,TMP≤2bar，过浓缩至浓度80.0-90.0g/L。
8.辅料添加及最终过滤
向超滤渗滤后样品中加入10％(w/w)聚山梨酯20母液，再使用20mM Tris-HAc,65mM Arg,120mM Trehalose，pH7.5缓冲液调节蛋白浓度至70.20～150.80mg/mL，即制备得到原液。之后进行除菌过滤，除菌过滤采用PES材质(0.22μm)过滤器过滤。原液聚山梨酯20的最终含量为0.02％(w/v)，辅料添加及除菌过滤的总回收率一般在80％以上。
9.原液
辅料添加及除菌过滤后的原液进行质量检测，纯度达到97％以上。
将原液储存在-40±5℃冰箱中。
实验例1：药效学研究
1.体外药效学
NVS451的活性分子是由人IL-17RA胞外段突变体和人IgG1Fc突变体两部分组成的双链融合蛋白，经CHO细胞重组表达获得。因此，该分子可表现出IL-17RA和Fc分子两方面的生物学功能特性。通过表面等离子共振(SPR)、体外靶细胞杀伤测定等分析技术对NVS451体外生物学活性开展系列研究，旨在阐明其体外药效相关属性。
1.1 IL-17A结合亲和力比较分析
NVS451(用量是75ug/ml)可与人IL-17A以高亲和力结合。用100nM-0.8nM的人IL-17A(Acrobiosystems,Cat:ILA-H5118)进行表面等离子共振，实验方法如下：
试剂配制
包被液：取1.06g的Na 2CO 3和0.84g NaHCO 3用超纯水充分溶解，用浓盐酸调其PH值为9.60后定容至200ml，用0.22滤膜过滤，放4℃保存；
10×TBS母液：取12.114g的Tris和43.83g的NaCl加入超纯水中充分溶解，用盐酸调PH值为7.55后定容至500ml，用0.22滤膜过滤，放4℃保存；
4×底物缓冲液母液：取7.16g的Na HPO·12H O和2.1g的柠檬酸用超纯水充分溶解，用NaOH调PH值为5.5后定容至100ml，用0.22滤膜过滤，放4℃保存。
实验操作
包被：
将IL-17A按照COA用超纯水重构，放置约30分钟左右，使其溶解充分，用室温下平衡好的包被液稀释IL-17A到30nM，后加入酶标板内，每孔100μl，封板膜封板放置在4℃过夜(约16h)。
洗板：
洗液现用现配，取100ml的10×TBS母液加入到900ml的超纯水稀释成1×TBS，再加入2.5ml的20％Tween 20，用配制好的洗液反复洗板4次，每孔300μl，拍干。
封闭：
现用现配，取2.5g的BSA用洗液稀释到50ml配制成5％BSA封闭液；在拍干的板内加入300μl的封闭液，37℃孵育1.5h。
洗板：
重复步骤2洗板。
加入样品：
稀释液现用现配，取5ml的封闭液用洗液稀释到50ml，配制成0.5％BSA样品稀释液，2倍梯度稀释样品到10μg/ml，将稀释好的样品加入到酶标板中，100μl/孔，设置背景对照(不包被+样品+二抗，包被+不加样品+二抗，不包被+不加样品+不加二抗)。封板膜封板37℃孵育1h。
洗板：
重复步骤2洗板。
二抗：
稀释液现用现配，取5ml的封闭液用洗液稀释到50ml，配制成0.5％BSA抗体稀释液，将辣根过氧化物酶标记的AffiniPure山羊抗人IgG和Fcγ片段特异性(min X Bov,Hrs,Ms Sr Prot)稀释成1:12000，每孔加入100μl，封板膜封板37℃孵育1h。
洗板:
重复步骤2洗板。
显色:
取12ml的4×底物缓冲液加入到36ml的超纯水中稀释成1×底物缓冲液后再加入38.5μl 3％H 2O 2和240μl 20mg/mL TMB(用DMSO溶解)，每孔加入200μl，37℃孵育20分钟。
终止:
在显色结束的板内每孔加入50μl 1mol/L硫酸。
读数:
450nm的波长下测量各孔的吸光度(OD值)，求其平均值。
数据处理：
将所得的OD值用GraphPad Prism 5对数据进行四参数拟合，并求出EC50。
(SPR)测试(结果如图3所示)，KD为1.8×10 -12M，亲和力高于同等条件下的苏金单抗(结果如图4所示)(已在国外批准上市(2015)的IL-17A单抗药物)和野生型人IL-17RA(wtIL-17RA，购自ACROBIOSYSTEMS公司)(结果如图5所示)。与IL-17A结合亲和力比较分析结果总结于表2。
表2.与IL-17A结合亲和力比较分析
Figure PCTCN2022100748-appb-000004
采用制备例1-4中相同的方法制备了IL-17RA胞外结构域具有3个突变(V302)和4个突变(V303)的IL-17RA融合蛋白(氨基酸序列分别如SEQ ID NO.6和SEQ ID NO.7所示)。
采用亲和ELISA方法检测V302和V303与IL-17A的亲和力。使用了两种试剂盒(R&D，Cat#:317-ILB-050；Peprotech，Cat#:900-K84)按照生产商提供的方法进行检测，结果显示(图19)，具有六个突变的NVS451与IL-17A亲和力百分比(上述两种试剂盒分别为776％和464％)显著高于V302和V303分子与IL-17A的亲和力。而V302和V303分子与IL-17A的亲和力与野生型人IL-17RA相当。
1.2不同种属来源的IL-17A的结合亲和力分析
在该研究中，比较在相同条件下4个物种(人、食蟹猴、小鼠、 大鼠)的IL-17A分子(KINGFISHER,货号分别为：RP0921H-025，RP1031Y-025，RP0355M-025；大鼠Biolegend，778704)与NVS451的结合情况，结果显示NVS451与4个不同物种IL-17A均可结合，且结合亲和力由高到低依次为：人>食蟹猴>大鼠>小鼠(KD见表3)。
表3.与不同种属IL-17A结合亲和力比较分析
Figure PCTCN2022100748-appb-000005
1.3 IL-17A、C、F、AF的结合亲和力分析
除了与人IL-17家族的IL-17A分子结合外，NVS451还与人源IL-17C、IL-17F、IL-17AF结合(结果分别见图6-8)。KD结果总结于表4。
在另一项研究中，比较了NVS451和野生型人IL-17RA与IL-17C、IL-17F(Acrobiosystems，ILC-H52H7，ILF-H4240)的亲和力大小，结果显示NVS451对人IL-17C、IL-17F的亲和力均高于野生型人IL-17RA(表5)。
表4 NVS451与人IL-17家族结合亲和力表征
Figure PCTCN2022100748-appb-000006
表5.NVS451和野生型IL-17RA与IL-17C、IL-17F的亲和力比较
Figure PCTCN2022100748-appb-000007
弱结合：图谱有响应信号，但强度低，无法量化读值。
N.A.：不适用
1.4生物学活性细胞实验
NVS451体外活性检测所使用的细胞系CCD-1070Sk(人成纤维上皮细胞系，ATCC CRL-2091)属于人成纤维上皮细胞系，其表面存在包括IL-17A、A/F和F在内的多种细胞因子受体。IL-17A能够和该细胞表面上的IL-17A受体IL-RA结合，刺激细胞产生细胞因子GRO-α，并且其含量可以用ELISA双抗夹心法准确检测到。当IL-17A和NVS451与CCD-1070Sk细胞共孵育时，NVS451能够通过与IL-17A的结合竞争抑制IL-17A与细胞表面上的IL-17A受体IL-17RA的结合，从而降低细胞上清液中GRO-α的分泌量。并且NVS451也能够基于同样的竞争抑制作用机制，通过与IL-17A/F、IL-17F结合，降低细胞上清液中GRO-α的分泌量。因此可以通过测定细胞GRO-α分泌量来测定NVS451对于包括IL-17A、IL-17A/F、IL-17F等多个靶点的体外活性作用。
1.4.1 NVS451在IL-17A诱导下的GRO-α因子抑制能力
通过测量IL-17A诱导的从人成纤维上皮细胞系CCD-1070Sk中释放GRO-α的抑制能力，分析了NVS451融合蛋白的体外相对效价，结果用半数抑制浓度IC 50值表示(表6)。
1、T75培养瓶内的人成纤维上皮细胞系CCD-1070Sk细胞用2ml 0.05％EDTA的胰酶消化，约2-5分钟，加入8ml完全培养基(10％FBS的EMEM基础培养基)中和，然后在1000rpm下离心5分钟，用完 全培养基重悬至细胞密度为1E5/ml，以100ul每孔，10000细胞每孔重悬于96孔板中，培养大约24小时，待细胞汇合度达到95％-100％开始给药实验。
2、细胞诱导及给药：所有药物的稀释均使用完全培养基，先将NVS451蛋白稀释至起始浓度为4ug/ml(终浓度为1ug/ml)，然后两倍梯度稀释9个点，以及最后一个0ug/ml浓度点，50ul每孔加入到细胞板中，然后将IL-17A稀释至浓度40ng/ml(终浓度为10ng/ml)，以50ul每孔加入每一个反应孔中，细胞给药培养作用24小时。
3、孵育24小时后，使用孔板离心机1500rpm离心5分钟，然后取上液100ul准备ELISA实验检测，此步骤是将3个复孔的细胞上清液混合稀释后，在ELISA实验中分别加入酶标板的3个复孔
4、ELISA分析
A、用PBS x 1稀释捕获抗体(鼠抗人GROα抗体，R&D Cat#DY275-05-840255)至浓度为0.25μg/ml，立即向每个ELISA微孔中加入100μl，包被平板并在室温下孵育过夜(24℃±4)。
B、清除各孔液体，每孔用300μl洗涤缓冲液洗涤板4次。最后一次用吸水纸彻底清理干净(洗涤缓冲液需要提前一天放置室温直至完全溶解)。
C、每孔加入300μl封闭缓冲液。在室温下((24℃±4)孵育至少1小时。
D、样品制备：
离心96孔CRL-2091细胞培养板，1000rpm离心5分钟。
将50μl的预孵育样品(sup)加入200μl样品稀释剂(样品现在以1:5稀释)稀释后准备上样。
E、标准品准备：
在稀释剂中制备1μg/ml的标准品系列稀释液，总共7个点稀释液和一个零点
F、清除各孔液体，每孔用300μl洗涤缓冲液洗涤板4次。最后一次用吸水纸彻底清理干净。
G、立即向每个孔中加入100μL标准品或样品，一式三份。在 室温下((24℃±4)，600rpm，孵育2小时。
H、清除各孔液体，每孔用300μl洗涤缓冲液洗涤板4次。最后一次用吸水纸彻底清理干净。
I、稀释剂中的稀释检测抗体(生物素化的山羊抗人GROα抗体,R&D Cat#DY275-05-840256)浓度为1.0μg/ml(初始浓度100μg/ml)。
J、在每个孔中加入100μl稀释的检测抗体。在室温下(24℃±4)，600rpm，孵育2小时。
K、清除各孔液体，每孔用300μl洗涤缓冲液洗涤板4次。最后一次用吸水纸彻底清理干净。
L、取6μL生物素偶联-HRP缀合物(1：2,000)，加入总体积为12ml样品稀释液(计算1个96孔板)。
M、每孔加入100μl。在室温下(24℃±4)，600转，孵育30分钟。
N、清除各孔液体，每孔用300μl洗涤缓冲液洗涤板4次。最后一次用吸水纸彻底清理干净。
O、每孔加入100μl底物溶液(ABTS底物提前30分钟放置室温，取出当次实验需要的用量)。在室温下分别孵育20分钟以进行显色。
P、使用酶标仪在405nm处监测颜色显色，波长校正设定在650nm。
同时检测目前市售IL-17A单克隆抗体苏金单抗(Cosentyx)在这一体外相对效价检测方法下的GRO-α的抑制能力，结果用半数抑制浓度IC 50值表示(表6)。结果显示出NVS451融合蛋白对IL-17A诱导的释放GRO-α细胞因子有抑制能力，并且较苏金单抗(Cosentyx)的IC 50值更低(图9)。
表6 NVS451和苏金单抗的体外活性效价：IL-17A诱导产生GRO-α的IC 50(μg/ml)
Figure PCTCN2022100748-appb-000008
Figure PCTCN2022100748-appb-000009
1.4.2 NVS451对IL-17A、IL-17A/F、IL-17F多种配体的体外活性效价比较
与上述实验相类似地，IL-17A/F与IL-17F诱导的人成纤维上皮细胞系CCD-1070Sk也能够产生GRO-α细胞因子，不同配体诱导作用下的NVS451融合蛋白体外效价结果用半数抑制浓度IC 50值表示(表7)。抑制作用S曲线分别见图10和图11。
表7 NVS451抑制人类皮肤成纤维细胞CCD-1070Sk GRO-a分泌检测结果
Figure PCTCN2022100748-appb-000010
2.体内药效学
银屑病是一种慢性炎症性皮肤病，以角质形成细胞过度增殖和异常分化为特点，由多种因素引起，发病机制尚未完全阐明，目前认为诸多免疫细胞和免疫相关细胞(包括树突状细胞、T细胞、内皮细胞等)，以及细胞因子参与了银屑病的发病和疾病状态的维持[1]，现有的应用较广泛的动物模型大致可分为药物诱导急性炎症模型、基因工程模型和异体移植模型。
咪喹莫特(Imiquimod，IMQ)诱导模型，IMQ是Toll样受体(Toll like receptor，TLR)7/8的激动剂，Van der Fits[2]等研究发现IMQ诱导的小鼠银屑病样皮损改变伴随着IL-23/IL-17轴的变化，与人类银屑病病理变化有很多相似之处，且具有操作简便、花费少等优点，因此用IMQ涂抹小鼠皮肤建立银屑病是目前国内外广泛应用的银屑病模型。但作为一种急性炎症性模型，其局限性为：(1)非特异性，不只在银屑病中，在其他疾病如***性红斑狼疮、特应性皮炎等皮损中也有类似的炎症表现[3]，IMQ诱导的皮肤炎症模型还被用于***性红斑狼疮的研究[4]；(2)治疗窗口期短，咪喹莫特外用可引起小 鼠脱水及体质量减轻，连续外用超过2周可能导致小鼠死亡[5]；(3)可重复性差，缺乏标准操作规程等。
异种皮肤移植免疫缺陷鼠(SCID)模型，是将人类皮肤移植到SCID小鼠上，注射患者的T细胞诱发银屑病症状，其遗传表型和致病过程最接近于人类银屑病，且因动物免疫缺陷，不受抗药抗体(anti-drag antibody，ADA)影响，是研究银屑病发病机理以及药物开发的最适合工具之一，而且最适合于在开始临床试验之前进行探索新型抗银屑病剂/治疗策略的临床前分析[6,7,8]。
2.1 NVS451在异种皮肤移植SCID小鼠银屑病模型中的药效评价(5，10，15mg/kg NVS451)。
将上述浓度的NVS451蛋白配置成20mM Tris-醋酸，65mM精氨酸，120mM海藻糖，0.02％吐温20，pH 7.5的制剂处方。
实验共使用60只SCID鼠(Envigo,耶路撒冷,以色列C.B-17/IcrHsd-scid-bg(米色-SCID)小鼠)，移植健康人皮肤于小鼠背部后，随机分为6组，每组10只小鼠：阴性对照组(阴性对照组是使用上述制剂处方去掉NVS451)、激素组(2mg/只动物***)、苏金单抗组(60mg/kg)和供试品高剂量(15mg/kg)、中剂量(10mg/kg)、低剂量(5mg/kg)组。阴性对照组、供试品组及苏金组皮下注射隔天给药共28天。激素组外用***每天2次，共28天。实验结束观察皮肤外观(结果见图12)、，并测量皮肤厚度。结果显示：NVS451对红肿、斑块和鳞屑均有一定程度改善作用，且与剂量呈正相关；15mg/kg剂量组与苏金组表皮厚度无统计学差异(P＞0.05)，该模型中NVS451具有与苏金相似的治疗效果。
表8.异种移植皮肤的目检结果
Figure PCTCN2022100748-appb-000011
表9 异种移植皮肤的组织学评估和厚度
Figure PCTCN2022100748-appb-000012
****与全部治疗组比较，-p＜0.01
**高剂量测试品与苏金单抗比较，-不显著的
***高剂量测试品和苏金单抗与低剂量和中剂量的比较，-p＜0.05，p＜0.05
2.2 NVS451(2.1中所述的制剂处方)在异种皮肤移植SCID小鼠银屑病模型中的药效评价(15，22.5，30，45，60mg/kg NVS451)
实验共使用100只SCID鼠(购于Envigo)，移植健康人皮肤于小鼠背部后，随机分为10组(见表10)。实验结束观察皮肤外观(结 果见图13)、取移植皮肤进行银屑病相关的组织学评估(结果见图14)，并测量皮肤厚度。结果显示，NVS451组均显示了治疗作用，其中15mg/kg一周两次、30mg/kg一周两次、22.5mg/kg一周两次和60mg/kg一周一次组，疗效类似，均为7/10的异种移植皮肤完全恢复且表皮厚度降低。但15mg/kg每周两次组，剩余3/10较其他治疗组显示了部分恢复，皮肤厚度为334±174μm，组织学评分为1.1(依据皮损表皮厚度，***上表皮变薄，角化过度，角化不全，粒细胞缺乏，Munro微脓肿，正常或异常网嵴延长，血管弯曲度，真皮***层单核细胞浸润等指标综合评估)。
表10.研究分组及剂量(辅料对照组为制剂处方去掉NVS451)
Figure PCTCN2022100748-appb-000013
表11.异种移植皮肤的组织学评估和厚度
Figure PCTCN2022100748-appb-000014
3.次要药效学
3.1 ADCC/CDC效应相关受体结合亲和力分析
NVS451发挥药效的作用机制不依赖于ADCC/CDC活性，而且Fc的细胞毒性作用ADCC/CDC可能带来不必要的免疫相关不良事件(irAE)，存在潜在的安全风险[9]。为避免产生该效应，NVS451的Fc部分突变了相关活性位点。在亲和力分析研究中，评估了与ADCC相关的7个CD分子及与CDC相关的C1q分子。结果表明，NVS451(75ug/ml)与ADCC相关的CD分子的结合亲和力处于不结合或低水平(KD大于10 -5M数量级，见表12)。在另一项研究中，与其他部分相同但具有天然IgG1Fc的IL-17AR融合蛋白(V301-wtFc)(75ug/ml)相比，NVS451与ADCC/CDC相关Fc受体亲和力显著降低(见表13)。
V301-wtFc的氨基酸序列如SEQ ID NO.17所示。
表12.NVS451与ADCC、CDC效应相关受体的亲和力分析
Figure PCTCN2022100748-appb-000015
表13 NVS451和V301-wtFc与ADCC、CDC效应相关受体亲和力分析
Figure PCTCN2022100748-appb-000016
Figure PCTCN2022100748-appb-000017
3.2 Fc片段区的ADCC/CDC功能活性评价
IgG1和IgG3亚型抗体的Fab区先结合靶细胞，其Fc部分再与NK等CTL细胞上的Fc受体(如FcγRIII)结合，之后可诱导产生ADCC。缺少膜表达IL-17的细胞模型无法有效在体外验证是否产生ADCC。因此，将可刺激ADCC的利妥昔单抗(Rituximab)的Fab段和NVS451的Fc段融合，构建RitxV301(即RitxNVS451)验证嵌合分子(轻链的氨基酸序列如SEQ ID NO.18所示，重链的氨基酸序列如SEQ ID NO.19所示)。以此对ADCC/CDC效应的去除进行验证(用GraphPad Prism 5分析软件分析实验数据，以抗体的浓度对数为x轴，对应的计算杀伤率值为y轴，选用四参数方程回归模拟，拟合抗体的计量效应曲线。所得数据经GraphPad Prism 5软件分析，阳性对照抗体(利妥昔单抗)符合四参数方程式y＝D+(A-D)/[1+(X/C)B]，在半对数坐标轴上成典型的S型曲线，R2≥0.95,计算EC50，通过EC50值判断ADCC和CDC效应))。研究结果显示(图15和16)：利妥昔单抗(罗氏(瑞士)在Raji细胞上表现了较强的ADCC和CDC杀伤效应，而RitxV301未显示出ADCC和CDC效应，说明NVS451的Fc区的设计使得RitxV301不能诱导NK-92MI CD16a细胞产生对Raji细胞的杀伤，并且NVS451的Fc区不能跟补体中的C1q分子结合，无法启动补体级联反应。
3.3 FcRn结合亲和力分析
Fc融合蛋白一般通过Fc的FcRn介导的循环提高目标蛋白药物的半衰期，改善药代动力学特征[10]。对FcRn的结合亲和力大小与药物在体内半衰期相关[11]。NVS451可与人、猴、大鼠的FcRn(acrobiosystems FCM-H5286，FCM-C5284，FCM-R5287)结合。同等条件下，其亲和力与苏金抗、野生型IgG1Fc(即V301-wtFc)均在相同水平(KD的数量级达10 -8M，见表14)，实验方法如下：。
试剂配制
运行试剂：含2mM KH 2PO 4,10mM Na 2HPO 4,137mM NaCl,2.7mM KCl，0.05％吐温-20(Tween-20)，pH调节至6.0；
His捕获试剂盒(货号：28-9950-56，GE)，其中包括：鼠抗His 抗体(1mg/mL)，固定试剂(10mM醋酸钠，pH 4.5)，再生试剂(甘氨酸盐酸，pH 1.5)；
氨基偶联试剂盒(货号：BR100050，GE)，其中包括：115mg N-羟基丁二酰亚胺(NHS)，750mg 1-乙基-(3-二甲基氨基丙基)碳二亚胺盐酸盐(EDC)和10.5mL 1M乙醇胺(pH 8.5)。将每管EDC和NHS分别加入10mL的去离子水，分装保存到-18℃至更低温度，保质期两个月。(参考GE氨基偶联指导手册《22-0510-62AG》)。
按照产品COA对IL-17 RA-wtFc(即V301-wtFc)蛋白，不同种属的FcRn蛋白进行溶解，以大于10μg每管的规格进行分装。避免反复冻融。
蛋白脱盐
使用脱盐柱及运行缓冲液(Running Buffer)将苏金单抗和IL-17 RA-wtFc蛋白脱盐。脱盐后的蛋白用UV-V进行浓度测定。脱盐后的蛋白以大于10μg每管的规格进行分装，并避免反复冻融。
芯片制备
将鼠抗His抗体用固定试剂(10mM醋酸钠，pH 4.5)稀释到50μg/mL，芯片每个通道使用约100μL鼠抗His抗体，固定两个通道约使用190μL固定试剂加入10μL鼠抗His抗体。首先，CM5芯片(biocore传感器芯片，GE)的表面用400mM EDC和100mM NHS以10μL/分钟的流速进行420s的活化。其次，将50μg/mL的鼠抗His抗体以10μL/分钟的流速注入到实验通道(FC4)约420s，固定量约为9000至14000RU。最后，芯片用1M乙醇胺以10μL/分钟进行420s封闭。参比通道(FC3)与试验通道(FC4)进行相同的操作。(参考GE的His捕获试剂盒指导手册《28-9974-71 AB》)。
捕获配体
将FcRn原液样品用运行试剂稀释至0.5μg/mL，并以10μL/分钟的流速注入到实验通道(FC4)约40RU。参比通道(FC3)不需要进行配体的捕获。
分析物多循环分析
将不同浓度的NVS451，苏金单抗和IL-17 RA-wtFc样品用相应运 行试剂进行稀释，将稀释后的样品依次以30μL/分钟的流速按照相应结合时间和解离时间注入到实验通道与参比通道。每一个浓度分析后，芯片需要用pH值为1.5的甘氨酸盐酸以30μL/分钟的流速再生60s，洗掉配体以及未解离的分析物。进行下一个浓度分析时，实验通道需要重新捕获相同量的配体。
其他
所有的操作步骤均在运行试剂中进行，SPR的分析试剂均需过滤脱气使用。
结果
数据分析:
使用Biacore 8K分析软件计算每个抗体的KD值。参比通道(FC3)用于背景的扣减。
在4批独立测试研究中，pH6.0酸性条件下NVS451(浓度:25nM-1.5625nM)可与人、食蟹猴、大鼠3不同种属的FcRn结合(图17)；且在pH7.4中性条件下(NVS451浓度100nM-1.5625nM)均不结合(图18)。
表14 人、大鼠、食蟹猴的FcRn受体亲和力比较
Figure PCTCN2022100748-appb-000018
实验例2：安全性评价
1.安全药理学
1.1食蟹猴安全药理试验
食蟹猴(购自北京中科灵瑞生物技术股份有限公司)安全药理试验伴随长毒进行，皮下注射实验例1的2.1节中的处方，NVS451剂量为15mg/kg、50mg/kg和150mg/kg(每周给药2次，连续给药4周，共给药9次，即分别于D1、D4、D8、D11、D15、D18、D22、D25和D29给药)。在本试验中，设置辅料对照组，使用实验例1的2.1节中的处方去掉NVS451，结合毒性指标观察进行了安全药理指标的检测，各组动物精神状态、行为活动等均未见异常；动物呼吸平稳，未见呼吸急促或减慢，也未见异常的胸式或腹式呼吸。在不同时间点(首次给药前(D-2)，以及首次给药后4h(±20分钟)、24h(±20分钟)、48h(±20分钟)、72h(±20分钟))进行各组动物的呼吸指标和心电图检查，均未见与给药相关的TV和RR，以及心率、P-R间期、QT间期、QRS时限、QRS电压、STe及QTcB值的规律性改变，心电图波形未见异常。于首次药前(D-2)，首次药后3～5小时(D1)，5次药后3～5小时(D15)，末次药后3～5小时(D29)以及恢复期结束前(D56)进行心电肢体II导联测定，观察心率、心电图波形、P-R间期、QRS时限、Q-T间期，未见异常，各时间点血压检测包括MBP、DBP和SBP检测均未见明显异常改变。故认为以15、30和150mg/kg的剂量皮下注射，供试品对食蟹猴中枢神经***、心血管***和呼吸***均未见明显影响。
1.2大鼠中枢神经***试验
GLP(良好实验室管理规范)条件下，通过功能组合观察法(FOB)评估NVS451单次皮下注射给药对SD大鼠(购买自北京维通利华实验动物技术有限公司(动物生产许可证：SCXK(京))中枢神经***功能的影响，剂量分别为30mg/kg(低剂量)、100mg/kg(中剂量)和300mg/kg(高剂量)。供试品高剂量组雄性动物在药后2h可见体温升高(辅料对照组(即制剂处方去掉NVS451)vs高剂量组：37.72±0.29℃ vs 38.40±0.14℃)；供试品高、中、低剂量组雌性动物在药后24h可见体温升高，辅料对照组vs高、中、低剂量组：37.60 ±0.28℃ vs 38.80±0.23、38.72±0.44、38.54±0.28℃)。上述体温的改变未见雌雄一致性，且未见规律性改变，认为与给药不相关。在笼内观察、手抓观察、开放环境观察、刺激性反应观察过程中，未见动物出现与给药相关的异常表现；未见与供试品相关的动物直立数、粪块数、前肢抓力、体温改变。结果表明，本试验条件下，NVS451对大鼠中枢神经***功能无影响。
2.药代动力学
2.1食蟹猴体内动力学
试验共用24只食蟹猴，分为4组(每组6只动物，雌雄各半)。单次皮下和静脉注射给药，组1至组4分别给予5(皮下注射)、15(皮下注射)、50(皮下注射)、15(静脉注射)mg/kg的NVS451，给药容量1mL/kg。
试验从动物后肢皮下静脉非给药部位采集药代血样(约1mL)至无抗凝剂的管中，1-3组动物采血时间点为：药前(前一天)、给药开始后1h、2h、4h、8h、24h(D2)、32h(D2)、48h(D3)、56h(D3)、72h(D4)、96h(D5)；第4组动物采血时间点为：药前(前一天)、给药开始后3分钟、1h、2h、6h、24h(D2)、32h(D2)、48h(D3)、56h(D3)、72h(D4)、96h(D5)。血样用于制备血清样本，并用于药代动力学分析(表15)。
表15.NVS451在食蟹猴体内的药代动力学参数
Figure PCTCN2022100748-appb-000019
Figure PCTCN2022100748-appb-000020
C max比＝C max中、高剂量均值/C max低剂量均值；
AUC比＝AUC last中、高剂量均值/AUC last低剂量均值；
F％＝(AUC last/剂量(SC))/(AUC last/剂量(IV))*100％。
试验结果表明：在5～50mg/kg剂量范围内，单次皮下注射给予食蟹猴后，NVS451在食蟹猴体内的血药浓度随剂量增加而增加；单次皮下注射和静脉注射给予食蟹猴后，性别间未观察到明显差异。
NVS451以5、15、50mg/kg的剂量单次皮下注射给予食蟹猴后，NVS451的平均C max和AUC last增长比例高于剂量增长比例。单次皮下注射给予5、15、50mg/kg剂量组动物NVS451的C max比值分别为1:6.12:38.06(雄)和1:5.46:34.29(雌)；AUC last比值分别为1:4.83:25.52(雄)和1:4.76:20.88(雌)。
经独立样本t检验表明，皮下注射和静脉注射各组的代谢动力学参数性别间均无统计学差异，NVS451在动物体内的代谢特征基本无明显性别差异。
在5～50mg/kg剂量范围内，NVS451在食蟹猴体内的T 1/2，各剂量组雌雄动物平均T 1/2在27.20～39.90之间；皮下注射各剂量组的T max基本一致，药物浓度均在4～8h达峰。
2.2大鼠体内动力学
试验共用48只SD大鼠，分为4组(每组12只动物，雌雄各半)。单次皮下和静脉注射给药，组1至组4分别给予10(皮下注射)、30(皮下注射)、100(皮下注射)、30(静脉注射)mg/kg的NVS451，给药容量2mL/kg。
试验从动物颈静脉采集药代血样(约0.4mL)至无抗凝剂的管中，1-3组动物采血时间点为：给药前(D-1)，给药开始后1h、2h、4h、8h、24h、32h、48h、56h、72h、96h；第4组动物采血时间点为：给药前(D-1)，给药开始后3min、1h、2h、6h、24h、32h、48h、56h、72h、96h。血样用于制备血清样本，并用于药代动力学分析(表16)。
表16.NVS451在SD大鼠体内的药代动力学参数表
Figure PCTCN2022100748-appb-000021
C max比＝C max中、高剂量均值/C max低剂量均值；
AUC比＝AUC last中、高剂量均值/AUC last低剂量均值；
F％＝(AUC last/剂量(SC))/(AUC last/剂量(IV))*100％。
试验结果表明：在10～100mg/kg剂量范围内，单次皮下注射给予SD大鼠后，NVS451在SD大鼠体内的血药浓度随剂量增加而增加；单次皮下注射和静脉注射给予SD大鼠后，性别间未观察到明显差异。
NVS451以10、30、100mg/kg的剂量单次皮下注射给予SD大鼠后，NVS451的平均Cmax和AUClast增长比例低于剂量增长比例。单次皮下注射给予10、30、100mg/kg剂量组动物NVS451的Cmax比值分别为1：2.03：6.56(雄)和1：1.62：6.04(雌)；AUClast比值分别为1：1.92：5.29(雄)和1：1.85：5.85(雌)。
皮下注射和静脉注射各组的代谢动力学参数性别间无明显差异，NVS451在动物体内的代谢特征基本无明显性别差异。
在10～100mg/kg剂量范围内，NVS451在SD大鼠体内的T1/2，各剂量组雌雄动物T1/2在51.46～69.48之间；皮下注射各剂量组的Tmax基本一致，药物浓度均在8～24h达峰。
3.分布和***
本试验主要采用TCA沉淀蛋白的方法结合SHPLC(分子排阻高效色谱法)对经同位素(125I)标记的注射用NVS451融合蛋白(NVS451)皮下注射给予SD大鼠后的组织分布及***特征进行了 研究。
试验共用36只动物，分为6组，每组6只动物，雌雄各半。以30mg/kg的剂量单次颈背部皮下注射给予供试品。125I标记后的供试品放化纯度为99.03％，比活性为0.09KBq/μg。1-4组动物分别于给药后4h、24h、96h和120h采集动物甲状腺、心、肺、肝、脾、肾、膀胱、生殖腺(卵巢、睾丸)、胃、小肠、脂肪、肌肉、脑、背部皮肤(非给药部位)、肠内容物、血清、尿液。5组动物给药后每天收集一次粪、尿，共收集5天。6组动物于注射后每小时收集1次排出胆汁，共收集8h。
试验结果表明：
1)大鼠皮下注射给予125I-NVS451后，药物主要分布在肠内容物、尿液、膀胱、背部皮肤等组织/器官中，而肌肉、脂肪、脑内较少分布。药物在绝大部分组织中的达峰时间在4h～24h，然后随着时间的延长浓度逐渐下降。
药物在背部皮肤(非给药部位)有一定的分布。其达峰时间为24h，4h与120h药物含量大致相等。与其他组织/器官相比，药物在背部皮肤浓度下降较为缓慢。
2)大鼠皮下注射给予125I-NVS451后放射性主要经肾***，5天后***掉给入药物的90％以上；胆汁***较少。
4.代谢
根据“ICH-S6(R1)生物制品的临床前安全性评价”指导原则，NVS451是大分子蛋白质药物，预期其在体内降解成肽和氨基酸，然后被***或重新用于体内蛋白质或肽的合成，因此未评估NVS451的代谢。
5.毒理学
选择食蟹猴和SD大鼠进行毒理学研究，NVS451与食蟹猴和大鼠IL-17A有交叉反应，为相关种属。试验均在GLP条件下进行，遵循现行的食品和药物管理局良好实验室管理规范(21CFR Part 58)，国家药品监督管理局(原国家食品药品监督管理总局)的《药物非临床研究质量管理规范》(局第34号令，2017年9月)。
6.单次给药毒性
6.1食蟹猴毒性研究
目的是评价NVS451单次皮下注射给予食蟹猴后观察14天内可能出现的急性毒性反应情况，设置45mg/kg、150mg/kg和450mg/kg三个剂量组。试验期间，各组动物均未见死亡或濒死。各给药组动物临床观察未见异常。与自身药前值和辅料对照组(制剂处方去掉NVS451)相比，各给药组动物体重、体温、心电图参数、血细胞计数、凝血指标、血生化、尿液检查均未见给药相关的异常改变。观察期结束(D15)，所有动物大体解剖均未见异常。结论：在本试验条件下，所有动物均未见死亡或濒死，未见给药相关的异常改变，最大耐受剂量(MTD)大于或等于450mg/kg。
6.2大鼠毒性研究
目的是评价NVS451单次皮下注射给予SD大鼠后观察14天内可能出现的急性毒性反应情况，设置90mg/kg、300mg/kg、900mg/kg三个剂量组。试验期间，各组动物均未见死亡或濒死，观察期结束(D15)，各组动物大体观察均未见异常改变。结论：本试验条件下，所有动物均未见死亡或濒死，动物最大耐受剂量(MTD)大于或等于900mg/kg。
7.重复给药毒性
7.1食蟹猴重复给药毒性研究
使用40只食蟹猴(20只/性别)，按性别随机分为4组(5只/性别/组)，分别为辅料对照组和供试品低、中、高剂量组。辅料对照组动物给予辅料对照品，2mL/kg，供试品低、中、高剂量组给予NVS451，给药剂量分别为15、50、150mg/kg，给药容量分别为0.2、0.67、2mL/kg，给药浓度为75mg/mL。所有动物经后肢皮下注射给药，每周给药2次，连续给药4周，共9次，即D1、D4、D8、D11、D15、D18、D22、D25和D29给药。试验期间，对动物进行临床观察、体重、体温、心电图、呼吸(呼吸频率和潮气量)、血压、眼科检查、血细胞计数、凝血功能、血液生化、尿液分析和免疫学指标(T淋巴细胞亚群、细胞因子、C反应蛋白、血清免疫球蛋白、补体、抗 药抗体)指标的检测；首次和第8次给药前后，对各组动物采血进行血药浓度测定。末次药后次日(D30)对部分动物(3只/性别/组)实施安乐死，4周恢复期结束(D57)对剩余动物实施安乐死；所有动物均进行骨髓涂片及阅片、大体解剖观察，主要脏器称重，计算相对脏器重量(脏体比和脏脑比)，并对40余种组织器官进行组织病理学检查。
结果：试验期间，各组动物均未见死亡或濒死，各组动物临床观察、体重、体温、眼科检查、尿液检查、血细胞计数、凝血功能(FIB除外)、血生化指标、淋巴细胞亚群、补体、细胞因子(除IL-17A)和免疫球蛋白均未见与给药相关的异常改变。动物心电图、呼吸和血压参数均未见给药相关的异常改变。给药局部肉眼观察未见红斑、水肿、硬结、破溃等异常反应。
试验期间，与同期同性别辅料对照组相比，5次药后次日(D16)，供试品高剂量组雄性动物可见CRP升高(193.0％)；末次药后次日(D30)，供试品低、中、高剂量组雄性动物可见CRP升高(70.5％、218.5％、246.9％)，供试品高剂量组雄性动物还可见FIB升高(56.4％)，差异具有统计学意义(P≤0.05)。停药4周后，以上改变均完全恢复。
7.1.1组织病理学检查：
试验过程中未发现动物死亡或濒死情况。
末次药后(D30)，15、50和150mg/kg剂量组动物的注射局部及腹股沟***可见与供试品相关改变，其中注射局部表现为真皮/皮下/肌层轻微～中度的单个核细胞性炎症，为供试品引起的刺激性反应；腹股沟***轻微～中度的副皮质区/髓索淋巴细胞数量增多，与注射局部单个核细胞性炎症相关，结合临床病理学相关指标未见明显改变，考虑为非不良反应。
4周恢复期结束(D57)，15和50mg/kg剂量组注射局部与供试品相关改变已完全恢复，150mg/kg剂量组注射局部与供试品相关改变已基本恢复。
7.2大鼠重复给药毒性研究
使用192只SD大鼠，雌雄各半，分性别区段按照体重随机分为 8组，1～4组主试验组的120只大鼠用作毒理研究(15只/性别/组)，5～8组卫星组的72只大鼠用于血清抗体和毒代动力学检测(6～10只/性别/组)。试验期间，辅料对照组动物给予NVS451白辅料溶液(4mL/kg)；供试品低、中、高剂量组分别给予NVS451，给药剂量分别为30、100、300mg/kg，给药容量分别为0.4、1.33、4mL/kg。所有动物均经颈背部皮下注射给药，每周给药2次，连续给药4周，共给药9次(即D1、D4、D8、D11、D15、D18、D22、D25和D29给药)，4周恢复期。
试验期间，主试验组动物主要进行临床观察，并对体重、食量、体温、血细胞计数、凝血功能、血液生化、眼科检查、尿液检查、T淋巴细胞亚群和细胞因子进行检测；卫星组动物于首次和第8次给药前后采血进行毒代动力学检测，并于不同时间点(首次给药前(D-1)、第15次给药前(D14)、末次给药前(D28)以及恢复期结束(D56))采血进行抗体测定。末次药后次日(D30)对主试验组前10只动物/性别/组实施安乐死，4周恢复期结束后(D57)对剩余动物实施安乐死。主试验组所有动物均进行大体解剖观察，主要脏器称重，计算相对脏器重量；并对辅料对照组、高剂量组动物40余种组织器官及低、中剂量组动物注射局部、进行组织病理学检查。
结果：试验期间，各组动物均未见死亡或濒死，临床观察未见异常。给药局部肉眼观察未见红斑、充血、肿胀、溃疡和硬结。各组动物眼科检查和尿液检查均未见异常；与同期同性别辅料对照组相比，各组动物体重、体温、食量、血细胞计数、凝血功能、血生化、T淋巴细胞亚群均未见与给药相关的异常改变。
7.2.1病理学检查：
本试验过程中无动物发生死亡。
安乐死动物大体观察，未见与供试品相关改变。
给药结束(D30)安乐死动物，30、100和300mg/kg剂量组注射局部可见与供试品相关的刺激性反应，表现为注射局部皮下和/或真皮轻微至中度单个核细胞性炎症。至4周恢复期结束，注射局部炎症基本完全恢复。
8.局部耐受毒性
局部耐受毒性试验伴随长毒开展，食蟹猴和SD大鼠均为每周给药两次(大鼠三个剂量组30、100和300mg/kg，食蟹猴三个剂量组15、50和150mg/kg)，连续给药4周，共9次(即D1、D4、D8、D11、D15、D18、D22、D25和D29给药)。试验期间，各组动物给药局部观察未见红斑、水肿、硬结、破溃等异常反应，局部耐受良好，未见毒性。
9.其他毒性
采用体外试管法，观察75mg/mL NVS451对人红细胞溶血及凝聚的影响(实验条件为常规条件)。自恒温箱温育后15分钟开始至3h观察结束时，供试品管中均可见试管上层液体无色澄明，管底红细胞下沉，经震荡后均匀分散，结果无溶血和凝聚发生。本试验条件下，75mg/mL的NVS451在体外对人红细胞无溶血作用，不引起人红细胞凝聚。
结论
1.药效学综合评估和结论
体外药效学：与IL-17A结合亲和力比较分析显示NVS451可与人IL-17A以高亲和力结合，亲和力高于同等条件下的苏金单抗和野生型人IL-17RA。除了与人IL-17家族的IL-17A分子结合外，NVS451还与IL-17C、IL-17F、IL-17AF结合。NVS451对人IL-17C、IL-17F的亲和力均高于野生型人IL-17RA。生物学活性细胞实验显示，NVS451融合蛋白显示出对IL-17A、IL-17A/F、IL-17F诱导的GRO-α细胞因子的抑制能力，并且对IL-17A诱导的GRO-α细胞因子的抑制能力较苏金单抗(Cosentyx)的IC 50值更低。
根据这些体外效价结果，NVS451主要通过抑制IL-17A和IL-17C、IL-17F、IL-17AF活性发挥作用。SPR实验显示的相互作用提示，在体内，推荐剂量的NVS451在银屑病患者中发挥主要抑制IL-17A，辅以抑制IL-17C、IL-17F、IL-17AF分子的临床活性。
亲和力分析研究表明NVS451与ADCC相关的CD分子的结合亲和力处于不结合或低水平，和天然IgG1Fc相比，其与ADCC/CDC 相关Fc受体亲和力显著降低，证明NVS451的ADCC结合位点被突变去除。与FcRn结合亲和力分析显示，NVS451可与人、猴、大鼠的FcRn结合。同等条件下，其亲和力与苏金单抗、野生型IgG1Fc均在相同水平，pH6.0酸性条件下NVS451可与人、食蟹猴、大鼠3不同种属的FcRn结合，且在pH7.4中性条件下均不结合。NVS451是一种携带人IgG1Fc结构域的可溶性抗体Fc融合蛋白，因此理论上能够与Fc受体相互作用，但NVS451发挥药效的作用机制不依赖于ADCC/CDC活性，且Fc的细胞毒性作用ADCC/CDC可能带来不必要的免疫相关副作用，存在潜在安全风险。为避免产生该效应，NVS451的Fc部分突变了相关活性位点。研究证实，和天然IgG1Fc相比，其与ADCC/CDC/ADCP相关Fc受体亲和力显著降低，且靶细胞杀伤实验(如图15和16所示)证实NVS451无ADCC/CDC活性作用。
体内药效学：在体内功能上，主要是在人T细胞驱动的银屑病模型中评估NVS451对银屑病的疗效。研究已证实，人类皮肤异种移植模型最适合于在开始临床试验之前进行探索新型抗银屑病剂/治疗策略的临床前分析，通过异种移植SCID鼠模型人皮肤组织学评估，包括评分和表皮厚度，显示了NVS451治疗人源化银屑病模型的疗效，在异种皮肤移植SCID小鼠银屑病模型中，NVS451以5mg/kg、10mg/kg和15mg/kg剂量隔天给药28天后，结果显示NVS451对红肿、斑块和鳞屑均有一定程度改善作用，且与剂量呈正相关；15mg/kg剂量组与苏金组表皮厚度无统计学差异(P＞0.05)，每周使用最高剂量的NVS451(60mg/kg)治疗一次与每周使用两次的低剂量治疗同样有效，最重要的是，每周使用NVS451治疗两次或每周使用高剂量治疗一次，显示出与苏金单抗的阳性对照相似的治疗效果。苏金单抗被认为是治疗银屑病最有前景的药物之一，这项研究为临床治疗的有效性提供了强有力的临床证据支持。
安全药理学：GLP条件下，NVS451单次皮下注射给药30mg/kg、100mg/kg和300mg/kg，结果显示NVS451对大鼠中枢神经***功能无影响。食蟹猴安全药理试验伴随长毒进行：以15、30和150mg/kg 的剂量皮下注射，供试品对食蟹猴中枢神经***、心血管***和呼吸***均未见明显影响。
2.药代和毒理学综合评估和结论
由于NVS451与食蟹猴与啮齿动物IL-17A有交叉反应，因此选择食蟹猴和大鼠作为毒性评价的相关物种。NVS451分别以15、50和150mg/kg的剂量重复皮下注射给予食蟹猴，每周2次，连续给药4周，共给药9次，各组动物均未见死亡或濒死，未见明显全身毒性反应，认为本试验的未见不良反应剂量(NOAEL)为150mg/kg。注射用NVS451融合蛋白分别以30、100和300mg/kg的剂量重复皮下注射给予SD大鼠，每周给药2次，连续给药4周，共给药9次，动物未见明显全身毒性反应，认为未见不良反应剂量(NOAEL)为300mg/kg。食蟹猴和大鼠毒代动力学表明，反复给药后，NVS451无蓄积。
在食蟹猴和大鼠体内进行了单剂量非GLP的药代动力学试验，以观察NVS451经静脉和皮下途径给药的药代动力学基本情况。NVS451单次皮下给药后在雄性和雌性食蟹猴体内的吸收药代动力学进行了评价，其药代动力学行为具有典型的免疫球蛋白分子的低血清清除率和半衰期，各个剂量组皮下给药96小时后均能检测到有效药物。在食蟹猴和SD大鼠的单次药代动力学试验中，代谢数据无明显性别差异，在5～50mg/kg剂量范围内，NVS451在食蟹猴体内的T1/2，各剂量组雌雄动物平均T1/2在27.20～39.90h之间。NVS451在SD大鼠体内的T1/2，在10～100mg/kg剂量范围内，各剂量组雌雄动物T1/2在51.46～69.48h之间。
3.非临床结论：
NVS451是一种选择性靶向IL-17A，IL-17C、IL-17F和IL-17AF异源二聚体细胞因子的高亲和力抗体Fc融合蛋白，结合数据表明NVS451与人和食蟹猴体内的预定靶点有很高的亲和力。
综上，本发明进行了项目的非临床数据总结，该数据提供了靶点特异性和作用方式的证据，每周使用NVS451治疗两次或每周使用高剂量治疗一次，显示出与苏金单抗的阳性对照相似的治疗效果。 NVS451在全面毒理学研究中耐受性良好。所有非临床评价研究为NVS451临床治疗的有效性和安全性提供了强有力的证据支持。
参考文献：
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Claims (25)

  1. 一种IL-17RA融合蛋白，其特征在于包括可操作地连接的、依次串联的信号肽、IL-17RA胞外结构域和IgG1恒定区。
  2. 根据权利要求1所述的IL-17RA融合蛋白，其中所述IL-17RA胞外结构域的氨基酸序列如SEQ ID NO.1、SEQ ID NO.2或SEQ ID NO.3所示。
  3. 根据权利要求1所述的IL-17RA融合蛋白，其中所述IgG1恒定区的氨基酸序列如SEQ ID NO.4所示。
  4. 根据权利要求1所述的IL-17RA融合蛋白，其中所述信号肽为IL-17-RA天然信号肽，其氨基酸序列如SEQ ID NO.5所示。
  5. 根据权利要求1所述的IL-17RA融合蛋白，其中所述IL-17RA胞外结构域与所述IgG1恒定区之间采用接头连接。
  6. 根据权利要求1所述的IL-17RA融合蛋白，其中所述IL-17RA融合蛋白的氨基酸序列如SEQ ID NO.6、SEQ ID NO.7或SEQ ID NO.8所示。
  7. 一种分离的、编码根据权利要求1-6中任一项所述IL-17RA融合蛋白的核酸。
  8. 一种表达载体，其含有与启动子有效连接的根据权利要求7所述的核酸。
  9. 一种宿主细胞，其含有根据权利要求8所述的表达载体。
  10. 根据权利要求9所述的宿主细胞，其中该宿主细胞的保藏编号为CGMCC 21011。
  11. 一种由根据权利要求1-6中任一项所述的IL-17RA融合蛋白形成的蛋白二聚体，该二聚体由两分子所述IL-17RA融合蛋白通过所述IgG1恒定区的半胱氨酸结合形成双链。
  12. 根据权利要求1-6中任一项所述的IL-17RA融合蛋白或根据权利要求11所述的蛋白二聚体在制备用于治疗银屑病,克罗恩病,斑块银屑病,胃肠炎,白塞病综合征,关节炎,葡萄膜炎,化脓性汗腺炎,扁平苔藓,副银屑病,哮喘,银屑病关节炎,肌腱炎,复发-缓解型多发性硬化,甲状腺相关性眼病,幼年型类风湿性关节炎,多发性硬化,狼疮肾炎,椎关节炎,强直性脊柱炎,类风湿关节炎,炎症性肠疾病,非酒精性脂肪肝,巨细胞性动脉炎,非放射学性轴性脊柱关节炎，寻常痤疮,三阴性乳腺肿瘤,多发性骨髓瘤，非小细胞肺癌,腺癌，结直肠癌，***癌，卡波济氏肉瘤,黑色素瘤,***和/或其他炎症性疾病的药物中的应用。
  13. 一种药物组合物，包含治疗有效量的根据权利要求1-6中任意一项所述的IL-17RA融合蛋白或根据权利要求11所述的蛋白二聚体作为活性成分和可药用的辅料。
  14. 根据权利要求13所述的药物组合物，其中所述可药用的辅料选自稀释剂、缓冲剂、保护剂、表面活性剂、抗氧化剂中的一种或多种。
  15. 根据权利要求14所述的药物组合物，其中所述缓冲剂选自组氨酸-醋酸缓冲液、Tris-醋酸缓冲液、盐酸缓冲液、磷酸缓冲液、醋酸缓冲液、组氨酸缓冲液，精氨酸缓冲液，琥珀酸缓冲液，柠檬酸缓冲液中的一种或多种。
  16. 根据权利要求14所述的药物组合物，其中所述保护剂选自海藻糖、吐温-20、吐温-80、蔗糖、氨基酸、多元醇、二糖、多糖中的一种或多种。
  17. 根据权利要求14所述的药物组合物，其中所述表面活性剂选自吐温-20、吐温-80、泊洛沙姆中的一种或多种。
  18. 根据权利要求13所述的药物组合物，其中单剂所述药物组合物包含5mg/ml-150mg/ml的所述IL-17RA融合蛋白或所述蛋白二聚体。
  19. 根据权利要求13所述的药物组合物，其中所述药物组合物为冻干剂或注射液的形式。
  20. 一种用于治疗银屑病,克罗恩病,斑块银屑病,胃肠炎,白塞病综合征,关节炎,葡萄膜炎,化脓性汗腺炎,扁平苔藓,副银屑病,哮喘,银屑病关节炎,肌腱炎,复发-缓解型多发性硬化,甲状腺相关性眼病,幼年型类风湿性关节炎,多发性硬化,狼疮肾炎,椎关节炎,强直性脊柱炎,类风湿关节炎,炎症性肠疾病,非酒精性脂肪肝,巨细胞性动脉炎,非放射学性轴性脊柱关节炎，寻常痤疮,三阴性乳腺肿瘤,多发性骨髓瘤，非小细胞肺癌,腺癌，结直肠癌，***癌，卡波济氏肉瘤,黑色素瘤,***和/或其他炎症性疾病的注射剂，包含权利要求13-19中任一项所述的药物组合物。
  21. 根据权利要求20所述的注射剂，其中所述注射剂为冻干粉剂的形式或液体制剂的形式。
  22. 根据权利要求20所述的注射剂，其中所述注射剂为皮下注射剂或静脉滴注剂。
  23. 根据权利要求21所述的注射剂，其中在所述液体制剂中，所述静脉制剂包含5mg/ml-150mg/ml的所述IL-17RA融合蛋白或所述蛋白二聚体、2-100mM的Tris-醋酸、10-250mM的精氨酸、50-500mM的海藻糖、0.01-5％的吐温-20。
  24. 根据权利要求21所述的注射剂，其中所述液体制剂是采用注射用水、缓冲盐水溶液、葡萄糖水溶液、氯化钠水溶液或乳酸林格氏液配制得到的。
  25. 根据权利要求21所述的注射剂，其中所述冻干粉剂是通过将所述液体制剂冷冻干燥制得的。
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CN116593718B (zh) * 2023-07-18 2023-09-15 军科正源（北京）药物研究有限责任公司 用于检测司库奇尤单抗抗药性抗体的试剂

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