WO2022194311A4 - Protéine de fusion fc d'anticorps d'il-17ra et son utilisation - Google Patents

Protéine de fusion fc d'anticorps d'il-17ra et son utilisation Download PDF

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WO2022194311A4
WO2022194311A4 PCT/CN2022/100748 CN2022100748W WO2022194311A4 WO 2022194311 A4 WO2022194311 A4 WO 2022194311A4 CN 2022100748 W CN2022100748 W CN 2022100748W WO 2022194311 A4 WO2022194311 A4 WO 2022194311A4
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fusion protein
nvs451
injection
buffer
pharmaceutical composition
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WO2022194311A3 (fr
WO2022194311A2 (fr
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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/fr
Publication of WO2022194311A3 publication Critical patent/WO2022194311A3/fr
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Definitions

  • the invention belongs to the field of medicine, and in particular relates to IL-17RA fusion protein, pharmaceutical composition, injection and application thereof.
  • Psoriasis commonly known as psoriasis, is a chronic autoimmune skin disease that is prone to relapse, which usually causes great physical and psychological damage to patients. WHO regards it as one of the major global health problems, with an incidence rate of 0.1% of the world's population. % to 3%. At present, there are 125 million psoriasis patients in the world. The overall prevalence rate in my country is 0.47%, and there are currently 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, and 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 pathogenesis is associated with dysregulation of the innate and adaptive immune systems, including dendritic cell activation and secretion of pro-inflammatory cytokines, leading to the development of psoriatic-characteristic skin inflammation.
  • 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) are the best-selling drugs.
  • 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
  • Th17 cells T17 helper cells
  • IL-17A pro-inflammatory cytokine IL-17A produced by Th17 cells and innate immune cells
  • inhibiting IL-17 may be a safer treatment option compared with other biologics.
  • the fully human IgG2 monoclonal antibody against IL-17A receptor (Brodalumab) marketed by Si Li Kang Company, and the fully human IgG4 monoclonal antibody against IL-17A (ixekizumab) of Eli Lilly and Company, together with etanercept and ustekinumab Anti-(IL-12/IL-23 antibody) head-to-head superiority comparative study was confirmed.
  • inhibitors developed against the IL-17A target can improve efficacy and safety compared with current inflammatory disease treatment drugs (such as TNF- ⁇ inhibitors), and are more effective against TNF- ⁇ inhibitors and IL-12 and IL-23 inhibitors are effective in psoriasis patients who are ineffective and more specific than IL-12 and IL-23 inhibitors.
  • current inflammatory disease treatment drugs such as TNF- ⁇ inhibitors
  • TNF- ⁇ inhibitors current inflammatory disease treatment drugs
  • IL-12 and IL-23 inhibitors are more effective against TNF- ⁇ inhibitors and IL-12 and IL-23 inhibitors are effective in psoriasis patients who are ineffective and more specific than IL-12 and IL-23 inhibitors.
  • secukinumab trade name Cosentyx
  • EMA European Medicines Agency
  • the present invention provides IL-17RA fusion protein, pharmaceutical composition, injection and application thereof.
  • the present invention provides:
  • An IL-17RA fusion protein which is characterized in that it comprises an operably linked signal peptide, an IL-17RA extracellular domain and an IgG1 constant region sequentially connected in series.
  • IL-17RA fusion protein according to (1), wherein the amino acid sequence of the IL-17RA fusion protein is as shown in SEQ ID NO.6, SEQ ID NO.7 or SEQ ID NO.8.
  • An expression vector comprising the nucleic acid according to (7) operably linked to a promoter.
  • the IL-17RA fusion protein according to any one of (1)-(6) or the protein dimer according to claim 11 is used for the treatment of psoriasis, Crohn's disease, plaque Lump psoriasis, gastroenteritis, Behcet's syndrome, arthritis, uveitis, hidradenitis suppurativa, lichen planus, parapsoriasis, asthma, psoriatic arthritis, tendonitis, relapsing-remitting Multiple sclerosis, thyroid-associated 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 neoplasms, multiple myeloma, non
  • 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 Pharmaceutically acceptable excipients.
  • composition according to (14), wherein the buffer is selected from histidine-acetic acid buffer, Tris-acetic acid buffer, hydrochloric acid buffer, phosphate buffer, acetate buffer, histidine buffer, one or more of arginine buffer, succinic acid buffer, and 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, polysaccharides or Various.
  • composition according to (14), wherein the surfactant is selected from one or more of Tween-20, Tween-80, and poloxamer.
  • 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 invention is a fully human antibody Fc fusion protein drug.
  • the present invention prolongs the half-life of the drug molecule by fusing IL-17RA with the Fc segment of the antibody, obtains longer-lasting drug activity, and reduces immunogenicity compared with antibody drugs.
  • the present invention found that selecting the Fc segment of IgG1, and further performing appropriate mutations on the Fc sequence, can greatly eliminate antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP) and complement Depends on cell-mediated cytotoxicity (CDC), and retains the effect of neonatal Fc receptor (FcRn)-mediated recycling in vivo, compared with the marketed IL-17A and IL-17RA antibodies, the side effects are low And more secure.
  • ADCC antibody-dependent cell-mediated cytotoxicity
  • ADCP antibody-dependent cellular phagocytosis
  • CDC complement Depends on cell-mediated cytotoxicity
  • the present invention enables the IL-17RA fusion protein to target IL-17A, IL-17C, IL-17F, IL-17A/F and other targets with high affinity by selecting and designing the sequence of IL-17RA.
  • 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 activities of various pro-inflammatory IL-17 cytokines, inhibiting Inflammatory signaling pathway, so 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 research, 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 experiment, GRO- ⁇ factor inhibitory ability, in vitro activity titer, in vivo potency, ADCC/CDC functional activity, FcRn binding affinity, safety pharmacology, pharmacokinetics
  • the activity and safety of the above-mentioned drugs were comprehensively evaluated in terms of medicine and toxicology.
  • Figure 1 shows the amino acid sequence of 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 between 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 between wild-type IL-17RA and human IL-17A protein in Experimental Example 1.
  • Fig. 6 shows the affinity measurement curve and fitting curve between IL-17RA fusion protein (NVS451) and human IL-17F protein in Experimental Example 1.
  • Fig. 7 shows the affinity measurement curve and fitting curve between 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 between IL-17RA fusion protein (NVS451) and human IL-17C protein in Experimental Example 1.
  • Fig. 10 shows the S-curve of GRO- ⁇ factor inhibitory effect of IL-17RA fusion protein (NVS451) on IL-17A/F induction in Experimental Example 1.
  • Fig. 11 shows the S-curve of GRO- ⁇ factor inhibitory effect induced by IL-17RA fusion protein (NVS451) on IL-17F in Experimental Example 1.
  • FIG. 12 shows the skin appearance of SCID mice after xenograft skin transplantation was treated with different drugs in Experimental Example 1.
  • FIG. 12 shows the skin appearance of SCID mice after xenograft skin transplantation was treated with different drugs in Experimental Example 1.
  • FIG. 13 shows the skin appearance of SCID mice after xenograft skin transplantation was treated with different drugs in Experimental Example 1.
  • FIG. 13 shows the skin appearance of SCID mice after xenograft skin transplantation was treated with different drugs in Experimental Example 1.
  • FIG. 14 shows skin pathological sections after xenograft skin transplantation SCID mice were treated with different drugs in Experimental Example 1.
  • FIG. 14 shows skin pathological sections after xenograft skin transplantation SCID mice were treated with different drugs in Experimental Example 1.
  • Fig. 15 shows a comparison of ADCC effects of RitxV301 and rituximab in Experimental Example 1.
  • FIG. 16 shows a 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. 18 shows the SPR analysis profiles of different concentrations of NVS451 and human FcRn under neutral conditions (pH 7.4) in Experimental Example 1.
  • Figure 19 shows the affinity comparison of IL-17RA fusion protein NVS451 (V301), V302 and V303 and IL-17A in Experimental Example 1; the concentration 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 Committee for the Collection of Microorganisms on November 23, 2020, and the preservation address: Chaoyang, Beijing No. 3, No. 1 Yard, Beichen West Road, District, Zip Code: 100101, and the deposit number is: CGMCC No.21011.
  • CGMCC General Microorganism Center
  • injection refers to: sterile solutions (including true solutions, emulsions and suspensions) made of drugs for injection into the body, and sterile solutions prepared before use (including true solutions, emulsions and suspensions), lyophilized powders or concentrated solutions.
  • intravenous infusion refers to a method of intravenously infusing a large amount of liquid containing drugs into the body through an infusion tube. Also known as “infusion”, “bit drip”, “static 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 in that it comprises an operably linked signal peptide, an IL-17RA extracellular domain and an IgG1 constant region sequentially connected in series.
  • the function of the signal peptide is mainly to guide the secretion of the target protein from the cytoplasm of the cell to the outside of the cell. Since the fusion protein has an IgG1 constant region, when it is secreted out of the cell, two molecules of the fusion protein form a double chain through the cysteine binding of the constant region and exert activity.
  • the present invention uses 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. Still 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 invention found that the IL-17RA fusion protein comprising these two mutants has improved thermostability and increased binding affinity to IL-17A compared with the wild type. The amino acid numbering is counted from the first position of the amino acid sequence of the extracellular domain of 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 invention finds that the IL-17RA fusion protein comprising the mutant has improved thermostability and improved binding affinity to IL-17A, IL-17C, IL-17F, IL-17A/F compared with comprising the wild type ; higher binding affinity to IL-17A than the mutants comprising the above-mentioned three and four mutations.
  • the present invention selects the constant region (Fc) of human IgG1 (Gene bank number: 3500) and IL-17RA to form a fusion protein.
  • the fusion protein not only retains the biological activity of the functional protein molecule, but because the Fc part has certain antibody characteristics and is stable, the fused protein has a longer circulation life and a longer half-life.
  • IgG isotypes include IgG1, IgG2, and IgG4, which elicit distinct ADCC, ADCP, and CDC effects that can have a major impact on target and non-target tissue toxicity.
  • 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 appropriately mutating the Fc sequence 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 a cysteine residue in the IgG1 hinge region with a serine residue to avoid 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 is well-defined by a single residue with a clear cleavage and 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 consecutive GSGs, one or more consecutive GGGGS, 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 (IL-17RA extracellular domain has three mutations, 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, in which the first 32 amino acids (bold) are the IL-17RA signal peptide, and the letters on the light gray background are recombined with 6 mutations (bold and underlined letters)
  • Asterisks (*) indicate potential glycosylation sites.
  • the mechanism of action of the IL-17RA fusion protein is to use the decoy receptor (IL-17RA-Fc) to compete with the natural receptor to bind to the IL-17 molecule, and to combine with 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 its receptor, but does not bind IL-17B, IL-17D, IL-
  • the combination of 17E can effectively block the biological activity of various pro-inflammatory IL-17 cytokines and inhibit the inflammatory signaling pathway, thereby more effectively relieving the symptoms of autoimmune diseases, and can obtain more effective drugs than single IL-17A target antibody drugs. Good therapeutic benefits, and better safety than IL-17RA monoclonal antibody.
  • the present invention also provides an isolated nucleic acid encoding the IL-17RA fusion protein according to the present invention.
  • the present invention uses molecular biology techniques to design the cDNA sequence of the fusion protein, and optimizes codons to express it in CHO cells.
  • the optimized DNA sequences encoding the amino acid sequences SEQ ID NO.1-8 are respectively shown as SEQ ID NO.9-16.
  • the present invention also provides an isolated mRNA transcribed from the DNA encoding the fusion protein described in the present invention.
  • the present invention also provides an expression vector, which contains 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 can facilitate the creation of high-yielding and stable cell lines.
  • the deposit number of the host cell is CGMCC 21011.
  • the host cell was constructed by using the CHO-S TM cell of Thermo Scientific Company, which can stably express the IL-17RA fusion protein of the present invention after construction.
  • the host cell 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, physical and chemical properties and biological functions; (2) It can grow on the wall , and can be cultured in suspension, and has a high ability to withstand shear stress and osmotic pressure; (3) has the ability to efficiently amplify and express recombinant genes, and the integration of foreign proteins is stable; (4) has the function of extracellular secretion of products , and rarely secretes its own endogenous protein, which is convenient for the separation and purification of downstream products; (5) It can be cultured in suspension or in serum-free medium to achieve high-density culture.
  • the CHO cells are the preferred system for the production of recombinant glycosyl proteins.
  • the host cell has been deposited in the General Microorganism Center (CGMCC) of the China Committee for the Collection of Microbial Cultures on November 23, 2020.
  • the preservation address is: No. 3, No. 1, Beichen West Road, Chaoyang District, Beijing, postcode: 100101, and the preservation 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 chains.
  • the IL-17RA fusion protein of the present invention has an IgG1 constant region, when it is secreted out of the cell, the two molecules of the fusion protein form a double chain through the cysteine in the constant region and exert 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 for the treatment of psoriasis, Crohn's disease, plaque psoriasis, Gastroenteritis, Behçet 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, spondylitis, ankylosing spondylitis, rheumatoid arthritis, inflammatory bowel disease, nonalcoholic fatty liver, giant cell arteritis, Non-radiological axial spondyloarthritis, acne vulgaris, triple-negative breast tumors, multiple myeloma, non-small cell lung cancer
  • the present invention is based on the binding affinity with IL-17A, biological activity cell experiment, GRO- ⁇ factor inhibitory ability, in vitro activity titer, in vivo potency, ADCC/CDC functional activity, FcRn binding affinity, safety pharmacology, pharmacokinetics
  • the drug activity and safety of IL-17AR fusion protein were comprehensively evaluated in terms of medicine and toxicology.
  • the IL-17RA fusion protein can target IL-17A, IL-17C, IL-17F, IL-17A/F and other targets with high affinity, thereby selectively blocking IL-17A , IL-17C, IL-17F and IL-17A/F bind to their receptors, thereby effectively blocking the biological activities of various pro-inflammatory IL-17 cytokines, inhibiting inflammatory signaling pathways, and thus more effectively relieving For the symptoms of autoimmune diseases, it can obtain better therapeutic benefits 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 .
  • Pharmaceutically acceptable auxiliary materials can be selected according to the dosage form used and actual needs.
  • the present invention deeply 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 excipients of the pharmaceutical composition are selected from one or more of diluents, buffers, protective agents, surfactants, and antioxidants.
  • the buffer is selected from the group consisting of histidine-acetate buffer, Tris-acetate buffer, hydrochloric acid buffer, phosphate buffer, acetate buffer, histidine buffer, arginine buffer, succinic acid buffer, One or more of the citrate buffer.
  • the protective agent is selected from one or more of trehalose, Tween-20, Tween-80, sucrose, amino acids (such as arginine), polyols, disaccharides, and polysaccharides.
  • trehalose and a combination of trehalose and arginine are most preferable.
  • the trehalose is present in the form of 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 made into a suitable dosage form as required.
  • the IL-17RA fusion protein is preferably in the form of freeze-dried preparation or injection.
  • the present invention also provides a method for treating psoriasis, Crohn's disease, plaque psoriasis, gastroenteritis, Behcet's syndrome, arthritis, uveitis, hidradenitis suppurativa, flat Lichen, parapsoriasis, asthma, psoriatic arthritis, tendinitis, relapsing-remitting multiple sclerosis, thyroid-associated eye disease, juvenile rheumatoid arthritis, multiple sclerosis, lupus nephritis, spondyloarthritis, Ankylosing spondylitis, rheumatoid arthritis, inflammatory bowel disease, nonalcoholic fatty liver disease, giant cell arteritis, nonradiographic axial spondyloarthritis, acne vulgaris, triple negative breast tumors, multiple myeloma, Injections for non-small cell lung cancer, adenocarcinoma, colorectal cancer, prostate cancer, Ka
  • the injection can be in the form of lyophilized powder or liquid preparation.
  • the injection is subcutaneous injection or intravenous infusion. Most preferred are subcutaneous injections.
  • the injection preferably comprises the IL-17RA fusion protein, Tris-acetic acid, arginine, trehalose, Tween-20 and a suitable solvent.
  • the injection comprises 5 mg/ml-150 mg/ml of the IL-17RA fusion protein or the protein dimer formed by the IL-17RA fusion protein, 2-100 mM Tris-acetic acid, 10-250 mM Arginine, 50-500 mM trehalose, 0.01-5% Tween-20 and suitable solvents.
  • the solvent can be a solvent commonly used in the preparation of injections.
  • a solvent commonly used in the preparation of injections.
  • water for injection buffered saline solution, dextrose aqueous solution, sodium chloride aqueous solution or lactated Ringer's solution, etc.
  • the liquid formulation is prepared using a solvent.
  • the liquid preparation can be prepared according to the prescription of the present invention by a method commonly used in the field of pharmacy.
  • Freeze-dried powders can be prepared by freeze-drying said liquid preparations.
  • the lyophilization process includes prefreezing, primary drying (sublimation) and secondary drying (desorption).
  • Pre-freezing includes lowering the temperature from 5°C to -40°C and maintaining it for an appropriate time; primary drying includes raising the temperature to -5 to 0°C and maintaining it for an appropriate time; secondary drying includes raising the temperature to 25°C to 30°C, and keep it 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 (amino acid numbering does not include the signal peptide sequence).
  • the linker peptide is GSG.
  • the IgG1 Fc domain uses IgG1, 2, 4 hybrids 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.
  • the pCHO1.0 mammalian cell expression vector from Thermo Scientific was used to construct the plasmid.
  • the pCHO1.0 expression vector contains two expression cassettes. Since only one expression cassette is required for the expression of NVS451 fusion protein, 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 synthetic 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, and the clones containing the correct size plasmid were inoculated and amplified, and a large number of plasmids were extracted.
  • Gene sequencing verified that the pCHO1.1-NVS451 expression vector was constructed correctly.
  • NVS451 fusion protein cell line selected CHO-S TM (cGMP-banked), which was derived from the Thermo Scientific company's Kit) cells as the original cell matrix.
  • NVS451 fusion protein expression plasmid pCHO1.1-NVS451 contains selection markers of puromycin and MTX.
  • pCHO1.1-NVS4510 was transferred into CHO-S cells by transfection reagent.
  • the transfection process was as follows: 50 ⁇ g circular expression plasmid pCHO1.1-NVS451 was diluted to 1.5ml volume by Opti PRO SFM, 50 ⁇ l Freestyle Max transfection reagent (GIBCO) was diluted to 1.5ml by Opti PRO SFM, and then the plasmid dilution and transfection Mix equal volumes of staining reagent diluent, add 30ml 1E6 cells/ml CHO-S cell suspension. After the transfected cells were cultured for 48 hours, puromycin and MTX were used as selection pressures for two-stage selection of stably transfected clone pools (Pools).
  • CHO-S lacks pac (adenylate cyclase) activity and only has basal DHFR (dihydrofolate reductase) activity
  • pac adenylate cyclase
  • DHFR basal DHFR
  • T3S1 and T3S4 were selected for the isolation of single clones (LDCs).
  • LDCs single clones
  • Monoclonal isolation adopts two rounds of limiting dilution to separate monoclonal, each round of cell plating concentration is less than 1 cell/well (according to statistical mathematical calculation method), and finally 5 candidate monoclonals are obtained: T3S4-7E3-6G5; T3S4- 7E3-3C3; T3S4-17G2-6E2; T3S4-8F2-6E10; T3S1-18E7-6C5.
  • the 70PDL (cell doubling time, equivalent to 70 passages) stability evaluation was performed on 5 candidate monoclonals. Based on the stability of expression stability, gene copy number, and mRNA transcription level, the monoclonal VAN301-T3S1- 18E7-6C5 was used as the main clone (the deposit number is CGMCC 21011), and VAN301-T3S4-17G2-6E2 was selected as the backup clone.
  • VAN301-T3S1-18E7-6C5 Use this monoclonal VAN301-T3S1-18E7-6C5 to establish a PCB (Primary Cell Bank), and on this basis, establish a GMP-conditioned MCB (Master Cell Bank) and WCB (Working Cell Bank).
  • PCB Primary Cell Bank
  • GMP-conditioned MCB Master Cell Bank
  • WCB Working Cell Bank
  • NVS451 fusion protein can be produced by 14 days fed-batch culture process.
  • Cell resuscitation Take a cell from the cell bank, resuscitate in a water bath at 37.0 ⁇ 0.5°C, transfer the seed solution to 10ml preheated Dynamis (containing 8mM L-Gln, 1:100ACA and 1g/L P188) immediately after thawing centrifuge at 300g for 5 minutes, discard the supernatant, then add 10ml of preheated Dynamis medium to resuspend the cells, and transfer the resuspended cell solution to a 125ml shake flask containing 28.0ml of Dynamis medium , the cell density is (0.15-0.35) ⁇ 10 6 cells/ml, and the cell viability is greater than 90%.
  • the subculture medium is Dynamis, and the initial seeding density of cells in subculture should be (0.40 ⁇ 0.10) ⁇ 10 6 cells/mL. After 3 days of culture, the cell density should reach (2.0 ⁇ 5.0) ⁇ 10 6 cells /mL can be subcultured, and the cell viability should be higher than 90.0% during the subculture process.
  • Reactor fed batch culture (Fed batch): The initial seeding density of cells was (0.40 ⁇ 0.10) ⁇ 10 6 cells/mL, and fed 2 ⁇ feed C+ (Efficient FeedC+, Cat#:A2503101, Gibco), during the culture process every day Detect the content of glucose and lactic acid, and add 450.0g/kg glucose mother solution to make up to 5.0g/L on the 3rd/5/7/9/11/13 days.
  • Harvest conditions for cell culture Harvest when the culture reaches day 14 or when the cell viability is lower than 80.0%, whichever comes first.
  • the cell culture harvest liquid was first clarified by two-stage deep filter membrane bag, then 1% Tween 80 and 0.3% tributyl phosphate were added to inactivate the virus, and then the target protein was captured by MabSelect SuRe affinity chromatography filler of GE Company , then use Millipore's Eshmuno CPX anion chromatography filler and GE's Capto Adhere cation chromatography filler to carry out two-step fine purification, then use ASAHI KASEI's BioEX nanofiltration membrane for nanofiltration, use Millipore's Pellicon 2, cut-off
  • the PES material with a molecular weight of 50KDa and the ultrafiltration membrane of the C flow channel are used for ultrafiltration and 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 ⁇ 100LMH (based on A1HC)
  • the maximum loading capacity of D0HC is 60L/m 2
  • the maximum loading capacity of A1HC is 140L/m 2
  • the pressure is controlled
  • the single-step recovery rate of depth filtration is generally around 90%.
  • Preliminary purification of the product was achieved by affinity chromatography using MabSelect SuRe media.
  • Use 50mM Tris-HAc, 150mM NaCl, pH 7.4 as the buffer solution for equilibration and washing after loading then use 20mM Tris, 1M NaCl, 0.5M Arg, pH8.6 for washing 2, and finally use 50mM glycine, pH3. 5 buffer to elute the target protein.
  • the eluate was neutralized to pH 7.8-8.2 with 1M Tris base.
  • Eshmuno CPX from Millipore was used as a cation chromatography filler. After affinity chromatography neutralization, the sample was adjusted to pH 6.3-6.7 with 1M HAc, and the adjusted sample was used as the loading sample for cation chromatography. After elution and gradient elution, it can effectively remove product-related impurities such as HCP, protein A, DNA, and some product analog impurities and fragments.
  • Capto Adere filler from GE was used as an anion chromatography filler to remove some impurities related to the product and process.
  • the cation chromatography eluate was first adjusted to pH 8.4-8.6 with 1M Tris base and water for injection, and the conductance was 19.0-23.0.
  • the sample after adjusting the pH and conductance was used as the sample for anion chromatography.
  • the samples eluted by anion chromatography were filtered by Asahi Kasei Nanofilter BioEX for virus removal.
  • the nanofiltration process mainly uses the different molecular sizes of viruses and protein products. Potential viruses are intercepted by the nanofiltration membrane, and the target protein flows through, thereby achieving the separation of the two.
  • the pre-filtration membrane can absorb 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 Micropore
  • BioEX Asahi Kasei
  • the intermediate product was concentrated using a Pellicon 2 (Millipore company) ultrafiltration membrane bag, and then the liquid was changed to a buffer system of 20 mM Tris-HAc, 65 mM Arg, 120 mM trehalose, and pH 7.5.
  • the material of the ultrafiltration membrane package 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 ; when concentrated, the inlet flux is 150-300LMH, the transmembrane pressure (TMP) ⁇ 1.5bar, and concentrated to a concentration of 18.0-22.0g/L; then change the liquid, change the liquid
  • the hourly inlet flux is 150-300LMH, TMP ⁇ 2bar, and the liquid replacement volume is ⁇ 5DV.
  • the inlet flux is 150-300LMH, TMP ⁇ 2bar, and the overconcentrated concentration reaches 80.0-90.0g/L.
  • the original solution after adding auxiliary materials and sterilizing and filtering is tested for quality, and the purity reaches more than 97%.
  • the active molecule of NVS451 is a double-chain fusion protein composed of human IL-17RA extracellular segment mutant and human IgG1Fc mutant, obtained through 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 (the dosage is 75ug/ml) can bind to human IL-17A with high affinity.
  • Coating solution Dissolve 1.06g of Na 2 CO 3 and 0.84g of NaHCO 3 in ultrapure water, adjust the pH value to 9.60 with concentrated hydrochloric acid, then set the volume to 200ml, filter with a 0.22 filter membrane, and store at 4°C;
  • 10 ⁇ TBS mother liquor Take 12.114g of Tris and 43.83g of NaCl and add them into ultrapure water to fully dissolve, adjust the pH value to 7.55 with hydrochloric acid, then adjust the volume to 500ml, filter with a 0.22 filter membrane, and store at 4°C;
  • 4 ⁇ substrate buffer mother liquor take 7.16g of Na HPO ⁇ 12HO and 2.1g of citric acid and fully dissolve them in ultrapure water, adjust the pH value to 5.5 with NaOH, then adjust the volume to 100ml, filter with a 0.22 filter membrane, put Store at 4°C.
  • IL-17A Reconstitute IL-17A with ultrapure water according to the COA, and let it stand for about 30 minutes to fully dissolve it. Dilute IL-17A to 30nM with the coating solution balanced at room temperature, and then add it to the microplate, 100 ⁇ l per well , Seal the plate with the sealing film and place it at 4°C overnight (about 16h).
  • the lotion is ready-to-use. Take 100ml of 10 ⁇ TBS mother solution and add it to 900ml of ultra-pure water to dilute it into 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 ready for use now. Take 5ml of the blocking solution and dilute it to 50ml with the washing solution to prepare a 0.5% BSA sample diluent. Dilute the sample to 10 ⁇ g/ml in a 2-fold gradient. Add the diluted sample to the microtiter plate. 100 ⁇ l/well, set background control (no coating + sample + secondary antibody, coating + no sample + secondary antibody, no coating + no sample + no secondary antibody). Seal the plate with film and incubate at 37°C for 1h.
  • the diluent is ready for use now, take 5ml of the blocking solution and dilute it to 50ml with the washing solution, and prepare it as a 0.5% BSA antibody dilution solution, and the AffiniPure goat anti-human IgG and Fc ⁇ fragment specificity (min X Bov, Hrs, Ms Sr Prot) were 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 hour.
  • 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 (amino acid sequences such as SEQ ID NO.6 and Shown in SEQ ID NO.7).
  • the affinity between V302 and V303 and IL-17A was detected by affinity ELISA.
  • Two kinds of kits (R&D, Cat#:317-ILB-050; Peprotech, Cat#:900-K84) were used to detect according to the method provided by the manufacturer, and the results showed ( Figure 19), NVS451 with six mutations and The percentage of IL-17A affinity (776% and 464% for the above two kits, respectively) was significantly higher than the affinity of V302 and V303 molecules to 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. No.: RP0921H-025, RP1031Y-025, RP0355M-025) of 4 species (human, cynomolgus monkey, mouse, rat) were compared under the same conditions; The binding of rat Biolegend, 778704) to NVS451, the results showed that NVS451 can bind to IL-17A of 4 different species, and the order of binding affinity from high to low is: human>cynomolgus monkey>rat>mouse (KD See Table 3).
  • NVS451 In addition to binding to IL-17A molecules of the human IL-17 family, NVS451 also binds to human IL-17C, IL-17F, and IL-17AF (results are shown in Figures 6-8, respectively). The KD results are summarized in Table 4.
  • Weak Binding The profile has a responsive signal, but the intensity is too low to quantify the readout.
  • the cell line CCD-1070Sk (human fibroblast epithelial cell line, ATCC CRL-2091) used in the in vitro activity detection of NVS451 belongs to the human fibroblast epithelial cell line, and there are multiple proteins including IL-17A, A/F and F on its surface.
  • IL-17A can combine with the IL-17A receptor IL-RA on the surface of the cell to stimulate the cell to produce the cytokine GRO- ⁇ , and its content can be accurately detected by the ELISA double-antibody sandwich method.
  • NVS451 When IL-17A and NVS451 were co-incubated with CCD-1070Sk cells, NVS451 could inhibit the binding of IL-17A to the IL-17A receptor IL-17RA on the cell surface by competing with IL-17A, thereby reducing the cell supernatant GRO- ⁇ secretion in the fluid. 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 mechanism of competitive inhibition. 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 IL-17A-induced inhibitory ability to release GRO- ⁇ from the human fibroblast epithelial cell line CCD-1070Sk, and the results were expressed by the half maximal inhibitory concentration IC50 value (Table 6) .
  • the human fibroblast epithelial cell line CCD-1070Sk cells in the T75 culture flask were digested with 2ml of 0.05% EDTA trypsin for about 2-5 minutes, and then neutralized by adding 8ml of complete medium (EMEM basal medium with 10% FBS) , then centrifuge at 1000rpm for 5 minutes, resuspend with complete medium to a cell density of 1E5/ml, resuspend 10000 cells per well in a 96-well plate with 100ul per well, and culture for about 24 hours until the cell confluency reaches 95%-100% start dosing experiments.
  • complete medium EMEM basal medium with 10% FBS
  • the concentration of the diluted detection antibody (biotinylated goat anti-human GRO ⁇ antibody, R&D Cat#DY275-05-840256) in the diluent is 1.0 ⁇ g/ml (initial concentration 100 ⁇ g/ml).
  • P. Use a microplate reader to monitor the color development at 405nm, and set the wavelength calibration at 650nm.
  • the human fibroblast epithelial cell line CCD-1070Sk induced by IL-17A/F and IL-17F can also produce GRO- ⁇ cytokines, and the in vitro titer results of NVS451 fusion protein induced by different ligands Expressed by half inhibitory concentration IC 50 value (Table 7).
  • the S-curves of inhibition are shown in Figure 10 and Figure 11, respectively.
  • Psoriasis is a chronic inflammatory skin disease characterized by excessive proliferation and abnormal differentiation of keratinocytes. 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 model and xenograft model.
  • Imiquimod (Imiquimod, IMQ) induced model IMQ is an agonist of Toll like receptor (TLR) 7/8, Van der Fits[2] found that IMQ induced psoriasis-like in mice
  • TLR Toll like receptor
  • IMQ is applied to the skin of mice to establish psoriasis It is a psoriasis model widely used at home and abroad.
  • the xenograft skin immunodeficiency mouse (SCID) model is to transplant human skin into SCID mice, and inject the patient's T cells to induce psoriasis symptoms. Its genetic phenotype and pathogenic process are closest to human psoriasis, and Due to animal immunodeficiency, it is not affected by anti-drag antibody (anti-drag antibody, ADA). It is one of the most suitable tools for studying the pathogenesis of psoriasis and drug development, and is most suitable for exploring new anti-drug antibodies before starting clinical trials. Preclinical Analysis of Psoriasis Agents/Treatment Strategies [6,7,8].
  • NVS451 protein concentration of NVS451 protein was configured into a formulation of 20mM Tris-acetic acid, 65mM arginine, 120mM 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, 10 in each group.
  • Mice negative control group (negative control group is to use above-mentioned preparation prescription to remove NVS451), hormone group (2mg/ animal dexamethasone), secukinumab group (60mg/kg) and test product high dose (15mg /kg), middle dose (10mg/kg), low dose (5mg/kg) groups.
  • Negative control group, test product group and Sujin group were subcutaneously administered every other day for a total of 28 days.
  • NVS451 has a certain degree of improvement effect on redness, plaque and scale, and it is positively correlated with the dose; there is no statistical difference in epidermal thickness between the 15mg/kg dose group and the Sujin group (P>0.05), and NVS451 in this model has Similar healing effect to Sujin.
  • the skin thickness was 334 ⁇ 174 ⁇ m
  • the histological score was 1.1 (according to the thickness of the epidermis of the lesion, the epidermis on the nipple was thinned, and the keratinized Hyperkeratosis, agranulocytosis, Munro microabscess, normal or abnormal reticular cristae elongation, vascular tortuosity, mononuclear cell infiltration in the papillary dermis and other indicators (comprehensive evaluation).
  • NVS451 does not depend on ADCC/CDC activity, and the cytotoxicity of Fc ADCC/CDC may bring unnecessary immune-related adverse events (irAE), which poses a potential safety risk [9]. To avoid this effect, the Fc part of NVS451 was mutated in the relevant active site.
  • affinity analysis study seven CD molecules associated with ADCC and the C1q molecule associated with CDC were evaluated. The results showed that the binding affinity of NVS451 (75ug/ml) to ADCC-related CD molecules was at no binding or at a low level (KD greater than 10 -5 M order of magnitude, see Table 12).
  • NVS451 had significantly lower affinity for ADCC/CDC-associated Fc receptors compared to the otherwise partially identical IL-17AR fusion protein (V301-wtFc) (75ug/ml) with native IgG1Fc (see Table 13 ).
  • amino acid sequence of V301-wtFc is shown in SEQ ID NO.17.
  • the Fab regions of IgG1 and IgG3 subtype antibodies first bind to target cells, and then their Fc parts bind to Fc receptors (such as Fc ⁇ RIII) on CTL cells such as NK, and then induce ADCC.
  • Fc receptors such as Fc ⁇ RIII
  • CTL cells such as NK
  • ADCC ADCC receptors
  • Cell models that lack membrane-expressing IL-17 cannot effectively test whether ADCC is produced in vitro. Therefore, the Fab segment of Rituximab (Rituximab) that can stimulate ADCC is fused with the Fc segment of NVS451 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).
  • Fc fusion proteins generally increase the half-life of target protein drugs and improve pharmacokinetic characteristics through Fc FcRn-mediated circulation [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 that of Sukinkang and wild-type IgG1Fc (ie V301-wtFc) (KD is on the order of 10 -8 M, see Table 14).
  • the experimental method is as follows: .
  • Running reagent containing 2mM KH 2 PO 4 , 10mM Na 2 HPO 4 , 137mM NaCl, 2.7mM KCl, 0.05% Tween-20 (Tween-20), pH adjusted to 6.0;
  • His capture kit (Catalog No.: 28-9950-56, GE), including: mouse anti-His antibody (1mg/mL), fixation 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 salt (EDC) and 10.5 mL of 1M ethanolamine (pH 8.5). Add 10mL of deionized water to each tube of EDC and NHS, and store in separate packages at -18°C or lower, with a shelf life of two months. (Refer to GE Amino Coupling Manual "22-0510-62AG").
  • IL-17 RA-wtFc i.e. V301-wtFc
  • FcRn proteins of different species i.e. V301-wtFc
  • concentration of desalted protein was determined by UV-V.
  • the desalted protein is divided into tubes with a size greater than 10 ⁇ g, and repeated freezing and thawing should be avoided.
  • CM5 chip biocore sensor chip, GE
  • FC4 experimental channel
  • 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").
  • the KD value of each antibody was 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 at pH 6.0 ( Figure 17); and at pH 7.4 Under neutral conditions (NVS451 concentration 100nM-1.5625nM) there is no binding (Figure 18).
  • the cynomolgus monkey purchased from Beijing Zhongke Lingrui Biotechnology Co., Ltd.
  • safety pharmacology test was carried out along with the long-term toxicity, and the subcutaneous injection of the prescription in Section 2.1 of Experimental Example 1, the dose of NVS451 was 15mg/kg, 50mg/kg and 150mg/kg. kg (administered 2 times a week, administered continuously for 4 weeks, administered 9 times in total, that is, administered on D1, D4, D8, D11, D15, D18, D22, D25 and D29 respectively).
  • Respiratory indicators and electrocardiogram examinations of animals in the group showed no drug-related TV and RR, and regular changes in heart rate, P-R interval, QT interval, QRS duration, QRS voltage, STe and QTcB values. See exception.
  • preparation prescription removes NVS451) vs high-dose group: 37.72 ⁇ 0.29°C vs 38.40 ⁇ 0.14°C); body temperature can be seen in female animals in the high-, medium-, and low-dose groups of the test product 24 hours after administration Elevated, 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-female consistency in the above-mentioned changes in body temperature, and no regular changes were seen, so it was considered to be irrelevant to the drug administration.
  • a total of 24 cynomolgus monkeys were used in the experiment, which were divided into 4 groups (6 animals in each group, half male and half male).
  • Single subcutaneous and intravenous injection administration groups 1 to 4 were given 5 (subcutaneous injection), 15 (subcutaneous injection), 50 (subcutaneous injection), 15 (intravenous injection) mg/kg of NVS451, and the administration volume was 1 mL/kg. kg.
  • the pharmacokinetic blood samples (about 1 mL) were collected from the non-administration part of the subcutaneous vein of the animal's hind limbs into a tube without anticoagulant. 2h, 4h, 8h, 24h (D2), 32h (D2), 48h (D3), 56h (D3), 72h (D4), 96h (D5); the blood collection time point of the fourth group of animals is: 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).
  • C max ratio mean value of medium and high doses of C max / mean value of low dose of C max ;
  • AUC ratio mean AUC last middle and high doses/AUC last mean low doses
  • test results show that: within the dose range of 5-50mg/kg, after a single subcutaneous injection in cynomolgus monkeys, the plasma concentration of NVS451 in cynomolgus monkeys increases with the dose; After the monkey, no significant difference was observed between sexes.
  • the average C max and AUC last increase ratio of NVS451 was higher than the dose increase ratio.
  • the ratios of Cmax of NVS451 in animals given subcutaneous injection of 5, 15, and 50mg/kg dose groups were 1:6.12:38.06 (male) and 1:5.46:34.29 (female); the ratios of AUC last were 1:4.83:25.52 (male) and 1:4.76:20.88 (female).
  • the independent sample t test showed that there was no statistical difference in the metabolic kinetic parameters between subcutaneous injection and intravenous injection groups, 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 is between 27.20 and 39.90; the T max of subcutaneous injection in each dose group is basically the same, The drug concentration reached its peak within 4-8 hours.
  • a total of 48 SD rats were used in the experiment, which were divided into 4 groups (12 animals in each group, half male and half male).
  • Single subcutaneous and intravenous injection administration groups 1 to 4 were given 10 (subcutaneous injection), 30 (subcutaneous injection), 100 (subcutaneous injection), 30 (intravenous injection) mg/kg of NVS451, and the administration volume was 2 mL/kg. kg.
  • pharmacokinetic blood samples (about 0.4 mL) were collected from the jugular vein of animals into tubes without anticoagulants.
  • the time points of blood collection for 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 the fourth group 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).
  • C max ratio mean value of medium and high doses of C max / mean value of low dose of C max ;
  • AUC ratio mean AUC last middle and high doses/AUC last mean low doses
  • test results showed that: within the dose range of 10-100 mg/kg, after a single subcutaneous injection to SD rats, the blood drug concentration of NVS451 in SD rats increased with the dose; No significant differences were observed between sexes.
  • the average Cmax and AUClast increase ratios of NVS451 were lower than the dose increase ratios.
  • the ratios of Cmax of NVS451 in animals given 10, 30, and 100 mg/kg dose groups by subcutaneous injection were 1: 2.03: 6.56 (male) and 1: 1.62: 6.04 (female); the ratios of AUClast were 1: 1.92: 5.29 (male). ) and 1:1.85:5.85 (female).
  • the T1/2 of NVS451 in SD rats, the T1/2 of male and female animals in each dose group is between 51.46-69.48; the Tmax of each dose group of subcutaneous injection is basically the same, and the drug concentration is between 8 ⁇ 24h to reach the peak.
  • the test article was given by subcutaneous injection at the back of the neck in a single 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.
  • Animals in groups 1-4 were collected thyroid gland, heart, lung, liver, spleen, kidney, bladder, gonad (ovary, testis), stomach, small intestine, fat, muscle, brain, Dorsal skin (not administration site), intestinal contents, serum, urine.
  • Feces and urine were collected from animals in the 5 groups once a day for a total of 5 days after administration.
  • Animals in the 6 groups were collected once an hour after injection to excrete bile for a total of 8 hours.
  • the drug After subcutaneous injection of 125I-NVS451 in rats, the drug is mainly distributed in intestinal contents, urine, bladder, back skin and other tissues/organs, while less distributed in muscle, fat and brain.
  • the peak time of the drug in most tissues is between 4h and 24h, and then the concentration gradually decreases with time.
  • the drug has a certain distribution on the back skin (non-administration site). Its peak time is 24h, and the drug content in 4h and 120h is roughly equal. Drug concentration decreased more slowly in dorsal skin than in other tissues/organs.
  • NVS451 is a macromolecular protein drug, which is expected to be degraded into peptides and amino acids in vivo, and then excreted or reused for protein or peptide synthesis in vivo , so the metabolism of NVS451 was not assessed.
  • NVS451 has cross-reaction with IL-17A in cynomolgus monkeys and rats, and is a related species. The tests were all carried out 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) "Drug Non-clinical Research Quality Management Practice "(Bureau Order No. 34, September 2017).
  • the purpose is to evaluate the acute toxic reaction that may occur within 14 days after a single subcutaneous injection of NVS451 is given to cynomolgus monkeys, and three dose groups of 45mg/kg, 150mg/kg and 450mg/kg are set up. During the experiment, no animals in each group were found dead or dying. There was no abnormality in clinical observation of animals in each administration group. Compared with the self-predrug value and the excipient control group (NVS451 was removed from the preparation prescription), there were no drug-related abnormalities in animal body weight, body temperature, electrocardiogram parameters, blood cell counts, coagulation indicators, blood biochemistry, and urine tests in each administration group Change.
  • the purpose is to evaluate the acute toxic reaction that may occur within 14 days after a single subcutaneous injection of NVS451 to SD rats, and set up three dose groups of 90mg/kg, 300mg/kg, and 900mg/kg. During the test period, no animals in each group were found dead or dying. At the end of the observation period (D15), no abnormal changes were found in the general observation of animals in each group. Conclusion: Under the test conditions, none of the animals died or died, and the maximum tolerated dose (MTD) of the animals was greater than or equal to 900mg/kg.
  • MTD maximum tolerated dose
  • 40 cynomolgus monkeys (20/sex) were used and randomly divided into 4 groups (5/sex/group) according to sex, which were respectively the auxiliary material control group and the low, middle and high dose groups of the test product.
  • the animals in the auxiliary material control group were given the auxiliary material reference substance, 2mL/kg, and the low, medium and high dose groups of the test product were given NVS451, the dosages were 15, 50, 150mg/kg, and the administration volumes were 0.2, 0.67, 2mL/kg, respectively. kg, the administration concentration is 75mg/mL.
  • mice All animals were administered subcutaneously in the hind limbs twice a week for 4 consecutive weeks, a total of 9 times, namely D1, D4, D8, D11, D15, D18, D22, D25 and D29.
  • body weight, body temperature, electrocardiogram, respiration (respiratory rate and tidal volume), blood pressure, eye examination, blood count, coagulation function, blood biochemistry, urinalysis and immunological indicators (T lymphocyte subsets) were carried out on the animals.
  • group cytokines, C-reactive protein, serum immunoglobulin, complement, anti-drug antibody) indicators; before and after the first and eighth administration, blood samples were taken from animals in each group to determine the blood drug concentration.
  • Some animals (3 animals/sex/group) were euthanized on the day after the last drug (D30), and the remaining animals were euthanized at the end of the 4-week recovery period (D57); bone marrow smears, film readings, and gross anatomical observations were performed on all animals , weighing the main organs, calculating relative organ weights (organ-to-body ratio and visceral-brain ratio), and performing histopathological examination on more than 40 kinds of tissues and organs.
  • mice in each group were found dead or dying. Animals in each group were clinically observed, body weight, body temperature, eye examination, urine examination, blood cell count, coagulation function (except FIB), blood biochemical indicators, lymphocyte subsets Group, complement, cytokines (except IL-17A) and immunoglobulin have no abnormal changes related to administration. There were no abnormal changes related to administration in the electrocardiogram, respiration and blood pressure parameters of the animals. There were no abnormal reactions such as erythema, edema, induration, ulceration and the like observed with the naked eye at the administration site.
  • the injection site and inguinal lymph nodes of the animals in the 15, 50 and 150 mg/kg dose groups showed changes related to the test product, and the injection site 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 paracortical/myeloid lymphocytes in the inguinal lymph nodes, which is related to the local mononuclear cell inflammation of the injection, and there is no obvious combination of clinical pathological indicators Changes are considered non-adverse reactions.
  • All animals were administered by subcutaneous injection on the back of the neck, administered twice a week for 4 consecutive weeks, a total of 9 administrations (i.e. D1, D4, D8, D11, D15, D18, D22, D25 and D29 administration ), a 4-week recovery period.
  • the animals in the main test group were mainly observed clinically, and the body weight, food intake, body temperature, blood cell count, coagulation function, blood biochemistry, eye examination, urine test, T lymphocyte subsets and cytokines were detected; Blood was collected before and after the first and eighth administration 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 at the end of the recovery period (D56)) blood was collected for antibody determination.
  • the first 10 animals/sex/group in the main test group were euthanized on the day after the last dose (D30), and the remaining animals were euthanized after the 4-week recovery period (D57).
  • the animals were euthanized, and the irritative reactions related to the test article were observed in the local injection of the 30, 100 and 300 mg/kg dose groups, showing mild to moderate mononuclear cell inflammation in the local subcutaneous and/or dermis of the injection.
  • the injection local inflammation was basically completely recovered.
  • the local tolerance toxicity test was carried out along with the long-term toxicity, and both cynomolgus monkeys and SD rats were administered twice a week (30, 100 and 300 mg/kg in three dose groups of rats, 15, 50 mg/kg in three dose groups of cynomolgus monkeys). and 150mg/kg), continuous administration for 4 weeks, a total of 9 times (ie D1, D4, D8, D11, D15, D18, D22, D25 and D29 administration). During the test period, no abnormal reactions such as erythema, edema, induration, ulceration, etc. were observed in the local observation of the animals in each group, and the local tolerance was good, and no toxicity was seen.
  • In vitro test tube method was used to observe the effect of 75mg/mL NVS451 on the hemolysis and aggregation of human erythrocytes (the experimental conditions were conventional conditions). From 15 minutes after incubation in the incubator to the end of 3 hours of observation, the upper layer of the test tube was colorless and clear, and the red blood cells at the bottom of the tube sank and dispersed evenly after shaking. No hemolysis and aggregation occurred. Under the test conditions, 75mg/mL NVS451 has no hemolytic effect on human red blood cells in vitro, and does not cause human red blood cell aggregation.
  • NVS451 can bind to human IL-17A with high affinity, which is higher than secukinumab and wild-type human IL-17RA under the same conditions.
  • 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 the ability to inhibit GRO- ⁇ cytokines induced by IL-17A, IL-17A/F, and IL-17F, and inhibited the GRO- ⁇ cytokines induced by IL-17A
  • the inhibitory ability is lower than the IC 50 value of secukinumab (Cosentyx).
  • NVS451 mainly acts by inhibiting the activities of IL-17A and IL-17C, IL-17F, and IL-17AF.
  • the interaction shown by the SPR experiment suggested that in vivo, the recommended dose of NVS451 exerted the main inhibition of IL-17A in psoriasis patients, supplemented by the clinical activity of inhibiting IL-17C, IL-17F, and IL-17AF molecules.
  • NVS451 binding affinity of NVS451 to ADCC-related CD molecules is non-binding or at a low level. Compared with natural IgG1Fc, its affinity with ADCC/CDC-related Fc receptors is significantly reduced, which proves that the ADCC binding site of NVS451 is mutated. . Binding affinity analysis with FcRn shows that NVS451 can bind with FcRn of human, monkey and rat. Under the same conditions, its affinity is at the same level as secukinumab and wild-type IgG1Fc.
  • NVS451 Under acidic conditions at pH 6.0, NVS451 can bind to FcRn of 3 different species of human, cynomolgus monkey, and rat, and at pH 7.4 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 action of NVS451 does not depend on ADCC/CDC activity, and the cytotoxicity of Fc ADCC /CDC may bring unnecessary immune-related side effects, and there are potential safety risks. To avoid this effect, the Fc part of NVS451 was mutated in the relevant active site.
  • NVS451 was administered at doses of 5 mg/kg, 10 mg/kg and 15 mg/kg every other day for 28 days, the results It shows that NVS451 has a certain degree of improvement effect on redness, plaque and scale, and it is positively correlated with the dose; there is no statistical difference in epidermal thickness between the 15mg/kg dose group and the Sujin group (P>0.05), and the highest dose of NVS451 is used every week (60mg/kg) once a week was as effective as a lower dose twice a week, and most importantly, a twice a week NVS451 or a higher dose once a week showed positive results with secukinumab Similar treatment effects were compared. Secukinumab is considered to be one of the most promising drugs for the treatment of psoriasis, and this study provides strong clinical evidence support for the effectiveness of clinical treatment.
  • NVS451 cross-reacts with cynomolgus monkey and rodent IL-17A
  • cynomolgus monkey and rat were chosen as relevant species for toxicity evaluation.
  • NVS451 was given to cynomolgus monkeys by repeated subcutaneous injection at doses of 15, 50 and 150 mg/kg, twice a week for 4 consecutive weeks, a total of 9 times of administration. No death or near death was observed in any animals in each group.
  • NOAEL no-observed adverse reaction dose of this test is considered to be 150 mg/kg.
  • the NVS451 fusion protein for injection was given to SD rats by repeated subcutaneous injection at doses of 30, 100 and 300 mg/kg, twice a week for 4 consecutive weeks, a total of 9 times of administration, and no obvious systemic toxicity was observed in the animals , the NOAEL is considered to be 300mg/kg. Toxicokinetics in cynomolgus monkeys and rats showed that NVS451 did not accumulate after repeated administration.
  • a single-dose non-GLP pharmacokinetic test was conducted in cynomolgus monkeys and rats to observe the basic pharmacokinetics of NVS451 administered by intravenous and subcutaneous routes.
  • the absorption pharmacokinetics of NVS451 after a single subcutaneous administration in male and female cynomolgus monkeys were evaluated, and its pharmacokinetic behavior has a low serum clearance rate and half-life typical of immunoglobulin molecules. Effective drugs can be detected after 96 hours of administration.
  • cynomolgus monkeys and SD rats there was no significant gender difference in the metabolic data.
  • the T1/2 of NVS451 in cynomolgus monkeys, male and female in each dose group The average T1/2 of the animals was between 27.20 and 39.90 hours.
  • the T1/2 of NVS451 in SD rats is within the dose range of 10-100mg/kg, and the T1/2 of male and female animals in each dose group is between 51.46-69.48h.
  • NVS451 is a high-affinity antibody Fc fusion protein selectively targeting IL-17A, IL-17C, IL-17F and IL-17AF heterodimeric cytokines.
  • the target has a high affinity.
  • the present invention has carried out the non-clinical data summary of the project, which provides evidence of target specificity and mode of action, and treatment with NVS451 twice a week or high dose once a week shows that it is more effective than Sekinin monotherapy.
  • the treatment effect was similar to that of the anti-positive control.
  • NVS451 was well tolerated in a comprehensive toxicology study. All non-clinical evaluation studies provide strong evidence support for the effectiveness and safety of NVS451 clinical treatment.

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Abstract

L'invention concerne une protéine de fusion Fc d'anticorps d'IL-17RA, une composition pharmaceutique, une injection et une utilisation de celles-ci. La protéine de fusion d'IL-17RA comprend des peptides de signal connectés séquentiellement et en série, opérationnels par connexion, un domaine extracellulaire d'IL-17RA et une région constante d'IgG1. La protéine de fusion d'IL-17RA selon la présente invention a une demi-vie étendue, possède une activité de médicament à action plus longue et une immunogénicité réduite par comparaison avec des médicaments à base d'anticorps. La conception de la présente invention permet à la protéine de fusion d'IL-17RA d'éliminer l'effet ADCC, ADCP et CDC, conserve le recyclage in vivo médié par les récepteurs Fc (FcRn) chez les nouveau-nés, et présente une réaction secondaire inférieure et est plus sûr comparé aux anticorps d'IL-17RA actuels disponibles dans le commerce.
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