WO2022194311A2 - 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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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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PCT/CN2022/100748
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WO2022194311A4 (fr
WO2022194311A3 (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 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

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.
PCT/CN2022/100748 2022-06-17 2022-06-23 Protéine de fusion fc d'anticorps d'il-17ra et son utilisation WO2022194311A2 (fr)

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