WO2019109819A1 - 一种hm-3融合蛋白及其应用 - Google Patents

一种hm-3融合蛋白及其应用 Download PDF

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WO2019109819A1
WO2019109819A1 PCT/CN2018/117188 CN2018117188W WO2019109819A1 WO 2019109819 A1 WO2019109819 A1 WO 2019109819A1 CN 2018117188 W CN2018117188 W CN 2018117188W WO 2019109819 A1 WO2019109819 A1 WO 2019109819A1
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tsl
fusion protein
group
ggggs
linker
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PCT/CN2018/117188
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English (en)
French (fr)
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黄瑞晶
范宝庆
李剑
马晓慧
王轶博
张莉华
曹小丹
李文蕾
王鹏银
陈妍
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天士力生物医药股份有限公司
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Priority to KR1020207012889A priority Critical patent/KR20200093530A/ko
Priority to CA3074755A priority patent/CA3074755A1/en
Priority to US16/768,619 priority patent/US11655308B2/en
Priority to AU2018378376A priority patent/AU2018378376B2/en
Priority to EP18884891.5A priority patent/EP3722305A4/en
Priority to JP2020530679A priority patent/JP7153726B2/ja
Publication of WO2019109819A1 publication Critical patent/WO2019109819A1/zh

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0019—Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0053—Mouth and digestive tract, i.e. intraoral and peroral administration
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A61P19/02—Drugs for skeletal disorders for joint disorders, e.g. arthritis, arthrosis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A61P19/08—Drugs for skeletal disorders for bone diseases, e.g. rachitism, Paget's disease
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/04—Linear peptides containing only normal peptide links
    • C07K7/08—Linear peptides containing only normal peptide links having 12 to 20 amino acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/30—Non-immunoglobulin-derived peptide or protein having an immunoglobulin constant or Fc region, or a fragment thereof, attached thereto

Definitions

  • the invention belongs to the field of biopharmaceutical technology, and more particularly to a long-acting HM-3 fusion protein molecule and application thereof.
  • Autoimmune diseases refer to diseases caused by the body's immune response to self-antigens and cause damage to their own tissues. Without timely and effective control, the consequences of autoimmune diseases are very serious, and ultimately even life-threatening. Common autoimmune diseases are: systemic lupus erythematosus, rheumatoid arthritis, scleroderma, hyperthyroidism, juvenile diabetes, primary platelet purpura, autoimmune hemolytic anemia, ulcerative colitis, and many types of skin. Disease, autoimmune liver disease, and so on.
  • RA Rheumatoid arthritis
  • RA is a chronic systemic autoimmune disease characterized by joint synovitis. Prolonged recurrent episodes of synovitis can lead to destruction of cartilage and bone in the joints, joint dysfunction, and even disability.
  • vascularlitis lesions involve various organs of the body, so this disease is also known as rheumatoid disease.
  • TNF ⁇ inhibitors are currently the mainstream biological agents for the treatment of rheumatoid arthritis; in 2013, the total sales of three TNF ⁇ inhibitors such as humira, enbrel and remicade were 28.108 billion US dollars, accounting for 84.58% of the total sales of the global rheumatoid market.
  • TNF inhibitors need to be combined with methotrexate to better perform, and some patients with rheumatoid arthritis are intolerant to methotrexate, resulting in the inability of these patients to use a combination regimen.
  • new blood vessels are spiral capillaries that protrude from the general blood vessels.
  • the human body can develop new blood vessels under certain conditions such as pregnancy.
  • new blood vessels appear, it will cause specific diseases.
  • angiogenic diseases such as cancer and wet macular degeneration.
  • Wet macular degeneration also known as neovascular macular degeneration, is characterized by the formation of choroidal neovascularization.
  • Osteoarthritis is a degenerative disease, which is caused by ageing, obesity, strain, trauma, joint congenital anomalies, joint deformities and other factors, such as degeneration of articular cartilage, joint edge and subchondral bone hyperplasia. Osteoarthrosis, degenerative arthritis, senile arthritis, hypertrophic arthritis, etc. Clinical manifestations include slow-developing joint pain, tenderness, stiffness, joint swelling, limited mobility, and joint deformities.
  • HM-3 is a polypeptide of 18 amino acid residues containing an arginine-glycine-aspartate sequence (RGD sequence), and its amino acid sequence is as follows:
  • HM-3 is a known structure and has been disclosed in CN1314705C, which has high affinity for integrin ⁇ v ⁇ 3, inhibits endothelial cell migration and angiogenesis by blocking the integrin ⁇ v ⁇ 3 signaling pathway and inhibiting the expression of VEGF and TNF ⁇ . Generate, thereby inhibiting RA synovial hyperplasia. Acute, subacute, and chronic inflammation tests in mice showed that HM-3 can simultaneously inhibit angiogenesis and inflammatory response, regulate the content of VEGF and TNF ⁇ in synovial tissue of collagen-type arthritis DBA/1 mice, and effectively alleviate RA symptoms. The treatment effect is better than methotrexate.
  • the HM-3 molecule has the advantages of clear efficacy and good safety, the in vivo half-life of the small peptide is only 27.66 ⁇ 7.37min. If the product is used clinically, it needs to be administered once or twice a day, greatly Limits clinical application.
  • modification or modification of the molecular structure is a common method for solving the problem of short half-life and continuous administration, and chemical modification is most widely used.
  • Chinese patent application CN102417540A discloses a polyethylene glycol modified HM-3:mPEG-SC-HM-3, which has a half-life much higher than that of HM-3.
  • PEG modification reduces immunogenicity and increases half-life by covering proteins, which results in PEG modification not applicable to all proteins. Some proteins cannot be modified normally because the modification sites are not exposed; some active sites of proteins are covered to cause activity decrease; Some protein modifications after conformational modification result in decreased activity or aggregation.
  • the PEG modified product must gradually degrade the drug protein by degrading the PEG molecule, but the low molecular weight PEG has nephrotoxicity, and the degradation mechanism of the high molecular weight PEG in vivo is unclear, which brings obvious drug risk to the PEG modified drug.
  • the PEG modification involves complex protein processing, and the length of the PEG molecule is different, the molecular weight of the PEG modified product is not uniform, which increases the production cost and reduces the homogeneity of the finished product, which brings problems to the industrialization.
  • the present invention provides an HM-3-Fc fusion protein and its use.
  • the present inventors have found that the preparation of a fusion protein of HM-3 and Fc can prolong the half-life of HM-3 while increasing the activity of the drug.
  • the present invention finds that the fusion protein exhibits higher biological activity and stability in vivo and in vitro, can significantly inhibit the inflammatory reaction, relieve symptoms of autoimmune diseases, neovascular diseases, osteoarthritis, and has long-acting plasma. half life.
  • the present invention discloses an HM-3-Fc fusion protein formed by linking an active polypeptide HM-3 to a human IgG-Fc fragment or an IgG-Fc mutant fragment.
  • the fusion protein of the present invention is prepared by simultaneously mutating a mutant of HM-3 and a human IgG-Fc fragment or an IgG-Fc fragment through a C-terminus or an N-terminus or a C-terminus and an N-terminus, and the ligation can be carried out through a ligation. Peptide linkage.
  • the fusion protein of the present invention can be described by the following structure:
  • n is selected from 1, 2, 3, 4 or 5.
  • Linker represents a linker peptide selected from the group consisting of:
  • a preferred linker peptide is the (GGGGS) a sequence, and the repeat number a is preferably 3, 4 or 5.
  • the IgG-Fc is a human IgG fragment of IgG1, IgG2, IgG3 or IgG4 or a mutant fragment thereof; among them, Fc of IgG2, IgG4 or a mutant thereof is preferred; and mutant mIgG4-Fc is most preferred.
  • the fusion protein of the present invention is preferably a fusion protein of the following sequence:
  • TSL-1 fusion protein HM-3-(GGGGS) 3 -IgG2-Fc;
  • TSL-2 fusion protein HM-3-(GGGGS) 3 -mIgG4-Fc;
  • TSL-3 fusion protein IgG2-Fc-(GGGGS) 3 -HM-3;
  • TSL-4 fusion protein mIgG4-Fc-(GGGGS) 3 -HM-3;
  • TSL-5 fusion protein HM-3-(GGGGS) 3 -IgG4-Fc
  • TSL-6 fusion protein IgG4-Fc-(GGGGS) 3 -HM-3
  • TSL-13 fusion protein HM-3-(GGGGS) 3 -mIgG4-Fc-(GGGGS) 3 -HM-3
  • TSL-14 fusion protein HM-3-HyFc
  • TSL-15 fusion protein mIgG4-Fc-G5-HM-3-G8-HM-3
  • TSL-16 fusion protein HyFc-(GGGGS) 3 -HM-3
  • TSL-17 fusion protein mIgG4-Fc-A (EAAAK) 4 A-HM-3
  • TSL-18 fusion protein mIgG4-Fc-(AP) 9 -HM-3
  • the fusion protein of the present invention can be produced by a synthetic method in which an HM-3 polypeptide and a Linker are linked by a covalent bond, and the other end of the Linker is linked to the IgG-Fc by a synthetic method.
  • the fusion protein of the invention can improve the action time of the drug in the body, prolong the half-life, and improve the compliance and compliance of the patient.
  • the present invention further provides a pharmaceutical composition comprising the fusion protein of the present invention, which is in the form of a pharmaceutically acceptable preparation selected from the group consisting of an injection, a capsule, a tablet, a pill, a nasal spray, or Aerosols are administered orally, intravenously, intravenously, subcutaneously or intramuscularly.
  • a pharmaceutically acceptable preparation selected from the group consisting of an injection, a capsule, a tablet, a pill, a nasal spray, or Aerosols are administered orally, intravenously, intravenously, subcutaneously or intramuscularly.
  • the invention further provides for the use of a fusion protein of the invention in the manufacture of a medicament for the treatment of an autoimmune disease.
  • the autoimmune disease is rheumatoid arthritis and has pharmacokinetic properties.
  • the invention further provides for the use of a fusion protein of the invention in the manufacture of a medicament for the treatment of neovascular diseases.
  • the neovascular disease is wet age-related macular degeneration, tumor metastasis.
  • the fusion protein of the present invention can be used as an ophthalmic drug, and has pharmacokinetic properties in the eye, particularly aqueous humor and vitreous.
  • the invention further provides for the use of a fusion protein of the invention in the manufacture of a medicament for the treatment of osteoarthritis.
  • the fusion protein of the present invention can protect chondrocytes and play a role in preventing and treating osteoarthritis through pathological changes of chondrocytes, and has pharmacokinetic properties.
  • the fusion protein of the present invention when prepared by genetic recombination, first synthesizes a DNA molecule encoding the fusion protein of the present invention, and the nucleotide sequence of the DNA is preferably the sequence of Sequence Listing 1.
  • the synthesized DNA molecule is prepared into a recombinant expression vector, preferably a mammalian cell expression vector, such as pcDNA3.4 (Invitrogen), through a plasmid.
  • a mammalian cell expression vector such as pcDNA3.4 (Invitrogen)
  • the present invention further transfects an expression vector into a host cell, preferably a mammalian expression cell, more preferably a HEK293 cell, a CHO cell.
  • a host cell preferably a mammalian expression cell, more preferably a HEK293 cell, a CHO cell.
  • the fusion protein of the present invention is isolated from the cell culture medium by expression culture of the host cell, and the fusion protein of the present invention can be obtained by purification.
  • the invention further provides for this a purification method of the fusion protein of the invention, preferably using a Protein A or Protein G affinity chromatography column.
  • FIG. 1 TSL-4 protein affinity capture map.
  • FIG. 1 Electrophoresis pattern after purification of TSL-5 and TSL-6.
  • Fig. 6 is a comparison diagram of the inhibitory effect of TSL-1 ⁇ 4 fusion protein on spleen lymphocyte proliferation.
  • Fig. 7 is a comparison diagram of the inhibition effect of TSL-13-18 fusion protein mouse spleen lymphocyte proliferation.
  • Fig. 8 is a comparative diagram showing the inhibitory effects of three Fc fragments and two directional spleen fusion proteins on spleen lymphocyte proliferation.
  • Fig. 10 is a graph showing the inhibition effect of proliferation of mouse spleen lymphocytes containing different amounts of HM-3 fusion protein.
  • Figure 11 is a graph showing the inhibitory effect of TSL-1 ⁇ 4 fusion protein on human macrophage U937 inflammatory factor TNF- ⁇ .
  • Figure 12 is a comparison of the inhibition rate of TSL-1 ⁇ 4 fusion protein on zebrafish angiogenesis.
  • FIG. 17 Effect of TSL-4 on arthritis scores in type II collagen-type arthritis mice.
  • Figure 20 is a graph showing the blood concentration time after a single subcutaneous injection of 12.5 mg/kg of TSL-4.
  • Figure 21 is a graph showing the plasma concentration time after a single subcutaneous injection of 37.5 mg/kg of TSL-4.
  • Figure 22 is a graph showing the plasma concentration time after a single subcutaneous injection of 4.17 mg/kg of TSL-4.
  • Figure 23 is a graph showing the mean plasma concentration time after a single subcutaneous injection of different doses of TSL-4 in rats.
  • Figure 24 is a graph of peak TSL-4 concentration (Cmax) in plasma versus dose administered.
  • Figure 25 is a graph showing the area under the curve of the drug and the dose administered.
  • Fig. 28 Radioactivity uptake pattern at 6 time points after injection of 89Zr-HM-3 into the left ankle joint cavity of arthritis model rats.
  • Fig. 29 The articular radioactivity uptake map was taken at 48h and 120h, respectively, after subcutaneous injection of 89Zr-HM-3 in arthritis model rats.
  • HM-3-Fc fusion protein consists of three parts, including an active part HM-3 polypeptide, a linker, and a human IgG-Fc fragment or an IgG-Fc mutant fragment; the specific design method is as follows:
  • Human IgG type antibodies have four subtypes depending on the heavy chain, which are IgG1, IgG2, IgG3, and IgG4.
  • the existing research results show that the four subtypes have different plasma half-life, cytotoxicity and other characteristics (see Table 1), and are directly related to the Fc fragment.
  • IgG3 has the shortest half-life in vivo, and its Fc fragment is not suitable as a chaperone to prolong the in vivo half-life of the protein of interest, and was first excluded by experiments.
  • the affinity of the Fc fragment for complement and Fc receptors determines antibody-dependent cytotoxicity (ADCC effect) and complement-dependent cytotoxicity (CDC effect) effector function.
  • IgG1 and IgG3 have the strongest ADCC and CDC effects.
  • the assay determined that the fusion protein was not suitable for construction as a healthy cell, and thus the Fc fragments of IgG1 and IgG3 were excluded after screening.
  • the target is normal somatic cells, only need to inhibit the signaling pathway, and extend the half-life in vivo as much as possible, so low or no ADCC effect and CDC effect are needed, according to which HM-3 fusion
  • the FC portion of the protein is more suitably selected for the Fc fragment of IgG2 or IgG4.
  • the present invention also contemplates the expansion of Fc fragment mutants of IgG2 and IgG4 within the screening range.
  • Linker is a polypeptide chain that links functional and chaperone proteins. Linker's sequence and length play a crucial role in the function of the fusion protein. The choice of Linker needs to consider the following aspects:
  • Linker 1) The length should be moderate, too short to ensure the relative independence of the space between proteins. Too long may increase the risk of Linker rupture and cause immunogenicity; 2) Linker's sequence cannot contain protease cleavage sites to avoid being cut off 3) Linker can choose natural Linker, manual Linker; 4) Linker can be divided into rigid Linker and flexible Linker.
  • GGGGS 1 (GGGGS) a is the most widely used flexible Linker, where a can be 1, 2, 3, 4, 5 or 6;
  • 2A(EAAAK) b A is an ⁇ -helical rigid Linker, where b can be 1, 2, 3, 4, 5 or 6;
  • 3(AP) c is a linear rigid Linker, where c can be from 1 to 18;
  • 4G d is a flexible Linker similar to (GGGGS) n , where d can be from 1 to 15.
  • N-terminal ligation refers to the attachment of a functional protein to the N-terminus of the Fc fragment
  • C-terminal ligation refers to the attachment of a functional protein to an Fc fragment.
  • TSL-1 fusion protein HM-3-(GGGGS) 3 -IgG2-Fc;
  • TSL-2 fusion protein HM-3-(GGGGS) 3 -mIgG4-Fc;
  • TSL-3 fusion protein IgG2-Fc-(GGGGS) 3 -HM-3;
  • TSL-4 fusion protein mIgG4-Fc-(GGGGS) 3 -HM-3;
  • TSL-5 fusion protein HM-3-(GGGGS) 3 -IgG4-Fc
  • TSL-6 fusion protein IgG4-Fc-(GGGGS) 3 -HM-3
  • TSL-13 fusion protein HM-3-(GGGGS) 3 -mIgG4-Fc-(GGGGS) 3 -HM-3
  • TSL-14 fusion protein HM-3-HyFc
  • TSL-15 fusion protein mIgG4-Fc-G5-HM-3-G8-HM-3
  • TSL-16 fusion protein HyFc-(GGGGS) 3 -HM-3
  • TSL-17 fusion protein mIgG4-Fc-A (EAAAK) 4 A-HM-3
  • TSL-18 fusion protein mIgG4-Fc-(AP) 9 -HM-3
  • the above 12 fusion proteins are the most representative candidate protein structures, and the cell expression and in vitro pharmacodynamic experiments are simultaneously performed, and the results of in vitro pharmacodynamic experiments are obtained. Through different comparison methods, various structural variables can be compared and selected. Optimal fusion protein structure. Then, through in vivo pharmacodynamics, pharmacokinetics, toxicology, etc., the possibility of drug formation was evaluated, and further developed into an innovative drug for treating autoimmune diseases such as rheumatoid arthritis.
  • the 12 fusion proteins are based on the same design route and use the same expression vector, so the transient transfection expression vector construction process is completely consistent, and the expression of mIgG4-Fc-(GGGGS) 3 -HM3 (TSL-4) fusion protein is introduced.
  • TSL-4 transient transfection expression vector construction process
  • the TSL-4 fusion protein is linked to an HM-3 molecule using the mIgG4-Fc fragment, and Linker uses (GGGGS) a , where a is 3.
  • amino acid sequences constituting the respective portions of TSL-4 were spliced in the order of mIgG4-Fc-(GGGGS) 3- HM3, and the sequence is shown in Table 12.
  • the above DNA sequence was fully synthesized by a biotechnology company, and ligated with the commercial expression vector pcDNA3.4-TOPO vector of Invitrogen to obtain the expression vector pcDNA3.4-mIgG4-Fc-(GGGGS) 3- HM3.
  • the expression vector was stored in E. coli DH5 ⁇ , sent to the laboratory in the form of a glycerol tube, and stored at -80 °C.
  • the E.coli DH5 ⁇ glycerol tube containing pcDNA3.4-mIgG4-Fc-(GGGGS) 3 -HM3 was thawed under sterile conditions and inoculated into 50 ml 100 ug/ml Amp-resistant LB medium according to the 1% inoculum. In a 250 ml shake flask, incubate at 37 ° C, 160 rpm overnight.
  • All vectors used for transfecting cells must be sterile and control endotoxin, so all expression vectors were prepared using the Roche Genopure plasmid Maxi Kit for plasmid extraction and plasmid processing.
  • d Purify the lysate. Use a speed of 12000 g or more at 4 ° C, centrifuge for 45 min or more, and carefully transfer the supernatant to the adsorption column.
  • the adsorption column was placed on a detoxified, sterile 50 ml round bottom high speed centrifuge tube, and 14 ml of elution buffer (Elution Buffer) preheated at 50 ° C was added, and gravity flowed.
  • Elution Buffer elution buffer
  • ThermoFisher's Expi293 Expression System is a commercial rapid protein preparation kit based on transient transfection of 293F cells (a screened HEK293 cell) for rapid acquisition of fusion proteins.
  • the preparation of the 12 fusion proteins used was carried out according to the following experimental protocol.
  • Transfected Expi293F cells were passaged at least three times from recovery. In the process of passage, according to the needs of the experiment, the scale of cultivation was expanded in turn.
  • the cell density should be 3-5 ⁇ 10 6 cells/ml, and the viability is greater than 95%.
  • the cell density was adjusted to 3 x 10 6 cells/ml, and the cell volume in each 2 L shake flask was adjusted to 680 ml.
  • plasmid DNA was reconstituted in 40 ml of Opti-MEM I Reduced Serum Medium and gently mixed.
  • ExpiFectamine 293 Reagent was added to Opti-MEM I Reduced Serum Medium to a volume of 40 ml. Gently mix and incubate for 5 min at room temperature (long incubation affects conversion efficiency).
  • the 12 fusion proteins are essentially Fc fusion proteins, they can be specifically captured by ProteinA affinity chromatography column. During the actual purification process, the purification parameters are completely identical. Therefore, the purification of a batch of TSL-4 protein is For example, the purification process of the fusion protein is described.
  • the method information is as follows:
  • the combined sample volume was approximately 138 ml, and the pH was adjusted to 4.12 to 7.0 with 20 mL of 1N NaOH. The eluent was changed from slightly turbid to clear. The measurement results are shown in Fig. 1.
  • the sample is proteinA eluent, the sample loading is 40ml;
  • peak 3 is the target protein peak, collect peak 3, start peak collection at 10 mAU, stop collecting at 10 mAu after peak;
  • Sample ultrafiltration concentration The sample of peak 3 was combined and concentrated by ultrafiltration.
  • the ultrafiltration membrane was selected to be 10 kDa, and the sample was concentrated to a protein concentration of more than 5 mg/ml, and then the sample was dispensed and stored in a refrigerator at -80 °C.
  • the initial combined sample volume of this batch is about 550 ml, the concentration is about 0.29 mg/ml, and finally concentrated to 27 ml, the concentration is about 5.53 mg/ml; the sample is divided and frozen.
  • the 20160308 batch electrophoresis purity was approximately 96.9%; as shown in Figure 4, the 20160308 batch HPLC-SEC purity was approximately 99.3%.
  • TSL-1 fusion protein HM-3-(GGGGS) 3 -IgG2-Fc;
  • TSL-2 fusion protein HM-3-(GGGGS) 3 -mIgG4-Fc;
  • TSL-3 fusion protein IgG2-Fc-(GGGGS) 3 -HM-3;
  • TSL-4 fusion protein mIgG4-Fc-(GGGGS) 3- HM-3.
  • TSL-5 fusion protein HM-3-(GGGGS) 3 -IgG4-Fc
  • TSL-6 fusion protein IgG4-Fc-(GGGGS)3-HM-3
  • TSL-13 fusion protein HM-3-(GGGGS) 3 -mIgG4-Fc-(GGGGS) 3 -HM-3
  • TSL-14 fusion protein HM3-HyFc
  • TSL-15 fusion protein mIgG4-Fc-G 5 -HM-3-G 8 -HM-3
  • TSL-16 fusion protein HyFc-(GGGGS) 3 -HM-3
  • TSL-17 fusion protein mIgG4-Fc-A (EAAAK) 4 A-HM-3
  • TSL-18 fusion protein mIgG4-Fc-(AP) 9 -HM-3
  • TSL-5 and 6 fusion proteins have serious dimer degradation problems, and a large amount of monomer exists, indicating that the fusion protein prepared by using IgG4-Fc cannot be stably formed. Dimer, thus eliminating both TSL-5 and TSL-6 fusion protein designs.
  • Example 7 Fusion protein mouse spleen lymphocyte proliferation inhibition experiment
  • mice were lethal to death, immediately immersed in 75% ethanol for 5 to 10 minutes, and the spleen was taken out in PBS on a clean bench.
  • the spleen was placed on a sterile cell sieve (200 mesh), and ground with a syringe head.
  • the PBS was continuously added in the middle, and the collected slurry was centrifuged (1000 rpm, 5 min), and the cells were washed 3 times with Tris-ammonium chloride solution. It was washed again with medium and then resuspended in medium. Finally, the cells were stained with trypan blue living cells, and the survival rate was over 95%.
  • 96-well plates were incubated for 48 h at 37 ° C in a 5% CO 2 incubator. 5 mg/mL of MTT was added to a 96-well plate at 20 ⁇ L per well, and incubation was continued for 4 hours in an incubator. Discard the medium in the 96-well plate, add 100 ⁇ L of DMSO to each well, and mix gently. The absorbance was measured with a microplate reader at a measuring wavelength of 570 nm and a reference wavelength of 630 nm.
  • PI Proliferation inhibition rate
  • a test is the absorbance of the dosing group, and A control is the absorbance of the negative control group.
  • the results obtained by the experiment were expressed as mean ⁇ SD, and a statistical T test was performed, *P ⁇ 0.05 was a significant difference, and **P ⁇ 0.01 was a very significant difference.
  • the efficacy of the fusion protein against rheumatoid arthritis was screened based on the results.
  • TSL-1-4 was tested.
  • the specific dosing scheme is shown in Table 2.
  • TSL-13-18 was tested.
  • the specific dosing scheme is shown in Table 3.
  • the P4 control group used the TSL-4 fusion protein as a control drug.
  • Figure 7 shows the inhibitory effect of each group of TSL-13-18 drugs on ConA-stimulated spleen cell proliferation.
  • the inhibition rate of spleen lymphocyte expansion in the P4 positive drug group and the solvent group was significantly increased (P ⁇ 0.05), while in the TSL-13-18 group, only TSL-17 and TSL-18 were used.
  • the concentration gradient of each sample was significantly higher than that of the ConA group (P ⁇ 0.05), but the inhibition rate was inferior to that of the P4 positive drug group.
  • Example 8 Single factor comparison of 12 alternative fusion protein structures
  • Example 6 it was found during the purification that the TSL-5 and TSL-6 fusion proteins were not naturally stable in maintaining the expected normal dimer state, so the two TSL-5 and TSL-6 were based on natural The human IgG4-Fc fragment fusion protein was eliminated, while the IgG4-Fc element was no longer a candidate fusion protein Fc fragment. Based on the results of mouse spleen lymphocyte proliferation inhibition experiments, the other 10 alternative fusion proteins were compared for a single structural factor.
  • the GGGGS*3 Linker was selected as the unified Linker. After excluding the native human IgG4-Fc, the three alternative Fc fragments, IgG2-Fc, mIgG4-Fc and HyFc, were combined to form six alternative fusions.
  • the proteins were TSL-1, 2, 3, 4, 14, and 16, respectively, and the optimal inhibition rates of each sample are shown in Table 4.
  • the number in the table is the inhibition rate of mouse spleen lymphocytes.
  • the C-terminal connection mode that is, HM-3 is attached to the C-terminus of the Fc fragment, and its pharmacological effect is significantly better than that of the N-terminal connection mode, so the N-terminal connection mode is excluded.
  • TSL-4 is slightly higher than that of TSL-3, so it is preliminarily believed that mIgG4-Fc is superior to IgG2-Fc, but IgG2-Fc is not eliminated, and TSL- is further compared in subsequent experiments. 3 and TSL-4.
  • TSL-4, TSL-17, and TSL-18 were used in a unified manner using the mIgG4-Fc and C-terminal ligation methods, and Linker was screened by comparing the in vitro efficacy.
  • the results of inhibition experiments on mouse spleen lymphocyte proliferation showed that the cell inhibition rates of TSL-17 and TSL-18 were lower than those of TSL-4, so it can be concluded that among the three types of Linker, A(EAAAK) n A and AP) n These two rigid Linkers are not suitable for the construction of HM-3 Fc fusion proteins.
  • the fusion protein molecule contains more HM-3, and the same dose will bring more potency.
  • TSL-13 was designed to link HM-3 at both ends of the Fc fragment
  • TSL-15 was designed to connect two HM-3s at the C-terminus of the Fc fragment.
  • HM-3 was linked to the C-terminus of Fc, and a single HM-3 molecule was ligated using GGGGS*3Linker flexible Linker to maximize the efficacy of HM-3, while IgG2-Fc and mIgG4-Fc Compared to the two Fc fragments, the mIgG4-Fc effect was slightly better, but not sufficient to eliminate IgG2-Fc, so the two protein structures TSL-3 and TSL-4 were further compared in subsequent experiments.
  • Example 9 In vitro drug screening of HM-3 fusion protein in human macrophage U937
  • the cell culture medium was collected, centrifuged, and the supernatant was taken, and the content of TNF- ⁇ in the supernatant was determined by ELISA.
  • the results obtained by the experiment were expressed as mean ⁇ SD, and a statistical T test was performed, *P ⁇ 0.05 was a significant difference, and **P ⁇ 0.01 was a very significant difference. According to the results, the efficacy of HM-3 fusion protein against rheumatoid arthritis was screened.
  • TNF ⁇ content in the supernatant of the high and low dose groups of HM-3 was 58.9 ⁇ 10.6pg/mL and 83.9 ⁇ 20.4pg/mL, respectively.
  • the content of TNF- ⁇ in the supernatant of mPEG-SC-HM-3 high and low dose groups was 45.6 ⁇ 5.9pg/mL and 57.3 ⁇ 2.7pg/mL, respectively. From the inhibition effect, TSL-4 was far superior. In the polypeptides HM-3 and mPEG-SC-HM-3.
  • Example 10 Screening of anti-angiogenic effects of HM-3 fusion protein in zebrafish
  • Transgenic vascular zebrafish were randomly selected from six-well plates in 30 wells, and HM-3 polypeptide, TSL-1 fusion protein, TSL-2 fusion protein, TSL-3 fusion protein and TSL- were separately administered intravenously.
  • 10 zebrafish were randomly selected from each group.
  • the inferior vasculature (SIVs) of vascular fluorescent transgenic zebrafish were observed by fluorescence microscope, photographed and maintained, and image analysis was performed using Nikon NIS-Elements D 3.10 advanced image processing software.
  • the intestinal vascular area (S) of the experimental group was calculated.
  • the angiogenesis inhibition rate is calculated as follows:
  • the positive control drug Avastin 500ng/tail group zebrafish intestinal vascular area (37853) compared with the normal control group (53193) p ⁇ 0.001 (compared with the normal control group, *p ⁇ 0.05, **p ⁇ 0.01, ***p ⁇ 0.001), and its angiogenesis inhibition rate was 29%, indicating that Avastin has obvious angiogenesis inhibitory effect.
  • HM-3 polypeptide, TSL-1, TSL-2, TSL-3 and TSL-4 fusion protein at 20 ng / tail dose, zebrafish intestinal blood vessel area were 52186, 39929, 42828, 40705 and 33869, respectively, with normal control In the group (53193), the angiogenesis inhibition rates were 2%, 25%, 19%, 23%, and 36%, respectively.
  • the results showed that the HM-3 polypeptide had no angiogenesis inhibitory effect at the injection dose of 20 ng/tail; the TSL-1, TSL-2, TSL-3 and TSL-4 fusion proteins were significant at the injection dose of 20 ng/tail.
  • the intestinal blood vessel area of the zebrafish were 42492, 37022, 32374, 38660 and 28297, respectively, compared with the normal control group ( Compared with 53193), the inhibition rate of angiogenesis was 20%, 30%, 39%, 27% and 47%, respectively, indicating that the five fusion proteins had significant angiogenesis inhibition at the injection dose of 66 ng/tail.
  • TSL-4 has the best inhibitory effect on angiogenesis.
  • Example 11 Chronic inflammation model of type II collagen-type arthritis in mice
  • mice Take Balb/c mice, first immunization, bovine type II collagen concentration is 4mg/mL, on the day of the test (0 days), complete Freund's adjuvant (CFA) and CII solution are mixed and emulsified in equal volume.
  • CFA complete Freund's adjuvant
  • CII solution CII solution
  • mice in the normal control group of Balb/c mice each mouse in each group was intramuscularly injected with 50 ⁇ L of emulsifier at the base of the tail to induce sensitization. After 21 days, the same dose of emulsifier was used for re-immunization at the base of the tail. Incomplete Freund's adjuvant (IFA) is used.
  • arthritis symptoms such as redness and swelling of the toe joints of the model mice showed successful modeling.
  • G6 (TSL-4, 50 mg/kg, once every 5 days for 3 times);
  • G7 (TSL-4, 25 mg/kg, once every 5 days for 3 times);
  • G8 (TSL-4, 25 mg/kg, once every 7 days for 2 times);
  • G9 (TSL-4, 25 mg/kg, once every 14 days, 1 time);
  • G10 (TSL-4, 12.5 mg/kg, once every 5 days for 3 times);
  • G11 (TSL-4, 12.5 mg/kg, once every 7 days for 2 times);
  • G12 (TSL-4, 12.5 mg/kg, once every 14 days, 1 time);
  • the normal control group and the model control group were injected with physiological saline subcutaneously, the dosage volume was 0.1 mL/10 g, and the administration was given once every other day for 8 times; the positive drug adalimumab group was subcutaneously injected with adalimumab 8 mg/kg.
  • the drug volume was 0.1 mL/10 g, and it was administered once every 2 weeks for 1 time. After 15 days of treatment, it was observed for another 15 days.
  • the specific dosage regimen is shown in Table 6.
  • Group Count only Dosage Frequency way Normal group (G1) 12 Saline 1 time/2d, 8 times SC Model group (G2) 12 Saline 1 time/2d, 8 times SC Adalimumab (G3) 8 8mg/kg 1 time / 14d, 1 time SC HM-3 (G4) 8 1.6mg/kg 2 times / d, 30 times IV mPEG-SC-HM-3(G5) 8 20mg/kg 1 time/2d, 8 times SC TSL-4 (G6) 8 50mg/kg 1 time/5d, 3 times SC TSL-4 (G7) 8 25mg/kg 1 time/5d, 3 times SC TSL-4 (G8) 8 25mg/kg 1 time / 7d, 2 times SC TSL-4 (G9) 8 25mg/kg 1 time / 14d, 1 time SC TSL-4 (G10) 8 12.5mg/kg 1 time/5d, 3 times SC TSL-4 (G11) 8 12.5mg/kg 1 time / 7d, 2 times SC TSL-4 (G12) 8
  • Mouse body weight measurement The body weight of each mouse was weighed using an electronic balance and measured every two days.
  • Foot thickness measurement The thickness of the left and right hind paws of each mouse was measured with a vernier caliper and measured every two days.
  • Ankle joint width measurement The width of the ankle joint of each mouse was measured with a vernier caliper and measured every two days.
  • AI score The severity of joint inflammation in each mouse was scored using the paw joint score. 0: no redness and swelling; 1: small toe joint swelling; 2: toe joints, toes are red and swollen; 3: ankles are red and swollen; 4: toe joints, toes, ankle joints are red and swollen. Score once every two days until the end of the experiment.
  • Blood was collected from the eyelids and serum was separated. After the animals were removed from the neck, the spleen and thymus were separated, weighed, and the spleen coefficient was calculated. The spleen and thymus were fixed in formalin fixative. From the lower end of the humerus, the upper edge of the external iliac crest was cut off, the weight of the entire foot including the ankle joint was weighed, and fixed in formalin fixative for histopathological examination.
  • TSL-4 Different administration regimens of TSL-4 have different therapeutic effects on the mouse type II collagen arthritis model, including G3 group (positive drug adalimumab group), G4 group (HM-3 group), G5 group.
  • mPEG-SC-HM-3 group G6 group (TSL-4, 50 mg/kg, once every 5 days for 3 times), G7 group (TSL-4, 25 mg/kg, once every 5 days, 3 times), G8 group (TSL-4, 25mg/kg, once every 7 days for 2 times), G10 group (TSL-4, 12.5mg/kg, 5 days for 3 times)
  • a better therapeutic effect, foot thickness, ankle width, foot circumference, arthritis score, spleen weight, foot weight and model group were significantly different ( * p ⁇ 0.05), of which G5 group (mPEG-SC) -HM-3 group), G6 group (TSL-4, 50 mg/kg, once every 5 days for 3 times), G7 group (TSL-4, 25 mg/kg, once every 5 days for 3 times) G8 group
  • G3 group positive drug adalimumab group
  • G5 mPEG-SC-HM-3 group
  • G6 group TSL-4, 50 mg/kg, once every 5 days for 3 times
  • G7 group TSL-4, 25 mg
  • G8 group TSL-4, 25mg/kg, 7 days for 2 times
  • the treatment effect is better than G3 group (positive drug adalimumab group)
  • the G4 group (HM-3 group) and the G10 group TSL-4, 12.5 mg/kg, once every 5 days for 3 times) were slightly less effective than the G3 group (positive drug adalimumab group).
  • the body weights of G3, G4, G5, G6, G7, G8, and G10 were 21.0 ⁇ 1.5, 20.9 ⁇ 1.5, 21.5 ⁇ 1.3, and 20.8 ⁇ 1.8. 21.1 ⁇ 1.6, 20.8 ⁇ 1.7, 21.5 ⁇ 1.5g, compared with the model group (G2 group, 19.5 ⁇ 1.6g), the increase is obvious;
  • the G3, G4, G5, G6, G7, G8, and G10 mice had a posterior degree of 3.04 ⁇ 0.11, 3.06 ⁇ 0.23, 2.94 ⁇ 0.13, 2.92 ⁇ 0.11, 2.94 ⁇ 0.07, 2.99 ⁇ 0.17, 3.15 ⁇ 0.07mm, compared with the model group (G2 group, 3.85 ⁇ 0.30mm), there is a very significant difference;
  • the G3, G4, G5, G6, G7, G8, and G10 groups had an ankle width of 4.23 ⁇ 0.08, 4.21 ⁇ 0.15, 3.99 ⁇ 0.12, 3.96. ⁇ 0.09, 3.98 ⁇ 0.08, 4.00 ⁇ 0.16, 4.26 ⁇ 0.13mm, compared with the model group (G2 group, 4.83 ⁇ 0.20mm), the increase is obvious;
  • the arthritis scores of G3, G4, G5, G6, G7, G8, and G10 mice were 7.6 ⁇ 0.9, 7.8 ⁇ 1.4, and 6.1 ⁇ , respectively.
  • the spleens of the G3 group, the G4 group, the G5 group, the G7 group, the G8 group, and the G10 group were 94.6 ⁇ 6.7 mg, 94.2 ⁇ 13.3 mg, 96.0 ⁇ 5.2 mg, and 95.1 ⁇ 9.5.
  • the G5 group, the G6 group, the G7 group, and the G8 were 174.6 ⁇ 11.7 mg, 176.2 ⁇ 8.9 mg, 176.4 ⁇ 9.7 mg, 177.1 ⁇ 7.8 mg, and the model group (G2).
  • the group, 219.3 ⁇ 20.6 mg) was significantly different.
  • the TSL-4 fusion protein of the present application can still reach the G4 group (one day in the case of administration once every 5 days (G6, G7, G10 group) or even once in 7 days (G8 group).
  • the same therapeutic effect was observed in the G5 group (administered once every two days), and the doses of the G4, G5, and G7 groups were equivalently converted in the doses, indicating that the half-life of the fusion protein of the present application was significantly prolonged.
  • TSL-4 was administered subcutaneously in a single back of 4.17, 12.5, 37.5 mg/kg, and blood was taken from the eyelids.
  • the sampling time points were: 0h, 0.5h, 1h, 3h, 5h, 6h, 7h, 8h, 10h, 12h, 16h, 20h, 24h, 28h, 41h, 53h, 65h, 77h.
  • the supernatant was centrifuged at 12,000 rpm/min for 200 ⁇ L, diluted with a PBS solution at a ratio of 1:3, and placed in an EP tube and stored in a refrigerator at -80 °C.
  • HM-3 monoclonal antibody was diluted with a coating solution (CBS) at a certain concentration, and added to an ELISA plate, and coated overnight.
  • CBS coating solution
  • Termination adding a quantitative termination of the liquid, and terminating the reaction.
  • the blood concentration-time curve results of a single subcutaneous injection of 4.17 mg/kg TSL-4 in rats are shown in Fig. 22, the blood concentration measurement results are shown in Table 9, and the pharmacokinetic parameters are shown in Table 10.
  • the mean plasma concentration-time curve of rats given a single subcutaneous injection of different doses of TSL-4 is shown in Figure 23.
  • the relationship between TSL-4 peak concentration (Cmax) in plasma and the dose administered is shown in Figure 24.
  • the relationship between AUC) and the dose administered is shown in Figure 25.
  • the comparison of the main pharmacokinetic parameters is shown in Table 10.
  • CL is 1.383 ⁇ 0.528 L/h/kg, 0.070759 ⁇ 0.0369 L/h/kg, and 0.0569 ⁇ 0.0336 L/h/kg
  • TSL-4 After a single subcutaneous injection of high, medium and low doses (37.5mg/kg, 12.5mg/kg, 4.17mg/kg) of TSL-4, TSL-4 showed a first-order kinetics in SD rats, consistent with linear drugs. Generational dynamics, the CT curve conforms to the two-compartment model. The T 1/2ke of TSL-4 in the high, medium and low dose groups was relatively long, indicating that TSL-4 was eliminated slowly in SD rats.
  • Type II collagen-induced arthritis model Rat modeling method 3 mL incomplete Freund's adjuvant (Chondrex Inc, lot number 160111) and 3 mL type II collagen (Chondrex Inc, lot number 160346) were emulsified on a homogenizer on ice until The emulsion is not dripped into the water.
  • Wistar rats (Shanghai Slack Laboratory Animals Co., Ltd., female, 130-150g, certificate number 2015000526387, 2015000527320), multiple injections were made in the tail (2 points, 0.15 mL of emulsion per point), and each rat was injected.
  • 0.3 mL of emulsion forms a local bulge on the skin surface. The incidence of the rats was observed after about 2 weeks, and the score was weighed.
  • Rats in arthritis model were given subcutaneously 89 Zr-HM-3 and free 89 Zr, respectively.
  • small-body PET/CT was used for standard CT and PET whole body scan; joint In the model rats, 89 Zr-HM-3 was administered to the left ankle joint cavity, and standard CT and PET whole body scans were performed at 1h, 24h, 48h, 120h, 192h, 360h using small animal PET/CT.
  • the radiographic uptake value SUV and %ID/g of the above tissues were obtained by delineating the heart, liver, lung, kidney, brain, ankle joint, spine bone and muscle of the arthritis model model with CT images.
  • Tissue collection includes 12 organs of heart, liver, spleen, lung, kidney, stomach, small intestine (near pylorus), pancreas, spine bone, muscle, gonad, brain.
  • the heart had the lowest %ID/g (0.01 ⁇ 0.01) at 1 h and the highest (1.02 ⁇ 0.12) at 24 h, then gradually decreased with time.
  • the lungs, left ankle joint cavity and right ankle joint cavity had the lowest radioactivity uptake %ID/g at 1 h, 0.04 ⁇ 0.06, 0.01 ⁇ 0.00, 0.01 ⁇ 0.00, respectively, and the highest at 48 h, 0.50 ⁇ 0.09, 0.52 ⁇ 0.09, respectively. 0.73 ⁇ 0.42, then gradually decreased with time; liver, kidney, and spine bones had the lowest %ID/g at 1 h, which were 0.00 ⁇ 0.00, 0.04 ⁇ 0.01, and 0.01 ⁇ 0.00, respectively.
  • the PET/CT scan was performed at 48 h after subcutaneous injection of 89Zr-HM-3 in the arthritis model rats, and the tissue gamma count was collected.
  • the renal radioactivity uptake %ID/g was the highest (2.60 ⁇ 0.09), followed by It is heart>liver>lung>spinal bone>left ankle joint cavity>right ankle joint cavity>muscle; arthritis model rats were injected with 89Zr-HM-3 subcutaneously for 120h, then PET/CT scan was performed first and then tissue gamma count was collected.
  • the renal radioactivity uptake %ID/g was the highest (3.72 ⁇ 0.70), followed by heart>liver>spine bone>lung>left ankle joint cavity>right ankle joint cavity>muscle.
  • the specific results are shown in Figure 27.
  • the left ankle joint cavity had the highest radioactivity uptake %ID/g (42.20 ⁇ 17.33), followed by heart>liver>kidney> Lung> right ankle joint cavity> spine bone> muscle; at 24h, the left ankle joint cavity radioactivity uptake %ID/g highest (6.50 ⁇ 3.06), followed by kidney>heart>liver>lung>right ankle joint cavity>spine bone> Muscle; at 48h, the left ankle joint cavity radioactivity uptake %ID / g highest (2.75 ⁇ 0.64), followed by kidney > heart > right ankle joint cavity > liver > lung > spine bone > muscle; 120h, renal radioactivity uptake %ID /g highest (2.78 ⁇ 0.44), followed by left ankle joint cavity>liver>right ankle joint cavity>spine bone>heart>lung>muscle; at 192h, kidney radioactivity uptake %ID/g highest (3.04 ⁇ 0.52), followed by It is left
  • the radioactivity uptake %ID/g of heart and lung at 1h was 0.59 ⁇ 0.94, 0.25 ⁇ 0.42, respectively, and the radioactivity uptake %ID/g was the highest at 24h. They were 1.15 ⁇ 0.14 and 0.71 ⁇ 0.39, respectively, and then gradually decreased with time; the left ankle joint cavity had the highest radioactivity uptake %ID/g at 1h (42.20 ⁇ 17.33), and then gradually decreased with time, at 360h.
  • the kidneys had the lowest radioactivity uptake %ID/g at 1 h (0.39 ⁇ 0.57), and the highest at 192 h (3.04 ⁇ 0.52), then gradually decreased with time; the spine bone had the lowest radioactivity uptake %ID/g at 1 h (0.10) ⁇ 0.17), then gradually increase with time, the highest at 360h (0.82 ⁇ 0.13).
  • the specific results are shown in Figure 28.
  • the brain's radioactive uptake %ID/g is very low at all time points, and the muscle radioactivity uptake %ID/g is close to or lower than the same time point, indicating that 89Zr-HM-3 is not easy to penetrate. Pass the blood brain barrier.
  • the half-life of 89Zr-HM-3 subcutaneous injection of arthritis model rats was 120.68 ⁇ 31.99h by DAS3.2.8 software non-compartmental model; 89Zr-HM-3 injected arthritis through the left ankle joint cavity.
  • the half-life of the model rats was 98.35 ⁇ 36.83h.
  • Example 14 Cellular protective effect of HM-3 fusion protein TSL-4 on hydrogen peroxide-induced bone tissue
  • Chondrocytes were cultured in T75 flasks for 3-4 days. When the degree of fusion reached 80%, the original culture solution was removed, and the cells were washed 2-3 times with sterile PBS, and then an appropriate amount of digestive juice was added, and the fresh culture solution was added after the cells were detached. The cell fluid containing the digestive juice is removed by centrifugation, and an appropriate amount of fresh culture medium is added to prepare a cell suspension of a suitable concentration for cell passage or cell experiment. Cell passage begins from the P1 generation until the end of the P5 generation, and each generation has a cryopreservation reserve. The cell algebra was P2-P5 for cell experiments.
  • a cell suspension having a density of 2.5*10 4 /mL was inoculated into a 96-well cell culture plate, and three groups were prepared, one group was a control group, and the other two groups were stimulated with hydrogen peroxide.
  • a cell-free medium was additionally used as a blank group. Each group is equipped with 4-5 sub-holes.
  • the cell culture plates were placed in an incubator for 24 hours. The next day, the culture solution was replaced with different experimental groups, each of which was administered in a volume of 100 ⁇ l and incubated for 24 or 48 hours, respectively.
  • each experimental group was added with 10 ⁇ L of 5 mg/ml MTT according to 1/10 of the cell culture volume, and the cells were incubated with the MTT in the incubator for 4 hours. The supernatant was then carefully discarded, followed by the addition of 200 ⁇ L of dimethyl sulfoxide (DMSO), and the absorbance was measured on a microplate reader at a test wavelength of 570 nm.
  • Cell relative activity ratio (%) calculation formula absorbance of the experimental group (per well) / control absorbance average and multiply by 100%. Mean ⁇ standard deviation for all experimental groups Said. Differences between each group were analyzed using Prism 7.0 software, one-way ANOVA test. *P ⁇ 0.05 was considered statistically significant, **P ⁇ 0.01 was a statistically significant difference, and ***P ⁇ 0.001 was a very statistically significant difference.
  • Rat chondrocytes article number: RAT-iCell-s003, Saibaikang.
  • Molding agent Hydrogen peroxide disinfectant (H 2 O 2 , hydrogen peroxide) (0.9M), batch number: 170928, Shandong Lierkang Medical Technology Co., Ltd.
  • MTT thiazole blue
  • DMSO D5879-500 ml, sigma company
  • the hydrogen peroxide experimental concentration range is 0.01, 0.1, 0.5, 1, 10 mM.
  • three modeling times were selected for 24, 48 and 72 hours, and two hydrogenation holes were provided for each dose of hydrogen peroxide.
  • the concentration range selected for TSL-4 is 0.1, 0.3, 1, 3, 9, 27 ⁇ M.
  • TSL-4 had an effect on the cellular activity of chondrocytes, two incubation times of 24, 48 and 72 hours were selected, and a total of two wells were provided for each dose of TSL-4 solution.
  • TSL-4 dose 24 hours 48 hours 72 hours 0 1.43 1.70 1.51 1 1.59 1.43 1.61 3 1.67 1.43 1.52 9 1.72 1.51 1.57 27 1.45 1.30 1.31
  • TSL-4 had no significant effect on cell viability at 24, 48 and 72 hours, and there was no significant difference between each dose of TSL-4 and the control group (0 ⁇ M TSL-4). Therefore, 24 hours was selected as the TSL-4 administration time. However, there was no difference in the dose of TSL-4 between 1 and 27 ⁇ M, so the effect of low dose of TSL-4 on cell viability should be examined.
  • TSL-4 Since each concentration of TSL-4 in the previous experiment had no effect on cell viability, it was diluted from 29.3 ⁇ M to 0.23 ⁇ M according to the 1:5 dilution ratio according to the TSL-4 original solution concentration (293 ⁇ M).
  • TSL-4 (0.23-29.3 ⁇ M) at each concentration was cultured for 24 hours with chondrocytes, and there was no significant change in cell viability, so 0.23, 1.17, 5.86, and 29.3 ⁇ M were selected as the concentration of TSL-4 administered in the subsequent experiments.
  • TSL-4 In order to detect the protective effect of TSL-4 on the chondrocyte protection of hydrogen peroxide damage, the experiment was divided into three groups, namely the control group (without hydrogen peroxide and TSL-4, only fresh culture medium), and the hydrogen peroxide module (only added with different Concentration of hydrogen peroxide cell culture medium) and TSL-4 administration group (containing hydrogen peroxide).
  • TSL-4 was incubated with chondrocytes for 24 hours with hydrogen peroxide.
  • the dose of hydrogen peroxide was chosen to be 0.1, 0.25, 0.5, 1 mM; the dose of TSL-4 was 0.23, 1.17, 5.86, 29.3 ⁇ M.
  • TSL-4 In the presence of different doses of hydrogen peroxide, the appropriate concentration of TSL-4 was incubated with chondrocytes for 24 hours. The relative activity of the cells in each dose group (relative to the absorbance of the control group) was used. As shown below.
  • TSL-4 can significantly improve the chondrocyte activity of hydrogen peroxide damage at 0.25 and 0.5 mM hydrogen peroxide concentration; in addition, 0.23 and 29.3 ⁇ M TSL-4 and hydrogen peroxide pair cells were stimulated with 0.1 mM hydrogen peroxide.
  • Significant inhibition of activity *P ⁇ 0.05; **P ⁇ 0.01; ***P ⁇ 0.001; ****P ⁇ 0.0001.
  • the optimum model concentration of hydrogen peroxide was determined to be between 0.1 and 1 mM.
  • concentration of hydrogen peroxide was 0.5 mM and 1 mM in this experiment, the cell activity ratio (%) decreased to nearly 5%. Left and right, this indicates that the hydrogen peroxide modeling is unstable, and it is easy to produce a difference in results. This may be due to the fact that the chondrocyte algebra (the chondrocyte algebra of this experiment is P4) is different, resulting in the deterioration of the chondrocyte antioxidant capacity. It is known that in chondrocytes in a 2D single cell culture environment, the cells are prone to fibrosis, and the expression of type II collagen and polysaccharide is reduced.
  • the concentration of hydrogen peroxide modeling should be less than 0.5 mM, and the cell viability ratio of chondrocytes stimulated by 0.25 mM hydrogen peroxide is about 70%, so A further concentration should be chosen between 0.25 and 0.5 mM hydrogen peroxide concentration, for example: a concentration of 0.35 mM hydrogen peroxide.
  • the optimal concentration of TSL-4 was 1.17 and 5.86 ⁇ M, which was statistically different (P ⁇ 0.05), and conversely, the concentrations of 0.23 and 29.3 ⁇ M.
  • the TSL-4 administration experiment has a significant inhibition of cell viability on the stimulation of chondrocyte activity by 0.1 mM hydrogen peroxide. Therefore, the second experiment was repeated, and the concentration of TSL-4 was selected to be 1.17 and 5.86 ⁇ M.
  • both 1.17 and 5.86 ⁇ M TSL-4 showed an improvement in the activity of chondrocytes damaged by hydrogen peroxide, but only in 0.35 mM hydrogen peroxide. Under conditions, 5.86 ⁇ M TSL-4 has a significant effect on improving the cell viability of chondrocytes.
  • One-way ANOVA was used to analyze the difference between the respective drug-administered groups and the corresponding concentration of the model group, and the expression of * was statistically significant, P ⁇ 0.05. Therefore, 5.86 ⁇ M TSL-4 could significantly improve the activity of chondrocytes damaged by hydrogen peroxide in the model group compared with 0.25-0.5 mM hydrogen peroxide for 24 hours.
  • the activity of chondrocytes was significantly improved, so the co-incubation of TSL-4 with hydrogen peroxide for 24 hours was the optimal administration time.
  • the concentration of hydrogen peroxide was between 0.25 and 0.5.
  • the incubation with TSL-4 significantly improved the activity of the cells.
  • the concentration of TSL-4 was improved at 1.17 and 5.86 ⁇ M.

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Abstract

本发明属于生物制药技术领域，更具体的说，涉及一种长效HM-3融合蛋白分子及其应用。本发明在HM-3分子序列的基础上，由活性多肽HM-3及其衍生物通过连接肽(Linker)或直接与人源Fc(IgG)片段或Fc(IgG)突变体片段连接，形成全新的分子实体。其通式为(HM-3) n-Linker-Fc(IgG)、Fc(IgG)-Linker-(HM-3) n、或者(HM-3) n-Linker-Fc(IgG)-Linker-(HM-3)n。本发明有效延长了HM-3半衰期，且成本低廉，克服了小肽成药性的关键难题。可作为一种治疗自身免疫疾病、新生血管疾病以及骨关节炎等极具潜力的药物。

Description

一种HM-3融合蛋白及其应用 技术领域
本发明属于生物制药技术领域，更具体的说，涉及一种长效HM-3融合蛋白分子及其应用。
背景技术
自身免疫性疾病是指机体对自身抗原发生免疫反应而导致自身组织损害所引起的疾病，如果不加以及时有效的控制，自身免疫性疾病的后果十分严重，最终甚至危害生命。常见的自身免疫疾病有：系统性红斑狼疮、类风湿性关节炎、硬皮病、甲状腺机能亢进、青少年糖尿病、原发性血小板紫癜、自身免疫性溶血性贫血、溃疡性结肠炎以及许多种皮肤病、自身免疫性肝病，等等。
类风湿关节炎(rheumatoid arthritis，RA)是一种以关节滑膜炎为特征的慢性全身性自身免疫性疾病。滑膜炎持久反复发作，可导致关节内软骨和骨的破坏，关节功能障碍，甚至残废。血管炎病变累及全身各个器官，故本病又称为类风湿病。
以类风湿关节炎为代表的炎性自身免疫性疾病，发病率和致残率高，我国患者逾千万人，是影响人类健康和生存质量的重大疾病。TNFα抑制剂是目前类风湿性关节炎治疗的主流生物制剂；2013年humira，enbrel，remicade等三款TNFα抑制剂的总销售额为281.08亿美元，占据全球类风湿市场总销售额的84.58％。然而，TNF抑制剂需与甲氨蝶呤联合使用才能更好地发挥疗效，而部分类风湿性关节炎患者对甲氨蝶呤不能耐受，导致此类患者不能使用联合治疗方案。此外，临床统计表明，采用TNFα抑制剂联合治疗方案后仍有约30％—40％的患者对该治疗方案无应答，未能达到主要治疗指标(症状缓解20％)。因此目前各大主流制药公司在研风湿关节炎治疗生物药物热门靶点除TNFα抑制剂外，还在积极进行JAK抑制剂，白细胞介素抑制剂、炎细胞浸润抑制剂亲环素以及整合素阻断剂的研究，这些药物可与TNFα抑制剂互为补充，为不同类型的类风湿性关节炎患者提供多种用药选择。
所谓新生血管，就是从一般血管新伸出的螺旋状毛细血管。人体在怀孕等一定条件下，可以出现新的血管，除此之外如果出现新生血管时，即会引发特定的疾病，这些疾病总称为“血管新生疾病”，如癌症、湿性黄斑变性等等。湿性黄斑变性又被称为新生血管性的黄斑变性，主要临床特征为形成了脉络膜新生血管。
骨关节炎为一种退行性病变，系由于增龄、肥胖、劳损、创伤、关节先天性异常、关节畸形等诸多因素引起的关节软骨退化损伤、关节边缘和软骨下骨反应性增生，又称骨关节病、退行性关节炎、老年性关节炎、肥大性关节炎等。临床表现为缓慢发展的关节疼痛、压痛、僵硬、关节肿胀、活动受限和关节畸形等。
HM-3是含有精氨酸-甘氨酸-天冬氨酸序列(RGD序列)的18个氨基酸残基的多肽，其氨基酸序列如下：
Ile-Val-Arg-Arg-Ala-Asp-Arg-Ala-Ala-Val-Pro-Gly-Gly-Gly-Gly-Arg-Gly-Asp。
HM-3为已知结构，在CN1314705C中已经公开，其对整合素αvβ3具有高度亲和性，通过阻断整合素αvβ3信号通路、抑制VEGF和TNFα的表达，从而抑制内皮细胞迁移和新生血管的生成，进而抑制RA滑膜增生。小鼠体内急性、亚急性、慢性炎症试验表明：HM-3可同时抑制血管新生和炎症反应、调节胶原型关节炎DBA/1小鼠滑膜组织VEGF和TNFα含量，有效的缓解RA症状，其治疗效果优于甲氨蝶呤。
虽然HM-3分子具有药效明确、安全性好的优势，该小肽的体内半衰期仅为27.66±7.37min，如该产品应用于临床，则需每日给药1次至2次，极大地限制了临床应用。在本领域中，对分子结构进行修饰或改造是解决半衰期较短、需连续给药问题的常用方法，其中以化学修饰应用最为广泛。
中国专利申请CN102417540A中公开了一种聚乙二醇修饰的HM-3：mPEG-SC-HM-3，其半衰期远远高于HM-3。
然而，PEG修饰技术本身存在种种问题。PEG修饰是通过覆盖蛋白降低免疫原性和提高半衰期的，这就造成PEG修饰并不适用于所有蛋白，有些蛋白由于修饰位点不暴露无法正常修饰；有些蛋白活性位点被覆盖造成活性下降；有些蛋白修饰后构象变化导致活性降低或者容易聚集。PEG修饰产物必须通过降解PEG 分子，逐步暴露药物蛋白，但低分子量的PEG有肾脏毒性，高分子量的PEG体内降解机理不清楚，这给PEG修饰药物带来明显的用药风险。另外，由于PEG修饰涉及复杂的蛋白处理过程，且PEG分子长度存在差异，造成PEG修饰产物的分子量不均一，增加生产成本的同时，降低了成品的均一性，给产业化带来难题。
为克服HM-3蛋白体内半衰期短、多肽合成成本高，不太适合工业化生产等问题，本发明提供一种HM-3-Fc融合蛋白及其应用。
发明内容
本发明发现，将HM-3和Fc制备成融合蛋白，可以延长HM-3半衰期，同时提高了药物活性。本发明发现，该融合蛋白，在体内外都体现出了更高的生物活性和稳定性，能够显著抑制炎症反应，缓解自身免疫疾病、新生血管疾病、骨关节炎的症状，同时具有长效血浆半衰期。
为此，本发明公开了一种HM-3-Fc融合蛋白，所述融合蛋白由活性多肽HM-3与人源IgG-Fc片段或IgG-Fc突变体片段连接形成。
本发明的融合蛋白，由HM-3和人源的IgG-Fc片段或IgG-Fc片段的突变体通过C端或N端或C端和N端同时连接，制备得到，其连接可以通过一个连接肽连接。
本发明的融合蛋白可以通过以下结构描述：
(HM-3) n-Linker-IgG-Fc、
IgG-Fc-Linker-(HM-3) n、
或
(HM-3) n-Linker-IgG-Fc-Linker-(HM-3) n；
其中n选自1，2，3，4或5。
其中Linker代表连接肽，所述连接肽选自：
①、(GGGGS) a，其中a为1，2，3，4，5或6；
②、A(EAAAK) bA，其中b为1，2，3，4，5或6；
③、(AP) c，其中c为1至18；
④、G d，其中d为1至15；
优选的连接肽为(GGGGS) a序列，重复数a优选3，4或5。
其中，所述IgG-Fc为人源的IgG1、IgG2、IgG3或IgG4的Fc片段或其突变体片段；其中优选的为IgG2、IgG4的Fc或其突变体；最优选为突变体mIgG4-Fc。
本发明的融合蛋白，优选以下序列的融合蛋白：
TSL-1融合蛋白：HM-3-(GGGGS) 3-IgG2-Fc；
TSL-2融合蛋白：HM-3-(GGGGS) 3-mIgG4-Fc；
TSL-3融合蛋白：IgG2-Fc-(GGGGS) 3-HM-3；
TSL-4融合蛋白：mIgG4-Fc-(GGGGS) 3-HM-3；
TSL-5融合蛋白：HM-3-(GGGGS) 3-IgG4-Fc
TSL-6融合蛋白：IgG4-Fc-(GGGGS) 3-HM-3
TSL-13融合蛋白：HM-3-(GGGGS) 3-mIgG4-Fc-(GGGGS) 3-HM-3
TSL-14融合蛋白：HM-3-HyFc
TSL-15融合蛋白：mIgG4-Fc-G5-HM-3-G8-HM-3
TSL-16融合蛋白：HyFc-(GGGGS) 3-HM-3
TSL-17融合蛋白：mIgG4-Fc-A(EAAAK) 4A-HM-3
TSL-18融合蛋白：mIgG4-Fc-(AP) 9-HM-3
本发明所述的融合蛋白，可以通过合成的方法制备，所述合成方法，是将HM-3多肽和Linker通过共价键连接，再通过合成方法Linker的另一端和IgG-Fc连接。
也可以通过基因重组的方法获得，如采用酵母、CHO、SP2/0、BHK和/或HEK293细胞进行表达；优选采用CHO细胞和/或HEK293细胞进行表达。
本发明所述的融合蛋白，可以提高药物在体内的作用时间，延长半衰期，提高患者的顺应性和依从性。
本发明进一步提供含有本发明的融合蛋白的药物组合物，所述药物组合物以适合药用的制剂形式存在，所述制剂形式选自：注射剂、胶囊、片剂、药丸、鼻喷剂、或气雾剂，给药方式包括口服、静脉注射、静脉滴注、皮下或肌肉注射。
本发明进一步提供使用本发明的融合蛋白在制备治疗自身免疫性疾病的药物中的应用。优选所述自身免疫性疾病为类风湿性关节炎,并具有药代动力学特性。
本发明进一步提供使用本发明的融合蛋白在制备治疗新生血管疾病的药物中的应用。优选所述新生血管疾病为湿性老年黄斑变性、肿瘤转移。
本发明的融合蛋白可以作为眼科用药，在眼部特别是房水及玻璃体具有药代动力学特性。
本发明进一步提供使用本发明的融合蛋白在制备治疗骨关节炎的药物中的应用。
本发明的融合蛋白可以对软骨细胞产生保护作用，并通过软骨细胞病理学改变，发挥预防和治疗骨关节炎的作用，同时具有药代动力学特性。
本发明所述的融合蛋白，在采用基因重组法制备时，先合成编码本发明融合蛋白的DNA分子，所述DNA的核苷酸序列优选的为序列表1的序列。
本发明在采用基因重组法制备时，将合成的DNA分子通过质粒制备成重组表达载体，优选哺乳动物细胞表达载体，如pcDNA3.4(Invitrogen公司)。
本发明进一步将表达载体转染宿主细胞，优选哺乳动物表达细胞，更优选HEK293细胞、CHO细胞。经对宿主细胞表达培养，从细胞培养液中分离出本发明的融合蛋白，经过纯化分离即可得到本发明的融合蛋白。本发明为此进一步提供本发明融合蛋白的纯化方法，优选地使用Protein A或Protein G亲和层析柱进行纯化。
附图说明
图1 TSL-4蛋白亲和捕获图。
图2 TSL-4蛋白的进一步分离纯化图。
图3 20160308批电泳图。
图4 20160308批HPLC-SEC图。
图5 TSL-5和TSL-6纯化后电泳图。
图6 TSL-1～4融合蛋白小鼠脾淋巴细胞增殖抑制效果对比图。
图7 TSL-13～18融合蛋白小鼠脾淋巴细胞增殖抑制效果对比图。
图8三种Fc片段、两种连接方向融合蛋白小鼠脾淋巴细胞增殖抑制效果对比图。
图9三种Linker融合蛋白小鼠脾淋巴细胞增殖抑制效果对比图。
图10含不同数量HM-3的融合蛋白小鼠脾淋巴细胞增殖抑制效果对比图。
图11 TSL-1～4融合蛋白对人巨噬细胞U937炎性因子TNF-α的抑制作用对比图。
图12 TSL-1～4融合蛋白对斑马鱼血管形成抑制率对比图。
图13 TSL-4对II型胶原型关节炎小鼠体重的影响图。
图14 TSL-4对II型胶原型关节炎小鼠足掌后度的影响图。
图15 TSL-4对II型胶原型关节炎小鼠足踝宽度的影响图。
图16 TSL-4对II型胶原型关节炎小鼠足周长的影响图。
图17 TSL-4对II型胶原型关节炎小鼠关节炎评分的影响图。
图18 TSL-4对II型胶原型关节炎小鼠脾脏以及胸腺重量的影响图。
图19 TSL-4对II型胶原型关节炎小鼠足重的影响图。
图20单次皮下注射12.5mg/kg的TSL-4后的血药浓度时间曲线图。
图21单次皮下注射37.5mg/kg的TSL-4后的血药浓度时间曲线图。
图22单次皮下注射4.17mg/kg的TSL-4后的血药浓度时间曲线图。
图23大鼠单次皮下注射不同剂量TSL-4后平均血药浓度时间曲线图。
图24血浆中TSL-4峰浓度(Cmax)与给药剂量的关系图。
图25药时曲线下面积与给药剂量的关系图。
图26关节炎模型大鼠皮下注射89Zr-HM-3后6个时间点PET/CT扫描放射性摄取图。
图27关节炎模型大鼠皮下注射89Zr-HM-3后分别在48h和120h时间点PET/CT扫描放射性摄取图。
图28关节炎模型大鼠左踝关节腔注射89Zr-HM-3后6个时间点放射性摄取图。
图29关节炎模型大鼠皮下注射89Zr-HM-3后分别在48h、120h时间点采集组织放射性摄取图。
具体实施方式
以下通过实施例进一步说明本发明。
实施例1，HM-3融合蛋白的分子设计
HM-3-Fc融合蛋白由3部分组成，包括活性部分HM-3多肽，linker，以及人源IgG-Fc片段或IgG-Fc突变体片段；其具体设计方法如下：
1、F C片段的选择
人IgG型抗体根据重链的不同，共有四种亚型，分别是IgG1，IgG2，IgG3，IgG4四种。已有的研究成果表明，四种亚型在血浆半衰期、细胞毒性等特征上均有所差异(见表1)，且与Fc片段直接相关。IgG3的体内半衰期最短，其Fc片段不太适宜作为伴侣分子延长目的蛋白的体内半衰期，经过试验首先被排除掉。而Fc片段对补体、Fc受体的亲和力决定了抗体依赖的细胞毒性(ADCC效应)以及补体依赖的细胞毒性的能力(CDC效应)效应功能，IgG1和IgG3具有最强的ADCC、CDC效应，经试验确定不适于构建为以健康细胞为靶点的融合蛋白，因此IgG1和IgG3的Fc片段均被筛选后排除。
基于所开发适应症为自身免疫性疾病，靶点为正常体细胞，仅需抑制信号通路，并尽可能延长体内半衰期，所以需要低的或者是没有ADCC效应以及CDC效应，据此HM-3融合蛋白FC部分更适宜选择IgG2或IgG4的Fc片段。而为了进一步改良天然Fc片段的特性，本发明也将IgG2和IgG4的Fc片段突变体扩大考虑在筛选范围之内。
表1 4种IgG亚型性质比较
Figure PCTCN2018117188-appb-000001
2、Linker的选择
Linker是连接功能蛋白和伴侣蛋白的多肽链，Linker的序列及长度对融合蛋白功能具有至关重要的作用。Linker的选择需要考虑如下几个方面：
1)长度要适中，太短无法保证蛋白之间空间上的相对独立，太长则可能增加Linker断裂、及引起免疫原性的风险；2)Linker的序列不能包含蛋白酶切位点，避免被切断；3)Linker可选择天然Linker、人工Linker；4)Linker可分为刚性Linker和柔性Linker。
经过大量实验本发明选择4种人工设计Linker作为验证备选：
①(GGGGS) a，是使用最为广泛的柔性Linker，其中a可以为1，2，3，4，5或6；
②A(EAAAK) bA，是α-螺旋刚性Linker，其中b可以为1，2，3，4，5或6；
③(AP) c，是直链刚性Linker，其中c可以为1至18；
④G d，是类似于(GGGGS) n的柔性Linker，其中d可以为1至15。
3.HM-3与Fc部分融合顺序
由于氨基酸序列方向的唯一性，两个蛋白质的融合存在两种连接方式，N－端连接指将功能蛋白连接到Fc片段的N－端，而C－端连接则指将功能蛋白连接到Fc片段的C－端。
4.备选融合蛋白结构设计
结合上述融合蛋白设计需要考虑的因素，至少可以产生超过300种融合蛋白结构，一一进行表达并验证药效是不现实的，因此从单因素比较的思路出发，首先选择最常用的(GGGGS) 3Linker，设计了如下六种分子：
TSL-1融合蛋白：HM-3-(GGGGS) 3-IgG2-Fc；
TSL-2融合蛋白：HM-3-(GGGGS) 3-mIgG4-Fc；
TSL-3融合蛋白：IgG2-Fc-(GGGGS) 3-HM-3；
TSL-4融合蛋白：mIgG4-Fc-(GGGGS) 3-HM-3；
TSL-5融合蛋白：HM-3-(GGGGS) 3-IgG4-Fc
TSL-6融合蛋白：IgG4-Fc-(GGGGS) 3-HM-3
随后在上述6种结构的基础上，通过单一因素替换，再引入HyFc、刚性Linker、重叠肽等设计思路，形成6种新的融合蛋白结构：
TSL-13融合蛋白：HM-3-(GGGGS) 3-mIgG4-Fc-(GGGGS) 3-HM-3
TSL-14融合蛋白：HM-3-HyFc
TSL-15融合蛋白：mIgG4-Fc-G5-HM-3-G8-HM-3
TSL-16融合蛋白：HyFc-(GGGGS) 3-HM-3
TSL-17融合蛋白：mIgG4-Fc-A(EAAAK) 4A-HM-3
TSL-18融合蛋白：mIgG4-Fc-(AP) 9-HM-3
以上氨基酸序列见序列表。
上述12种融合蛋白作为最具代表性的备选蛋白结构，同步进行细胞表达、体外药效学实验，获得体外药效实验结果，通过不同的比较方式，可以对各个结构变量进行比较，筛选出最佳融合蛋白结构。随后通过体内药效学，药代动力学，毒理学等评价其成药的可能性，从而进一步开发成治疗类风湿关节炎等自身免疫性疾病的创新药物。
实施例2：载体的构建
12种融合蛋白基于相同的设计途径、使用相同的表达载体，因此瞬时转染表达载体构建过程完全一致，以mIgG4-Fc-(GGGGS) 3-HM3(TSL-4)融合蛋白为例，介绍表达载体的构建过程。
TSL-4融合蛋白采用mIgG4-Fc片段与一个HM-3分子相连，Linker采用(GGGGS) a，其中a为3。
将组成TSL-4各个部分的氨基酸序列，按照mIgG4-Fc-(GGGGS) 3-HM3的顺序进行拼接后，序列见序列表12。
mIgG4-Fc-(GGGGS) 3-HM3融合蛋白对应的DNA序列见序列表1。
为了适应CHO或HEK293等真核细胞分泌表达，添加必要的Kozak序列及信号肽，获得完整的mIgG4-Fc-(GGGGS) 3-HM3表达序列，DNA序列见序列表2。
委托生物技术公司全合成上述DNA序列，并通过亚克隆与invitrogen公司的商业化表达载体pcDNA3.4-TOPO vector进行连接，获得表达载体pcDNA3.4-mIgG4-Fc-(GGGGS) 3-HM3。表达载体保存于E.coli DH5α，以甘油管的形式，寄送到实验室，-80℃保存。
实施例3：表达载体保存菌株的长期保存
在无菌条件下，将含有pcDNA3.4-mIgG4-Fc-(GGGGS) 3-HM3的E.coli DH5α甘油管解冻，按照1％接种量，接种到含50ml 100ug/ml Amp抗性LB培养基的250ml摇瓶中，37℃，160rpm震荡过夜培养。
随后在无菌条件下，向菌液中加入25ml无菌60％甘油，充分混匀后，按1ml/支，将菌液分装至无菌的1.5ml离心管中，完成甘油管的制备，-80℃长期保存。
实施例4，表达载体的制备
所有用于转染细胞的载体必须无菌、且控制内毒素，因此所有表达载体的制备，均选用Roche公司的Genopure plasmid Maxi Kit进行质粒大提，并对质粒进行无菌处理。
(1)质粒提取前一天早上，从-80℃中取出一支甘油管，在无菌条件下，按照1％接种量，将菌液接种至含5ml 100ug/ml Amp抗性LB培养基的试管中，37℃，160rpm震荡培养6～8h。
(2)在无菌条件下，将试管中的菌液全部接入含500ml 100ug/ml Amp抗性LB培养基的2L摇瓶，37℃，160rpm震荡过夜培养。
(3)使用Roche公司的Genopure plasmid Maxi Kit进行质粒大提。方法如下：
a.在4℃下，3000-5000g离心5-10min，收集500ml的E.coli菌体。加入24ml重悬缓冲液(Suspension Buffer，含有RNase)，充分重悬菌体。
b.加入24ml裂解缓冲液(Lysis Buffer)，颠倒混匀6-8次，室温静置2-3分钟。
c.加入24ml中和缓冲液(Neutralization Buffer)，立即颠倒混匀6-8次，出现絮状沉淀。
d.净化溶菌液。在4℃使用12000g以上速度，离心45min以上，小心将上清移至吸附柱。
e.在吸附柱上套上试剂盒中自带的纸环，放置在锥形瓶口，在吸附材料上滴加6ml的平衡缓冲液(Equilibration Buffer)。
f.将e中得到的清洁菌裂解液加入吸附柱中，靠重力自然流过。弃流穿液。
g.吸附柱中加入12ml冲洗缓冲液(Wash Buffer)，靠重力自然流过，弃流穿液。重复两次。
h.在超净台中，将吸附柱放置在去内毒素、无菌50ml圆底高速离心管上，加入50℃预热的14ml洗脱缓冲液(Elution Buffer)，重力流过。
i.加入20ml异丙醇，沉淀质粒DNA，在4℃使用大于15000g离心30min，超净台中小心弃上清。(此步需要特别注意，有时质粒并未形成固体沉淀，而是形成粘稠液体处于离心管底部，使用移液管出去上层大部分液体，然后小心将剩余上清移入一个无菌尖底离心管，如15ml离心管或50ml离心管，尽量不要吸到含质粒粘稠液体。随后，观察尖底离心管，将底部不小心吸到的含质粒粘稠液体吸回圆底离心管。)
j.使用4ml预冷的75％乙醇，拧紧离心管盖，旋转离心管，让75％乙醇湿润整个离心管内部，一方面将可能残余在管壁的质粒洗下，另一方面对整个离心管内壁除菌。在4℃使用大于15000g离心10min。重复一次。
k.在超净台中，倒掉75％乙醇，用10ul移液器小心吸掉残余液体，将离心管倒置在吸水纸上干燥约20分钟。
l.在超净台中，使用200-400ul无菌ddH2O，完全浸泡固体质粒，拧紧离心管盖，4℃倾斜静置离心管过夜，让质粒完全溶解。超净台中将质粒移入无菌1.5ml离心管中，取2ul用于浓度检测。根据浓度检测结果，使用无菌ddH2O将质粒浓度调整至1ug/ul，使用无菌0.22μm滤膜过膜除菌，并取5ul质粒接种于含有5ml无抗性LB培养基的试管中，37℃过夜震荡培养，确认无菌。至此完成无菌质粒的制备。4℃或-20℃保存质粒DNA.
至此，完成表达载体的制备工作。
实施例5，融合蛋白快速表达
ThermoFisher公司的Expi293 Expression System是基于293F细胞(一种经过筛选的HEK293细胞)瞬时转染的商业化的快速蛋白制备试剂盒，用于融合蛋白的快速获取。所用12种融合蛋白的制备均遵循下述实验方案进行。
1.根据Expi293 Expression System使用说明，使用2L摇瓶进行实验时，每瓶细胞最终体积为800ml。
2.转染的Expi293F细胞，从复苏算起，最少进行三次传代。传代过程中根据实验需要，依次扩大培养规模。
3.瞬时转染前一天，按照2×10 6cells/ml活细胞密度接入总体积为1200ml Expi293 Expression Medium，37℃，8％CO2，125rpm震荡培养。
4.瞬时转染当天，先对前一天培养的细胞计数，细胞密度应为3-5×10 6cells/ml，活率大于95％。调整细胞密度为3×10 6cells/ml，每个2L摇瓶中的细胞体积调整至680ml。
5.800ug质粒DNA复溶于40ml的Opti-MEM I Reduced Serum Medium，轻柔混匀。
6.2.16mL的ExpiFectamine 293 Reagent加入Opti-MEM I Reduced Serum Medium，定容至40ml。轻柔混匀，室温孵育5min(长时间孵育影响转化效率)。
7.上述两种溶液混合，轻柔混匀，室温孵育20-30min。完成质粒-转染试剂混合液准备工作。
8.将80ml质粒-转染试剂混合液加入第4步的细胞培养液中，共760ml。
9.37℃，8％CO 2，125rpm震荡培养18h。
10.加入4mL的ExpiFectamine 293 Transfection Enhancer 1和40ml的ExpiFectamine 293 Transfection Enhancer 2。至此，总体积为804ml。
11.37℃，8％CO 2，125rpm震荡培养。
12.转染后第六天结束发酵，取样使用免疫比浊仪检测产量，并进行蛋白纯化。
实施例6，融合蛋白的纯化
由于12种融合蛋白本质上均为Fc融合蛋白，可以用ProteinA亲和层析柱进行特异性捕获，实际纯化过程中，发现纯化参数完全一致，因此此处以某一批TSL-4蛋白的纯化为例，介绍融合蛋白的纯化流程。
1.样品预处理：20160308批1.60L发酵液采用Beckman J×25离心机，500ml离心杯，7500rmp，20min,4℃，所获得上清液约1.46L用于下一步protein A捕获；
2.目的蛋白亲和捕获：
色谱柱信息如下
Figure PCTCN2018117188-appb-000002
方法信息如下：
1)首先用500ml的0.2M NaOH进行灭菌，流速10ml/min；
2)20mM PB，0.15M NaCl，pH 7.0缓冲液平衡色谱柱，体积约为1000ml，流速20ml/min；
3)上样：样品预先调节pH至中性，流速20ml/min；
4)20mM PB，0.15M NaCl，pH 7.0缓冲液冲洗色谱柱，约800ml，流速20ml/min；
5)50mM柠檬酸-柠檬酸钠，0.15M NaCL pH3.0缓冲液洗脱目的蛋白，起峰20mAu开始收集，峰后20mAu停止收集；流速20ml/min；
6)色谱柱最后用500ml 0.2M NaOH溶液清洗色谱柱，用ddH 2O水冲洗至中性后，用20％乙醇保存色谱柱；
合并样品体积约138ml，用1N NaOH 20ml调节pH 4.12至7.0，洗脱液由略混浊变至澄清。测定结果如图1所示。
3.凝胶层析进行进一步的分离纯化
色谱柱参数：
Figure PCTCN2018117188-appb-000003
方法信息：
1)用0.5M NaOH 300ml对色谱柱进行灭菌，流速10ml/min，后用超纯水冲洗至约中性；
2)用PBS缓冲液，pH 7.4平衡色谱柱，平衡体积约为1500ml，流速10ml/min；
3)上样，样品为proteinA洗脱液，上样量40ml；
4)收集样品，峰3为目的蛋白峰，收集峰3，起峰10mAU开始收集，峰后10mAu停止收集；
5)最后用0.1M NaOH保存色谱柱，流速10ml/min。
测定结果如图2所示。
4.样品超滤浓缩：对峰3的样品进行合并超滤浓缩，超滤膜选用10kDa，样品浓缩至目的蛋白浓度大于5mg/ml，然后分装样品，于-80℃冰箱保存。本批次起始合并样品体积约550ml，浓度约0.29mg/ml，最终浓缩至27ml，浓度约为5.53mg/ml；分装样品，冻存。
5.最终样品的纯度
如图3所示，20160308批电泳纯度约为96.9％；如图4所示，20160308批HPLC-SEC纯度约为99.3％。
遵循相同的纯化流程，成功表达并制备了12种融合蛋白：
TSL-1融合蛋白：HM-3-(GGGGS) 3-IgG2-Fc；
TSL-2融合蛋白：HM-3-(GGGGS) 3-mIgG4-Fc；
TSL-3融合蛋白：IgG2-Fc-(GGGGS) 3-HM-3；
TSL-4融合蛋白：mIgG4-Fc-(GGGGS) 3-HM-3。
TSL-5融合蛋白：HM-3-(GGGGS) 3-IgG4-Fc
TSL-6融合蛋白：IgG4-Fc-(GGGGS)3-HM-3
TSL-13融合蛋白：HM-3-(GGGGS) 3-mIgG4-Fc-(GGGGS) 3-HM-3
TSL-14融合蛋白：HM3-HyFc
TSL-15融合蛋白：mIgG4-Fc-G 5-HM-3-G 8-HM-3
TSL-16融合蛋白：HyFc-(GGGGS) 3-HM-3
TSL-17融合蛋白：mIgG4-Fc-A(EAAAK) 4A-HM-3
TSL-18融合蛋白：mIgG4-Fc-(AP) 9-HM-3
在蛋白制备过程中，如图5所示，发现TSL-5、6两种融合蛋白存在严重的二聚体降解问题，大量以单体形式存在，说明使用IgG4-Fc制备的融合蛋白不能稳定形成二聚体，因此淘汰TSL-5和TSL-6两种融合蛋白设计方案。
实施例7：融合蛋白小鼠脾淋巴细胞增殖抑制实验
实验方法：将小鼠眼眶放血致死，立即放入75％乙醇浸泡5～10min，于超净工作台中取出脾脏放入PBS中。将脾脏置于无菌细胞筛上(200目)，用注射器头研磨，中途需要不断加入PBS，收集研磨液离心(1000转/分，5min)，用Tris-氯化铵溶液洗涤细胞3次，再用培养基洗涤一遍，然后用培养基重悬。最后将细胞进行台盼蓝活细胞染色，存活率95％以上。调整活细胞浓度为2×10 6个/mL，于96孔板每孔加入细胞100μL，同时加入ConA(刀豆蛋白，刺激脾细胞增殖)与药物，每组设置6个复孔。
将96孔板在37℃，5％CO2培养箱中孵育48h。向96孔板中加入5mg/mL的MTT，每孔20μL，于培养箱中继续培养4h。弃去96孔板中的培养液，每孔加入100μL DMSO，轻轻混匀。用酶标仪于测量波长570nm，参比波长630nm处测定吸光值。
按照公式计算增殖抑制率(Proliferation inhibition rate,PI)：
Figure PCTCN2018117188-appb-000004
其中A test为加药组的吸光值，A control为阴性对照组的吸光值。试验得到的结果以mean±SD表示，并进行统计T检验，*P<0.05为显著性差异，**P<0.01为极显著性差异。根据结果筛选融合蛋白抗类风湿关节炎的药效。
第一轮实验，检测TSL-1-4，具体加药方案见表2。
表2给药方案
Figure PCTCN2018117188-appb-000005
Figure PCTCN2018117188-appb-000006
实验结果：如图6所示，为筛选HM-3融合蛋白TSL-1、TSL-2、TSL-3、TSL-4的抗类风湿关节炎活性，实验中采用小鼠脾淋巴细胞增殖实验对HM-3融合蛋白TSL-1、TSL-2、TSL-3、TSL-4进行初步筛选，结果显示HM-3、mPEG-SC-HM-3、HM-3融合蛋白TSL-1、TSL-2、TSL-3、TSL-4的最佳抑制率分别为17.9％±8.8％、40.5％±9.3％、39.6％±9.4％、34.1％±11.7％、61.4％±1.6％、63.3％±11.0％，其中HM-3融合蛋白TSL-3、TSL-4抑制率高于阳性对照组，且和阴性组比较具有极显著差异。
第二轮实验，检测TSL-13-18,具体加药方案见表3。P4对照组使用TSL-4融合蛋白作为对照药。
表3给药方案
Figure PCTCN2018117188-appb-000007
实验结果
图7表示TSL-13-18各组药物对ConA刺激的脾细胞增殖的抑制作用。P4阳性药组、溶剂组较ConA组，小鼠脾淋巴细胞扩增抑制率均有显著提高(P<0.05)，而TSL-13～18各组药物中，仅TSL-17和TSL-18两个样品的各浓度梯度较ConA组在小鼠脾淋巴细胞扩增抑制率上有显著提高(P<0.05)，但抑制幅度不及P4阳性药组。
实施例8：12种备选融合蛋白结构单因素比较
首先，实施例6中已经提到，在纯化过程中发现，由于TSL-5和TSL-6融合蛋白不能稳定保持预期的正常二聚体状态，因此TSL-5和TSL-6这两种基于天然人源IgG4-Fc片段融合蛋白被淘汰，同时IgG4-Fc这一元件不再作为备选融合蛋白Fc片段。基于小鼠脾淋巴细胞增殖抑制实验结果，其余10种备选融合蛋白进行单一结构因素比较。
1、Fc片段及连接方向比较
选择GGGGS*3 Linker作为统一Linker，在排除天然人源IgG4-Fc后，共有IgG2-Fc、mIgG4-Fc和HyFc 这3种备选Fc片段，结合两种连接方向，共形成6种备选融合蛋白，分别是TSL-1、2、3、4、14、16，各样品的最佳抑制率见表4所示。
表4三种Fc片段、两种连接方向融合蛋白小鼠脾淋巴细胞增殖实验最佳抑制率比较
Figure PCTCN2018117188-appb-000008
注，表格中数字为小鼠脾淋巴细胞抑制率
如图8所示，比较数据可以得到如下结果：
1)基于HyFc构建的融合蛋白体外药效显著低于对照样品TSL-4融合蛋白，因此淘汰HyFc作为伴侣分子延长HM-3药效的资格。
2)C端连接方式，即HM-3连接于Fc片段的C端，其药效显著优于N端连接方式，因此排除N端连接方式。
3)同为C端连接方式，TSL-4的药效稍高于TSL-3，因此初步认为mIgG4-Fc要优于IgG2-Fc，但暂不淘汰IgG2-Fc，待后续实验进一步比较TSL-3和TSL-4。
2、Linker单因素比较
如图9所示，TSL-4、TSL-17、TSL-18统一使用mIgG4-Fc和C端连接方式，通过比较体外药效，筛选Linker。小鼠脾淋巴细胞增殖抑制实验结果显示，TSL-17和TSL-18的细胞抑制率均低于TSL-4，因此可以得出结论，三种类型的Linker中，A(EAAAK) nA和(AP) n这两种刚性Linker并不适用于构建HM-3的Fc融合蛋白。
3、单一HM-3和多HM-3融合蛋白比较
理论上，融合蛋白分子中包含更多的HM-3，相同给药量的情况下将带来更强的药效。基于此种思路，TSL-13被设计为Fc片段两端连接HM-3，TSL-15被设计为Fc片段C端连接2个HM-3。
如图10所示，通过小鼠脾淋巴细胞增殖抑制实验，统一使用mIgG4-Fc和GGGGS*3Linker，比较TSL-2、4、13、15的体外药效，分析多个HM-3对药效的作用。
比较后发现，TSL-13、15的药效均低于TSL-4蛋白，多个HM-3并未体现出提升药效的价值，因此淘汰包含多个HM-3的融合蛋白设计方案。
经过多轮比较，可以确定HM-3连接于Fc的C端，使用GGGGS*3Linker柔性Linker连接单一HM-3分子，能最大限度的保持HM-3的药效，而IgG2-Fc和mIgG4-Fc两种Fc片段相比，mIgG4-Fc效果稍好，但不足以淘汰IgG2-Fc，因此TSL-3和TSL-4这两种蛋白结构在后续实验中进一步比较。
实施例9：HM-3融合蛋白在人巨噬细胞U937的抑制反应体外药效筛选
实验方法：人巨噬细胞U937在含10％胎牛血清和双抗的RPMl-1640培养基中于37℃、5％CO 2的恒温培养箱中培养，每2d左右换液1次。取处于对数生长期的U937细胞进行收集、重悬，调整细胞浓度为5×10 5个/mL，于96孔板中加入细胞100μL/孔，n＝3，过夜，次日，用LPS(1μg/mL)进行诱导，与此同时，给予药物进行治疗，分别分为阳性对照药组adalimumab，HM-3多肽组，mPEG-SC--HM-3多肽组、HM-3融合蛋白(TSL-1、TSL-2、TSL-3、TSL-4)组，具体给药方案见表5。
表5给药方案
Figure PCTCN2018117188-appb-000009
在药物作用48小时后，收集细胞培养液，离心，取上清，用ELISA方法测定上清液中TNF-α的含量。试验得到的结果以mean±SD表示，并进行统计T检验，*P<0.05为显著性差异，**P<0.01为极显著性差异。根据结果筛选HM-3融合蛋白抗类风湿关节炎的药效。
实验结果：如图11所示，为筛选HM-3融合蛋白TSL-1、TSL-2、TSL-3、TSL-4的抗类风湿关节炎活性，实验中采用ELISA方法检测经LPS诱导的巨噬细胞上清液中TNF-α的含量，以此确定HM-3融合蛋白TSL-1、TSL-2、TSL-3、TSL-4的抗类风湿关节炎活性。结果显示HM-3融合蛋白TSL-3、TSL-4中剂量(9μM)表现出最佳抑制效果，细胞上清液中TNF-α含量为38.6±12.9pg/mL和22.2±8.9pg/mL，与阴性组(77.6±19.6pg/mL)比较具有极显著差异；此外，HM-3高、低剂量组细胞上清液中TNFα含量分别为58.9±10.6pg/mL、83.9±20.4pg/mL。mPEG-SC-HM-3高、低剂量组细胞上清液中TNF-α含量分别为45.6±5.9pg/mL、57.3±2.7pg/mL，从抑制效果来看，TSL-4要远远优于多肽HM-3和mPEG-SC-HM-3。
实施例10：HM-3融合蛋白在斑马鱼体内抗血管形成作用药效筛选
实验方法：随机选取转基因血管荧光斑马鱼于六孔板中，每孔30尾，分别静脉注射给予HM-3多肽、TSL-1融合蛋白、TSL-2融合蛋白、TSL-3融合蛋白和TSL-4融合蛋白20ng/尾和66ng/尾剂量，阳性对照药Avastin 500ng/尾剂量，注射体积均为20nL/尾；注射20nL/尾缓冲液的斑马鱼为溶剂对照组，不作任何处理斑马鱼为正常对照组。处理24h后，每组随机选取10尾斑马鱼，使用荧光显微镜观察血管荧光转基因斑马鱼的肠下血管(SIVs)，拍照并保持图片，使用尼康NIS-Elements D 3.10高级图像处理软件进行图像分析，计算实验组肠下血管面积(S)。血管形成抑制率计算公式如下：
Figure PCTCN2018117188-appb-000010
用方差分析和Dunnett’s T-检验进行统计学分析，结果用Mean±SE表示，p<0.05表明具有显著性差异。
实验结果：
如图12所示，阳性对照药Avastin 500ng/尾组斑马鱼肠下血管面积(37853)与正常对照组(53193)比较p<0.001(与正常对照组比较，*p<0.05，**p<0.01，***p<0.001)，其血管形成抑制率为29％，说明Avastin具有明显的血管形成抑制作用。
HM-3多肽、TSL-1、TSL-2、TSL-3和TSL-4融合蛋白在20ng/尾剂量时，斑马鱼肠下血管面积分别为52186、39929、42828、40705和33869，与正常对照组(53193)比较，血管形成抑制率分别为2％、25％、19％、23％和36％。实验结果说明HM-3多肽在注射剂量为20ng/尾时无血管形成抑制作用；TSL-1、TSL-2、TSL-3和TSL-4融合蛋白在注射剂量为20ng/尾时均具有明显的血管形成抑制作用，其中TSL-4的血管形成抑制作用最好。
HM-3、TSL-1、TSL-2、TSL-3和TSL-4在66ng/尾剂量时，对斑马鱼肠下血管面积分别为42492、37022、32374、38660和28297，与正常对照组(53193)相比，其血管形成抑制率分别为20％、30％、39％、27％和47％，说明5种融合蛋白在注射剂量为66ng/尾时均具有明显的血管形成抑制作用，其中TSL-4的血管形成抑制作用最好。
实施例11：小鼠II型胶原型关节炎慢性炎症模型
实验方法：取Balb/c小鼠，首次免疫，牛II型胶原浓度为4mg/mL，试验当天(0天)，将完全弗氏佐剂(CFA)与CII溶液等体积混匀、乳化，除Balb/c小鼠正常对照组外，其他各组每只小鼠于其尾根部皮内注射乳化剂50μL进行致敏，21天后以相同剂量的乳化剂于尾根部进行再次免疫，此次佐剂用不完全弗氏佐剂(IFA)。大约在试验第29天，造模小鼠足趾关节出现红肿等关节炎症状表明造模成功。
治疗方法
实验第30d造模组动物随机分为：
G1(正常对照组)；
G2(模型组)；
G3(阳性药阿达木单抗组)；
G4(HM-3组)；
G5(mPEG-SC-HM-3组)；
G6(TSL-4，50mg/kg，5天给药一次，共3次)；
G7(TSL-4，25mg/kg，5天给药一次，共3次)；
G8(TSL-4，25mg/kg，7天给药一次，共2次)；
G9(TSL-4，25mg/kg，14天给药一次，共1次)；
G10(TSL-4，12.5mg/kg，5天给药一次，共3次)；
G11(TSL-4，12.5mg/kg，7天给药一次，共2次)；
G12(TSL-4，12.5mg/kg，14天给药一次，共1次)；
共12组，除G1(正常对照组)和G2(模型组)老鼠12只外，其余每组8只。正常对照组及模型对照组均皮下注射生理盐水，给药体积为0.1mL/10g，隔天给予一次，共给予8次；阳性药阿达木单抗组皮下注射阿达木单抗8mg/kg，给药体积为0.1mL/10g，2周给药一次，共1次，在治疗15天完成后再进行观察15天。具体给药方案见表6。
表6给药方案
组别 只数 给药剂量 频数 途径
正常组(G1) 12 生理盐水 1次/2d，8次 SC
模型组(G2) 12 生理盐水 1次/2d，8次 SC
Adalimumab(G3) 8 8mg/kg 1次/14d，1次 SC
HM-3(G4) 8 1.6mg/kg 2次/d，30次 IV
mPEG-SC-HM-3(G5) 8 20mg/kg 1次/2d，8次 SC
TSL-4(G6) 8 50mg/kg 1次/5d，3次 SC
TSL-4(G7) 8 25mg/kg 1次/5d，3次 SC
TSL-4(G8) 8 25mg/kg 1次/7d，2次 SC
TSL-4(G9) 8 25mg/kg 1次/14d，1次 SC
TSL-4(G10) 8 12.5mg/kg 1次/5d，3次 SC
TSL-4(G11) 8 12.5mg/kg 1次/7d，2次 SC
TSL-4(G12) 8 12.5mg/kg 1次/14d，1次 SC
关节炎指标评价
小鼠体重测定：用电子天平称量每只小鼠体重，隔两天测量一次。
足掌厚度测定：用游标卡尺测定每只小鼠的左右后足足掌厚度，隔两天测量一次。
足踝关节宽度测定：用游标卡尺测定每只小鼠左右足踝关节的宽度，隔两天测量一次。
关节炎指数(arthritis index，AI)评分：采用足爪关节评分法对每只小鼠关节炎症严重程度进行评分。0：无红肿；1：足小趾关节红肿；2：趾关节、足趾均红肿；3：踝关节以下均红肿；4：趾关节、足趾、踝关节均红肿。隔两天评分一次，到实验结束为止。
病理学指标评价
眼眶采血，分离血清。脱颈处死动物后，分离脾脏、胸腺，称重，计算脾脏系数，脾脏和胸腺置福尔马林固定液中固定。从胫骨下端内、外踝上缘连线处切断，称量包括踝关节在内整个足爪的重量，并置福尔马林固定液中固定以进行组织病理学检查。
实验结果：TSL-4不同给药方案对小鼠II型胶原性关节炎模型具有不同的治疗效果，其中G3组(阳性药阿达木单抗组)、G4组(HM-3组)、G5组(mPEG-SC-HM-3组)、G6组(TSL-4，50mg/kg，5天给药一次，共3次)、G7组(TSL-4，25mg/kg，5天给药一次，共3次)、G8组(TSL-4，25mg/kg，7天给药一次，共2次)、G10组(TSL-4，12.5mg/kg，5天给药一次，共3次)表现出较好的治疗效果，足厚度、足踝宽度、足周长、关节炎评分、脾脏重量、足重量和模型组比较均具有显著性差异( *p<0.05)，其中G5组(mPEG-SC-HM-3组)、G6组(TSL-4，50mg/kg，5天给药一次，共3次)、G7组(TSL-4，25mg/kg，5天给药一次，共3次)、G8组(TSL-4，25mg/kg，7天给药一次，共2次)治疗效果优于G3组(阳性 药阿达木单抗组)，但是两者比较无显著性差异，根据足厚度、足踝宽度、足周长、关节炎评分可以发现在治疗早期G3组(阳性药阿达木单抗组)效果优于其他组，在治疗中期和后期G5组(mPEG-SC-HM-3组)、G6组(TSL-4，50mg/kg，5天给药一次，共3次)、G7组(TSL-4，25mg/kg，5天给药一次，共3次)、G8组(TSL-4，25mg/kg，7天给药一次，共2次)治疗效果优于G3组(阳性药阿达木单抗组)，另外G4组(HM-3组)和G10组(TSL-4，12.5mg/kg，5天给药一次，共3次)的治疗效果略低于G3组(阳性药阿达木单抗组)，和G2组(模型组)比较具有显著性差异( *p<0.05)，和G3组(阳性药阿达木单抗组)相比无显著性差异，由体重动态曲线和体外对正常细胞的毒性实验可以发现，各个治疗组无明显的毒副作用。
1.TSL-4对II型胶原型关节炎小鼠体重的影响
如图13所示，实验第60天结束时，G3组，G4组，G5组,G6组,G7组，G8组，G10组的体重21.0±1.5、20.9±1.5、21.5±1.3、20.8±1.8、21.1±1.6、20.8±1.7、21.5±1.5g，与模型组(G2组，19.5±1.6g)相比，增加明显；
2.TSL-4对II型胶原型关节炎小鼠足掌后度的影响
如图14所示，实验第60天结束时，G3组，G4组，G5组,G6组,G7组，G8组，G10组的小鼠足掌后度3.04±0.11、3.06±0.23、2.94±0.13、2.92±0.11、2.94±0.07、2.99±0.17、3.15±0.07mm，与模型组(G2组，3.85±0.30mm)相比，有极显著差异；
3.TSL-4对II型胶原型关节炎小鼠足踝宽度的影响
如图15所示，实验第60天结束时，G3组，G4组，G5组,G6组,G7组，G8组，G10组的足踝宽度4.23±0.08、4.21±0.15、3.99±0.12、3.96±0.09、3.98±0.08、4.00±0.16、4.26±0.13mm，与模型组(G2组，4.83±0.20mm)相比，增加明显；
4.TSL-4对II型胶原型关节炎小鼠足周长的影响
如图16所示，实验第60天结束时，G3组，G4组，G5组,G6组,G7组，G8组，G10组的足周长分别为11.42±0.20、11.41±0.53、10.88±0.28、10.80±0.26、10.87±0.14、10.97±0.32、11.64±0.19mm，模型组(G2组，13.62±0.64mm)相比，有极显著差异；
5.TSL-4对II型胶原型关节炎小鼠关节炎评分的影响
如图17所示，实验第60天结束时，G3组，G4组，G5组,G6组,G7组，G8组，G10组小鼠关节炎评分分别为7.6±0.9、7.8±1.4、6.1±1.2、6.5±1.1、6.6±1.1、7.1±0.6、8.6±1.8,与模型组小鼠关节炎评分(G2组，11.5±1.7)比较有极显著性差异；
6.TSL-4对II型胶原型关节炎小鼠脾脏以及胸腺的影响
如图18所示，实验第60天结束时，G3组，G4组，G5组,G7组，G8组，G10组的脾脏94.6±6.7mg、94.2±13.3mg、96.0±5.2mg、95.1±9.5mg、95.8±11.4mg、97.5±5.1mg，与模型组(G2组，114.6±14.5mg)相比，有显著差异；小鼠胸腺除模型组(39.9±8.9mg)与正常对照组胸腺重量(25.9±6.2mg)比较具有显著性差异(*p<0.05)外，其余各组与与模型组比较均无显著性差异。
7.TSL-4对II型胶原型关节炎小鼠足重的影响
如图19所示，实验第60天结束时，G5组，G6组，G7组，G8足重174.6±11.7mg、176.2±8.9mg、176.4±9.7mg、177.1±7.8mg，与模型组(G2组，219.3±20.6mg)相比，有显著差异。
由以上结论可知，本申请TSL-4融合蛋白在5天给药一次(G6、G7、G10组)、甚至是7天给药一次(G8组)的情况下，仍然可以达到与G4组(一天给药2次)、G5组(两天给药一次)一样的治疗效果，且在用药剂量上等摩尔换算G4，G5，G7组用药量相当，说明本申请的融合蛋白半衰期明显延长。
实施例12：TSL-4药代动力学分析
1、SD大鼠给药方式
(1)购买18只SD大鼠，雌雄各半，放于动物适应性饲养一周。
(2)分成3组，雌雄各半，并记录其体重。
(3)TSL-4分别以4.17、12.5、37.5mg/kg单次背部皮下给药，眼眶取血，取样时间点分别是：0h、0.5h、1h、3h、5h、6h、7h、8h、10h、12h、16h、20h、24h、28h、41h、53h、65h、77h。12000rpm/min 离心取上清200μL，按照1:3的比例用PBS溶液进行稀释，放入EP管内置于-80℃冰箱储存。
(4)通过双抗夹心ELISA法测定生物样品。
2、血药浓度检测方式
双抗夹心ELISA法基本程序的确立
(1)包被，将HM-3单克隆抗体用包被液(CBS)以一定浓度稀释，加入酶标板，包被过夜。
(2)洗板，将包被液弃去，用PBST洗涤3次，拍干。
(3)封闭，加入封闭液，37.5℃封闭，重复洗板过程，拍干。
(4)加入待测样品，酶标板中加入待测样品，设置复孔，37.5℃孵育，重复洗板过程，拍干。
(5)加入二抗，加入按照比例稀释的二抗，37.5℃孵育。重复洗板过程，拍干。
(6)加入酶标二抗反应，加入利用5％脱脂奶粉溶液按照一定比例稀释的HRP酶标二抗，37.5℃孵育。重复洗板过程，拍干。
(7)加入底物，加入一定体积L的TMB底物，避光反应。
(8)终止，加入定量终止液体，终止反应。
(9)检测，用酶标仪读取OD450nm。
3、实验结果
3.1 SD大鼠单次皮下注射12.5mg/kg的TSL-4的药代动力学研究结果
大鼠单次皮下注射给予12.5mg/kg的TSL-4后，血药浓度-时间曲线结果见图20，血药浓度测定结果见表7，药代动力学参数结果见表10。
表7单次皮下注射12.5mg/kg的TSL-4后的血药浓度(单位:μg/mL)
Figure PCTCN2018117188-appb-000011
NA表示低于检测下限
3.2 SD大鼠单次皮下注射37.5mg/kg的TSL-4后药代动力学实验结果
大鼠单次皮下注射给予37.5mg/kg的TSL-4后血药浓度-时间曲线结果见图21，血药浓度测定结果见表8，药代动力学参数结果见表10。
表8单次皮下注射37.5mg/kg的TSL-4后的血药浓度(单位:μg/mL)
Figure PCTCN2018117188-appb-000012
NA表示低于检测下限
3.3 SD大鼠单次皮下注射低剂量TSL-4后药代动力学实验结果
大鼠单次皮下注射给予4.17mg/kg的TSL-4后血药浓度-时间曲线结果见图22，血药浓度测定结果见表9，药代动力学参数结果见表10。
表9单次皮下注射4.17mg/kg的TSL-4后的血药浓度(单位:μg/mL)
Figure PCTCN2018117188-appb-000013
Figure PCTCN2018117188-appb-000014
NA表示低于检测下限
3.4 SD大鼠单次皮下注射给予不同剂量的TSL-4后药代动力学参数比较
大鼠单次皮下注射给予不同剂量TSL-4后平均血药浓度-时间曲线见图23，血浆中TSL-4峰浓度(Cmax)与给药剂量的关系见图24，药时曲线下面积(AUC)与给药剂量的关系见图25，主要药代动力学参数比较结果见表10。
表10主要药代动力学参数比较结果(n＝6)
Figure PCTCN2018117188-appb-000015
以上结果表明，SD大鼠单次皮下注射高、中、低剂量(37.5mg/kg，12.5mg/kg，4.17mg/kg)的
TSL-4后，各剂量组的AUC 0-∞分别为9006.668±3859.519、147501.4±28351.84、176110±39596.45μg/L·h，与给药剂量成良好的线性关系(R 2＝0.9421)；Cmax分别为1.531±0.122、14.045±3.182、37.863±8.396μg/mL，与给药剂量成良好的线性关系(R 2＝0.9729)；吸收半衰期T 1/2ka分别为：2.824±0.697h、3.788±0.848h、4.478±0.223h；消除半衰期T 1/2ke分别为30.710±3.100h、32.321±4.935h、34.356±1.578h，达峰时间T max分别为7.4±0.55h、7±0.707h、7±0.707h。CL分别为1.383±0.528L/h/kg、0.070759±0.0369L/h/kg、0.0569±0.0336L/h/kg。
单次皮下注射高、中、低剂量(37.5mg/kg，12.5mg/kg，4.17mg/kg)的TSL-4后，TSL-4在SD大鼠体内呈一级动力学过程，符合线性药代动力学特征，C-T曲线符合二房室模型。高、中、低剂量组的TSL-4的T 1/2ke均相对较长，说明TSL-4在SD大鼠体内消除较为缓慢。
实施例13：TSL-4组织分布
实验目的：
(1)通过小动物PET/CT活体扫描研究关节炎模型大鼠单次皮下注射 89Zr-HM-3后的踝关节和全身其余组织的分布。
(2)通过离体组织伽马计数检测研究关节炎模型大鼠单次皮下注射 89Zr-HM-3后的踝关节和全身其余组织的分布。
(3)通过小动物PET/CT活体扫描研究关节炎模型大鼠单次踝关节腔注射 89Zr-HM-3后的踝关节和全身其余组织的分布。
实验方法：
Ⅱ型胶原诱导的关节炎模型大鼠造模方法：3mL不完全弗氏佐剂(Chondrex Inc，批号160111)和3mLⅡ型胶原(Chondrex Inc，批号160346)在冰上用匀浆器进行乳化，直至乳剂滴入水中不散。在Wistar大鼠(上海斯莱克实验动物有限责任公司，雌性，130-150g，合格证号2015000526387、2015000527320)尾巴皮内多点注射(2点，每点注射0.15mL乳剂)，每只大鼠注射0.3mL乳剂，使皮肤表面形成局部鼓包。约2周后观察大鼠发病情况，称重评分。
表11模型发病评分标准
评分 观察标准
0 未见任何明显的红肿
1 仅局限于跗骨或者踝关节出现红斑和轻度肿胀
2 红斑和轻度肿胀从踝关节延伸至跗骨
3 红斑和轻度肿胀从踝关节延伸至跖骨关节
4 踝关节、足部、脚趾均呈现出红斑和严重的肿胀
关节炎模型大鼠分别经皮下给予 89Zr-HM-3、游离 89Zr后，在1h、12h、24h、48h、120h、192h时间点使用小动物PET/CT进行标准CT和PET全身扫描；关节炎模型大鼠经左踝关节腔给予 89Zr-HM-3后，在1h、24h、48h、120h、192h、360h使用小动物PET/CT进行标准CT和PET全身扫描。结合CT图像勾画关节炎模型大鼠心脏、肝脏、肺脏、肾脏、脑、踝关节腔、脊柱骨、肌肉，获得上述组织的放射性摄取值SUV和％ID/g。
关节炎模型大鼠经皮下给予 89Zr-HM-3后分别在48h、120h时间点先进行标准CT和PET全身扫描，然后立即通过微灌注方式采集踝关节腔液以及全血、心、肝、脾、肺、肾、胃、小肠(近幽门处)、胰腺、脊椎骨、肌肉、性腺、脑组织，称重后置于放免管中采用伽马计数检测关节腔液、全血和组织中的放射性摄取值。通过γ计数结果计算关节炎模型大鼠的血液和组织中的放射性摄取值％ID/g。
表12动物的分组、给药及采样方案
Figure PCTCN2018117188-appb-000016
a：给药后1h、12h、24h、48h多床位全身扫描(每个床位PET扫描10min)，120h、192h多床位全身扫描(每个床位PET扫描15min)，360h多床位全身扫描(每个床位PET扫描20min)。
b：组织采集包括心脏、肝脏、脾脏、肺脏、肾脏、胃、小肠(近幽门处)、胰腺、脊柱骨、肌肉、性腺、脑12个脏器。
c：关节炎模型大鼠扫描前10min进行麻醉，具体麻醉方法为使用3-5％异氟烷、0.5-2L/min空气诱导SD大鼠麻醉，使用1-3％异氟烷、0.5-1L/min空气维持SD大鼠麻醉。实验过程中，于原始记录表格上准确记录动物体重、初始注射剂量及测量时间、注射时间、残留剂量及测量时间、小动物PET/CT不同时间点扫描时间等详细信息。
实验结果
1.受试物标记
表13 89Zr-HM-3质控实验结果
批号 16121501 16121502 L17011101
性状 澄清透明 澄清透明 澄清透明
pH 7.2 7.2 7.2
放射性化学纯(％) 90.40 90.40 92.71
化学纯度(％) 98.37 98.37 98.09
表14 89Zr-HM-3体外稳定性实验结果-1
批号 1h 4h 8h 9h
16121501 90.40％ 90.01％ 90.74％ 90.11％
16121502 90.40％ 90.01％ 90.74％ 90.11％
表15 89Zr-HM-3体外稳定性实验结果-2
批号 1h 4h 7h
L17011101 91.03％ 90.73％ 90.89％
如表13-15所示，批号为16121501、16121502、L17011101的 89Zr-HM-3质控符合实验要求。
2.PET/CT扫描实验
表16关节炎模型大鼠皮下注射 89Zr-HM-3后6个时间点PET/CT扫描放射性摄取％ID/g
Figure PCTCN2018117188-appb-000017
Figure PCTCN2018117188-appb-000018
如表16所示，关节炎模型大鼠皮下注射89Zr-HM-3后1h时，肾脏放射性摄取％ID/g最高(0.04±0.01)，其次是肺脏>左踝关节腔＝右踝关节腔＝脊柱骨>心脏＝肌肉>肝脏；12h时，心脏放射性摄取％ID/g最高(0.90±0.04)，其次肾脏>肝脏>肺脏>左踝关节腔>右踝关节腔>脊柱骨>肌肉；24h时，肾脏放射性摄取％ID/g最高(1.28±0.05)，其次是心脏>肝脏>左踝关节腔>右踝关节腔>肺脏>脊柱骨>肌肉；48h时，肾脏放射性摄取％ID/g最高(2.08±0.19)，其次是心脏>右踝关节腔>肝脏>左踝关节腔>肺脏>脊柱骨>肌肉；120h时，肾脏放射性摄取％ID/g最高(3.89±0.08)，其次是脊柱骨>肝脏>心脏>右踝关节腔>左踝关节腔>肺脏>肌肉；192h时，肾脏放射性摄取％ID/g最高(4.42±0.11)，其次是脊柱骨>肝脏>右踝关节腔>左踝关节腔>心脏>肺脏>肌肉。
关节炎模型大鼠皮下注射89Zr-HM-3后，心脏在1h时放射性摄取％ID/g最低(0.01±0.01)，在24h时最高(1.02±0.12)，然后随着时间的延长逐渐降低；肺脏、左踝关节腔、右踝关节腔在1h时放射性摄取％ID/g最低，分别为0.04±0.06、0.01±0.00、0.01±0.00，在48h时最高，分别是0.50±0.09、0.52±0.09、0.73±0.42，然后随着时间的延长逐渐降低；肝脏、肾脏、脊柱骨在1h时放射性摄取％ID/g最低，分别是0.00±0.00、0.04±0.01、0.01±0.00，随着时间的延长逐渐增加，在192h时最高，分别是1.02±0.27、4.42±0.11、1.02±0.05；肌肉在1h时放射性摄取％ID/g最低(0.01±0.01)，在120h最高(0.14±0.04)。具体结果见图26。
表17关节炎模型大鼠皮下注射 89Zr-HM-3后分别在48h和120h时间点PET/CT扫描放射性摄取％ID/g
Figure PCTCN2018117188-appb-000019
如表17所示，关节炎模型大鼠皮下注射89Zr-HM-3后48h时间点先进行PET/CT扫描然后采集组织伽马计数，肾脏放射性摄取％ID/g最高(2.60±0.09)，其次是心脏>肝脏>肺脏>脊柱骨>左踝关节腔>右踝关节腔>肌肉；关节炎模型大鼠皮下注射89Zr-HM-3后120h时间点先进行PET/CT扫描然后采集组织伽马计数，肾脏放射性摄取％ID/g最高(3.72±0.70)，其次是心脏>肝脏>脊柱骨>肺脏>左踝关节腔>右踝关节腔>肌肉。具体结果见图27。
表18关节炎模型大鼠左踝关节腔注射 89Zr-HM-3后6个时间点放射性摄取％ID/g
Figure PCTCN2018117188-appb-000020
Figure PCTCN2018117188-appb-000021
如表18所示，关节炎模型大鼠左踝关节腔注射89Zr-HM-3后1h时，左踝关节腔放射性摄取％ID/g最高(42.20±17.33)，其次是心脏>肝脏>肾脏>肺脏>右踝关节腔>脊柱骨>肌肉；24h时，左踝关节腔放射性摄取％ID/g最高(6.50±3.06)，其次是肾脏>心脏>肝脏>肺脏>右踝关节腔>脊柱骨>肌肉；48h时，左踝关节腔放射性摄取％ID/g最高(2.75±0.64)，其次是肾脏>心脏>右踝关节腔>肝脏>肺脏>脊柱骨>肌肉；120h时，肾脏放射性摄取％ID/g最高(2.78±0.44)，其次是左踝关节腔>肝脏>右踝关节腔>脊柱骨>心脏>肺脏>肌肉；192h时，肾脏放射性摄取％ID/g最高(3.04±0.52)，其次是左踝关节腔>肝脏>脊柱骨>右踝关节腔>心脏>肺脏>肌肉；360h时，肾脏放射性摄取％ID/g最高(2.70±0.54)，其次是左踝关节腔>脊柱骨>肝脏>右踝关节腔>心脏>肺脏>肌肉。
关节炎模型大鼠左踝关节腔注射89Zr-HM-3后，心脏、肺脏在1h时放射性摄取％ID/g分别为0.59±0.94、0.25±0.42，在24h时放射性摄取％ID/g最高，分别为1.15±0.14、0.71±0.39，然后随着时间的延长逐渐降低；左踝关节腔在1h时放射性摄取％ID/g最高(42.20±17.33)，然后随着时间的延长逐渐降低，在360h时最低(1.69±0.29)；右踝关节腔、肌肉在1h时放射性摄取％ID/g最低，分别为0.11±0.18、0.06±0.10，在48h时最高，分别为0.73±0.05、0.19±0.03，然后随着时间的延长逐渐降低；肝脏在1h时放射性摄取％ID/g最低(0.40±0.65)，在24h时放射性摄取％ID/g最高(0.88±0.21)，然后随着时间的延长轻微降低；肾脏在1h时放射性摄取％ID/g最低(0.39±0.57)，192h时最高(3.04±0.52)，然后随着时间的延长逐渐降低；脊柱骨在1h时放射性摄取％ID/g最低(0.10±0.17)，然后随着时间的延长逐渐增加，在360h时最高(0.82±0.13)。具体结果见图28。
表19关节炎模型大鼠皮下注射游离 89Zr后6个时间点放射性摄取％ID/g
Figure PCTCN2018117188-appb-000022
如表19所示，2h时，心脏放射性％ID/g最高(1.08)，其次是肾脏>肝脏＝脊柱骨>肺脏>右踝关节腔>肌肉>左踝关节腔；12h、24h、48h、120h、192h时，脊柱骨放射性％ID/g均最高，分别为2.43、3.64、4.05、5.35、5.38，均远高于相同时间点其他组织。
3.伽马计数实验
表20关节炎模型大鼠皮下注射 89Zr-HM-3后分别在48h、120h时间点采集组织放射性摄取％ID/g
Figure PCTCN2018117188-appb-000023
如表20所示，关节炎模型大鼠皮下注射89Zr-HM-3后48 h时采集全血和组织，全血放射性摄取％ID/g最高(2.32±0.20)，其次是肺脏>肾脏>性腺>肝脏>脾脏>心脏>肠>脊柱骨>关节腔液>胰腺>胃>肌肉；实验动物皮下注射89Zr-HM-3后120h时采集全血和组织，肾脏放射性摄取％ID/g最高(2.16±1.94)，其次是全血>肺脏>性腺>脾脏>关节腔液>肝脏>肠>脊柱骨>胰腺>心脏>胃>肌肉。具体结果见图29。
4.结论
实验结果显示，游离89Zr在给药12h后主要分布于骨骼，89Zr在12h、24h、48h、120h、196h时间点时骨骼中的放射性摄取％ID/g远高于其他组织，表明89Zr-HM-3在关节炎模型大鼠体内基本比较稳定；脑在全部时间点放射性摄取％ID/g均很低，接近或低于相同时间点肌肉放射性摄取％ID/g，表明89Zr-HM-3不易透过血脑屏障。关节炎模型大鼠分别皮下注射、左踝关节腔注射89Zr-HM-3后，心脏放射性摄取在24h时均最高，然后随着时间的延长逐渐降低，踝关节腔(非给药部位)放射性摄取在48h时均最高，然后随着时间的延长逐渐降低，肾脏、肝脏的放射性摄取随着时间的延长逐渐升高或者维持较高的水平。
表21关节炎模型大鼠分别皮下注射和左踝关节腔注射 89Zr-HM-3后在心脏的半衰期h
实验编号 G1-F-01 G1-F-02 G1-F-03 G4-F-01 G4-F-02 G4-F-03
T 1/2 153.17 89.21 119.66 56.84 127.11 111.09
如表21所示，通过DAS3.2.8软件非房室模型分析89Zr-HM-3经皮下注射关节炎模型大鼠的半衰期为120.68±31.99h；89Zr-HM-3经左踝关节腔注射关节炎模型大鼠的半衰期为98.35±36.83h。
实施例14：HM-3融合蛋白TSL-4对双氧水损伤的骨组织细胞保护作用
1、实验方法
所有细胞实验均在无菌的生物安全柜内进行操作，操作平台在使用前后都要进行无菌(UV照射和75％医用酒精)处理，细胞废液要收集在废液桶，实验中用到的废弃移液管和移液枪头投放到高压灭菌袋内进行后期高压灭菌消毒。
软骨细胞在T75培养瓶培养3-4天。当融合度达到80％后，移去原培养液，加入无菌PBS清洗细胞2-3次，然后加入适量的消化液，待细胞脱壁之后再加入新鲜培养液。离心移去含有消化液的细胞液，再加入适量的新鲜培养液配成适宜浓度的细胞悬浮液，用于细胞传代或细胞实验。细胞传代从P1代开始，直至P5代结束，每代都有冻存保留。细胞代数为P2-P5用于细胞实验。
按照200μL/孔的接种量，将密度为2.5*10 4个/mL的细胞悬浮液接种在96孔细胞培养板里，共配制3组，一组为对照组，另两组给予双氧水刺激。另配不含细胞的培养液作为空白组。每组配4-5个副孔。将细胞培养板放置于培养箱孵育24小时。第二天将培养液替换为不同实验组，每组给药体积为100μl，分别孵育24或48小时。
按实验计划结束孵育后，每个实验组按照细胞培养体积的1/10分别加入10μL的5mg/ml MTT,让细胞跟MTT在培养箱孵育4小时。之后小心弃上清液，接着加入200μL二甲基亚砜(DMSO)，混匀后在酶标仪上以试验波长为570 nm测定吸光度。细胞相对活性比率(％)计算公式：实验组的吸光度(每孔)/对照组吸光度平均值再乘以100％。所有实验组均以均数±标准差
Figure PCTCN2018117188-appb-000024
表示。每组之间的差异用Prism7.0软件分析，one-way ANOVA检验。*P<0.05为有统计学差异，**P<0.01为显著统计学差异，***P<0.001为极显著统计学差异。
2、主要实验材料
(1)大鼠软骨细胞，货号：RAT-iCell-s003，赛百康公司。
(2)造模剂：
Figure PCTCN2018117188-appb-000025
过氧化氢消毒液(H 2O 2，双氧水)(0.9M)，批号：170928，山东利尔康医疗科技股份有限公司。
(3)细胞培养液体系：原代软骨细胞基础培养基+10％胎牛血清(FBS)+1％细胞培养添加剂+1％青霉素/链霉素(PS)(货号：PriMed-iCell-020，赛百康公司)
(4)细胞传代相关试剂：PBS无菌生理盐水(pH＝7.2)(货号：C0221A，碧云天公司)；消化液：含0.02％EDTA+0.25％胰蛋白酶溶液(货号：25200056，Thermo fisher scientific公司)；常用热灭活无菌血清(货号：10100147，gibco公司)。
(5)MTT相关试剂：MTT(噻唑蓝)(货号：M2128-500mg，sigma公司)；DMSO(D5879-500ml，sigma公司)。
3、实验结果
实验1，双氧水造模时间和剂量优化
双氧水实验浓度范围为0.01，0.1，0.5，1，10mM。为了确定双氧水造模时间和剂量，选择24，48和72小时3个造模时间，每个剂量的双氧水共配备两个检测孔。
表22，不同双氧水造模时间和剂量的MTT检测值
Figure PCTCN2018117188-appb-000026
Figure PCTCN2018117188-appb-000027
上述结果表明，软骨细胞在双氧水刺激后24，48和72小时的细胞活性改变相似；细胞活性随着双氧水浓度升高而降低。且1和10mM双氧水浓度在这三个时间点都比对照组(0mM双氧水)显著降低，故选用24小时为双氧水造模时间。另外选择双氧水的剂量应是与对对照组的相对活性为0.5左右为最佳。实验结果显示双氧水剂量应选择在0.1-1mM之间。
实验2，HM-3融合蛋白TSL-4剂量优化
TSL-4选择的浓度范围是0.1，0.3，1，3，9，27μM。
为了确定TSL-4对软骨细胞的细胞活性有无影响，选择24，48和72小时3个孵育时间，每个剂量的TSL-4溶液共配备两个检测孔。
表23，不同TSL-4给药时间和剂量MTT检测值
TSL-4给药剂量(μM) 24小时 48小时 72小时
0 1.43 1.70 1.51
1 1.59 1.43 1.61
3 1.67 1.43 1.52
9 1.72 1.51 1.57
27 1.45 1.30 1.31
上述结果表明，TSL-4在24，48和72小时对细胞活性没有显著的影响，且各个剂量的TSL-4与对照组(0μM TSL-4)没有显著差异。故选择24小时作为TSL-4给药时间。但TSL-4剂量在1-27μM之间没有差异，所以应再检测低剂量的TSL-4对细胞活性的影响。
表24，低剂量TSL-4给药24小时后MTT检测值
TSL-4给药剂量(μM) 24小时
0 1.23
0.1 1.26
0.3 1.2
1 1.15
3 1.08
上述结果表明，TSL-4在0.1-3μM之间对细胞活性的影响没有明显的差异。
由于前期实验各个浓度的TSL-4均对细胞活性没有影响，故根据TSL-4原溶液浓度(293μM)，按照1：5稀释比例从29.3μM稀释至0.23μM。
表25，TSL-4使用剂量给药24小时后MTT检测值
TSL-4给药剂量(μM) 24小时
0 1.68
0.23 1.59
1.17 1.4
5.86 1.39
29.3 1.24
同样各个浓度的TSL-4(0.23-29.3μM)与软骨细胞培养24小时，对细胞活性没有较大的变化，所以选择0.23，1.17，5.86，29.3μM作为后续实验中TSL-4给药浓度。
实验3，检测TSL-4对双氧水损伤的软骨细胞保护作用
为了检测TSL-4对双氧水损伤的软骨细胞保护作用，实验一共分为三组，分别为对照组(不含双氧水和TSL-4，只加入新鲜培养液)，双氧水造模组(只加入含有不同浓度的双氧水细胞培养液)和TSL-4给药组(含双氧水)。为了检测TSL-4对双氧水损伤软骨细胞的保护作用，TSL-4与双氧水一起与软骨细胞 培养24小时。双氧水剂量选择为0.1，0.25，0.5,1mM；TSL-4剂量为0.23，1.17，5.86，29.3μM。
在不同剂量双氧水存在的条件下，适量浓度的TSL-4与软骨细胞培养24小时，各个给药组的细胞相对活性(与对照组的吸光度相对百分比)用
Figure PCTCN2018117188-appb-000028
如下表示。
表26，不同双氧水和TSL-4对软骨细胞活性的影响
Figure PCTCN2018117188-appb-000029
上述结果表明，在0.25和0.5mM双氧水浓度下1.17或5.86μM TSL-4可以显著的改善双氧水损伤的软骨细胞活性；另外在0.1mM双氧水刺激下，0.23和29.3μM TSL-4与双氧水组对细胞活性有显著抑制作用*P<0.05；**P<0.01；***P<0.001；****P<0.0001。
在实施例14的实验1中，测定双氧水的最适造模浓度为0.1-1mM之间，本实验选择双氧水的浓度为0.5mM和1mM时时候，细胞活性比率(％)均下降到接近5％左右，这说明双氧水造模不稳定，易产生结果差异，这有可能是软骨细胞的代数(本次实验的软骨细胞代数为P4)不同，造成软骨细胞抗氧化能力变差所致，由查文献所知，软骨细胞在2D单细胞培养环境下，细胞易成纤维化，其表达的II型胶原蛋白和多糖均减少。为了准确检测出TSL-4抗双氧水损伤软骨细胞的保护作用，根据上述实验结果，双氧水造模浓度应该低于0.5mM，而0.25mM浓度的双氧水刺激软骨细胞的细胞活性比率为70％左右，所以应该在0.25和0.5mM双氧水浓度之间再选择一个浓度，例如：0.35mM浓度的双氧水。另外当双氧水浓度为0.25或0.5mM时，TSL-4的最适给药浓度在1.17和5.86μM时候，有统计学差异(P<0.05)，而与之相反的是，0.23和29.3μM浓度的TSL-4给药实验对0.1mM双氧水刺激软骨细胞活性竟有较显著的抑制细胞活性。故第二次重复实验，TSL-4给药浓度选为1.17和5.86μM。
在0.25,0.35,0.5mM造模剂双氧水存在的条件下，1.17和5.86μM的TSL-4与软骨细胞培养24小时，各个给药组的细胞相对活性(与对照组的吸光度相对百分比)用
Figure PCTCN2018117188-appb-000030
如下表示。
表27，双氧水和TSL-4对软骨细胞活性的影响重复试验
Figure PCTCN2018117188-appb-000031
由上述结果得出，在三个剂量的(0.25，0.35，0.5mM)双氧水刺激下，1.17和5.86μM TSL-4都显示出双氧水损伤的软骨细胞的活性具有改善作用，但只有在0.35mM双氧水条件下，5.86μM TSL-4对软骨细胞具有显著改善细胞活性的作用。用one-way ANOVA分析各个给药组与相应浓度的模型组的差异对比，用*代表具有统计学差异，P<0.05。所以5.86μM TSL-4在与0.25-0.5mM双氧水共同培养24小时要比模型组能显著改善双氧水损伤软骨细胞的活性。
综上所述，在TSL-4与双氧水共同孵育情况下，对软骨细胞活性有较显著的改善作用，所以TSL-4与双氧水的共同孵育24小时为最佳给药时间。双氧水的造模浓度在0.25-0.5之间，与TSL-4共同孵育有较显著改善细胞活性的作用；而TSL-4给药浓度在1.17和5.86μM条件下有较强的细胞活性改善作用。

Claims (10)

  1. 一种融合蛋白，由活性多肽HM-3与人源IgG-Fc片段或IgG-Fc突变体片段连接形成，连接方式为：
    HM-3通过连接肽或直接与人源IgG-Fc片段或IgG-Fc突变体片段N端连接，或与C端连接，或C、N端同时连接，其通式为(HM-3) n-Linker-IgG-Fc、IgG-Fc-Linker-(HM-3) n、或
    (HM-3) n-Linker-IgG-Fc-Linker-(HM-3) n；其中n选自1，2，3，4或5。
  2. 根据权利要求1所述的融合蛋白，其特征在于，所述的连接肽选自：
    ①、(GGGGS) a，其中a为1，2，3，4，5或6；
    ②、A(EAAAK) bA，其中b为1，2，3，4，5或6；
    ③、(AP) c，其中c为1至18；
    ④、G d，其中d为1至15；
  3. 根据权利要求2所述的融合蛋白，其特征在于，所述的连接肽为(GGGGS) a序列，重复数a选自：3，4或5。
  4. 根据权利要求1所述的融合蛋白，其特征在于，所述的IgG-Fc序列为人源IgG1、IgG2、IgG3或IgG4的Fc或其突变体。
  5. 根据权利要求1所述的融合蛋白，其特征在于，所述融合蛋白选自：
    TSL-1融合蛋白：HM-3-(GGGGS) 3-IgG2-Fc；
    TSL-2融合蛋白：HM-3-(GGGGS) 3-mIgG4-Fc；
    TSL-3融合蛋白：IgG2-Fc-(GGGGS) 3-HM-3；
    TSL-4融合蛋白：mIgG4-Fc-(GGGGS) 3-HM-3；
    TSL-13融合蛋白：HM-3-(GGGGS) 3-mIgG4-Fc-(GGGGS) 3-HM-3；
    TSL-14融合蛋白：HM-3-HyFc；
    TSL-15融合蛋白：mIgG4-Fc-G5-HM-3-G8-HM-3；
    TSL-16融合蛋白：HyFc-(GGGGS) 3-HM-3；
    TSL-17融合蛋白：mIgG4-Fc-A(EAAAK) 4A-HM-3；
    TSL-18融合蛋白：mIgG4-Fc-(AP) 9-HM-3。
  6. 根据权利要求1-5任一项权利要求所述的融合蛋白，其特征在于，采用基因重组技术制备，其中采用的表达细胞选自：酵母、CHO、SP2/0、BHK和/或HEK293。
  7. 含有权利要求1所述融合蛋白的药物组合物，所述药物组合物以适合药用的制剂形式存在，所述制剂形式选自：注射剂、胶囊、片剂、药丸、鼻喷剂、或气雾剂，给药方式包括口服、静脉注射、静脉滴注、皮下或肌肉注射。
  8. 权利要求1所述的融合蛋白在制备治疗自身免疫性疾病，治疗新生血管疾病，治疗骨关节炎的药物中的应用。
  9. 权利要求8所述自身免疫性疾病为类风湿性关节炎。
  10. 权利要求8所述新生血管疾病为湿性老年黄斑变性。
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