WO2017129064A1 - 抗补体因子D的人源化Fab和人源化抗体及其用途 - Google Patents

抗补体因子D的人源化Fab和人源化抗体及其用途 Download PDF

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WO2017129064A1
WO2017129064A1 PCT/CN2017/071914 CN2017071914W WO2017129064A1 WO 2017129064 A1 WO2017129064 A1 WO 2017129064A1 CN 2017071914 W CN2017071914 W CN 2017071914W WO 2017129064 A1 WO2017129064 A1 WO 2017129064A1
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sequence
amino acid
variable region
chain variable
light chain
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PCT/CN2017/071914
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French (fr)
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柯潇
雷刚
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成都康弘生物科技有限公司
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/40—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against enzymes
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/55—Fab or Fab'
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value

Definitions

  • the present invention relates to the field of biotechnology, and in particular to a humanized Fab and humanized antibody against complement factor D and uses thereof.
  • Age-related macular degeneration also known as age-related macular degeneration (AMD)
  • AMD age-related macular degeneration
  • AMD age-related macular degeneration
  • Age-related macular degeneration mainly manifests as a decrease in the ability of retinal pigment epithelial cells to phagocytosis and digestion of the extracellular disk membrane. As a result, the residual membrane bodies that have not been completely digested are retained in the basal cell cytoplasm and excreted outside the cell. Deposited on the Bruch film to form a glass film. This change is more pronounced due to the structural and functional specificity of the macula.
  • Vitreous hernia is also seen in the elderly with normal vision, but the subsequent pathological changes lead to macular degeneration. Or causing Bruch membrane rupture, choroidal capillaries through the ruptured Bruch membrane into the RPE and under the retinal nerve epithelium, forming choroidal neovascularization. Due to the abnormal structure of the neovascular wall, blood vessels leak and hemorrhage, which leads to a series of secondary pathological changes. Age-related macular degeneration mostly occurs in people over the age of 45, and its prevalence increases with age, which is an important disease for blindness in the elderly. AMD blindness accounts for about 8.7% of global blindness. There are about 30 million AMD patients worldwide, and about 500,000 people are blinded by AMD every year.
  • Complement is a group of activated and enzymatically active proteins present in normal human and animal serum and tissue fluids. Its role can assist and supplement specific antibodies, mediating immune lysis and hemolysis.
  • the complement system is a multi-molecular system consisting of more than 30 soluble proteins, membrane-bound proteins and complement receptors. According to its biological functions, it can be divided into three types: complement intrinsic components, complement regulatory components and complement receptors (CR). There are three main pathways for the regulation of the complement system: the classical pathway, the alternative pathway, and the lectin pathway. Different regulatory pathways affect the production of different complement proteins and play different roles. The role. Abnormal activation of the complement system often causes an overreaction of the body's immune system, causing damage to the body's own normal tissues.
  • the complement system can cause local inflammation, tissue damage, and upregulation of angiogenic factors.
  • Diseases associated with excessive activation of complement include rheumatoid arthritis, senile dementia, systemic lupus erythematosus, ischemic reperfusion, acute myocardial infarction, Acute respiratory distress syndrome, etc.
  • LBL pathway the alternative pathway and the lectin pathway (LBL pathway) are independent activation mechanisms of antibody-mediated responses and are the most important pathogenic mechanism in the pathogenesis of AMD [Paul A. et, al. The Immune System and AMD, Curr Ophthalmol Rep (2014) 2:14–19], in which complement factor D (EC: 3.4.21.66) is an important component that initiates activation of alternative pathways and belongs to the trypsin family.
  • Complement factor D is present mainly in activated form in serum, and active factor D (D) acts on C3bB, possibly cleavage of factor B into Ba and Bb at the arginine-lysine bond at positions 234-235 Two fragments to initiate the replacement A cascade of activation reactions of pathways.
  • antibodies against complement D are species-specific, and anti-complement factor antibodies (AFD; FCFD4514S; Lampalizumab) have weak inhibition against the alternative complement pathway (AP) of dogs or sheep.
  • AP alternative complement pathway
  • One of the objects of the present invention is to provide a humanized antigen-binding fragment Fab against human complement factor D or an anti-human complement factor D humanized antibody which has better affinity and better complement to human complement factor D. Stability and biological activity.
  • the invention provides a humanized antibody against F-factor D or an antigen-binding fragment thereof, comprising a light chain variable region and a heavy chain variable region, wherein the light chain variable region comprises the amino acid sequence ITSTDIDDDX 1 N, amino acid Sequence GGX 2 TLRP and amino acid sequence X 3 QSDS X 4 PYX 5 , X 1 is V, M or L, X 2 is N or A, X 3 is M or L, X 4 is L or Y, and X 5 is I or T; the heavy chain variable region contains amino acid sequence 1, sequence 2 and amino acid sequence Y 1 GGY 2 NY 3 , said Y 1 is A or E, Y 2 is S, V or P, and Y 3 is N, Y , F, H, S or R; when X 1 , X 2 , X 3 , X 4 and X 5 are different, the following conditions are satisfied: X 1 is M, X 2 is N, X 3 is L, and
  • the humanized antibody or antigen-binding fragment Fab thereof wherein X 1 is M, X 2 is A, X 3 is L, X 3 is L, and X 5 is T, Y 1 is not A, Y 2 is not V and Y 3 is not N or Y 1 is not E, Y 2 is not P and Y 3 is not N.
  • X 1 , X 2 , X 3 , X 4 , Y 1 , Y 2 and Y 3 satisfy the above conditions at the same time.
  • the humanized antibody or antigen-binding fragment Fab thereof preferably, wherein X 1 is V, X 2 is N, X 3 is L, X 4 is L, and X 5 is I, Y 1 is not E, Y 2 Not V and Y 3 are not S.
  • X 1 , X 2 , X 3 , X 4 , X 5 , Y 1 , Y 2 and Y 3 satisfy the above conditions at the same time.
  • the humanized antibody or antigen-binding fragment Fab thereof preferably, wherein X 1 is V, X 2 is N, X 3 is L, X 4 is Y, and X 5 is T, Y 1 is not E, Y 2 Not V and Y 3 are not N or H, or Y 1 is not E, Y 2 is not P and Y 3 is not N.
  • X 1 , X 2 , X 3 , X 4 , X 5 , Y 1 , Y 2 and Y 3 satisfy the above conditions at the same time.
  • the humanized antibody or antigen-binding fragment Fab thereof preferably, when X 1 is V, X 2 is N, X 3 is M, X 4 is L, and X 5 is T, Y 1 is not E, Y 2 Not P and Y 3 are not N.
  • X 1 , X 2 , X 3 , X 4 , X 5 , Y 1 , Y 2 and Y 3 satisfy the above conditions at the same time.
  • the humanized antibody or antigen-binding fragment Fab thereof preferably, when X 1 is V, X 2 is A, X 3 is L, X 4 is L, and X 5 is T, Y 1 is not E, Y 2 Not V and Y 3 are not N.
  • X 1 , X 2 , X 3 , X 4 , X 5 , Y 1 , Y 2 and Y 3 satisfy the above conditions at the same time.
  • the humanized antibody or antigen-binding fragment Fab thereof preferably, wherein X 1 is L, X 2 is A, X 3 is L, X 4 is L, and X 5 is T, Y 1 is not E, Y 2 Not V and Y 3 are not N.
  • X 1 , X 2 , X 3 , X 4 , X 5 , Y 1 , Y 2 and Y 3 satisfy the above conditions at the same time.
  • the humanized antibody or antigen-binding fragment Fab thereof against complement factor D comprises the following heavy chain variable region and light chain variable region sequence: the amino acid sequence of the light chain variable region is sequence 3 in the sequence listing, Any one of 5, 8, 13, 15, and 17; the amino acid sequence of the heavy chain variable region is any one of the sequences 19, 21, 23, 25, 27, 29, 31, 33 in the sequence listing .
  • KH001 the amino acid sequence of the light chain variable region is sequence 3 in the sequence listing, the amino acid sequence of the heavy chain variable region is the sequence 33 in the sequence listing;
  • KH002 the amino acid sequence of the light chain variable region is the sequence 8 in the sequence listing, the amino acid sequence of the heavy chain variable region is the sequence 33 in the sequence listing;
  • KH003 the amino acid sequence of the light chain variable region is sequence 3 in the sequence listing, the amino acid sequence of the heavy chain variable region is the sequence 21 in the sequence listing;
  • KH004 the amino acid sequence of the light chain variable region is sequence 3 in the sequence listing, the amino acid sequence of the heavy chain variable region is the sequence 31 in the sequence listing;
  • KH005 the amino acid sequence of the light chain variable region is sequence 3 in the sequence listing, the amino acid sequence of the heavy chain variable region is the sequence 19 in the sequence listing;
  • KH006 the amino acid sequence of the light chain variable region is sequence 3 in the sequence listing, the amino acid sequence of the heavy chain variable region is the sequence 23 in the sequence listing;
  • KH007 the amino acid sequence of the light chain variable region is sequence 3 in the sequence listing, the amino acid sequence of the heavy chain variable region is the sequence 25 in the sequence listing;
  • KH008 the amino acid sequence of the light chain variable region is sequence 3 in the sequence listing, the amino acid sequence of the heavy chain variable region is the sequence 27 in the sequence listing;
  • KH009 the amino acid sequence of the light chain variable region is sequence 3 in the sequence listing, the amino acid sequence of the heavy chain variable region is the sequence 29 in the sequence listing;
  • KH010 the amino acid sequence of the light chain variable region is the sequence 13 in the sequence listing, the amino acid sequence of the heavy chain variable region is the sequence 33 in the sequence listing;
  • KH011 the amino acid sequence of the light chain variable region is the sequence 15 in the sequence listing, the amino acid sequence of the heavy chain variable region is the sequence 33 in the sequence listing;
  • KH012 the amino acid sequence of the light chain variable region is the sequence 13 in the sequence listing, and the amino acid sequence of the heavy chain variable region is the sequence 21 in the sequence listing;
  • KH013 the amino acid sequence of the light chain variable region is the sequence 13 in the sequence listing, the amino acid sequence of the heavy chain variable region is the sequence 31 in the sequence listing;
  • KH014 the amino acid sequence of the light chain variable region is the sequence 13 in the sequence listing, the amino acid sequence of the heavy chain variable region is the sequence 23 in the sequence listing;
  • KH015 the amino acid sequence of the light chain variable region is the sequence 13 in the sequence listing, the amino acid sequence of the heavy chain variable region is the sequence 25 in the sequence listing;
  • KH016(XY1252) the amino acid sequence of the light chain variable region is the sequence 13 in the sequence listing, and the amino acid sequence of the heavy chain variable region is the sequence 29 in the sequence listing;
  • KH017 the amino acid sequence of the light chain variable region is the sequence 15 in the sequence listing, and the amino acid sequence of the heavy chain variable region is the sequence 31 in the sequence listing;
  • KH018 the amino acid sequence of the light chain variable region is the sequence 15 in the sequence listing, the amino acid sequence of the heavy chain variable region is the sequence 19 in the sequence listing;
  • KH019 the amino acid sequence of the light chain variable region is the sequence 15 in the sequence listing, the amino acid sequence of the heavy chain variable region is the sequence 23 in the sequence listing;
  • KH020 the amino acid sequence of the light chain variable region is the sequence 15 in the sequence listing, the amino acid sequence of the heavy chain variable region is the sequence 25 in the sequence listing;
  • KH021 the amino acid sequence of the light chain variable region is the sequence 15 in the sequence listing, the amino acid sequence of the heavy chain variable region is the sequence 27 in the sequence listing;
  • KH022 the amino acid sequence of the light chain variable region is the sequence 15 in the sequence listing, the amino acid sequence of the heavy chain variable region is the sequence 29 in the sequence listing;
  • KH024 the amino acid sequence of the light chain variable region is the sequence 17 in the sequence listing, the amino acid sequence of the heavy chain variable region is the sequence 19 in the sequence listing;
  • KH027 the amino acid sequence of the light chain variable region is sequence 5 in the sequence listing, and the amino acid sequence of the heavy chain variable region is the sequence 31 in the sequence listing;
  • KH028 The amino acid sequence of the light chain variable region is sequence 5 in the sequence listing, and the amino acid sequence of the heavy chain variable region is sequence 25 in the sequence listing.
  • the CH1 of the Fd fragment of the Fab may be a human constant region CH1 of any type (IgG, IgA, IgM, IgE, IgD) or subclass (IgG1, IgG2, IgG3, IgG4, IgM1, IgM2, IgA1, IgA2);
  • the light chain constant region may be of any type (kappa type or lambda type), subtype ( ⁇ 1, ⁇ 2, ⁇ 3, ⁇ 4), or allotropy ( ⁇ m(1), ⁇ m(2), ⁇ m(3)) Human constant region of the light chain.
  • the Fab consists of a heavy chain fragment consisting of a heavy chain variable region and a heavy chain constant region, and a light chain consisting of a light chain variable region and a light chain constant region.
  • the amino acid sequence of the heavy chain constant region is preferably identical to the amino acid sequence of the human antibody heavy chain constant region, wherein the amino acid sequence of the light chain constant region is preferably identical to the human antibody light chain constant region amino acid sequence; wherein the heavy chain is constant
  • the amino acid sequence of the region is sequence 39; the amino acid sequence of the light chain constant region is sequence 41.
  • the heavy chain constant region may be of any type (IgG, IgA, IgM, IgE, IgD) or a subclass (IgG1, IgG2, IgG3, IgG4, IgM1, IgM2) a human constant region of IgA1, IgA2);
  • the light chain constant region may be of any type (kappa type or lambda type), a subtype ( ⁇ 1, ⁇ 2, ⁇ 3, ⁇ 4), or an isotropic ( ⁇ m(1), Human constant region of the ⁇ m(2), ⁇ m(3)) light chain.
  • the antibody consists of a heavy chain or a light chain, the amino acid sequence of the heavy chain constant region is identical to the amino acid sequence of the human antibody heavy chain constant region, the amino acid sequence of the light chain constant region and the human antibody light chain constant region amino acid sequence the same.
  • the amino acid sequence of the human antibody heavy chain constant region is preferably sequence 43, and the human antibody light chain constant region amino acid sequence is preferably Is the sequence 41.
  • the present invention still further provides an antibody selected from any one of the following:
  • KH-A its light chain sequence is sequence 45, and its heavy chain sequence is sequence 47;
  • KH-B its light chain sequence is sequence 49, and its heavy chain sequence is sequence 51;
  • KH-C its light chain sequence is sequence 45, and its heavy chain sequence is sequence 53;
  • KH-D its light chain sequence is sequence 49, and its heavy chain sequence is sequence 55;
  • KH-E its light chain sequence is sequence 57, and its heavy chain sequence is sequence 59;
  • KH-F its light chain sequence is sequence 57 and its heavy chain sequence is sequence 61.
  • the invention also provides a polynucleotide encoding the humanized antibody or antigen-binding fragment Fab of any of the above.
  • the above polynucleotide preferably the coding sequence of the light chain variable region of the antibody in the humanized antibody or antigen-binding fragment Fab is sequence 4, sequence 6, sequence 9, sequence 14, sequence 16 and sequence in the sequence listing In any one of 18, the coding sequence of the heavy chain variable region is any one of sequence 20, sequence 22, sequence 24, sequence 26, sequence 28, sequence 30, sequence 32, and sequence 34 in the sequence listing.
  • the coding sequence of the heavy chain constant region of the Fab is as shown in SEQ ID NO: 40 in the Sequence Listing; the light chain constant region coding sequence of the Fab is as shown in SEQ ID NO: 42 in the Sequence Listing;
  • the coding sequence for the constant region of the chain is set forth in SEQ ID NO: 44 in the Sequence Listing; the coding sequence for the constant region of the light chain of the antibody is shown as SEQ ID NO: 42 in the Sequence Listing.
  • the invention further provides a polynucleotide selected from any of the following:
  • KH-A its light chain sequence is sequence 46, and its heavy chain sequence is sequence 48;
  • KH-B its light chain sequence is sequence 50, and its heavy chain sequence is sequence 52;
  • KH-C its light chain sequence is sequence 46, and its heavy chain sequence is sequence 54;
  • KH-D its light chain sequence is sequence 50, and its heavy chain sequence is sequence 56;
  • KH-E its light chain sequence is sequence 58, and its heavy chain sequence is sequence 60;
  • KH-F its light chain sequence is sequence 58, and its heavy chain sequence is sequence 62.
  • the present invention still further provides the use of the above humanized antibody or antigen-binding fragment Fab, or the above polynucleotide, which is selected from any of the following uses:
  • the present invention relates to a human complement factor D (CFD)-related disease which is caused by an increase in human complement factor D; preferably rheumatoid arthritis, myocardial infarction, reperfusion injury or eye disease; wherein the eye disease is more preferably age-related macular degeneration Or diabetic retinopathy.
  • age-related macular degeneration AMD includes non-exudative (such as meta-advanced AMD or map-like atrophy (GA)) and exudative (such as wet AMD (choroidal neovascularization (CNV)) AMD, diabetic retinopathy (DR), endophthalmitis, and uveitis.
  • the complement-related eye condition is meta-stage dry AMD.
  • the present invention still further provides a pharmaceutical composition
  • a pharmaceutical composition comprising an adjuvant and an active ingredient, the active ingredient comprising at least one of the following: the humanized antibody or antigen-binding fragment Fab of any of the above, or any of the above One more Nucleotide; the excipient is a pharmaceutically acceptable carrier or excipient.
  • the present invention still further provides a method for producing the humanized Fab or antibody against the complement factor D, which comprises the steps of: transfecting or transforming a recombinant vector containing the gene encoding the antibody into a host cell, and performing expression under suitable conditions; purification.
  • the host cell is a eukaryotic cell or a prokaryotic cell, preferably a mammalian cell, a bacterium, a yeast, an insect cell or the like.
  • mammalian cells include, but are not limited to, 293 cells, CHO cells, SP20 cells, NSO cells, COS cells, BHK cells or PerC6 cells, etc.
  • the methods of transfecting cells include, but are not limited to, electroporation, lipids Body-mediated method, calcium-mediated method, etc.
  • a preferred expression of the antibody is to genetically amplify the recombinant vector in a host cell that has been stably transfected to increase the expression level of the corresponding recombinant protein, for example, with a recombinant containing dihydrofolate reductase (DHFR).
  • DHFR dihydrofolate reductase
  • the concentration of methotrexate (MTX) can be added to the cell culture medium to amplify the number of copies of the recombinant vector in the host cell.
  • the protein concentration in the culture solution can be determined by enzyme-linked immunosorbent assay (ELISA) or other methods.
  • ELISA enzyme-linked immunosorbent assay
  • purification can be performed by Protein G affinity chromatography; IgG protein can be purified by Protein A affinity chromatography.
  • KH023 the amino acid sequence of the light chain variable region is the sequence 17 in the sequence listing, the amino acid sequence of the heavy chain variable region is the sequence 21 in the sequence listing;
  • KH025 the amino acid sequence of the light chain variable region is the sequence 17 in the sequence listing, and the amino acid sequence of the heavy chain variable region is the sequence 25 in the sequence listing;
  • KH026 the amino acid sequence of the light chain variable region is sequence 5 in the sequence listing, the amino acid sequence of the heavy chain variable region is the sequence 21 in the sequence listing;
  • KH029 the amino acid sequence of the light chain variable region is the sequence 5 in the sequence listing, the amino acid sequence of the heavy chain variable region is the sequence 19 in the sequence listing;
  • KH030 the amino acid sequence of the light chain variable region is the sequence 5 in the sequence listing, the amino acid sequence of the heavy chain variable region is the sequence 33 in the sequence listing;
  • KH031 the amino acid sequence of the light chain variable region is sequence 7 in the sequence listing, the amino acid sequence of the heavy chain variable region is the sequence 33 in the sequence listing;
  • KH032 the amino acid sequence of the light chain variable region is the sequence 10 in the sequence listing, the amino acid sequence of the heavy chain variable region is the sequence 33 in the sequence listing;
  • KH033 the amino acid sequence of the light chain variable region is the sequence 11 in the sequence listing, the amino acid sequence of the heavy chain variable region is the sequence 35 in the sequence listing;
  • KH034 the amino acid sequence of the light chain variable region is the sequence 11 in the sequence listing, and the amino acid sequence of the heavy chain variable region is the sequence 36 in the sequence listing;
  • KH035 the amino acid sequence of the light chain variable region is the sequence 12 in the sequence listing, the amino acid sequence of the heavy chain variable region is the sequence 35 in the sequence listing;
  • KH036 the amino acid sequence of the light chain variable region is sequence 17 in the sequence listing, the amino group of the heavy chain variable region The acid sequence is the sequence 33 in the sequence listing;
  • KH037 the amino acid sequence of the light chain variable region is the sequence 13 in the sequence listing, the amino acid sequence of the heavy chain variable region is the sequence 19 in the sequence listing;
  • KH038 the amino acid sequence of the light chain variable region is the sequence 13 in the sequence listing, the amino acid sequence of the heavy chain variable region is the sequence 27 in the sequence listing;
  • KH039 the amino acid sequence of the light chain variable region is the sequence 15 in the sequence listing, and the amino acid sequence of the heavy chain variable region is the sequence 21 in the sequence listing;
  • KH040 the amino acid sequence of the light chain variable region is the sequence 17 in the sequence listing, and the amino acid sequence of the heavy chain variable region is the sequence 31 in the sequence listing;
  • KH000 The amino acid sequence of the light chain variable region is the sequence 37 in the sequence listing, and the amino acid sequence of the heavy chain variable region is the sequence 38 in the sequence listing.
  • KH111 the light chain variable region amino acid sequence is sequence 11
  • the heavy chain variable region amino acid sequence is sequence 33.
  • nucleotide sequence of coding sequence 3 is sequence 4; nucleotide sequence of coding sequence 5 is sequence 6; nucleotide sequence of coding sequence 8 is sequence 9; the nucleotide sequence of coding sequence 13 is sequence 14; the nucleotide sequence of coding sequence 15 is sequence 16; and the nucleotide sequence of coding sequence 17 is sequence 18.
  • nucleotide sequence of coding sequence 11 is sequence 63; nucleotide sequence of coding sequence 19 is sequence 20; nucleotide sequence of coding sequence 21 is sequence 22; the nucleotide sequence of the coding sequence 23 is the sequence 24; the nucleotide sequence of the coding sequence 25 is the sequence 26; the nucleotide sequence of the coding sequence 27 is the sequence 28; the nucleotide sequence of the coding sequence 29 is the sequence 30;
  • the nucleotide sequence of coding sequence 31 is sequence 32; the nucleotide sequence of coding sequence 33 is sequence 34; and the nucleotide sequence of coding sequence 29 is sequence 30.
  • nucleotide sequence encoding the sequence 39 is the sequence 40; and the nucleotide sequence encoding the sequence 43 is the sequence 44.
  • nucleotide sequence of coding sequence 41 is sequence 42.
  • the amino acid sequence of the KH102 light chain is sequence 106
  • the nucleotide sequence of coding sequence 106 is sequence 107
  • the heavy chain amino acid sequence is sequence 108
  • the nucleotide sequence encoding sequence 108 is sequence 109.
  • the invention also provides an antigen-binding fragment A or antigen-binding fragment B derived from the Fab.
  • the antigen-binding fragment A is a Fab', F(ab')2, Fv, a heavy chain variable region, a light chain variable region, a polypeptide fragment selected from the heavy chain variable region, or a light chain selected from the Fab derived from the Fab.
  • a polypeptide fragment of a variable region is a Fab, Fab', F(ab')2, Fv (antibody variable region fragment), a heavy chain variable region, a light chain variable region derived from the antibody, A polypeptide fragment selected from the heavy chain variable region or a polypeptide fragment selected from the light chain variable region.
  • F(ab')2 consists of a pair of light chains and a pair of heavy chains slightly larger than Fd (called Fd'). Pepsin hydrolyzes IgG molecules to produce this fragment, which contains two Fabs that bind two epitopes. Bit.
  • Fd' contains approximately 235 amino acid residues including VH, CH1 and hinge regions.
  • Fv consists of a light chain variable region (VL) and a heavy chain variable region (VH), which are bound together by non-covalent bonds and have a molecular weight of about 1/6 of the intact antibody molecule, with a single antigen binding site.
  • Fv includes ScFv (single Chain antibody), DsFv (disulfide-stabilized antibody), and the like.
  • ScFv is a combination of VH and VL with a suitable oligonucleotide (linker) to express it as a single peptide chain.
  • DsFv introduces a cysteine into the appropriate part of the light chain variable region and the heavy chain variable region to form a disulfide-fixed Fv segment, which has been proved to have better binding ability and stability than ScFv.
  • the antibody fragment of the present invention may be, for example, Fab, Fab', F(ab')2, scFv, (scFv)2, dAb, complementarity determining region (CDR) fragment, linear antibody, single chain antibody molecule, minibody, diabody Or a multispecific antibody formed from an antibody fragment.
  • Another aspect of the invention is the use of these humanized antibodies or antibody fragments thereof (e.g., antigen-binding fragments) for the preparation of a medicament or composition for preventing and/or treating a disorder associated with excessive or uncontrolled complement activation.
  • They include complement activation during cardiopulmonary bypass surgery; acute myocardial infarction, aneurysm, stroke, hemorrhagic shock, crush injury, multiple organ failure, intestinal ischemia, or other events that cause ischemia-reperfusion injury Complement activation.
  • Complement activation has been shown to be associated with inflammatory conditions such as severe burns, endotoxemia, septic shock, adult respiratory distress syndrome, hemodialysis; anaphylactic shock, severe asthma, angioedema, Crohn's disease , sickle cell anemia, glomerulonephritis and pancreatitis after streptococcal infection.
  • the condition can be the result of an adverse drug reaction, a drug allergy, an IL-2 induced vascular leak syndrome, or a radiographic (comparative) contrast agent allergy. It also includes autoimmune diseases such as systemic lupus erythematosus, myasthenia gravis, rheumatoid arthritis, Alzheimer's disease and multiple sclerosis.
  • complement activation is also associated with ocular diseases (its pathology involves all ocular conditions and diseases of the complement including the classical and alternative complement pathways) such as, for example, but not limited to, macular degeneration diseases such as age-related macular degeneration at all stages (AMD) ) including dry and wet (non-exudative and exudative) forms, diabetic retinopathy and other ischemic-related retinopathy, choroidal neovascularization (CNV), uveitis, diabetic macular edema, pathological myopia, von Hippel-Lindau disease, ocular histoplasmosis, central retinal vein occlusion (CRVO), corneal neovascularization, and retinal neovascularization.
  • macular degeneration diseases such as age-related macular degeneration at all stages (AMD) ) including dry and wet (non-exudative and exudative) forms, diabetic retinopathy and other ischemic-related retinopathy
  • age-related macular degeneration includes non-exudative (such as intermediate dry AMD or geographic atrophy (GA)) and exudative (such as wet AMD (choroidal neovascularization (CNV)).
  • AMD diabetic retinopathy
  • CNV choroidal neovascularization
  • AMD diabetic retinopathy
  • DR endophthalmitis
  • uveitis in addition non-exudative AMD may include the presence of hard glass enamel, soft glass enamel, map-like atrophy, and/or pigment agglomeration.
  • the present invention and the prior art antibodies may have differences in antibody and antigen binding epitopes, and the present invention has higher affinity, stability, and biological activity.
  • Still another object of the present invention is to provide a genetic material comprising a recombinant vector, a recombinant strain, a recombinant cell line, a recombinant virus or an expression cassette containing the gene.
  • the recombinant vector is a prokaryotic expression vector or a eukaryotic expression vector expressing the antibody or Fab or antigen-binding fragment.
  • the recombinant strain is Escherichia coli carrying the gene.
  • the recombinant cell line may be a transgenic cell line or a fusion cell line, wherein the transgenic cell line may be a mammalian cell line, preferably a CHO cell, which is transfected with the anti-complement D humanized antibody or Fab or antigen-binding fragment encoding gene of the present invention. , or 293 cells and sublines thereof; the fusion cell line may be a hybridoma cell that secretes an anti-complement D humanized antibody of the invention.
  • the recombinant virus is a recombinant adenovirus carrying a gene or a recombinant adeno-associated virus or the like.
  • the expression cassette is a DNA molecule, which is followed by three fragments from upstream to downstream: a promoter, the antibody or Fab which is transcribed by the promoter, or the antigen-binding fragment Encoding genes and terminators.
  • a recombinant vector containing the antibody, or the Fab, or the antigen-binding fragment-encoding gene can be transfected or transformed into a host cell to express the corresponding protein to obtain the antibody or Fab, or the antigen-binding fragment.
  • the host cell may be a eukaryotic cell or a prokaryotic cell including, but not limited to, mammalian cells, bacteria, yeast, insect cells, and the like.
  • mammalian cells that can be used for large-scale expression of proteins, such as 293 cells, CHO cells, SP20 cells, NSO cells, COS cells, BHK cells, or PerC6 cells.
  • methods for transfecting cells including but not limited to: electroporation, liposome-mediated methods, calcium-mediated methods, and the like.
  • a preferred expression method of the antibody or Fab, or the antigen-binding fragment is to genetically amplify a recombinant vector in a host cell that has been stably transfected to increase the expression level of the corresponding recombinant protein, for example, After the recombinant vector of dihydrofolate reductase (DHFR) is stably transfected into a host cell lacking DHFR, the concentration of methotrexate (MTX) can be added to the cell culture medium to amplify the number of copies of the recombinant vector in the host cell.
  • DHFR dihydrofolate reductase
  • MTX methotrexate
  • the protein concentration in the culture solution can be determined by enzyme-linked immunosorbent assay (ELISA) or other methods.
  • ELISA enzyme-linked immunosorbent assay
  • purification can be performed by Protein G affinity chromatography; IgG protein can be purified by Protein A affinity chromatography.
  • Still another object of the present invention is to provide a pharmaceutical composition
  • a pharmaceutical composition comprising an adjuvant and an active ingredient, the active ingredient comprising at least one of the following: the antibody, the Fab, the antigen-binding fragment, The gene, and the genetic material; the excipient is a pharmaceutically acceptable carrier or excipient.
  • the active ingredient of the pharmaceutical composition may also be any of the above-described Fabs or antibodies, or any of the above-described antigen-binding fragments, or any of the above-described genes, or any of the above-mentioned genetic materials.
  • any of the following substances in the treatment of a disease associated with complement D is also within the scope of the invention: the antibody, the Fab, the antigen-binding fragment, the gene, the genetic material, and Said pharmaceutical composition.
  • the invention provides a kit comprising an antibody of the invention or a fragment thereof (e.g., an antigen-binding fragment).
  • the invention provides kits comprising an antibody of the invention or a fragment thereof (e.g., an antigen-binding fragment) and instructions for use.
  • the invention provides a kit comprising a first container containing a composition comprising one or more antibodies or antigen-binding fragments thereof of the invention; and a second container containing a buffer.
  • the humanized complement factor D antibody PCR introduction site mutation and the screening of the mutant antibody described in the present invention are completed by conventional gene recombination technology and immunological technology based on antigen-antibody interaction, and specific experimental method steps such as "Molecular Cloning” > Third edition (Joseph Sambrook, Science Press, August 1, 2002) and similar experimental manuals.
  • the hemolytic suppression IC50 involved in the examples was obtained by inputting the measured OD value (OD405) into the graphpad prism5 software.
  • Each sequence consists of a variable region and a constant region sequence, wherein: the synthetic light chain variable region sequence is sequence 63, the heavy chain variable region sequence is sequence 34, and the light chain constant region is sequence 42, heavy chain
  • the constant region is sequence 40.
  • the construction of the humanized Fab expression vector was carried out by PCR site-directed mutagenesis. The specific steps were as follows: the synthetic light heavy chain sequence (plasmid) or the obtained mutant Fab plasmid was used as a template, and the light and heavy chain constructs of each Fab expression vector were respectively contained. Four primers were amplified by three PCRs. First, two complementary inner primers containing the same base mutation were used for PCR amplification with the primers on both sides, and the PCR product was used as a template. The mutated humanized Fab antibody light heavy chain fragment is finally obtained.
  • the primers for light chain amplification are as follows:
  • the primers on both sides are: sequence 64 and sequence 65
  • the intermediate mutation primers are: sequence 66 and sequence 67; sequence 68 and sequence 69; sequence 70 and sequence 71; sequence 72 and sequence 73; sequence 74 and sequence 75; sequence 76 and sequence 77.
  • the primers on both sides are: sequence 78 and sequence 79
  • the intermediate mutation primers are: sequence 80 and sequence 81; sequence 82 and sequence 83; sequence 84 and sequence 85; sequence 86 and sequence 87; sequence 88 and sequence 89; sequence 90 and sequence 91; sequence 92 and sequence 93; Sequence 95; sequence 96 and sequence 97.
  • the light chain and heavy chain fragments obtained above were ligated and inserted into a pTLR vector capable of simultaneously expressing the light and heavy chains of the Fab protein by restriction enzyme ligation (the modified pET22b(+) vector can express Fab in Escherichia coli. In the antibody).
  • the light chain DNA containing the enzyme cleavage sites NdeI and EcoRI at both ends and the heavy chain fragment DNA containing the restriction sites of NotI and XhoI, respectively, are prepared by PCR using the above primers.
  • the DNA was cloned into the corresponding restriction endonuclease sites in the pTLR vector (modified pET22b(+) vector), ie, the light chain DNA was inserted between the restriction endonuclease NdeI and EcoRI sites, respectively.
  • Each heavy chain fragment (Fd) DNA was inserted between the restriction endonuclease NotI and XhoI sites, respectively, to construct several Fab expression vectors.
  • a DNA fragment (sequence 98) is artificially synthesized, which in turn contains a T7 terminator (T7 terminator), a T7 promoter (T7promoter), a lactose operon. (lac operator), ribosome binding site (RBS) sequence, and upper and lower ends containing SalI and NotI restriction sites, pET22b(+) vector and synthetic DNA sequence were digested with Sal I and Not I, respectively.
  • T 4 ligase NEB connected after conversion, a conventional sequencing method of screening monoclonal pick the correct transformation vector.
  • the heavy chain variable region fragment obtained above was subjected to codon optimization (Suzhou Jinweizhi Co., Ltd.), and the heavy chain fragment was spliced together with the constant region by overlapping PCR to obtain a heavy chain sequence.
  • the light chain and heavy chain sequences obtained above were sequentially recombined into the expression plasmid Freedom pCHO1.0 (Life technologies), and the constructed recombinant expression plasmid was transfected into CHO-S cells to express full-length antibodies.
  • the specific operations are as follows: (1) The light chain will amplify the light chain codon optimized template (Suzhou Jinweizhi Co., Ltd.) with primers containing appropriate restriction sites and signal peptide sequences, and the amplification primers are: sequence 99 and sequence 100. .
  • the obtained PCR product was digested with AvrII enzyme (product of NEB) and BstZ17I (product of NEB), and then ligated with the same double-cut Freedom pCHO1.0 large fragment using T4 ligase (product of Takara) to obtain pCHO. -L.
  • the heavy chain needs to be spliced together with the artificially synthesized IgG4 constant region (Suzhou Jinweizhi Co., Ltd.) by overlap extension PCR, and the number of overlapping nucleotides is 40, and then recombined into pCHO-L.
  • a primer containing the appropriate restriction site and signal peptide sequence is used to amplify the heavy chain template, and the inner complementary complementary PCR primers are designed at the variable region and the constant boundary, respectively, and PCR amplification is performed with the primers on both sides, and then the PCR product is obtained.
  • PCR amplification of the primers on both sides finally obtained the spliced humanized heavy chain PCR product.
  • the amplification primers are as follows:
  • the primers on both sides are: sequence 101, sequence 102 and sequence 103
  • the intermediate primers are: sequence 104 and sequence 105.
  • the spliced PCR product was digested with EcoRV enzyme (NEB company product) and PacI enzyme (NEB company product), and then recombined into the same double-digested expression plasmid pCHO-L to obtain pCHO-HL, which was digested and sequenced. The identification is correct.
  • the Fab expression vector constructed above was transformed into E. coli competent cell BL21(DE3) (Tiangen), and ampicillin resistant 2-YT plate (peptone 1.6%, yeast extract 1%, NaCl 0.5%, agar powder 1.5%). ). The next day, the plate with the appropriate colony density was selected and the monoclonal colonies were picked to inoculate 5 ml of ampicillin-resistant 2-YT liquid medium, and cultured overnight, at 37 ° C, 220 rpm, 16 h. The next day, 5 ml of the bacterial solution was inoculated into 500 ml of ampicillin-resistant 2-YT liquid medium, and cultured at 33 ° C, 220 rpm to an OD 600 of 0.6.
  • IPTG was added to the medium to a final concentration of 50 ⁇ M to induce expression of different Fab proteins with an induction time of 3 h.
  • the culture solution after the induction of expression was centrifuged at 4500 rpm, 10 ° C for 15 min, the supernatant was discarded, and 40 ml of pre-cooled TES solution (Tris 24.23 g, sucrose 171.2 g, EDTA 0.186 g, dissolved in 1000 ml of deionized water) was used for precipitation.
  • Post-protein loading continue to wash the column with the balance solution after the end of the sample, and then directly elute with the eluent (0.1M Gly-HCl, pH 2.5), collect the eluted sample, and pre-buffer the Tris buffer before collecting the sample.
  • the liquid to sample volume ratio of 1:9 was added to 1.0M Tris-HCl (pH 9.0) in the sample collection tube, and the pH was quickly adjusted to 7.0.
  • the collected liquid was the target protein. After the protein was analyzed by SDS-PAGE, the purity was The protein sample was subjected to concentration measurement, and finally stored at -80 ° C after dispensing, to obtain a Fab protein with higher purity.
  • the full-length antibody protein expression process comprises the steps of transfecting the above-prepared antibody IgG recombinant expression vector into CHO cells; using Puromycin (invitrogen) and MTX (Sigma) to select an engineered cell line highly expressing anti-complement factor D protein, Expand the culture of the cell line, and finally separate and purify the full length from the cell culture medium.
  • antibody Specifically include:
  • Host cell CHO-S cells were selected and electroporated using Cell line Nucleofector kit V transfection reagent (Lonza) using the expression vector plasmid constructed by linearization described above. After transfection, the 6-well plate was placed in a 37 ° C, 8% CO 2 shaker for 48 h.
  • Two-stage stepwise pressurization screening was performed on the polyclonal cells obtained by transfection.
  • the antibiotic concentration was set to 30P. /500M, 50P/1000M, culture until the survival rate is higher than 90%, after the initial screening to obtain the resistant cell bank, enter the shake flask culture for 14 days batch evaluation, select the target protein with higher expression and better activity.
  • the pool was subjected to monoclonal screening, and 100 to 150 monoclonal clones were obtained, and high-yield positive monoclonal cells were selected to expand the culture. These cell lines are used to produce antibody proteins.
  • the CHO-S engineering cell strain was cultured in a CHO-S CD Medium (Life Technologies) medium at 37 ° C, 8% CO 2 shaker for 13 days, centrifuged at 2000 rpm, and the supernatant was collected, followed by MabSelect SuRe affinity layer.
  • the anti-complement factor D antibody was isolated and purified by a column (GE).
  • the complement bypass pathway activity was measured by the ACH50 method, and rabbit red blood cells (homemade) were washed 3 times in GVB/Mg-EGTA Buffer and resuspended to 6 ⁇ 10 8 /ml.
  • Different concentrations of anti-Factor D antigen-binding Fab or antibody were prepared with GVB/Mg-EGTA Buffer at concentrations of 44.44 nM, 22.22 nM, 11.11 nM, 5.56 nM, 2.78 nM, 1.39 nM, respectively.
  • Example 5 Analysis of thermal stability of different Fab proteins based on real-time PCR
  • the protein dissociates the exposed fluorophore at a certain temperature to excite the fluorescent signal, and continuously collects the change of the fluorescence signal intensity of the protein during continuous heating by a quantitative PCR instrument (MJ Research) to draw a melting curve of the protein, and calculates The Tm value of the protein, the magnitude of which positively reflects the ability of the protein to be thermostable.
  • concentration of the anti-Factor D antigen-binding fragment Fab was 0.4 ⁇ g/uL using 1 ⁇ PBS solution, and the 5-fold diluted SYPRO dye (Cat No. S6650, lift technologies) was added in the dark, and the total reaction volume was 50 ⁇ l. ⁇ PBS solution to make up.
  • the melting curve experiment was run on the real-time quantitative PCR instrument. The melting curve was set to 25 ° C and gradually heated to 95 ° C. The reading was performed every 0.5 ° C. The instrument was equipped with software option monitor 3 for Tm analysis.
  • the differential scanning microcalorimetry can scan the thermal fusion changes during thermal denaturation of proteins with temperature, and finally fit various thermodynamic parameters ( ⁇ , entropy, free energy) including half the denaturation temperature T m . Wait).
  • DSC differential scanning microcalorimetry
  • ⁇ entropy, free energy
  • T m denaturation temperature
  • differential scanning calorimetry typically yields three thermodynamic half-denaturation temperatures for the CH2, CH3, and Fab regions of the antibody.
  • the Fc segment consisting of CH2 and CH3 is relatively fixed, and thus the stability of the Fab region has an important influence on the stability of the antibody.
  • the thermal denaturation process of antibodies KH-A, KH-F, and KH102 between 25-100 °C was scanned at a temperature increase rate of 1 °C/min using a Malvern Microcal Auto VP-Capillary DSC instrument and analyzed with the Origin program attached to the instrument.
  • the data fits thermodynamic parameters such as T m values.
  • the sample was placed in PBS buffer, filtered at pH 7.4, 0.22 ⁇ m filter, and the concentration was 1 mg/mL.
  • Set the DSC automatic cleaning program on the software side clean the syringe with the DECON washing solution, and thoroughly clean the syringe, automatic sample line and sample cell with ultrapure water. The whole process is completed in about 30 minutes.
  • Ni-NTA Biosensor was selected as a biosensor for detection by using the Octet QK protein interaction platform; CFD (monkey CFD or rat CFD) at a suitable concentration (eg, 10 ⁇ g/ml) was used as a detection antigen; at 200 nM, 100 nM, 50nM, 25nM, 12.25nM, 6.25nM, 0nM full-length antibody protein detection antibody and antigen binding, the program is set to: Baseline 120s, Loding 700s, Baseline 120s, Association 700S, Dissociation 1500s.
  • Ni-NTA Biosensor was selected as the biosensor for detection; CFD (monkey CFD or rat CFD) at a concentration of 10 ug/ml was used as the detection antigen; at 500 nM, 250 nM, 125 nM, 62.5 nM, 31.25 nM, 15.625 nM, 7.8125 nM, 0 nM The concentration gradient of the anti-CFD Fab protein was detected to detect the binding of the Fab protein to the antigen.
  • the program settings are: Baseline 120s, Loding 700s, Baseline 120s, Association 700S, Dissociation 1500s. Antibody affinity KD was calculated from the combined curves.
  • Table 5 shows that the Fab or antibody of the present invention has a higher binding to monkey CFD.
  • Table 6 shows that the antibody of the present invention has a certain binding force to rat CFD.
  • the complement bypass pathway activity was measured by the ACH50 method, and rabbit red blood cells (home-made) were washed 3 times in GVB/Mg-EGTA Buffer (4 ° C, 1600 rpm centrifugation), and then diluted with GVB/Mg-EGTA to 1 ⁇ 109 cells/ The cell suspension of ml was placed on ice to cool. Different concentrations of anti-Factor D antigen-binding full-length antibody were prepared by GVB/Mg-EGTA Buffer with concentration gradients of 23mg/ml, 20mg/ml, 15mg/ml, 10mg/ml, 5mg/L, 1mg/ml, respectively.
  • the concentration of the factor D-binding fragment Fab was 1 mg/ml, 50 ⁇ l of the sample and 50 ⁇ l of rat serum (home-made) were mixed, shaken at room temperature for 30 s, and incubated at room temperature for 7 min; then 30 ⁇ l of red blood cell suspension was added, and shaken at 4 ° C for 30 s, 37 Incubate for 40 min at °C; remove and centrifuge (4 ° C, 2000 rpm, 3 min); transfer 80 ul supernatant to 96-well plate and measure OD at 405 nm. Data are expressed as a percentage relative to hemolysis induced when no antibody is added. No activity was detected at 1.5 mg/ml for KH-000.
  • the initial binding of the CFD protein of pigs and guinea pigs obtained by transient transfection was initially evaluated using the OctetQK protein interaction platform.
  • the C-terminus of the pig (GeneID: 396877) and guinea pig (GeneID: 100724693) gene sequences was fused 10 ⁇ . His tag is optimized and synthesized by Nanjing Kingsray Biotechnology Co., Ltd.
  • the synthetically optimized gene was inserted into the expression vector pCHO1.0, and the plasmid was electroporated into host cell DG44 according to Cell line Nucleofector kit V transfection reagent (Lonza).
  • the specific method is as follows: full-length antibody KH-A, KH-F kinetic analysis selects Protein A as a biosensor for detection; firstly binds the antibody to the protein A biosensor at a suitable concentration (eg, 10 ⁇ g/ml), and tests The transient transfection supernatant of pig and guinea pig CFD was combined with the antibody.
  • the program was set to: Baseline 120s, Loding 700s, Baseline 120s, Association 700S, Dissociation 1500s.
  • Negative control was set to CHO-negative cell culture without untransfected plasmid liquid.
  • the kinetic analysis of Fab antibody KH027 and KH021 selected Ni-NTA as the biosensor for detection; firstly, the transfection supernatant was separately bound to the Ni-NTA biosensor, and then the transient transfer of different species was tested using 10 ⁇ g/ml Fab antibody.
  • the combination of the supernatant and the antibody was set to: Baseline 120s, Loding 700s, Baseline 120s, Association 700S, Dissociation 1500s.
  • the activity of the antibody is affected according to the environment of the different buffer system.
  • the activity of the antibody in the human vitreous humor is simulated by taking the rabbit vitreous humor instead of the human vitreous humor, and the activity of the antibody is initially evaluated.
  • Ni-NTA Biosensor was selected as the biosensor for detection by using the OctetQK protein interaction platform; 10 ⁇ g/ml human CFD protein was used as the detection antigen; and 125 nM, 0 nM was used as the anti-CFD full-length antibody KH-A.
  • the concentration, using vitreous humor as a detection matrix, was tested against the kinetics of CFD full-length antibodies in vitreous humor.
  • the program is set to: Baseline 120s, Loding 700s, Baseline 120s, Association 700S, Dissociation1500s.
  • the initial activity of the antibody was determined based on the kinetic binding curve.
  • Table 11 shows that the antibody KH-A has good binding activity to human CFD in rabbit vitreous humor.

Abstract

本发明公开了一种抗补体因子D的人源化Fab和人源化抗体及其用途，该人源化Fab和人源化抗体含有轻链可变区和重链可变区。

Description

抗补体因子D的人源化Fab和人源化抗体及其用途 技术领域
本发明涉及生物技术领域，具体涉及一种抗补体因子D的人源化Fab和人源化抗体及其用途。
背景技术
老年性黄斑变性又称年龄相关性黄斑变性(AMD)，是一种由视网膜中心区的细胞或组织退化和血管增生而引起的疾病，可分为干性AMD和湿性AMD两种。老年性黄斑变性主要表现为视网膜色素上皮细胞对视细胞外节盘膜吞噬消化能力下降，结果使未被完全消化的盘膜残余小体潴留于基底部细胞原浆中，并向细胞外排出，沉积于Bruch膜，形成玻璃膜疣。由于黄斑部结构与功能上的特殊性，此种改变更为明显。玻璃膜疣也见于正常视力的老年人，但由此继发的种种病理改变后，则导致黄斑部变性发生。或者引起Bruch膜本断裂，脉络膜毛细血管通过破裂的Bruch膜进入RPE下及视网膜神经上皮下，形成脉络膜新生血管。由于新生血管壁的结构异常，导致血管的渗漏和出血，进而引发一系列的继发性病理改变。老年性黄斑变性大多发生于45岁以上，其患病率随年龄增长而增高，是当前老年人致盲的重要疾病。AMD致盲约占全球致盲的8.7％，全球约有3000万AMD患者，每年约有50万人因为AMD而致盲。美国2004年报告的AMD的患病率为1.47％，约有175万AMD患者，而到2020年，推测将会有高达300万的AMD患者。我国邹海东等作的一项调查显示，大于50岁以上的人群的AMD的患病率为15.5％。其中湿性AMD占11.9％。按照联合国的人口数据资料，我国在2005年大于50岁以上的人口数为接近3亿。随着人口老龄化，AMD的患病率将显著增加。
补体是存在于正常人和动物血清与组织液中的一组经活化后具有酶活性的蛋白质。其作用可辅助和补充特异性抗体，介导免疫溶菌、溶血作用。补体系统是由30余种可溶性蛋白、膜结合性蛋白和补体受体组成的多分子系统，根据其生物学功能，可分为补体固有成分、补体调控成分和补体受体(CR)三类，补体系统的调控主要有三个途径：经典途径(Classical pathway)、旁路途径(Alternative pathway)和凝集素(MBL)途径(Lectin pathway)，不同的调控途径会影响产生不同的补体蛋白，并发挥不同的作用。补体系统非正常激活常常会引起人体免疫系统的过度反应，造成人体自身正常组织的损伤。补体系统可以导致局部的炎症，组织损伤以及血管生成因子的上调，与补体过度激活相关的疾病有类风湿性关节炎、老年性痴呆、系统性红斑狼疮、缺血性再灌注、急性心肌梗死、急性呼吸窘迫综合征等。近期研究表明，旁路途径(Alternative pathway)和凝集素(MBL)途径(Lectin pathway)是抗体介导反应的独立激活机制，是AMD病因中最主要的致病机制[Paul A.et,al.The Immune System and AMD,Curr Ophthalmol Rep(2014)2:14–19]，其中补体因子D(complement factor D，EC:3.4.21.46)是启动替代途径激活的重要成分，属于胰蛋白酶家族。补体因子D在血清中主要以活化形式而存在，具有活性的D因子(D)作用于C3bB，可能在第234-235位的精氨酸-赖氨酸键处将B因子裂解为Ba和Bb两个片段，从而启动替代 途径的级联活化反应。
现有技术中抗补体D的抗体存在着种属特异性，抗补体因子抗体(AFD；FCFD4514S；Lampalizumab)针对狗或羊的补体旁路途径(alternative complement pathway，简称AP)有微弱的抑制，而针对兔子、大鼠、小鼠、豚鼠、仓鼠或猪的AP没有抑制，而对于人或猴具有相应较强的抑制(Kelly M,et,al.Complement Inhibition in Cynomolgus Monkeys by Anti-Factor D Antigen-Binding Fragment for the Treatment of an Advanced Form of Dry Releated Macular Degeneration,J Pharmacol Exp Ther 351:527-537,December 2014)。
发明内容
本发明的目的之一是提供抗人补体因子D的人源化抗原结合片段Fab或抗人补体因子D人源化抗体，该Fab或抗体与人补体因子D有较好的亲和力、具有更好的稳定性和生物活性。
本发明一方面提供一种抗补体因子D的人源化抗体或其抗原结合片段Fab，含有轻链可变区和重链可变区，其中轻链可变区含有氨基酸序列ITSTDIDDDX1N、氨基酸序列GGX2TLRP和氨基酸序列X3QSDS X4PYX5,所述X1为V、M或L，X2为N或A，X3为M或L，X4为L或Y,X5为I或T；重链可变区含有氨基酸序列1、序列2和氨基酸序列Y1GGY2NY3，所述Y1为A或E，Y2为S、V或P，Y3为N、Y、F、H、S或R；X1、X2、X3、X4、X5不同时满足下述条件：X1为M、X2为N、X3为L、X4为L、X5为T。
所述的人源化抗体或其抗原结合片段Fab，优选所述X1为M、X2为A、X3为L、X3为L和X5为T时，Y1不为A、Y2不为V和Y3不为N或Y1不为E，Y2不为P和Y3不为N。X1、X2、X3、X4、Y1、Y2和Y3同时满足上述条件。
所述人源化抗体或其抗原结合片段Fab，优选所述X1为V、X2为N、X3为L、X4为L和X5为I时，Y1不为E、Y2不为V和Y3不为S。X1、X2、X3、X4、X5、Y1、Y2和Y3同时满足上述条件。
所述人源化抗体或其抗原结合片段Fab，优选所述X1为V、X2为N、X3为L、X4为Y和X5为T时，Y1不为E、Y2不为V和Y3不为N或H，或Y1不为E、Y2不为P和Y3不为N。X1、X2、X3、X4、X5、Y1、Y2和Y3同时满足上述条件。
所述人源化抗体或其抗原结合片段Fab，优选所述X1为V、X2为N、X3为M、X4为L和X5为T时，Y1不为E、Y2不为P和Y3不为N。X1、X2、X3、X4、X5、Y1、Y2和Y3同时满足上述条件。
所述人源化抗体或其抗原结合片段Fab，优选所述X1为V、X2为A、X3为L、X4为L和X5为T时，Y1不为E、Y2不为V和Y3不为N。X1、X2、X3、X4、X5、Y1、Y2和Y3同时满足上述条件。
所述人源化抗体或其抗原结合片段Fab，优选所述X1为L、X2为A、X3为L、X4为L和X5为T时，Y1不为E、Y2不为V和Y3不为N。X1、X2、X3、X4、X5、Y1、Y2和Y3同时满足上述条件。
所述抗补体因子D的人源化抗体或其抗原结合片段Fab，包含如下重链可变区和轻链可变区序列：所述轻链可变区的氨基酸序列为序列表中序列3、5、8、13、15、17中的任一种；所述重链可变区的氨基酸序列为序列表中序列19、21、23、25、27、29、31、33中的任一种。
所述的抗补体因子D的人源化抗体或其抗原结合片段Fab，其特征在于，所述抗原结合片段Fab选自下述任一种：
KH001：所述轻链可变区的氨基酸序列为序列表中序列3，所述重链可变区的氨基酸序列为序列表中序列33；
KH002：所述轻链可变区的氨基酸序列为序列表中序列8，所述重链可变区的氨基酸序列为序列表中序列33；
KH003：所述轻链可变区的氨基酸序列为序列表中序列3，所述重链可变区的氨基酸序列为序列表中序列21；
KH004：所述轻链可变区的氨基酸序列为序列表中序列3，所述重链可变区的氨基酸序列为序列表中序列31；
KH005：所述轻链可变区的氨基酸序列为序列表中序列3，所述重链可变区的氨基酸序列为序列表中序列19；
KH006：所述轻链可变区的氨基酸序列为序列表中序列3，所述重链可变区的氨基酸序列为序列表中序列23；
KH007：所述轻链可变区的氨基酸序列为序列表中序列3，所述重链可变区的氨基酸序列为序列表中序列25；
KH008：所述轻链可变区的氨基酸序列为序列表中序列3，所述重链可变区的氨基酸序列为序列表中序列27；
KH009：所述轻链可变区的氨基酸序列为序列表中序列3，所述重链可变区的氨基酸序列为序列表中序列29；
KH010：所述轻链可变区的氨基酸序列为序列表中序列13，所述重链可变区的氨基酸序列为序列表中序列33；
KH011：所述轻链可变区的氨基酸序列为序列表中序列15，所述重链可变区的氨基酸序列为序列表中序列33；
KH012：所述轻链可变区的氨基酸序列为序列表中序列13，所述重链可变区的氨基酸序列为序列表中序列21；
KH013：所述轻链可变区的氨基酸序列为序列表中序列13，所述重链可变区的氨基酸序列为序列表中序列31；
KH014：所述轻链可变区的氨基酸序列为序列表中序列13，所述重链可变区的氨基酸序列为序列表中序列23；
KH015：所述轻链可变区的氨基酸序列为序列表中序列13，所述重链可变区的氨基酸序列为序列表中序列25；
KH016(XY1252)：所述轻链可变区的氨基酸序列为序列表中序列13，所述重链可变区的氨基酸序列为序列表中序列29；
KH017：所述轻链可变区的氨基酸序列为序列表中序列15，所述重链可变区的氨基酸序列为序列表中序列31；
KH018：所述轻链可变区的氨基酸序列为序列表中序列15，所述重链可变区的氨基酸序列为序列表中序列19；
KH019：所述轻链可变区的氨基酸序列为序列表中序列15，所述重链可变区的氨基酸序列为序列表中序列23；
KH020：所述轻链可变区的氨基酸序列为序列表中序列15，所述重链可变区的氨基酸序列为序列表中序列25；
KH021：所述轻链可变区的氨基酸序列为序列表中序列15，所述重链可变区的氨基酸序列为序列表中序列27；
KH022：所述轻链可变区的氨基酸序列为序列表中序列15，所述重链可变区的氨基酸序列为序列表中序列29；
KH024：所述轻链可变区的氨基酸序列为序列表中序列17，所述重链可变区的氨基酸序列为序列表中序列19；
KH027：所述轻链可变区的氨基酸序列为序列表中序列5，所述重链可变区的氨基酸序列为序列表中序列31；
KH028：所述轻链可变区的氨基酸序列为序列表中序列5，所述重链可变区的氨基酸序列为序列表中序列25。
所述Fab的Fd片段的CH1可以是任意类型(IgG、IgA、IgM、IgE、IgD)或亚类(IgG1、IgG2、IgG3、IgG4，IgM1、IgM2、IgA1、IgA2)的人源恒定区CH1；所述轻链恒定区可为任意类型(κ型或λ型)、亚型(λ1、λ2、λ3、λ4)、或同种异性(κm(1)、κm(2)、κm(3))轻链的人源恒定区。
所述Fab由重链片段和轻链组成，所述重链片段由重链可变区和重链恒定区组成，所述轻链由轻链可变区和轻链恒定区组成。其中所述重链恒定区的氨基酸序列优选与人抗体重链恒定区的氨基酸序列相同，所述轻链恒定区的氨基酸序列优选与人抗体轻链恒定区氨基酸序列相同；其中所述重链恒定区的氨基酸序列为序列39；所述轻链恒定区的氨基酸序列为序列41。
所述抗补体因子D的人源化抗体中，所述重链恒定区可以是任意类型(IgG、IgA、IgM、IgE、IgD)或亚类(IgG1、IgG2、IgG3、IgG4，IgM1、IgM2、IgA1、IgA2)的人源恒定区；所述轻链恒定区可为任意类型(κ型或λ型)、亚型(λ1、λ2、λ3、λ4)、或同种异性(κm(1)、κm(2)、κm(3))轻链的人源恒定区。
所述抗体由重链或轻链组成，所述重链恒定区的氨基酸序列与人抗体重链恒定区的氨基酸序列相同，所述轻链恒定区的氨基酸序列与人抗体轻链恒定区氨基酸序列相同。所述人抗体重链恒定区的氨基酸序列优选为序列43，所述人抗体轻链恒定区氨基酸序列优选 为序列41。
本发明更进一步提供选自下述任一种的抗体：
KH-A：其轻链序列为序列45，其重链序列为序列47；
KH-B：其轻链序列为序列49，其重链序列为序列51；
KH-C：其轻链序列为序列45，其重链序列为序列53；
KH-D：其轻链序列为序列49，其重链序列为序列55；
KH-E：其轻链序列为序列57，其重链序列为序列59；
KH-F：其轻链序列为序列57，其重链序列为序列61。
本发明还提供一种多核苷酸，其编码上述任一所述人源化抗体或抗原结合片段Fab。
上述多核苷酸，优选所述人源化抗体或抗原结合片段Fab中所述抗体的轻链可变区的编码序列为序列表中序列4、序列6、序列9、序列14、序列16和序列18中的任一种，重链可变区的编码序列为序列表中序列20、序列22、序列24、序列26、序列28、序列30、序列32、序列34中的任一种。
上述多核苷酸中优选所述Fab的重链恒定区的编码序列如序列表中序列40所示；所述Fab的轻链恒定区编码序列如序列表中序列42所示；所述抗体的重链恒定区的编码序列如序列表中序列44所示；所述抗体的轻链恒定区的编码序列如序列表中序列42所示。
本发明跟进一步提供选自下述任一种多核苷酸：
KH-A：其轻链序列为序列46，其重链序列为序列48；
KH-B：其轻链序列为序列50，其重链序列为序列52；
KH-C：其轻链序列为序列46，其重链序列为序列54；
KH-D：其轻链序列为序列50，其重链序列为序列56；
KH-E：其轻链序列为序列58，其重链序列为序列60；
KH-F：其轻链序列为序列58，其重链序列为序列62。
本发明还进一步提供上述人源化抗体或抗原结合片段Fab，或上述多核苷酸的用途，其选自以下任一种用途：
d1)在制备预防和/或治疗人补体因子D相关疾病药物中的用途，或
d2)在制备中和人补体因子D产品中的用途，或
d3)在制备定性或定量检测人补体因子D的试剂盒中的用途。
本发明涉及人补体因子D(CFD)相关疾病为由人补体因子D升高引起的疾病；优选类风湿性关节炎、心肌梗死，再灌注损伤或眼病；其中眼病更优选为年龄相关性黄斑变性或糖尿病性视网膜病。其中老年性黄斑变性(AMD)包括非渗出性(例如中期干性AMD或地图样萎缩(GA))和渗出性(例如湿性AMD(脉络膜新血管形成(CNV))AMD、糖尿病视网膜病(DR)、眼内炎(endophthalmitis)和葡萄膜炎。在一个例子中，所述补体相关眼状况是中期干性AMD。
本发明还更进一步提供一种药物组合物，含有辅料和活性成分，所述活性成分含有下述物质中的至少一种：上述任一所述人源化抗体或抗原结合片段Fab，或上述任一所述多 核苷酸；所述辅料为药学上可接受的载体或赋形剂。
本发明还进一步提供所述抗补体因子D的人源化Fab或抗体的生产方法，具体步骤为：将含所述抗体编码基因的重组载体转染或转化宿主细胞，在适宜条件下进行表达、纯化。其中，所述宿主细胞为真核细胞或原核细胞，优选为哺乳动物细胞、细菌、酵母、昆虫细胞等。其中所述哺乳动物细胞包括但不限于293细胞、CHO细胞、SP20细胞、NS0细胞、COS细胞、BHK细胞或PerC6细胞等，其中所述转染细胞的方法包括但不限于：电穿孔，脂质体介导法，钙介导法等。
一种较佳的所述抗体表达方式是在已经稳定转染的宿主细胞中对重组载体进行基因扩增，以提高相应重组蛋白的表达量，例如用含有二氢叶酸还原酶(DHFR)的重组载体稳定转染缺乏DHFR的宿主细胞后，可在细胞培养液中添加氨甲蝶呤(MTX)的浓度以扩增重组载体在宿主细胞中的拷贝数量。
含有所述编码基因序列的抗体表达后，可用酶联免疫吸附法(ELISA)或其他方法测定培养液中的蛋白浓度。对于Fab片段，可用Protein G亲和层析法提纯；IgG蛋白可用Protein A亲和层析法提纯。
相关序列对应如下：
KH023：所述轻链可变区的氨基酸序列为序列表中序列17，所述重链可变区的氨基酸序列为序列表中序列21；
KH025：所述轻链可变区的氨基酸序列为序列表中序列17，所述重链可变区的氨基酸序列为序列表中序列25；
KH026：所述轻链可变区的氨基酸序列为序列表中序列5，所述重链可变区的氨基酸序列为序列表中序列21；
KH029：所述轻链可变区的氨基酸序列为序列表中序列5，所述重链可变区的氨基酸序列为序列表中序列19；
KH030：所述轻链可变区的氨基酸序列为序列表中序列5，所述重链可变区的氨基酸序列为序列表中序列33；
KH031：所述轻链可变区的氨基酸序列为序列表中序列7，所述重链可变区的氨基酸序列为序列表中序列33；
KH032：所述轻链可变区的氨基酸序列为序列表中序列10，所述重链可变区的氨基酸序列为序列表中序列33；
KH033：所述轻链可变区的氨基酸序列为序列表中序列11，所述重链可变区的氨基酸序列为序列表中序列35；
KH034：所述轻链可变区的氨基酸序列为序列表中序列11，所述重链可变区的氨基酸序列为序列表中序列36；
KH035：所述轻链可变区的氨基酸序列为序列表中序列12，所述重链可变区的氨基酸序列为序列表中序列35；
KH036：所述轻链可变区的氨基酸序列为序列表中序列17，所述重链可变区的氨基 酸序列为序列表中序列33；
KH037：所述轻链可变区的氨基酸序列为序列表中序列13，所述重链可变区的氨基酸序列为序列表中序列19；
KH038：所述轻链可变区的氨基酸序列为序列表中序列13，所述重链可变区的氨基酸序列为序列表中序列27；
KH039：所述轻链可变区的氨基酸序列为序列表中序列15，所述重链可变区的氨基酸序列为序列表中序列21；
KH040：所述轻链可变区的氨基酸序列为序列表中序列17，所述重链可变区的氨基酸序列为序列表中序列31；
KH000：所述轻链可变区的氨基酸序列为序列表中序列37，所述重链可变区的氨基酸序列为序列表中序列38。
KH111：所述轻链可变区氨基酸序列为序列11，所述重链可变区氨基酸序列为序列33。
部分轻链可变区的氨基酸和核苷酸的对应关系：编码序列3的核苷酸序列为序列4；编码序列5的核苷酸序列为序列6；编码序列8的核苷酸序列为序列9；编码序列13的核苷酸序列为序列14；编码序列15的核苷酸序列为序列16；编码序列17的核苷酸序列为序列18。
部分重链可变区的氨基酸和核苷酸的对应关系：编码序列11的核苷酸序列为序列63；编码序列19的核苷酸序列为序列20；编码序列21的核苷酸序列为序列22；编码序列23的核苷酸序列为序列24；编码序列25的核苷酸序列为序列26；编码序列27的核苷酸序列为序列28；编码序列29的核苷酸序列为序列30；编码序列31的核苷酸序列为序列32；编码序列33的核苷酸序列为序列34；编码序列29的核苷酸序列为序列30。
部分重链恒定区的氨基酸和核苷酸的对应关系：编码序列39的核苷酸序列为序列40；编码序列43的核苷酸序列为序列44。
部分轻链恒定区的氨基酸和核苷酸的对应关系：编码序列41的核苷酸序列为序列42。
KH102轻链的氨基酸序列为序列106，编码序列106的核苷酸序列为序列107；其重链氨基酸序列为序列108，编码序列108的核苷酸序列为序列109。
本发明还提供由所述Fab衍生的抗原结合片段A或抗原结合片段B。抗原结合片段A为由所述Fab衍生的Fab’、F(ab’)2、Fv、重链可变区、轻链可变区、选自重链可变区的多肽片段或选自轻链可变区的多肽片段；抗原结合片段B为由所述抗体衍生的Fab、Fab’、F(ab’)2、Fv(抗体可变区片段)、重链可变区、轻链可变区、选自重链可变区的多肽片段或选自轻链可变区的多肽片段。
上述F(ab′)2由一对轻链和一对略大于Fd的重链(称为Fd′)组成，胃蛋白酶水解IgG分子产生此片段，它包含两个Fab，可结合2个抗原表位。Fd′约含有235个氨基酸残基，包括VH、CH1和绞链区。Fv由轻链可变区(VL)和重链可变区(VH)组成，二者以非共价键结合在一起，分子量约为完整抗体分子的1/6，具有单一抗原结合位点。Fv包括ScFv(单 链抗体)、DsFv(二硫键稳定抗体)等。ScFv是将VH和VL用一段适当的寡聚核苷酸(接头，linker)连起来，使之表达为单一的肽链。DsFv是在轻链可变区和重链可变区适当部位各引入一个半胱氨酸，形成一个二硫键固定的Fv段，经证实其结合能力及稳定性均优于ScFv。
本发明的抗体片段可以是例如Fab、Fab’、F(ab’)2、scFv、(scFv)2、dAb、互补决定区(CDR)片段、线性抗体、单链抗体分子、微型抗体、双抗体、或自抗体片段形成的多特异性抗体。
本发明的另一方面是这些人源化抗体或其抗体片段(例如抗原结合片段)制备用于预防和/或治疗与过度的或不受控制的补体活化有关的病症的药物或组合物的用途。它们包括心肺旁路手术期间的补体活化；急性心肌梗死、动脉瘤、中风、出血性休克、挤压伤、多器官功能衰竭、肠缺血或其它引起缺血的事件后缺血-再灌注损伤所致补体活化。补体活化已经显示出与炎性状况有关，诸如重度烧伤、内毒素血症、感染性休克、成人呼吸窘迫综合征、血液透析；过敏性休克、严重的哮喘、血管性水肿、克罗恩氏病、镰状细胞贫血、链球菌感染后肾小球肾炎和胰腺炎。所述病症可以是不良药物反应、药物变态反应、IL-2诱导的血管渗漏综合征或放射摄影(对比)造影剂变态反应的结果。它还包括自身免疫病，诸如系统性红斑狼疮、重症肌无力、类风湿性关节炎、阿尔茨海默氏病和多发性硬化。更重要的是补体活化还与眼疾病有关(其病理涉及补体包括经典和旁路补体途径的所有眼状况和疾病)，诸如例如但不限于黄斑变性性疾病诸如所有阶段的老年性黄斑变性(AMD)包括干性和湿性(非渗出性和渗出性)形式、糖尿病视网膜病和其它缺血相关视网膜病、脉络膜新血管形成(CNV)、葡萄膜炎、糖尿病黄斑水肿、病理性近视、von Hippel-Lindau病、眼的组织胞浆菌病、视网膜中央静脉阻塞(CRVO)、角膜新血管形成、和视网膜新血管形成。其中老年性黄斑变性(AMD)包括非渗出性(例如中期(intermediate)干性AMD或地图样萎缩(geographic atrophy，GA))和渗出性(例如湿性AMD(脉络膜新血管形成(CNV))AMD、糖尿病视网膜病(DR)、眼内炎和葡萄膜炎，另外非渗出性AMD可包括硬玻璃疣、软玻璃疣、地图样萎缩和/或色素结块的存在。
本发明与现有技术的抗体可能存在抗体和抗原结合表位上的差异，本发明具有较高亲和力、稳定性、以及生物活性。
本发明的又一个目的是提供下述遗传材料：含有所述基因的重组载体、重组菌、重组细胞系、重组病毒或表达盒。
所述重组载体为表达所述抗体或Fab或抗原结合片段的原核表达载体或真核表达载体。所述重组菌为携带有所述基因的大肠杆菌。所述重组细胞系可为转基因细胞系或融合细胞系，其中转基因细胞系可为转入本发明的抗补体D人源化抗体或Fab或抗原结合片段编码基因的哺乳动物细胞系，优选CHO细胞系，或293细胞及其亚系；融合细胞系可为分泌本发明的抗补体D人源化抗体的杂交瘤细胞。所述重组病毒为携带有所述基因的重组腺病毒或重组腺相关病毒等。所述表达盒是一个DNA分子，从上游至下游依次为如下三个片段：启动子、由所述启动子启动转录的所述抗体或Fab，或所述抗原结合片段的 编码基因和终止子。
将含所述抗体，或所述Fab，或所述抗原结合片段编码基因的重组载体转染或转化宿主细胞，即可表达相应的蛋白，获得所述抗体或Fab，或所述抗原结合片段。所述宿主细胞可以是真核细胞，也可以是原核细胞，它们包含但不限于哺乳动物细胞、细菌、酵母、昆虫细胞等。可用于蛋白质大规模表达的哺乳动物细胞有多种，如293细胞、CHO细胞、SP20细胞、NS0细胞、COS细胞、BHK细胞或PerC6细胞等。转染细胞的方法有多种，其中包括但不限于：电穿孔，脂质体介导法，钙介导法等。
一种较佳的所述抗体或Fab，或所述抗原结合片段的表达方式是在已经稳定转染的宿主细胞中对重组载体进行基因扩增，以提高相应重组蛋白的表达量，例如用含有二氢叶酸还原酶(DHFR)的重组载体稳定转染缺乏DHFR的宿主细胞后，可在细胞培养液中添加氨甲喋啶(MTX)的浓度以扩增重组载体在宿主细胞中的拷贝数量。
含有所述编码基因序列组合的Fab或IgG表达后，可用酶联免疫吸附法(ELISA)或其他方法测定培养液中的蛋白浓度。对于Fab片段，可用Protein G亲和层析法提纯；IgG蛋白可用Protein A亲和层析法提纯。
本发明的又一个目的是提供药物组合物，该药物组合物含有辅料和活性成分，所述活性成分含有下述物质中的至少一种：所述抗体，所述Fab，所述抗原结合片段，所述基因，和所述遗传材料；所述辅料为药学上可接受的载体或赋形剂。该药物组合物的活性成分也可只为上述任一Fab或抗体，或上述任一所述抗原结合片段，或上述任一所述基因，或上述遗传材料中的任一种。
下述物质中的任一种在治疗补体D相关疾病中的应用也属于本发明的保护范围：所述抗体，所述Fab，所述抗原结合片段，所述基因，所述遗传材料，和所述药物组合物。
另一方面，本发明提供试剂盒，其包含本发明的抗体或其片段(例如抗原结合片段)。本发明提供试剂盒，其包含本发明的抗体或其片段(例如抗原结合片段)和使用指令。本发明提供试剂盒包含装有组合物的第一容器，该组合物包含本发明的一种或多种抗体或其抗原结合片段；和装有缓冲剂的第二容器。
具体实施方式
下述实施例中所用的材料、试剂等，如无特殊说明，均可从商业途径得到。
本发明描述的人源化补体因子D抗体PCR引入位点突变及突变抗体的筛选是通过常规基因重组技术及基于抗原抗体相互作用的免疫学技术所完成，具体实验方法步骤如<<分子克隆>>第三版(Joseph Sambrook，科学出版社，2002年8月1日)及类似实验手册所记载。实施例中涉及到的溶血抑制IC50是将测得的OD值(OD405)输入graphpad prism5软件获得。
下面结合具体实施例，进一步阐述本发明。应理解，这些实施例仅用于说明本发明而不用于限制本发明的范围。
实施例1、抗原结合片段Fab表达
(一)轻链、重链的获得
通过人工基因合成的方法，合成人源化的抗体Fab轻链和重链序列(苏州金唯智生物有限公司)。每条序列均由可变区和恒定区序列组成，其中：合成的轻链可变区序列为序列63，重链可变区序列为序列34，轻链的恒定区为序列42，重链的恒定区为序列40。
(二)人源化Fab载体的构建
使用PCR定点突变的方法进行人源化Fab表达载体的构建，具体步骤为：以合成的轻重链序列(质粒)或者已获得的突变Fab质粒为模板，每个Fab表达载体的轻重链构建分别含有4条引物，分3次PCR进行扩增，首先应用两个互补的含有相同碱基突变的内侧引物分别同两侧引物进行PCR扩增，再以此PCR产物为模板，两侧引物进行PCR扩增最终获得突变的人源化Fab抗体轻重链片段。
轻链扩增的引物如下：
两侧引物分别为：序列64和序列65
中间突变引物为：序列66与序列67；序列68与序列69；序列70与序列71；序列72与序列73；序列74与序列75；序列76与序列77。
重链扩增的引物如下：
两侧引物分别为：序列78和序列79
中间突变引物为：序列80与序列81；序列82与序列83；序列84与序列85；序列86与序列87；序列88与序列89；序列90与序列91；序列92与序列93；序列94与序列95；序列96与序列97。
将上述获得的轻链和重链片段通过酶切连接的方法分别连接插入到可同时表达Fab蛋白轻链和重链的pTLR载体(经过改造的pET22b(+)载体，可在大肠杆菌中表达Fab抗体)中。具体操作为，使用上述引物通过PCR的方法制备两端分别含有酶切位点NdeI和EcoRI的轻链DNA，以及两端分别含有酶切位点NotI和XhoI的各重链片段DNA，将这两个DNA分别克隆入pTLR载体(经过改造的pET22b(+)载体)中的相应限制性内切酶位点之间，即轻链DNA分别插入到限制性内切酶NdeI和EcoRI位点之间，各重链片段(Fd)DNA分别插入到限制性内切酶NotI和XhoI位点之间，构建数个Fab表达载体。
对pET22b(+)载体进行改造获得上述pTLR载体的具体方法如下：首先通过人工合成一段DNA片段(序列98)，该片段依次含有T7终止子(T7terminator)，T7启动子(T7promoter)、乳糖操纵子(lac operator)、核糖体结合位点(RBS)序列,以及上下两端包含有SalI和NotI酶切位点，pET22b(+)载体和合成的DNA序列分别用Sal I和Not I双酶切后，T4连接酶(NEB)连接后转化，常规方法挑单克隆测序筛选出正确改造的载体。
(三)抗体IgG重组表达载体构建
将上述所获得的重链可变区片段经过密码子优化后(苏州金唯智有限公司)，以重叠PCR将重链片段与恒定区拼接在一起，获得重链序列。将上述获得的轻链和重链序列分别依次重组至表达质粒Freedom pCHO1.0(Life technologies)中，将构建好的重组表达质粒转染CHO-S细胞，表达全长抗体。
具体操作如下所述：(1)轻链将含有适当酶切位点和信号肽序列的引物扩增轻链密码子优化模板(苏州金唯智有限公司)，扩增引物为：序列99和序列100。获得的PCR产物以AvrII酶(NEB公司产品)和BstZ17I酶(NEB公司产品)双酶切后，使用T4连接酶(Takara公司产品)与同样双酶切的Freedom pCHO1.0大片段相连接获得pCHO-L。2)重链需以重叠延伸PCR将可变区与人工合成的IgG4恒定区(苏州金唯智有限公司)拼接在一起，重叠的核苷酸数量为40个，再重组至pCHO-L。含有适当酶切位点和信号肽序列的引物扩增重链模板，在可变区与恒定区分界处设计内侧互补的重叠PCR引物，分别同两侧引物进行PCR扩增，再以此PCR产物为模板，两侧引物进行PCR扩增最终获得拼接的人源化重链PCR产物。扩增引物如下：
两侧引物为：序列101，序列102和序列103
中间引物为：序列104和序列105。
将拼接的PCR产物以EcoRV酶(NEB公司产品)和PacI酶(NEB公司产品)酶切后再重组至同样双酶切的表达质粒pCHO-L中获得pCHO-HL，并经鉴酶切与测序鉴定正确。
实施例2 Fab周质蛋白的表达
将上述构建的Fab表达载体分别转化大肠杆菌感受态细胞BL21(DE3)(Tiangen)，涂氨苄青霉素抗性2-YT平板(蛋白胨1.6％，酵母提取物1％，NaCl 0.5％，琼脂粉1.5％)。第二天选择合适菌落密度的板挑取单克隆菌落接种5ml氨苄青霉素抗性2-YT液体培养基,过夜培养，条件为：37℃，220rpm，16h。次日，将5ml菌液接种到500ml氨苄青霉素抗性2-YT液体培养基，33℃，220rmp培养至OD600为0.6。在培养基中加入IPTG至终浓度为50μM，诱导不同Fab蛋白表达，诱导时间为3h。将诱导表达结束后的培养液在4500rpm，10℃条件下，离心15min，弃上清，沉淀用40ml预冷的TES溶液(Tris24.23g，蔗糖171.2g，EDTA0.186g，溶于1000ml去离子水，调整pH值到8.0)充分重悬菌体沉淀；重悬后再加入66ml预冷的1/5TES溶液到重悬后的菌液中，冰上孵育40min；冰浴结束后，13000rpm，4℃，离心10min；离心后收集上清，上清即为含有Fab蛋白的细胞周质提取液。周质提取液过G-25(GE公司，17-0034-01)柱脱盐处理后，准备Protein G(GE公司17-0618-04)预装柱，平衡液(20mM磷酸缓冲液pH 6.5)平衡后蛋白上样，上样结束后继续用平衡液清洗柱子，然后用洗脱液(0.1M Gly-HCl，pH2.5)直接洗脱，收集洗脱样品，并预先在收集样品前按照Tris缓冲液与样品体积比1：9加入1.0M Tris-HCl(PH9.0)于样品收集管里，快速调PH7.0，收集到的液体即为目的蛋白，蛋白经过SDS-PAGE分析纯度后，对蛋白样品进行浓度测定，最后分装后保存于-80℃，得到纯度较高的Fab蛋白。
实施例3 不同全长抗体的表达
全长抗体蛋白表达过程包括以下步骤：将上述构建的抗体IgG重组表达载体转染CHO细胞；利用Puromycin(invitrogen公司)和MTX(Sigma公司)联合筛选高表达抗补体因子D蛋白的工程细胞株，扩大培养该细胞株，最后从细胞培养液中分离纯化全长 抗体。具体包括：
(1)表达载体的转染
选择宿主细胞CHO-S细胞，使用线性化上述构建的表达载体质粒按Cell line Nucleofector kit V转染试剂(Lonza公司)进行电转染。转染后6孔板置于37℃、8％CO2摇床培养48h。
(2)CHO-S工程细胞株的建立
对转染所得的多克隆细胞进行两个阶段的逐步加压初筛。第一阶段两个pool梯度设置为10P/100M，20P/200M(P＝μg/mL Puromycin，M＝nM MTX)，细胞活率达到85％即进行第二阶段加压筛选，抗生素浓度设置为30P/500M，50P/1000M，培养至活率高于90％即完成初筛得到抗性细胞库后，进入摇瓶培养14天批次评估，选择目的蛋白表达量较高、活性较优的稳定表达pool进行单克隆筛选，获得了100～150个单克隆，选择高产的阳性单克隆细胞扩大培养。应用这些细胞株生产抗体蛋白。
(3)全长抗体的分离制备
利用CHO-S CD Medium(Life technologies公司)培养基，在37℃、8％CO2摇床培养上述CHO-S工程细胞株13天，2000rpm离心后，收集上清，再用MabSelect SuRe亲和层析柱(GE公司)分离纯化得到抗补体因子D抗体。
实施例4.不同Fab和抗体抑制补体旁路途径溶血实验
采用ACH50法测定补体旁路途径活性，将家兔红细胞(自制)在GVB/Mg-EGTA Buffer中洗涤3次，并重悬至6×108/ml。用GVB/Mg-EGTA Buffer配制不同浓度的抗因子D抗原结合片段Fab或抗体，浓度分别为44.44nM、22.22nM、11.11nM、5.56nM、2.78nM、1.39nM。将抗体50μl与20％的人血清(Complement C1q-Depleted Serum,Human，234401-1ml，Calbiochem)50μl等体积混匀，室温孵育7min；再加入30μl的红细胞悬液，4℃振荡30s，37℃孵育30min；2000rpm，4℃离心3min，取80μl上清转移至96孔酶标板，405nm测定吸光值。数据表述成相对于在不加抗体时诱导的溶血的百分比。同时设置血清溶解对照组(不加抗体)、无血清对照组(不加人血清)以及质控对照组(以去离子水代替人血清，不加抗体)。
表1抗因子D的抗原结合片段Fab补体旁路途径活性结果
Figure PCTCN2017071914-appb-000001
Figure PCTCN2017071914-appb-000002
表2抗因子D的抗体补体旁路途径活性结果
Figure PCTCN2017071914-appb-000003
实施例5.基于荧光定量PCR分析不同Fab蛋白热稳定性实验
蛋白(抗体)在一定温度下解离暴露荧光基团进而激发出荧光信号，通过荧光定量PCR仪(MJ Research)连续收集蛋白在连续升温过程中荧光信号强度的变化绘制出蛋白的熔解曲线，计算出该蛋白的Tm值，该值的大小正向反映的是蛋白热稳定性的能力。首先使用1×PBS溶液配制抗因子D抗原结合片段Fab浓度为0.4μg/uL，避光加入5倍稀释的SYPRO染料(Cat No.S6650,lift technologies)，总反应体积为50μl，不足部分使用1×PBS溶液补足。实时定量PCR仪上运行熔解曲线实验，熔解曲线设置为25℃逐渐升温至95℃，每0.5℃读数一次，使用仪器自带软件option monitor3进行Tm值分析。
表3抗因子D的Fab蛋白热稳定性
Figure PCTCN2017071914-appb-000004
Figure PCTCN2017071914-appb-000005
实施例6.基于差示扫描量热技术(DSC)分析不同抗体蛋白热稳定性实验
通过差示扫描微量热技术(DSC)能够随温度扫描蛋白质热变性过程中的热熔的变化，并最终拟合得到包括半数变性温度Tm在内的各种热力学参数(焓、熵、自由能等)。对于全长抗体分子来说，差示扫描量热技术通常能够更为精确得到抗体CH2、CH3及Fab区的三个热力学半数变性温度。一般来说，由CH2、CH3组成的Fc区段相对固定，因而Fab区的稳定性对于抗体的稳定性具有重要的影响。利用马尔文公司Microcal Auto VP-Capillary DSC仪器以1℃/分钟的升温速度扫描25-100℃之间抗体KH-A、KH-F、以及KH102的热变性过程，并以仪器附带的Origin程序分析数据拟合出Tm值等热力学参数。样品配置于PBS缓冲液中，pH 7.4，0.22μm滤膜过滤，浓度1mg/mL。在软件端设定DSC自动清洗程序，以DECON洗涤液清洗后用超纯水彻底清洗进样针、自动进样管路及样品池，整个流程大约30min完成；设定扫描参数：扫描温度范围20-100℃，扫描速度60℃/h，其中先扫描缓冲液平衡仪器4次，再以1X PBS缓冲液为对照依次扫描待测抗体分子。每个样品的扫描时间平均约2h。扫描结束后以DSC自动清洗程序彻底清洗仪器。将原始扫描数据扣除缓冲液对照并以各样品的摩尔浓度归一化，再利用Origin软件将基线拉平，以Non-two-state model拟合数据进行Fab对应区Tm值结果分析。
表4抗因子D的抗体Fab对应区蛋白热稳定性
Figure PCTCN2017071914-appb-000006
实施例7.抗体不同种属动力学实验
通过利用OctetQK蛋白质相互作用平台，选择Ni-NTA Biosensor作为检测用生物传感器；以适合的浓度(如：10μg/ml)的CFD(猴子CFD或大鼠CFD)作为检测抗原；以200nM、100nM、50nM、25nM、12.25nM、6.25nM、0nM全长抗体蛋白检测抗体与抗原的结合情况，程序设置为：Baseline 120s，Loding 700s，Baseline 120s，Association 700S，Dissociation 1500s。选择Ni-NTA Biosensor作为检测用生物传感器；以10ug/ml浓度的CFD(猴子CFD或大鼠CFD)作为检测抗原；以500nM、250nM、125nM、62.5nM、31.25nM、15.625nM、7.8125nM、0nM为抗CFD Fab蛋白检测浓度梯度，检测Fab蛋白与抗原的结合情况。程序设置为：Baseline 120s，Loding 700s，Baseline 120s，Association 700S，Dissociation 1500s。根据结合的曲线计算抗体亲和力KD。
表5.不同Fab或抗体与猴子CFD的亲和力结果
Figure PCTCN2017071914-appb-000007
Figure PCTCN2017071914-appb-000008
表5表明本发明Fab或抗体同猴子CFD具有较高的结合。
表6.不同抗体与大鼠CFD的亲和力结果
Figure PCTCN2017071914-appb-000009
表6表明本发明抗体与大鼠CFD具有一定的结合力。
实施例8.不同抗体或Fab抑制大鼠血清致兔红细胞溶血实验
采用ACH50法测定补体旁路途径活性，将家兔红细胞(自制)在GVB/Mg-EGTA Buffer中洗涤3次(4℃，1600rpm离心)，计数后用GVB/Mg-EGTA稀释至1×109cells/ml的细胞悬液，放置于冰上冷却。用GVB/Mg-EGTA Buffer配制不同浓度的抗因子D抗原结合全长抗体，浓度梯度分别为23mg/ml、20mg/ml、15mg/ml、10mg/ml、5mg/L、1mg/ml，而抗因子D结合片段Fab的浓度为1mg/ml，将50μl的样品和50μl的大鼠血清(自制)混匀，室温振荡30s，室温孵育7min；再加入30μl的红细胞悬液，4℃振荡30s，37℃孵育40min；拿出后冷冻离心(4℃，2000rpm，3min)；转移80ul上清至96孔板，405nm测OD值。数据表述成相对于在不加抗体时诱导的溶血的百分比。KH-000在1.5mg/ml未检测到活性。
表7.全长抗体抑制大鼠血清致兔红细胞溶血实验结果
样品 IC50
KH-A 3.860mg/ml
KH-F 3.956mg/ml
表8.Fab抗体抑制大鼠血清致兔红细胞溶血实验结果
Fab样品浓度(mg/ml) 抑制率(％)
KH021(1.5mg/ml) 3.191
KH027(1.0mg/ml) 28.210
实施例9.不同抗体或Fab对豚鼠或猪的动力学实验
使用OctetQK蛋白质相互作用平台初步对瞬时转染获得的猪和豚鼠的CFD蛋白进行初步的结合情况进行评估，首先将猪(GeneID：396877)、豚鼠(GeneID：100724693)基因序列的C端融合10×His tag由南京金斯瑞生物科技有限公司优化并合成。然后将合成优化后的基因插入到表达载体pCHO1.0中，质粒按Cell line Nucleofector kit V转染试剂(Lonza公司)进行电转染宿主细胞DG44，转染后6孔板置于37℃、5％CO2摇床培养48h后，1000g，离心3分钟，使用上清液进行蛋白动力学分析。具体方法如下进行：全长抗体KH-A，KH-F动力学分析选择Protein A作为检测用生物传感器；首先以适合的浓度(如：10μg/ml)将抗体结合至protein A生物传感器上，测试猪和豚鼠CFD的瞬时转染上清同抗体的结合情况，程序设置为：Baseline 120s，Loding 700s，Baseline 120s，Association 700S，Dissociation 1500s。阴性对照设置为未转染质粒的CHO阴性细胞培养 液。Fab抗体KH027和KH021动力学分析选择Ni-NTA作为检测用生物传感器；首先将转染上清液分别结合至Ni-NTA生物传感器上，然后使用10μg/ml的Fab抗体测试不同种属的瞬时转染上清同抗体的结合情况，程序设置为：Baseline 120s，Loding 700s，Baseline120s，Association 700S，Dissociation 1500s。
表9.不同Fab或抗体与豚鼠CFD的解离常数
Figure PCTCN2017071914-appb-000010
表10.不同Fab或抗体与猪CFD的解离常数
Figure PCTCN2017071914-appb-000011
表9和表10结果表明，抗体及Fab与豚鼠或猪的CFD具有良好的结合力。
实施例10.抗体在兔子玻璃体液中动力学测试实验
抗体的活性根据不同的缓冲体系环境会受到影响，通过采取兔子玻璃体液代替人玻璃体液来模拟抗体在人玻璃体液中的活性，对抗体的活性进行初步评价。在此实验中，通过利用OctetQK蛋白质相互作用平台，选择Ni-NTA Biosensor作为检测用生物传感器；以10μg/ml人CFD蛋白作为检测抗原；以125nM，0nM为抗CFD全长抗体KH-A的检测浓度，以玻璃体液作为检测基质，对抗CFD全长抗体在玻璃体液中的动力学进行检测。程序设置为：Baseline 120s，Loding 700s，Baseline 120s，Association 700S，Dissociation1500s。根据动力学结合曲线判定抗体的初步活性。
表11.抗体KH-A与人CFD在兔玻璃体液中的解离常数
Figure PCTCN2017071914-appb-000012
表11显示，抗体KH-A在兔子玻璃体液中同人CFD具有良好的结合活性。

Claims (22)

  1. 一种抗补体因子D的人源化抗体或其抗原结合片段Fab，含有轻链可变区和重链可变区，其中轻链可变区含有氨基酸序列ITSTDIDDDX1N、氨基酸序列GGX2TLRP和氨基酸序列X3QSDS X4PYX5，所述X1为V、M或L，X2为N或A，X3为M或L，X4为L或Y,X5为I或T；重链可变区含有氨基酸序列1、序列2和氨基酸序列Y1GGY2NY3，所述Y1为A或E，Y2为S、V或P，Y3为N、Y、F、H、S或R；所述X1、X2、X3、X4、X5不同时满足下述条件：X1为M、X2为N、X3为L、X4为L、X5为T。
  2. 根据权利要求1所述抗补体因子D的人源化抗体或其抗原结合片段Fab，包含如下重链可变区和轻链可变区序列：所述轻链可变区的氨基酸序列为序列表中序列3、5、8、13、15、17中的任一种；所述重链可变区的氨基酸序列为序列表中序列19、21、23、25、27、29、31、33中的任一种。
  3. 根据权利要求2所述抗补体因子D的人源化抗体或其抗原结合片段Fab，其特征在于所述抗原结合片段Fab选自下述任一种：
    KH001：所述轻链可变区的氨基酸序列为序列表中序列3，所述重链可变区的氨基酸序列为序列表中序列33；
    KH002：所述轻链可变区的氨基酸序列为序列表中序列8，所述重链可变区的氨基酸序列为序列表中序列33；
    KH003：所述轻链可变区的氨基酸序列为序列表中序列3，所述重链可变区的氨基酸序列为序列表中序列21；
    KH004：所述轻链可变区的氨基酸序列为序列表中序列3，所述重链可变区的氨基酸序列为序列表中序列31；
    KH005：所述轻链可变区的氨基酸序列为序列表中序列3，所述重链可变区的氨基酸序列为序列表中序列19；
    KH006：所述轻链可变区的氨基酸序列为序列表中序列3，所述重链可变区的氨基酸序列为序列表中序列23；
    KH007：所述轻链可变区的氨基酸序列为序列表中序列3，所述重链可变区的氨基酸序列为序列表中序列25；
    KH008：所述轻链可变区的氨基酸序列为序列表中序列3，所述重链可变区的氨基酸序列为序列表中序列27；
    KH009：所述轻链可变区的氨基酸序列为序列表中序列3，所述重链可变区的氨基酸序列为序列表中序列29；
    KH010：所述轻链可变区的氨基酸序列为序列表中序列13，所述重链可变区的氨基酸序列为序列表中序列33；
    KH011：所述轻链可变区的氨基酸序列为序列表中序列15，所述重链可变区的氨基酸序列为序列表中序列33；
    KH012：所述轻链可变区的氨基酸序列为序列表中序列13，所述重链可变区的氨基酸序列为序列表中序列21；
    KH013：所述轻链可变区的氨基酸序列为序列表中序列13，所述重链可变区的氨基酸序列为序列表中序列31；
    KH014：所述轻链可变区的氨基酸序列为序列表中序列13，所述重链可变区的氨基酸序列为序列表中序列23；
    KH015：所述轻链可变区的氨基酸序列为序列表中序列13，所述重链可变区的氨基酸序列为序列表中序列25；
    KH016：所述轻链可变区的氨基酸序列为序列表中序列13，所述重链可变区的氨基酸序列为序列表中序列29；
    KH017：所述轻链可变区的氨基酸序列为序列表中序列15，所述重链可变区的氨基酸序列为序列表中序列31；
    KH018：所述轻链可变区的氨基酸序列为序列表中序列15，所述重链可变区的氨基酸序列为序列表中序列19；
    KH019：所述轻链可变区的氨基酸序列为序列表中序列15，所述重链可变区的氨基酸序列为序列表中序列23；
    KH020：所述轻链可变区的氨基酸序列为序列表中序列15，所述重链可变区的氨基酸序列为序列表中序列25；
    KH021：所述轻链可变区的氨基酸序列为序列表中序列15，所述重链可变区的氨基酸序列为序列表中序列27；
    KH022：所述轻链可变区的氨基酸序列为序列表中序列15，所述重链可变区的氨基酸序列为序列表中序列29；
    KH024：所述轻链可变区的氨基酸序列为序列表中序列17，所述重链可变区的氨基酸序列为序列表中序列19；
    KH027：所述轻链可变区的氨基酸序列为序列表中序列5，所述重链可变区的氨基酸序列为序列表中序列31；
    KH028：所述轻链可变区的氨基酸序列为序列表中序列5，所述重链可变区的氨基酸序 列为序列表中序列25。
  4. 根据权利要求2或3所述抗补体因子D的人源化抗体或其抗原结合片段Fab，其特征在于所述Fab由重链片段和轻链组成，所述重链片段由重链可变区和重链恒定区的CH1和部分铰链区组成，所述轻链由轻链可变区和轻链恒定区组成。
  5. 根据权利要求4所述抗补体因子D的人源化抗体或其抗原结合片段Fab，其特征在于所述重链恒定区的CH1氨基酸序列与人抗体重链恒定区CH1的氨基酸序列相同，所述轻链恒定区的氨基酸序列与人抗体轻链恒定区氨基酸序列相同。
  6. 根据权利要求5所述抗补体因子D的人源化抗体或其抗原结合片段Fab，其特征在于其中所述重链恒定区CH1和部分铰链区的氨基酸序列为序列39；所述轻链恒定区的氨基酸序列为序列41。
  7. 根据权利要求2或3所述抗补体因子D的人源化抗体或其抗原结合片段Fab，其特征在于所述抗体由重链或轻链组成，所述重链恒定区的氨基酸序列与人抗体重链恒定区的氨基酸序列相同，所述轻链恒定区的氨基酸序列与人抗体轻链恒定区氨基酸序列相同。
  8. 根据权利要求7所述抗补体因子D的人源化抗体或其抗原结合片段Fab，其特征在于所述人抗体重链恒定区的氨基酸序列为序列43，所述人抗体轻链恒定区氨基酸序列为序列41。
  9. 根据权利要求8所述抗补体因子D的人源化抗体或其抗原结合片段Fab，其特征在于所述抗体选自下述任一种：
    KH-A：其轻链序列为序列45，其重链序列为序列47；
    KH-B：其轻链序列为序列49，其重链序列为序列51；
    KH-C：其轻链序列为序列45，其重链序列为序列53；
    KH-D：其轻链序列为序列49，其重链序列为序列55；
    KH-E：其轻链序列为序列57，其重链序列为序列59；
    KH-F：其轻链序列为序列57，其重链序列为序列61。
  10. 由权利要求1-9中任一所述抗补体因子D的人源化抗体或其抗原结合片段Fab衍生的抗原结合片段，其特征在于：所述衍生抗原结合片段为Fab’、F(ab’)2、Fv、重链可变区、轻链可变区、选自重链可变区的多肽片段或选自轻链可变区的多肽片段。
  11. 一种多核苷酸，其编码权利要求1-10中任一所述人源化抗体、抗原结合片段Fab、或前述人源化抗体、抗原结合片段Fab衍生的抗原结合片段。
  12. 根据权利要求11所述的多核苷酸，其特征在于：所述人源化抗体或抗原结合片段Fab中所述抗体的轻链可变区的编码序列为序列表中序列4、序列6、序列9、序列14、序列16和序列18中的任一种，重链可变区的编码序列为序列表中序列20、序列22、序列24、序列26、 序列28、序列30、序列32、序列34中的任一种。
  13. 根据权利要求12所述的多核苷酸，其特征在于：所述Fab的重链恒定区的编码序列如序列表中序列40所示；所述Fab的轻链恒定区编码序列如序列表中序列42所示。
  14. 根据权利要求12所述的多核苷酸，其特征在于：所述抗体的重链恒定区的编码序列如序列表中序列44所示；所述抗体的轻链恒定区的编码序列如序列表中序列42所示。
  15. 根据权利要求14所述的多核苷酸，其特征在于：抗体选自下述任一种多核苷酸：
    KH-A：其轻链序列为序列46，其重链序列为序列48；
    KH-B：其轻链序列为序列50，其重链序列为序列52；
    KH-C：其轻链序列为序列46，其重链序列为序列54；
    KH-D：其轻链序列为序列50，其重链序列为序列56；
    KH-E：其轻链序列为序列58，其重链序列为序列60；
    KH-F：其轻链序列为序列58，其重链序列为序列62。
  16. 下述遗传材料：含有权利要求12-15中任一所述多核苷酸的重组载体、重组菌、重组细胞系、重组病毒或表达盒。
  17. 根据权利要求16所述的遗传材料，其特征在于：所述重组载体为表达所述Fab或抗体或抗原结合片段的原核表达载体或真核表达载体；所述重组菌为携带有所述基因的大肠杆菌；所述重组细胞系为转入所述基因的哺乳动物细胞系，优选CHO细胞系，或293细胞及其亚系；所述重组病毒为携带有所述基因的重组腺病毒或重组腺相关病毒。
  18. 权利要求1-10中任一所述人源化抗体或抗原结合片段Fab，或权利要求11-15中任一所述多核苷酸，或权利要求16-17任一所述遗传材料的用途：
    d1)在制备预防和/或治疗人补体因子D相关疾病药物中的用途，或
    d2)在制备中和人补体因子D产品中的用途，或
    d3)在制备定性或定量检测人补体因子D的试剂盒中的用途。
  19. 根据权利要求18所述的用途，其特征在于所述人补体因子D相关疾病为由人补体因子D升高引起的疾病；优选眼病、风湿性关节炎、心肌梗死或再灌注损伤。
  20. 根据权利要求19所述的用途，其特征在于所述眼病为年龄相关性黄斑变性或糖尿病性视网膜病。
  21. 根据权利要求20所述的用途，其特征在于所述老年性黄斑变性为非渗出性黄斑变性。
  22. 一种药物组合物，含有辅料和活性成分，所述活性成分含有下述物质中的至少一种：权利要求1-10中任一所述人源化抗体或抗原结合片段Fab，或权利要求11-15中任一所述多核苷酸，或权利要求16-17任一所述遗传材料；所述辅料为药学上可接受的载体或赋形剂。
PCT/CN2017/071914 2016-01-28 2017-01-20 抗补体因子D的人源化Fab和人源化抗体及其用途 WO2017129064A1 (zh)

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US11155610B2 (en) 2014-06-28 2021-10-26 Kodiak Sciences Inc. Dual PDGF/VEGF antagonists
US11066465B2 (en) 2015-12-30 2021-07-20 Kodiak Sciences Inc. Antibodies and conjugates thereof
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