CN107522783A - A kind of anti-interleukin-17 A antibody, its preparation method and application - Google Patents

A kind of anti-interleukin-17 A antibody, its preparation method and application Download PDF

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Publication number
CN107522783A
CN107522783A CN201710916535.7A CN201710916535A CN107522783A CN 107522783 A CN107522783 A CN 107522783A CN 201710916535 A CN201710916535 A CN 201710916535A CN 107522783 A CN107522783 A CN 107522783A
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antibody
chain variable
light chain
variable district
present
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CN107522783B (en
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朱向阳
贾慧峰
于海佳
俞玲
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Zhejiang Huahai Biotechnology Co ltd
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Bio Pharmaceutical Technology (shanghai) Co Ltd
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Priority to PCT/CN2018/073457 priority patent/WO2019061962A1/en
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70503—Immunoglobulin superfamily
    • C07K14/7051—T-cell receptor (TcR)-CD3 complex
    • 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
    • C07K16/24—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against cytokines, lymphokines or interferons
    • C07K16/244—Interleukins [IL]
    • 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
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/21—Immunoglobulins specific features characterized by taxonomic origin from primates, e.g. man
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/565—Complementarity determining region [CDR]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
    • C07K2317/62—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising only variable region components
    • C07K2317/622—Single chain antibody (scFv)
    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide

Abstract

The invention provides a kind of antibody, its preparation method and the application of anti-interleukin-17 A (IL 17A).Specifically, the invention provides a kind of new anti-IL 17A monoclonal antibodies.The antibody of the present invention can combine IL 17A antigens with high specificity, and it has very high affinity and very low immunogenicity, and for preparing prevention or treating the medicine of the related for example various inflammation of disease of IL 17A or autoimmune disease.

Description

A kind of anti-interleukin-17 A antibody, its preparation method and application
Technical field
The present invention relates to field of medicaments, more particularly to antibody, its preparation for targeting interleukin-17 A (also referred to as IL-17) Methods and applications.
Background technology
So far, existing 6 members of IL-17 families are found：IL-17A(IL-17)、IL-17B、IL-17C、IL- 17D、IL-17E(IL-25)、IL-17F.These interleukin-17 cell factors can be incorporated on corresponding acceptor, so as to be situated between Lead different inflammatory reactions.
IL-17A is initially found to be the CD4 by activating+T cell is secreted.This category feature secretes IL-17A T cell Subgroup is referred to as Th17 cells.In addition to Th17 cells, cytotoxicity CD8+T cell (Tc17), gamma delta T cells, NKT T cell (NKT-17) and B cell can also express IL-17A under given conditions.Innate immune cells, including monocyte, neutrality Granulocyte, NK and lymphoid tissue induction sample (Lti-like) cell can also produce IL-17A.Recently, at one On being found in the research of Trypanosoma cruzi infection, B cell can also produce IL-17A.Some nonimmune cells, such as enteron aisle Pan Shi are thin Born of the same parents and enterocyte can also stress in the case of produce IL-17A.Because the distribution of Th17 cells in vivo is most wide, and There is wide application in inflammatory reaction, so, it is generally recognized that it or IL-17A main source cell.And produce IL-17A's The anti-infectious immunity that innate cells participate in host mainly as the early stage defense cell of body reacts.
IL-17A is to be by the homodimer of disulfide bond, molecular weight by two chains of 155 amino acid 35kDa.The signal peptide (AA) and 123 amino acid chain regions that IL-17 structure is made up of 23 amino acid are formed.
The acceptor of IL-17 combination I type cell surfaces is referred to as IL-17R, wherein there is at least three kinds：IL-17RA, IL-17RB and IL-17RC.IL-17A is combined IL-17RA and IL-17RC acceptors with IL-17F in the form of homodimer or heterodimer Compound carrys out transduction signal, and participates in body autoimmune disease, various inflammatory reactions and the reaction of host's anti-infectious immunity. IL-17C combination IL-17RA and IL-17RE receptor complex activate downstream signal, promote body anti-infectious immunity, autoimmunity Disease and inflammatory reaction.IL-17B is found that IL-17RB can be combined, but signal is still unclear downstream.IL-17RB also can be with IL-17RA forms receptor complex mediation IL-17E and causes II type immune responses.Il-17E, which is also reported, can promote tumour thin The apoptosis of born of the same parents.IL-17D acceptor and downstream signal and orphan receptor IL-17RD part and downstream signal are still unclear at present Chu.
IL-17A mainly induces the signal activation of the non-haematological origin cell including epithelial cell and stroma cell. The inflammation factor and chemotactic factor (CF) of IL-17A induced expressions can promote the recruitment of panimmunity cell, so as to exempt to itself Epidemic disease plays facilitation.Research finds, IL-17A and IL-17F, and its Major Secretory T cell subgroup Th17 cells, a variety of Also played an important role in autoimmune disease, among these including autoimmune disease, such as rheumatoid arthritis (rheumatoid arthritis, RA) and multiple sclerosis (multiple sclerosis, MS), and inflammatory bowel disease (inflammatory boweldisease, IBD), psoriasis (psoriasis), systemic loupus erythematosus (systemic Lupus erythematosus, SLE) and type i diabetes (type1diabetes, T1D).
IL-17A and IL-17F mainly expresses the inflammation factor and chemotactic factor (CF) to play its rush by inducing target cell Enter the function of inflammatory reaction.IL-17A is combined with cell surface receptor IL-17RA, is recruited IL-17RC and is formed heterodimer, is situated between Lead downstream signaling pathway.IL-17 can activate TRAF6 (TNF-receptor associated factor after being combined with its acceptor 6).IL-17 and IL-1 and TNF shares identical transcription pathway, and it can activate NF-kB and 3 MAP (mitrogen-activated protein) Enzyme, including ERK1, ERK2, JNK, p38.These paths are found in synovial cell and cartilage cell.
Therefore, in view of IL-17A is acted in all kinds of relevant diseases and function, this area still needs exploitation and is suitable to treatment The anti-IL-17A specific antibodies of the improvement of patient.
The content of the invention
The object of the invention just there is provided a kind of anti-IL-17A antibody, preparation method and use.
The first aspect of the present invention, there is provided a kind of weight chain variable district of antibody, described weight chain variable district include following Three complementary determining region CDR：
SEQ ID NO:CDR1 shown in 7,
SEQ ID NO:CDR2 shown in 8, and
SEQ ID NO:CDR3 shown in 9.
In another preference, any one amino acid sequence also includes optionally past adding in above-mentioned amino acid sequence Add, lack, modify and/or substitute at least one (such as 1-3, preferably 1-2, more preferably 1) amino acid and can retain The derived sequence of IL-17A binding affinities.
In another preference, the weight chain variable district also includes people Yuan FR areas or mouse Yuan FR areas.
In another preference, the weight chain variable district has SEQ ID NO:Amino acid sequence shown in 1.
In another preference, the weight chain variable district has SEQ ID NO:Amino acid sequence shown in 5.
The second aspect of the present invention, there is provided a kind of heavy chain of antibody, described heavy chain have such as first aspect present invention Described weight chain variable district.
In another preference, the heavy chain of described antibody also includes heavy chain constant region.
In another preference, described heavy chain constant region behaviour source, mouse source or rabbit source.
The third aspect of the present invention, there is provided a kind of light chain variable district of antibody, described light chain variable district include following Three complementary determining region CDR：
SEQ ID NO:CDR1' shown in 10,
Amino acid sequence is GAT CDR2', and
SEQ ID NO:CDR3' shown in 11.
In another preference, any one amino acid sequence also includes optionally past adding in above-mentioned amino acid sequence Add, lack, modify and/or substitute at least one (such as 1-3, preferably 1-2, more preferably 1) amino acid and can retain The derived sequence of IL-17A binding affinities.
In another preference, the light chain variable district also includes people Yuan FR areas or mouse Yuan FR areas.
In another preference, the light chain variable district has SEQ ID NO:Amino acid sequence shown in 2.
In another preference, the light chain variable district has SEQ ID NO:Amino acid sequence shown in 6.
The fourth aspect of the present invention, there is provided a kind of light chain of antibody, described light chain, which has, such as weighs third party of the present invention Light chain variable district described in face.
In another preference, the light chain of described antibody also includes constant region of light chain.
In another preference, described constant region of light chain behaviour source, mouse source or rabbit source.
The fifth aspect of the present invention, there is provided a kind of antibody, the antibody have：
(1) weight chain variable district as described in the first aspect of the invention；And/or
(2) light chain variable district as described in third aspect present invention；
Or the antibody has：Heavy chain as described in respect of the second aspect of the invention；And/or such as fourth aspect present invention institute The light chain stated.
In another preference, the EC of the adhesion of the antibody on human IL-17A albumen (preferably wild type)50For 5- 50ng/ml。
In another preference, the EC of the adhesion of the antibody on human IL-17A albumen (preferably wild type)50For 12.6ng/ml。
In another preference, the antibody is selected from：Animal sources antibody, chimeric antibody, humanized antibody or its combination.
In another preference, described antibody is double-chain antibody or single-chain antibody.
In another preference, described antibody is monoclonal antibody.
In another preference, described antibody is the monoclonal antibody of part or full humanization.
In another preference, the weight chain variabl area sequence such as SEQ ID NO. of the antibody:Shown in 1 or 5；And/or
The light-chain variable sequence of described antibody such as SEQ ID NO.:Shown in 2 or 6.
In another preference, the weight chain variabl area sequence such as SEQ ID NO. of the antibody:Shown in 1；It is and described The light-chain variable sequence of antibody such as SEQ ID NO.:Shown in 2.
In another preference, the weight chain variabl area sequence such as SEQ ID NO. of the antibody:Shown in 5；It is and described The light-chain variable sequence of antibody such as SEQ ID NO.:Shown in 6.
In another preference, described antibody is drug conjugates form.
The sixth aspect of the present invention, there is provided a kind of recombinant protein, described recombinant protein have：
(i) weight chain variable district as described in the first aspect of the invention, such as heavy chain as described in respect of the second aspect of the invention, this hair As described in light chain variable district described in the bright third aspect, the light chain as described in fourth aspect present invention or fifth aspect present invention Antibody；And
(ii) sequence label of optional assistance expression and/or purifying.
In another preference, described sequence label includes 6His labels.
In another preference, described recombinant protein (or polypeptide) includes fusion protein.
In another preference, described recombinant protein is monomer, dimer or polymer.
The seventh aspect of the present invention, there is provided a kind of CAR constructions, the monoclonal antibody antigen of described CAR constructions The scFV sections of calmodulin binding domain CaM are to specifically bind to IL-17A land, and the scFv has such as first aspect present invention Described weight chain variable district and the light chain variable district as described in third aspect present invention.
The eighth aspect of the present invention, there is provided a kind of immunocyte of restructuring, described immunocyte expression external source as CAR constructions described in seventh aspect present invention.
In another preference, described immunocyte is selected from the group：NK cells, T cell.
In another preference, described immunocyte comes from people or non-human mammal (such as mouse).
The ninth aspect of the present invention, there is provided a kind of antibody drug conjugates, described antibody drug conjugates contain：
(a) antibody moiety, the antibody moiety are selected from the group：Weight chain variable district as described in the first aspect of the invention, such as Heavy chain described in second aspect of the present invention, the light chain variable district as described in third aspect present invention, such as fourth aspect present invention institute The antibody described in light chain or fifth aspect present invention stated or its combination；With
(b) it is selected from the group with the coupling moiety of antibody moiety coupling, the coupling moiety：Detectable, medicine Thing, toxin, cell factor, radionuclide, enzyme or its combination.
In another preference, described antibody moiety is carried out even with described coupling moiety by chemical bond or joint Connection.
The tenth aspect of the present invention, there is provided a kind of purposes of active component, the active component are selected from the group：Such as this hair Weight chain variable district described in bright first aspect, heavy chain as described in respect of the second aspect of the invention, as described in third aspect present invention Light chain variable district, the light chain as described in fourth aspect present invention or the antibody as described in fifth aspect present invention, such as present invention the 6th Recombinant protein described in aspect, the immunocyte as described in eighth aspect present invention, the antibody as described in ninth aspect present invention Drug conjugates or its combination, the active component are used for (a) and prepare detection reagent or kit；And/or (b) prepares prevention And/or the medicine for the treatment of IL-17A relevant diseases.
In another preference, the IL-17A relevant diseases are selected from the group：Inflammation, autoimmune disease or its combination； Preferably autoimmune disease.
In another preference, described disease is selected from the group：It is psoriasis, psoriatic arthritis, ankylosing spondylitis, more Hair property sclerosis, inflammatory arthritis or its combination, it is therefore preferable to inflammatory arthritis.
In another preference, described inflammatory arthritis is selected from the group：Osteoarthritis, rheumatoid arthritis, rheumatism Property arthritis or its combination, it is therefore preferable to rheumatic arthritis.
In another preference, described antibody is drug conjugates (ADC) form.
In another preference, described detection reagent or kit are used to diagnose IL-17A relevant diseases.
In another preference, the detection reagent or kit are used to detect IL-17A albumen in sample.
In another preference, described detection reagent is detection lug.
The eleventh aspect of the present invention, there is provided a kind of pharmaceutical composition, described pharmaceutical composition contain：
(i) active component, the active component are selected from the group：Weight chain variable district as described in the first aspect of the invention, such as Heavy chain described in second aspect of the present invention, the light chain variable district as described in third aspect present invention, such as fourth aspect present invention institute The antibody described in light chain or fifth aspect present invention stated, such as recombinant protein as described in sixth aspect present invention, the present invention the Immunocyte described in eight aspects, the antibody drug conjugates as described in ninth aspect present invention or its combination；And
(ii) pharmaceutically acceptable carrier.
In another preference, described pharmaceutical composition is liquid formulation.
In another preference, described pharmaceutical composition is injection.
The twelveth aspect of the present invention, there is provided a kind of polynucleotides, described polynucleotide encoding are selected from the group more Peptide：
(1) weight chain variable district as described in the first aspect of the invention, such as heavy chain as described in respect of the second aspect of the invention, this hair As described in light chain variable district described in the bright third aspect, the light chain as described in fourth aspect present invention or fifth aspect present invention Antibody；Or
(2) recombinant protein as described in sixth aspect present invention；
(3) the CAR constructions as described in seventh aspect present invention.
The thirteenth aspect of the present invention, there is provided a kind of carrier, described carrier contain such as the twelfth aspect of the present invention institute The polynucleotides stated.
In another preference, described carrier includes：Bacterial plasmid, bacteriophage, yeast plasmid, plant cell virus, Mammalian cell virus such as adenovirus, retrovirus or other carriers.
The fourteenth aspect of the present invention, there is provided a kind of genetically engineered host cell, described host cell contain The present invention the 13rd aspect as described in carrier or genome in integration just like the twelfth aspect of the present invention as described in polynucleotides.
A kind of the fifteenth aspect of the present invention, there is provided IL- in vitro detection (including diagnostic or nondiagnostic) sample The method of 17A albumen, methods described include step：
(1) in vitro, the sample is contacted with the antibody as described in fifth aspect present invention；
(2) detect whether to form antigen-antibody complex, wherein forming compound means that in sample IL-17A eggs be present In vain.
The sixteenth aspect of the present invention, there is provided a kind of detection plate, described detection plate include：Substrate (supporting plate) and survey Strip, described test-strips contain the antibody as described in fifth aspect present invention or the immune idol as described in ninth aspect present invention Join thing.
The seventeenth aspect of the present invention, there is provided a kind of kit, the kit include：
(1) first container, the antibody as described in fifth aspect present invention is contained in first container；And/or
(2) second container, the secondary antibody containing the anti-antibody as described in fifth aspect present invention in the second container；
Or the kit contain the present invention the 16th in terms of as described in detection plate.
The eighteenth aspect of the present invention, there is provided a kind of preparation method of recombinant polypeptide, methods described include：
(a) under conditions suitable for the expression, host cell of the culture as described in fourteenth aspect of the present invention；
(b) recombinant polypeptide is isolated from culture, described recombinant polypeptide is anti-as described in fifth aspect present invention Body or the recombinant protein as described in sixth aspect present invention.
The nineteenth aspect of the present invention, there is provided a kind of method of IL-17A relevant diseases, methods described include：To needs Object apply antibody as described in fifth aspect present invention, the antibody-drug conjugates of the antibody or antibody as described in expressing CAR-T cells or its combination.
It should be understood that within the scope of the present invention, above-mentioned each technical characteristic of the invention and have in below (eg embodiment) It can be combined with each other between each technical characteristic of body description, so as to form new or preferable technical scheme.As space is limited, exist This no longer tires out one by one states.
Embodiment
The present inventor by extensive and in-depth study, by a large amount of screenings, unexpectedly obtain it is a kind of have it is extremely excellent Affinity and specific anti-IL-17A monoclonal antibodies, the humanized antibody obtained based on the antibody.Antibody of the present invention IL-17A antigens can be combined with high specificity, and it has very high affinity, and (ELISA determines its EC50About 15.4ng/ml), And the combination of IL-17A and IL-17 acceptors is significantly inhibited, and for mammal in itself without visible toxic side effect.Herein On the basis of complete the present invention.
Term
As used herein, term " conjugate " is to refer to and the soluble recepter of targeted integration or its fragment or its is similar Thing, or antibody or its fragment or its analog." IL-17A conjugates " of the present invention, referring to can specific recognition IL-17A And the antibody combined with IL-17A or its fragment or its analog.
As used herein, term " giving " and " processing " refer to exogenous drugs, therapeutic agent, diagnosticum or composition application In animal, people, subject, cell, tissue, organ or biofluid." giving " and " processing " can refer to treatment, drug metabolism is moved Mechanics, diagnosis, research and experimental method.The processing of cell include contact of contact and reagent of the reagent with cell with fluid, Contact of the fluid with cell." giving " and " processing " is still meant that by reagent, diagnosis, binding compositions or by another cell External and ex vivo treatment." processing " refers to treatment processing, prevention or preventative when applied to people, animal or study subject Measure, research and diagnosis；Including IL-17A conjugates and human or animal, subject, cell, tissue, physiological compartment or physiology stream The contact of body.
As used herein, term " treatment ", which is showed, gives the interior or topical therapeutic agent of patient, includes any one of the present invention IL-17A conjugates and combinations thereof, the patient has one or more disease symptomses, and the known therapeutic agent is to these Symptom has therapeutic action.Generally, with effectively alleviate the amount of the therapeutic agent of one or more disease symptomses (therapeutically effective amount) to Give patient.
As used herein, term " optional " " optionally " mean event described later or situation can occur but It is not required to occur.For example, " optionally including 1-3 heavy chain of antibody variable region " refers to that the heavy chain of antibody variable region of particular sequence can It can be 1,2 or 3 to have but be not required.
Antibody
As used herein, term " antibody " refers to immunoglobulin, is by two identical heavy chains and two identical light chains Four peptide chain structures being formed by connecting by interchain disulfide bond.The amino acid of immunoglobulin heavy chain constant region forms and put in order Difference, therefore its antigenicity is also different.Accordingly, immunoglobulin can be divided into five classes, or the not isotype for immunoglobulin, That is IgM, IgD, IgG, IgA and IgE, corresponding to different immunoglobulin like protein heavy chain constant region be referred to as α, δ, ε, γ and μ.IgG represents most important one kind in immunoglobulin, and due to chemical constitution and biological function difference, it can be divided into 4 again Subclass：IgG1, IgG2, IgG3 and IgG4.Light chain is divided into κ or λ chains by the difference of constant region.The Asia of different immunoglobulin like protein Unit structure and 3-d modelling are known to those skilled in the art.
Heavy chain of antibody and light chain are very big close to the sequence variation of about 110 amino acid of N-terminal, are variable region (V areas)；It is close Remaining amino acid sequence of C-terminal is relatively stable, is constant region (C areas).Variable region includes 3 hypervariable regions (HVR) and 4 sequence phases To conservative FR areas (FR).4 FR amino acid sequence is relatively conservative, does not participate in association reaction directly.Determine 3 hypervariable regions Determine the specificity of antibody, also known as complementarity-determining region (CDR).Every light chain variable district (LCVR) and weight chain variable district (HCVR) By 3 CDR regions and 4 FR district's groups into, the order being arranged in order from aminoterminal to c-terminus is FR1, CDR1, FR2, CDR2, FR3、CDR3、FR4.3 CDR regions of light chain, i.e. light chain hypervariable region (LCDR), refer to LCDR1, LCDR2 and LCDR3；3 of heavy chain CDR region, i.e. heavy chain hypervariable region (HCDR), refer to HCDR1, HCDR2 and HCDR3.The described antibody of invention or antigen-binding fragment The cdr amino acid residue in LCVR and HCVR areas meets known Kabat coding rules (LCDR1-3, HCDR2- in quantity and position 3), or kabat and chothia coding rule (HCDR1) is met.Four FR areas in native heavy and light chain variable district are big It is in beta sheet configuration in cause, is connected by three CDR for forming connection ring, part β-pleated sheet structure can be formed in some cases.Often CDR in bar chain is by FR areas firmly against the antigen-binding site that together form antibody together and with the CDR of another chain. Be which Amino acid profile FR or CDR region domain can be determined by the amino acid sequence for the antibody for comparing same type.It is constant Area does not participate in the combination of antibody and antigen directly, but they show different effector functions, such as participate in the dependence of antibody In the cytotoxicity of antibody.
As used herein, term " antigen-binding fragment ", the Fab fragments with antigen-binding activity, Fab ' fragments, F are referred to (ab ') 2 fragment, or single Fv fragments.Fv antibody contains heavy chain of antibody variable region, light chain variable district, but does not have constant region, and has There is the minimum antibody fragment of whole antigen binding sites.In general, the polypeptide that Fv antibody is also included between VH and VL domains connect Head, and the structure needed for antigen binding can be formed.
As used herein, term " antigenic determinant " refers to discontinuous on antigen, by antibody of the present invention or antigen binding fragment The three dimensions site of section identification.
The present invention not only includes complete antibody, in addition to the fragment with immunocompetent antibody or antibody and other sequences Arrange the fusion protein formed.Therefore, present invention additionally comprises the fragment of the antibody, derivative and analog.
In the present invention, antibody includes mouse, the chimeric, humanization prepared by the technology known to those skilled in the art Or full people antibody.Recombinant antibodies, such as chimeric and humanization monoclonal antibody, including people's and inhuman portion Point, DNA recombinant technique well known in the art can be used.
As used herein, term " monoclonal antibody " refers to the antibody of the clones secrete derived from individual cells source.Monoclonal Antibody is high degree of specificity, for single epitope.Described cell is probably eucaryon, protokaryon or bacteriophage gram Grand cell line.
As used herein, term " chimeric antibody " is V areas gene by murine antibody and the C areas gene splicing of human antibody For mosaic gene, carrier, the antibody molecule of transfection host cell expression are inserted into.Both the height for having remained parent murine antibody is special Property and affinity, enable its people source Fc section effectively mediating biologic effector function again.
As used herein, term " humanized antibody ", it is a kind of anti-variable region forms of modification of mouse of the present invention, has and be derived from The CDR region of (or being substantially derived from) non-human antibody (preferably mouse monoclonal antibody), and substantially from human antibody sequence FR areas and constant region；The CDR region sequence that mouse resists is grafted onto on different types of human germline antibody's frame sequence.Because CDR Sequence is responsible for most antibody-antigene interaction, it is possible to it is specific natural to express simulation by construction of expression vector The recombinant antibodies of existing antibody characteristic.
In the present invention, antibody can be monospecific, bispecific, tri-specific or more multiple specifics.
In the present invention, antibody of the invention also includes its conservative variant, refers to the amino acid sequence with antibody of the present invention Row are compared, and have at most 10, preferably at most 8, more preferably at most 5, most preferably at most 3 amino acid by property it is similar or Similar amino acid is replaced and forms polypeptide.These conservative variation's polypeptides carry out amino acid substitution preferably based on Table A and produced It is raw.
Table A
Initial residue Representational substitution Preferable substitution
Ala(A) Val；Leu；Ile Val
Arg(R) Lys；Gln；Asn Lys
Asn(N) Gln；His；Lys；Arg Gln
Asp(D) Glu Glu
Cys(C) Ser Ser
Gln(Q) Asn Asn
Glu(E) Asp Asp
Gly(G) Pro；Ala Ala
His(H) Asn；Gln；Lys；Arg Arg
Ile(I) Leu；Val；Met；Ala；Phe Leu
Leu(L) Ile；Val；Met；Ala；Phe Ile
Lys(K) Arg；Gln；Asn Arg
Met(M) Leu；Phe；Ile Leu
Phe(F) Leu；Val；Ile；Ala；Tyr Leu
Pro(P) Ala Ala
Ser(S) Thr Thr
Thr(T) Ser Ser
Trp(W) Tyr；Phe Tyr
Tyr(Y) Trp；Phe；Thr；Ser Phe
Val(V) Ile；Leu；Met；Phe；Ala Leu
Anti- IL-17A antibody
As used herein, term " IL-17A " generally refers to natural or restructuring human il-17 A, and human il-17 A Non-human homologue.Unless otherwise directed, otherwise (such as it is for human il-17 A using the molecular weight of IL-17A homodimer 30KDa) calculate IL-17A molar concentration.
As used herein, term " human il-17 A (huIL-17A) " refer to the human il-17 A albumen number of logging in NP-002180 and AAT22064 mature form (i.e. residue 24-155), and its natural variant and polymorphism.
The present invention provides a kind of for IL-17A high specific and the antibody of high-affinity, and it includes heavy chain and light chain, The heavy chain contains weight chain variable district (VH) amino acid sequence, and the light chain contains light chain variable district (VL) amino acid sequence.
Preferably, the respective CDR of weight chain variable district (VH) amino acid sequence and light chain variable district (VL) amino acid sequence is selected From the following group：
a1)SEQ ID No.:7；
a2)SEQ ID No.:8；
a3)SEQ ID No.:9；
a4)SEQ ID No.:10；
a5)GAT；
a6)SEQ ID No.:11；
A7) any one amino acid sequence by addition, missing, modification and/or substitutes at least in above-mentioned amino acid sequence The sequence with IL-17A binding affinities of one (such as 1-5,1-3, preferably 1-2, more preferably 1) amino acid.
It is described by adding, lacking, modifying and/or substituting at least one amino acid sequence institute shape in another preference Into sequence be preferably that homology is at least 80%, preferably at least 85%, more preferably at least 90%, most preferably at least 95% Amino acid sequence.
The antibody of the present invention can be double-strand or single-chain antibody, and can be selected from animal sources antibody, chimeric antibody, people Source antibody, more preferably humanized antibody, people-animal chimeric antibody, more preferably full humanized antibody.
Antibody derivatives of the present invention can be single-chain antibody, and/or antibody fragment, such as：Fab、Fab’、(Fab’)2 Or other known antibody derivatives etc. in the field, and IgA, IgD, IgE, IgG and IgM antibody or other hypotypes is anti- Any one or a few in body.
Wherein, the animal is preferably mammal, such as mouse.
Antibody of the present invention can be target human il-17 A mouse source antibody, chimeric antibody, humanized antibody, CDR graftings and/ Or the antibody of modification.
In one preferred embodiment of the invention, above-mentioned SEQ ID No.:7th, any one or a few sequence in 8 and 9, Or they through the sequence with IL-17A binding affinities for addition, lack, modifying and/or substituting at least one amino acid, Positioned at weight chain variable district (VH) CDR region.
In one preferred embodiment of the invention, above-mentioned SEQ ID No.:10th, amino acid sequence:GAT and SEQ ID No.:At least one amino acid is added, lacks, modifies and/or substituted to any one or a few sequence or their processes in 11 Sequence with IL-17A binding affinities, positioned at light chain variable district (VL) CDR region.
In a kind of more preferred embodiment of the present invention, VH CDR1, CDR2, CDR3 are separately selected from SEQ ID No.:7th, in 8 and 9 any one or a few sequence or they by adding, lacking, modifying and/or substituting at least one amino The sequence with IL-17A binding affinities of acid；VL CDR1, CDR2, CDR3 are separately selected from SEQ ID No.:10、 Amino acid sequence:GAT and SEQ ID No.:In 11 any one or a few sequence or they by addition, missing, modification and/ Or the sequence with IL-17A binding affinities of at least one amino acid of substitution.
In the above of the present invention, the addition, missing, modification and/or the amino acid quantity substituted, preferably no more than The 40% of initial amino acid sequence total amino acid quantity, more preferably more preferably less than 35%, more preferably 1-33%, 5- 30%, more preferably 10-25%, more preferably 15-20%.
In the present invention, the amino acid quantity of the addition, missing, modification and/or substitution is typically 1,2,3,4 or 5, Preferably 1-3, it is more preferably 1-2, is most preferably 1.
The preparation of antibody
Any method for being suitable to produce monoclonal antibody can be used in producing anti-IL-17A antibody of the invention.For example, can So that animal is immunized with connection or naturally occurring IL-17A homodimers or its fragment.Suitable immunity inoculation side can be used Method, including adjuvant, immunostimulant, repetition booster immunization inoculation, can use one or more approach.
The IL-17 of any suitable form can serve as immunogene (antigen), and special to IL-17A for generation is inhuman Antibody, screen the biological activity of the antibody.Excite the ripe human il-17 A that immunogene can be total length, including it is natural same Source dimer, or the peptide containing single/multiple epitope.Immunogene can be used alone, or with one or more known in the art Immunogenicity reagents recombination uses.Immunogene can be purified by natural origin, or be produced in the cell of genetic modification.Compile The DNA of code immunogene can be genome or non genome (such as cDNA) on source.Suitable heredity can be used to carry Body surface reaches the DNA of encoding immunogens, and the carrier includes but is not limited to adenovirus vector, gland relevant viral vector, baculoviral Carrier, material and non-virus carrier.
The illustrative methods for producing the anti-human IL-17A antibody of the present invention are described in embodiment 1.
Humanized antibody can be selected from any kind of immunoglobulin, including IgM, IgD, IgG, IgA and IgE.At this In invention, antibody is IgG antibody, uses IgG1 hypotypes.By using the biological characteristis screening antibodies described in Examples below The optimization of required constant domain sequence is easily achieved, to produce required biological activity.
Equally, any sort light chain can use in this paper Compounds and methods for.Specifically, κ, λ chain or its change Body can be used in the Compounds and methods for of the present invention.
The illustrative methods of the anti-human IL-17A antibody of the humanization present invention are described in embodiment 5.
The sequence of antibody of the present invention or the DNA molecular of its fragment can use routine techniques, for example utilize PCR amplifications or gene The methods of group library screening, obtains.In addition, can also be merged the coded sequence of light chain and heavy chain, single-chain antibody is formed.
Once obtain relevant sequence, it is possible to obtain relevant sequence in large quantity with recombination method.This is typically will It is cloned into carrier, then is transferred to cell, then by conventional method from the host cell after propagation isolated relevant sequence.
In addition, relevant sequence can be also synthesized with artificial synthesized method, when especially fragment length is shorter.Generally, lead to After first synthesizing multiple small fragments, the very long fragment of sequence can be obtained by being then attached again.Then the DNA sequence dna can be introduced this In field in known various existing DNA moleculars (or such as carrier) and cell.
The invention further relates to include above-mentioned appropriate DNA sequence dna and the carrier of appropriate promoter or control sequence.This A little carriers can be used for converting appropriate host cell, allow it to marking protein.
Host cell can be prokaryotic, such as bacterial cell；Or low eukaryotic, such as yeast cells；It is or high Deng eukaryotic, such as mammalian cell.Preferable zooblast includes (but being not limited to)：CHO-S、CHO-K1、HEK-293 Cell.
Heretofore described can be carried out the step of converting host cell with recombinant DNA with technology well known in the art.Obtain The transformant obtained can use conventional method culture, the polypeptide of the coded by said gene of the transformant expression present invention.According to host used Cell, cultivated under suitable conditions with conventional medium.
Generally, under conditions of antibody expression of the present invention is adapted to, the host cell of culture conversion gained.Then with routine Immunoglobulin purification step, such as albumin A-Sepharose, hydroxyapatite chromatography, gel electrophoresis, dialysis, ion exchange layer The routine well known to those skilled in the art such as analysis, hydrophobic chromatography, sieve chromatography or affinity chromatography isolates and purifies means and purified To the antibody of the present invention.
Gained monoclonal antibody can be identified with conventional meanses.For example the binding specificity of monoclonal antibody is available immune Precipitation or external binding tests (such as radioimmunoassay (RIA) or enzyme linked immunosorbent assay (ELISA) (ELISA)) determine.
Using
The invention provides the purposes of antibody of the present invention, for example, for prepare diagnostic preparation or prepare for prevent and/or Treat the medicine of the related diseases of IL-17A.The disease related IL-17A includes inflammation disease, autoimmune disease etc., bag Include but be not limited to psoriasis, psoriatic arthritis, ankylosing spondylitis, multiple sclerosis, inflammatory bowel disease (such as Chron Disease, ulcerative colitis etc.), osteoarthritis, rheumatoid arthritis (RA), rheumatic arthritis or osteoporosis, inflammatory Fibrosis (such as chorionitis, pulmonary fibrosis and hardening), asthma (including allergic asthma), allergy and cancer.
Pharmaceutical composition
Present invention also offers a kind of composition.In preference, described composition is pharmaceutical composition, and it contains The antibody stated or its active fragment or its fusion protein or its ADC or corresponding CAR-T cells, and pharmaceutically acceptable load Body.Generally, these materials can be formulated in nontoxic, inert and pharmaceutically acceptable aqueous carrier medium, wherein pH 5-8 is ordinarily be about, preferably pH is about 6-8, although pH value can have with the property and illness to be treated that are formulated material Changed.The pharmaceutical composition prepared can be administered by conventional route, including (but being not limited to)：In knurl, Intraperitoneal, intravenous or part administration.
Antibody of the present invention can also express the cell therapy being used in the cell by nucleotide sequence, such as, institute State antibody and be used for Chimeric antigen receptor T cell immunotherapy (CAR-T) etc..
The pharmaceutical composition of the present invention can be directly used for combining IL-17A protein moleculars, thus can be used for preventing and treating Disease related IL-17A.In addition, other therapeutic agents can be also used simultaneously.
The pharmaceutical composition of the present invention contain safe and effective amount (such as 0.001-99wt%, preferably 0.01-90wt%, more Good ground 0.1-80wt%) above-mentioned monoclonal antibody (or its conjugate) of the invention and pharmaceutically acceptable carrier or tax Shape agent.This kind of carrier includes (but being not limited to)：Salt solution, buffer solution, glucose, water, glycerine, ethanol, and combinations thereof.Medicine system Agent should match with administering mode.The pharmaceutical composition of the present invention can be made into injection form, such as with physiological saline or contain There are glucose and the aqueous solution of other assistant agents to be prepared by conventional method.Pharmaceutical composition such as injection, solution are preferably sterile Under the conditions of manufacture.The dosage of active component is therapeutically effective amount, such as the mg/kg of about 1 microgram/kg body weight-about 5 daily Body weight.In addition, the polypeptide of the present invention can be also used together with other therapeutic agents.
It is that the pharmaceutical composition of safe and effective amount is applied to mammal during using pharmaceutical composition, the wherein safety Effective dose typically at least about 10 micrograms/kg body weight, and 50 mg/kg body weight are in most cases no more than about, compared with Good ground dosage is the mg/kg body weight of about 10 micrograms/kg body weight-about 20.Certainly, specific dosage is also contemplated that administration way The factors such as footpath, patient health situation, within the scope of these are all skilled practitioners technical ability.
Detection applications and kit
The antibody of the present invention can be used for detection to apply, such as detecting sample, so as to provide diagnostic message.
In the present invention, used sample (sample) includes cell, tissue samples and biopsy specimen.The art that the present invention uses Language " biopsy " should include the biopsy of all kinds well known by persons skilled in the art.Therefore the biopsy used in the present invention can wrap Include the tissue samples for example prepared by the puncture of endoscopic procedures or organ or needle puncture biopsy.
The sample used in the present invention includes fixed or preservation cell or tissue sample.
Present invention also offers a kind of kit for referring to the antibody (or its fragment) containing the present invention, at one of the present invention In preference, described kit also includes container, operation instructions, buffer etc..In preference, antibody of the invention can To be fixed on detection plate.
Main advantages of the present invention
(a) antibody of the present invention has excellent bioactivity and specificity, and (ELISA is determined with very high adhesion Its EC50Up to it is about 10-15ng/ml).In addition, there is IL-17A good binding affinity, can be used as targetting IL-17A Antibody.
(b) compared with the antibody of mouse source, humanized antibody of the present invention not only have with the more preferable affinities of IL-17A, and have There is lower immunogenicity.
(c) antibody of the present invention can significantly inhibit the combination of IL-17A and IL-17 acceptors, and not have in itself for mammal Visible toxic side effect.
With reference to specific embodiment, the present invention is expanded on further.It should be understood that these embodiments are merely to illustrate the present invention Rather than limitation the scope of the present invention.The experimental method of unreceipted actual conditions in the following example, generally according to conventional strip Part, such as Sambrook et al., molecular cloning：Laboratory manual (New York:Cold Spring Harbor Laboratory Press, 1989) condition described in, or according to the condition proposed by manufacturer.Unless otherwise indicated, it is no Then percentage and number are percentage by weight and parts by weight.
1 anti-human IL-17A of embodiment mouse monoclonal antibody --- the preparation method of original antibodies
1.1 prepare the hybridoma for producing mouse resource monoclonal antibody
The IL-17A albumen of employment is subcutaneously exempted from as after antigen, with adjuvant emulsion to BALB/c mouse progress multiple spot first Epidemic disease, immune serum potency is monitored, takes the splenocyte of mouse to be merged with myeloma (Sp2/0) cell after reaching requirement, Screened by HAT, the hybridoma polyclonal cells of acquisition.
The screening technique of 1.2 indirect ELISAs --- hybridoma
The polyclonal of high specific combination is filtered out using ELISA detection method, monoclonal culture is carried out, reuses The monoclonal cell strain that ELISA method screening high specific combines；Have by HT1080 cells to IL-6 release experiments, screening The monoclonal cell strain of cell function effect, affinity and half-life period then are analyzed with Biacore methods, finally gives expression IL-17A monoclonal cell.
Test material：
Recombinant human IL-17A, Sino Biological, 12047-HNAS
Test method：
Human IL-17A are configured to 1 μ g/ml coating buffers with CBS, 50 μ L/ holes add ELISA Plate, 2~8 DEG C of coatings 12 Wrapper sheet raffinate is discarded more than hour, adds 3% milk, per the μ L of hole 200, room temperature is closed 1 hour.Added per hole and be no less than 200 μ L PBST wash 1 time, hybridoma supematant is diluted to 100 μ g/ml, then 10 times of dilutions, 10 gradients, 100 μ L/ holes add ELISA Plate. Incubation at room temperature 1 hour, the PBST no less than 200 μ L is added per hole, 3% milk-PBST is added after washing 4 times and dilutes 25000 times The sheep anti-mouse igg Fc (being purchased from Jackson companies) of HRP couplings, 100 μ L/ holes sample-adding.After incubation at room temperature 1 hour, add not per hole Less than 200 μ L PBST, wash 6 times, pat dry.TMB nitrite ions are added, per the μ L of hole 100.Room temperature reaction adds 2M H after 5 minutes2SO4 Terminating reaction, 50 μ L/ holes.The ELISA Plate of stopped reaction is put on ELIASA, 450nm wavelength is read and reads absorbance OD450 values.
Result of the test：
The hybridoma of table 1. compares human IL-17a binding activity
As can be seen from Table 1, in numerous antibody filtered out, mouse source antibody and people caused by hybridoma 14F10/9F6 IL-17a binding activity is higher.
The anti-IL-17A antibody V- gene orders clone of embodiment 2
Based on 5 ' RACE technologies, measure encodes the DNA sequences by the hybridoma 14F10/9F6 mouse antibody variable regions expressed Row.In short, with the RACE of SMART 5 ' synthetic agent box (TAKARA, NO.634859) by manufacturer's directions for use prepare heavy chain and The gene specific cDNA of light chain.PCR primer is analyzed by agarose gel electrophoresis.The variable region size of both heavy chain and light chain is about For 500 base-pairs.The stripe size for reacting gained is suitably expanded into PCR primer and is cloned into carrier pEASY-Blunt In Simple plasmids (the full formula gold in Beijing, No.CB111-02), and it is transformed into Stellar competent escherichia coli cells In (TAKARA, No.636763).By using the bacterium colony PCR screening and clonings of general M13 primers forward or backwards, from each anti- 2-3 should be selected to clone to analyze for DNA sequencing.With Expasy-translate tool (http://web.expasy.org/ Translate/ each sequencing reaction result each cloned) is analyzed.Sequencing result shows the anti-IL17A of 14F10/9F6 expression Antibody V region sequences are as follows：
IL17-HC5SEQ ID NO：1
QVQLQQSGPEVVRPGVSVKISCKGSGYTFTDYAMHWVKQSHAKSLEWIGVIGPYNDNTNYNQKFKGKAT MTVDKSSSTAYMEIARLTSEDSAIYYCARGERYFDYWGQGTTLTVSS
IL17-LC5SEQ ID NO：2
DIQMTQSSSYLSVSLGGRVTITCRASDHINNWLAWYQQKPGNAPRLLIYGATSLETGVPSRFSGSGSGK DYTLSITSLQTEDVATYYCQQYWTIPLTFGAGTKLELK
Wherein, underscore CDR1', CDR2', CDR3'(SEQ ID NO.:10th, amino acid sequence:GAT and SEQ ID NO.:11)。
Table 2:The anti-IL-17A antibody CDR sequence in mouse source
Embodiment 3 is built and chimeric antibody expression
The preparation of 3.1 chimeric antibodies
It is connected, is come with human IgG1 and k constant regions respectively by the mouse 14F10/9F6VH and VL area cDNA for cloning PCR Build chimeric heavy chain and light chain.With 5 ' and 3 ' ends of PCR primer modification Mouse cDNA array, the design of primers increases as each chain Add suitable targeting sequencing, and increase the restriction site for making it possible to be cloned on existing recombinant antibodies expression vector pHB-Fc. The preparation method of pHB-Fc plasmid vectors is as follows：With pcDNA/HA-FLAG (Accession#:FJ524378) carrier is the plasmid that sets out, Human IgG1 or k constant-region sequences are added behind restriction endonuclease EcoRI, it is huge to add the mankind before restriction endonuclease HindIII Cell virus (HCMV) promotes subsequence (Accession#:X17403), behind ampicillin resistance gene, HCMV promotions Son above adds Chinese hamster glutamine synthetase gene (Accession#:X03495).
Host cell used in protein expression is available from the CHO-K1 cells (Cat#CCL-61) of ATCC companies.The cell passes through Cross a series of domestication steps, tame into can be carried out in serum free medium (EX-CELLTM302) suspension culture CHO-K1 it is thin Born of the same parents.Using the cell, the method turned by electricity, the light chain built and heavy chain recombinant expression plasmid are transferred to cell.It is put into training Support case culture 3-5 days.The antibody concentration from CHO-K1 transfection supernatants is measured with indirect ELISA.The CHO- of this display transfection K1 cells secrete about 30mg/L chimeric antibody.
Novartis humanization anti-IL-17 antibody (Novartis mAb) as positive control according to US 7,807, The humanized sequence that 155B2 (AIN 457) is provided is cloned, and is transiently transfected and expressed.
The measure of 3.2 chimeric antibodies
Test material：
Recombinant human IL-17A, Sino Biological, 12047-HNAS
Test method：
Method replaces hybridoma supematant, the rabbit anti-human igg's Fc antibody (Lip river being coupled with HRP with embodiment 1 with chimeric antibody Sun one hundred is difficult to understand to lead to experiment material center) combined work with restructuring human IL-17A instead of the sheep anti-mouse igg Fc of HRP couplings, measure Property.
Result of the test：
The chimeric antibody of table 3. compares human IL-17A binding activity
Sample EC50(ng/ml)
Positive control (Novartis mAb) 35.11
Negative control (PBS) -
Chimeric antibody 29.98
As a result prove, compared with mouse source antibody and positive control, the IL-17A of chimeric antibody and people have stronger adhesion.
The different genera immunological cross-reaction of the chimeric antibody of embodiment 4
In the present embodiment, the IL-17A of anti-IL-17A antibody and different genera antigen-anti-is determined using ELSIA methods Body binding ability.
Test material：
Recombinant human IL-17A, Sino Biological, 12047-HNAS
Recombinant mouse IL-17A, Sino Biological, PBV10159R-010
Recombinant marmoset IL-17A, Sino Biological, 90287-CNAE-100
Test method：
Method replaces hybridoma supematant, the rabbit anti-human igg's Fc antibody (Lip river being coupled with HRP with embodiment 1 with chimeric antibody Positive one hundred logical experiment material center difficult to understand) instead of the sheep anti-mouse igg Fc of HRP couplings, determine and human IL-17A, mouse respectively IL-17A and marmoset IL-17A binding activity.
Result of the test：
The chimeric antibody of table 4. and mouse IL-17A combination results
Sample EC50(ng/ml)
Positive control (Novartis mAb) -
Negative control (PBS) -
Chimeric antibody -
The chimeric antibody of table 5. and human IL-17A combination results
Sample EC50(ng/ml)
Positive control (Novartis mAb) 35.11
Negative control (PBS) -
Chimeric antibody 29.98
The chimeric antibody of table 6. and marmoset IL-17A combination results
Sample EC50(ng/ml)
Positive control (Novartis mAb) 34.1
Negative control (PBS) -
Chimeric antibody 26.39
By chimeric antibody of the present invention it can be seen from table 4-6 in addition to having combination with human il-17 A, while have with marmoset IL-17A With reference to, and with the IL-17A of mouse without combination, and the affinity of chimeric antibody and people, marmoset IL-17A be superior to it is positive right According to.
The preparation of the humanized antibody of embodiment 5
The humanization of antibody uses following methods.In short, by the variable chain sequences of antibody and NCBI Protein Data Banks In available sequences compare, by identifying and analyzing, finally determine be suitably in thereon build CDR transplanting heavy chain and light chain people Framework.
Based on follow-up test and screening, a kind of preferable FR areas are from hu-VH (SEQ ID NO：3), light chain hu- VL(SEQ ID NO：4) humanization FR areas：
hu-VH SEQ ID NO：3
QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYAMHWVRQAPGQRLEWMGWINAGNGNTKYSQKFQGRVT ITRDTSASTAYMELSSLRSEDTAVYYCAR
hu-VL SEQ ID NO：4
DIQMTQSPSSLSASVGDRVTITCRASQGISNSLAWYQQKPGKAPKLLLYAASRLESGVPSRFSGSGSGT DYTLTISSLQPEDFATYYCQQYYSTP
Important amino acid residue in the amino acid residue guarded during repacking according to human antibody FR areas and antibody FR areas, Design improvement site, humanization mutation design is carried out to the variable region of antibody, is expanded using round pcr and builds humanization point and dashed forward Become antibody expressing plasmid.By humanization point mutation antibody expressing plasmid respectively through CHO-K1 (ATC C, NO.CCL-61) cell point Do not express, obtain humanized antibody albumen after purification.Utilize ELISA, acceptor combination Inhibition test, Biacore and cytoactive Detection etc., obtains a kind of very excellent humanized antibody of performance.
VH the and VL sequences of humanized antibody are respectively such as SEQ ID NO.:Shown in 5 and 6：
IL17-HC5-1D4SEQ ID NO：5
QVQLVQSGAEVKKPGASVKVSCKASGYTFTDYAMHWVRQAPGQGLEWMGVIGPYNDNTNYAQKFQGRVT MTVDTSTSTVYMELSSLRSEDTAIYYCARGERYFDYWGQGTTVTVSS
IL17-LC5-1C5SEQ ID NO：6
DIQMTQSPSTLSASVGDRVTITCRASDHINNWLAWYQQKPGKAPKLLIYGATSLETGVPSRFSGSGSGT EYTLTISSLQPDDFATYYCQQYWTIPLTFGQGTKLEIK
Test result indicates that compared with the antibody of mouse source, humanized antibody has more preferable affinity and specificity, sees implementation Example 6.
The measure of the humanized antibody of embodiment 6
Test material：
Recombinant human IL-17A, Sino Biological, 12047-HNAS
Test method：
Method replaces hybridoma supematant, the rabbit anti-human igg's Fc antibody being coupled with HRP with embodiment 1 with humanized antibody (the logical experiment material center difficult to understand of Luoyang one hundred) replaces the sheep anti-mouse igg Fc of HRP couplings, and measure is combined with restructuring human IL-17A Activity.
Result of the test：
The humanized antibody of table 7. compares human IL-17A binding activity
As a result show, process is humanization modified, and the present inventor is unexpectedly obtained to human IL-17A binding activity not Only do not decline, there is the humanized antibody further improved on the contrary.The EC of mouse source antibody50Value is about 3 times of humanized antibody.Together Shi Faxian, compared to positive control antibodies Novartis mAb, the EC of antibody of the present invention50Value is lower, has with human il-17 A more Strong binding activity.
The compatibility detection of the Humanized monoclonal antibodies of embodiment 7
In the present embodiment, Ag-Ab binding kineticses and affinity are determined using BIACORE methods.
Test material：
Recombinant human IL-17A, Sino Biological, 12047-HNAS
Amino coupled kit, GE, BR-1000-50
HBS-EP (10X), GE, BR-1006-69
Human Antibody Captrue Kit, GE, BR-1008-39
Test method：
With amino coupled kit, amino coupled fixes Human on Sereis S Sensor Chip CM5 chips Antibody Capture Antiboy, Anti-Human Capture-CM5 chips.20~30min of equilibrium at room temperature, by chip Load instrument.Dilute antigen with level pad, dilute antigen 1 0nM 5 concentration gradients of initial dilution, and set 2 it is zero dense Spend (i.e. level pad) and a repetition concentration (generally least concentration repeats).Antibody samples are diluted with level pad To experimental work concentration, 2~8 DEG C of sealings are standby.After the completion of sample analysis, from corresponding analysis program to data analysis, really Recognize without obvious reference binding, from Kinetics, 1:1binding modle, Fitting Analysis, obtain the dynamic of sample Mechanics parameter.
Result of the test：
The humanized antibody of table 8. and human IL-17a affinity testing results
Sample Ka(1/Ms) Kd(1/s) KD(M)
Positive control (Novartis mAb) 1.46E+06 1.61E-04 1.11E-10
Chimeric antibody 1.04E+06 1.01E-04 9.70E-11
Humanized antibody 9.56E+06 1.81E-04 1.90E-11
Shown with human il-17 A affinity constant (KD (M)) result, the affinity of humanized antibody of the present invention with it is embedding and Affinity of antibody is suitable, and nearly an order of magnitude all higher than positive control antibodies, has stronger affinity.
In addition, the present inventor also examines to humanized antibody cellular level bioactivity, antigen-binding specificity, internal drug effect Survey is determined, and finds the humanized antibody of the present invention and has good bioactivity and specificity, and can significantly improve IL- The symptom of 17A relevant diseases.
All it is incorporated as referring in this application in all documents that the present invention refers to, it is independent just as each document It is incorporated as with reference to such.In addition, it is to be understood that after the above-mentioned instruction content of the present invention has been read, those skilled in the art can To be made various changes or modifications to the present invention, these equivalent form of values equally fall within the model that the application appended claims are limited Enclose.
Sequence table
<110>The rich biological medicine technology of China（Shanghai）Co., Ltd
<120>A kind of anti-interleukin-17 A antibody, its preparation method and application
<130> P2017-0956
<160> 11
<170> PatentIn version 3.5
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<212> PRT
<213>Artificial sequence (artificial sequence)
<400> 6
Asp Ile Gln Met Thr Gln Ser Pro Ser Thr Leu Ser Ala Ser Val Gly
1 5 10 15
Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Asp His Ile Asn Asn Trp
20 25 30
Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile
35 40 45
Tyr Gly Ala Thr Ser Leu Glu Thr Gly Val Pro Ser Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Thr Glu Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro
65 70 75 80
Asp Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Trp Thr Ile Pro Leu
85 90 95
Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys
100 105
<210> 7
<211> 8
<212> PRT
<213>Mouse (Mus musculus)
<400> 7
Gly Tyr Thr Phe Thr Asp Tyr Ala
1 5
<210> 8
<211> 8
<212> PRT
<213>Mouse (Mus musculus)
<400> 8
Ile Gly Pro Tyr Asn Asp Asn Thr
1 5
<210> 9
<211> 9
<212> PRT
<213>Mouse (Mus musculus)
<400> 9
Ala Arg Gly Glu Arg Tyr Phe Asp Tyr
1 5
<210> 10
<211> 6
<212> PRT
<213>Mouse (Mus musculus)
<400> 10
Asp His Ile Asn Asn Trp
1 5
<210> 11
<211> 9
<212> PRT
<213>Mouse (Mus musculus)
<400> 11
Gln Gln Tyr Trp Thr Ile Pro Leu Thr
1 5

Claims (10)

1. a kind of weight chain variable district of antibody, it is characterised in that described weight chain variable district includes following three complementary determining regions CDR：
SEQ ID NO:CDR1 shown in 7,
SEQ ID NO:CDR2 shown in 8, and
SEQ ID NO:CDR3 shown in 9.
2. a kind of heavy chain of antibody, it is characterised in that described heavy chain has weight chain variable district as claimed in claim 1.
3. a kind of light chain variable district of antibody, it is characterised in that described light chain variable district includes following three complementary determining regions CDR：
SEQ ID NO:CDR1' shown in 10,
Amino acid sequence is GAT CDR2', and
SEQ ID NO:CDR3' shown in 11.
4. a kind of light chain of antibody, it is characterised in that described light chain has light chain variable district as claimed in claim 3.
5. a kind of antibody, it is characterised in that the antibody has：
(1) weight chain variable district as claimed in claim 1；And/or
(2) light chain variable district as claimed in claim 3；
Or the antibody has：Heavy chain as claimed in claim 2；And/or light chain as claimed in claim 4.
6. antibody according to claim 5, it is characterised in that the antibody is selected from：Animal sources antibody, chimeric antibody, people Source antibody or its combination.
7. antibody according to claim 5, it is characterised in that the weight chain variabl area sequence of the antibody such as SEQ ID NO.:Shown in 1 or 5；And/or
The light-chain variable sequence of described antibody such as SEQ ID NO.:Shown in 2 or 6.
8. a kind of recombinant protein, it is characterised in that described recombinant protein has：
(i) weight chain variable district as claimed in claim 1, heavy chain as claimed in claim 2, as claimed in claim 3 light Chain variable region, light chain as claimed in claim 4 or antibody as claimed in claim 5；And
(ii) sequence label of optional assistance expression and/or purifying.
A kind of 9. CAR constructions, it is characterised in that the scFV of the monoclonal antibody antigen calmodulin binding domain CaM of described CAR constructions Section is to specifically bind to IL-17A land, and the scFv have weight chain variable district as claimed in claim 1 and Light chain variable district as claimed in claim 3.
10. a kind of purposes of active component, the active component are selected from the group：As claimed in claim 1 weight chain variable district, such as Heavy chain, light chain variable district as claimed in claim 3 described in claim 2, such as light chain as claimed in claim 4, right It is required that antibody, recombinant protein as claimed in claim 8 or its combination described in 5, it is characterised in that the active component is used Detection reagent or kit are prepared in (a)；And/or (b) prepares the medicine of prevention and/or treatment IL-17A relevant diseases.
CN201710916535.7A 2017-09-30 2017-09-30 Antibody against interleukin 17A, preparation method and application thereof Active CN107522783B (en)

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