CN103588882A - Anti-idiotype antibody for human CD22 antibody, and application thereof - Google Patents

Anti-idiotype antibody for human CD22 antibody, and application thereof Download PDF

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CN103588882A
CN103588882A CN201210286457.4A CN201210286457A CN103588882A CN 103588882 A CN103588882 A CN 103588882A CN 201210286457 A CN201210286457 A CN 201210286457A CN 103588882 A CN103588882 A CN 103588882A
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antibody
sequence
people
heavy chain
sequence table
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CN103588882B (en
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梁瑞安
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CHINA ANTIBODY PHARMACY Co Ltd
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Abstract

The invention discloses an anti-idiotype antibody for a human CD22 antibody, and an application thereof. The anti-idiotype antibody for the human CD22 antibody comprises three antibody heavy chain complementary determining regions and three antibody light chain complementary determining regions, wherein amino acid sequences of the three antibody heavy chain complementary determining regions are shown as 31-35 sites, 50-66 sites and 99-106 sites of a sequence 1 in a sequence table respectively; and amino acid sequences of the three antibody light chain complementary determining regions are shown as 154-168 sites, 184-190 sites and 223-231 sites of a sequence 1 in the sequence table. The anti-idiotype antibody for the human CD22 antibody can inhibit combination of the human CD22 antibody with a natural ligand of human CD22 antigen.

Description

Antiidiotypic antibody and application thereof for people CD22 antibody
Technical field
The present invention relates to antiidiotypic antibody and application thereof for people CD22 antibody.
Background technology
It is not a kind of novelty or complicated theory that using monoclonal antibody (MAb) carries out targeted therapy.Yet the enforcement of this theory relates to the integration of a plurality of subject knowledge technology, comprise antibody molecule design, clone is optimized, and industrialization is produced, and purifies, formulation optimization and each special quality control detect, and the monoclonal antibody medicine of being produced to guarantee can reach the clinical effectiveness of expection.Up to the present, existing up to a hundred the therapeutic monoclonal antibody products for various indications are in each clinical experimental stage.In contrast should, in a large number based on or the theory and the technique means that are derived from antibody all along with the development of monoclonal antibody industry, make rapid progress, to the range of application of expansion currently available products.
One of them derivative, based on immune network theory, is first proposed in 1974 by Niels Jerne (referring to JerneNK:1974.Ann Immunol 125C:373-389).Jerne proposes, and immunity system is one and interacts to regulate the immunoreactive network of control by antiidiotypic antibody.This understanding was further expanded other purposes that is applied to antiidiotypic antibody afterwards.Antiidiotypic antibody is also called Ab2 conventionally, is for the immune antibody Ab1(antibody that to be immunity system produce for exotic antigen) and its idiotope (the unique antigen recognition bunch in antibody molecule surface) is there is to the antibody of specific recognition adsorptive power.Ab2 can be divided into three different classifications: 1) Ab2 α identifies antigen-binding site (ABS) idiotope in addition on original Ab1 antibody; 2) Ab2 β identifies the site of antigen-binding site and simulates its corresponding space structure, thereby forms " the interior image " of exotic antigen; 3) Ab2 γ identifies the site of antigen-binding site equally, but the structure of exotic antigen is not simulated.
It is generally acknowledged, the most attractive is Ab2 β antibody in Ab2 Antibody types, especially when attempting that antigen development is for the effective vaccine of autoantigen or inertia antigen as an alternative with Ab2 β antibody, for example, for the tumor vaccine of tumour specific antigen or tumor associated antigen, and some directeds toward bacteria, the vaccine of the pathogenic agent such as virus and parasite.But the Ab2 antibody of other types is also very important, they can be used for developing relevant detection method, and assistance has production and the clinical potency assessment of the therapeutic Ab1 antibody of pharmaceutical use.
People CD22 antigen maturation or malignant B cell surface expression (
Figure BDA00002003756200011
et al.1989.Leucocyte Typing IV:White cell differentiation antigens.New York, Oxford University Press.P.63-64).CD22 is a kind of Molecular regulator (Hatta et al.1999.Immunogenetics 49:280-286) that prevents immunity system radical response or autoimmune disease.
SM03 is by the derivative a kind of anti-CD22 chimeric antibody (Yang Lei etc. of mouse source antibody RFB4.2006。Recombinate structure and the evaluation of anti-B cell lymphoma chimeric antibody.Chinese Journal of New Drugs, 15 3 phases of volume: 186-192), and entered clinical experimental stage (Li et al.2012.Landes BioScience J 4 (2): 256-266) in the treatment of non Ho lymphoma.Because SM03 monoclonal antibody be take mature B cell as target and suppresses its function, this monoclonal antibody product application has expanded to the treatment of other autoimmune disease indication, especially rheumatoid arthritis (RA) and systemic lupus erythematous (SLE).
The humanization modified further application that advances anti-CD22 antibody that SM03 monoclonal antibody is used framework to reinvent technology to carry out (beam Ruian, etc.2006。Application antibody " framework is reinvented " technique construction humanized antibody fSM03.Chinese Journal of New Drugs, 15 21 phases of volume: 1832-1836).Through humanization modified SM03 monoclonal antibody, be named as SM06.SM03 and SM06 be for the same site on people CD22 antigen, and have similar avidity.Yet in aminoacid sequence and structure, SM03 only has antigen recognition site identical with SM06, this same section consists of their complementary determining region (CDR) sequences separately.
SM03 and SM06 can specific recognition adsorb people CD22 antigen, and in conjunction with rear antibody-antigenic complex, obvious internalization can occur.This characteristic makes the biological activity assay of SM03 and SM06 monoclonal antibody product become very difficult.Other also has similar difficulty for antibody class product that can internalization antigen, constant chain for example, CD33 etc.In addition, CLINICAL PHARMACOKINETIS STUDY ON needs a sane method of convenience to measure free SM03, SM06(and RFB4) and their various derivatives (scFv single-chain antibody, Fab, Diabody, immunotoxin, medicine combination etc.) content, in order to assess its serum half-life.Because solubility Free form CD2 2 is not general, and exogenous CD22 is more unstable, and a kind of suitable antiidiotypic antibody for SM03 and derivative thereof just seems and has using value.
Summary of the invention
A technical problem to be solved by this invention is to provide the antiidiotypic antibody for people CD22 antibody, this antiidiotypic antibody specificity is for people CD22 antibody (" CD22 antibody family "), can adsorb " CD22 antibody family ", its absorption fragment is to " CD22 antibody family " antibody molecule variable region tool specific reaction, and its absorption segment is specifically to " CD22 antibody family " antibody molecule antigen-binding site (ABS) tool specific reaction.This antiidiotypic antibody can prevent the natural part people CD22 of people CD22 antibody and its antigen to be combined.
The CD22 of people described in the present invention antibody is and contains following 3 heavy chain of antibody complementary determining regions and 3 light chain of antibody complementary determining regions and the antibody that can be combined with people CD22 antigen-specific: heavy chain CDR 1sequence be IYDMS, heavy chain CDR 2sequence be YISGGGTTYYPDTVKG, heavy chain CDR 3sequence be HSGYGSSYGVLFAY, light chain CDR 1sequence be RASQDISNYLN, light chain CDR 2sequence be YTSILHS, light chain CDR 3sequence be QQGNTLPWT.Described people CD22 antibody comprises mouse source antibody (as RFB4), chimeric antibody (as SM03), people's antibody and humanized antibody (as SM06) and their various derivative are as scFv single-chain antibody, double antibody, bi-specific antibody, antibodies body and other various forms antibody fusion proteins etc., general designation " CD22 antibody family ".
Antiidiotypic antibody for people CD22 antibody provided by the present invention, it comprises 3 heavy chain of antibody complementary determining regions and 3 light chain of antibody complementary determining regions (6 frame regions in Fig. 3); The aminoacid sequence of described 3 heavy chain of antibody complementary determining regions is respectively as the 31-35 position (CDR of sequence in sequence table 1 1), 50-66 position (CDR 2) and 99-106 position (CDR 3), the aminoacid sequence of described 3 light chain of antibody complementary determining regions is respectively as the 154-168 position (CDR of sequence in sequence table 1 1), 184-190 position (CDR 2) and 223-231 position (CDR 3).
Wherein, above-mentioned 6 complementary determining regions formation of this antiidiotypic antibody and the structure of the idiotype part specific combination of people CD22 antibody.Following embodiment 1 experimental results show that, the single-chain antibody that contains above-mentioned 6 complementary determining regions can with 3 kinds of people CD22 antibody (RFB4, SM03 and SM06) specific binding, the sequence same section of these 3 kinds of people CD22 antibody only has 3 heavy chain of antibody complementary determining regions and 3 light chain of antibody complementary determining regions.Described people CD22 antibody for containing 3 light chain of antibody complementary determining regions shown in sequence 11-13 in 3 heavy chain of antibody complementary determining regions shown in sequence 8-10 in ordered list and sequence table can with the antibody of people CD22 specific binding, as RFB4, SM03 and SM06.Wherein, sequence 8 is CDR 1sequence, sequence 9 is CDR 2sequence, sequence 10 is CDR 3sequence, sequence 10 is CDR 1sequence, sequence 11 is CDR 2sequence, sequence 12 is CDR 3sequence.
Further, the described antiidiotypic antibody for people CD22 antibody comprises variable region of heavy chain and variable region of light chain; The aminoacid sequence of described variable region of heavy chain is as the 1-116 position of sequence in sequence table 1, and the aminoacid sequence of described light chain chain variable region is as the 131-240 position of sequence in sequence table 1.
Further, the described antiidiotypic antibody for people CD22 antibody specifically can be:
A) aminoacid sequence of heavy chain is the sequence 2 in sequence table, and the aminoacid sequence of light chain is the antiidiotype mouse IgG 2a/kappa immunoglobulin molecules IdmG2a/k that comprises light CH sequence of the following embodiment 3 of the sequence 3(in sequence table);
B) aminoacid sequence except described 3 heavy chain of antibody complementary determining regions in the sequence in sequence table 2 is carried out to the replacement of amino-acid residue, disappearance or interpolation, and/or the aminoacid sequence except described 3 light chain of antibody complementary determining regions in the sequence in sequence table 3 is carried out to the replacement of amino-acid residue, the antiidiotypic antibody for people CD22 antibody that disappearance or interpolation obtain, be preferably the aminoacid sequence except the variable region of heavy chain of 1-117 position in described sequence 2 is carried out to the replacement of amino-acid residue, disappearance or interpolation, and/or the aminoacid sequence except the light variable region of 1-111 position in described sequence 3 is carried out to the replacement of amino-acid residue, the antiidiotypic antibody for people CD22 antibody that disappearance or interpolation obtain,
C) single-chain antibody shown in the sequence in sequence table 1 (single-chain antibody shown in Fig. 3 in following embodiment 1 and 2), or in sequence table, aminoterminal or the carboxyl terminal of sequence 1 adds the histidine-tagged fusion rotein obtaining;
D) by the sequence in sequence table 1, the aminoacid sequence except described 3 heavy chain of antibody complementary determining regions and described 3 light chain of antibody complementary determining regions carries out the antiidiotypic antibody for people CD22 antibody that replacement, disappearance or the interpolation of amino-acid residue obtain, and is preferably the connection peptides sequence shown in the 117-130 position of sequence 1 in described sequence table is carried out to the antiidiotypic antibody for people CD22 antibody that replacement, disappearance or the interpolation of amino-acid residue obtain;
E) aminoacid sequence of heavy chain is the sequence 6 in sequence table; The aminoacid sequence of light chain is the IdGmFdA of the following embodiment 5 of the sequence 3(in described sequence table, and what Figure 14 showed is the heavy chain amino acid sequence of IdGmFdA);
F) aminoacid sequence of heavy chain is the sequence 7 in sequence table; The aminoacid sequence of light chain is the IdGmFdG of the following embodiment 5 of the sequence 3(in described sequence table, and what Figure 15 showed is the heavy chain amino acid sequence of IdGmFdG);
G) aminoacid sequence of heavy chain is the sequence 4 in sequence table; The aminoacid sequence of light chain is the IdGmD of the following embodiment 5 of the sequence 3(in described sequence table, and what Figure 12 showed is the heavy chain amino acid sequence of IdGmD);
H) aminoacid sequence of heavy chain is the sequence 5 in sequence table; The aminoacid sequence of light chain is the IdDmD of the following embodiment 5 of the sequence 3(in described sequence table, and what Figure 13 showed is the heavy chain amino acid sequence of IdDmD).
Wherein, sequence 1 in sequence table is comprised of 240 amino-acid residues, sequence 2 in sequence table is comprised of 447 amino-acid residues, sequence 3 in sequence table is comprised of 219 amino-acid residues, sequence 4 in sequence table is comprised of 502 amino-acid residues, sequence 5 in sequence table is comprised of 408 amino-acid residues, and the sequence 6 in sequence table is comprised of 305 amino-acid residues, and the sequence 7 in sequence table is comprised of 264 amino-acid residues.
Wherein, antiidiotypic antibody for people CD22 antibody can be expressed as membrane albumen by variform, these forms comprise: cross-film IgD antibody (IdGmD of following embodiment 5 and IdDmD), or with the single-chain antibody scFv of glycoprotein A (IdGmFdA of following embodiment 5) or GPI protein fusion (IdGmFdG of following embodiment 5), Fab, Fab ', F (ab ') 2.
Above-mentioned any nucleic acid molecule for the antiidiotypic antibody of people CD22 antibody of encoding belongs to protection scope of the present invention.
Wherein, described nucleic acid molecule can be DNA, as cDNA, genomic dna or recombinant DNA; Described nucleic acid molecule can be also RNA, as mRNA or hnRNA etc.
Described nucleic acid molecule specifically can be following 1)-6) in arbitrary described gene:
1) encoding sequence of the heavy chain of above-mentioned a) described antibody is the sequence 9 in sequence table, and the above-mentioned a) encoding sequence of the light chain chain of described antibody is the sequence 10 in sequence table;
2) above-mentioned c) encoding sequence of described antibody is the sequence 8 in sequence table;
3) above-mentioned e) described antibody the encoding sequence of heavy chain be the sequence 13 in sequence table, above-mentioned e) encoding sequence of light chain chain of described antibody is the sequence 10 in sequence table;
4) above-mentioned f) described antibody the encoding sequence of heavy chain be the sequence 14 in sequence table, above-mentioned f) encoding sequence of light chain chain of described antibody is the sequence 10 in sequence table;
5) above-mentioned g) described antibody the encoding sequence of heavy chain be the sequence 11 in sequence table, above-mentioned g) encoding sequence of light chain chain of described antibody is the sequence 10 in sequence table;
6) above-mentioned h) described antibody the encoding sequence of heavy chain be the sequence 12 in sequence table, above-mentioned h) encoding sequence of light chain chain of described antibody is the sequence 10 in sequence table.
Wherein, the sequence 1 in sequence table is comprised of 240 amino-acid residues, the DNA encoding of the sequence 8 in sequence table, and sequence 8 is comprised of 720 Nucleotide; Sequence 2 in sequence table is comprised of 447 amino-acid residues, the DNA encoding of the sequence 9 in sequence table, and sequence 9 is comprised of 1344 Nucleotide; Sequence 3 in sequence table is comprised of 219 amino-acid residues, the DNA encoding of the sequence 10 in sequence table, and sequence 10 is comprised of 660 Nucleotide; Sequence 4 in sequence table is comprised of 502 amino-acid residues, the DNA encoding of the sequence 11 in sequence table, and sequence 11 is comprised of 1509 Nucleotide; Sequence 5 in sequence table is comprised of 408 amino-acid residues, the DNA encoding of the sequence 12 in sequence table, and sequence 12 is comprised of 1227 Nucleotide; Sequence 6 in sequence table is comprised of 305 amino-acid residues, the DNA encoding of the sequence 13 in sequence table, and sequence 13 is comprised of 918 Nucleotide; Sequence 7 in sequence table is comprised of 264 amino-acid residues, the DNA encoding of the sequence 14 in sequence table, and sequence 14 is comprised of 795 Nucleotide.
The biomaterial of following a1, a2 or a3 also belongs to protection scope of the present invention:
A1. the expression cassette, recombinant vectors, recombinant microorganism or the recombinant cell lines that contain above-mentioned any nucleic acid molecule;
A2. express above-mentioned any recombinant expression vector, recombinant microorganism or recombinant cell lines for the antiidiotypic antibody of people CD22 antibody;
A3. express recombinant expression vector, recombinant microorganism or the recombinant cell lines of any gene.
In above-mentioned biomaterial, expression cassette described in a1, refer to and can in host cell, express above-mentioned any DNA for the antiidiotypic antibody of people CD22 antibody, this DNA not only can comprise above-mentioned any promotor for the antiidiotypic antibody genetic transcription of people CD22 antibody of startup, also can comprise and stop above-mentioned any terminator for the antiidiotypic antibody genetic transcription of people CD22 antibody.Further, described expression cassette also can comprise enhancer sequence.Recombinant microorganism described in a2 and a3 specifically can be yeast, bacterium, algae and fungi.Recombinant cell lines described in a2 and a3 does not comprise the reproductive material of plant, specifically can be surface of cell membrane and expresses above-mentioned any recombinant cell lines (cell strain) for the antiidiotypic antibody of people CD22 antibody.
The present invention also provides above-mentioned any following a plurality of purposes for the antiidiotypic antibody of people CD22 antibody:
B1. detect the reagent of people CD22 antibody concentration, its activeconstituents is above-mentioned any antiidiotypic antibody for people CD22 antibody;
B2. the reagent that detects the human antimouse antibody reaction being caused by people CD22 antibody, its activeconstituents is above-mentioned any antiidiotypic antibody for people CD22 antibody;
B3. the reagent that detects the anti-chimeric antibody reaction of the people who is caused by people CD22 antibody, its activeconstituents is above-mentioned any antiidiotypic antibody for people CD22 antibody;
B4. detect the reagent of the anti-human antibody response of people being caused by people CD22 antibody, its activeconstituents is above-mentioned any antiidiotypic antibody for people CD22 antibody;
B5. detect the reagent of the antibody-mediated Complement Dependent cytotoxic reaction of people CD22, its activeconstituents is that surface of cell membrane is expressed above-mentioned any recombinant cell lines for the antiidiotypic antibody of people CD22 antibody;
B6. the reagent that detects the antibody-mediated antibody-dependant cell mediated cell poison reaction of people CD22, its activeconstituents is that surface of cell membrane is expressed above-mentioned any recombinant cell lines for the antiidiotypic antibody of people CD22 antibody;
B7. detect the bioactive reagent of people CD22 antibody, its activeconstituents is that surface of cell membrane is expressed above-mentioned any recombinant cell lines for the antiidiotypic antibody of people CD22 antibody;
B8. above-mentioned any antiidiotypic antibody for people CD22 antibody detects the application in people CD22 antibody concentration reagent in preparation;
B9. above-mentioned any antiidiotypic antibody for people CD22 antibody detects the application in the human antimouse antibody reaction reagent being caused by people CD22 antibody in preparation;
B10. above-mentioned any antiidiotypic antibody for people CD22 antibody detects the application in the anti-chimeric antibody reaction reagent of people being caused by people CD22 antibody in preparation;
Above-mentioned any antiidiotypic antibody for people CD22 antibody of b11 detects the application in the anti-human antibody response reagent of people being caused by people CD22 antibody in preparation;
B12. surface of cell membrane is expressed above-mentioned any reconstitution cell for the antiidiotypic antibody of people CD22 antibody and is tied up to preparation and detect the application in the antibody-mediated Complement Dependent cytotoxic reaction reagent of people CD22;
B13. surface of cell membrane is expressed above-mentioned any reconstitution cell for the antiidiotypic antibody of people CD22 antibody and is tied up to preparation and detect the application in the antibody-mediated antibody-dependant cell mediated cell poison reaction reagent of people CD22;
B14. surface of cell membrane is expressed above-mentioned any reconstitution cell for the antiidiotypic antibody of people CD22 antibody and is tied up to preparation and detect the application in people CD22 antibody bioactive agents;
B15. detect people CD22 antibody to the penetrance of tumour cell and/or adsorptivity reagent, its activeconstituents is arbitrary described antibody in claim 1-3.
B16. above-mentioned any antiidiotypic antibody for people CD22 antibody detects people CD22 antibody to the application in the penetrance of tumour cell and/or adsorptivity reagent in preparation.
Wherein, above-mentioned b1 and b8 specifically can be and using above-mentioned any antiidiotypic antibody for people CD22 antibody as diagnostic reagent, monitor in immunotherapy the content of people CD22 antibody in blood samples of patients, the pharmacokinetics behavior of the monitoring people CD22 antibody drug of injecting.Can detect as follows " CD22 antibody family " (people CD22 antibody) anti-body contg in sample serum:
1) with the coated ELISA enzyme plate of above-mentioned any antiidiotypic antibody for people CD22 antibody, above-mentioned any comprises scFv single-chain antibody for people CD22 antibody, or complete IgG, or mouse IgG 2a/kappa kind type, and other isotype;
2) on enzyme plate, add test serum sample;
3) on enzyme plate, add anti-human Fc specific antibody such as peroxidase labelling such two anti-and;
4) according to two of absorption, resist how much record the anti-human CD22 anti-body contg in test serum.
In above-mentioned b7 and b14, surface of cell membrane is expressed above-mentioned any recombinant cell lines for the antiidiotypic antibody of people CD22 antibody can be by antibody-mediated, there is Complement Dependent cytotoxic reaction (CDC) or antibody-dependant cell mediated cell poison reaction (ADCC), in order to the biological activity of quick estimation people CD22 antibody (as SM03 or SM06), thereby reach the object of quality control.Utilize surface of cell membrane to express above-mentioned any recombinant cell lines for the antiidiotypic antibody of people CD22 antibody, the detection method by Complement Dependent cytotoxic reaction evaluator CD22 antibody biologic activity, specifically can comprise following steps:
1) surface of cell membrane being expressed to above-mentioned any recombinant cell lines and anti-CD22 antibody for the antiidiotypic antibody of people CD22 antibody hatches jointly;
2) in mixture, add complement proteins;
3) hatch the effect of measuring its Complement Dependent cellulotoxic effect (CDC) after suitable time.
Utilize surface of cell membrane to express above-mentioned any recombinant cell lines for the antiidiotypic antibody of people CD22 antibody, the detection method by antibody-dependant cell mediated cell toxic effect evaluator CD22 antibody biologic activity, specifically can comprise following steps:
1) surface of cell membrane being expressed to above-mentioned any recombinant cell lines and anti-CD22 antibody for the antiidiotypic antibody of people CD22 antibody hatches jointly;
2) add peripheral blood lymphocytes (PBMC);
3) hatch the effect of measuring its antibody-dependant cell mediated cell toxic effect (ADCC) after suitable time.
Above-mentioned b2-b4, b9-b11 specifically can be using above-mentioned any antiidiotypic antibody for people CD22 antibody as HAHA, the positive control in the anti-antibody immune response researchs such as HACA or HAMA.Because these anti-antibodys react the clinical effectiveness (Gruber van Haarlem et al.2000.Cancer Res.60:1921-1926) that may affect patient, the anaphylaxis of inclusive NAND expection is relevant, cause significant pharmacokinetics behavior change and affect in the body of injection of antibodies medicine distributing, treatment option that therefore can antagonist immunotherapy to its monitoring produces material impact.The method that the anti-chimeric antibody reaction of the people who is caused by people CD22 antibody in the individuality of chimeric or Humanized anti-human CD22 monoclonal antibody (HACA) or the anti-human antibody response of people (HAHA) have been injected in detection provided by the present invention comprises following steps:
1) from accepting the individuality of injection, collect serum sample;
2) coated CD22 antigen on ELISA enzyme plate, the anti-CD22 free antibodies that simultaneously adds sensitive concentration, the serum sample that adds different multiples dilution, does not add the hole of serum sample as negative control, adds the hole of the anti-CD22 idiotype antibody of finite concentration as positive control;
3) after cleaning, add the anti-human IgG Fc bis-of HRP mark anti-, detect the absorption signal of the anti-CD22 antibody of sensitive concentration to CD22 antigen;
4) existence that detects anti-CD22 antiidiotypic antibody in serum sample whether, as there is the generation of having proved HACA in individuality or HAHA reaction in anti-CD22 antiidiotypic antibody.
In above-mentioned b15 and b16, can be by above-mentioned any antiidiotypic antibody for people CD22 antibody (antiidiotype mouse IgG 2a/kappa immunoglobulin molecules of CH sequence as light in comprising of following embodiment 3,) application tumor tissue section immunohistochemical analysis, research " CD22 antibody family " medicine is to the penetrance of tumour cell and/or adsorptivity.
The invention provides the anti-human CD22 antibody of a species specificity identification and (comprise mouse source antibody (as RFB4), chimeric antibody (as SM03), people's antibody and humanized antibody (as SM06) and their various derivative are as scFv single-chain antibody, double antibody, bi-specific antibody, antibodies body and other various forms antibody fusion proteins etc., general designation " CD22 antibody family ") antiidiotypic antibody of antigen-binding site (ABS), for the antiidiotypic antibody of people CD22 antibody.Antiidiotypic antibody for people CD22 antibody provided by the invention, can be applicable to concentration and the biological activity of identification and assessment " CD22 antibody family " antibody, and for " CD22 antibody family " anti-body contg of clinical quantitative analysis serum.The present invention also can be applicable to detect the human antimouse antibody reaction (HAMA) that in " CD22 antibody family " monoclonal antibody medicine clinical trial, patient may occur, the anti-chimeric antibody reaction of people (HACA) and the anti-human antibody response of people (HAHA).Meanwhile, also provide the method that can express for the clone of the antiidiotypic antibody of people CD22 antibody that builds; And the Complement Dependent cytotoxic reaction (CDC) that this type of cell is mediated for detection of " CD22 antibody family " monoclonal antibody product and/or antibody-dependant cell mediated cell poison reaction (ADCC), realized biological activity assay and the assessment of monoclonal antibody product.
Accompanying drawing explanation
Fig. 1 expresses the phage #1-#3 of scFv single-chain antibody to mouse source CD22 antibody (RFB4), the chimeric CD22 antibody of people mouse (SM03), the specific adsorption of humanization CD22 antibody (SM06).
Fig. 2 A is expressed heavy chain complementary determining region (CDR) sequence of phage #1-#3.
Fig. 2 B is the expressed light chain CDR sequence of phage #1-#3.
Fig. 3 is to RFB4, SM03, and SM06 has the scFv sequence of complete sequence that the phage #3 of specific recognition absorption shows.Institute's frame region is for supplementing determining area.ScFv single-chain antibody is configured to variable region of heavy chain-catenation sequence-variable region of light chain.Connection aminoacid sequence used is G4-S-G-S-G-S-S-G4.
Fig. 4 is that the capacitive scFv that phage #3 expresses can suppress the absorption of SM03 antibody to Raji cell in flow cytometry experiment.Three peaks are from left to right respectively blank, SM03+scFv(phage#3) and SM03.
Lymphoma Pharmacokinetic Profile (the 360mg/m that the SM03 that Fig. 5 records for the capacitive scFv that adopts phage #3 to express treats 2dosage group, intravenous injection, weekly, successive administration 4 weeks).
Fig. 6 can DNA amplification carrier for expression mouse IgG 2a/kappa immunoglobulin (Ig) antiidiotypic antibody IdmG2a/'s.
Fig. 7 be IdmG2a/k purification antibody reduction and non-reduced situation under SDS-PAGE electrophorogram.In figure, 1 and 2 is reduction electrophoresis result, and 3 is molecular weight standard (BenchMark tMmolecular weight of albumen standard, American I nvitrogen company product, batch: 688736), 4 and 5 is non-reduced electrophoresis result.
Fig. 8 is that IdmG2a/k can be by SM03 specific recognition, and other antibody can not adsorb (CD20 antibody (hAnti-CD20), CD147 antibody (hAnti-CD147), TNF antibody (Infliximab)).
Fig. 9 is that Flow Cytometry result shows that antiidiotype mouse IgG antibody I dmG2a/k can effectively prevent the CD22 antigen of SM03 absorption Raji cell surface.
Figure 10 is mouse source CD22 antibody RFB4, and chimeric antibody SM03 and the humanized antibody SM06 that uses framework to reinvent technology can be adsorbed onto with the SM03 antibody competition of HRP mark antiidiotype mouse IgG antibody I dmG2a/k
The patients with SLE Pharmacokinetic Profile of Figure 11 for using SM03 monoclonal antibody product to treat.The Plasma Concentration of SM03 is recorded by ELISA method, and IdmG2a/k is as solid phase capture antibodies.
Figure 12 is for merging the CH3 region of the cross-film region sequence of mouse source IgD and " mouse source antiidiotype IgG antibody " cross-film that obtains for the heavy chain amino acid sequence of the antiidiotypic antibody IdGmD of people CD22 antibody.
Figure 13 is for obtaining IgG2a heavy chain the heavy chain amino acid sequence of the antiidiotypic antibody IdDmD for people CD22 antibody of cross-film with the replacement of mouse source IgD sequence.
Figure 14 is the heavy chain amino acid sequence of the antiidiotypic antibody IdGmFdA for people CD22 antibody of the form expression cross-film by membrane Fab-glycoprotein fusion rotein.
Figure 15 be with Fab-GPI fixedly the form of fusion rotein express the heavy chain amino acid sequence of the antiidiotypic antibody IdGmFdG for people CD22 antibody of " mouse source antiidiotype IgG " antibody.
Figure 16 is expression on the transfectional cell mesentery of the various fusion rotein forms of antiidiotype mouse IgG antibody.
Five pictures are from top to bottom followed successively by the SP2/0 cell system that does not add SM03, adding SM03 is the SP2/0 cell system of 1 μ g/ml to final concentration, adding SM03 is the recombined engineering cell system of the expression IdDmD of 0.1 μ g/ml and 1 μ g/ml to final concentration, add SM03 to final concentration be 0.1 μ g/ml, the recombined engineering cell system of the expression IdGmFdA of 0.1 μ g/ml and 1 μ g/ml, add SM03 to final concentration be 0.1 μ g/ml, the recombined engineering cell system of the expression IdGmFdG of 0.1 μ g/ml and 1 μ g/ml.
Figure 17 is the antibody-mediated comparison (three pictures are from top to bottom followed successively by IdGmD, IdGmFdA, IdGmFdG) of expressing the Complement Dependent cytotoxic reaction (CDC) of various antiidiotype mouse IgG antibody fusion protein transfectional cell series for cross-film of SM03
Embodiment
For the ease of more fully understanding the present invention, describe, some basic definitions are as follows in this declaration definition:
" immunoglobulin (Ig) " this definition herein refers to and consists of one or more polypeptide chain, most of by the related gene coded protein molecule of immunoglobulin (Ig).The immunoglobulin gene coding having cracked comprises kappa, lamda, alpha, gamma (IgG1, IgG2, IgG3, IgG4), delta, these constant region genes of epsilon and mu, and countless immune globulin variable region genes.Complete immunoglobulin (Ig) " light chain " (by 214 Amino acid profiles, the about 25Kd of molecular weight) consists of kappa or the lamda constant region genes encoding of N-terminal variable region gene (approximately 110 amino acid) and hydroxyl terminal.Similarly, complete immunoglobulin (Ig) " heavy chain " is (by 446 Amino acid profiles, the about 50Kd of molecular weight) by variable region gene, encoded in (approximately 116 amino acid) and describe one of them constant region genes encoding formation above, as gamma constant region gene (approximately 300 amino acid) coding forms IgG molecule.
Unless specialize, " antibody " used herein this definition be widely used for referring to complete antibody molecule with and derivative.These derivatives have at least comprised one section of variable region segment from light chain immunoglobulin or heavy chain, and comprise such as F (ab ') 2, Fab, Fab ', Fd, Fabc, scFv, double antibody, monospecific antibody light chain, monospecific antibody heavy chain, the chimeric fusions of antibody chain, the antibody molecule segment that bispecific antibody and other similar molecules are such.
" chimeric antibody " used herein this definition refers to the protein molecular being formed by the immunoglobulin light variable region of heavy chain of inhuman generic kind biology and human immunoglobulin molecule constant region Recombinant design.
" humanization " used herein this definition only refers to that the part from the complementary determining region, immunoglobulin molecules variable region (CDR) of inhuman generic kind biology retains, remaining main variable region part and all constant region part are all come the source for people, the protein molecular forming through Recombinant design.
" idiotype " used herein this definition refers on antibody molecule and determines the specific special segment of antigen.Idiotype segment is positioned at monoclonal antibody part, for it description conventionally the common participation based on antibody molecule heavy chain light chain form antigen-binding site.
" isotype " used herein this definition refers to that the antigen-specific by immunoglobulin molecules determines, according to heavy chain classification and subclass, and light chain type immunoglobulin molecules type and the hypotype thereof of being divided.For example, four of IgG isotypes are IgG 1, IgG 2, IgG 3, IgG 4.
" CD22 antibody family " described herein is derivative by mouse source antibody RFB4.Its derivative chimeric antibody (SM03) is the same with original mouse source antibody with humanized antibody (SM06), for the B site tool specificity on people CD22 antigen.Use the clinical trial of SM03 treatment B cell lymphoma and other autoimmune diseases to start to walk.In clinical practice, in order to meet the demand of measuring clinically " CD22 antibody family " product (as the SM03 monoclonal antibody) content of injecting in serum, build and qualitative a kind of antiidiotypic antibody, as ELISA reagent, measure " the CD22 antibody family " product concentration in patients serum.In addition, the antiidiotypic antibody producing can also be used in and in ELISA method, weigh HACA or HAHA reaction, also or in contrast antibody and as the competitive antibody in diagnostic reagent estimation patients serum.
The antiidiotypic antibody of single-chain antibody form is by the mouse through SM03 immunity, and produces by the method for phage display.Concrete grammar is for to extract spleen cell with it from injecting the mouse of anti-CD22 chimeric antibody SM03, and separating mRNA, then degeneracy flank variable region primer amplification weight chain variable region sequence.According to standard step, gained variable region sequences is introduced to scFv single-chain antibody phage display library afterwards.Through several take turns the screening carried out with SM03 and RFB4 antibody and eliminate after, select for SM03 RFB4 and the specific phage of SM06 tool, and the corresponding variable region sequences of selected phage display.In showing the phage of antibody variable region sequence, to select mouse source, chimeric and humanization " CD22 antibody family " has the displaying phage of high-affinity.
Antiidiotypic antibody sequence is expressed with the form of scFv single-chain antibody occlusion body at first in intestinal bacteria, understands afterwards sex change and refolding; The scFv single-chain antibody of tool activity is used as ELISA reagent and detects the serum SM03 content in clinical trial.Concise and to the point step is as follows, use exactly the coated ELISA enzyme plate of antiidiotype scFv single-chain antibody (scFv) of refolding, to after patients serum's dilution of SM03 treatment, add again, through hatching, clean, SM03 antibody in patients serum will be combined with coated antiidiotype scFv single-chain antibody, and its specific binding can develop the color with goat anti-human igg-Fc specific antibody (U.S. Jackson ImmunoResearch company product) of HPR mark.Yet antiidiotype scFv single-chain antibody has unsettled tendency, and the mode producing from bacterium occlusion body can cause the diversity of protein denaturation and refolding, makes detected result consistence not high.In addition, the unstable of antiidiotype scFv single-chain antibody, makes its storage and batch preparation of checking subsequently become very difficult.
And on the other hand, well-known, complete immunoglobulin molecules can keep the stability of several months and even several years under suitable condition of storage, can there is not considerable change in its antibody activity and quality yet.In order to create the stable and consistent detection method of result, estimate the CD22 antibody in serum, and HACA and HAHA reaction, the variable region sequences of antiidiotype scFv single-chain antibody is used to build a kind of intactly immunoglobulin molecules.Because the chimeric antibody in " CD22 antibody family " and humanized antibody all have the constant region sequence of human IgG1's heavy chain and kappa light chain, they therefore can by ELISA conventionally the anti-human IgG Fc of HRP mark specific antibody used (or similarly combination) identify.Therefore, designed antiidiotypic antibody immunoglobulin molecules should not cause with meeting the human IgG constant region sequence of cross reaction.Mouse IgG 2a/kappa antibody (mouse source antiidiotype IgG antibody) constant region sequence not can with anti-human Fc antibody generation cross reaction, so be selected to build complete antiidiotypic antibody immunoglobulin molecules.It should be noted that other isotype or also can adopt from the constant region sequence of other species.According to designed antiidiotypic antibody immunoglobulin (Ig), produced the express cell system that productive rate reaches 30ug/ml.Owing to producing expression vector that express cell system adopts with the DHFR gene that can increase, this productive rate can further increase according to general amplification-cloning process in case there is a need.Yet, present stage productive rate enough meet to have produced the mouse source antiidiotype IgG antibody of consistent batch, and detect for pharmacokinetic and HACA or HAHA.Concise and to the point step is as follows, is exactly with the coated ELISA enzyme plate of mouse source antiidiotype IgG antibody, then adds the patients serum of use " CD22 antibody family " antibody drug treatment, through hatching, can not be cleaned totally with the albumen of " mouse source antiidiotype IgG antibody " absorption.And anti-CD22 antibody in serum can be specifically and be coated with " mouse source antiidiotype IgG antibody " combination, and develop the color with HRP mark goat anti-human igg Fc specific antibody.Because the mouse source constant region sequence of " mouse source antiidiotype IgG antibody " can't detect the HRP traget antibody generation cross reaction of use, can't there is the problem of high background signal in this detection method.This detection method empirical tests has high performance reproducibility, sensitivity and specificity.Same method is applied to identification and the avidity of estimation " CD22 antibody family " by expansion.Application " mouse source antiidiotype IgG antibody ", as positive control, is set up the Biacore analytical procedure of HACA and the HAHA of serum sample, and realizing the immune response of patients serum's anti-antibody also becomes possibility.In addition, " mouse source antiidiotype IgG antibody " also can be employed tumor tissue section's immunohistochemical analysis, and research " CD22 antibody family " medicine is to the penetrance of tumour cell and adsorptivity.The generation of " mouse source antiidiotype IgG antibody " that can specific binding target antibody, for security and the pharmacokinetic property of the target antibody of assessment injection in high sensitivity medicine on clinical meaning provides raw material reagent.Therefore constructed " the mouse source antiidiotype IgG antibody " of the present invention obtain the diagnostic test reagent that card is worth for a kind of tool important practical, for studying the patient clinical sample of accepting SM03 immunotherapy.
One aspect of the present invention is the resistance inhibitor action supplying method " CD22 antibody family " being adsorbed its antigen for detecting " mouse source antiidiotype IgG antibody ".Another aspect is for detecting " mouse source antiidiotype IgG antibody ", to catch and detect the ability supplying method of adsorbed idiotype antibody.Darker one deck implication is for detecting " mouse source antiidiotype IgG antibody " adsorptive power supplying method to its idiotype antibody (anti-CD22 antibody).Yet, be also for detecting " CD22 antibody family " content supplying method in serum sample on the other hand.The present invention points to too one and detects HAMA with antibody that this patent is described, the method for HACA and HAHA reaction.
Another kind of application of the present invention is to utilize antiidiotypic antibody to build non-internalization cross-film to express the special absorption segment of SM03-engineering cell system.The clone of building can be used for setting up assessment SM03 monoclonal antibody and derivative thereof as RFB4, the detection method of the biological effects such as SM06.The foundation of this method means with the detection that similar approach realizes the antibody biological effectiveness that absorption can internalization surface antigen and is of universal significance.
Below test illustration listed for purposes of the present invention is described, but do not represent this type of application that is confined to of the present invention.
Experimental technique in following embodiment, if no special instructions, is ordinary method.
In following embodiment, material used, reagent etc., if no special instructions, all can obtain from commercial channels.
Embodiment 1, the antiidiotypic antibody for people CD22 antibody existing with single-chain antibody form
The present embodiment uses phage display library technology to obtain the variable region weight chain-ordering of anti-SM03 antiidiotypic antibody, further prepares the antiidiotype single-chain antibody for people CD22 antibody---the soluble scFv that phage #3 expresses.Concrete steps are as follows:
1, utilize the mouse of injection SM03 immunization to prepare phage display library
Within approximately 6 weeks, large female Balb/c mouse is by immunization, method is according to standard immunoassay inoculation step (publishing referring to < < In Antibodies:A Laboratory Manual > > 1988 Nian You U.S. Cold Spring Harbor Laboratory) peritoneal injection 100ug SM03 monoclonal antibody, and with 200uL complete Freund's adjuvant (U.S. Sigma-Aldrich company product) emulsification.For the second time and for the third time immunization is carried out respectively after being separated by 14 days and 35 days, method is peritoneal injection 100ugSM03 monoclonal antibody (China Antibody Pharmacy Co., Ltd.), coordinates 200uL incomplete Freund's adjuvant to carry out emulsification (U.S. Sigma-Aldrich company product).
Measure after the SM03 antibody titers in mice serum, the mouse spleen cell of immunization after 39 days is used to separated full RNA and preparation cDNA(is used Superscript II test kit, American I nvitrogen company product).Then according to synthetic degenerated primer in pertinent literature, utilize pcr amplification immune globulin variable region gene sequence (Cheng et al.2005.Biochem Biophys Res Commun 338:1654-1660).Subsequently, according to the recombinant phages antibody system product guide of U.S. Amersham company, build variable region segment single-chain antibody (scFv) phage display library.
With reference to pertinent literature, carry out after the amplification of variable region segment scFv single-chain antibody phage display library and filobactivirus, with SM03 monoclonal antibody and mouse source CD22 monoclonal antibody RFB4(Ancell, Cat:171-820) screen and eliminate (McWhirter et al.2006.PNAS 103:1041-1046).Concise and to the point step is as follows, by the SM03 of same amount (100ug/ml) or mouse source RFB4 bicarbonate buffer (15mM Na for antibody 2cO 3, 35mM NaHCO 3) be coated with, with the method screening concentration of biological screening, be 10 12phage.Through the incubated at room of 2 hours slight concussion conditions, 0.1M glycine-hydrochloride buffer of 100uL for the scFv phage adsorbed, pH2.2 is through the wash-out of hatching of 10 minutes, then uses the 1M Tris-HCl of 10uL, the neutralization buffer neutralization of pH8.0.This screening process repeats four times, and the phage titre before and after the each screening of record.
The phage surviving in screening process is through rescue, its absorption specificity is assessed by the method for phage-ELISA, use anti-CD22 antibody RFB4, SM03 and SM06(China Antibody Pharmacy Co., Ltd.), and other control antibodies is further screened as antigen.Method is briefly described as follows, and in 96 hole ELISA enzyme plates, is coated with respectively RFB4, SM03, and SM06, chimeric anti-TNF antibodies and BSA(are used the coated damping fluid of the supercarbonate of 50uL pH9.6, and package amount is 1ug; After 4 ℃ of overnight incubation, with the borate buffer of 200uL pH8.0, rinse; Again equally with borate buffer 37 ℃ of sealings 1 hour), to SM03, have three phage clones (supernatant liquor of 100uL) of the highest absorption avidity to be added in hole 37 ℃ and hatch 1 hour.Through the flushing of the borate buffer of 5 pH8.0, the anti-M13 mouse antibodies of HRP mark (Amersham company product) adds after 1:3000 dilution.Hatch after 1 hour through 37 ℃, (10mgOPD is dissolved in 10mL containing the citric acid phosphoric acid salt buffer of 8uL30% hydrogen peroxide solution, pH5.0) develops the color to use O-Ursol D (OPD) reaction solution of 100uL.The selection result as shown in Figure 1, selected three scFv displaying phages (phage #1-#3) can be adsorbed RFB4, SM03 and SM06(mouse source, chimeric and humanized CD22 antibody) but can not adsorb other antibody (anti-TNF antibodies (infliximab)) or reference protein (BSA).Due to RFB4, the sequence same section of SM03 and SM06 only has the CDR part on target antibody, or antigen-binding site, the result shows that selected three scFv show that phage all partly has specificity to the idiotype of SM03.
2, scFv that can specific adsorption SM03 antigen-binding portion thereof shows the mensuration of phage variable region sequences
The scFv DNA sequences encoding of selected displaying phage checks order by mulberry lattice sequencing.The DNA sequence dna of corresponding antibody variable region is very similar, has only in the fragmentary position of framework segment or CDR3 segment and has part difference.Fig. 2 has shown the CDR segment sequence in the weight chain-ordering that selected phage shows, 3 CDR of the heavy chain of the scFv that phage #1-#3 shows are identical, the CDR1 of the light chain of the scFv that phage #1-#3 shows is identical with CDR2, and having the expressed light chain CDR3 region of phage #2 only has an amino acid (underscore mark in Fig. 2).
The phage #3 of the scFv sequence of 3, showing can prevent SM03 antibody absorption Raji cell
Because phage #3 has demonstrated relatively high absorption avidity, its single stranded sequence is responded into DNA encoding and uses molecule clone technology to build scFv bacterial expression vector.The scFv sequence of complete sequence that phage #3 shows is as Fig. 3.Sequence 8 in the scFv sequence encoding DNA(sequence table that phage #3 shows) be connected into pET3a carrier (Novagen Cat:69418) carrier and be transfected into BL21(DE3) pLys competent cell expresses.The aminoterminal of scFv is connected with a His-tag gene and has been convenient to the purification after expression.Through IPTG abduction delivering, the occlusion body that contains scFv is through collecting, and sex change (using containing 6M Guanidinium hydrochloride 20mM sodium phosphate, 0.5M sodium-chlor, the damping fluid of pH7.4), folds and purify by the description of product with HItrap Chelating HP separator column.Concise and to the point step is as follows, contains the scFv of His-tag after sex change, at Ni 2+ exist in situation, to be adsorbed onto HItrap Chelating HP separator column.Use progressively reduces the Guanidinium hydrochloride damping fluid of concentration according to gradient, and (containing 20mM sodium phosphate, 0.5M sodium-chlor, pH7.4) rinses until do not have residual Guanidinium hydrochloride to retain.With the imidazole buffer of the 5-40mM of several column volumes, (containing 20mM sodium phosphate, 0.5M sodium-chlor, pH7.4) rinses again.Eluted sample merges, and albumen wherein can detect by the method for SDS-PAGE electrophoresis.Determined amino acid sequence result shows, after purifying, gained albumen is that the aminoterminal of the 1st amino acids residue of sequence 1 in sequence table connects the fusion rotein that His-tag obtains, this fusion rotein is the antiidiotype single-chain antibody for people CD22 antibody, the soluble scFv of expressing hereinafter referred to as phage #3.
The soluble scFv that phage #3 expresses can be used to prevent the CD22 antigen of SM03 antibody absorption Raji cell surface expression.Test concise and to the point step as follows: Raji cell and SM03 antibody that PBS was cleaned are hatched jointly; the expressed soluble scFv of phage #3 that simultaneously adds different concns; by the Raj i cell that only adds SM03 antibody in contrast, do not add the Raji cell of any reagent as blank (blank); After half an hour, incubated at room and PBS cleaned, in all samples, add the fluorescently-labeled goat anti-human igg Fc specific antibody of 1:50 dilution anti-as two, incubated at room uses Beckman system of fluorescence analysis to analyze its fluorescent signal by cleaning half an hour.Result is as Fig. 4, show to add 100ug/ml group, the result of its flow cytometry shows that obvious fluorescent signal declines, and shows that the capacitive scFv that phage #3 expresses can effectively must suppress the CD22 antigen that SM03 antibody is adsorbed onto Raji cell surface.
Experimental example 2, the soluble scFv that utilizes embodiment 1 pnagus medius #3 to express carry out the pharmacokinetic in SM03 clinical trial
The specificity of the capacitive scFv expressing due to embodiment 1 pnagus medius #3 to SM03, therefore can be used for developing corresponding detection method and measure the antibody concentration in the patients serum who accepts anti-CD22 antibody treatment, particularly assist the required pharmacokinetic of clinical practice.The capacitive scFv wrapper sheet 96 hole ELISA enzyme plates of expressing with embodiment 1 pnagus medius #3.Through BSA sealing, clean, each blood sampling point serum of patient of accepting SM03 treatment adds in hand-hole after dilution.Through 37 ℃, hatch and clean with abundant for 2 hours, HRP mark goat-anti people Fc antibody (Jackson Immunoresearch company product) adds in hand-hole after 1:4000 dilution.Enzyme plate after 1 hour 37 ℃ are hatched, cleans 5 times again, and with the colour developing of TMB nitrite ion, color signal is according to standard ELISA detection method 450nm wavelength readings, and calculates the SM03 concentration of being caught with this.
Fig. 5 has shown the typical drug-time curve of SM03, and method therefor as mentioned above.Patients serum's sample is from the Malignant Lymphoma of accepting anti-CD22 antibody SM03 treatment, 380mg/m 2dosage group, 1 time weekly, successive administration 4 weeks.Serum sample is from the different time points collection of administration front and back.
Embodiment 3, the antiidiotypic antibody IdmG2a/k for people CD22 antibody existing with mouse IgG 2a/kappa immunoglobulin molecules form
The capacitive scFv preparation that embodiment 1 pnagus medius #3 expresses needs microbial culture, and abduction delivering and occlusion body are collected, the step of the series of complexes such as the folding and His-tag purification of protein denaturation.In addition the more unstable and difficult storage of scFv.In order to produce, both retain the capacitive scFv absorption specificity that embodiment 1 pnagus medius #3 expresses, can be purified by standard step again and the stable molecule easily storing, soluble scFv variable region of heavy chain (VH) coding DNA that embodiment 1 pnagus medius #3 expresses and variable region of light chain (VK) encoding sequence are with pcr amplification and the expression vector pIdmkappa-VK and the pIdmIgG-VH that use molecule clone technology to be integrated into can to increase, and it has also comprised the constant region partial sequence (as shown in Figure 6) of mouse source kappa light chain and IgG2a heavy chain.
This construction process that can increase expression vector is that dissolubility scFv variable region of heavy chain (VH) coding DNA and variable region of light chain (VK) encoding sequence in coding example 1 are connected to can the increase constant region of expression vector of corresponding mouse source kappa light chain and IgG2a heavy chain separately.Its mouse source kappa light chain expression vector pIdmkappa-VK is the expression vector of an about 10Kb.It stems from carrier pSVgpt (Mulligan & Berg 1981.PNAS 78:2072-2076), the IgG enhanser (Enhancer) that it has comprised a upstream, one is used for inserting the BamHI/BglII of VK section and the cloning site of XbaI, and the gene order of the mouse kappa constant region of light chain of the intron of ining succession (intron).In the downstream of constant region of light chain gene order, placed a hygromycin selective marker with SV40 promoter expression.The construction process of light chain expression vector is as follows: the gpt selective marker of pSVgpt is replaced with to hygromycin selective marker, obtain recombinant vectors pSVhyg.Between the EcoRI of pSVhyg and the recognition site of BamHI, insert the Ig enhanser of 319bp and before BamHI site, insert an XbaI restriction enzyme enzyme recognition site, obtain recombinant vectors pSVhyg-Ig.The kappa enhanser that inserts 199bp between the BamHI of pSVhyg-Ig and SacI recognition site, obtains recombinant vectors pSVhyg-Ig-kappa.Between the restriction enzyme SacI of pSVhyg-Ig-kappa and BamHI recognition site, insert the mouse source kappa constant region of light chain fragment of the intron of ining succession (intron) of 1.2Kb, between BamHI and XbaI restriction endonuclease sites, be connected into the VK sequence that phage #3 shows, obtain mouse source kappa light chain expression vector pIdmkappa-VK.Wherein, the Ig enhanser nucleotide sequence of 319bp is the 1st to the 319th deoxynucleotide from the 5 ' end of GeneBank Accession Number:K01901.The kappa enhanser nucleotide sequence of 199bp is the 1st to 199 deoxynucleotides from the 5 ' end of GeneBank Accession Number:K01325.The nucleotide sequence of the intron mouse endogenous light chain constant region fragment of 1.2Kb is the 1st to 1209 deoxynucleotides from the 5 ' end of GeneBank Accession Number:J00241.
The construction process of mouse IgG 2a heavy chain expression carrier pIdmIgG-VH is roughly similar, be similarly and be derived from carrier pSVgpt(Mulligan & Berg 1981.PNAS 78:2072-2076) 10Kb size expression vector, the IgG enhanser (Enhancer) that it has comprised a upstream, Xho I and a Hind III cloning site that is used for inserting VH section, and the gene order of the mouse IgG 2a CH of the intron of ining succession (intron).In the downstream of weight chain constant area gene sequence, placed a gpt selective marker with SV40 promoter expression.The construction process of this carrier is as follows: between the EcoRI of pSVgpt and the recognition site of XhoI, insert the Ig enhanser of 319bp, obtain recombinant vectors pSVgpt-Ig.Between the restriction enzyme HindIII of pSVgpt-Ig and BamHI recognition site, insert the mouse IgG 2a CH fragment of the intron of ining succession (intron) of 2Kb, between XhoI and HindIII restriction endonuclease sites, be connected into the VH sequence that phage #3 shows, obtain mouse IgG 2a heavy chain expression carrier pIdmIgG-VH.Wherein, the Ig enhanser of 319bp and nucleotide sequence are the 1st to the 319th deoxynucleotides from the 5 ' end of GeneBank Accession Number:K01901.The nucleotide sequence of the intron mouse source CH fragment of ining succession of 2Kb is the 1st to 2009 deoxynucleotides from the 5 ' end of GeneBank Accession Number:J00228.
This expression vector can be used to the multiple mammalian host cell line of transfection, include, but are not limited to Chinese hamster ovary (CHO) clone, mouse myeloma cell line SP2/0 or NS0, young hamster kidney (BHK) clone, human embryos kidney cell line 293 (HEK293), African green monkey kidney COS clone etc.In the present embodiment, select SP2/0 engineering cell system as host, used the method for electric shock perforation transfection to carry out transfection by standard step.Through methotrexate, select, the clone of survival is used to check its antibody expression.Select positive colony to increase, through the amplification of number wheel, the recombined engineering clone of expressing antibody is exaggerated cultivation, and its " mouse source antiidiotype IgG antibody " (hereinafter referred to as IdmG2a/k) producing is also collected and purifies.Meanwhile, the RNA that expresses the recombined engineering clone of antibody is also extracted, and through RT-PCR method and Sang Ge sequencing, obtains the cDNA encoding sequence of the weight chain of IdmG2a/k.Through comparison, IdmG2a/k, the VH of the capacitive scFv that its weight chain variable region sequence and embodiment 1 pnagus medius #3 express, VL segment sequence is identical, and the while is with the constant region of mouse IgG 2a heavy chain and kappa light chain.Heavy chain DNA sequences encoding for the antiidiotypic antibody IdmG2a/k of people CD22 antibody is the sequence 9 in sequence table, the aminoacid sequence of sequence 2 in code sequence list; The light chain DNA sequences encoding of IdmG2a/k is the sequence 10 in sequence table, the aminoacid sequence of sequence 3 in code sequence list.
Express the recombined engineering clone of IdmG2a/k through amplification culture, it is expressed the antibody process producing and extracts cells and supernatant, with albumin A separator column, by standard step, purifies.The antibody of purifying can be in 4 ℃ of preservations in PBS damping fluid.The SDS-PAGE electrophoresis pattern of the purification antibody that Fig. 7 has shown IdmG2a/k in reduction and non-reduced situation.
Purified can being used for by the coated ELISA enzyme plate of standard step of IdmG2a/k of recombined engineering expression of cell lines.
On ELISA enzyme connecting plate, every hole adds the IdmG2a/k of 50uL 10ug/ml, and 4 ℃ of night incubation are coated with.To the enzyme plate being coated with, every hole adds the 60uLSM03 antibody of different concns and other irrelevant control antibodies, as anti-CD20 antibodies Rituximab (Mabthera, company of Switzerland Roche Group product), anti-CD147 antibody or anti-TNF antibodies Infliximab(Lei Ke， Switzerland Cilag AG company).Through 1.5 hours incubated at room, with PBS, clean after 3 times, add the goat-anti people Fc antibody (Jackson Immunoresearch company product) of the HRP mark of 1:5000 dilution.After 45 minutes incubated at room, its absorption can develop the color and use 450nm wavelength readings with OPD again.Result shows, " mouse source antiidiotype IgG antibody " IdmG2a/k of recombined engineering expression of cell lines can only specific adsorption SM03, but not other control antibodies (as shown in Figure 8).
In order to assess the ability of the IdmG2a/k of recombined engineering expression of cell lines and the CD22 antigenic competition under state of nature absorption SM03 antibody, use Raji(mankind's Burkitt lymphoma) clone naturally expresses CD22 antigen as surface and originates and carried out flow cytometry experiment.Concise and to the point step is as follows, and 0.5 * 10 6the mouse source antiidiotype IgG antibody I dmG2a/k of the SM03 antibody of individual Raji cell and 1ug/ml and different concns is hatched jointly, incubation conditions is that 4 ℃ of the PBS-FA damping fluids (containing 1% foetal calf serum FBS, the PBS solution of 0.01% sodiumazide) of 200uL are hatched 30 minutes.With PBS, clean after 3 times, add the anti-human Fc antibody of fluorescent mark (Jackson Immunoresearch company product) of 50 times of dilutions and continue 4 ℃ and hatch 30 minutes.With PBS, clean after 3 times again, with the PBS-FA damping fluid containing 0.5% formalin, fix.Through fixing cell, with PBS, hang, use the FACScan analytical system of U.S. Becton Dickenson company to carry out Flow Cytometry Analysis.Result demonstration, antiidiotypic antibody IdmG2a/k can effectively according to dosage suppress SM03 and be adsorbed onto its natural part (Fig. 9).
Embodiment 4, utilize " the CD22 antibody family " content in the IdmG2a/k monitoring clinical practice of embodiment 3
1, the total antigen-binding site sequence of the IdmG2a/k of embodiment 3 energy specific binding " CD22 antibody family ", can adsorb mouse source, chimeric and humanized anti-CD22 antibody specifically.
Competitive adsorption detection method further shows, the adsorption site of antiidiotypic antibody is positioned at antigen-binding site (ABS) sequence, is mouse source antibody RFB4, the unique publicly-owned sequence of chimeric antibody SM03 and humanized antibody SM06.The method that competitive adsorption detects is briefly as follows, and on ELISA enzyme connecting plate, every hole adds the IdmG2a/k of the embodiment 3 of 50uL 10ug/ml, and 4 ℃ of night incubation are coated with.Inferior daily PBS cleans after 3 times, seals 2 hours, then clean 5 times with PBS by the 3%BSA solution room temperature of 200uL.SM03 antibody is labeled as SM03-HRP traget antibody (by Tian Jian bio tech ltd, Tianjin mark) with HRP.SM03-HRP traget antibody after 1:4000 dilution, the competition antibody with different concns, comprises RFB4, SM03, SM06 and some other irrelevant control antibodies (SM09, N005, N009) are mixed.Mixed antibody adds the ELISA enzyme connecting plate of the IdmG2a/k that has been coated with embodiment 3.SM03-HRP traget antibody is to developing the color with TMB nitrite ion after coated antiidiotypic antibody adsorption levels.
Result shows, RFB4, SM03 and SM06 can equivalence with SM03-HRP traget antibody competitive adsorption to the IdmG2a/k of embodiment 3, other irrelevant antibody does not have competitive adsorption ability (as shown in figure 10), the IdmG2a/k that shows embodiment 3 is that specific adsorption arrives RFB4, the consensus sequence of SM03 and SM06, but not other site, this consensus sequence is their antigen recognition site (ABS).Wherein, control antibodies SM09, N005, N009(China Antibody Pharmacy Co., Ltd.), for non-CD22 antigen, also not with anti-CD22 antibody generation cross reaction.
2, utilize IdmG2a/k exploitation assessment SM03 clinical experiment Chinese medicine dynamic (dynamical) standard detecting method of generation of embodiment 3
Along with " mouse source antiidiotype IgG antibody " adsorbs specific confirmation to " CD22 antibody family " antigen-binding site, a kind of can estimate in clinical experiment RFB4 in patients serum, the ELISA detection method of SM03 and SM06 anti-body contg have been developed.Following illustration has been described the detection method performance history of estimation SM03 serum content, and this method also can promote the use of other member of " CD22 antibody family ".Concise and to the point step is as described below, and on 96 hole ELISA enzyme connecting plates, every hole adds the IdmG2a/k of the embodiment 3 of 0.4ug/ml, and coated condition is every hole 50uLPBS, pH7.4,4 ℃ of overnight incubation; Inferior daily 1%BSA solution room temperature sealing 2 hours.Accept patients serum's sample of SM03 treatment after dilution certain multiple, add with the multiple hole of external source SM03 standard substance of different concns, diluent is selected the blank human serum PBS impact on background with removal serum containing 0.1%.Every hole adds after the sample of 50uL incubated at room 2 hours, and after cleaning, at the HRP mark goat-anti people Fc antibody (Jackson Immunoresearch company product) that adds 1:4000 dilution, incubated at room is cleaned after 1 hour and added TMB to develop the color.Color signal is read plate record with the visible ray of 450nm wavelength.Accepting the time dependent situation of SM03 antibody concentration residual in Antybody therapy peripheral blood in patients can be by obtaining with comparing of external source SM03 standard substance.
Figure 11 has shown a typical pharmacokinetics behavior of accepting the patient of SM03 treatment, and detection method used is previously described developed ELISA detection method.
Embodiment 5, estimate the novel detection method of anti-internalization antigen-antibody biological effectiveness
One, build the engineering cell system that cross-film is expressed antiidiotypic antibody absorption segment
In storing the process of manufacture of therapeutic antibody, in the mode of quality control, assess the biological activity of antibody, take SM03 as example, have two important consideration: the ability of adsorption property (being read as in detail absorption avidity and specificity) and antibody-mediated biologically (as ADCC or CDC).The latter estimates by the mode that biological effectiveness detects conventionally.Due to the equal adsorbable CD22 antigen of " CD22 antibody family " antibody, and after absorption, internalization phenomenon can be there is very soon, SM03 antibody and other anti-CD22 antibody, mediation CDC reaction smoothly in can not testing in vitro." CD22 antibody family " antibody is expressed after positive cell surface being adsorbed onto CD22, can not stop time enough at cell surface and fully act on complement (being assumed to be C1q complement) by its Fc part, consequently can not correctly cause and cause apoptotic chain reaction.This is likely that the anti-CD22 antibody as SM03 does not observe the reason of the CDC reaction that can measure.For other, for meeting, there is the antibody of quick internalization phenomenon surface antigen, as CD33, Lewis(Y) antigen, constant chain (CD74), this explanation also can be convinced.In order to confirm that the detection method of antibody adsorption property (specificity and avidity) has proved that the suction sheet consisting of antibody weight chain variable region wrecks folded, sequence, match all correctly, but can not provide the contingent protein variant of antibody Fc part or deletion condition.And these disappearances or variation may hinder in antibody body and bring into play its correct biological effectiveness as the used time.Therefore, exploitation be take cell detection as basic universal test method, estimates to take meeting the antibody biological function that the antigen of quick internalization phenomenon is target occurs is desirable.
In the present invention, therefore the absorption segment of antiidiotypic antibody is redesigned, and can be non-internalization membrane albumen at cell surface expression.This innovation completes by following several modes:
1, antiidiotypic antibody is expressed in the mode of cross-film IgD
A. the antiidiotypic antibody IdGmD for people CD22 antibody that obtains cross-film is merged in the cross-film region sequence of mouse source IgD and the CH3 region of " mouse source antiidiotype IgG antibody "
For the aminoacid sequence in the mouse YuanIgD cross-film district (TM) of merging with antiidiotype IgG antibody if Figure 12 is with as shown in the sequence of underscore.
In order to build, can express IgG2a-TM (IgD) protein gene of fusion, chemosynthesis the DNA sequence dna of one section of coding " mouse source antiidiotype IgG antibody " part carboxyl terminal sequence.This section of DNA sequence degeneracy two restriction enzyme point of contact BsrG1 and EagI, be cloned in corresponding original mouse IgG 2a constant region sequence assisting.。This section of sequence encoding original mouse IgG 2a and the IgD(TM of 41 amino acid lengths) merge segment.This sequence is cloned in former IgG2a sequence (Fig. 6), has replaced one section of sequence of IgG2a CH3 territory carboxyl terminal to make it not only comprise the TM sequence that former sequence has also increased mouse source IgD.Expressed albumen should be the permeable membrane segment (the complete VH-CH1-hinge-CH2-CH3-TM of being configured to) (heavy chain as shown in figure 12) of the heavy chain fusion mouse source IgD of IgG2a immunoglobulin (Ig), by the antiidiotypic antibody called after IdGmD for people CD22 antibody of this cross-film.
Select SP2/0 engineering cell system as host, use the method for electric shock perforation transfection to carry out transfection by standard step.Through methotrexate, select, the clone of survival is used to check its antibody expression.Select positive clone to increase, through the amplification of number wheel, the recombined engineering clone of expressing antibody is exaggerated cultivation, and the cross-film antibody I dGmD that it produces is also collected and purifies.Meanwhile, the RNA that expresses the recombined engineering clone of antibody is also extracted, and through RT-PCR method and Sang Ge sequencing, obtains the cDNA encoding sequence of the weight chain of IdGmD.Heavy chain DNA sequences encoding for the antiidiotypic antibody IdGmD of people CD22 antibody is the sequence 11 in sequence table, the aminoacid sequence of sequence 4 in code sequence list (aminoacid sequence of Figure 12); The light chain DNA sequences encoding of IdGmD is the sequence 10 in sequence table, the aminoacid sequence of sequence 3 in code sequence list.
B. IgG2a heavy chain is replaced obtaining the antiidiotypic antibody IdDmD for people CD22 antibody of cross-film by mouse source IgD sequence
The constant region sequence of original " mouse source antiidiotype IgG antibody " is replaced by the mouse source IgD sequence of membrane form completely.With the mouse source IgD complete amino acid sequence of permeable membrane segment TM sequence as shown in figure 13.
The mouse source IgD cDNA sequence of coding membrane form is according to the synthetic method chemosynthesis of standard oligonucleotide.Introduce restriction site (XhoI and EagI) and be cloned into the corresponding position of former mouse IgG 2a expression vector to facilitate.This sequence is cloned into corresponding position (Fig. 6) in former IgG2a expression vector, has replaced the constant region sequence of mouse IgG 2a with membrane IgD protein sequence, obtains the recombinant expression vector pIdDmD of IdDmD.Expressed albumen comprises the permeable membrane segment (morphosis is VH-CH1-hinge-CH3-TM) of the heavy chain band IgD of IgD immunoglobulin (Ig).It should be noted that mouse source IgD does not have CH2 region, very not large thereby its structure and IgG or people source IgD produce, as (cross-film district underscore mark) shown in Figure 13.
Because just illustration experiment, only has the expression vector dna plasmid process restriction endonuclease linearize of the antiidiotype IgD-TM of (b) part description.With mouse myeloma cell line SP2/0 as transfection host cell.According to pertinent literature explanation, SP2/0 cell can only produce endogenous Ig β and can not produce Ig α.Owing to expressing the acting in conjunction that needs Ig α and Ig β on the film of IgD immunoglobulin molecules, the expression vector of Ig α is also together transfected into SP2/0 host cell according to standard electric blow hole transfection method with the expression vector pIdDmD of IgD-TM fusion rotein.Transfection the cell of plasmid with selecting according to DHFR system standard method containing the selective medium of methotrexate.Through selecting the cell of survival, by the method that cell ELISA detects, test its expressed antiidiotype specificity in film surface.Test concise and to the point step as follows, the SM03 antibody of 50uL 10ug/ml joins 10 6transfectional cell (first rinsing 3 times with PBS).4 ℃, the cell that has mixed antibody is hatched after 1 hour and rinsed 3 times with PBS.Then add the HRP mark goat anti-human igg Fc antibody through 1:1000 dilution of 50uL, hatch 1 hour for 4 ℃.With PBS, rinse after 1 time, add 50uL colour developing, the cell of the anti-SM03 IgD positive on film surface has color reaction.After 10 minutes incubated at room, add the diluted acid termination reaction of 50uL 0.18M.Antibody-cell mixture is centrifugal, collect supernatant liquor and read light absorption ratio with wavelength 450nm.
Select positive clone to increase, through the amplification of number wheel, the recombined engineering clone of expressing antibody is exaggerated cultivation, and the cross-film antibody I dDmD that it produces is also collected and purifies.Meanwhile, the RNA that expresses the recombined engineering clone of antibody is also extracted, and through RT-PCR method and Sang Ge sequencing, obtains the cDNA encoding sequence of the weight chain of IdDmD.Heavy chain DNA sequences encoding for the antiidiotypic antibody IdDmD of people CD22 antibody is the sequence 12 in sequence table, the aminoacid sequence of sequence 5 in code sequence list; The light chain DNA sequences encoding of IdDmD is the sequence 10 in sequence table, the aminoacid sequence of sequence 3 in code sequence list.
Result shows, at the anti-SM03IgD of Membrane surface expression, needs Ig α to be transfected into murine myeloma cell simultaneously.The result of flow cytometry experiment (this experimental technique is with the flow cytometry of following step 2) has been confirmed the Membrane surface expression (Figure 16) of IgD.
2, by the form of membrane Fab-glycoprotein fusion rotein, express the antiidiotypic antibody IdGmFdA for people CD22 antibody of cross-film
Yu“Shu source, glycoprotein A cross-film region antiidiotype IgG antibody from Surface of Erythrocytes " mouse IgG 2a hinge area carboxyl terminal merges mutually.This antibody fragment merges expection meeting expresses to be fixed on the Fab mode of cell surface by glycoprotein A cross-film district on transfectional cell surface.
The glycoprotein A segment (comprising cross-film region sequence) that is used for merging anti-SM03 antibody fragment as Figure 14 with as shown in the aminoacid sequence of underscore.
Gene order is directly merged in Ig2a antibody hinge region, mouse source and encoded after last amino acid whose gene order, the cross-film district that expressed albumen should be blended in glycoprotein A for " mouse source antiidiotype IgG antibody " heavy chain Fd region and bag inner region (segment composition-formed is: VH-CH1-hinge area-glycoprotein A cross-film district+bag inner region, Figure 14).
The expression vector of expressing " mouse source antiidiotype IgG antibody " Fd segment-glycoprotein A fusion rotein at surface of cell membrane is through having built.Brief description is as follows, the DNA sequences encoding that mouse IgG 2a antibody CH1-hinge area segment is blended in glycoprotein A carboxyl end segment (comprising cross-film region sequence) according to standard oligonucleotide synthesis method through chemosynthesis gained.The sequence of synthesized has comprised point of contact in in-frame XhoI and EagI.Composition sequence utilizes the molecular biology clone technology of standard to insert the corresponding site in anti-SM03IgG2a antibody expression vector (Fig. 6) gene order, replace the CH2-CH3 regional code sequence in former IgG2a antibody sequence, obtained the recombinant expression vector pIdGmFdA of IdGmFdA.
The expression vector pIdGmFdA plasmid DNA of " mouse source antiidiotype IgG antibody " Fab segment-glycoprotein A fusion rotein is transfected into mouse SP2/0 host cell through linearize.Cell through transfection plasmid utilizes DHFR gene Selection system to carry out positive colony selection with methotrexate according to standard method.Select positive clone to increase, through the amplification of number wheel, the recombined engineering clone of expressing antibody is exaggerated cultivation, and the cross-film antibody I dGmFdA that it produces is also collected and purifies.Meanwhile, the RNA that expresses the recombined engineering clone of antibody is also extracted, and through RT-PCR method and Sang Ge sequencing, obtains the cDNA encoding sequence of the weight chain of IdGmFdA.Heavy chain DNA sequences encoding for the antiidiotypic antibody IdGmFdA of people CD22 antibody is the sequence 13 in sequence table, the aminoacid sequence of sequence 6 in code sequence list; The light chain DNA sequences encoding of IdGmFdA is the sequence 10 in sequence table, the aminoacid sequence of sequence 3 in code sequence list.
The survival positive colony of selecting is used for measuring the cell surface expression situation of its antiidiotype Fab-glycoprotein A fusion rotein, and method therefor is still described before cell ELISA detection method.The cell detecting successfully at cell surface expression fusion rotein through ELISA further carries out flow cytometry: SM03 antibody is as primary antibodie, and the fluorescently-labeled goat anti-human igg Fc of FITC antibody (Jackson Immunoresearch company product) is as detecting antibody (two is anti-).Brief description is as follows, and 5 * 10 5the SM03 of individual transfectional cell and 0.01,0.1 or 1 μ g is jointly hatched in the PBS-FA of 100uL damping fluid (containing 1% foetal calf serum FCS, the PBS solution of 0.05% sodiumazide).Incubation conditions is 4 ℃, 30 minutes time, cleans 3 times to remove non-absorption antibody afterwards with PBS.SM03 is the detection assessment with fluorescence FITC mark goat anti-human igg's Fc antibody (Jackson Immunoresearch company product) to the adsorptive capacity of transfectional cell.With hatching after 30 minutes through 4 ℃ containing the 100uLPBS-FA damping fluid of 20 times of dilution fluorescent-labeled antibody, with PBS, rinse cell 3 times, its fluorescent signal is analyzed by FACScan system.Result shows, the cell surface expression (as shown in figure 16) that antiidiotype Fab-glycoprotein A fusion rotein IdGmFdA can at transfected into bone marrow oncocyte be effectively.
3, with Fab-GPI fixedly the form of fusion rotein express the antiidiotypic antibody IdGmFdG for people CD22 antibody of " mouse source antiidiotype IgG " antibody
GPI segment is derivative by the ldl receptor being attached on DAF hydrophobic region, and this segment directly merges after " mouse source antiidiotype IgG antibody " IgG2a hinge area.Fusion rotein expection is to fix by GPI, and the Fab segment mode that is attached to cell surface is expressed.
By be attached to the derivative GPI segment aminoacid sequence of ldl receptor on DAF hydrophobic region in Figure 15 with as shown in the aminoacid sequence of underscore.
Its DNA sequences encoding is directly merged after last amino acid gene order of coding mouse IgG 2a antibody hinge region; " mouse source antiidiotype IgG antibody " heavy chain Fd part that should be expressed albumen merges with GPI-DAF segment and (merges segment and be configured to VH-CH1-GPI-DAF, as shown in figure 15).
The expression vector of expressing above-mentioned fusion rotein has built.Brief description is as follows, the DNA sequence dna of coding mouse IgG 2a CH1-hinge area and GPI-DAF fusion segment according to the synthetic method of standard oligonucleotide through chemosynthesis gained.In sequence, comprise point of contact (shown in underscore) in in-frame XhoI and EagI.Composition sequence utilizes the molecular biology clone technology of standard to insert the corresponding site in " mouse source antiidiotype IgG antibody " expression vector (Fig. 6) gene order, replace the CH2-CH3 regional code sequence in former IgG2a antibody sequence, obtained the recombinant expression vector pIdGmFdG of IdGmFdG.
PIdGmFdG plasmid DNA is transfected into mouse SP2/0 host cell through linearize.Cell through transfection plasmid utilizes DHFR gene Selection system to carry out positive colony selection with methotrexate according to standard method.Select positive clone to increase, through the amplification of number wheel, the recombined engineering clone of expressing antibody is exaggerated cultivation, and the cross-film antibody I dGmFdG that it produces is also collected and purifies.Meanwhile, the RNA that expresses the recombined engineering clone of antibody is also extracted, and through RT-PCR method and Sang Ge sequencing, obtains the cDNA encoding sequence of the weight chain of IdGmFdG.Heavy chain DNA sequences encoding for the antiidiotypic antibody IdGmFdG of people CD22 antibody is the sequence 14 in sequence table, the aminoacid sequence of sequence 7 in code sequence list; The light chain DNA sequences encoding of IdGmFdG is the sequence 10 in sequence table, the aminoacid sequence of sequence 3 in code sequence list.
The survival positive colony of selecting is used for measuring the cell surface expression situation of its antiidiotype Fab-glycoprotein A fusion rotein, and method therefor is still described before cell ELISA detection method.The cell detecting successfully at cell surface expression fusion rotein through ELISA further carries out flow cytometry: SM03 antibody is as primary antibodie, the fluorescently-labeled goat-anti people of FITC FcIgG antibody (Jackson Immunoresearch company product) is as detecting antibody (two is anti-), and concrete grammar is with the flow cytometry of above-mentioned steps 2).Result shows, the cell surface expression (as shown in figure 16) that " mouse source antiidiotype IgG antibody " Fab segment-GPI fusion rotein IdGmFdG can at transfected into bone marrow oncocyte be effectively.
Two, set up the cell detection method in order to assessment " CD22 antibody family " biological effectiveness
By the differing appearance type transfectional cell at the corresponding fusion rotein of cell surface expression (IdDmD, IdGmFdA and IdGmFdG) of above-mentioned steps one, calibrated cell concn is 2 * 10 6individual/milliliter.On 96 orifice plates, every hole adds 50uL cell.Guinea pig serum (Guinea Pig Serum, the GPS) lyophilized powder of Canada Cedarlane company is mixed with 100% GPS solution with the substratum of 1 milliliter.In being added with the hole of transfectional cell, add the SM03 antibody containing the 50uL different concns of 10%GPS.Through 2 hours, 37 ℃, hatching under 5% carbon dioxide conditions, every hole adds the CCK-8(cell counting kit-8 of U.S. Dojindo Molecular Technology Corp. of 20uL) reagent.After 3 hours, according to the description of product, by 450nm visible wavelength, read Cell viability.Result shows, can be for the transfectional cell at cell surface expression " mouse source antiidiotype IgG antibody " Fab segment-glycoprotein A fusion rotein IdGmFdA and " mouse source antiidiotype IgG antibody " Fab segment-GPI-DAF fusion rotein IdGmFdG such as " CD22 antibody family " product that SM03 is such, effectively mediation Complement Dependent cytotoxic reaction (CDC reaction, as shown in figure 17).Transfectional cell at cell surface expression antiidiotype IgD-TM segment protein I dDmD is not had to obvious CDC effect.May still can there is internalization phenomenon for the IgD of cell surface in reason, so that blocked CDC reaction after adsorbing with " CD22 antibody family " product; Or because expression amount on the film of antiidiotype-TM segment albumen does not reach the threshold value that causes complement activation very little and.In any case, three kinds of different transfectional cells in cell surface expression " mouse source antiidiotype IgG antibody " absorption fragment, there are two kinds can according to dosage effectively mediate SM03 and cause CDC and kill and wound, shown that this strategy can be as the suitable biological detection method of exploitation, thus the general governing principle that correct assessment can internalization antigen-antibody biological effectiveness.
Figure IDA00002003756800041
Figure IDA00002003756800051
Figure IDA00002003756800061
Figure IDA00002003756800071
Figure IDA00002003756800081
Figure IDA00002003756800091
Figure IDA00002003756800101
Figure IDA00002003756800111
Figure IDA00002003756800121
Figure IDA00002003756800141
Figure IDA00002003756800151

Claims (10)

1. an antibody, comprises 3 heavy chain of antibody complementary determining regions and 3 light chain of antibody complementary determining regions; The aminoacid sequence of described 3 heavy chain of antibody complementary determining regions is respectively as 31-35 position, 50-66 position and the 99-106 position of sequence in sequence table 1, and the aminoacid sequence of described 3 light chain of antibody complementary determining regions is respectively as 154-168 position, 184-190 position and the 223-231 position of sequence in sequence table 1.
2. antibody according to claim 1, is characterized in that: described antibody comprises variable region of heavy chain and variable region of light chain; The aminoacid sequence of described variable region of heavy chain is as the 1-116 position of sequence in sequence table 1, and the aminoacid sequence of described light chain chain variable region is as the 131-240 position of sequence in sequence table 1.
3. antibody according to claim 1, is characterized in that: described antibody be following a)-h) in a kind of:
A) aminoacid sequence of heavy chain is the sequence 2 in sequence table, and the aminoacid sequence of light chain is the sequence 3 in sequence table;
B) aminoacid sequence except described 3 heavy chain of antibody complementary determining regions in the sequence in sequence table 2 is carried out to the replacement of amino-acid residue, disappearance or interpolation, and/or the aminoacid sequence except described 3 light chain of antibody complementary determining regions in the sequence in sequence table 3 is carried out to the replacement of amino-acid residue, the antiidiotypic antibody for people CD22 antibody that disappearance or interpolation obtain, be preferably the aminoacid sequence except the variable region of heavy chain of 1-117 position in described sequence 2 is carried out to the replacement of amino-acid residue, disappearance or interpolation, and/or the aminoacid sequence except the light variable region of 1-111 position in described sequence 3 is carried out to the replacement of amino-acid residue, the antiidiotypic antibody for people CD22 antibody that disappearance or interpolation obtain,
C) single-chain antibody shown in the sequence in sequence table 1, or in sequence table, aminoterminal or the carboxyl terminal of sequence 1 adds the histidine-tagged fusion rotein obtaining;
D) by the sequence in sequence table 1, the aminoacid sequence except described 3 heavy chain of antibody complementary determining regions and described 3 light chain of antibody complementary determining regions carries out the antiidiotypic antibody for people CD22 antibody that replacement, disappearance or the interpolation of amino-acid residue obtain, and is preferably the connection peptides sequence shown in the 117-130 position of sequence 1 in described sequence table is carried out to the antiidiotypic antibody for people CD22 antibody that replacement, disappearance or the interpolation of amino-acid residue obtain;
E) aminoacid sequence of heavy chain is the sequence 6 in sequence table; The aminoacid sequence of light chain is the sequence 3 in described sequence table;
F) aminoacid sequence of heavy chain is the sequence 7 in sequence table; The aminoacid sequence of light chain is the sequence 3 in described sequence table;
G) aminoacid sequence of heavy chain is the sequence 4 in sequence table; The aminoacid sequence of light chain is the sequence 3 in described sequence table;
H) aminoacid sequence of heavy chain is the sequence 5 in sequence table; The aminoacid sequence of light chain is the sequence 3 in described sequence table.
4. the nucleic acid molecule of arbitrary described antibody in coding claim 1-3.
5. nucleic acid molecule according to claim 4, described nucleic acid molecule is following 1)-6) in arbitrary described gene:
1) in claim 3, a) encoding sequence of the heavy chain of described antibody is the sequence 9 in sequence table, and in claim 3, a) encoding sequence of the light chain chain of described antibody is the sequence 10 in sequence table;
2) c in claim 3) encoding sequence of described antibody is the sequence 8 in sequence table;
3) e in claim 3) described antibody the encoding sequence of heavy chain be the sequence 13 in sequence table, e in claim 3) encoding sequence of light chain chain of described antibody is the sequence 10 in sequence table;
4) f in claim 3) described antibody the encoding sequence of heavy chain be the sequence 14 in sequence table, f in claim 3) encoding sequence of light chain chain of described antibody is the sequence 10 in sequence table;
5) g in claim 3) described antibody the encoding sequence of heavy chain be the sequence 11 in sequence table, g in claim 3) encoding sequence of light chain chain of described antibody is the sequence 10 in sequence table;
6) h in claim 3) described antibody the encoding sequence of heavy chain be the sequence 12 in sequence table, h in claim 3) encoding sequence of light chain chain of described antibody is the sequence 10 in sequence table.
6.1), 2) or 3) biomaterial:
1) expression cassette, recombinant vectors, recombinant microorganism or the recombinant cell lines that contain nucleic acid molecule described in claim 4 or 5;
2) express recombinant expression vector, recombinant microorganism or the recombinant cell lines of arbitrary described antibody in claim 1-3;
3) recombinant expression vector, recombinant microorganism or the recombinant cell lines of nucleic acid molecule described in expression claim 5.
7. surface of cell membrane is expressed the recombinant cell lines of arbitrary described antibody in claim 1-3.
8.1) any reagent-5):
1) detect the reagent of people CD22 antibody concentration, its activeconstituents is arbitrary described antibody in claim 1-3;
2) detect the human antimouse antibody reaction reagent being caused by people CD22 antibody, its activeconstituents is arbitrary described antibody in claim 1-3;
3) detect the anti-chimeric antibody reaction reagent of people being caused by people CD22 antibody, its activeconstituents is arbitrary described antibody in claim 1-3;
4) detect the anti-human antibody response reagent of people being caused by people CD22 antibody, its activeconstituents is arbitrary described antibody in claim 1-3;
5) detect people CD22 antibody to the penetrance of tumour cell and/or adsorptivity reagent, its activeconstituents is arbitrary described antibody in claim 1-3.
9.1) any reagent-3):
1) detect the bioactive reagent of people's CD22 antibody, its activeconstituents for its activeconstituents be recombinant cell lines claimed in claim 7;
2) detect the antibody-mediated Complement Dependent cytotoxic reaction reagent of people CD22, its activeconstituents for its activeconstituents be recombinant cell lines claimed in claim 7;
3) detect the reagent of the antibody-mediated antibody-dependant cell mediated cell poison reaction of people CD22, its activeconstituents for its activeconstituents be recombinant cell lines claimed in claim 7.
10.1) the arbitrary application-8):
1) in claim 1-3, arbitrary described antibody detects the application in CD22 antibody concentration reagent in preparation;
2) reconstitution cell claimed in claim 7 ties up to preparation and detects the application in CD22 antibody bioactive agents;
3) in claim 1-3, arbitrary described antibody detects the application in the human antimouse antibody reaction reagent being caused by people CD22 antibody in preparation;
4) in claim 1-3, arbitrary described antibody detects the application in the anti-chimeric antibody reaction reagent of people being caused by people CD22 antibody in preparation;
5) in claim 1-3, arbitrary described antibody detects the application in the anti-human antibody response reagent of people being caused by people CD22 antibody in preparation;
6) reconstitution cell claimed in claim 7 ties up to preparation and detects the application in the antibody-mediated Complement Dependent cytotoxic reaction reagent of people CD22;
7) reconstitution cell claimed in claim 7 ties up to preparation and detects the application in the antibody-mediated antibody-dependant cell mediated cell poison reaction reagent of people CD22;
8) in claim 1-3, arbitrary described antibody detects people CD22 antibody to the application in the penetrance of tumour cell and/or adsorptivity reagent in preparation.
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WO2022262783A1 (en) * 2021-06-16 2022-12-22 西安宇繁生物科技有限责任公司 Anti-cd22 fully human antibody or antigen binding fragment thereof, preparation method therefor, and use thereof

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