CN103588882B - For antiidiotypic antibody and the application thereof of people CD22 antibody - Google Patents

For antiidiotypic antibody and the application thereof of people CD22 antibody Download PDF

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CN103588882B
CN103588882B CN201210286457.4A CN201210286457A CN103588882B CN 103588882 B CN103588882 B CN 103588882B CN 201210286457 A CN201210286457 A CN 201210286457A CN 103588882 B CN103588882 B CN 103588882B
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antibody
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people
heavy chain
cell
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CN103588882A (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 the antiidiotypic antibody for people CD22 antibody and application thereof.3 heavy chain of antibody complementary determining regions and 3 light chain of antibody complementary determining regions should be comprised for the antiidiotypic antibody of people CD22 antibody; The aminoacid sequence of described 3 heavy chain of antibody complementary determining regions is respectively as the 31-35 position of sequence in sequence table 1,50-66 position and 99-106 position, and the aminoacid sequence of described 3 light chain of antibody complementary determining regions is respectively as the 154-168 position of sequence in sequence table 1,184-190 position and 223-231 position.This antiidiotypic antibody specificity, for people CD22 antibody, can prevent people CD22 antibody and its natural part people CD22 antigen to be combined.

Description

For antiidiotypic antibody and the application thereof of people CD22 antibody
Technical field
The present invention relates to the antiidiotypic antibody for people CD22 antibody and application thereof.
Background technology
Using monoclonal antibody (MAb) carries out the theory that targeted therapy is not a kind of novelty or complexity.But the enforcement of this theory relates to the integration of multiple subject knowledge technology, comprise antibody molecule design, clone is optimized, and industrialization is produced, and purifies, formulation optimization and each special quality control detect, to guarantee that produced monoclonal antibody medicine can reach the clinical effectiveness of expection.Up to the present, existing up to a hundred are in each clinical experimental stage for the therapeutic monoclonal antibody product of various indication.Correspond, in a large number based on or be derived from the theory of antibody and technique means is all maked rapid progress along with the development of monoclonal antibody industry, to expanding the range of application of currently available products.
One of them derivative, based on immune network theory, first proposed (referring to JerneNK:1974.Ann Immunol 125C:373-389) by Niels Jerne in 1974.Jerne proposes, and immunity system is one and is interacted by antiidiotypic antibody and carry out the immunoreactive network of regulable control.This understanding is afterwards by further expansive approach other purposes to antiidiotypic antibody.Antiidiotypic antibody is also called Ab2 usually, is the antibody produced for exotic antigen for immune antibody Ab1(and immunity system) and its idiotope (antibody molecule surface unique antigen recognition bunch) is had to the antibody of specific recognition adsorptive power.Ab2 can be divided into three different classifications: 1) Ab2 α identifies the idiotope on original Ab1 antibody beyond antigen-binding site (ABS); 2) Ab2 β identifies the site of antigen-binding site and simulates its corresponding space structure, thus is formed " the interior image " of exotic antigen; 3) Ab2 γ identifies the site of antigen-binding site equally, but does not simulate the structure of exotic antigen.
It is generally acknowledged, most magnetism be Ab2 β antibody in Ab2 Antibody types, especially when attempt with Ab2 β antibody as an alternative the effective vaccine of antigen development for autoantigen or inertia antigen time, such as 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, assist production and the clinical potency assessment with the therapeutic Ab1 antibody of pharmaceutical use.
People CD22 antigen ripe or malignant B cell surface expression ( 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) preventing immunity system radical response or autoimmune disease.
SM03 is by the derivative anti-CD22 chimeric antibody of the one (Yang Lei etc. of mouse source antibody RFB4.2006。To recombinate the structure of anti-B cell lymphoma chimeric antibody and qualification.Chinese Journal of New Drugs, 15 volume 3 phase: 186-192), and in the treatment of non Ho lymphoma, entered clinical experimental stage (Li et al.2012.Landes BioScience J 4 (2): 256-266).Be target due to SM03 monoclonal antibody with mature B cell and suppress 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).
To SM03 monoclonal antibody use framework-reengineering technology to carry out humanization modified advance anti-CD22 antibody further application (beam Ruian, etc.2006。Application antibody " framework-reengineering " technique construction humanized antibody fSM03.Chinese Journal of New Drugs, 15 volume 21 phase: 1832-1836).SM06 is named as through humanization modified SM03 monoclonal antibody.SM03 and SM06 for the same site on people CD22 antigen, and has similar avidity.But on aminoacid sequence and structure, SM03 with SM06 only has antigen recognition site identical, this same section is by they respective complementary determining region (CDR) Sequence composition.
SM03 and SM06 specific recognition can adsorb people CD22 antigen, and after combining, obvious internalization can occur Antibody-antigen complexes.This characteristic makes to become very difficult to the biological activity assay of SM03 and SM06 monoclonal antibody product.Other such as, for the antibody class product of internalizing antigen can also have similar difficulty, constant chain, CD33 etc.In addition, the method that CLINICAL PHARMACOKINETIS STUDY ON needs convenience sane is to measure free SM03, SM06(and RFB4) and their various derivatives (scFv single-chain antibody, Fab, Diabody, immunotoxin, drug conjugates 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 to have 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 race "), can adsorb " CD22 antibody race ", its absorption fragment is to " CD22 antibody race " antibody molecule variable region tool specific reaction, and its absorption segment is specifically to " CD22 antibody race " antibody molecule antigen-binding site (ABS) tool specific reaction.This antiidiotypic antibody can prevent people CD22 antibody and its natural part people CD22 antigen to be combined.
The CD22 of people described in the present invention antibody is containing, for example lower 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, antibody binding pair and other various forms antibody fusion proteins etc., be referred to as " CD22 antibody race ".
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 of this antiidiotypic antibody form the structure with the idiotype part specific combination of people CD22 antibody.The experiment of following embodiment 1 proves, single-chain antibody containing above-mentioned 6 complementary determining regions can with 3 kinds of people CD22 antibody (RFB4, SM03 and SM06) specific bindings, 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 comprising weight chain constant-region sequences of the following embodiment 3 of sequence 3(in sequence table);
B) aminoacid sequence in the sequence 2 in sequence table except described 3 heavy chain of antibody complementary determining regions is carried out the replacement of amino-acid residue, disappearance or interpolation, and/or the aminoacid sequence in the sequence 3 in sequence table except described 3 light chain of antibody complementary determining regions is carried out the replacement of amino-acid residue, the antiidiotypic antibody for people CD22 antibody that disappearance or interpolation obtain, be preferably the replacement aminoacid sequence in described sequence 2 except the variable region of heavy chain of 1-117 position being carried out amino-acid residue, disappearance or interpolation, and/or the aminoacid sequence in described sequence 3 except the light variable region of 1-111 position is carried out the replacement of amino-acid residue, the antiidiotypic antibody for people CD22 antibody that disappearance or interpolation obtain,
C) single-chain antibody (in following embodiment 1 and 2 single-chain antibody shown in Fig. 3) shown in sequence 1 in sequence table, or in sequence table, the aminoterminal of sequence 1 or carboxyl terminal add the histidine-tagged fusion rotein obtained;
D) aminoacid sequence of the sequence 1 in sequence table except described 3 heavy chain of antibody complementary determining regions and described 3 light chain of antibody complementary determining regions is carried out the antiidiotypic antibody for people CD22 antibody that the replacement of amino-acid residue, disappearance or interpolation obtain, preferably the connection peptides sequence shown in the 117-130 position of sequence 1 in described sequence table is carried out the antiidiotypic antibody for people CD22 antibody that the replacement of amino-acid residue, disappearance or interpolation obtain;
E) aminoacid sequence of heavy chain is the sequence 6 in sequence table; The aminoacid sequence of light chain be the IdGmFdA of the following embodiment 5 of sequence 3(in described sequence table, Figure 14 display be 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 be the IdGmFdG of the following embodiment 5 of sequence 3(in described sequence table, Figure 15 display be 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 be the IdGmD of the following embodiment 5 of sequence 3(in described sequence table, Figure 12 display be 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 be the IdDmD of the following embodiment 5 of sequence 3(in described sequence table, Figure 13 display be the heavy chain amino acid sequence of IdDmD).
Wherein, sequence 1 in sequence table is made up of 240 amino-acid residues, sequence 2 in sequence table is made up of 447 amino-acid residues, sequence 3 in sequence table is made up of 219 amino-acid residues, sequence 4 in sequence table is made up of 502 amino-acid residues, sequence 5 in sequence table is made up of 408 amino-acid residues, and the sequence 6 in sequence table is made up of 305 amino-acid residues, and the sequence 7 in sequence table is made up 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 and IdDmD of following embodiment 5), 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.
Any one nucleic acid molecule for the antiidiotypic antibody of people CD22 antibody above-mentioned 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, above-mentioned a) described in the encoding sequence of light chain chain of antibody be sequence 10 in sequence table;
2) encoding sequence of above-mentioned c) described antibody is the sequence 8 in sequence table;
3) above-mentioned e) described antibody the encoding sequence of heavy chain be sequence 13 in sequence table, the encoding sequence of the light chain chain of above-mentioned e) described antibody is the sequence 10 in sequence table;
4) above-mentioned f) described antibody the encoding sequence of heavy chain be sequence 14 in sequence table, the encoding sequence of the light chain chain of above-mentioned f) described antibody is the sequence 10 in sequence table;
5) above-mentioned g) described antibody the encoding sequence of heavy chain be sequence 11 in sequence table, the encoding sequence of the light chain chain of above-mentioned g) described antibody is the sequence 10 in sequence table;
6) above-mentioned h) described antibody the encoding sequence of heavy chain be sequence 12 in sequence table, the encoding sequence of the light chain chain of above-mentioned h) described antibody is the sequence 10 in sequence table.
Wherein, the sequence 1 in sequence table is made up of 240 amino-acid residues, and by the DNA encoding of the sequence 8 in sequence table, sequence 8 is made up of 720 Nucleotide; Sequence 2 in sequence table is made up of 447 amino-acid residues, and by the DNA encoding of the sequence 9 in sequence table, sequence 9 is made up of 1344 Nucleotide; Sequence 3 in sequence table is made up of 219 amino-acid residues, and by the DNA encoding of the sequence 10 in sequence table, sequence 10 is made up of 660 Nucleotide; Sequence 4 in sequence table is made up of 502 amino-acid residues, and by the DNA encoding of the sequence 11 in sequence table, sequence 11 is made up of 1509 Nucleotide; Sequence 5 in sequence table is made up of 408 amino-acid residues, and by the DNA encoding of the sequence 12 in sequence table, sequence 12 is made up of 1227 Nucleotide; Sequence 6 in sequence table is made up of 305 amino-acid residues, and by the DNA encoding of the sequence 13 in sequence table, sequence 13 is made up of 918 Nucleotide; Sequence 7 in sequence table is made up of 264 amino-acid residues, and by the DNA encoding of the sequence 14 in sequence table, sequence 14 is made up of 795 Nucleotide.
The biomaterial of following a1, a2 or a3 also belongs to protection scope of the present invention:
A1. the expression cassette containing any one nucleic acid molecule above-mentioned, recombinant vectors, recombinant microorganism or recombinant cell lines;
A2. any one recombinant expression vector for the antiidiotypic antibody of people CD22 antibody above-mentioned, recombinant microorganism or recombinant cell lines is expressed;
A3. the recombinant expression vector of any one gene, recombinant microorganism or recombinant cell lines is expressed.
In above-mentioned biomaterial, expression cassette described in a1, refer to and can express any one DNA for the antiidiotypic antibody of people CD22 antibody above-mentioned in host cell, this DNA not only can comprise startup any one promotor for the antiidiotypic antibody genetic transcription of people CD22 antibody above-mentioned, also can comprise and stop any one terminator for the antiidiotypic antibody genetic transcription of people CD22 antibody above-mentioned.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 any one recombinant cell lines (cell strain) for the antiidiotypic antibody of people CD22 antibody above-mentioned.
Present invention also offers any one following multiple purposes for the antiidiotypic antibody of people CD22 antibody above-mentioned:
B1. detect the reagent of people CD22 antibody concentration, its activeconstituents is any one antiidiotypic antibody for people CD22 antibody above-mentioned;
B2. detect the reagent of the human antimouse antibody reaction caused by people CD22 antibody, its activeconstituents is any one antiidiotypic antibody for people CD22 antibody above-mentioned;
B3. detect the reagent of the anti-chimeric antibody reaction of the people caused by people CD22 antibody, its activeconstituents is any one antiidiotypic antibody for people CD22 antibody above-mentioned;
B4. detect the reagent of the people anti-human antibody reaction caused by people CD22 antibody, its activeconstituents is any one antiidiotypic antibody for people CD22 antibody above-mentioned;
B5. detect the reagent of the antibody-mediated complement dependent cellular poison reaction of people CD22, its activeconstituents is that surface of cell membrane expresses any one recombinant cell lines for the antiidiotypic antibody of people CD22 antibody above-mentioned;
B6. detect the reagent of the antibody-mediated antibody-dependant cell mediated cell poison reaction of people CD22, its activeconstituents is that surface of cell membrane expresses any one recombinant cell lines for the antiidiotypic antibody of people CD22 antibody above-mentioned;
B7. detect the reagent of people CD22 antibody bioactive, its activeconstituents is that surface of cell membrane expresses any one recombinant cell lines for the antiidiotypic antibody of people CD22 antibody above-mentioned;
B8. any one antiidiotypic antibody for people CD22 antibody above-mentioned detects the application in people CD22 antibody concentration reagent in preparation;
B9. any one antiidiotypic antibody for people CD22 antibody above-mentioned detects the application in the human antimouse antibody reaction reagent caused by people CD22 antibody in preparation;
B10. any one antiidiotypic antibody for people CD22 antibody above-mentioned detects the application in the anti-chimeric antibody reaction reagent of people caused by people CD22 antibody in preparation;
B11 any one antiidiotypic antibody for people CD22 antibody above-mentioned detects the application in the people anti-human antibody reaction reagent caused by people CD22 antibody in preparation;
B12. surface of cell membrane is expressed any one reconstitution cell for the antiidiotypic antibody of people CD22 antibody above-mentioned and is tied up to the application prepared and detect in the antibody-mediated complement dependent cellular poison reaction reagent of people CD22;
B13. surface of cell membrane is expressed any one reconstitution cell for the antiidiotypic antibody of people CD22 antibody above-mentioned and is tied up to the application prepared and detect in the antibody-mediated antibody-dependant cell mediated cell poison reaction reagent of people CD22;
B14. surface of cell membrane is expressed any one reconstitution cell for the antiidiotypic antibody of people CD22 antibody above-mentioned and is tied up to the application prepared and detect in people CD22 antibody bioactive reagent;
B15. detect penetrance and/or the adsorptivity reagent of people CD22 antibodies on tumor cell, its activeconstituents is arbitrary described antibody in claim 1-3.
B16. any one antiidiotypic antibody application in the penetrance and/or adsorptivity reagent of preparation detection people CD22 antibodies on tumor cell for people CD22 antibody above-mentioned.
Wherein, above-mentioned b1 and b8 specifically can be using any one antiidiotypic antibody for people CD22 antibody above-mentioned as diagnostic reagent to monitor the content of people CD22 antibody in blood samples of patients in immunotherapy, monitoring inject the pharmacokinetics behavior of people CD22 antibody drug." CD22 antibody race " (people CD22 antibody) anti-body contg in Sample serum can be detected as follows:
1) with any one antiidiotypic antibody bag for people CD22 antibody above-mentioned by ELISA enzyme plate, above-mentioned any one comprise 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, test serum sample is added;
3) add on enzyme plate the anti-human Fc specific antibody of such as peroxidase labelling such two anti-and;
4) according to the two anti-anti-human CD22 anti-body contgs how much recorded in test serum of absorption.
In above-mentioned b7 and b14, surface of cell membrane expresses any one recombinant cell lines for the antiidiotypic antibody of people CD22 antibody above-mentioned can by antibody-mediated, there is complement dependent cellular poison reaction (CDC) or antibody-dependant cell mediated cell poison reaction (ADCC), in order to estimate the biological activity of people CD22 antibody (as SM03 or SM06) fast, thus reach the object of quality control.Utilize surface of cell membrane to express any one recombinant cell lines for the antiidiotypic antibody of people CD22 antibody above-mentioned, by the detection method of complement dependent cellular poison reaction evaluator CD22 antibody biologic activity, specifically can comprise following steps:
1) surface of cell membrane is expressed any one recombinant cell lines for the antiidiotypic antibody of people CD22 antibody above-mentioned and anti-CD22 antibody is hatched jointly;
2) complement proteins is added in the mixture;
3) effect of its complement dependent cellular toxic effect (CDC) is measured after hatching suitable time.
Utilize surface of cell membrane to express any one recombinant cell lines for the antiidiotypic antibody of people CD22 antibody above-mentioned, by the detection method of antibody-dependant cell mediated cell toxic effect evaluator CD22 antibody biologic activity, specifically can comprise following steps:
1) surface of cell membrane is expressed any one recombinant cell lines for the antiidiotypic antibody of people CD22 antibody above-mentioned and anti-CD22 antibody is hatched jointly;
2) peripheral blood lymphocytes (PBMC) is added;
3) effect of its antibody-dependant cell mediated cell toxic effect (ADCC) is measured after hatching suitable time.
Above-mentioned b2-b4, b9-b11 specifically can be any one antiidiotypic antibody for people CD22 antibody above-mentioned as the positive control in the anti-antibody immune response researchs such as HAHA, HACA or HAMA.Because the reaction of these anti-antibodys may affect the clinical effectiveness (Gruber van Haarlem et al.2000.Cancer Res.60:1921-1926) of patient, the anaphylaxis of inclusive NAND expection is correlated with, cause significant pharmacokinetics behavior change and affect the distribution in vivo of injection of antibodies medicine, therefore the treatment option of antagonist immunotherapy can produce material impact to its monitoring.The method that anti-chimeric antibody reaction (HACA) of people or people anti-human antibody reaction (HAHA) caused by people CD22 antibody in the individuality of chimeric or Humanized anti-human CD22 monoclonal antibody has been injected in detection provided by the present invention comprises following steps:
1) serum sample is collected from the individuality accepting injection;
2) wrap on ELISA enzyme plate by CD22 antigen, add the anti-CD22 free antibodies of sensitive concentration simultaneously, add the serum sample of different multiples dilution, the hole not adding serum sample, as negative control, adds the hole of the anti-CD22 idiotype antibody of finite concentration as positive control;
3) the anti-human igg Fc bis-adding HRP mark after cleaning resists, and detects the absorption signal of anti-CD22 antibody to CD22 antigen of sensitive concentration;
4) existence detecting anti-CD22 antiidiotypic antibody in serum sample whether, as anti-CD22 antiidiotypic antibody exists the generation of then having proved HACA or HAHA reaction in individuality.
In above-mentioned b15 and b16, can by any one antiidiotypic antibody for people CD22 antibody above-mentioned (as the antiidiotype mouse IgG 2a/kappa immunoglobulin molecules comprising weight chain constant-region sequences of following embodiment 3,) application tumor tissue section immunohistochemical analysis, the penetrance of research " CD22 antibody race " drug on tumor 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, antibody binding pair and other various forms antibody fusion proteins etc., be referred to as " CD22 antibody race ") antiidiotypic antibody of antigen-binding site (ABS), namely 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 race " antibody, and for " the CD22 antibody race " anti-body contg in clinical quantitative analysis serum.The present invention also can be applicable to detect human antimouse antibody reaction (HAMA) that in " CD22 antibody race " monoclonal antibody medicine clinical trial, patient may occur, anti-chimeric antibody reaction (HACA) of people and people anti-human antibody reaction (HAHA).Meanwhile, the method for the clone building the antiidiotypic antibody can expressed for people CD22 antibody is additionally provided; And this type of cell is used for complement dependent cellular poison reaction (CDC) and/or antibody-dependant cell mediated cell poison reaction (ADCC) that detect the mediation of " CD22 antibody race " monoclonal antibody product, achieve biological activity assay and the assessment of monoclonal antibody product.
Accompanying drawing explanation
Fig. 1 be express scFv single-chain antibody phage #1-#3 to mouse source CD22 antibody (RFB4), people mouse is fitted together to CD22 antibody (SM03), the specific adsorption of humanization CD22 antibody (SM06).
Heavy chain complementary determining region (CDR) sequence of Fig. 2 A expressed by phage #1-#3.
The CDR sequence of Fig. 2 B expressed by phage #1-#3.
Fig. 3 is the scFv sequence complete sequence that the phage #3 having specific recognition absorption to RFB4, SM03, SM06 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 SM03 antibody to the absorption of Raji cell in flow cytometry experiment.Three peaks are from left to right respectively blank, SM03+scFv(phage#3) and SM03.
Fig. 5 is the Lymphoma Pharmacokinetic Profile (360mg/m of the SM03 treatment that the capacitive scFv adopting phage #3 to express records 2dosage group, intravenous injection, once in a week, successive administration 4 weeks).
Fig. 6 be express mouse IgG 2a/kappa immunoglobulin (Ig) antiidiotypic antibody IdmG2a/ can DNA amplification carrier.
Fig. 7 is the SDS-PAGE electrophorogram of purification antibody under reduction and non-reduced situation of IdmG2a/k.In figure, 1 and 2 is reduction electrophoresis result, and 3 is molecular weight standard (BenchMark tMprotein Marker, American I nvitrogen Products, batch: 688736), 4 and 5 is non-reduced electrophoresis result.
Fig. 8 is that IdmG2a/k can 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 display antiidiotype mouse IgG antibody I dmG2a/k can effectively prevent SM03 to adsorb the CD22 antigen of Raji cell surface.
Figure 10 is that the SM03 antibody competition that the humanized antibody SM06 of mouse source CD22 antibody RFB4, chimeric antibody SM03 and utilization framework-reengineering technology can mark with HRP is adsorbed onto antiidiotype mouse IgG antibody I dmG2a/k
Figure 11 uses SM03 monoclonal antibody product to carry out the patients with SLE Pharmacokinetic Profile for the treatment of.The Plasma Concentration of SM03 is recorded by ELISA method, and IdmG2a/k is as solid phase capture antibody.
Figure 12 is the cross-film that obtained by the CH3 area merges of the transmembrane domain of mouse source IgD and " the mouse source antiidiotype IgG antibody " heavy chain amino acid sequence for the antiidiotypic antibody IdGmD of people CD22 antibody.
Figure 13 is the heavy chain amino acid sequence replacing IgG2a heavy chain mouse source IgD sequence to obtain the antiidiotypic antibody IdDmD for people CD22 antibody of cross-film.
Figure 14 is the heavy chain amino acid sequence being expressed the antiidiotypic antibody IdGmFdA for people CD22 antibody of cross-film by the form of membrane Fab-glycoprotein fusion rotein.
Figure 15 is the heavy chain amino acid sequence of the antiidiotypic antibody IdGmFdG for people CD22 antibody fixing form expression " mouse source antiidiotype IgG " antibody of fusion rotein with Fab-GPI.
Figure 16 be the various fusion rotein form of antiidiotype mouse IgG antibody transfectional cell mesentery on expression.
Five pictures are from top to bottom followed successively by the SP2/0 cell system not adding SM03, add the SP2/0 cell system that SM03 is 1 μ g/ml to final concentration, add the recombined engineering cell system that SM03 is the expression IdDmD of 0.1 μ g/ml and 1 μ g/ml to final concentration, adding SM03 to final concentration is 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, adding SM03 to final concentration is 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) expressing complement dependent cellular poison reaction (CDC) of various antiidiotype mouse IgG antibody fusion protein transfectional cell series for cross-film of SM03
Embodiment
Describe for the ease of more fully understanding the present invention, some basic definitions are as follows in this declaration definition:
" immunoglobulin (Ig) " this definition herein refers to and is made up of one or more polypeptide chain, and major part is by the related gene coded protein molecule of immunoglobulin (Ig).The immunoglobulin gene coding cracked comprises kappa, lamda, alpha, gamma (IgG1, IgG2, IgG3, IgG4), these constant region gene of delta, epsilon and mu, and countless immune globulin variable region genes.Complete immunoglobulin (Ig) " light chain " (by 214 Amino acid profiles, molecular weight is about 25Kd) is made up of kappa or the lamda constant region gene coding of N-terminal variable region gene (about 110 amino acid) and hydroxyl terminal.Similarly, complete immunoglobulin (Ig) " heavy chain " is (by 446 Amino acid profiles, molecular weight is about 50Kd) to be encoded (about 116 amino acid) by variable region gene and one of them constant region gene described is encoded and formed above, as gamma constant region gene (about 300 amino acid) is encoded formation IgG molecule.
Unless specifically stated otherwise, " antibody " this definition used herein is widely used for referring to complete antibody molecule and its derivative.These derivatives at least contain 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, antibody chain chimeric fusion, bispecific antibody and the such antibody molecule segment of other similar molecules.
" chimeric antibody " used herein this definition refers to the protein molecular of immunoglobulin light variable region of heavy chain by inhuman generic kind biology and human immunoglobulin molecules's constant region Recombinant design.
" humanization " used herein this definition refers to and only retains from the part of the immunoglobulin molecules variable region complementary determining area (CDR) of inhuman generic kind biology, remaining main variable region portion and all constant region all come for people source, through the protein molecular of Recombinant design.
" idiotype " used herein this definition refers to and antibody molecule determines the specific special segment of antigen.Idiotype segment is positioned at monoclonal antibody part, and the description for it forms antigen-binding site based on the common participation of antibody molecule heavy chain light chain usually.
" isotype " used herein this definition refers to and is determined by the antigen-specific of immunoglobulin molecules, according to heavy chain classification and subclass, and light chain type the immunoglobulin molecules type of dividing and hypotype thereof.Such as, four isotypes of IgG are IgG 1, IgG 2, IgG 3, IgG 4.
" CD22 antibody race " described herein is derived by mouse source antibody RFB4.It the chimeric antibody (SM03) that derives the same with original murine antibody with humanized antibody (SM06), for the B site tool specificity on people CD22 antigen.The clinical trial of using SM03 to treat B cell lymphoma and other autoimmune diseases starts to walk.In clinical practice, in order to meet the demand measuring " CD22 antibody race " product (as the SM03 monoclonal antibody) content injected in serum clinically, build and qualitative a kind of antiidiotypic antibody, measure " the CD22 antibody race " product concentration in patients serum as ELISA reagent.In addition, the antiidiotypic antibody produced can also be used in ELISA method weighs HACA or HAHA reaction, also or in contrast antibody and the competitive antibody that is used as in diagnostic reagent estimation patients serum.
The antiidiotypic antibody of single chain antibody format by the mouse through SM03 immunity, and produces by the method for phage display.Concrete grammar is extract spleen cell with it from the mouse of the anti-CD22 chimeric antibody SM03 of injection, and separating mRNA, then degeneracy flank variable region primers amplification weight chain variable region sequence.According to standard step, gained variable region sequences is introduced scFv single chain antibody phage afterwards and show storehouse.Take turns after the screening carried out with SM03 and RFB4 antibody eliminates through several, select for the specific phage of SM03, RFB4 and SM06 tool, and the corresponding variable region sequences of selected phage display.In the phage of showing antibody variable sequences, select mouse source, chimeric and humanization " CD22 antibody race " has the displaying phage of most high-affinity.
Antiidiotypic antibody sequence is expressed with the form of scFv single-chain antibody occlusion body at first in intestinal bacteria, understands sex change and refolding afterwards; The scFv single-chain antibody of tool activity is used as the serum SM03 content in ELISA reagent detection clinical trial.Concise and to the point step is as follows, use antiidiotype scFv single-chain antibody (scFv) bag of refolding by ELISA enzyme plate exactly, add after patients serum's dilution of again SM03 being treated, through hatching, cleaning, SM03 antibody in patients serum will with the antiidiotype scFv single-chain antibody of bag quilt be combined, its specific binding can with goat anti-human igg-Fc specific antibody (the U.S. Jackson ImmunoResearch Products) colour developing of HPR mark.But antiidiotype scFv single-chain antibody has unstable tendency, and the mode produced 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 a checking batch preparation subsequently become very difficult.
And on the other hand, as everyone knows, complete immunoglobulin molecules can keep the stability of several months and even several years under suitable condition of storage, and its antibody activity and quality also considerable change can not occur.In order to create the stable and detection method that result is consistent to 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 one intactly immunoglobulin molecules.Because the chimeric antibody in " CD22 antibody race " and humanized antibody all have the constant-region sequences of human IgG1's heavy chain and kappa light chain, they therefore can by ELISA HRP usually used mark anti-human igg Fc specific antibody (or similar combination) identify.Therefore, designed antiidiotypic antibody immunoglobulin molecules should with the human IgG constant-region sequences that can cause cross reaction.Can not there is cross reaction with anti-human Fc antibodies in mouse IgG 2a/kappa antibody (mouse source antiidiotype IgG antibody) constant-region sequences, so be selected to build complete antiidiotypic antibody immunoglobulin molecules.It should be noted that other isotype or the constant-region sequences from other species also can adopt.According to designed antiidiotypic antibody immunoglobulin (Ig), create the express cell system that productive rate reaches 30ug/ml.Owing to producing the expression vector that adopts of express cell system with the DHFR gene that can increase, this productive rate can increase according to general amplification-cloning process in case there is a need further.But, the present stage productive rate enough satisfied mouse source antiidiotype IgG antibody producing consistent batch, and detect for pharmacokinetic and HACA or HAHA.Concise and to the point step is as follows, uses mouse source antiidiotype IgG antibody bag by ELISA enzyme plate exactly, then adds the patients serum of use " CD22 antibody race " antibody drug treatment, and through hatching, the albumen do not adsorbed with " mouse source antiidiotype IgG antibody " can be cleaned up.And anti-CD22 antibody in serum can specifically with bag quilt " mouse source antiidiotype IgG antibody " combine, and mark the colour developing of goat anti-human igg Fc specific antibody with HRP.Due to the HRP traget antibody generation cross reaction that the mouse source constant-region sequences of " mouse source antiidiotype IgG antibody " can't detect, 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 by expansive approach to the estimation identification of " CD22 antibody race " and avidity.Application " mouse source antiidiotype IgG antibody " is as positive control, and set up the Biacore analytical procedure of HACA and HAHA of serum sample, 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, the penetrance of research " CD22 antibody race " drug on tumor cell and adsorptivity.Can the generation of " mouse source antiidiotype IgG antibody " of specific binding target antibody, on clinical meaning, assessment injection target antibody Drug safety and pharmacokinetic property provide source chemicals in high sensitivity." mouse source antiidiotype IgG antibody " constructed by the present invention therefore obtains the diagnostic test reagent that card is worth for a kind of tool important practical, for studying the patient clinical sample accepting SM03 immunotherapy.
One aspect of the present invention is for detecting " mouse source antiidiotype IgG antibody " to " CD22 antibody race " the resistance inhibitor action supplying method to its Antigen adsorption.Another aspect catches for detecting " mouse source antiidiotype IgG antibody " and detect the ability supplying method of adsorbed idiotype antibody.Darker one deck implication is detect " mouse source antiidiotype IgG antibody " adsorptive power supplying method to its idiotype antibody (anti-CD22 antibody).But, be also for detecting " CD22 antibody race " content supplying method in serum sample on the other hand.The present invention points to one too and carries out detection HAMA with antibody described by this patent, the method for HACA and HAHA reaction.
Another kind application of the present invention utilizes antiidiotypic antibody to build non-internalization cross-film to express SM03-Specific adsorption segment engineering cell system.Clone of building can be used for set up assessment SM03 monoclonal antibody and derivative as the detection method of the biological effects such as RFB4, SM06.The foundation of this method means that realizing absorption by similar approach can be of universal significance in the detection of antibody biological effectiveness of internalizing surface antigen.
Below test illustration for illustrating listed by purposes of the present invention, but do not represent and be of the present inventionly confined to this type of and apply.
Experimental technique in following embodiment, if no special instructions, is ordinary method.
Material used in following embodiment, reagent etc., if no special instructions, all can obtain from commercial channels.
Embodiment 1, the antiidiotypic antibody for people CD22 antibody existed with single chain antibody format
The present embodiment uses phage display library technology to obtain the variable region weight chain-ordering of anti-SM03 antiidiotypic antibody, prepares the antiidiotype single-chain antibody for people CD22 antibody further---the soluble scFv that phage #3 expresses.Concrete steps are as follows:
1, the mouse of injection SM03 immunization is utilized to prepare phage display library
About 6 weeks large female Balb/c mouse are by immunization, method is according to standard immunoassay inoculation step (being published by U.S. Cold Spring Harbor Laboratory for 1988 referring to " InAntibodies:A Laboratory Manual ") peritoneal injection 100ug SM03 monoclonal antibody, and with 200uL complete Freund's adjuvant (Sigma-Aldrich's product) emulsification.Second time and for the third time immunization are 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 (Sigma-Aldrich's product).
After measuring the SM03 antibody titers in mice serum, the Mouse spleen cells of immunization after 39 days is used to be separated full RNA and preparation cDNA(use Superscript II test kit, American I nvitrogen Products).Then synthesize degenerated primer according in pertinent literature, utilize pcr amplification immune globulin variable region gene sequence (Cheng et al.2005.BiochemBiophys Res Commun 338:1654-1660).Subsequently, according to the recombinant phages antibody system product guide of Amersham company of the U.S., build variable region segment single-chain antibody (scFv) phage display library.
Carry out the amplification of segment scFv single chain antibody phage displaying storehouse, variable region and filobactivirus with reference to pertinent literature after, with SM03 monoclonal antibody and mouse source CD22 monoclonal antibody RFB4(Ancell, Cat:171-820) carry out screening superseded (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 antibody bicarbonate buffer (15mM Na 2cO 3, 35mM NaHCO 3) carry out bag quilt, be 10 with the method screening concentration of biological screening 12phage.The incubated at room of slight concussion condition through 2 hours, the 0.1M glycine-HCI damping fluid of the scFv phage 100uL adsorbed, pH2.2 through 10 minutes hatch wash-out, then use 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 each screening of record.
Screening process survival phage through rescue, its absorption specificity method of phage-ELISA is assessed, use anti-CD22 antibody RFB4, SM03 and SM06(China Antibody Pharmacy Co., Ltd.), and other control antibodies is screened further as antigen.Method is briefly described as follows, and in 96 hole ELISA enzyme plates, wraps respectively by RFB4, SM03, SM06, and chimeric anti-TNF antibodies and BSA(use the supercarbonate bag of 50uL pH9.6 to be buffered liquid, and package amount is 1ug; After 4 DEG C of overnight incubation, rinse with the borate buffer of 200uL pH8.0; 1 hour is closed at 37 DEG C equally again with borate buffer), there are three phage clones (supernatant liquor of 100uL) of the highest absorption avidity to be added in hole 37 DEG C to SM03 and hatch 1 hour.Through the flushing of the borate buffer of 5 pH8.0, the anti-M13 mouse antibodies (Amersham Products) of HRP mark adds after 1:3000 dilution.After 37 DEG C hatch 1 hour, O-Ursol D (OPD) reaction solution of 100uL (10mgOPD is dissolved in the citrate phosphate buffer of 10mL containing 8uL30% hydrogen peroxide solution, pH5.0) is used to develop the color.The selection result as shown in Figure 1, three selected scFv show that phage (phage #1-#3) can adsorb RFB4, SM03 and SM06(mouse source, chimeric and humanized CD22 antibody) but other antibody (anti-TNF antibodies (infliximab)) or reference protein (BSA) can not be adsorbed.Sequence same section due to RFB4, SM03 and SM06 only has the CDR part on target antibody, or antigen-binding site, the result shows that three selected scFv show that phage all has specificity to the idiotype part of SM03.
2, the scFv of specific adsorption SM03 antigen-binding portion thereof the mensuration of phage variable region sequences can be shown
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 and has part variation in the fragmentary position of framework segment or CDR3 segment.Fig. 2 illustrates the CDR fragment 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, CDR1 with CDR2 of the light chain of the scFv that phage #1-#3 shows is identical, and there is an amino acid (in Fig. 2 underscore mark) in the light chain CDR3 region had only expressed by phage #2.
The phage #3 of the scFv sequence of 3, showing can prevent SM03 antibody to adsorb Raji cell
Because phage #3 shows relatively high absorption avidity, its single stranded sequence is responded into DNA encoding and uses molecule clone technology to construct scFv bacterial expression vector.The scFv sequence 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 expressing.Through IPTG abduction delivering, the occlusion body containing 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, containing the scFv of His-tag after sex change, at Ni 2+ there is situation under be adsorbed onto HItrap Chelating HP separator column.The guanidinium-hydrochloride buffer (containing 20mM sodium phosphate, 0.5M sodium-chlor, pH7.4) that use progressively reduces concentration according to gradient carries out rinsing until do not have residual Guanidinium hydrochloride to retain.Rinse with the imidazole buffer of the 5-40mM of several column volume (containing 20mM sodium phosphate, 0.5M sodium-chlor, pH7.4) 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 the fusion rotein that the aminoterminal of the 1st amino acids residue of sequence 1 in sequence table connects His-tag and 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 SM03 antibody to adsorb the CD22 antigen of Raji cell surface expression.Test concise and to the point step as follows: Raji cell cleaned for PBS and SM03 antibody are jointly hatched; add the soluble scFv expressed by phage #3 of different concns simultaneously; to only add the Raj i cell of SM03 antibody in contrast, do not add the Raji cell of any reagent as blank (blank); Through half an hour incubated at room and PBS cleaning after, the fluorescently-labeled goat anti-human igg Fc specific antibody adding 1:50 dilution in all samples resists as two, through cleaning after incubated at room half an hour, uses Beckman system of fluorescence analysis to analyze its fluorescent signal.Result as Fig. 4, show to add 100ug/ml group, the result of its flow cytometry shows obvious fluorescent signal and declines, and the capacitive scFv that display phage #3 expresses can effectively must suppress SM03 antibody to be adsorbed onto the CD22 antigen of Raji cell surface.
Experimental example 2, the soluble scFv utilizing embodiment 1 pnagus medius #3 to express carry out the pharmacokinetic in SM03 clinical trial
Because the capacitive scFv of embodiment 1 pnagus medius #3 expression is to the specificity of SM03, therefore can be used for developing corresponding detection method to measure the antibody concentration in the patients serum accepting anti-CD22 antibody treatment, particularly assist the pharmacokinetic needed for clinical practice.With the capacitive scFv wrapper sheet 96 hole ELISA enzyme plate that embodiment 1 pnagus medius #3 expresses.Close through BSA, cleaning, each blood sampling point serum of patient accepting SM03 treatment adds in hand-hole after dilution.Hatch 2 hours and fully cleaning through 37 DEG C, HRP marks sheep anti-human Fc antibodies (Jackson Immunoresearch Products) and adds in hand-hole after 1:4000 dilution.Enzyme plate, again after 1 hour 37 DEG C hatch, cleans 5 times, and with the colour developing of TMB nitrite ion, color signal according to standard ELISA detection method 450nm wavelength readings, and calculates caught SM03 concentration with this.
Fig. 5 illustrates the typical drug-time curve of SM03, and method therefor is described above.Patients serum's sample from accept anti-CD22 antibody SM03 treat Malignant Lymphoma, 380mg/m 2dosage group, 1 time weekly, successive administration 4 weeks.Serum sample is from the different time points collection before and after administration.
Embodiment 3, the antiidiotypic antibody IdmG2a/k for people CD22 antibody existed 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, and protein denaturation folds and the step of the series of complex such as His-tag purification.In addition, scFv is more unstable and not easily store.In order to produce the capacitive scFv absorption specificity both retaining embodiment 1 pnagus medius #3 and express, can be purified by standard step again and the stable molecule easily stored, 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 pcr amplification expression vector pIdmkappa-VK and pIdmIgG-VH using molecule clone technology to be integrated into can to increase, it further comprises the constant region sequence (as shown in Figure 6) of mouse source kappa light chain and IgG2a heavy chain.
This construction process that can increase expression vector dissolubility scFv variable region of heavy chain (VH) coding DNA in encoding example 1 and variable region of light chain (VK) encoding sequence is connected to separately corresponding mouse source kappa light chain and IgG2a heavy chain can to increase the constant region of expression vector.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), it contains the IgG enhanser (Enhancer) of a upstream, a cloning site being used for BamHI/BglII and XbaI inserting VK section, and the gene order of the mouse kappa constant region of light chain of intron of ining succession (intron).In the downstream of light chain constant region gene sequence, 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 hygromycin selective marker, obtains recombinant vectors pSVhyg.Between the recognition site of EcoRI and BamHI of pSVhyg, insert the Ig enhanser of 319bp and insert an XbaI restriction enzyme enzyme recognition site before BamHI site, obtaining recombinant vectors pSVhyg-Ig.Between BamHI and the SacI recognition site of pSVhyg-Ig, insert the kappa enhanser of 199bp, obtain recombinant vectors pSVhyg-Ig-kappa.The mouse source kappa constant region of light chain fragment of the intron of ining succession (intron) of 1.2Kb is inserted between the restriction enzyme SacI and BamHI recognition site of pSVhyg-Ig-kappa, 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 of the 5 ' end from GeneBank Accession Number:K01901.The kappa enhanser nucleotide sequence of 199bp is the 1 to 199 deoxynucleotide of the 5 ' end from GeneBankAccession Number:K01325.The nucleotide sequence of the intron mouse endogenous light chain constant segments of 1.2Kb is the 1 to 1209 deoxynucleotide of the 5 ' end from GeneBank Accession Number:J00241.
The construction process of mouse IgG 2a heavy chain expression vector pIdmIgG-VH is roughly similar, be similarly and be derived from carrier pSVgpt(Mulligan & Berg 1981.PNAS 78:2072-2076) 10Kb size expression vector, it contains the IgG enhanser (Enhancer) of a upstream, Xho I and Hind III cloning site being used for inserting VH section, and the gene order of the mouse IgG 2a CH of 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: the Ig enhanser inserting 319bp between the recognition site of EcoRI and XhoI of pSVgpt, obtains recombinant vectors pSVgpt-Ig.The mouse IgG 2a heavy chain constant region segment of the intron of ining succession (intron) of 2Kb is inserted between the restriction enzyme HindIII and BamHI recognition site of pSVgpt-Ig, between XhoI and HindIII restriction endonuclease sites, be connected into the VH sequence that phage #3 shows, obtain mouse IgG 2a heavy chain expression vector pIdmIgG-VH.Wherein, the Ig enhanser of 319bp and nucleotide sequence are the 1st to the 319th deoxynucleotides of the 5 ' end from GeneBank Accession Number:K01901.The nucleotide sequence of the intron mouse source heavy chain constant region segment of ining succession of 2Kb is the 1 to 2009 deoxynucleotide of the 5 ' end from 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 embryonic kidney cells is 293 (HEK293), African green monkey kidney COS clone etc.In the present embodiment, select SP2/0 engineering cell system as host, use the method for electric shock perforation transfection to carry out transfection by standard step.Select through methotrexate, 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 expressing antibody is exaggerated cultivates, and its " mouse source antiidiotype IgG antibody " (hereinafter referred to as IdmG2a/k) produced also is collected and purifies.Meanwhile, the RNA expressing 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, the VH of IdmG2a/k, the capacitive scFv that its weight chain variable region sequence and embodiment 1 pnagus medius #3 express, VL fragment sequence is identical, simultaneously 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 polynucleotide; The light chain DNA sequences encoding of IdmG2a/k is the sequence 10 in sequence table, the aminoacid sequence of sequence 3 in polynucleotide.
Express the recombined engineering clone of IdmG2a/k through amplification culture, it is expressed the antibody produced and passes through extraction cells and supernatant, purifies by standard step with albumin A separator column.The antibody of purifying can in 4 DEG C of preservations in PBS damping fluid.Fig. 7 shows the SDS-PAGE electrophoresis pattern of purification antibody under reduction and non-reduced situation of IdmG2a/k.
The IdmG2a/k of recombined engineering expression of cell lines is purified can be used for by standard step bag by ELISA enzyme plate.
On ELISA enzyme connecting plate, every hole adds the IdmG2a/k of 50uL 10ug/ml, and 4 DEG C of night incubation carry out bag quilt.To bag by complete enzyme plate, every hole adds 60uLSM03 antibody and other the irrelevant control antibody of different concns, as anti-CD20 antibodies Rituximab (Mabthera, Roche Group of Switzerland Products), anti-CD147 antibody or anti-TNF antibodies Infliximab(class gram, Cilag AG company of Switzerland).Through 1.5 hours incubated at room, after cleaning 3 times with PBS, add the sheep anti-human Fc antibodies (Jackson Immunoresearch Products) of the HRP mark of 1:5000 dilution.Again after 45 minutes incubated at room, its absorption can develop the color with OPD and use 450nm wavelength readings.Result shows, and " 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 the CD22 antigenic competition assessed under the IdmG2a/k of recombined engineering expression of cell lines and state of nature adsorbs the ability of SM03 antibody, using Raji(mankind's Burkitt lymphoma) clone naturally expresses CD22 antigenic source as surface and carried out flow cytometry experiment.Concise and to the point step is as follows, and 0.5 × 10 6the SM03 antibody of individual Raji cell and 1ug/ml and the mouse source antiidiotype IgG antibody IdmG2a/k of different concns are hatched jointly, incubation conditions is that the PBS-FA damping fluid (containing 1% foetal calf serum FBS, the PBS solution of 0.01% sodiumazide) 4 DEG C of 200uL hatches 30 minutes.After cleaning 3 times with PBS, add 50 times of fluorescent mark anti-human Fc antibodies (Jackson Immunoresearch Products) diluted and continue 4 DEG C and hatch 30 minutes.After cleaning 3 times with PBS again, fix with the PBS-FA damping fluid containing 0.5% formalin.Hang with PBS through fixing cell, use the FACScan analytical system of Becton Dickenson company of the U.S. to carry out Flow Cytometry Analysis.Result shows, and antiidiotypic antibody IdmG2a/k can according to dosage suppress SM03 to be adsorbed onto its natural part (Fig. 9) effectively.
Embodiment 4, " the CD22 antibody race " content utilized in the IdmG2a/k monitoring clinical practice of embodiment 3
1, the antigen-binding portion bit sequence that IdmG2a/k energy specific binding " CD22 antibody race " of embodiment 3 is total, can adsorb mouse source specifically, chimeric and humanized anti-CD22 antibody.
Competitive adsorption detection method shows further, and the adsorption site of antiidiotypic antibody is positioned at antigen-binding site (ABS) sequence, is the uniquely publicly-owned sequence of mouse source antibody RFB4, 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 DEG C of night incubation carry out bag quilt.After secondary daily PBS cleans 3 times, close 2 hours by the 3%BSA solution room temperature of 200uL, then clean 5 times with PBS.SM03 antibody HRP is labeled as SM03-HRP traget antibody (being marked by Tian Jian bio tech ltd, Tianjin).SM03-HRP traget antibody after 1:4000 dilution, the competition antibody with different concns, comprises RFB4, SM03, SM06 and the mixing of some other irrelevant control antibody (SM09, N005, N009).Mixed antibody adds bag by the ELISA enzyme connecting plate of the IdmG2a/k of embodiment 3.SM03-HRP traget antibody to bag quilt antiidiotypic antibody adsorption levels after can develop the color with TMB nitrite ion.
Result shows, RFB4, SM03 and SM06 can equivalence with SM03-HRP traget antibody competitive adsorption to the IdmG2a/k of embodiment 3, other irrelevant antibodies does not then have competitive adsorption ability (as shown in Figure 10), show that the IdmG2a/k of embodiment 3 is that specific adsorption arrives RFB4, the consensus sequence of SM03 and SM06, but not other site, this consensus sequence i.e. 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, the standard detecting method of the IdmG2a/k exploitation assessment SM03 clinical experiment pharmacokinetic of embodiment 3 is utilized
Along with " mouse source antiidiotype IgG antibody " adsorbs specific confirmation to " CD22 antibody race " antigen-binding site, develop the ELISA detection method that one can estimate RFB4, SM03 and SM06 anti-body contg in patients serum in clinical experiment.Following illustration describes the detection method performance history of estimation SM03 serum content, other member that this method also can promote the use of " CD22 antibody race ".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 wrapping by condition is every hole 50uLPBS, pH7.4,4 DEG C of overnight incubation; Secondary daily 1%BSA solution room temperature closes 2 hours.The patients serum's sample accepting SM03 treatment, after dilution certain multiple, adds with the multiple hole of the external source SM03 standard substance of different concns, diluent select containing 0.1% blank human serum PBS to remove the impact of serum on background.Every hole to add after the sample of 50uL incubated at room 2 hours, and after cleaning, mark sheep anti-human Fc antibodies (JacksonImmunoresearch Products) at the HRP adding 1:4000 dilution, incubated at room clean after 1 hour and added TMB and develops the color.The visible ray of color signal 450nm wavelength reads plate record.Accept the time dependent situation of SM03 antibody concentration residual in Antybody therapy peripheral blood in patients to be obtained by the comparison with external source SM03 standard substance.
Figure 11 illustrates the representative pharmacokinetic behavior that accepts 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-internalizing antigen antibody biological effectiveness
One, the engineering cell system that cross-film expresses antiidiotypic antibody absorption segment is built
In the process storing manufacture of therapeutic antibody, the biological activity of antibody is assessed in the mode of quality control, for SM03, there are two important consideration: the ability of adsorption property (being read as absorption avidity and specificity in detail) and antibody-mediated biologically (as ADCC or CDC).The latter estimates by the mode that biological effectiveness detects usually.Due to all adsorbable CD22 antigen of " CD22 antibody race " antibody, and internalization phenomenon, SM03 antibody and other anti-CD22 antibody after absorption, can be there is very soon, mediation CDC reaction smoothly in can not testing in vitro." CD22 antibody race " antibody is after being adsorbed onto the positive cell surface of CD22 expression, time enough can not be stopped at cell surface fully to act on complement (being assumed to be C1q complement) by its Fc part, so that correctly can not 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 the antibody that rapid internalization phenomenon surface antigen can occur, 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) demonstrates the suction sheet be made up of antibody weight chain variable region and wrecks folded, sequence, pairing is all correct, but can not provide the contingent protein variant of antibody Fc portion or deletion condition.And these disappearances or variation may hinder during effect in antibody body and play its correct biological effectiveness.Therefore, develop the universal test method based on cell detection, to estimate with the antigen that rapid internalization phenomenon can occur be target, and antibody biological function 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 is completed by following several mode:
1, antiidiotypic antibody is expressed in the mode of cross-film IgD
A. the transmembrane domain of mouse source IgD and the CH3 area merges of " mouse source antiidiotype IgG antibody " are obtained the antiidiotypic antibody IdGmD for people CD22 antibody of cross-film
For the aminoacid sequence in IgD cross-film district, mouse source (TM) that merges with antiidiotype IgG antibody as shown in the sequence with underscore in Figure 12.
In order to build IgG2a-TM (IgD) protein gene can expressing fusion, the chemosynthesis DNA sequence dna of one section of coding " mouse source antiidiotype IgG antibody " part carboxyl-termini sequences.This section of DNA sequence degeneracy two restriction enzyme point of contact BsrG1 and EagI, assisting to be cloned in corresponding original mouse IgG 2a constant-region sequences.。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), the one section of sequence that instead of IgG2a CH3 territory carboxyl terminal make it not only comprise TM sequence that former sequence also add mouse source IgD.Expressed albumen should be permeable membrane segment (complete be configured to VH-CH1-hinge-CH2-CH3-TM) (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.Select through methotrexate, 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 expressing antibody is exaggerated cultivates, and the cross-film antibody I dGmD that it produces also is collected and purifies.Meanwhile, the RNA expressing 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 (aminoacid sequence of Figure 12) of sequence 4 in polynucleotide; The light chain DNA sequences encoding of IdGmD is the sequence 10 in sequence table, the aminoacid sequence of sequence 3 in polynucleotide.
B. IgG2a heavy chain is replaced by mouse source IgD sequence the antiidiotypic antibody IdDmD for people CD22 antibody obtaining cross-film
The constant-region sequences of original " mouse source antiidiotype IgG antibody " is replaced by the mouse source IgD sequence of membrane form completely.Mouse source IgD complete amino acid sequence with permeable membrane segment TM sequence as shown in figure 13.
The method chemosynthesis that the mouse source IgD cDNA sequence of encoding membrane form is synthesized according to 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, instead of the constant-region sequences of mouse IgG 2a, obtain the recombinant expression vector pIdDmD of IdDmD with membrane IgD protein sequence.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, thus its structure and IgG or people source IgD produce, as (cross-film district underscore marks) shown in Figure 13 not very large.
Because just illustrative experiment, the expression vector dna plasmid of the antiidiotype IgD-TM only having (b) part to describe is through restriction endonuclease linearize.With mouse myeloma cell line SP2/0 as transfection host cell.Illustrate according to pertinent literature, SP2/0 cell can only produce endogenous Ig β and can not produce Ig α.Owing to the film of IgD immunoglobulin molecules being expressed the acting in conjunction needing Ig α and Ig β, 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.The cell of transfection plasmid to be selected according to DHFR system standard method with the selective medium containing methotrexate.Method through selecting the cell cell ELISA of survival to detect tests the antiidiotype specificity expressed by its 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).3 times are rinsed with PBS after 4 DEG C, the cell being mixed with antibody is hatched 1 hour.Then the HRP through 1:1000 dilution adding 50uL marks goat anti-human igg Fc antibody, hatches 1 hour for 4 DEG C.After rinsing 1 time with PBS again, 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.By centrifugal for antibody-cell mixture, collect supernatant liquor wavelength 450nm and read light absorption ratio.
Select positive clone to increase, through the amplification of number wheel, the recombined engineering clone expressing antibody is exaggerated cultivates, and the cross-film antibody I dDmD that it produces also is collected and purifies.Meanwhile, the RNA expressing 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 polynucleotide; The light chain DNA sequences encoding of IdDmD is the sequence 10 in sequence table, the aminoacid sequence of sequence 3 in polynucleotide.
Result shows, and needs Ig α to be transfected into murine myeloma cell at the anti-SM03IgD of Membrane surface expression simultaneously.The result of flow cytometry experiment (this experimental technique is with the flow cytometry of following step 2) confirms the Membrane surface expression (Figure 16) of IgD.
2, the antiidiotypic antibody IdGmFdA for people CD22 antibody of cross-film is expressed by the form of membrane Fab-glycoprotein fusion rotein
Glycoprotein A trans-membrane region from Surface of Erythrocytes is merged mutually with " mouse source antiidiotype IgG antibody " mouse IgG 2a hinge area carboxyl terminal.This antibody fragment merges expection can be expressed in the Fab mode being fixed on cell surface by glycoprotein A cross-film district on transfectional cell surface.
Be used for merging the glycoprotein A segment (comprising transmembrane domain) of anti-SM03 antibody fragment as shown in the aminoacid sequence of the band underscore of Figure 14.
Gene order is directly merged after last amino acid whose gene order is encoded in Ig2a antibody hinge region, mouse source, expressed albumen should for " mouse source antiidiotype IgG antibody " heavy chain Fd area merges be in the cross-film district of glycoprotein A 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 " mouse source antiidiotype IgG antibody " Fd segment-glycoprotein A fusion rotein is expressed through having built at surface of cell membrane.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 transmembrane domain) according to standard oligonucleotide synthetic method through chemosynthesis gained.Synthesized sequence contains point of contact in in-frame XhoI and EagI.Composition sequence utilizes the molecular biology clone technology of standard to insert corresponding site in anti-SM03IgG2a antibody expression vector (Fig. 6) gene order, instead of the CH2-CH3 regional code sequence in former IgG2a antibody sequence, obtain 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 methotrexate through transfected plasmids utilizes DHFR gene Selection system to carry out positive colony selection according to standard method.Select positive clone to increase, through the amplification of number wheel, the recombined engineering clone expressing antibody is exaggerated cultivates, and the cross-film antibody I dGmFdA that it produces also is collected and purifies.Meanwhile, the RNA expressing 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 polynucleotide; The light chain DNA sequences encoding of IdGmFdA is the sequence 10 in sequence table, the aminoacid sequence of sequence 3 in polynucleotide.
The survival positive colony selected is used for measuring the cell surface expression situation of its antiidiotype Fab-glycoprotein A fusion rotein, and method therefor is still cell ELISA detection method described before.Detect through ELISA and successfully carry out flow cytometry further at the cell of cell surface expression fusion rotein: SM03 antibody is as primary antibodie, and FITC fluorescently-labeled goat anti-human igg Fc antibody (Jackson Immunoresearch Products) is as detecting antibody (two resist).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 damping fluid (containing 1% foetal calf serum FCS, the PBS solution of 0.05% sodiumazide) of 100uL.Incubation conditions is 4 DEG C, 30 minutes time, cleans 3 times to remove non-adsorbed antibody afterwards with PBS.SM03 marks the check and evaluation of goat anti-human igg Fc antibody (JacksonImmunoresearch Products) with fluorescence FITC to the adsorptive capacity of transfectional cell.After hatching 30 minutes with the 100uLPBS-FA damping fluid containing 20 times of dilution fluorescent-labeled antibody through 4 DEG C, rinse cell 3 times with PBS, its fluorescent signal FACScan system is analyzed.Result shows, and antiidiotype Fab-glycoprotein A fusion rotein IdGmFdA can effectively at the cell surface expression (as shown in figure 16) of transfected into bone marrow oncocyte system.
3, the antiidiotypic antibody IdGmFdG for people CD22 antibody of form expression " mouse source antiidiotype IgG " antibody of fusion rotein is fixed with Fab-GPI
GPI segment is derived by the ldl receptor be 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 being attached to cell surface is expressed.
The GPI segment aminoacid sequence derived by the ldl receptor be attached on DAF hydrophobic region is as shown in the aminoacid sequence with underscore in Figure 15.
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, and the method that the DNA sequence dna that coding mouse IgG 2a CH1-hinge area and GPI-DAF merge segment synthesizes according to standard oligonucleotide is through chemosynthesis gained.Point of contact in in-frame XhoI and EagI (shown in underscore) is comprised in sequence.Composition sequence utilizes the molecular biology clone technology of standard to insert corresponding site in " mouse source antiidiotype IgG antibody " expression vector (Fig. 6) gene order, instead of the CH2-CH3 regional code sequence in former IgG2a antibody sequence, obtain the recombinant expression vector pIdGmFdG of IdGmFdG.
PIdGmFdG plasmid DNA is transfected into mouse SP2/0 host cell through linearize.Cell methotrexate through transfected plasmids utilizes DHFR gene Selection system to carry out positive colony selection according to standard method.Select positive clone to increase, through the amplification of number wheel, the recombined engineering clone expressing antibody is exaggerated cultivates, and the cross-film antibody I dGmFdG that it produces also is collected and purifies.Meanwhile, the RNA expressing 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 polynucleotide; The light chain DNA sequences encoding of IdGmFdG is the sequence 10 in sequence table, the aminoacid sequence of sequence 3 in polynucleotide.
The survival positive colony selected is used for measuring the cell surface expression situation of its antiidiotype Fab-glycoprotein A fusion rotein, and method therefor is still cell ELISA detection method described before.Detect through ELISA and successfully carry out flow cytometry further at the cell of cell surface expression fusion rotein: SM03 antibody is as primary antibodie, FITC fluorescently-labeled goat-anti people FcIgG antibody (Jackson Immunoresearch Products) is as detecting antibody (two resist), and concrete grammar is with the flow cytometry of above-mentioned steps 2).Result shows, and " mouse source antiidiotype IgG antibody " Fab segment-GPI fusion rotein IdGmFdG can effectively at the cell surface expression (as shown in figure 16) of transfected into bone marrow oncocyte system.
Two, the cell detection method in order to assess " CD22 antibody race " biological effectiveness is set up
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 Cedarlane company of Canada is mixed with the GPS solution of 100% with the substratum of 1 milliliter.The SM03 antibody of the 50uL different concns containing 10%GPS is added in the hole being added with transfectional cell.Through 2 hours, 37 DEG C, hatching under 5% carbon dioxide conditions, every hole adds the CCK-8(cell counting kit-8 of Dojindo Molecular Technology Corp. of the U.S. of 20uL) reagent.After 3 hours, read Cell viability according to the description of product by 450nm visible wavelength.Result shows, such " the CD22 antibody race " product of such as SM03 can 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, (CDC reacts the reaction of effective mediate complement dependent cells poison, as shown in figure 17).Obvious CDC effect is not then had to the transfectional cell at cell surface expression antiidiotype IgD-TM segment protein I dDmD.Still may can there is internalization phenomenon for the IgD of cell surface with " CD22 antibody race " product in reason, so that blocked CDC reaction after adsorbing; Or cause the threshold value of complement activation because expression amount on the film of antiidiotype-TM segment albumen does not reach very little and.In any case, three kinds of different transfectional cells of fragment are adsorbed at cell surface expression " mouse source antiidiotype IgG antibody ", have two kinds according to dosage effectively can mediate SM03 initiation CDC to kill and wound, showing this strategy can as exploitation suitable biological detection method, thus correct assessment can the general governing principle of internalizing antigen antibody biological effectiveness.

Claims (9)

1. an antibody, 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.
2. antibody according to claim 1, is characterized in that: described antibody is following a)-f) in one:
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) single-chain antibody shown in sequence 1 in sequence table, or in sequence table, the aminoterminal of sequence 1 or carboxyl terminal add the histidine-tagged fusion rotein obtained;
C) 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;
D) 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;
E) 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;
F) 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.
3. the nucleic acid molecule of antibody described in coding claim 1 or 2.
4. nucleic acid molecule according to claim 3, described nucleic acid molecule is following 1)-6) in arbitrary described gene:
1) in claim 2, a) encoding sequence of the heavy chain of described antibody is the sequence 9 in sequence table, in claim 2 a) described in the encoding sequence of light chain of antibody be sequence 10 in sequence table;
2) in claim 2, b) encoding sequence of described antibody is the sequence 8 in sequence table;
3) in claim 2 c) described antibody the encoding sequence of heavy chain be sequence 13 in sequence table, in claim 2, e) encoding sequence of the light chain of described antibody is the sequence 10 in sequence table;
4) in claim 2 d) described antibody the encoding sequence of heavy chain be sequence 14 in sequence table, in claim 2, f) encoding sequence of the light chain of described antibody is the sequence 10 in sequence table;
5) in claim 2 e) described antibody the encoding sequence of heavy chain be sequence 11 in sequence table, in claim 2, g) encoding sequence of the light chain of described antibody is the sequence 10 in sequence table;
6) in claim 2 f) described antibody the encoding sequence of heavy chain be sequence 12 in sequence table, in claim 2, h) encoding sequence of the light chain of described antibody is the sequence 10 in sequence table.
5.1), 2) or 3) biomaterial:
1) expression cassette containing nucleic acid molecule described in claim 3 or 4, recombinant vectors, recombinant microorganism or recombinant cell lines;
2) recombinant expression vector of antibody described in claim 1 or 2, recombinant microorganism or recombinant cell lines is expressed;
3) recombinant expression vector of nucleic acid molecule described in claim 4, recombinant microorganism or recombinant cell lines is expressed.
6. surface of cell membrane expresses the c of claim 2)-f) in the recombinant cell lines of arbitrary described antibody.
7.1) any one reagent-2):
1) detect the reagent of people CD22 antibody concentration, its activeconstituents is antibody described in claim 1 or 2;
2) detect penetrance and/or the adsorptivity reagent of people CD22 antibodies on tumor cell, its activeconstituents is antibody described in claim 1 or 2;
Described people CD22 antibody is RFB4, SM03 or SM06.
8.1) any one reagent-2):
1) detect the complement dependent cellular poison reaction reagent that people CD22 is antibody-mediated, its activeconstituents for its activeconstituents be recombinant cell lines according to claim 6;
2) 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 according to claim 6;
Described people CD22 antibody is RFB4, SM03 or SM06.
9.1) the arbitrary application-4):
1) antibody described in claim 1 or 2 detects the application in CD22 antibody concentration reagent in preparation;
2) reconstitution cell according to claim 6 ties up to the application in the antibody-mediated complement dependent cellular poison reaction reagent of preparation detection people CD22;
3) reconstitution cell according to claim 6 ties up to the application in the antibody-mediated antibody-dependant cell mediated cell poison reaction reagent of preparation detection people CD22;
4) application of antibody described in claim 1 or 2 in the penetrance and/or adsorptivity reagent of preparation detection people CD22 antibodies on tumor cell;
Described people CD22 antibody is RFB4, SM03 or SM06.
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