CN103588882B - Anti-idiotypic antibody against human CD22 antibody and application thereof - Google Patents

Abstract

The invention discloses an anti-idiotype antibody aiming at a human CD22 antibody and application thereof. The anti-idiotype antibody against human CD22 antibody comprises 3 antibody heavy chain complementarity determining regions and 3 antibody light chain complementarity determining regions; the amino acid sequences of the 3 antibody heavy chain complementarity determining regions are respectively shown as 31-35 th, 50-66 th and 99-106 th positions of the sequence 1 in the sequence table, and the amino acid sequences of the 3 antibody light chain complementarity determining regions are respectively shown as 154 th and 168 th positions, 184 th and 190 th positions and 223 th and 231 th positions of the sequence 1 in the sequence table. The anti-idiotype antibody is specific to a human CD22 antibody, and can inhibit the binding of a human CD22 antibody and a natural ligand of the human CD22 antigen.

Description

Anti-idiotypic antibody against human CD22 antibody and application thereof

technical field

The present invention relates to anti-idiotypic antibody against human CD22 antibody and application thereof.

Background technique

Targeted therapy using monoclonal antibodies (MAbs) is not a novel or complicated concept. However, the implementation of this concept involves the integration of multi-disciplinary knowledge and technologies, including antibody molecular design, cell line optimization, industrial production, purification, formulation optimization and various quality control tests to ensure that the produced monoclonal antibody drugs can achieve the expected clinical effect. So far, hundreds of therapeutic monoclonal antibody products for various indications are in various stages of clinical trials. Correspondingly, a large number of concepts and technical means based on or derived from antibodies are changing with the development of the monoclonal antibody industry, in order to expand the application range of existing products.

One of the derivatives is based on the immune network theory, first proposed by Niels Jerne in 1974 (see JerneNK: 1974. Ann Immunol 125C:373-389). Jerne proposed that the immune system is a network that regulates and controls immune responses through the interaction of anti-idiotypic antibodies. This insight was later extended to other uses of anti-idiotypic antibodies. Anti-idiotypic antibodies, also commonly called Ab2, are antibodies that target the immune antibody Ab1 (that is, antibodies produced by the immune system against foreign antigens) and have specific recognition and adsorption capabilities for its idiotopes (unique antigen recognition clusters on the surface of antibody molecules). Ab2 can be divided into three different categories: 1) Ab2α recognizes unique bits other than the antigen-binding site (ABS) on the original Ab1 antibody; 2) Ab2β recognizes the site of the antigen-binding site and mimics its corresponding spatial structure, thereby forming a foreign antigen. "Inner image"; 3) Ab2γ also recognizes the site of the antigen-binding site, but does not mimic the structure of the foreign antigen.

It is generally believed that the Ab2β antibody is the most attractive of the Ab2 antibody class, especially when trying to use the Ab2β antibody as a surrogate antigen to develop effective vaccines against self-antigens or inert antigens, such as tumor vaccines against tumor-specific antigens or tumor-associated antigens , and some vaccines against pathogens such as bacteria, viruses, and parasites. However, other types of Ab2 antibodies cannot be ignored, and they can be used to develop relevant detection methods to assist in the production and clinical potency evaluation of therapeutic Ab1 antibodies with medicinal value.

The human CD22 antigen is expressed on the surface of mature or malignant B cells ( et al. 1989. Leucocyte Typing IV: White cell differentiation antigens. New York, Oxford University Press. P. 63-64). CD22 is a regulatory molecule that protects against immune system overreaction or autoimmune disease (Hatta et al. 1999. Immunogenetics 49:280-286).

SM03 is an anti-CD22 chimeric antibody derived from the mouse antibody RFB4 (Yang Lei et al. 2006. Construction and identification of recombinant anti-B-cell lymphoma chimeric antibody. Chinese Journal of New Drugs, Volume 15, Issue 3: 186-192) , and has entered the clinical trial stage in the treatment of non-Ho's lymphoma (Li et al.2012.Landes BioScience J 4(2):256-266). Since the SM03 mAb targets mature B cells and inhibits their function, the application of this mAb product has been expanded to the treatment of other autoimmune disease indications, especially rheumatoid arthritis (RA) and systemic lupus erythematosus (SLE) ).

The humanized transformation of SM03 monoclonal antibody using framework remodeling technology has further promoted the application of anti-CD22 antibody (Liang Ruian, et al. 2006. Application of antibody "framework remodeling" technology to construct humanized antibody fSM03. Chinese Journal of New Drugs, 15 Issue 21:1832-1836). The humanized SM03 monoclonal antibody was named SM06. SM03 and SM06 target the same site on the human CD22 antigen with similar affinities. However, in amino acid sequence and structure, only the antigen recognition part of SM03 and SM06 is identical, and this identical part is composed of their respective complementarity determining region (CDR) sequences.

Both SM03 and SM06 can specifically recognize and adsorb human CD22 antigen, and the antibody-antigen complex will be obviously internalized after binding. This feature makes it very difficult to detect the biological activity of SM03 and SM06 mAb products. Other antibody products targeting internalizable antigens have similar difficulties, such as invariant chains, CD33, and so on. In addition, clinical pharmacokinetic studies require a convenient and robust method for the determination of free SM03, SM06 (and RFB4) and their various derivatives (scFv single-chain antibody, Fab, diabodies, immunotoxins, drug conjugates, etc. etc.) to assess its serum half-life. Since soluble free CD22 is not common and exogenous CD22 is relatively unstable, a suitable anti-idiotypic antibody against SM03 and its derivatives appears to be of great application value.

Contents of the invention

A technical problem to be solved by the present invention is to provide anti-idiotypic antibodies against human CD22 antibodies. The anti-idiotypic antibodies are specific for human CD22 antibodies ("CD22 antibody family") and can adsorb "CD22 antibody family". It has a specific reaction to the variable region of the antibody molecule of the "CD22 antibody family", and its adsorption fragment has a specific reaction to the antigen binding site (ABS) of the antibody molecule of the "CD22 antibody family". The anti-idiotypic antibody can inhibit the binding of human CD22 antibody to its natural ligand human CD22 antigen.

The human CD22 antibodies described in the present invention all contain the following three antibody heavy chain complementarity determining regions and three antibody light chain complementarity determining regions, and can specifically bind to human CD22 antigen: the sequence of heavy chain CDR ₁ is IYDMS The sequence of CDR ₂ of the heavy chain is YISGGGTTYYPDTVKG, the sequence of CDR ₃ of the heavy chain is HSGYGSSYGVLFAY, the sequence of CDR ₁ of the light chain is RASQDISNYLN, the sequence of CDR ₂ of the light chain is YTSILHS, and the sequence of CDR ₃ of the light chain is QQGNTLPWT. The human CD22 antibodies include murine antibodies (such as RFB4), chimeric antibodies (such as SM03), human antibodies, and humanized antibodies (such as SM06) and their various derivatives such as scFv single-chain antibodies, diabodies, Bispecific antibodies, antibody conjugates and other forms of antibody fusion proteins are collectively referred to as "CD22 antibody family".

The anti-idiotypic antibody against human CD22 antibody provided by the present invention includes 3 antibody heavy chain complementarity determining regions and 3 antibody light chain complementarity determining regions (6 boxed regions in Figure 3); the 3 anti The amino acid sequences of the complementarity determining regions of the heavy chain are respectively as 31-35 (CDR ₁ ), 50-66 (CDR ₂ ) and 99-106 (CDR ₃ ) of Sequence 1 in the Sequence Listing, the three The amino acid sequences of the complementarity determining regions of the light chain of the antibody are respectively as 154-168 (CDR ₁ ), 184-190 (CDR ₂ ) and 223-231 (CDR ₃ ) of Sequence 1 in the Sequence Listing.

Wherein, the above six complementarity determining regions of the anti-idiotype antibody form a structure that specifically binds to the idiotype part of the human CD22 antibody. The experiment in Example 1 below proves that the single-chain antibody containing the above six complementarity-determining regions can specifically bind to three kinds of human CD22 antibodies (RFB4, SM03 and SM06), and the sequences of these three kinds of human CD22 antibodies are only 3 There are three complementarity-determining regions of antibody heavy chains and three complementarity-determining regions of antibody light chains. The human CD22 antibody is specific for human CD22 and contains three antibody heavy chain complementarity determining regions shown in sequences 8-10 in the sequence listing and three antibody light chain complementarity determining regions shown in sequences 11-13 in the sequence listing. sex-binding antibodies such as RFB4, SM03 and SM06. Among them, sequence 8 is a sequence of CDR ₁ , sequence 9 is a sequence of CDR ₂ , sequence 10 is a sequence of CDR ₃ , sequence 10 is a sequence of CDR ₁ , sequence 11 is a sequence of CDR ₂ , and sequence 12 is a sequence of CDR ₃ .

Further, the anti-idiotypic antibody against human CD22 antibody includes a heavy chain variable region and a light chain variable region; the amino acid sequence of the heavy chain variable region is as in the 1-116th position of Sequence 1 in the Sequence Listing. The amino acid sequence of the light chain variable region is as the 131-240th position of sequence 1 in the sequence listing.

Further, the anti-idiotypic antibody against human CD22 antibody can specifically be:

a) The amino acid sequence of the heavy chain is sequence 2 in the sequence listing, and the amino acid sequence of the light chain is sequence 3 in the sequence listing (the anti-idiotypic murine IgG2a/kappa immune system comprising the constant region sequence of the light and heavy chains in Example 3 below globulin molecule IdmG2a/k);

b) Substituting, deleting or adding amino acid residues to the amino acid sequence in Sequence 2 in the sequence listing except for the three antibody heavy chain complementarity determining regions, and/or excluding the above-mentioned amino acid residues in Sequence 3 in the sequence listing The anti-idiotypic antibody against the human CD22 antibody obtained by substituting, deleting or adding amino acid residues to the amino acid sequences outside the complementarity determining regions of the light chains of the three antibodies is preferably the heavy sequence 2 except for positions 1-117. Substitution, deletion or addition of amino acid residues in the amino acid sequence outside the chain variable region, and/or substitution of amino acid residues in the amino acid sequence except for the light variable region at positions 1-111 in the sequence 3, Deletion or addition of resulting anti-idiotypic antibodies against human CD22 antibodies;

c) the single-chain antibody shown in Sequence 1 in the sequence listing (single-chain antibody shown in Figure 3 in Examples 1 and 2 below), or adding histidine to the amino-terminal or carboxyl-terminal of Sequence 1 in the sequence listing Fusion protein obtained by acid labeling;

d) Substitution, deletion or addition of amino acid residues to the amino acid sequence of Sequence 1 in the sequence listing except for the three antibody heavy chain complementarity determining regions and the three antibody light chain complementarity determining regions against human CD22 The anti-idiotype antibody of the antibody is preferably an anti-idiotype antibody against human CD22 antibody obtained by substituting, deleting or adding amino acid residues to the connecting peptide sequence shown in the 117-130 position of Sequence 1 in the sequence listing ;

e) The amino acid sequence of the heavy chain is sequence 6 in the sequence listing; the amino acid sequence of the light chain is sequence 3 in the sequence listing (IdGmFdA in Example 5 below, Figure 14 shows the heavy chain amino acid sequence of IdGmFdA) ;

f) The amino acid sequence of the heavy chain is sequence 7 in the sequence listing; the amino acid sequence of the light chain is sequence 3 in the sequence listing (IdGmFdG in Example 5 below, Figure 15 shows the heavy chain amino acid sequence of IdGmFdG) ;

g) The amino acid sequence of the heavy chain is sequence 4 in the sequence listing; the amino acid sequence of the light chain is sequence 3 in the sequence listing (IdGmD in Example 5 below, Figure 12 shows the heavy chain amino acid sequence of IdGmD) ;

h) The amino acid sequence of the heavy chain is sequence 5 in the sequence listing; the amino acid sequence of the light chain is sequence 3 in the sequence listing (IdDmD in Example 5 below, Figure 13 shows the heavy chain amino acid sequence of IdDmD) .

Among them, sequence 1 in the sequence listing consists of 240 amino acid residues, sequence 2 in the sequence listing consists of 447 amino acid residues, sequence 3 in the sequence listing consists of 219 amino acid residues, sequence 4 in the sequence listing It consists of 502 amino acid residues, sequence 5 in the sequence listing consists of 408 amino acid residues, sequence 6 in the sequence listing consists of 305 amino acid residues, and sequence 7 in the sequence listing consists of 264 amino acid residues.

Among them, the anti-idiotypic antibody against human CD22 antibody can be expressed as an attached protein in various forms, and these forms include: transmembrane IgD antibody (IdGmD and IdDmD in the following Example 5), or with glycoprotein A (the following implementation IdGmFdA of Example 5) or GPI protein fusion (IdGmFdG of Example 5 below) single-chain antibody scFv, Fab, Fab', F(ab') ₂ .

Nucleic acid molecules encoding any of the above-mentioned anti-idiotypic antibodies against human CD22 antibodies belong to the protection scope of the present invention.

Wherein, the nucleic acid molecule can be DNA, such as cDNA, genomic DNA or recombinant DNA; the nucleic acid molecule can also be RNA, such as mRNA or hnRNA.

The nucleic acid molecule can specifically be the gene described in any of the following 1)-6):

1) The coding sequence of the heavy chain of the antibody described in a) above is sequence 9 in the sequence listing, and the coding sequence of the light chain of the antibody described in a) above is sequence 10 in the sequence listing;

2) The coding sequence of the antibody described in c) above is sequence 8 in the sequence listing;

3) The coding sequence of the heavy chain of the antibody described in e) above is sequence 13 in the sequence listing, and the coding sequence of the light chain of the antibody described in e) above is sequence 10 in the sequence listing;

4) The coding sequence of the heavy chain of the antibody described in f) above is sequence 14 in the sequence listing, and the coding sequence of the light chain of the antibody described in f) above is sequence 10 in the sequence listing;

5) The coding sequence of the heavy chain of the antibody described in g) above is sequence 11 in the sequence listing, and the coding sequence of the light chain of the antibody described in g) above is sequence 10 in the sequence listing;

6) The coding sequence of the heavy chain of the antibody described in h) above is sequence 12 in the sequence listing, and the coding sequence of the light chain of the antibody described in h) above is sequence 10 in the sequence listing.

Among them, sequence 1 in the sequence listing consists of 240 amino acid residues, encoded by the DNA of sequence 8 in the sequence listing, and sequence 8 consists of 720 nucleotides; sequence 2 in the sequence listing consists of 447 amino acid residues , encoded by the DNA of sequence 9 in the sequence listing, sequence 9 consists of 1344 nucleotides; sequence 3 in the sequence listing consists of 219 amino acid residues, encoded by the DNA of sequence 10 in the sequence listing, and sequence 10 consists of 660 nucleotides; sequence 4 in the sequence listing consists of 502 amino acid residues, encoded by the DNA of sequence 11 in the sequence listing, and sequence 11 consists of 1509 nucleotides; sequence 5 in the sequence listing consists of 408 amino acid residues, encoded by the DNA of sequence 12 in the sequence listing, sequence 12 consists of 1227 nucleotides; sequence 6 in the sequence listing consists of 305 amino acid residues, encoded by the DNA of sequence 13 in the sequence listing For encoding, sequence 13 consists of 918 nucleotides; sequence 7 in the sequence listing consists of 264 amino acid residues, encoded by the DNA of sequence 14 in the sequence listing, and sequence 14 consists of 795 nucleotides.

The biological materials of the following a1, a2 or a3 also belong to the protection scope of the present invention:

a1. Expression cassettes, recombinant vectors, recombinant microorganisms or recombinant cell lines containing any of the above nucleic acid molecules;

a2. A recombinant expression vector, recombinant microorganism or recombinant cell line expressing any of the above-mentioned anti-idiotypic antibodies against human CD22 antibodies;

a3. A recombinant expression vector, recombinant microorganism or recombinant cell line expressing any gene.

In the above-mentioned biological materials, the expression cassette described in a1 refers to the DNA capable of expressing any of the above-mentioned anti-idiotypic antibodies against human CD22 antibodies in host cells, and the DNA may not only include the activation of any of the above-mentioned anti-human CD22 antibodies The promoter for the transcription of the anti-idiotypic antibody gene can also include a terminator for terminating any of the above-mentioned anti-idiotype antibody gene transcription for human CD22 antibodies. Further, the expression cassette may also include an enhancer sequence. The recombinant microorganisms described in a2 and a3 can specifically be yeast, bacteria, algae and fungi. The recombinant cell lines described in a2 and a3 do not include plant propagation materials, and can specifically be recombinant cell lines (cell strains) expressing any of the above-mentioned anti-idiotypic antibodies against human CD22 antibodies on the cell membrane surface.

The present invention also provides the following multiple uses of any of the above-mentioned anti-idiotypic antibodies against human CD22 antibodies:

b1. The reagent for detecting the concentration of human CD22 antibody, the active ingredient of which is any one of the above-mentioned anti-idiotypic antibodies against human CD22 antibody;

b2. A reagent for detecting the human anti-mouse antibody reaction caused by human CD22 antibody, the active ingredient of which is any of the above-mentioned anti-idiotypic antibodies against human CD22 antibody;

b3. A reagent for detecting the human anti-chimeric antibody response caused by human CD22 antibody, the active ingredient of which is any of the above-mentioned anti-idiotypic antibodies against human CD22 antibody;

b4. The reagent for detecting the human anti-human antibody reaction caused by human CD22 antibody, the active ingredient of which is any one of the above-mentioned anti-idiotypic antibodies against human CD22 antibody;

b5. A reagent for detecting complement-dependent cytotoxicity mediated by human CD22 antibody, the active ingredient of which is a recombinant cell line expressing any of the above-mentioned anti-idiotypic antibodies against human CD22 antibody on the surface of the cell membrane;

b6. A reagent for detecting antibody-dependent cell-mediated cytotoxicity mediated by human CD22 antibody, the active ingredient of which is a recombinant cell line expressing any of the above-mentioned anti-idiotypic antibodies against human CD22 antibody on the surface of the cell membrane;

b7. A reagent for detecting the biological activity of human CD22 antibody, the active ingredient of which is a recombinant cell line expressing any of the above-mentioned anti-idiotypic antibodies against human CD22 antibody on the surface of the cell membrane;

b8. Application of any of the above-mentioned anti-idiotypic antibodies against human CD22 antibody in the preparation of reagents for detecting the concentration of human CD22 antibody;

b9. Application of any of the above-mentioned anti-idiotypic antibodies against human CD22 antibodies in the preparation and detection of human anti-mouse antibody reaction reagents caused by human CD22 antibodies;

b10. The application of any of the above-mentioned anti-idiotypic antibodies against human CD22 antibodies in the preparation and detection of human anti-chimeric antibody reaction reagents caused by human CD22 antibodies;

b11 Application of any of the above-mentioned anti-idiotypic antibodies against human CD22 antibodies in the preparation and detection of human anti-human antibody reaction reagents caused by human CD22 antibodies;

b12. The application of any recombinant cell line expressing any of the above-mentioned anti-idiotypic antibodies against human CD22 antibody on the surface of the cell membrane in the preparation and detection of human CD22 antibody-mediated complement-dependent cytotoxicity reagents;

b13. The application of the recombinant cell line expressing any of the above-mentioned anti-idiotypic antibodies against human CD22 antibody on the surface of the cell membrane in the preparation and detection of antibody-dependent cell-mediated cytotoxicity mediated by human CD22 antibody;

b14. The application of the recombinant cell line expressing any of the above-mentioned anti-idiotypic antibodies against human CD22 antibody on the surface of the cell membrane in the preparation of reagents for detecting the biological activity of human CD22 antibody;

b15. A reagent for detecting the penetration and/or adsorption of human CD22 antibody to tumor cells, the active ingredient of which is the antibody of any one of claims 1-3.

b16. Use of any of the above-mentioned anti-idiotypic antibodies against human CD22 antibodies in the preparation of reagents for detecting the penetration and/or adsorption of human CD22 antibodies to tumor cells.

Among them, the above-mentioned b1 and b8 can specifically be to use any of the above-mentioned anti-idiotypic antibodies against human CD22 antibodies as diagnostic reagents to monitor the content of human CD22 antibodies in the blood of patients during immunotherapy, and to monitor the pharmacokinetics of injected human CD22 antibody drugs. dynamic behavior. The "CD22 antibody family" (human CD22 antibody) antibody content in the sample serum can be detected according to the following methods:

1) Coat the ELISA plate with any of the above-mentioned anti-idiotypic antibodies against human CD22 antibodies, any of the above-mentioned antibodies against human CD22 includes scFv single-chain antibodies, or intact IgG, or mouse IgG2a/kappa types, and other isotypes;

2) Add the serum sample to be tested on the microtiter plate;

3) Add secondary antibodies such as peroxidase-labeled anti-human Fc-specific antibodies and;

4) Measure the anti-human CD22 antibody content in the serum to be tested according to the amount of the adsorbed secondary antibody.

In b7 and b14 above, the recombinant cell line expressing any of the above-mentioned anti-idiotypic antibodies against human CD22 antibody on the cell membrane surface can undergo complement-dependent cytotoxicity (CDC) or antibody-dependent cell-mediated cytotoxicity through antibody-mediated (ADCC), used to quickly estimate the biological activity of human CD22 antibodies (such as SM03 or SM06), so as to achieve the purpose of quality control. Using the recombinant cell line expressing any of the above-mentioned anti-idiotypic antibodies against human CD22 antibody on the surface of the cell membrane, the detection method for evaluating the biological activity of human CD22 antibody through complement-dependent cytotoxicity may specifically include the following steps:

1) Incubate the recombinant cell line expressing any of the above-mentioned anti-idiotypic antibodies against human CD22 antibody on the surface of the cell membrane with the anti-CD22 antibody;

2) Add complement proteins to the mixture;

3) After incubating for an appropriate time, measure the effect of its complement-dependent cytotoxic effect (CDC).

Using the recombinant cell line expressing any of the above-mentioned anti-idiotypic antibodies against human CD22 antibody on the surface of the cell membrane, the detection method for evaluating the biological activity of human CD22 antibody through antibody-dependent cell-mediated cytotoxicity may specifically include the following steps:

1) Incubate the recombinant cell line expressing any of the above-mentioned anti-idiotypic antibodies against human CD22 antibody on the surface of the cell membrane with the anti-CD22 antibody;

2) Add peripheral blood mononuclear cells (PBMC);

3) Measure the effect of antibody-dependent cell-mediated cytotoxicity (ADCC) after incubation for an appropriate time.

The above-mentioned b2-b4, b9-b11 can specifically be any one of the above-mentioned anti-idiotypic antibodies against human CD22 antibody as a positive control in the research of anti-antibody immune response such as HAHA, HACA or HAMA. Because these anti-antibody responses may affect the patient's clinical outcome (Gruber van Haarlem et al.2000. Cancer Res.60:1921-1926), or be associated with unexpected allergic reactions, resulting in significant changes in pharmacokinetic behavior and Affects the distribution of injected antibody drugs in the body, so their monitoring will have an important impact on the treatment options of antibody immunotherapy. The method for detecting human anti-chimeric antibody response (HACA) or human anti-human antibody response (HAHA) caused by human CD22 antibody in an individual injected with chimeric or humanized anti-human CD22 monoclonal antibody provided by the present invention comprises Follow the steps below:

1) collection of serum samples from individuals receiving injections;

2) Coat the CD22 antigen on the ELISA microtiter plate, add a sensitive concentration of anti-CD22 free antibody at the same time, add serum samples diluted in different times, and the wells without serum samples are used as negative controls, and add a certain concentration of anti-CD22 idiotype antibody. well as a positive control;

3) After washing, add HRP-labeled anti-human IgG Fc secondary antibody to detect the adsorption signal of the sensitive concentration of anti-CD22 antibody to CD22 antigen;

4) Detect the presence or absence of anti-CD22 anti-idiotypic antibodies in serum samples, such as the presence of anti-CD22 anti-idiotypic antibodies, which supports the occurrence of HACA or HAHA reactions in individuals.

In the above-mentioned b15 and b16, any of the above-mentioned anti-idiotypic antibodies against human CD22 antibodies (such as the anti-idiotype mouse IgG2a/kappa immunoglobulin molecule containing the light and heavy chain constant region sequences of the following Example 3) can be applied Immunohistochemical analysis of tumor tissue sections to study the penetration and/or adsorption of "CD22 antibody family" drugs to tumor cells.

The present invention provides a specific recognition anti-human CD22 antibody (including murine antibody (such as RFB4), chimeric antibody (such as SM03), human antibody, and humanized antibody (such as SM06) and their various derivatives Such as scFv single-chain antibodies, diabodies, bispecific antibodies, antibody conjugates and other forms of antibody fusion proteins, etc., collectively referred to as "CD22 antibody family") anti-idiotypic antibodies against the antigen-binding site (ABS), that is, against human CD22 Anti-idiotypic antibodies for antibodies. The anti-idiotypic antibody against human CD22 antibody provided by the present invention can be applied to identify and evaluate the concentration and biological activity of "CD22 antibody family" antibodies, and be used for clinical quantitative analysis of the content of "CD22 antibody family" antibodies in serum. The invention can also be applied to detect possible human anti-mouse antibody reaction (HAMA), human anti-chimeric antibody reaction (HACA) and human anti-human antibody reaction (HAHA) in clinical trials of "CD22 antibody family" monoclonal antibody drugs. At the same time, it also provides a method for constructing a cell line capable of expressing an anti-idiotypic antibody against human CD22 antibody; and using such cells to detect complement-dependent cytotoxicity (CDC) mediated by monoclonal antibody products of the "CD22 antibody family" And/or antibody-dependent cell-mediated cytotoxicity (ADCC), to achieve the detection and evaluation of the biological activity of monoclonal antibody products.

Description of drawings

Figure 1 shows the specific adsorption of phage #1-#3 expressing scFv single-chain antibody to murine CD22 antibody (RFB4), human-mouse chimeric CD22 antibody (SM03), and humanized CD22 antibody (SM06).

Figure 2A is the heavy chain complementarity determining region (CDR) sequence expressed by phage #1-#3.

Figure 2B shows the light chain CDR sequences expressed by phage #1-#3.

Figure 3 is the complete sequence of the scFv sequence displayed by phage #3 which has specific recognition and adsorption for RFB4, SM03 and SM06. The framed area is the supplementary decision region. The scFv single-chain antibody is composed of heavy chain variable region-connecting sequence-light chain variable region. The linker amino acid sequence used was G4-S-G-S-G-S-S-G4.

Figure 4 shows that the compatible scFv expressed by phage #3 can inhibit the adsorption of SM03 antibody to Raji cells in the flow cytometry experiment. The three peaks from left to right are blank control, SM03+scFv (phage#3) and SM03.

Figure 5 shows the pharmacokinetic performance of lymphoma patients treated with SM03 measured by the capacitive scFv expressed by phage #3 (360mg/ ^m2 dose group, intravenous injection, once a week, continuous administration for 4 weeks).

Fig. 6 is an expandable DNA vector expressing murine IgG2a/kappa immunoglobulin anti-idiotypic antibody IdmG2a/.

Fig. 7 is the SDS-PAGE electrophoresis diagram of the purified antibody of IdmG2a/k under reduced and non-reduced conditions. In the figure, 1 and 2 are the results of reduction electrophoresis, 3 is the molecular weight standard (BenchMark ^TM protein molecular weight standard, product of Invitrogen, USA, batch: 688736), 4 and 5 are the results of non-reduction electrophoresis.

Figure 8 shows that IdmG2a/k can be specifically recognized by SM03, while other antibodies cannot be adsorbed (CD20 antibody (hAnti-CD20), CD147 antibody (hAnti-CD147), TNF antibody (Infliximab)).

Figure 9 shows the results of flow cytometry showing that the anti-idiotypic mouse IgG antibody IdmG2a/k can effectively inhibit SM03 from adsorbing the CD22 antigen on the surface of Raji cells.

Figure 10 shows that the mouse CD22 antibody RFB4, the chimeric antibody SM03 and the humanized antibody SM06 using framework remodeling technology can compete with the HRP-labeled SM03 antibody to adsorb to the anti-idiotypic mouse IgG antibody IdmG2a/k

Figure 11 shows the pharmacokinetic performance of patients with lupus erythematosus treated with SM03 monoclonal antibody products. The plasma concentration of SM03 was measured by ELISA method, and IdmG2a/k was used as a solid-phase capture antibody.

Figure 12 is the heavy chain amino acid sequence of the transmembrane anti-idiotype antibody IdGmD for human CD22 antibody obtained by fusing the transmembrane region sequence of mouse IgD with the CH3 region of "mouse anti-idiotype IgG antibody".

Figure 13 is the amino acid sequence of the heavy chain of the transmembrane anti-idiotypic antibody IdDmD against human CD22 antibody obtained by substituting the IgG2a heavy chain with the mouse IgD sequence.

Figure 14 is the heavy chain amino acid sequence of the anti-idiotypic antibody IdGmFdA for human CD22 antibody expressed transmembrane in the form of membrane-attached Fab-glycoprotein fusion protein.

Figure 15 is the heavy chain amino acid sequence of the anti-idiotypic antibody IdGmFdG of the human CD22 antibody expressed in the form of a Fab-GPI immobilized fusion protein of the "mouse anti-idiotype IgG" antibody.

Figure 16 shows the expression of various fusion protein forms of anti-idiotypic murine IgG antibodies on the mesangial membrane of transfected cells.

The five pictures from top to bottom are the SP2/0 cell system without adding SM03, the SP2/0 cell system adding SM03 to a final concentration of 1 μg/ml, adding SM03 to a final concentration of 0.1 μg/ml and 1 μg/ml The recombinant engineering cell system expressing IdDmD, the recombinant engineering cell system expressing IdGmFdA adding SM03 to the final concentration of 0.1 μg/ml, 0.1 μg/ml and 1 μg/ml, adding SM03 to the final concentration of 0.1 μg/ml, 0.1 μg /ml and 1μg/ml recombinant engineering cell system expressing IdGmFdG.

Figure 17 is the comparison of SM03 antibody-mediated complement-dependent cytotoxicity (CDC) against transmembrane-expressing various anti-idiotypic murine IgG antibody fusion protein transfection cell lines (the three pictures from top to bottom are IdGmD, IdGmD, IdGmFdA, IdGmFdG)

Detailed ways

In order to facilitate a more comprehensive understanding of the description of the present invention, some basic definitions are defined here as follows:

The definition of "immunoglobulin" herein refers to protein molecules composed of one or more polypeptide chains, most of which are encoded by immunoglobulin-related genes. The immunoglobulin gene codes that have been deciphered include kappa, lamda, alpha, gamma (IgG1, IgG2, IgG3, IgG4), delta, epsilon and mu these constant region genes, and countless immunoglobulin variable region genes. The complete immunoglobulin "light chain" (consisting of 214 amino acids, with a molecular weight of about 25Kd) is encoded by an amino-terminal variable region gene (about 110 amino acids) and a hydroxyl-terminal kappa or lamda constant region gene. Similarly, the complete immunoglobulin "heavy chain" (consisting of 446 amino acids, molecular weight about 50Kd) is composed of variable region genes encoding (about 116 amino acids) and one of the constant region genes described above, such as gamma constant Region genes (approximately 300 amino acids) encode the constituent IgG molecules.

As used herein, the definition of "antibody" is used broadly to refer to whole antibody molecules as well as derivatives thereof, unless otherwise specified. These derivatives contain at least a fragment of the variable region from an immunoglobulin light chain or heavy chain, and include such as F(ab')2, Fab, Fab', Fd, Fabc, scFv, diabody, single antibody light chain , single antibody heavy chains, antibody chain chimeric fusions, bispecific antibodies, and other similar molecules such as antibody molecule fragments.

As used herein, the definition of "chimeric antibody" refers to a protein molecule designed by recombining the variable regions of immunoglobulin light and heavy chains from non-human species and the constant regions of human immunoglobulin molecules.

The definition of "humanization" used herein refers to the retention of only part of the complementarity-determining regions (CDRs) of the variable regions of immunoglobulin molecules from non-human organisms, and the remaining part of the main variable regions and all the constant regions Some of them come from human sources and are recombinantly designed protein molecules.

As used herein, the definition of "idiotype" refers to the particular segment of an antibody molecule that determines its specificity for an antigen. The idiotype fragment is located in the Fab part of the antibody, and its description is usually based on the joint participation of the heavy and light chains of the antibody molecule to form the antigen-binding site.

The definition of "isotype" used herein refers to the immunoglobulin molecule type and its subtype according to the class and subclass of the heavy chain and the type of the light chain, determined by the antigen specificity of the immunoglobulin molecule . For example, the four isotypes of IgG are IgG ₁ , IgG ₂ , IgG ₃ , IgG ₄ .

The "CD22 antibody family" described herein is derived from the murine antibody RFB4. Its derived chimeric antibody (SM03) and humanized antibody (SM06) are the same as the original mouse antibody, and are specific for the B site on the human CD22 antigen. Clinical trials using SM03 to treat B-cell lymphoma and other autoimmune diseases have already begun. In clinical practice, in order to meet the needs of clinical determination of the content of injected "CD22 antibody family" products (such as SM03 monoclonal antibody) in serum, an anti-idiotypic antibody was constructed and characterized as an ELISA reagent to detect Concentration of "CD22 antibody family" products. In addition, the generated anti-idiotypic antibodies can also be used in ELISA methods to measure HACA or HAHA responses, or as control antibodies and as diagnostic reagents to evaluate competing antibodies in patient sera.

Anti-idiotypic antibodies in the form of single chain antibodies were produced by phage display from mice immunized with SM03. The specific method is to extract spleen cells from mice injected with anti-CD22 chimeric antibody SM03, and isolate mRNA, and then degenerate flanking variable region primers to amplify light and heavy chain variable region sequences. The resulting variable region sequences were then introduced into the scFv single-chain antibody phage display library according to standard procedures. After several rounds of screening and elimination with SM03 and RFB4 antibodies, phages specific to SM03, RFB4 and SM06 were selected, and the selected phages displayed the corresponding variable region sequences. Among phages displaying antibody variable region sequences, the displaying phages with the highest affinity for murine, chimeric and humanized "CD22 antibody families" were selected.

The anti-idiotypic antibody sequence was initially expressed in Escherichia coli in the form of scFv single-chain antibody inclusion bodies, and then denatured and refolded; the active scFv single-chain antibody was used as an ELISA reagent to detect serum SM03 levels in clinical trials. The brief steps are as follows, that is, to coat the ELISA plate with the refolded anti-idiotypic scFv single-chain antibody (scFv), and then add the serum of the patient treated with SM03 after dilution, after incubation and washing, the SM03 antibody in the patient's serum will be Combined with the coated anti-idiotypic scFv single-chain antibody, its specific binding can be developed with HPR-labeled goat anti-human IgG-Fc specific antibody (product of Jackson ImmunoResearch, USA). However, anti-idiotypic scFv single-chain antibodies tend to be unstable, and the way they are produced from bacterial inclusion bodies can lead to protein denaturation and refolding diversity, making the detection results inconsistent. In addition, the instability of anti-idiotypic scFv scFv makes its storage and subsequent preparation of validation batches very difficult.

On the other hand, it is well known that intact immunoglobulin molecules can maintain stability for months or even years under suitable storage conditions, and their antibody activity and quality will not change significantly. To create a robust and consistent assay to estimate CD22 antibodies in serum, as well as HACA and HAHA responses, the variable region sequences of anti-idiotypic scFv single-chain antibodies were used to construct an intact immunoglobulin molecule. Since the chimeric antibodies and humanized antibodies in the "CD22 antibody family" all have the constant region sequences of human IgG1 heavy chain and kappa light chain, they can be labeled with anti-human IgG Fc-specific antibody (or similar combination) identified. Therefore, the designed anti-idiotypic antibody immunoglobulin molecules should not carry human IgG constant region sequences that would cause cross-reactivity. Murine IgG2a/kappa antibody (murine anti-idiotype IgG antibody) constant region sequences do not cross-react with anti-human Fc antibodies and were therefore selected for the construction of complete anti-idiotype antibody immunoglobulin molecules. It should be noted that other isotypes or constant region sequences from other species may also be used. Based on the designed anti-idiotypic antibody immunoglobulin, an expressing cell line with a yield of 30ug/ml was generated. Since the expression vector used to generate the expression cell line has an amplifiable DHFR gene, this yield can be further increased according to the general amplification-cloning method if necessary. However, the current yield is sufficient to produce consistent batches of murine anti-idiotypic IgG antibodies for pharmacokinetic studies and HACA or HAHA assays. The brief steps are as follows, that is, coat the ELISA plate with mouse anti-idiotype IgG antibody, then add the serum of patients treated with "CD22 antibody family" antibody drug, and after incubation, it will not adsorb with "mouse anti-idiotype IgG antibody" The protein will be washed clean. The anti-CD22 antibody in the serum will specifically bind to the coated "mouse anti-idiotypic IgG antibody", and the HRP-labeled goat anti-human IgG Fc specific antibody will be used for color development. Since the mouse constant region sequence of the "mouse anti-idiotypic IgG antibody" does not cross-react with the HRP-labeled antibody used for detection, this detection method does not have the problem of high background signal. This assay has been proven to be highly reproducible, sensitive and specific. The same approach was extended to estimate the recognition and affinity of the "CD22 antibody family". Using "mouse anti-idiotypic IgG antibody" as a positive control, the Biacore analysis method of HACA and HAHA in serum samples is established, and it is also possible to realize anti-antibody immune response in patient serum. In addition, "mouse anti-idiotypic IgG antibody" can also be used for immunohistochemical analysis of tumor tissue sections to study the penetration and adsorption of "CD22 antibody family" drugs to tumor cells. The generation of "mouse anti-idiotypic IgG antibody" that can specifically bind to the target antibody provides raw material reagents for evaluating the safety and pharmacokinetic properties of injection target antibody drugs with high sensitivity in clinical sense. The "mouse-derived anti-idiotypic IgG antibody" constructed by the present invention is therefore proved to be a diagnostic test reagent with important practical value, which is used to study clinical samples of patients receiving SM03 immunotherapy.

One aspect of the present invention is to provide a method for detecting the inhibitory effect of "mouse anti-idiotypic IgG antibody" on the adsorption of antigen by "CD22 antibody family". Another aspect is to provide a method for detecting the ability of the "mouse anti-idiotype IgG antibody" to capture and detect the adsorbed idiotype antibody. A deeper meaning is to provide a method for detecting the adsorption capacity of "mouse anti-idiotype IgG antibody" to its idiotype antibody (anti-CD22 antibody). However, on the other hand, it also provides a method for detecting the content of "CD22 antibody family" in serum samples. The present invention is also directed to a method for detecting HAMA, HACA and HAHA reactions using the antibodies described in this patent.

Another application of the present invention is to use the anti-idiotypic antibody to construct a non-internalized transmembrane-expressing SM03-specific adsorption fragment engineered cell line. The established cell lines can be used to establish detection methods for evaluating the biological efficacy of SM03 monoclonal antibody and its derivatives such as RFB4 and SM06. The establishment of this method means that it is of general significance to use similar methods to detect the biological efficacy of antibodies that adsorb and internalize surface antigens.

The following experimental illustrations are listed to illustrate the use of the present invention, but do not represent that the present invention is limited to such applications.

The experimental methods in the following examples are conventional methods unless otherwise specified.

The materials and reagents used in the following examples can be obtained from commercial sources unless otherwise specified.

Example 1. Anti-idiotypic antibodies against human CD22 antibodies in the form of single-chain antibodies

In this example, the variable region light and heavy chain sequences of the anti-SM03 anti-idiotypic antibody were obtained by using phage display library technology, and the anti-idiotypic single-chain antibody against human CD22 antibody—the soluble scFv expressed by phage #3 was further prepared. Specific steps are as follows:

1. Preparation of phage display library by injecting SM03 immunized mice

About 6 weeks old female Balb/c mice were immunized by intraperitoneal injection of 100ug SM03 monoclonal antibody according to the standard immunization procedure (see "InAntibodies: A Laboratory Manual" published by Cold Spring Harbor Laboratory, USA in 1988), and treated with 200uL complete Freund's adjuvant (product of Sigma-Aldrich, USA) was emulsified. The second and third immunizations were carried out after 14 days and 35 days respectively. The method was intraperitoneal injection of 100ug SM03 monoclonal antibody (China Antibody Pharmaceutical Co., Ltd.), emulsified with 200uL incomplete Freund's adjuvant (US Sigma- Aldrich product).

After measuring the SM03 antibody titer in mouse serum, mouse spleen cells 39 days after immunization were used to isolate total RNA and prepare cDNA (using Superscript II kit, product of Invitrogen, USA). Then degenerate primers were synthesized according to the relevant literature, and the immunoglobulin variable region gene sequence was amplified by PCR (Cheng et al. 2005. Biochem Biophys Res Commun 338: 1654-1660). Subsequently, a phage display library of variable region fragment single-chain antibody (scFv) was constructed according to the product guidelines of the recombinant phage antibody system of Amersham, USA.

After amplifying the variable region fragment scFv single-chain antibody phage display library and filamentous phage referring to relevant literature, the SM03 monoclonal antibody and mouse CD22 monoclonal antibody RFB4 (Ancell, Cat: 171-820) were used to screen and eliminate (McWhirter et al. al. 2006. PNAS 103:1041-1046). The brief steps are as follows, the same content (100ug/ml) of SM03 or mouse RFB4 antibody is coated with bicarbonate buffer (15mM Na ₂ CO ₃ , 35mM NaHCO ₃ ), and the screening concentration is 10 ¹² by biological screening method of bacteriophages. After 2 hours of incubation at room temperature under mild shaking conditions, the adsorbed scFv phage was eluted with 100uL of 0.1M glycine-hydrochloric acid buffer, pH2.2, incubated for 10 minutes, and then 10uL of 1M Tris-HCl, pH8.0 Neutralization buffer neutralizes. This screening process was repeated four times, and the phage titers before and after each screening were recorded.

The phages that survived the screening process were rescued, and their adsorption specificity was evaluated by phage-ELISA method, and anti-CD22 antibodies RFB4, SM03 and SM06 (China Antibody Pharmaceutical Co., Ltd.), and other control antibodies were used as antigens for further screening. The method is briefly described as follows. RFB4, SM03, SM06, chimeric anti-TNF antibody and BSA were respectively coated on a 96-well ELISA plate (using 50uL bicarbonate coating buffer pH9.6, the coating volume was 1ug ; After overnight incubation at 4°C, wash with 200uL borate buffer solution at pH 8.0; then block with borate buffer solution at 37°C for 1 hour), the three phage clones with the highest adsorption affinity to SM03 (100uL supernatant solution) was added to the wells and incubated at 37°C for 1 hour. After 5 times of washing with borate buffer solution with pH 8.0, HRP-labeled anti-M13 mouse antibody (product of Amersham Company) was added after dilution of 1:3000. After incubation at 37°C for 1 hour, 100 uL of O-p-phenylenediamine (OPD) reaction solution (10 mg OPD dissolved in 10 mL of citrate-phosphate buffer containing 8 uL of 30% hydrogen peroxide solution, pH 5.0) was used for color development. The screening results are shown in Figure 1. The three selected scFv displaying phages (phage #1-#3) can all adsorb RFB4, SM03 and SM06 (mouse, chimeric and humanized CD22 antibodies) but cannot adsorb other Antibody (anti-TNF antibody (infliximab)) or control protein (BSA). Since RFB4, SM03 and SM06 share only the CDR part of the target antibody, or the antigen-binding site, this result indicates that the three selected scFv displaying phages are all specific to the idiotype part of SM03.

2. Determination of the variable region sequence of the scFv displaying phage that can specifically adsorb the SM03 antigen-binding part

The scFv-encoding DNA sequences of selected displaying phages were sequenced by Sanger sequencing. The DNA sequences corresponding to the variable regions of the antibodies are very similar, with only some differences in the sporadic positions of the framework fragments or CDR3 fragments. Figure 2 shows the CDR fragment sequence in the light and heavy chain sequences displayed by the selected phages. The three CDRs of the heavy chain of the scFv displayed by phage #1-#3 are identical, and the scFv displayed by phage #1-#3 The CDR1 and CDR2 of the light chain are identical except for one amino acid in the CDR3 region of the light chain expressed by phage #2 (underlined in Figure 2).

3. The displayed scFv sequence of phage #3 can inhibit the adsorption of SM03 antibody to Raji cells

Because phage #3 showed relatively high adsorption affinity, its single-strand sequence was restored to DNA code and the scFv bacterial expression vector was constructed by molecular cloning technology. The complete sequence of the scFv sequence displayed by phage #3 is shown in Figure 3. The DNA encoding the scFv sequence displayed by phage #3 (sequence 8 in the sequence listing) was ligated into the pET3a vector (Novagen Cat: 69418) and transfected into BL21 (DE3) pLys induction cells for expression. There is a His-tag gene attached to the amino terminal of the scFv to facilitate purification after expression. After induction of expression with IPTG, the inclusion bodies containing scFv were collected, denatured (using a buffer containing 6M guanidine hydrochloride, 20mM sodium phosphate, 0.5M sodium chloride, pH7.4), folded and separated by HItrap Chelating HP column according to the product instructions Purify. The brief steps are as follows. After the scFv containing His-tag is denatured, it is adsorbed to the HItrap Chelating HP separation column in the presence of Ni ² +. Wash with guanidine hydrochloride buffer solution (containing 20 mM sodium phosphate, 0.5 M sodium chloride, pH 7.4) with gradually decreasing concentration until no residual guanidine hydrochloride remains. Then wash with several bed volumes of 5-40mM imidazole buffer (containing 20mM sodium phosphate, 0.5M sodium chloride, pH 7.4). The eluted samples were combined, and the proteins in them could be detected by SDS-PAGE electrophoresis. The results of amino acid sequence determination show that the protein obtained after purification is a fusion protein obtained by connecting the amino terminal of the first amino acid residue of sequence 1 in the sequence listing with His-tag, and the fusion protein is the anti-idiotype of the human CD22 antibody Single chain antibody, hereinafter referred to as soluble scFv expressed by phage #3.

Soluble scFv expressed by phage #3 can be used to inhibit the adsorption of SM03 antibody to CD22 antigen expressed on the surface of Raji cells. The brief steps of the experiment are as follows: Raji cells washed with PBS were incubated with SM03 antibody, and different concentrations of soluble scFv expressed by phage #3 were added at the same time. Raj i cells only added with SM03 antibody were used as a control, and Raji cells without any reagents were added. The cells were used as a blank control (blank); after half an hour of incubation at room temperature and washing with PBS, a 1:50 dilution of fluorescently labeled goat anti-human IgG Fc specific antibody was added to all samples as a secondary antibody, and after half an hour of incubation at room temperature, it was washed. The fluorescence signal was analyzed by Beckman fluorescence analysis system. The results are shown in Figure 4, which shows that in the group added with 100ug/ml, the results of flow cytometry showed a significant decrease in fluorescence signal, indicating that the compatible scFv expressed by phage #3 can effectively inhibit the adsorption of SM03 antibody to the surface of Raji cells CD22 antigen.

Experimental example 2, using the soluble scFv expressed by phage #3 in Example 1 to conduct pharmacokinetic research in SM03 clinical trials

Due to the specificity of the compatible scFv expressed by phage #3 in Example 1 to SM03, it can be used to develop a corresponding detection method to determine the antibody concentration in the serum of patients receiving anti-CD22 antibody treatment, especially to assist clinical practice. required pharmacokinetic studies. A 96-well ELISA plate was coated with the compatible scFv expressed by phage #3 in Example 1. After blocking and washing with BSA, the serum of each blood collection point of patients receiving SM03 treatment was diluted and added to the wells. After incubation at 37°C for 2 hours and thorough washing, HRP-labeled goat anti-human Fc antibody (product of Jackson Immunoresearch) was diluted 1:4000 and added to the wells. After incubation at 37°C for 1 hour, the microtiter plate was washed 5 times, developed with TMB chromogenic solution, and the chromogenic signal was read at a wavelength of 450 nm according to the standard ELISA detection method, and the captured SM03 concentration was calculated accordingly.

Figure 5 shows a typical drug-time profile of SM03 using the method described above. Serum samples of patients were obtained from patients with malignant lymphoma who were treated with anti-CD22 antibody SM03, 380mg/ ^m2 dose group, once a week, for 4 consecutive weeks. Serum samples were collected at various time points before and after dosing.

Example 3. Anti-idiotypic antibody IdmG2a/k for human CD22 antibody in the form of murine IgG2a/kappa immunoglobulin molecules

The preparation of compatible scFv expressed by phage #3 in Example 1 requires a series of complex steps such as bacterial culture, induced expression and collection of inclusion bodies, protein denaturation and folding, and His-tag purification. Furthermore, scFvs are less stable and difficult to store. In order to produce a molecule that not only retains the adsorption specificity of the soluble scFv expressed by phage #3 in Example 1, but also can be purified by standard procedures and is stable and easy to store, the variable region of the heavy chain of the soluble scFv expressed by phage #3 in Example 1 (VH) coding DNA and light chain variable region (VK) coding sequences were amplified by PCR and integrated into the amplifiable expression vectors pIdmkappa-VK and pIdmIgG-VH using molecular cloning techniques, which also contained the murine kappa light chain And the partial sequence of constant region of IgG2a heavy chain (as shown in Figure 6).

The method for constructing the amplifiable expression vector is to link the coding DNA of the heavy chain variable region (VH) and the light chain variable region (VK) coding sequence of the soluble scFv in Example 1 to the corresponding mouse kappa light chain and IgG2a The heavy chain amplifies the constant region of the expression vector. Its murine kappa light chain expression vector pIdmkappa-VK is an expression vector of about 10Kb. It is derived from the vector pSVgpt (Mulligan&Berg 1981. PNAS 78:2072-2076), which contains an upstream IgG enhancer (Enhancer), a cloning site for inserting BamHI/BglII and XbaI of the VK segment, and even Gene sequence of mouse kappa light chain constant region with intron. Downstream of the light chain constant region gene sequence, a hygromycin selectable marker expressed from the SV40 promoter was placed. The construction method of the light chain expression vector is as follows: the gpt selection marker of pSVgpt is replaced with the hygromycin selection marker to obtain the recombinant vector pSVhyg. A 319bp Ig enhancer was inserted between the EcoRI and BamHI recognition sites of pSVhyg and an XbaI restriction endonuclease recognition site was inserted before the BamHI site to obtain the recombinant vector pSVhyg-Ig. A 199bp kappa enhancer was inserted between the BamHI and SacI recognition sites of pSVhyg-Ig to obtain the recombinant vector pSVhyg-Ig-kappa. Insert a 1.2Kb murine kappa light chain constant region fragment connected with an intron (intron) between the restriction endonuclease SacI and BamHI recognition sites of pSVhyg-Ig-kappa, and insert BamHI and XbaI restriction endonucleases The VK sequence displayed by phage #3 was connected between the sites to obtain the murine kappa light chain expression vector pIdmkappa-VK. Among them, the 319bp Ig enhancer nucleotide sequence is the 1st to 319th deoxynucleotides from the 5' end of GeneBank Accession Number: K01901. The 199bp kappa enhancer nucleotide sequence is the 1st to 199th deoxynucleotides from the 5' end of GeneBank Accession Number: K01325. The nucleotide sequence of the 1.2Kb intron murine light chain constant region fragment is from the 1st to 1209th deoxynucleotides at the 5' end of GeneBank Accession Number: J00241.

The construction method of the murine IgG2a heavy chain expression vector pIdmIgG-VH is roughly similar, and it is also a 10Kb expression vector derived from the vector pSVgpt (Mulligan & Berg 1981. PNAS 78:2072-2076), which contains an upstream IgG enhancer (Enhancer ), a Xho I and Hind III cloning site for inserting the VH segment, and the gene sequence of the mouse IgG2a heavy chain constant region connected to the intron (intron). Downstream of the heavy chain constant region gene sequence, a gpt selectable marker expressed with the SV40 promoter was placed. The construction method of this vector is as follows: a 319bp Ig enhancer is inserted between the EcoRI and XhoI recognition sites of pSVgpt to obtain the recombinant vector pSVgpt-Ig. Insert a 2Kb intron-linked murine IgG2a heavy chain constant region fragment between the restriction endonuclease HindIII and BamHI recognition sites of pSVgpt-Ig, between the XhoI and HindIII restriction endonuclease sites The VH sequence displayed by phage #3 was interlinked to obtain the murine IgG2a heavy chain expression vector pIdmIgG-VH. Among them, the 319bp Ig enhancer and nucleotide sequence are the 1st to 319th deoxynucleotides from the 5' end of GeneBank Accession Number: K01901. The 2Kb nucleotide sequence of the murine heavy chain constant region fragment connected to the intron is deoxynucleotides 1 to 2009 from the 5' end of GeneBank Accession Number: J00228.

This expression vector can be used to transfect a variety of mammalian host cell lines, including but not limited to Chinese hamster ovary (CHO) cell lines, mouse myeloma cell lines SP2/0 or NS0, baby hamster kidney (BHK) Cell lines, human embryonic kidney cell line 293 (HEK293), African green monkey kidney COS cell line, etc. In this example, the SP2/0 engineering cell line was selected as the host, and the transfection was carried out according to the standard steps by using the electroporation transfection method. After methotrexate selection, surviving clones were tested for antibody expression. Positive clones were selected for amplification. After several rounds of amplification, the recombinant engineered cell lines expressing antibodies were amplified and cultivated, and the "mouse-derived anti-idiotypic IgG antibodies" (hereinafter referred to as IdmG2a/k) produced by them were also collected and analyzed. Purify. At the same time, the RNA of the recombinant engineering cell line expressing the antibody was also extracted, and the cDNA coding sequence of the light and heavy chains of IdmG2a/k was obtained through RT-PCR method and Sanger sequencing method. Verified by comparison, IdmG2a/k, its light and heavy chain variable region sequence is the same as the VH and VL fragment sequences of the compatible scFv expressed by phage #3 in Example 1, and has the mouse IgG2a heavy chain and kappa light chain at the same time constant region. The heavy chain coding DNA sequence of the anti-idiotypic antibody IdmG2a/k for human CD22 antibody is sequence 9 in the sequence listing, and the amino acid sequence of sequence 2 in the coding sequence listing; the light chain coding DNA sequence of IdmG2a/k is sequence listing Sequence 10 encodes the amino acid sequence of Sequence 3 in the sequence listing.

The recombinant engineered cell line expressing IdmG2a/k was scaled up and cultured, and the antibody produced by the expression was extracted from the cell culture supernatant and purified with a protein A separation column according to standard procedures. Purified antibodies can be stored in PBS buffer at 4°C. Fig. 7 shows the SDS-PAGE electrophoresis patterns of the purified antibody of IdmG2a/k under reduced and non-reduced conditions.

The purified IdmG2a/k expressed by the recombinant engineering cell line can be used to coat ELISA plates according to standard procedures.

Add 50uL of 10ug/ml IdmG2a/k to each well of the ELISA plate and incubate overnight at 4°C for coating. For the coated ELISA plate, add different concentrations of 60uLSM03 antibody and other non-related control antibodies to each well, such as anti-CD20 antibody Rituximab (Rituximab, Swiss Roche Pharmaceutical Company), anti-CD147 antibody or anti-TNF antibody Infliximab (class grams, Cilag AG, Switzerland). After incubation at room temperature for 1.5 hours and washing with PBS three times, a 1:5000 dilution of HRP-labeled goat anti-human Fc antibody (product of Jackson Immunoresearch) was added. After another 45 minutes of incubation at room temperature, the adsorption can be visualized with OPD and read at a wavelength of 450 nm. The results showed that the "mouse anti-idiotypic IgG antibody" IdmG2a/k expressed by the recombinant engineered cell line could only specifically adsorb SM03, but not other control antibodies (as shown in Figure 8).

To assess the ability of IdmG2a/k expressed by recombinantly engineered cell lines to compete with the native CD22 antigen for adsorption of SM03 antibody, flow cytometry was performed using the Raji (human Burkitt's lymphoma) cell line as a source of surface natively expressed CD22 antigen experiment. The brief steps are as follows: 0.5×10 ⁶ Raji cells were incubated with 1ug/ml of SM03 antibody and different concentrations of mouse-derived anti-idiotypic IgG antibody IdmG2a/k. The incubation condition was 200uL of PBS-FA buffer (containing 1% fetal Bovine serum FBS, 0.01% sodium azide in PBS) and incubated at 4°C for 30 minutes. After washing with PBS for 3 times, a 50-fold diluted fluorescently labeled anti-human Fc antibody (product of Jackson Immunoresearch) was added and incubated at 4°C for 30 minutes. After washing three times with PBS, the cells were fixed with PBS-FA buffer containing 0.5% formalin. The fixed cells were suspended in PBS, and analyzed by flow cytometry using the FACScan analysis system of Becton Dickenson Company, USA. The results showed that the anti-idiotypic antibody IdmG2a/k could effectively inhibit the adsorption of SM03 to its natural ligand in doses (Fig. 9).

Example 4. Using the IdmG2a/k of Example 3 to monitor the content of "CD22 antibody family" in clinical practice

1. The IdmG2a/k of Example 3 can specifically bind to the common antigen-binding site sequence of the "CD22 antibody family", and can specifically adsorb murine, chimeric and humanized anti-CD22 antibodies.

The competitive adsorption detection method further showed that the adsorption site of the anti-idiotypic antibody was located in the antigen binding site (ABS) sequence, which was the only public sequence of the murine antibody RFB4, chimeric antibody SM03 and humanized antibody SM06. The method of competitive adsorption detection is briefly as follows. Add 50uL 10ug/ml of the IdmG2a/k of Example 3 to each well of the ELISA enzyme-linked plate, and incubate overnight at 4°C for coating. After washing 3 times with PBS the next day, block with 200 uL of 3% BSA solution at room temperature for 2 hours, and then wash 5 times with PBS. The SM03 antibody was labeled with HRP as SM03-HRP labeled antibody (labeled by Tianjin Tianjian Biotechnology Co., Ltd.). 1:4000 diluted SM03-HRP labeled antibody, mixed with different concentrations of competing antibodies, including RFB4, SM03, SM06 and some other non-related control antibodies (SM09, N005, N009). The mixed antibodies were added to the ELISA plate coated with the IdmG2a/k of Example 3. After the SM03-HRP-labeled antibody absorbs the coated anti-idiotypic antibody, it can be developed with TMB chromogenic solution.

The results show that RFB4, SM03 and SM06 can equivalently compete with the SM03-HRP labeled antibody to adsorb to the IdmG2a/k of Example 3, while other non-related antibodies have no competitive adsorption capacity (as shown in Figure 10), indicating that the embodiment The IdmG2a/k of 3 is specifically adsorbed to the consensus sequence of RFB4, SM03 and SM06, but not to other sites, and this consensus sequence is their antigen recognition site (ABS). Among them, the control antibodies SM09, N005, and N009 (China Antibody Pharmaceutical Co., Ltd.) were directed against non-CD22 antigens and did not cross-react with anti-CD22 antibodies.

2. Using the IdmG2a/k of Example 3 to develop and evaluate the standard detection method for pharmacokinetics in SM03 clinical trials

With the confirmation of the adsorption specificity of the "mouse anti-idiotypic IgG antibody" to the antigen binding site of the "CD22 antibody family", an ELISA assay has been developed to estimate the RFB4, SM03 and SM06 antibody levels in the serum of patients in clinical trials method. The following example describes the development of an assay to estimate serum levels of SM03, which can also be generalized to other members of the "CD22 antibody family". The brief steps are as follows. Add 0.4ug/ml of IdmG2a/k of Example 3 to each well of a 96-well ELISA plate, and the coating conditions are 50uLPBS per well, pH7.4, and incubate overnight at 4°C; use it the next day 1% BSA solution blocked for 2 hours at room temperature. Serum samples from patients receiving SM03 treatment were diluted by a certain number of times, and added to duplicate wells with different concentrations of exogenous SM03 standard products. The diluent was selected to contain 0.1% blank human serum in PBS to remove the influence of serum on the background. Add 50uL of sample to each well and incubate at room temperature for 2 hours. After washing, add 1:4000 diluted HRP-labeled goat anti-human Fc antibody (product of Jackson Immunoresearch Company), incubate at room temperature for 1 hour, wash and add TMB for color development. The chromogenic signal was recorded with visible light at a wavelength of 450nm. The change over time of the residual SM03 antibody concentration in the peripheral blood of patients receiving antibody treatment can be obtained by comparing with the exogenous SM03 standard.

Figure 11 shows the typical pharmacokinetic behavior of a patient treated with SM03 using the ELISA assay developed previously described.

Example 5. Novel detection method for estimating the biological efficacy of anti-internalization antigen antibody

1. Construction of engineered cell lines transmembranely expressing adsorbed fragments of anti-idiotypic antibodies

In the process of storage and production of therapeutic antibodies, the biological activity of antibodies is evaluated by quality control. Taking SM03 as an example, there are two important considerations: adsorption properties (interpreted in detail as adsorption affinity and specificity) and antibody-mediated biology Ability to react (eg ADCC or CDC). The latter is usually estimated by means of biological efficacy assays. Since all antibodies of the "CD22 antibody family" can adsorb CD22 antigen, and internalization will occur soon after adsorption, SM03 antibody and other anti-CD22 antibodies cannot successfully mediate CDC reaction in vitro experiments. After the "CD22 antibody family" antibody is adsorbed to the surface of CD22-positive cells, it will not stay on the cell surface for a sufficient time to allow its Fc part to fully interact with complement (assumed to be C1q complement), so that it cannot properly initiate apoptosis. chain reaction. This is likely the reason why no measurable CDC response was observed with anti-CD22 antibodies like SM03. This explanation is also convincing for other antibodies against surface antigens that undergo rapid internalization, such as CD33, Lewis (Y) antigen, invariant chain (CD74). The detection method used to confirm the adsorption properties (specificity and affinity) of the antibody proves that the adsorption fragments composed of the variable region of the light and heavy chains of the antibody are folded, sorted, and paired correctly, but they cannot provide information about possible protein variations or Missing situation. These deletions or mutations may prevent the antibody from exerting its correct biological function when acting in vivo. Therefore, it is desirable to develop universal cell-based assays to assess the biological function of antibodies targeting antigens that undergo rapid internalization.

The adsorption moiety of the anti-idiotypic antibody of the present invention is therefore redesigned so that it can be expressed on the cell surface as a non-internalized appendage protein. This innovation is accomplished in several ways:

1. Express the anti-idiotypic antibody in the form of transmembrane IgD

a. The transmembrane region sequence of mouse IgD was fused with the CH3 region of "mouse anti-idiotype IgG antibody" to obtain the transmembrane anti-idiotype antibody IdGmD against human CD22 antibody

The amino acid sequence of the murine IgD transmembrane region (TM) used for fusion with the anti-idiotypic IgG antibody is shown as the underlined sequence in Figure 12 .

In order to construct an IgG2a-TM (IgD) protein gene capable of expressing fusion, a DNA sequence encoding part of the carboxy-terminal sequence of the "mouse-derived anti-idiotypic IgG antibody" was chemically synthesized. This DNA sequence is degenerated with two restriction sites, BsrG1 and EagI, to facilitate its cloning into the corresponding original murine IgG2a constant region sequence. . This sequence encodes a fusion fragment of 41 amino acids of original murine IgG2a and IgD(TM). This sequence was cloned into the original IgG2a sequence (Figure 6), replacing a sequence at the carboxy-terminal of the IgG2a CH3 domain so that it not only contained the original sequence but also added the TM sequence of murine IgD. The expressed protein should be the heavy chain of IgG2a immunoglobulin fused to the transmembrane fragment of mouse IgD (the complete structure is VH-CH1-hinge-CH2-CH3-TM) (heavy chain is shown in Figure 12), and the span The membrane anti-idiotypic antibody against human CD22 antibody was named IdGmD.

The SP2/0 engineering cell line was selected as the host, and the transfection was carried out according to the standard steps by using the electroporation transfection method. After methotrexate selection, surviving clones were tested for antibody expression. Positive clones were selected for amplification. After several rounds of amplification, the recombinant engineered cell lines expressing antibodies were amplified and cultured, and the transmembrane antibody IdGmD produced by them was also collected and purified. At the same time, the RNA of the recombinant engineering cell line expressing the antibody was also extracted, and the cDNA coding sequence of the light and heavy chains of IdGmD was obtained through RT-PCR method and Sanger sequencing method. The DNA sequence encoding the heavy chain of the anti-idiotypic antibody IdGmD for human CD22 antibody is sequence 11 in the sequence listing, and the amino acid sequence of sequence 4 in the encoding sequence listing (the amino acid sequence in Figure 12); the DNA sequence encoding the light chain of IdGmD is sequence Sequence 10 in the list encodes the amino acid sequence of Sequence 3 in the sequence list.

b. Replace IgG2a heavy chain with mouse IgD sequence to obtain transmembrane anti-idiotypic antibody IdDmD against human CD22 antibody

The constant region sequence of the original "murine anti-idiotypic IgG antibody" is completely replaced with the membrane-attached murine IgD sequence. The complete amino acid sequence of murine IgD with the TM sequence of the transmembrane fragment is shown in Figure 13 .

The murine IgD cDNA sequence encoding the membrane-attached form was chemically synthesized according to standard oligonucleotide synthesis methods. Restriction points (XhoI and EagI) were introduced to facilitate cloning into the corresponding parts of the original mouse IgG2a expression vector. This sequence was cloned into the corresponding part of the original IgG2a expression vector (Figure 6), and the constant region sequence of murine IgG2a was replaced by the membrane-attached IgD protein sequence to obtain the recombinant expression vector pIdDmD of IdDmD. The expressed protein includes the heavy chain of IgD immunoglobulin with IgD transmembrane fragment (morphological structure is VH-CH1-hinge-CH3-TM). It is worth noting that murine IgD does not have a CH2 region, so its structure is very different from IgG or human IgD, as shown in Figure 13 (the transmembrane region is underlined).

Because it is just an example experiment, only the anti-idiotypic IgD-TM expression vector DNA plasmid described in part (b) was linearized with endonucleases. The mouse myeloma cell line SP2/0 was used as the host cell for transfection. According to relevant literature, SP2/0 cells can only produce endogenous Igβ but not Igα. Since the membrane expression of IgD immunoglobulin molecules requires the joint action of Igα and Igβ, the expression vector of Igα is also transfected into SP2/0 host cells together with the expression vector pIdDmD of IgD-TM fusion protein according to the standard electroporation transfection method. The cells transfected with the plasmid were selected with the selective medium containing methotrexate according to the standard method of DHFR system. The selected surviving cells were tested for the specificity of the anti-idiotype expressed on their membrane surface by cell ELISA detection method. The brief steps of the experiment are as follows, 50uL of 10ug/ml SM03 antibody was added to 10 ⁶ transfected cells (washed 3 times with PBS first). The cells mixed with the antibody were incubated at 4°C for 1 hour and washed 3 times with PBS. Then add 50uL of 1:1000 diluted HRP-labeled goat anti-human IgG Fc antibody and incubate at 4°C for 1 hour. After washing once with PBS, add 50uL for color development, and the anti-SM03 IgD positive cells on the membrane surface will have a color reaction. After 10 minutes of incubation at room temperature, 50uL of 0.18M dilute acid was added to stop the reaction. The antibody-cell mixture was centrifuged, and the supernatant was collected to read the absorbance at a wavelength of 450 nm.

Positive clones were selected for amplification. After several rounds of amplification, the recombinant engineering cell lines expressing antibodies were amplified and cultured, and the transmembrane antibody IdDmD produced by them was also collected and purified. At the same time, the RNA of the recombinant engineering cell line expressing the antibody was also extracted, and the cDNA coding sequence of the light and heavy chains of IdDmD was obtained through RT-PCR method and Sanger sequencing method. The heavy chain coding DNA sequence of the anti-idiotypic antibody IdDmD for human CD22 antibody is sequence 12 in the sequence listing, encoding the amino acid sequence of sequence 5 in the sequence listing; the light chain coding DNA sequence of IdDmD is sequence 10 in the sequence listing, encoding Amino acid sequence of sequence 3 in the sequence listing.

The results showed that expression of anti-SM03 IgD on the membrane surface required simultaneous transfection of Igα into mouse myeloma cells. The results of the flow cytometry experiment (the experimental method is the same as the flow cytometry analysis in step 2 below) confirmed the membrane surface expression of IgD ( FIG. 16 ).

2. Express the transmembrane anti-idiotypic antibody IdGmFdA against human CD22 antibody in the form of membrane-attached Fab-glycoprotein fusion protein

The transmembrane region of glycoprotein A from the surface of the red blood cell membrane is fused to the carboxy-terminal of the mouse IgG2a hinge region of the "mouse anti-idiotypic IgG antibody". This fusion of antibody fragments is expected to be expressed on the surface of transfected cells as a Fab anchored to the cell surface by the glycoprotein A transmembrane domain.

The glycoprotein A fragment (including the transmembrane region sequence) used to fuse the anti-SM03 antibody fragment is shown in the underlined amino acid sequence in FIG. 14 .

The gene sequence is directly fused behind the gene sequence encoding the last amino acid in the mouse Ig2a antibody hinge region, and the expressed protein should be "mouse anti-idiotypic IgG antibody" heavy chain Fd region fused to the transmembrane region of glycoprotein A and Inner bag region (fragment form: VH-CH1-hinge region-glycoprotein A transmembrane region+inner bag region, Figure 14).

The expression vector expressing the Fd fragment-glycoprotein A fusion protein of the "mouse anti-idiotype IgG antibody" on the surface of the cell membrane is constructed. A brief description is as follows. The coding DNA sequence of the murine IgG2a antibody CH1-hinge region fragment fused to the carboxy-terminal fragment of glycoprotein A (including the transmembrane region sequence) was chemically synthesized according to the standard oligonucleotide synthesis method. The synthesized sequence contains in-frame XhoI and EagI incision points. The synthetic sequence was inserted into the corresponding site in the gene sequence of the anti-SM03IgG2a antibody expression vector (Figure 6) using standard molecular biology cloning techniques, replacing the CH2-CH3 region coding sequence in the original IgG2a antibody sequence, and the recombinant expression vector pIdGmFdA of IdGmFdA was obtained .

"Mouse anti-idiotypic IgG antibody" Fab fragment-glycoprotein A fusion protein expression vector pIdGmFdA plasmid DNA was transfected into mouse SP2/0 host cells by linearization. Cells transfected with plasmids were selected for positive clones with methotrexate using the DHFR gene selection system according to standard methods. The positive clones were selected for amplification, and after several rounds of amplification, the recombinant engineered cell lines expressing antibodies were amplified and cultivated, and the transmembrane antibody IdGmFdA produced by them was also collected and purified. At the same time, the RNA of the recombinant engineering cell line expressing the antibody was also extracted, and the cDNA coding sequence of the light and heavy chains of IdGmFdA was obtained through RT-PCR method and Sanger sequencing method. The heavy chain coding DNA sequence of the anti-idiotypic antibody IdGmFdA for human CD22 antibody is sequence 13 in the sequence listing, encoding the amino acid sequence of sequence 6 in the sequence listing; the light chain coding DNA sequence of IdGmFdA is sequence 10 in the sequence listing, encoding Amino acid sequence of sequence 3 in the sequence listing.

The selected surviving positive clones were used to determine the cell surface expression of their anti-idiotype Fab-glycoprotein A fusion protein, and the method used was still the cell ELISA detection method described above. The cells that successfully express the fusion protein on the cell surface detected by ELISA were further analyzed by flow cytometry: SM03 antibody was used as the primary antibody, and FITC fluorescently labeled goat anti-human IgG Fc antibody (product of Jackson Immunoresearch Company) was used as the detection antibody (secondary antibody). The brief instructions are as follows, 5×10 ⁵ transfected cells were co-incubated with 0.01, 0.1 or 1 μg of SM03 in 100uL of PBS-FA buffer (1% fetal calf serum FCS, 0.05% sodium azide in PBS solution) . The incubation condition was 4°C for 30 minutes, and then washed 3 times with PBS to remove non-adsorbed antibodies. The adsorption amount of SM03 to transfected cells was evaluated by detection of fluorescent FITC-labeled goat anti-human IgG Fc antibody (product of Jackson Immunoresearch). After incubating at 4°C for 30 minutes with 100uL PBS-FA buffer solution containing 20-fold diluted fluorescently-labeled antibody, the cells were washed with PBS for 3 times, and the fluorescence signals were analyzed with the FACScan system. The results showed that the anti-idiotype Fab-glycoprotein A fusion protein IdGmFdA can be effectively expressed on the cell surface of the transfected myeloma cell line (as shown in FIG. 16 ).

3. The anti-idiotypic antibody IdGmFdG against human CD22 antibody expressing "mouse anti-idiotype IgG" antibody in the form of Fab-GPI immobilized fusion protein

The GPI fragment is derived from the LDL receptor attached to the hydrophobic region of DAF, and this fragment is fused directly after the IgG2a hinge region of the "murine anti-idiotypic IgG antibody". Fusion proteins are expected to be expressed as Fab fragments attached to the cell surface via GPI immobilization.

The amino acid sequence of the GPI fragment derived from the LDL receptor attached to the hydrophobic region of DAF is shown as the underlined amino acid sequence in FIG. 15 .

The coding DNA sequence is fused directly behind the last amino acid gene sequence of the mouse IgG2a antibody hinge region; the expressed protein should be the fusion of the Fd part of the heavy chain of the "mouse anti-idiotypic IgG antibody" and the GPI-DAF fragment (the fusion fragment consists of is VH-CH1-GPI-DAF, as shown in Figure 15).

The expression vector expressing the above fusion protein has been constructed. A brief description is as follows. The DNA sequence encoding the fusion fragment of mouse IgG2a CH1-hinge region and GPI-DAF was chemically synthesized according to the standard oligonucleotide synthesis method. The sequence contains in-frame XhoI and EagI incision points (underlined). The synthetic sequence was inserted into the corresponding site in the gene sequence of the "mouse anti-idiotypic IgG antibody" expression vector (Figure 6) using standard molecular biology cloning techniques, replacing the CH2-CH3 region coding sequence in the original IgG2a antibody sequence, and obtained The recombinant expression vector pIdGmFdG of IdGmFdG.

The pIdGmFdG plasmid DNA was linearized and transfected into mouse SP2/0 host cells. Cells transfected with plasmids were selected for positive clones with methotrexate using the DHFR gene selection system according to standard methods. The positive clones were selected for amplification, and after several rounds of amplification, the recombinant engineered cell lines expressing antibodies were amplified and cultured, and the transmembrane antibodies IdGmFdG produced were also collected and purified. At the same time, the RNA of the recombinant engineering cell line expressing the antibody was also extracted, and the cDNA coding sequence of the light and heavy chains of IdGmFdG was obtained through RT-PCR method and Sanger sequencing method. The heavy chain coding DNA sequence of the anti-idiotypic antibody IdGmFdG for human CD22 antibody is sequence 14 in the sequence listing, encoding the amino acid sequence of sequence 7 in the sequence listing; the light chain coding DNA sequence of IdGmFdG is sequence 10 in the sequence listing, encoding Amino acid sequence of sequence 3 in the sequence listing.

The selected surviving positive clones were used to determine the cell surface expression of their anti-idiotype Fab-glycoprotein A fusion protein, and the method used was still the cell ELISA detection method described above. The cells that successfully express the fusion protein on the cell surface detected by ELISA were further analyzed by flow cytometry: SM03 antibody was used as the primary antibody, and FITC fluorescently labeled goat anti-human FcIgG antibody (product of Jackson Immunoresearch Company) was used as the detection antibody (secondary antibody). The specific method Same as flow cytometric analysis in step 2 above). The results showed that the "mouse anti-idiotypic IgG antibody" Fab fragment-GPI fusion protein IdGmFdG can be effectively expressed on the cell surface of the transfected myeloma cell line (as shown in Figure 16).

2. Establish a cell detection method for evaluating the biological efficacy of the "CD22 antibody family"

Transfect cells with different expression types that express the corresponding fusion proteins (IdDmD, IdGmFdA, and IdGmFdG) on the cell surface in step 1 above, and the corrected cell concentration is 2×10 ⁶ cells/ml. Add 50uL of cells to each well of a 96-well plate. The lyophilized powder of Guinea Pig Serum (GPS) from Cedarlane Company in Canada was prepared into 100% GPS solution with 1 ml of culture medium. Add 50uL of different concentrations of SM03 antibodies containing 10% GPS to the wells containing the transfected cells. After 2 hours of incubation at 37°C and 5% carbon dioxide, 20uL of CCK-8 (cell counting kit-8) reagent from Dojindo Molecular Technology Company of the United States was added to each well. After 3 hours, read the cell viability by 450nm visible light wavelength according to the product instructions. The results show that "CD22 antibody family" products such as SM03 can target the Fab fragment of "murine anti-idiotype IgG antibody" expressed on the cell surface-glycoprotein A fusion protein IdGmFdA and the "murine anti-idiotype IgG antibody" Fab fragment- The cells transfected with the GPI-DAF fusion protein IdGmFdG can effectively mediate the complement-dependent cytotoxic reaction (CDC reaction, as shown in FIG. 17 ). However, there was no obvious CDC effect on transfected cells expressing anti-idiotypic IgD-TM fragment protein IdDmD on the cell surface. The reason may be that the IgD on the cell surface will still be internalized after being adsorbed by the "CD22 antibody family" products, so as to block the CDC reaction; or because the expression of the anti-idiotype-TM fragment protein on the membrane is too small Reach a critical value that triggers complement activation. However, two of the three different transfected cells expressing the adsorbed fragment of the "murine anti-idiotypic IgG antibody" on the cell surface were dose-effective in mediating SM03-induced CDC killing, showing that this strategy can be used as an effective method for developing suitable biological agents. general guidelines for the correct assessment of the biological efficacy of antibodies to internalizing antigens.

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.