CN114014931A - Fab fragment of human anti-human CD23 antibody, pharmaceutical composition and application thereof - Google Patents

Abstract

The invention discloses a Fab fragment of a humanized anti-human CD23 antibody, a pharmaceutical composition and application thereof. Comprises a light chain V region and a heavy chain V region, wherein the amino acid sequence of the light chain V region is shown as SEQ ID NO.1, and the amino acid sequence of the heavy chain V region is shown as SEQ ID NO. 2. The Fab fragment can specifically recognize human CD23 and has high affinity, and can be used for regulating the activity of CD23, thereby controlling and mediating cell adhesion, regulating the release of IgE and histamine, and also protecting B cells from apoptosis and regulating the growth of bone marrow cells by regulating the activity of CD 23. Fab fragments of human anti-human CD23 antibody can be used for preparing medicine for treating lymph related diseases.

Description

Fab fragment of human anti-human CD23 antibody, pharmaceutical composition and application thereof

Technical Field

The invention relates to the technical field of biological detection, and particularly relates to a Fab fragment of a humanized anti-human CD23 antibody, a pharmaceutical composition and application thereof.

Background

CD23, also known as fceri, is a low affinity receptor for IgE in humans and vertebrates, i.e., the type II receptor for IgE; it is a transmembrane glycoprotein widely distributed on the cell membrane surfaces of B cells, eosinophilic granulocytes (EOS), activated monocytes/macrophages, platelets, follicular dendritic cells, Langerhans cells, epidermal keratinocytes, thymic epithelial cells, partial T cells, natural killer cells and the like, and has a molecular weight of 45 kDa.

CD23 is a differentiation antigen on the surface of human B lymphocytes. CD23 belongs to typical II type transmembrane glycoprotein, and is composed of 321 amino acids, including intracellular N-terminal composed of 23 amino acid residues, transmembrane region composed of 21 amino acid residues, and C-terminal extracellular region composed of 277 amino acid residues; the carboxyl terminal is positioned outside the cell, and the amino terminal is positioned inside the cell, belonging to the C-type animal lectin superfamily.

The human CD23 gene is located on chromosome 19 and is a single copy gene, and there are two types, and since the 6 amino acids at the amino terminal of the encoded CD23 molecule are different due to their different promoter genes, two types of CD23 are formed: CD23a and CD23 b. Cell membrane surface CD23 can be cleaved by itself or by proteolytic enzymes into soluble fragments of varying sizes, called soluble CD23(sCD23), which are mostly still IgE-binding and exhibit multiple functions unrelated to IgE.

CD23 is a multifunctional receptor molecule/cytokine that is involved in regulating IgE synthesis, promoting B cell activation, proliferation and differentiation, and promoting T cell maturation by mediating B-T cell interactions through binding to membrane CD21, enhancing antigen presentation from B cells to T cells.

At present, the specific activation process of B lymphocytes and the IgE production regulation mechanism are not clear, and the provision of Fab fragments of a human anti-human CD23 antibody has important significance for clinical diagnosis and scientific research.

In view of this, the invention is particularly proposed.

Disclosure of Invention

The present invention aims to provide a Fab fragment of a human anti-human CD23 antibody, a pharmaceutical composition and an application thereof to solve the above technical problems.

The invention is realized by the following steps:

the invention provides a Fab fragment of a humanized anti-human CD23 antibody, which comprises a light chain V region and a heavy chain V region, wherein the amino acid sequence of the light chain V region is shown as SEQ ID NO.1, and the amino acid sequence of the heavy chain V region is shown as SEQ ID NO. 2.

In a preferred embodiment of the present invention, the nucleotide sequence of the gene of the light chain V region is shown in SEQ ID NO. 3.

In a preferred embodiment of the present invention, the nucleotide sequence of the gene of the heavy chain V region is shown in SEQ ID NO. 4.

The invention also provides an expression vector containing a nucleotide sequence encoding a light chain V region and/or a nucleotide sequence encoding a heavy chain V region.

The invention also provides a host cell, which contains the expression vector.

In a preferred embodiment of the present invention, the host cell is Escherichia coli or a eukaryotic cell.

The invention also provides a pharmaceutical composition, which comprises the Fab fragment of the humanized anti-human CD23 antibody.

The invention also provides application of the Fab fragment of the humanized anti-human CD23 antibody in preparation of medicaments for treating the following diseases, wherein the diseases are any one of the following diseases:

the neoplastic disease is selected from recurrent Hodgkin's disease; drug resistant hodgkin's disease; high grade, low grade and intermediate grade non-hodgkin's lymphoma; b cell chronic lymphocytic leukemia; plasma cell-like lymphocytic lymphoma; mantle cell lymphoma; follicular lymphoma; diffuse large cell lymphoma; burkitt's lymphoma; AIDS-related lymphoma; monocytic B-cell lymphoma; angioimmunoblastic lymphadenopathy.

The invention provides a medicament for treating lymph related diseases, which comprises Fab fragments of the human anti-human CD23 antibody.

In a preferred embodiment of the present invention, the above-mentioned medicament for treating lymph related diseases further comprises pharmaceutically acceptable additives or adjuvants, and preferably, the dosage form of the pharmaceutical composition is selected from tablets, pills, powders, suspensions, gels, emulsions, creams, granules, nanoparticles, capsules, suppositories, injections, sprays and injections.

The invention has the following beneficial effects:

the Fab fragment of the humanized anti-human CD23 antibody provided by the invention is fully humanized and has no Fc segment, can specifically identify human CD23 and has higher affinity, and can be used for regulating the activity of CD23, thereby controlling mediated cell adhesion, regulating the release of IgE and histamine, and preventing B cells from apoptosis and regulating the growth of bone marrow cells by regulating the activity of CD 23. Fab fragments of human anti-human CD23 antibody can be used for preparing medicine for treating lymph related diseases.

Drawings

In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings needed to be used in the embodiments will be briefly described below, it should be understood that the following drawings only illustrate some embodiments of the present invention and therefore should not be considered as limiting the scope, and for those skilled in the art, other related drawings can be obtained according to the drawings without inventive efforts.

FIG. 1 is a PAGE picture of protein fragments of recombinant human CD 23.

Detailed Description

The invention provides a Fab fragment of a humanized anti-human CD23 antibody, which comprises a light chain V region and a heavy chain V region, wherein the amino acid sequence of the light chain V region is shown as SEQ ID NO.1, and the amino acid sequence of the heavy chain V region is shown as SEQ ID NO. 2.

The human anti-human CD23 antibody provided by the invention is selected from a natural human immunoglobulin gene library, and Fab fragments of the anti-human CD23 antibody are obtained by screening from the library through ELISA sequencing analysis and the like. Through expression, purification and identification, the Fab fragment of the humanized anti-human CD23 antibody provided by the invention can specifically recognize human CD23, and the affinity of the Fab fragment of the humanized anti-human CD23 antibody and human CD23 is higher.

In a preferred embodiment of the present invention, the nucleotide sequence of the gene of the light chain V region is shown in SEQ ID NO. 3.

In a preferred embodiment of the present invention, the nucleotide sequence of the gene of the heavy chain V region is shown in SEQ ID NO. 4.

The invention also provides an expression vector containing a nucleotide sequence encoding a light chain V region and/or a nucleotide sequence encoding a heavy chain V region.

Alternatively, the expression vector may contain both a nucleotide sequence encoding a light chain V region and a nucleotide sequence encoding a heavy chain V region. Optionally, a nucleotide sequence encoding a light chain V region is used as an insert, and a promoter and a terminator are respectively disposed at both ends of the insert; the nucleotide sequence encoding the heavy chain V region has a corresponding promoter and terminator disposed at both ends. The expression vector is also provided with a corresponding resistance screening site and a corresponding enzyme cutting site.

The invention also provides a host cell, which contains the expression vector.

In a preferred embodiment of the present invention, the host cell is Escherichia coli or a eukaryotic cell. Alternatively, the host cell includes, but is not limited to, JM 109.

The invention also provides a pharmaceutical composition, which comprises the Fab fragment of the humanized anti-human CD23 antibody.

Alternatively, a Fab fragment of the above human anti-human CD23 antibody is used as an active ingredient.

Alternatively, the pharmaceutical composition is used for the targeted regulation of the activity of CD23, and the pharmaceutical composition includes, but is not limited to, mediating cell adhesion, regulating the release of IgE and histamine, controlling B cells from apoptosis, regulating bone marrow cell growth, and the like.

Alternatively, the pharmaceutical composition can be used for treating B lymphocyte related diseases.

The invention also provides application of the Fab fragment of the humanized anti-human CD23 antibody in preparation of medicaments for treating the following diseases, wherein the diseases are any one of the following diseases:

the neoplastic disease is selected from recurrent Hodgkin's disease; drug resistant hodgkin's disease; high grade, low grade and intermediate grade non-hodgkin's lymphoma; b cell chronic lymphocytic leukemia; plasma cell-like lymphocytic lymphoma; mantle cell lymphoma; follicular lymphoma; diffuse large cell lymphoma; burkitt's lymphoma; AIDS-related lymphoma; monocytic B-cell lymphoma; angioimmunoblastic lymphadenopathy.

Alternatively, the Fab fragment of the humanized anti-human CD23 antibody can be used for treating diseases mediated by toll IgE, such as ulcerative colitis and Crohn's disease.

Alternatively, the Fab fragment of the above human anti-human CD23 antibody may be used alone, or in combination with an immunosuppressive agent, such as an antibody, e.g., a steroid or anti-lymphocyte antibody, or in combination with a tolerance-inducing agent, an anti-autoimmune agent, or an anti-inflammatory agent. For example, a CD4+ T cell inhibitor such as an anti-CD 4 antibody, a TNF antagonist such as an anti-CD 8 antibody or an anti-TNF antibody, and the like.

Alternatively, the pharmaceutical compositions provided by the present invention may be particularly useful for subcutaneous, intramuscular or intravenous administration.

The invention provides a medicament for treating lymph related diseases, which comprises Fab fragments of the human anti-human CD23 antibody.

In a preferred embodiment of the present invention, the above-mentioned medicament for treating lymph related diseases further comprises pharmaceutically acceptable additives or adjuvants, and preferably, the dosage form of the pharmaceutical composition is selected from tablets, pills, powders, suspensions, gels, emulsions, creams, granules, nanoparticles, capsules, suppositories, injections, sprays and injections.

Fab fragments of the humanized anti-human CD23 antibodies provided by the present invention can be lyophilized for storage and reconstituted in an appropriate carrier prior to use. Lyophilization and reconstitution can result in varying degrees of loss of antibody activity, and thus the amount used needs to be adjusted to compensate for this.

In order to make the objects, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products available commercially.

The features and properties of the present invention are described in further detail below with reference to examples.

Example 1

This example provides a method for screening Fab fragments of human anti-human CD23 antibody. It includes:

(1) and (5) constructing an antibody screening library. Specifically, 270 healthy people are selected, 5ml of peripheral anticoagulation blood is taken respectively, lymphocytes are separated by Ficoll-Paque, and the lymphocytes are mixed and used for constructing an antibody library to extract total RNA. The total RNA is reverse transcribed into cDNA by oligo (dT)16 by a reverse transcription kit of the whole gene, and PCR amplification of immunoglobulin gamma, kappa and lambda chain genes is carried out by upstream and downstream primers of conserved sequences of human CD23 light and heavy chain variable regions.

After the PCR product is purified, the kappa chain and lambda chain products are cut by double enzyme. The products of the cleaved kappa chain and the lambda chain are connected with a human immunoglobulin Fab expression vector pFab-His2, and then are transformed into JM109 competent cells to form a light chain library. Respectively digesting and modifying the light chain library and the gamma chain PCR product by BamH I and Hind III, performing ligation reaction, and then electrically transducing the product into Escherichia coli JM109 to form 8 × 10⁸The antibody library of the independent clone titer of (1) was subjected to enzyme digestion electrophoresis using 1ul of DNA.

(2) Recombinant expression of a protein fragment of human CD 23.

The anti-CD 23 receptor binds to CD23 expressed on hematopoietic cells.

Firstly, synthesizing cDNA required for coding human CD23 protein fragment, and amplifying gene fragment of human CD23 protein fragment by PCR; the gene fragment is subjected to enzyme digestion purification by BamH I and Hind III, then is connected with pET19b (Novagen) vector, is transformed into Escherichia coli JM109, the obtained positive clone is subjected to sequence analysis, a plasmid with a correct sequence is transferred into an expression host bacterium BL21star (DE3) plysS competent cell (Novagen), is induced and expressed by isopropyl-beta-D-thiogalactoside (IPTG), an inclusion body protein is extracted, and is subjected to protein renaturation (an inclusion body protein purification method according to Novagen), wherein the molecular weight of the inclusion body protein is 28kDa (figure 1). The specificity and sensitivity of the recombinant protein were tested by spotting 2. mu.g of purified protein fragment of human CD23 on nitrocellulose.

The results show that the protein fragment of recombinant human CD23 can specifically react with the anti-human CD23 antibody.

(3) Screening of human immunoglobulin G against human CD23

Take 8X 10⁸The antibody library DNA10ng of independent clone titer is transformed into 100 mul of Escherichia coli JM109, bacterial liquid is coated on a flat plate, the culture is carried out for 7 hours at 37 ℃, a nitrocellulose membrane with the diameter of 82mM is coated on the flat plate when the clone diameter is about 0.3mM, the clone is completely transferred to a membrane, the membrane is placed on an LB flat plate containing 1.0mM IPTG, the induced expression is carried out for 6 hours at 30 ℃, and then lysozyme, DNase and bovine serum albumin are used for carrying out the bacteriolysis on the membrane; the dark positive clones on the membrane were then screened by colony blotting.

Taking 10ng of DNA from an antibody library, transforming 100 mu lJM109 escherichia coli, picking 3 single clones obtained by transformation into 2ml of a culture medium containing ampicillin, adding IPTG when OD600 is 0.6-0.8 to enable the final concentration to be 0.1mM, inducing expression at 30 ℃ for 10-12 hours, centrifuging at 14000rpm for 2 minutes to collect bacteria, adding 100 mu l of PBS containing 1mM PMSF into bacterial precipitates, suspending the bacteria, ultrasonically crushing at 4 ℃, centrifuging at 14000rpm for 10 minutes, taking supernatant obtained after ultrasonic lysis of 100 mu l/hole bacteria or CD23 Ab 1 diluted by bacterial liquid or CD23 Ab 1 diluted by 3% BSA, adding an enzyme linked immunosorbent assay plate coated with 0.5 mu g of natural CD23(10 ng/hole), carrying out enzyme linked immunosorbent assay reaction, taking HRP-labeled anti-human IgG Fab as a secondary antibody, and developing color by using o-phenylenediamine.

Screening to obtain 1 positive clone which can be specifically combined with the protein fragment of the human CD 23.

(4) Characterization of heavy and light chain genes of Fab fragment of anti-human CD23 antibody

Taking positively cloned plasmids, carrying out restriction enzyme digestion by restriction enzymes respectively to obtain light chain and heavy chain genes, connecting with sequencing vectors CV-2 and CV-1 modified by enzyme digestion, transforming escherichia coli JM109, extracting plasmid DNA containing the light chain or heavy chain genes respectively, carrying out sequencing by taking M13Reverse as a primer, calculating an amino acid sequence by Vector NTI 10 software, carrying out homology analysis by IgBlast, and dividing CDRs and FRs of light chain variable regions and heavy chain variable regions of positive clones according to a Kabat system.

The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention, and various modifications and changes may be made by those skilled in the art. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Sequence listing

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Claims (10)

1. The Fab fragment of the humanized anti-human CD23 antibody is characterized by comprising a light chain V region and a heavy chain V region, wherein the amino acid sequence of the light chain V region is shown as SEQ ID NO.1, and the amino acid sequence of the heavy chain V region is shown as SEQ ID NO. 2.

2. The Fab fragment of human anti-human CD23 antibody according to claim 1, wherein the light chain V region gene has the nucleotide sequence shown in SEQ ID No. 3.

3. The Fab fragment of human anti-human CD23 antibody according to claim 2, wherein the nucleotide sequence of the gene of the heavy chain V region is shown in SEQ ID No. 4.

4. An expression vector comprising a nucleotide sequence encoding a light chain V region and/or a nucleotide sequence encoding a heavy chain V region.

5. A host cell comprising the expression vector of claim 4.

6. The host cell of claim 5, wherein the host cell is E.coli or a eukaryotic cell.

7. A pharmaceutical composition comprising a Fab fragment of the human anti-human CD23 antibody of any one of claims 1 to 3.

8. Use of a Fab fragment of the human anti-human CD23 antibody according to any one of claims 1 to 3 for the preparation and use as a medicament for the treatment of any one of the following diseases:

the neoplastic disease is selected from recurrent Hodgkin's disease; drug resistant hodgkin's disease; high grade, low grade and intermediate grade non-hodgkin's lymphoma; b cell chronic lymphocytic leukemia; plasma cell-like lymphocytic lymphoma; mantle cell lymphoma; follicular lymphoma; diffuse large cell lymphoma; burkitt's lymphoma; AIDS-related lymphoma; monocytic B-cell lymphoma; angioimmunoblastic lymphadenopathy.

9. A medicament for treating a lymphoid-related disease comprising a Fab fragment of a human anti-human CD23 antibody according to any one of claims 1-3.

10. The medicament of claim 9, wherein the medicament for treating the lymph related disease further comprises pharmaceutically acceptable additives or adjuvants, and preferably the dosage form of the pharmaceutical composition is selected from the group consisting of tablets, pills, powders, suspensions, gels, emulsions, creams, granules, nanoparticles, capsules, suppositories, injections, sprays and injections.

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