CN114106180A

CN114106180A - Monoclonal antibody and application thereof

Info

Publication number: CN114106180A
Application number: CN202111514203.9A
Authority: CN
Inventors: 张娟; 黄林华; 谢家骏; 泮明珠; 南丽迪
Original assignee: China Pharmaceutical University
Current assignee: China Pharmaceutical University
Priority date: 2021-12-13
Filing date: 2021-12-13
Publication date: 2022-03-01
Anticipated expiration: 2041-12-13
Also published as: CN114106180B

Abstract

The invention belongs to the technical field of biology, and particularly relates to a CD24 monoclonal antibody and application thereof. The invention discloses a monoclonal antibody with the bioactivity of neutralizing human leukocyte differentiation antigen CD24 and a sequence of the monoclonal antibody. The CD24 monoclonal antibody can be specifically combined with CD24, and can be used for treating diseases related to CD24 over-expression, abnormal expression and uncontrolled expression, or used for biological detection such as ELISA, Western blot, flow cytometry and the like. The invention also provides a CD24 monoclonal antibody with engineered cysteine, which can be used for preparing a uniform antibody conjugate.

Description

Monoclonal antibody and application thereof

Technical Field

The invention belongs to the technical field of biology, and particularly relates to a CD24 monoclonal antibody and application thereof.

Background

The human leukocyte differentiation antigen CD24 (clustericofdiferentiation 24) is a tumor marker that has been under intense study in recent years. Current research shows that the CD24 molecule is a highly glycosylated sialoglycoprotein with an apparent molecular weight between 30 and 70 kDa. CD24 contains 33 amino acid residues of the core protein skeleton structure, has 16 potential O-or N-glycosylation sites, and is highly homologous with mouse thermostable antigen; the mature polypeptide can be anchored to the surface of cell membrane by carboxyl-terminal phosphatidylinositol (GPI).

Under physiological conditions, the CD24 molecule is expressed at low levels only on immature B cells, mature granulocytes, and a few epithelial and neural cells; when the organism is in a pathological state, the surface of most malignant tumor cells can detect CD24 molecules with high expression level, and the expression level is closely related to the occurrence and development of tumors.

P-selectin is expressed on the surface of activated platelets and endothelial cells, and CD24 is one of its ligands. The high expression of CD24 molecule can enhance the adhesion of tumor cell to blood platelet and endothelial cell, and promote the recurrence and metastasis of tumor. Of course, as a cell membrane surface signaling molecule, CD24 is capable of mediating the proliferation, adhesion, metastasis, and invasion of tumor cells through a variety of mechanisms of action. The research further finds that the CD24 molecule expressed by the tumor cell can be combined with the inhibitory receptor siglec10 on the surface of the macrophage, and the phagocytosis of the macrophage on the tumor is inhibited. Therefore, the CD24 is expected to be a new target for researching tumorigenesis mechanisms and developing related diagnostic reagents.

The Antibody Drug Conjugate (ADC) delivers the drug to a tumor region by means of the targeting property of the antibody, so that the off-target effect of the drug can be obviously reduced, and the treatment window is improved. Currently marketed ADC drugs are free cysteines via random surface lysines or reduced four interchain disulfides using conventional non-specific coupling methods. This results in a highly heterogeneous mixture of ADCs that not only presents manufacturing repeatability challenges, but also significantly reduces the therapeutic index. Junutula et al (Nat Biotechnol 2008) obtain a uniform antibody drug conjugate by introducing engineered cysteine in an antibody constant region and specifically coupling drug molecules to a mutation site, and also provide a new idea for the development of subsequent homogeneous ADCs.

Disclosure of Invention

Based on the background, the invention provides a CD24 monoclonal antibody, wherein the heavy chain and light chain variable region amino acid sequences and CDR regions thereof are shown as SEQ ID No. 1-8. The constant region is human IgG1 type, so that the immunogenicity is reduced and the expression is easier.

The invention also provides a CD24 chimeric antibody sequence with engineered cysteine, which can be used for preparing a uniform antibody conjugate, wherein the engineered sites are that the amino acid of the heavy chain is cysteine according to EU numbering 239, and the light chain is cysteine according to EU numbering 211. The specific amino acid sequence is shown in SEQ ID No. 9-10.

The monoclonal antibody of the invention can be specifically combined with CD24, and can be used for preparing medicaments for treating diseases related to CD24 abnormal expression or excessive expression and uncontrolled expression; or used for preparing products for detecting CD24 expression by Western blot, ELISA and flow cytometry.

Advantageous effects

1. The invention provides a brand-new CD24 chimeric antibody, which is not reported in documents, and the monoclonal antibody can be specifically combined with CD24, and can be used for preparing a medicament for treating diseases related to CD24 abnormal expression or excessive expression and uncontrolled; or used for preparing products for detecting CD24 expression by Western blot, ELISA and flow cytometry.

2. The constant region of the monoclonal antibody of the invention is human IgG1 type, compared with the murine monoclonal antibody, the immunogenicity to the human body is reduced, and the monoclonal antibody is favorable for large-scale expression in eukaryotic cells. In addition, the invention utilizes the thought of cysteine coupling, introduces the engineered cysteine residue on the CD24 antibody molecule by an overlap-PCR method, and the cysteine residue can generate Michael addition reaction with the drug with maleimide group, and can be used for preparing the uniform CD24 antibody-drug site-specific conjugate.

Drawings

FIG. 1 shows the results of SDS-PAGE and Western blot for the CD24 monoclonal antibody. A: non-reduction electrophoresis of CD24 monoclonal antibody; b: reduction electrophoresis; c: western blot results, Lane1 non-reduction electrophoresis and Lane2 reduction electrophoresis; m: marker, the same as below.

FIG. 2 is an agarose electrophoresis of CD24 monoclonal antibody heavy chain with engineered cysteine introduced, where A is the heavy chain mutation site front and back PCR product; b is an overlap-PCR product.

FIG. 3 is an agarose electrophoresis of the light chain of the CD24 monoclonal antibody with engineered cysteines introduced, where A is the light chain mutation site forepart PCR product; b is a rear-segment PCR product of the light chain mutation site; c is an overlap-PCR product.

FIG. 4 shows ELISA detection of the binding of CD24 monoclonal antibody to recombinant human CD24 antigen.

FIG. 5 shows the detection of the binding of the CD24 monoclonal antibody to the tumor CD24 antigen by Western Blot. A: lane1 is BEL-7402 cell lysate; b: lane1 is MDA-MB-468 cell lysate; c: lane1 is HCT-116 cell lysate.

FIG. 6 is a flow cytometry analysis of the binding of the CD24 monoclonal antibody to tumor cells, wherein A is BEL-7402, B is HT-29, C is HepG2, and D is MDA-MB-468.

Detailed Description

EXAMPLE 1 preparation of CD24 monoclonal antibody

1. Preparation of hybridoma cells

1) After dissolving the recombinant protein GST-hCD24 with PBS to a final concentration of 1mg/ml, the mixture was mixed with a rapid adjuvant in equal volume and six weeks old BALB/c mice. The immunization protocol was performed according to the QuickAntibody instructions.

2) ICR mice (SPF grade, purchased from the university of promiscuous, promiscuous) peritoneal macrophages were taken one day prior to fusion and resuspended in HAT medium (purchased from GIBCO, containing xanthine, aminopterin, and thymidine). Inoculating to 96-well plate, culturing at 37 deg.C in 5% CO2 incubator for 24 h.

Mouse myeloma cell line SP20-Ag14 was cultured in a flask of ordinary DMEM complete medium (containing 15% FBS and purchased from GIBCO) to allow the cells to grow well. 250ml of each fusion preparation was grown at a density of 2 xl 0⁶cells/m1 are healthy cells in the middle of logarithmic growth.

Immunized mice were sacrificed and spleens were removed after infiltration with alcohol. Spleen was placed on a cell screen and cut into pieces with sterile surgical scissors. After spleen grinding by squeezing with the plunger, cells were passed through a mesh into a 50ml centrifuge tube by rinsing with 5ml DMEM incomplete medium. Spleen cell suspension was collected for fusion.

3) Fusion

A. 20ml of serum-free DMEM medium and 1ml of PEG1450 (from Sigma) were prepared and incubated at 37 ℃.

B. Myeloma cells and spleen cells were collected, centrifuged at 1500rpm at room temperature for 5min, washed twice with serum-free medium, and then suspended in 10ml of serum-free medium. Will be 5X 10⁸Spleen cells and 1X 10⁸Myeloma cells were mixed well, centrifuged at 1500rpm for 5min, and the supernatant was discarded. Gently tap the bottom of the tube to loosen the cells.

C. Taking out the culture medium and PEG preheated at 37 deg.C, placing the cells in 37 deg.C water bath, slowly dripping 1ml of PEG within 1min, and stopping with serum-free culture medium in time. The supernatant was discarded by centrifugation and the cells were resuspended in 50ml HAT medium and gently mixed.

D. Cells were added to 5 96-well plates, 100. mu.l per well, which had been plated with feeder cells. The mixture was cultured at 37 ℃ in a 5% CO2 incubator for 4 days. According to the growth state of cells, half-changing the HAT culture solution for 1-3 times before screening to avoid the culture medium in the holes from yellowing.

E. After 7 days after the fusion, the whole medium was changed to the medium containing HT. After 24h, cell culture supernatant was aspirated and positive clones were screened by indirect ELISA.

2. Cloning of antibody heavy and light chain variable region gene

1) Collection 10⁷A number of hybridoma cells in log phase,extracting total RNA by using an RNA extraction kit, dissolving in 20-50 mu l of RNase-free water, and storing at-70 ℃. The first strand cDNA is synthesized through reverse transcription with total RNA as template and the reaction is performed according to the product instruction.

2) The polymerase is PrimerSTAR high fidelity polymerase. Based on the antibody variable region FR1 and CH1 genes, an upstream and a downstream degenerate primer are designed and synthesized, wherein the sequences of a light chain VLF (upstream primer) and a VLB (downstream primer) and heavy chain VHF1, VHF2 and VHB primers are as follows:

VLF：gg gag ctc gay att gtg mts acm car wct mca；

VLB：ggt gca tgc gga tac agt tgg tgc agc atc；

VHF1:ctt ccg gaa ttc sar gtn mag ctg sag tc

VHF2:ctt ccg gaa ttc sar gtn mag ctg sag tcw gg

VHB:gga aga tct ata gac aga tgg ggg tgt cgt ttt ggc

(degenerate codon specification: r ═ a, g; y ═ c, t; m ═ a, c; s ═ c, g; w ═ a, t)

The reaction volume was 50. mu.l, and the reaction conditions were: 5min at 94 ℃; circulating for 30 times at 94 deg.C for 30s, 58 deg.C for 1min, and 72 deg.C for 1 min; 10min at 72 ℃.

Construction of full-Length antibody to CD24

1) The antibody light chain variable region and the human Ig kappa chain constant region are spliced through overlap-PCR, and the antibody heavy chain variable region and the human IgG1 constant region are spliced to obtain the CD24 monoclonal antibody cG 7.

2) The recombinant plasmid of light and heavy chains of the cG7 encoding the CD24 monoclonal antibody is transfected into HEK293 cells according to the proportion of 2:1, after 5 days of culture, supernatant is collected by centrifugation at 4 ℃, the CD24 monoclonal antibody is obtained after purification through a protein A column, the molecular weight and purity of the CD24 monoclonal antibody are detected through SDS-PAGE, and the type of the CD24 antibody is detected by using a goat anti-human IgG antibody (Wuhan Sanying). The results are shown in FIG. 1. A: non-reduction electrophoresis of CD24 monoclonal antibody; b: reduction electrophoresis; c: western blot detection of antibody types, Lane1 non-reducing electrophoresis and Lane2 reducing electrophoresis; m: marker, the same as below.

Example 2 preparation of CD24 monoclonal antibody and engineering of cysteine:

serine (TCA) at position 239 (EU numbering) of heavy chain of parent CD24 monoclonal antibody cG7 was mutated to cysteine (TGC) by overlap-PCR, and the results are shown in fig. 1. A: lane1 is the heavy chain mutation site front segment PCR product (805bp), Lane2 is the heavy chain mutation site rear segment PCR product (656 bp); lane1 is the heavy chain Overlap-PCR product (1421 bp).

Valine (GTC) at position 211 (EU numbering) of the light chain was mutated to cysteine (TGC). The CD24 monoclonal antibody cG7SV with engineered cysteine was obtained. The electrophoresis results of the PCR products upstream and downstream of the mutation site and the overlap-PCR product are shown in FIG. 2. A: lane1 is the light chain mutation site forepart PCR product (728 bp); b: lane1 is the light chain mutation site post-segment PCR product (65 bp); lane1 is the light chain Overlap-PCR product (763 bp).

Example 3ELISA to detect binding of CD24 monoclonal antibody to recombinant human CD24 protein:

the binding ability of the antibody to the antigen can be preliminarily judged by ELISA. Here we immobilized recombinant human CD24 protein on an enzyme strip and added a series of concentration gradients of CD24 antibody for detection.

1. Firstly, activating an enzyme strip by ultraviolet irradiation for 30 minutes;

2. adding 100 μ L of recombinant human CD24 protein with concentration of 0.5ug/mL into each well, using BSA as negative control, and coating overnight at 4 deg.C;

3. adding 200 μ L PBST, washing for 3 times, each for 5min, adding 200 μ L5% skimmed milk, and sealing at 37 deg.C for 2 hr;

4. discarding skimmed milk, adding 200 μ L PBS, washing for 3 times, each time for 5min, adding 0.08-2.5 nM CD24 monoclonal antibody as primary antibody, setting isotype control, and incubating at 37 deg.C for 2 h;

5. removing primary antibody, washing with 200 μ L PBST for 5 times (5 min each time), adding 100 μ L goat anti-human IgG (Wuhan Sanying, 1:20000 dilution), and incubating at 37 deg.C for 1 h;

6. absorbing the secondary antibody, washing for 5 times by PBST, adding 100 mu L of TMB color development solution, and incubating for 15-20 min at room temperature in a dark place;

7. the reaction was stopped by adding 50. mu.L of 2M dilute sulfuric acid, and the values of OD450 and OD630 were measured by a microplate reader.

The experiment was repeated 3 times, and the results are shown in fig. 4, with an EC50 value of 0.1524nM for cG7 and an EC50 value of 0.1530nM for cG7 SV. The CD24 monoclonal antibody of the invention has the ability of specifically binding to CD24 protein and can be applied to ELISA experiments.

Example 4Western Blot to detect binding of CD24 monoclonal antibody to tumor CD24 antigen:

1. after being separated by 10 percent SDS-PAGE, the tumor cell lysate is transferred to a PVDF membrane and is incubated for 2 hours at 37 ℃ by 5 percent skimmed milk powder for sealing;

2. rinsing with TBST for 5min, adding CD24 monoclonal antibody (diluted with 5% skimmed milk powder) with final concentration of 1 μ g/mL as primary antibody, and incubating at 37 deg.C for 2 h;

3. discarding the primary antibody, rinsing with TBST for 3 times, each time for 10min, adding goat anti-human IgG antibody (biologies, diluted 1: 50000) labeled with HRP as secondary antibody, and incubating at 37 deg.C for 1 h;

4. ECL chemiluminescence color development liquid (Holliving being next, A, B liquid is mixed according to the ratio of 1: 1) is uniformly dripped on the surface of the PVDF film and is placed in an exposure instrument for exposure. The results are shown in FIG. 5, A: lane1 is BEL-7402 cell lysate, M is marker (the same below); b: lane1 is MDA-MB-468 cell lysate; c: lane1 is HCT-116 cell lysate. The CD24 positive cell (BEL7402, MDA-MB-468) lysate shows a band at the corresponding position, but the negative cell strain (HCT-116) does not show a band, which indicates that the CD24 monoclonal antibody has the capability of specifically binding to tumor CD24 protein and can be applied to Western blot detection.

Example 5 flow cytometry detection of binding of the CD24 monoclonal antibody to tumor cells:

1. human tumor cell strains HT-29, BEL-7402 and HepG2 of high expression CD24 are cultured in a 1640 culture medium containing 10% FBS at 37 ℃ under the condition of 5% CO 2; the MDA-MB-468 cell line was cultured in L-15 medium containing 10% FBS at 37 ℃ under 100% air condition.

2. Digesting well-grown tumor cells with pancreatin, adding fresh culture medium, resuspending, counting, and taking 2 x 10 per tube⁵The cells were centrifuged at 1500rpm for 3min at 4 ℃ and the supernatant was discarded. Wash twice with 500ul of pre-cooled PBS containing 2% FBS.

3. Adding 250 μ L of 100nM CD24 monoclonal antibody as primary antibody, resuspending cells, adding 100nM irrelevant antibody as isotype control, and incubating at 4 deg.C for 1h to allow binding of antibody and cells;

centrifuging at 1500rpm for 3min at 4.4 deg.C, and discarding the supernatant. Wash twice with 500ul of pre-cooled PBS containing 2% FBS.

5. Adding 250 μ L FITC labeled goat anti-human IgG antibody (Wuhan Sanying, 1:100 dilution) as secondary antibody, resuspending cells, and incubating at 4 deg.C for 1 h;

centrifuging at 1500rpm for 3min at 6.4 deg.C, and discarding the supernatant. Wash twice with 500ul of pre-cooled PBS containing 2% FBS. Add 300. mu.L PBS for resuspension and use flow cytometry for detection. As shown in FIG. 6, the CD24 monoclonal antibody of the present invention can effectively bind to tumor cells with high expression of CD24, and has good tumor cell targeting ability.

Sequence listing

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Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu

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Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr

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Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val

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Asp Lys Arg Val Glu Pro Lys Ser Cys Gln Gly Ala Ile Gly Leu Pro

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Gly Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Cys Val

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Claims

1. A CD24 monoclonal antibody is characterized in that the heavy chain and light chain variable regions thereof respectively comprise amino acid sequences of CDR 1-3 regions shown in SEQ ID Nos. 2-4 and SEQ ID Nos. 6-8.

2. The monoclonal antibody to CD24, wherein the structure of the monoclonal antibody comprises the amino acid sequences of the heavy and light chain variable regions shown in SEQ ID No.1 and SEQ ID No. 5.

3. The monoclonal antibody to CD24, wherein the monoclonal antibody has a structure comprising amino acid sequences of the heavy and light chain constant regions shown in SEQ ID No.9 and SEQ ID No. 10; or an amino acid sequence having at least 80% homology to said sequence, and the heavy chain has cysteine at amino acid position 239 in accordance with EU numbering and the light chain has cysteine at amino acid position 211 in accordance with EU numbering.

4. An engineered antibody comprising heavy and light chain amino acid sequences identical to the amino acid sequences of claim 3; the genetic engineering antibody comprises: human-murine chimeric antibodies; or a humanized antibody; or a functional fragment of an antibody, Fab; or is a single chain antibody; or an antibody functional fragment VH-L fused with a heavy chain variable region and a complete light chain; or a functional fragment of an antibody comprising an arrangement, tandem or combination of one or more CDRs of the heavy and light chains; or the antibody-like functional fusion protein obtained by connecting, splicing and fusing the antibody, the antibody functional fragment and other various proteins or polypeptides.

5. A nucleotide sequence encoding the monoclonal antibody of any one of claims 1 to 3 or the engineered antibody of 4.

6. An expression vector comprising a nucleic acid sequence encoding the nucleic acid sequence of claim 5.

7. A host cell comprising the expression vector of claim 6.

8. An antibody conjugate comprising the monoclonal antibody according to claims 1 to 3 or the genetically engineered antibody according to claim 4 as a targeting moiety conjugated to a radionuclide or a chemical drug or a toxin or an oligonucleotide or a nitric oxide donor.

9. Use of the monoclonal antibody of claims 1-3 or the engineered antibody of claim 4 or the antibody conjugate of claim 8 in the preparation of an anti-tumor medicament.