CN116836288B

CN116836288B - Anti-human CD55 engineering antibody and application thereof

Info

Publication number: CN116836288B
Application number: CN202311091973.6A
Authority: CN
Inventors: 林家旺; 李子旸; 李原; 周珊; 赵雨萌; 陶欣勇; 郝军凤; 刘红芹; 屈浩; 廖晓龙
Original assignee: Suzhou Weidu Biotechnology Co ltd Tianjin Branch
Current assignee: Suzhou Vdo Biotech Co ltd; Suzhou Weidu Biotechnology Co ltd Tianjin Branch
Priority date: 2023-08-29
Filing date: 2023-08-29
Publication date: 2023-11-03
Anticipated expiration: 2043-08-29
Also published as: CN116836288A

Abstract

The invention provides an anti-human CD55 engineering antibody and application thereof, wherein the amino acid sequence of CDR1 of a heavy chain variable region is shown as SEQ ID NO. 1; the amino acid sequence of CDR2 of the heavy chain variable region is shown as SEQ ID NO. 2; the amino acid sequence of CDR3 of the heavy chain variable region is shown in SEQ ID NO. 3. The light chain variable region gene and the heavy chain variable region gene of the anti-human CD55 monoclonal antibody and the expression products thereof are recombined to express the generated anti-CD 55 antibody, and can be specifically combined with human CD55 molecules.

Description

Anti-human CD55 engineering antibody and application thereof

Technical Field

The invention belongs to the field of biological medicine, and particularly relates to an anti-human CD55 engineering antibody and application thereof.

Background

Complement is part of nonspecific humoral immunity and plays a variety of roles in the body's clearance of pathogens and anti-tumor immune mechanisms. CD55 is a known complement regulatory protein, and has been studied mainly for its role in inflammation, protein-lost enteropathy, malaria and other diseases, and with the rise of tumor immunotherapy, the study of CD55 protein in cancer has been attracting attention.

Advances in immunotherapy have significantly improved survival prognosis in advanced tumor patients, and metastatic colon cancer patients with microsatellite highly unstable (MSI-H) and mismatch repair dysfunction (UIMR) exhibit high remission rates for treatment with inhibitors of the programmed death receptor (PD-1) and its ligand, programmed death receptor ligand-1 (PD-U), whereas more than about 85% of colorectal cancers are microsatellite highly stable (MSS) type, which results in limited beneficiaries using PD-1/PD-L1 inhibitors. Thus, there is a need to find new immune checkpoints and to explore new directions for tumor immunotherapy.

CD55 protein is not expressed on the surface of normal colon mucosa epithelial cell membrane and in cytoplasm, but is highly expressed on the surface of colon cancer tissue cell membrane and in cytoplasm. The increased expression of CD55 protein in colon cancer is related to poor prognosis of colon cancer patients, and is an independent risk factor affecting the total postoperative survival time of III-IV colon cancer patients. Targeting CD55 protein may be a new therapeutic approach for colon cancer patients.

Disclosure of Invention

In view of the above, the present invention aims to overcome the defects in the prior art and provides an anti-human CD55 engineering antibody and application thereof.

In order to achieve the above purpose, the technical scheme of the invention is realized as follows:

the invention provides an anti-human CD55 engineering antibody, which comprises a heavy chain variable region and a light chain variable region;

the complementarity determining regions of the heavy chain variable region include CDR1, CDR2 and CDR3; the amino acid sequence of CDR1 of the heavy chain variable region is shown as SEQ ID NO. 1; the amino acid sequence of CDR2 of the heavy chain variable region is shown as SEQ ID NO. 2; the amino acid sequence of CDR3 of the heavy chain variable region is shown as SEQ ID NO. 3;

the complementarity determining regions of the light chain variable region include CDR1, CDR2 and CDR3; the amino acid sequence of CDR1 of the light chain variable region is shown as SEQ ID NO. 4; the amino acid sequence of CDR2 of the light chain variable region is shown as SEQ ID NO. 5; the amino acid sequence of CDR3 of the light chain variable region is shown as SEQ ID NO. 6.

Further, the framework regions of the heavy chain variable region include FR1, FR2, FR3 and FR4; the amino acid sequence of FR1 of the heavy chain variable region is shown as SEQ ID NO. 7; the amino acid sequence of FR2 of the heavy chain variable region is shown as SEQ ID NO. 8; the amino acid sequence of FR3 of the heavy chain variable region is shown as SEQ ID NO. 9; the amino acid sequence of FR4 of the heavy chain variable region is shown as SEQ ID NO. 10;

the framework regions of the light chain variable region comprise FR1, FR2, FR3 and FR4; the amino acid sequence of FR1 of the light chain variable region is shown as SEQ ID NO. 11; the amino acid sequence of FR2 of the light chain variable region is shown as SEQ ID NO. 12; the amino acid sequence of FR3 of the light chain variable region is shown as SEQ ID NO. 13; the amino acid sequence of FR4 of the light chain variable region is shown as SEQ ID NO. 14.

Further, the amino acid sequence of the heavy chain variable region of the engineering antibody is shown as SEQ ID NO.15, and the amino acid sequence of the light chain variable region is shown as SEQ ID NO. 16.

The invention also provides a nucleic acid molecule which codes for the anti-human CD55 engineering antibody.

Further, the nucleotide sequence of the nucleic acid molecule encoding the heavy chain variable region of the anti-human CD55 engineering antibody is shown as SEQ ID NO. 17, and the nucleotide sequence encoding the light chain variable region is shown as SEQ ID NO. 18.

The invention also provides an expression vector comprising the nucleic acid molecule.

The invention also provides application of the anti-human CD55 engineering antibody in preparing a medicament for treating intraepithelial neoplasia.

The invention also provides application of the anti-human CD55 engineering antibody in preparation of a reagent for diagnosing intraepithelial neoplasia.

The invention also provides application of the anti-human CD55 engineering antibody in preparing a medicament for treating colon cancer; the application of the anti-human CD55 engineering antibody in preparing a reagent for diagnosing colon cancer.

The invention also provides an immunoassay kit which contains the anti-human CD55 engineering antibody and/or the expression vector.

Compared with the prior art, the invention has the following advantages:

the anti-human CD55 engineering antibody can be used for diagnosing and treating colon cancer.

The light chain variable region gene and the heavy chain variable region gene of the anti-human CD55 monoclonal antibody and the expression products thereof are recombined to express the generated anti-CD 55 antibody, and can be specifically combined with human CD55 molecules.

Drawings

FIG. 1 is a purified electrophoresis diagram of an anti-human CD55 engineering antibody according to the embodiment 3 of the present invention: wherein the left side is reduction electrophoresis, and the right side is non-reduction electrophoresis;

FIG. 2 is a graph showing the activity detection of the anti-human CD55 engineering antibody according to the invention in example 4: wherein, graph A is the result of human peripheral blood and graph B is the result of red blood cells;

FIG. 3 is a graph showing the activity detection of an anti-human CD55-PE engineered antibody according to example 6 of the present invention: wherein panel A is the human peripheral blood results and panel B is the red blood cell results.

Detailed Description

Unless defined otherwise, technical terms used in the following examples have the same meaning as commonly understood by one of ordinary skill in the art to which the present invention pertains. The test reagents used in the following examples, unless otherwise specified, are all conventional biochemical reagents; the experimental methods are conventional methods unless otherwise specified.

The present invention will be described in detail with reference to examples.

EXAMPLE 1 cloning of the light and heavy chain variable region Gene of mouse anti-human CD55 antibody

1. RNA extraction: using the Trizol one-step method, 1) hybridoma cells were taken at about 1X 10 ⁶ Adding 1ml of Trizol, blowing and mixing uniformly, and standing at room temperature for 10 minutes; 2) Adding 0.2ml of chloroform, shaking vigorously for 15 seconds, and standing at room temperature for 2-3 minutes; 3) Centrifugation at 12000rpm,4℃for 15 min; 4) Taking the supernatant, adding 0.5ml of isopropanol, and standing for 15 minutes at room temperature; 5) Centrifugation at 12000rpm,4℃for 15 min; 6) The supernatant was discarded, 1ml of 75% ethanol was added for washing, and the mixture was centrifuged at 7500rpm at 4℃for 5 minutes; 7) Discarding supernatant, air drying the precipitate, and adding 30 μLDEPC water for dissolution;

2. reverse transcription to cDNA (40 μl): reverse transcription reaction is carried out by using a TransScript All-in-one First-Strand cDNA Synthesis SuperMix for qPCR kit, 1.0 mug of total RNA is taken, 4 mug of TransScript All-in-one SuperMix for qPCR mug, 4 mug of gDNA remover, and DEPC water are supplemented to 20 mug, and after reaction is carried out at 42 ℃ for 15min, incubation is carried out at 85 ℃ for 5min, and the obtained product is preserved at 20 ℃;

3. PCR amplification of the light and heavy chain variable region genes of CD55 antibodies

Light chain variable region gene PCR amplification reaction System (50. Mu.l): designing universal degenerate primers: the upstream primer 5'-GACATT GTG CTC ACC CAG WCT SMH-3' (SEQ ID NO: 19), the downstream primer 5'-CCG TTAGAT CTC CAR BTT KGT SCS-3' (SEQ ID NO: 20); using cDNA as a template, and amplifying the pfu DNA polymerase with high fidelity; the PCR cycle was carried out at 94℃for 5 minutes; 94℃for 30 seconds, 55℃for 30 seconds, 72℃for 30 seconds, 30 cycles total; finally, the mixture is extended for 10 minutes at 72 ℃;

heavy chain variable region gene PCR amplification reaction System (50. Mu.l): an upstream primer 5'-CAG GTS MARCTG CAGSAG TCW GG-3' (SEQ ID NO: 21); the downstream primer 5'-TGA GGA GAC KGT GAC HGT GGT SCC-3' (SEQ ID NO: 22); using cDNA as a template, and amplifying the pfu DNA polymerase with high fidelity; the PCR cycle was carried out at 94℃for 5 minutes; 94 ℃,30 seconds, 55 ℃,30 seconds, 72 ℃,30 seconds, 35 cycles total; finally, the mixture is extended for 10 minutes at 72 ℃;

4. construction of a sequencing vector: pClone007 Blunt Simple Vector Kit is purchased from a family of organisms; the PCR product of the light and heavy chain variable region gene is recovered, is connected with a pClone007 Blunt Simple Vector carrier, is transformed by calcium chloride according to a conventional method, is screened for positive clones at an ampicillin concentration of 100 mu g/ml, is sent to a sequencing system, and completely accords with the characteristics of a plurality of conserved framework amino acids of an antibody in a protein database, and the sequence is an antibody gene sequence. Named pClone007-VH and pClone007-VL, respectively.

Example 2 construction of engineering antibody expression vectors pcDNA3.4-H and pcDNA3.4-L

Primers for amplifying heavy chain variable region (PDH-F and PDH-R), primers for amplifying light chain variable region (PDL-F and PDL-R), primers for amplifying heavy chain vector (ZTH-F and ZTH-R) and primers for amplifying light chain vector (ZTL-F and ZTL-R) were designed and synthesized based on the sequences of light and heavy chain variable region and the sequences of construction vector pcDNA3.4.

PDH-F：

5’-TTCCAGGTTCCACTGGTGACCAGGTCCAACTTCAGCAGTCTGG-3’ （SEQ ID NO:23）

PDH-R：

5’-GATGGGGGTGTCGTTTTGGCTGCAGAGACAGTGATCAGAGTCCC-3’ （SEQ ID NO:24）

PDL-F：

5’-TTCCAGGTTCCACTGGTGACGACATTGTGCTGACACAGTCTCCT-3’ （SEQ ID NO:25）

PDL-R：

5’-ACAGTTGGTGCAGCATCAGCCCGTTTGATTTCCAGCTTGG-3’（SEQ ID NO:26）

ZTH-F：

5’-GCCAAAACGACACCCCCA-3’ （SEQ ID NO:27）

ZTH-R：

5’-GTCACCAGTGGAACCTGGAACC-3’（SEQ ID NO:28）

ZTL-F：

5’-GCTGATGCTGCACCAACTGTAT-3’（SEQ ID NO:29）

ZTL-R：

5’-GTCACCAGTGGAACCTGGAACC-3’（SEQ ID NO:30）

Light chain variable region gene PCR amplification reaction System (50. Mu.l): an upstream primer PDL-F; a downstream primer PDL-R; using pClone007-VL as a template, and amplifying the pfu DNA polymerase with high fidelity; the PCR cycle was carried out at 94℃for 5 minutes; 94℃for 30 seconds, 58℃for 60 seconds, 72℃for 30 seconds, 30 cycles total; finally, the extension is carried out at 72 ℃ for 10 minutes.

Heavy chain variable region gene PCR amplification reaction System (50. Mu.l): an upstream primer PDH-F; a downstream primer PDH-R; using pClone007-VH as a template, and amplifying the pfu DNA polymerase with high fidelity; the PCR cycle was carried out at 94℃for 5 minutes; 94℃for 30 seconds, 58℃for 60 seconds, 72℃for 30 seconds, 30 cycles total; finally, the extension is carried out at 72 ℃ for 10 minutes.

Amplification of light chain linearization vector: an upstream primer ZTL-F; a downstream primer ZTL-R; using pcDNA-CD15-L (synthetic sequence, containing mouse IgG1 constant region nucleotide sequence) as template, amplifying with high-fidelity pfu DNA polymerase; the PCR cycle was carried out at 94℃for 5 minutes; 94℃for 30 seconds, 58℃for 60 seconds, 72℃for 4 minutes, 30 cycles in total; finally, the mixture is extended for 10 minutes at 72 ℃;

and respectively recovering PCR products, respectively recombining the recovered light chain variable region PCR products and heavy chain variable region PCR products with the recovered linearization carrier products by using recombinase, converting DH5 alpha clone strains after recombination, picking single bacterial colony for amplification culture, carrying out sample feeding sequencing, and sequencing correctly for transfecting CHO cells.

Amplification of heavy chain linearization vector: an upstream primer ZTH-F; a downstream primer ZTH-R; using pcDNA-CD15-H (synthetic sequence, containing mouse IgG1 constant region nucleotide sequence) as template, amplifying with high-fidelity pfu DNA polymerase; the PCR cycle was carried out at 94℃for 5 minutes; 94℃for 30 seconds, 58℃for 60 seconds, 72℃for 4 minutes, 30 cycles in total; finally, the mixture is extended for 10 minutes at 72 ℃;

EXAMPLE 3 expression and purification of an anti-human CD55 engineering antibody

1. Expression of engineered antibody CD55 antibody: taking out the prepared CHO cells from an incubator, preparing two 15ml sterile centrifuge tubes, adding 5ml SPM culture medium and 100 mug sterile plasmid DNA (containing light chain expression vector and heavy chain expression vector) into one of the two sterile centrifuge tubes, and gently beating and uniformly mixing; taking the other separation tube, adding 5ml of SPM and 320 μl of transfection reagent, lightly blowing and mixing; transferring all liquid in the centrifuge tube containing the transfection reagent into the centrifuge tube containing the plasmid, and lightly blowing and uniformly mixing; standing for 5 minutes at room temperature to prepare a plasmid-carrier compound; taking out cells from the constant temperature shaking table, adding the prepared plasmid-carrier complex while shaking, and returning to CO ₂ Shake culturing in a constant temperature shaking table; supplementing auxiliary materials on the 1 st and 5 th days, and harvesting cell supernatants on the 12 th day;

2. purification of anti-human CD55 engineered antibodies: purifying the cell supernatant collected in the previous step by using a Protein A affinity column, loading a sample after PBS balances the column, eluting by using glycine with the pH value of 3.0, replacing the solution by using a G25 column for eluting pure antibody, replacing the solution by PBS, sampling for electrophoresis test, and freezing at-20 ℃ after subpackaging, wherein the result is shown in the figure 1, and the result of reduction and non-reduction of the CD55 engineering antibody pure product (the left side is reduction electrophoresis, the right side is non-reduction electrophoresis).

Example 4 Activity assay of anti-human CD55 engineering antibodies

1. Detecting peripheral blood of normal people: adding 100 μl of normal anticoagulated peripheral blood into each tube, adding different amounts of engineering antibody CD55, and incubating at room temperature in dark place for 30 min; adding 2ml of hemolysin, reacting at room temperature in a dark place for 10 minutes, centrifuging at 1000 rpm for 5 minutes, and discarding the supernatant; adding 2ml of cold PBS buffer, re-suspending, centrifuging at 1000 rpm for 5 minutes, and discarding the supernatant; then 0.5 mug of APC marked mouse secondary antibody is added, and incubated for 30 minutes at room temperature and in dark place; adding 2ml of cold PBS buffer, re-suspending, centrifuging at 1000 rpm for 5 minutes, and discarding the supernatant; adding a matched antibody CD45-PerCP, reacting for 20 minutes, adding 2ml of cold PBS buffer solution, resuspending, centrifuging for 5 minutes at 1000 revolutions per minute, and discarding the supernatant; 250 μl PBS buffer was added and the flow cytometer detected, the results are shown in FIG. 2.

2. Erythrocyte detection: peripheral blood 1: after 100 dilutions, 100 μl of diluted peripheral blood was added to each tube, and different amounts of engineering antibody CD55 were added, and incubated at room temperature for 30 minutes in the absence of light; centrifuging at 1000 rpm for 5min, and discarding supernatant; adding 2ml of cold PBS buffer, re-suspending, centrifuging at 1000 rpm for 5 minutes, and discarding the supernatant; then 0.5 mug of APC marked mouse secondary antibody is added, and incubated for 30 minutes at room temperature and in dark place; adding 2ml of cold PBS buffer, re-suspending, centrifuging at 1000 rpm for 5 minutes, and discarding the supernatant; after adding 250 μl of PBS buffer solution and detecting by flow cytometry, as shown in FIG. 2, after binding CD55 to lymphocytes and erythrocytes, adding a fluorescent secondary antibody, a fluorescent signal can be detected, which indicates that the CD55 engineering antibody can bind to CD55 on the surfaces of lymphocytes and erythrocytes, the activity is normal, and the average fluorescence intensity is reduced with the decrease of the addition of the antibody.

EXAMPLE 5 PE labelling of anti-human CD55 engineering antibodies

Reducing the anti-human CD55 engineering antibody by using a reducing agent, uniformly mixing the reducing agent with the activated PE, stirring at 25 ℃, and reacting for 1 hour; and purifying by using an S200 increment purification column to obtain the PE marked CD55 engineering antibody.

EXAMPLE 6 Activity assay of anti-human CD55-PE engineered antibody

1. Detecting peripheral blood of normal people: adding 100 μl of normal human anticoagulated peripheral blood into each tube, adding different amounts of engineering antibodies CD55-PE (1.0 μg, 0.5 μg, 0.25, 0.13 μg, 0.06 μg, 0.03 μg), adding antibodies CD45-PerCP, and incubating at room temperature in dark place for 30 min; adding 2ml of hemolysin, reacting at room temperature in a dark place for 10 minutes, centrifuging at 1000 rpm for 5 minutes, and discarding the supernatant; adding 2ml of cold PBS buffer, centrifuging at 1000 rpm for 5 minutes, and discarding the supernatant; 300 μl PBS buffer was added and the flow cytometer detected, the results are shown in FIG. 3.

2. Erythrocyte detection: peripheral blood 1: after 100 dilutions, 100 μl of diluted peripheral blood is added into each tube, different amounts of engineering antibody CD55-PE are respectively added, and the mixture is incubated for 30 minutes at room temperature in a dark place; centrifuging at 1000 rpm for 5min, and discarding supernatant; adding 2ml of cold PBS buffer, re-suspending, centrifuging at 1000 rpm for 5 minutes, and discarding the supernatant; after the addition of 250. Mu.l of PBS buffer and detection by flow cytometry, as shown in FIG. 3, a fluorescent signal was detected after binding of CD55-PE to lymphocytes and erythrocytes, indicating that the CD55-PE engineered antibody could bind to CD55 on the surfaces of lymphocytes and erythrocytes with normal activity and that the average fluorescence intensity decreased with decreasing antibody addition.

The foregoing description of the preferred embodiments of the invention is not intended to be limiting, but rather is intended to cover all modifications, equivalents, alternatives, and improvements that fall within the spirit and scope of the invention.

Claims

1. An anti-human CD55 engineered antibody, characterized in that: the engineered antibody comprises a heavy chain variable region and a light chain variable region;

2. The anti-human CD55 engineering antibody according to claim 1, wherein: the framework regions of the heavy chain variable region comprise FR1, FR2, FR3 and FR4; the amino acid sequence of FR1 of the heavy chain variable region is shown as SEQ ID NO. 7; the amino acid sequence of FR2 of the heavy chain variable region is shown as SEQ ID NO. 8; the amino acid sequence of FR3 of the heavy chain variable region is shown as SEQ ID NO. 9; the amino acid sequence of FR4 of the heavy chain variable region is shown as SEQ ID NO. 10;

3. The anti-human CD55 engineering antibody according to claim 1, wherein: the amino acid sequence of the heavy chain variable region of the engineering antibody is shown as SEQ ID NO.15, and the amino acid sequence of the light chain variable region is shown as SEQ ID NO. 16.

4. A nucleic acid molecule characterized in that: the nucleic acid molecule encodes the anti-human CD55 engineered antibody of any one of claims 1-3.

5. The nucleic acid molecule of claim 4, wherein: the nucleotide sequence of the nucleic acid molecule encoding the heavy chain variable region of the anti-human CD55 engineering antibody is shown as SEQ ID NO. 17, and the nucleotide sequence encoding the light chain variable region is shown as SEQ ID NO. 18.

6. An expression vector, characterized in that: the expression vector comprises the nucleic acid molecule of claim 4 or 5.

7. Use of an anti-human CD55 engineering antibody according to any one of claims 1-3, characterized in that: the application of the anti-human CD55 engineering antibody in preparing a reagent for diagnosing colon cancer.

8. An immunoassay kit, characterized in that: the kit contains the anti-human CD55 engineering antibody of any one of claims 1-3 and/or the expression vector of claim 6.