CN113278074A

CN113278074A - anti-CEACAM 5 nano antibody

Info

Publication number: CN113278074A
Application number: CN202110397962.5A
Authority: CN
Inventors: 单鸿; 李丹; 梅超明; 杨帆
Original assignee: Fifth Affiliated Hospital of Sun Yat Sen University
Current assignee: Fifth Affiliated Hospital of Sun Yat Sen University
Priority date: 2020-08-04
Filing date: 2020-08-04
Publication date: 2021-08-20
Anticipated expiration: 2040-08-04
Also published as: CN113105548B; CN113105548A; CN113278074B; CN112028997A; CN113234164B; CN113105549B; CN113201070B; CN113201070A; CN112028997B; CN113105549A; CN113234164A

Abstract

The invention provides a nano antibody. The antibody has the amino acid sequence of SEQ ID NO:1, comprising 4 FRs and 3 CDRs. The nano antibody can specifically target human CEACAM5 protein, and has the characteristics of small molecular weight, high water solubility, high tolerance, high stability, high antigen binding property, low immunogenicity and the like.

Description

anti-CEACAM 5 nano antibody

Technical Field

The invention relates to the field of biotechnology, in particular to a nano antibody and a preparation method and application thereof, and the nano antibody can be applied to research and development of CEACAM5 protein detection reagents and therapeutic antibodies.

Background

Carcinoembryonic antigen (CEA) is a protein found in colon cancer and fetal intestinal tissues, is a marker for tumor detection in clinical early, generates a small amount of CEA in normal human somatic cells, is expressed in various organs of malignant tumor patients, can be used for diagnosis and identification of tumors, but the diagnosis of the tumor standard lacks specificity. CEA is high and mainly appears in various tumors such as pancreatic cancer, colorectal cancer, breast cancer, gastric cancer, small cell lung cancer and the like, and the diagnosis sensitivity of CEA in the serum of a tumor patient is closely related to the existence of lymph nodes of the tumor, the size of the tumor and whether the tumor has metastasis of other parts.

In 1993, Hamers-Casterman et al reported on nature that a naturally occurring light chain-deficient antibody, i.e., a heavy chain antibody, was present in camelid serum in large amounts. Single domain heavy chain antibodies refer to genetically engineered antibodies consisting of only heavy chain antibody variable regions, also known as VHH antibodies or nanobodies (Nb). In contrast to ordinary antibodies, camelid heavy chain antibodies lack a CH1 region between the heavy chain variable region and the hinge region, except for the absence of a light chain. The constant region CH1 is the site of anchoring to the light chain, is present in the genome of nanobody, but is spliced out during mRNA formation, and nanobody has antigen specificity and high affinity with only 3 CDRs, whereas ordinary antibodies require 6 CDRs, which are the smallest unit known at present to bind the target antigen. Compared with the common antibody, the nano antibody has the advantages of small molecular weight, simple structure, easy gene modification, small volume, good antigen specificity, strong tissue penetration and high stability, and has wide application prospect in the aspects of diagnosis and treatment of diseases. Based on this, we developed a novel nano antibody against CEACAM5 in order to further improve the function of the antibody.

Disclosure of Invention

The invention aims to provide a nano antibody aiming at CEACAM5, and simultaneously provides a coding sequence and a preparation method of the nano antibody.

The inventor of the application adopts alpaca immunity and phage display technology to combine mutually, and develops the high-affinity nano antibody targeting CEACAM 5.

In one aspect, the invention features a nanobody. According to an embodiment of the present invention, the nanobody has an amino acid sequence of SEQ ID NO: 1. the nano-antibody according to the embodiment of the invention is an antibody specifically targeting CEACAM5 protein, can bind to CEACAM5 protein in native conformation, and has the unique properties of (1) high water solubility, high tolerance, high stability, high antigen binding property, low immunogenicity and stronger tissue penetration compared with a common antibody.

According to the embodiment of the invention, the amino acid sequence of the nanobody is shown as SEQ ID NO. 1, and the framework regions are FR1 shown as SEQ ID NO. 2, FR2 shown as SEQ ID NO. 4, FR3 shown as SEQ ID NO. 6, FR4 shown as SEQ ID NO. 8; the complementarity determining regions are CDR1 shown in SEQ ID NO. 3, CDR2 shown in SEQ ID NO. 5 and CDR3 shown in SEQ ID NO. 7.

The nano-antibody according to the embodiment of the invention is an antibody specifically targeting CEACAM5 protein, can be combined with CEACAM5 protein in a natural conformation, and has high water solubility, high tolerance, high stability, high antigen binding property, low immunogenicity and stronger tissue penetration.

In yet another aspect, the invention features a nucleic acid. According to an embodiment of the invention, the nucleic acid is: nucleic acid encoding the nanobody described above or a complementary sequence thereof. The nucleic acid according to the embodiment of the invention specifically encodes the aforementioned nanobody, which can specifically target CEACAM5, bind to CEACAM5 in native conformation, and has high water solubility, high tolerance, high stability, high antigen binding property, low immunogenicity and strong tissue penetration.

According to an embodiment of the invention, the nucleic acid has the sequence of SEQ ID NO: 9.

The protein encoded by the above-described nucleic acid according to the embodiment of the present invention has a remarkably high affinity activity to CEACAM5, and has a structure typical of nanobodies, i.e., composed of a framework region (FR1, FR2, FR3 and FR4) and a complementarity determining region (CDR1, CDR2 and CDR 3).

It is to be noted that, with respect to the nucleic acids mentioned in the claims and the specification of the present invention, those skilled in the art should understand that any one or two of the complementary double strands are actually included. For convenience, in the claims and specification, although only one strand is given in most cases, the complementary strand is actually disclosed. In addition, the gene sequence in the present application includes a DNA form or an RNA form, and one is disclosed, which means that the other is also disclosed.

In yet another aspect, the present invention provides an expression vector comprising the above-described nucleic acid.

In yet another aspect, the present invention provides a host cell comprising the above-described expression vector.

Has the advantages that:

compared with the traditional antibody preparation technology, the alpaca immune and phage display technology are combined with each other, the advantages of the phage display technology are fully utilized, namely (1) larger library capacity, the screening operation is simple, and the flux is high; (2) the high efficiency of panning makes it possible to successfully select the phage which exists in trace but has high affinity, and can be enriched by infecting bacteria; (3) can directly obtain antibody gene, is convenient for further constructing various genetic engineering antibodies, and can also be used for some antibodies which are difficult to prepare, such as weak immunogen, toxic antigen and the like, and humanized antibodies. Compared with the traditional antibody, the obtained nano antibody has the advantages of high water solubility, high tolerance, high stability, high antigen binding property, low immunogenicity and strong tissue penetration.

The invention has the following advantages: the method comprises the steps of immunizing alpaca with recombinant CEACAM5 protein, establishing a nano antibody gene library aiming at CEACAM5 by utilizing alpaca peripheral blood lymphocytes, coupling CEACAM5 on an enzyme label plate in a test, screening an immune nano antibody gene library (alpaca heavy chain antibody phage display gene library) by using an antigen in the form of the nano antibody gene library by using a phage display technology, thus obtaining a nano antibody gene aiming at CEACAM5 specificity, transferring the gene into escherichia coli, and establishing a nano antibody strain capable of being efficiently expressed in the escherichia coli.

Drawings

FIG. 1 shows the ELISA detection result of CEACAM5 antibody in the serum of alpaca after immunization in the example.

FIG. 2 is a diagram showing the result of SDS-PAGE electrophoresis detecting the expression of CEACAM5 nanobody in the examples.

FIG. 3 shows ELISA assay results of purified CEACAM5 nanobody affinity of example.

Detailed Description

The present invention is further described below by way of specific examples, but the present invention is not limited to the examples.

Example 1

Construction of Nanobody library aiming at CEACAM5 protein

The recombinant CEACAM5 protein is mixed with Freund's adjuvant to immunize alpaca for 4 times. After 4 times of immunization, extracting 50mL of alpaca peripheral blood lymphocytes and extracting total RNA; the VHH region of the antibody was then cloned by the Nest-PCR technique and the cloned product was inserted into phage by homologous recombination to obtain a phage display library.

Example 2

Affinity panning and post-panning library amplification

(1) Affinity panning

1) Diluting the recombinant CEACAM5 protein with a carbonate buffer solution with the pH value of 9.6 to the final concentration of 5 mu g/mL, adding the diluted protein into enzyme-labeled holes according to 100 mu L/hole, coating 8 holes on each target molecule, and coating overnight at 4 ℃;

2) discarding the coating solution, washing with PBS for 3 times, adding 300 mu L of 3% BSA-PBS blocking solution into each hole, and blocking for 1h at 37 ℃;

3) washing with PBS for 3 times, adding 100 muL phage library, and incubating for 1h at 37 ℃;

5) unbound phage were aspirated, washed 6 times with PBST and 2 times with PBS;

6) adding 100 mu L Gly-HCl eluent, incubating for 8min at 37 ℃, and eluting specifically combined phage; transferring the eluent into a 1.5mL sterile centrifuge tube, and quickly neutralizing with 10 μ L Tris-HCl neutralization buffer;

7) taking 10 mu L for gradient dilution, determining the titer, calculating the elutriation recovery rate, mixing the rest eluates, performing amplification and purification for the next round of affinity elutriation, and changing the elutriation conditions, wherein the elutriation conditions are shown in table 1:

TABLE 1 affinity panning conditions

Number of rounds

Antigen coating concentration (mug/mL)

Sealing liquid

Input amount of library (cfu)

Integration time (h)

Concentration of PBST

Number of PBST washes

1

5

BSA-PBS

2×10¹¹

1

0.1%

6

(2) Amplification of post-panning libraries

1) Mixing elutriation eluate with 5mL of E.coli TG1 culture in early stage of logarithmic growth, standing at 37 deg.C for 30min, and shake culturing at 220r/min for 30 min; centrifuging at 1000g for 15min, removing supernatant, resuspending with 500 μ L2 XYT and spreading on 200mm 2 XYT-GA plate;

2) scraping bacteria with 10ml 2 XYT liquid culture medium, adding 500 μ L suspension into 50ml 2 XYT liquid culture medium, and shaking at 37 deg.C for 30 min; adding M13K07 helper phage according to the proportion of cell: phase =1:20, standing at 37 ℃ for 30min, and performing shaking culture at 220r/min for 30 min; the culture is subpackaged in a centrifuge tube, the temperature is 25 ℃, the speed is 5000 r/min, the time is 10 min, the cell sediment is resuspended in 50mL of 2 XYT-AK liquid culture medium, and the shaking culture is carried out overnight at the temperature of 30 ℃ and the speed of 230 r/min;

3) centrifuging the overnight culture at 4 deg.C and 10000 r/min for 20 min, transferring the supernatant to a new centrifuge tube, adding 1/5 volume of PEG-NaCl, mixing, and standing at 4 deg.C for more than 2 hr;

4) removing supernatant at 4 ℃, 10000 r/min, 20 min, suspending the precipitate in 1mL PBS, adding 1/5 volume of PEG/NaCl, mixing uniformly, and placing at 4 ℃ for more than 1 h;

5) and (4) removing the supernatant at 12000 r/min for 2min, suspending the precipitate in 200 mu L PBS to obtain an amplification product, and determining the titer for the next round of panning or analysis.

Example 3

Identification and analysis of specific phage clones

(1) Rescue of phagemids

1) Randomly picking 96 monoclonals from a plate with the titer of the eluate in the first round of panning by using a sterilized toothpick, inoculating the monoclonals into 1mL of 2 XYT-A, and carrying out shaking culture at 37 ℃ and 220r/min for 8 hours;

2) taking 200 muL of the culture, and pressing cell: phase =1: adding M13K07 bacteriophage at a ratio of 20, standing at 37 deg.C for 15min, and performing shake culture at 220r/min for 45 min;

3) 2 XYT-AK with the volume of 800 muL is supplemented, and the shaking culture is carried out overnight at the temperature of 30 ℃;

4) centrifuging at 12000 rpm for 2min the next day, taking the supernatant, and using the supernatant for monoclonal ELISA identification;

(2) identification of Positive phage clones

1) Diluting the recombinant CEACAM5 protein with a carbonate buffer solution with the pH value of 9.6 to the final concentration of 2 mu g/mL, adding the diluted protein into an enzyme-labeled hole according to 100 mu L/hole, and coating overnight at 4 ℃;

2) discarding the coating solution, washing for 3 times by PBST, adding 5% skimmed milk of 300 mu L into each hole, and sealing for 1h at 37 ℃;

3) PBST is washed for 3 times, 50 mu L of phage culture solution supernatant and 50 mu L of 5% skimmed milk are added into each hole, and incubation is carried out for 1h at 37 ℃;

4) PBST is washed for 5 times, and horseradish peroxidase-labeled anti-M13 antibody (diluted by 5% skimmed milk powder at a ratio of 1: 10000) is added into the PBST for acting for 1 hour at 37 ℃;

5) PBST wash plate 6 times. Adding TMB color developing solution for color development, 100 μ L/well, 37 deg.C, 7 min, adding stop solution to stop reaction, 50 μ L/well, and measuring optical density at 450 nm.

(3) Sequence analysis of Positive phage clones

Positive clones were subjected to sequencing analysis.

Example 4

Expression and purification of CEACAM5 nano antibody in prokaryotic expression system

1) Cloning VHH fragments of different clone strains obtained by the previous sequencing analysis into a BL21 (DE3) prokaryotic expression vector;

2) LB plates (50 ug/ml kanamycin) were plated;

3) selecting a single colony, inoculating the single colony in 15mL LB culture solution containing kanamycin, and carrying out shake culture at 37 ℃ overnight;

4) inoculating 1mL of overnight strain into 300 mL of LB culture medium, performing shake culture at 37 ℃, adding IPTG (isopropyl-beta-thiogalactoside) when the OD value reaches 0.6-1, and performing shake culture at 16 ℃ for 16-20 h;

5) centrifuging to collect bacteria, and crushing the bacteria to obtain an antibody crude extract;

6) purifying antibody protein by nickel column ion affinity chromatography, eluting with imidazole gradient elution method, eluting with low concentration imidazole eluate (30 mmol) to remove impurity band, and eluting with high concentration imidazole eluate (200 mmol, 500 mmol) to obtain protein with purity of above 90%.

Through the method, the CEACAM5 nano antibody B95 is obtained.

B95 amino acid sequence (SEQ ID NO:1)

EVQVVESGGGLVQAGGSLKLSCAASFDTFSIHAMGWYRQVPGKQRELVAAITSSGGTNYADSVKGRFTISRDNAKNTVYLQMNSLKPGDTAVYYCNEPFRLFDWGQGTQVTVSS

FR1: EVQVVESGGGLVQAGGSLKLSCAAS (SEQ ID NO:2)

CDR1: FDTFSIHA (SEQ ID NO:3)

FR2: MGWYRQVPGKQRELVAA (SEQ ID NO:4)

CDR2: ITSSGGT (SEQ ID NO:5)

FR3: NYADSVKGRFTISRDNAKNTVYLQMNSLKPGDTAVYYC (SEQ ID NO:6)

CDR3: NEPFRLFD (SEQ ID NO:7)

FR4: WGQGTQVTVSS (SEQ ID NO:8)

The overall nucleotide sequence:

gaggtgcaggtggtggagtctgggggaggcttggtgcaggctggggggtctctgaaactctcctgtgcagcctcttttgacacgttcagtatacatgccatgggctggtaccgccaggttccagggaagcagcgcgaattggtcgcagctattactagtagtggcggcacaaactacgcagactccgtgaagggccgattcaccatctccagagacaacgccaagaacacggtgtatctgcaaatgaacagcctgaaacctggagacacagccgtctattactgtaatgaaccgtttcgcctcttcgactggggccaggggacccaggtcaccgtctcctcagcgcaccacagcgaagaccccagc (SEQ ID NO:9)。

sequence listing

<110> secondary fifth Hospital of Zhongshan university

<120> anti-CEACAM 5 nano antibody

<160> 9

<170> SIPOSequenceListing 1.0

<210> 1

<211> 114

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 1

Glu Val Gln Val Val Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly

1 5 10 15

Ser Leu Lys Leu Ser Cys Ala Ala Ser Phe Asp Thr Phe Ser Ile His

20 25 30

Ala Met Gly Trp Tyr Arg Gln Val Pro Gly Lys Gln Arg Glu Leu Val

35 40 45

Ala Ala Ile Thr Ser Ser Gly Gly Thr Asn Tyr Ala Asp Ser Val Lys

50 55 60

Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr Leu

65 70 75 80

Gln Met Asn Ser Leu Lys Pro Gly Asp Thr Ala Val Tyr Tyr Cys Asn

85 90 95

Glu Pro Phe Arg Leu Phe Asp Trp Gly Gln Gly Thr Gln Val Thr Val

100 105 110

Ser Ser

<210> 2

<211> 25

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 2

Glu Val Gln Val Val Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly

1 5 10 15

Ser Leu Lys Leu Ser Cys Ala Ala Ser

20 25

<210> 3

<211> 8

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 3

Phe Asp Thr Phe Ser Ile His Ala

1 5

<210> 4

<211> 17

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 4

Met Gly Trp Tyr Arg Gln Val Pro Gly Lys Gln Arg Glu Leu Val Ala

1 5 10 15

Ala

<210> 5

<211> 7

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 5

Ile Thr Ser Ser Gly Gly Thr

1 5

<210> 6

<211> 38

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 6

Asn Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn

1 5 10 15

Ala Lys Asn Thr Val Tyr Leu Gln Met Asn Ser Leu Lys Pro Gly Asp

20 25 30

Thr Ala Val Tyr Tyr Cys

35

<210> 7

<211> 8

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 7

Asn Glu Pro Phe Arg Leu Phe Asp

1 5

<210> 8

<211> 11

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 8

Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser

1 5 10

<210> 9

<211> 366

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 9

gaggtgcagg tggtggagtc tgggggaggc ttggtgcagg ctggggggtc tctgaaactc 60

tcctgtgcag cctcttttga cacgttcagt atacatgcca tgggctggta ccgccaggtt 120

ccagggaagc agcgcgaatt ggtcgcagct attactagta gtggcggcac aaactacgca 180

gactccgtga agggccgatt caccatctcc agagacaacg ccaagaacac ggtgtatctg 240

caaatgaaca gcctgaaacc tggagacaca gccgtctatt actgtaatga accgtttcgc 300

ctcttcgact ggggccaggg gacccaggtc accgtctcct cagcgcacca cagcgaagac 360

cccagc 366

Claims

1. A nano antibody aiming at CEACAM5 is characterized in that the amino acid sequence of the nano antibody is shown as SEQ ID NO. 1;

the framework regions are FR1 shown in SEQ ID NO. 2, FR2 shown in SEQ ID NO. 4, FR3 shown in SEQ ID NO. 6 and FR4 shown in SEQ ID NO. 8;

the complementarity determining regions are CDR1 shown in SEQ ID NO. 3, CDR2 shown in SEQ ID NO. 5 and CDR3 shown in SEQ ID NO. 7.

2. A nucleic acid, wherein said nucleic acid is: a nucleic acid encoding the antibody of claim 1 or a complementary sequence thereof (SEQ ID NO: 9).

3. An expression vector comprising the nucleic acid of claim 2.

4. A host cell comprising the expression vector of claim 3.