CN108218989B

CN108218989B - anti-PD-1 nano antibody PD-1/Nb18 and preparation method and application thereof

Info

Publication number: CN108218989B
Application number: CN201711223594.2A
Authority: CN
Inventors: 卢小玲; 莫凤珍; 汤卓然
Original assignee: Guangxi Medical University
Current assignee: Guangxi Medical University
Priority date: 2017-11-29
Filing date: 2017-11-29
Publication date: 2020-08-18
Anticipated expiration: 2037-11-29
Also published as: CN108218989A

Abstract

The invention discloses an anti-PD-1 nano antibody PD-1/Nb18 and a preparation method and application thereof. The nano antibody PD-1/Nb18 provided by the invention comprises an antigenic determinant complementary region and a framework region; the antigenic determinant complementary region of the nano antibody PD-1/Nb18 consists of CDR1, CDR2 and CDR 3; through the nano antibody PD-1/Nb18 nucleotide sequence and the host cell disclosed by the invention, the nano antibody PD-1/Nb18 can be efficiently expressed in escherichia coli, is applied to the research and development of a PD-1 molecular detection reagent, and is used for preparing a tumor inhibitor or a tumor cell inhibitor and preparing a medicine for inhibiting PD-1 activity and promoting T cell proliferation.

Description

anti-PD-1 nano antibody PD-1/Nb18 and preparation method and application thereof

Technical Field

The invention relates to an anti-PD-1 nano antibody PD-1/Nb18 in the field of biomedicine and a preparation method and application thereof.

Background

Programmed death receptor 1 (PD-1) is a transmembrane glycoprotein molecule which activates the surface expression of T cells, belongs to a member of the CD28 superfamily, has negative regulation effect on the proliferation of T cells and plays an important regulation effect in immune response. PD-1 is expressed predominantly in activated T cells and B cells, and is a surface receptor for activated T cells, and PD-1 has two ligands, apoptosis-ligand 1(PD-L1, also known as B7-H1) and apoptosis-ligand 1(PD-L2, also known as B7-DC). The tumor microenvironment in the body can induce infiltrated T cells to highly express PD-1 molecules, and the tumor cells can highly express ligands PD-L1 and PD-L2 of PD-1, so that after PD-1 channels in the tumor microenvironment continuously activate PD-L1 to be linked with PD-1, the function of the T cells is inhibited, and signals for attacking tumors cannot be sent to an immune system. The PD-1 inhibitor can block the combination of PD-1 and PD-L1, block negative regulation signals, and restore the activity of T cells, thereby enhancing the immune response. Highly specific, low-reactivity anti-PD-1 monoclonal antibodies have been confirmed in several clinical trials, such as the drug manufactured by Shimadong under the name Keytruda (Pembrolizumab) and the drug manufactured by Shinobao under the name Opdivo (Nivolumab) nivolumab.

However, there are many problems in the application of antibody drugs, such as long development cycle and high production cost; difficult to mass produce; poor stability, easy degradation and high storage cost; is easy to be polluted, and the maintenance cost is high; and has immunogenicity and the like, thereby limiting the application range of the medicine in clinic.

The nano antibody technology is an antibody engineering revolution which is carried out by biomedical scientists by combining molecular biology technology with the concept of nano particle science on the basis of traditional antibodies, thereby developing the latest and smallest antibody molecules. In 1993, Hamers et al reported that a camelid has a heavy chain antibody with a naturally deleted light chain and heavy chain constant region 1(CH1) in it, and the variable region was cloned to obtain a single domain antibody consisting of only one heavy chain variable region, called VHH (variable domain of heavyain of heavy-chain antibody), which has now been renamed as "Nanobody" (Nb). The nano antibody has the smallest antigen binding fragment with complete function, and the crystal structure of the nano antibody is elliptic, the diameter of the nano antibody is 2.5nm, and the length of the nano antibody is 4 nm. Nb has a plurality of unique properties, is very suitable for genetic modification, and shows wide application prospects in the aspects of accurate diagnosis, targeted treatment and the like of diseases. The nano antibody is much simpler in chemical composition and shape than an antibody, has no chemical hydrophobicity, is stronger in heat resistance and acid and alkali resistance, is easier to combine with each other or other compounds, can be encoded by a single gene, and is easy to synthesize by microorganisms. The nano antibody has good tolerance to the environment, high conformational stability, smaller molecular weight and better clinical treatment effect, and meanwhile, the small protein molecules are easier to synthesize and have lower price. The unique property of the nano antibody enables the nano antibody to show wider application prospects in the aspects of accurate diagnosis of diseases, immune targeted therapy and the like.

Disclosure of Invention

The technical problem to be solved by the invention is how to prepare the medicine for effectively treating the tumor.

In order to solve the technical problem, the invention firstly provides a nano antibody PD-1/Nb 18.

In a first aspect of the invention, there is provided a nanobody, referred to as PD-1/Nb18, comprising an epitope-complementing region CDR and a framework region FR; the CDR of the antigenic determinant complementary region of the nanobody consists of CDR1, CDR2 and CDR 3; the amino acid sequence of the CDR1 is the 23 rd to 32 th amino acid of SEQ ID No.8 in the sequence table; the amino acid sequence of the CDR2 is amino acid 49-56 of SEQ ID No.8 in the sequence table; the amino acid sequence of the CDR3 is 94-112 th amino acid of SEQ ID No.8 in the sequence table.

Preferably, the framework region FR of the nanobody consists of FR1, FR2, FR3 and FR 4; wherein the amino acid sequence of FR1 is the amino acid at the 1 st to 22 th positions of SEQ ID No.8 in the sequence table; the amino acid sequence of the FR2 is the 33 th-48 th amino acid of SEQ ID No.8 in the sequence table; the amino acid sequence of FR3 is 57-93 th amino acid of SEQ ID No.8 in the sequence table; the amino acid sequence of the FR4 is the 113 th and 123 th amino acids of SEQ ID No.8 in the sequence table.

Preferably, the amino acid sequence of the nano antibody PD-1/Nb18 is shown as SEQ ID No.8 in the sequence table.

The invention also correspondingly provides a VHH chain of the nano antibody of PD-1, which comprises an amino acid sequence of a framework region FR and a complementarity determining region CDR, wherein the framework region FR is selected from the following FR: SEQ ID NO: FR1 as shown in 1, SEQ ID NO: FR2 as shown in 2, SEQ ID NO: FR3 as shown in SEQ ID NO: FR4 shown in FIG. 4; the CDR is selected from the amino acid sequence of CDR of the following group: SEQ ID NO: 5, CDR1 shown in SEQ ID NO: 6, CDR2 shown in SEQ ID NO: CDR3 shown in FIG. 7.

Preferably, the VHH chain of the nanobody of PD-1 has the amino acid sequence of SEQ ID NO: 8.

In order to facilitate the purification of the nano antibody PD-1/Nb18, the amino terminal or the carboxyl terminal of the protein shown by the amino acids 1 to 123 of the SEQ ID No.8 in the sequence table can be connected with the label shown in the table 1.

TABLE 1 sequence of tags

Label (R)	Residue of	Sequence of
			Poly-Arg	5-6 (typically 5)	RRRRR
Poly-His	2-10 (generally 6)	HHHHHH
			FLAG	8	DYKDDDDK
Strep-tag II	8	WSHPQFEK
			c-myc	10	EQKLISEEDL
HA	9	YPYDVPDYA

On the other hand, the nano antibody PD-1/Nb18 can be synthesized artificially, or can be obtained by synthesizing the coding gene and then carrying out biological expression. The coding gene of the nano antibody PD-1/Nb18 can be obtained by deleting one or more codons of amino acid residues in a DNA sequence shown in SEQ ID No.9 in a sequence table, and/or carrying out missense mutation of one or more base pairs, and/or connecting a coding sequence of a label shown in the table 1 at the 5 'end and/or the 3' end of the coding gene.

In order to solve the technical problems, the invention also provides a biological material related to the nano antibody PD-1/Nb 18.

The biological material related to the nano antibody PD-1/Nb18 provided by the invention is any one of B1) to B12):

B1) nucleic acid molecules encoding the nanobody PD-1/Nb 18;

B2) an expression cassette comprising the nucleic acid molecule of B1);

B3) a recombinant vector comprising the nucleic acid molecule of B1);

B4) a recombinant vector comprising the expression cassette of B2);

B5) a recombinant microorganism comprising the nucleic acid molecule of B1);

B6) a recombinant microorganism comprising the expression cassette of B2);

B7) a recombinant microorganism containing the recombinant vector of B3);

B8) a recombinant microorganism containing the recombinant vector of B4);

B9) a transgenic animal cell line comprising the nucleic acid molecule of B1);

B10) a transgenic animal cell line comprising the expression cassette of B2);

B11) a transgenic animal cell line containing the recombinant vector of B3);

B12) a transgenic animal cell line comprising the recombinant vector of B4).

In the above biological material, the nucleic acid molecule may be DNA, such as cDNA, genomic DNA or recombinant DNA; the nucleic acid molecule may also be RNA, such as mRNA or hnRNA, etc.

In the above biomaterial, the expression cassette containing a nucleic acid molecule encoding the nanobody PD-1/Nb18 described in B2), also called PD-1/Nb18 gene expression cassette, refers to DNA capable of expressing the nanobody PD-1/Nb18 in a host cell, and the DNA may include not only a promoter for initiating transcription of the nanobody PD-1/Nb18 gene, but also a terminator for terminating transcription of the nanobody PD-1/Nb18 gene. Further, the expression cassette may also include an enhancer sequence.

The existing expression vector can be used for constructing a recombinant vector containing the nano antibody PD-1/Nb18 gene expression cassette.

In the above biological material, the vector may be a plasmid, a cosmid, a phage, or a viral vector.

In the above biological material, the recombinant vector may be a recombinant vector obtained by introducing the nucleic acid molecule of B1) into pComb 3. In one embodiment of the invention, B3) the recombinant vector is a recombinant vector pComb3-PD-1/Nb18 obtained by introducing a coding gene (nucleotide sequence is 1-369 th nucleotide of SEQ ID No.9 in a sequence table) of the nano antibody PD-1/Nb18 into pComb3, and the recombinant vector pComb3-PD-1/Nb18 expresses the nano antibody PD-1/Nb18 shown in SEQ ID No. 8.

In the above biological material, the microorganism may be yeast, bacteria, algae or fungi.

In the above biological material, the transgenic animal cell line does not include propagation material; the recombinant microorganism may be a recombinant microorganism obtained by introducing the nucleic acid molecule of B1) into Escherichia coli WK 6.

The nucleotide sequence of the nanobody PD-1/Nb18 of the invention B1) can be easily mutated by a person of ordinary skill in the art using known methods, such as directed evolution and point mutation. Those artificially modified nucleotides having 75% or more than 75% identity with the nucleotide sequence of the nanobody PD-1/Nb18 of the invention B1), as long as the nucleotides encoding the nanobody PD-1/Nb18 and having the nanobody PD-1/Nb18 activity, are derived from the nucleotide sequence of the invention and are identical to the sequence of the invention.

The term "identity" as used herein refers to sequence similarity to a native nucleic acid sequence. "identity" includes a nucleotide sequence having 75% or more, or 85% or more, or 90% or more, or 95% or more identity to the nucleotide sequence of the present invention encoding the protein represented by SEQ ID No. 8. Identity can be assessed visually or by computer software. Using computer software, the identity between two or more sequences can be expressed in percent (%), which can be used to assess the identity between related sequences.

The above-mentioned identity of 75% or more may be 75%, 80%, 85%, 90% or 95% or more.

In the above biological material, the nucleic acid molecule of B1) is 1) or 2) or 3) described below:

1) the nucleotide sequence is a cDNA molecule or a DNA molecule of SEQ ID No.9 in a sequence table;

2) a cDNA molecule or a genome DNA molecule which has 75 percent or more than 75 percent of identity with the nucleotide sequence defined by 1) and codes the nano antibody PD-1/Nb 18;

3) hybridizing with the nucleotide sequence defined in 1) under strict conditions, and encoding the cDNA molecule or genome DNA molecule of the nano antibody PD-1/Nb 18.

In order to solve the technical problems, the invention also provides a derivative antibody of the nano antibody PD-1/Nb 18.

The derivative antibody of the nano antibody PD-1/Nb18 provided by the invention is a) or b) or c) or d) or e):

a) a single-chain antibody containing the nano antibody PD-1/Nb 18;

b) a fusion antibody comprising a) said single chain antibody;

c) a fusion antibody containing the nano antibody PD-1/Nb 18;

d) fab containing the nanobody PD-1/Nb 18;

e) an intact antibody containing the Nanobody PD-1/Nb 18.

In order to solve the technical problems, the invention also provides a preparation method of the nano antibody PD-1/Nb 18.

The preparation method of the nano antibody PD-1/Nb18 provided by the invention comprises the steps of introducing a nucleic acid molecule for coding the nano antibody PD-1/Nb18 into a receptor cell to obtain a transgenic cell for expressing the nano antibody PD-1/Nb18, and culturing the transgenic cell to obtain the nano antibody PD-1/Nb 18.

In the preparation method of the nano antibody PD-1/Nb18, the nucleotide sequence of the nucleic acid molecule for coding the nano antibody PD-1/Nb18 is shown as SEQ ID No.9 in a sequence table.

In the preparation method of the nano antibody PD-1/Nb18, the receptor cell can be a microbial cell, such as Escherichia coli, and specifically can be Escherichia coli WK 6.

In order to solve the technical problem, the invention also provides any one of the following applications A1-A8:

a1 and application of the nano antibody PD-1/Nb18 in preparation of tumor inhibitors or tumor cell inhibitors;

a2, the application of the biological material in preparing tumor inhibitors or tumor cell inhibitors;

a3, and the application of the derivative antibody of the nano antibody in preparing a tumor inhibitor or a tumor cell inhibitor;

a4 and the application of the preparation method of the nano antibody PD-1/Nb18 in preparing tumor inhibitors or tumor cell inhibitors;

a5 and application of the nano antibody PD-1/Nb18 in preparing products for inhibiting PD-1 activity or promoting T cell proliferation;

a6, the application of the biological material in preparing products for inhibiting PD-1 activity or promoting T cell proliferation;

a7, and application of the derivative antibody in preparing products for inhibiting PD-1 activity or promoting T cell proliferation;

a8 and application of the preparation method of the nano antibody PD-1/Nb18 in preparation of products for inhibiting PD-1 activity or promoting T cell proliferation.

The product may be a medicament.

The primer pair for amplifying the nucleic acid molecule of the amino acid sequence shown in SEQ ID No.8 in the coding sequence list or any fragment of the amino acid sequence also belongs to the protection scope of the invention.

The invention provides an anti-PD-1 nano antibody, a nucleotide sequence and a host cell for encoding the nano antibody, and a preparation method and application thereof. The nano antibody can be efficiently expressed in escherichia coli, is applied to the research and development of PD-1 molecular detection reagents, and is used for preparing tumor inhibitors or tumor cell inhibitors and medicines for inhibiting PD-1 activity and promoting T cell proliferation.

Drawings

FIG. 1 is a DNA electrophoresis diagram of a nanobody, and DNA bands from left to right of a gel hole are respectively: the first path is a molecular marker of 2000bp, the second path is a PCR product, and the PCR product band is about 400 bp;

FIG. 2 is an electrophoresis diagram of SDS-PAGE of PD-1 nanobody PD-1/Nb18 after purification by nickel column resin gel affinity chromatography; lane M represents protein molecular weight Marker in KDa;

FIG. 3A shows the results of the binding experiment of the Nanobody PD-1/Nb18 to 293T cells that are not transfected with PD-1; FIG. 3B shows the results of the binding experiment of the Nanobody PD-1/Nb18 to 293T cells transfected with PD-1.

Detailed Description

The present invention is described in further detail below with reference to specific embodiments, which are given for the purpose of illustration only and are not intended to limit the scope of the invention.

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

Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.

The E.coli WK6 in the examples described below was obtained from Guangxi medical university after agreement with the Wanyakun laboratory of the institute of Life sciences of southeast university, and the biomaterial was only used for repeating the experiments related to the present invention, and was not used for other purposes.

Example 1 preparation of Nanobodies

The invention provides 1 camel-derived nano antibody, which is named as PD-1/Nb18, and the amino acid sequence of the nano antibody PD-1/Nb18 is shown as SEQ ID No.8 in a sequence table and is coded by a nucleotide sequence of SEQ ID No. 9.

The nucleotide electrophoresis diagram of the nano antibody PD-1/Nb18 is shown in figure 1, wherein the first channel is a molecular marker of 2000bp, the second channel is a PCR product, and the PCR product band is about 400 bp.

The DNA fragment between the PstI and NotI recognition sequences of the vector pComb3 (product of Biovector) was replaced with the DNA molecule shown in SEQ ID No.9, and the other sequences were not changed, resulting in a recombinant vector pComb3-PD-1/Nb18, pComb3-PD-1/Nb18 differing from pComb3 only in that the DNA fragment between the PstI and NotI recognition sequences of pComb3 was replaced with the DNA molecule shown in SEQ ID No. 9. The recombinant vector pComb3-PD-1/Nb18 expresses a nano antibody PD-1/Nb18 shown in SEQ ID No. 8. The pComb3-PD-1/Nb18 is introduced into Escherichia coli WK6 to obtain recombinant strain WK6-pComb3-PD-1/Nb 18.

The specific preparation steps of the nano antibody are as follows:

(1) coating WK6-pComb3-PD-1/Nb18 on LB plate containing ampicillin and glucose (in the LB plate, the concentrations of ampicillin and glucose are 100. mu.g/mL and 20mg/mL, respectively), and culturing overnight (10-14 hours) at 25-37 ℃;

(2) selecting single colony, inoculating into 5mL LB culture solution containing ampicillin (in LB culture solution, ampicillin concentration is 100 μ g/mL), and shake culturing at 25-37 deg.C overnight (10-14 hr);

(3) inoculating the overnight cultured culture solution in the step (2) into a fresh TB culture solution according to a ratio of 1: 300-1: 350, carrying out shake culture at 25-37 ℃ until the OD value reaches 0.6-1.0, adding IPTG (isopropyl-beta-D-1/Nb 18) to obtain a WK6-pComb3-PD-1/Nb18 culture solution, enabling the concentration of IPTG in the WK6-pComb3-PD-1/Nb18 culture solution to be 1mM, and carrying out culture on the WK6-pComb3-PD-1/Nb18 culture solution on a shake table (the rotation speed of a shake table is 220 plus 250rpm) at 20-30 ℃ overnight (10-14 hours) to obtain a WK6-pComb3-PD-1/Nb18 induction solution;

(4) centrifuging the induction liquid WK6-pComb3-PD-1/Nb18 obtained in the step (3) at 4 ℃, and collecting thalli;

(5) obtaining a crude antibody extract by an infiltration method in the literature (Zhu M, Hu Y, Li G, Ou W, Mao P, Xin S, Wan Y: Combining magnetic nanoparticles with biologically functionalized nanoparticles for rapid and sensitive detection of antibiotics H3N2.nanoscale Res Lett 2014,9: 528.);

(6) nanobody PD-1/Nb18 was prepared by nickel column ion affinity chromatography in the literature (Zhu M, Hu Y, Li G, Ou W, Mao P, Xin S, Wan Y: Combining magnetic nanoparticles with biologically functionalized nanoparticles for rapid and sensitive detection of antibiotics H3N2.nanoscale Res Lett 2014,9: 528.). The SDA-PAGE electrophoresis chart of the nano antibody PD-1/Nb18 is respectively shown in figure 2, and the size of the nano antibody PD-1/Nb18 is about 15 KDa. The result shows that the purity of the nano antibody PD-1/Nb18 obtained by the method can reach more than 90%.

Example 2 determination of binding ratio of Nanobody PD-1/Nb18 to PD-1

Determination of binding rate of nano antibody PD-1/Nb18 and PD-1 (direct method)

The binding rate of the nano antibody PD-1/Nb18 and PD-1 is detected by 293T cells stably transferring PD-1, the nano antibody PD-1/Nb18(1 mu g) of the example 1 is added into 1-6 × 10⁶The 293T cells were incubated at 4 ℃ for 20-40min in the absence of light, washed with PBS 2 times, then incubated with 5. mu.l of PE anti-HA tag antibody (abcam, Clone:20B12) at 4 ℃ for 20-40min, washed with PBS 2 times, and then the samples were subjected to BACKMAN flow cytometer, and the results are shown in FIG. 3B, and 293T cells which were not transfected with PD-1 were used as a control in FIG. 3A. FIG. 3A is the percent binding of the blank control and PD-1 nanobody PD-1/Nb18, respectively, to 293T cells that are not transfected with PD-1; FIG. 3B is the percent binding of 293T cells stably transfected with PD-1 by the blank control and the PD-1 nanobody PD-1/Nb18, respectively; in FIGS. 3A and 3B, the horizontal axis represents fluorescence intensity (PE) and the vertical axis represents percentage by number (% of Max), S2 represents a blank, and S1 represents PD-1 nanobody PD-1/Nb 18. The results show that the PD-1 nano antibody PD-1/Nb18 can be well combined with 293T cells stably transferring PD-1.

Finally, it should be noted that: the above examples are only intended to illustrate the technical solution of the present invention, but not to limit it; although the present invention has been described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that: the technical solutions described in the foregoing embodiments may still be modified, or some technical features may be equivalently replaced; and such modifications or substitutions do not depart from the spirit and scope of the corresponding technical solutions of the embodiments of the present invention.

SEQUENCE LISTING

<110> Guangxi university of medical science

<120> anti-PD-1 nano antibody PD-1/Nb18 and preparation method and application thereof

<130>GY17100593

<160>9

<170>PatentIn version 3.3

<210>1

<211>22

<212>PRT

<213> Artificial sequence

<220>

<221>CONFLICT

<222>(1)..(22)

<223>FR1

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Leu Gln Glu Ser Gly Gly Gly Ser Val Gln Ser Gly Gly Ser Leu Arg

1 5 10 15

Leu Thr Cys Ala Ala Ser

20

<210>2

<211>16

<212>PRT

<213> Artificial sequence

<220>

<221>CONFLICT

<222>(1)..(16)

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<400>2

Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val Ala Ser Ile

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<210>3

<211>37

<212>PRT

<213> Artificial sequence

<220>

<221>CONFLICT

<222>(1)..(37)

<223>FR3

<400>3

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

1 5 10 15

Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu Gln Pro Glu Asp Thr Ala

20 25 30

Met Tyr Tyr Cys Ala

35

<210>4

<211>11

<212>PRT

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<220>

<221>CONFLICT

<222>(1)..(11)

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<400>4

Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser

1 5 10

<210>5

<211>10

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<221>CONFLICT

<222>(1)..(10)

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Gly Tyr Thr Tyr Ser Asn Tyr Tyr Val Gly

1 510

<210>6

<211>8

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Asp Thr Leu Gly Tyr Thr Arg Tyr

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<210>7

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Leu Gln Glu Ser Gly Gly Gly Ser Val Gln Ser Gly Gly Ser Leu Arg

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ctgcaggagt ctgggggagg ctcggtgcag tccggagggt ctctgagact cacctgtgca 60

gcctctggat acacctacag taactactac gtgggctggt tccgccaggc tccaggaaag 120

gagcgcgagg gggtcgcaag tattgatact cttggttata caagatacgc agactccgtg 180

aaggggcgat tcaccatctc ccgtgacaac gccaagaaca cgctgtatct gcaaatgaac 240

agcctgcaac cggaggacac ggccatgtat tactgtgcgg cccctcgttc tccttactac 300

cggggtcaga cgttctggga gggggcgtat aactactggg gccaggggac ccaggtcacc 360

gtctcctca 369

Claims

1. A nanobody comprising an epitope-complementing region and a framework region; the method is characterized in that:

the antigenic determinant complementary region of the nanobody consists of CDR1, CDR2 and CDR 3;

the amino acid sequence of the CDR1 is the 23 rd to 32 th amino acid of SEQ ID No.8 in the sequence table;

the amino acid sequence of the CDR2 is amino acid 49-56 of SEQ ID No.8 in the sequence table;

the amino acid sequence of the CDR3 is amino acid 94-112 of SEQ ID No.8 in the sequence table;

the amino acid sequence of the nano antibody is shown as SEQ ID No.8 in a sequence table.

2. The biological material related to the nanobody of claim 1, which is any one of B1) to B12):

B1) a nucleic acid molecule encoding the nanobody of claim 1;

B2) an expression cassette comprising the nucleic acid molecule of B1);

B3) a recombinant vector comprising the nucleic acid molecule of B1);

B4) a recombinant vector comprising the expression cassette of B2);

B5) a recombinant microorganism comprising the nucleic acid molecule of B1);

B6) a recombinant microorganism comprising the expression cassette of B2);

B7) a recombinant microorganism containing the recombinant vector of B3);

B8) a recombinant microorganism containing the recombinant vector of B4);

B9) a transgenic animal cell line comprising the nucleic acid molecule of B1);

B10) a transgenic animal cell line comprising the expression cassette of B2);

B11) a transgenic animal cell line containing the recombinant vector of B3);

B12) a transgenic animal cell line containing the recombinant vector of B4);

the nucleic acid molecule is:

the nucleotide sequence is cDNA molecule or DNA molecule of SEQ ID No.9 in the sequence table.

3. The nanobody derivative antibody of claim 1, which is a) or b) or c) or d) or e):

a) a single chain antibody comprising the nanobody of claim 1;

b) a fusion antibody comprising a) said single chain antibody;

c) a fusion antibody comprising the nanobody of claim 1;

d) a Fab comprising the nanobody of claim 1;

e) an intact antibody comprising a nanobody according to claim 1.

4. The method for preparing the nanobody of claim 1, which comprises introducing a nucleic acid molecule encoding the nanobody of claim 1 into a recipient cell to obtain a transgenic cell expressing the nanobody, and culturing the transgenic cell to obtain the nanobody, wherein the nucleotide sequence of the nucleic acid molecule encoding the nanobody of claim 1 is represented by SEQ id No.9 of the sequence listing.

5. The method of claim 4, wherein: the recipient cell is a microbial cell.

6. Any one of the following uses A1-A4:

a1, use of the nanobody of claim 1 for binding to a T cell expressing PD-1;

use of a2, the biomaterial of claim 2, for binding to a T cell expressing PD-1;

use of a3, the derivatized antibody of claim 3, for binding to a T cell expressing PD-1;

use of A4, the method of claim 4, in binding to a T cell expressing PD-1.