CN108218988A - Nano antibody PD-1/Nb52 of anti-PD-1 and preparation method and application - Google Patents

Abstract

The invention discloses anti-PD-1 nanobody PD-1/Nb52 and its preparation method and application. The nanobody provided by the present invention comprises an epitope complementary region and a framework region of the Nanobody PD‑1/Nb52; the epitope complementary region of the Nanobody PD‑1/Nb52 consists of CDR1, CDR2 and CDR3; by The nucleotide sequence and host cell of the Nanobody PD-1/Nb52 disclosed in the present invention, the Nanobody PD-1/Nb52 can be efficiently expressed in Escherichia coli, and is applied to the research and development of PD-1 molecular detection reagents, preparation of inhibitory PD‑1 activity and the preparation of tumor suppressors or tumor cell suppressors and drugs that promote T cell proliferation.

Description

Anti-PD-1 nanobody PD-1/Nb52 and its preparation method and application

technical field

The invention relates to anti-PD-1 nanobody PD-1/Nb52 in the field of biomedicine and its preparation method and application.

Background technique

Programmed death receptor 1 (programmed death 1, PD-1) is a transmembrane glycoprotein molecule expressed on the surface of activated T cells, which is a member of the CD28 superfamily and negatively regulates the proliferation of T cells. play an important regulatory role. PD-1 is mainly expressed in activated T cells and B cells, and is a surface receptor of activated T cells. PD-1 has two ligands, programmed cell death-ligand 1 (PD-L1, Also known as B7-H1) and programmed cell death-ligand 1 (PD-L2, also known as B7-DC). The tumor microenvironment in the body will induce infiltrating T cells to highly express PD-1 molecules, and tumor cells will highly express PD-1 ligands PD-L1 and PD-L2, resulting in the continuous activation of PD-1 pathway in the tumor microenvironment. After -L1 is combined with PD-1, the function of T cells is inhibited, and it cannot send a signal to the immune system to attack the tumor. PD-1 inhibitors can block the combination of PD-1 and PD-L1, block negative regulatory signals, and restore the activity of T cells, thereby enhancing the immune response. Anti-PD-1 monoclonal antibodies with high specificity and low reactivity have been affirmed in many clinical trials, such as the drug named Keytruda (Pembrolizumab) produced by Merck & Co., and the drug named Opdivo (Nivolumab) nivolumab produced by Bristol-Myers Squibb. medicine.

However, there are many problems in the application of antibody drugs, such as long research and development cycle, high production cost, difficulty in large-scale production, poor stability and easy degradation, high storage cost, easy contamination, high maintenance cost, and immunogenicity, etc. its scope of clinical application.

Nanobody technology is the latest and smallest antibody molecule developed by biomedical scientists on the basis of traditional antibodies, using molecular biology technology combined with the concept of nanoparticle science to carry out an antibody engineering revolution. In 1993, Hamers et al. reported that there are heavy chain antibodies naturally missing the light chain and heavy chain constant region 1 (CH1) in camels, and their variable regions were cloned to obtain single domain antibodies consisting of only one heavy chain variable region, called VHH (variable domain of heavychain of heavy-chain antibody), has been renamed as "nanobody" (nanobody, Nb). Nanobodies are the smallest antigen-binding fragments with complete functions, and their crystal structures are elliptical, 2.5nm in diameter and 4nm long. Nb has many unique properties and is very suitable for genetic modification, showing broad application prospects in the precise diagnosis and targeted therapy of diseases. Nanobodies are much simpler than antibodies in chemical composition and shape, do not have chemical hydrophobicity, have stronger heat resistance and acid and alkali resistance, are easier to combine with each other or with other compounds, can be encoded by a single gene, and are easily synthesized by microorganisms . Nanobodies have good tolerance to the environment, have high conformational stability, and have smaller molecular weights, which have better clinical therapeutic effects. At the same time, these small protein molecules are easier to synthesize and lower in price. The unique properties of nanobodies make them more promising in the precise diagnosis of diseases and targeted immune therapy.

Contents of the invention

The technical problem to be solved by the present invention is how to prepare drugs for effectively treating tumors.

In order to solve the above technical problems, the present invention firstly provides nanobodies.

The first aspect of the present invention provides a nanobody called PD-1/Nb52, which comprises an epitope complementary region CDR and a framework region FR; the epitope complementary region CDR of the nanobody consists of CDR1 , CDR2 and CDR3; the amino acid sequence of the CDR1 is the 23-32 amino acid of SEQ ID No.8 in the sequence listing; the amino acid sequence of the CDR2 is the 49-56 of SEQ ID No.8 in the sequence listing Amino acid; the amino acid sequence of the CDR3 is amino acid 94-112 of SEQ ID No. 8 in the sequence listing.

Preferably, the framework region FR of the nanobody is composed of FR1, FR2, FR3 and FR4; wherein the amino acid sequence of FR1 is amino acid 1-22 of SEQ ID No.8 in the sequence listing; the amino acid sequence of FR2 is It is the 33-48 amino acid of SEQ ID No.8 in the sequence listing; the amino acid sequence of the FR3 is the 57-93 amino acid of SEQ ID No.8 in the sequence listing; the amino acid sequence of the FR4 is SEQ ID No.8 in the sequence listing. Amino acids 113-123 of ID No.8.

Preferably, the amino acid sequence of the Nanobody PD-1/Nb52 is shown in SEQ ID No.8 in the sequence listing.

The present invention also provides a VHH chain of a PD-1 nanobody PD-1/Nb52 correspondingly, including a framework region FR and an epitope complementary region CDR, and the framework region FR is selected from the amino acid sequence of the FR of the following group: SEQ FR1 shown in ID NO: 1, FR2 shown in SEQ ID NO: 2, FR3 shown in SEQ ID NO: 3, FR4 shown in SEQ ID NO: 4; the CDR of the complementary region of the antigenic determinant is selected from the following group Amino acid sequences of the CDRs: CDR1 shown in SEQ ID NO:5, CDR2 shown in SEQ ID NO:6, and CDR3 shown in SEQ ID NO:7.

Preferably, the VHH chain of the PD-1 Nanobody PD-1/Nb52 has the amino acid sequence shown in SEQ ID NO:8.

In order to facilitate the purification of the Nanobody PD-1/Nb52, the amino-terminal or carboxyl-terminal of the protein shown in amino acids 1-123 of SEQ ID No. 8 in the sequence listing can be linked with a label as shown in Table 1 .

Table 1. Sequence of tags

Label Residues sequence Poly-Arg 5-6 (usually 5) RRRRR Poly-His 2-10 (usually 6) HHHHHH FLAG 8 DYKDDDDK Strep-tag II 8 WSHPQFEK c-myc 10 EQKLISEEDL HA 9 YPYDVPDYA

In another aspect of the present invention, the Nanobody PD-1/Nb52 can be synthesized artificially, or its coding gene can be firstly synthesized and then biologically expressed. The coding gene of the nanobody PD-1/Nb52 can be obtained by deleting the codon of one or several amino acid residues in the DNA sequence shown in SEQ ID No.9 in the sequence listing, and/or adding one or several bases The right missense mutation, and/or the coding sequence of the tag shown in Table 1 is connected at its 5' end and/or 3' end.

To solve the above technical problems, the present invention also provides biological materials related to the Nanobody PD-1/Nb52.

The biological material related to the Nanobody PD-1/Nb52 provided by the present invention is any one of B1) to B12):

B1) a nucleic acid molecule encoding said Nanobody PD-1/Nb52;

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

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

B4) a recombinant vector containing the expression cassette described in B2);

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

B6) a recombinant microorganism containing the expression cassette described in B2);

B7) a recombinant microorganism containing the recombinant vector described in B3);

B8) a recombinant microorganism containing the recombinant vector described in B4);

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

B10) a transgenic animal cell line containing the expression cassette described in B2);

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

B12) A transgenic animal cell line containing the recombinant vector described in B4).

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

Among the above biological materials, the expression cassette containing the nucleic acid molecule encoding the Nanobody PD-1/Nb52 described in B2), also known as the PD-1/Nb52 gene expression cassette, refers to the ability to express the Nanobody in a host cell The DNA of PD-1/Nb52, the DNA may not only include a promoter that initiates the transcription of the nanobody PD-1/Nb52 gene, but also includes a terminator that terminates the transcription of the nanobody PD-1/Nb52 gene. Further, the expression cassette may also include an enhancer sequence.

An existing expression vector can be used to construct a recombinant vector containing the nanobody PD-1/Nb52 gene expression cassette.

In the above biological materials, the vector can be a plasmid, a cosmid, a phage or a viral vector.

In the above biological material, the recombinant vector can be a recombinant vector obtained by introducing the nucleic acid molecule of B1) into pComb3. In one embodiment of the present invention, B3) the recombinant vector is the encoding gene of the Nanobody PD-1/Nb52 (the nucleotide sequence is the 1st-369th core of SEQ ID No.9 in the sequence listing nucleotide) into the recombinant vector pComb3-PD-1/Nb52 obtained in pComb3, and the recombinant vector pComb3-PD-1/Nb52 expresses the Nanobody PD-1/Nb52 shown in SEQ ID No.8.

In the above biological materials, the microorganisms can be yeast, bacteria, algae or fungi.

In the above biological materials, the transgenic animal cell line does not include propagation materials; the recombinant microorganism can be a recombinant microorganism obtained by introducing the nucleic acid molecule of B1) into Escherichia coli WK6.

Those skilled in the art can easily use known methods, such as directed evolution and point mutation methods, to mutate the nucleotide sequence of the Nanobody PD-1/Nb52 described in B1) of the present invention. Those artificially modified nucleotides having 75% or more identity to the nucleotide sequence of the Nanobody PD-1/Nb52 in B1) of the present invention, as long as they encode the Nanobody PD-1/Nb52 Nb52 and having nanobody PD-1/Nb52 activity are all derived from the nucleotide sequence of the present invention and are equivalent to the sequence of the present invention.

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

The identity of 75% or more may be 75%, 80%, 85%, 90% or more.

Among the above biological materials, the nucleic acid molecules described in B1) are the following 1) or 2) or 3):

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

2) A cDNA molecule or genomic DNA molecule that has 75% or more identity to the nucleotide sequence defined in 1) and encodes the Nanobody PD-1/Nb52;

3) Hybridizing with the nucleotide sequence defined in 1) under stringent conditions, and encoding the cDNA molecule or genomic DNA molecule of the Nanobody PD-1/Nb52.

To solve the above technical problems, the present invention also provides derivative antibodies of the nanobody PD-1/Nb52.

The derivative antibody of the Nanobody PD-1/Nb52 provided by the present invention is the following a) or b) or c) or d) or e):

a) a single chain antibody containing said Nanobody PD-1/Nb52;

b) a fusion antibody containing the single-chain antibody described in a);

c) a fusion antibody comprising said Nanobody PD-1/Nb52;

d) a Fab containing said Nanobody PD-1/Nb52;

e) Whole antibodies comprising said Nanobody PD-1/Nb52.

In order to solve the above technical problems, the present invention also provides a preparation method of the Nanobody PD-1/Nb52.

The preparation method of the Nanobody PD-1/Nb52 provided by the present invention comprises introducing the nucleic acid molecule encoding the Nanobody PD-1/Nb52 into a recipient cell to obtain a transgene expressing the Nanobody PD-1/Nb52 cells, culturing the transgenic cells to obtain the nanobody PD-1/Nb52.

In the above preparation method of Nanobody PD-1/Nb52, the nucleotide sequence of the nucleic acid molecule encoding the Nanobody PD-1/Nb52 is shown in SEQ ID No.9 in the sequence listing.

In the above method for preparing nanobody PD-1/Nb52, the recipient cell may be a microbial cell, such as Escherichia coli, specifically Escherichia coli WK6.

In order to solve the problems of the technologies described above, the present invention also provides any application in the following A1-A8:

A1, the application of the Nanobody PD-1/Nb52 in the preparation of tumor suppressors or tumor cell suppressors;

A2, the application of the biological material in the preparation of tumor suppressor or tumor cell suppressor;

A3, the application of the derivative antibody of the nanobody PD-1/Nb52 in the preparation of tumor suppressor or tumor cell suppressor;

A4. Application of the preparation method of the nanobody PD-1/Nb52 in the preparation of tumor suppressors or tumor cell suppressors;

A5. Application of the Nanobody PD-1/Nb52 in the preparation of products that inhibit PD-1 activity or promote T cell proliferation;

A6. The application of the biological material in the preparation of products that inhibit PD-1 activity or promote T cell proliferation;

A7. The application of the derivative antibody in the preparation of products that inhibit PD-1 activity or promote T cell proliferation;

A8. Application of the preparation method of the Nanobody PD-1/Nb52 in the preparation of products that inhibit PD-1 activity or promote T cell proliferation.

The above-mentioned product may be a medicament.

The pair of primers for amplifying the nucleic acid molecule encoding the amino acid sequence shown in SEQ ID No. 8 in the sequence list or any fragment amino acid sequence also falls within the protection scope of the present invention.

The invention provides an anti-PD-1 nanobody PD-1/Nb52, a nucleotide sequence encoding the nanobody PD-1/Nb52 and a host cell, as well as a preparation method and application thereof. The nanobody PD-1/Nb52 can be highly expressed in Escherichia coli, and is applied to the development of PD-1 molecular detection reagents, the preparation of tumor inhibitors or tumor cell inhibitors, and the preparation of drugs that inhibit PD-1 activity and promote T cell proliferation .

Description of drawings

Figure 1 is the DNA electrophoresis image of nanobodies. The DNA bands in the gel wells from left to right are: the first lane is the molecular marker of 2000bp, the second lane is the PCR product, and the PCR product band is about 400bp;

Figure 2 is the electrophoresis of the SDS-PAGE of the PD-1 nanobody PD-1/Nb52 purified by nickel column resin gel affinity chromatography; lane M represents the protein molecular weight Marker, in KDa;

Figure 3A is the result of the binding experiment of Nanobody PD-1/Nb52 and 293T cells not transfected with PD-1; Figure 3B is the result of the binding experiment of Nanobody PD-1/Nb52 and 293T cells transfected with PD-1.

Detailed ways

The present invention will be further described in detail below in conjunction with specific embodiments, and the given examples are only for clarifying the present invention, not for limiting the scope of the present invention.

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

The materials and reagents used in the following examples can be obtained from commercial sources unless otherwise specified.

The Escherichia coli WK6 in the following examples is approved by the Wan Yakun Laboratory of the Institute of Life Sciences of Southeast University, and the public can obtain the biological material from Guangxi Medical University. This biological material is only used for repeating the relevant experiments of the present invention and cannot be used as Use for other purposes.

Embodiment 1, the preparation of nanobody

The present invention provides a nanobody derived from camel, its name is PD-1/Nb52, the amino acid sequence of the nanobody PD-1/Nb52 is shown in SEQ ID No.8 in the sequence listing, represented by SEQ ID No. 9 nucleotide sequence encoding.

The nucleotide electrophoresis pattern of the nanobody PD-1/Nb52 is shown in Figure 1, in which the first lane is a 2000bp molecular marker, and the rest of the wells are PCR products, and the PCR product band is about 400bp.

Replace the DNA fragment between the PstI and NotI recognition sequences of the vector pComb3 (Biovector product) with the DNA molecule shown in SEQID No.9, and keep the other sequences unchanged to obtain the recombinant vector pComb3-PD-1/Nb52, pComb3-PD- The only difference between 1/Nb52 and pComb3 is that the DNA segment between the PstI and NotI recognition sequences of pComb3 is replaced by the DNA molecule shown in SEQ ID No.9. The recombinant vector pComb3-PD-1/Nb52 expresses the Nanobody PD-1/Nb52 shown in SEQ ID No.8. The pComb3-PD-1/Nb52 was introduced into Escherichia coli WK6 to obtain the recombinant strain WK6-pComb3-PD-1/Nb52.

The specific preparation steps of nanobodies are as follows:

(1) Spread WK6-pComb3-PD-1/Nb52 on an LB plate containing ampicillin and glucose (in the LB plate, the concentrations of ampicillin and glucose are 100 μg/mL and 20 mg/mL, respectively), 25-37 Cultivate overnight (10-14 hours);

(2) Select a single colony and inoculate it in 5 mL of LB culture solution containing ampicillin (in the LB culture solution, the concentration of ampicillin is 100 μg/mL), and cultivate overnight (10-14 hours) on a shaker at 25-37° C.;

(3) Inoculate 1 mL of the overnight culture solution in step (2) into 300-350 mL of TB culture solution, culture on a shaker at 25-37°C until the OD value reaches 0.6-1, then add IPTG to obtain WK6-pComb3-PD- 1/Nb52 culture solution, so that the concentration of IPTG in the WK6-pComb3-PD-1/Nb52 culture solution is 1 mM, put the WK6-pComb3-PD-1/Nb52 culture solution at 20-30 ° C on a shaker (the Cultivate overnight (10-14 hours) at a rotational speed of 220-250rpm to obtain WK6-pComb3-PD-1/Nb52 induction solution;

(4) Centrifuge the WK6-pComb3-PD-1/Nb52 induction solution in step (3) at 4°C to collect the bacteria;

(5) Using literature (Zhu M, Hu Y, Li G, Ou W, Mao P, Xin S, Wan Y: Combining magnetic nanoparticles with biotinylated nanoparticles for rapid and sensitive detection of influenza H3N2.Nanoscale Res Lett 2014,9:528.) The infiltration method in the method was used to obtain the antibody crude extract;

(6) Using literature (Zhu M, Hu Y, Li G, Ou W, Mao P, Xin S, Wan Y: Combining magnetic nanoparticles with biotinylated nanoparticles for rapid and sensitive detection of influenza H3N2.Nanoscale Res Lett 2014,9:528.) Nanobody PD-1/Nb52 was prepared by nickel column ion affinity chromatography. The SDA-PAGE electrophoresis images of nanobody PD-1/Nb52 are shown in Figure 2, respectively, and the size of nanobody PD-1/Nb52 is about 15KDa. The results show that the purity of the nanobody PD-1/Nb52 obtained by the above method can reach more than 90%.

Example 2, Determination of Nanobody and PD-1 Binding Rate

Determination of the binding rate of Nanobody PD-1/Nb52 to PD-1 (direct method)

293T cells stably transfected with PD-1 were used to detect the binding rate of nanobody PD-1/Nb52 to PD-1, and the nanobody PD-1/Nb52 (1 μg) in Example 1 was added to 1-6×10 ⁶ above-mentioned 293T cells Incubate at 4°C in the dark for 20-40min, wash twice with PBS, add 5μl PE anti-HA tag antibody (abcam, Clone: 20B12) and incubate at 4°C for 20-40min, wash twice with PBS, put the sample on BACKMAN flow cytometry Cytometry, the results are shown in Figure 3B, and 293T cells not transfected with PD-1 were used as a control, as shown in Figure 3A. Figure 3A is the binding percentage of blank control and PD-1 nanobody PD-1/Nb52 respectively to 293T cells not transfected with PD-1; Figure 3B is the stable transformation of blank control and PD-1 nanobody PD-1/Nb52 respectively The binding percentage of 293T cells of PD-1; the horizontal axis in Figure 3A and 3B is the fluorescence intensity (PE), the vertical axis is the percentage of the number (% of Max), S2 represents the blank control, and S1 represents the PD-1 nanobody PD-1 /Nb52. From the results shown in the figure, it can be seen that PD-1 nanobody PD-1/Nb52 can well bind to 293T cells stably transfected with PD-1.

Finally, it should be noted that: the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those of ordinary skill in the art should understand that: it can still be Modifications are made to the technical solutions described in the foregoing embodiments, or equivalent replacements are made to some of the technical features; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the spirit and scope of the technical solutions of the various embodiments of the present invention.

SEQUENCE LISTING

<110> Guangxi Medical University

<120> Anti-PD-1 nanobody PD-1/Nb52 and its preparation method and application

<130> GY17100594

<160> 9

<170> PatentIn version 3.3

<210> 1

<211> 22

<212> PRT

<213> Artificial sequence

<220>

<221> CONFLICT

<222> (1)..(22)

<223>FR1

<400> 1

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

1 5 10 15

<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 Asp Lys

1 5 10 15

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

20 25 30

Met Tyr Tyr Cys Ala

35

<210> 4

<211> 11

<212> PRT

<213> Artificial sequence

<220>

<221> CONFLICT

<222> (1)..(11)

<223>FR4

<400> 4

Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser

1 5 10

<210> 5

<211> 10

<212> PRT

<213> Artificial sequence

<220>

<221> CONFLICT

<222> (1)..(10)

<223> CDR1

<400> 5

Gly Tyr Thr Tyr Ser Asn Tyr Tyr Met Gly

1 5 10

<210> 6

<211> 8

<212> PRT

<213> Artificial sequence

<220>

<221> CONFLICT

<222> (1)..(8)

<223> CDR2

<400> 6

Asp Thr Leu Gly Tyr Thr Arg Tyr

1 5

<210> 7

<211> 19

<212> PRT

<213> Artificial sequence

<220>

<221> CONFLICT

<222> (1)..(19)

<223> CDR3

<400> 7

Ala Pro Arg Ser Pro Tyr Tyr Arg Gly Gln Thr Phe Trp Glu Gly Ala

1 5 10 15

Tyr Asn Tyr

<210> 8

<211> 123

<212> PRT

<213> Artificial sequence

<400> 8

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 Gly Tyr Thr Tyr Ser Asn Tyr Tyr Met Gly

20 25 30

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

35 40 45

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

50 55 60

Thr Ile Ser Arg Asp Asn Asp Lys Asn Thr Leu Tyr Leu Gln Met Ser

65 70 75 80

Ser Leu Gln Pro Glu Asp Thr Ala Met Tyr Tyr Cys Ala Ala Pro Arg

85 90 95

Ser Pro Tyr Tyr Arg Gly Gln Thr Phe Trp Glu Gly Ala Tyr Asn Tyr

100 105 110

Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser

115 120

<210> 9

<211> 369

<212>DNA

<213> Artificial sequence

<400> 9

ctgcaggagt ctggaggagg ctcggtgcag tccggagggt ctctgagact cacctgtgca 60

gcctctggat aacacctacag taactactac atgggctggt tccgccaggc tccaggaaag 120

gagcgcgagg gggtcgcaag tattgatact cttggttata caagatacgc agactccgtg 180

aaggggcgat tcaccatctc ccgtgacaac gataagaaca cgctgtatct gcaaatgagc 240

agcctgcaac cggaggacac ggccatgtat tactgtgcgg cccctcgttc tccttatactac 300

cggggtcaga cgttctggga gggggcgtat aactactggg gccaggggac ccaggtcacc 360

gtctcctca 369

Claims (10)

1. nano antibody includes antigenic determinant complementary region；It is characterized in that：

The antigenic determinant complementary region of the nano antibody is made of CDR1, CDR2 and CDR3；

The amino acid sequence of the CDR1 is the 23-32 amino acids of SEQ ID No.8 in sequence table；

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

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

2. nano antibody according to claim 1, it is characterised in that：The nano antibody is by antigenic determinant complementation Area and the skeleton district's groups into.

3. nano antibody according to claim 1 or 2, it is characterised in that：The amino acid sequence of the nano antibody such as sequence In list shown in SEQ ID No.8.

Any one of 4. it is B1 with claims 1 or 2 or the 3 relevant biomaterials of nano antibody) to B12)：

B1 the nucleic acid molecules of claims 1 or 2 or 3 nano antibodies) are encoded；

B2) contain B1) expression cassettes of the nucleic acid molecules；

B3) contain B1) recombinant vectors of the nucleic acid molecules；

B4) contain B2) recombinant vector of the expression cassette；

B5) contain B1) recombinant microorganisms of the nucleic acid molecules；

B6) contain B2) recombinant microorganism of the expression cassette；

B7) contain B3) recombinant microorganism of the recombinant vector；

B8) contain B4) recombinant microorganism of the recombinant vector；

B9) contain B1) the transgenetic animal cell systems of the nucleic acid molecules；

B10) contain B2) the transgenetic animal cell system of the expression cassette；

B11) contain B3) the transgenetic animal cell system of the recombinant vector；

B12) contain B4) the transgenetic animal cell system of the recombinant vector.

5. biomaterial according to claim 4, it is characterised in that：B1) nucleic acid molecules for it is following 1) or 2) or 3)：

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

2) nucleotide sequence with 1) limiting has 75% or more than 75% homogeneity, and encodes described in claims 1 or 2 or 3 The cDNA molecules or genomic DNA molecule of nano antibody；

3) nucleotide sequence hybridization with 1) restriction, and encode claims 1 or 2 or 3 nano antibodies under strict conditions CDNA molecules or genomic DNA molecule.

6. the derivative antibody of claims 1 or 2 or 3 nano antibodies, for it is following a) or b) or c) or d) or e)：

A) single-chain antibody containing claims 1 or 2 or 3 nano antibodies；

B) contain the fusion antibody of a) single-chain antibody；

C) fusion antibody containing claims 1 or 2 or 3 nano antibodies；

D) Fab containing claims 1 or 2 or 3 nano antibodies；

E) complete antibody containing claims 1 or 2 or 3 nano antibodies.

7. the preparation method of claims 1 or 2 or 3 nano antibodies, including claims 1 or 2 or 3 nanometers will be encoded The nucleic acid molecules of antibody import the transgenic cell that recipient cell obtains expressing the nano antibody, and it is thin to cultivate the transgenosis Born of the same parents obtain the nano antibody.

8. according to the method described in claim 7, it is characterized in that：Described coding claims 1 or 2 or 3 nano antibodies Nucleic acid molecules nucleotide sequence as shown in SEQ ID No.9 in sequence table.

9. according to the method described in claim 7, it is characterized in that：The recipient cell is microbial cell.

10. any one of following A 1-A8 purposes：

The application of A1, claims 1 or 2 or 3 nano antibodies in tumor inhibitor or inhibiting tumour cells agent is prepared；

Application of any biomaterial in tumor inhibitor or inhibiting tumour cells agent is prepared in A2, claim 4-5；

Application of the derivative antibody in tumor inhibitor or inhibiting tumour cells agent is prepared described in A3, claim 6；

The application of A4,7 or 8 the method for claim in tumor inhibitor or inhibiting tumour cells agent is prepared；

A5, claims 1 or 2 or 3 nano antibodies inhibit PD-1 activity in preparation or T cell are promoted to be proliferated answering in product With；

Any biomaterial is in preparing inhibition PD-1 activity or promoting T cell proliferation product in A6, claim 4-5 Using；

Derivative antibody described in A7, claim 6 is preparing the application in inhibiting PD-1 activity or promoting T cell proliferation product；

A8, claim 7 or 8 or 9 the methods are preparing the application in inhibiting PD-1 activity or promoting T cell proliferation product.