CN106336460B

CN106336460B - Anti-human PD-1 protein antibody and coding gene and application thereof

Info

Publication number: CN106336460B
Application number: CN201510415116.6A
Authority: CN
Inventors: 华绍炳
Original assignee: Hangzhou Beiyi Pharmaceutical Co ltd
Current assignee: Hangzhou Beiyi Pharmaceutical Co ltd
Priority date: 2015-07-15
Filing date: 2015-07-15
Publication date: 2021-02-12
Anticipated expiration: 2035-07-15
Also published as: CN106336460A

Abstract

The invention discloses an anti-human PD-1 protein antibody, a coding gene and an application thereof, wherein the antibody comprises a heavy chain variable region and a light chain variable region, and the amino acid sequences of three hypervariable regions of the heavy chain variable region, namely CDRH1, CDRH2 and CDRH3, are respectively as follows: GGSFSGYYWS, EINHSGSTNYNPSLKS, GSPDSSRARGYYMDV, respectively; the amino acid sequences of three hypervariable regions CDRL1, CDRL2 and CDRL3 of the light chain variable region are RASQGIRNDLG, AASSLQS, LQHNSYPL respectively. The invention constructs a human single-chain antibody library, screens the single-chain antibody by adopting ribosome display technology, and the obtained anti-human PD-1 protein antibody can be specifically combined with human PD-1 protein, has high affinity, can be used for detecting cells expressing PD-1, can also be used as a fully human anti-human PD-1 antibody for treating human diseases, and effectively prevents and treats tumor diseases.

Description

Anti-human PD-1 protein antibody and coding gene and application thereof

Technical Field

The invention relates to the technical field of genetic engineering, in particular to an anti-human PD-1 protein antibody and a coding gene and application thereof.

Background

Mammalian T cells play an extremely important role in the immune system. T cells rely on Antigen Presenting Cells (APCs) or other molecules to digest foreign or harmful antigens and re-present them in an antigen form that can be recognized by T cells, so that T cells are activated. This process is closely regulated by many molecules, some of which promote T cell activation to ensure that the immune system is sufficiently immune to invading pathogens; some molecules play a role in inhibition, preventing the immune system from being over-activated.

There is a population of proteins on T cells involved in regulation, designated the CD28 Receptor Family (Receptor Family), including: CD28, CTLA-4, PD-1 (also known as CD279), ICOS and BTLA (Okazaki et al, curr. Opin. Immunol, 2002, 14: 391779-. In this family, CD28 was the first protein to be found to have a helper receptor function, and helper receptors within the family fall into two broad categories, one being the activating receptor responsible for transmitting stimulatory signals and the other being the inhibitory receptor transmitting inhibitory signals. On Antigen Presenting Cells (APC), there is a corresponding ligand molecule belonging to the B7 family.

Among the inhibitory receptors of T cells, the two most attractive receptors are Cytotoxic T Lymphocyte Antigen 4 (CTLA-4) and Programmed cell Death Protein-1 (PD-1, also known as CD 279). The ligands corresponding to CTLA-4 on antigen presenting cells are known to be B7-1 and B7-2; and the ligands corresponding to PD-1 are PD-L1 and PD-L2. Human PD-1 is a 55kDa type I transmembrane protein, the coding gene of which belongs to the immunoglobulin (Ig) gene superfamily (superfamily) (Agata et al. IntImmunol, 1996, 8: 765-772). Many studies have shown that binding of PD-L1 or PD-L2 to PD-1 inhibits activation of T cells (Freeman et al J Exp Med, 2000, 192: 1027-. PD-1 is expressed mainly in immune cells such as T cells, B cells and NK cells, but rarely in other tissues such as muscle, epidermis and nerve cells. Furthermore, high expression of PD-1 is often closely associated with activation of immune cells. These molecules are now referred to as "immune checkpoint" receptors and ligands.

However, the delicate and complex immune system is also subject to failure, which may be the primary cause of many diseases. For example, when the suppressive immunity function is abnormally weakened, the overreacting immune system may be hostile and begin to attack the own cell tissue, causing autoimmune diseases. PD-1 deleted or mutated mice develop a variety of autoimmune diseases, such as: autoimmune cardiomyopathy, lupus-like syndrome with rheumatoid arthritis and nephritis, encephalomyelitis, and rheumatoid arthritis (Nishimura et al Immunity, 1999, 11: 141-.

Under normal circumstances, the immune system has an "immune surveillance" mechanism that can recognize and destroy cancer cells. However, cancer cells also have "immune evasion" mechanisms, one of which is the expression of B7 family ligands outside self cancer cells, which inhibit the anti-tumor effects of T cells by interacting with T cell inhibitory receptors. Researchers found that high expression of PD-1 in Tumor-encapsulating Lymphocytes (TILs) and high expression of PD-L1 were closely related to various tumors such as lung Cancer, liver Cancer, stomach Cancer, breast Cancer, ovarian Cancer, pancreatic Cancer, melanoma, and throat Cancer (Konishi et al Clin Cancer Res, 2004, 10: 5094-.

The high expression of PD-1 and PD-L1 in these tumor cells often results in poor prognosis in these tumor patients. Scientists have therefore attempted to understand the mechanisms by which cancer cells evade immune surveillance, and to develop new therapies for these pathways that render cancer cells unable to suppress the immune response and thereby enhance the immune system's ability to fight cancer itself. A simple schematic of the activation process of T cells is shown in figure 1.

The high expression of PD-1 and PD-L1 is not only associated with the escape of cancer cells from immunity and thus to tumors, but also with the infection and amplification of viruses in humans. For example, Hepatitis B Virus (HBV) and Hepatitis C Virus (HCV) can cause high expression of PD-L1 in liver cells, thereby activating the PD-1 signaling pathway of T-effector cells, resulting in massive depletion of T cells and persistent infection of the virus (Boni et al.J Virol, 2007, 81: 4215-.

Thus, obtaining antibodies that neutralize human PD-1 and/or PD-L1 appears to be of great interest for the detection and treatment of a variety of diseases, such as the treatment of tumors and chronic viral infections (Blank et al cancer Immunol, 2005, 54: 307-314; Okazaki et al Intimuol, 2007, 19: 813-824).

Disclosure of Invention

The invention provides an anti-human PD-1 protein antibody, a coding gene and application thereof, wherein the antibody has high affinity with human PD-1 and can be used for detecting and treating human diseases.

The anti-human PD-1 protein antibody comprises a heavy chain variable region and a light chain variable region, wherein the amino acid sequences of three hypervariable regions of a heavy chain variable region, namely CDRH1, CDRH2 and CDRH3, are respectively as follows:GGSFSGYYWS、EINHSGSTNYNPSLKS、GSPDSSRARGYYMDV；

CDRH1 is located at positions 26-35 of the heavy chain variable region, CDRH2 is located at positions 50-65 of the heavy chain variable region, and CDRH3 is located at positions 98-112 of the heavy chain variable region;

the amino acid sequences of three hypervariable regions of a light chain variable region of the light chain variable region are respectively CDRL1, CDRL2 and CDRL3RASQGIRNDLG、AASSLQS、LQHNSYPL。

CDRL1 is located at position 24-34 of the light chain variable region; CDRL2 is located at positions 50-56 of the light chain variable region, and CDRL3 is located at positions 89-96 of the light chain variable region.

The amino acid sequences of the three hypervariable regions vary greatly over the variable regions of the antibody molecule, while the amino acid sequences of the regions between the hypervariable regions vary less. These three hypervariable regions are sterically complementary to antigenic determinants, and are therefore also referred to as Complementarity Determining Regions (CDRs), with the CDRs of the different heavy and light chains determining the specificity of the antibody for the antigen.

Preferably, the amino acid sequence of the heavy chain variable region is shown in SEQ ID No.1, and the amino acid sequence of the light chain variable region is shown in SEQ ID No. 2.

The antibody may be a whole antibody or an antigen-binding portion of a whole antibody. The whole antibody is preferably IgG4 type; the antigen binding portion is preferably a Fab fragment, Fab 'fragment, F (ab')₂Fragments or single chain antibodies, more preferably single chain antibodies.

The antigen binding part not only reserves the area capable of being specifically bound with the antigen, but also avoids the side effect caused by the antigenicity of the Fc fragment; the single-chain antibody has the advantages of easy penetration into tumor tissue, increased medicine concentration, small immunogenicity, short half-life period in vivo circulation, easy elimination, easy connection with toxin or enzyme gene to directly obtain immunotoxin or enzyme-labeled antibody, etc.

Antigen binding portions can be prepared by recombinant DNA techniques or by enzymatic/chemical cleavage of whole antibodies. The preparation method of the anti-human papilloma virus L1 protein single-chain antibody comprises the following steps: a human single-chain antibody library is constructed by adopting a method disclosed by Chinese patent document with publication number CN 1444651A, and an anti-human PD-1 protein antibody is screened from the human single-chain antibody library.

The invention also provides the coding gene of the anti-human PD-1 protein antibody, wherein the nucleotide sequence of the coding gene of the heavy chain variable region is shown as SEQ ID No.3, and the coding gene of the light chain variable region is shown as SEQ ID No. 4.

The invention also provides a recombinant vector or an expression system containing the coding gene. The original vector of the recombinant vector is pACT2 or pET27b or a full-length antibody expression vector.

The anti-human PD-1 protein antibody can be used for preparing anti-tumor drugs or detecting PD-1 expression cells. The anti-human PD-1 protein antibody can be specifically combined with human PD-1 protein, wherein the full-length recombinant human anti-human PD-1 antibody can be used for detecting cells expressing PD-1, and can also be used for preventing and treating tumor diseases.

Compared with the prior art, the invention has the beneficial effects that:

the invention obtains the anti-human PD-1 protein antibody by constructing a human single-chain antibody library and screening the single-chain antibody by adopting a ribosome display technology, wherein the antibody is a fully human anti-PD-1 protein antibody, can be specifically combined with human PD-1 protein, has high affinity with the PD-1 protein, can be used for detecting cells expressing PD-1, can also be used for treating human diseases, and can effectively prevent and treat tumor diseases.

Drawings

FIG. 1 is a schematic diagram showing the process of activating T cells by binding PD-1 protein antibody to PD-1.

FIG. 2 is a schematic diagram showing the construction of DNA sequences of fully human antibody libraries (T7-VH-Vkappa-Ckappa and T7-VH-Vlambda-Ckappa) for ribosome display according to the present invention.

FIG. 3 is a schematic diagram of the process for screening single-chain antibodies using ribosome display technology according to the present invention.

FIG. 4 is a schematic diagram of the regeneration process of the single-chain antibody expression system in the process of screening single-chain antibodies using ribosome display technology according to the present invention.

FIG. 5 shows the ELISA detection results of anti-human PD-1 single-chain antibody # HPD1-A and purified human PD-1 protein.

FIG. 6 shows the results of the full-length antibody QAV122-A against human PD-1 and the nonspecific antibody human IgG inducing the secretion of gamma interferon from human peripheral blood mononuclear cells.

FIG. 7 shows the results of inhibition of hepatoma cell Hep3B/PD-L1 transfected with PD-L1 in SCID mice by anti-human PD-1 full-length antibody QAV122-A, nonspecific antibody human IgG and PBS control group (arrow indicates the time of antibody drug injection).

Detailed Description

The present invention will be described in further detail with reference to the accompanying drawings and specific embodiments.

Example 1 construction of fully human Single chain antibody library for ribosome display

Ribosome display DNA sequence construction process using fully human antibody libraries (T7-VH-Vkappa-Ckappa and T7-VH-Vlambda-Ckappa) is shown in FIG. 2.

The specific construction process is as follows:

1. amplifying to obtain human antibody heavy chain and light chain variable region DNA

Poly A + RNA (purchased from Clontech) from human bone marrow, human fetal liver, human spleen and human peripheral blood leukocytes was used as a template for reverse transcription of poly A + RNA into cDNA using oligo (dT) and random primers (purchased from Clontech) using a reverse transcriptase kit (purchased from Clontech) according to the protocol guidelines provided by the Clontech kit.

The above cDNA was used as a template, and PCR amplification was performed using a series of primers recognizing the human antibody heavy chain variable region (VH) and light chain variable region (VL) genes to obtain the DNA sequences of all heavy chain variable regions and light chain variable regions in human antibodies. A series of primers recognizing the variable region genes of the heavy and light chains of the human antibody have the following sequences:

the first group is 5' -end primers (SEQ ID Nos. 11-17) for amplifying human antibody heavy chain variable region (VH) genes, comprising:

VH1b：5’-CTATAGGAACAGACCACCATGCAGGTGCAGCTGCAGGAGTC(C/G)G-3’；

VH2b：5’-CTATAGGAACAGACCACCATGCAGGTACAGCTGCAGCAGTCA-3’；

VH3b：5’-CTATAGGAACAGACCACCATGCAGGTGCAGCTACAGCAGTGGG-3’；

VH4b：5’-CTATAGGAACAGACCACCATGGAGGTGCAG CTG(G/T)TGGAG(A/T)C(C/T)-3’；

VH5b：5’-CTATAGGAAC AGACCACCATGCAGGTCCAGCT(G/T)GT(A/G)CAGTCTGG-3’；

VH6b：5’-CTATAGGAACAGACCACCATGCAG(A/G)TCACCTTGAAGGAGTCTG-3’；

VH7b：5’-CTATAGGAACAGACCACCATGCAGGTGCAGCTGGTG(C/G)A(A/G)TCTGG-3’；

and the second group is 3' -end primers (SEQ ID No. 18-23) for amplifying human antibody heavy chain variable region (VH) genes, which comprise:

VH1f：5’-GCCGCCTGATCCACCACCGCCTGAGGAGAC(A/G)GTGACCAGGGTG-3’；

VH2f：5’-GCCGCCTGATCCACCACCGCCTGAGGAGACGGTGACCAGGGTT-3’；

VH3f：5’-GCCGCCTGATCCACCACCGCCTGAAGAGACGGTGACCATTGT-3’；

VH4f：5’-GCCGCCTGATCCACCACCGCCTGAGGAGACGGTGACCGTGGTCC-3’；

VH5f：5’-GCCGCCTGATCCACCACCGCCGGTTGGGGCGGATGCACTCC-3’；

VH6f：5’-GCCGCCTGATCCACCACCGCC(C/G)GATGGGCCCTTGGTGGA(A/G)GC-3’；

and a third group of primers (SEQ ID Nos. 24 to 32) for amplifying a variable region (V.lambda.) gene of a human antibody lambda-light chain, comprising:

VL1b：5’-GGCAGCGGTGGTGGAGGCAGTCAGTCTGT(C/G)(C/G/T)TGACGCAGCCGCC-3’；

VL2b：5’-GGCAGCGGTGGTGGAGGCAGTTCCTATG(A/T)GCTGAC(A/T)CAGCCAC-3’；

VL3b：5’-GGCAGCGGTGGTGGAGGCAGTTCCTATGAGCTGA(C/T)(A/G)CAGC(C/T)ACC-3’；

VL4b：5’-GGCAGCGGTGGTGGAGGCAGTCAGCCTGTGCTGACTCA(A/G)(C/T)C-3’；

VL5b：5’-GGCAGCGGTGGTGGAGGCAGTCAG(A/G/T)CTGTGGTGAC(C/T)CAGGAGCC-3’；

VL6b：5’-GGCAGCGGTGGTGGAGGCAGTCAGCC(A/T)G(G/T)GCTGACTCAGCC(A/C)CC-3’；

VL7b：5’-GGCAGCGGTGGTGGAGGCAGTTCCTCTGAGCTGA(C/G)TCAGGA(C/G)CC-3’；

VL8b：5’-GGCAGCGGTGGTGGAGGCAGTCAGTCTG(C/T)(C/T)CTGA(C/T)TCAGCCT-3’；

VL9b：5’-GGCAGCGGTGGTGGAGGCAGTAATTTTATGCTGACTCAGCCCC-3’；

and a fourth group of primers (SEQ ID Nos. 33 to 34) for amplifying a variable region (V.lambda.) gene of a human antibody lambda-light chain, comprising:

VL1f：5’-AGATGGTGCAGCCACAGTTCGTAGGACGGT(C/G)A(C/G)CTTGGTCC-3’；

VL2f：5’-AGATGGTGCAGCCACAGTTCGGAGGACGGTCAGCTGGGTGC-3’；

and a fifth group of primers (SEQ ID Nos. 35 to 38) for amplifying a human antibody k-light chain variable region (Vk) gene at the 5' -end, comprising:

Vκ1b：5’-GGCAGCGGTGGTGGAGGCAGTGACATCC(A/G)G(A/G/T)TGACCCAGTCTCC-3’；

Vκ2b：5’-GGCAGCGGTGGTGGAGGCAGTGAAATTGT(A/G)(A/T)TGAC(A/G)CAGTCTCC-3’；

Vκ3b：5’-GGCAGCGGTGGTGGAGGCAGTGATATTGTG(A/C)TGAC(C/G/T)CAG(A/T)CTCC-3’；

Vκ4b：5’-GGCAGCGGTGGTGGAGGCAGTGAAACGACACTCACGCAGTCTC-3’；

sixth group, 3' -end primer (SEQ ID No.39) for amplifying human antibody k-light chain constant region (Vk) gene:

Cκf：5’-CTCTAGAACACTCTCCCCTGTTGAAGCTCTTTGTGACGGGCGAGCTCAGGCCCTGATGGGT GACTTCGCAGGCGTAGACTTTG-3’。

when the heavy chain variable region (VH) in the human antibody is amplified, a first group of primers and a second group of primers are combined, namely 42 PCR reactions are carried out; when the lambda-light chain variable region (V lambda) in the human antibody is amplified, 18 PCR reactions are carried out by using the combination of the third group of primers and the fourth group of primers; when a kappa light chain variable region (Vkappa) in a human antibody is amplified, 4 PCR reactions are performed by using the combination of the fifth primer set and the sixth primer set.

The first group of primers comprises a partial T7 promoter sequence, a Kozak sequence (CCACC) and a translation initiation coding sequence ATG; the fourth and sixth set of primers contained the sequence of the human kappa light chain constant region (underlined); the second, third and fifth primers contain linker peptide sequences (underlined) for linking the heavy chain variable region and the light chain variable region of the antibody.

During amplification, the PCR reaction system and the reaction conditions are the same, and the PCR reaction system is as follows:

the components are mixed evenly and then placed in a PCR instrument for reaction. The reaction conditions are as follows: melting at 94 ℃ for 1 min, annealing at 50 ℃ for 1 min, extension at 72 ℃ for 2.5 min, and cycling 30 times.

2. Sequences linking the T7 promoter sequence, the linker peptide sequence and the kappa light chain constant region

A T7 promoter sequence, a Kozak sequence and a translation initiation coding ATG are added at the 5' -end of the heavy chain variable region; and a linker peptide sequence is added to the 3' -end thereof. Adding a connecting peptide sequence at the 5' -end of a variable region of the lambda light chain; while the invariant region of the kappa light chain was added at its 3' -end. The end of the 3' -terminal kappa light chain constant region does not contain a translation termination codon (stop codon).

(1) Combination T7 promoter sequence-human antibody heavy chain variable region (VH) sequence-linker peptide sequence

Taking human antibody heavy chain variable region DNA obtained by PCR (first group and second group of primers) in the step 1 as a template, and respectively taking a primer 7 and a primer 8 as an upstream primer and a downstream primer, wherein the sequences are as follows:

the forward primer (primer 7, i.e., T7Kz, SEQ ID No.40, underlined T7 promoter sequence):

5’-GCAGCTAATACGACTCACTATAGGAACAGACCACCATG-3’；

the downstream primer (primer 8, SEQ ID No.41, for the linker peptide reverse strand sequence):

5’-ACTGCCTCCACCACCGCTGCCACCTCCGCCAGATCCTCCGCCGCCTGATCCACCACCGCC-3’；

PCR amplification was carried out under the same conditions and system as in step 1 (step 1 refers to the first part of this example, the part of the human antibody heavy and light chain variable region DNA amplified). After completion of the reaction, the DNA sequence of the heavy chain variable region of the human antibody contained the T7 promoter and Kozak sequence at the 5 '-end and a peptide reverse-chain sequence (i.e., T7-Kozak-VH-linker) at the 3' -end.

(2) Combinatorial linker peptide sequences-human antibody lambda light chain variable region (V.lambda.) sequences-and sequences of kappa light chain constant region (Ckappa)

The invariant sequence of the kappa chain was obtained from the human kappa light chain DNA amplification obtained in step 1 using the following two primers:

Cκb(SEQ ID No.42)：5’-CGAACTGTGGCTGCACCATCTGT-3’；

Cκf(SEQ ID No.39)

the template DNA for PCR was human kappa light chain DNA obtained from step 1. The conditions and system of the PCR reaction were the same as those in step 1. The kappa chain constant region (Ck) DNA sequence (SEQ ID No.43) obtained after amplification;

SEQ ID No.43 is as follows: 5'-CGAACTGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGTTCTAGAG-3', respectively;

the human antibody lambda light chain variable region (V lambda) DNA obtained by PCR (third and fourth group primers) in step 1 is used as a template, and a primer 9 and a Ckappa f (SEQ ID No.39) are respectively used as an upstream primer and a downstream primer, and the sequences are as follows:

upstream primer (primer 9, SEQ ID No.44, linker peptide cis-strand sequence):

5’-GGCGGTGGTGGATCAGGCGGCGGAGGATCTGGCGGAGGTGGCAGCGGTGGTGGAGGCAGT-3’；

the kappa chain constant region (C kappa) DNA sequence (SEQ ID No.43) was mixed and PCR amplification was performed under the same reaction conditions and system as in step 1.

After the reaction is completed, a human antibody lambda light chain variable region (V lambda) DNA sequence-kappa chain constant region (C kappa) DNA sequence (i.e., linker peptide-V lambda-C kappa) containing a 5' -linker peptide cis chain sequence is obtained.

3. Construction of ribosome display human Single chain antibody Gene library

Mixing the PCR products obtained in the above steps, namely: the human antibody heavy chain variable region DNA (T7-Kozak-VH-connecting peptide) containing the T7 promoter, the Kozak sequence and the connecting peptide sequence obtained by PCR amplification in the step 2(1), the DNA sequence (connecting peptide-V lambda-C kappa) of the connecting peptide-human antibody lambda light chain variable region DNA (V lambda) -kappa chain constant region (C kappa) in the step 2(2), and the kappa chain variable region (V kappa) and invariant region (C kappa) DNA products (connecting peptide-V kappa-C kappa) obtained by PCR amplification of the fifth group and the sixth group of primers in the step 1 are mixed, and PCR amplification is carried out by taking the mixed DNA as a template and taking the primer 7(T7Kz, SEQ ID No.40) and the primer Ckf (SEQ ID No.39) as an upstream primer and a downstream primer respectively, and the reaction conditions and the system are the same as the step 1.

As shown in fig. 2, after the reaction was completed, a human single chain antibody (scFv) DNA library for ribosome display comprising T7 promoter-Kozak sequence-ATG human antibody heavy chain variable region DNA sequence (VH), linker peptide DNA sequence, light chain variable region DNA sequence, and kappa light chain constant region DNA sequence (ck) was obtained. The human single-chain antibody does not contain a translation termination codon (stop codon) at the end of the 3' -terminal kappa light chain constant region. In ribosome translation, a protein-ribosome-mRNA complex is formed without falling off from mRNA or releasing translated protein or polypeptide in the absence of a terminator.

Example 2 ribosome display screening of fully human Single chain antibodies binding to PD-1 protein

The process of ribosome display screening for single chain antibodies is shown in FIG. 3. The scFv single-chain Antibody library DNA prepared in example 1 above, which does not contain a terminator, is transcribed and translated in vitro to produce a complex ("ARM complex") formed by Antibody-Ribosome (Ribosome) -mRNA.

The antibodies on the ARM complex are screened with the antigen immobilized on a solid phase. After eluting the non-specific ARM complex, obtaining DNA capable of combining with antigen and antibody by using an RT-PCR method. The full-length DNA for ribosome display is obtained by adding T7 promoter again by PCR method.

(1) Biotin coupling of PD-1 protein to avidin magnetic beads

PD-1 protein was purchased from Beijing Yiqiao Shenzhou Biotechnology Co., Ltd; EZ-link^TMsulfo-NHS-LC-biotin was purchased from Pierce; avidin magnetic beads (Dynabeads M-280 streptavidin) were purchased from Dynal.

PD-1 protein (phosphate buffered saline PBS, pH 7.5) was mixed with sulfo-NHS-LC-biotin (mixing ratio: 25. mu.g protein: 1. mu.g sulfo-NHS-LC-biotin), reacted at 25 ℃ for 30 minutes at room temperature, and reacted at 4 ℃ overnight with 2X 500 ml PBS, pH 7.5.

The PD-1 protein coupled with biotin can be used for coupling to avidin magnetic beads. 50 microliters of avidin magnetic beads (Dynabeads M-280 streptavidin) were washed 3 times with 300 microliters of 0.1M phosphate buffer (pH 7.5), and then resuspended in 50 microliters of PBS.

5. mu.g of biotin-coupled PD-1 protein was mixed with avidin magnetic beads (mixing ratio of protein to magnetic beads: 10. mu.g: 1 mg), and reacted at room temperature of 25 ℃ for 30 minutes. After 3 washes with 300. mu.l PBS, the PD-1 protein-coupled magnetic beads were resuspended in 50. mu.l PBS (containing 0.02% sodium azide SodiumAzide).

(2) Preparation of an "ARM Complex" library

Rabbit blood erythrocyte Lysate (Rabbit Reticulocyte Lysate TNT for T7) of the T7 System Combined transcription and translation (TNT) was purchased from Promega; AMV Reverse Transcriptase (AMV Reverse Transcriptase) was purchased from Promega; RNase-free DNase I (RNase-free DNase I) was purchased from Roche.

Incubate at 30 ℃ for 60 minutes.

After incubation, 14. mu.L of 10 XDnase I buffer (400mM Tris-HCl, pH7.5, 60mM MgCl) was added₂100mM NaCl) and 200 units of RNase-free DNase I, H₂O to 140. mu.L, and incubation at 30 ℃ for another 30 minutes was continued to digest the DNA. Then, 210. mu.L of cold (4 ℃) PBS (containing 5mM Mg Acetate) was added to generate the "ARM complex" library.

(3) PD-1 protein screening ARM complex library

Mixing 150 μ L of the ARM complex library obtained in the step (2) with 5 μ L of magnetic beads coupled with PD-1 protein, and slowly shaking the mixture at 4 ℃ for 2 hours; then, the beads were washed 3 times with 100 μ L cold PBS; add 20. mu.L of sterile water without RNase.

(4) In situ RT-PCR recovery of scFv antibody DNA

Since the mRNA sequence at the 3 ' -end of the above-mentioned screened ARM complex is occupied by ribosomes staying at the 3 ' -end, reverse transcription is performed during the recovery process using a primer (RT1) located at about 60 base positions upstream of the 3 ' -end of the mRNA. The process is shown in figure 4. RT-PCR kits were purchased from Roche.

The sequence of the RT1 primer (SEQ ID No.45) is as follows: RT 1: 5'-ACTTCGCAGGCGTAGAC-3', respectively;

in the RT-PCR reaction, the primer (Kz1) at the other end was used with a sequence containing Kozac sequence and ATG upstream of the mRNA. The sequence of primer Kz1 is as follows (SEQ ID No. 46): kz 1: 5'-GAACAGACCACCATG-3', respectively;

the RT-PCR reaction conditions were as follows:

the reaction conditions were as follows:

1 cycle: 48 ℃ for 45 minutes; 94 ℃ for 2 minutes; 35 cycles: 30 seconds at 94 ℃; 54 ℃ for 1 minute; at 68 ℃ for 2 minutes; finally, at 68 ℃, 7 minutes; then maintained at 4 ℃.

(5) Regeneration of full-Length scFv antibody DNA

The DNA product obtained in the above step (4) is shorter than the original DNA and does not contain the T7 promoter sequence. Full-length scFv antibody DNA was regenerated by PCR reaction using primer T7Kz (SEQ ID No.40) and primer Ckf (SEQ ID No. 39).

The reaction conditions are as follows:

the reaction conditions were as follows:

30 cycles: 30 seconds at 94 ℃; 54 ℃ for 1 minute; at 68 ℃ for 2 minutes; finally, at 68 ℃, 7 minutes; then maintained at 4 ℃.

The above-mentioned steps (2) to (5) were repeated for 3 cycles, the final PCR product was cloned into a plasmid vector using a PCR cloning kit (purchased from Life Technologies), and the finally obtained scFv single-chain antibody DNA was sequenced.

After the multi-step and multi-cycle enrichment and analysis, a single-chain antibody with specificity to human PD-1 protein is obtained, the number is # hPD1-A, an ABI automatic sequencer is used for sequencing and analyzing the single-chain antibody, and the result shows that:

the DNA sequence of the heavy chain variable region of # hPD1-A is shown in SEQ ID No. 3;

the amino acid sequence of the heavy chain variable region of # hPD1-A (SEQ ID No.1) is: QVQLQQWGAGLLKPSETLSLTCAVYGGSFSGYYWSWIRQPPGKGLEWIGEINHSGSTNYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARGS PDSSRARGYYMDVWGKGTTVTVSSGSASAPT；

Wherein, the underlined parts are three hypervariable regions of CDRH1, CDRH2 and CDRH3(SEQ ID Nos. 5-7) in sequence;

the DNA sequence of the light chain variable region of # hPD1-A is shown in SEQ ID No. 4;

the light chain variable region amino acid sequence of # hPD1-A (SEQ ID No.2) is: DIRLTQSPSSLSASVGDRVTITCRASQGIRNDLGWYQQKPGKAPKRLIYAASSLQSGVPSRFSGSGSATDFTLTISSLQPEDFATYYCLQHNSYPLTFGGGTKLEIK；

Wherein, the underlined parts are three hypervariable regions of CDRL1, CDRL2 and CDRL3(SEQ ID Nos. 8-10) in sequence.

EXAMPLE 3 Single chain antibody expression and purification

Cloning the coding gene of the single-chain antibody # hPD1-A into an expression vector pET27b (+) to construct pET27b-HPD1 a;

pET27b-HPD1a was transformed into the expression bacterium E.coli BL21(DE3) and expression was induced with IPTG (0.5mM) according to the method provided by Novagen; in the expressed target protein, the N end of the scFv is a pelB sequence, and the pelB sequence can secrete the expressed scFv into the periplasmic cavity (periplastmic space) of BL21(DE 3); the C end of the scFv contains an HSV tag and a 6 XHis tag, which facilitates the purification of the target protein;

the single-chain antibody of the anti-human PD-1 protein is conveniently obtained by separation and purification with a Ni-NTA column by the method provided by Qiagen company.

Example 4 ELISA testing of specificity of Single chain antibodies to human PD-1 protein

Recombinant human PD-1 protein (hPD1) was purchased from Beijing Yiqian Shenzhou Biotechnology, Inc. The C-terminal of the recombinant PD-1 protein carries a His tag and an Fc fragment of human IgG 1.

The ELISA test method was as follows:

(1) coating 96-well plate with hPD1 protein, and standing at 2-8 deg.C overnight;

(2) sealing the coated 96-pore plate by using SuperBlock;

(3) serially diluting single-chain antibody # hPD1-A in 0.02% BSA, adding into 96-well coated with hPD1 protein, and binding with hPD1 protein;

(4) cleaning a 96-well plate, adding a 5000-fold diluted antibody of a mouse anti-HSV marker to detect the combined single-chain antibody;

(5) cleaning a 96-well plate, and adding a 10000-fold diluted goat anti-mouse IgG antibody-horseradish peroxidase conjugate;

(6) after the 96-pore plate is finally cleaned, horse radish peroxidase substrate TMB reagent is applied for color development treatment;

(7) the reaction was stopped with 0.5M sulfuric acid and the absorption spectrum at 450nm was measured.

The results are shown in FIG. 5, which shows that the single-chain antibody # hPD1-A can effectively bind to hPD1 protein.

Example 5 affinity of full-Length anti-human PD-1 antibody to human PD-1

The Qingdao Ishualong Biotech Co., Ltd is entrusted to prepare the full-length recombinant human anti-human PD-1 protein antibody QAV122-A (the buffer solution is a conventional phosphate buffer solution, and the concentration is 1.0 mg/ml). The full-length QAV122-A is of the IgG4 type and has a variable region sequence identical to that of the single chain antibody # hPD 1-A. Human PD-1 protein (catalog No. 10377-H03H) was purchased from Beijing Yiqian Shenzhou Biotechnology, Inc., and is expressed by human cells.

BIAcore applying principle of Surface Plasmon Resonance (SPR)^TMThe T-200 instrument (GE Life Sciences) essentially employs conventional methods to detect the affinity constant of QAV122-A with PD-1 protein. The detection buffer was HBSN buffer (10mM HEPES pH 7.4, 0.15M NaCl, 3mM EDTA, 0.005% v/v surfactant P20, GE Healthcare).

Association rates (K)_on) And Dissociation rates (K)_off) Calculated according to the Langmuir affinity model (BIA Evaluation software, GE Life Sciences). Equilibrium affinity constant (K)_D) Is K_off/K_onThe ratio of (a) to (b).

The results are as follows:

TABLE 1 QAV122-A antibody affinity constants

Antibodies	K_on(M^-1，s^-1)	K_off(s)	K_D(M)
				QAV122-A	1.26×10⁵	8.93×10^-4	7.09×10^-9

Example 6 full-Length anti-human PD-1 antibody activates secretion of human Primary Peripheral Blood Mononuclear Cells (PBMC) Gamma Interferon

1. Establishment of Stable cell lines

HEK293 cells were derived from American ATCC (Rockville, Maryland). A cDNA encoding human PD-L1 (NCBI Access # NM-014143) was synthesized by Nanjing Kinshire and cloned into pcDNA3.1/Hygromycin plasmid.

The plasmid DNA with PD-L1 described above was transfected into HEK293 cells using conventional cell transfection techniques and screened in medium containing 200. mu.g/ml of Hygromycin (Sigma). The HEK293/PD-L1 cells after screening were cultured by limiting Dilution (Limited Dilution) to obtain a cell line (Fuller SA et al, 2001.Current Protocols of molecular biology, Chapter 11, section 11.8). The cell line expressing PD-L1 was analyzed by anti-PD-L1 antibody (catalog No. 17-5983, eBioscience, Inc., USA, San Diego) in combination with flow cytometry FACS, and cell lines expressing high amounts of PD-L1 were selected.

2. Anti-human PD-1 antibody activates secretion of gamma interferon from human PBMC

The majority (about 60-70%) of PBMC cells, human primary peripheral blood mononuclear cells, are T cells, which express PD-1 molecules on their surface. After pretreatment with anti-CD 3 antibody, T cells are activated to secrete interferon gamma when co-cultured with anti-PD-1 antibody and cells expressing PD-L1 on the surface.

PBMC were derived from peripheral blood of healthy donors and prepared by Ficoll Lymphocyte Separation Medium (Sigma-Aldrich, USA, Histopaque-1077) density gradient centrifugation method according to the supplier's method.

PBMC cells prepared as described above were first pretreated with anti-CD 3 monoclonal antibody (40ng/ml) (eBioscience, Calif., USA, catalog No. 16-0037) for 3 days; then, pretreated PBMC cells (1X 10)⁴) And HEK293/PD-L1 cell (3X 10) described above⁴) Co-culture in 96-well, equal cell culture plates for 18 hours in medium containing 1.0nM of QAV122-A, an anti-PD-1 antibody, or 1.0nM of a non-specific human IgG antibody (Sigma, USA); after 18 hours of culture, the culture supernatant was analyzed using a gamma interferon (gamma-IFN) ELISA kit (Beckton-Dickinson, USA). Secretion of interferon gamma is a very important indicator of T cell activation.

As shown in FIG. 6, anti-human PD-1 antibody can activate T cells of PBMC and cause T cells to efficiently secrete interferon-gamma, compared with non-specific human IgG.

Example 7 anti-human PD-1 antibody activates PBMC of human peripheral blood mononuclear cells and inhibits tumor growth in animal models

Human hepatoma tumor cell line Hep3B was derived from american ATCC (Rockville, Maryland). A cDNA encoding human PD-L1 (NCBI Access # NM-014143) was synthesized by Nanjing Kinshire and cloned into pcDNA3.1/Hygromycin plasmid.

The above plasmid DNA with PD-L1 was transfected into Hep3B cells using conventional cell transfection techniques, and selected for 14 days in a medium containing 200. mu.g/ml of Hygromycin (Sigma Co.); the selected Hep3B/PD-L1 cell line was cultured by dilution to obtain a cell line (Fuller SA et al, 2001.Current Protocols of Molecular biology. Chapter 11, section 11.8). The cell line expressing PD-L1 was analyzed by anti-PD-L1 antibody (catalog No. 17-5983, eBioscience, Inc., USA, San Diego) in combination with flow cytometry FACS, and human hepatoma tumor cell line Hep3B/PD-L1 with high expression level of PD-L1 was selected.

Tumors were induced in SCID mice according to the methods described in the literature (Pollack VA et al 1999, "Inhibition of Epidermal growth factor receptor phosphorylation in human carbonic mas with CP-358: Dynamics of receptor Inhibition in situ and receptors effects in therapy," J.Pharmacol. exp.Ther.291: 739-748 ").

Human liver cancer tumor cell line Hep3B/PD-L1 (about 3x 10)⁶Individual cells) and 50% Matrigel (Beckton-Dickinson, usa) preparations were subcutaneously injected into SCID mice (experimental animal technology ltd, viton, beijing) of 6-7 week males to induce tumors; after 15 days of tumor cell inoculation, tumors formed to about 100-200mm³In size, 5X 10 is injected into the tumor⁵Primary human peripheral blood mononuclear cells PBMC (PBMC cells from three healthy donors after mixing).

Three days after PBMC inoculation, these SCID mice were divided into three groups, and (1) anti-PD-1 antibody (QAV122-A), (2) non-specific human IgG protein (Sigma, USA), (3) phosphate buffered saline PBS was injected subcutaneously into the SCID mice. The antibody dose was 10mg/kg, and the same dose was re-injected every 10 days for a total of three injections. The size of the tumor was determined by measuring two diameters of the tumor with a vernier caliper and calculating the volume of the tumor (length x [ width ] according to the method described in the literature ("Protocols for screening chemical agents and natural products against biological systems." Cancer chemiher. Rep.3: 1-104.)) according to the method described in the literature]²)/2。

FIG. 7 shows the tumor size versus time after three days of PBMC inoculation, after subcutaneous injection of anti-PD-1 antibody (QAV122-A) or non-specific human IgG protein or PBS, respectively, into the SCID mice described above.

The detection result shows that the anti-PD-1 antibody QAV122-A has stronger tumor inhibition effect on tumor cells compared with a control group. After 40 days of anti-PD-1 antibody QAV122-A treatment, the tumor size was only about 50% of the control group.

Claims

1. The anti-human PD-1 protein antibody comprises a heavy chain variable region and a light chain variable region, and is characterized in that the amino acid sequences of three hypervariable regions of the heavy chain variable region, namely CDRH1, CDRH2 and CDRH3, are respectively as follows:GGSFSGYYWS、EINHSGSTNYNPSLKS、GSPDSSRARGYYMDV(ii) a The amino acid sequences of three hypervariable regions of a light chain variable region of the light chain variable region are respectively CDRL1, CDRL2 and CDRL3RASQGIRNDLG、AASSLQS、LQHNSYPL。

2. The anti-human PD-1 protein antibody according to claim 1, wherein the amino acid sequence of the heavy chain variable region is represented by SEQ ID No.1, and the amino acid sequence of the light chain variable region is represented by SEQ ID No. 2.

3. The anti-human PD-1 protein antibody according to claim 2, wherein said anti-human PD-1 protein antibody is a whole antibody.

4. The anti-human PD-1 protein antibody of claim 3, wherein the whole antibody is of the IgG4 type.

5. The encoding gene of the anti-human PD-1 protein antibody according to claim 2, wherein the nucleotide sequence of the heavy chain variable region encoding gene is shown in SEQ ID No.3, and the light chain variable region encoding gene is shown in SEQ ID No. 4.

6. A recombinant vector comprising the coding gene of claim 5.

7. An expression system comprising the encoding gene of claim 5.

8. The use of the anti-human PD-1 protein antibody according to any one of claims 1 to 4 for the preparation of an antitumor medicament, wherein the tumor is liver cancer.