CN113336848B

CN113336848B - anti-PD-1 antibody and application thereof

Info

Publication number: CN113336848B
Application number: CN202110152527.6A
Authority: CN
Inventors: 董坚; 孙亚如; 杨圣; 聂紫; 李文荣
Original assignee: Shanghai Life Medical Science & Technology Co ltd
Current assignee: Shanghai Life Medical Science & Technology Co ltd
Priority date: 2021-02-03
Filing date: 2021-02-03
Publication date: 2022-08-19
Anticipated expiration: 2041-02-03
Also published as: CN113336848A

Abstract

The invention discloses an anti-PD-1 antibody and a preparation method thereof. Specifically, the invention discloses a PD-1 antibody or an antigen-binding fragment thereof, which comprises an antibody heavy chain variable region shown as SEQ ID NO. 2 and an antibody light chain variable region shown as SEQ ID NO. 4. The anti-PD-1 antibody has high specificity and strong affinity, can effectively block the combination of PD-1 and PD-L1, and activates immune cells; in addition, the antibody of the invention can be prepared into a secretory antibody, is expressed on a chimeric antigen receptor immune cell, and plays an anti-tumor role together with the chimeric antigen receptor.

Description

anti-PD-1 antibody and application thereof

Technical Field

The invention relates to the field of biotechnology and antibodies, in particular to an anti-PD-1 antibody and application thereof in anti-tumor treatment.

Background

Immune checkpoint molecules (immunecheckpoint) are inhibitory regulatory molecules in the immune system that are important for maintaining self-tolerance, preventing autoimmune responses, and minimizing tissue damage by controlling the time and intensity of immune responses. The immune check point molecule is expressed on immune cells, and can inhibit the function of the immune cells, so that an organism cannot generate effective anti-tumor immune response, and tumors form immune escape. The tumor-associated immune checkpoint molecules are mainly: PD1, CTLA4, TIM3, TIGIT, LAG3, and other B7 and CD28 family molecules.

Protein programmed death 1(PD-1) is a 55kDa type I transmembrane protein that is part of the Ig gene superfamily, and PD-1 is expressed on activated B cells, T cells, and myeloid cells. There are two ligands for PD-1, PD-L1 and PD-L2, which, upon binding to PD-1, reduce T cell activation. PD-L1 is highly expressed in a variety of human tumors. The interaction between PD-1 and PD-L1 results in a decrease in tumor-infiltrating lymphocytes, a decrease in T cell receptor-mediated proliferation, and immune evasion of cancerous cells. Immunosuppression can be reversed by inhibiting the local interaction of PD-1 with PD-L1 and the effect is additive when the interaction of PD-1 with PD-L2 is also blocked. The immune checkpoint inhibitor is developed to block the action between tumor cells expressing immune checkpoints and immune cells, so as to block the inhibition of tumor cells on immune cells and obviously improve the activity of immune cells to inhibit tumors. The immune checkpoint inhibitor acts on the immune system of a patient to activate T cells, is suitable for treating various tumors, and becomes a universal therapy for the tumors.

Accordingly, there is a need in the art to develop agents that efficiently recognize PD-1 and methods of using such agents.

Disclosure of Invention

The present invention is directed to an anti-PD-1 antibody that effectively recognizes PD-1 molecules and its use in anti-tumor therapy.

In a first aspect of the present invention, there is provided a heavy chain variable region of an antibody, comprising the following three complementarity determining regions CDRs:

a CDR1 as set forth in SEQ ID No. 5;

a CDR2 as set forth in SEQ ID No. 6; and

CDR3 as shown in SEQ ID NO. 7.

In another preferred embodiment, the heavy chain variable region further comprises a human FR region or a murine FR region.

In another preferred embodiment, the heavy chain variable region has an amino acid sequence as shown in SEQ ID No. 2.

In a second aspect of the present invention, there is provided an antibody light chain variable region comprising the following three complementarity determining regions CDRs:

a CDR 1' as set forth in SEQ ID No. 8;

a CDR 2' as shown in SEQ ID No. 9; and

CDR 3' as shown in SEQ ID No. 10.

In another preferred embodiment, the light chain variable region further comprises an FR region of human or murine origin.

In another preferred embodiment, the light chain variable region has the amino acid sequence shown in SEQ ID No. 4.

In a third aspect of the invention, there is provided a PD-1 antibody, or antigen-binding fragment thereof, comprising an antibody heavy chain variable region according to the first aspect of the invention, and an antibody light chain variable region according to the second aspect of the invention;

alternatively, the antibody or antigen-binding fragment thereof comprises a heavy chain having the antibody heavy chain variable region of the first aspect of the invention, and a light chain having the antibody light chain variable region of the second aspect of the invention.

In another preferred example, the heavy chain variable region of the antibody or antigen binding fragment thereof has the amino acid sequence shown in SEQ ID No. 2, and the light chain variable region has the amino acid sequence shown in SEQ ID No. 4.

In another preferred embodiment, the heavy chain further comprises a heavy chain constant region.

In another preferred embodiment, the heavy chain constant region is of human or murine origin.

In another preferred embodiment, the light chain of the antibody further comprises a light chain constant region.

In another preferred embodiment, the light chain constant region is of human or murine origin.

In another preferred embodiment, the antibody is a double-chain antibody or a single-chain antibody.

In another preferred embodiment, the antibody is a full-length protein, or an antigen-binding fragment of an antibody.

In another preferred embodiment, the antibody is a monoclonal antibody.

In another preferred embodiment, the antibody is a partially or fully humanized monoclonal antibody.

In another preferred embodiment, the antibody further comprises a linker peptide between the heavy chain variable region and the light chain variable region.

In a fourth aspect of the present invention, there is provided a secreted antibody targeting PD-1, which comprises the antibody heavy chain variable region of the first aspect of the present invention and the antibody light chain variable region of the first aspect of the present invention, and which is expressed in and secreted extracellularly by immune cells.

In another preferred example, the heavy chain variable region of the secretory antibody has an amino acid sequence as shown in SEQ ID No. 2, and the light chain variable region has an amino acid sequence as shown in SEQ ID No. 4.

In another preferred embodiment, the surface of the immune cell can also express a chimeric antigen receptor.

In another preferred embodiment, the immune cell is a T cell, an NK cell, or a combination thereof.

In another preferred embodiment, the immune cell is a chimeric antigen receptor T cell (CAR-T cell).

In another preferred embodiment, the chimeric antigen receptor is selected from the group consisting of: CEA, Mesothelin, Claudin 18.2, GPC3, NKG2D, MUC1, or a combination thereof.

In a fifth aspect, the present invention provides a polynucleotide molecule encoding the PD-1 antibody or antigen-binding fragment thereof according to the third aspect of the present invention, the heavy chain variable region according to the first aspect of the present invention, the light chain variable region according to the second aspect of the present invention, or the secreted PD-1-targeted antibody according to the fourth aspect of the present invention.

In another preferred embodiment, the polynucleotide molecule encodes the heavy chain variable region of the PD-1 antibody, and the nucleotide sequence thereof is shown in SEQ ID NO. 1.

In another preferred embodiment, the polynucleotide molecule encodes the light chain variable region of the PD-1 antibody, and the nucleotide sequence thereof is shown in SEQ ID NO. 3.

In a sixth aspect of the invention, there is provided a vector comprising a polynucleotide molecule according to the fifth aspect of the invention.

In another preferred embodiment, the carrier is selected from the group consisting of: DNA, RNA, a plasmid, a eukaryotic expression vector, a prokaryotic expression vector, a lentiviral vector, an adenoviral vector, a retroviral vector, a transposon, or a combination thereof.

In another preferred embodiment, the vector is a eukaryotic expression vector.

In a seventh aspect of the invention, there is provided an engineered cell comprising a vector according to the sixth aspect of the invention, or having integrated into the chromosome an exogenous polynucleotide molecule according to the fifth aspect of the invention, or expressing a PD-1 antibody or antigen-binding fragment thereof according to the third aspect of the invention, or expressing a secreted antibody targeting PD-1 according to the fourth aspect of the invention.

In another preferred embodiment, the cell is a eukaryotic cell or a prokaryotic cell.

In another preferred embodiment, the cell is an immune cell.

In an eighth aspect of the invention, there is provided a pharmaceutical composition comprising a PD-1 antibody or antigen-binding fragment thereof according to the third aspect of the invention, a secreted antibody targeting PD-1 according to the fourth aspect of the invention, or an engineered cell according to the seventh aspect of the invention, and a pharmaceutically acceptable carrier, diluent or excipient.

In a ninth aspect of the present invention, there is provided a use of the PD-1 antibody or antigen-binding fragment thereof according to the third aspect of the present invention, the secreted antibody targeting PD-1 according to the fourth aspect of the present invention, the polynucleotide molecule according to the fifth aspect of the present invention, the vector according to the sixth aspect of the present invention, the engineered cell according to the seventh aspect of the present invention, or the pharmaceutical composition according to the eighth aspect of the present invention, for the preparation of a medicament or a formulation for the prevention and/or treatment of cancer or tumor.

In another preferred embodiment, the cancer or tumor is selected from the group consisting of: a hematologic tumor, a lymphoma, a solid tumor, or a combination thereof.

In another preferred embodiment, the hematological tumor is selected from the group consisting of: acute Myeloid Leukemia (AML), Multiple Myeloma (MM), Chronic Lymphocytic Leukemia (CLL), Acute Lymphoblastic Leukemia (ALL), diffuse large B-cell lymphoma (DLBCL), or a combination thereof.

In another preferred embodiment, the lymphoma is selected from the group consisting of: hodgkin Lymphoma (HL), diffuse large B-cell lymphoma (DLBCL), Follicular Lymphoma (FL), chronic lymphocytic white blood Cells (CLL), Small Lymphocytic Lymphoma (SLL), Marginal Zone Lymphoma (MZL), Mantle Cell Lymphoma (MCL), Burkitt's Lymphoma (BL), and other complex B-cell non-hodgkin lymphomas.

In another preferred embodiment, the solid tumor is selected from the group consisting of: gastric cancer, gastric cancer peritoneal metastasis, liver cancer, kidney tumor, lung cancer, small intestine cancer, bone cancer, prostate cancer, colorectal cancer, breast cancer, large intestine cancer, cervical cancer, ovarian cancer, lymphatic cancer, nasopharyngeal cancer, adrenal gland tumor, bladder tumor, non-small cell lung cancer (NSCLC), brain glioma, endometrial cancer, testicular cancer, colorectal cancer, urinary tract tumor, thyroid cancer, or a combination thereof.

In a tenth aspect of the invention, there is provided a method of making a PD-1 antibody or antigen-binding fragment thereof according to the third aspect of the invention, comprising culturing a cell comprising a polynucleotide encoding the antibody or antigen-binding fragment thereof under conditions suitable for production of the antibody or antigen-binding fragment thereof, and recovering the antibody or antigen-binding fragment from the cell or culture.

In an eleventh aspect of the invention, there is provided a method of making an engineered cell according to the seventh aspect of the invention, comprising the steps of: transducing the polynucleotide molecule of the fifth aspect of the invention or the vector of the sixth aspect of the invention into a cell, thereby obtaining the engineered cell.

In another preferred embodiment, the cell is an immune cell.

In a twelfth aspect of the invention, there is provided a method for preventing and/or treating a disease, comprising administering to a subject in need thereof a therapeutically effective amount of the PD-1 antibody or antigen-binding fragment thereof according to the third aspect of the invention, the engineered cell according to the seventh aspect of the invention, or the pharmaceutical composition according to the eighth aspect of the invention.

In another preferred embodiment, the disease is cancer or a tumor.

In a thirteenth aspect of the invention, there is provided an immunodetection point inhibitor comprising an antibody or antigen-binding fragment thereof according to the third aspect of the invention, and a pharmaceutically acceptable carrier, diluent or excipient.

It is to be understood that within the scope of the present invention, the above-described features of the present invention and those specifically described below (e.g., in the examples) may be combined with each other to form new or preferred embodiments. Not to be reiterated herein, but to the extent of space.

Drawings

FIG. 1 binding of post-diabody mouse serum to CHO-PD-1 cells as detected by FACS.

FIG. 2 binding of mouse serum to CHO-PD-1 cells after triple immunization as detected by FACS.

FIG. 3 wells positive for screening results by FACS detection.

FIG. 4 positive subclone detection results by FACS detection.

FIG. 5 flow assay results for PD1-05-4C3-1D12-1C8 blocking the binding of PD-1 to PD-L1.

FIG. 6 results of subtype PD1-05-4C3-1D12-1C8 antibody by flow analysis.

FIG. 7 expression identification of PD1-05-4C3-1D12-1C8 recombinant antibody by flow analysis.

FIG. 8, PD1-05-4C3-1D12-1C8 antibody heavy chain expression vector map.

FIG. 9 is a PD1-05-4C3-1D12-1C8 antibody light chain expression vector map.

Detailed Description

The present inventors have made extensive and intensive studies and, for the first time, have unexpectedly developed an anti-PD-1 monoclonal antibody. Experiments prove that the anti-PD-1 monoclonal antibody has high binding specificity and strong affinity with PD-1, and can effectively block the binding of PD-1 and PD-L1. The anti-PD-1 monoclonal antibody can be used as an immunodetection point inhibitor to activate immune cells and improve the activity of an organism to kill tumors, and can also be used for preparing a pharmaceutical composition for treating cancers/tumors. In addition, when the antibody of the invention is prepared into a secretory antibody, the antibody can be expressed on a chimeric antigen receptor immune cell, and the chimeric antigen receptor can play an anti-tumor role in combination, thereby providing a new method for treating tumors.

Term(s)

In order that the invention may be more readily understood, certain terms are first defined. As used in this application, each of the following terms shall have the meaning given below, unless explicitly specified otherwise herein. Other definitions are set forth throughout the application.

The term "about" refers to a value or composition that is within an acceptable error range for the particular value or composition as determined by one of ordinary skill in the art, which will depend in part on how the value or composition is measured or determined. For example, as used herein, the expression "about 100" includes 99 and 101 and all values in between (e.g., 99.1, 99.2, 99.3, 99.4, etc.).

As used herein, the term "comprising" or "includes" can be open, semi-closed, and closed. In other words, the term also includes "consisting essentially of …," or "consisting of ….

Immunodetection point

As used herein, the term "immunodetection point" is a regulatory molecule that plays an inhibitory role in the immune system, and in particular refers to the protein programmed death 1(PD-1) molecule.

The antibody or the antigen binding fragment thereof can be specifically bound with PD-1 expressed on the surface of an immune cell, effectively block the binding of the PD-1 and a ligand PD-L1 expressed on the surface of a tumor cell, relieve the inhibition of the PD-1 on the immune cell and activate the immune cell. Therefore, the antibody or the antigen binding fragment thereof can be used as an immunodetection point inhibitor or used for preparing an anti-tumor medicament.

Antibodies

As used herein, the term "antibody" is an heterotetrameric glycan protein of about 150000 daltons with identical structural features, consisting of two identical light chains (L) and two identical heavy chains (H). Each light chain is linked to a heavy chain by one covalent disulfide bond, while the number of disulfide bonds varies between heavy chains of different immunoglobulin isotypes. Each heavy and light chain also has regularly spaced intrachain disulfide bonds. Each heavy chain has at one end a variable region (VH) followed by a plurality of constant regions. Each light chain has a variable domain (VL) at one end and a constant domain at the other end; the constant region of the light chain is opposite the first constant region of the heavy chain, and the variable region of the light chain is opposite the variable region of the heavy chain. Particular amino acid residues form the interface between the variable regions of the light and heavy chains.

As used herein, the term "variable" means that certain portions of the variable regions in an antibody differ in sequence, which results in the binding and specificity of each particular antibody for its particular antigen. However, the variability is not evenly distributed throughout the antibody variable region. It is concentrated in three segments called Complementarity Determining Regions (CDRs) or hypervariable regions in the light and heavy chain variable regions. The more conserved portions of the variable regions are called Framework Regions (FR). The variable regions of native heavy and light chains each comprise four FR regions, in a substantially β -sheet configuration, connected by three CDRs (CDR1, CDR2, and CDR3) that form a connecting loop, and in some cases may form part of a β -sheet structure. The CDRs in each chain are held close together by the FR region and form the antigen binding site of the antibody with the CDRs of the other chain (see Kabat et al, NIH Publ. No.91-3242, Vol I, 647-669 (1991)). The constant regions are not directly involved in the binding of antibodies to antigens, but they exhibit different effector functions, such as participation in antibody-dependent cytotoxicity of antibodies.

The "light chains" of vertebrate antibodies (immunoglobulins) can be assigned to one of two distinct classes (termed kappa and lambda) based on the amino acid sequence of their constant regions. Immunoglobulins can be assigned to different classes based on the amino acid sequence of their heavy chain constant regions. There are mainly 5 classes of immunoglobulins: IgA, IgD, IgE, IgG and IgM, some of which can be further divided into subclasses (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgA and IgA 2. The heavy chain constant regions corresponding to different classes of immunoglobulins are referred to as α, δ, ε, γ, and μ, respectively. The subunit structures and three-dimensional configurations of different classes of immunoglobulins are well known to those skilled in the art.

The variable regions of the heavy and/or light chains of the antibodies of the invention are of particular interest, since at least some of them are involved in binding to an antigen. Thus, the invention includes those molecules having the light and heavy chain variable regions of a monoclonal antibody with CDRs that are more than 90% (preferably more than 95%, most preferably more than 98%) homologous to the CDRs identified herein.

As used herein, the terms "heavy chain variable region" and "HV" are used interchangeably.

As used herein, the term "light chain variable region" is used interchangeably with "LV".

As used herein, the term "variable region" is used interchangeably with "Complementary Determining Region (CDR)".

Secreted antibodies

As used herein, the term "secreted antibody" refers to a PD-1 antibody expressed on an immune cell, which antibody is not immobilized on the cell membrane and can be released from the immune cell outside the cell to reach other sites in the body.

The invention provides a secretory antibody targeting PD-1, which comprises a heavy chain variable region (CDR) as shown in SEQ ID No.:2, and an antibody heavy chain variable region as set forth in SEQ ID No.:4, and the secreted antibody is expressed in an immune cell. The surface of the immune cell can simultaneously express a chimeric antigen receptor.

In another preferred embodiment, the chimeric antigen receptor includes (but is not limited to): CEA, Mesothelin, Claudin 18.2, GPC3, NKG2D, MUC1, and the like.

Polynucleotide molecules and vectors

The invention also provides polynucleotide molecules encoding the above antibodies or fragments or fusion proteins thereof. The polynucleotide of the present invention may be in the form of DNA or RNA. The form of DNA includes cDNA, genomic DNA or artificially synthesized DNA. The DNA may be single-stranded or double-stranded. The DNA may be the coding strand or the non-coding strand. The sequence of the coding region encoding the mature polypeptide may be identical to SEQ ID No.:1 and 3 are identical or degenerate variants. As used herein, "degenerate variant" means in the present invention a variant that encodes a polypeptide having the same amino acid sequence as the polypeptide of the present invention, but which has an amino acid sequence identical to SEQ ID No.:1 and 3, or a variant thereof.

Polynucleotides encoding the mature polypeptides of the invention include: a coding sequence encoding only the mature polypeptide; the coding sequence for the mature polypeptide and various additional coding sequences; the coding sequence (and optionally additional coding sequences) as well as non-coding sequences for the mature polypeptide.

The term "polynucleotide encoding a polypeptide" may include a polynucleotide encoding the polypeptide, and may also include additional coding and/or non-coding sequences.

The present invention also relates to polynucleotides which hybridize to the above-described sequences and which have at least 50%, preferably at least 70%, and more preferably at least 80% identity between the two sequences. The present invention particularly relates to polynucleotides which hybridize under stringent conditions to the polynucleotides of the invention. In the present invention, "stringent conditions" mean: (1) hybridization and elution at lower ionic strength and higher temperature, such as 0.2 XSSC, 0.1% SDS,60 ℃; or (2) adding denaturant during hybridization, such as 50% (v/v) formamide, 0.1% calf serum/0.1% Ficoll, 42 deg.C, etc.; or (3) hybridization only when the identity between two sequences is at least 90% or more, preferably 95% or more. And, a polypeptide encoded by the hybridizable polynucleotide hybridizes to SEQ ID No.:2 and 4 have the same biological functions and activities.

The full-length nucleotide sequence of the antibody of the present invention or a fragment thereof can be obtained by a PCR amplification method, a recombinant method, or an artificial synthesis method. One possibility is to use synthetic methods to synthesize the sequence of interest, especially when the fragment length is short. Generally, fragments with long sequences are obtained by first synthesizing a plurality of small fragments and then ligating them. Alternatively, the coding sequence for the heavy chain and an expression tag (e.g., 6His) can be fused together to form a fusion protein.

Once the sequence of interest has been obtained, it can be obtained in large quantities by recombinant methods. This is usually done by cloning it into a vector, transferring it into a cell, and isolating the relevant sequence from the propagated host cell by conventional methods. The biomolecules (polynucleotides, proteins, etc.) to which the present invention relates include biomolecules in an isolated form. At present, DNA sequences encoding the proteins of the present invention (or fragments or derivatives thereof) have been obtained completely by chemical synthesis. The DNA sequence may then be introduced into various existing DNA molecules (or vectors, for example) and cells known in the art. Furthermore, mutations can also be introduced into the protein sequences of the invention by chemical synthesis.

The invention also relates to a vector comprising a suitable DNA sequence as described above and a suitable promoter or control sequence. These vectors may be used to transform an appropriate host cell so that it can express the protein.

The host cell may be a prokaryotic cell, such as a bacterial cell; or lower eukaryotic cells, such as yeast cells; or higher eukaryotic cells, such as mammalian cells. Representative examples are: escherichia coli, streptomyces; bacterial cells of salmonella typhimurium; fungal cells such as yeast; insect cells of Drosophila S2 or Sf 9; CHO, COS7, 293 cells, T cells, NK cells, and the like.

Pharmaceutical composition

The invention also provides a pharmaceutical composition comprising an antibody according to the third aspect of the invention, a secreted antibody according to the fourth aspect of the invention or an engineered cell according to the seventh aspect of the invention, and a pharmaceutically acceptable carrier, excipient or diluent. Generally, these materials will be formulated in a non-toxic, inert and pharmaceutically acceptable aqueous carrier medium, wherein the pH is generally from about 5 to about 8, preferably from about 6 to about 8, although the pH will vary depending on the nature of the material being formulated and the condition being treated. The formulated pharmaceutical compositions may be administered by conventional routes including, but not limited to: oral, respiratory, intratumoral, intraperitoneal, intravenous, or topical administration.

The pharmaceutical composition of the present invention comprises a safe and effective amount (e.g., 0.001-99 wt%, preferably 0.01-90 wt%, more preferably 0.1-80 wt%) of the antibody (or conjugate thereof), chimeric antigen receptor, or chimeric antigen receptor T cell of the present invention as described above, and a pharmaceutically acceptable carrier or excipient. Such vectors include (but are not limited to): saline, buffer, glucose, water, glycerol, ethanol, and combinations thereof. The pharmaceutical preparation should be compatible with the mode of administration. The pharmaceutical composition of the present invention can be prepared in the form of injection, for example, by a conventional method using physiological saline or an aqueous solution containing glucose and other adjuvants. Pharmaceutical compositions such as injections, solutions are preferably manufactured under sterile conditions. The amount of active ingredient administered is a therapeutically effective amount, for example from about 1 microgram per kilogram of body weight to about 10 milligrams per kilogram of body weight per day. In addition, the pharmaceutical compositions of the present invention may also be used with other therapeutic agents.

When using pharmaceutical compositions, a safe and effective amount of the immunomer is administered to the mammal, wherein the safe and effective amount is generally at least about 10 micrograms/kg body weight, and in most cases no more than about 8 mg/kg body weight, preferably the dose is from about 10 micrograms/kg body weight to about 1 mg/kg body weight. Of course, the particular dosage will depend upon such factors as the route of administration, the health of the patient, and the like, and is within the skill of the skilled practitioner.

The main advantages of the invention are:

(1) the antibody of the invention has high specificity and strong affinity, and can be prepared in large scale, and the quality of the monoclonal antibody is easy to control.

(2) The antibody of the invention can be used as an immunodetection point inhibitor to activate immunocytes and improve the activity of an organism to kill tumors, and can be used for preparing monoclonal antibody medicaments, double-antibody medicaments or multifunctional antibodies.

(3) The antibody of the invention can be used for preparing a reagent for diagnosing the vitality of immune cells.

(4) The antibody of the invention can be prepared into a secretory type, is expressed on a chimeric antigen receptor immune cell, and plays an anti-tumor role by combining the chimeric antigen receptor.

The invention will be further illustrated with reference to the following specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. Experimental procedures without specific conditions noted in the following examples, molecular cloning is generally performed according to conventional conditions such as Sambrook et al: the conditions described in the Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the manufacturer's recommendations. Unless otherwise indicated, percentages and parts are by weight.

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

Example 1

Screening of PD-1 antibodies

The main technical scheme of the embodiment is as follows:

1. mice were given three DNA immunizations +1 cell blast immunizations two weeks apart. Wherein, the DNA refers to human PD-1DNA, and the cells used are CHO-PD-1 (i.e., CHO cells expressing PD-1).

2. FACS screening was performed using CHO-PD-1 (PD-1 expressing CHO cells) cells.

The following experimental procedures were followed:

1. immunization of mice

Experimental mice were immunized according to the protocol of table 1:

table 1 time protocol for immunization of mice

Mouse ID	One need not	Secondary drug	Sanwu (Chinese character of 'Sanwu')	Ballistic immunization
					PD1-A-01	2019/6/25	2019/7/9		2019/7/23
PD1-A-02	2019/6/25	2019/7/9
					PD1-A-03	2019/6/25	2019/7/9
PD1-A-04	2019/6/25	2019/7/9	2019/7/23
					PD1-A-05	2019/6/25	2019/7/9	2019/7/23	2019/9/13
PD1-A-06	2019/6/25	2019/7/9	2019/7/23
					PD1-A-07	2019/6/25	2019/7/9
PD1-A-08	2019/6/25	2019/7/9	2019/7/23
					PD1-A-09	2019/6/25	2019/7/9	2019/7/23
PD1-A-10	2019/6/25	2019/7/9	2019/7/23	2019/8/19
					PD1-A-11	2019/9/16	2019/9/30	2019/10/14
PD1-A-12	2019/9/16	2019/9/30	2019/10/14
					PD1-A-13	2019/9/16	2019/9/30	2019/10/14
PD1-A-14	2019/9/16	2019/9/30	2019/10/14
					PD1-A-15	2019/9/16	2019/9/30	2019/10/14

Wherein: firstly, the method avoids: each mouse was immunized with 60 μ g DNA (intramuscular injection); II, exemption: each mouse was immunized with 60 μ g of DNA; and (3) three-step (I): each mouse was immunized with 60 μ g of DNA; impact immunization: using 1X 10 ⁷ And (3) performing tail vein injection impact immunization on CHO-PD-1 cells.

2. Serum detection

(1) FACS detection of hyperimmune serum

The sera from the mice after the second immunization were tested for binding to CHO-PD-1 by FACS.

Experimental materials: CHO-PD-1+ mouse diabatic serum + goat anti-mouse IgGFc-FITC.

Appropriate amount of CHO-PD-1 cells were dispensed into 1.5ml EP tubes, centrifuged, resuspended in 50. mu.l of 1:100 diluted mouse serum, allowed to stand at 4 ℃ for 15 minutes, centrifuged, resuspended in 50. mu.l of goat anti-mouse IgG Fc-FITC diluted in PBS 1:500, allowed to stand at 4 ℃ for 15 minutes, centrifuged to replace the supernatant, resuspended in 200. mu.l of PBS, and FACS flow analysis was performed.

The results are shown in FIG. 1. The results show that mouse PD1-A-01, 05, 08, 10 can be subject to ballistic immune fusion.

(2) FACS detection of triple-immune serum

The sera of mice after the triple immunization were tested for binding to CHO-PD-1 by FACS.

Experimental materials: CHO-PD-1+ mouse triaimmune serum + goat anti-mouse IgGFc-FITC.

The results are shown in FIG. 2. The results show that mouse PD1-A-11, 13, 14, 15 can be subjected to impact immune fusion.

3. Fusion of

After 4 days of the mouse shock immunization, the B lymphocytes of the mouse were fused with myeloma cells, respectively, to obtain corresponding hybridoma cells.

4. Post-fusion screening

(1) Primary screening: each mouse was plated in 5 separate 96-well plates (12 × 8 wells, 1-12 for row number, a-H for column number, as in table 1), numbered 1, 2, 3,4, 5, respectively, for HAT screening. Table 2 shows the HAT prescreening results.

Table 2 HAT prescreening results:

mouse ID	Preliminary screening positive
		PD1-A-01	Is free of
PD1-A-10	Is composed of
		PD1-A-05	1E7,2B11,3D12,4A3,4A6,4A9,4C3,4D5,5A5,5C8

(2) FACS detection screening

The wells that were initially screened positive were further screened using FACS detection. The results are shown in FIG. 3. The results showed that all of PD1-05-1E7,2B11,3D12,4A3,4a6,4a9,4C3,4D5,5a5,5C8 were positive. Thus, the positive wells were subcloned.

5. Subcloning of the established strain

The positive subclones were summarized in Table 3 below by FACS detection:

TABLE 3 summary of positive subclones

The subclone assay results are shown in FIG. 4.

Example 2

Detection of blocking Effect of Positive subclone antibody

The effect of positive subclones in blocking the binding of PD-1 to PD-L1 was tested as follows:

(1) CHO-PD-1 cells were aliquoted into 1.5mL EP tubes.

(2) Centrifuging at 4000rpm for 5min, and discarding the supernatant.

(3) The experimental group was used to resuspend cells using 50. mu.L hybridoma supernatant, the control group was used to resuspend cells using 50. mu.L PBS, and the cells were allowed to stand at 4 ℃ for 15 min.

(4) 50 μ L of PD-L1-hFc solution with the concentration of 50 μ g/mL, 5 μ g/mL and 0.5 μ g/mL is respectively mixed with the cell suspension and is kept stand for 15min at 4 ℃.

(5) Centrifuging at 4000rpm for 5min, and discarding the supernatant.

(6) 50 μ L of a mixture of 1:250 diluted Goat anti-mouseIgG-FITC and 1:500 diluted Goat anti-human IgG Dylight650 secondary antibody was resuspended in the above cell pellet and allowed to stand at 4 ℃ for 15 min.

(7) Centrifuging at 4000rpm for 5min, and discarding the supernatant.

(8)300 μ L PBS resuspended cells and flow analyzed.

The flow analysis results are shown in fig. 5. The result shows that the subclone PD1-05-4C3-1D12-1C8 has the function of blocking the combination of PD-1 and PD-L1. In addition, from the above results, it can be seen that Goat anti-mouseIgG-FITC has cross reaction with hFc, and the cross reaction is more obvious when the concentration of hFc is higher, but the cross reaction does not affect the detection of blocking reaction.

Example 3

Construction of recombinant PD-1 antibody

First, the subclone PD1-05-4C3-1D12-1C8 identified in example 2 was subjected to antibody sequence and subtype analysis.

The experimental procedure was as follows:

(1) CHO-PD-1 cells were aliquoted into 1.5mL EP tubes.

(2) Centrifuging at 4000rpm for 5min, and discarding the supernatant.

(3) 50. mu.L of hybridoma supernatant was taken and the cells were resuspended and allowed to stand at 4 ℃ for 15 min.

(4) Centrifuging at 4000rpm for 5min, and discarding the supernatant.

(5) 50 μ L of 1:200 diluted Goat anti-mouseigG1-FITC, Goat anti-mouseigG2a-FITC and Goat anti-mouseigG2b-FITC was resuspended in the cells and allowed to stand at 4 ℃ for 15 min.

(6) Centrifuging at 4000rpm for 5min, and discarding the supernatant.

(7)300 μ L PBS resuspended cells and flow analyzed.

The flow analysis results are shown in fig. 6. The results show that: the subtype of the PD1-05-4C3-1D12-1C8 antibody is IgG2 b.

Antibody gene sequencing was performed using the Sanger method of capillary electrophoresis.

Details regarding PD1-05-4C3-1D12-1C8 antibody are summarized in table 4 below:

TABLE 4 subtype and Gene sequence of PD1-05-4C3-1D12-1C8 antibody

Subclone number	Subtype of cell	Heavy chain gene	Light chain gene
				PD1-05-4C3-1D12-1C8	IgG2b	IGHV1-18	IGKV12-98

IGHV 1-18: the sequence is shown in SEQ ID No. 1(DNA) and SEQ ID No. 2 (amino acid).

IGKV 12-98: the sequence is shown in SEQ ID No. 3(DNA) and SEQ ID No. 4 (amino acid).

Expression verification of recombinant antibodies:

the nucleotide sequences encoding the heavy chain variable region and the light chain variable region of the PD1-05-4C3-1D12-1C8 antibody are respectively cloned into a pCAG eukaryotic expression vector to obtain recombinant expression vectors for respectively expressing the heavy chain variable region (figure 8) and the light chain variable region (figure 9) of the antibody.

The light and heavy chain expression vectors were co-transfected into 293T cells, and the supernatants were collected for FACS validation.

The FACS validation experiment included the following steps:

(1) CHO-PD-1 cells were aliquoted into 1.5mL EP tubes.

(2) Centrifuging at 4000rpm for 5min, and discarding the supernatant.

(3) 50 μ L of the cell transfection supernatant of 293T was taken and resuspended, and left to stand at 4 ℃ for 15 min.

(4) Centrifuging at 4000rpm for 5min, and discarding the supernatant.

(5) 50 μ L of 1:200 diluted Goat anti-mouseIgG-FITC was resuspended in the cells and allowed to stand at 4 ℃ for 15 min.

(6) Centrifuging at 4000rpm for 5min, and discarding the supernatant.

(7)300 μ L PBS resuspended cells and flow analyzed.

The flow analysis results are shown in FIG. 7. The result shows that the supernatant signal of the recombinant antibody transfection of PD1-05-4C3-1D12-1C8 is better. The recombinant antibody can effectively recognize the PD-1 antigen in CHO-PD-1 cells, and the result shows that the antibody can activate immune cells in vivo by recognizing the PD-1 antigen under a physiological state and blocking the combination of PD-1 and PD-L1.

The DNA sequence and amino acid sequence of the PD1-05-4C3-1D12-1C8 antibody are shown in the following table:

all documents referred to herein are incorporated by reference into this application as if each were individually incorporated by reference. Furthermore, it should be understood that various changes and modifications of the present invention can be made by those skilled in the art after reading the above teachings of the present invention, and these equivalents also fall within the scope of the present invention as defined by the appended claims.

Sequence listing

<110> Shanghai Lai Fisher medical science and technology Co Ltd

<120> anti-PD-1 antibody and use thereof

<130> P2020-1793

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atgggatgga gctggatctt tctcttcttc ctgtcaggaa ctgcaggtgt cctctctgag 60

gtcctgctgc aacagtctgg acctgagctg gtgaagcctg gggcttcagt gaagatatcc 120

tgcaaggctt ctggatacac attctctgac aacaacatag actgggtgag gcagagccat 180

ggaaagagcc ttgagtggat tggagatatt aatcctagca atggtggtgt tatctacaaa 240

cagaatttcg aggggaaggc catattgact gtagacaagt cctccagcac agcctacatg 300

gagctccgca gcctgacatc tgaggacact tcagtctatt attgtgcaag aactaggtcc 360

gatggtatgg actactgggg tcaaggaacc tcagtcaccg tctcctca 408

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atcacatgcc tggcaagtca gaccattggt acatggttag catggtatca gcagaaacca 180

gggaaatctc ctcagctcct gatttatgat gcaaccagtt tgacagatgg ggtcccatca 240

aggttcagtg gtagtggatc tggcacaaag ttttctttca agatcagcag cctacaggct 300

gaagattttg taagttatta ctgtcaacaa ttttacagta ctccattcac gttcggctcg 360

gggacaaagt tggaaataaa a 381

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Gln Leu Leu Ile Tyr Asp Ala Thr Ser Leu Thr Asp Gly Val Pro Ser

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Arg Phe Ser Gly Ser Gly Ser Gly Thr Lys Phe Ser Phe Lys Ile Ser

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Ser Leu Gln Ala Glu Asp Phe Val Ser Tyr Tyr Cys Gln Gln Phe Tyr

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Ser Thr Pro Phe Thr Phe Gly Ser Gly Thr Lys Leu Glu Ile Lys

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1 5

Claims

1. A PD-1 antibody or antigen-binding fragment thereof, wherein the heavy chain variable region of said antibody or antigen-binding fragment thereof comprises the following three complementarity determining regions CDRs:

a CDR1 as set forth in SEQ ID No. 5;

a CDR2 as set forth in SEQ ID No. 6; and

a CDR3 as set forth in SEQ ID No. 7; and

the light chain variable region of the antibody or antigen-binding fragment thereof comprises the following three complementarity determining regions CDRs:

a CDR 1' as shown in SEQ ID No. 8;

a CDR 2' as set forth in SEQ ID No. 9; and

CDR 3' as shown in SEQ ID No. 10.

2. The antibody or antigen-binding fragment thereof of claim 1, wherein the heavy chain variable region of the antibody or antigen-binding fragment thereof has an amino acid sequence as set forth in SEQ ID No. 2 and the light chain variable region has an amino acid sequence as set forth in SEQ ID No. 4.

3. The antibody or antigen-binding fragment thereof of claim 1, wherein the heavy chain of the antibody or antigen-binding fragment thereof further comprises a heavy chain constant region; the light chain of the antibody or antigen-binding fragment thereof further comprises a light chain constant region.

4. The antibody or antigen-binding fragment thereof of claim 1, wherein the antibody is a double-chain antibody or a single-chain antibody.

5. A secreted antibody targeting PD-1, wherein said secreted antibody comprises the heavy chain variable region and the light chain variable region of claim 1, and wherein said secreted antibody is expressed in and secreted extracellularly by immune cells.

6. A polynucleotide molecule encoding the PD-1 antibody or antigen-binding fragment thereof according to claim 1 or the secreted antibody targeted to PD-1 according to claim 5.

7. A vector comprising the polynucleotide molecule of claim 6.

8. An engineered cell comprising the vector of claim 7, or having integrated into its chromosome an exogenous polynucleotide molecule of claim 6, or expressing the PD-1 antibody or antigen-binding fragment thereof of claim 1, or expressing the secreted antibody targeting PD-1 of claim 5.

9. The cell of claim 8, wherein the cell is an immune cell and the cell surface simultaneously expresses a chimeric antigen receptor.

10. A pharmaceutical composition comprising the PD-1 antibody of claim 1, the secreted antibody targeted to PD-1 of claim 5, or the engineered cell of claim 8, and a pharmaceutically acceptable carrier, diluent, or excipient.