Antibodies that bind to PD-1

Abstract

The present invention discloses antibodies that bind to PD-1, comprising one or more CDRs selected from those having the amino acid sequences set forth in SEQ ID NOs: 13, 14, 15, 16, 17, and 18. These antibodies have high affinity and a slow dissociation rate for PD-1, and possess the activity of neutralizing PD-1 in vitro. The antibodies of the present invention may be full-length antibodies or antigen-binding portions thereof, and can be used for applications such as detecting PD-1.

Description

Detailed Description of the Invention

[0001] [Technical field] The present invention belongs to the field of biotechnology and relates to an antibody that binds to PD-1. [Background technology] Programmed death receptor-1 (PD-1) is an immune inhibitory receptor expressed in activated T cells, B cells, and myeloid cells, and belongs to the CD28 immunoglobulin superfamily. PD-1 is a 55 kDa type I transmembrane glycoprotein that contains an Ig variable structural domain for ligand binding and a cytoplasmic tail for binding to signaling molecules. The PD-1 cytoplasmic tail contains two tyrosine-based signaling modules, ITIM (immunoreceptor tyrosine-based inhibitory motif) and ITSM (immunoreceptor tyrosine-based switch motif).

[0002] Two ligands of PD-1, PD-L1 and PD-L2, have been shown to be constitutively expressed or induced in various cell types, including non-hematopoietic tissues and various tumors. PD-L1 is expressed on B cells, T cells, myeloid cells, and dendritic cells, but also on peripheral cells (e.g., microvascular endothelial cells) and non-lymphoid organs (e.g., heart, lung, etc.). In contrast, PD-L2 is only present on macrophages and dendritic cells.

[0003] The role of PD-1 in cancer has been established in the literature. The interaction of PD-1 with its ligands has been shown to negatively regulate antigen receptor signaling and reduce T cell responses. The mechanism by which PD-1 weakly regulates T cell responses is similar to that of CTLA-4, but there are also differences, as both molecules regulate an overlapping set of signaling proteins. PD-L1 is abundant in various human tumors (it is inducible by IFNγ in most PD-1 negative tumor cell lines). Many studies to date have shown that the interaction between PD-1 and PD-L1 reduces lymphocytes invading tumors, reduces T cell receptor-mediated proliferation, and evades cancer cell immunity. Blocking the interaction between PD-1 and PD-L1 has been shown to increase T cell proliferation and cell factor production, and improve the immunity of tumor-specific CD8+ T cells, thus contributing to the elimination of tumor cells by the immune system.

[0004] Immunohistochemical characterization of biological tissues has already detected PD-1 (on tumor-infiltrating lymphocytes) and / or PD-L1 (on tumor cells) in many human primary tumors, including lesions from lung, liver, ovarian, cervical, skin, colon, glioma, bladder, breast, kidney, esophageal, gastric, oral squamous cell, urothelial and pancreatic cancers, and head and neck tumors. Expression of PD-1 ligands on tumor cells in a variety of tumor types is associated with poor prognosis in cancer patients.

[0005] In summary, the PD-1 / PD-L1 pathway is a well-documented target in the development of antibody therapies to treat cancer, and therefore, we must be aware of PD-1 agents and methods of using such agents. Summary of the Invention The present invention provides antibodies or fragments thereof that bind to PD-1.

[0006] In order to solve the above technical problems, one aspect of the present invention provides an antibody or antibody fragment comprising one, two, three, four, five or six CDRs selected from the amino acid sequences shown in SEQ ID NOs:13, 14, 15, 16, 17 and 18, or CDRs having one, two or three conservative amino acid substitutions compared thereto.

[0007] In one embodiment, the antibody or antibody fragment binds to PD-1.

[0008] In one embodiment, the antibody or antibody fragment binds to human PD-1 (hPD-1).

[0009] In some embodiments, the antibody or antibody fragment blocks the binding of hPD-1 to hPD-L1 (human PD-L1) and / or disrupts the downstream signaling pathway mediated by hPD-1 / hPD-L1.

[0010] In one embodiment, the antibody or antibody fragment encodes the nucleotide sequence of SEQ ID NO:13 as shown at positions 73-114 of SEQ ID NO:2.

[0011] In one embodiment, the antibody or antibody fragment according to any one of the above encodes the nucleotide sequence of SEQ ID NO:14 as shown in positions 160-177 of SEQ ID NO:2.

[0012] In one embodiment, the antibody or antibody fragment according to any one of the above encodes the nucleotide sequence of SEQ ID NO:15 as shown at positions 271-303 of SEQ ID NO:2.

[0013] In one embodiment, the antibody or antibody fragment according to any one of the above encodes the nucleotide sequence of SEQ ID NO:16 as shown in positions 91-108 of SEQ ID NO:1.

[0014] In one embodiment, the antibody or antibody fragment according to any one of the above encodes the nucleotide sequence of SEQ ID NO:17 as shown at positions 148-192 of SEQ ID NO:1.

[0015] In one embodiment, the antibody or antibody fragment according to any one of the above encodes the nucleotide sequence of SEQ ID NO:18 as shown at positions 292-327 of SEQ ID NO:1.

[0016] In one embodiment, the antibody or antibody fragment according to any one of the above, wherein the antibody or antibody fragment comprises one, two or three heavy chain CDRs having the amino acid sequences set forth in SEQ ID NOs: 16, 17 and 18; and / or The antibody or antibody fragment comprises one, two or three light chain CDRs having the amino acid sequences shown in SEQ ID NOs:13, 14 and 15.

[0017] In one embodiment, the antibody or antibody fragment according to any one of the above, comprises a light chain CDR having the amino acid sequence shown in SEQ ID NO:13, 14 and 15, and a heavy chain CDR having the amino acid sequence shown in SEQ ID NO:16, 17 and 18.

[0018] In certain embodiments, the antibody or antibody fragment is an isolated antibody or antibody fragment.

[0019] In one embodiment, the antibody or antibody fragment according to any one of the above, wherein the antibody or antibody fragment comprises a heavy chain variable region comprising an amino acid sequence as set forth in SEQ ID NO:7, or an amino acid sequence that is at least 90%, at least 95%, at least 98%, or at least 99% identical to the amino acid sequence as set forth in SEQ ID NO:7, or an amino acid sequence that has one or more conservative amino acid substitutions compared to the amino acid sequence as set forth in SEQ ID NO:7, and / or a light chain variable region comprising an amino acid sequence as set forth in SEQ ID NO:8, or an amino acid sequence that is at least 90%, at least 95%, at least 98%, or at least 99% identical to the amino acid sequence as set forth in SEQ ID NO:8, or an amino acid sequence that has one or more conservative amino acid substitutions compared to the amino acid sequence as set forth in SEQ ID NO:8.

[0020] In one embodiment, the antibody or antibody fragment according to any one of the above, wherein the antibody or antibody fragment comprises a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO:7 and a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO:8.

[0021] In one embodiment, the antibody or antibody fragment described in any one of the above encodes the nucleotide sequence of SEQ ID NO:7 as shown in SEQ ID NO:1 and encodes the nucleotide sequence of SEQ ID NO:8 as shown in SEQ ID NO:2.

[0022] In one embodiment, in the antibody or antibody fragment described in any one of the above, the antibody or antibody fragment further comprises a human γ4 heavy chain constant region or a variant thereof, and / or a human κ light chain constant region or a variant thereof.

[0023] In order to solve the above technical problems, the present invention provides a heavy chain comprising an amino acid sequence as set forth in SEQ ID NO:71, an amino acid sequence having at least 90% identity with the amino acid sequence as set forth in SEQ ID NO:71, or an amino acid sequence having one or more conservative amino acid substitutions compared to the amino acid sequence as set forth in SEQ ID NO:71, and and a light chain comprising an amino acid sequence as set forth in SEQ ID NO:72, an amino acid sequence having at least 90% identity to the amino acid sequence as set forth in SEQ ID NO:72, or an amino acid sequence having one or more conservative amino acid substitutions compared to the amino acid sequence as set forth in SEQ ID NO:72.

[0024] In one embodiment, the antibody or antibody fragment according to any one of the above, wherein the antibody or antibody fragment comprises a heavy chain as set forth in SEQ ID NO:71 and / or a light chain as set forth in SEQ ID NO:72.

[0025] In one embodiment, the antibody or antibody fragment according to any one of the above, wherein the antibody or antibody fragment comprises a heavy chain as set forth in SEQ ID NO:71 and a light chain as set forth in SEQ ID NO:72.

[0026] In one embodiment, the antibody or antibody fragment described in any one of the above encodes the nucleotide sequence of SEQ ID NO:71 as shown in SEQ ID NO:73 and encodes the nucleotide sequence of SEQ ID NO:72 as shown in SEQ ID NO:74.

[0027] In one embodiment, the antibody or antibody fragment according to any one of the above, said antibody or antibody fragment is a human antibody or human antibody fragment.

[0028] In one embodiment, the antibody or antibody fragment according to any one of the above, wherein the antibody fragment is a Fab, Fv or scFv antibody.

[0029] In one embodiment, the antibody or antibody fragment according to any one of the above, wherein the antibody or antibody fragment is a monoclonal antibody (including a full-length monoclonal antibody), a polyclonal antibody or a multispecific antibody (e.g., a bispecific antibody).

[0030] In one embodiment, the antibody or antibody fragment according to any one of the above, wherein the variant comprises at most 20, at most 10, at most 5, or at most 3 conservative amino acid substitutions.

[0031] In order to solve the above technical problem, another aspect of the present invention further provides a variant of the antibody or antibody fragment described in any one of the above, which comprises one, two or three conservative amino acid substitutions and has a similar function to the antibody or antibody fragment.

[0032] In order to solve the above technical problem, in another aspect of the present invention there is further provided a biomaterial, which is B1) or B2) as follows: B1) a nucleic acid molecule encoding an antibody or antibody fragment or variant according to any one of the above, (The nucleic acid molecule may be DNA, such as cDNA, genomic DNA, or recombinant DNA, or may be RNA, such as mRNA or hnRNA.) B2) An expression cassette comprising the nucleic acid molecule according to B1), vector , recombinant cell or recombinant microorganism.

[0033] This biomaterial relates to and can be applied, for example, for the production of the above-mentioned antibodies or antibody fragments or variants.

[0034] In one embodiment, the present invention provides a molecule comprising: 1) a nucleic acid fragment, such as: A nucleic acid fragment as set forth at positions 91-108 of SEQ ID NO:1, A nucleic acid fragment set forth at positions 148-192 of SEQ ID NO:1, A nucleic acid fragment as set forth at positions 292-327 of SEQ ID NO:1, A nucleic acid fragment set forth at positions 73-114 of SEQ ID NO:2, A nucleic acid fragment as set forth at positions 160-177 of SEQ ID NO:2, A nucleic acid fragment as set forth at positions 271-303 of SEQ ID NO:2; A nucleic acid fragment as set forth in SEQ ID NO:1, A nucleic acid fragment as set forth in SEQ ID NO:2, A nucleic acid fragment as set forth in SEQ ID NO:73, or A nucleic acid fragment as set forth in SEQ ID NO:74, 2) a nucleic acid fragment which hybridizes with a nucleic acid fragment as defined in 1) and encodes an antibody or antibody fragment or variant according to any one of the above, 3) A nucleic acid fragment having 90% or more identity to a nucleic acid fragment limited to 1) or 2) and encoding an antibody or antibody fragment or variant according to any one of the above.

[0035] The nucleic acid molecule can be applied to any part of the biomaterial.

[0036] The term "identity" as used herein can be assessed by eye or by computer software (e.g., the software programs described in Current Protocols in Molecular Biology by Ausubel et al. eds. (2007)). When a site in the compared sequences is occupied by the same base or amino acid, the molecules are identical at that site. Identity between two or multiple sequences can be expressed as a percentage (%), which is applicable to assessing identity between related sequences. A polynucleotide or amino acid sequence having a particular percentage (e.g., 90%, 95%, 98% or 99%) of "sequence identity" with another sequence means that, when comparing the sequences, that percentage of bases or amino acids are the same in the two sequences being compared.

[0037] In certain embodiments, treatment with an anti-PD-1 antibody or antibody fragment can be combined with radiation therapy, which induces the death of cancer cells and improves the availability of tumor antigens to present and activate immune cells.

[0038] In other embodiments, treatment with the anti-PD-1 antibody or antibody fragment may be combined with surgery to remove the cancer cells being treated.

[0039] In other embodiments, the anti-PD-1 antibodies or antibody fragments can be combined with therapies that can synergize with PD-1 blockade, including targeted drugs to remove hormones or inhibit angiogenesis, or targeted drugs to proteins active in tumor cells, both of which can increase tumor cell death and improve the effectiveness of immune stimulatory tumor antigens. When combined with anti-PD-1 antibodies or antibody fragments, improved T cell activation can prolong immune control of cancer.

[0040] In some embodiments, the anti-PD-1 antibody or antibody fragment can be combined with another therapeutic antibody for treating cancer or infectious disease. As a non-limiting embodiment, a combination of anti-PD-1 with an antibody targeting Her2 / neu or targeting the EGF receptor is provided. In another non-limiting embodiment, the anti-PD-1 antibody or antibody fragment is combined with a therapy targeting VEGF or its receptor. In another embodiment, the anti-PD-1 antibody or antibody fragment is combined with an anti-CTLA-4 antibody. In yet another non-limiting embodiment, the anti-PD-1 antibody is combined with an antibody targeting RSV.

[0041] In another aspect of the invention there is further provided a method for preparing an antibody or antibody fragment or variant according to any one of the above, said method comprising the steps of: a. Preparing any one of the above antibodies or antibody fragments or variants thereof by culturing a recombinant cell containing a nucleic acid molecule encoding said antibody or antibody fragment or variant thereof in a culture medium under conditions allowing expression of said antibody or antibody fragment or variant.

[0042] In one embodiment, the method further comprises: b. recovering said antibody or antibody fragment or variant from said recombinant cell or medium.

[0043] In another aspect of the invention there is further provided the use of an antibody or antibody fragment or variant according to any one of the above claims in the preparation of any one of the following products: (1) Products that detect PD-1, (2) Products that stimulate or enhance immune responses; (3) Products that treat cancer.

[0044] In one embodiment, the product that detects PD-1 is a product that detects hPD-1, for example a product that detects expression of hPD-1 in specific cells, tissues, or serum in vitro, or a product that detects hPD-1 in vivo.

[0045] In one embodiment, in a product for stimulating or enhancing an immune response, immune cells are contacted with the antigen and said antibody or antibody fragment or variant, thereby stimulating or enhancing an immune response to the antigen.

[0046] In one embodiment, the immune response is a T cell-mediated immune response.

[0047] In some embodiments, the product for treating cancer is selected from the group consisting of melanoma (e.g., malignant metastatic melanoma), renal cancer (e.g., clear cell carcinoma), prostate cancer (e.g., hormonally refractory prostate adenocarcinoma), pancreatic adenocarcinoma, breast cancer, colon cancer, lung cancer (e.g., non-small cell lung cancer), esophageal cancer, squamous cell carcinoma of the head and neck, liver cancer, ovarian cancer, cervical cancer, thyroid cancer, spongioblastoma, glioma, leukemia, lymphoma, and other malignant tumors.

[0048] In another aspect of the invention, there is further provided a pharmaceutical composition comprising an antibody or antibody fragment or variant as defined in any one of the above as a therapeutically active ingredient, and a pharma- ceutically acceptable excipient, diluent or carrier, or a mixture thereof.

[0049] In another aspect of the invention there is further provided a method of treating cancer, such as the cancers mentioned above, comprising administering to an individual in need thereof an effective amount of an antibody or antibody fragment or variant or pharmaceutical composition as described in the invention, said individual may be (for example) a rodent, canine, porcine, primate or human.

[0050] In one embodiment, the antibodies or antibody fragments provided by the present invention bind to hPD-1 and exhibit a number of favorable properties, including:

[0051] 1. The antibody or antibody fragment provided by the present invention can bind to hPD-1 with high affinity and regulate immune responses. Specifically, the antibody or antibody fragment of the present invention can bind to hPD-1 with a KD of about 20 pM or less.

[0052] 2. The antibody or antibody fragment provided by the present invention has a titer of about 1.98 × 10 6 1 / Ms or higher k 結合 It can bind to hPD-1 at

[0053] 3. The antibody or antibody fragment provided by the present invention has a titer of about 4.11 × 10 -5 k equal to or less than 1 / s 解離 The dissociation constant may be measured by biolayer optical interferometry (e.g., ForteBio Octet method) or by surface plasmon resonance technology (e.g., Biacore).

[0054] 4. The antibody or antibody fragment provided by the present invention has an IC of about 1 nM or less. 50 The binding of hPD-L1 to hPD-1 can also be blocked by any method known in the art, such as FACS, to measure the degree of ligand binding blockade and obtain IC 50 can be calculated.

[0055] 5. The antibody or antibody fragment provided by the present invention can block the binding of hPD-1 and hPD-L1, thereby disrupting the downstream signaling pathway mediated by hPD-1 / hPD-L1. BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 is a graph of dose-dependent binding of anti-hPD-1 antibodies to purified hPD-1 in a protein ELISA.

[0056] Figure 2 shows the k binding and k dissociation curves of CL1 antibody measured by Biolayer Interferometry. Each curve represents the dynamics of the binding of CL1 antibody to hPD-1 at various concentrations.

[0057] Figure 3: Detection of blocking of hPD-1 / hPD-L1 binding by CL1 antibody by flow cytometry.

[0058] [Figure 4] Detection of the functional activity of anti-hPD-1 antibody by PD-1 / PD-L1 Blockade Bioassay. [Mode for carrying out the invention] The experimental methods used in the examples described below are all conventional methods unless otherwise stated.

[0059] Materials, reagents and the like used in the examples described below are all available from commercial sources unless otherwise specified.

[0060] The present invention will be further described below in conjunction with specific examples. It should be understood that the following examples are not intended to limit the scope of the present invention, but are merely intended to illustrate the present invention. [Abbreviations and definitions] Unless otherwise explained, the following terms shall have the meanings set forth below. Other terms and abbreviations have the meanings commonly accepted in the art.

[0061] "Antibody" refers to any form of antibody that exhibits the required biological activity (e.g., inhibiting binding of a ligand to its receptor or inhibiting receptor signaling by a ligand). Thus, "antibody" is used in its broadest sense and expressly includes, but is not limited to, monoclonal antibodies (including full-length monoclonal antibodies), polyclonal and multispecific antibodies (e.g., bispecific antibodies), fully human, humanized, primatized, chimeric antibodies, single chain antibodies, and the like.

[0062] An "antibody fragment" is a portion of an antibody, e.g., F(ab')2, F(ab)2, Fab', Fab, Fv, scFv, etc. Regardless of structure, an antibody fragment binds with the same antigen that is recognized by the intact antibody. The term "antibody fragment" includes aptamers, enantiomers, and bivalent antibodies. The term "antibody fragment" further includes any synthetic or genetically engineered protein that performs the action of an antibody by binding to a specific antigen to form a complex.

[0063] A "Fab fragment" is composed of one light chain and the CH1 and variable regions of one heavy chain. The heavy chain of a Fab molecule cannot form disulfide bonds with other heavy chain molecules.

[0064] The "Fc region" contains two heavy chain fragments containing the CH2 and CH3 structural domains of an antibody, which are held together by hydrophobic interaction of the CH3 structural domain by two or multiple disulfide bonds.

[0065] The "Fv region" comprises the variable regions from both the heavy and light chains, but lacks the constant regions.

[0066] A "single-chain Fv antibody" (or "scFv antibody") is an antibody fragment comprising the VH and VL structural domains of an antibody, wherein these structural domains are present in a single polypeptide chain. Generally, the Fv polypeptide further comprises a polypeptide linker between the VH and VL structural domains which enables the scFv to form the required structure for antigen binding. For a general review of scFvs, see U.S. Patent No. 6,423,538.

[0067] Those skilled in the art will appreciate that the types of antibody heavy chains include gamma, mu, alpha, delta or epsilon (γ, μ, α, δ, ε) and also include several subclasses (e.g., γ1-γ4). Depending on the nature of the chain, the "type" of the antibody is determined as IgG, IgM, IgA, IgD or IgE, respectively. For example, immunoglobulin subclasses (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgG5, etc., have already been well characterized and are known to have conferred functional specificities. Any immunoglobulin type is covered by the claims of the present invention. In one embodiment, the type of immunoglobulin molecule is IgG. IgG generally comprises two identical light chain polypeptides with a molecular weight of about 23,000 daltons and two identical heavy chain polypeptides with a molecular weight of about 53,000-70,000. The four chains are connected by disulfide bonds in a "Y" configuration, with the light chains starting at the mouth of the "Y" and continuing through the variable regions to surround the heavy chains. IgG4 forms of antibodies are a subclass of IgG, and their heavy chains are of the γ4 subtype. In one embodiment, the antibody of the invention is an IgG4.

[0068] "Hypervariable region" are those antibody amino acid residues responsible for antigen binding. The hypervariable region comprises amino acid residues from the "complementarity determining regions" or "CDRs" defined by sequence alignment. "Framework" or "FR" residues are those variable domain residues other than the hypervariable region residues as herein defined.

[0069] The antibodies of the present invention may be derived from any animal, including birds and mammals. Preferably, the antibodies are of human, murine, donkey, rabbit, goat, camel, llama, horse or chicken origin. In other embodiments, the variable regions may be of condricthoid origin (e.g., from shark). A "human antibody" is an antibody whose amino acid sequence corresponds to that of a human antibody and / or prepared by any technique for preparing human antibodies as set forth herein. This definition expressly excludes humanized antibodies that contain non-human antigen-binding residues.

[0070] An "isolated antibody" is an antibody that has been separated from all or part of the components of its natural environment. Contaminant components of its natural environment are substances that would interfere with the diagnostic or therapeutic application of the antibody, including enzymes, hormones, and other proteinaceous or non-proteinaceous solutes. In some embodiments, the antibody is purified to the extent that: (1) the antibody is more than 95% by weight, e.g., more than 99% by weight, as measured by the Lowry method; (2) sufficient to obtain at least 15 residues of N-terminal or internal amino acid sequence by a rotating cup sequenator; or (3) as determined to be homogeneous by Coomassie blue or silver stained SDS-PAGE under reducing or non-reducing conditions. An isolated antibody includes the antibody in its original site within a recombinant cell, since it is free of at least one component of the antibody's natural environment. An isolated antibody is generally prepared by at least one purification step. In certain embodiments, the purity of the isolated antibodies is at least about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, about 99%, or a range between any two of these values ​​(including the endpoints), or any value therein.

[0071] A "nucleic acid" or "polynucleotide" is a polymeric molecule composed of the single nucleotides adenine (a), cytosine (c), guanine (g), thymine (t) (or uracil (u) in RNA), e.g., DNA, RNA, or modifications thereof. A nucleic acid molecule may be a naturally occurring nucleic acid molecule, or a synthetic nucleic acid molecule, or a combination of one or more naturally occurring nucleic acid molecules and one or more synthetic nucleic acid molecules. Examples of nucleic acids include, but are not limited to, a gene or gene fragment (e.g., a probe, primer, EST or SAGE label), exon, intron, messenger RNA (mRNA), transfer RNA, ribosomal RNA, ribozyme, cDNA, dsRNA, siRNA, miRNA, recombinant polynucleotides, branched polynucleotides, plasmids, carriers, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes, and primers.

[0072] An "isolated nucleic acid molecule" is a nucleic acid molecule that has been identified and separated from at least one contaminating nucleic acid molecule. An isolated nucleic acid molecule is different from the form or environment in which it is found in nature. Thus, an isolated nucleic acid molecule is different from the nucleic acid molecule as it exists in its natural cell. However, isolated nucleic acid molecules include nucleic acid molecules contained in cells that normally express the antibody, e.g., the chromosomal site at which the nucleic acid molecule is located is different from the chromosomal site at which it exists in natural cells.

[0073] A "monoclonal antibody" is an antibody derived from a group of essentially homogenous antibodies, each of which is identical. Monoclonal antibodies are highly specific, being able to target a single antigenic site. This is contrary to conventional (polyclonal) antibody preparations, which typically contain several different antibodies against several different determinants (epitopes), with each of the various monoclonal antibodies targeting only a single determinant on the antigen.

[0074] "Immune cells" include cells that are linked to hematopoiesis and function in the immune response. Immune cells include B and T lymphocytes, natural killer cells, monocytes, macrophages, eosinophils, mast cells, basophils, and granulocytes.

[0075] As used herein, a "variant" of a sequence is a sequence that differs from the shown sequence in one or more amino acid residues, yet retains the biological activity of the resulting molecule.

[0076] An "amino acid" is an organic compound that contains both an amino group and a carboxyl group, such as an α-amino acid, and can be encoded by a nucleic acid, either as such or in precursor form. A single amino acid is encoded by a nucleic acid that consists of three nucleotides (a so-called codon or base triplet). The ability to encode the same amino acid by different codons is called the "degeneracy of the genetic code." Amino acids include natural and unnatural amino acids. Naturally occurring amino acids include alanine (three letter code: ala, one letter code: A), arginine (arg, R), asparagine (asn, N), aspartic acid (asp, D), cysteine ​​(cys, C), glutamine (gln, Q), glutamic acid (glu, E), glycine (gly, G), histidine (his, H), isoleucine (ile, I), leucine (leu, L), lysine (lys, K), methionine (met, M), phenylalanine (phe, F), proline (pro, P), serine (ser, S), threonine (thr, T), tryptophan (trp, W), tyrosine (tyr, Y) and valine (val, V).

[0077] A "conservatively substituted variant" or "conservatively substituted amino acid substitution" is an amino acid substitution known to those skilled in the art, which generally does not alter the biological activity of the resulting molecule. In general, those skilled in the art recognize that single amino acid substitutions in non-essential regions of a polypeptide do not fundamentally alter the biological activity. Conservative substitutions may be made with amino acids containing side chains with similar chemical properties, such as: 1) aliphatic side chains: glycine, alanine, valine, leucine, and isoleucine; 2) aliphatic hydroxyl side chains: serine and threonine; 3) amide-containing side chains: asparagine and glutamine; 4) aromatic side chains: phenylalanine, tyrosine, and tryptophan; 5) alkaline side chains: lysine, arginine, and histidine; and 6) acidic side chains: aspartic acid and glutamic acid.

[0078] The term "about" as used herein means that a numerical value is within an acceptable error range of a specific value as determined by one of ordinary skill in the art, the numerical portion being determined by how it is measured or determined (i.e., the limitations of the measurement system). Alternatively, "about" may mean a range of at most ±20%, such as a range of ±10%, ±5% or ±1%. Unless otherwise stated, when a specific value appears in the present application and claims, the meaning of "about" should be within an acceptable error range of the specific value.

[0079] "Specific" binding, when referring to a ligand / receptor, antibody / antigen, or other binding pair, is a binding reaction that determines whether or not a protein, such as PD-1, is present in a heterogeneous population of proteins and / or other biological reagents. Thus, under given conditions, a particular ligand / antigen binds to a particular receptor / antibody and does not bind in significant amounts to other proteins in the sample. Specific binding can be described by an equilibrium dissociation constant (KD), where a smaller KD means a tighter binding. Methods for determining whether two molecules are specifically bound include, for example, equilibrium dialysis, surface plasmon resonance, biolayer optical interferometry, and the like, and are well known in the art. Antibodies that "specifically bind" to PD-1 include antibodies with an equilibrium dissociation constant KD with PD-1 of about 100 nM or less, about 10 nM or less, about 5 nM or less, or about 1 nM or less.

[0080] As used herein, the term "biomaterial" refers to a nucleic acid molecule, an expression cassette, a recombinant vector , recombinant cells or recombinant microorganisms.

[0081] "Administration" and "treatment", when referring to an animal, human, experimental subject, cell, tissue, organ, or biological fluid, refer to contacting an exogenous drug, therapeutic, diagnostic, or composition with the animal, human, subject, cell, tissue, organ, or biological fluid. "Administration" and "treatment" may refer, for example, to therapeutic, pharmacokinetic, diagnostic, research, and experimental methods. Cell therapy includes contacting a reagent with a cell and contacting a reagent with a flowing fluid, where the flowing fluid contacts the cell. "Administration" and "treatment" further refer to in vitro and ex vivo treatment of cells, for example, with a reagent, diagnostic, binding composition, or with other cells.

[0082] The terms "inhibition" or "treat" or "treatment" as used herein include alleviating the progression of symptoms associated with a disease and / or reducing the severity of impending or anticipated symptoms of said disease. The terms further include alleviating existing symptoms, preventing other symptoms, and alleviating or preventing the potential causes of these symptoms. Thus, the terms indicate that a beneficial result has already been imparted to a vertebrate subject, such as a human, suffering from a disease.

[0083] The term "therapeutically effective amount" or "effective amount" as used herein refers to an amount of an anti-PD-1 antibody or fragment thereof that, when administered alone or in combination with other therapeutic agents to a cell, tissue, or subject, effectively prevents or alleviates the disease or condition being treated. A therapeutically effective amount further refers to an amount of the antibody or fragment thereof sufficient to alleviate a symptom, for example, treating, curing, preventing, or alleviating an associated medical condition, or improving the treatment, cure, prevention, or alleviation rate of the disease. The effective amount for a particular subject can vary depending on a variety of factors, for example, the disease being treated, the overall physical condition of the patient, the route and dose of administration, and the severity of side effects. An effective amount may be the maximum dose or dosing regimen that avoids significant side effects or toxic effects. When administering an active ingredient administered alone to an individual, the therapeutically effective amount refers to that ingredient alone. When administered in combination, regardless of whether administered in combination, sequentially, or simultaneously, the therapeutically effective amount is the combined amount of active ingredients that produces a therapeutic effect. A therapeutically effective amount reduces symptoms by at least 10%, typically at least 20%, preferably at least 30%, more preferably at least 40%, and most preferably at least 50%. In one embodiment, an effective amount of anti-PD-1 antibody or fragment thereof is between 0.01 mg and 1 g. In one embodiment, an effective amount of anti-PD-1 antibody or fragment thereof is between 10 mg and 750 mg. The antibodies described herein can be administered by any conventional route, such as intravenous or subcutaneous injection.

[0084] Monoclonal antibodies Monoclonal antibodies against PD-1 can be prepared using knowledge and skill in the art, and antibodies or antibody fragments can be isolated from scFv yeast display libraries.

[0085] Conventional methods allow the easy isolation and sequencing of DNA encoding a monoclonal antibody. Once isolated, the DNA can be placed into an expression vehicle, and the vehicle can be transfected into a host cell, such as an E. coli cell, a monkey COS cell, a Chinese hamster ovary (CHO) cell, or a myeloma cell in which no immunoglobulin is otherwise produced, such that synthesis of the monoclonal antibody is achieved in the recombinant host cell. Recombinant preparation of antibodies is described in more detail below.

[0086] Chimeric antibodies Antibody DNA can also be modified, for example, by replacing the coding sequence for human heavy and light chain constant structural domains with homologous murine sequences, or by covalently linking a sequence encoding an immunoglobulin with all or part of a sequence encoding a non-immunoglobulin substance (e.g., a protein structural domain). Typically, the constant structural domains of an antibody or the variable structural domains of one antigen-binding site of the replaced antibody are replaced with the non-immunoglobulin substance, resulting in a bivalent chimeric antibody that contains one antigen-binding site specific for an antigen and another antigen-binding site specific for a different antigen.

[0087] Antibody purification If recombinant techniques are used, the antibody can be produced intracellularly, in the periplasmic space, or directly secreted into the medium. If the antibody is produced intracellularly, in the periplasmic space, the cells are slurry freeze-thawed for about 30 minutes in the presence of sodium acetate (pH 3.5), EDTA, and phenylmethylsulfonyl fluoride (PMSF), and centrifugation is used to remove cell debris. If the antibody is secreted into the medium, the supernatant from the expression system can first be concentrated using a commercially available protein concentration filter (e.g., Amicon or Millipore Pellicon ultrafiltration unit). In any of the above steps, protease inhibitors (e.g., PMSF) can be used to inhibit protein hydrolysis, and antibiotics can be used to prevent the growth of external contaminants.

[0088] For example, antibody compositions can be prepared from cells by hydroxyapatite, gel electrophoresis, dialysis and affinity chromatography, with affinity chromatography being the preferred purification technique. The application of Protein A as an affinity ligand depends on the type and isotype of the immunoglobulin Fc region present in the antibody. Protein A can be applied to purify antibodies based on human gamma 1, gamma 2 or gamma 4 heavy chains (Lindmark et al., 1983, J. Immunol. Meth. 62:1-13). The matrix on which the affinity ligand relies is typically agarose, although other matrices may be utilized. Mechanically stable matrices such as controlled pore glass and poly(styrene-divinylbenzene) allow for faster obtainable flow rates and shorter processing times compared to agarose. If the antibody contains a CH3 structural domain, it can be purified by Bakerbond ABX™ resin. Depending on the antibody to be recovered, other protein purification techniques may be used, such as, for example, separation on an ion exchange column, ethanol precipitation, reverse phase HPLC, chromatography on an anion or cation exchange resin (e.g., a polyaspartic acid column), chromatographic focusing, and SDS-PAGE.

[0089] Therapeutic Applications of the Antibodies and Antibody Fragments of the Invention The antibodies or antigen-binding fragments of the invention that specifically bind to PD-1 are applicable to stimulating or enhancing an immune response. The antibodies and antibody fragments of the invention are particularly applicable to treating subjects suffering from diseases that can be treated by enhancing a T cell-mediated immune response. In one embodiment, the subject includes a human patient in need of an enhanced immune response.

[0090] In one embodiment, the present invention further provides a method of treating a disease by enhancing a T cell-mediated immune response, the method comprising administering to a patient in need thereof an effective therapeutic amount of an antibody or antigen-binding fragment described in the present invention. In one embodiment, the disease treated by enhancing the T cell-mediated immune response is cancer.

[0091] cancer The antibodies or antigen-binding fragments of the present invention can be applied to treat cancer (i.e., inhibiting the proliferation or survival of tumor cells). In some embodiments, cancers whose proliferation can be inhibited by the antibodies of the present invention include cancers that are generally sensitive to immunotherapy, but also include cancers that have not been implicated in immunotherapy to date. In some embodiments, non-limiting forms for treating cancer include melanoma (e.g., malignant metastatic melanoma), kidney cancer (e.g., clear cell carcinoma), prostate cancer (e.g., hormonally refractory prostate adenocarcinoma), pancreatic adenocarcinoma, breast cancer, colon cancer, lung cancer (e.g., non-small cell lung cancer), esophageal cancer, head and neck squamous cell carcinoma, liver cancer, ovarian cancer, cervical cancer, thyroid cancer, spongioblastoma, glioma, leukemia, lymphoma, and other malignant tumors. In addition, the present invention includes refractory or recurrent cancers whose proliferation can be inhibited by the antibodies or antigen-binding fragments of the present invention.

[0092] Non-therapeutic Applications of Antibodies and Antibody Fragments of the Invention There are already commercial anti-PD-1 antibodies for non-therapeutic applications, such as Mab1086 anti-hPD-1 monoclonal antibody sold by R&D Systems of Minneapolis, MN, USA, which can be applied to flow cytometry, Western Blot and ELISA. The antibodies of the present invention can be applied to any of the non-therapeutic purposes provided by Mab1086.

[0093] The antibodies of the present invention can be applied in diagnostic assays, for example to detect the expression of PD-1 in specific cells, tissues or serum. For diagnostic applications, the antibodies are generally marked (directly or indirectly) with a detectable moiety. Many markers are available, generally divided into biotin, fluorochromes, radioactive nucleotides, enzymes, iodine and biosynthetic markers.

[0094] The antibodies of the invention can further be applied to in vivo diagnostic assays. Generally, they are coupled to a radionuclide (e.g., 111 In, 99 Tc, 4 C. 125 I, 3 H, 32 P, 35 S 18 F) Mark the antibodies.

[0095] Pharmaceutical Compositions The present invention further provides pharmaceutical compositions, comprising an effective amount of an antibody or antibody fragment, and a pharma- ceutically acceptable carrier.

[0096] In certain embodiments, the term "pharmaceutically acceptable carrier" refers to a substance approved by a government regulatory agency or listed in other generally recognized pharmacopeias for use in animals (especially humans). Further, a "pharmaceutically acceptable carrier" generally refers to any type of non-toxic solid, semi-solid or liquid filler, diluent, encapsulating material or formulation additive.

[0097] The term "carrier" refers to a therapeutic diluent, adjuvant, excipient, or carrier used with an active ingredient. Such pharmaceutical carriers may be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable, or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil, and the like. In some embodiments, the carrier may be water when the pharmaceutical composition is administered intravenously. Saline solutions, aqueous glucose solutions, and glycerin solutions may be used as liquid carriers, particularly for injectable solutions. Examples of suitable pharmaceutical carriers are described in Remington's Pharmaceutical Sciences by EW Martin, which is incorporated herein by reference. Such compositions provide a suitable dosage form for the patient by combining a clinically effective amount of the antibody or antibody fragment with a suitable carrier. The formulation should be adapted to the mode of administration. The formulation may be packaged in ampoules, disposable syringes, or multiple dose vials made of glass or plastic.

[0098] In some embodiments, the composition is prepared by conventional processes into a pharmaceutical composition suitable for intravenous injection into humans. The composition for intravenous administration is generally a solution in a sterile or other water-permeable buffer. The pharmaceutical composition may further include a solubilizer and a local anesthetic, such as lidocaine, to alleviate pain at the injection site. Generally, the active ingredient is provided singly or in admixture in unit dose form, for example in a sealed container (e.g., an ampoule or sachet) that indicates the amount of active agent in the form of a dry lyophilized powder or water-free concentrate. When the composition is administered by infusion, the composition may be packaged separately in an infusion bottle containing sterile medical water or saline. When the composition is administered by injection, an ampoule of sterile water for injection or saline may be used so that the active ingredients can be mixed before administration.

[0099] The antibody or antibody fragment of the present invention includes its salt forms. Pharmaceutically acceptable salts include those derived from salts formed with anions such as hydrochloric acid, phosphoric acid, acetic acid, oxalic acid, tartaric acid, and the like, and those derived from salts formed with cations such as sodium, potassium, ammonium, calcium, ferric hydroxide, isopropylamine, triethylamine, 2-ethylaminoethanol, histidine, procaine, and the like. [Reagents or materials] hPD-1 is a product of Beijing Yiqiao Shenzhou Technology Co., Ltd., with catalog number 10377-H03H-B.

[0100] hIgG is a product of Genscript Biotech Co., Ltd., catalog number A01006.

[0101] Jurkat cells expressing hPD-1 are a product of Promega, catalog no. It's J1252.

[0102] Unmarked hPD-1 is a product of Beijing Yiqiao Shenzhou Technology Co., Ltd., with catalog number 10084-HNAH.

[0103] PE-marked goat anti-human Fc is a product of Thermofisher, catalog number 12-4998-82.

[0104] CHO-K1 cells are a product of Promega, catalog number J1252.

[0105] F-12 is a product of Thermofisher and has catalog number 11765062.

[0106] The Bio-Glo Luciferase Assay System is a product of Promega and has the catalog number G7940. Example 1: Preparation of anti-hPD-1 antibody and its sequence determination First, human VL and VH cDNAs were prepared from human lymphocyte mRNA, and an scFv yeast display library was prepared using the cDNAs. The CL1 and CL1-related antibodies of the present invention were isolated by screening the scFv yeast display library. Methodologies for preparing and screening such libraries are already known in the art. Methods and reagents specifically adapted for preparing and screening antibody display libraries can be found in literature such as U.S. Patent No. 6,423,538 to Wittrup et al.

[0107] The DNA sequences encoding the scFv variable regions expressed in yeast were determined by gene sequencing. Each sequencing reaction used for each cloning was analyzed by Seqman. The sequences are disclosed in the sequence listing and listed in Table 1.

[0108] [Table 1] TIFF0007641910000002.tif249169TIFF0007641910000003.tif249169TIFF0007641910 000004.tif249169TIFF0007641910000005.tif249169TIFF0007641910000006.tif25016 9TIFF0007641910000007.tif250169TIFF0007641910000008.tif249169TIFF0007641910 000009.tif249169TIFF0007641910000010.tif248169TIFF0007641910000011.tif24169

[0109] Positions 73-114 of SEQ ID NO:2 encode the nucleotide sequence of SEQ ID NO:13.

[0110] Positions 160-177 of SEQ ID NO:2 encode the nucleotide sequence of SEQ ID NO:14.

[0111] Positions 271-303 of SEQ ID NO:2 encode the nucleotide sequence of SEQ ID NO:15.

[0112] Positions 91-108 of SEQ ID NO:1 encode the nucleotide sequence of SEQ ID NO:16.

[0113] Positions 148-192 of SEQ ID NO:1 encode the nucleotide sequence of SEQ ID NO:17.

[0114] Positions 292-327 of SEQ ID NO:1 encode the nucleotide sequence of SEQ ID NO:18.

[0115] Example 2: Generation and expression of CL1 antibody and related antibodies The VL and VH region DNA of the PCR-cloned CL1 antibody was linked to the human κ light chain constant region and IgG4 constant region (γ4 heavy chain constant region), respectively, to produce a light chain and a heavy chain. A DNA sequence was generated. The sequence was modified at the 5' and 3' ends with PCR primers designed to add appropriate leader sequences to each strand and then cloned into an existing recombinant antibody expression vehicle. Sequence analysis demonstrated correct construction of the vehicle. 40 μg of expression plasmid was added to 700 μL of 10 total cells. 7 The cells were homogenously mixed with 100% CHO, placed in a 0.4 cm electroporation cup, and placed in an electroporator, and the electroporation parameters (voltage 300 V, electric pulse time 17 ms, square wave) were set to complete the electroporation. After completing the electroporation, the cells were cultured in 8 mL of growth medium for 3 days. The supernatant was measured for transfected antibody concentration by ELISA, and the engineered cell line with high expression and good cell growth was finally determined.

[0116] Cells were grown in CD-CHO (Gibco) in shake flasks for 12 days. Antibodies in cell supernatants were purified by Protein A affinity chromatography, washing, elution with 1M acetic acid and neutralization with 3M Tris. Finally, the buffer was changed to 100 mM acetic acid adjusted to pH 5.5 with 1M Tris to obtain CL1 antibody.

[0117] The construction and expression of CL5, CL9, CL2, CL7, CL6 and CL20 were similarly carried out using methods similar to those described above.

[0118] The amino acid sequences of the purified CL1, CL5, CL9, CL2, CL7, CL6 and CL20 antibodies are as expected.

[0119] Example 3: Analysis of the affinity of anti-hPD-1 antibodies to hPD-1 Enzyme-linked immunosorbent assay (ELISA) was used to measure the binding of antibodies to hPD-1. hPD-1 was immobilized on a 96-well plate by overnight incubation at 4℃. After coating, the plate was washed five times with PBST. Different concentrations of dilutions of antibodies CL1, CL5, CL9, CL2, CL7, CL6, CL20 and hIgG were prepared in PBST and incubated with the immobilized hPD-1 for 1 h at room temperature. After binding, Plates were washed five times with PBST, incubated for 1 h at room temperature with PBST containing goat anti-mouse IgG marked with peroxidase (HRP) diluted to 1 / 10000, washed again and developed with TMB.

[0120] The results of the ELISA are shown in Figure 1. The half-maximal effective concentration (EC 50 ) represents the binding affinity, and the results are shown in Table 2.

[0121] As is evident from the experimental results, the anti-hPD-1 antibodies CL1, CL5, CL9, CL2, CL7, Both CL6 and CL20 bind to hPD-1, and among them, EC2 bound to hPD-1 of CL150 The affinity of CL5 was about 0.208 nM, and the affinity of CL6 was the highest. Clearly superior to CL7, CL6 and CL20.

[0122] Example 4: Kinetic analysis of antibody CL1 binding to hPD-1 To further characterize the binding properties of the antibody, antibody CL1 was profiled by Biolayer Optical Interferometry on an Octet system (ForteBio, Menlo Park, CA) to reveal the binding kinetics and calculate the equilibrium binding constant. This was performed by coupling the hPD-1 fusion protein with biotin to a streptavidin biosensor.

[0123] To immobilize hPD-1 on the streptavidin biosensor substrate, hPD-1 must be biotinylated. First, a 10 mg / ml biotin solution was prepared by dissolving 1.0 mg of biotin in 100 μl of dimethyl sulfoxide, and then 10 μl of biotin solution was added for every milliliter of hPD-1 (1.8 mg / ml), and the reaction was gently stirred and incubated at room temperature in the dark for 2 hours to obtain hPD-1-biotin. A PD-10 column containing Sephadex G-25M (Pharmacia, Catalog No. 17-0851-01) was equilibrated with 25 ml of cold PBS and 2 ml of hPD-1-biotin was loaded per column. The column was eluted with 10×1 ml of cold PBS. The eluent was collected and read at OD280 to obtain purified hPD-1-biotin, which was stored at -80°C until use.

[0124] Streptavidin biosensors were pre-wetted in wells containing PBS for 10 min and then equilibrated with PBS for 2 min. The biosensors were coupled with hPD-1-biotin by immersing the biosensors in a 20 μg / mL solution of hPD-1-biotin in PBS for 5 min and equilibrating the biosensors with PBS for 2 min. The binding status of anti-PD-1 antibodies was observed by placing the biosensors in wells containing various concentrations of antibody (0.125-2 nM, antibody with SDS-PAGE purity >99%) and monitored for 30 min. Dissociation was monitored after transferring the biosensors to PBS and monitored for 90 min. The observed binding and dissociation rates (k 結合 and k 解離 ) was fitted to calculate the equilibrium binding constant KD.

[0125] The results of the kinetic study are shown in Table 2 and FIG.

[0126] As can be seen from the experimental results, the KD of the CL1 antibody binding to hPD-1 is approximately 0.0207 nM, and the strong affinity is due to its much faster binding rate and slower dissociation rate than ordinary antibodies.

[0127] Example 5: Study of blocking antibody CL1 on hPD-1 / hPD-L1 binding Blockade of ligand binding was studied by flow cytometry. Jurkat cells expressing hPD-1 were plated in a 96-well plate and various concentrations of antibodies (CL1 or hIgG (as a negative control)) and unmarked hPD-L1 at a concentration of 1 μg / mL were added. The mixture was placed on ice for 30 min, washed three times with FACS buffer (PBS containing 0.3% BSA) and incubated with PE-marked goat anti-human Fc for another 30 min on ice. The cells were washed again three times with FACS buffer and analyzed by flow cytometer. Data were analyzed by nonlinear regression method in Prism software to determine IC 50 The values ​​were calculated, and the results are shown in Table 2 and FIG.

[0128] As shown in the experimental results, the anti-PD-1 antibody CL1 was able to completely inhibit the binding of hPD-L1 to hPD-1 on Jurkat cells in a dose-dependent manner, and the IC 50 The value is approximately 0.77 nM.

[0129] [Table 2]

[0130] Example 6: Kinetic analysis of CL1-related antibodies A series of antibodies related to the CL1 antibody were analyzed by Biolayer Optical Interferometry as described in Example 4. The amino acid sequences of the VL and VH regions of the related antibodies are shown in the Sequence Listing. The equilibrium binding constants KD of each related antibody are shown in Table 3.

[0131] [Table 3]

[0132] As is evident from the results in Table 3, when the VH and VL are the same, the affinity of the scFv format antibody to hPD-1 is lower than that of the IgG4 format antibody. When the same antibody format is used, i.e., both are IgG4 or scFv, the VH amino acid sequence of antibody CL1 is left unchanged, but after substituting the VL of antibody CL1 (substituting with the VL of antibody CL5, the VL of antibody CL9, the VL of antibody CL2, or the VL of antibody CL7), the affinity is reduced in all cases, and the affinity is most reduced after substituting with the VL of CL9. It is clear that the VL of antibody CL1 plays an important role in the binding of CL1 to hPD-1, and when the amino acid sequence is changed, the conformation is changed, which may affect the binding to hPD-1.

[0133] Example 7: Analysis of the functional activity of anti-hPD-1 antibodies To detect the functional activity of the anti-hPD-1 antibodies, in vitro detection of the inhibitory ability of the antibodies on hPD-1 activity was performed.

[0134] Anti-hPD-1 antibody blocks the hPD-1 / hPD-L1 interaction and inhibits the proliferation and proliferation of Jurkat cells. Activates immune signal pathways downstream of TCR.

[0135] A reporter gene is a gene whose expression product is highly detectable. Its coding sequence is fused with a gene expression regulatory sequence to form a chimeric gene, which is expressed under the control of the regulatory sequence, and the upstream signal is indirectly detected by detecting the expression product. The PD-1 / PD-L1 Blockade Bioassay system is applied to detect the biological activity of anti-hPD-1 monoclonal antibody. Its main principle is that PD-L1 and T cell receptor activator protein (TCR activator) are stably expressed on the cell membrane of CHO-K1 cell line, and mimic antigen presenting cells (APCs) are generated. Jurkat is a human T lymphocyte line with PD-1 expression on the cell membrane and transfected with a Luciferase reporter gene carrier with NFAT promoter. When the two cells were incubated together, the TCR activator on the CHO-K1 cells bound to the TCR on the surface of Jurkat cells, inducing the downstream signaling pathway and ultimately activating the expression of the reporter gene; at the same time, the interaction of PD-1 / PD-L1 blocked this activation effect. After the addition of anti-PD-1 monoclonal antibody, the interaction of PD-1 / PD-L1 was blocked, the expression of the luciferase reporter gene was restored, and a fluorescent signal was generated, with the signal value being positively correlated with the amount of anti-PD-1 monoclonal antibody added.

[0136] Anti-hPD-1 antibodies were detected and evaluated by co-incubating the cells with the antibody as described below. CHO-K1 cells were resuspended in F-12 + 10% FBS medium (containing 200 μg / ml Hygromycin B and 250 μg / ml G418) at a cell density of 4.0 × 10 5 Adjust the cells / ml and inoculate 96-well cell plates at 100 μl per well. Keep the 96-well plates at 37°C and 5% CO.2 The plates were then placed in a 35°C incubator and incubated overnight. 96-well microtiter plates containing 125 μl of antibodies (CL1, CL5, and hIgG) were serially diluted 2.5-fold in triplicate along the plate in detection diluent (RPMI medium containing 1% fetal bovine serum (FBS)). Jurkat cells were resuspended in detection diluent to a cell density of 1.25x10 6 The 96-well plate was removed and the medium was discarded, after which 40 μl of the prepared control (hIgG) and detection samples (CL1, CL5) were added to the 96-well plate, and 40 μl of Jurkat cells were added to the 96-well plate. The 96-well plate was incubated at 37° C., 5% CO 2 After placing the plates in a 35°C incubator for 6 h, they were then placed at room temperature for 10 min, after which 80 μl of Bio-Glo Luciferase Assay System reagent was added per well and placed at room temperature for 30 min away from light. The fluorescence values ​​of each well of the 96-well plate were read using the autofluorescence detection function of a multifunctional microplate reader. The data of the control and target samples were curve-fitted using a 4-parameter model using SoftMax Pro 6.5.1 software, and the EC values ​​were calculated using standard methods. 50 The results are shown in Table 2 and FIG.

[0137] As can be seen from the results in Table 2 and Figure 4, in the presence of anti-hPD-1 antibodies, PD-1 / PD-L1 interaction was blocked, the TCR downstream signal pathway of the inhibited Jurkat cells was reactivated, the expression of the luciferase reporter gene was restored, and fluorescent signals were generated, and the inhibition of PD-1 / PD-L1 interaction by anti-hPD-1 antibodies was dose-dependent. Furthermore, the inhibitory abilities of antibodies CL1 and CL5 on hPD-1 activity EC 50 were approximately 0.269 μg / ml and 0.932 μg / ml, respectively, indicating that CL1 had a stronger inhibitory ability on hPD-1 activity, again proving that the functional activity of antibody CL1 is superior to that of CL5 at the cellular level, which is also consistent with the results of Example 3.

[0138] Example 8: Analysis and comparison of binding kinetics of antibodies CL1 and CL20 with hPD-1 The binding kinetics of antibodies CL1 and CL20 to hPD-1 were analyzed and compared using surface plasmon resonance technology in a Biacore system (GE, Boston, MA).

[0139] Detection was performed using a Protein A chip, and antibody CL1 or antibody CL20 at a concentration of 5 μg / ml was passed through the experimental channel at a flow rate of 10 μl / min, and captured for 10 s so that the capture amount was about 200 RU, and then the flow rate was adjusted to 30 μl / min, and different concentrations of PD-1 (0.625 nM, 1.25 nM, 2.5 nM, 5 nM, 10 nM) were added in sequence, with an association time of 180 s and a dissociation time of 1200 s. The binding and dissociation curves were obtained by fitting with a 1:1 Langmuir binding model, and the binding kinetic parameters of antibody CL1 and antibody CL20 with hPD-1 (k binding: association rate, k dissociation: dissociation rate, KD: binding dissociation equilibrium constant) were obtained. The results of the kinetic study are shown in Table 4. The KD of antibody CL1 binding to hPD-1 is approximately 0.606 nM, and the KD of antibody CL20 binding to hPD-1 is approximately 4.434 nM, making the affinity of antibody CL1 to hPD-1 approximately 7-fold higher than that of antibody CL20.

[0140] [Table 4] [Brief description of the drawings]

[0141] [Figure 1] 1 is a graph of dose-dependent binding of anti-hPD-1 antibodies to purified hPD-1 in a protein ELISA. [Diagram 2] 1 is a graph showing the k binding and k dissociation of CL1 antibody measured by Biolayer Interferometry, where each curve represents the kinetic process of the binding of CL1 antibody with hPD-1 at various concentrations. [Diagram 3]Detection of blocking of hPD-1 / hPD-L1 binding by CL1 antibody using flow cytometry. [Figure 4] Detection of the functional activity of anti-hPD-1 antibody by PD-1 / PD-L1 Blockade Bioassay.

Claims

1. An anti-PD-1 antibody or an antibody fragment thereof, The anti-PD-1 antibody or antibody fragment thereof is characterized in that it comprises a light chain CDR having the amino acid sequences shown in SEQ ID NOs: 13, 14, and 15, and a heavy chain CDR having the amino acid sequences shown in SEQ ID NOs: 16, 17, and 18.

2. The anti-PD-1 antibody or antibody fragment thereof according to claim 1, wherein the anti-PD-1 antibody or antibody fragment thereof is an isolated anti-PD-1 antibody or antibody fragment thereof.

3. The anti-PD-1 antibody or antibody fragment thereof is a heavy chain variable region comprising an amino acid sequence as set forth in SEQ ID NO:7, or an amino acid sequence having at least 90% identity with the amino acid sequence as set forth in SEQ ID NO:7, or an amino acid sequence having one or more conservative amino acid substitutions compared to the amino acid sequence as set forth in SEQ ID NO:7; and / or 3. The anti-PD-1 antibody or antibody fragment thereof according to claim 1 or 2, comprising a light chain variable region containing an amino acid sequence set forth in SEQ ID NO: 8, or an amino acid sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO: 8, or an amino acid sequence having one or more conservative amino acid substitutions compared to the amino acid sequence set forth in SEQ ID NO:

8.

4. An anti-PD-1 antibody or antibody fragment thereof comprising a heavy chain variable region and a light chain variable region, the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO:7, The light chain variable region comprises the amino acid sequence shown in SEQ ID NO:

8.

5. The anti-PD-1 antibody or antibody fragment thereof according to any one of claims 1 to 4, further comprising a human γ4 heavy chain constant region or a mutant thereof, and / or a human κ light chain constant region or a mutant thereof.

6. 2. The anti-PD-1 antibody or antibody fragment thereof of claim 1, comprising a heavy chain comprising an amino acid sequence set forth in SEQ ID NO: 71, or an amino acid sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO: 71, or an amino acid sequence having one or more conservative amino acid substitutions compared to the amino acid sequence set forth in SEQ ID NO: 71, and a light chain comprising an amino acid sequence set forth in SEQ ID NO: 72, or an amino acid sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO: 72, or an amino acid sequence having one or more conservative amino acid substitutions compared to the amino acid sequence set forth in SEQ ID NO:

72.

7. An anti-PD-1 antibody or antibody fragment thereof comprising a heavy chain and a light chain, the heavy chain comprises the amino acid sequence set forth in SEQ ID NO: 71, The light chain of the anti-PD-1 antibody or antibody fragment thereof comprises an amino acid sequence shown in SEQ ID NO:

72.

8. B1) A nucleic acid molecule encoding the anti-PD-1 antibody or antibody fragment thereof according to any one of claims 1 to 7; or B2) A biological material related to the anti-PD-1 antibody or its antibody fragment according to any one of claims 1 to 7, which is an expression cassette, a recombinant vector, a recombinant cell, or a recombinant microorganism comprising the nucleic acid molecule according to B1).

9. The nucleic acid molecule according to B1) is 1) A DNA molecule such as: A nucleic acid fragment shown in positions 91-108 of SEQ ID NO:1, A nucleic acid fragment shown in positions 148-192 of SEQ ID NO:1, A nucleic acid fragment as shown in positions 292-327 of SEQ ID NO:1, A nucleic acid fragment shown in positions 73-114 of SEQ ID NO:2, A nucleic acid fragment shown in positions 160-177 of SEQ ID NO:2, A nucleic acid fragment shown at positions 271-303 of SEQ ID NO:2, A nucleic acid fragment as set forth in SEQ ID NO: 1, A nucleic acid fragment as set forth in SEQ ID NO:2, A nucleic acid fragment set forth in SEQ ID NO: 73, or A nucleic acid fragment set forth in SEQ ID NO: 74, 2) A nucleic acid fragment that hybridizes with the nucleic acid fragment defined in 1) and encodes an anti-PD-1 antibody or antibody fragment thereof according to any one of claims 1 to 7; 3) A nucleic acid fragment having 90% or more identity to a DNA molecule defined in 1) or 2) above, encoding an anti-PD-1 antibody or antibody fragment thereof according to any one of claims 1 to 7; 9. The biomaterial according to claim 8, comprising a nucleic acid fragment as shown in 1), 2) or 3) above.

10. A method for preparing the anti-PD-1 antibody or antibody fragment thereof according to any one of claims 1 to 7, comprising: Culturing a recombinant cell comprising the nucleic acid molecule of claim 9 in a medium under conditions allowing expression of the nucleic acid molecule, thereby preparing the anti-PD-1 antibody or antibody fragment thereof.

11. A pharmaceutical composition comprising the anti-PD-1 antibody or antibody fragment thereof according to any one of claims 1 to 7, and a pharma- ceutically acceptable excipient, diluent or carrier, or a mixture thereof.

12. (1) A product that detects PD-1, (2) Products that stimulate or enhance immune responses; (3) Products for treating cancer; Use of an anti-PD-1 antibody or antibody fragment thereof according to any one of claims 1 to 7 in the preparation of any one of the above products.

13. The pharmaceutical composition of claim 11 , wherein the pharmaceutical composition is used to treat cancer.

14. The pharmaceutical composition of claim 13, wherein the cancer is melanoma, renal cancer, prostate cancer, pancreatic adenocarcinoma, breast cancer, colon cancer, lung cancer, esophageal cancer, head and neck squamous cell carcinoma, liver cancer, ovarian cancer, cervical cancer, thyroid cancer, spongioblastoma, glioma, leukemia or lymphoma.

15. The pharmaceutical composition according to claim 13 , wherein the cancer is malignant metastatic melanoma, clear cell carcinoma, hormonally refractory prostate adenocarcinoma, or non-small cell lung cancer.

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