Anti-pd-l2 nanobodies and uses thereof

Abstract

The present application relates to an anti-PD-L2 nanobody and derivatives and uses thereof. Specifically, the present application provides an anti-PD-L2 nanobody, a gene sequence encoding the nanobody, a corresponding expression vector and a host cell capable of expressing the nanobody, and a production method of the nanobody of the present application. The present application also provides fusion proteins simultaneously having the PD-L2 nanobody of the present application. The anti-PD-L2 nanobody and fusion proteins of the present application have high affinity for PD-L2 and can effectively block the binding of PD-L2 to the receptor PD-1.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biomedical or biopharmaceutical technology, more particularly relates to an anti-PD-L2 nanobody and its application. BACKGROUND

[0002] PD-1 (Programmed Death-1), also known as CD279 (Cluster of Differentiation 279), is an important immunosuppressive molecule, an immunoglobulin superfamily, and a 288-amino acid residue membrane protein. It was first cloned from the apoptotic mouse T cell hybridoma 2B4.11.

[0003] PD-1 is an immune checkpoint that prevents autoimmunity through two mechanisms. First, it promotes apoptosis (programmed cell death) of antigen-specific T cells in lymph nodes. Second, it reduces the apoptosis of regulatory T cells (anti-inflammatory, suppressive T cells). By down-regulating the immune system's response to the body's cells, and by suppressing T cell inflammatory activity, it regulates the immune system and promotes self-tolerance. This can prevent autoimmune diseases, but it can also prevent the immune system from killing cancer cells. Immune regulation targeting PD-1 is of great significance in fighting tumors, fighting infections, fighting autoimmune diseases, and organ transplant survival.

[0004] PD-1 has at least two ligands, PD-L1 and PD-L2.

[0005] PD-L2 is the second ligand of PD-1, which is expressed on DC cells, macrophages, human primary nasal epithelial cells, Th2 cells, activated T cells, and some B cell subsets. On human T cells, PD-L2 acts as a negative regulator of T cell activity, inhibiting cell proliferation, IL-2 and IFN-γ production through interaction with PD-1. Treatment with PD-L2-Ig can reduce the TC-1 lung tumor burden in mice and increase survival, which may be through depletion of T cells expressing high levels of PD-1.

[0006] The successful application of monoclonal antibodies in the detection and biological targeting therapy of cancer has brought about a revolution in tumor treatment. However, traditional monoclonal antibodies (150 kD) have a large molecular weight and are difficult to penetrate tissues, resulting in a low effective concentration in the tumor area and insufficient treatment effect; traditional antibodies have high immunogenicity, and modified antibodies are difficult to achieve the original affinity. In addition, the development cycle of fully humanized traditional antibodies is long, the production cost is high, and the stability is not enough, and many other factors limit their application and popularization in clinical practice.

[0007] Nanobodies are currently the smallest antibody molecules, with a molecular weight of 1 / 10 of ordinary antibodies. In addition to the antigen reactivity of monoclonal antibodies, nanobodies also have some unique functional properties, such as small molecular weight, strong stability, good solubility, easy expression, weak immunogenicity, strong penetration, strong targeting, simple humanization, low preparation cost, etc., which almost perfectly overcome the defects of traditional antibodies such as long development cycle, low stability, and harsh storage conditions.

[0008] However, there is currently a lack of satisfactory nanobodies against PD-L2 in the art. Therefore, there is an urgent need in the art to develop new effective specific nanobodies against PD-L2. SUMMARY

[0009] The purpose of the present application is to provide a new class of effective specific nanobodies against PD-L2.

[0010] In a first aspect of the present application, a complementarity determining region CDR of an anti-PD-L2 nanobody VHH chain is provided, the CDR of the VHH chain consisting of:

[0011] a CDR1 having an amino acid sequence as set forth in SEQ ID NO: 5n+1;

[0012] a CDR2 having an amino acid sequence as set forth in SEQ ID NO: 5n+2; and

[0013] a CDR3 having an amino acid sequence as set forth in SEQ ID NO: 5n+3;

[0014] wherein each n is independently 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 13.

[0015] In another preferred embodiment, the n is 2 or 11.

[0016] In another preferred embodiment, the CDR1, CDR2, and CDR3 are separated by a framework region FR1, FR2, FR3, and FR4 of the VHH chain.

[0017] In a second aspect of the present application, a VHH chain of an anti-PD-L2 nanobody is provided, the VHH chain of the anti-PD-L2 nanobody comprising the CDR1, CDR2, and CDR3 as described in the first aspect of the present application.

[0018] In another preferred embodiment, the amino acid sequence of the VHH chain of the anti-PD-L2 nanobody is as set forth in SEQ ID NO: 5n+4, 71, 73, 75, or 77;

[0019] wherein n is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or 13;

[0020] wherein any one of the above amino acid sequences further comprises a derivative sequence optionally having 1-8 (preferably 1-5, more preferably 1-3) amino acid residues added, deleted, modified and / or substituted, and capable of retaining the PD-L2 binding affinity of the PD-L2 Nanobody.

[0021] In another preferred embodiment, n is 2 or 11.

[0022] In another preferred embodiment, the amino acid sequence of the VHH chain of the anti-PD-L2 Nanobody is as set forth in SEQ ID NO: 14, 59, 71, 73, 75 or 77.

[0023] In a third aspect of the present application, there is provided an anti-PD-L2 Nanobody, which is a Nanobody against a PD-L2 epitope and has a VHH chain of the anti-PD-L2 Nanobody as described in the second aspect of the present application.

[0024] In a fourth aspect of the present application, there is provided a Nanobody fusion protein having a structure as set forth in Formula I from N-terminus to C-terminus:

[0025] Z1-Z2-L-Z3 (Formula I)

[0026] In the formula,

[0027] Z1 is a VHH chain of the anti-PD-L2 Nanobody as described in the second aspect of the present application;

[0028] Z2 is an Fc fragment of an immunoglobulin;

[0029] L is a linker sequence;

[0030] Z3 is an immunomodulatory molecule moiety.

[0031] In another preferred embodiment, the immunoglobulin is IgG1, IgG2, IgG3, IgG4.

[0032] In another preferred embodiment, the amino acid sequence of Z2 is as set forth in SEQ ID NO: 79.

[0033] DKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 79, IgG1 Fc fragment amino acid sequence)

[0034] In another preferred embodiment, the amino acid sequence of Z2 is identical or substantially identical to the amino acid sequence set forth in SEQ ID NO: 79.

[0035] In another preferred embodiment, L has an amino acid sequence selected from the group consisting of GGGGS, (GGGGS)2, (GGGGS)3, (GGGGS)4, (GGGGS)5, or a combination thereof.

[0036] In another preferred embodiment, the amino acid sequence of L is set forth in SEQ ID NO: 80.

[0037] GGGGSGGGGSGGGGSGGGGSG (SEQ ID NO: 80)

[0038] In another preferred embodiment, the amino acid sequence of L is identical or substantially identical to the amino acid sequence set forth in SEQ ID NO: 80.

[0039] In another preferred embodiment, the immunomodulatory molecule is a TGFβRII extracellular domain.

[0040] In another preferred embodiment, the amino acid sequence of Z3 is set forth in SEQ ID NO: 81.

[0041] IPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWR KNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPD (SEQ ID NO: 81, TGFβRII extracellular domain)

[0042] In another preferred embodiment, the amino acid sequence of Z3 is identical or substantially identical to the amino acid sequence set forth in SEQ ID NO: 81.

[0043] In another preferred embodiment, the substantially identical is at most 50 (preferably 1-20, more preferably 1-10, more preferably 1-5, most preferably 1-3) amino acids are not identical, wherein the not identical includes substitution, deletion or addition of amino acids.

[0044] In another preferred embodiment, the substantially identical is that the amino acid sequence has at least 70%, at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the corresponding amino acid sequence.

[0045] In a fifth aspect of the present application, a polynucleotide encoding a protein selected from the group consisting of a CDR region of an anti-PD-L2 Nanobody VHH chain as described in the first aspect of the present application, a VHH chain of an anti-PD-L2 Nanobody as described in the second aspect of the present application, an anti-PD-L2 Nanobody as described in the third aspect of the present application, or a Nanobody fusion protein as described in the fourth aspect of the present application is provided.

[0046] In another preferred embodiment, the polynucleotide has a nucleotide sequence as set forth in SEQ ID NO: 5n, 72, 74, 76 or 78;

[0047] wherein the n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14.

[0048] In another preferred embodiment, the polynucleotide comprises DNA or RNA.

[0049] In a sixth aspect of the present application, an expression vector containing the polynucleotide as described in the fifth aspect of the present application is provided.

[0050] In a seventh aspect of the present application, a host cell containing the expression vector as described in the sixth aspect of the present application, or having integrated into its genome the polynucleotide as described in the fifth aspect of the present application is provided.

[0051] In another preferred embodiment, the host cell comprises a prokaryotic cell or a eukaryotic cell.

[0052] In another preferred embodiment, the host cell is selected from the group consisting of E. coli, a yeast cell, a mammalian cell.

[0053] In an eighth aspect of the present application, there is provided a method of producing an anti-PD-L2 Nanobody, comprising the steps of:

[0054] (a) culturing the host cell of the sixth aspect of the present application under conditions suitable for production of a Nanobody, thereby obtaining a culture comprising said anti-PD-L2 Nanobody; and

[0055] (b) isolating or recovering said anti-PD-L2 Nanobody from said culture.

[0056] In another preferred embodiment, said anti-PD-L2 Nanobody has an amino acid sequence as set forth in SEQ ID NO: 5n+4, 71, 73, 75, or 77;

[0057] wherein said n is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 13.

[0058] In a ninth aspect of the present application, there is provided an immunoconjugate comprising:

[0059] (a) a VHH chain of an anti-PD-L2 Nanobody of the second aspect of the present application, an anti-PD-L2 Nanobody of the third aspect of the present application, or a Nanobody fusion protein of the fourth aspect of the present application; and

[0060] (b) a conjugating moiety selected from the group consisting of a detectable label, a drug, a toxin, a cytokine, a radionuclide, or an enzyme.

[0061] In another preferred embodiment, said conjugating moiety is a drug or a toxin.

[0062] In another preferred embodiment, said conjugating moiety is a detectable label.

[0063] In another preferred embodiment, said conjugate is selected from the group consisting of a fluorescent or luminescent label, a radioactive label, an MRI (magnetic resonance imaging) or CT (computed tomography) contrast agent, or an enzyme capable of producing a detectable product, a radionuclide, a biological toxin, a cytokine (such as IL-2, etc.), an antibody, an antibody Fc fragment, an antibody scFv fragment, a gold nanoparticle / nanorod, a viral particle, a liposome, a nanomagnetic particle, a prodrug-activating enzyme (e.g., DT-diaphorase (DTD) or biphenyl hydrolase-like protein (BPHL)), a chemotherapeutic agent (e.g., cisplatin), or any form of nanoparticle, etc.

[0064] In another preferred embodiment, said immunoconjugate comprises a multivalent (e.g., bivalent) VHH chain of an anti-PD-L2 Nanobody of the second aspect of the present application, an anti-PD-L2 Nanobody of the third aspect of the present application, or a Nanobody fusion protein of the fourth aspect of the present application.

[0065] In another preferred embodiment, the multivalent refers to a plurality of repeats of the VHH chain of the anti-PD-L2 Nanobody as described in the second aspect of the present application, the anti-PD-L2 Nanobody as described in the third aspect of the present application, or the Nanobody fusion protein as described in the fourth aspect of the present application in the amino acid sequence of the immunoconjugate.

[0066] In a tenth aspect of the present application, there is provided the use of the anti-PD-L2 Nanobody as described in the third aspect of the present application or the Nanobody fusion protein as described in the fourth aspect of the present application for the preparation of (a) a reagent for detecting a PD-L2 molecule; (b) a medicament for treating a tumor.

[0067] In another preferred embodiment, the detecting comprises flow detection, cellular immunofluorescence detection.

[0068] In an eleventh aspect of the present application, there is provided a pharmaceutical composition comprising:

[0069] (i) the complementarity determining region (CDR) of the anti-PD-L2 Nanobody VHH chain as described in the first aspect of the present application, the VHH chain of the anti-PD-L2 Nanobody as described in the second aspect of the present application, or the anti-PD-L2 Nanobody as described in the third aspect of the present application, the Nanobody fusion protein as described in the fourth aspect of the present application, or the immunoconjugate as described in the ninth aspect of the present application; and

[0070] (ii) a pharmaceutically acceptable carrier.

[0071] In another preferred embodiment, the pharmaceutical composition is in the form of an injection.

[0072] In another preferred embodiment, the pharmaceutical composition is for use in the preparation of a medicament for treating a tumor selected from the group consisting of gastric cancer, liver cancer, leukemia, kidney tumor, lung cancer, small intestine cancer, bone cancer, prostate cancer, colorectal cancer, breast cancer, large intestine cancer, prostate cancer, cervical cancer, lymphoma, adrenal gland tumor, bladder tumor, or a combination thereof.

[0073] In a twelfth aspect of the present application, there is provided the use of one or more of the anti-PD-L2 Nanobody as described in the third aspect of the present application or the Nanobody fusion protein as described in the fourth aspect of the present application:

[0074] (i) for detecting a human PD-L2 molecule;

[0075] (ii) for flow detection;

[0076] (iii) for cellular immunofluorescence detection;

[0077] (iv) for treating a tumor; and

[0078] (v) for tumor diagnosis.

[0079] In another preferred embodiment, the use is non-diagnostic and non-therapeutic.

[0080] In a thirteenth aspect of the present application, there is provided a recombinant protein, said recombinant protein having:

[0081] (i) the sequence of a heavy chain variable region VHH as described in the second aspect of the present application or the sequence of a Nanobody as described in the third aspect of the present application, or the Nanobody fusion protein as described in the fourth aspect of the present application; and

[0082] (ii) an optional tag sequence to facilitate expression and / or purification.

[0083] In another preferred embodiment, the tag sequence comprises: a 6His tag, a HA tag, a Flag tag, a Fc tag, or a combination thereof.

[0084] In another preferred embodiment, the recombinant protein specifically binds to PD-L2 protein.

[0085] In a fourteenth aspect of the present application, there is provided the use of a VHH chain as described in the second aspect of the present application, a Nanobody as described in the third aspect of the present application, a Nanobody fusion protein as described in the fourth aspect of the present application, or an immunoconjugate as described in the ninth aspect of the present application, for the manufacture of a medicament, a reagent, an assay plate or a kit;

[0086] wherein the reagent, assay plate or kit is used for: detecting PD-L2 protein in a sample;

[0087] wherein the medicament is used for treating or preventing a tumor expressing PD-L2 protein (i.e. PD-L2 positive).

[0088] In another preferred embodiment, the tumor comprises: gastric cancer, lymphoma, liver cancer, leukemia, kidney tumor, lung cancer, small intestine cancer, bone cancer, prostate cancer, colorectal cancer, breast cancer, colon cancer, prostate cancer, adrenal tumor, or a combination thereof.

[0089] In a fifteenth aspect of the present application, there is provided a method for detecting PD-L2 protein in a sample, comprising the steps of:

[0090] (1) contacting the sample with a Nanobody as described in the third aspect of the present application or a Nanobody fusion protein as described in the fourth aspect of the present application;

[0091] (2) detecting whether an antigen-antibody complex is formed, wherein the formation of the complex indicates the presence of PD-L2 protein in the sample.

[0092] In another preferred embodiment, the detecting comprises qualitative detection and quantitative detection.

[0093] In a sixteenth aspect of the application, there is provided a method of treating a disease, comprising administering to a subject in need thereof a Nanobody according to the third aspect of the application, a Nanobody fusion protein according to the fourth aspect of the application or an immunoconjugate according to the ninth aspect of the application.

[0094] In another preferred embodiment, the subject comprises a mammal.

[0095] In another preferred embodiment, the mammal is a human.

[0096] It should be understood that, within the scope of the present application, the above technical features of the present application and the technical features specifically described hereinafter (e.g. in the examples) can be combined with each other to form new or preferred technical solutions. Due to the limited space, they will not be listed one by one here. BRIEF DESCRIPTION OF DRAWINGS

[0097] Figure 1 Binding activity of purified anti-PD-L2 antibodies to CHO-hPD-L2 cells is shown.

[0098] Figure 2 Blocking effect of purified anti-PD-L2 antibodies on the binding of PD-L2 to PD-1 is shown.

[0099] Figure 3 Binding activity of D-Na-96 humanized antibodies to CHO-hPD-L2 cells is shown.

[0100] Figure 4 Blocking effect of D-Na-96 humanized antibodies on the binding of PD-L2 to PD-1 is shown. DETAILED DESCRIPTION

[0101] The present inventors have made extensive and in-depth research, and through a large number of screenings, developed an anti-PD-L2 Nanobody for the first time. Experimental results show that the PD-L2 Nanobody, its mutants and humanized antibodies obtained by the present application have high affinity for PD-L2 and can effectively block the binding of PD-L2 to PD-1. On this basis, the present application is completed.

[0102] TERMS

[0103] For the purposes of the present invention, certain technical and scientific terms are specifically defined below. Unless specifically defined herein, all other technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The description herein is given for the purposes of describing particular embodiments only and is not intended to limit the present invention which is limited only by the claims appended hereto.

[0104] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. As used herein, the term "about," when used in reference to a particular recited numerical value, means that the value can vary from the recited value by not more than 1%. For example, as used herein, the expression "about 100" includes all values between 99 and 101 (e.g., 99.1, 99.2, 99.3, 99.4, etc.).

[0105] The three letter and one letter codes for amino acid residues are in accordance with the IUPAC-IUBMB standards as described in J. Biol. Chem., 243, p. 3558 (1968).

[0106] As used herein, the term "treatment" refers to the administration of an internal or external therapeutic agent, including the monoclonal antibodies against human enterovirus 2C protein of the present invention and compositions thereof, to a patient having one or more symptoms of a disease, where the therapeutic agent is known to have a therapeutic effect on the symptoms. Typically, the patient is administered the therapeutic agent in an amount effective to alleviate one or more symptoms of the disease (therapeutically effective amount).

[0107] As used herein, the term "optional" or "optionally" means that the subsequently described event or circumstance can, but need not, occur. For example, "optionally comprising 1-3 antibody heavy chain variable regions" means that the antibody heavy chain variable region of the particular sequence can, but need not, have one, two, or three.

[0108] The "sequence identity" as described herein means the extent of identity between two nucleic acid or two amino acid sequences when optimally aligned and compared with appropriate mutations, such as substitutions, insertions or deletions. The sequence identity between the sequences described herein and the sequences having identity thereto can be at least 85%, 90% or 95%, preferably at least 95%. Non-limiting examples include 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%.

[0109] Nanobodies of the present invention

[0110] As used herein, the terms "nanobody of the invention", "anti-PD-L2 nanobody of the invention", "PD-L2 nanobody of the invention" are used interchangeably and refer to a nanobody which specifically recognizes and binds to PD-L2, including human PD-L2. Particularly preferred are nanobodies whose amino acid sequence of the VHH chain is as set forth in SEQ ID NO: 14, 59, 71, 73, 75 or 77.

[0111] As used herein, the term "antibody" or "immunoglobulin" is a heterotetrameric glycoprotein of about 150,000 daltons, comprising two identical light (L) chains and two identical heavy (H) chains. Each light chain is linked to a heavy chain by one covalent disulfide bond, while the number of disulfide bonds between heavy chains varies among the different immunoglobulin isotypes. There are also interchain disulfide bonds within each heavy and light chain. Each heavy chain has at one end a variable region (VH) followed by a number of constant regions. Each light chain has a variable region (VL) at one end and a constant region at its other end; the light chain's constant region is aligned with the first constant region of the heavy chain, and the light chain's variable region is aligned with the variable region of the heavy chain. Particular amino acid residues are believed to form an interface between the light chain and heavy chain variable regions.

[0112] As used herein, the terms "single domain antibody (VHH)", "nanobody" have the same meaning and refer to the variable region of the heavy chain of an antibody, which is cloned to construct a single domain antibody (VHH) consisting of only one heavy chain variable region, which is the smallest antigen binding fragment with full functionality. Usually, the variable region of the heavy chain of an antibody naturally lacking a light chain and a heavy chain constant region 1 (CH1) is cloned to construct a single domain antibody (VHH) consisting of only one heavy chain variable region.

[0113] As used herein, the term "variable" refers to the fact that certain portions of the variable domains differ extensively in sequence among antibodies and are used in the binding and specificity of each particular antibody for its particular antigen. However, the variability is not evenly distributed throughout the variable domains of antibodies; it is concentrated in three segments called complementarity determining regions (CDRs) or hypervariable regions both in the light chain and the heavy chain variable domains. The more highly conserved portions of the variable domains are called the framework regions (FRs). The variable domains of the heavy and light chains each comprise four FR regions, largely

[0114] As known to those skilled in the art, immunoconjugates and fusion expression products include conjugates formed by the combination of a drug, toxin, cytokine, radionuclide, enzyme, and other diagnostic or therapeutic molecule with an antibody or fragment thereof of the present application. The present application also includes cell surface markers or antigens bound to the anti-PD-L2 protein antibodies or fragments thereof.

[0115] As used herein, the term "heavy chain variable region" is used interchangeably with "V H " and "HCVR."

[0116] As used herein, the term "variable region" is used interchangeably with "complementarity determining region" (CDR).

[0117] In a preferred embodiment of the present application, the heavy chain variable region of the antibody comprises three complementarity determining regions, CDR1, CDR2, and CDR3.

[0118] In a preferred embodiment of the present application, the heavy chain of the antibody comprises the heavy chain variable region described above and a heavy chain constant region.

[0119] In the present application, the terms "antibody of the present application," "protein of the present application," or "polypeptide of the present application" are used interchangeably and refer to a polypeptide that specifically binds to a PD-L2 protein, such as a protein or polypeptide having a heavy chain variable region. They can or can not contain the initial methionine.

[0120] The present application also provides other proteins or fusion expression products having the antibody of the present application. Specifically, the present application includes any protein or protein conjugate and fusion expression product (i.e., immunoconjugates and fusion expression products) having a heavy chain comprising a variable region, as long as the variable region is identical or at least 90% homologous, preferably at least 95% homologous, to the heavy chain variable region of the antibody of the present application.

[0121] Generally, the antigen binding properties of an antibody can be described by three specific regions located in the heavy chain variable region, called the variable regions (CDRs), which are interspaced by four framework regions (FRs), the amino acid sequences of which are relatively conserved and do not directly participate in the binding reaction. These CDRs form loop structures, which are spatially close to each other through the beta sheet formed by the FRs in between. The CDRs on the heavy chain and the corresponding CDRs on the light chain constitute the antigen binding site of the antibody. It can be determined which amino acids constitute the FR or CDR regions by comparing the amino acid sequences of antibodies of the same type.

[0122] The variable regions of the heavy chains of the antibodies of the present application are of particular interest because at least a portion of them are involved in binding to the antigen. Thus, the present application includes molecules having the variable region of the heavy chain of the antibody with the CDRs, provided that the CDRs have more than 90% (preferably more than 95%, most preferably more than 98%) homology with the CDRs identified herein.

[0123] The present application includes not only intact antibodies, but also fragments of the antibodies that are immunologically active or fusions of the antibodies with other sequences. Thus, the present application also includes fragments, derivatives and analogs of the antibodies.

[0124] As used herein, the terms "fragment", "derivative" and "analog" refer to polypeptides that substantially retain the same biological function or activity of the antibodies of the present application. The fragments, derivatives or analogs of the polypeptides of the present application can be (i) polypeptides having one or more conservative or non-conservative amino acid substitutions (preferably conservative amino acid substitutions) of the amino acid residues, where such substituted amino acid residues can or can not be encoded by the genetic code, or (ii) polypeptides having a substitution group at one or more amino acid residues, or (iii) polypeptides formed by fusing the mature polypeptide to another compound such as a compound that increases the half-life of the polypeptide, e.g., polyethylene glycol, or (iv) polypeptides formed by fusing additional amino acid sequences to the polypeptide, such as leader sequences or secretion sequences or sequences or proteins that are used to purify the polypeptide or prosequences, or fusion proteins with a 6His tag. These fragments, derivatives and analogs are within the purview of one of ordinary skill in the art in light of the teachings herein.

[0125] The antibodies of the present application refer to polypeptides having PD-L2 protein binding activity, including the CDR regions described above. The term also includes variants of polypeptides having the CDR regions described above that have the same function as the antibodies of the present application. These variants include, but are not limited to, deletion, insertion and / or substitution of one or more (typically 1-50, preferably 1-30, more preferably 1-20, most preferably 1-10) amino acids, and addition of one or several (typically 20 or fewer, preferably 10 or fewer, more preferably 5 or fewer) amino acids at the C-terminus and / or the N-terminus. For example, in the art, substitution of similar or identical amino acids typically does not change the function of the protein. Also, for example, addition of one or several amino acids at the C-terminus and / or the N-terminus typically does not change the function of the protein. The term also includes active fragments and active derivatives of the antibodies of the present application.

[0126] Variants of the polypeptides include homologous sequences, conservative variants, allelic variants, natural mutants, induced mutants, proteins encoded by DNA that hybridizes to the DNA encoding the antibodies of the application under conditions of high or low stringency, and polypeptides or proteins obtained using antisera against the antibodies of the application.

[0127] The application also provides other polypeptides, such as fusion proteins comprising a nanobody or fragment thereof. In addition to the nearly full-length polypeptides, the application also includes fragments of the nanobodies of the application. Typically, the fragments have at least about 50 contiguous amino acids of the antibodies of the application, preferably at least about 50 contiguous amino acids, more preferably at least about 80 contiguous amino acids, and most preferably at least about 100 contiguous amino acids.

[0128] In the present application, "conservative variants of the antibodies of the application" are polypeptides in which up to 10, preferably up to 8, more preferably up to 5, and most preferably up to 3 amino acids of the amino acid sequence of the antibodies of the application are replaced by amino acids of similar or analogous properties. These conservative variant polypeptides are preferably generated by making amino acid replacements according to Table 1.

[0129] Table A

[0130] Original residue Representative substitutions Preferred substitutions Ala (A) Val; Leu; lie Val Arg (R) Lys; Gin; Asn Lys Asn (N) Gin; His; Lys; Arg Gin Asp (D) Glu Glu Cys (C) Ser Ser Gin (Q) Asn Asn Glu (E) Asp Asp Gly (G) Pro; Ala Ala His (H) Asn; Gin; Lys; Arg Arg lie (I) Leu; Val; Met; Ala; Phe Leu Leu (L) lie; Val; Met; Ala; Phe lie Lys (K) Arg; Gin; Asn Arg Met (M) Leu; Phe; lie Leu Phe (F) Leu; Val; lie; Ala; Tyr Leu Pro (P) Ala Ala Ser (S) Thr Thr Thr (T) Ser Ser Trp (W) Tyr; Phe Tyr Tyr (Y) Trp; Phe; Thr; Ser Phe Val (V) lie; Leu; Met; Phe; Ala Leu

[0131] The application also provides polynucleotide molecules encoding the above antibodies or fragments thereof or fusion proteins thereof. The polynucleotides of the application can be in the form of DNA or RNA. The DNA form includes cDNA, genomic DNA, or artificially synthesized DNA. The DNA can be single-stranded or double-stranded. The DNA can be the coding strand or the non-coding strand.

[0132] Polynucleotides encoding the mature polypeptides of the application include: coding sequences that encode only the mature polypeptides; coding sequences that encode the mature polypeptides and various additional coding sequences; coding sequences that encode the mature polypeptides (and optional additional coding sequences) and non-coding sequences.

[0133] The term "polynucleotide encoding a polypeptide" can be a polynucleotide that includes only the polynucleotide encoding the polypeptide, or a polynucleotide that also includes additional coding and / or non-coding sequences.

[0134] The present application also relates to polynucleotides that hybridize to the above sequences and have at least 50%, preferably at least 70%, more preferably at least 80% identity between the two sequences. The present application particularly relates to polynucleotides that hybridize to the polynucleotides of the present application under stringent conditions. In the present application, "stringent conditions" means: (1) hybridization and washing under low ionic strength and high temperature, such as 0.2 x SSC, 0.1% SDS, 60°C; or (2) hybridization with the addition of denaturant, such as 50% (v / v) formamide, 0.1% calf serum / 0.1% Ficoll, 42°C, etc.; or (3) hybridization only occurs when the identity between the two sequences is at least 90%, more preferably 95% or more. Furthermore, the polypeptide encoded by the hybridizable polynucleotide has the same biological function and activity as the mature polypeptide.

[0135] The nucleotide full-length sequence of the antibody of the present application or fragments thereof can be obtained by PCR amplification, recombination or artificial synthesis. One possible method is to synthesize the relevant sequence by artificial synthesis, especially when the length of the fragment is relatively short. Generally, a long fragment of the sequence can be obtained by first synthesizing a plurality of small fragments and then ligating them together. In addition, the coding sequence of the heavy chain can be fused with an expression tag (such as 6His) to form a fusion protein.

[0136] Once the relevant sequence is obtained, the relevant sequence can be obtained in large quantities by recombination. This is usually done by cloning it into a vector, then into cells, and then separating the relevant sequence from the proliferated host cells by conventional methods. The biomolecules (nucleic acids, proteins, etc.) involved in the present application include biomolecules that exist in an isolated form.

[0137] At present, the DNA sequence encoding the protein (or fragment thereof, or derivative thereof) of the present application can be obtained entirely by chemical synthesis. The DNA sequence can then be introduced into various existing DNA molecules (or vectors, etc.) and cells known in the art. In addition, mutations can be introduced into the protein sequence of the present application by chemical synthesis.

[0138] Table B Summary of Nanobody Sequences of the Present Invention

[0139]

[0140]

[0141]

[0142]

[0143]

[0144]

[0145] The present application also relates to vectors comprising the appropriate DNA sequences described above and appropriate promoters or control sequences. These vectors can be used to transform appropriate host cells to enable them to express the proteins.

[0146] The host cells can be prokaryotic cells, such as bacterial cells; or lower eukaryotic cells, such as yeast cells; or higher eukaryotic cells, such as mammalian cells. Representative examples of such host cells are: E. coli, Streptomyces; bacterial cells of Salmonella typhimurium; fungal cells such as yeast; insect cells such as Drosophila S2 or Sf9; animal cells such as CHO, COS7, 293 cells, etc.

[0147] Transformation of host cells with recombinant DNA can be performed using conventional techniques well known to those skilled in the art. When the host is a prokaryote, such as E. coli, the transformation of the host cell can be effected by the CaCl2method, which is well known in the art. Another method is the use of MgCl2. If necessary, the transformation can also be performed by electroporation. When the host is a eukaryote, the DNA can be transferred into the host cell using a DNA transfer method such as calcium phosphate co-precipitation, conventional mechanical methods such as microinjection, electroporation, liposome packaging, etc.

[0148] The transformants obtained can be cultured in conventional media using conventional procedures to express the polypeptides encoded by the genes of the present application. The medium used to culture the host cells is dependent on the host cell used and can be selected from various conventional media. The culture conditions, such as temperature, pH and the like, are those under which the host cell grows optimally. The selected promoter is induced after the host cell has reached an appropriate cell density using an appropriate method (e.g., temperature shift or chemical induction). The cells are then cultured for an additional period.

[0149] The recombinant polypeptides in the above methods can be expressed intracellularly, on the cell membrane, or secreted from the cell. If desired, the recombinant proteins can be isolated and purified from the cell using their physical, chemical and other properties by various separation methods. These methods are well known to those skilled in the art. Examples of these methods include, but are not limited to: conventional renaturation treatment, treatment with protein precipitants (salting-out method), centrifugation, osmotic lysis, ultra-treatment, ultra-centrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, high performance liquid chromatography (HPLC) and other various liquid chromatography techniques and combinations of these methods.

[0150] The antibodies of the present application can be used alone or in combination or conjugation with detectable labels (for diagnostic purposes), therapeutic agents, PK (protein kinase) modifying moieties or any combination of these.

[0151] Detectable labels for diagnostic purposes include, but are not limited to, fluorescent or luminescent labels, radioactive labels, MRI (magnetic resonance imaging) or CT (computerized tomography) contrast agents, or enzymes capable of producing a detectable product.

[0152] Therapeutic agents that can be conjugated or coupled to the antibodies of the present application include, but are not limited to, 1. radionuclides; 2. biological toxins; 3. cytokines such as IL-2 and the like; 4. gold nanoparticles / nanorods; 5. viral particles; 6. liposomes; 7. nanomagnetic particles; 8. prodrug-activating enzymes (e.g., DT-diaphorase (DTD) or benzyl-hydrolyzing enzyme-like protein (BPHL)); 9. chemotherapeutic agents (e.g., cisplatin) or any form of nanoparticles and the like.

[0153] Fusion protein of the present application

[0154] As described herein, "fusion protein of the present application" refers to a bifunctional fusion protein having both the anti-PD-L2 nanobody of the first aspect of the present application and an immunomodulatory molecule moiety.

[0155] In the present application, a fusion protein is provided, the nanobody fusion protein having a structure as shown in Formula I from N-terminus to C-terminus:

[0156] Z1-Z2-L-Z3 (Formula I)

[0157] In the formula,

[0158] Z1 is a VHH chain of the anti-PD-L2 nanobody as described in the second aspect of the present application;

[0159] Z2 is an Fc fragment of an immunoglobulin;

[0160] L is a linker sequence;

[0161] Z3 is an immunomodulatory molecule moiety.

[0162] Preferably, the immunoglobulin can be IgG1, IgG2, IgG3, IgG4, or the like.

[0163] In a preferred embodiment, the immunoglobulin is IgG1, and the amino acid sequence of Z2 is as shown in SEQ ID NO: 79. In other embodiments, the amino acid sequence of Z2 is identical or substantially identical to the amino acid sequence as shown in SEQ ID NO: 79.

[0164] In the present application, the L is a flexible amino acid linker. Preferably, L has an amino acid sequence selected from the group consisting of GGGGS, (GGGGS)2, (GGGGS)3, (GGGGS)4, (GGGGS)5, or a combination thereof.

[0165] In a preferred embodiment, the amino acid sequence of L is set forth in SEQ ID NO: 80. In other embodiments, the amino acid sequence of L is identical or substantially identical to the amino acid sequence set forth in SEQ ID NO: 80.

[0166] In an embodiment of the present application, the immunomodulatory molecule is a TGFβRII extracellular domain. Preferably, the amino acid sequence of Z3 is set forth in SEQ ID NO: 81. In other embodiments, the amino acid sequence of Z3 is identical or substantially identical to the amino acid sequence set forth in SEQ ID NO: 81.

[0167] In the present application, substantially identical means that at most 50 (preferably 1-20, more preferably 1-10, more preferably 1-5, most preferably 1-3) amino acids are not identical, wherein the not identical includes substitution, deletion or addition of amino acids.

[0168] Preferably, substantially identical means that the sequence identity of the amino acid sequence to the corresponding amino acid sequence is at least 70%, at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%.

[0169] TGFβ is a key inducer of epithelial-mesenchymal-transition (EMT). Meanwhile, TGFβ has a strong immunosuppressive effect in the tumor microenvironment, which further has important regulatory effects on tumor occurrence, development, metastasis and drug resistance.

[0170] Therefore, in an embodiment of the present application, TGFβ receptor II is selected as the immunomodulatory molecule in the fusion protein. The fusion protein of the present application has the advantages of high dual-target binding affinity and strong specificity, thereby further enhancing the anti-tumor immune function.

[0171] Pharmaceutical composition

[0172] The present application also provides a composition. Preferably, the composition is a pharmaceutical composition comprising the antibody or active fragment thereof or fusion protein thereof as described above, and a pharmaceutically acceptable carrier. Generally, these substances can be formulated in a non-toxic, inert and pharmaceutically acceptable aqueous carrier medium, wherein the pH is generally about 5-8, preferably about 6-8, although the pH value can vary depending on the nature of the substance to be formulated and the condition to be treated. The prepared pharmaceutical composition can be administered by conventional routes, including but not limited to intratumoral, intraperitoneal, intravenous, or local administration.

[0173] The pharmaceutical composition of the present application can be directly used in combination with PD-L2 protein molecules, and thus can be used for treating tumors. In addition, other therapeutic agents can also be used simultaneously.

[0174] The pharmaceutical composition of the present application 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 nanobody (or conjugate thereof) as described above, and a pharmaceutically acceptable carrier or excipient. Such carriers include but are not limited to saline, buffer, glucose, water, glycerol, ethanol, and combinations thereof. The pharmaceutical preparation should be matched with the administration method. The pharmaceutical composition of the present application can be prepared in the form of a needle, for example, by conventional methods using physiological saline or an aqueous solution containing glucose and other adjuvants. The pharmaceutical composition such as a needle, a solution is preferably manufactured under sterile conditions. The amount of active ingredient administered is a therapeutically effective amount, for example, about 10 μg / kg body weight to about 50 mg / kg body weight per day. In addition, the polypeptide of the present application can also be used with other therapeutic agents.

[0175] When using the pharmaceutical composition, a safe and effective amount of the immunoconjugate is administered to a mammal, wherein the safe and effective amount is generally at least about 10 μg / kg body weight, and in most cases does not exceed about 50 mg / kg body weight, preferably the dose is about 10 μg / kg body weight to about 10 mg / kg body weight. Of course, the specific dose should also take into account the administration route, the patient's health status, etc., which are within the skill of the skilled physician.

[0176] Labeled nanobody

[0177] In a preferred embodiment of the present application, the nanobody carries a detectable label. More preferably, the label is selected from the group consisting of an isotope, a colloidal gold label, a colored label or a fluorescent label.

[0178] The colloidal gold labeling can be carried out by methods known to those skilled in the art. In a preferred embodiment of the present application, the anti-PD-L2 nanobody is labeled with colloidal gold to obtain a colloidal gold-labeled nanobody.

[0179] The anti-PD-L2 nanobody of the present application has good specificity and high titer.

[0180] Detection method

[0181] The present application also relates to a method for detecting PD-L2 protein. The steps of the method are roughly as follows: obtaining a cell and / or tissue sample; dissolving the sample in a medium; detecting the level of PD-L2 protein in the dissolved sample.

[0182] In the detection method of the present application, the sample used is not particularly limited, and a representative example is a cell-containing sample present in a cell preservation solution.

[0183] Kit

[0184] The present application also provides a kit containing the antibody (or fragment thereof) or detection plate of the present application. In a preferred embodiment of the present application, the kit further comprises a container, instructions for use, a buffer, etc.

[0185] The present application also provides a detection kit for detecting the level of PD-L2, which comprises an antibody that recognizes PD-L2 protein, a lysis medium for dissolving the sample, and general reagents and buffers required for detection, such as various buffers, detection labels, detection substrates, etc. The detection kit can be an in vitro diagnostic device.

[0186] Application

[0187] As described above, the nanobody of the present application has wide biological and clinical application value, and its application involves diagnosis and treatment of diseases related to PD-L2, basic medical research, biological research, and many other fields. A preferred application is for clinical diagnosis and targeted therapy against PD-L2.

[0188] The main advantages of the present application include:

[0189] 1) The nanobody of the present application is highly specific to human PD-L2 protein with correct spatial structure.

[0190] 2) The nanobody of the present application has strong affinity.

[0191] 3) The nanobody of the present application is easy to produce.

[0192] 4) Inhibition of the PD-1 / PD-L2 pathway based on targeting and neutralizing TGF-β in the tumor microenvironment can restore T cell activity, enhance immune response, and more effectively improve the effect of inhibiting tumor occurrence and development.

[0193] The application will be further described in connection with specific examples. It should be understood that these examples are only used to illustrate the application and not used to limit the scope of the application. The experimental methods in the following examples, if not otherwise specified, are generally carried out according to the conventional conditions, for example, the conditions described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or the conditions suggested by the manufacturer. Unless otherwise specified, the percentages and parts are weight percentages and weight parts.

[0194] Example 1: Construction of Nanobody Library

[0195] 1.1 Animal immunization

[0196] One mg of human PD-L2 antigen (purchased from AcroBiosystems) was mixed with an equal volume of Freund's adjuvant, and two llamas were immunized once a week for a total of 4 times to stimulate B cells to express antigen-specific nanobodies. After the end of the 4 times of immunization, 50 ml of peripheral blood of the llamas was extracted, and lymphocytes were obtained by using lymphocyte separation medium. Total RNA was extracted by using Trizol (purchased from Invitrogen) RNA extraction reagent. The total cDNA of the llamas was obtained by reverse transcription using a cDNA synthesis kit (purchased from Invitrogen).

[0197] 1.2 Nanobody gene amplification

[0198] In the first round of PCR, IgG2 and IgG3 sequences were amplified from the cDNA:

[0199] Table 1. Primers for the first round of PCR

[0200] Name Sequence (5' to 3') SEQ ID NO: Upstream primer GTCCTGGCTGCTCTTCTACAAGG 82 Downstream primer GGTACGTGCTGTTGAACTGTTCC 83

[0201] The PCR product was subjected to agarose gel electrophoresis, and the fragment at 750 bp was recovered by cutting the gel and used for the second round of VHH sequence amplification. The primers for the second round of PCR amplification were as follows:

[0202] Table 2. Primers for the second round of PCR

[0203] Name Sequence (5' to 3') SEQ ID NO: Upstream primer CTAGTGCGGCCGCcTGGAGACGGTGACCTGGGT 84 Downstream primer CGCGGATCCCAGGTGCAGCTGCAGGAGTCTGGRGGAGG 85

[0204] The second round of PCR product was used as a template for the third round of PCR to add homologous arms to the VHH gene. The primers for the third round of PCR amplification were as follows:

[0205] Table 3. Primers for the third round of PCR

[0206]

[0207] The target fragment was recovered by PCR purification kit (purchased from QIAGEN).

[0208] 1.3 Library construction

[0209] The linearized yeast display vector and the PCR product of the third round were mixed and then electroporated into S. cerevisiae (purchased from ATCC) to construct the anti-PD-L2 nanobody library from two animals and determine the library capacity, which was 4.47 x 1010 7 and 4.14 x 1010 7 .

[0210] Example 2: Screening of PD-L2 nanobodies

[0211] 2.1 Biotinylation labeling of human PD-L2 protein

[0212] An appropriate volume of double distilled water was used to dissolve human PD-L2 protein (purchased from AcroBiosystems), and then biotin was dissolved and mixed with the protein solution according to the product instructions of the biotin labeling kit (purchased from Thermo). The mixture was incubated at 4°C for 2 hours. The excess biotin was removed by a desalting column (purchased from Thermo), and the pretreatment of the desalting column and the collection of the sample were performed according to the product instructions.

[0213] 2.2 MACS enrichment of yeast capable of specific binding to PD-L2

[0214] The VHH library constructed in Example 1 was inoculated into SD-CAA expansion medium (1 L of SD-CAA expansion medium containing 6.7 g of YNB, 5 g of tyrosine, 13.62 g of Na2HPO4·12H2O, 7.44 g of NaH2PO4, and 2% glucose), and the inoculated yeast cell number was >10 x library capacity (the initial expansion concentration = 0.5 OD 600 / ml), and the culture was incubated at 30°C and 225 rpm overnight. 10 x library capacity of yeast cells were centrifuged at 3000 rpm for 5 min (the following centrifugation operations were the same), and the culture medium was removed. The yeast cells were resuspended in SD-CAA induction medium, and the initial concentration was adjusted to 0.5 OD 600 / ml, and the induction was performed overnight. The library concentration after induction was determined, 10 x library capacity of yeast cells were centrifuged to remove the culture medium. The yeast cells were resuspended in 50 ml of washing solution (PBS + 0.5% BSA + 2 mM EDTA), and the supernatant was removed by centrifugation. The yeast cells were resuspended in 10 ml of washing solution.

[0215] Add biotin-labeled PD-L2 protein (final concentration 100 mM), incubate at room temperature for 30 min, centrifuge to collect yeast cells, and wash the yeast with 50 ml of washing solution for 3 times. Resuspend the yeast cells with 5 ml of washing solution, and add 200 μl of SA magnetic beads (purchased from Milttenyi), and incubate for 10 min by inverting. Wash the yeast and magnetic bead mixture with washing solution for 3 times, and add the mixture to a LS purification column (purchased from Milttenyi). Place the LS purification column on a magnetic stand, and wash to remove non-specifically bound yeast cells. Remove the purification column from the magnetic stand, and elute the yeast with washing solution. Centrifuge the eluted yeast, and transfer to 200 ml of SD-CAA expansion medium for expansion.

[0216] 2.3 Flow cytometry sorting for high affinity yeast cells

[0217] Inoculate the MACS-enriched yeast cells in SD-CAA expansion medium, and the initial expansion concentration is 0.5 OD 600 / ml. Shake the flask at 30°C and 225 rpm overnight. Resuspend the yeast cells with SD-CAA induction medium (1 L of SD-CAA induction medium contains 6.7 g of YNB, 5 g of tyrosine, 13.62 g of Na2HPO4·12H2O, 7.44 g of NaH2PO4, and 2% of galactose, 2% of raffinose, and 0.1% of glucose), and the initial concentration is 0.5 OD 600 / ml, and induce overnight. Add 1:200 diluted anti-c-Myc mouse antibody (purchased from Thermo) and 100 nM biotin-labeled PD-L2 antigen, and incubate at room temperature for 10 min. Wash the yeast with PBS for 3 times, add 1:500 diluted goat anti-mouse IgG (H+L) Alexa Fluor Plus 488 fluorescent antibody (purchased from Invitrogen) and streptavidin APC conjugate fluorescent antibody (purchased from Invitrogen), and incubate at 4°C in the dark for 15 min. Resuspend the cells with 2 ml of PBS, and use a BD FACSAriaII instrument to sort to obtain yeast with high binding capacity to the PD-L2 antigen.

[0218] 2.4 Retrieval of PD-L2 nanobody candidate molecule antibody genes

[0219] The yeast with high binding capacity to the PD-L2 antigen obtained by MACS and FACS enrichment is cultured in SD-CAA expansion medium at 30°C and 225 rpm overnight, and the yeast plasmid is extracted according to the yeast plasmid extraction kit (purchased from Tiangen). The plasmid is transformed into Top10 competent cells (purchased from Tiangen) by electroporation, and plated on ampicillin-resistant plates, and incubated at 37°C overnight. Pick single colonies for sequencing to obtain VHH gene sequences.

[0220] Example 3: Construction and expression purification of heavy chain antibody

[0221] 3.1 Construction of antibody genes into pCDNA3.1 expression vector

[0222] The VHH gene sequence and human IgG1 (LALA mutation) Fc segment were connected, and homologous recombination was used to construct in pCDNA3.1 vector linearized by EcoR I / Not I double enzyme digestion (purchased from Vazyme), and the process was according to the product instruction. The homologous recombination product was transformed into Top10 competent cells, coated on ampicillin resistant plates, incubated at 37°C overnight, and single colonies were picked for sequencing and plasmid extraction.

[0223] 3.2 Cell transfection and protein purification

[0224] ExpiCHO TM Expression system kit (purchased from Thermo), the extracted plasmid was transformed into Expi-CHO cells, the transfection method was according to the product instruction, and the supernatant was collected after 5 days of cell culture. The target protein was purified by protein A magnetic bead (purchased from Jin Suri) sorting method.

[0225] The magnetic beads were resuspended (1-4 times the volume of the magnetic beads) with an appropriate volume of binding buffer (PBS + 0.1% Tween 20, pH 7.4) and added to the sample to be purified, incubated at room temperature for 1 hour, with gentle shaking during the incubation. The sample was placed on a magnetic stand (purchased from Beaver), and the supernatant was discarded. The magnetic beads were washed with binding buffer 3 times. According to 3-5 times the volume of the magnetic beads, elution buffer (0.1M sodium citrate, pH 3.2) was added, and the sample was shaken at room temperature for 5-10 min. The sample was placed back on the magnetic stand, and the elution buffer was collected and transferred to a collection tube containing neutralization buffer (1M Tris, pH 8.54) and mixed well to obtain the target protein.

[0226] Example 4: Purified anti-PD-L2 antibody binds to human PD-L2

[0227] CHO cells overexpressing human PD-L2 (CHO-hPD-L2 cells) were generated by transfecting pCHO1.0 vector (purchased from Invitrogen) cloned with human PD-L2 cDNA (purchased from Sino Biological). The expanded CHO-hPD-L2 cells were adjusted to a cell density of 2x10 6Cells were harvested and adjusted to 1 x 106cells / ml, 100 μl / well added to 96-well flow plate, centrifuged for standby. Purified PD-L2 antibody was diluted with PBS, 1000 nM as a start, 3-fold dilution for 12 points, 100 μl / well of the above diluted sample was added to the above 96-well flow plate with cells, 4°C incubated for 30 min, washed twice with PBS. 100 μl / well of goat F(ab')2 anti-human IgG-Fc (PE) (purchased from Abeam) diluted 100 times with PBS was added, 4°C incubated for 30 min, washed twice with PBS. 100 μl / well of PBS resuspended cells was added, and detected on a CytoFlex (Bechman) flow cytometer and the corresponding MFI was calculated.

[0228] In the determination experiment of the above method, the experimental results are as shown in Table 2, all the purified samples and CHO-hPD-L2 cells of the present application have binding activity. Figure 1

[0229] Example 5: PD-L2 antibody affinity determination

[0230] ForteBio affinity determination was performed according to the existing method (Estep, P, et al. Determination of antibody-antigen affinity and epitope binning based on high-throughput method. MAbs, 2013. 5(2): p. 270-8). Briefly, the sensor was equilibrated in the analysis buffer for 30 min, then the baseline was established by detecting on-line for 60 s, and the purified antibody obtained as described above was loaded on the AHQ sensor. The sensor was then placed in 100 nM PD-L2 antigen for 5 min, and then transferred to PBS for dissociation for 5 min. The kinetics was analyzed using a 1:1 binding model.

[0231] Table 4. Affinity of candidate molecules

[0232]

[0233]

[0234] Example 6: Purified anti-PD-L2 antibody blocks PD-L2 binding to PD-1

[0235] CHO cells overexpressing human PD-1 (CHO-hPD-1 cells) were generated by transfecting pCHO1.0 vector (purchased from Invitrogen) with human PD-1 cDNA (purchased from Sino Biological). The expanded culture of CHO-hPD-1 cells was adjusted to a cell density of 2 x 106cells / ml, 100 μl / well was added to a 96-well flow plate, and centrifuged for standby. The purified PD-L2 antibody was diluted with PBS, 1000 nM as a start, 3-fold dilution for 12 points, 100 μl / well of the above diluted sample was added to the above 96-well flow plate with cells, 4°C incubated for 30 min, washed twice with PBS. 100 μl / well of goat F(ab')2 anti-human IgG-Fc (PE) (purchased from Abeam) diluted 100 times with PBS was added, 4°C incubated for 30 min, washed twice with PBS. 100 μl / well of PBS resuspended cells was added, and detected on a CytoFlex (Bechman) flow cytometer and the corresponding MFI was calculated. 6 ​The purified mutant samples were diluted with PBS, 3-fold dilution from 1000 nM for 12 points, and 60 μl / well of the diluted samples were added to a 96-well sample dilution plate, and 60 μl / well of biotinylated human PD-L2 protein (purchased from AcroBiosystems) was added, with a final concentration of 1 μg / ml, and incubated with the purified samples at 4°C for 30 min. 100 μl / well of the co-incubated samples were added to the above-mentioned 96-well flow plate with cells, and incubated at 4°C for 30 min, and washed twice with PBS. 100 μl / well of APC goat anti-mouse IgG (minimum x-reactivity) antibody (purchased from Biolegend) diluted 100 times with PBS was added, and incubated at 4°C for 30 min, and washed twice with PBS. 100 μl / well of PBS-resuspended cells was added, and detected on a CytoFlex (Bechman) flow cytometer and the corresponding MFI was calculated.

[0236] In the determination experiment of the above method, the experimental results are as shown in Table 3, and all the purified samples of the present application can block the binding of PD-L2 to PD-1. Figure 2

[0237] Example 7: Construction of PD-L2 antibody humanization

[0238] In order to reduce the immunogenicity of monoclonal antibodies in the human body, the D-NA-96 and D-Ye-29 antibodies were humanized. The humanization method used the VHH humanization universal framework grafting method, and the mutation of part of the amino acids of the antibody framework 2 (framework 2) was completed according to the method reported in the literature (Vincke, C., et al., General strategies for humanizing camelid single-domain antibodies and for identifying a universal humanized nanobody scaffold. J Biol Chem 284(5):3273-3284).

[0239] In this study, IMGT (http: / / www.imgt.org) was used to evaluate the humanization level of D-NA-96, D-Ye-29 and humanized sequences, and the results are shown in Table 5. The humanization level of all humanized samples is higher than 80%, which meets the requirements of later drug development.

[0240] Table 5. Homology of D-NA-96 / D-Ye-29 humanized sequences and human

[0241] Number Germline Homology D-Na-96 IGHV3-48*03 69.40％ HZ-D-Na-96-1 IGHV3-48*03 84.70％ HZ-D-Na-96-2 IGHV3-48*03 83.70％ HZ-D-Na-96-3 IGHV3-48*03 80.60％ D-Ye-29 IGHV3-23*01 71.1％ HZ-D-Ye-29 IGHV3-23*01 80.40％

[0242] ​The protein construction, expression, and purification methods were the same as in Example 3. Protein purity was determined using HPLC. The HPLC method was as follows: mobile phase: 150 mM Na₂HPO₄·12H₂O, pH 7.0; chromatographic conditions: detection wavelength: 280 nm, column temperature: 25 °C, flow rate: 0.35 ml / min, detection time: 20 min; Zenix-C SEC-300 column (SEPAX 4.6 × 300 mm, 3 μm).

[0243] Table 6. Purity test results of D-Na-96 / D-Ye-29 humanized antibody

[0244] Number Monomer ratio (%) D-Na-96 100 HZ-D-Na-96-1 96.53 HZ-D-Na-96-2 99.92 HZ-D-Na-96-3 98.84 D-Ye-29 100 HZ-D-Ye-29 98.80

[0245] Example 8: Binding of D-Na-96 humanized sample with human PD-L2

[0246] This experiment tested the binding activity of the purified humanized D-Na-96 sample with CHO-hPD-L2 cells, using the same experimental method as in Example 4.

[0247] Experimental results are as follows Figure 3 As shown, the humanized D-Na-96 sample exhibited good binding activity with CHO-hPD-L2 cells, with levels comparable to those of D-Na-96.

[0248] Example 9: Determination of Affinity for Humanized D-Na-96 / D-Ye-29 Samples

[0249] This experiment tested the binding affinity of the purified humanized D-Na-96 / D-Ye-29 sample to human PD-L2. The experimental method was the same as in Example 5.

[0250] The experimental results are shown in Table 7. The humanized D-Na-96 / D-Ye-29 sample has good binding activity with human PD-L2 protein.

[0251] Table 7. Affinity of D-Na-96 / D-Ye-29 humanized samples

[0252] Number KD (M) kon (1 / Ms) kdis (1 / s) D-Na-96 2.50E-09 2.62E+05 6.55E-04 HZ-D-Na-96-1 1.68E-09 3.44E+05 5.77E-04 HZ-D-Na-96-2 1.58E-09 3.23E+05 5.12E-04 HZ-D-Na-96-3 1.99E-09 2.97E+05 5.90E-04 D-Ye-29 1.93E-09 1.95E+05 3.76E-04 HZ-D-Ye-29 4.49E-09 3.86E+05 1.73E-03

[0253] Example 10: D-Na-96 humanized sample blocks PD-L2 and PD-1 binding

[0254] This experiment tested the ability of purified humanized D-Na-96 samples to block the binding of PD-L2 and PD-1. The experimental method was the same as in Example 6.

[0255] Experimental results are as follows Figure 4As shown, all of the humanized samples of the present application can block the binding of PD-L2 to PD-1, and the blocking level is comparable to D-Na-96.

[0256] All documents referred to in this disclosure are incorporated herein by reference as if each individual document were incorporated by reference. In addition, it is to be understood that the application can be carried out by specifically different embodiments and that each disclosed embodiment can be implemented with or without the corresponding use of the other embodiments. Other embodiments will occur to those skilled in the art upon consideration of this disclosure and can be had without departing from the spirit of the application. Therefore, the scope of the application should be determined only by the narrowest of the claims that can be supported.

Claims

1. An anti-PD-L2 Nanobody, characterized in that, The complementarity determining regions CDR of the VHH chain of the anti-PD-L2 Nanobody consists of: (i) a CDR1 of an amino acid sequence as set forth in SEQ ID NO: 6; a CDR2 of an amino acid sequence as set forth in SEQ ID NO: 7; and a CDR3 of an amino acid sequence as set forth in SEQ ID NO: 8; or (ii) a CDR1 of an amino acid sequence as set forth in SEQ ID NO: 11; a CDR2 of an amino acid sequence as set forth in SEQ ID NO: 12; and a CDR3 of an amino acid sequence as set forth in SEQ ID NO: 13; or (iii) a CDR1 of an amino acid sequence as set forth in SEQ ID NO: 61; a CDR2 of an amino acid sequence as set forth in SEQ ID NO: 62; and a CDR3 of an amino acid sequence as set forth in SEQ ID NO:

63. The CDR1, CDR2 and CDR3 are separated by framework regions FR1, FR2, FR3 and FR4 of the VHH chain.

2. The anti-PD-L2 Nanobody of claim 1, wherein The amino acid sequence of the VHH chain of the anti-PD-L2 Nanobody is as set forth in SEQ ID NO: 9, 14, 64, 71, 73, 75.

3. The anti-PD-L2 Nanobody of claim 1, wherein The Nanobody fusion protein has a structure as set forth in Formula I from N-terminus to C-terminus:

4. A Nanobody fusion protein, characterized in that, Z1-Z2-L-Z3 (Formula I) In the formula, Z1 is the VHH chain of the anti-PD-L2 Nanobody as set forth in claim 1; Z2 is an Fc fragment of an immunoglobulin; L is a linker sequence; Z3 is an immunomodulatory molecule moiety. The immunoglobulin is IgG1, IgG2, IgG3, IgG4.

5. The nanobody fusion protein of claim 4, wherein, The amino acid sequence of Z2 is as set forth in SEQ ID NO:

79.

6. The nanobody fusion protein of claim 4, wherein, The L has an amino acid sequence selected from the group consisting of GGGGS, (GGGGS)2, (GGGGS)3, (GGGGS)4, (GGGGS)5, or a combination thereof.

7. The nanobody fusion protein of claim 4, wherein, The amino acid sequence of the L is as set forth in SEQ ID NO:

80.

8. The nanobody fusion protein of claim 4, wherein, The polynucleotide encodes the anti-PD-L2 Nanobody as set forth in claim 1, or the Nanobody fusion protein as set forth in claim 4.

9. A polynucleotide comprising a nucleic acid sequence encoding a polypeptide of claim 1. The polynucleotide has a nucleotide sequence as set forth in SEQ ID NO: 10, 15, 65, 72, 74, 76.

10. The polynucleotide of claim 9, wherein The expression vector contains the polynucleotide as set forth in claim 9.

11. An expression vector, characterized in that, The host cell contains the expression vector as set forth in claim 11, or the polynucleotide as set forth in claim 9 is integrated into the genome of the host cell.

12. A host cell, characterized in that, The method comprises the steps of:

13. A method of producing an anti-PD-L2 Nanobody, characterized in that, (a) culturing the host cell as set forth in claim 12 under suitable conditions for producing the Nanobody, thereby obtaining a culture containing the anti-PD-L2 Nanobody; and (b) isolating or recovering the anti-PD-L2 Nanobody from the culture. The immunoconjugate contains:

14. An immunoconjugate, comprising, (a) the anti-PD-L2 Nanobody as set forth in claim 1, or the Nanobody fusion protein as set forth in claim 4; and (b) a conjugating moiety selected from the group consisting of a detectable label, a cytokine. The immunoconjugate contains:

15. An immunoconjugate, comprising, ​ (a) the anti-PD-L2 Nanobody of claim 1, or the Nanobody fusion protein of claim 4; and (b) a conjugating moiety selected from the group consisting of: a radionuclide, an enzyme.

16. Use of an anti-PD-L2 Nanobody according to claim 1 or a Nanobody fusion protein according to claim 4, characterized in that, for the preparation of (a) a reagent for detecting a PD-L2 molecule; (b) a medicament for treating a tumor; said tumor selected from the group consisting of: gastric cancer, liver cancer, kidney tumor, lung cancer, small intestine cancer, bone cancer, prostate cancer, colorectal cancer, breast cancer, large intestine cancer, cervical cancer, adrenal gland tumor, bladder tumor, or a combination thereof.

17. A pharmaceutical composition comprising, in combination, a compound of any one of claims 1-16 and a pharmaceutically acceptable carrier. comprising: (i) the anti-PD-L2 Nanobody of claim 1, the Nanobody fusion protein of claim 4, or the immunoconjugate of claim 14 or 15; and (ii) a pharmaceutically acceptable carrier.

18. Use of one or more of the anti-PD-L2 Nanobody of claim 1 or the Nanobody fusion protein of claim 4: (i) for detecting a PD-L2 molecule; (ii) for flow cytometry; and (iii) for cellular immunofluorescence; and, said use is non-diagnostic and non-therapeutic.

19. A recombinant protein, characterized in that, said recombinant protein has: (i) the sequence of the anti-PD-L2 Nanobody of claim 1, or the Nanobody fusion protein of claim 4; and (ii) a tag sequence to facilitate expression and / or purification.

20. Use of an anti-PD-L2 Nanobody according to claim 1, a Nanobody fusion protein according to claim 4, or an immunoconjugate according to claim 14 or 15, characterized in that, for the preparation of a detection plate or a kit; wherein said detection plate or kit is used for: detecting a PD-L2 protein in a sample.

21. A method for detecting PD-L2 protein in a sample for a purpose other than disease diagnosis, characterized by, said method comprising the steps of: (1) contacting a sample with the anti-PD-L2 Nanobody of claim 1 or the Nanobody fusion protein of claim 4; (2) detecting whether an antigen-antibody complex is formed, wherein the formation of the complex indicates the presence of a PD-L2 protein in the sample.

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