Anti-urokinase plasminogen activator receptor antibody and application thereof

Abstract

The invention belongs to the technical field of biology, and provides an anti-urokinase plasminogen activator receptor antibody or an antigen binding fragment thereof and application. The antibody or the antigen binding fragment thereof comprises a heavy chain variable region and a light chain variable region, the heavy chain variable region comprises heavy chain complementarity determining regions HCDR1, HCDR2 and HCDR3, and the light chain variable region comprises light chain complementarity determining regions LCDR1, LCDR2 and LCDR3. The antibody or the antigen binding fragment of the antibody can be combined with human uPAR protein in a high affinity mode, and anti-tumor specific immune response can be triggered.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biotechnology, and relates to an anti-urokinase plasminogen activator receptor antibody and uses thereof. BACKGROUND

[0002] uPA (urokinase-type plasminogen activator) is a proteolytic enzyme that promotes the fibrinolytic process. Its main function is to promote the conversion of plasminogen to plasmin, which is responsible for the degradation of fibrin, dissolving blood clots and tissue fibers. In addition to its role in the fibrinolytic system, uPA is also associated with cell migration, invasion and tumor growth and spread. uPAR is the receptor for uPA, which exists on the surface of cell membranes. It binds to uPA to form a complex, promoting the activity of uPA on the cell surface, thereby regulating cell migration, invasion and fusion. uPAR is also associated with cell adhesion, signal transduction and other biological processes such as inflammation. Studies have shown that the uPA-uPAR system composed of urokinase-type plasminogen activator (uPA), urokinase-type plasminogen activator receptor (uPAR) and plasminogen activator inhibitor (PAI) plays an important role in the occurrence and development of tumors.

[0003] uPAR is a high-affinity receptor for urokinase-type plasminogen activator (uPA, also known as PLAU) and is anchored to the cell membrane by a glycosylphosphatidylinositol (GPI) anchor. The extracellular region includes D1, D2, D3 domains, and the D1-D2 junction can be cleaved to produce D1 and D2-D3, where D1 alone can also bind to uPA, but the affinity is at least 1500 times lower than that of the complete uPAR binding. D2-D3 has the same chemotactic properties as uPA and can bind to the GPCR protein FPRL1 to transmit chemotactic signals. uPAR can also be detached from the membrane as a whole to form soluble suPAR, which is believed to be a marker for tumor prognosis.

[0004] As a ligand, uPA, initially an inactive zymogen pro-uPA, can be activated upon binding to uPAR, forming a disulfide-linked, two-chain structure with activity. uPAR binding to uPA leads to plasminogen activation and degradation of various ECM proteins, the extracellular matrix is no longer tightly connected, giving tumor cells the opportunity to migrate and invade, and at the same time, the release of ECM-related growth factors by cells not only enhances the expression of uPA and uPAR in turn, but also induces tumor angiogenesis and epithelial mesenchymal transition, in addition, it can also regulate cell proliferation and prevent cell apoptosis, and plays an important role in the occurrence and development of tumors. In addition, uPAR can not only promote the degradation of extracellular matrix proteins by binding to uPA, but also bind to integrin α5β1, α3β1 or αvβ3 to form a complex and vitronection to mediate the activation of RAS / RAF / MEK / ERK, PI3K / AKT and other cell signal pathways. The above effects not only can enhance the proliferation, migration and invasion ability of tumor cells, but also can inhibit tumor cell apoptosis and promote tumor tissue angiogenesis.

[0005] Recent evidence suggests that uPAR-activated signaling pathways contribute to cancer cell evasion and reduced cytotoxic effects of anticancer drugs. The elevated expression of the uPAR (PLAUR)-encoding gene in cancer can be regulated by different mechanisms. Transcription factors such as Sp1, NF-κB, TCF, and hypoxia-inducible factor 1α are often activated by different types of cancer-related signaling pathways, which bind to the cis-acting elements upstream of the uPAR gene, triggering its high expression in cancer. In the uPA-uPAR system, it slows down the growth of mouse melanoma by inhibiting the expression of uPA, uPAR, MMP-2 and MMP-9, etc.

[0006] Urokinase plasminogen activator receptor (uPAR) is expressed on a variety of cell types, including macrophages, neutrophils, endothelial cells, and importantly, on many cancer cells. uPAR expression is significantly upregulated in malignant tissues compared to normal tissues, with particularly high expression observed in pancreatic ductal adenocarcinoma (PDAC), triple-negative breast cancer, colorectal cancer, and glioblastoma. Critically, uPAR expression is strongly associated with poor clinical outcomes across multiple cancer types, making it an attractive prognostic biomarker and therapeutic target. The limited expression of the receptor in normal tissues (mainly restricted to immune cells at sites of inflammation) provides a valuable therapeutic window for antibody-based targeting strategies.

[0007] The standard of care for many uPAR-expressing cancers remains inadequate, with limited treatment options and poor long-term outcomes. Current treatment modalities, including surgery, chemotherapy, and radiotherapy, provide limited benefit, particularly for advanced disease, highlighting a critical unmet need for new therapeutic strategies. Numerous studies have confirmed the close relationship between uPAR-uPA and various malignant tumors, and targeted intervention can be performed at multiple links according to the mechanism of action. Currently, there is no uPAR-uPA-targeted drug on the market. SUMMARY

[0008] The technical problem to be solved by the present application is to provide an isolated antibody or antigen-binding fragment thereof binding to human urokinase plasminogen activator receptor (uPAR).

[0009] In a first aspect, the present application provides an anti-uPAR antibody or antigen-binding fragment thereof having binding specificity to human urokinase plasminogen activator receptor (uPAR), wherein the antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising heavy chain complementarity determining regions HCDR1, HCDR2 and HCDR3, and a light chain variable region comprising light chain complementarity determining regions LCDR1, LCDR2 and LCDR3; wherein, (1) the amino acid sequence of the HCDR1 is shown as SEQ ID NO: 2, the amino acid sequence of the HCDR2 is shown as SEQ ID NO: 10, the amino acid sequence of the HCDR3 is shown as SEQ ID NO: 4, the amino acid sequence of the LCDR1 is shown as SEQ ID NO: 6, the amino acid sequence of the LCDR2 is shown as SEQ ID NO: 7, the amino acid sequence of the LCDR3 is shown as SEQ ID NO: 8; or (1) the amino acid sequence of the HCDR1 is shown as SEQ ID NO: 2, the amino acid sequence of the HCDR2 is shown as SEQ ID NO: 10, the amino acid sequence of the HCDR3 is shown as SEQ ID NO: 12, the amino acid sequence of the LCDR1 is shown as SEQ ID NO: 6, the amino acid sequence of the LCDR2 is shown as SEQ ID NO: 7, the amino acid sequence of the LCDR3 is shown as SEQ ID NO: 8; or (3) the amino acid sequence of the HCDR1 is shown as SEQ ID NO: 14, the amino acid sequence of the HCDR2 is set forth in SEQ ID NO: 10, the amino acid sequence of the HCDR3 is set forth in SEQ ID NO: 4, the amino acid sequence of the LCDR1 is set forth in SEQ ID NO: 6, the amino acid sequence of the LCDR2 is set forth in SEQ ID NO: 7, the amino acid sequence of the LCDR3 is set forth in SEQ ID NO: 8; or (4) the amino acid sequence of the HCDR1 is set forth in SEQ ID NO: 14, the amino acid sequence of the HCDR2 is set forth in SEQ ID NO: 10, the amino acid sequence of the HCDR3 is set forth in SEQ ID NO: 12, the amino acid sequence of the LCDR1 is set forth in SEQ ID NO: 17, the amino acid sequence of the LCDR2 is set forth in SEQ ID NO: 7, the amino acid sequence of the LCDR3 is set forth in SEQ ID NO: 8.

[0010] One of skill in the art can determine the sequence of the CDRs from the heavy chain variable region sequences and light chain variable region sequences shown, using methods known in the art. The methods known in the art can be the Kabat scheme, the AbM scheme, the Chothia scheme, or the Contact scheme. The sequence of the CDR regions obtained using different schemes can vary for the same variable region sequence, which is known in the art. And one of skill in the art can understand that the CDR regions obtained using different schemes all fall within the scope of the present application.

[0011] In some embodiments, the anti-uPAR antibody or antigen binding fragment thereof comprises a heavy chain variable region and a light chain variable region, wherein the amino acid sequence of the heavy chain variable region is set forth in SEQ ID NO: 9, or has at least 90% sequence identity, such as at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity, to the sequence set forth in SEQ ID NO: 9; and the amino acid sequence of the light chain variable region is set forth in SEQ ID NO: 5, or has at least 90% sequence identity, such as at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity, to the sequence set forth in SEQ ID NO: 5; or the heavy chain variable region is set forth in SEQ ID NO: 11, or has at least 90% sequence identity, such as at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity, to the sequence set forth in SEQ ID NO: 11; and the amino acid sequence of the light chain variable region is set forth in SEQ ID NO: 5, or has at least 90% sequence identity, such as at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity, to the sequence set forth in SEQ ID NO: 5; or the heavy chain variable region is set forth in SEQ ID NO: 13, or has at least 90% sequence identity, such as at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity, to the sequence set forth in SEQ ID NO: 13; and the amino acid sequence of the light chain variable region is set forth in SEQ ID NO: 5, or has at least 90% sequence identity, such as at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity, to the sequence set forth in SEQ ID NO: 5; or the heavy chain variable region is set forth in SEQ ID NO: 15, or has at least 90% sequence identity, such as at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity, to the sequence set forth in SEQ ID NO: 15; and the amino acid sequence of the light chain variable region is set forth in SEQ ID NO: 16, or has at least 90% sequence identity, such as at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity, to the sequence set forth in SEQ ID NO: 16.

[0012] In some embodiments, in any of the above-described antibodies or antigen binding fragments thereof, the antibody or antigen binding fragment thereof further comprises a heavy chain constant region, a light chain constant region, an Fc region, or a combination thereof.

[0013] In some embodiments, in any of the above-described antibodies or antigen binding fragments thereof, the light chain constant region is a kappa chain or a lambda chain constant region.

[0014] In some embodiments, in any of the antibodies or antigen-binding fragments thereof described above, the heavy chain constant region is selected from the IgG, IgM, IgA, IgE, or IgD class.

[0015] In some embodiments, in any of the antibodies or antigen-binding fragments thereof described above, the heavy chain constant region is a heavy chain constant region selected from the IgGl, IgG2, IgG3, or IgG4 subclass.

[0016] In some embodiments, in any of the antibodies or antigen-binding fragments thereof described above, the antibody or antigen-binding fragment thereof further comprises a human IgGl heavy chain constant region or a variant thereof, and / or a human kappa light chain constant region or a variant thereof.

[0017] In some embodiments, in any of the antibodies or antigen-binding fragments thereof described above, the antibody or antigen-binding fragment thereof further comprises a heavy chain constant region having an amino acid sequence as set forth in SEQ ID NO: 18, and a light chain constant region having an amino acid sequence as set forth in SEQ ID NO: 19.

[0018] In some embodiments, in any of the antibodies or antigen-binding fragments thereof described above, the antibody or antigen-binding fragment thereof is a chimeric antibody, a humanized antibody.

[0019] In some embodiments, in any of the antibodies or antigen-binding fragments thereof described above, the antigen-binding fragment is a F(ab')2, F(ab)2, Fab', Fab, Fv, or scFv.

[0020] In some embodiments, in any of the antibodies or antigen-binding fragments thereof described above, the antibody or antigen-binding fragment thereof is a monoclonal antibody (including a full-length monoclonal antibody), a polyclonal antibody, or a multispecific antibody (e.g., bispecific antibody).

[0021] In a second aspect, the present application provides a biological material selected from (a)~(c) below (a) a nucleic acid molecule encoding any of the antibodies or antigen-binding fragments thereof described above.

[0022] In some embodiments, the nucleic acid molecule can be a DNA, such as a cDNA, a genomic DNA, or a recombinant DNA. In other embodiments, the nucleic acid molecule can also be an RNA, such as an mRNA or an hnRNA, etc.

[0023] In some embodiments, the nucleic acid molecule is an isolated nucleic acid molecule.

[0024] (b) a recombinant vector comprising the nucleic acid molecule described above.

[0025] The vector can be an expression vector. In some embodiments, the vector is a eukaryotic expression vector, a prokaryotic expression vector, an artificial chromosome, a phage vector, etc.

[0026] (c) a recombinant cell comprising the above nucleic acid molecule and / or the above recombinant vector.

[0027] The recombinant cell is transformed or infected with the nucleic acid molecule or the recombinant vector of the present application. In some embodiments, the host cell can be any prokaryotic or eukaryotic cell, such as a bacterial or an insect, fungal, plant or animal cell. In some embodiments, the host cell is prokaryotic, such as E. coli. In other embodiments, the host cell is eukaryotic, such as a 293 cell, a CHO cell, a yeast cell, or a plant cell. In some embodiments, the host cell is other cell suitable for producing an antibody or antigen-binding fragment thereof.

[0028] In a third aspect, the present application provides a method of producing an antibody or antigen-binding fragment thereof of the present application, the method comprising culturing a recombinant cell comprising a nucleic acid molecule encoding the antibody or antigen-binding fragment thereof of the present application under conditions suitable for expression of the antibody. In some embodiments, the method further comprises recovering the antibody or antigen-binding fragment thereof from the recombinant cell or the culture medium.

[0029] In a fourth aspect, the present application provides a composition comprising the antibody or antigen-binding fragment thereof of the present application or the biomaterial of the present application.

[0030] In some embodiments, the above composition is a pharmaceutical composition, further comprising a pharmaceutically acceptable carrier.

[0031] In a fifth aspect, the present application provides use of any of the above-mentioned antibody or antigen-binding fragment thereof, any of the above-mentioned biomaterial and / or any of the above-mentioned composition in the manufacture of any of the following products: (a) a product for detecting uPAR; (b) a product for stimulating or enhancing an immune response; (c) a product for preventing and / or treating a disease associated with abnormal expression of uPAR; (d) a product for killing or inhibiting the growth of a cell expressing uPAR in vitro or in vivo.

[0032] In some embodiments, the disease associated with abnormal expression of uPAR is cancer.

[0033] In some embodiments, the disease associated with abnormal expression of uPAR is melanoma, pancreatic cancer, pancreatic ductal adenocarcinoma, triple-negative breast cancer, colorectal cancer, or glioblastoma.

[0034] The antibody or antigen-binding fragment thereof provided by the present application binds to human uPAR and exhibits many excellent properties: it can bind to human uPAR protein with high affinity, binds to MDA-MB-231 with high affinity, can induce an anti-tumor specific immune response, and provides a new direction for the treatment of cancer. BRIEF DESCRIPTION OF DRAWINGS

[0035] Figure 1 Binding activity of supernatant of anti-human uPAR chimeric antibody to human uPAR protein (primary screening).

[0036] Figure 2 Binding activity of anti-human uPAR chimeric antibody to human uPAR protein.

[0037] Figure 3 Binding activity of anti-human uPAR chimeric antibody to MDA-MB-231 cell line endogenously expressing human uPAR.

[0038] Figure 4 Results of antibody-dependent cell killing activity of anti-human uPAR chimeric antibody on MDA-MB-231 cell line endogenously expressing human uPAR.

[0039] Figure 5 Results of antibody-dependent cell killing activity of anti-human uPAR antibody on MDA-MB-231 cell line endogenously expressing human uPAR. DETAILED DESCRIPTION

[0040] The present application will be further described with reference to the following specific examples. It should be understood that the following examples are illustrative only and are not intended to limit the scope of the present application. Unless otherwise indicated, the techniques utilized in the examples are routine procedures, or are performed by following the manufacturer's instructions or protocols. Reagents and biological materials used in the examples are commercially available unless otherwise indicated.

[0041] Abbreviations and Definitions Unless otherwise indicated, the following terms shall have the following meanings. Other terms or abbreviations have their commonly understood meanings in the art.

[0042] "Antibody" refers to any form of an antibody that exhibits a desired biological activity (e.g., inhibits binding of a ligand to its receptor or inhibits ligand-induced receptor signaling by inhibiting ligand-induced receptor signaling). Thus, "antibody" is used in the broadest sense and specifically encompasses, but is not limited to, monoclonal antibodies (including full length monoclonal antibodies), polyclonal antibodies, and multispecific antibodies (e.g., bispecific antibodies), fully human, humanized, primatized, chimeric, single-chain antibodies, and the like.

[0043] "Antigen binding fragment" refers to a portion of an antibody, such as F(ab')2, F(ab)2, Fab', Fab, Fv, scFv, and the like. Regardless of structure, an antibody fragment binds to the same antigen recognized by the intact antibody. The term "antigen binding fragment" includes aptamers, mirror images, and diabodies. The term "antigen binding fragment" also includes any synthetic or genetically engineered protein that functions as an antibody by forming a complex with a particular antigen.

[0044] "Fab fragment" consists of one light chain and the CH1 and variable region of one heavy chain. The heavy chain of a Fab molecule cannot form a disulfide bond with another heavy chain molecule.

[0045] "Fc region" contains two heavy chain fragments comprising the CH2 and CH3 domains of an antibody. The two heavy chain fragments are held together by two or more disulfide bonds and by hydrophobic interactions of the CH3 domains.

[0046] "Fv region" comprises variable regions from both the heavy and light chains, but lacks constant regions.

[0047] "Single-chain Fv antibody" (or "scFv antibody") refers to an antibody fragment that comprises the VH and VL domains of an antibody, wherein these domains are present in a single polypeptide chain. Generally, the Fv polypeptide further comprises a polypeptide linker between the VH and VL domains that enables the scFv to form the desired structure for antigen binding. For a review of scFv see U.S. Patent No. 6,423,538.

[0048] Those skilled in the art will understand that the class of an antibody heavy chain includes gamma, mu, alpha, delta, or epsilon (γ, μ, α, δ, ε), with some subclasses thereof (e.g., γ1-γ4). The class of an antibody is determined by the nature of the constant domain of the heavy chain. Immunoglobulin subclasses (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, etc., have been well characterized and the functional specificities conferred thereby are known. All immunoglobulin classes are within the scope of the present disclosure. In some embodiments, the immunoglobulin molecule is of the IgG class. IgG generally comprises two identical light polypeptide chains of molecular weight approximately 23,000 daltons and two identical heavy polypeptide chains of molecular weight approximately 53,000-70,000. The four chains are linked by disulfide bonds in a "Y" configuration, with the light chain beginning at the "Y" mouth and continuing around the heavy chain through the variable region. Antibodies of the IgG1 form are a subclass of IgG, whose heavy chain is of the γ1 subtype. In some embodiments, the antibodies of the present disclosure are of the IgG1 class.

[0049] As used herein, the term "heavy chain constant region" includes an amino acid sequence derived from an immunoglobulin heavy chain. A polypeptide comprising a heavy chain constant region comprises at least one of the following: a CH1 domain, a hinge (e.g., an upper hinge region, an intermediate hinge region, and / or a lower hinge region) domain, a CH2 domain, a CH3 domain, or a variant or fragment thereof. For example, an antigen binding polypeptide for use in the present application can comprise: a polypeptide chain comprising a CH1 domain; a polypeptide chain comprising a CH1 domain, at least a portion of a hinge domain, and a CH2 domain; a polypeptide chain comprising a CH1 domain and a CH3 domain; a polypeptide chain comprising a CH1 domain, at least a portion of a hinge domain, and a CH3 domain; or a polypeptide chain comprising a CH1 domain, at least a portion of a hinge domain, a CH2 domain, and a CH3 domain. In another embodiment, a polypeptide of the present application comprises a polypeptide chain comprising a CH3 domain. Furthermore, an antibody for use in the present application can lack at least a portion of a CH2 domain (e.g., all or a portion of a CH2 domain). As noted above, one of ordinary skill in the art will understand that a heavy chain constant region can be modified such that it differs in amino acid sequence from a naturally occurring immunoglobulin molecule.

[0050] "Hypermutation region" refers to antibody amino acid residues responsible for antigen binding. The hypermutation region comprises amino acid residues from the "complementarity determining regions" or "CDRs" as defined by sequence alignment. "Framework" residues or "FR" residues are variable domain residues other than the hypermutation region residues defined herein.

[0051] An "isolated antibody" is one which has been separated from all or part of its natural environment. Contaminant components of its natural environment are materials which would interfere with diagnostic or therapeutic uses for the antibody, and can include enzymes, hormones, and other proteinaceous or nonproteinaceous solutes. In some embodiments, the antibody is purified (1) to greater than 95% by weight of antibody, such as 99% by weight, as determined by the Lowry method, (2) to a degree sufficient to obtain at least 15 residues of N-terminal or internal amino acid sequence by use of a spinning cup sequenator, or (3) to homogeneity by SDS-PAGE under reducing or nonreducing conditions in conjunction with staining or by other suitable means. An isolated antibody includes an antibody in situ within recombinant cells since at least one component of the antibody's natural environment will not be present. Ordinarily, an isolated antibody will be prepared by at least one purification step. In some embodiments, the isolated antibody 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 (inclusive) or any value therein.

[0052] A "nucleic acid" or "polynucleotide" refers to a polymeric molecule composed of individual nucleotides: adenine (a), cytosine (c), guanine (g), thymine (t) (or uracil (u) in RNA), such as DNA, RNA or modifications thereof. A nucleic acid molecule can be a natural nucleic acid molecule or a synthetic nucleic acid molecule or a combination of one or more natural 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 (for example, a probe, primer, EST or SAGE tag), an exon, an intron, messenger RNA (mRNA), transfer RNA, ribosomal RNA, ribozymes, cDNA, dsRNA, siRNA, miRNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes and primers.

[0053] An "isolated nucleic acid molecule" is a nucleic acid molecule that has been identified and separated from at least one contaminant nucleic acid molecule. An isolated nucleic acid molecule is distinguished from a nucleic acid molecule in its natural state or environment. Thus, an isolated nucleic acid molecule is distinguished from a nucleic acid molecule in its natural cellular environment. However, an isolated nucleic acid molecule includes a nucleic acid molecule contained in cells that are commonly expressing the antibody, for example, the nucleic acid molecule is in a different chromosomal location than in the natural cell.

[0054] The term "identity" as used herein can be assessed by eye or by computer software such as the software programs described in Ausubel et al. eds. (2007) in Current Protocols in Molecular Biology. A molecule is identical at a position when that position in the compared sequences is occupied by the same base or amino acid. Identity between two or more sequences can be expressed as a percentage (%) which can be used to assess identity between related sequences. A polynucleotide sequence or amino acid sequence has a certain percentage (e.g., 90%, 95%, 98%, or 99%) of "sequence identity" to another sequence if the percentage of bases or amino acids that are the same in the two sequences when aligned is that percentage.

[0055] A "monoclonal antibody" refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations that can be present in minor amounts. Monoclonal antibodies are highly specific, being directed against a single antigenic site.

[0056] The term "chimeric antibody" refers to an antibody in which a portion of the heavy and / or light chain is derived from one source or species, while the remainder of the heavy and / or light chain is derived from a different source or species.

[0057] "Immune cells" include cells that have hematopoietic origin and function in the immune response. Immune cells include B lymphocytes, T lymphocytes, natural killer cells, monocytes, macrophages, eosinophils, mast cells, basophils, and granulocytes.

[0058] A "variant" sequence as used herein refers to a sequence that differs from the indicated sequence at one or more amino acid residues but retains the biological activity of the resulting molecule.

[0059] An "amino acid" refers to an organic compound containing both an amino group and a carboxyl group, such as an alpha-amino acid, which can be encoded by a nucleic acid, either directly or in the form of a precursor. A single amino acid is encoded by a nucleic acid composed of three nucleotides (a so-called codon or base triplet). The same amino acid can be encoded by different codons, a phenomenon known as "degeneracy of the genetic code." Amino acids include natural amino acids and unnatural amino acids. Natural 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 (gin, 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).

[0060] A "conservatively substituted variant" or "conservative amino acid substitution" refers to amino acid substitutions that are known to one of skill in the art to generally not alter the biological activity of the resulting molecule. Generally, a single amino acid substitution in a non-essential region of a polypeptide is generally recognized as not altering the biological activity by those skilled in the art. Conservative substitutions can be made, for example, by replacing a member of one of the following groups of chemically similar amino acids: 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) basic side chains: lysine, arginine, and histidine; 6) acidic side chains: aspartic acid and glutamic acid.

[0061] The term "about" as used herein, is intended to refer to a range of values that are acceptable to one of ordinary skill in the art as determined by the particular value, and depends in part on how the value is measured or determined (i.e., the limitations of the measurement system). Alternatively, "about" can mean a range of up to ±20%, such as ±10%, ±5% or ±1%. Unless otherwise stated, where a particular value appears in the application and claims, the meaning of "about" should be assumed to be within an acceptable error range of that particular value.

[0062] "Specific" binding, when referring to a ligand / receptor, antibody / antigen, or other binding pair, refers to the determination of the presence or absence of a binding reaction of a protein, e.g., LAIR1, in a heterogeneous population of proteins and / or other biological agents. Thus, under the specified conditions, a particular ligand / antigen binds to a particular receptor / antibody and does not bind in significant amounts to other proteins present in the sample.

[0063] The term "encodes" when applied to a polynucleotide refers to a polynucleotide that is referred to as "encoding" a polypeptide if, in its natural state or when manipulated by methods known to those skilled in the art, it can be transcribed and / or translated to produce a polypeptide and / or fragments thereof. The anti-sense strand is the complement of such a nucleic acid and the coding sequence can be deduced therefrom.

[0064] "Administering" and "treatment" when used in reference to an animal, human, subject, cell, tissue, organ, or biological fluid means contacting the exogenous pharmaceutical, therapeutic, diagnostic, or composition with the animal, human, subject, cell, tissue, organ, or biological fluid. "Administering" and "treatment" can refer to, for example, therapeutic methods, pharmacokinetic methods, diagnostic methods, research methods, and experimental methods. Treating a cell includes contacting the agent with the cell and contacting the agent with a fluid in which the cell is in contact. "Administering" and "treatment" also mean, for example, in vitro and ex vivo treatment of cells by agents, diagnostic agents, binding compositions, or by other cells.

[0065] The term "treatment" includes amelioration or cessation of a disorder or symptoms thereof. Treatment includes inhibition, e.g., reduction in the overall frequency of occurrence of a disorder or symptoms thereof.

[0066] The term "prevention" includes avoidance of the initial of a disorder or symptoms thereof.

[0067] The term "therapeutically effective amount" or "effective amount" as used herein refers to the amount of an anti-uPAR antibody or antigen-binding fragment thereof, when administered to a cell, tissue, or subject either alone or in combination with another therapeutic agent, that is effective to prevent or alleviate the disease or condition to be treated. A therapeutically effective dose further refers to that amount of the antibody or antigen-binding fragment thereof sufficient to result in a reduction of symptoms, e.g., a therapeutic, curative, prophylactic, or palliative treatment of the relevant medical condition, or an increase in the rate of therapeutic, curative, prophylactic, or palliative treatment of the condition. The effective amount for a particular subject can vary depending on a variety of factors, e.g., the disease to be treated, the overall health status of the patient, the method route and dose of administration, and the severity of side effects. The effective amount can be the maximum dose or administration regimen that avoids significant side effects or toxic effects. When administered to an individual, a therapeutically effective amount refers to the individual component when administered alone. When administered in combination, a therapeutically effective amount refers to the amount of the combination of active ingredients that produces a therapeutic effect, regardless of whether it is administered jointly, consecutively, or simultaneously.

[0068] Pharmaceutical compositions The present application also provides pharmaceutical compositions. Such compositions comprise an effective amount of an antibody or antigen-binding fragment thereof and a pharmaceutically acceptable carrier.

[0069] In some embodiments, the term "pharmaceutically acceptable carrier" refers to a material that is approved by a regulatory agency of the government or listed in the other generally recognized pharmacopeia for use in animals, and more particularly in humans. Further, a "pharmaceutically acceptable carrier" would generally be any type of nontoxic solid, semi-solid or liquid filler, diluent, encapsulating material or formulation auxiliary of whatever means of introduction is used.

[0070] The term "carrier" refers to a diluent, adjuvant, excipient, or vehicle with which the active ingredient is administered in therapy. Such a pharmaceutical carrier can be a sterile liquid, such as water and oils, including those of

[0071] In some embodiments, the pharmaceutical compositions of the present application can be administered by any suitable route known in the art, including, but not limited to: oral, nasal, intradermal, subcutaneous, intravenous, intramuscular, intrabronchial, intrapleural, intraperitoneal, intraarterial, lymphatic, and / or cerebrospinal.

[0072] In some embodiments, the compositions are formulated in accordance with routine procedures as pharmaceutical compositions adapted for intravenous injection to humans. Typically, compositions for intravenous administration are solutions in sterile isotonic aqueous buffer. A pharmaceutical composition can also include a solubilizing agent and a local anesthetic such as lidocaine to ease pain at the site of the injection. Generally, the ingredients are supplied either separately or mixed together in unit dosage form, for example, as a dry lyophilized powder or water free concentrate in a hermetically sealed container such as an ampule or sachette indicating the quantity of active agent. Where the composition is to be administered by infusion, it can be dispensed with an infusion bottle containing sterile pharmaceutical grade water or saline. Where the composition is administered by injection, an ampule of sterile water for injection or saline can be provided so that the ingredients can be mixed prior to administration.

[0073] The antibody or antigen-binding fragment thereof of the present application includes a salt form thereof. The pharmaceutically acceptable salt includes a salt formed from an anion such as hydrochloric acid, phosphoric acid, acetic acid, oxalic acid, tartaric acid, and the like, and a salt formed from a cation such as sodium, potassium, ammonium, calcium, ferric hydroxide, isopropylamine, triethylamine, 2-ethylaminoethanol, histidine, procaine, and the like.

[0074] Example 1: Production of Chimeric Antibody Against Human uPAR 1. Designing Anti-Human uPAR Chimeric Antibody Variable Region Sequence by Screening and Combinatorial Mutagenesis New chimeric antibodies against human uPAR were generated by modifying ATN-658 (reference patent CN101022830A). The structure of known anti-human uPAR Fab (ATN-658) and human uPAR complex (PDB ID: 4K24) was used to design chimeric antibodies against human uPAR by screening and combinatorial mutagenesis. First, the CDR regions were selected by combining the CDRs labeled according to both Kabat and IMGT encoding methods, and the amino acid residues within 5A from human uPAR or the amino acid residues that may bind to human uPAR after mutation were selected. Single-point saturation mutagenesis scanning or two-point combinatorial mutagenesis scanning was performed on these amino acid residues using MOE and Bioluminate to generate new anti-human uPAR antibody sequences. The cell supernatant of the new anti-human uPAR antibody was filtered through a 0.22 μm membrane and then subjected to kinetic evaluation using Carterra LSA. The mutation sites with the highest affinity improvement were selected for combination to generate double-mutation / three-mutation / four-mutation combinatorial mutation chimeric antibodies against human uPAR. The chimeric antibodies against human uPAR are numbered and the mutation conditions and sequences are shown in Table 1 and Table 2 (for example: K30ER represents K mutation to R at position 30E according to KABATA encoding), wherein the position number is derived from the results of KABATA numbering on the AbRSA website.

[0075] Table 1: Numbering and Mutation Conditions of Chimeric Antibodies Against Human uPAR

[0076] Table 2: CDR Sequences and Variable Region Sequences of Chimeric Antibodies Against Human uPAR

[0077] 2. Preparation of Chimeric Antibodies Against Human uPAR The light and heavy chain variable regions (Table 2) were constructed into human constant regions (IgG1 / K, Table 3), respectively, to construct the corresponding chimeric antibodies, and the sequences were verified by sequencing to be correct. The corresponding nucleic acids encoding the chimeric antibodies were expressed using Expi293 cells and purified using Protein A magnetic beads or packing material, according to the following methods: Expi293 cells expressing chimeric antibodies: Expi293 cells (Thermo, Cat. No. A14635CN) were diluted to 2.5 x 10 6 cells / mL and incubated at 37°C, 8% CO2, 120 rpm. The next day, the viable cell density and viability were determined, and the cell transfection density should be 5 x 10 6 cells / mL, and the cell viability should be greater than 95%. PEI / plasmid complex preparation: PEI (1 mg / mL, Polysciences, Cat. No. 24765-1) was inverted and mixed. The plasmids expressing light and heavy chain antibodies were diluted with OPM-293 CD05 medium (Shanghai Oupu Mai Biological Technology Co., Ltd., Cat. No. 81075-001) to a total of 1 μg / mL, and the volume of the medium used to dilute the plasmids was 1 / 20 of the transfection volume. The plasmids expressing light and heavy chain antibodies were mixed at a ratio of 1:1.5. PEI reagent was diluted with OPM-293 CD05 medium, and the volume of the medium used to dilute the PEI was 1 / 20 of the transfection volume. The PEI reagent was gently inverted and mixed at room temperature for 5 minutes. The diluted PEI reagent was added to the diluted plasmids, and the mixture was gently inverted and mixed. The PEI / plasmid complex was incubated at room temperature for 15 minutes. For 24-well plate culture, the solution was added dropwise to the 24-well plate and mixed well. After transfection, the 24-well plate was sealed and incubated at 37°C, 8% CO2, 220 rpm. For shake flask culture, the solution was slowly added to the transfer shake flask, and the shake flask was gently rotated during the addition. After transfection, the shake flask was incubated at 37°C, 8% CO2, 120 rpm. On the second day after transfection (24 hours after transfection), 10% OPM-293 ProFeed (Shanghai Oupu Mai Biological Technology Co., Ltd., Cat. No. F081918) was added to the shake flask, and the shake flask was gently rotated during the addition. The shake flask was then returned to the incubator for further culture for 5-7 days. The 24-well plate was centrifuged at 400g, and the expression supernatant was transferred to a new 24-well plate to harvest the supernatant. 200 μl of the expression supernatant was filtered using a 0.22 μm filter to harvest the filtered expression supernatant.

[0078] Protein A magnetic beads purification of antibody: Add 1 ml equilibration buffer (PBS, pH 7.4) and 300 μl rProtein A Magarose Beads (Rigao, Cat No: SM003025) suspension to No.1 24-well plate, add 2 ml expression supernatant to No.2 24-well plate, add 2 ml washing buffer (PBS, pH 7.4) to No.3 and No.4 plates, add 0.5 ml elution buffer (0.1 M Glycin, pH 3.0) to No.5 plate, and put them into P24 24-channel automatic purification instrument (Rigao, Cat No: P241001) in order. Set the program as follows: Load: position 1; Get Beads: position 1, time 60 s; Binding: position 2, time 30 min; Washing: position 3 / 4, time 300 s; Elution: position 5, time 10 min; Unload: position 4. After the program is completed, add 50 μl 1 M Tris-Hcl pH 8.5 neutralization buffer to No.5 plate to neutralize the antibody.

[0079] Protein A column purification of antibody: Prepare the gravity chromatography column, open the upper cover of the gravity chromatography column, place the gasket at the bottom of the gravity column, and press tightly. Prepare the filler Protein A (Cytiva, Cat No: 17549801), accurately calculate the required filler suspension liquid volume from the target filler volume and the suspension ratio of the filler, required filler suspension liquid volume = target filler volume / suspension ratio of the filler. Vortex the filler thoroughly to ensure that the filler is completely suspended. Add the filler suspension liquid to the bottom of the gravity chromatography column. Add at least 10 CV of equilibration buffer PBS to the gravity chromatography column. After equilibration, detect the pH of the outlet. If the target pH 7.4 is not reached, continue to add the equilibration buffer until the target pH 7.4 is reached. Slowly add a certain volume of sample to the gravity chromatography column. Add at least 10 CV of elution buffer to the gravity chromatography column. Slowly add 5 CV of elution buffer (0.1 M Glycin, pH 3.0) to the gravity chromatography column, and incubate for 3-5 minutes to collect the eluate. Repeat the elution step as needed. Neutralization: adjust the pH to the target pH 5-6 with neutralization buffer (1 M Tris). Determine the protein concentration with a Nanodrop. Replace the storage buffer of the antibody with PBS by ultrafiltration.

[0080] Table 3 Constant region sequences

[0081] Example 2: Functional identification of anti-human uPAR chimeric antibody 1. Preliminary screening of anti-human uPAR chimeric antibody by surface plasmon resonance The binding activity of anti-human uPAR chimeric antibody supernatant to human uPAR was assessed using Carterra LSA via surface plasmon resonance (SPR) for rapid kinetic screening. First, an anti-human IgG capture layer was prepared on an HC30M chip (Carterra, catalog number 4279) via primary amine conjugation. In short, 200 mM MEDC and 50 mM sulfonyl... The chip surface was activated for 10 minutes with a 1:1 mixture of NHS (Cytiva, catalog number: BR-1000-50), followed by conjugation of anti-human-Fc IgG (Cytiva, catalog number: 29-2346-00) at 20 µg / mL in 10 mM sodium acetate buffer (Cytiva, catalog number: 29-2346-00) at pH 5.0 for 10 minutes. Unconjugated spaces on the chip surface were blocked for 7 minutes with 1 M ethanolamine hydrochloride at pH 8.5. The filtered cell expression supernatant was diluted 1:10 with 1×HBS-EP+ buffer, 0.05% P20, pH 7.4 (10 mM HEPES pH 7.4, 150 mM NaCl, 3 mM EDTA, 0.05% Tween 20; Cytiva, catalog number: BR-1006-69). For capture kinetics, the antibody group was captured in the diluted filtered cell expression supernatant for 10 minutes using a prepared anti-human-Fc IgG surface and a 96-channel printhead (96PH). Then, purified recombinant antigen (human uPAR, Acro Biosystems catalog number UPR-H52H4) was injected onto the antibody group in eight 3-fold dilution series starting at 300 nM using a single flow cell (SFC). Each injection consisted of a 3-minute binding phase and a 7-minute dissociation phase. The surface was regenerated between antigens using a regeneration buffer of 10 mM glycine hydrochloride, pH 1.5. Using Carterra Kinetics software, the obtained sensor maps were fitted to a 1:1 binding model to estimate the binding rate constant (ka), dissociation rate constant (kd), and dissociation equilibrium constant (KD). The results are as follows: Figure 1 As shown in Table 4.

[0082] Table 4. Binding activity of anti-human uPAR chimeric antibody supernatant to human uPAR protein (initial screening)

[0083] The results showed that, compared with ATN-658, the anti-human uPAR chimeric antibody exhibited enhanced binding activity to human uPAR protein.

[0084] 2. Affinity detection of anti-human uPAR chimeric antibodies via surface plasmon resonance. The dissociation equilibrium constant (KD) of the selected anti-human uPAR chimeric antibodies in the initial screening was determined by surface plasmon resonance (SPR) using a Biacore 8K microarray to analyze binding activity. The CM5 chip (Cytiva, catalog number: 29149603) was used. The 8 channels were first activated for 420 seconds with an activator (a mixture of 200 mM MEDC and 50 mM NHS in equal proportions, Cytiva, catalog number: BR-1000-50) at a flow rate of 10 μL / min. Anti-human FcIgG (Cytiva, catalog number: 29-2346-00) was diluted to 20 μg / mL with 10 mM NaAc (pH 5.0) and simultaneously injected into the chip at a rate of 10 μL / min. 8 channels, and Fc1 flows sequentially through the 8 channels. At Fc2, the injection time was 420 s, and the response value reached approximately 8000, enabling anti-human FcIgG to couple to the chip via the amino group. Finally, the chip was blocked with 1M ethanolamine hydrochloride for 420 s at a flow rate of 10 μL / min. The antibody expression supernatant was purified using Protein A magnetic beads to obtain anti-human uPAR chimeric antibody. The antibody was diluted to 2 μg / ml and then injected onto the CM5 chip via anti-human FcIgG. Antibody was injected into the flow cell Fc2 of each channel at a binding time of 60 s and a flow rate of 10 μL / min. Next, the analyte antigen (human uPAR, AcroBiosystems, catalog number UPR-H52H4) was diluted to 100 nM with 1×HBS-EP+ buffer, 0.05% P20, pH 7.4 (10 mM HEPES pH 7.4, 150 mM NaCl, 3 mM EDTA, 0.05% Tween 20; Cytiva, catalog number: BR-1006-69), followed by a 2-fold serial dilution. Two zero concentrations were set to remove background signals and flow through the Fc2 of each channel of the chip. The injection rate was 30 μL / min, the sample binding time was 180 s, and the dissociation time was 420 s. After each binding and dissociation, the chip surface was regenerated with 10 mM glycine at pH 1.5 for 30 s at an injection rate of 30 μL / min. After subtracting the reference channel (flow cell 1) and the two zero-concentration signals, the original data were fitted using the 1:1 interaction model built into Biacore8K to calculate the binding rate constant (ka), dissociation rate constant (kd), and dissociation equilibrium constant (KD). The results are as follows: Figure 2 As shown in Table 5.

[0085] Table 5. Binding activity of anti-human uPAR chimeric antibodies against human uPAR protein.

[0086] Results showed that the binding activity of anti-human uPAR chimeric antibodies to human uPAR protein was improved to varying degrees compared with ATN-658, and HC-ATN-658-5 (heavy chain Y54R) was the most improved. The binding activity of all combination mutants of anti-human uPAR chimeric antibodies to human uPAR protein was improved, and HC-ATN-658-15 (heavy chain Y54R, G98M), HC-ATN-658-16 (heavy chain Y54R, S28H), HC-ATN-658-24 (heavy chain Y54R, G98M, S28H, light chain K30ER) were significantly improved. Among all anti-human uPAR chimeric antibodies, HC-ATN-658-24 (heavy chain Y54R, G98M, S28H, light chain K30ER) had the highest binding activity to human uPAR protein.

[0087] 3. Binding activity of anti-human uPAR chimeric antibodies to triple-negative breast cancer MDA-MB-231 cell line The triple-negative breast cancer MDA-MB-231 cell line (Nanjing Keye Biotechnology Co., Ltd., Catalog No: CBP60382) endogenously expressing human uPAR was prepared to 1.5 x 10 5Cells were resuspended in 100 μL / well of 96-well V-bottom plate, and centrifuged, then the supernatant was discarded. The chimeric antibodies HC-ATN-658-5, HC-ATN-658-15, HC-ATN-658-16, HC-ATN-658-24 against human uPAR were diluted to the initial working concentration of 150 nM with MACS buffer, and then diluted 3-fold gradient. The gradient-diluted antibodies were added to the resuspended cells in the well plate at 100 μL / well, mixed by blowing, and incubated at 4°C for 1 hour. After incubation, the cells were centrifuged and washed with 200 μl of MACS buffer. The AlexaFlour-647 labeled secondary antibody Alexa Fluor® 647 AffiniPure Goat Anti-Human IgG, Fcγfragment specific (Jackson, 109-605-190) was diluted 1:1000 with MACS buffer, and 100 μL of the secondary antibody diluent was added to each well, the cell pellet was resuspended and mixed by blowing, and incubated at 4°C for about 30 minutes. After incubation, the cells were centrifuged and washed with MACS buffer for three times, and then 100 μL of MACS buffer was added to resuspend the cells in the well plate. The M3 Median APC-H was read by flow cytometry (CytoFLEX), and the experimental data was analyzed by Graphpad Prism 8.0 software, taking the logarithm of the antibody concentration as the x-axis and the corresponding M3 Median APC-H value as the y-axis, and using a four-parameter equation regression model to fit the antibody dose-effect curve, and the EC50 value was calculated. 50 The results are shown in Figure 3 and Table 6.

[0088] Table 6 Binding activity of anti-human uPAR chimeric antibodies to MDA-MB-231 cell line

[0089] The results showed that, compared with ATN-658, the anti-human uPAR chimeric antibodies HC-ATN-658-5, HC-ATN-658-15, HC-ATN-658-16, and HC-ATN-658-24 had stronger binding activity to the human uPAR endogenously expressing cell line MDA-MB-231. Among them, HC-ATN-658-24 had the strongest binding activity to the human uPAR endogenously expressing cell line MDA-MB-231.

[0090] 4. Antibody-dependent cell ADCC killing activity of the human uPAR endogenously expressing cell line MDA-MB-231 (reporter gene test) It was found in clinical studies that anti-uPAR antibodies can clear tumor cells through ADCC effect. Anti-uPAR antibody-dependent FcyRIIIa activation was determined by incubating Jurkat-human FcyRIIIa (158V)-NFAT with endogenous human uPAR-expressing cell line MDA-MB-231 cells. The Fab end of the uPAR antibody binds to the target uPAR on the target cells MDA-MB-231, and the Fc end binds to the FcyRIIIa receptor on the effector cells, thereby activating the NFAT signaling pathway in the effector cells. The luciferase produced by the activation of the NFAT pathway was quantified to reflect the ADCC activity of the antibody, and the specific method is as follows: MDA-MB-231 cells 300 g centrifugation for 5 minutes, the cell concentration was adjusted to 1.2 x 10 6 cells / mL. 50 μL per well was inoculated in a white opaque plate as target cells. The chimeric anti-uPAR antibody of the application, positive control antibody, isotype (Biyuntong Technology Co., Ltd., Catalog No: 00K0W3003) was diluted with DMEM medium to a concentration of 40 μg / ml, and then diluted 4 times gradient. Eight concentration gradients were collected. Jurkat-human FcyRIIIa (158V)-NFAT (GM-Bio Technology (Shanghai) Co., Ltd., GM-C05619) cells were centrifuged at 300 g for 5 minutes, and the supernatant was discarded. The cell concentration was adjusted to 1.5 x 10 6 cells / mL. Take 100 μL cells and add to each culture well containing target cells, and add 50 μL of the above gradient-diluted anti-uPAR antibody to each well. Induce overnight at 37°C, 5% CO2, and equilibrate at room temperature for at least 15 minutes. Add 100 μL luciferase substrate solution (Vazyme, DD1203) to each well, mix, and react at room temperature for 5 minutes in the dark. Use the enzyme label instrument to read the relative light unit value on the cell culture plate. Use Graphpad Prism 8.0 software to analyze the test data, with the logarithm of the anti-uPAR antibody concentration as the x-axis and the corresponding RLU value as the y-axis. Select the four-parameter equation regression model to fit the dose-effect curve of the anti-uPAR antibody.

[0091] The ADCC effect of anti-uPAR antibody is shown in Figure 4 and Table 7. Compared with ATN-658, anti-human uPAR antibody HC-ATN-658-5, HC-ATN-658-15, HC-ATN-658-16, HC-ATN-658-24 has significantly enhanced ADCC killing activity on endogenous human uPAR-expressing cell line MDA-MB-231.

[0092] Table 7 ADCC activity of anti-human uPAR antibodies on MDA-MB-231 cell line

[0093] 5. ADCC effect induced by healthy human PBMC and anti-uPAR antibodies The ADCC effect of anti-uPAR antibodies was detected with PBMC as effector cells and MDA-MB-231 cells as target cells. The PBMC of healthy donors (Allcells) were recovered from cryopreservation and adjusted to a cell concentration of 2x10 6 cells / mL with 1640 complete medium containing 200 IU / mL IL-2 (Peprotech, item number: 200-02) and cultured overnight in a 37°C, 5% CO2 incubator. The next day, the unadherent PBMC were aspirated, centrifuged at 300 g for 5 minutes, and the supernatant was discarded. The cell concentration was adjusted to about 3x10 6 cells / mL by adding 1640 medium. The anti-uPAR antibodies and hIgG1 isotype (Bing Biological Technology Co., Ltd., item number: B117901) were diluted to 2 times the working concentration, with an initial concentration of 0.8 μg / mL and a 5-fold gradient dilution. The collected MDA-MB-231 cells were centrifuged at 300 g for 5 minutes, the supernatant was discarded, and the cell concentration was adjusted to 2x10 6 cells / mL with DPBS (gibco, item number: C14190500CP). The MDA-MB-231 cells were labeled with CellTrace™ Violet (Invitrogen, item number: C34557) according to the instructions, and the CellTrace™ Violet-labeled MDA-MB-231 cells were adjusted to a cell concentration of about 1.5x10 5 cells / mL with 1640 medium. 50 μL of CellTrace™ Violet-labeled MDA-MB-231 cells, 50 μL of PBMC, and 100 μL of diluted anti-uPAR antibodies were added to each well of a U-bottom 96-well plate, mixed well, and incubated in a 37°C, 5% CO2 incubator for about 5 hours. 2 μL of PI (Invitrogen, item number: 006990-50) dye was added to each well, mixed, and incubated at room temperature in the dark for 5 minutes to label dead cells. The percentage of PI-positive cells in CellTrace™ Violet-positive cells was read by flow cytometry, and the lysis% was calculated as the proportion of CellTrace™ Violet-labeled PI-positive cells to calculate the ADCC effect induced by the antibodies. The ADCC effect of anti-uPAR antibodies is shown in Table 7. Figure 5The anti-human uPAR antibody HC-ATN-658-24 showed a significantly higher ADCC killing activity against the endogenously human uPAR expressing cell line MDA-MB-231 compared to ATN-658.

Claims

1. An anti-uPAR antibody or its antigen-binding fragment, wherein, The antibody or its antigen-binding fragment comprises a heavy chain variable region and a light chain variable region. The heavy chain variable region includes heavy chain complementarity-determining regions HCDR1, HCDR2, and HCDR3, and the light chain variable region includes light chain complementarity-determining regions LCDR1, LCDR2, and LCDR3. The amino acid sequence of HCDR1 is shown in SEQ ID NO:

14. The amino acid sequence of HCDR2 is shown in SEQ ID NO:

10. The amino acid sequence of HCDR3 is shown in SEQ ID NO:

4. The amino acid sequence of LCDR1 is shown in SEQ ID NO:

6. The amino acid sequence of LCDR2 is shown in SEQ ID NO:

7. The amino acid sequence of LCDR3 is shown in SEQ ID NO: 8; or The amino acid sequence of HCDR1 is shown in SEQ ID NO:

14. The amino acid sequence of HCDR2 is shown in SEQ ID NO:

10. The amino acid sequence of HCDR3 is shown in SEQ ID NO:

12. The amino acid sequence of LCDR1 is shown in SEQ ID NO:

17. The amino acid sequence of LCDR2 is shown in SEQ ID NO:

7. The amino acid sequence of LCDR3 is shown in SEQ ID NO:

8.

2. The antibody or its antigen-binding fragment according to claim 1, wherein, The heavy chain variable region is as shown in SEQ ID NO: 13, or has at least 90% sequence identity with the sequence shown in SEQ ID NO: 13; the amino acid sequence of the light chain variable region is as shown in SEQ ID NO: 5, or has at least 90% sequence identity with the sequence shown in SEQ ID NO: 5; or The heavy chain variable region is as shown in SEQ ID NO: 15, or has at least 90% sequence identity with the sequence shown in SEQ ID NO: 15; the amino acid sequence of the light chain variable region is as shown in SEQ ID NO: 16, or has at least 90% sequence identity with the sequence shown in SEQ ID NO:

16.

3. The antibody or antigen-binding fragment thereof according to claim 2, wherein the antibody or antigen-binding fragment thereof further comprises a heavy chain constant region, a light chain constant region, an Fc region, or a combination thereof; wherein, The light chain constant region is a κ chain or λ chain constant region; the heavy chain constant region is selected from IgG, IgM, IgA, IgE or IgD categories.

4. The antibody or its antigen-binding fragment according to claim 3, wherein, The antibody or its antigen-binding fragment further comprises a heavy chain constant region and a light chain constant region, the amino acid sequence of the heavy chain constant region being shown in SEQ ID NO: 18, and the amino acid sequence of the light chain constant region being shown in SEQ ID NO:

19.

5. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 4, wherein the antigen-binding fragment is F(ab')2, F(ab)2, Fab', Fab, Fv or scFv.

6. A biological material selected from (a) to (c) of the following. (a) A nucleic acid molecule encoding an antibody or an antigen-binding fragment thereof as described in any one of claims 1 to 5; (b) A recombinant vector comprising the nucleic acid molecule described in (a); (c) A recombinant cell comprising (a) the nucleic acid molecule and / or (b) the recombinant vector.

7. A method for preparing an antibody or antigen-binding fragment thereof according to any one of claims 1 to 5, the method comprising, when suitable for antibody expression, culturing recombinant cells comprising a nucleic acid molecule encoding an antibody or antigen-binding fragment thereof according to any one of claims 1 to 5.

8. A composition comprising the antibody or antigen-binding fragment thereof as described in any one of claims 1 to 5 or the biological material as described in claim 6.

9. The use of the antibody or antigen-binding fragment thereof according to any one of claims 1 to 5, the biomaterial according to claim 6, and / or the composition according to claim 8 in the preparation of any of the following products: (a) Products tested for uPAR; (b) Products that stimulate or enhance the immune response; (c) Products for the prevention and / or treatment of diseases associated with abnormal uPAR expression; (d) Products that kill or inhibit the growth of uPAR-expressing cells in vitro or in vivo.

10. The application according to claim 9, wherein, The diseases associated with abnormal uPAR expression are cancers, selected from melanoma, pancreatic cancer, pancreatic ductal adenocarcinoma, triple-negative breast cancer, colorectal cancer, or glioblastoma.

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