Anti-urokinase plasminogen activator receptor antibodies and uses thereof

Abstract

This invention belongs to the field of biotechnology and provides an antibody or its antigen-binding fragment against the urokinase plasminogen activator receptor and its applications. The antibody or its antigen-binding fragment of this invention 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 antibody or its antigen-binding fragment of this invention can bind with high affinity to human uPAR protein and can induce an anti-tumor specific immune response.

Description

[0001] This application is a divisional application of Chinese Patent Application No. 202511831970.0, filed on December 8, 2025, entitled "Anti-urokinase plasminogen activator receptor antibody and its use". Technical Field

[0002] This invention belongs to the field of biotechnology and relates to anti-urokinase plasminogen activator receptor antibodies and their uses. Background Technology

[0003] uPA (urokinase-type plasminogen activator) is a proteolytic enzyme that promotes fibrinolysis. Its main function is to promote the conversion of plasminogen into plasmin, which is responsible for degrading fibrin, dissolving thrombi 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 a receptor for uPA, located on the cell membrane surface. It binds to uPA, forming a complex that promotes uPA activity on the cell surface, thereby regulating cell migration, invasion, and fusion. uPAR is also involved in biological processes such as cell adhesion, signal transduction, and inflammation. Studies have shown that the uPA-uPAR system, composed of urokinase-type plasminogen activator (uPA), urokinase-type plasminogen activator receptor (uPAR), and urokinase-type plasminogen activator inhibitor (PAI), plays an important role in the development and progression of tumors.

[0004] uPAR is a high-affinity receptor for urokinase-type plasminogen activator (uPA, also known as PLAU), anchored to the cell membrane via glycosylphosphatidylinositol (GPI). Its extracellular region includes D1, D2, and D3 domains. The D1-D2 junction can be cleaved to produce D1 and D2-D3. Individual D1 can also bind to uPA, but its affinity is at least 1500 times lower than that of intact uPAR. D2-D3 exhibits chemotactic properties similar to uPA and can bind to the GPCR protein FPRL1 to transmit chemotactic signals. uPAR can also detach entirely from the membrane, forming soluble suPAR; this soluble form is considered a potential indicator of tumor prognosis.

[0005] As a ligand, uPA initially exists as an inactive pro-uPA zymogen. Upon binding to uPAR, it is activated, forming an active disulfide-linked double-stranded structure. The binding of uPAR to uPA leads to plasminogen activation and degradation of various ECM proteins, resulting in looser extracellular matrix connections and creating an opportunity for tumor cell metastasis. Simultaneously, it induces the release of ECM-related growth factors, which not only enhance the expression of uPA and uPAR but also induce tumor angiogenesis and epithelial-mesenchymal transition. Furthermore, it regulates cell proliferation and prevents apoptosis, playing a crucial role in tumor development and progression. In addition, uPAR can promote extracellular matrix protein degradation by binding to uPA and can also bind to integrins α5β1, α3β1, or αvβ3 to form complexes and vitronection, mediating the activation of cell signaling pathways such as RAS / RAF / MEK / ERK and PI3K / AKT. These effects not only enhance tumor cell proliferation, migration, and invasion but also inhibit tumor cell apoptosis and promote tumor tissue angiogenesis.

[0006] Recent evidence suggests that uPAR-activated signaling pathways help cancer cells evade and reduce the cytotoxic effects of anticancer drugs. The increased expression of the gene encoding uPAR (PLAUR) in cancer may be regulated by various mechanisms. Transcription factors, such as Sp1, NF-κB, TCF, and hypoxia-inducible factor 1α, are frequently activated by different types of cancer-related signaling pathways, binding to cis-acting elements upstream of the uPAR gene and triggering its high expression in cancer. In the uPA-uPAR system, it slows the growth of mouse melanoma by inhibiting the expression of uPA, uPAR, MMP-2, and MMP-9.

[0007] Urokinase plasminogen activator receptor (uPAR) is expressed in multiple cell types, including macrophages, neutrophils, and endothelial cells, and importantly, in many cancer cells. Compared to normal tissues, uPAR expression is significantly upregulated in malignant tissues, with particularly high expression observed in pancreatic ductal adenocarcinoma (PDAC), triple-negative breast cancer, colorectal cancer, and glioblastoma. Crucially, uPAR expression is strongly associated with poor clinical outcomes in multiple cancer types, making it an attractive prognostic biomarker and therapeutic target. The limited expression of the receptor in normal tissues (primarily confined to immune cells at sites of inflammation) provides a valuable therapeutic window for antibody-based targeting strategies.

[0008] Many uPAR-expressing cancers still suffer from inadequate standards of care, limited treatment options, and poor long-term outcomes. Current treatments, including surgery, chemotherapy, and radiation therapy, offer limited benefit, particularly for advanced disease, highlighting a critical unmet need for novel treatment strategies. Numerous studies have confirmed the close association of uPAR-uPA with various malignancies and suggest the possibility of targeted intervention at multiple stages based on its mechanism of action; currently, there are no marketed drugs targeting uPAR-uPA. Summary of the Invention

[0009] The technical problem to be solved by the present invention is to provide an isolated antibody or antigen-binding fragment thereof that binds to human urokinase plasminogen activator receptor (uPAR).

[0010] In a first aspect, the present invention provides an anti-uPAR antibody or its antigen-binding fragment having binding specificity to human urokinase plasminogen activator receptor (uPAR), 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 comprising heavy chain complementarity-determining regions HCDR1, HCDR2, and HCDR3, and the light chain variable region comprising light chain complementarity-determining regions LCDR1, LCDR2, and LCDR3; wherein... (1) The amino acid sequence of HCDR1 is shown in SEQ ID NO: 2. 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 (2) The amino acid sequence of HCDR1 is shown in SEQ ID NO: 2. 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: 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 (3) 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 (4) 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.

[0011] Those skilled in the art can determine the CDR sequence based on the illustrated heavy chain and light chain variable region sequences using methods known in the art. These known methods may include the Kabat scheme, the AbM scheme, the Chothia scheme, or the Contact scheme. It is well known in the art that CDR sequences obtained using different schemes for the same variable region sequence will differ. Furthermore, those skilled in the art will understand that CDR regions obtained using different schemes are all within the scope of protection of this invention.

[0012] In some embodiments, the anti-uPAR antibody or its antigen-binding fragment comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region is as shown in SEQ ID NO: 9, or has at least 90% sequence identity with the sequence shown in SEQ ID NO: 9, 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%, or at least 99% sequence identity; 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, 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%, or at least 99% sequence identity; or The heavy chain variable region is as shown in SEQ ID NO: 11, or has at least 90% sequence identity with the sequence shown in SEQ ID NO: 11, 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%, or at least 99% sequence identity; 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, 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%, or at least 99% sequence identity; or 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, 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%, or at least 99% sequence identity; 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, 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%, or at least 99% sequence identity; 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, 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%, or at least 99% sequence identity; 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, 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%, or at least 99% sequence identity.

[0013] In some embodiments, any of the antibodies or antigen-binding fragments described above further comprises a heavy chain constant region, a light chain constant region, an Fc region, or a combination thereof.

[0014] In some embodiments, in any of the antibodies or antigen-binding fragments described above, the light chain constant region is a κ chain or λ chain constant region.

[0015] In some embodiments, in any of the antibodies or antigen-binding fragments described above, the heavy chain constant region is selected from the IgG, IgM, IgA, IgE or IgD class.

[0016] In some embodiments, in any of the antibodies or antigen-binding fragments described above, the heavy chain constant region is a heavy chain constant region selected from the IgG1, IgG2, IgG3 or IgG4 subclasses.

[0017] In some embodiments, any of the antibodies or antigen-binding fragments described above further comprise a human IgG1 heavy chain constant region or a variant thereof, and / or a human κ light chain constant region or a variant thereof.

[0018] In some embodiments, any of the antibodies or antigen-binding fragments described above further comprise 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.

[0019] In some embodiments, in any of the antibodies or antigen-binding fragments described above, the antibody or antigen-binding fragment is a chimeric antibody or a humanized antibody.

[0020] In some embodiments, the antigen-binding fragment of any of the antibodies or antigen-binding fragments described above is F(ab')2, F(ab)2, Fab', Fab, Fv, or scFv.

[0021] In some embodiments, in any of the antibodies or antigen-binding fragments described above, the antibody or antigen-binding fragment is a monoclonal antibody (including full-length monoclonal antibodies), a polyclonal antibody, or a multispecific antibody (e.g., a bispecific antibody).

[0022] In a second aspect, the present invention provides a biomaterial selected from (a) to (c) below. (a) A nucleic acid molecule that encodes any of the antibodies or antigen-binding fragments described above.

[0023] In some embodiments, the nucleic acid molecule may be DNA, such as cDNA, genomic DNA, or recombinant DNA. In other embodiments, the nucleic acid molecule may also be RNA, such as mRNA or hnRNA.

[0024] In some implementations, the nucleic acid molecule is an isolated nucleic acid molecule.

[0025] (b) A recombinant vector containing the aforementioned nucleic acid molecules.

[0026] 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 bacteriophage vector, etc.

[0027] (c) Recombinant cells containing the above-mentioned nucleic acid molecules and / or the above-mentioned recombinant vectors.

[0028] The recombinant cells are obtained by transforming or infecting host cells with the nucleic acid molecules or recombinant vectors of the present invention. In some embodiments, the host cell can be any prokaryotic or eukaryotic cell, such as bacterial or insect, fungal, plant, or animal cells. In some embodiments, the host cell is prokaryotic, such as *Escherichia coli*. In other embodiments, the host cell is eukaryotic, such as 293 cells, CHO cells, yeast cells, or plant cells. In some embodiments, the host cell is another cell suitable for preparing antibodies or their antigen-binding fragments.

[0029] In a third aspect, the present invention provides a method for preparing the antibody or antigen-binding fragment thereof described in the present invention, the method comprising culturing recombinant cells containing a nucleic acid molecule encoding the antibody or antigen-binding fragment thereof of the present invention, in a manner suitable for antibody expression.

[0030] In some embodiments, the method further includes: recovering the antibody or its antigen-binding fragment from the recombinant cells or culture medium.

[0031] In a fourth aspect, the present invention provides a composition comprising the antibody described herein or an antigen-binding fragment thereof, or the biological material of the present invention.

[0032] In some embodiments, the above composition is a pharmaceutical composition and also includes a pharmaceutically acceptable carrier.

[0033] In a fifth aspect, the present invention provides the use of any of the antibodies or antigen-binding fragments thereof described above, any of the biological materials described above, and / or any of the compositions described above 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.

[0034] In some implementations, the disease associated with abnormal uPAR expression is cancer.

[0035] In some specific embodiments, the diseases associated with abnormal uPAR expression are melanoma, pancreatic cancer, pancreatic ductal adenocarcinoma, triple-negative breast cancer, colorectal cancer, or glioblastoma.

[0036] The antibody or its antigen-binding fragment provided by this invention binds to human uPAR and exhibits many excellent properties: it can bind with high affinity to human uPAR protein and with high affinity to MDA-MB-231, which can trigger a tumor-specific immune response, providing a new direction for cancer treatment. Attached Figure Description

[0037] Figure 1 To assess the binding activity of anti-human uPAR chimeric antibody supernatant to human uPAR protein (initial screening).

[0038] Figure 2 This demonstrates the binding activity of the anti-human uPAR chimeric antibody to the human uPAR protein.

[0039] Figure 3 To assess the binding activity of the anti-human uPAR chimeric antibody with the endogenous human uPAR-expressing MDA-MB-231 cell line.

[0040] Figure 4 The results show the antibody-dependent cell-killing activity of the anti-human uPAR chimeric antibody against the endogenous human uPAR-expressing MDA-MB-231 cell line.

[0041] Figure 5 The results show the antibody-dependent cell-killing activity of anti-human uPAR antibody against the endogenous human uPAR-expressing MDA-MB-231 cell line. Detailed Implementation

[0042] The present invention will be further described below with reference to specific embodiments. It should be understood that the following embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Unless otherwise specified, the techniques used in the embodiments are conventional practices in the art, or experimental methods recommended by the reagent kit and instrument manufacturers. Unless otherwise specified, the reagents and biological materials used in the embodiments are commercially available.

[0043] Abbreviations and Definitions Unless otherwise stated, the following terms shall have the meanings described below. Other terms or abbreviations shall have meanings known in the art.

[0044] "Antibody" refers to any form of antibody that exhibits a desired biological activity (e.g., inhibiting the binding of a ligand to its receptor or by inhibiting receptor signal transduction induced by a ligand). Therefore, "antibody" is used in its broadest sense and explicitly includes, but is not limited to, monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies and multispecific antibodies (e.g., bispecific antibodies), fully human, humanized, primate-derived, chimeric antibodies, single-chain antibodies, etc.

[0045] "Antigen-binding fragment" refers to a portion of an antibody, such as F(ab')2, F(ab)2, Fab', Fab, Fv, scFv, etc. Regardless of its structure, the antibody fragment binds to the same antigen recognized by the intact antibody. The term "antigen-binding fragment" includes aptamers, mirror isoforms, and bivalent antibodies. The term "antigen-binding fragment" also includes any synthetic or genetically engineered protein that functions as an antibody by binding to a specific antigen to form a complex.

[0046] A "Fab fragment" consists of a light chain, a heavy chain (CH1), and a variable region. The heavy chain of a Fab molecule cannot form a disulfide bond with another heavy chain molecule.

[0047] The “Fc region” contains two heavy chain segments with CH2 and CH3 domains containing the antibody. The two heavy chain segments are held together by two or more disulfide bonds and through the hydrophobic interaction of the CH3 domain.

[0048] The “Fv region” contains variable regions from both the heavy and light chains, but lacks constant regions.

[0049] A "single-chain Fv antibody" (or "scFv antibody") is an antibody fragment containing both VH and VL domains of the antibody, wherein these domains are contained within a single polypeptide chain. Generally, Fv polypeptides also contain a polypeptide linker between the VH and VL domains, which allows the scFv to form the desired structure for antigen binding. For a review of scFv, see U.S. Patent No. 6,423,538.

[0050] Those skilled in the art will understand that antibody heavy chains are classified as gamma, mu, alpha, delta, or epsilon (γ, μ, α, δ, ε), with some subclasses (e.g., γ1-γ4). The properties of this chain determine the "type" of the antibody, namely IgG, IgM, IgA, IgD, or IgE. Immunoglobulin subclasses (isotypes), such as IgG1, IgG2, IgG3, IgG4, etc., have been well characterized and their assigned functional specificities are known. All immunoglobulin types are within the scope of protection disclosed in this invention. In some embodiments, the immunoglobulin molecule is of the IgG type. IgG typically comprises two identical light chain polypeptides with a molecular weight of approximately 23,000 Daltons and two identical heavy chain polypeptides with a molecular weight of approximately 53,000-70,000. These four chains are linked by disulfide bonds in a "Y" configuration, wherein the light chain begins at the "Y" port and continues to surround the heavy chain through a variable region. Antibodies in the form of IgG1 are a subclass of IgG, with their heavy chain being the γ1 subtype. In some embodiments, the antibody disclosed in this invention is IgG1.

[0051] As used herein, the term "heavy chain constant region" includes an amino acid sequence derived from the immunoglobulin heavy chain. A polypeptide containing a heavy chain constant region comprises at least one of the following: a CH1 domain, a hinge (e.g., an upper hinge region, a middle 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 used in this invention may comprise: a polypeptide chain containing a CH1 domain; a polypeptide chain containing at least a portion of a CH1 domain, a hinge domain, and a CH2 domain; a polypeptide chain containing both a CH1 domain and a CH3 domain; a polypeptide chain containing at least a portion of a CH1 domain, a hinge domain, and a CH3 domain; or a polypeptide chain containing at least a portion of a CH1 domain, a hinge domain, a CH2 domain, and a CH3 domain. In another embodiment, the polypeptide of this invention comprises a polypeptide chain containing a CH3 domain. Furthermore, antibodies used in this invention may lack at least a portion of the CH2 domain (e.g., all or part of the CH2 domain). As described above, those skilled in the art will understand that the heavy chain constant region can be modified to differ from naturally occurring immunoglobulin molecules in its amino acid sequence.

[0052] The "hypervariant region" refers to the antibody amino acid residues responsible for antigen binding. The hypervariant region contains the following amino acid residues: amino acid residues from the "complementarity-determining region" or "CDR" defined by sequence alignment. "Framework" residues or "FR" residues are variable domain residues other than those defined in the hypervariant region.

[0053] "Isolated antibody" is an antibody separated from all or part of its natural environmental components. The contaminating components of its natural environment are substances that could interfere with the diagnostic or therapeutic application of the antibody and may include enzymes, hormones, and other proteolysinic or non-proteolysinic substances. In some embodiments, the antibody is purified to the extent that: (1) more than 95% by weight, such as more than 99% by weight, as determined by the Lowry method; (2) sufficient to obtain at least 15 residues of the N-terminal or internal amino acid sequence using a twist-cup sequencer; or (3) determined to be homogeneous by SDS-PAGE stained with Coomassie blue or silver under reducing or non-reducing conditions. Isolated antibodies include antibodies in situ within recombinant cells, since at least one component of the antibody's natural environment will be absent. Isolated antibodies are typically prepared by at least one purification step. In some embodiments, the purity of 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 ​​(including the endpoint) or any value therein.

[0054] "Nucleic acid" or "polynucleotide" refers to a polymer molecule composed of a single nucleotide: adenine (a), cytosine (c), guanine (g), thymine (t) (or uracil (u) in RNA), such as DNA, RNA, or modifications thereof. Nucleic acid molecules can be natural or synthetic nucleic acid molecules, or a combination of one or more natural nucleic acid molecules with one or more synthetic nucleic acid molecules. Examples of nucleic acids include, but are not limited to: genes or gene fragments (e.g., probes, primers, EST or SAGE tags), exons, introns, messenger RNA (mRNA), transfer RNA, ribosomal RNA, ribonuclease, 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.

[0055] "Isolated nucleic acid molecules" are nucleic acid molecules that have been identified and separated from at least one contaminating nucleic acid molecule. Isolated nucleic acid molecules differ from their naturally occurring form or environment. Therefore, isolated nucleic acid molecules are distinct from nucleic acid molecules present in their natural cells. However, isolated nucleic acid molecules include nucleic acid molecules contained in cells that normally express antibodies, for example, where the chromosomal location of the nucleic acid molecule differs from its chromosomal location in natural cells.

[0056] The term "identity" as used in this article can be evaluated by the naked eye or by computer software (such as the software program described in Current Protocols in Molecular Biology by Ausubel et al. eds. (2007)). When the positions in the compared sequences are occupied by the same bases or amino acids, the molecules are identical at that position. Identity between two or more sequences can be expressed as a percentage (%), which can be used to evaluate the identity between related sequences. "Sequence identity" of a polynucleotide or amino acid sequence with another sequence at a certain percentage (e.g., 90%, 95%, 98%, or 99%) means that when the sequences are aligned, that percentage of bases or amino acids are the same in the two compared sequences.

[0057] "Monoclonal antibody" refers to an antibody derived from a substantially homogeneous group of antibodies, the individual antibodies constituting the group being identical. Monoclonal antibodies are highly specific, targeting a single antigenic site. Furthermore, unlike conventional (polyclonal) antibody preparations, which typically include multiple different antibodies targeting multiple different determinants (epitopes), each monoclonal antibody targets only a single determinant on the antigen.

[0058] The term "chimeric antibody" refers to an antibody in which a portion of the heavy chain and / or light chain originates from one source or species, while the remainder of the heavy chain and / or light chain originates from a different source or species.

[0059] "Immune cells" include cells that have a hematopoietic origin and play a role in the immune response. Immune cells include: B lymphocytes, T lymphocytes, natural killer cells, monocytes, macrophages, eosinophils, mast cells, basophils, and granulocytes.

[0060] The sequence “variant” used in this article refers to a sequence that differs from the sequence shown at one or more amino acid residues but retains the biological activity of the resulting molecule.

[0061] Amino acids are organic compounds that contain both amino and carboxyl groups, such as α-amino acids, which can be encoded by nucleic acids directly or in their precursor form. A single amino acid is encoded by a nucleic acid consisting of three nucleotides (so-called codons or base triplets). The fact that the same amino acid can be encoded by different codons is called "degeneracy of the genetic code." Amino acids include both natural and non-natural 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 (gln, Q), glutamic acid (glu, E), glycine (gly, G), histidine (his, H), isoleucine (ile, I), leucine (leu, L), lysine (lys, K), methionine (met, M), phenylalanine (phe, F), proline (pro, P), serine (ser, S), threonine (thr, T), tryptophan (trp, W), tyrosine (tyr, Y), and valine (val, V).

[0062] "Variations of conserved substitutions" or "conserved amino acid substitutions" refer to amino acid substitutions known to those skilled in the art that such substitutions generally do not alter the biological activity of the resulting molecule. Generally, it is generally accepted by those skilled in the art that a single amino acid substitution in a non-essential region of a polypeptide does not substantially alter its biological activity. Conserved substitutions can be made by amino acid substitutions with chemically similar side chains, such as: 1) aliphatic side chains: glycine, alanine, valine, leucine, and isoleucine; 2) aliphatic hydroxyl side chains: serine and threonine; 3) amide-containing side chains: asparagine and glutamine; 4) aromatic side chains: phenylalanine, tyrosine, and tryptophan; 5) basic side chains: lysine, arginine, and histidine; 6) acidic side chains: aspartic acid and glutamic acid.

[0063] As used herein, the term "about" means a numerical value within an acceptable margin of error for a specific value determined by a person skilled in the art, the numerical value depending in part on how it is measured or determined (i.e., the limits of the measurement system). Alternatively, "about" may mean a range of up to ±20%, such as ±10%, ±5%, or ±1%. Unless otherwise stated, when a specific value appears in this application and claims, the meaning of "about" should be assumed to be within an acceptable margin of error for that specific value.

[0064] When referring to ligand / receptor, antibody / antigen, or other binding pairs, "specific" binding means determining the presence of a binding reaction of the protein, such as LAIR1, within a heterogeneous population of proteins and / or other biological reagents. Therefore, under specified conditions, a particular ligand / antigen binds to a specific receptor / antibody and does not bind in significant amounts to other proteins present in the sample.

[0065] When applied to polynucleotides, the term "encoding" refers to a polynucleotide that, if in its natural state or when manipulated by methods known to those skilled in the art, can be transcribed and / or translated to produce mRNA containing a polypeptide and / or fragments thereof, is called "encoding" a polypeptide. The antisense strand is the complement of this nucleic acid, and the coding sequence can be deduced from it.

[0066] When "giving" and "treating" are used to refer to animals, humans, experimental subjects, cells, tissues, organs, or biological fluids, it means contacting an exogenous drug, therapeutic agent, diagnostic agent, or composition with the animal, human, subject, cell, tissue, organ, or biological fluid. "Giving" and "treating" can refer to, for example, methods of treatment, pharmacokinetic methods, diagnostic methods, research methods, and experimental methods. Treating cells includes contacting a reagent with cells and contacting a reagent with a fluid, wherein the fluid contacts the cells. "Giving" and "treating" also mean, for example, in vitro and ex vivo treatment of cells by means of a reagent, diagnostic agent, conjugated composition, or other cells.

[0067] The term "treatment" refers to the improvement or cessation of a condition or its symptoms. Treatment includes suppression, such as reducing the overall frequency of attacks of a condition or its symptoms.

[0068] The term "prevention" includes avoiding the initial stages of a disease or its symptoms.

[0069] As used herein, the term "therapeutic effective dose" or "effective dose" refers to the amount of an anti-uPAR antibody or its antigen-binding fragment, administered alone or in combination with another therapeutic agent, that effectively prevents or alleviates the disease or condition to be treated. A therapeutic effective dose further refers to the amount of the antibody or its antigen-binding fragment sufficient to cause symptom relief, such relief being, for example, treatment, cure, prevention, or alleviation of an associated medical condition, or an increase in the rate of treatment, cure, prevention, or alleviation of the symptom. The effective dose for a specific subject can vary depending on a variety of factors, such as the disease to be treated, the patient's overall health condition, the route and dosage of administration, and the severity of side effects. An effective dose may be the maximum dose or administration regimen that avoids significant side effects or toxicity. When administered to an individual as a single active ingredient, the therapeutic effective dose refers to that single ingredient. When administered in combination, the therapeutic effective dose refers to the combined amount of active ingredients that produce a therapeutic effect, regardless of whether they are administered in combination, continuously, or simultaneously.

[0070] Pharmaceutical Composition The present invention also provides pharmaceutical compositions. Such compositions comprise an effective dose of an antibody or an antigen-binding fragment thereof, and a pharmaceutically acceptable carrier.

[0071] In some implementations, the term "pharmaceuticalally acceptable carrier" refers to a substance approved by a government regulatory agency or listed in another recognized pharmacopoeia for use in animals (particularly for humans). Furthermore, "pharmaceuticalally acceptable carrier" will generally be any type of non-toxic solid, semi-solid, or liquid filler, diluent, encapsulating material, or formulation adjuvant.

[0072] The term "carrier" refers to a diluent, adjuvant, excipient, or carrier used in conjunction with an active ingredient for therapeutic purposes. Such drug carriers can be sterile liquids, such as water and oils, including petroleum, animal, plant, or synthetic oils, such as peanut oil, soybean oil, mineral oil, sesame oil, etc. In some embodiments, the carrier can be water when the drug composition is administered intravenously. Saline solutions, glucose solutions, and glycerol solutions can also be used as liquid carriers, particularly for injectable solutions. Examples of suitable drug carriers are described in EW Martin's Remington's Pharmaceutical Sciences, which are incorporated herein by reference. Such compositions will contain a clinically effective dose of an antibody or antibody fragment, along with a suitable carrier, to provide a dosage form suitable for the patient. The formulation should be suitable for the mode of administration. The formulation can be packaged in ampoules, disposable syringes, or multi-dose vials made of glass or plastic.

[0073] In some embodiments, the pharmaceutical compositions of the present invention may 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 fluid administration.

[0074] In some embodiments, the composition is formulated into a pharmaceutical composition suitable for intravenous injection into the human body according to conventional procedures. Compositions for intravenous administration are typically solutions in sterile isotonic buffer solutions. The pharmaceutical composition may also contain a solubilizer and a local anesthetic such as lidocaine to relieve pain at the injection site. Generally, the active ingredient is supplied individually or in combination in unit doses, such as as a dry lyophilized powder or anhydrous concentrate in a sealed container (such as an ampoule or sachet) indicating the amount of active agent. When the composition is administered by infusion, it can be dispensed using an infusion bottle containing sterile pharmaceutical-grade water or saline. When the composition is administered by injection, ampoules of sterile water or saline for injection can be used, allowing the active ingredient to be mixed before administration.

[0075] The antibodies or antigen-binding fragments thereof of the present invention include their salt forms. Pharmaceutically acceptable salts include those derived from anions such as hydrochloric acid, phosphoric acid, acetic acid, oxalic acid, tartaric acid, etc., and those derived from cations such as sodium, potassium, ammonium, calcium, ferric hydroxide, isopropylamine, triethylamine, 2-ethylaminoethanol, histidine, procaine, etc.

[0076] Example 1: Generation of chimeric antibodies against human uPAR 1. Screening and combination mutation design of variable region sequences for anti-human uPAR chimeric antibodies Novel chimeric antibodies against human uPAR were generated by modifying ATN-658 (referencing patent CN101022830A). Chimeric anti-human uPAR antibodies were designed using the known structure of the anti-human uPAR Fab (ATN-658) complex with human uPAR (PDB ID: 4K24). First, the CDR region was selected by combining CDRs encoded using both Kabat and IMGT methods. Amino acid residues within 5 Å of human uPAR in the CDR region, or those that might bind to human uPAR after mutation, were selected. These amino acid residues were scanned for single-point saturation mutations or two-point combined mutations using MOE and Bioluminate to generate new anti-human uPAR antibody sequences. The cell expression supernatant of the new anti-human uPAR antibodies was filtered through a 0.22 μm membrane and kinetics were evaluated using Carterra LSA. The mutation sites with the highest affinity enhancement were selected for combination to generate double / triple / quadruple mutant chimeric anti-human uPAR antibodies. The chimeric antibody numbers, mutation information, and sequences of anti-human uPAR are shown in Tables 1 and 2 (e.g., K30ER indicates that the K at position 30E of the KABATA encoding is mutated to R). The position numbers are obtained from the KABATA numbering results of the AbRSA website.

[0077] Table 1. Numbering and mutation status of chimeric antibodies against human uPAR

[0078] Table 2. CDR and variable region sequences of chimeric antibodies against human uPAR

[0079] 2. Preparation of chimeric antibodies against human uPAR The light and heavy chain variable regions (Table 2) were constructed onto the human constant region (IgG1 / K, Table 3) to construct the corresponding chimeric antibodies, and the sequences were verified to be correct by sequencing.

[0080] The corresponding nucleic acid encoding the chimeric antibody was expressed in Expi293 cells and purified using Protein A magnetic beads or packing material, as follows: Expi293 cells expressing chimeric antibody: One day before transfection, Expi293 cells (Thermo, catalog number: A14635CN) were diluted to 2.5 × 10⁻⁶. 6 Cells / mL were cultured at 37°C in an 8% CO2 shaker at 120 rpm. On the second day, viable cell density and survival rate were measured; the cell transfection density should be 5 × 10⁶ cells / mL. 6 Cells / mL, cell viability >95%. Preparation of PEI / plasmid complex: Mix PEI (1 mg / mL, Polysciences, catalog number: 24765-1) by inverting. Dilute the plasmids expressing light and heavy chain antibodies separately with OPM-293CD05 medium (Shanghai Aopumai Biotechnology Co., Ltd., catalog number: 81075-001), with a total plasmid volume of 1 μg / mL. The volume of the medium used to dilute the plasmids should be 1 / 20 of the transfection volume. Mix gently, resulting in a light-to-heavy chain antibody plasmid ratio of 1:1.5. Dilute the PEI reagent with OPM-293CD05 medium, with a volume of the medium used to dilute the PEI of 1 / 20 of the transfection volume. Mix gently by inverting and incubate at room temperature for 5 minutes. Add the diluted PEI reagent to the diluted plasmids and mix gently by inverting. Incubate the PEI / plasmid complex at room temperature for 15 minutes. For 24-well plate culture, add the solution dropwise to the 24-well plate and shake well. After transfection, seal the 24-well plate and incubate at 37℃ and 8% CO2 on a shaker at 220 rpm. For shake flask culture, slowly add the solution dropwise to the transfer shake flask, gently rotating the flask during the addition process. After transfection, incubate the shake flask at 37℃ and 8% CO2 on a shaker at 120 rpm. On the second day after transfection (24 hours post-transfection), add 10% OPM-293 ProFeed (Shanghai Aopumai Biotechnology Co., Ltd., catalog number: F081918) to the shake flask, gently rotating the flask during the addition process. Then, return the shake flask to the shaker and continue incubation for 5-7 days. Centrifuge the 24-well plate at 400g and transfer the expression supernatant to a new 24-well plate to harvest the supernatant. Filter 200 μl of the expression supernatant through a 0.22 μm filter membrane to harvest the filtered expression supernatant.

[0081] Protein A magnetic bead purification of antibodies: Add 1 ml of equilibration buffer (PBS, pH 7.4) and 300 μl of Protein A Magarose Beads (Tiandi Renhe, catalog number: SM003025) suspension to well 1 of a 24-well plate; add 2 ml of expression supernatant to well 2 of a 24-well plate; add 2 ml of washing buffer (PBS, pH 7.4) to wells 3 and 4 respectively; add 0.5 ml of elution buffer (0.1 M glycine, pH 3.0) to well 5. Place the plates sequentially into a P24 24-channel automated purification system (Tiandi Renhe, catalog number: P241001). 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 finishes running, add 50 μl of 1M Tris-HCl pH 8.5 neutralizing solution to plate 5 to neutralize the antibody.

[0082] Protein A column purification of antibodies: Prepare a gravity chromatography column. Open the column cap and place the gasket at the bottom of the column, pressing it firmly. Prepare the packing material, Protein A (Cytiva, catalog number: 17549801). Calculate the required packing suspension volume precisely based on the target packing volume and the packing suspension ratio: Required packing suspension volume = Target packing volume / Packing suspension ratio. Vortex the packing material thoroughly to ensure complete suspension. Add the packing suspension to the bottom of the gravity chromatography column. Add at least 10 CV of equilibration buffer (PBS) to the column. After equilibration, check the outlet pH. If the target pH of 7.4 is not reached, continue adding equilibration buffer until the target pH of 7.4 is reached. Slowly add a certain volume of sample to the column. Add at least 10 CV of eluent to the column. Slowly add 5 CV of elution buffer (0.1M Glycin, pH 3.0) to the column and incubate for 3-5 minutes. Collect the eluent. Repeat the elution step as needed. Neutralization: Adjust the pH to the target pH of 5-6 using neutralization buffer (1M Tris). Determine protein concentration using Nanodrop. Replace the antibody-containing buffer with PBS via ultrafiltration.

[0083] Table 3. Sequence of Constant Regions

[0084] Example 2: Functional identification of chimeric antibodies against human uPAR 1. Initial screening of anti-human uPAR chimeric antibodies via surface plasmon resonance Rapid kinetic screening was performed using Carterra LSA to assess the binding activity of anti-human uPAR chimeric antibody supernatant to human uPAR via surface plasmon resonance (SPR). First, an anti-human IgG capture layer was prepared on an HC30M chip (Carterra, catalog number 4279) via primary amine coupling. Briefly, the chip surface was activated for 10 min with a 1:1 mixture of 200 mM MEDC and 50 mM sulfo-NHS (Cytiva, catalog number: BR-1000-50), followed by coupling with anti-human-Fc IgG (Cytiva, catalog number: 29-2346-00) at 20 µg / mL for 10 min in 10 mM sodium acetate buffer (Cytiva, catalog number: 29-2346-00) at pH 5.0. Unconjugated spaces on the chip surface were blocked with 1 M ethanolamine hydrochloride at pH 8.5 for 7 min. 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.

[0085] Table 4. Binding activity of anti-human uPAR chimeric antibody supernatant to human uPAR protein (initial screening)

[0086] The results showed that, compared with ATN-658, the anti-human uPAR chimeric antibody exhibited enhanced binding activity to human uPAR protein.

[0087] 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, and the binding activity was analyzed. Channels 1-8 of the CM5 chip (Cytiva, catalog number: 29149603) were first activated for 420 s 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 channels 1-8 of the chip at a rate of 10 μL / min, sequentially flowing through channels Fc1-Fc2 of the eight channels. The injection time was 420 s, and the response value reached approximately 8000, indicating that the anti-human FcIgG was coupled to the chip via amino groups. Finally, the chip was blocked with 1M ethanolamine hydrochloride for 420 seconds at a flow rate of 10 μL / min. The antibody expression supernatant was purified with Protein A magnetic beads to obtain anti-human uPAR chimeric antibody. The antibody was diluted to 2 μg / ml and then bound to the CM5 chip with anti-human FcIgG. The antibody was injected into the flow cell Fc2 of each channel for 60 seconds at 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.

[0088] Table 5. Binding activity of anti-human uPAR chimeric antibodies against human uPAR protein.

[0089] The results showed that, compared with ATN-658, the anti-human uPAR chimeric antibodies enhanced the binding activity of human uPAR protein to varying degrees, with HC-ATN-658-5 (heavy chain Y54R) showing the greatest enhancement. All combined mutant anti-human uPAR chimeric antibodies enhanced the binding activity of human uPAR protein, with HC-ATN-658-15 (heavy chain Y54R, G98M), HC-ATN-658-16 (heavy chain Y54R, S28H), and HC-ATN-658-24 (heavy chain Y54R, G98M, S28H, light chain K30ER) showing the most significant enhancement. Among all anti-human uPAR chimeric antibodies, HC-ATN-658-24 (heavy chain Y54R, G98M, S28H, light chain K30ER) exhibited the highest enhancement in binding activity to human uPAR protein.

[0090] 3. Binding activity of anti-human uPAR chimeric antibody with triple-negative breast cancer MDA-MB-231 cell line The triple-negative breast cancer cell line MDA-MB-231 (Nanjing Kebai Biotechnology Co., Ltd., CBP60382) expressing endogenous human uPAR was cultured in MACS buffer (Baiying Biotechnology, catalog number: 20241220) at a concentration of 1.5 × 10⁻⁶ cells / mL. 5Cells / well were placed in 96-well V-bottom plates, and the supernatant was discarded after centrifugation. Chimeric antibodies against human uPAR (HC-ATN-658-5, HC-ATN-658-15, HC-ATN-658-16, HC-ATN-658-24), positive control antibody ATN-658, and negative control antibody Anti-HEL Human IgG1 (E356D / M358L)-Kappa (Baiying Biotechnology) were diluted to an initial working concentration of 150 nM with MACS buffer, and then serially diluted 3-fold with MACS buffer. Cells were resuspended in the serially diluted antibody at 100 μL / well, mixed by pipetting, and incubated at 4°C for 1 hour. After incubation, cells were centrifuged and washed with 200 μL of MACS buffer. The Alexa Fluor® 647 AffiniPure Goat Anti-Human IgG, Fcγfragment specific (Jackson, 109-605-190) labeled with Alexa Flour-647 was diluted 1:1000 with MACS buffer. 100 μL of the secondary antibody dilution was added to each well to resuspend cell clumps, and the mixture was pipetted and incubated at 4°C for approximately 30 minutes. After incubation, the cells were centrifuged, washed three times with MACS buffer, and then resuspended in 100 μL of MACS buffer per well. M3 Median APC-H values ​​were read using a flow cytometer (CytoFLEX). The experimental data were analyzed using Graphpad Prism 8.0 software, with the logarithm of antibody concentration on the x-axis and the corresponding M3 Median APC-H value on the y-axis. A four-parameter regression model was used to fit the antibody dose-response curve and calculate the EC50. 50 The result is as follows Figure 3 As shown in Table 6.

[0091] Table 6. Binding activity of anti-human uPAR chimeric antibody against MDA-MB-231 cell line.

[0092] 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 exhibited stronger binding activity against the endogenous human uPAR-expressing cell line MDA-MB-231. Among them, HC-ATN-658-24 showed the strongest binding activity against the endogenous human uPAR-expressing cell line MDA-MB-231.

[0093] 4. Antibody-dependent cell ADCC killing activity of endogenously expressed human uPAR cell line MDA-MB-231 (reporter gene assay) Clinical studies have shown that anti-uPAR antibodies can eliminate tumor cells through ADCC. Jurkat-human FcγRⅢa (158V)-NFAT was used to incubate MDA-MB-231 cells, which endogenously express human uPAR, to determine anti-uPAR antibody-dependent FcγRⅢa activation. The Fab terminus of the uPAR antibody binds to the target uPAR on target cells MDA-MB-231, while its Fc terminus binds to the FcγRⅢa receptor on effector cells, thereby activating the NFAT signaling pathway in effector cells. The ADCC activity of the antibody was reflected by quantifying the luciferase produced by NFAT pathway activation. The specific method is as follows: MDA-MB-231 cells were centrifuged at 300 g for 5 minutes, and the cell concentration was adjusted to 1.2 × 10⁻⁶ cells using DMEM medium. 6 Cells / mL. 50 μL of each cell was seeded into a white opaque plate as target cells. The chimeric anti-uPAR antibody, positive control antibody, and isotype (Baiying Biotechnology Co., Ltd., catalog number: 00K0W3003) were diluted with DMEM medium to a concentration of 40 μg / mL, and then serially diluted 4-fold (8 concentration gradients) with culture medium. Jurkat-human FcγRⅢa(158V)-NFAT (Jiman Biotechnology (Shanghai) Co., Ltd., GM-C05619) cells were collected, centrifuged at 300 g for 5 minutes, the supernatant was discarded, and DMEM medium was added to adjust the cell concentration to 1.5 × 10⁻⁶ cells / mL. 6 Cells / mL. Add 100 μL of cells to each well containing target cells, and simultaneously add 50 μL of the serially diluted anti-uPAR antibody to each well. Induce overnight at 37°C and 5% CO2, and equilibrate at room temperature for at least 15 minutes. Add 100 μL of luciferase substrate solution (Vazyme, DD1203) to each well, mix, and incubate in the dark at room temperature for 5 minutes. Read the relative light units (RLU) values ​​on the cell culture plate using a microplate reader. Analyze the experimental data using Graphpad Prism 8.0 software, with the logarithm of the anti-uPAR antibody concentration as the x-axis and the corresponding RLU value as the y-axis. Use a four-parameter regression model to fit the dose-response curve of the anti-uPAR antibody.

[0094] Anti-uPAR antibody ADCC effect such as Figure 4 As shown in Table 7, compared with ATN-658, the anti-human uPAR antibodies HC-ATN-658-5, HC-ATN-658-15, HC-ATN-658-16, and HC-ATN-658-24 significantly enhanced the killing activity against the endogenous human uPAR-expressing cell line MDA-MB-231 ADCC.

[0095] Table 7. Anti-human uPAR antibody activity against ADCC in MDA-MB-231 cell line

[0096] 5. ADCC effect induced by PBMCs and anti-uPAR antibodies in healthy individuals PBMCs were used as effector cells, and MDA-MB-231 cells were used as target cells to detect the ADCC effect of anti-uPAR antibodies. Cryopreserved healthy donor PBMCs (Allcells) were resuscitated and their concentration adjusted to 2 × 10⁻⁶ cells using 1640 complete medium containing 200 IU / mL IL-2 (Peprotech, catalog number: 200-02). 6 Cells / mL were cultured overnight at 37 ℃ in a 5% CO2 incubator. The next day, non-adherent PBMCs were harvested, centrifuged at 300 g for 5 minutes, the supernatant was discarded, and 1640 medium was added to adjust the cell concentration to approximately 3 × 10⁻⁶ cells / mL. 6 Cells / mL. Anti-uPAR antibody and hIgG1 isotype (Baiying Biotechnology Co., Ltd., catalog number: B117901) were diluted in 1640 medium to 2 times the working mass concentration, starting at 0.8 μg / mL, with 5-fold serial dilutions. After centrifuging the collected MDA-MB-231 cells at 300 g for 5 minutes, the supernatant was discarded, and the cell concentration was adjusted to 2 × 10⁻⁶ cells / mL with DPBS (gibco, catalog number: C14190500CP). 6 Cells / mL were added to label MDA-MB-231 cells with CellTrace™ Violet (Invitrogen, catalog number: C34557) according to the manufacturer's instructions. The concentration of CellTrace™ Violet-labeled MDA-MB-231 cells was then adjusted to approximately 1.5 × 10⁻⁶ cells / mL using 1640 medium. 5 Cells / mL. Add 50 μL of CellTrace™ Violet-labeled MDA-MB-231, 50 μL of PBMC, and 100 μL of diluted anti-uPAR antibody to each well of a U-bottom 96-well plate. Mix well and incubate at 37 ℃ with 5% CO2 for approximately 5 hours. Add 2 μL of PI (Invitrogen, catalog number: 006990-50) dye to each well, mix, and incubate at room temperature in the dark for 5 minutes to label dead cells. Flow cytometry was used to read the percentage of PI-positive cells out of CellTrace™ Violet-positive cells. The antibody-induced ADCC effect was calculated using lysis% = the proportion of CellTrace™ Violet-labeled PI-positive cells.

[0097] Anti-uPAR antibody ADCC effect such as Figure 5As shown. Compared with ATN-658, the anti-human uPAR antibodies HC-ATN-658-16 and HC-ATN-658-24 showed significantly enhanced killing activity against the endogenous human uPAR-expressing cell line MDA-MB-231 ADCC.

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. (1) The amino acid sequence of HCDR1 is shown in SEQ ID NO:

2. 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 (2) The amino acid sequence of HCDR1 is shown in SEQ ID NO:

2. 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:

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.

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: 9, or has at least 90% sequence identity with the sequence shown in SEQ ID NO: 9; 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: 11, or has at least 90% sequence identity with the sequence shown in SEQ ID NO: 11; 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.

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) Reagents for detecting uPAR; (b) Medications for the prevention and / or treatment of diseases associated with abnormal uPAR expression; in, The diseases associated with abnormal uPAR expression are cancers, specifically melanoma, pancreatic cancer, triple-negative breast cancer, colorectal cancer, or glioblastoma.

10. The application according to claim 9, wherein, The pancreatic cancer mentioned is pancreatic ductal adenocarcinoma.

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