Upar antibody and use thereof

Abstract

Provided are a uPAR antibody and a use in the treatment of cancer and immune-related diseases. The selected uPAR antibody has strong affinity, good stability, and strong blocking capability, can significantly mediate the killing capability of immune cells against tumor cells, and can be used for the diagnosis, treatment, and prognosis of tumors expressing uPAR and immune-related diseases.

Description

uPAR antibodies and uses thereof TECHNICAL FIELD

[0001] The present application relates to the field of antibody technology, in particular to uPAR antibodies or antigen-binding fragments, and their use in the treatment of cancer and aging-related diseases. BACKGROUND

[0002] With the development of the field of biomedical research, antibody drugs have become an important means in the immunotherapy of cancer and other diseases. Monoclonal antibody preparations have made positive progress in the treatment of tumors, transplant rejection, and autoimmune diseases. Antibody drugs targeting PD-1 / PDL-1, HER2, CD20, and other targets are widely used in the treatment of cancer, infectious diseases, and autoimmune diseases, and are favored by the pharmaceutical market due to their high specificity and low adverse reaction rate. However, while antibody drugs are rapidly developing, the number of covered targets is quite limited. Although PD-1 / PDL-1 antibody targets have achieved excellent results in first-line treatment, the clinical treatment results of many patients with solid tumors show that the drug response rate is low, only about 25%. HER2 antibody drugs have made positive progress in the treatment of HER2-positive breast cancer and gastric cancer patients, but are limited by the low HER2 positivity rate, and other antibody drugs targeting other targets are needed to fill the gap. Finding new antibody drug targets and expanding new clinical indications for antibody drugs are important needs for current development.

[0003] uPAR is mainly expressed on the surface of monocytes and macrophages, and is lowly expressed or not expressed on the surface of most normal cells, but shows a significant increase during tissue remodeling, wound healing, stress, and inflammatory immune response. At the same time, high expression of uPAR has been detected in various tumor tissues such as gastric cancer, breast cancer, and ovarian cancer, and abnormal expression of uPAR is a prerequisite for tumor invasion and metastasis. In the tumor microenvironment, uPAR plays an immunosuppressive role. uPAR plays an important role in tumor proliferation, apoptosis, angiogenesis, invasion, and metastasis by interacting with protein ligands such as uPA, integrin, and vitronectin, thereby participating in the occurrence and development of tumors. Studies have shown that knocking out uPAR using the CRISPR / Cas9 system successfully inhibited human cancer cell proliferation. Another study showed that uPAR expression is related to the expression of matrix metalloproteinases and phosphorylated ERK proteins in oral squamous cell carcinoma, and silencing uPAR can reduce the expression of related proteins and reduce cell proliferation. The uPAR-uPA complex promotes plasminogen activation, affects extracellular matrix remodeling, regulates the release of related growth factors, and plays a promoting role in cancer cell migration.

[0004] Immunity is a key factor involved in many diseases such as cancer, atherosclerosis, aging, and the like. Abnormal immunity causes inflammation imbalance, which is one of the important reasons for aging. In a research article related to aging, by comparing the aging cells and the normal cells adjacent to the aging cells, it is found that the uPAR protein is up-regulated on the aging cells, and it is determined that uPAR is a protein widely induced on the surface of the aging cells. A CAR-T cell therapy targeting uPAR is proposed, and the expression of the aging-related secretions of the mice is reversed, which has a positive effect on improving the survival period of the mice. uPAR becomes a specific target for targeting the aging cells, and the anti-aging therapy for selectively eliminating the aging cells becomes possible.

[0005] In addition, the expression level of uPAR is related to poor prognosis of patients. In a breast cancer research, the expression levels of uPAR, uPA, MMP and other proteins in the blood of patients are detected to reflect the activation level of the plasminogen system, and the correlation analysis between the expression levels and the disease progression of the patients is performed. It is found that the patients with higher uPAR detection are more likely to have cancer recurrence. In a research on melanoma, it is found that the expression level of uPAR in the vesicles secreted by immune cells is significantly related to the survival period of the patients. The stronger the expression of uPAR is, the shorter the survival period of the patients is, which proves the potential of uPAR as a prognostic indicator.

[0006] Therefore, uPAR is expected to become an ideal molecular target for tumor targeted therapy. Screening of high-activity antibodies targeting uPAR has important significance for developing tumor immunotherapy, expanding new clinical indications of antibody drugs, and optimizing early clinical diagnosis and prognosis evaluation schemes for patients. In view of the above, the present application is proposed.

[0007] SUMMARY

[0008] In view of the above technical problems, the present application proves that uPAR can inhibit the function of T cells, and a series of antibodies or antigen-binding fragments thereof capable of high-affinity binding to uPAR and neutralizing / blocking the activity of uPAR are developed through intensive research and development. Experimental results prove that the uPAR antibodies have strong affinity, good stability, strong blocking ability to the binding of uPA or VTN to uPAR, and can significantly inhibit the self-renewal ability of tumor cells, and can be used for diagnosis, prevention, treatment and prognosis of diseases related to uPAR expression (such as cancer and aging-related diseases), thereby achieving the following purposes of the present application.

[0009] Therefore, the present application includes the following aspects: the first aspect of the present application is to provide an antibody or antigen binding fragment thereof against uPAR; the second aspect of the present application is to provide a polynucleotide encoding the antibody or antigen binding fragment thereof, a corresponding recombinant vector, a host cell, or a kit or a pharmaceutical composition comprising the same; the third aspect of the present application is to provide an application of the antibody or antigen binding fragment thereof in preventing or treating tumors and aging-related diseases; the fourth aspect of the present application is to provide an application of the antibody or antigen binding fragment thereof in detecting or diagnosing tumors and aging-related diseases; the fifth aspect of the present application is to provide an application of the antibody or antigen binding fragment thereof in prognostic evaluation of tumors and aging-related diseases; the sixth aspect of the present application is to provide a method for preparing the uPAR antibody or antigen binding fragment thereof; the seventh aspect of the present application is to provide a method for diagnosing, preventing, treating or prognostic evaluation of tumors and aging-related diseases; and the eighth aspect of the present application is to provide an application of uPAR in T cell inhibition or an application of uPAR inhibitor in T cell activation.

[0010] To achieve the above-mentioned purposes, the present application adopts the following technical solutions.

[0011] The first aspect of the present application provides an antibody or antigen binding fragment thereof comprising a heavy chain variable region and a light chain variable region, comprising 3 CDRs in the heavy chain variable region amino acid sequence of any one of SEQ ID NO. 4, 12, 20, 28, 36, 44, 52, 60, 68, 76, 84, 92, 100, 101, 110 or 111 and 3 CDRs in the light chain variable region amino acid sequence of any one of SEQ ID NO. 8, 16, 24, 32, 40, 48, 56, 64, 72, 80, 88, 96, 105, 106, 115 or 116; or a variant having single or multiple CDRs with no more than 2 amino acid changes per CDR region.

[0012] Preferably, the CDRs are based on the residue ranges of different encoding rules as follows:

[0013] Further, when the antibody HCDRs are encoded according to the IMGT encoding rule, the antibody or antigen binding fragment comprises the following CDR sequences:

[0014] i. the HCDR1 has an amino acid sequence as set forth in SEQ ID NO. 1, 9, 17, 25, 33, 41, 49, 57, 65, 73, 81, 89, 97, or 107; ii. the HCDR2 has an amino acid sequence as set forth in SEQ ID NO. 2, 10, 18, 26, 34, 42, 50, 58, 66, 74, 82, 90, 98, or 108; iii. the HCDR3 has an amino acid sequence as set forth in SEQ ID NO. 3, 11, 19, 27, 35, 43, 51, 59, 67, 75, 83, 91, 99, or 109; iv. the LCDR1 has an amino acid sequence as set forth in SEQ ID NO. 5, 13, 21, 29, 37, 45, 53, 61, 69, 77, 85, 93, 102, or 112; v. the LCDR2 has an amino acid sequence as set forth in SEQ ID NO. 6, 14, 22, 30, 38, 46, 54, 62, 70, 78, 86, 94, 103, or 113; vi. the LCDR3 has an amino acid sequence as set forth in SEQ ID NO. 7, 15, 23, 31, 39, 47, 55, 63, 71, 79, 87, 95, 104, or 114;

[0015] or a variant of any of the above i-vi having a single or multiple amino acid changes in the six CDR regions, no more than 2 amino acid changes per CDR region.

[0016] Further, the HCDR1 / HCDR2 / HCDR3 combination can comprise one or more groups selected from the following:

[0017] 1) HCDR1 / HCDR2 / HCDR3:

[0018] SEQ ID NO. 1 / SEQ ID NO. 2 / SEQ ID NO. 3, SEQ ID NO. 9 / SEQ ID NO. 10 / SEQ ID NO. 11, SEQ ID NO. 17 / SEQ ID NO. 18 / SEQ ID NO. 19, SEQ ID NO. 25 / SEQ ID NO. 26 / SEQ ID NO. 27, SEQ ID NO. 33 / SEQ ID NO. 34 / SEQ ID NO. 35, SEQ ID NO. 41 / SEQ ID NO. 42 / SEQ ID NO. 43, SEQ ID NO. 49 / SEQ ID NO. 50 / SEQ ID NO. 51, SEQ ID NO. 57 / SEQ ID NO. 58 / SEQ ID NO. 59, SEQ ID NO. 65 / SEQ ID NO. 66 / SEQ ID NO. 67, SEQ ID NO. 73 / SEQ ID NO. 74 / SEQ ID NO. 75, SEQ ID NO. 81 / SEQ ID NO. 82 / SEQ ID NO. 83, SEQ ID NO. 89 / SEQ ID NO. 90 / SEQ ID NO. 91, SEQ ID NO. 97 / SEQ ID NO. 98 / SEQ ID NO. 99, or SEQ ID NO. 107 / SEQ ID NO. 108 / SEQ ID NO. 109;

[0019] 2) or the HCDR of (1) above comprising one or more amino acid substitutions, deletions, or insertions of no more than 2 amino acids;

[0020] the combination of LCDR1 / LCDR2 / LCDR3 comprises one or more groups selected from:

[0021] 1) LCDR1 / LCDR2 / LCDR3 is:

[0022] SEQ ID NO. 5 / SEQ ID NO. 6 / SEQ ID NO. 7, SEQ ID NO. 13 / SEQ ID NO. 14 / SEQ ID NO. 15, SEQ ID NO. 21 / SEQ ID NO. 22 / SEQ ID NO. 23, SEQ ID NO. 29 / SEQ ID NO. 30 / SEQ ID NO. 31, SEQ ID NO. 37 / SEQ ID NO. 38 / SEQ ID NO. 39, SEQ ID NO. 45 / SEQ ID NO. 46 / SEQ ID NO. 47, SEQ ID NO. 53 / SEQ ID NO. 54 / SEQ ID NO. 55, SEQ ID NO. 61 / SEQ ID NO. 62 / SEQ ID NO. 63, SEQ ID NO. 69 / SEQ ID NO. 70 / SEQ ID NO. 71, SEQ ID NO. 77 / SEQ ID NO. 78 / SEQ ID NO. 79, SEQ ID NO. 85 / SEQ ID NO. 86 / SEQ ID NO. 87, SEQ ID NO. 93 / SEQ ID NO. 94 / SEQ ID NO. 95, SEQ ID NO. 102 / SEQ ID NO. 103 / SEQ ID NO. 104, or SEQ ID NO. 112 / SEQ ID NO. 113 / SEQ ID NO. 114;

[0023] 2) HCDR of (1) above containing one or more amino acid substitutions, deletions or insertions of no more than 2 amino acids;

[0024] In some embodiments, the antibody or antigen-binding fragment thereof is specifically the table sequences in the examples.

[0025] In some embodiments, the uPAR antibody or its antigen-binding fragment of this application further includes a heavy chain variable region VH sequence and a light chain variable region VL sequence, wherein the heavy chain variable region VH sequence has at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with an amino acid sequence selected from the group consisting of: SEQ ID NO.4, 12, 20, 28, 36, 44, 52, 60, 68, 76, 84, 92, 100, 101, 110, or 111; the light chain variable region (VL) sequence has at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with an amino acid sequence selected from the group consisting of: SEQ ID NO.8, 16, 24, 32, 40, 48, 56, 64, 72, 80, 88, 96, 105, 106, 115, or 116.

[0026] Furthermore, the antibody is further connected to a coupling portion, which is selected from one or more of radionuclides, drugs, toxins, cytokines, enzymes, fluorescein, carrier proteins, lipids, and biotin; wherein, the antibody and the coupling portion may be selectively linked by a linker, preferably the linker being a peptide or polypeptide.

[0027] Furthermore, the antibody or antigen-binding fragment may be selected from monoclonal antibodies, polyclonal antibodies, antiserum, chimeric antibodies, humanized antibodies, and human antibodies;

[0028] More preferably, the antibody is selected from multispecific antibodies, single-chain Fv (scFv), single-chain antibodies, anti-idiotype (anti-Id) antibodies, biantibodies, microantibodies, nanobodies, single-domain antibodies, Fab fragments, F(ab') fragments, disulfide-linked bispecific Fv (sdFv), and intracellular antibodies.

[0029] In a second aspect, this application also provides a nucleic acid encoding an antibody fragment or an antigen-binding fragment of the first aspect thereof, said nucleic acid encoding any of the antibodies or antigen-binding fragments described above.

[0030] The third aspect of this application provides a vector containing the nucleic acid of the second aspect, and an optional regulatory sequence;

[0031] Furthermore, the recombinant vector can be a cloning vector or an expression vector, without limitation;

[0032] Furthermore, the regulatory sequence may be selected from leader sequences, polyadenylated sequences, propeptide sequences, promoters, signal sequences, transcription terminators, or any combination thereof, without limitation.

[0033] The fourth aspect of this application provides a host cell containing the nucleic acid of the second aspect or the vector of the third aspect.

[0034] Furthermore, the host cells include, but are not limited to, mammalian cells such as yeast cells, Chinese hamster ovary cells, and human embryonic kidney cells, or other cells suitable for preparing antibodies or their antigen-binding fragments.

[0035] The fifth aspect of this application provides a pharmaceutical composition comprising one or more of the antibodies or antigen-binding fragments, polynucleotides, recombinant vectors, or host cells described above.

[0036] Preferably, the composition further comprises a pharmaceutically acceptable carrier or excipient.

[0037] A sixth aspect of this application provides a kit characterized in that it comprises one or more of the antibodies or antigen-binding fragments, polynucleotides, recombinant vectors, and host cells described above, and is contained in a suitable container.

[0038] The seventh aspect of this application provides a method for preparing a uPAR-binding antibody or an antigen-binding fragment thereof of the first aspect, the method comprising the steps of expressing a vector of the third aspect in a host cell culture to generate the antibody; and recovering antibody molecules from the cell culture.

[0039] The eighth aspect of this application provides the use of an antibody or antigen-binding fragment thereof comprising the first aspect in any of the following aspects:

[0040] a) Application in the prevention and treatment of uPAR expression-related diseases (cancer and immune-related diseases);

[0041] b) Application in the preparation of drugs for the prevention and treatment of uPAR expression-related diseases (cancer and immune-related diseases);

[0042] c) Application in the diagnosis of uPAR expression-related diseases (cancer and immune-related diseases);

[0043] d) Application in the preparation of diagnostic kits for uPAR expression-related diseases (cancer and immune-related diseases);

[0044] e) Application in prognostic assessment of uPAR expression-related diseases (cancer and immune-related diseases);

[0045] f) Application in the preparation of prognostic assessment kits for uPAR expression-related diseases (cancer and immune-related diseases);

[0046] g) Application in T cell activation;

[0047] h) Application in the preparation of T cell activation products.

[0048] Furthermore, the uPAR expression-related diseases include cancer and age-related diseases.

[0049] The ninth aspect of this application provides a pharmaceutical composition comprising the uPAR antibody or antigen-binding fragment thereof described in the first aspect, and a pharmaceutically optional pharmaceutical carrier.

[0050] The tenth aspect of this application provides a method for treating uPAR expression-related diseases, particularly methods for treating cancer and aging-related diseases, comprising a method of administering an effective amount of an antibody of the first aspect or an antigen-binding fragment thereof, or a pharmaceutical composition of the ninth aspect, to an individual in need of treatment.

[0051] The eleventh aspect of this application provides a CAR comprising the antibody or antigen-binding fragment thereof of the first aspect described above, particularly a humanized scFv fragment; in some preferred aspects, the CAR is derived from the VH2+VL1 combination of AB4 or the VH1+VL2 combination of AB20, that is, the antibody or antigen-binding fragment thereof comprises HCDR1 / HCDR2 / HCDR3 as shown in SEQ IN NO. 97-99 and LCDR1 / LCDR2 / LCDR3 as shown in SEQ IN NO. 102-104, or HCDR1 / HCDR2 / HCDR3 as shown in SEQ IN NO. 107-109 and LCDR1 / LCDR2 / LCDR3 as shown in SEQ IN NO. 112-114; in some more preferred aspects, the amino acid sequence of the heavy chain variable region is SEQ ID NO. 101 and the amino acid sequence of the light chain variable region is SEQ ID NO. 105; or the amino acid sequence of the heavy chain variable region comprises SEQ ID NO. 110 and the amino acid sequence of the light chain variable region comprises SEQ ID NO. 116.

[0052] The twelfth aspect of this application provides a CAR-T cell comprising the aforementioned CAR.

[0053] The thirteenth aspect of this application is to provide the use of uPAR in T cell suppression, or the use of uPAR inhibitors in T cell activation.

[0054] Advantages of this application: The uPAR antibody of this application has a strong affinity for uPAR, which facilitates rapid recognition and stable binding of the uPAR antibody to uPAR-positive tumor cells; the good stability of the binding to uPAR helps to prolong the action time of the antibody drug and improve its efficacy; moreover, the antibody has a strong blocking ability, which can effectively block uPAR binding protein ligands (including uPA and VTN) and inhibit uPAR biological function. In addition, the uPAR antibody of this application can specifically inhibit uPAR-positive tumor cells (such as THP-1), improve the tumor immune microenvironment, and the corresponding antibody drug has the advantage of high specificity compared with other drugs, reducing the probability of off-target effects and improving drug safety. Attached Figure Description

[0055] To more clearly illustrate the technical solutions in the specific embodiments of this application or the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of this application. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0056] Figure 1. Results of co-culture of ascites myeloid cells from gastric cancer patients and T cells from healthy individuals;

[0057] Figure 2. Results of co-culturing THP-1 cells (normal cells and uPAR knockout cells) with healthy human T cells;

[0058] Figure 3. Results of mouse serum immunotiter detection;

[0059] Figure 4. Screening results of antibody affinity in supernatant detected by ELISA;

[0060] Figure 5. Screening results of antibody affinity in supernatant detected by FACS;

[0061] Figure 6. Screening results of supernatant antibody blocking efficiency detection;

[0062] Figure 7. Preparation and identification of anti-uPAR monoclonal antibodies: FACS detection results of antibody affinity;

[0063] Figure 8. SPR affinity test results of AB4 anti-uPAR monoclonal antibody;

[0064] Figure 9. SPR affinity assay results of AB17-AB22 anti-uPAR monoclonal antibody;

[0065] Figure 10. SPR affinity detection results of anti-uPAR monoclonal antibodies such as AB23-AB31;

[0066] Figure 11. Preparation and identification of anti-uPAR monoclonal antibodies: Results of antibody blocking uPA-uPAR binding;

[0067] Figure 12. Preparation and identification of anti-uPAR monoclonal antibody: Results of supernatant chimeric antibody blocking VTN-uPAR binding;

[0068] Figure 13. Detection results of T cell killing regulated by anti-uPAR monoclonal antibody;

[0069] Figure 14. Identification results of the binding region of chimeric antibodies in the supernatant;

[0070] Figure 15. Results of ADCC mediated by anti-uPAR monoclonal antibody;

[0071] Figure 16. Detection results of species reaction of AB4 and AB20 monoclonal antibodies;

[0072] Figure 17. Schematic diagram of CAR structural strategies for constructing different combinations of humanized VH and VL sequences of AB4 and AB20;

[0073] Figure 18. Detection of the binding ability of CARs with different combinations of humanized VH and VL sequences of AB4 and AB20 to humanized recombinant uPAR protein;

[0074] Figure 19. Comparison of the recognition and binding ability of humanized VH2+VL1 combination of AB4 and humanized VH1+VL2 combination of AB20 on recombinant human uPAR protein.

[0075] Invention Details

[0076] This application discloses a method for separating an antibody or its antigen-binding fragment. Those skilled in the art can refer to this document to implement its application. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included within this application. The preparation method and application of this application have been described through preferred embodiments. Those skilled in the art can obviously modify or appropriately change and combine the preparation method and application described herein without departing from the content, spirit, and scope of this application to implement and apply the technology of this application. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains.

[0077] The following terms or definitions are provided merely to aid in understanding this application. These definitions should not be construed as having a scope less than that understood by those skilled in the art.

[0078] Unless otherwise defined below, all technical and scientific terms used in the specific embodiments of this application are intended to have the same meaning as commonly understood by those skilled in the art. While it is believed that the following terms will be well understood by those skilled in the art, the following definitions are set forth to better explain this application.

[0079] As used in this application, the terms “comprising,” “including,” “having,” “containing,” or “involving” are inclusive or open-ended and do not exclude other unlisted elements or method steps. The term “consisting of” is considered a preferred embodiment of the term “comprising.” If a group is defined below as comprising at least a certain number of embodiments, this should also be understood to disclose a group that preferably consists only of those embodiments.

[0080] When referring to a singular noun, the indefinite or definite article used, such as "a" or "a kind of," "the," includes the plural form of the noun.

[0081] The terms "approximately" and "generally" indicate an accuracy range that, as would be understood by those skilled in the art, still guarantees the technical effect of the features in question. This term typically indicates a deviation from the indicated value of ±10%, preferably ±5%.

[0082] Terms such as "or more," "at least," and "more than," for example, "at least one," should be understood to include, but are not limited to, at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100 or 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000 or more of the stated values. This also includes any larger numbers or fractions in between. Conversely, the term "not exceeding" includes every value less than the stated values. For example, "no more than 100 nucleotides" includes 100, 99, 98, 97, 96, 95, 94, 93, 92, 91, 90, 89, 88, 87, 86, 85, 84, 83, 82, 81, 80, 79, 78, 77, 76, 75, 74, 73, 72, 71, 70, 69, 68, 67, 66, 65, 64, 63, 62, 61, 60, 59, 58, 57, 56, 55, 54, 53, 52, 51, 50, 49, 48, 47, 46, 45, 44, 43, 42, 41, 40, 39, 38, 37, 36, 35, 34, 33, 32, 31, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, and 0 nucleotides. Also includes any smaller numbers or fractions in between.

[0083] The terms "multiple," "at least two," "two or more," and "at least the second" should be understood to include, but are not limited to, at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, and 70. 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100 or 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000 or more. Also includes any larger numbers or fractions in between.

[0084] Terminology definition:

[0085] As used herein, the term "antibody" refers to a polypeptide of the immunoglobulin family that can bind nonvalently, reversibly, and specifically to a corresponding antigen. For example, a naturally occurring IgG antibody is a tetramer comprising at least two heavy (H) chains and two light (L) chains linked by disulfide bonds. Each heavy chain contains a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region. The heavy chain constant region contains three domains: CH1, CH2, and CH3. Each light chain contains a light chain variable region (abbreviated herein as VL) and a light chain constant region. The light chain constant region contains one domain: CL. The VH and VL regions can be further subdivided into hypervariable regions called complementarity-determining regions (CDRs), which are interspersed with more conserved regions called framework regions (FRs). Each VH and VL consists of three CDRs and four FRs arranged in the following order from the amino terminus to the carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. The variable regions of the heavy and light chains contain binding domains that interact with antigens. The constant regions of an antibody can mediate the binding of immunoglobulins to host tissues or factors, including different cells of the immune system (e.g., effector cells) and the first component (Clq) of the classical complement system. "Antibody" includes, but is not limited to, monoclonal antibodies, human antibodies, humanized antibodies, camelid antibodies, chimeric antibodies, and anti-idiotype (anti-Id) antibodies (including, for example, anti-Id antibodies against the antibodies of this disclosure). Antibodies can belong to any isotype / class (e.g., IgG, IgE, IgM, IgD, IgA, and IgY) or subclass (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2).

[0086] Antibodies contain globular regions of heavy or light chain polypeptides called "domains." Domains may contain peptide rings, typically three to four rings, which are stabilized, for example, by β-sheets and / or intrachain disulfide bonds. Domains are generally referred to as "constant" or "variable" based on the relative lack of sequence variation within the domain of different class members in the case of "constant" domains, or significant variation within the domain of different class members in the case of "variable" domains. Antibody or polypeptide "domains" are generally referred to interchangeably in the art as antibody or polypeptide "regions."

[0087] Antibodies can be classified into five classes based on the amino acid sequence of the constant region of the antibody heavy chain: IgA, IgD, IgE, IgG, and IgM. Several isotypes within these classes can be further subdivided into subclasses, such as IgG1, IgG2, IgG3, IgA1, and IgA2. The constant structural domains of the heavy chain corresponding to different types of immunoglobulins are designated α, δ, ε, γ, and μ, respectively. Antibodies can be classified into κ and λ regions based on the different constant regions (CL) of the antibody light chain. Within the full-length light and heavy chains, the variable and constant regions are typically linked by "J" regions of approximately 12 or more amino acids, and the heavy chain also includes "D" regions of approximately 10 or more amino acids.

[0088] The term "monoclonal antibody" refers to a preparation of an antibody molecule having a single amino acid composition, without specifying the method of its preparation. Monoclonal antibodies or their immunologically active fragments can be produced using hybridoma technology, recombinant technology, phage display technology, synthetic technology, or other production technologies known in the art. The methods for preparing monoclonal antibodies described in this application include in vitro culture of hybridoma cells or preparation via DNA recombination technology. Monoclonal antibodies are highly specific, targeting a single antigenic site. Each monoclonal antibody targets a single determinant on the antigen.

[0089] The term “antigen” is an entity (e.g., a protein entity or peptide) that is specifically bound to an immunoglobulin or antibody (or its antigen-binding fragment).

[0090] The term "fragment" refers to a portion or part of an antibody or antibody chain that contains fewer amino acid residues than a complete or whole antibody or antibody chain, wherein the portion preferably retains at least one, preferably most or all, of the functions normally associated with that portion when it is present in the complete antibody. Fragments can be obtained by chemically or enzymatically treating a complete or whole antibody or antibody chain. Fragments can also be obtained through recombinant processes.

[0091] The term "variable" indicates that certain portions of the variable region in an antibody differ sequentially, contributing to the binding and specificity of various specific antibodies to their respective antigens. However, variability is not uniformly distributed throughout the entire variable region of an antibody. It is concentrated in three segments within the variable regions of the light and heavy chains, known as complementarity-determining regions (CDRs) or hypervariable regions. The more conserved portions of the variable region are called framework regions (FRs). The variable regions of the native heavy and light chains each contain four FRs, which are generally β-sheet configurations linked by three CDRs forming a linking loop, and in some cases, partially β-sheet structures. The CDRs in each chain are tightly packed together via the FR regions and, together with the CDRs of the other chain, form the antigen-binding site of the antibody (see Kabat et al., NIH Publ. No. 91-3242, Vol. I, pp. 647-669 (1991)). Constant regions do not directly participate in antibody-antigen binding, but they exhibit different effector functions, such as participating in antibody-dependent cytotoxicity.

[0092] The terms "complementarity-determining domain" or "complementarity-determining region" ("CDR") are interchangeable terms for the hypervariable regions of the VL and VH. A CDR is the target protein binding site of an antibody chain carrying the specific antibody for that target protein. Each VL or VH contains three CDRs (CDR1-3, numbered sequentially from the N-terminus), totaling approximately 15-20% of the variable domain. CDRs can be designated by their region and sequence. For example, "VHCDR1" or "HCDR1" both refer to the first CDR of the heavy chain variable region. CDRs are structurally complementary to the epitope of the target protein and are therefore directly responsible for binding specificity. The remaining extensions of the VL or VH (the so-called framework regions) exhibit less variation in their amino acid sequence (Kuby, *Immunology*, 4th ed., Chapter 4, WH Freeman & Co., New York, 2000).

[0093] In a given light chain variable region or heavy chain variable region amino acid sequence, the precise amino acid sequence boundaries of each CDR can be determined using any or a combination of many known antibody CDR assignment systems, including, for example: Chothia (Chothia et al. (1989) Nature 342:877-883, Al-Lazikani et al., “Standard conformations for the canonical structures of immunoglobulins”, Journal of Molecular Biology, 273, 927-948 (1997)) based on antibody three-dimensional structure and CDR loop topology; Kabat (Kabat et al., Sequences of Proteins of Immunological Interest, 4th edition, USDepartment of Health and Human Services, National Institutes of Health (1987)) based on antibody sequence variability; AbM (University of Bath); Contact (University College London); and the International ImMunoGeneTics. The database (IMGT) and the North CDR definition based on affinity propagation clustering using a large number of crystal structures.

[0094] However, it should be noted that the boundaries of the CDRs for the variable region of the same antibody may differ based on different assignment systems. That is, the CDR sequences for the variable region of the same antibody may differ under different assignment systems. For example, the residue ranges of CDR regions using Kabat and IMGT numbers under different assignment systems are shown in the table below.

[0095] CDR residue ranges defined under different assignment systems:

[0096] Therefore, when referring to antibodies defined by a specific CDR sequence as defined in this application, the scope of said antibody also includes antibodies whose variable region sequence contains the specific CDR sequence, but whose claimed CDR boundaries differ from those defined in this application due to the application of different schemes (e.g., different assignment system rules or combinations).

[0097] The CDR of the antibody in this application can be determined artificially by any method or combination thereof in the art. Unless otherwise stated, in this application, the term "CDR" or "CDR sequence" covers a CDR sequence determined in any of the foregoing methods.

[0098] Antibodies may include, for example, monoclonal antibodies, recombinant antibodies, monospecific antibodies, multispecific antibodies (including bispecific antibodies), human antibodies, engineered antibodies, humanized antibodies, chimeric antibodies, immunoglobulins, synthetic antibodies, tetramer antibodies comprising two heavy chain and two light chain molecules, antibody light chain monomers, antibody heavy chain monomers, antibody light chain dimers, antibody heavy chain dimers, antibody light chain-antibody heavy chain pairs, intracellular antibodies, antibody fusions (sometimes referred to herein as “antibody conjugates”), heteroconjugated antibodies, single-domain antibodies, monovalent antibodies, single-chain antibodies or single-chain Fv (scFv), camel-derived antibodies, affinity molecules, Fab fragments, F(ab')2 fragments, disulfide-linked Fv (sdFv), anti-idiotypic (anti-Id) antibodies (including, for example, anti-anti-Id antibodies), microantibodies, domain antibodies, synthetic antibodies (sometimes referred to herein as “antibody mimics”), and any of the above antigen-binding fragments.

[0099] The term "antigen-binding fragment" refers to one or more portions of an antibody that retain the ability to specifically interact with an epitope of an antigen (e.g., through binding, steric hindrance, stabilization / destabilization, spatial distribution). Examples of binding fragments include, but are not limited to: single-chain Fv (scFv), disulfide-linked Fv (sdFv), Fab fragments, F(ab') fragments (i.e., monovalent fragments consisting of VL, VH, CL, and CH1 domains); F(ab)2 fragments (i.e., bivalent fragments comprising two Fab fragments linked by a disulfide bridge at the hinge region); Fd fragments consisting of VH and CH1 domains; Fv fragments consisting of VL and VH domains of a single arm of the antibody; dAb fragments consisting of VH domains (Ward et al., Nature 341:544-546, 1989); and isolated complementarity-determining regions (CDRs) or other epitope-binding fragments of the antibody. Furthermore, although the two domains VL and VH of the Fv fragment are encoded by separate genes, these two domains can be linked using recombination methods by enabling them to become synthetic linkers of a single protein chain in which the VL and VH regions are used to form a monovalent molecule (referred to as a single-chain Fv (“scFv”); see, for example, Bird et al., Science 242:423-426, 1988; and Huston et al., Proceedings of the National Academy of Sciences 85:5879-5883, 1988). Such single-chain antibodies are also intended to be covered within the term “antigen-binding fragment”. These antigen-binding fragments are obtained using conventional techniques known to those skilled in the art, and their applicability is screened in the same manner as that of intact antibodies.

[0100] Antigen-binding fragments can also be incorporated into single-domain antibodies, large antibodies, micro antibodies, nanobodies, intracellular antibodies, biantibodies, triantibodies, tetraantibodies, v-NARs, and bi-scFvs (see, for example, Hollinger and Hudson, Nature Biotechnology 23:1126-1136, 2005). Antigen-binding fragments can be grafted onto scaffolds based on peptides such as fibronectin type III (Fn3) (see U.S. Patent No. 6,703,199, which describes a fibronectin peptide monoclonal antibody). Antigen-binding fragments can be incorporated into single-chain molecules comprising a pair of tandem Fv segments (VH-CH1-VH-CH1) forming a pair of antigen-binding regions together with a complementary light chain peptide (Zapata et al., Protein Engineering 8:1057-1062, 1995; and U.S. Patent No. 5,641,870).

[0101] The term "chimeric antibody" refers to an antibody whose variable and constant regions originate from different species. Specifically, the variable region of a chimeric antibody is derived from antibodies of a specific species or belonging to a specific antibody class or subclass, with the corresponding sequence being identical or homologous, while the constant region of the chain is derived from an antibody of another species or belonging to another antibody class or subclass. The chimeric antibody involved in this application is derived from the chimera of a heavy / light chain variable region from a murine antibody and a heavy / light chain constant region from a human antibody.

[0102] The term "humanized antibody" refers to a chimeric antibody containing amino acid residues from a non-human HVR and amino acid residues from a human FR. Specifically, the variant regions of the non-human antibody that bind to a specific antigen are retained, while the remaining sequences are replaced with human antibody sequences. The purpose of humanization is to reduce the risk of an immune response (immunogenicity) from the non-human antibody to the human immune system, while preserving the binding specificity and affinity of the original antibody. In some embodiments, the humanized antibody will contain substantially all of at least one, typically two, variable domains, where all or substantially all of the HVRs (e.g., CDRs) correspond to the sequence of the non-human antibody, and all or substantially all of the FRs correspond to the sequence of the human antibody.

[0103] The term "CAR" refers to chimeric antigen receptor, which is an artificial receptor molecule prepared through genetic engineering technology. It can enable immune effector cells (such as T cells) to have specificity for a specific target antigen epitope, thereby enhancing the function of T cells in recognizing antigen signals and activating.

[0104] The term "affinity" refers to the strength of the interaction between an antibody and an antigen at a single antigenic site. Within each antigenic site, the variable region of the antibody "arm" interacts with the antigen at many sites through weak non-covalent forces; the more interactions, the stronger the affinity.

[0105] In this document, the term "competition" refers to the situation where antigen-binding proteins (e.g., neutralizing antigen-binding proteins or neutralizing antibodies) compete for the same epitope. This means that the competition between antigen-binding proteins is determined by an assay in which the antigen-binding protein to be detected (e.g., an antibody or its immunologically functional fragment) prevents or inhibits (e.g., reduces) the specific binding of a reference antigen-binding protein (e.g., a ligand or reference antibody) to the common antigen.

[0106] As used herein, the term "variant" refers to a heavy chain variable region or light chain variable region that has been modified by at least one, for example, substitution, deletion, or addition of one, two, or three amino acids, such that the modified antigen-binding protein containing the heavy chain or light chain variant substantially retains the biological characteristics of the original antigen-binding protein. In one embodiment, the antigen-binding protein containing the modified heavy chain variable region or light chain variable region sequence retains 70%, 80%, 90%, or 100% of the biological characteristics of the original antigen-binding protein. It should be understood that each heavy chain variable region or light chain variable region can be modified alone or in combination with another heavy chain variable region or light chain variable region. The antigen-binding protein of this disclosure comprises a heavy chain variable region amino acid sequence that is 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% homologous to the heavy chain variable region amino acid sequence described herein. The antigen-binding protein disclosed herein comprises a light chain variable region amino acid sequence that is 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% homologous to the amino acid sequence of the light chain variable region described herein. The percentage of homology may be over the entire heavy chain variable region and / or the entire light chain variable region, or the percentage of homology may be limited to the framework region, with the sequence corresponding to the CDR having 100% identity with the CDR disclosed herein within the heavy chain variable region and / or light chain variable region. As used herein, the term "CDR variant" refers to a CDR that has been modified by at least one, for example, substitution, deletion, or addition of one, two, or three amino acids, such that the modified antigen-binding protein containing the CDR variant substantially retains the biological characteristics of the unmodified antigen-binding protein. In one embodiment, the antigen-binding protein containing the variant CDR retains 60%, 70%, 80%, 90%, or 100% of the biological characteristics of the unmodified antigen-binding protein. It should be understood that each CDR that can be modified can be modified alone or in combination with another CDR. In one implementation, the modification is a substitution, particularly a conservative substitution.

[0107] As used herein, the term "vector" refers to a nucleic acid molecule capable of amplifying another nucleic acid linked to it through transformation. This term includes vectors that function as self-replicating nucleic acid structures as well as vectors that bind to the genome of a host cell that has already been introduced therein. Some vectors are capable of directing the expression of nucleic acids operatively linked to them. Such vectors are referred to herein as "expression vectors."

[0108] The term "host cell" refers to a cell to which foreign nucleic acids have been introduced, including the progeny of such cells. It is capable of expressing the foreign nucleic acids intracellularly or on the cell membrane, or releasing them extracellularly.

[0109] The term “subject” includes both humans and non-human animals. Non-human animals include all vertebrates, such as mammals and non-mammals, including non-human primates, sheep, dogs, cattle, chickens, amphibians, and reptiles. Unless otherwise specified, the terms “patient” or “subject” are used interchangeably herein.

[0110] The term "neutralization" refers to the suppression of viral infection in host cells, as demonstrated by the absence of viral gene expression. Without being limited by any one theory, the mechanism of neutralization by a specific antibody can involve blocking the interaction between viral capsid proteins and cell surface receptors or disrupting any stage of the entry and transport process before the viral genome is delivered to the host cell nucleus.

[0111] As used herein, on the one hand, the term “treatment” for any disease or condition means to alleviate said disease or condition (i.e., to slow or stop or reduce the development of at least one symptom of the disease or its clinical symptoms). On the other hand, “treatment” means to alleviate or improve at least one physical parameter, including those that the patient may not be able to discern. In yet another aspect, “treatment” means to regulate a disease or condition physically (e.g., stabilizing identifiable symptoms), physiologically (e.g., stabilizing physical parameters), or both.

[0112] The term "kit" is used to refer to a combination of reagents and other materials that facilitate sample analysis. In some embodiments, the immunoassay kits described herein include suitable antigens, a binder containing a detectable portion, and a detection reagent. A system for amplifying the signal generated by the detectable portion may or may not be included in the kit. Furthermore, in other embodiments, the kit includes, but is not limited to, components such as devices for sample collection, sample tubes, supports, trays, racks, plates, plates, user instructions for the kit, solutions or other chemical reagents, and sample and / or control samples for standardization and normalization.

[0113] The term "pharmaceutically acceptable" means that the carrier, diluent, excipient and / or salt thereof are chemically and / or physically compatible with other components in the formulation and are physiologically compatible with the recipient.

[0114] The term "pharmaceutically acceptable carriers and / or excipients" refers to carriers and / or excipients that are pharmacologically and / or physiologically compatible with the subject and the active agent, and are well known in the art (see, for example, Remington's Pharmaceutical Sciences. Edited by Gennaro AR, 19th ed. Pennsylvania: Mack Publishing Company, 1995), and includes, but is not limited to, pH adjusters, surfactants, adjuvants, and ionic strength enhancers. For example, pH adjusters include, but are not limited to, phosphate buffers; surfactants include, but are not limited to, cationic, anionic, or nonionic surfactants, such as Tween-80; and ionic strength enhancers include, but are not limited to, sodium chloride.

[0115] The uPAR targeted by the antibody in this application:

[0116] uPAR, a urokinase-type plasminogen activator receptor, is primarily expressed on the surface of monocytes and macrophages. While it is expressed at low levels or absently on the surface of most normal cells, it shows a significant increasing trend during tissue remodeling, wound healing, stress, and inflammatory immune responses. Furthermore, high expression of uPAR has been detected in various tumor tissues, including gastric cancer, breast cancer, and ovarian cancer, and abnormal uPAR expression is a prerequisite for tumor invasion and metastasis. In the tumor microenvironment, uPAR plays an immunosuppressive role. uPAR interacts with protein ligands such as uPA, integrins, and glassin, playing a crucial role in tumor proliferation, apoptosis, angiogenesis, invasion, and metastasis, thus participating in tumorigenesis and development. Studies have shown that knocking out uPAR using the CRISPR / Cas9 system successfully inhibits the proliferation of human cancer cells. Other studies have shown that uPAR expression is correlated with the expression of matrix metalloproteinases and phosphorylated ERK proteins in oral squamous cell carcinoma; silencing uPAR can attenuate the expression of related proteins and reduce cell proliferation. The uPAR-uPA complex promotes plasminogen activation, influences extracellular matrix remodeling, and regulates the release of related growth factors, thus playing a promoting role in cancer cell migration. Studies have also shown that uPAR protein is upregulated in senescent cells, confirming it as a widely induced protein on the surface of senescent cells. This has led to the development of CAR-T cell therapy targeting uPAR, which has positively impacted the reversal of aging-related secretion expression in mice and improved mouse survival. Furthermore, uPAR expression levels are associated with poor patient prognosis. In a breast cancer study, authors measured the expression of uPAR, uPA, and MMP proteins in patients' blood to reflect the activation level of the plasminogen system and analyzed its correlation with disease progression. They found that patients with higher uPAR levels were more prone to cancer recurrence. A study on melanoma found that uPAR expression levels in vesicles secreted by immune cells were significantly correlated with patient survival; stronger uPAR expression was associated with shorter survival, demonstrating uPAR's potential as a prognostic indicator. Therefore, uPAR is expected to become an ideal molecular target for tumor targeted therapy. Screening for antibodies that can target uPAR is of great significance for developing new targets for tumor immunotherapy, expanding new clinical indications for antibody drugs, and optimizing early clinical diagnosis and prognostic assessment for patients.

[0117] Therefore, the term "uPAR expression-related disease" as used herein is defined as a disease in which uPAR-positive cells promote disease progression (e.g., promote tumor cell invasion and metastasis, inhibit T cell proliferation and secretion of cytotoxic factors), or uPAR-positive cells are identified as senescent cells and can mediate inflammation. In short, regardless of whether uPAR plays a promoting role in disease or serves as a biomarker for identification, this application defines it as a uPAR expression-related disease, including but not limited to various cancers and immune (especially aging)-related diseases.

[0118] 1. The antibody or antigen-binding fragment against uPAR in this application

[0119] The terms “antibody against uPAR,” “anti-uPAR antibody,” “uPAR antibody,” or “antibody that binds to uPAR” are used interchangeably herein and refer to an antibody or antigen-binding fragment of this application that is capable of binding to the uPAR protein with sufficient affinity, thereby enabling the antibody to be used for the diagnosis, prevention, treatment, and prognosis of uPAR.

[0120] The antibody and antigen-binding fragment of this application bind to uPAR with high affinity and specificity. In some embodiments, the antibody of this application is a blocking antibody or a neutralizing antibody. In some embodiments, the blocking antibody or neutralizing antibody can be used to prevent and / or treat individuals with uPAR-related diseases.

[0121] In some implementations, based on the different analytical methods for CDR defined above, the uPAR antibody or antigen-binding fragment of this application is identified as a sequence of the following type:

[0122] It may contain three CDRs from any of the heavy chain variable region amino acid sequences shown in SEQ ID NO.4, 12, 20, 28, 36, 44, 52, 60, 68, 76, 84, 92, 100, 101, 110 or 111;

[0123] And three CDRs in the amino acid sequence of the light chain variable region shown in any of SEQ ID NO. 8, 16, 24, 32, 40, 48, 56, 64, 72, 80, 88, 96, 105, 106, 115 or 116;

[0124] Or variants that have one or more CDRs with no more than two amino acid variations in each CDR region, similar to the light and heavy chain CDR regions mentioned above.

[0125] In some implementations, when antibody HCDRs are encoded according to the IMGT encoding rules, the antibody or antigen-binding fragment contains the following CDR sequence:

[0126] i. The amino acid sequence of HCDR1 is as shown in SEQ ID NO. 1, 9, 17, 25, 33, 41, 49, 57, 65, 73, 81, 89, 97 or 107;

[0127] ii. The amino acid sequence of the HCDR2 is as shown in SEQ ID NO.2, 10, 18, 26, 34, 42, 50, 58, 66, 74, 82, 90, 98 or 108;

[0128] iii. The amino acid sequence of the HCDR3 is shown in SEQ ID NO. 3, 11, 19, 27, 35, 43, 51, 59, 67, 75, 83, 91, 99 or 109;

[0129] iv. The amino acid sequence of said LCDR1 is as shown in SEQ ID NO. 5, 13, 21, 29, 37, 45, 53, 61, 69, 77, 85, 93, 102 or 112;

[0130] v. The amino acid sequence of the LCDR2 is as shown in SEQ ID NO. 6, 14, 22, 30, 38, 46, 54, 62, 70, 78, 86, 94, 103 or 113;

[0131] vi. The amino acid sequence of the LCDR3 is as shown in SEQ ID NO. 7, 15, 23, 31, 39, 47, 55, 63, 71, 79, 87, 95, 104 or 114;

[0132] Alternatively, variants with one or more CDRs having no more than two amino acid variations per CDR region, similar to the six CDR regions of i-vi described above.

[0133] In some implementations, the combination of HCDR1 / HCDR2 / HCDR3 may comprise one or more groups selected from the following:

[0134] The combination of HCDR1 / HCDR2 / HCDR3 includes one or more groups selected from the following:

[0135] 1) HCDR1 / HCDR2 / HCDR3:

[0136] SEQ ID NO.1 / SEQ ID NO.2 / SEQ ID NO.3, SEQ ID NO.9 / SEQ ID NO.10 / SEQ ID NO.11, SEQ ID NO.17 / SEQ ID NO.18 / SEQ ID NO.19, SEQ ID NO.25 / SEQ ID NO.26 / SEQ ID NO.27, SEQ ID NO.33 / SEQ ID NO.34 / SEQ ID NO.35, SEQ ID NO.41 / SEQ ID NO.42 / SEQ ID NO.43, SEQ ID NO.49 / SEQ ID NO.50 / SEQ ID NO.51, SEQ ID NO.57 / SEQ ID NO.58 / SEQ ID NO.59, SEQ ID NO65 / SEQ ID NO.66 / SEQ ID NO.67, SEQ ID NO.73 / SEQ ID NO.74 / SEQ ID NO.75, SEQ ID NO.81 / SEQ ID NO.82 / SEQ ID NO.83, SEQ ID NO.89 / SEQ ID NO.90 / SEQ ID NO.91, SEQ ID NO.97 / SEQ ID NO.98 / SEQ ID NO.99 or SEQ ID NO.107 / SEQ ID NO.108 / SEQ ID NO.109;

[0137] 2) or HCDR of (1) above containing one or more amino acid substitutions, deletions or insertions of no more than two amino acids;

[0138] The combination of LCDR1 / LCDR2 / LCDR3 includes one or more groups selected from the following:

[0139] 1) LCDR1 / LCDR2 / LCDR3 are:

[0140] SEQ ID NO.5 / SEQ ID NO.6 / SEQ ID NO.7, SEQ ID NO.13 / SEQ ID NO.14 / SEQ ID NO.15, SEQ ID NO.21 / SEQ ID NO.22 / SEQ ID NO.23, SEQ ID NO.29 / SEQ ID NO.30 / SEQ ID NO.31, SEQ ID NO.37 / SEQ ID NO.38 / SEQ ID NO.39, SEQ ID NO.45 / SEQ ID NO.46 / SEQ ID NO.47, SEQ ID NO.53 / SEQ ID NO.54 / SEQ ID NO.55, SEQ ID NO.61 / SEQ ID NO.62 / SEQ ID NO.63, SEQ ID NO69 / SEQ ID NO.70 / SEQ ID NO.71, SEQ ID NO.77 / SEQ ID NO.78 / SEQ ID NO.79, SEQ ID NO.85 / SEQ ID NO.86 / SEQ ID NO.87, SEQ ID NO.93 / SEQ ID NO.94 / SEQ ID NO.95, SEQ ID NO.102 / SEQ ID NO.103 / SEQ ID NO.104 or SEQ ID NO.112 / SEQ ID NO.113 / SEQ ID NO.114;

[0141] 2) or LCDRs of (1) above containing one or more amino acid substitutions, deletions or insertions of no more than two amino acids.

[0142] In some specific embodiments, the uPAR-binding antibody or its antigen-binding fragment of this application further includes a heavy chain variable region VH sequence and a light chain variable region VL sequence. The heavy chain variable region VH sequence has at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with an amino acid sequence selected from the group consisting of: SEQ ID NO. 4, 12, 20, 28, 36, 44, 52, 60, 68, 76, 84, 92, 100, 101, 110, or 111; the light chain variable region VL sequence has at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with an amino acid sequence selected from the group consisting of: SEQ ID NO. NO.8, 16, 24, 32, 40, 48, 56, 64, 72, 80, 88, 96, 105, 106, 115 or 116; preferably, the amino acid changes do not occur in the CDR region.

[0143] In some embodiments, the antibody or antigen-binding fragment of this application further includes an Fc region derived from IgG, such as IgG1, IgG2, IgG3, or IgG4.

[0144] In some embodiments, the amino acid changes in the above-mentioned amino acid homology include amino acid substitution, insertion, or deletion. Preferably, the amino acid changes described herein are amino acid substitutions, preferably conservative substitutions. A conservative substitution is the substitution of one amino acid with another amino acid of the same class, such as the substitution of one acidic amino acid with another acidic amino acid, one basic amino acid with another basic amino acid, or one neutral amino acid with another neutral amino acid. Exemplary substitutions are shown in the table below (amino acid substitutions).

[0145] In a preferred embodiment, the amino acid changes described herein occur in regions outside the CDR (e.g., in the FR). More preferably, the amino acid changes described herein occur in the Fc region. In some embodiments, an anti-uPAR antibody comprising one or more mutated Fc domains is provided, which enhances or weakens the antibody's binding to the FcRn receptor, for example, at acidic pH compared to neutral pH.

[0146] In some embodiments, the antibody or antigen-binding fragment may be further linked to a conjugation portion selected from one or more radionuclides, drugs, toxins, cytokines, enzymes, luciferins, carrier proteins, lipids, and biotin.

[0147] In some embodiments, the antibody may be selectively linked to the conjugation portion via a linker, preferably a peptide or polypeptide.

[0148] In some embodiments, the antibody or antigen-binding fragment may be selected from monoclonal antibodies, polyclonal antibodies, antiserum, chimeric antibodies, humanized antibodies, and human antibodies; more preferably, the antibody may be selected from multispecific antibodies, single-chain Fv (scFv), single-chain antibodies, anti-idiotype (anti-Id) antibodies, biantibodies, microantibodies, nanobodies, single-domain antibodies, Fab fragments, F(ab') fragments, disulfide-linked bispecific Fv (sdFv), and intracellular antibodies.

[0149] In some embodiments, the antibodies or antigen-binding fragments described herein can be generated through recombinant expression. The aforementioned nucleic acids encoding variable regions of the light and heavy chains, optionally linked to constant regions, can be inserted into an expression vector. The vector containing the nucleic acid encoding the antibody described herein is itself an aspect of this application. The light and heavy chains can be cloned into the same or different expression vectors. The nucleic acid encoding the antibody chain described herein can be operatively linked to one or more control sequences in the expression vector to ensure expression of the antibody chain. Expression control sequences include, but are not limited to, promoters (e.g., naturally associated or heterologous promoters), signal sequences, enhancer elements, and transcription termination sequences. Preferably, the expression control sequences are eukaryotic promoter systems in vectors capable of transforming or transfecting eukaryotic host cells. Such vectors can be incorporated into suitable hosts, thereby maintaining the host under conditions suitable for high-level expression of the nucleotide sequence and for the collection and purification of antibodies.

[0150] 2. The nucleic acid, the vector containing it, and the host cell of this application.

[0151] This application provides a nucleic acid encoding any of the above uPAR antibodies or their antigen-binding fragments or any strand thereof. In one embodiment, a vector comprising said nucleic acid is provided. In one embodiment, the vector is an expression vector. In one embodiment, a host cell comprising said nucleic acid or said vector is provided. In one embodiment, the host cell is eukaryotic. In another embodiment, the host cell is selected from yeast cells, mammalian cells, or other cells suitable for preparing antibodies or their antigen-binding fragments. In another embodiment, the host cell is prokaryotic.

[0152] The nucleic acid involved in this application is the nucleic acid encoding an antibody against uPAR protein or its antigen-binding fragment or its VH or VL domain; it is understood that any nucleic acid that can encode the above-mentioned antibody or its antigen-binding fragment or its VH or VL domain is within the scope of this application.

[0153] The vector of this application containing one or more nucleic acids encoding the antibodies described herein may be a cloning vector or an expression vector, and there is no limitation herein.

[0154] In one implementation, the vector is an expression vector, such as a eukaryotic expression vector. Vectors include, but are not limited to, viruses, plasmids, granules, λ phages, or yeast artificial chromosomes (YACs).

[0155] In one embodiment, the vector contains an optional regulatory sequence; in some embodiments, the regulatory sequence may be selected from a leader sequence, a polyadenylated sequence, a propeptide sequence, a promoter, a signal sequence, a transcription terminator, or any combination thereof, without limitation.

[0156] The host cell containing the expression vector in this application may be a yeast cell, a mammalian cell, or other cell suitable for preparing antibodies or their antigen-binding fragments. In some embodiments, suitable host cells include prokaryotic microorganisms such as *Escherichia coli*. Host cells may also be eukaryotic microorganisms such as filamentous fungi or yeast, or various eukaryotic cells such as insect cells. Vertebrate cells may also be used as hosts. For example, mammalian cell lines modified for suspension growth may be used. Examples of useful mammalian host cell lines include the SV40-transformed monkey kidney CV1 line (COS-7); human embryonic kidney lines (HEK293 or 293F cells), 293 cells, young hamster kidney cells (BHK), monkey kidney cells (CV1), African green monkey kidney cells (VERO-76), human cervical cancer cells (HELA), canine kidney cells (MDCK), Buffalo rat liver cells (BRL 3A), human lung cells (W138), human liver cells (Hep G2), Chinese hamster ovary cells (CHO cells), CHOS cells, NSO cells, and myeloma cell lines such as Y0, NSO, P3X63, and Sp2 / 0. For a review of mammalian host cell lines suitable for protein production, see, for example, Yazaki and Wu, Methods in Molecular Biology, Vol. 248 (edited by BKCLo, Humana Press, Totowa, NJ), pp. 255-268 (2003). In a preferred embodiment, the host cell is a CHO cell or a 293 cell.

[0157] Vectors containing the polynucleotide sequences of interest (e.g., heavy and light chain coding sequences and expression control sequences) described herein can be transferred into host cells using well-known methods, which vary depending on the type of cell host. For example, calcium chloride transfection is commonly used for prokaryotic cells, while calcium phosphate treatment, electroporation, lipid transfection, bio-projectiles, or virus-based transfection can be used for other cell hosts. (See Green and Sambrook, Molecular Cloning: A Laboratory Manual (Cold Spring Harbor, 4th ed., 2012). Other methods for transforming mammalian cells include the use of polybrene, protoplast fusion, liposomes, electroporation, and microinjection (see Sambrook et al., ibid.). To produce transgenic animals, the transgene can be microinjected into fertilized oocytes or integrated into the genome of embryonic stem cells, and the nuclei of these cells are transferred into enucleated oocytes.

[0158] 3. Preparation, production, and purification of the antibody or antigen-binding fragment of this application.

[0159] The method for preparing an antibody or an antigen-binding fragment thereof according to this application may include expressing the vector described herein in a host cell culture to generate the antibody and recovering the antibody from the cell culture.

[0160] In some embodiments, the method may include transferring a vector containing one or more nucleic acids encoding an antibody against uPAR as described above, or an antigen-binding fragment thereof, or an antibody chain thereof, into a host cell as described herein; culturing the host cell culture under conditions that allow nucleic acid expression; and recovering the expressed antibody against uPAR or the antigen-binding fragment thereof. Any suitable method known in the art may be employed.

[0161] This application provides a method for preparing uPAR antibodies or antigen-binding fragments, wherein the method includes culturing a host cell containing a nucleic acid encoding the antibody or an expression vector containing the nucleic acid under conditions suitable for expressing a nucleic acid encoding the uPAR antibody or antigen-binding fragment, and optionally isolating the antibody or antigen-binding fragment. In one embodiment, the method further includes recovering and purifying the corresponding antibody or antigen-binding fragment from the host cell (or host cell culture medium).

[0162] In some embodiments, the antibodies or antigen-binding fragments prepared herein can be purified using known existing techniques such as high-performance liquid chromatography (HPLC), ion-exchange chromatography, gel electrophoresis, affinity chromatography, size exclusion chromatography, etc. The actual conditions used to purify a specific protein also depend on factors such as net charge, hydrophobicity, and hydrophilicity, which are obvious to those skilled in the art. The purity of the antibodies of this application can be determined by any of a variety of well-known analytical methods, including size exclusion chromatography, gel electrophoresis, high-performance liquid chromatography, etc.

[0163] In some embodiments, the uPAR antibodies provided herein can be identified, screened, or characterized for their physical / chemical properties and / or biological activity using a variety of assays known in the art. On one hand, the antigen-binding activity of the antibodies of this application can be tested, for example, by known methods such as ELISA, Western blotting, etc. Binding to uPAR can be determined using methods known in the art; in some embodiments, SPR, flow cytometry, or biofilm interferometry can be used to determine the binding of the uPAR antibodies of this application to uPAR.

[0164] 4. The pharmaceutical composition, therapeutic application, and treatment method of this application.

[0165] In some embodiments, this application provides compositions comprising any uPAR antibody or antigen-binding fragment described herein, preferably pharmaceutical compositions that may be used for prophylactic or therapeutic purposes.

[0166] In one embodiment, the composition further comprises a pharmaceutical excipient. In one embodiment, the composition (e.g., a pharmaceutical composition) comprises the uPAR antibody or antigen-binding fragment of this application, and a combination of one or more other therapeutic agents (e.g., small molecule drugs).

[0167] In some embodiments, the uPAR antibody or its antigen-binding fragment described in this application can be formulated and / or administered as a pharmaceutical composition comprising an active therapeutic antibody reagent and various other pharmaceutically acceptable components. Preferred forms depend on the intended route of administration and therapeutic application. Depending on the desired formulation, the composition may also contain pharmaceutically acceptable, non-toxic carriers or diluents, which are defined as carriers commonly used to formulate pharmaceutical compositions for administration to animals or humans. Diluents are selected so as not to affect the bioactivity of the composition. Examples of such diluents are distilled water, physiological phosphate-buffered saline, Ringer's solution, glucose solution, and Hank's solution. Furthermore, the pharmaceutical composition or formulation may also include other carriers, adjuvants, or non-toxic, non-therapeutic, non-immunogenic stabilizers, etc.

[0168] In some embodiments, the pharmaceutical compositions of the uPAR antibody or its antigen-binding fragment described in this application may further include large, slowly metabolizing macromolecules, such as proteins, polysaccharides like chitosan, polylactic acid, polyglycolic acid and copolymers (e.g., agarose, cellulose, etc.), polymeric amino acids, amino acid copolymers, and lipid aggregates (e.g., oil droplets or liposomes). Furthermore, these carriers can be used as immunostimulants (i.e., adjuvants).

[0169] In one embodiment, the pharmaceutical composition of this application may further comprise more than one active ingredient required for the specific indication to be treated, preferably those active ingredients having complementary activities that do not adversely affect each other. For example, it is desirable to also provide other anti-infective active ingredients, such as other antibodies, anti-infective agents, small molecule drugs, or immunomodulators. The active ingredients are suitably combined in an amount effective for the intended use.

[0170] The term "pharmaceutical carrier" can include any and all physiologically compatible solvents, dispersion media, isotonic agents, and absorption delay agents. Pharmaceutical carriers suitable for this application can be sterile liquids, such as water and oils, including those of petroleum, animal, plant, or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil, etc. Water is a preferred carrier when the pharmaceutical composition is administered intravenously. Saline solutions, aqueous dextran, and glycerol solutions can also be used as liquid carriers, particularly for injectable solutions. Suitable excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glyceryl monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol, etc. For information on the use and applications of excipients, see also "Handbook of Pharmaceutical Excipients," 5th edition, R.C. Rowe, P.J. Seskey, and S.O. Wen, Pharmaceutical Press, London, Chicago. If desired, the compositions may also contain small amounts of wetting agents or emulsifiers, or pH buffers. These compositions may be in the form of solutions, suspensions, emulsions, tablets, pills, capsules, powders, sustained-release formulations, etc. Oral formulations may contain standard pharmaceutical carriers and / or excipients, such as pharmaceutical-grade mannitol, lactose, starch, magnesium stearate, saccharin.

[0171] In one embodiment, this application also provides a method for preventing or treating uPAR expression-related diseases or ailments (particularly including cancer or immune-related diseases) in a subject, comprising the step of administering the above-described pharmaceutical composition to the subject.

[0172] "Subjects with uPAR expression-related diseases" includes subjects with conditions such as cancer or immune-related diseases. Preventative agents can be administered before the onset of symptoms characteristic of the related disease in order to stop the disease or, alternatively, delay its progression.

[0173] 5. Application of uPAR in suppressor T cells (as per this application)

[0174] This application confirms that uPAR-positive cells can suppress T cell function. Specifically, through co-culturing myeloid cells derived from ascites of gastric cancer patients with T cells from healthy individuals, the results showed that uPAR-positive cells, compared to uPAR-positive cells, could inhibit T cell function. low Myeloid cells and T cells co-culture group, uPAR high In the co-culture group of myeloid cells and T cells, the expression levels of GranzymB, TNF-α, Perforin, IFN-γ, and IL-2 in T cells were decreased, indicating that uPAR... high Myeloid cells inhibit the expression of effector factors by T cells; in addition, this application also co-cultured THP-1 cells knocked out by uPAR with T cells from healthy individuals. The results showed that, compared with the THP-1-WT cell co-culture group, the T cells co-cultured with T cells in the THP-1-uPAR-KO cell co-culture group showed increased levels of GranzymB, TNF-α, Perforin, IFN-γ and IL-2, indicating that uPAR can inhibit the expression of effector factors by T cells.

[0175] Therefore, this application also relates to the application of uPAR in T cell suppression, or the application of uPAR inhibitors in T cell activation.

[0176] 6. Application of this application in disease diagnosis and prognostic assessment, and the corresponding reagent kit.

[0177] The antibody or antigen-binding fragment provided in this application can be used to detect the presence or abundance of uPAR in biological samples, and then to diagnose the aforementioned diseases related to uPAR expression, or to evaluate the prognosis of these patients.

[0178] The antibodies or antigen-binding fragments provided in this application can be conveniently incorporated into a kit, thus facilitating the detection of uPAR. uPAR proteins in vivo or in vitro within biological fluids or tissues can be detected by the antibodies or antigen-binding fragments provided in this application.

[0179] The biological samples may include, but are not limited to: liquid samples from the test individual, such as urine, saliva, cerebrospinal fluid, blood, serum and the like; or solid or semi-solid samples from the test individual, such as tissue, feces and the like.

[0180] When used herein, the term "detection" includes quantitative or qualitative detection. Exemplary detection methods may involve immunohistochemistry, immunocytochemistry, flow cytometry (e.g., FACS), magnetic beads with antibody molecules, and ELISA assays. In some embodiments, the anti-antibody or its antigen-binding fragment of this application may be coupled to a detectable label such as luciferase or biotinylate for direct or indirect immunoassays such as FACS, IHC, and ELISA in a liquid or solid phase, including competitive or non-competitive assays.

[0181] 7. Other applications of the antibody or its antigen-binding fragment in this application

[0182] It is understood that, based on the fundamental properties of the neutralizing / blocking active antibody of this application, the antibody or its antigen-binding fragment of this application can also be used as an evaluation reference and thus applied in the process of screening other neutralizing antibodies.

[0183] The technical solutions of this application will be clearly and completely described below with reference to the embodiments. Obviously, the described embodiments are only some embodiments of this application, not all embodiments. Based on the embodiments of this application, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this application. Example

[0184] The technical solutions of this application will now be clearly and completely described with reference to the accompanying drawings. Obviously, the described embodiments are only some, not all, of the embodiments of this application. Based on the embodiments of this application, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this application.

[0185] Example 1: uPAR-positive cells inhibit T cell function

[0186] 1. Co-culture of myeloid cells derived from ascites in gastric cancer (GC) patients with T cells from healthy individuals

[0187] a. Isolation of ascites cells from GC patients: Ascites fluid from GC patients was placed into 50 mL centrifuge tubes and centrifuged at 1200 rpm for 5 min. After discarding the supernatant, 5 mL of erythrocyte lysis buffer was added to resuspend the cell pellet. The pellet was mixed by pipetting and incubated at room temperature for 5 min to observe the lysis. Once the suspension was clear, 15 mL of PBS was added, and the pellet was centrifuged at 1200 rpm for 5 min. The supernatant was discarded. Then, the cells were resuspended in RPMI 1640 medium containing 10% fetal bovine serum (containing 100 U / mL penicillin and 100 μg / mL streptomycin).

[0188] b. Sorting of myeloid cells from ascites fluid: Flow cytometry was used to analyze the expression of uPAR in various myeloid cells from ascites fluid. In this example, the expression of uPAR in macrophages (Mac), granulocyte-like myeloid immunosuppressive cells (G-MDSC), and monocyte-like myeloid immunosuppressive cells (M-MDSC) was mainly analyzed. The results are shown in Figure 1A, indicating that all types of myeloid cells expressed uPAR. Further sorting of myeloid cells (CD45+CD11b+) into uPAR-expressing cells was performed. high and uPAR low The cells were used for subsequent co-culture experiments.

[0189] c. Co-culture of myeloid cells and T cells: Myeloid cells (CD45+CD11b+) are cultured with T cells from healthy individuals (cell count 1×10⁻⁶). 4 T cells were co-cultured at different ratios (0.25:1, 1:1, and 4:1), and the expression of T cell effector factors (GranzymB, TNF-α, Perforin, IFN-γ, and IL-2) was detected by flow cytometry. The results are shown in Figures 1B-F. (The text abruptly ends here, likely due to an incomplete translation or missing information.) low Myeloid cells and T cells co-culture group, uPAR high In the co-culture group of myeloid cells and T cells, the expression levels of GranzymB, TNF-α, Perforin, IFN-γ, and IL-2 in T cells were decreased, indicating that uPAR... high Myeloid cells suppress the expression of effector factors by T cells.

[0190] 2. Co-culture of uPAR knockout THP-1 cells (THP-1-uPAR-KO) with healthy human T cells

[0191] a. Construction of uPAR knockout cells of THP-1: uPAR was knocked out in THP-1 (containing GFP tag) using CRISPR gene editing technology. The results are shown in Figure 2A. The uPAR expression of THP-1-uPAR-KO cells was negative, indicating that uPAR knockout THP-1 cells were successfully constructed.

[0192] b. Co-culture of THP-1 cells with healthy human T cells: Normal THP-1 cells (THP-1-WT) and uPAR-knockout THP-1 cells were co-cultured with healthy human T cells (cell number 1×10⁻⁶). 4T cells were co-cultured with T cells at different ratios (0.25:1, 1:1, and 4:1), and the expression of T cell effector factors (GranzymB, TNF-α, Perforin, IFN-γ, and IL-2) was detected by flow cytometry. The results are shown in Figures 2B-F. Compared with the THP-1-WT cell co-culture group, the T cells in the THP-1-uPAR-KO cell co-culture group showed increased levels of GranzymB, TNF-α, Perforin, IFN-γ, and IL-2, indicating that uPAR can inhibit the expression of T cell effector factors.

[0193] Example 2 Monoclonal Antibody Preparation and Screening

[0194] 1. Animal immunization

[0195] a. Mouse Immunization: Six- to eight-week-old BalB / C (G1-TB1) and SJL (G1-TB2) mice were immunized using different immunization methods and cycles, employing recombinant uPAR protein and uPAR-overexpressing cell lines. A first booster immunization was administered two weeks after the initial immunization, followed by a second booster immunization three weeks later. One week after each booster immunization, orbital blood samples were collected for serum immunotiter testing.

[0196] b. Serum titer detection: After diluting the obtained mouse immune serum, the specific binding of uPAR recombinant protein and uPAR overexpressing cells was detected by ELISA. Mice with high serum titers were selected for subsequent fusion and screening. The ELISA results are shown in Figure 3. A high signal value in mouse serum diluted >1:10000 was considered a good immune response and could be used for subsequent antibody preparation.

[0197] 2. Hybridoma screening

[0198] Antibody molecule screening is performed using an automated high-throughput hybridoma antibody discovery platform (Beckman’s FXP liquid workstation and iQue plus high-throughput screening platform) that integrates robotic arms and high-throughput antibody sorting equipment into a single workflow.

[0199] a. Based on the serum titer test results from the aforementioned steps, the selected mice were boosted with immunization. Three days later, the spleen and lymph nodes were harvested. Using optimized conditions, the ground cells were electrofused with mouse myeloma cells (sp2 / 0). After culturing in HAT and HT media for 10-14 days, 323 hybridoma clones (numbered #1-323) were obtained. Subsequently, the supernatant from these hybridoma clones was used for preliminary screening.

[0200] b. Initial screening was performed using ELISA to screen for binding to human proteins. Positive clones were then selected and cultured for secondary screening using FACS on MDA-MB-231 cell lines overexpressing uPAR.

[0201] Based on the binding results, clones with strong binding ability were selected and subcloned by the parent clone through multiple rounds of limiting dilution. After 7 days of subcloning, the hybridoma supernatant and the MDA-MB-231 cell line overexpressing uPAR were incubated and the binding was screened using ELISA and FACS assays. The detection results are shown in Figures 4 and 5.

[0202] c. The high-affinity hybridoma supernatant obtained above was further tested using ligand and receptor blocking assays to evaluate whether the hybridoma supernatant could block the binding of uPAR to its ligand uPA (and / or other ligands). The specific test results are shown in Figure 6.

[0203] d. Through the above steps, the monoclonal cells with strong binding ability and blocking activity were finally expanded, cultured, and cryopreserved for later use. A total of 31 hybridoma supernatants were screened and assigned corresponding antibody numbers, as shown in the table below.

[0204] Example 3 Antibody preparation, purification and performance identification

[0205] a. Antibody sequence capture: Total RNA was extracted from monoclonal cells using the Trizol method and cDNA was obtained by reverse transcription using the 5' RACE method. The heavy and light chain variable region sequences were amplified and obtained. The heavy and light chain sequences were cloned into a vector, Sanger sequencing was performed, and biological analysis was conducted to finally obtain effective antibody heavy and light chain variable region sequences.

[0206] b. Antibody preparation and purification: The effective antibody heavy chain and light chain variable region sequences were constructed into the PTT5 expression vector modified by Maiwei Biotechnology, and the chimeric antibody was expressed and purified using the HEK293 mammalian cell system.

[0207] c. Antibody Affinity Identification: In this embodiment, the affinity of antibodies AB#1-31 for binding to MDA-MB-231 cells was detected by flow cytometry (Figure 7). Additionally, the affinity of some antibodies was detected using surface ionosphere resonance (SPR) technology (Figures 8-10).

[0208] d. Detection of antibody blocking activity: Both uPA and VTN have been reported to bind to uPAR for signal transduction. To verify whether the antibodies in this application have blocking activity, this embodiment uses a chimeric receptor reporter cell system to detect the ability of antibodies to block the binding of uPA (Figure 11) or VTN (Figure 12) to uPAR. The results show that most of the antibodies in this application can block the binding of uPA to uPAR, or the binding of VTN to uPAR.

[0209] e. Cell-killing ability detection: This application first detected the expression of uPAR by THP-1 cells and constructed uPAR-knockout THP-1 cells (hereinafter referred to as THP-1KO cells). THP-1 cells without uPAR knockout were referred to as THP-1WT cells. A co-culture system of THP-1 (WT or KO) cells and T cells was established, and the antibody described in this application was added to the co-culture system. The results showed that most uPAR antibodies could enhance the killing effect of T cells on THP-1 uPAR-positive cells (Figure 13).

[0210] f. Identification of antibody-binding epitopes: uPAR has three domains, D1, D2, and D3. The D1-D2 linker can be cleaved by proteases (e.g., uPA51, plasmin 53, and MMPs54) to produce a soluble D1 fragment and a membrane-bound or detachable D2-D3 fragment. This cleavage phenomenon is common in physiological states. Commercial uPAR antibodies (Vim5) can only bind to the D1 domain of uPAR; therefore, they cannot bind to the cleaved uPAR. Based on this, this application constructed a truncated uPAR reporter cell line according to the three domains of uPAR and detected the binding sites of the corresponding antibodies to uPAR (Figure 14). It can be seen that the binding regions of the antibodies in this application are different from those of commercial antibodies.

[0211] g. Antibody-mediated ADCC activity detection: Antibody-dependent cell-mediated cytotoxicity (ADCC) is one way antibodies exert their effects. Antibodies recruit effector cells such as natural killer cells to kill target cells by specifically binding to antigens on the surface of target cells. The cytotoxic activity of monoclonal antibodies can be verified by ADCC experiments. In this example, THP-1-GFP cells (cells stably expressing the GFP fluorescent group) and PBMC cells derived from healthy individuals were co-cultured, and monoclonal antibodies (AB20 and AB26 were exemplified in this application) were added to the co-culture system. After 20 hours of co-culture, 7-AAD staining was used to label live and dead cells in the co-culture system, and flow cytometry was used to analyze double-positive cells (double-positive cells are THP-1 killed cells). Statistical analysis of the ADCC activity of monoclonal antibodies was performed (Figure 15). The results show that both AB20 and AB26 monoclonal antibodies have ADCC activity.

[0212] h. Monoclonal Antibody Species Reactivity Assay: To verify the species specificity of monoclonal antibodies, the binding of monoclonal antibodies to human or cynomolgus monkey uPAR proteins was detected using an ELISA assay. In this example, monoclonal antibodies AB4 and AB20 were tested, and the results are shown in Figure 16. Both antibodies AB4 and AB20 bound to human uPAR proteins; in addition, AB4 also bound to cynomolgus monkey uPAR proteins.

[0213] In summary, the antibodies AB12, AB19, AB20, and AB23 obtained in this application exhibit strong affinity; AB26 can more effectively block uPA binding to uPAR; AB6, AB9, and AB12 can more effectively block VTN binding to uPAR; antibodies AB4, AB7, AB9, AB12, and AB16 can recognize the D2 domain of uPAR, while antibodies AB19, AB20, AB23, AB24, and AB29 can recognize the D2+D3 domain of uPAR. All antibodies in this application can recognize the cleaved uPAR. Based on the above screening, the highly active antibodies and their sequence information (IMGT annotation) obtained in this application are shown in the table below.

[0214] Example 4: Humanized Design and Performance Verification

[0215] a. In this embodiment, antibodies AB4 and AB20 were further humanized. Two humanized sequences were optimized for the heavy and light chains of each monoclonal antibody. The specific optimized sequences are as follows:

[0216] b. The humanized VH and VL sequences of antibodies AB4 and AB20 were converted into nucleic acid sequences and combined into scFv sequences with different combinations and sequences (VH1+VL1, VH1+VL2, VH2+VL1, VH2+VL2, VL1+VH1, VL1+VH2, VL2+VH1, VL2+VH2), which were then constructed on a CAR backbone vector. The construction strategy is shown in Figure 17.

[0217] c. CAR structures with different combinations of scFv sequences were expressed in HEK293T cells. HEK293T cells were then incubated with human recombinant uPAR protein (containing a his tag) for 1 h. The binding of HEK293T cells to human uPAR protein was then detected using anti-his-APC antibody.

[0218] The test results are shown in Figure 18. The results show that the humanized sequences of AB4 or AB20 can bind to human uPAR recombinant protein. Among them, the binding ability of the VH2+VL1 combination of AB4 and the VH1+VL2 combination of AB20 is the best in the same class.

[0219] d. Comparison of the VH2+VL1 combination of AB4 and the VH1+VL2 combination of AB20: HEK293T cells expressing the VH2+VL1 combination of AB4 and the VH1+VL2 combination of AB20 were incubated with different concentrations of human recombinant uPAR protein to compare the sensitivity of the two to uPAR recognition and binding.

[0220] The test results are shown in Figure 19. It can be seen that the humanized VH1+VL2 combination of AB20 is more sensitive in recognizing uPAR.

[0221] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of this application, and are not intended to limit them. Although this application has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features therein. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of this application.

Claims

1. A uPAR antibody or antigen-binding fragment thereof, characterized in that, a heavy chain variable region amino acid sequence as set forth in SEQ ID NO. 4, 12, 20, 28, 36, 44, 52, 60, 68, 76, 84, 92, 100, 101, 110, or 111 and three CDRs of a light chain variable region amino acid sequence as set forth in SEQ ID NO. 8, 16, 24, 32, 40, 48, 56, 64, 72, 80, 88, 96, 105, 106, 115, or 116; or a variant thereof having a single or multiple CDR changes of no more than 2 amino acids per CDR region; Preferably, when the antibody HCDRs are encoded according to the IMGT numbering, the antibody or antigen binding fragment comprises the following CDR sequences: i. the HCDR1 amino acid sequence is as set forth in SEQ ID NO. 1, 9, 17, 25, 33, 41, 49, 57, 65, 73, 81, 89, 97, or 107; ii. the HCDR2 amino acid sequence is as set forth in SEQ ID NO. 2, 10, 18, 26, 34, 42, 50, 58, 66, 74, 82, 90, 98, or 108; iii. the HCDR3 amino acid sequence is as set forth in SEQ ID NO. 3, 11, 19, 27, 35, 43, 51, 59, 67, 75, 83, 91, 99, or 109; iv. the LCDR1 amino acid sequence is as set forth in SEQ ID NO. 5, 13, 21, 29, 37, 45, 53, 61, 69, 77, 85, 93, 102, or 112; v. the LCDR2 amino acid sequence is as set forth in SEQ ID NO. 6, 14, 22, 30, 38, 46, 54, 62, 70, 78, 86, 94, 103, or 113; vi. the LCDR3 amino acid sequence is as set forth in SEQ ID NO. 7, 15, 23, 31, 39, 47, 55, 63, 71, 79, 87, 95, 104, or 114; or a variant thereof having a single or multiple CDR changes of no more than 2 amino acids per CDR region.

2. The antibody or antigen binding fragment of claim 1, wherein: the combination of HCDR1 / HCDR2 / HCDR3 comprises one or more combinations selected from the group consisting of: 1) HCDR1 / HCDR2 / HCDR3: SEQ ID NO. 1 / SEQ ID NO. 2 / SEQ ID NO. 3, SEQ ID NO. 9 / SEQ ID NO. 10 / SEQ ID NO. 11, SEQ ID NO. 17 / SEQ ID NO. 18 / SEQ ID NO. 19, SEQ ID NO. 25 / SEQ ID NO. 26 / SEQ ID NO. 27, SEQ ID NO. 33 / SEQ ID NO. 34 / SEQ ID NO. 35, SEQ ID NO. 41 / SEQ ID NO. 42 / SEQ ID NO. 43, SEQ ID NO. 49 / SEQ ID NO. 50 / SEQ ID NO. 51, SEQ ID NO. 57 / SEQ ID NO. 58 / SEQ ID NO. 59, SEQ ID NO. 65 / SEQ ID NO. 66 / SEQ ID NO. 67, SEQ ID NO. 73 / SEQ ID NO. 74 / SEQ ID NO. 75, SEQ ID NO. 81 / SEQ ID NO. 82 / SEQ ID NO. 83, SEQ ID NO. 89 / SEQ ID NO. 90 / SEQ ID NO. 91, SEQ ID NO. 97 / SEQ ID NO. 98 / SEQ ID NO. 99, SEQ ID NO. 105 / SEQ ID NO. 106 / SEQ ID NO. 107, SEQ ID NO. 113 / SEQ ID NO. 114 / SEQ ID NO. 115, or SEQ ID NO. 121 / SEQ ID NO. 122 / SEQ ID NO.

123. NO. 17 / SEQ ID NO. 18 / SEQ ID NO. 19, SEQ ID NO. 25 / SEQ ID NO. 26 / SEQ ID NO. 27, SEQ ID NO. 33 / SEQ ID NO. 34 / SEQ ID NO. 35, SEQ ID NO. 41 / SEQ ID NO. 42 / SEQ ID NO. 43, SEQ ID NO. 49 / SEQ ID NO. 50 / SEQ ID NO. 51, SEQ ID NO. 57 / SEQ ID NO. 58 / SEQ ID NO. 59, SEQ ID NO. 65 / SEQ ID NO. 66 / SEQ ID NO. 67, SEQ ID NO. 73 / SEQ ID NO. 74 / SEQ ID NO. 75, SEQ ID NO. 81 / SEQ ID NO. 82 / SEQ ID NO. 83, SEQ ID NO. 89 / SEQ ID NO. 90 / SEQ ID NO. 91, SEQ ID NO. 97 / SEQ ID NO. 98 / SEQ ID NO. 99, or SEQ ID NO. 107 / SEQ ID NO. 108 / SEQ ID NO. 109; 2) or the HCDR of (1) above containing one or more amino acid substitutions, deletions, or insertions of no more than 2 amino acids; the combination of the LCDR1 / LCDR2 / LCDR3 comprises one or more groups selected from: 1) LCDR1 / LCDR2 / LCDR3 is: SEQ ID NO. 5 / SEQ ID NO. 6 / SEQ ID NO. 7, SEQ ID NO. 13 / SEQ ID NO. 14 / SEQ ID NO. 15, SEQ SEQ ID NO. 21 / SEQ ID NO. 22 / SEQ ID NO. 23, SEQ ID NO. 29 / SEQ ID NO. 30 / SEQ ID NO. 31, SEQ ID NO. 37 / SEQ ID NO. 38 / SEQ ID NO. 39, SEQ ID NO. 45 / SEQ ID NO. 46 / SEQ ID NO. 47, SEQ ID NO. 53 / SEQ ID NO. 54 / SEQ ID NO. 55, SEQ ID NO. 61 / SEQ ID NO. 62 / SEQ ID NO. 63, SEQ ID NO. 69 / SEQ ID NO. 70 / SEQ ID NO. 71, SEQ ID NO. 77 / SEQ ID NO. 78 / SEQ ID NO. 79, SEQ ID NO. 85 / SEQ ID NO. 86 / SEQ ID NO. 87, SEQ ID NO. 93 / SEQ ID NO. 94 / SEQ ID NO. 95, SEQ ID NO. 102 / SEQ ID NO. 103 / SEQ ID NO. 104, or SEQ ID NO. 112 / SEQ ID NO. 113 / SEQ ID NO. 114; 2) or HCDR of (1) above containing one or more amino acid substitutions, deletions or insertions of no more than 2 amino acids; Preferably, the antibody or antigen binding fragment comprises a heavy chain variable region and a light chain variable region, the sequences selected from the group consisting of: a) the amino acid sequence of the heavy chain variable region comprises an amino acid sequence selected from the group consisting of SEQ ID NO. 4, 12, 20, 28, 36, 44, 52, 60, 68, 76, 84, 92, 100, 101, 110, or 111, or has at least 70%, 80%, 90%, 95%, 99%, or 100% sequence identity to a sequence in the group; b) the amino acid sequence of the light chain variable region comprises an amino acid sequence selected from the group consisting of SEQ ID NO. 8, 16, 24, 32, 40, 48, 56, 64, 72, 80, 88, 96, 105, 106, 115, or 116, or has at least 70%, 80%, 90%, 95%, 99%, or 100% sequence identity to a sequence in the group.

3. The antibody or antigen binding fragment of any one of claims 1-2, characterized in that, The antibody or antigen binding fragment is further linked to a coupling moiety selected from one or more of a radionuclide, a drug, a toxin, a cytokine, an enzyme, a fluorophore, a carrier protein, a lipid, and biotin; the antibody or antigen binding fragment is linked to the coupling moiety, optionally through a linker; preferably, the linker is a peptide or polypeptide; More preferably, the antibody or antigen-binding fragment is selected from the group consisting of a monoclonal antibody, a polyclonal antibody, an antiserum, a chimeric antibody, a humanized antibody, and a human antibody; preferably, the antibody is selected from the group consisting of a multispecific antibody, a single-chain Fv (scFv), a single-chain antibody, an anti-idiotypic (anti-Id) antibody, a diabody, a minibody, a nanobody, a single-domain antibody, a Fab fragment, a F(ab’) fragment, a disulfide-linked bispecific Fv (sdFv), and an intrabody.

4. An isolated polynucleotide, comprising, The polynucleotide encodes the antibody or antigen-binding fragment of any one of claims 1-3.

5. A recombinant vector comprising the polynucleotide of claim 4, and optionally a regulatory sequence; Preferably, the recombinant vector is a cloning vector or an expression vector; More preferably, the regulatory sequence is selected from the group consisting of a leader sequence, a polyadenylation sequence, a propeptide sequence, a promoter, a signal sequence, a transcription terminator, or any combination thereof.

6. A host cell, characterized in that, comprising the recombinant vector of claim 5; Preferably, the host cell is a prokaryotic cell or a eukaryotic cell; More preferably, the host cell includes, but is not limited to, a yeast cell, a Chinese hamster ovary cell, a human embryonic kidney cell.

7. A pharmaceutical composition, characterized by, comprising one or more of the antibody or antigen-binding fragment, the polynucleotide, the recombinant vector, and the host cell of any one of claims 1-3; Preferably, the pharmaceutical composition is a CAR or a CAR-T cell; More preferably, the composition further comprises a pharmaceutically acceptable carrier or excipient.

8. Any one of the following applications of the antibody or antigen-binding fragment of any one of claims 1-3: a) use in preventing, treating uPAR expression related diseases (cancer and immune related diseases); b) use in preparing a medicament for preventing, treating uPAR expression related diseases (cancer and immune related diseases); c) use in diagnosing uPAR expression related diseases (cancer and immune related diseases); d) use in preparing a kit for diagnosing uPAR expression related diseases (cancer and immune related diseases); e) use in prognostic evaluation of uPAR expression related diseases (cancer and immune related diseases); f) use in preparing a kit for prognostic evaluation of uPAR expression related diseases (cancer and immune related diseases); g) use in T cell activation; h) use in preparing a product for T cell activation.

9. A method of treating uPAR positive associated diseases (cancer and immune related diseases) characterized in that, by administering to a subject an effective amount of the antibody or antigen-binding fragment thereof of any one of claims 1-3, or the pharmaceutical composition of claim 7.

10. Use of uPAR in T cell inhibition, or use of a uPAR inhibitor in T cell activation.

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