Antibodies targeting uPAR and molecular conjugates thereof

Abstract

The present invention relates to antibodies and molecular conjugates targeting the uPAR receptor, in particular to antibody-drug conjugates (ADC) of said antibodies and their use in delivering active agents to cells and tissues expressing uPAR. The present invention also relates to the use of said ADCs in the treatment of diseases involving cells expressing uPAR, such as certain cancers.

Description

Technical Field

[0001] This invention relates to antibodies and molecular conjugates targeting the uPAR receptor, particularly antibody-drug conjugates (ADCs) of said antibodies and their use in delivering active agents to cells and tissues expressing uPAR. The invention further relates to the use of said ADCs in treating diseases involving cells expressing uPAR, such as certain cancers. Background of the Invention

[0003] Urokinase-type plasminogen activator receptor (uPAR), also known as CD87, is a glycolipid-anchored membrane protein component of the plasminogen activation system. Due to its aberrant overexpression, its pathophysiological role in almost all aggressive human cancers, and its poor prognosis, it has become a recognized tumor target. The receptor's remarkable expression profile—present in both tumor cells and the associated tumor microenvironment, at baseline levels almost undetectable in normal tissues, and possessing highly efficient endocytic capacity—has prompted a shift in therapeutic strategies from functional inhibition to cytotoxic therapies that selectively eliminate uPAR-expressing cells through targeted cytotoxic attacks (Metrangolo et al., 2021).

[0004] One of the treatment strategies investigated involves the use of antibody-drug conjugates (ADCs). ADCs are an emerging and rapidly developing class of anticancer therapies that have achieved significant clinical and regulatory milestones. Currently, more than 10 FDA-approved ADCs are marketed for oncological diseases, with hundreds more undergoing advanced clinical and preclinical evaluations. ADCs are designed as prodrugs, chemically linked to a tumor-selective monoclonal antibody (mAb) and a cytotoxic moiety. Upon binding to and subsequently internalizing with a target cell receptor, they are selectively delivered to the target cell, thereby achieving selective tumor killing.

[0005] This approach gives cytotoxic agents a more favorable therapeutic spectrum and effectively limits off-target toxicity to healthy tissues, a typical drawback of standard chemotherapy and radiotherapy (Dumontet C et al., 2023).

[0006] There are currently two examples of anti-uPAR ADCs (Lourenço et al., 2023; Harel et al., 2019), both of which are related to the treatment of triple-negative breast cancer.

[0007] Currently, most cancer types have treatment options. However, due to the lack of specificity in these treatments, the efficacy is often unsatisfactory, or accompanied by harmful side effects. Therefore, there is an urgent need to develop more specific and potent treatment methods, especially for highly aggressive cancer types, such as those characterized by uPAR receptor overexpression. Invention Overview

[0009] This invention relates to novel antibodies or antigen-binding fragments thereof targeting the uPAR receptor, and antibody-drug conjugates (ADCs) based thereon. The ADCs disclosed herein have demonstrated high affinity, cross-species reactivity, and applicability, making them suitable as therapeutic agents for a variety of aggressive uPAR-positive malignancies, particularly pancreatic ductal adenocarcinoma (PDAC), for which there is no unmet medical need. PDAC is the cancer with the highest receptor expression levels, both in cancerous and stromal tissues, and exhibits the greatest difference in expression between tumor and normal tissues. Although there is ongoing evidence suggesting that uPAR plays a pro-invasive role in the progression of PDAC, no uPAR-targeting ADCs for this tumor have been approved or are currently in preclinical or clinical trials (Kumar et al., 2022).

[0010] Therefore, one aspect of the present invention is an antibody or antigen-binding fragment thereof that binds to the urokinase-type plasminogen activator receptor (uPAR), wherein the antibody is selected from the following: a. An antibody or its antigen-binding fragment, containing i. An immunoglobulin light chain comprising the amino acid sequence of SEQ ID NO: 5 or a sequence having at least 90% sequence identity with it, or consisting of the amino acid sequence of SEQ ID NO: 5 or a sequence having at least 90% sequence identity with it, and ii. An immunoglobulin heavy chain comprising the amino acid sequence of SEQ ID NO: 10 or a sequence having at least 90% sequence identity with it, or consisting of the amino acid sequence of SEQ ID NO: 10 or a sequence having at least 90% sequence identity with it; b. An antibody or its antigen-binding fragment, containing i. The variable region of the immunoglobulin light chain, comprising the amino acid sequence of SEQ ID NO: 6 or a sequence having at least 90% sequence identity with it, or consisting of the amino acid sequence of SEQ ID NO: 6 or a sequence having at least 90% sequence identity with it, and ii. The variable region of the immunoglobulin heavy chain, comprising the amino acid sequence of SEQ ID NO: 11 or a sequence having at least 90% sequence identity with it, or consisting of the amino acid sequence of SEQ ID NO: 11 or a sequence having at least 90% sequence identity with it; c. An antibody or its antigen-binding fragment, containing i. The variable region of the immunoglobulin light chain, comprising the amino acid sequence of SEQ ID NO: 35 as complementarity-determining region 1 (CDR1), the amino acid sequence of SEQ ID NO: 36 as CDR2, and the amino acid sequence of SEQ ID NO: 37 as CDR3, and ii. The variable region of the immunoglobulin heavy chain, comprising the amino acid sequence of SEQ ID NO: 38 as complementarity-determining region 1 (CDR1), the amino acid sequence of SEQ ID NO: 39 as CDR2, and the amino acid sequence of SEQ ID NO: 40 as CDR3; d. An antibody or its antigen-binding fragment, containing i. The variable region of the immunoglobulin light chain, comprising the amino acid sequence of SEQ ID NO: 7 as complementarity-determining region 1 (CDR1), the amino acid sequence of SEQ ID NO: 8 as CDR2, and the amino acid sequence of SEQ ID NO: 9 as CDR3, and ii. The variable region of the immunoglobulin heavy chain, comprising the amino acid sequence of SEQ ID NO: 12 as complementarity-determining region 1 (CDR1), the amino acid sequence of SEQ ID NO: 13 as CDR2, and the amino acid sequence of SEQ ID NO: 14 as CDR3; e. An antibody or an antigen-binding fragment thereof from any of a, b, c, or d; f. An antibody or a chimeric version of an antigen-binding fragment thereof from any of a, b, c, or d; g. An antibody or its antigen-binding fragment, containing i. An immunoglobulin light chain variable region comprising an amino acid sequence selected from SEQ ID NO: 53-57 or a sequence having at least 90% sequence identity with it, or consisting of an amino acid sequence selected from SEQ ID NO: 53-57 or a sequence having at least 90% sequence identity with it, and ii. The variable region of the immunoglobulin heavy chain, comprising an amino acid sequence selected from SEQ ID NO: 47-51 or a sequence having at least 90% sequence identity with it, or consisting of an amino acid sequence selected from SEQ ID NO: 47-51 or a sequence having at least 90% sequence identity with it.

[0011] Another aspect of the invention is an antibody or antigen-binding fragment thereof that binds to urokinase-type plasminogen activator receptor (uPAR), wherein said antibody is selected from the following: a. An antibody or its antigen-binding fragment, containing i. An immunoglobulin light chain comprising the amino acid sequence of SEQ ID NO: 15 or a sequence having at least 90% sequence identity with it, or consisting of the amino acid sequence of SEQ ID NO: 15 or a sequence having at least 90% sequence identity with it, and ii. An immunoglobulin heavy chain comprising the amino acid sequence of SEQ ID NO: 20 or a sequence having at least 90% sequence identity with it, or consisting of the amino acid sequence of SEQ ID NO: 20 or a sequence having at least 90% sequence identity with it; b. An antibody or its antigen-binding fragment, containing iii. An immunoglobulin light chain variable region comprising the amino acid sequence of SEQ ID NO: 16 or a sequence having at least 90% sequence identity with it, or consisting of the amino acid sequence of SEQ ID NO: 16 or a sequence having at least 90% sequence identity with it, and iv. The variable region of the immunoglobulin heavy chain, comprising the amino acid sequence of SEQ ID NO: 21 or a sequence having at least 90% sequence identity with it, or consisting of the amino acid sequence of SEQ ID NO: 21 or a sequence having at least 90% sequence identity with it; c. An antibody or its antigen-binding fragment, containing v. Variable regions of the immunoglobulin light chain, comprising the amino acid sequence of SEQ ID NO: 17 as complementarity-determining region 1 (CDR1), the amino acid sequence of SEQ ID NO: 18 as CDR2, and the amino acid sequence of SEQ ID NO: 19 as CDR3, and vi. The variable region of the immunoglobulin heavy chain, comprising the amino acid sequence of SEQ ID NO: 22 as complementarity-determining region 1 (CDR1), the amino acid sequence of SEQ ID NO: 23 as CDR2, and the amino acid sequence of SEQ ID NO: 24 as CDR3; d. A humanized version of an antibody or its antigen-binding fragment from any of a, b, or c; e. An antibody or a chimeric version of an antigen-binding fragment of any one of a, b, or c.

[0012] Another aspect of the invention is an antibody or antigen-binding fragment thereof that binds to urokinase-type plasminogen activator receptor (uPAR), wherein said antibody is selected from the following: a. An antibody or its antigen-binding fragment, containing vii. Immunoglobulin light chains comprising the amino acid sequence of SEQ ID NO: 25 or a sequence having at least 90% sequence identity with it, or consisting of the amino acid sequence of SEQ ID NO: 25 or a sequence having at least 90% sequence identity with it, and viii. Immunoglobulin heavy chain comprising the amino acid sequence of SEQ ID NO: 30 or a sequence having at least 90% sequence identity with it, or consisting of the amino acid sequence of SEQ ID NO: 30 or a sequence having at least 90% sequence identity with it; b. An antibody or its antigen-binding fragment, containing ix. Variable region of immunoglobulin light chain, comprising the amino acid sequence of SEQ ID NO: 26 or a sequence having at least 90% sequence identity with it, or consisting of the amino acid sequence of SEQ ID NO: 26 or a sequence having at least 90% sequence identity with it, and x. The variable region of the immunoglobulin heavy chain, comprising the amino acid sequence of SEQ ID NO: 31 or a sequence having at least 90% sequence identity with it, or consisting of the amino acid sequence of SEQ ID NO: 31 or a sequence having at least 90% sequence identity with it; c. An antibody or its antigen-binding fragment, containing xi. The variable region of the immunoglobulin light chain, comprising the amino acid sequence of SEQ ID NO: 27 as complementarity-determining region 1 (CDR1), the amino acid sequence of SEQ ID NO: 28 as CDR2, and the amino acid sequence of SEQ ID NO: 29 as CDR3, and xii. The variable region of the immunoglobulin heavy chain, comprising the amino acid sequence of SEQ ID NO: 32 as complementarity-determining region 1 (CDR1), the amino acid sequence of SEQ ID NO: 33 as CDR2, and the amino acid sequence of SEQ ID NO: 34 as CDR3; d. A humanized version of an antibody or its antigen-binding fragment from any of a, b, or c; e. An antibody or a chimeric version of an antigen-binding fragment of any one of a, b, or c.

[0013] Other aspects of the invention include antibody-drug conjugates comprising an antibody or an antigen-binding fragment thereof as defined herein, and further comprising an active agent, and optionally comprising a connector for linking the antibody or the antigen-binding fragment thereof to the active agent.

[0014] Other aspects of the invention include a method for treating a disease characterized by cells expressing and / or overexpressing uPAR, the method comprising administering to a subject an antibody as defined above, an ADC as defined above, or a pharmaceutical composition comprising an antibody or ADC as defined above. Attached Figure Description

[0015] Figure 1 Topological structure of mAb-binding epitopes on the uPAR receptor. Surface representation of the uPAR semi-open conformation (PDB code: 1YWH). Epitopes FL1 (D2 domain), FL2 (D1 domain), and FL3 (D3 domain) are highlighted in the figure.

[0016] Figure 2 Internalization of α-uPAR mAb in uPAR-positive U937 leukemia cells. The histogram shows the internalization of iodine-125 ( 125 I) The internalization fraction of the labeled α-uPAR mAb in the reference uPAR-positive leukemia cell line U937, quantified by a radioactive internalization assay after incubation at 37°C for 4 hours (see Examples). The cross-reactive FL1 mAb with the highest degree of internalization was selected as the lead antibody candidate for ADC development. NC: Negative control.

[0017] Figure 3 : A schematic diagram representing the connection between FL1 and DBCO-PEG4-VC-PAB-DMEA-PNU-159682.

[0018] The FL1-based PNU ADC comprises two linker-payload portions that are click-coupled to conserved N-glycosylation sites of the Fc fragment of the mAb, as described in the Examples section. The FL1 linked to DBCO-PEG4-VC-PAB-DMEA-PNU-159682 is also referred to herein as the FL1-PNU ADC.

[0019] Figure 4 FL1-PNU-ADC exhibited robust and selective in vitro cytotoxicity in uPAR-expressing PDAC cells. 。

[0020] Dose-response curves showed the in vitro antitumor activity of FL1-PNU ADC in the target cell group compared to unmodified Ab, NC (negative control), aTNP-PNU ADC, and free payload. Cell viability was assessed by colorimetric assay (MTS assay) after incubation with serially diluted test compounds for 76 h (unmodified Ab and free toxin) and 120 h (ADC), expressed as a percentage of untreated control cells. Free toxin showed indiscriminate activity across the entire cell group, unaffected by test concentration and uPAR status, while unconjugated FL1 showed no antiproliferative activity. In contrast, the derived FL1-PNU conjugate exhibited robust cytotoxic activity in all receptor-positive cells with a clear concentration-dependent characteristic, with mean EC50 values ​​in the picomolar range. In contrast, uPAR null KPC cells were essentially unaffected, similar to cells treated with the negative control ADC aTNP-PNU, confirming the on-target antitumor activity of FL1-PNU. In both cases, nonspecific cell killing was observed only at the highest concentration; nevertheless, the selective activity of FL1-PNU still has a fairly broad therapeutic window.

[0021] Figure 5 In vitro cytotoxicity in other relevant uPAR-positive tumors.

[0022] In addition to PDAC cells, the efficacy of FL1-PNU ADC was also tested in other relevant uPAR-positive cancer cell types, including the leukemia cell line U937, glioblastoma cell line U87, triple-negative breast cancer cell line MDA-MB-231, and mesothelioma cell line REN-luc. Cells were incubated with serially diluted FL1-PNU ADC and the negative control aTNP-FL1 ADC. Cell viability was assessed by colorimetric assay (MTS assay) after 120 hours and expressed as a percentage relative to untreated control cells. Treatment with FL1-PNU ADC produced a potent and dose-dependent killing response in all tested cell types with a large window of specificity, as evidenced by the non-responsiveness of the a-TNP-based non-targeted control ADC.

[0023] Figure 6 In vivo efficacy of FL1-PNU-ADC in xenograft and orthotopic PDAC models 。

[0024] Human PDAC AsPC1 cells were subcutaneously transplanted into Balb-c nude mice. When the tumor reached 50-120 mm... 3Mice were randomly divided into three groups of 11-12 each, and subsequently received three cycles of weekly intravenous (iv) doses of 0.75 mg / kg of the ADC product, including FL1-PNU, the unbound control ADC aTNP-PNU, or a PBS mediator control. Animals were followed up for a total of 62 days, with tumor volume and body weight measured three times weekly. Figure 6 A). Compared to the PBS group and the negative control (NC) ADC group, which were euthanized due to excessive tumor burden, the FL1-PNU treatment group showed tumor volume reduction after the second dose, and all but one mouse achieved complete tumor clearance upon cessation of treatment. Five animals experienced palpable small tumor recurrence at approximately 48 days, but their condition subsequently stabilized until the end of the observation period. Six out of twelve mice were completely cured and showed no signs of recurrence throughout the follow-up period.

[0025] Therefore, compared with the two control groups, the antitumor effect of FL1-PNU translated into a very significant long-term survival benefit, with 9 mice surviving to the endpoint. Figure 6 B). No weight loss was observed in any group. Figure 6 C).

[0026] Figure 6 Figure D shows the treatment regimen design and timeline for the orthotopic PDAC model. KPC2 cancer cells expressing luciferase were injected orally into the pancreatic head of female C567BL / 6 mice (n = 8–10). Tumor growth was monitored using IVIS imaging. Mice were randomly assigned to groups and treatment began on day 14 post-tumor implantation, with the weekly treatment regimen identical to Figure A), consisting of two doses (0.75 mg / kg ADC).

[0027] Figure 6 E shows the tumor images obtained by each treatment group at the endpoint (day 28).

[0028] Figure 6 F shows the average tumor weight (in grams) at the endpoint (day 28) for each treatment group.

[0029] Figure 6 G shows mouse body weight measured twice weekly and expressed relative to initial body weight. FL1-PNU ADC was able to induce a significant anti-tumor effect, with a final tumor weight reduction of 3-fold and 2.2-fold, respectively, compared to the mediator and isotype control ADC treatment groups, and no body weight loss was observed during treatment. p ≤ 0.05, p ≤ 0.001, ns: no statistical significance (univariate ANOVA, GraphPad Prism 9). Two studies have shown that anti-uPAR FL1-PNU ADCs have very potent and selective efficacy in various PDAC models, with a generally favorable tolerability profile.

[0030] Figure 7 FL1-PNU remodels inhibitory TEM and promotes immune regulation. We performed immunophenotypic analysis to investigate the effects of ADC on the immune microenvironment, including major myeloid and lymphoid markers. Significant increases in both T lymphocytes and B lymphocytes were confirmed in FL1-PNU ADC-treated samples, while almost no increase was observed in the PBS-treated group. For myeloid (CD11b) + The cell population, which comprised nearly 90% of immune cells in the control sample, was reduced to almost half in the FL1-PNU treatment group. Similar reductions were also observed in Ly6G. + / Ly6C- lo / in (The percentage corresponding to neutrophils / polymorphonuclear myeloid-derived suppressor cells (PMN-MDSC)) which constitute the dominant myeloid cell population in the mediator-treated samples. Ly6C hi Cells (identified as monocytes or macrophages with classic inflammatory phenotypes, absent in tumors treated with the medium) were significantly amplified in FL1-PNU-ADC treated samples. p ≤ 0.05; P ≤ 0.01; p ≤ 0.001 (one-way ANOVA, GraphPad Prism 9).

[0031] Figure 8 In vitro efficacy comparison of FL1-PNU, 2G10-PNU, and ATN658-PNU conjugates. To compare the efficacy of ADCs containing FL1, 2G10, and ATN658, these antibodies were conjugated using the same linker payload and conjugation method as described in the examples. Figure 4 As shown, the efficacy of the conjugate was evaluated on uPAR-positive TNBC cells MDA-MB-231 four days after treatment. FL1-PNU appeared to be more potent than 2G10-PNU and ATN658-PNU, confirming FL1 as a superior anti-uPAR mAb candidate in ADC form.

[0032] Figure 9In vitro efficacy comparison of FL1-PNU, FL2-PNU, FL3-PNU, and 2G10-PNU conjugates. To compare the efficacy of ADCs containing FL1, FL2, and FL3 mAbs with 2G10, these antibodies were conjugated using the same linker payload and conjugation method as described in the examples. Figure 4 As shown, the efficacy of the conjugates was evaluated on uPAR-positive TNBC cells MDA-MB-231 three days after treatment. FL1-PNU appeared to be more potent than all other conjugates tested, including 2G10-PNU, confirming FL1 as a superior anti-uPAR mAb candidate in ADC form. FL2-PNU appeared to be more potent than 2G10 and FL3-PNU, respectively.

[0033] Figure 10 In vitro efficacy comparison of FL1-PNU with humanized candidates HC1-LC4-PNU and HC5-LC3-PNU. To compare the efficacy of ADCs containing FL1 with ADCs containing humanized mAb candidates (containing VH1 (SEQ ID NO: 47) and VL4 (SEQ ID NO: 56), referred to as HC1-LC4, and containing VH5 (SEQ ID NO: 51) and VL3 (SEQ ID NO: 55), referred to as HC5-LC3), these antibodies were conjugated using the same linker payload and conjugation method as described in the examples for PNU ADCs. Figure 4 As shown, the efficacy of the conjugates was evaluated on uPAR-positive TNBC cells MDA-MB-231 three days after treatment. Hyhel-PNU served as a non-targeted negative control mAb. HC1-LC4-PNU and HC5-LC3-PNU exhibited similar potency and appeared to be more effective than the parental FL1-PNU based on mouse FL1.

[0034] Figure 11 In vitro efficacy of DAR4 FL1-MMAE. Cells were reacted with serially diluted ADCs (0.2–1.2 x 10⁻⁶). -5 Incubate with µM) for 120 hours and then... Figure 4 The cell viability was assessed. FL1-MMAE induced a dose-specific uPAR response, with EC50 values ​​of 3.7 nM and 0.36 nM for MDA-MB-231 and HT-1080, respectively.

[0035] Figure 12Analysis of uPAR expression in cultured fibroblasts, M2-polarized BMDM, and normal mouse skin. (A) Flow cytometry analysis of surface uPAR levels in human 1BR3.G, mouse NIH3T3 fibroblasts, and M2-differentiated BMDM. Bone marrow progenitor cells were isolated from the pelvis, femur, and tibia of FVB / N mice (8–10 weeks old). The collected cells were then cultured in 96-well plates (100 μL per well, 5 × 10⁻⁶ cells / well). 4 M2-like macrophages were differentiated in DMEM (10% FBS, 1% P / S) containing 20 ng / mL mM-CSF (Biotechne) for 4 days, followed by stimulation with 20 ng / mL mM-CSF and 20 ng / mL mIL-4 (Biotechne) for 2 days. For flow cytometry analysis, cells were incubated on ice for 1 hour in the dark with a pre-determined saturation concentration of A647-labeled FL1. After staining, samples were stained with 1 µg / mL 7-AAD (Invitrogen, #1890506) to exclude cell death and were finally collected on a BD LSR Fortessa 20X (BD Biosciences). The positive signal of uPAR detected on the surface is expressed as median fluorescence intensity (MFI). (B) Immunohistochemical staining of uPAR in normal mouse skin. MuPAR immunostaining was performed on the skin of naïve C57BL / 6 mice using rabbit anti-smuPAR pAb P47. Weak uPAR staining was detected in sparse, resting fibroblasts in the skin (black arrows). Scale bar, 100 μm.

[0036] Figure 13 FL1-PNU targets uPAR-positive immune cells and stromal cells, and induces a bystander effect to kill uPAR-negative cancer cells. (A) M2-polarized BMDM, human and mouse fibroblast lines 1BR3.G and NIH3T3, after three days of exposure to FL1-PNU and aTNP-PNU ADC, were assessed for cell viability by MTS assay (n = 3, ± SD). (B) Schematic diagram of the established transwell co-culture system. uPAR... KOKPC2 cancer cells were seeded in 6-well transwell plates, while uPAR-positive fibroblasts (1BR3.G or NIH3T) were seeded in the upper transwell permeable insert chambers with a membrane pore size of 0.4 μm. 4 nM of FL1-PNU and aTNP-PNU ADC [EC80 values ​​of 1BR3.G and NIH3T3, (C)] was added to the upper chamber, and viable cells in both chambers were quantified after 72 hours of incubation. The percentage of cell viability relative to aTNP-PNU-treated cells (as a control) is shown in (D) (n = 2). Invention Details

[0038] The inventors of this paper provide a series of alternative anti-uPAR antibodies and their antibody-drug conjugates that exhibit improved ligand affinity and efficacy compared to prior art anti-uPAR antibodies, and also show improved internalization and in vivo efficacy compared to other anti-uPAR antibodies.

[0039] anti-uPAR antibody

[0040] The anti-uPAR antibody disclosed herein can be any immunoglobulin class, including IgG, IgM, IgD, IgE, IgA, and any subclasses thereof. IgG subclasses are also well known in the art, including but not limited to human IgG1, IgG2, IgG3, and IgG4. In one embodiment, the anti-uPAR antibody is an IgG monoclonal antibody. In one embodiment, the anti-uPAR antibody is IgG1κ. In one embodiment, the anti-uPAR antibody is an antigen-binding fragment.

[0041] In one embodiment of this disclosure, the FL1 antibody described herein comprises a light chain variable region containing an amino acid of SEQ ID NO: 6 (i.e., the variable region of SEQ ID NO: 5) and a heavy chain variable region containing an amino acid of SEQ ID NO: 11 (i.e., the variable region of SEQ ID NO: 10).

[0042] In one embodiment of this disclosure, the FL1 antibody described herein may comprise a light chain containing an amino acid of SEQ ID NO: 5 or composed of SEQ ID NO: 5 and a heavy chain containing an amino acid of SEQ ID NO: 10 or composed of SEQ ID NO: 10.

[0043] In one embodiment of this disclosure, the anti-uPAR antibody as defined herein comprises: a. An immunoglobulin light chain variable region comprising the amino acid sequence of SEQ ID NO: 6 or composed of the amino acid sequence of SEQ ID NO: 6; and / or b. Containing the amino acid sequence of SEQ ID NO: 11 or the variable region of the immunoglobulin heavy chain consisting of the amino acid sequence of SEQ ID NO: 11.

[0044] In one embodiment of this disclosure, the antibody that binds to uPAR as defined herein comprises: a. An immunoglobulin light chain containing the amino acid sequence of SEQ ID NO: 5; and / or b. An immunoglobulin heavy chain containing the amino acid sequence of SEQ ID NO: 10.

[0045] In one embodiment of this disclosure, the antibody that binds to uPAR as defined herein comprises: a. An immunoglobulin light chain consisting of the amino acid sequence of SEQ ID NO: 5; and b. An immunoglobulin heavy chain consisting of the amino acid sequence of SEQ ID NO: 10.

[0046] In some implementations, the antibody against uPAR or its antigen-binding fragment is characterized in that the variable regions of the heavy and light chains of the antibody contain multiple complementarity-determining regions (CDRs).

[0047] Complementarity-determining regions (CDRs) were predicted according to the IMGT numbering scheme defined by Lefranc et al. (2005) using an online antibody region-specific alignment (AbRSA) tool (see references). Alternatively, CDRs were predicted according to the scheme defined by Kabat et al. (see references for Kabat et al. (1983), Kabat et al. (1991), and Wu and Kabat (2008)) using the computerized Kabat numbering program published by Dunbar and Deane (2016).

[0048] Therefore, in one embodiment of this disclosure, the uPAR-binding antibody FL1 described herein may comprise an immunoglobulin light chain variable region comprising CDR1, CDR2, and CDR3 containing the amino acid sequences of SEQ ID NO: 7, 8, and 9, respectively; and may also comprise an immunoglobulin heavy chain variable region comprising CDR1, CDR2, and CDR3 containing the amino acid sequences of SEQ ID NO: 12, 13, and 14, respectively.

[0049] In one embodiment of this disclosure, the antibody that binds to uPAR as defined herein comprises: a. An immunoglobulin light chain containing the amino acid sequence of SEQ ID NO: 15; and / or b. An immunoglobulin heavy chain containing the amino acid sequence of SEQ ID NO: 20.

[0050] In one embodiment of this disclosure, the antibody that binds to uPAR as defined herein comprises: a. An immunoglobulin light chain consisting of the amino acid sequence of SEQ ID NO: 15; and b. An immunoglobulin heavy chain consisting of the amino acid sequence of SEQ ID NO: 20.

[0051] In one embodiment of this disclosure, the antibody binding to uPAR may be further characterized as comprising an immunoglobulin light chain variable region comprising CDR1, CDR2, and CDR3 containing the amino acid sequences of SEQ ID NO: 17, 18, and 19, respectively; and also comprising an immunoglobulin heavy chain variable region comprising CDR1, CDR2, and CDR3 containing the amino acid sequences of SEQ ID NO: 22, 23, and 24, respectively.

[0052] In one embodiment of this disclosure, the antibody that binds to uPAR as defined herein comprises: a. An immunoglobulin light chain containing the amino acid sequence of SEQ ID NO: 25; and / or b. An immunoglobulin heavy chain containing the amino acid sequence of SEQ ID NO: 30.

[0053] In one embodiment of this disclosure, the antibody that binds to uPAR as defined herein comprises: a. An immunoglobulin light chain consisting of the amino acid sequence of SEQ ID NO: 25; and b. An immunoglobulin heavy chain consisting of the amino acid sequence of SEQ ID NO: 30.

[0054] In one embodiment of this disclosure, the antibody binding to uPAR may be further characterized as: comprising an immunoglobulin light chain variable region comprising CDR1, CDR2, and CDR3 containing the amino acid sequences of SEQ ID NO: 27, 28, and 29, respectively; and further comprising an immunoglobulin heavy chain variable region comprising CDR1, CDR2, and CDR3 containing the amino acid sequences of SEQ ID NO: 32, 33, and 34, respectively.

[0055] In one embodiment of this disclosure, the antibody that binds to uPAR as defined herein comprises: a. The variable region of the immunoglobulin light chain, comprising the amino acid sequence of SEQ ID NO: 53 or a sequence having at least 90% sequence identity with it, or consisting of the amino acid sequence of SEQ ID NO: 53 or a sequence having at least 90% sequence identity with it; the variable region of the immunoglobulin heavy chain, comprising the amino acid sequence of SEQ ID NO: 47 or a sequence having at least 90% sequence identity with it, or consisting of the amino acid sequence of SEQ ID NO: 47 or a sequence having at least 90% sequence identity with it; b. Variable region of immunoglobulin light chain, comprising the amino acid sequence of SEQ ID NO: 53 or a sequence having at least 90% sequence identity with it, or consisting of the amino acid sequence of SEQ ID NO: 53 or a sequence having at least 90% sequence identity with it; variable region of immunoglobulin heavy chain, comprising the amino acid sequence of SEQ ID NO: 48 or a sequence having at least 90% sequence identity with it, or consisting of the amino acid sequence of SEQ ID NO: 48 or a sequence having at least 90% sequence identity with it; c. The variable region of the immunoglobulin light chain, comprising the amino acid sequence of SEQ ID NO: 53 or a sequence having at least 90% sequence identity with it, or consisting of the amino acid sequence of SEQ ID NO: 53 or a sequence having at least 90% sequence identity with it; the variable region of the immunoglobulin heavy chain, comprising the amino acid sequence of SEQ ID NO: 49 or a sequence having at least 90% sequence identity with it, or consisting of the amino acid sequence of SEQ ID NO: 49 or a sequence having at least 90% sequence identity with it; d. The variable region of the immunoglobulin light chain, comprising the amino acid sequence of SEQ ID NO: 53 or a sequence having at least 90% sequence identity with it, or consisting of the amino acid sequence of SEQ ID NO: 53 or a sequence having at least 90% sequence identity with it; the variable region of the immunoglobulin heavy chain, comprising the amino acid sequence of SEQ ID NO: 50 or a sequence having at least 90% sequence identity with it, or consisting of the amino acid sequence of SEQ ID NO: 50 or a sequence having at least 90% sequence identity with it; e. The variable region of the immunoglobulin light chain, comprising the amino acid sequence of SEQ ID NO: 53 or a sequence having at least 90% sequence identity with it, or consisting of the amino acid sequence of SEQ ID NO: 53 or a sequence having at least 90% sequence identity with it; the variable region of the immunoglobulin heavy chain, comprising the amino acid sequence of SEQ ID NO: 51 or a sequence having at least 90% sequence identity with it, or consisting of the amino acid sequence of SEQ ID NO: 51 or a sequence having at least 90% sequence identity with it; f. The variable region of the immunoglobulin light chain, comprising the amino acid sequence of SEQ ID NO: 54 or a sequence having at least 90% sequence identity with it, or consisting of the amino acid sequence of SEQ ID NO: 54 or a sequence having at least 90% sequence identity with it; the variable region of the immunoglobulin heavy chain, comprising the amino acid sequence of SEQ ID NO: 47 or a sequence having at least 90% sequence identity with it, or consisting of the amino acid sequence of SEQ ID NO: 47 or a sequence having at least 90% sequence identity with it; g. The variable region of the immunoglobulin light chain, comprising the amino acid sequence of SEQ ID NO: 54 or a sequence having at least 90% sequence identity with it, or consisting of the amino acid sequence of SEQ ID NO: 54 or a sequence having at least 90% sequence identity with it; the variable region of the immunoglobulin heavy chain, comprising the amino acid sequence of SEQ ID NO: 48 or a sequence having at least 90% sequence identity with it, or consisting of the amino acid sequence of SEQ ID NO: 48 or a sequence having at least 90% sequence identity with it; h. The variable region of the immunoglobulin light chain, comprising the amino acid sequence of SEQ ID NO: 54 or a sequence having at least 90% sequence identity with it, or consisting of the amino acid sequence of SEQ ID NO: 54 or a sequence having at least 90% sequence identity with it; the variable region of the immunoglobulin heavy chain, comprising the amino acid sequence of SEQ ID NO: 49 or a sequence having at least 90% sequence identity with it, or consisting of the amino acid sequence of SEQ ID NO: 49 or a sequence having at least 90% sequence identity with it. i. The variable region of the immunoglobulin light chain, comprising the amino acid sequence of SEQ ID NO: 54 or a sequence having at least 90% sequence identity with it, or consisting of the amino acid sequence of SEQ ID NO: 54 or a sequence having at least 90% sequence identity with it; the variable region of the immunoglobulin heavy chain, comprising the amino acid sequence of SEQ ID NO: 50 or a sequence having at least 90% sequence identity with it, or consisting of the amino acid sequence of SEQ ID NO: 50 or a sequence having at least 90% sequence identity with it; j. An immunoglobulin light chain variable region comprising the amino acid sequence of SEQ ID NO: 54 or a sequence having at least 90% sequence identity with it, or consisting of the amino acid sequence of SEQ ID NO: 54 or a sequence having at least 90% sequence identity with it; an immunoglobulin heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 51 or a sequence having at least 90% sequence identity with it, or consisting of the amino acid sequence of SEQ ID NO: 51 or a sequence having at least 90% sequence identity with it; k. The variable region of the immunoglobulin light chain, comprising the amino acid sequence of SEQ ID NO: 55 or a sequence having at least 90% sequence identity with it, or consisting of the amino acid sequence of SEQ ID NO: 55 or a sequence having at least 90% sequence identity with it; the variable region of the immunoglobulin heavy chain, comprising the amino acid sequence of SEQ ID NO: 47 or a sequence having at least 90% sequence identity with it, or consisting of the amino acid sequence of SEQ ID NO: 47 or a sequence having at least 90% sequence identity with it; 1. An immunoglobulin light chain variable region comprising the amino acid sequence of SEQ ID NO: 55 or a sequence having at least 90% sequence identity with it, or consisting of the amino acid sequence of SEQ ID NO: 55 or a sequence having at least 90% sequence identity with it; an immunoglobulin heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 48 or a sequence having at least 90% sequence identity with it, or consisting of the amino acid sequence of SEQ ID NO: 48 or a sequence having at least 90% sequence identity with it; m. Variable region of immunoglobulin light chain, comprising the amino acid sequence of SEQ ID NO: 55 or a sequence having at least 90% sequence identity with it, or consisting of the amino acid sequence of SEQ ID NO: 55 or a sequence having at least 90% sequence identity with it; variable region of immunoglobulin heavy chain, comprising the amino acid sequence of SEQ ID NO: 49 or a sequence having at least 90% sequence identity with it, or consisting of the amino acid sequence of SEQ ID NO: 49 or a sequence having at least 90% sequence identity with it; n. Variable region of immunoglobulin light chain, comprising the amino acid sequence of SEQ ID NO: 55 or a sequence having at least 90% sequence identity with it, or consisting of the amino acid sequence of SEQ ID NO: 55 or a sequence having at least 90% sequence identity with it; variable region of immunoglobulin heavy chain, comprising the amino acid sequence of SEQ ID NO: 50 or a sequence having at least 90% sequence identity with it, or consisting of the amino acid sequence of SEQ ID NO: 50 or a sequence having at least 90% sequence identity with it; o. The variable region of the immunoglobulin light chain, comprising the amino acid sequence of SEQ ID NO: 55 or a sequence having at least 90% sequence identity with it, or consisting of the amino acid sequence of SEQ ID NO: 55 or a sequence having at least 90% sequence identity with it; the variable region of the immunoglobulin heavy chain, comprising the amino acid sequence of SEQ ID NO: 51 or a sequence having at least 90% sequence identity with it, or consisting of the amino acid sequence of SEQ ID NO: 51 or a sequence having at least 90% sequence identity with it; p. Variable region of immunoglobulin light chain, comprising the amino acid sequence of SEQ ID NO: 56 or a sequence having at least 90% sequence identity with it, or consisting of the amino acid sequence of SEQ ID NO: 56 or a sequence having at least 90% sequence identity with it; variable region of immunoglobulin heavy chain, comprising the amino acid sequence of SEQ ID NO: 47 or a sequence having at least 90% sequence identity with it, or consisting of the amino acid sequence of SEQ ID NO: 47 or a sequence having at least 90% sequence identity with it; q. The variable region of the immunoglobulin light chain, comprising the amino acid sequence of SEQ ID NO: 56 or a sequence having at least 90% sequence identity with it, or consisting of the amino acid sequence of SEQ ID NO: 56 or a sequence having at least 90% sequence identity with it; the variable region of the immunoglobulin heavy chain, comprising the amino acid sequence of SEQ ID NO: 48 or a sequence having at least 90% sequence identity with it, or consisting of the amino acid sequence of SEQ ID NO: 48 or a sequence having at least 90% sequence identity with it; r. Variable region of immunoglobulin light chain, comprising the amino acid sequence of SEQ ID NO: 56 or a sequence having at least 90% sequence identity with it, or consisting of the amino acid sequence of SEQ ID NO: 56 or a sequence having at least 90% sequence identity with it; variable region of immunoglobulin heavy chain, comprising the amino acid sequence of SEQ ID NO: 49 or a sequence having at least 90% sequence identity with it, or consisting of the amino acid sequence of SEQ ID NO: 49 or a sequence having at least 90% sequence identity with it; s. The variable region of the immunoglobulin light chain, comprising the amino acid sequence of SEQ ID NO: 56 or a sequence having at least 90% sequence identity with it, or consisting of the amino acid sequence of SEQ ID NO: 56 or a sequence having at least 90% sequence identity with it; the variable region of the immunoglobulin heavy chain, comprising the amino acid sequence of SEQ ID NO: 50 or a sequence having at least 90% sequence identity with it, or consisting of the amino acid sequence of SEQ ID NO: 50 or a sequence having at least 90% sequence identity with it; t. The variable region of the immunoglobulin light chain, comprising the amino acid sequence of SEQ ID NO: 56 or a sequence having at least 90% sequence identity with it, or consisting of the amino acid sequence of SEQ ID NO: 56 or a sequence having at least 90% sequence identity with it; the variable region of the immunoglobulin heavy chain, comprising the amino acid sequence of SEQ ID NO: 51 or a sequence having at least 90% sequence identity with it, or consisting of the amino acid sequence of SEQ ID NO: 51 or a sequence having at least 90% sequence identity with it; u. Variable region of immunoglobulin light chain, comprising the amino acid sequence of SEQ ID NO: 57 or a sequence having at least 90% sequence identity with it, or consisting of the amino acid sequence of SEQ ID NO: 57 or a sequence having at least 90% sequence identity with it; variable region of immunoglobulin heavy chain, comprising the amino acid sequence of SEQ ID NO: 47 or a sequence having at least 90% sequence identity with it, or consisting of the amino acid sequence of SEQ ID NO: 47 or a sequence having at least 90% sequence identity with it; v. Variable region of immunoglobulin light chain, comprising the amino acid sequence of SEQ ID NO: 57 or a sequence having at least 90% sequence identity with it, or consisting of the amino acid sequence of SEQ ID NO: 57 or a sequence having at least 90% sequence identity with it; variable region of immunoglobulin heavy chain, comprising the amino acid sequence of SEQ ID NO: 48 or a sequence having at least 90% sequence identity with it, or consisting of the amino acid sequence of SEQ ID NO: 48 or a sequence having at least 90% sequence identity with it; w. The variable region of the immunoglobulin light chain, comprising the amino acid sequence of SEQ ID NO: 57 or a sequence having at least 90% sequence identity with it, or consisting of the amino acid sequence of SEQ ID NO: 57 or a sequence having at least 90% sequence identity with it; the variable region of the immunoglobulin heavy chain, comprising the amino acid sequence of SEQ ID NO: 49 or a sequence having at least 90% sequence identity with it, or consisting of the amino acid sequence of SEQ ID NO: 49 or a sequence having at least 90% sequence identity with it; x. The variable region of the immunoglobulin light chain, comprising the amino acid sequence of SEQ ID NO: 57 or a sequence having at least 90% sequence identity with it, or consisting of the amino acid sequence of SEQ ID NO: 57 or a sequence having at least 90% sequence identity with it; the variable region of the immunoglobulin heavy chain, comprising the amino acid sequence of SEQ ID NO: 50 or a sequence having at least 90% sequence identity with it, or consisting of the amino acid sequence of SEQ ID NO: 50 or a sequence having at least 90% sequence identity with it; or y. The variable region of the immunoglobulin light chain, comprising the amino acid sequence of SEQ ID NO: 57 or a sequence having at least 90% sequence identity with it, or consisting of the amino acid sequence of SEQ ID NO: 57 or a sequence having at least 90% sequence identity with it; the variable region of the immunoglobulin heavy chain, comprising the amino acid sequence of SEQ ID NO: 51 or a sequence having at least 90% sequence identity with it, or consisting of the amino acid sequence of SEQ ID NO: 51 or a sequence having at least 90% sequence identity with it.

[0056] In one embodiment of this disclosure, the antibody binding to uPAR comprises an immunoglobulin light chain variable region comprising or consisting of the amino acid sequence of SEQ ID NO: 56; and an immunoglobulin heavy chain variable region comprising or consisting of the amino acid sequence of SEQ ID NO: 47.

[0057] In one embodiment of this disclosure, the antibody binding to uPAR comprises an immunoglobulin light chain variable region comprising or consisting of the amino acid sequence of SEQ ID NO: 55; and an immunoglobulin heavy chain variable region comprising or consisting of the amino acid sequence of SEQ ID NO: 51.

[0058] In one embodiment of this disclosure, the antibody binding to uPAR comprises a light chain (LC) IgK isotype constant domain. In another embodiment of this disclosure, the antibody binding to uPAR comprises a light chain (LC) constant domain containing or consisting of an amino acid sequence selected from or composed ...

[0059] In one embodiment of this disclosure, the antibody that binds to uPAR comprises a heavy chain (HC) IgG1 constant domain. In another embodiment of this disclosure, the antibody that binds to uPAR comprises a heavy chain (HC) constant domain that comprises or consists of the amino acid sequence of SEQ ID NO: 52.

[0060] Surface plasmon resonance (SPR) studies were conducted to determine the interaction between specific immobilized antibodies and soluble recombinant uPAR. WT Real-time binding kinetics between them. Using single-point alanine scanning mutagenesis, the binding epitopes of FL1, FL2, and FL3 on uPAR were determined via surface plasmon resonance, such as... Figure 1 As shown. Table 1 also lists another anti-uPAR mAb (called 2G10) for reference and comparison. This indicates the reported affinity of immobilized uPAR for the interaction of the Fab fragment of 2G10 in solution (Harel et al., 2019).

[0061] As can be directly seen in Table 1 below, compared with 2G10, FL1, FL2, and FL3 all exhibit better (i.e., lower) K values ​​for human uPAR (marked as huPAR in Table 1). D Values. Due to a lack of cross-species responsiveness, FL3 and 2G10 did not list K values ​​for mouse uPAR (muPAR). D value.

[0062] Table 1 – Characteristics of the selected monoclonal anti-uPAR antibodies.

[0063]

[0064] FL2 and FL3 exhibit similar high affinity, especially FL3, as well as cross-reactivity of FL2, and are able to promote target-mediated internalization upon binding to a unique uPAR epitope. Figure 2 Therefore, it is considered a valuable mAb candidate for targeted mediators for uPAR-targeted therapy (including but not limited to ADCs).

[0065] The data in this paper demonstrate that anti-uPAR antibodies targeting the D1-D3 domains of uPAR provide a surprisingly strong interaction between the antibody and the receptor. Furthermore, it is confirmed that anti-uPAR antibodies targeting the D2 domain of uPAR are extensively internalized, making them highly suitable and efficient for use in ADCs.

[0066] In one embodiment, the anti-uPAR antibody disclosed herein may be a humanized antibody or a mouse antibody that specifically binds to the uPAR receptor. Furthermore, the antibody disclosed herein specifically binds to at least one of the D1 (SEQ ID NO: 2), D2 (SEQ ID NO: 3), or D3 (SEQ ID NO: 4) domains of the uPAR receptor.

[0067] In one embodiment, the antibody FL1 described herein binds to the D2 domain (SEQ ID NO: 3) of the uPAR receptor. More specifically, in one embodiment, the antibody FL1 described herein may be characterized as binding to a conformational epitope on the D2 domain comprising amino acid residues 8 (S), 10 (D), 12 (S), 15 (R), and 17 (R) of the uPAR D2 domain (SEQ ID NO: 3), or composed of the foregoing. This epitope may also be defined as the amino acid residue Ser of full-length human uPAR (SEQ ID NO: 1). 122 Asp 124 Ser 126 Arg 129 and Arg 131 Therefore, one embodiment of this disclosure is an antibody or antigen-binding fragment thereof targeting uPAR, the antibody or antigen-binding fragment thereof binding to the conformational epitope Ser of full-length human uPAR (SEQ ID NO: 1). 122 Asp 124 Ser 126 Arg 129 and Arg 131 The antibody may bind to, or bind to, amino acid residues 8 (S), 10 (D), 12 (S), 15 (R), and 17 (R) or a conformational epitope comprising the uPAR D2 domain (SEQ ID NO: 3). Another embodiment of this disclosure is an isolated antibody or antigen-binding fragment thereof that binds to, an isolated antibody or antigen-binding fragment thereof, to, an amino acid residue 8 (S), 10 (D), 12 (S), 15 (R), and 17 (R) or a conformational epitope comprising the uPAR D2 domain (SEQ ID NO: 3).

[0068] In one embodiment, the antibody FL2 described herein binds to the D1 domain (SEQ ID NO: 2) of the uPAR receptor. More specifically, in one embodiment, the antibody FL2 described herein can be characterized as binding to the conformational epitope Leu of the uPAR D1 domain (SEQ ID NO: 2). 19 Asp 22 Combine.

[0069] In one embodiment, the antibody FL3 described herein binds to the D3 domain (SEQ ID NO: 4) of the uPAR receptor. More specifically, in one embodiment, the antibody FL3 described herein can be characterized as binding to the linear epitope Asp of the uPAR D3 domain (SEQ ID NO: 4). 84 Leu 85 Combine.

[0070] The data presented in this article indicate that anti-uPAR antibodies binding to epitopes in uPAR domain 2 exhibit particularly high levels of internalization, which can also be seen from... Figure 2 This can be seen from the data. In addition, we also used the fluorescently labeled A647-FL1 (A647 refers to Alexa Fluor). TM -647 (Thermo Fisher Scientific) assessed uPAR-dependent uptake and lysosomal transport in mouse uPAR-positive KPC cells using confocal microscopy to verify the lack of FL1 uptake in uPAR knockout control cells (unpublished results).

[0071] It is generally accepted and understood in the art that the CDR is primarily responsible for antigen recognition of the antibodies disclosed herein. Therefore, in the preferred embodiment, any sequence variations are located outside the CDR. All variant antibodies and antigen-binding fragments disclosed herein retain the ability to bind efficiently to uPAR (SEQ ID NO: 1). Residues 1 to 22 of SEQ ID NO: 1 constitute the signal peptide.

[0072] Methods for generating antibodies are well known in the art. For example, antibodies can be generated by any of a variety of methods, including inducing in vivo production of antibody molecules, screening immunoglobulin libraries, or generating monoclonal antibody molecules using cultured cell lines. These methods include, but are not limited to, hybridoma technology, human B-cell hybridoma technology, and Epstein-Barr (EBV) hybridoma technology.

[0073] Similarly, antibody fragments can also be obtained using methods known in the art. For example, antibody fragments according to the invention can be prepared by proteolytic hydrolysis of antibodies with various enzymes, or by expressing DNA encoding the fragment in *Escherichia coli* or mammalian cells (e.g., Chinese hamster ovary cell culture or other protein expression systems). Alternatively, antibody fragments can also be obtained by digesting intact antibodies with pepsin or papain using conventional methods.

[0074] "Antibody" includes essentially complete antibody molecules, mouse antibodies, chimeric antibodies, humanized antibodies, human antibodies, single-chain antibodies, bispecific antibodies, antibody heavy chains, antibody light chains, homodimers and heterodimers of antibody heavy chains and / or light chains, as well as their antigen-binding fragments and derivatives.

[0075] "Antigen-binding fragment" refers to an antibody functional fragment that can bind to uPAR.

[0076] In one embodiment, the anti-uPAR antibody of this disclosure is selected from mouse antibodies, chimeric antibodies, human antibodies, humanized antibodies, humanized antigen-binding fragments, Fab fragments, Fab' fragments, F(ab')2 fragments, Fv fragments, single-chain antibodies (SCA) (e.g., scFv), variable portions of their heavy and / or light chains, or Fab miniantibodies, wherein these fragments or modified antibodies may be derived from mouse antibodies, chimeric antibodies, human antibodies, or humanized antibodies.

[0077] In one implementation, the anti-uPAR antibody is a humanized or fully human monoclonal antibody or its antigen-binding fragment.

[0078] In one embodiment, the anti-uPAR antibody disclosed herein is a recombinant antibody.

[0079] Using antibody fragments instead of whole antibodies offers several advantages. The smaller size of the fragments allows for improved pharmacological properties, such as better tissue penetration. Furthermore, antigen-binding fragments can be expressed and secreted in E. coli or other non-mammalian host cells, enabling easy, large-scale production of these fragments.

[0080] Fab is a fragment containing a monovalent antigen-binding fragment of an antibody molecule. It can be produced by digesting an intact antibody with papain or by other specific proteolytic methods, thereby generating a light chain and a portion of the heavy chain.

[0081] F(ab')2 is a fragment of an antibody that can be obtained by treating an intact antibody with pepsin or other specific proteolytic methods to produce a divalent antigen-binding fragment without subsequent reduction; F(ab')2 is a dimer formed by two Fab fragments linked by two disulfide bonds.

[0082] Fv is a genetically engineered fragment containing both light and heavy chain variable regions, expressed as two strands.

[0083] Single-chain antibodies (SCAs) are genetically engineered molecules containing variable regions in both the light and heavy chains, which are linked together by suitable polypeptide linkers to form gene-fused single-chain molecules, including scFv.

[0084] Polypeptides, polynucleotides, carriers, and host cells

[0085] One embodiment of this disclosure is a separate polynucleotide encoding any polypeptide disclosed herein, specifically a separate polynucleotide encoding the amino acid sequence of any one of SEQ ID NO: 5, 6, 10, and 11.

[0086] In one embodiment, the polynucleotide comprises SEQ ID NO: 41 and / or SEQ ID NO: 42, which encode SEQ ID NO: 5 and SEQ ID NO: 10, respectively.

[0087] In one embodiment, this disclosure provides an isolated polynucleotide comprising SEQ ID NO: 41, and optionally, the polynucleotide further comprising SEQ ID NO: 42.

[0088] One embodiment of this disclosure is a separate polynucleotide encoding any polypeptide disclosed herein, specifically a separate polynucleotide encoding an amino acid sequence of any one of SEQ ID NO: 15, 16, 20, and 21.

[0089] In one embodiment, the polynucleotide comprises SEQ ID NO: 43 and / or SEQ ID NO: 44, which encode SEQ ID NO: 15 and SEQ ID NO: 20, respectively.

[0090] In one embodiment, this disclosure provides an isolated polynucleotide comprising SEQ ID NO: 43, and optionally, the polynucleotide further comprising SEQ ID NO: 44.

[0091] One embodiment of this disclosure is a separate polynucleotide encoding any polypeptide disclosed herein, specifically a separate polynucleotide encoding an amino acid sequence of any one of SEQ ID NO: 25, 26, 30, and 31.

[0092] In one embodiment, the polynucleotide comprises SEQ ID NO: 45 and / or SEQ ID NO: 46, which encode SEQ ID NO: 25 and SEQ ID NO: 30, respectively.

[0093] In one embodiment, this disclosure provides an isolated polynucleotide comprising SEQ ID NO: 45, and optionally, the polynucleotide further comprising SEQ ID NO: 46.

[0094] In one embodiment, the polypeptide is an isolated polypeptide.

[0095] One embodiment of this disclosure is an isolated polynucleotide encoding any polypeptide disclosed herein, specifically an isolated polynucleotide encoding an antibody or antigen-binding fragment thereof comprising: i. The variable region of the immunoglobulin light chain, comprising an amino acid sequence selected from SEQ ID NO: 53-57 or a sequence having at least 90% sequence identity with it, or consisting of an amino acid sequence selected from SEQ ID NO: 53-57 or a sequence having at least 90% sequence identity with it, and ii. The variable region of the immunoglobulin heavy chain, comprising an amino acid sequence selected from SEQ ID NO: 47-51 or a sequence having at least 90% sequence identity with it, or consisting of an amino acid sequence selected from SEQ ID NO: 47-51 or a sequence having at least 90% sequence identity with it.

[0096] One embodiment of this disclosure is a vector, such as an expression vector, which contains a polynucleotide as defined herein.

[0097] In one embodiment of this disclosure, the vector is a mammalian expression vector.

[0098] In one embodiment of this disclosure, the vector is a plasmid vector, such as a plasmid vector selected from the pD2610-v13 (ATUM), pSV and pCMV series of plasmid vectors.

[0099] In one embodiment of this disclosure, the vector is a viral vector, such as a viral vector selected from adenovirus vectors, lentivirus vectors, adeno-associated virus vectors, herpesvirus vectors, vaccinia virus vectors, poxvirus vectors, baculovirus vectors, and oncolytic virus vectors.

[0100] Another embodiment of this disclosure is a host cell containing polynucleotides and / or vectors as defined herein.

[0101] In one embodiment of this disclosure, the host cell containing the polynucleotide and / or vector described herein is selected from CHO (Chinese hamster ovary) cells, COS (derived from CV-1 (ape) and carrying SV40 genetic material) cells, HEK (human embryonic kidney) cells, and HeLa (Henrietta Lacks) cells.

[0102] In one implementation, the host cell is a recombinant host cell.

[0103] Antibody-drug conjugates (ADCs) containing humanized anti-uPAR antibodies.

[0104] Antibody-drug conjugates are a type of highly potent biopharmaceutical designed for targeted therapy. They are highly specific and particularly suitable for treating cancers that are difficult to target with other therapies.

[0105] The inventors have demonstrated through data that anti-uPAR ADCs are a powerful tool for treating cancers characterized by uPAR overexpression. Therefore, the data presented indicate that anti-uPAR ADC treatment results in a significantly greater reduction in overall cell viability compared to non-targeting aTNP-based ADCs.

[0106] A particularly preferred embodiment of this disclosure is an antibody-drug conjugate (ADC) comprising: a. Antibodies or their antigen-binding fragments as defined in this article. b. Surfactants, and c. (Optional) Connector for connecting a) to b).

[0107] The antibodies defined herein may be independently selected from the antibodies FL1, FL2 and / or FL3 described herein, or their antigen-binding fragments.

[0108] In one embodiment of this disclosure, the antibody-drug conjugate (ADC) as defined herein comprises: a. An antibody or its antigen-binding fragment, comprising: i) Containing the amino acid sequence of SEQ ID NO: 6 or the variable region of the immunoglobulin light chain composed of the amino acid sequence of SEQ ID NO: 6, and / or ii) Contains the amino acid sequence of SEQ ID NO: 11 or the variable region of the immunoglobulin heavy chain consisting of the amino acid sequence of SEQ ID NO: 11; b. Surfactants, and c. (Optional) Connector for connecting a) to b).

[0109] In one embodiment of this disclosure, the antibody-drug conjugate (ADC) as defined herein comprises: a. An antibody or its antigen-binding fragment, comprising: i) An immunoglobulin light chain containing the amino acid sequence of SEQ ID NO: 5 or composed of the amino acid sequence of SEQ ID NO: 5; and / or ii) An immunoglobulin heavy chain containing the amino acid sequence of SEQ ID NO: 10 or consisting of the amino acid sequence of SEQ ID NO: 10; b. Surfactants, and c. (Optional) Connector for connecting a) to b).

[0110] In one embodiment of this disclosure, the antibody-drug conjugate (ADC) as defined herein comprises: a. An antibody or its antigen-binding fragment, comprising: i) An immunoglobulin light chain consisting of the amino acid sequence of SEQ ID NO: 5; and ii) An immunoglobulin heavy chain consisting of the amino acid sequence of SEQ ID NO: 10; b. Surfactants, and c. (Optional) Connector for connecting a) to b).

[0111] In another embodiment of this disclosure, the antibody-drug conjugate (ADC) as defined herein comprises: a. An antibody or its antigen-binding fragment, comprising: i) Containing the amino acid sequence of SEQ ID NO: 16 or the variable region of the immunoglobulin light chain composed of the amino acid sequence of SEQ ID NO: 16; and / or ii) Contains the amino acid sequence of SEQ ID NO: 21 or an immunoglobulin heavy chain variable region consisting of the amino acid sequence of SEQ ID NO: 21; b. Surfactants, and c. (Optional) Connector for connecting a) to b).

[0112] In one embodiment of this disclosure, the antibody-drug conjugate (ADC) as defined herein comprises: a. An antibody or its antigen-binding fragment, comprising: i) An immunoglobulin light chain containing the amino acid sequence of SEQ ID NO: 15 or composed of the amino acid sequence of SEQ ID NO: 15; and / or ii) An immunoglobulin heavy chain containing the amino acid sequence of SEQ ID NO: 20 or consisting of the amino acid sequence of SEQ ID NO: 20; b. Surfactants, and c. (Optional) Connector for connecting a) to b).

[0113] In one embodiment of this disclosure, the antibody-drug conjugate (ADC) as defined herein comprises: a. An antibody or its antigen-binding fragment, comprising: i) An immunoglobulin light chain consisting of the amino acid sequence of SEQ ID NO: 15; and ii) An immunoglobulin heavy chain consisting of the amino acid sequence of SEQ ID NO: 20; b. Surfactants, and c. (Optional) Connector for connecting a) to b).

[0114] In another embodiment of this disclosure, the antibody-drug conjugate (ADC) as defined herein comprises: a. An antibody or its antigen-binding fragment, comprising: i) Containing the amino acid sequence of SEQ ID NO: 26 or the variable region of an immunoglobulin light chain composed of the amino acid sequence of SEQ ID NO: 26; and / or ii) Contains the amino acid sequence of SEQ ID NO: 31 or an immunoglobulin heavy chain variable region consisting of the amino acid sequence of SEQ ID NO: 31; b. Surfactants, and c. (Optional) Connector for connecting a) to b).

[0115] In one embodiment of this disclosure, the antibody-drug conjugate (ADC) as defined herein comprises: a. An antibody or its antigen-binding fragment, comprising: i) An immunoglobulin light chain containing the amino acid sequence of SEQ ID NO: 25 or composed of the amino acid sequence of SEQ ID NO: 25; and / or ii) An immunoglobulin heavy chain containing the amino acid sequence of SEQ ID NO: 30 or composed of the amino acid sequence of SEQ ID NO: 30. b. Surfactants, and c. (Optional) Connector for connecting a) to b).

[0116] In one embodiment of this disclosure, the antibody-drug conjugate (ADC) as defined herein comprises: a. An antibody or its antigen-binding fragment, comprising: i) An immunoglobulin light chain consisting of the amino acid sequence of SEQ ID NO: 25; and ii) An immunoglobulin heavy chain consisting of the amino acid sequence of SEQ ID NO: 30; b. Surfactants, and c. (Optional) Connector for connecting a) to b).

[0117] In one embodiment of this disclosure, the antibody-drug conjugate (ADC) as defined herein comprises: a. An antibody or its antigen-binding fragment, comprising: i) Containing the amino acid sequence of SEQ ID NO: 56 or the variable region of an immunoglobulin light chain composed of the amino acid sequence of SEQ ID NO: 56; and / or ii) Containing the amino acid sequence of SEQ ID NO: 47 or the immunoglobulin heavy chain variable region consisting of the amino acid sequence of SEQ ID NO: 47; b. Surfactants, and c. (Optional) Connector for connecting a) to b).

[0118] In one embodiment of this disclosure, the antibody-drug conjugate (ADC) as defined herein comprises: a. An antibody or its antigen-binding fragment, comprising: i) Containing the amino acid sequence of SEQ ID NO: 55 or the variable region of an immunoglobulin light chain composed of the amino acid sequence of SEQ ID NO: 55; and / or ii) Contains the amino acid sequence of SEQ ID NO: 51 or an immunoglobulin heavy chain variable region consisting of the amino acid sequence of SEQ ID NO: 51; b. Surfactants, and c. (Optional) Connector for connecting a) to b).

[0119] Surfactant

[0120] The ADC disclosed herein comprises an active agent, such as a drug, that can be delivered intracellularly to cells expressing uPAR. The active agent may be, for example, a therapeutic agent, a radioisotope, or a detectable label. In a preferred embodiment, the active agent is a therapeutic agent.

[0121] In one embodiment, the active agent may be a radioactive isotope or contain a radioactive isotope. The radioactive isotope can be used as a radiation emitter for treating diseased tissue or for diagnostic purposes. In one embodiment, the radioactive isotope may be derived from… 60 Co、 89 Sr、 90 Y、 99m Tc, 131 I, 137 Cs、 153 Sm or 223 Rd consists of or contains 60 Co、 89 Sr、 90 Y、 99m Tc, 131 I, 137 Cs、 153 Sm or 223 Rd. In one embodiment of this disclosure, the radioactive isotope may be combined with a chelating agent such as DOTA or EDTA or other chelating agents known in the art.

[0122] In one implementation, the active agent is a therapeutic agent. Categories of therapeutic agents include DNA cross-linking agents, DNA alkylating agents, DNA strand scission agents, anthracyclines, antimetabolites, antimicrotubule / antimitotic agents, histone deacetylase inhibitors, kinase inhibitors, metabolic inhibitors, peptide antibiotics, immune checkpoint inhibitors, platinum-based antitumor agents, topoisomerase inhibitors, DNA or RNA polymerase inhibitors, nucleotide-based agents, and cytotoxic antibiotics.

[0123] In a preferred embodiment, the activator is a cytotoxic agent that allows for the effective killing of cells expressing uPAR.

[0124] In one implementation, the active agent is a chemotherapeutic agent.

[0125] In one embodiment, the active agent is a DNA cross-linking agent, such as a DNA cross-linking agent selected from cisplatin or cisplatin derivatives such as carboplatin or oxaliplatin, mitomycin C (MMC), pyrrolobenzodiazepine (PBD), and dipyrrolobenzodiazepine derivatives such as SGD-1882 or any of these derivatives.

[0126] In one embodiment of this disclosure, the active agent is a DNA alkylating agent, such as a DNA alkylating agent selected from nitrogen mustards such as tri(2-chloroethyl)amine, pyridinobenzodiazepine or pyridinobenzodiazepine derivatives, indolinobenzodiazepine dimer, and Duocarmycin SA or any derivative thereof.

[0127] In one embodiment, the active agent is a DNA strand-breaking agent, such as a DNA strand-breaking agent selected from calicheamicin and hamiltrone or any derivative thereof.

[0128] In one embodiment, the active agent is an anthracene ring, such as an anthracene ring selected from daunorubicin, doxorubicin, epirubicin, idarubicin and PNU-159682 or any derivative thereof.

[0129] In one embodiment, the active agent is PNU-159682 or a derivative thereof.

[0130] In one embodiment, the active agent is an antimetabolite, such as an antimetabolite selected from folic acid antagonists like methotrexate, purine antimetabolites like 6-mercaptopurine or 6-thioguanine or fludarabine phosphate or pentostatin or cladribine, and pyrimidine antimetabolites like 5-fluorouracil or 5-fluorodeoxyuridine or cytarabine or gemcitabine or any derivative thereof.

[0131] In one embodiment, the active agent is an antimitotic agent, such as a derivative of auristatin or dolastatin, such as monomethylauristatin E (MMAE), monomethylauristatin F (MMAF), etc.; taxanes such as paclitaxel or docetaxel, etc.; vinca alkaloids such as vinblastine, vincristine, vindesine, or vinorelbine, etc.; mayatansinoid, colchicine, and podophyllotoxin, or any derivative thereof.

[0132] In one embodiment, the active agent is monomethyloritidine E (MMAE) or a derivative thereof.

[0133] In one embodiment, the active agent is a histone deacetylase inhibitor, such as a histone deacetylase inhibitor selected from trichostatin A, vorinostat, belinostat, panabiostat, givinostat, resminostat, abexinostat, quisinostat, rocilinostat, practinostat, CHR-3996, valproic acid, butyric acid, phenylbutyric acid, entinostat, tacedinaline, 4SC202, mocetinostat, romidepsin, nicotinamide, sirtinol, cambinol, and EX-527 or any derivative thereof.

[0134] In one embodiment, the active agent is a kinase inhibitor, such as a kinase inhibitor selected from genistein, lavendustin C, PP1-AG1872, PP2-AG1879, SU6656, CGP77675, PD166285, imatinib, erlotinib, gefitinib, lavendustin A, cetuximab, UCS15A, herbimycin A, and radicicol, or any derivative thereof.

[0135] In one embodiment, the active agent is a metabolic inhibitor, such as a NAMPT inhibitor. Examples of NAMPT inhibitors include APO866, GMX-1777, GMX-1778, ATG-019, and OT-82, or any derivatives thereof.

[0136] In one implementation, the active agent is an immune checkpoint inhibitor, such as a PD-1 inhibitor or a PD-L1 inhibitor. Examples of PD-1 inhibitors include pembrolizumab, nivolumab, cimiplimab, JTX-4014, spartalizumab, camrelizumab, sintilimab, tislelizumab, toripalimab, dostarlimab, AMP-224, and AMP-514. Examples of PD-L1 inhibitors include atezolizumab, avelumab, durvalumab, KN035, CK-301, AUNP12, CA-170, and BMS-986189, or any derivatives thereof.

[0137] In one embodiment, the active agent is a platinum-based antitumor agent, such as a platinum-based antitumor agent selected from lipoplatin, cisplatin, carboplatin, oxaliplatin, nedaplatin, picoplatin, phenanthriplatin, satraplatin, and triplatinum tetranitrate or any derivative thereof.

[0138] In one embodiment, the active agent is a topoisomerase inhibitor, such as a topoisomerase inhibitor selected from camptothecin or its derivatives such as topotecan, belototecan, lurtotecan, irinotecan, SN-38, exatecan, and Dxd or any derivative thereof.

[0139] In one embodiment, the active agent is a DNA or RNA polymerase inhibitor, such as a polymerase inhibitor selected from amanitin or α-amanitin or derivatives thereof, actinomycin D and aphidicolin or any derivatives thereof.

[0140] In one embodiment, the active agent is a nucleotide-based agent, such as an RNA-oligonucleotide or a DNA-oligonucleotide, such as siRNA or miRNA.

[0141] Each antibody molecule may contain one or more drug units. The ratio of the number of drug molecules relative to each antibody is expressed as the drug-to-antibody ratio (DAR). In one embodiment, the DAR is between 1 and 10, for example between 2 and 8, for example between 2 and 6, for example 2 or 4, for example 2.

[0142] connector

[0143] Stable linker between antibody and active agent is an important aspect of ADC technology. Linkers can be based on chemical motifs, including disulfides, hydrazones, or peptides (cleavable) or thioethers (non-cleavable), and control the distribution and delivery of the cytotoxic agent to target cells. Both cleavable and non-cleavable linkers have been proven safe in preclinical and clinical trials.

[0144] The type of linker (cleavable or non-cleavable) imparts specific properties to the delivered drug. For example, a cleavable linker can be cleaved, for instance, by enzymes in the target cell, leading to the effective intracellular release of the active agent, such as a cytotoxic agent. In contrast, ADCs containing non-cleavable linkers lack a drug release mechanism and must rely on mechanisms such as targeted antibody degradation to release the drug. Furthermore, as those skilled in the art will understand, linker composition can influence key factors such as the solubility and pharmacokinetic properties of the ADC as a whole.

[0145] In a preferred embodiment of this disclosure, the uPAR-targeting ADC disclosed herein includes a linker that connects an antibody to an active agent.

[0146] In one embodiment of this disclosure, the connector may be pyrolytic or non-pyrolytic.

[0147] Cleavable groups include disulfide bonds, amide bonds, substituted amide bonds in the form of peptide bonds, thioamide bonds, ester bonds, thioester bonds, vicinal diol bonds, or hemiacetals. These groups or other cleavable bonds may include enzymatically cleavable bonds, such as peptide bonds (cleaved by peptidases), phosphate bonds (cleaved by phosphatases), nucleic acid bonds (cleaved by endonucleases), and sugar bonds (cleaved by glycosidases).

[0148] In another embodiment of this disclosure, the connector is a cleavable connector that allows the active agent to be released intracellularly into the target cells.

[0149] In a further embodiment, the linker is a peptide linker. The choice of peptide sequence is crucial for the success of the conjugate. In some embodiments, the linker is stable to serum proteases but is cleaved by lysosomal enzymes in the target cells.

[0150] In a further embodiment, the linker is a linker containing a peptide that can be enzymatically cleaved, such as a linker containing a peptide that can be cleaved by cathepsins. The cathepsin can be one of several types of cathepsins, a group of lysosomal proteases.

[0151] In another embodiment of this disclosure, the connector comprises or is composed of a dipeptide, such as valine-citrulline (VC) or valine-alanine (VA).

[0152] In one embodiment, the linker comprises or is composed of a dipeptide, such as valine-citrulline (VC) or valine-alanine (VA), which may be further linked to other structural elements via amide bonds. Valine-citrulline-based linkers in which the citrulline carboxyl group is modified to have a substituted amide function can be cleaved by lysosomal cathepsins, while valine-alanine-based linkers in which the alanine carboxyl group is modified to have a substituted amide function can be cleaved by other lysosomal proteases, including other cathepsins.

[0153] In yet another embodiment of this disclosure, the antibody-drug conjugate as defined herein further comprises a spacer, such as a spacer comprising p-aminobenzoic acid, p-aminobenzyl (PAB), p-aminobenzylcarbamate (PABC), p-aminobenzoyloxycarbonyl, or polyethylene glycol (PEG).

[0154] In one embodiment of this disclosure, the antibody-drug conjugate as defined herein comprises p-aminobenzylcarbamate (PABC).

[0155] In one embodiment of this disclosure, the antibody-drug conjugate as defined herein comprises p-aminobenzyl (PAB).

[0156] In one embodiment of this disclosure, the antibody-drug conjugate as defined herein comprises one or more polyethylene glycol (PEG), such as one PEG, two PEGs, three PEGs, four PEGs, or five PEGs. In one embodiment of this disclosure, the antibody-drug conjugate as defined herein comprises PEG spacers, such as PEG2, PEG3, PEG4, or PEG5.

[0157] In another embodiment of this disclosure, the antibody-drug conjugate as defined herein further comprises a linker group, such as a linker group comprising or consisting of: maleimide and hexanoic acid (MC), N-hydroxysuccinimide, a reactive linker group pointing to a modified or unmodified protein-binding carbohydrate, a peptide sequence required for an enzymatic reaction, an azide or an alkyne, or derived therefrom by reaction with an antibody or a chemically or enzymatically generated derivative thereof.

[0158] In one embodiment of this disclosure, the ADC further comprises a linker entity. This linker entity may, for example, link an antibody and a cleavable linker, wherein the linker entity is a reaction product between an antibody functionalized to have a specific reactive moiety and another moiety functionalized on the cleavable linker to have a matching reactive moiety. Such matching chemical reactivity is well known in the field of chemistry and in one embodiment may take the form of alkyne / azide click chemistry, such as SPAAC or CuAAC type reactivity. In another embodiment, the reactive linker group comprises or consists of maleimide and hexanoic acid (MC), wherein the maleimide preferably reacts with cysteine ​​thiol during coupling. In other embodiments, the linker group comprises or consists of: N-hydroxysuccinimide, a reactive linker group pointing to a modified or unmodified protein-binding carbohydrate, a peptide sequence required for an enzymatic reaction, an azide, or an alkyne, or derived from these by reaction with an antibody or a chemically or enzymatically generated derivative thereof.

[0159] In one embodiment of this disclosure, the ADC comprises an antibody targeting uPAR (e.g., any of FL1, FL2, and / or FL3 as defined herein, including humanized variants thereof) and a linker-drug complex. In one embodiment, the linker-drug complex comprises or is composed of one or more of the following: the cytotoxic agent PNU-159682, a spacer (e.g., PEG3, PEG4, PAB, and DMEA), a cathepsin-cleavable linker (e.g., valine-citrulline, VC), and a linker group (e.g., DBCO). In one embodiment of this disclosure, the drug-linker may comprise or be composed of DBCO-PEG4-VC-PAB-DMEA-PNU-159682, with the following structural formula: .

[0160] In another embodiment, the cytotoxic agent is an antimitotic cytotoxic agent, such as monomethylolpropionate E (MMAE), and the drug-linker portion of the ADC within the scope of this invention may comprise or consist of DBCO-PEG3-VC-PAB-MMAE. In one embodiment of this disclosure, the ADC comprises an antibody targeting uPAR as defined herein, and a linker-drug complex DBCO-(PEG)n-VC-PAB-DMEA-PNU159682 or DBCO-(PEG)n-VC-PAB-MMAE, where n represents the length of the polyethylene glycol spacer and can take any value between 1 and 6, preferably 3 or 4, such as DBCO-PEG4-VC-PAB-DMEA-PNU159682 or DBCO-PEG3-VC-PAB-MMAE. In one embodiment, the linker-drug complex contained in the ADC described herein may also be identified by CAS Registry Numbers 2259318-56-2 or 2754384-60-4. These linker-drug complexes have a spacer (p-aminobenzyl), a cathepsin-cleavable linker (valine-citrulline (VC) dipeptide), and a linker group containing dibenzocyclooctylamine (DBCO), sometimes also called aza-dibenzocyclooctylene (ADIBO), which can undergo a SPAAC-type reaction with an azide-functionalized antibody or fragment thereof to form a stable conjugate. DBCO as used herein may refer to CAS: 1255942-06-3, and the DBCO-PEG4 combination may refer to CAS 1537170-85-6. In one embodiment, the SPAAC-type reaction yields one or more regioisomers, all of which are within the scope of this disclosure.

[0161] As used herein, the term "regioisomer" refers to a positional isomer, a class of structural isomers in which the position or substituent changes on the parent structure. In this document, the term "regioisomer" inherently includes all regioisomers, whether pure regioisomers or mixtures of two or more regioisomers, without departing from the chemical structure of the azide-functionalized DBCO-PEG4-VC-PAB-DMEA-PNU159682. The regioisomers identified herein preferably refer to the following positional and / or structural isomers (I) and (II) of the DBCO moiety:

[0162] Wherein Ab represents the anti-uPAR antibody or its antigen-binding fragment, and R represents the linker portion connected to the cytotoxic drug, preferably PEG4-VC-PAB-DMEA-PNU159682 or PEG3-VC-PAB-MMAE.

[0163] In one embodiment, the ADC targeting uPAR in this disclosure comprises or consists of the following: a. The antibodies defined in this article include: i) An immunoglobulin light chain consisting of the amino acid sequence of SEQ ID NO: 5; and ii) An immunoglobulin heavy chain consisting of the amino acid sequence of SEQ ID NO: 10; b. VC connector, c. DBCO or ADIBO linker group d. PAB or PABC spacers, and e. PNU159682 surfactant, The ADC may optionally further include PEG, such as PEG4.

[0164] In one embodiment, the ADC targeting uPAR in this disclosure comprises or consists of the following: a. The antibodies defined in this article include: i) Containing the amino acid sequence of SEQ ID NO: 6 or the variable region of an immunoglobulin light chain composed of the amino acid sequence of SEQ ID NO: 6; and / or ii) Contains the amino acid sequence of SEQ ID NO: 11 or the variable region of the immunoglobulin heavy chain consisting of the amino acid sequence of SEQ ID NO: 11; b. VC connector, c. DBCO or ADIBO linker group d. PAB or PABC spacers, and e. PNU159682 surfactant, The ADC may optionally further include PEG, such as PEG4.

[0165] In one embodiment, the ADC targeting uPAR in this disclosure comprises or consists of the following: a. The antibodies defined in this article include: i) Containing the amino acid sequence of SEQ ID NO: 56 or the variable region of an immunoglobulin light chain composed of the amino acid sequence of SEQ ID NO: 56; and / or ii) Containing the amino acid sequence of SEQ ID NO: 47 or the immunoglobulin heavy chain variable region consisting of the amino acid sequence of SEQ ID NO: 47; b. VC connector, c. DBCO or ADIBO linker group d. PAB or PABC spacers, and e. PNU159682 surfactant, The ADC may optionally further include PEG, such as PEG4.

[0166] In one embodiment, the ADC targeting uPAR in this disclosure comprises or consists of the following: a. The antibodies defined in this article include: i) Containing the amino acid sequence of SEQ ID NO: 55 or the variable region of an immunoglobulin light chain composed of the amino acid sequence of SEQ ID NO: 55; and / or ii) Contains the amino acid sequence of SEQ ID NO: 51 or an immunoglobulin heavy chain variable region consisting of the amino acid sequence of SEQ ID NO: 51; b. VC connector, c. DBCO or ADIBO linker group d. PAB or PABC spacers, and e. PNU159682 surfactant, The ADC may optionally further include PEG, such as PEG4.

[0167] In one embodiment, the ADC disclosed herein may also contain a DMEA spacer and / or group, particularly in those embodiments where the cytotoxic agent is PNU-159682.

[0168] Therapeutic uses

[0169] The uPAR-targeted ADCs described herein can be used to deliver active agents (e.g., therapeutic agents or cytotoxic agents) to cells expressing uPAR and similar proteins, thereby treating a range of diseases and conditions characterized by the expression or overexpression of said proteins.

[0170] Therefore, one embodiment of this disclosure is an antibody or antibody-drug conjugate against uPAR as defined herein, used as a drug.

[0171] Another embodiment of this disclosure is a method for treating a disease characterized by the expression and / or overexpression of uPAR in the cells of a subject, the method comprising administering an antibody or antibody-drug conjugate as defined herein to the subject.

[0172] The urokinase receptor has an optimal tumor-selective expression profile and is present in the matrix components of most invasive cancers, making it an attractive and versatile target for cytotoxic-based targeted therapies such as antibody-drug conjugates.

[0173] In one embodiment of this disclosure, the method is a method as defined herein, wherein a disease characterized by cells expressing and / or overexpressing uPAR is cancer.

[0174] In one embodiment of this disclosure, the method is a method as defined herein, wherein the cancer is selected from pancreatic adenocarcinoma (PAAD), such as pancreatic ductal adenocarcinoma (PDAC), bladder urothelial carcinoma (BLCA), invasive breast carcinoma (BRCA), cervical squamous cell carcinoma and endometrial adenocarcinoma (CESC), cholangiocarcinoma (CHOL), colonic adenocarcinoma (COAD), esophageal cancer (ESCA), glioblastoma, glioblastoma multiforme (GBM), head and neck squamous cell carcinoma (HNSCC), leukemia, acute myeloid leukemia (LAML), ovarian serous cystadenocarcinoma (OV), rectal adenocarcinoma (READ), sarcoma (SARC), cutaneous melanoma (SKCM), and gastric adenocarcinoma (STAD).

[0175] In one embodiment of this disclosure, the cancer is selected from pancreatic cancer, breast cancer, colon cancer, gastric cancer, esophageal cancer, hepatocellular carcinoma, prostate cancer, bladder cancer, mesothelioma, fibrosarcoma, and squamous cell carcinoma.

[0176] In one embodiment of this disclosure, the cancer is breast cancer, such as selected from triple-negative breast cancer (TNBC), basal-like breast cancer (BLBC), HER-2 breast cancer, Luminal A breast cancer, and Luminal B breast cancer.

[0177] In one embodiment of this disclosure, the cancer is preferably pancreatic cancer, such as pancreatic ductal adenocarcinoma.

[0178] In one embodiment of this disclosure, the cancer is mesothelioma, such as pleural mesothelioma, peritoneal mesothelioma, and pericardial mesothelioma.

[0179] In one embodiment of this disclosure, the cancer is leukemia, such as acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL), and chronic myeloid leukemia (CML), or subtypes of these leukemias.

[0180] In one embodiment of this disclosure, the method is a method as defined herein, wherein the cancer is a metastatic cancer.

[0181] In one embodiment of this disclosure, the method is a method as defined herein, wherein the cancer is a solid tumor.

[0182] In one embodiment of this disclosure, the method is a method as defined herein, wherein the cancer is glioblastoma.

[0183] One embodiment of this disclosure is a pharmaceutical composition comprising an effective amount of an antibody or antibody-drug conjugate as defined herein, and a pharmaceutically acceptable buffer, diluent, carrier, adjuvant, or excipient.

[0184] As used herein, "therapeutic effective amount," "effective amount," or "therapeuticly effective" refers to an amount that produces a therapeutic effect under a given condition and administration regimen. This is a predetermined mass of the active ingredient, calculated to produce the desired therapeutic effect when used in combination with the required additives and diluents (i.e., carriers or administration media). Furthermore, it also refers to an amount sufficient to reduce (more preferably, prevent) clinically significant deficiencies in host activity, function, and response. Alternatively, a therapeutically effective amount refers to an amount sufficient to improve a clinically significant condition in the host. Those skilled in the art will understand that the amount of a compound used may vary depending on its specific activity. A suitable dosage may comprise a predetermined amount of the active composition, calculated to produce the desired therapeutic effect when combined with the required diluents.

[0185] The ADC disclosed herein can be formulated into any type of pharmaceutical composition known in the art suitable for delivering the ADC.

[0186] The pharmaceutical composition may be prepared using methods known in the art that have sufficient storage stability and are suitable for human and / or animal administration. For example, the pharmaceutical composition may be lyophilized, such as by freeze-drying, spray drying, spray cooling, or particle preparation using supercritical particle formation technology.

[0187] "Pharmaceutical acceptable" means a substance that is non-toxic and does not diminish the efficacy of an ADC. Such pharmaceutically acceptable buffers, carriers, or excipients are well known in the art (see Remington's PharmaceuticalSciences, 18th edition, A. R Gennaro, Ed., Mack Publishing Company (1990) and handbook of Pharmaceutical Excipients, 3rd edition, A. Kibbe, Ed., Pharmaceutical Press (2000), the contents of which are incorporated herein by reference).

[0188] The term "buffer solution" refers to an aqueous solution containing an acid-base mixture for the purpose of stabilizing pH. Pharmaceutically acceptable buffer solutions are well known in the art.

[0189] The term "diluent" refers to an aqueous or non-aqueous solution intended to dilute the pharmaceutical preparation.

[0190] The term "adjuvant" refers to any compound added to a formulation to enhance the biological effect of the pharmaceutical agent of the present invention. Adjuvants can be one or more of zinc, copper, or silver salts with different anions, such as, but not limited to, fluorides, chlorides, bromides, iodides, thiocyanates, sulfites, hydroxides, phosphates, carbonates, lactates, glycolates, citrates, borates, tartrates, and acetates with different acyl group compositions. Adjuvants can also be cationic polymers, such as cationic cellulose ethers, cationic cellulose esters, deacetylated hyaluronic acid, chitosan, cationic dendritic polymers, cationic synthetic polymers (e.g., polyvinylimidazole), and cationic polypeptides (e.g., polyhistidine, polylysine, polyarginine, and peptides containing these amino acids).

[0191] Excipients can be one or more of carbohydrates, polymers, lipids, and minerals. Examples of carbohydrates include lactose, glucose, sucrose, mannitol, and cyclodextrin, which are added to the composition, for example, to facilitate lyophilization. Examples of polymers include starch, cellulose ethers, cellulose, carboxymethyl cellulose, hydroxypropyl methyl cellulose, hydroxyethyl cellulose, ethyl hydroxyethyl cellulose, alginate, carrageenan, hyaluronic acid and its derivatives, polyacrylic acid, polysulfonates, polyethylene glycol / polyethylene oxide, polyethylene oxide / polypropylene oxide copolymers, polyvinyl alcohol / polyvinyl acetate with different degrees of hydrolysis, and polyvinylpyrrolidone. All these polymers can have different molecular weights and are added to the composition, for example, to control viscosity, achieve bioadhesion, or protect lipids from chemical and protein hydrolytic degradation. Examples of lipids include fatty acids, phospholipids, monoglycerides, diglycerides, and triglycerides, ceramides, sphingolipids, and glycolipids, all of which can have different acyl chain lengths and saturations; egg lecithin, soy lecithin, hydrogenated egg lecithin, and hydrogenated soy lecithin are added to the composition for reasons similar to polymers. Examples of minerals include talc, magnesium oxide, zinc oxide, and titanium dioxide, which are added to the composition to obtain benefits such as reducing liquid aggregation or improving pigment properties.

[0192] The ADC or pharmaceutical composition containing an ADC disclosed herein may be administered via any suitable route known to those skilled in the art. Therefore, possible routes of administration include parenteral (intravenous, subcutaneous, and intramuscular), topical, ocular, nasal, pulmonary, buccal, oral, vaginal, and rectal administration. Additionally, administration via implantation is also possible.

[0193] In a preferred embodiment, the pharmaceutical compositions can be administered parenterally, such as intravenously, intra-articularly, intra-arterially, intraperitoneally, intrathecally, intracardiacly, intrasternally, intracranially, intramuscularly, or subcutaneously, or via infusion techniques. They are conveniently used in the form of sterile aqueous solutions, which may contain other substances, such as sufficient salts or glucose, to make the solution isotonic with blood. If necessary, the aqueous solution should be appropriately buffered.

[0194] In one embodiment of this disclosure, the method is a method as defined herein, wherein the antibody-drug conjugate is administered via parenteral administration, such as intravenous, intra-articular, intra-articular, intraperitoneal, intrathecal, intraventricular, intrasternal, intracranial, intramuscular, or subcutaneous administration, or via infusion techniques.

[0195] Formulations suitable for parenteral administration include aqueous and non-aqueous sterile injectable solutions, which may contain antioxidants, buffers, bacteriostatic agents, and solutes to make the formulation isotonic with the target recipient's blood; and aqueous and non-aqueous sterile suspensions, which may contain suspending agents and thickeners. The formulations may be in single-dose or multi-dose packaging, such as sealed ampoules and vials, and may be stored in a lyophilized (freeze-dried) state, requiring only immediate addition of a sterile liquid carrier, such as water for injection, before use. Immediate injectable solutions and suspensions may be formulated from the aforementioned types of sterile powders, granules, and tablets.

[0196] In one embodiment of this disclosure, the method is a method as defined herein, wherein the antibody-drug conjugate or antibody is administered intravenously.

[0197] In one embodiment of this disclosure, the method is a method as defined herein, wherein the antibody-drug conjugate or antibody is administered subcutaneously.

[0198] In one embodiment of this disclosure, the method is a method as defined herein, wherein an antibody-drug conjugate or antibody is administered in combination with one or more other agents (e.g., one or more other therapeutic agents).

[0199] In one embodiment of this disclosure, the ADC or antibody of this disclosure is administered together with other reagents and / or therapeutic agents that can improve the functional efficiency of the ADC, such as established or novel drugs that can increase lysosomal membrane permeability, thereby facilitating the entry of molecules from inside the lysosome into the cytoplasm, or drugs that can increase blood-brain barrier permeability.

[0200] In one embodiment of this disclosure, the ADC or antibody described herein can be administered in combination with a variety of anticancer drugs, such as antimetabolites, alkylating agents, anthracyclines and other cytotoxic antibiotics, vinca derivatives, antimicrotubule / antimitotic agents, histone deacetylase inhibitors, kinase inhibitors, peptide antibiotics, immune checkpoint inhibitors, platinum-based antitumor drugs, etoposide, taxanes, topoisomerase inhibitors, antiproliferative immunosuppressants, corticosteroids, sex hormones and hormone antagonists, cytotoxic antibiotics and other therapeutic agents.

[0201] The data in this study demonstrate that FL1-PNU-ADCs provide significant immunomodulatory functions in the tumor microenvironment, including the stromal compartment, after treatment. This is the first reported uPAR-targeting ADC with this complementary mechanism.

[0202] In one embodiment of the invention, the ADCs or antibodies described herein induce immunomodulation of tumor-associated cells (e.g., interstitial compartments of pancreatic cancer) as part of an anticancer therapy. The immunomodulatory effects observed with FL1-PNU ADCs enable their use in combination with other immunotherapies, and in a preferred embodiment, they can be used in combination with immune checkpoint inhibitors to obtain more effective ADC therapy.

[0203] Therefore, in one embodiment of this disclosure, the method is a method as defined herein, wherein cells expressing uPAR exhibit uPAR overexpression.

[0204] In one embodiment of this disclosure, the method is a method as defined herein, wherein the cells expressing uPAR are tumor cells.

[0205] In one embodiment of this disclosure, the method is a method as defined herein, wherein the cells expressing uPAR are tumor-associated cells.

[0206] Tumor-associated cells include, but are not limited to, activated fibroblasts, myofibroblasts, neovascularized cells and infiltrating cells of macrophage-monocyte lineage or other leukocyte types, as well as cells of the stromal tissue surrounding the tumor, which are particularly important in cancers such as pancreatic cancer.

[0207] In one embodiment of this disclosure, the method is a method as defined herein, wherein an antibody or antibody-drug conjugate induces cell death and / or inhibits the growth and / or proliferation of cells expressing uPAR.

[0208] In one embodiment of this disclosure, the method is a method as defined herein, wherein an antibody or antibody-drug conjugate induces cells expressing uPAR to release free cytotoxins, thereby leading to cell death and / or inhibition of growth and / or proliferation of neighboring cancer cells. In this disclosure, this may be referred to as the "bystander effect."

[0209] In one embodiment of this disclosure, the method is a method as defined herein, wherein the treatment is amenable or curative.

[0210] Another embodiment of this disclosure is a method for inhibiting tumor progression in a subject, comprising administering to the subject an antibody or antibody-drug conjugate or pharmaceutical composition as defined herein.

[0211] Another embodiment of this disclosure is a method for inhibiting, reducing, or eliminating a subject's ability to metastasize to a tumor, comprising administering to the subject an antibody or antibody-drug conjugate or pharmaceutical composition as defined herein.

[0212] Another embodiment of this disclosure is a kit containing an antibody or antibody-drug conjugate or pharmaceutical composition as defined herein, optionally also containing means and / or instructions for use for administering the antibody-drug conjugate or pharmaceutical composition to a subject.

[0213] In one embodiment, this disclosure relates to the antibody-drug conjugate or pharmaceutical composition described herein for the manufacture of a medicament for treating a disease (e.g., cancer) characterized by cells expressing uPAR.

[0214] We do not wish to be bound by theory, but believe that the specific binding of antibodies to the D2 and / or D1 domains of uPAR can enable species cross-reactivity, as demonstrated by the antibody candidates FL1 and FL2 tested in this paper.

[0215] Example

[0216] Antibody

[0217] All anti-uPAR mAbs and isotype-matched negative control mAbs against trinitrophenol (aTNP) were prepared internally using standard hybridoma technology. Additionally, a recombinant HyHEL-10 mouse IgG1 Kappa isotype control antibody (Sydlabs, #PA007154.m1) was also used as a non-targeted negative control mAb. FL1 and FL2, which exhibit cross-reactivity to human and mouse uPAR, were obtained by immunizing uPAR-deficient mice with recombinant purified soluble human and mouse uPAR, respectively.

[0218] Antibody 2G10 was produced internally using state-of-the-art recombinant technology based on the antibody sequence disclosed in WO 2019 / 113248 A1 (University of California), while the recombinant mouse ATN-658 was purchased from Creative Biolabs (#:PABL-355).

[0219] AntimuPAR pAb P47 was previously described in H. Solberg et al. 2001.

[0220] All interactions studied (e.g., surface plasmon resonance measurements) were performed on a Biacore 3000 (Biacore, Sweden) with a run buffer of 10 mM HEPES, 150 mM NaCl, 3 mM EDTA, and 0.005% (v / v) surfactant P-20 at pH 7.4. Purified anti-uPAR mAb (1 μg / ml) was used... N -hydroxysuccinimide / N- Ethyl -N'-[3-(diethylamino)propyl]carbodiimide was covalently immobilized on a carboxymethylated dextran matrix (CM5 sensor chip), resulting in an immobilized mAb amount of 400-1000 RU. To determine the kinetics of the mAb-uPAR interaction, purified human uPAR was serially diluted 2-fold (0.4-200 nM in run buffer) and analyzed at 50 l / min at 20 °C. To verify the reproducibility of the analytical results, 50 nM uPAR was repeatedly tested at the end of each experiment. After each run, the sensor chip was regenerated by two consecutive injections of 0.1 M acetic acid (dissolved in 0.5 M NaCl). Subsequently, the binding and dissociation phases were fitted to a bimolecular interaction model using BIAevaluation 4.1 software (Biacore), thereby deriving the kinetic rate constants from these real-time interaction analyses. k on and k off .

[0221] To determine the functional epitopes of mAb FL1, FL2, and FL3 of this disclosure, their binding kinetics with a large number of purified uPAR mutants carrying identified single-site alanine substitutions throughout the entire sequence of the mature protein (except for cysteine ​​residues) were analyzed.

[0222] uPAR mutants were tested at 10, 30, and 90 nM. Intact soluble uPAR (residues 23-305 of SEQ ID NO: 1) and immobilized mAb (approximately 7 fmol / mm) were compared. 2 The kinetic constants of the interaction were determined at 20 °C at a flow rate of 50 µl / min, as described above. Functional hotspot residues in the epitopes of these mAbs were defined as residues at positions 1–4, where alanine substitution significantly affects the dissociation rate constant determined by surface plasmon resonance. k off The impact is greatest (see above).

[0223] Humanized antibodies

[0224] Humanized FL1 variants were prepared using a fusion antibody method. CDRs were identified using a combined IMGT / Kabat CDR numbering system. Five human frame sequences derived from mature human IgG were used as the "receptor" frames for the FL1 LCDR and HCDR sequences. Briefly, a BLAST search algorithm was used to search a human IgG sequence database for comparison with the mouse VH domain, and candidate sequences were selected from the top 200 BLAST results based on a combination of frame homology, preservation of key frame residues, and typical loop structures. Therefore, the humanized sequence was expected to be non-immunogenic and retain the typical structure of the CDR loop. Subsequently, the VH and VL domains were synthesized within frames of the human IgG1 constant domain sequence (SEQ ID NO: 52) and the IgK isotype constant region sequence (SEQ ID NO: 59), respectively. Codon optimization was performed on the entire heavy and light chain sequences, and the DNA sequences were validated by DNA sequencing analysis. A total of 25 humanized variants and 1 chimeric antibody (with a mouse variable domain and a human Ig constant domain) were expressed as positive controls, all using a CHO-based transient expression system (see Table 2 below). The antibodies were then purified from the cell culture supernatant using standard chromatography techniques. The purified antibodies were subsequently characterized and ranked based on yield, binding affinity (to muPAR and huPAR), purity, monodispersity, and endotoxin levels. Ten humanized variants were ultimately selected as leading candidate antibodies for further ADC development. Preliminary ADC testing was performed on MDA-MB-231 cells. Figure 10 Results for two representative candidate antibodies are shown: HC1-LC4-PNU (containing VH1 (SEQ ID NO: 47) and VL4 (SEQ ID NO: 56)) and HC5-LC3-PNU (containing VH5 (SEQ ID NO: 51) and VL3 (SEQ ID NO: 55)).

[0225] Table 2: Heavy chain variable region (VH) and light chain variable region (VL) of humanized FL1 antibody. VH1 = SEQ ID NO:47, VH2 = SEQ ID NO:48, VH3 = SEQ ID NO:49, VH4 = SEQ ID NO:50, VH5 = SEQ ID NO:51, VL1 = SEQ ID NO:53, VL2 = SEQ ID NO:54, VL3 = SEQ ID NO:55, VL4 = SEQ ID NO:56, VL5 = SEQ ID NO:57.

[0226]

[0227] cell lines

[0228] All cancer cells in Table 3 were purchased from the American Type Culture Collection (ATCC). Mouse PDAC cell lines KPC1 and KPC2, and the uPAR gene relative knockout clone PLAUR (named uPAR in this paper) were also used. KO (Gifted by Professor Matthew J. Flick.) Parental KPC cells were derived from KPC ( Kras G12D / + ， p53 R172H / + Elas CreER / + Primary tumors in mice, compared to uPAR KO Cloning was performed using a CRISPR RNA-guided Fokl nuclease strategy, as described in Yang Y. et al. 2019. The fibroblast cell lines used for the bystander effect were mouse NIH3T3 (ATCC) and human 1BR3.6 (European Certified Cell Culture Collection, ECACC) cells. The cell lines were cultured at 37°C in a 5% CO2 incubator using the supplier-recommended medium supplemented with 10% fetal bovine serum and 1% penicillin / streptomycin; additionally, 2.5% horse serum was added to the MIA PaCa-2 medium formulation.

[0229] Table 3 – uPAR-positive cell groups detected

[0230] M2-polarized macrophages are derived from bone marrow-derived macrophages, such as... Figure 12 As shown, and see (HJ Jürgensen et al. 2019).

[0231] Fluorescent and radioactive labeling

[0232] According to the manufacturer's instructions, use the mouse IgG1 labeling kit (Thermo Scientific). TM FL1 labeled with Alexa Fluor-647 (A647) was generated using #A20186. Briefly, sodium bicarbonate buffer containing 1 mg / ml mAb at pH 8.3 was incubated with a 5-fold molar excess of A647 reactive dye (dissolved in DMSO, 10 mg / ml) at room temperature with gentle shaking for 1 hour in the dark, followed by gel filtration purification using the PD-10 desalting column provided in the kit. The degree of labeling (DOL) of the A647 conjugate was characterized by absorbance measurements according to the supplier's protocol.

[0233] FL1 mAb was performed as described by Engelholm et al. (2003).125 I-labeling was performed, followed by SDS-page analysis and visualization on a phosphorescent imager (Fuji Fla 3000).

[0234] Radiometric assessment of anti-uPAR mAb uptake in uPAR-positive leukemia cells U937

[0235] Three copies were inoculated into a 24-well plate (10). 5 Cells in (cells / well) and 125 I-labeled a-uPAR mAb was incubated at 37°C for 4 hours to induce endocytosis. 125 I-collagen was used as the non-binding control (NC). After incubation, cells were washed three times with ice-cold 1×PBS to remove excess dye, and then briefly treated with 0.25% trypsin-EDTA containing 50 μg / mL proteinase K to remove surface-bound mAbs. Cells were collected by centrifugation and resuspended in FACS buffer. The radioactivity of the precipitate (corresponding to the endocytic fraction) was measured in a γ counter (Nucleocounter 3000), and the results are expressed as counts per minute (CPM).

[0236] confocal microscope

[0237] Microscopic-based qualitative assessment of A647-labeled FL1 uptake was performed on adherent KPC cells seeded in triplicate at 5000 cells / well in 24-well plates. Adherent cells were seeded on coverslips. After incubation at 37°C for 4 hours, the cells were washed three times with ice-cold 1X PBS to remove unbound antibodies. Live cells were then incubated for 15 minutes at room temperature with 1X PBS containing 1 µg / ml A488-WGA (Invitrogen, #W11261) for surface staining, followed by three more washes. Cells were then fixed with 4% PFA at room temperature for 15 minutes and stained with ProLong PBS containing DAPI as a nuclear counterstain. TM Gold Anti-Fading Preservative Tablets (Thermo Scientific) TM Mount the slide (#P36941) and allow it to dry overnight. Acquire images using a 40X oil immersion microscope on a Zeiss LSM 800 or LSM900 confocal microscope.

[0238] To investigate antibody lysosomal transport, cells were pre-incubated with 100 μM of E64d protease inhibitor (Sigma-Aldrich, #330005) at 37 °C for 1 hour prior to assay.

[0239] Histology and Immunohistochemistry

[0240] Tissue sections were dewaxed (using tissue clearing agent (Sakura Finetek #1466) and decreasing concentrations of ethanol), rinsed with tap water, and stained with uPAR as follows: After dewaxing, tissue sections were treated with 5 μg / mL proteinase K at 37 °C for 15 min for antigen retrieval. After washing, the samples were incubated in 1% H2O2 for 15 min to block endogenous peroxidase, followed by washing with tap water and 0.5% TBS-T. Then, the sections were diluted 1:1000 with primary antibody (rabbit anti-smuPAR pAb P47, 0.5 μg / ml) in antibody dilution buffer (Dako, #S3022) and incubated overnight at 4 °C. Finally, detection was performed using the following secondary antibody: Envision-HRP anti-rabbit antibody (#4003, Dako). After incubation at room temperature for 45 min, the chromogenic substrate DAB (Origene, #C09-12) was added and incubated for another 5 min. Finally, after rinsing, the sections were counterstained with Mayer hematoxylin for 30 seconds, followed by dehydration, drying, and mounting, as described above. All stained sections were scanned using a NanoZoomer-XR digital slide scanner C12000-01 (Hamamatsu) and analyzed using NDP.view2 Plus software.

[0241] Generation and characterization of site-specific DAR2 and DAR4 glycan-linked ADCs

[0242] Site-specific glycan-linked PNU-159682-ADC was developed using a copper-free click chemistry approach with the help of GlyCLICK. TM The conjugation technique utilizes a commercially available click-reactive linker-toxin DBCO-PEG4-VC-PAB-DMEA-PNU-159682 (Levena) to produce ( Figure 3 This method follows a strain-promoted alkyne-azidocycloaddition reaction (SPAAC) well known to those skilled in the art. Azido-modified mAbs were produced using the GlyCLICK Azido Activation Kit (Genovis) according to the manufacturer's protocol. Briefly, antibodies buffer-exchanged in 1X TBS were deglycosylated with immobilized GlycINATOR© to the N-acetylglucosamine (GlcNAc) moiety within the IgGFc N-glycan, followed by azido activation of the exposed GlcNAc with GalT (Y289L) and UDP-GalNAz. After overnight incubation at 30°C, the cells were centrifuged using a 2 ml desalted column (40 KMWCO; Thermo Scientific). TMUnbound UDP-GalNAz was removed by purification (#87768). Prior to conjugation, successful azide modification was confirmed by Coomassie brilliant blue staining on a reducing SDS-PAGE gel. The adapter-loador (dissolved in a mixture of 80% propylene glycol (PG) and 20% N,N-dimethylformamide (DMF)) was added with a 15-fold molar excess of Az-modified mAb (dissolved in TBS) at a ratio of 3:1, and the reaction was incubated overnight with gentle shaking at 25°C. SDS-PAGE analysis was then performed, and the conjugate was purified using a Zeba centrifugation desalting column, or incubated for another 24 hours (if conjugation was incomplete). The concentration, purity, and drug-antibody ratio (DAR) of the ADC product were further determined by UV-Vis spectroscopy and MALDI-TOF. Based on FL1, FL2, FL3, HC1-LC4, HC5-LC3, 2G10, ATN658, or aTNP, confirm successful coupling and the expected DAR value of 2 in the ADC construct.

[0243] Similarly, the generation of glycan-linked DAR4 MMAE-ADCs also employs a similar site-specific glycan conjugation method, using SiteClick. TM Antibody azide modification kit (Invitrogen, ##S20026) was used, and the procedure was performed according to the manufacturer's instructions. In short, the terminal galactose residues on the N-linked sugar of the antibody Fc region were first removed by β-galactosidase cleavage (in 1XTBS), followed by azide activation catalyzed by 1,4-galactosyltransferase (Gal-T) of the terminal GlcNAc residues using UDP-GalNAz. After overnight incubation at 30°C, the antibody was centrifuged using a 2 ml desalted column (40K MWCO; Thermo Scientific). TM Purification was performed to remove unbound UDP-GalNAz. Successful azide modification was confirmed by reducing SDS-PAGE analysis using Coomassie Brilliant Blue staining. Finally, the azide-modified antibody was conjugated with a 15-fold molar excess of the linker-loaded DBCO-(PEG)3-VC-PAB-MMAE (MedChemExpress, #HY-111012, CAS: 2754384-60-4) (dissolved in a mixture of 80% propylene glycol (PG) and 20% N,N-dimethylformamide (DMF)) at a ratio of 3:1, and the reaction was incubated overnight with gentle shaking at 25°C. Conjugation was confirmed by SDS-PAGE analysis, and the conjugate was purified using a Zeba centrifugal desalting column. DAR values ​​were determined by UV-Vis spectroscopy, confirming that both FL1- and Hyhel-based ADCs achieved the expected DAR value of 4.

[0244] In vitro cytotoxicity assay

[0245] In 100 µl of complete growth medium, add to triplicate portions at 3 x 10⁻⁶ per well. 4 Cells were seeded at 1 / ml in 96-well plates with eight serially diluted 4-fold ADC conjugates (FL1-PNU, FL2-PNU, FL3-PNU, HC1-LC4-PNU, HC5-LC3-PNU, 2G10-PNU, ATN658-PNU, FL1-MMAE and NC-PNU and MMAE-NC ADC) and (if applicable) unconjugated FL1 (naked FL1) and free toxin (PNU). Control wells received only PBS-added medium. After culturing at 37°C, 5% CO2 for 3 or 5 days, cells were incubated for 1 hour with 15 µl of CellTiter 96® Aqueous Single Solution Cell Proliferation Assay Kit (MTS, Promega) and then cell viability was assessed. The absorbance of the plates was then measured at 490 nm and 650 nm (for background subtraction) using a Spectra Max Plus microplate reader (Molecular Devices). Cell viability was calculated as a percentage of untreated control samples. Figure 4 , Figure 5 (FL1-PNU) and Figure 11 (FL1-MMAE) shows that, compared to the non-targeted NC-PNU ADC, the FL1-based ADC exhibits stronger, uPAR-dependent cytotoxicity (except in uPAR knockout cells). This effect was observed in all uPAR-expressing cell lines tested. Furthermore, the FL1-based ADC FL1-PNU appears to be more effective than 2G10-PNU and ATN658-PNU. Figure 8 The humanized candidates HC1-LC4-PNU and HC5-LC3-PNU appear to be more effective than the parental FL1-PNU based on mouse FL1. Figure 11 ).

[0246] In vivo efficacy of FL1-PNU in the PDAC model

[0247] The efficacy of FL1-PNU was tested in xenograft and allogeneic mouse models of PDAC using the cross-reactivity of FL1-PNU. Relevant data can be found in [link to relevant data]. Figure 6 Xenografts derived from the AsPC-1 cell line were grafted at a rate of 2.5 x 10⁻⁶ cells per mouse. 6 A concentration of 1 cell / 100 µl sterile PBS was subcutaneously transplanted into the right upper quadrant of 7-10 week old female BALB / cAnNRj-Foxn1 nu / nu mice (JANVIER LABS). The tumor volume was increased to 80-150 mm. 3Animals were randomly divided into groups of 12 based on average tumor size and body weight, and were injected weekly via tail vein for three consecutive weeks with either the test drug (0.75 mg / ml) or a PBS-mediated control. Tumor volume (mm²) was recorded two to three times weekly. 3 = Length × Width 2 / 2) and body weight, and closely monitor mice for any signs of discomfort or abnormal behavior. At the preset endpoint (tumor volume exceeding 1000 mm), 3 or diameter exceeding 12 Mice were euthanized when they exhibited the following symptoms: tumor ulceration exceeding 8 mm (length); weight loss of approximately 15%; or abnormal behavior. 8-week-old wild-type C57BL / 6 mice (Jackson Laboratories) were injected orally into the pancreas with a solution containing 5 × 10⁻⁶ mmol / L. 4 Luc-KPC2 cells were injected with 30 µl of sterile PBS and then subjected to a pre-defined surgical procedure for syngeneic transplantation studies. Two weeks post-surgery, D-luciferase (PerkinElmer, #122799) was administered intraperitoneally, and tumor growth was qualitatively monitored using an IVIS Spectrum imaging system (PerkinElmer). Mice with no detectable luciferase activity (2 / 30) were excluded from the study. The remaining mice (28 / 30) were randomly assigned to three treatment groups (n = 8–10), with equal mean luciferase activity in each group, and were administered the drug twice as described above. The mice were then sacrificed on day 28 post-tumor implantation. Treatment efficacy was assessed by comparing tumor weight at the end of the study.

[0248] Immunophenotypic analysis

[0249] At harvest, mice were euthanized and tumors were removed. Spleens from tumor-free mice were collected simultaneously as controls for proper staining. Briefly, the dissected tumors were gently rinsed with ice-cold 1X PBS, and any attached healthy tissue was removed with a sterile scalpel. The tumors were then minced with a razor blade and placed in 6-well plates on ice for enzymatic digestion in 2–3 ml of digestion buffer consisting of RPMI 1640 medium, 5 mM CaCl2, 1.5 mg / ml collagenase D (11088858001, Roche), and 100 μg / ml DNase I. The samples were then incubated at 37°C for 35 minutes with gentle shaking every 10 minutes to promote digestion. The spleens were physically ground into sterile culture dishes and placed on ice until the tumors were completely digested. The isolated single-cell suspension was then rinsed with ice-cold medium to neutralize enzyme activity, filtered through a 70 μm cell sieve, and collected by centrifugation. Subsequently, following the manufacturer's instructions, the cell pellet was resuspended in 5 ml of ACK lysis buffer (Gibco).TM Red blood cell lysis was performed in (#A1049201). After washing with ice-cold 1X PBS, cells were counted and 2×10⁻⁶ cells were added. 6 100 μl of cells per cell were aliquoted into the wells of a 96-well polypropylene round-bottom plate (Corning® 96, #3879), collected by centrifugation, and used for subsequent staining. Live-dead cell staining was performed first by resuspending the cell pellet in 100 µl of diluted Zombie Aqua ether. TM (1:400, diluted in 1X PBS) and then incubated at room temperature in the dark for 20 minutes. A mixture of live and heat-inactivated spleen cells was used as a live-dead cell monostain control (1:800). Immunostaining was then performed using universal lymphoid and myeloid cell differentiation groups. The following anti-mouse antibodies / dyes (purchased from BD Biosciences unless otherwise specified) were used: CD45-BUV395 (1:800), TCR β-Pacific blue (BioLegend, 1:500), CD19-PE-Cy7 (1:800), CD11b-PerCP-eFluor 710 (Invitrogen, 1:1600), Ly6C-PE (1:800), and Ly6G-APC-H7 (1:400). Use 100 µl of freshly prepared antibody master mix in FACS staining buffer (SB, 1X PBS, 1% BSA) (containing True-Stain Monocyte Blocker for Fc receptor blockade). TM Samples were stained with (BioLegend, #426102) and incubated at 4°C in the dark for 20 minutes. Single-stain controls were prepared using UltraComp eBeads (Invitrogen, #01-2222-42). After surface staining and two SB washes, samples and corresponding eBeads were fixed in paraformaldehyde and stored at 4°C in the dark until detection. Samples were collected using Becton Dickinson LSL Fortessa and FACSDiva 8.0.1 software at the UNC Flow Cytometry Core Laboratory. At least 10 samples were collected for both cell samples and beads. 6 and 10 4 Each event / pipe. Using FlowJo TM Software v10.8.1 is used for data analysis.

[0250] FL-1-PNU targets uPAR-positive immune and stromal cells and induces in vitro bystander cytotoxicity.

[0251] Transwell co-culture assay

[0252] uPAR-positive fibroblasts, human 1BR3.G cells, and mouse NIH3T3 cells were co-cultured with uPAR in dual-chamber transwell clear culture dishes. KO KPC2 cancer cells were used to assess the bystander activity of ADCs. A total of 7.5 × 10⁻⁶ cells were collected. 4 uPAR KO KPC2 cells were seeded into the lower chamber of a 6-well cell culture plate, and 1.5 × 10⁶ cells were added. 5 Fibroblasts were seeded into the upper transwell insert chamber (Corning, #3450), which has a microscopically transparent polyester membrane with a pore size of 0.4 μm and is specifically designed for drug delivery studies. 24 hours post-seeding, FL1-PNU or aTNP-PNUADC (4 nM) was added to the upper insert chamber. After 72 hours of incubation, the cells in both chambers were separated, and live / dead cells were stained with Solution-13 AO-DAPI staining agent (ChemoMetec, #910-3013). Cell viability was then measured using a NucleoCounter NC-3000 (ChemoMetec).

[0253] Given the confirmed negative impact of dysplastic TMEs on PDAC resistance, targeting cancer, matrix, and immunosuppressive components may enhance treatment response. Therefore, we investigated the activity of FL1-PNU against M2-differentiated bone marrow-derived macrophages (M2-BMDM) and human and mouse fibroblast lines (i.e., 1BR3.G and NIH3T3). These cells served as in vitro models of tumor-associated macrophages and activated cancer-associated fibroblasts (CAFs), which dominate PDAC TMEs and are known to upregulate uPAR compared to normal quiescent cells. Figure 12 ).

[0254] Bone marrow progenitor cells were isolated from the pelvis, femur, and tibia of FVB / N mice (8-10 weeks old). The collected cells were then cultured in 96-well plates (100 μL per well, containing 5 × 10⁶ cells / well). 4Cells were differentiated into M2-like macrophages for 4 days in DMEM (10% FBS, 1% P / S) medium containing 20 ng / mL mM-CSF (Biotechne), followed by stimulation for another 2 days with 20 ng / mL mM-CSF and 20 ng / mL mIL-4 (Biotechne). For flow cytometry analysis, cells were incubated on ice for 1 hour in the dark with a predetermined saturation concentration of A647-labeled FL1. After staining, samples were stained with 1 µg / mL 7-AAD (Invitrogen, #1890506) to exclude cell death, and finally collected on a BD LSR Fortessa 20X (BD Biosciences).

[0255] Compared to aTNP-PNU, both M2-BMDM and fibroblasts showed dose- and uPAR-dependent responses to FL1-PNU after three days of ADC exposure. Figure 13 A).

[0256] Next, we evaluated whether FL1-PNU-generated uPAR-mediated matrix / immune targeting could also promote the killing of uPAR-negative cancer cells through bystander cytotoxicity. To this end, we used a two-compartment Transwell culture dish, seeding uPAR-positive fibroblasts into the upper permeable insert chamber, and then... KO KPC2 cancer cells were seeded into the lower layer of wells. Figure 13 B). In this system, any potential bystander factors can diffuse through the porous membrane to the bottom compartment. Based on the results of single culture, we selected a FL1-PNU concentration of 4 nM to achieve minimal targeting of uPAR-null cells and maximum cytotoxicity (EC80) to uPAR-positive fibroblasts. Figure 13 C). Both monoculture and transwell coculture were treated with FL1-PNU and aTNP-PNU ADC, and cell viability was assessed after three days of incubation. As expected, under monoculture conditions, direct FL1-PNU treatment significantly affected the viability of uPAR-positive fibroblasts compared to aTNP-PNU ADC, but had little effect on uPAR-positive fibroblasts. KO KPC2 cell viability was not affected. However, in the co-culture system, we observed a significant decrease in both uPAR-positive and uPAR-negative cell populations after exposure to FL1-PNU. Figure 13 D). These results indicate that free PNU diffuses from FL1-PNU-targeted uPAR-positive fibroblasts into the culture medium, leading to the diffusion of adjacent uPAR cells. KO Cell killing, these uPARs KOCells were sensitive to PNU itself (data not shown), but not to uPAR-ADC.

[0257] These data suggest that FL1-PNU can effectively target the matrix and immune cells in a uPAR-dependent manner. This leads to drug release and, through a bystander effect, kills uPAR-negative tumor cells that are sensitive to cytotoxins.

[0258] Results and Conclusions

[0259] Internalization data in uPAR-expressing U937 cells showed that all tested mAbs FL1, FL2, and FL3 were significantly internalized, supporting their potential as ADC candidates. FL1 exhibited the best internalization and was therefore selected as the lead candidate. Importantly, no uptake was observed in receptor-deficient cells (i.e., KPC knockout cells), confirming the uPAR-mediated internalization process (unpublished results).

[0260] Subsequently, we evaluated the in vitro and in vivo efficacy of an FL1-PNU-based ADC containing the potent anthracycline derivative PNU159682 in relevant preclinical PDAC models. We also evaluated the in vitro efficacy of FL1 conjugated with a payload possessing a different cytotoxic mechanism (i.e., the antimitotic cytotoxic MMAE).

[0261] In in vitro experiments, various human and mouse cell lines with different uPAR levels were examined, demonstrating that these cells were highly sensitive to the antitumor activity of FL1-ADC, and that this sensitivity was uPAR- and concentration-dependent, with EC50 values ​​in the low nanomolar / picomolar range and a broad selective therapeutic window. Figure 4 , 5 (and 11). In fact, neither uPAR-null cells nor cells exposed to the non-targeted ADC (aTNP-PNU) responded, further confirming that effective FL1-based a-uPAR ADCs possess specific cytotoxic activity. The cytotoxicity was demonstrated to stem from the selective delivery of the cytotoxic fraction, as unconjugated FL1 does not have antitumor activity.

[0262] Similarly, FL1-ADCs based on humanized FL1 variants also exhibited significant in vitro cytotoxicity. For example... Figure 10 As shown, these humanized FL1-ADCs appear to be more active than the parental FL1-based ADCs.

[0263] In addition to PDAC cells, FL1-PNU also showed strong activity against uPAR-positive tumor-associated macrophages and CAFs in in vitro models. Figure 12 and Figure 13Directly targeting these cells can help alleviate fibrosis and immunosuppressive barriers, and weaken tumor-stromal interactions that drive PDAC progression and metastasis. Furthermore, as our transwell co-culture experiments have shown, uPAR-positive cells, when targeted by FL1-PNU, can mediate a bystander effect, which can further enhance antitumor responses by promoting drug distribution in tumors with heterogeneous uPAR expression, such as PDAC.

[0264] To better simulate the biological complexity of human diseases, we further validated the in vivo efficacy of FL1 in different PDAC mouse models. The simplest model included a xenograft immunodeficiency model based on AsPC-1, one of the sensitive human cell lines studied. Three treatment groups consisted of a uPAR-targeted ADC, a non-specific ADC, and a PBS-mediated control group, with optimized treatment regimens to ensure tolerability. Compared to the two control groups whose treatment was discontinued due to excessive tumor burden, FL1-PNU rapidly and significantly reduced tumor volume in all but one of the tested mice, with half of the mice achieving complete cure after treatment cessation. Figure 6 A). Five mice experienced tumor recurrence, developing small lumps later in the study, but their condition remained stable until the end of follow-up. Importantly, FL1-PNU-induced antitumor activity significantly prolonged overall survival. Figure 6 B), at the study endpoint, 9 out of 12 mice were still alive and there was no significant weight loss associated with treatment. Figure 6 C).

[0265] To validate these encouraging findings in a more biologically relevant PDAC model and in a context that preserves immunological integrity, we replicated this study using a definitively confirmed syngeneic in situ model established from KPC cells in an immune-functional host. Figure 6 D). Only two doses were required for FL1-PNU to exert a significant uPAR-dependent antitumor effect, confirmed by a significant reduction in tumor volume in treated mice compared to two control groups. Figure 6 E and 6F). Notably, no weight loss or clinically obvious toxicity was observed in the mice, indicating that FL1-PNU was well tolerated under the experimental treatment regimen. Figure 6 G). Histological evaluation of major animal organs such as the liver and kidneys further confirmed this, with no obvious morphological abnormalities detected.

[0266] Importantly, immunological analysis of syngeneic in situ tumors showed significant infiltration of T lymphocytes and inflammatory cells after FL1-PNU treatment. Figure 8In stark contrast, the PBS group was almost entirely lacking in these immune cell components, consistent with the immunologically “cold” nature of the PDAC tumor microenvironment (TME). Furthermore, in the mediator sample, myeloid cells (CD11b) were present. + The number of neutrophils and myeloid-derived suppressor cells (which belong to and dominate the suppressive immune repertoire of the tumor stroma) was almost halved in the FL1-PNU treatment group. These data suggest that the tumorigenic activity of FL1-PNU triggers broad immunomodulation, activating both adaptive and innate immunity, which in turn enhances the efficacy of ADCs. Simultaneously, targeting the myeloid-dominated suppressive microenvironment can desensitize immune tolerance and promote activated antitumor immune responses. Since uPAR is highly expressed on these cells, the observed reduction may actually stem from the direct targeting of these cells by the ADC, suggesting that currently effective ADCs possess a potent uPAR-specific stroma-targeting effect.

[0267] The potency of FL1-PNU was compared with that of an ADC containing two known antibodies, 2G10 and ATN658, conjugated with the same adapter payload. Figure 8 It can be seen that the FL1-PNU appears to be more effective than the 2G10-PNU and ATN658-PNU, confirming that FL1 is a superior anti-uPAR mAb candidate in ADC mode.

[0268] Similarly, we compared the in vitro efficacy of ADCs containing FL1, FL2, and FL3 mAbs with that of ADCs containing the known antibody 2G10. All these antibodies were conjugated to the same linker payload. Figure 9 It can be seen that the efficiency of FL1-PNU appears to be higher than all other conjugates tested, including 2G10-PNU, which confirms that FL1 is a superior anti-uPAR mAb candidate in ADC mode. FL2-PNU appears to be more efficient than 2G10 and FL3-PNU, respectively.

[0269] Based on the presented results, particularly the antiproliferative effects of FL1-based ADCs in various in vitro and in vivo models, including the PDAC model, we believe that conjugates containing FL1 and a payload (e.g., PNU159682 or MMAE), such as the effective FL1-DBCO-PEG4-VC-PAB-DMEA-PNU-159682 or FL1-DBCO-(PEG)3-VC-PAB-MMAE modalities in the embodiments of this application, are particularly suitable for treating cancers expressing uPAR, especially PDAC. ADCs containing a humanized version of FL1 are also considered particularly suitable for treating cancers expressing uPAR.

[0270] Furthermore, given that FL1-PNU conjugates have demonstrated similar robust and selective in vitro efficacy in uPAR-positive U937, U87, MDA-MB-231, and REN-Luc cells, as well as FL1-MMAE ADCs in uPAR-positive HT-1080 cells, we evaluated the therapeutic value of this ADC in other aggressive therapeutic indications (leukemia, glioblastoma, triple-negative breast cancer, fibrosarcoma, and mesothelioma, respectively), and laid a solid foundation for its application in all existing uPAR-positive tumor indications (e.g., as a pan-cancer therapy).

[0271] Sequence Overview

[0272] References

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[0290] Wu, T.T., Kabat, E. A. (2008) “Pillars article: an analysis of thesequences of the variable regions of Bence Jones proteins and myeloma lightchains and their implications for antibody complementarity.” J. Exp. Med.132, 211-250. J. Immunology 180, 7057-7096.

[0291] Dunbar, J., Deane, C. M. (2016) “ANARCI: antigen receptor numberingand receptor classification.” Bioinformatics, 32, 298-300.

Claims

1. An antibody or an antigen-binding fragment thereof that binds to urokinase-type plasminogen activator receptor (uPAR), wherein the antibody is selected from the following: a. An antibody or its antigen-binding fragment, containing i. An immunoglobulin light chain comprising the amino acid sequence of SEQ ID NO: 5 or a sequence having at least 90% sequence identity with it, or consisting of the amino acid sequence of SEQ ID NO: 5 or a sequence having at least 90% sequence identity with it, and ii. An immunoglobulin heavy chain comprising the amino acid sequence of SEQ ID NO: 10 or a sequence having at least 90% sequence identity with it, or consisting of the amino acid sequence of SEQ ID NO: 10 or a sequence having at least 90% sequence identity with it; b. An antibody or its antigen-binding fragment, containing i. The variable region of the immunoglobulin light chain, comprising the amino acid sequence of SEQ ID NO: 6 or a sequence having at least 90% sequence identity with it, or consisting of the amino acid sequence of SEQ ID NO: 6 or a sequence having at least 90% sequence identity with it, and ii. The variable region of the immunoglobulin heavy chain, comprising the amino acid sequence of SEQ ID NO: 11 or a sequence having at least 90% sequence identity with it, or consisting of the amino acid sequence of SEQ ID NO: 11 or a sequence having at least 90% sequence identity with it; c. An antibody or its antigen-binding fragment, containing i. The variable region of the immunoglobulin light chain, comprising the amino acid sequence of SEQ ID NO: 35 as complementarity-determining region 1 (CDR1), the amino acid sequence of SEQ ID NO: 36 as CDR2, and the amino acid sequence of SEQ ID NO: 37 as CDR3, and ii. The variable region of the immunoglobulin heavy chain, comprising the amino acid sequence of SEQ ID NO: 38 as complementarity-determining region 1 (CDR1), the amino acid sequence of SEQ ID NO: 39 as CDR2, and the amino acid sequence of SEQ ID NO: 40 as CDR3; d. An antibody or its antigen-binding fragment, containing i. The variable region of the immunoglobulin light chain, comprising the amino acid sequence of SEQ ID NO: 7 as complementarity-determining region 1 (CDR1), the amino acid sequence of SEQ ID NO: 8 as CDR2, and the amino acid sequence of SEQ ID NO: 9 as CDR3, and ii. The variable region of the immunoglobulin heavy chain, comprising the amino acid sequence of SEQ ID NO: 12 as complementarity-determining region 1 (CDR1), the amino acid sequence of SEQ ID NO: 13 as CDR2, and the amino acid sequence of SEQ ID NO: 14 as CDR3; e. A humanized version of an antibody or antigen-binding fragment of any of a, b, c, or d; f. A chimeric version of an antibody or antigen-binding fragment of any of a, b, c, or d; g. An antibody or its antigen-binding fragment, containing i. An immunoglobulin light chain variable region comprising an amino acid sequence selected from SEQ ID NO: 53-57 or a sequence having at least 90% sequence identity with it, or consisting of an amino acid sequence selected from SEQ ID NO: 53-57 or a sequence having at least 90% sequence identity with it, and ii. The variable region of the immunoglobulin heavy chain, comprising an amino acid sequence selected from SEQ ID NO: 47-51 or a sequence having at least 90% sequence identity with it, or consisting of an amino acid sequence selected from SEQ ID NO: 47-51 or a sequence having at least 90% sequence identity with it.

2. An antibody or an antigen-binding fragment thereof that binds to urokinase-type plasminogen activator receptor (uPAR), wherein the antibody is selected from the following: a. An antibody or its antigen-binding fragment, containing i. An immunoglobulin light chain comprising the amino acid sequence of SEQ ID NO: 15 or a sequence having at least 90% sequence identity with it, or consisting of the amino acid sequence of SEQ ID NO: 15 or a sequence having at least 90% sequence identity with it, and ii. An immunoglobulin heavy chain comprising the amino acid sequence of SEQ ID NO: 20 or a sequence having at least 90% sequence identity with it, or consisting of the amino acid sequence of SEQ ID NO: 20 or a sequence having at least 90% sequence identity with it; b. An antibody or its antigen-binding fragment, containing i. The variable region of the immunoglobulin light chain, comprising the amino acid sequence of SEQ ID NO: 16 or a sequence having at least 90% sequence identity with it, or consisting of the amino acid sequence of SEQ ID NO: 16 or a sequence having at least 90% sequence identity with it, and ii. The variable region of the immunoglobulin heavy chain, comprising the amino acid sequence of SEQ ID NO: 21 or a sequence having at least 90% sequence identity with it, or consisting of the amino acid sequence of SEQ ID NO: 21 or a sequence having at least 90% sequence identity with it; c. An antibody or its antigen-binding fragment, containing i. The variable region of the immunoglobulin light chain, comprising the amino acid sequence of SEQ ID NO: 17 as complementarity-determining region 1 (CDR1), the amino acid sequence of SEQ ID NO: 18 as CDR2, and the amino acid sequence of SEQ ID NO: 19 as CDR3, and ii. The variable region of the immunoglobulin heavy chain, comprising the amino acid sequence of SEQ ID NO: 22 as complementarity-determining region 1 (CDR1), the amino acid sequence of SEQ ID NO: 23 as CDR2, and the amino acid sequence of SEQ ID NO: 24 as CDR3; d. A humanized version of an antibody or antigen-binding fragment of any of a, b, or c; e. A chimeric version of an antibody or antigen-binding fragment of any of a, b, or c.

3. An antibody or an antigen-binding fragment thereof that binds to urokinase-type plasminogen activator receptor (uPAR), wherein the antibody is selected from the following: a. An antibody or its antigen-binding fragment, containing i. An immunoglobulin light chain comprising the amino acid sequence of SEQ ID NO: 25 or a sequence having at least 90% sequence identity with it, or consisting of the amino acid sequence of SEQ ID NO: 25 or a sequence having at least 90% sequence identity with it, and ii. An immunoglobulin heavy chain comprising the amino acid sequence of SEQ ID NO: 30 or a sequence having at least 90% sequence identity with it, or consisting of the amino acid sequence of SEQ ID NO: 30 or a sequence having at least 90% sequence identity with it; b. An antibody or its antigen-binding fragment, containing i. The variable region of the immunoglobulin light chain, comprising the amino acid sequence of SEQ ID NO: 26 or a sequence having at least 90% sequence identity with it, or consisting of the amino acid sequence of SEQ ID NO: 26 or a sequence having at least 90% sequence identity with it, and ii. The variable region of the immunoglobulin heavy chain, comprising the amino acid sequence of SEQ ID NO: 31 or a sequence having at least 90% sequence identity with it, or consisting of the amino acid sequence of SEQ ID NO: 31 or a sequence having at least 90% sequence identity with it; c. An antibody or its antigen-binding fragment, containing i. The variable region of the immunoglobulin light chain, comprising the amino acid sequence of SEQ ID NO: 27 as complementarity-determining region 1 (CDR1), the amino acid sequence of SEQ ID NO: 28 as CDR2, and the amino acid sequence of SEQ ID NO: 29 as CDR3, and ii. The variable region of the immunoglobulin heavy chain, comprising the amino acid sequence of SEQ ID NO: 32 as complementarity-determining region 1 (CDR1), the amino acid sequence of SEQ ID NO: 33 as CDR2, and the amino acid sequence of SEQ ID NO: 34 as CDR3; d. A humanized version of an antibody or antigen-binding fragment of any of a, b, or c. e. A chimeric version of an antibody or antigen-binding fragment of any of a, b, or c.

4. The antibody according to any one of the preceding claims, wherein the antibody is: a. An antibody or its antigen-binding fragment, containing i. The variable region of the immunoglobulin light chain, comprising the amino acid sequence of SEQ ID NO: 56 or a sequence having at least 90% sequence identity with it, or consisting of the amino acid sequence of SEQ ID NO: 56 or a sequence having at least 90% sequence identity with it, and ii. An immunoglobulin heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 47 or a sequence having at least 90% sequence identity with it, or consisting of the amino acid sequence of SEQ ID NO: 47 or a sequence having at least 90% sequence identity with it; or b. An antibody or its antigen-binding fragment, containing i. The variable region of the immunoglobulin light chain, comprising the amino acid sequence of SEQ ID NO: 55 or a sequence having at least 90% sequence identity with it, or consisting of the amino acid sequence of SEQ ID NO: 55 or a sequence having at least 90% sequence identity with it, and ii. The variable region of the immunoglobulin heavy chain, comprising the amino acid sequence of SEQ ID NO: 51 or a sequence having at least 90% sequence identity with it, or consisting of the amino acid sequence of SEQ ID NO: 51 or a sequence having at least 90% sequence identity with it.

5. The antibody according to any one of the preceding claims, wherein any sequence variation is located outside the complementarity-determining region.

6. The antibody according to any one of the preceding claims, wherein the antibody is selected from mouse antibodies, chimeric antibodies, human antibodies, humanized antibodies, humanized antigen-binding fragments, Fab fragments, Fab' fragments, F(ab')2 fragments, Fv, single-chain antibodies (SCA) such as scFv, variable portions of their heavy and / or light chains, and Fab microantibodies.

7. The antibody according to any one of the preceding claims, wherein the antibody is a monoclonal antibody.

8. The antibody according to any one of the preceding claims, wherein the antibody is a humanized or fully human monoclonal antibody or an antigen-binding fragment thereof.

9. The antibody according to any one of the preceding claims, wherein the antibody is a recombinant antibody.

10. The antibody according to any one of the preceding claims, wherein the antibody is an immunoglobulin antibody, such as IgG, IgM, IgD, IgE, IgA and any subclass thereof, such as IgG1, IgG2, IgG3 or IgG4.

11. The antibody according to any one of the preceding claims, wherein the antibody comprises a light chain (LC) constant domain, the light chain constant domain comprising an amino acid sequence selected from SEQ ID NO: 58-59 or a sequence having at least 90% sequence identity with it, or consisting of an amino acid sequence selected from SEQ ID NO: 58-59 or a sequence having at least 90% sequence identity with it.

12. The antibody according to any one of the preceding claims, wherein the antibody comprises a heavy chain (HC) constant domain, the heavy chain constant domain comprising the amino acid sequence of SEQ ID NO: 52 or a sequence having at least 90% sequence identity with it, or consisting of the amino acid sequence of SEQ ID NO: 52 or a sequence having at least 90% sequence identity with it.

13. The antibody according to claim 1, wherein the antibody does not bind to SEQ ID NO: 2 and / or does not bind to SEQ ID NO:

4.

14. The antibody according to claim 1, wherein the antibody binds to a conformational epitope of uPAR domain D2 (SEQ ID NO: 3), the conformational epitope comprising, or consisting of, amino acid residues 8 (S), 10 (D), 12 (S), 15 (R), and 17 (R).

15. Antibody-drug conjugates (ADCs), comprising: a. The antibody as defined in any of the preceding claims, b. Surfactants, and c. Optional connector for connecting a) to b).

16. The antibody-drug conjugate of claim 15, wherein the active agent is selected from therapeutic agents, cytotoxic agents, radioisotopes, and detectable markers.

17. The antibody-drug conjugate according to any one of claims 15 to 16, wherein the active agent is a cytotoxic agent.

18. The antibody-drug conjugate according to any one of claims 15 to 17, wherein the active agent is a therapeutic agent, for example selected from antimicrotubule / antimitotic agents, DNA cross-linking agents, DNA alkylating agents, DNA strand breaking agents, anthracyclines, antimetabolites, histone deacetylase inhibitors, kinase inhibitors, metabolic inhibitors, peptide antibiotics, immune checkpoint inhibitors, platinum-based antitumor drugs, topoisomerase inhibitors, DNA or RNA polymerase inhibitors, nucleotide-based agents, and therapeutic agents of cytotoxic antibiotics.

19. The antibody-drug conjugate according to any one of claims 15 to 18, wherein the active agent is an antimitotic agent, for example selected from: derivatives of oliquistatin or dolalastatin, such as monomethyloliquistatin E (MMAE), monomethyloliquistatin F (MMAF), etc.; taxanes such as paclitaxel or docetaxel, etc.; vinca alkaloids such as vincristine, vinblastine, vinorelbine, or vinorelbine, etc.; mayatansinoid, colchicine, and podophyllotoxin.

20. The antibody-drug conjugate of claim 19, wherein the active agent is monomethylolpropionate E (MMAE).

21. The antibody-drug conjugate according to any one of claims 15 to 18, wherein the active agent is a DNA cross-linking agent, such as a DNA cross-linking agent selected from cisplatin or cisplatin derivatives such as carboplatin or oxaliplatin, mitomycin C (MMC), pyrrolobenzodiazepine and dipyrrolobenzodiazepine derivatives such as SGD-1882.

22. The antibody-drug conjugate according to any one of claims 15 to 18, wherein the active agent is a DNA alkylating agent, such as a nitrogen mustard such as tri(2-chloroethyl)amine, pyridobenzodiazepine or pyridobenzodiazepine derivatives, indolebenzodiazepine dimer and Duocarmycin SA.

23. The antibody-drug conjugate according to any one of claims 15 to 18, wherein the active agent is a DNA strand breaking agent, such as a DNA strand breaking agent selected from calichimycin and hamidone.

24. The antibody-drug conjugate according to any one of claims 15 to 18, wherein the active agent is an anthracene ring, such as anthracene rings selected from daunorubicin, doxorubicin, epirubicin, idarubicin and PNU-159682.

25. The antibody-drug conjugate according to claim 24, wherein the active agent is PNU-159682.

26. The antibody-drug conjugate according to any one of claims 15 to 18, wherein the active agent is an antimetabolite, for example selected from folic acid antagonists such as methotrexate, purine antimetabolites such as 6-mercaptopurine or 6-thioguanine or fludarabine phosphate or pentostatin or cladribine, and pyrimidine antimetabolites such as 5-fluorouracil or 5-fluorodeoxyuridine or cytarabine or gemcitabine.

27. The antibody-drug conjugate according to any one of claims 15 to 18, wherein the active agent is a histone deacetylase inhibitor, such as a histone deacetylase inhibitor selected from trogostatin A, vorinostat, belinstat, pabistat, gilvestat, remistat, ibestat, quinolostat, rocilinostat, pranostat, CHR-3996, valproic acid, butyric acid, phenylbutyric acid, entenolol, acetyldinarin, 4SC202, mocetinostat, romidesin, nicotinamide, citinol, cambinol, and EX-527.

28. The antibody-drug conjugate according to any one of claims 15 to 18, wherein the active agent is a kinase inhibitor, such as a kinase inhibitor selected from genistein, fumonisin C, PP1-AG1872, PP2-AG1879, SU6656, CGP77675, PD166285, imatinib, erlotinib, gefitinib, fumonisin A, cetuximab, UCS15A, atrazine A, and rhizobacterin.

29. The antibody-drug conjugate according to any one of claims 15 to 18, wherein the active agent is a metabolic inhibitor, such as a NAMPT inhibitor selected from APO866, GMX-1777, GMX-1778, ATG-019 and OT-82.

30. The antibody-drug conjugate according to any one of claims 15 to 18, wherein the active agent is an immune checkpoint inhibitor, such as a PD-1 inhibitor selected from pembrolizumab, nivolumab, cimiprimab, JTX-4014, stalilimumab, camrelizumab, sintilimab, tislelizumab, toripalimab, dotalimab, AMP-224, and AMP-514; or a PD-L1 inhibitor selected from atezolizumab, avelumab, durvalumab, KN035, CK-301, AUNP12, CA-170, and BMS-986189.

31. The antibody-drug conjugate according to any one of claims 15 to 18, wherein the active agent is a platinum-based antitumor agent, such as a platinum-based antitumor agent selected from aliplatin, cisplatin, carboplatin, oxaliplatin, nedaplatin, pyridine, phenanthriplatin, cetplatin and triplatinium tetranitrate.

32. The antibody-drug conjugate according to any one of claims 15 to 18, wherein the active agent is a topoisomerase inhibitor, such as a topoisomerase inhibitor selected from camptothecin or its derivatives, such as topotecan, belotetane, letopotecan, irinotecan, SN-38, eczetane, and Dxd.

33. The antibody-drug conjugate according to any one of claims 15 to 18, wherein the active agent is a DNA or RNA polymerase inhibitor, such as a polymerase inhibitor selected from amatoxins or α-amatoxins or derivatives thereof, actinomycin D, and afidimycin.

34. The antibody-drug conjugate according to any one of claims 15 to 33, wherein the active agent comprises a drug selected from... 60 Co、 89 Sr、 90 Y、 99m Tc, 131 I, 137 Cs、 153 Sm or 223 Radioactive isotopes of Rd.

35. The antibody-drug conjugate according to any one of claims 15 to 34, wherein the drug-to-antibody ratio (DAR) is 1 to 10, for example 2 to 8, for example 2 to 6, for example 2 or 4, for example 2.

36. The antibody-drug conjugate according to any one of claims 15 to 34, wherein the drug-to-antibody ratio (DAR) is 2 to 6, for example 2 or 4, for example 2.

37. The antibody-drug conjugate according to any one of claims 15 to 36, wherein the antibody-drug conjugate comprises a linker selected from cleavable linkers and non-cleavable linkers, and optionally, the linker is a peptide linker.

38. The antibody-drug conjugate according to any one of claims 15 to 37, wherein the linker comprises or is composed of a dipeptide, such as valine-citrulline (VC) or valine-alanine (VA).

39. The antibody-drug conjugate according to any one of claims 15 to 38, wherein the antibody-drug conjugate further comprises a spacer, for example, a spacer comprising p-aminobenzoic acid, p-aminobenzyl (PAB), p-aminobenzylcarbamate (PABC), p-aminobenzoyloxycarbonyl or polyethylene glycol (PEG).

40. The antibody-drug conjugate according to any one of claims 15 to 39, wherein the antibody-drug conjugate further comprises a linker group, for example comprising or consisting of the following: dibenzocyclooctylene, including amine-functionalized dibenzocyclooctylene, optionally modified with PEG such as PEG3 or PEG4, maleimide, N-hydroxysuccinimide, a reactive linker group pointing to a modified or unmodified protein-binding carbohydrate, a peptide sequence required for an enzymatic reaction, an azide or alkyne, or derived from these by reaction with an antibody or a chemically or enzymatically generated derivative thereof.

41. The antibody-drug conjugate according to any one of claims 15 to 40, wherein, The antibody-drug conjugate prior to antibody conjugation contains the following molecular structures: 。 42. The antibody-drug conjugate according to any one of claims 15 to 18 and 34 to 41, wherein the antibody-drug conjugate comprises or is composed of the following: a. An antibody as defined in any one of claims 1 to 14, b. VC connector, c. A dibenzocyclooctylamine (DBCO) linker, optionally N-functionalized with PEG4. d. PAB or PABC spacers, and e. PNU-159682 as an active agent.

43. The antibody-drug conjugate according to any one of claims 15 to 18 and 34 to 42, wherein the antibody-drug conjugate comprises CAS identifier 2259318-56-2.

44. The antibody-drug conjugate according to any one of claims 15 to 18 and 34 to 43, wherein the antibody-drug conjugate comprises the antibody as defined in claim 1 and DBCO-PEG4-VC-PAB-DMAE-PNU159682.

45. The antibody-drug conjugate according to any one of claims 15 to 18 and 34 to 44, wherein the antibody-drug conjugate comprises the antibody as defined in claim 1 and ADIBO-PEG4-VC-PAB-DMAE-PNU159682 linked by a triazole moiety.

46. ​​The antibody-drug conjugate according to any one of claims 15 to 18 and 34 to 45, wherein the antibody-drug conjugate comprises, or is composed of, the following structures or regioisomers thereof: The wavy line represents any chemical bond with the antibody.

47. The antibody-drug conjugate according to any one of claims 15 to 40, wherein, The antibody-drug conjugate prior to antibody conjugation contains the CAS identifier 2754384-60-4.

48. The antibody according to any one of claims 1 to 14 or the antibody-drug conjugate according to any one of claims 15 to 47, used as a drug.

49. A pharmaceutical composition comprising an antibody according to any one of claims 1 to 14 or an antibody-drug conjugate according to any one of claims 15 to 47, and a pharmaceutically acceptable buffer, diluent, carrier, adjuvant, or excipient.

50. The antibody according to any one of claims 1 to 14, or the antibody-drug conjugate according to any one of claims 15 to 47, or the pharmaceutical composition according to claim 49, for treating a disease characterized by cells expressing uPAR, such as cancer characterized by cells expressing uPAR.

51. The antibody, antibody-drug conjugate, or composition used according to claim 50, wherein the disease characterized by cells expressing uPAR is cancer.

52. The antibody, antibody-drug conjugate, or composition used according to claim 51, wherein the cancer is selected from pancreatic adenocarcinoma (PAAD) such as pancreatic ductal adenocarcinoma (PDAC), bladder urothelial carcinoma (BLCA), invasive breast carcinoma (BRCA), cervical squamous cell carcinoma and endometrial adenocarcinoma (CESC), cholangiocarcinoma (CHOL), colonic adenocarcinoma (COAD), esophageal cancer (ESCA), glioblastoma multiforme (GBM), head and neck squamous cell carcinoma (HNSCC), acute myeloid leukemia (LAML), ovarian serous cystadenocarcinoma (OV), rectal adenocarcinoma (READ), sarcoma (SARC), cutaneous melanoma (SKCM), and gastric adenocarcinoma (STAD).

53. The antibody, antibody-drug conjugate, or composition used according to claim 51, wherein the cancer is selected from pancreatic cancer, breast cancer, colon cancer, gastric cancer, esophageal cancer, hepatocellular carcinoma, prostate cancer, bladder cancer, mesothelioma, fibrosarcoma, and squamous cell carcinoma.

54. The antibody, antibody-drug conjugate, or composition used according to claim 51, wherein the cancer is breast cancer, for example selected from triple-negative breast cancer (TNBC), basal-like breast cancer (BLBC), HER-2 breast cancer, Luminal A breast cancer, and Luminal B breast cancer.

55. The antibody, antibody-drug conjugate, or composition used according to claim 51, wherein the cancer is pancreatic cancer, such as pancreatic ductal adenocarcinoma.

56. The antibody, antibody-drug conjugate, or composition used according to claim 51, wherein the cancer is mesothelioma, for example selected from pleural mesothelioma, peritoneal mesothelioma, and pericardial mesothelioma.

57. The antibody, antibody-drug conjugate, or composition used according to any one of claims 51 to 56, wherein the cancer is a metastatic cancer.

58. The antibody, antibody-drug conjugate, or composition used according to any one of claims 50 to 57, wherein the administration is parenteral, such as intravenous, intra-articular, intra-articular, intraperitoneal, intrathecal, intraventricular, intrasternal, intracranial, intramuscular, or subcutaneous administration, or administration by infusion technique.

59. The antibody, antibody-drug conjugate, or composition used according to any one of claims 50 to 58, wherein the administration is intravenous.

60. The antibody, antibody-drug conjugate, or composition used according to any one of claims 50 to 59, wherein the antibody, antibody-drug conjugate, or composition is administered in combination with one or more other agents, such as one or more other therapeutic agents.

61. The antibody, antibody-drug conjugate, or composition used according to any one of claims 50 to 60, wherein the cells expressing uPAR exhibit uPAR overexpression, optionally wherein the cells expressing uPAR are tumor cells and / or tumor-associated cells.

62. The antibody, antibody-drug conjugate, or composition used according to any one of claims 50 to 61, wherein the antibody, antibody-drug conjugate, or composition induces cell death and / or inhibits the growth and / or proliferation of cells expressing uPAR.

63. The antibody, antibody-drug conjugate, or composition used according to any one of claims 50 to 61, wherein the antibody, antibody-drug conjugate, or composition induces the release of free cytotoxins from cells expressing uPAR, resulting in cell death and / or inhibiting the growth and / or proliferation of adjacent cancer cells.

64. The antibody, antibody-drug conjugate, or composition used according to any one of claims 50 to 63, wherein the treatment is amenable or curative.

65. A method for inhibiting tumor progression in a subject using an antibody according to any one of claims 1 to 14, an antibody-drug conjugate according to any one of claims 15 to 47, or a pharmaceutical composition according to claim 49.

66. A method for inhibiting, reducing, or eliminating the metastatic ability of tumors expressing uPAR in a subject, using an antibody according to any one of claims 1 to 14, an antibody-drug conjugate according to any one of claims 15 to 47, or a pharmaceutical composition according to claim 49.

67. A kit comprising an antibody according to any one of claims 1 to 14, an antibody-drug conjugate according to any one of claims 15 to 47, or a pharmaceutical composition according to claim 49, optionally further comprising a device for administering the antibody or antibody-drug conjugate to a subject and / or instructions for use.

68. The antibody according to any one of claims 1 to 14, the antibody-drug conjugate according to any one of claims 15 to 47, or The pharmaceutical composition according to claim 49 is used to manufacture a medicament for treating diseases characterized by cells expressing uPAR, such as cancer.

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