Anti-urokinase plasminogen activator receptor antibodies and methods of use thereof

Abstract

Antibodies that specifically bind to the human urokinase-type plasminogen activator receptor (uPAR) are provided. In some cases, the antibodies have cross reactivity with one or more non-human animal uPAR polypeptides, such as non-human primate uPAR, e.g., cynomolgus monkey uPAR. Fusion proteins and conjugates comprising the antibodies of the disclosure are also provided. Methods of treating conditions associated with uPAR expression and / or activity using the antibodies, fusion proteins and conjugates of the present disclosure are also provided. In some embodiments, the condition associated with uPAR expression and / or activity is cancer. Non-limiting examples of such cancers include those characterized by cancer cells expressing uPAR on its surface, cancers characterized by stromal cells in a tumor microenvironment expressing uPAR on its surface, and / or the like.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of U.S. Provisional Patent Application No. 63 / 413,530, filed on October 5, 2022, which is incorporated herein by reference in its entirety.

[0003] Incorporation by Reference into the Sequence Listing provided as a Sequence Listing XML file

[0004] The Sequence Listing is hereby provided as a Sequence Listing XML, UCSF-667WO_SEQ_LIST, created on October 4, 2023 and having a size of 53,857 bytes. The contents of the Sequence Listing XML are incorporated herein by reference in their entirety.

[0005] introduction

[0006] The key feature of tumor cells is their enhanced ability to degrade the extracellular matrix (ECM), allowing tumor cell movement, invasion and metastasis. Urokinase-type plasminogen activator receptor (uPAR) is an integral membrane protein that is tethered to the plasma membrane via a glycosylphosphatidylinositol (GPI) anchor. This well-studied receptor participates in the binding of various partners (such as urokinase-type plasminogen activator (uPA), vitronectin (VN) and transmembrane receptors) to regulate a variety of cellular processes, including extracellular proteolysis, angiogenesis, cell adhesion, migration and downstream signaling events (1). Many studies have shown that overexpression of uPAR is tumor-specific (2,3), making it an outstanding biomarker for identifying tumor aggressiveness (4-6) and an attractive target for cancer treatment, particularly breast cancer (8-11) (7).

[0007] There is increasing evidence that uPAR and HER2 are co-amplified in both breast cancer in situ and metastatic breast cancer, and they work synergistically to cause the tumor to progress toward the beginning of the metastatic phenotype (12, 13). In addition, the use of RNAi with anti-HER2 antibodies to downregulate uPAR induced a synergistic effect in inhibiting breast cancer cell growth, highlighting the potential of combination therapy as an effective treatment for breast cancer (14). Although clinical results have shown that FDA-approved anti-HER2 antibodies are effective for metastatic HER2-positive breast cancer, several mechanisms of resistance to anti-HER2 therapies have been identified (15, 16). In addition, due to the lack of HER2 expression, HER2 is not an effective target for patients with triple-negative breast cancer (TNBC) (9), so it is necessary to develop new treatment strategies. Multiple groups have developed a series of antagonists, such as recombinant antibodies (rAb), small molecules and peptides, to block the interaction of uPAR with its partner (17-22). Some of these uPAR-targeting agents have also been designed as novel preclinical immunotherapies (17, 23, 24), diagnostic imaging tools (17, 25, 26), and drug delivery vehicles (24), validating uPAR as a potential therapeutic target. Summary of the invention

[0008] Antibodies that specifically bind to human urokinase-type plasminogen activator receptor (uPAR) are provided. In some cases, the antibody has cross-reactivity with one or more non-human animal uPAR polypeptides (such as non-human primate uPAR, for example, cynomolgus monkey uPAR). Fusion proteins and conjugates comprising antibodies of the present disclosure are also provided. Methods for treating conditions associated with uPAR expression and / or activity using antibodies, fusion proteins and conjugates of the present disclosure are also provided. In some embodiments, the condition associated with uPAR expression and / or activity is cancer. Non-limiting examples of such cancers include those cancers characterized by cancer cells expressing uPAR on their surface, cancers characterized by stromal cells in a tumor microenvironment expressing uPAR on their surface, and / or the like. BRIEF DESCRIPTION OF THE DRAWINGS

[0009] Figures 1A to 1D :A) Mouse immune campaign pipeline. B) Using Beacon TMWorkflow for accelerated discovery of cross-reactive anti-uPAR antibodies of the platform. C) Each ASC is cultured in a separate nanopen to allow secreted antibodies to accumulate. After culturing the cells for 1-2 hours, anti-mouse IgG (H+L) coated beads are input into the channel together with fluorescently labeled uPAR (AF488-human suPAR, green; and AF647-cyno uPAR, red). Since the secreted antibodies are fixed on the beads, antigen recognition allows time-dependent accumulation of fluorescent signals directly above each nanopen. D) Increased signals in the AF488 and AF647 channels are observed between T0 and T11, indicating that the antibodies are able to recognize human uPAR / cyno uPAR.

[0010] Figure 2 : Binding curve of the initial antibody to MDA-MB-231 cells expressing human uPAR on the cell surface. MFI represents median fluorescence intensity.

[0011] FIG. 3A to FIG. 3B :(A) Cross-reactivity profile of 12 antibody candidates evaluated by ELISA, showing that they have cross-reactivity with human uPAR and cyno uPAR, and 2G10 and 3C6 are specific binders of human uPAR. (B) Antibody candidates can elicit MI CD16a effector cells performed ADCC against MDA-MB-231 cells in a dose-dependent manner. Two-way ANOVA followed by post hoc Tukey test revealed significant activity relative to human IgG1 (huIgG1). * = p ≤ 0.001.

[0012] Figure 4 : Eight selected antibody candidates were able to induce dose-dependent cell death in MBA-MB-231 in the presence of human PBMCs from three healthy donors. Two-way ANOVA followed by post hoc Tukey test revealed significant activity relative to human IgG1 (huIgG1). * = p ≤ 0.05, ** = p ≤ 0.001.

[0013] Figure 5 A to Figure 5C: A) In the presence of an anti-human FcFab conjugated to cytotoxic MMAE by a cathepsin-cleavable linker, dose-dependent cytotoxicity of candidates 3159, 8163, 11857 ​​and 3595 was observed. B) The therapeutic efficacy of new antibody candidates was determined in an orthotopic mouse model of human breast cancer using MDA-MB-231 cells. Compared with untreated controls, animals showed reduced tumor size, and after 21 days of treatment, significant tumor growth inhibition was observed in animals treated with 11857. C) Throughout the 30-day treatment, significant impairment of tumor growth rate was observed for antibody-treated animals, and 11857 ​​was the most active agent. Data are shown as mean ± standard deviation. Statistical analysis was two-way ANOVA, with post hoc multiple comparisons using Dunnett's test. *p<0.05; **p<0.01; ***p<0.001.

[0014] Figure 6 A to Figure 6 C: A) Molecular surface representation of the human uPAR-ATF-SMB complex. The uPA N-terminal fragment (ATF) is shown as a gray ribbon diagram, and the vitronectin (VN) SMB domain is shown as a blue ribbon diagram. (PDB ID: 3BT1). Mutation variants between human uPAR and cyno uPAR are highlighted in yellow. B) BLI traces identify non-overlapping epitopes between each lead antibody and 2G10 that binds to the uPA recognition site. Further association steps show competitive blocking of VN binding by each antibody candidate. C) BLI competition assays reveal that candidates 8163 and 3159 have different binding sites, and candidate 11857 ​​has partially overlapping epitopes with candidates 8163 and 3159.

[0015] Figure 7 : Proposed binding model of a new antibody candidate against uPAR, highlighting its inhibitory effect on vitronectin binding and a different binding epitope compared to 2G10. Antibodies 3159 and 8163 recognize different epitopes on uPAR, and 11857 ​​has a partially overlapping epitope with antibodies 3159 and 8163.

[0016] FIG. 8A to FIG. 8B : A) Recombinant human suPAR expressed and characterized by SDS-PAGE and immunoblotting. B) Further characterization by LC-MS / MS showed 59.7% coverage of the complete protein sequence (SEQ ID NO: 55).

[0017] Fig. 9 : Antibody titers from each animal monitored throughout the 60-day immunization campaign using recombinant human uPAR as the immunogen, showing the production of anti-uPAR antibodies.

[0018] Fig.10 : Eight selected lead antibody candidates were able to block the adhesion of MDA-MB-231 cells to vitronectin in a dose-responsive manner.

[0019] Fig.11 : BLI competition assay between candidate 3159 and vitronectin in reverse order showed the ability of 3159 to block the binding of vitronectin to uPAR.

[0020] Fig.12 : BLI competition assays for the lead antibody and 3C6 discovered by phage display showed that they have different binding epitopes.

[0021] Fig.13 : At different time points, 89 PET / CT sections obtained from mice infected with Zr-DFO-3159 antibody.

[0022] Fig.14 : At different time points, 89 Maximum intensity projections acquired from mice stained with Zr-DFO-3159 antibody.

[0023] Fig.15 : At different time points, 89 PET / CT sections obtained from mice infected with Zr-DFO-11857 ​​antibody.

[0024] Fig.16 : At different time points, 89 Maximum intensity projections acquired from mice stained with Zr-DFO-11857 ​​antibody.

[0025] Fig.17 : Tumor time-activity curves including SUV mean data obtained from 4 tumors in the 3159 cohort and 3 tumors in the 11857 ​​cohort.

[0026] Fig.18 : SUV mean data obtained by region of interest analysis of tumor and various normal tissues from mice in the 3159 cohort.

[0027] Fig.19 : SUV mean data obtained by region of interest analysis of tumors and various normal tissues from mice in the 11857 ​​cohort.

[0028] Fig. 20 : Antitumor assessment depicting the fold change in volume of UMUC3 tumors.

[0029] Fig.21 .Anti-tumor evaluation depicting volume changes in UMUC3 tumors. DETAILED DESCRIPTION

[0030] Before describing the antibodies and methods of the present disclosure in more detail, it should be understood that the antibodies and methods are not limited to the specific embodiments described and thus may of course vary. It should also be understood that the terminology used herein is only for the purpose of describing specific embodiments and is not intended to be limiting, as the scope of the antibodies and methods is limited only by the appended claims.

[0031] Where a range of values ​​is provided, it is understood that each intervening value between the upper and lower limits of the range, to the tenth of the unit of the lower limit and any other stated or intervening value in the stated range is encompassed in the antibodies and methods unless the context clearly dictates otherwise. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges and are also encompassed in the antibodies and methods, subject to any specific exclusions in the stated ranges. Where a stated range includes one or both limits, ranges excluding either or both of those included limits are also encompassed in the antibodies and methods.

[0032] Certain ranges are given herein where the term "about" precedes a numerical value. The term "about" is used herein to provide literal support for the exact number that follows it, as well as numbers that are close to or approximately the number that follows the term. In determining whether a number is close to or approximately a specifically recited number, the close or approximate unrecited number may be a substantial equivalent of the number that is specifically recited in the context in which it appears.

[0033] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the antibodies and methods belong. Although any antibodies and methods similar or equivalent to those described herein can also be used in the practice or testing of the antibodies and methods, representative exemplary antibodies and methods are now described.

[0034] All publications and patents cited in this specification are incorporated herein by reference as if each individual publication or patent was specifically and individually indicated to be incorporated by reference and is incorporated herein by reference to disclose and describe the materials and / or methods in connection with which the publication is cited. The citation of any publication is for its disclosure prior to the filing date and should not be construed as an admission that the antibodies and methods of the present invention are not entitled to antedate such publications, as the publication dates provided may be different from the actual publication dates which may need to be independently confirmed.

[0035] It is noted that, as used herein and in the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. It should also be noted that the claims can be drafted to exclude any optional elements. Thus, this statement is intended to serve as antecedent basis for use of exclusive terminology such as "only," "only" and the like in connection with the recitation of claim elements, or use of a "negative" limitation.

[0036] It should be understood that, for the sake of clarity, certain features of the antibodies and methods described in the context of separate embodiments may also be provided in a single embodiment in combination. Conversely, for the sake of brevity, the various features of the antibodies and methods described in the context of a single embodiment may also be provided individually or in any suitable subcombination. All combinations of embodiments are specifically included by the present disclosure and are disclosed herein as if each and every combination were individually and explicitly disclosed to the extent that such combinations include operable processes and / or compositions. In addition, all subcombinations listed in the embodiments describing such variables are also specifically included by the antibodies and methods of the present invention and are disclosed herein as if each and every such subcombination were individually and explicitly disclosed herein.

[0037] Those skilled in the art will appreciate upon reading this disclosure that each individual embodiment described and illustrated herein has discrete components and features that can be readily separated or combined with the features of any other several embodiments without departing from the scope or spirit of the method of the present invention. Any narrated method can be performed in the order of events recited or in any other order that is logically possible.

[0038] Anti-UPAR antibodies

[0039] The present disclosure provides anti-urokinase plasminogen activator receptor (uPAR) antibodies. uPAR (UniProtQ03405-human) is also called CD87, encoded by the PLAUR gene, and belongs to the lymphoid antigen-6 superfamily. uPAR was first identified as a cell surface receptor for urokinase plasminogen activator (uPA). The mature uPAR molecule is a single-chain membrane glycoprotein receptor composed of 313 amino acid residues and is anchored to the cell membrane by a glycosylphosphatidylinositol (GPI) bond; it contains 3 homologous domains, i.e., D1, D2 and D3, with a total molecular weight of 55-60 kDa. uPAR mediates a variety of biological processes, such as plasminogen activation, proteolysis, cell signal transduction and adhesion. Under normal physiological conditions, uPAR is usually expressed at low levels. In the process of tissue remodeling, wound healing, inflammation and embryogenesis, uPAR is transiently expressed at high levels and participates in the process of extracellular matrix (ECM) degradation, thrombolysis, cell invasion and migration.

[0040] uPAR has the multiple functional effects related to tumor progression, including tumor proliferation and apoptosis, metastasis, angiogenesis, multidrug resistance (MDR) and prognosis. The analysis of tumor samples has been shown to express high uPAR in most solid tumor tissues, including but not limited to breast cancer, lung cancer, bladder cancer, ovarian cancer, prostate cancer, liver cancer, colon cancer, pancreatic cancer and gastric cancer, as well as glioma and several hematological malignancies. In addition, uPAR is expressed at high levels on stromal cells (such as vascular endothelial cells, tumor-associated fibroblasts and tumor-associated macrophages) in the tumor microenvironment, and its expression level is closely related to the survival of the patient with tumor and the aggressiveness of the tumor.

[0041] In certain embodiments, the antibodies of the present disclosure specifically bind to human urokinase-type plasminogen activator receptor (uPAR) and compete for binding to human uPAR with antibodies having one, two, three, four, five, or all six complementary determining regions (CDRs) of one or more of the anti-uPAR antibodies designated herein as antibodies 3159, 8163, 11857, or 3595. In some embodiments, such antibodies comprise one, two, three, four, five, or all six CDRs of an antibody designated herein as antibodies 3159, 8163, 11857, or 3595. In some embodiments, such antibodies comprise VDRs that are identical to those of an antibody designated herein as antibodies 3159, 8163, 11857, or 3595. H and / or V L or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 100% identical to a variable heavy chain (V H ) polypeptide and / or variable light chain (V L ) polypeptide.

[0042] Antibodies 3159, 8163, 11857 ​​and 3595 are selected from a large number of identified anti-human uPAR antibodies based at least in part on their ability to have cross-reactivity with cynomolgus monkey uPAR. Compared with other species, cynomolgus monkeys (cyno) are genetically similar to humans and are the most relevant non-human primate models for preclinical studies in the development of antibody drugs. In addition, as shown in the following experimental section, these unique cross-reactive antibodies show antibody-dependent cellular toxicity (ADCC), ADC cytotoxicity and inhibitory effects on cell adhesion for human cancer cells. As shown herein, these antibodies also show therapeutic efficacy in reducing tumor growth in the in situ animal model of human cancer, and provide a binding model for these antibodies, which shows their unique binding epitopes that cause activity for uPAR.

[0043] The V values ​​of 3159, 8163, 11857 ​​and 3595 antibodies are provided in Table 1 below. H Peptide and V L Amino acid sequence of the polypeptide. The CDR sequences according to the Kabat definition are underlined.

[0044] Table 1 – Amino acid and nucleotide sequences

[0045]

[0046]

[0047]

[0048]

[0049]

[0050]

[0051]

[0052]

[0053]

[0054]

[0055]

[0056] According to some embodiments, the antibodies of the present disclosure specifically bind to human uPAR and comprise antibodies containing one, two, three, four, five or all six CDRs of the antibody designated herein as antibody 3159, or compete with the antibody for binding to human uPAR. The CDR sequences can be defined according to Kabat. In certain embodiments, such antibodies comprise: V H Polypeptide, the V H The polypeptide comprises a V sequence similar to that of the antibody designated herein as antibody 3159. H The polypeptide has an amino acid sequence that is 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 100% identical; V L Polypeptide, the V L The polypeptide comprises a V sequence similar to that of the antibody designated herein as antibody 3159. L The polypeptide has an amino acid sequence that is 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 100% identical; or both. According to some embodiments, the V sequence of the antibody referred to herein as antibody 3159 is 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 100% identical; or both. H Peptide, V L Such antibodies have V H Peptide, V L One or more amino acid substitutions (eg, one or more conservative amino acid substitutions) are included in one or more framework regions of one or both polypeptides.

[0057] According to certain embodiments, the antibodies of the present disclosure specifically bind to human uPAR and include antibodies containing one, two, three, four, five or all six CDRs of the antibody designated herein as antibody 8163, or compete with the antibody for binding to human uPAR. The CDR sequences can be defined according to Kabat. In certain embodiments, such antibodies include: V H Polypeptide, the V H The polypeptide comprises a V sequence similar to that of the antibody designated herein as antibody 8163. H The polypeptide has an amino acid sequence that is 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 100% identical; VL Polypeptide, the V L The polypeptide comprises a V sequence similar to that of the antibody designated herein as antibody 8163. L The polypeptide has an amino acid sequence that is 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 100% identical; or both. According to some embodiments, the V sequence of the antibody referred to herein as antibody 8163 is 50% identical to that of the antibody. H Peptide, V L Such antibodies have V H Peptide, V L One or more amino acid substitutions (eg, one or more conservative amino acid substitutions) are included in one or more framework regions of one or both polypeptides.

[0058] According to some embodiments, the antibodies of the present disclosure specifically bind to human uPAR and comprise antibodies containing one, two, three, four, five or all six CDRs of an antibody designated herein as antibody 11857, or compete with the antibody for binding to human uPAR. The CDR sequences can be defined according to Kabat. In certain embodiments, such antibodies comprise: a VH polypeptide comprising a VH polypeptide having 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 100% identity to a VH polypeptide of an antibody designated herein as antibody 11857. an amino acid sequence; a VL polypeptide comprising an amino acid sequence that is 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 100% identical to the VL polypeptide of an antibody designated herein as antibody 11857; or both. According to some embodiments, the VL polypeptide of an antibody designated herein as antibody 11857 ​​comprises an amino acid sequence that is 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 100% identical to the VL polypeptide of an antibody designated herein as antibody 11857. H Peptide, V L Such antibodies have V H Peptide, V L One or more amino acid substitutions (eg, one or more conservative amino acid substitutions) are included in one or more framework regions of one or both polypeptides.

[0059] According to some embodiments, the antibodies of the present disclosure specifically bind to human uPAR and comprise antibodies containing one, two, three, four, five or all six CDRs of an antibody designated herein as antibody 3595, or compete with such antibody for binding to human uPAR. CDR sequences can be defined according to Kabat. In certain embodiments, such antibodies comprise: a VH polypeptide comprising a VH polypeptide having 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 100% identity to a VH polypeptide of an antibody designated herein as antibody 3595. an amino acid sequence; a VL polypeptide comprising an amino acid sequence that is 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 100% identical to the VL polypeptide of an antibody designated herein as antibody 3595; or both. According to some embodiments, the VL polypeptide of an antibody designated herein as antibody 3595 is 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 100% identical to the VL polypeptide of an antibody designated herein as antibody 3595. H Peptide, V L Such antibodies have V H Peptide, V L One or more amino acid substitutions (eg, one or more conservative amino acid substitutions) are included in one or more framework regions of one or both polypeptides.

[0060] According to some embodiments, the CDRs are defined according to the Kabat numbering system. In certain embodiments, the CDRs may be defined according to the IMGT numbering system.

[0061] In certain embodiments, there is provided a V relative to that shown in Table 1 H and / or V L The antibody variants of amino acid sequence have one or more amino acid substitutions. The interested site for substitution mutagenesis includes one or more CDR and / or one or more framework regions (FR). Conservative substitutions are shown in the following table under the title of "preferred substitution". More substantial changes are provided in the following table under the title of "exemplary substitution", and are further described below with reference to the amino acid side chain category. Amino acid substitutions can be introduced into the interested antibody, and the product is screened for desired activity (for example, retained / improved antigen binding, reduced immunogenicity, improved developability, improved manufacturability and or the like).

[0062]

[0063]

[0064] Amino acids can be grouped according to common side chain properties:

[0065] (1) Hydrophobic: norleucine, Met, Ala, Val, Leu, Ile;

[0066] (2) Neutral hydrophilic: Cys, Ser, Thr, Asn, Gln;

[0067] (3) Acidic: Asp, Glu;

[0068] (4) Basic: His, Lys, Arg;

[0069] (5) Residues that affect chain orientation: Gly, Pro;

[0070] (6) Aromatic: Trp, Tyr, Phe.

[0071] Non-conservative substitutions entail exchanging a member of one of these classes for another class.

[0072] Methods are available for measuring the affinity of anti-human uPAR antibodies for human uPAR using direct binding or competitive binding assays. In direct binding assays, the equilibrium binding constant (K) can be measured using candidate anti-human uPAR antibodies conjugated to a fluorophore or radioisotope, or candidate anti-human uPAR antibodies comprising an N-terminal or C-terminal epitope tag. D ) for detection by labeled antibodies. If a marker or label is not feasible or desired, a competitive binding assay can be used to determine the half-maximal inhibitory concentration (IC 50 ), i.e., the amount of unlabeled candidate anti-human uPAR antibody at which 50% of the maximal signal of the labeled competitor can be detected. The measured IC 50 Calculate K D Ligand depletion will be more pronounced when measuring high affinity interactions at lower concentration ranges and can be avoided or minimized by reducing the amount of human uPAR added to the experiment or by increasing the binding reaction volume.

[0073] It can be easily determined whether an antibody of the present disclosure "competes" for binding to an antigen with a second antibody using competitive binding assays known in the art. For example, a competitive antibody can be identified via an antibody competition assay. For example, a sample of a first antibody can be bound to a solid support. Then, a sample of a second antibody suspected of being able to compete with such a first antibody is added. One of the two antibodies is labeled. If the labeled antibody and the unlabeled antibody bind to separate and discrete sites on the antigen, the labeled antibody will bind to the same level regardless of whether there is a suspected competing antibody. However, if the sites of interaction are identical or overlapping, the unlabeled antibody will compete, and the amount of the labeled antibody bound to the antigen will be reduced. If the unlabeled antibody is present in excess, very little (if any) labeled antibody will bind.

[0074] For the purposes of this disclosure, competing antibodies are those that reduce the binding of antibodies to antigens by about 50% or more, about 60% or more, about 70% or more, about 80% or more, about 85% or more, about 90% or more, about 95% or more, or about 99% or more. The details of the program for carrying out such competitive determinations are known and can be found in, for example, Harlow and Lane, Antibodies, A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1988, 567-569, 1988, ISBN 0-87969-314-2. Such determinations can be quantitatively determined using purified antibodies. A standard curve can be established by titrating an antibody against itself, i.e., using the same antibody for both marker and competitor. Unlabeled competing antibodies can be titrated to inhibit the ability of labeled antibodies to bind to plates. Results can be plotted, and the concentration required for the desired degree of binding inhibition can be compared.

[0075] Human uPAR polypeptides that can be used to determine whether an antibody of the disclosure competes with a second antibody for binding to human uPAR are set forth in UniProt Q03405.

[0076] The term "antibody" may include antibodies or immunoglobulins of any isotype (e.g., IgG (e.g., IgG1, IgG2, IgG3 or IgG4), IgE, IgD, IgA, IgM, etc.), whole antibodies (e.g., antibodies composed of a tetramer, which in turn is composed of two dimers of heavy and light chain polypeptides); single-chain antibodies (e.g., scFv); fragments of antibodies that retain specific binding to cell surface molecules of target cells (e.g., fragments of whole antibodies or single-chain antibodies), including but not limited to single-chain Fv (scFv), Fab, (Fab')2, (scFv')2, and diabodies; chimeric antibodies; monoclonal antibodies, human antibodies, humanized antibodies (e.g., humanized whole antibodies, humanized half antibodies, or humanized antibody fragments, such as humanized scFv); and fusion proteins comprising an antigen-binding portion of an antibody and a non-antibody protein. In some embodiments, the antibody is selected from IgG, Fv, single-chain antibodies, scFv, Fab, (Fab')2, F(ab') or Fab'. The antibody can be detectably labeled, for example, with an in vivo imaging agent, a radioisotope, an enzyme that produces a detectable product, a fluorescent protein, etc. The antibody can be further conjugated to other moieties, such as members of a specific binding pair, e.g., biotin (a member of a biotin-avidin specific binding pair), etc.

[0077] The variable region of an immunoglobulin light or heavy chain consists of a "framework" region (FR) interrupted by three hypervariable regions, which are also called "complementarity determining regions" or "CDRs". The extent of the framework region and CDR can be defined based on databases known in the art. See, for example, "Sequences of Proteins of Immunological Interest," E. Kabat et al., Sequences of proteins of immunological interest, 4th edition, USDept. Health and Human Services, Public Health Services, Bethesda, MD (1987), Lefranc et al., IMGT, the international ImMunoGeneTics information Nucl.Acids Res., 2005, 33: D593-D597 (www.imgt.org / textes / IMGTScientificChart / ) and / or V Base at vbase.mrc-cpe.cam.ac.uk / ). The sequences of the framework regions of different light chains or heavy chains are relatively conserved among species. The framework region of an antibody, i.e., the combined framework region of the light chain and the heavy chain, is used to position and align the CDR. The CDR is primarily responsible for binding to the epitope of the antigen.

[0078] Any anti-human uPAR antibody of the present disclosure may be a monoclonal antibody. As used herein, the term "monoclonal antibody" refers to an antibody composition having a homogeneous antibody population. The term is not limited by the manner in which it is made. The term encompasses whole immunoglobulin molecules, as well as Fab molecules, F(ab')2 fragments, Fv fragments, single-chain fragment variable (scFv), fusion proteins comprising the antigen-binding portion of an antibody and non-antibody proteins, and other molecules that exhibit the immunological binding properties of a parent monoclonal antibody molecule. Methods for preparing monoclonal antibodies are known in the art and will be described more fully below.

[0079] Any anti-human uPAR antibody disclosed herein may be a recombinant antibody or a modified antibody, such as a chimeric antibody, a deimmunized antibody, and / or an antibody produced in vitro. The term "recombinant" antibody or "modified" antibody as used herein is intended to include all antibodies prepared, expressed, produced, or isolated by recombinant means, such as (i) antibodies expressed by one or more recombinant expression vectors transfected into a host cell; (ii) antibodies isolated from a recombinant combinatorial antibody library; (iii) antibodies isolated from an animal (e.g., a mouse) that is transgenic for human immunoglobulin genes; or (iv) antibodies prepared, expressed, produced, or isolated by any other means involving splicing human immunoglobulin gene sequences to other DNA sequences. Such recombinant antibodies include, for example, chimeric antibodies, deimmunized antibodies, and / or antibodies produced in vitro.

[0080] Any anti-human uPAR antibody of the present disclosure can be isolated. "Isolated" means that the antibody is separated from all or some of the components that accompany it in nature. "Isolated" also refers to a state in which the antibody is separated from all or some of the components that accompany it during its manufacture (e.g., chemical synthesis, recombinant expression, culture medium, etc. and / or the like).

[0081] Any anti-human uPAR antibody of the present disclosure may comprise a degree and / or pattern of glycosylation that is different from the degree and / or pattern of glycosylation of an antibody produced in nature (e.g., produced in an animal (e.g., produced in a human). For example, an anti-human uPAR antibody of the present disclosure may be a recombinant antibody (e.g., a monoclonal antibody) expressed by one or more recombinant expression vectors transfected into a host cell, wherein the expressed recombinant anti-human uPAR antibody comprises a different degree of glycosylation, a different pattern of glycosylation, or both, compared to the degree and / or pattern of glycosylation of the antibody when produced in nature (e.g., when produced in an animal in response to immunization with a human uPAR antigen).

[0082] In some embodiments, the anti-human uPAR antibodies of the present disclosure comprise a heavy chain comprising an Fc region, and the Fc region is conserved on the V H is heterologous - that is, the Fc region comprises an amino acid sequence (e.g., one or more amino acid substitutions, deletions and / or insertions), one or more post-translational modifications and / or the like, such that the Fc region and V H The antibodies of the combination do not exist in nature, for example, unlike anti-human uPAR antibodies produced in animals in response to immunization with human uPAR antigens.

[0083] In certain embodiments, one or more amino acid modifications may be introduced into the Fc region of an antibody provided herein, thereby producing an Fc region variant. The Fc region variant may comprise a mouse Fc region sequence (e.g., IgG1, IgG2a, or IgG2b) comprising an amino acid modification (e.g., substitution) at one or more amino acid positions. The Fc region variant may comprise a human Fc region sequence (e.g., human IgG1, IgG2, IgG3, or IgG4 Fc region) comprising an amino acid modification (e.g., substitution) at one or more amino acid positions (e.g., an IgG4 isotype comprising an S228P mutation).

[0084] In certain embodiments, the Fc region is mutated to increase its affinity for FcRn at pH 6.0, and thus extend the antibody half-life. Antibodies with enhanced affinity for FcRn include those with substitutions of one or more of Fc region residues 252, 253, 254, 256, 428, 434, including the so-called YTE mutation with M252Y / S254T / T256E substitutions (Dall'Acqua et al., J Immunol. 169: 5171-5180 (2002)) or LS mutations M428L / N434S (Zalevsky et al., Nat Biotechnol. 28 (2): 157–159 (2010)).

[0085] When referring to antibodies, the phrases "specifically binds," "specific for," "immunoreactive," and "immunoreactivity," and "antigen binding specificity" refer to a binding reaction with an antigen that is highly preferential to the antigen or a fragment thereof such that the presence of the antigen is determined in the presence of a heterogeneous population of antigens (e.g., proteins and other biologics, e.g., in a sample). Thus, under specified immunoassay conditions, a specified antibody binds to a specific human uPAR antigen and does not bind to other antigens present in the sample in significant amounts. Specific binding to an antigen under such conditions may require an antibody that is selected for its specificity for a particular antigen. For example, an anti-human uPAR antibody may specifically bind to the human uPAR antigen and not exhibit comparable binding to other proteins present in the sample (e.g., not exhibiting detectable binding to other proteins present in the sample).

[0086] In some embodiments, if the antibodies of the present disclosure are expressed at, for example, greater than or equal to about 10 5 M -1 Affinity or K a The antibody "specifically binds" to the human uPAR antigen if it binds or associates with the human uPAR antigen (i.e., has an equilibrium association constant for the specific binding interaction of units of 1 / M). In certain embodiments, the antibody binds or associates with the human uPAR antigen with an affinity greater than or equal to about 10 6 M -1 , 10 7 M -1 , 10 8 M -1 , 10 9 M -1 , 10 10 M -1 , 10 11 M -1 , 10 12 M -1 or 10 13 M -1 K a Binds to human uPAR. "High affinity" binding means binding with an affinity of at least 10 7 M -1 , at least 10 8 M -1 , at least 10 9 M -1 , at least 10 10 M -1 , at least 10 11 M -1 , at least 10 12 M-1 , at least 10 13 M -1 or higher K a Alternatively, affinity can be defined as having units of M (e.g., 10 -5 M to 10 -13 The equilibrium dissociation constant (K) of a specific binding interaction of D ). In some embodiments, specific binding means that the antibody binds to less than or equal to about 10 -5 M, less than or equal to about 10 -6 M, less than or equal to about 10 -7 M, less than or equal to about 10 -8 M, or less than or equal to about 10 -9 M, 10 -10 M, 10 -11 M, or 10 -12 M or smaller K D Binding to human uPAR. The binding affinity of the antibody to human uPAR can be readily determined using conventional techniques, such as by biolayer interferometry (BLI); competitive ELISA (enzyme-linked immunosorbent assay); equilibrium dialysis; surface plasmon resonance (SPR) technology (e.g., BIAcore 2000 instrument, using the general procedures outlined by the manufacturer); radioimmunoassay; and / or the like.

[0087] In certain embodiments, the antibodies of the present disclosure cross-react with non-human uPAR. For example, the anti-human uPAR antibodies of the present disclosure can cross-react with non-human primate uPAR. In a non-limiting example, the non-human primate uPAR is cynomolgus monkey uPAR. Also as an example, the anti-human uPAR antibodies of the present disclosure can cross-react with rodent uPAR. In some cases, the rodent uPAR is mouse uPAR.

[0088] If an antibody of the present disclosure is specific for two different antigens or antigenic determinants (as defined herein) (e.g., uPAR from two different species of mammals such as human and cynomolgus monkey), then the antibody is said to be "cross-reactive" to these two different antigens or antigenic determinants. In certain embodiments, when the two antigens are specific for uPAR, the two antigens are cross-reactive. 50 and / or K DWhen the affinity for Ag1 and the affinity for Ag2 are in a similar range, the antibody that binds to antigen 1 (Ag1) is "cross-reactive" with antigen 2 (Ag2). According to some embodiments, the monoclonal antibody that binds to Ag1 is cross-reactive with Ag2 when the ratio of the affinity for Ag1 to the affinity for Ag2 is equal to or less than 10 (<10) and equal to or greater than 0.1 (>0.1), which means that the difference in affinity for Ag1 and Ag2 is no more than 10 times (affinity in monovalent K D The antibodies of the present disclosure may have a ratio of affinity for human uPAR to affinity for cynomolgus monkey uPAR that is equal to or less than 10 (<10) and equal to or greater than 0.1 (>0.1), which means that the affinity for human uPAR and cynomolgus monkey uPAR differs by no more than 10-fold (affinity in monovalent K D Such antibodies can be used, for example, in toxicology studies in cynomolgus monkeys, as the toxicity profile observed in cynomolgus monkeys will correlate with potential adverse effects expected in humans.

[0089] "Epitope" is a site on an antigen that binds to an antibody. An epitope can be formed by continuous or non-continuous amino acids juxtaposed by the folding (e.g., tertiary folding) of a protein. An epitope formed by continuous amino acids is usually retained when exposed to a denaturing solvent, while an epitope formed by folding is usually lost when treated with a denaturing solvent. An epitope usually includes at least 3, and more usually, at least 5 or 8 to 10 amino acids in a linear or spatial conformation. Methods for determining the spatial conformation of an epitope include, for example, x-ray crystallography and two-dimensional nuclear magnetic resonance. See, for example, Epitope Mapping Protocols in Methods in Molecular Biology, Vol. 66, edited by Glenn E. Morris (1996). Several commercial laboratories provide epitope mapping services. An epitope bound by an antibody with immunoreactivity to human uPAR can reside on the surface of, for example, human uPAR, so that such epitopes are considered to be accessible to the surface of human uPAR, accessible to solvents, and / or exposed to the surface of human uPAR.

[0090] According to some embodiments, the anti-uPAR antibody of the present disclosure is a humanized antibody. As used herein, a humanized antibody is a recombinant polypeptide derived from a non-human (e.g., rabbit, rodent, etc.) antibody, and has been modified to include at least a portion of the framework and / or constant region of a human antibody. Humanized antibodies also encompass chimeric antibodies and CDR-grafted antibodies, in which different regions can be derived from different species. A chimeric antibody can be an antibody comprising a variable region from any source connected to a human constant region (e.g., a human Fc domain). Therefore, in a chimeric antibody, the variable region can be non-human, and the constant region is human. A CDR-grafted antibody is an antibody comprising a CDR from a non-human "donor" antibody, which is connected to a framework region from a human "acceptor" antibody. For example, an antibody of the present disclosure in the form of scFv can be connected to a human constant region (e.g., an Fc domain) to make a human immunoglobulin.

[0091] Typically, humanized antibodies produce a reduced immune response in a human host compared to a non-humanized version of the same antibody. Antibodies can be humanized using a variety of techniques, including, for example, CDR grafting, veneer or surface resurfacing, chain shuffling, etc. In certain embodiments, framework substitutions are identified by simulating the interactions of CDR and framework residues to identify framework residues that are important for antigen binding and sequence comparison, thereby identifying abnormal framework residues at specific positions.

[0092] Therefore, any antibody described herein can be humanized using available methods. Rabbit CDR or mouse CDR are substituted into the human variable domain framework and can result in retaining its correct spatial orientation, wherein, for example, the human variable domain framework adopts the same or similar conformation as the rabbit or mouse variable framework originated from the CDR. This can be achieved by obtaining human variable domains from human antibodies, and the framework sequence of the human antibody has a high degree of sequence identity with the rabbit or mouse variable framework domain derived from the CDR. Heavy chain and light chain variable framework regions can be derived from the same or different human antibody sequences. The human antibody sequence can be the sequence of a naturally occurring human antibody, or can be the consensus sequence of several human antibodies.

[0093] After identifying the complementary determining regions of the rabbit or mouse donor immunoglobulin and the appropriate human acceptor immunoglobulin, the next step is to determine which residues (if any) from these components should be replaced to optimize the properties of the resulting humanized antibody. Generally, the replacement of human amino acid residues with rabbit or mouse should be minimized because the introduction of rabbit or mouse residues will increase the risk of the antibody eliciting a human anti-rabbit antibody (HARA) or human anti-mouse antibody (HAMA) response in the human body. Methods for determining immune responses recognized in the art can be performed to monitor HARA or HAMA responses in specific patients or during clinical trials. Patients administered humanized antibodies can be given an immunogenicity assessment at the beginning of the therapy and throughout the administration of the therapy. For example, HARA or HAMA responses are measured by detecting antibodies to humanized therapeutic agents in serum samples from patients using methods known to those skilled in the art, including surface plasmon resonance technology (BIACORE) and / or solid phase ELISA analysis. In many embodiments, the subject humanized antibody does not substantially induce a HARA response in a human subject.

[0094] Based on its possible influence on CDR conformation and / or binding to antigen, select certain amino acids from human variable region framework residues for substitution. The non-natural juxtaposition of rabbit or mouse CDR region and human variable framework region may lead to non-natural conformational restrictions, unless corrected by the substitution of certain amino acid residues, otherwise it will lead to the loss of binding affinity. The selection of amino acid residues for substitution can be determined in part by computer modeling. Computer hardware and software for generating three-dimensional images of immunoglobulin molecules are known in the art. Generally, molecular models are generated from the parsed structure of immunoglobulin chains or their domains. The amino acid sequence similarity of the chain to be modeled is compared with the chain or domain of the parsed three-dimensional structure, and the chain or domain showing the maximum sequence similarity is selected as the starting point for building a molecular model. Chains or domains sharing at least 50% sequence identity are selected for modeling, and preferably those chains or domains sharing at least 60%, 70%, 80%, 90% or higher sequence identity are selected for modeling. The parsed starting structure is modified to allow differences between the actual amino acids in the immunoglobulin chain or domain being modeled and those in the starting structure. The modified structures are then assembled into composite immunoglobulins.Finally, the model is refined by energy minimization and by verifying that all atoms are within appropriate distances from each other and that bond lengths and angles are within chemically acceptable limits.

[0095] When the framework residues as defined by, for example, Kabat constitute the structural loop residues as defined by, for example, Chothia, the amino acid present in rabbit or mouse antibody can be selected for substitution into humanized antibodies. The residue "adjacent to CDR region" is included in the position directly adjacent to one or more CDRs in the primary sequence of humanized immunoglobulin chain, for example, in the amino acid residues in the position directly adjacent to the CDR as defined by Kabat or as defined by Chothia (see, for example, Chothia and Lesk JMB 196:901 (1987)). These amino acids may interact with the amino acid in CDR especially, and if selected from acceptor, donor CDR can be distorted and affinity can be reduced. In addition, adjacent amino acids can directly interact with antigens (Amit et al., Science, 233:747 (1986)), and it may be desirable to select these amino acids from donors to keep contacting with all antigens providing affinity in the original antibody. Methods that can be used to humanize any of the antibodies described herein include, but are not limited to, those described in: Williams, D., Matthews, D. & Jones, T. Humanising Antibodies by CDR Grafting. Antibody Engineering 319–339 (2010) doi: 10.1007 / 978-3-642-01144-3_21; Kuramochi, T., Igawa, T., Tsunoda, H. & Hattori, K. Humanization and simultaneous optimization of monoclonal antibody. Methods Mol. Biol. 1060, 123–37 (2014); Hwang, WY, Almagro, JC, Buss, TN, Tan, P. & Foote, J. Use of human germline genes in a CDR homology-based approach to antibody humanization. Methods 36, 35–42 (2005); Lo, BKAntibodyhumanization by CDR grafting. Methods Mol. Biol. 248, 135–59 (2004); and Lefranc, M.-PP, Ehrenmann, F., Ginestoux, C., Giudicelli, V. & Durouux, P. databases and tools for antibody engineering and humanization. Methods Mol. Biol. 907, 3–37 (2012); the disclosure of which is incorporated herein by reference in its entirety for all purposes.

[0096] Bispecific Antibodies

[0097] Bispecific antibodies are also provided. In certain embodiments, the bispecific antibodies of the present disclosure comprise a first antigen binding domain comprising a V domain of any of the anti-human uPAR antibodies of the present disclosure (including any of such antibodies described above). H Peptide-V L Polypeptide pairs. The bispecific antibody may comprise a second antigen binding domain that specifically binds to a human uPAR polypeptide bound by the first antigen binding domain. In certain embodiments, the bispecific antibody comprises a second antigen binding domain that specifically binds to an antigen other than uPAR.

[0098] Bispecific antibodies of the present disclosure include antibodies and bispecific antibody fragments having full-length antibody structures. "Full length" as used herein refers to an antibody having two full-length antibody heavy chains and two full-length antibody light chains. Full-length antibody heavy chains (HC) are composed of well-known heavy chain variable domains and constant domains VH, CH1, CH2 and CH3. Full-length antibody light chains (LC) are composed of well-known light chain variable domains and constant domains VL and CL. Full-length antibodies may lack C-terminal lysine in one or two heavy chains. The term "Fab arm" refers to a heavy chain: light chain pair that specifically binds to an antigen.

[0099] For example, full-length bispecific antibodies can be produced by introducing substitutions at the heavy chain CH3 interface in each half molecule, using Fab arm exchange (or half molecule exchange) between two monospecific bivalent antibodies, to facilitate the formation of heterodimers of two antibody half molecules with different specificities in vitro or using coexpression in a cell-free environment. The Fab arm exchange reaction is the result of the dissociation-association of disulfide bonds and CH3 domains. The heavy chain disulfide bonds in the hinge region of the parental monospecific antibody are reduced. The resulting free cysteine ​​of one of the parental monospecific antibodies forms an inter-heavy chain disulfide bond with the cysteine ​​residue of the second parental monospecific antibody molecule, and at the same time, the CH3 domain of the parental antibody is released and re-formed by dissociation-association. The CH3 domain of the Fab arm can be engineered to facilitate heterodimerization rather than homodimerization. The resulting product is a bispecific antibody with two Fab arms or half molecules, each of which binds to a different epitope.

[0100] The "knob-in-hole" strategy (see, e.g., WO 2006 / 028936) can be used to generate full-length bispecific antibodies. In short, selected amino acids that form the interface of the CHS domain in human IgG can be mutated at positions that affect the interaction of the CH3 domains to promote the formation of heterodimers. Amino acids with small side chains (holes) are introduced into the heavy chain of an antibody that specifically binds to a first antigen, and amino acids with large side chains (knobs) are introduced into the heavy chain of an antibody that specifically binds to a second antigen. After co-expression of the two antibodies, heterodimers are formed due to the preferential interaction of the heavy chain with the "hole" with the heavy chain with the "knob". Exemplary CH3 substitution pairs that form a knob and hole are (represented as modified position in the first CH3 domain of the first heavy chain / modified position in the second CH3 domain of the second heavy chain): T366Y7F405A, T366W / F405W, F405W / Y407A, T394W / Y407T, T3945 / Y407A, T366W / T394S, F405W / T394S and T366W / T366S_L368A_Y407V.

[0101] Other strategies can be used, such as by replacing positively charged residues at one CH3 surface and negatively charged residues at the second CH3 surface, using electrostatic interactions to promote heavy chain heterodimerization, as described in US2010 / 0015133; US2009 / 0182127; US2010 / 028637 or US2011 / 0123532. In other strategies. Heterodimerization can be promoted by the following substitutions (represented as modified positions in the first CH3 domain of the first heavy chain / modified positions in the second CH3 domain of the second heavy chain): L351 Y_F405A_Y407V T394W, T366I_K392M_T394W / F405A_Y407V, T366L_K392M_T394W / F405A_Y407V, L351Y_Y407A, T366A_K409F, L351Y_Y407A / T366V_K409F, Y407A / T366A_K409F or T350V_L351Y_F405A_Y407V / T350V_T366L_K392L_T394W, as described in US2012 / 0149876 or US2013 / 0195849.

[0102] Single-chain bispecific antibodies are also provided. In some embodiments, the single-chain bispecific antibodies of the present disclosure are bispecific scFvs. Details about bispecific scFvs can be found in, for example, Zhou et al. (2017) J Cancer 8(18): 3689-3696.

[0103] Methods that can be used to generate multispecific (e.g., bispecific) antibodies from the antibodies described herein include, but are not limited to, Ellerman, D. (2019). "Bispecific T-cell engagers: Towards understanding variables influencing the in vitro potency and tumor selectivity and their modulation to enhance their efficacy and safety." Methods 154: 102-117; Brinkmann, U. and RE Kontermann (2017). "The making of bispecific antibodies." mAbs 9(2): 182-212; and Suurs, FV et al., (2019). "A review of bispecific antibodies and antibody constructs in oncology and clinical challenges." Pharmacol Ther 201: 103-119; the disclosures of which are incorporated herein by reference in their entirety for all purposes.

[0104] Fusion Protein

[0105] Fusion proteins are also provided. In certain embodiments, the fusion proteins of the present disclosure include chains of any one of the anti-uPAR antibodies of the present disclosure fused to heterologous sequences of amino acids. The heterologous sequences of amino acids may be fused to the C-terminus of the antibody chain or the N-terminus of the antibody chain. In certain embodiments, the fusion proteins of the present disclosure include heterologous sequences at the C-terminus of the antibody chain and heterologous sequences at the N-terminus of the antibody chain, wherein the heterologous sequences may be the same sequence or different sequences. "Heterologous" as used in the context of nucleic acids or polypeptides generally means that the nucleic acid or polypeptide is from a source different from the source to which the nucleic acid or polypeptide is associated or connected (e.g., molecules of different sequences, sources of different species, etc.), so that the nucleic acid or polypeptide is a nucleic acid or polypeptide not found in nature. For example, in a fusion protein, a light chain polypeptide and a reporter polypeptide (e.g., GFP, red fluorescent protein (e.g., mCherry), luciferase, etc.) are referred to as "heterologous" to each other. Similarly, the CDR from a mouse antibody and the constant region from a human antibody are "heterologous" to each other.

[0106] The chains of anti-human uPAR antibodies can be fused to any interested heterologous sequence. Interested heterologous sequences include, but are not limited to, albumin, transferrin, XTEN, homologous amino acid polymers, proline-alanine-serine polymers, elastin-like peptides, or any combination thereof. In some aspects, the heterologous polypeptide increases the stability and / or serum half-life of the antibody after it is administered to an individual in need thereof, compared to the same antibody not fused to the heterologous sequence.

[0107] In certain embodiments, the fusion protein of the present disclosure comprises a single chain antibody, for example, a V or V fragment of any of the anti-human uPAR antibodies of the present disclosure. H Peptide-V L The anti-human uPAR antibodies of the present disclosure include any of the single-chain antibodies described above.

[0108] According to some embodiments, when the fusion protein comprises a single chain antibody (e.g., any of the single chain antibodies of the present disclosure, including any of the scFv described herein), the fusion protein is a chimeric antigen receptor (CAR) comprising the single chain antibody, a transmembrane domain, and an intracellular signaling domain.

[0109] CAR of the present disclosure may include one or more joint sequences between various domains. "Variable region connection sequence" is an amino acid sequence that connects the heavy chain variable region to the light chain variable region and provides a spacer function compatible with the interaction of two sub-binding domains, so that the resulting polypeptide retains the same specific binding affinity for the target molecule as an antibody comprising the same light chain and heavy chain variable regions. A non-limiting example of a variable region connection sequence is a serine-glycine joint, such as a serine-glycine joint including the amino acid sequence GGGGSGGGGSGGGGS (G4S) 3 (SEQ ID NO: 54). In some aspects, the joint separates one or more heavy or light chain variable domains, hinge domains, transmembrane domains, costimulatory domains, and / or primary signaling domains. In a specific embodiment, CAR includes one, two, three, four, or five or more joints. In a specific embodiment, the length of the joint is about 1 to about 25 amino acids, about 5 to about 20 amino acids, or about 10 to about 20 amino acids or any intermediate length of amino acids. In some embodiments, the linker is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or more amino acids in length.

[0110] In some embodiments, the antigen binding domain of CAR is followed by one or more spacer domains, which move the antigen binding domain away from the effector cell surface (e.g., the surface of T cells expressing CAR) to achieve appropriate cell / cell contact, antigen binding and / or activation. The spacer domain (and any other spacer domains, joints and / or the like described herein) can be derived from natural sources, synthetic sources, semi-synthetic sources or recombinant sources. In certain embodiments, the spacer domain is a part of an immunoglobulin, including but not limited to one or more heavy chain constant regions, such as CH2 and CH3. The spacer domain may include a naturally occurring immunoglobulin hinge region or an amino acid sequence of an altered immunoglobulin hinge region. In one embodiment, the spacer domain includes CH2 and / or CH3 of IgG1, IgG4 or IgD. The exemplary spacer domains suitable for CAR described herein include hinge regions (such as CD8α and CD4) derived from the extracellular region of type 1 membrane proteins, which may be wild-type hinge regions from these molecules or variants thereof. In some aspects, the hinge domain includes a CD8α hinge region. In some embodiments, the hinge is a PD-1 hinge or a CD152 hinge.

[0111] "Transmembrane domain" (Tm domain) is a CAR portion that fuses the extracellular binding portion and the intracellular signaling domain and anchors CAR to the plasma membrane of a cell (e.g., immune effector cell). The TM domain can be derived from a natural source, a synthetic source, a semi-synthetic source, or a recombinant source. In some embodiments, the Tm domain is derived from an alpha chain or beta chain (e.g., including at least its transmembrane region or its functional portion) of a T cell receptor, CD35, CD3ζ, CD3γ, CD3δ, CD4, CD5, CD8α, CD9, CD16, CD22, CD27, CD28, CD33, CD37, CD45, CD64, CD80, CD86, CD134, CD137, CD152, CD154, or PD-1.

[0112] In one embodiment, CAR includes a Tm domain derived from CD8α. In some aspects, CAR includes a Tm domain derived from CD8α and a short oligopeptide or polypeptide linker connecting the Tm domain of CAR to the intracellular signaling domain, such as a length of 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids. For example, a glycine-serine linker can be used as such a linker.

[0113] The "intracellular signaling" domain of CAR refers to a part of CAR that is involved in transducing signals from CAR bound to target molecules / antigens to the interior of immune effector cells to trigger effector cell functions, such as activation, cytokine production, proliferation and / or cytotoxic activity, including the release of cytotoxic factors to CAR-bound target cells, or other cell responses triggered by target molecules / antigens bound to extracellular CAR domains. Therefore, the term "intracellular signaling domain" refers to a protein portion that transduces effector function signals and guides cells to perform specific functions. In terms of using a truncated portion of an intracellular signaling domain, such a truncated portion can be used to replace the full-length intracellular signaling domain as long as it transduces effector function signals. The term intracellular signaling domain is intended to include any truncated portion of an intracellular signaling domain sufficient for transducing effector function signals.

[0114] The signal generated by the T cell receptor (TCR) alone is not enough to fully activate T cells, and secondary or costimulatory signals are also required. Therefore, T cell activation is mediated by two different types of intracellular signaling domains: a primary signaling domain that initiates antigen-dependent primary activation through TCR (e.g., TCR / CD3 complex) and a costimulatory signaling domain that acts in an antigen-independent manner to provide a secondary or costimulatory signal. Therefore, the CAR of the present disclosure may include an intracellular signaling domain that includes one or more "costimulatory signaling domains" and a "primary signaling domain".

[0115] The primary signaling domain regulates the primary activation of the TCR complex in a stimulatory manner or in an inhibitory manner. The primary signaling domain that acts in a stimulatory manner may include a signaling motif known as an immunoreceptor tyrosine-based activation motif (or "ITAM"). Non-limiting examples of ITAM-containing primary signaling domains suitable for CARs of the present disclosure include those derived from FcRγ, FcRβ, CD3γ, CD3δ, CD3ε, CD3ζ, CD22, CD79α, CD79β, and CD66δ. In certain embodiments, CAR includes a CD3ζ primary signaling domain and one or more costimulatory signaling domains. The intracellular primary signaling domain and the costimulatory signaling domain are operably connected to the carboxyl end of the transmembrane domain.

[0116] In some embodiments, CAR includes one or more costimulatory signaling domains to enhance the efficacy and amplification of immune effector cells (e.g., T cells) expressing CAR. As used herein, the term "costimulatory signaling domain" or "costimulatory domain" refers to the intracellular signaling domain of a costimulatory molecule or its active fragment. In a specific embodiment, the example costimulatory molecules suitable for CAR include TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, CARD11, CD2, CD7, CD27, CD28, CD30, CD40, CD54 (ICAM), CD83, CD134 (OX40), CD137 (4-1BB), CD278 (ICOS), DAP10, LAT, KD2C, SLP76, TRIM and ZAP70. In some embodiments, the CAR comprises one or more co-stimulatory signaling domains selected from the group consisting of 4-1BB (CD137), CD28, and CD134, and a CD3 zeta primary signaling domain.

[0117] CAR of the present disclosure may include any kind of suitable domain, including but not limited to leader sequence; hinge, spacer and / or linker domain; transmembrane domain; costimulatory domain; signaling domain (e.g., CD3ζ domain); ribosomal skipping element; restriction enzyme sequence; reporter protein domain; and / or the like. Non-limiting examples of such domains that may be included in CAR of the present disclosure include those provided in Table 6 below. As will be understood by those of ordinary skill in the art, the amino acid sequence of one or more of the domains shown in Table 6 (e.g., linker, hinge, transmembrane, costimulatory, signaling, ribosomal skipping element; restriction enzyme sequence; reporter protein, etc.) can be modified as needed, such as for improved functions of CAR, etc.

[0118] In certain aspects, the CAR of the present disclosure includes a single-chain antibody (e.g., any scFv of the present disclosure) bound to human uPAR; a transmembrane domain of a polypeptide selected from the group consisting of: CD4, CD8α, CD154, and PD-1; one or more intracellular costimulatory signaling domains of a polypeptide selected from the group consisting of: 4-1BB (CD137), CD28, and CD134; and an intracellular signaling domain of a polypeptide selected from the group consisting of: FcRγ, FcRβ, CD3γ, CD3δ, CD3ε, CD3ζ, CD22, CD79α, CD79β, and CD66δ. Such CARs may further include a spacer domain between the antigen binding portion and the transmembrane domain, such as a CD8α hinge.

[0119] According to some embodiments, a CAR is provided, which comprises, from N-terminus to C-terminus, a variable heavy chain (V H ) polypeptides, linkers, variable light chains (V L ), CD8 hinge region (which in some embodiments is an extended CD8 hinge region), CD8 transmembrane domain, 4-1BB costimulatory domain and CD3ζ signaling domain. According to certain embodiments, a CAR is provided, which comprises a variable light chain (V L) of an antibody described herein from N-terminus to C-terminus. L ) polypeptide, linker, variable heavy chain (V H ), CD8 hinge region (which in some embodiments is an extended CD8 hinge region), CD8 transmembrane domain, 4-1BB costimulatory domain and CD3ζ signaling domain. In certain embodiments, a CAR is provided, which comprises a variable heavy chain (V H ) polypeptides, linkers, variable light chains (V L ), CD28 hinge region, CD28 transmembrane domain, 4-1BB costimulatory domain and CD3ζ signaling domain. According to some embodiments, a CAR is provided, wherein the CAR comprises a variable light chain (V L) of an antibody described herein from N-terminus to C-terminus. L ) polypeptide, linker, variable heavy chain (V H ), CD28 hinge region, CD28 transmembrane domain, 4-1BB costimulatory domain and CD3ζ signaling domain. Any of the CARs disclosed herein may be included in V H The domain at the N-terminus of the polypeptide. For example, a leader sequence (e.g., a GM-CSFR leader sequence) can be present at the N-terminus of the CAR of the present disclosure.

[0120] As required, the CAR of the present disclosure may include one or more additional domains. Non-limiting examples of such additional domains include ribosome jumping elements, enzymatic domains (e.g., domains with nuclease activity such as restriction endonuclease activity), domains capable of detecting CAR (e.g., reporter protein domains (e.g., fluorescent proteins (e.g., eGFP, mCherry or the like), luminescent proteins and / or the like)), etc. For example, in certain embodiments, there is provided a CAR comprising a ribosome jumping element, a restriction enzyme domain and / or a reporter protein domain.

[0121] According to some embodiments, the CAR of the present disclosure is provided by a single polypeptide. In certain embodiments, the CAR of the present disclosure is provided by two or more polypeptides. When the CAR is provided by two or more polypeptides, the CAR can be provided in any useful multi-polypeptide format, including universal CAR formats, such as biotin-binding immune receptor (BBIR) formats (see, e.g., Urbanska K, Powell DJ. Development of a novel universal immune receptor for antigen targeting to infinity and beyond. Oncoimmunology. 2012; 1(5):777-779. doi:10.4161 / onci.19730, and Urbanska K, Lanitis E, Poussin M et al., A universal strategy for adoptive immunotherapy of cancer through use of a novel T cell antigen receptor. 2013; 72(7):1844-1852. doi:10.1158 / 0008-5472.CAN-11-3890.A); switchable CAR formats with peptide new epitopes (PNEs) (see, e.g., Kim et al., (2015) J Am Chem Soc. 2015; 137(8):2832-2835; Ma et al., (2016) Proc Natl Acad Sci 113(4):E450-8; Rodgers et al., (2016) Proc Natl Acad Sci. 113(4):E459-E468; Viaud et al., (2018) Proc Natl Acad Sci 115(46):E10898-E10906); SUPRA CAR format with leucine zipper (see, e.g., Cho et al., (2018) Cell 173(6):1426-1438.e11); CAR-T adapter molecule (CAM)-based format with FITC folate (see, e.g., Lee et al., (2019) Cancer Res. 79(2):387-396; and Lu et al., (2019) Front Oncol. 9:151); anti-FITC-folate adapter format (see, e.g., Chu et al., (2018) Biosci Trends.12(3):298-308); anti-FITC antibody adapter CAR format (see, e.g., Tamada et al., (2012) Clin Cancer Res. 18(23):6436-6445); Fc-targeted (e.g., anti-CD16) CAR + anti-tumor antibody format (see, e.g., Kudo et al., (2014) Cancer Res. 74(1):93-103); and the like. .

[0122] Conjugate

[0123] The present disclosure also provides conjugates. According to some embodiments, the conjugates of the present disclosure include any one of the antibodies or fusion proteins of the present disclosure, and a pharmaceutical agent conjugated to the antibody or fusion protein. The term "conjugated" generally refers to a covalent or non-covalent (usually covalent) chemical connection that tightly associates a molecule of interest with a second molecule of interest. In certain embodiments, the pharmaceutical agent conjugated to the antibody or fusion protein is a chemotherapeutic agent, a toxin, a radiation sensitizer, a radioisotope (e.g., a therapeutic radioisotope), a detectable marker, or a half-life extension portion.

[0124] According to some embodiments, the agent is a therapeutic agent, such as a chemotherapeutic agent. As used herein, a "therapeutic agent" is a physiologically or pharmacologically active substance that can produce a desired biological effect in a target site of an animal such as a mammal or a human being. The therapeutic agent can be any inorganic compound or an organic compound. Examples include, but are not limited to, peptides, proteins, nucleic acids (including siRNA, miRNA and DNA), polymers and small molecules. The therapeutic agent can reduce, inhibit, weaken, reduce, prevent or stabilize the development or process of a disease, illness or cell growth in an animal such as a mammal or a human being. The therapeutic agent of interest includes an agent that can affect the function of the cell / tissue to which the conjugate is bound via the specific binding of the antibody portion of the conjugate to the antigen. When the function of the cell / tissue is pathological, an agent that reduces the function of the cell / tissue can be used. In some aspects, the conjugate of the present disclosure includes an agent that reduces the function of the target cell / tissue by inhibiting cell proliferation and / or killing the cell / tissue. Such agents can vary and include cell growth inhibitors and cytotoxic agents, for example, agents that can kill target cell tissues when they are internalized into the target cell or when they are not internalized into the target cell.

[0125] In certain embodiments, the therapeutic agent is a cytotoxic agent selected from the group consisting of enediynes, lexitropsins, duocarmycins, taxanes, puromycins, dolastatins, maytansinoids, and vinca alkaloids. In some embodiments, the cytotoxic agent is paclitaxel, docetaxel, CC-1065, CPT-11 (SN-38), topotecan, doxorubicin, morpholino-doxorubicin, rhizoxin, cyanomorpholino-doxorubicin, dolastatin-10, echinomycin, combretin, calicheamicin, maytansine, maytansine DM1, maytansine DM4, DM-1, auristatin or other dolastatin derivatives such as auristatin E or auristatin F, AEB (AEB-071), AEVB (5-benzoylvaleric acid-AE ester), AEFP (antibody-endostatin fusion protein), MMAE (monomethyl auristatin E), MMAF (monomethyl auristatin F), pyrrolobenzodiazepines. type (PBD), eleutherobin, fusobacterium or any combination thereof.

[0126] According to some embodiments, the agent is a toxin, such as a protein toxin selected from the group consisting of hemiasterlin and hemiasterlin analogs such as HTI-286 (see, e.g., USPN 7,579,323; WO 2004 / 026293; and USPN 8,129,407, the entire disclosure of which is incorporated herein by reference), abrin, strychnine, cypermethrin, diphtheria toxin, frog dart toxin, botulinum toxin, Shiga toxin, endotoxin, Pseudomonas exotoxin, Pseudomonas endotoxin, tetanus toxin, pertussis toxin, anthrax toxin, cholera toxin, falcarinol, fumonisin B1, fumonisin B2, aflatoxin, maurotoxin, agitoxin, charybdotoxin, margatoxin, slotoxin, scyllatoxin, hefutoxin, calciseptine, taicatoxin, calcicludine, geldanamycin, gelonin, lotaustralin, ocratoxin A, patulin, ricin, strychnine, trichothecenes, zearalenone and tetrodotoxin. Enzymatically active toxins and fragments thereof that can be used include diphtheria A chain, non-binding active fragments of diphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain, abrin A chain, modeccin A chain, α-sarcin, Aleurites fordii protein, Dianthus caryophyllus protein, Phytolacca americana protein (PAPI, PAPII and PAP-S), Momordica charantia inhibitor, Curcin, crotonin, sapaonaria officinalis inhibitor, gelonin, mitogellin, restrictocin, phenomycin,

[0127] Enomycin and trichothecenes.

[0128] In certain embodiments, the agent is a radiation sensitizer. As used herein, a "radiation sensitizer" is an agent that enhances the ability of radiation to kill tumor cells. Non-limiting examples of radiation sensitizers that can be conjugated to antibodies or fusion proteins include cisplatin, 5-fluorouracil (5-FU), AZD7762, selumetinib, and the like.

[0129] In certain embodiments, the agent is a radioactive isotope, for example, which can be used for therapy and / or detection (e.g., imaging). Non-limiting examples of radioactive isotopes that can be conjugated to an antibody or fusion protein include, but are not limited to 225 Ac,111 Ag 114 Ag 71 As 72 As 77 As 211 And 198 I 199 I 212 Hello 213 Hello 75 Bro 76 Bro 11 C、 13 C、 55 Co. 62 Cu、 64 Cu、 67 Cu、 165 Dy 166 Dy 169 Err 18 F、 19 F、 52 Fe 59 Fe 66 Ga 67 Ga 68 Ga 72 Ga 154-158 God 157 God 159 God 166 Ho 120 I 121 I 123 I 124 I 125 I 131 I 110 in 111 in 113m in 194 Ir 81m Cr 177 sun 51 Mn 52 Mn 99 For 13 N 15 N 15 Or 17 Or 32 Q 33 Q 211 Pb、 212 Pb、 109 pd 149 PM 151 PM 142 Mr. 143 Mr. 191 PT. 193m PT. 195mPt, 223 Ra, 142 Rb, 186 Re, 188 Re, 189 Re, 105 Rh, 47 Sc, 75 Se, 153 Sm, 117m Sn, 121 Sn, 83 Sr. 89 Sr. 161 Tb, 94 Tc, 99 Tc, 99m Tc, 227 Th, 201 Tl, 172 Tm, 127 Te, 90 Y. 169 Yb, 175 Yb, 133 X and 89 Zr.

[0130] In certain embodiments, the radioisotope is conjugated to the antibody or fusion protein via a chelator, such as a bifunctional chelator. The bifunctional chelator may comprise a metal chelating moiety and a reactive functional group, the metal chelating moiety binding the radioisotope in a stable coordination complex, the reactive functional group covalently attached to a targeting moiety, such as any antibody or fusion protein of the present disclosure, so that the radioisotope can be appropriately directed to a desired molecular target in vivo. Examples of bifunctional chelators that can be used to conjugate the antibody or fusion protein of the present disclosure to the radioisotope include those described in Price & Orvig (2014) Chem. Soc. Rev. 43: 260; and Brechbiel (2008) Q J Nucl Med Mol Imaging 52 (2): 166-173.

[0131] According to some embodiments, the radioisotope is a therapeutic radioisotope. In certain embodiments, the radioisotope is an alpha emitting radioisotope, such as 225 Ac, 211 At 212 Bi / 212 Pb, 213 Bi, 223 Ra or 227 In other embodiments, the radioisotope is a beta-negative emitting radioisotope, such as 32 P. 33 P. 67 Cu,90 Y. 131 I or 177 Lu.

[0132] According to some embodiments, the medicament is a labeling agent. "Labeling agent" (or "detectable marker") means that the medicament can detectably label antibodies or fusion proteins so that the antibodies or fusion proteins can be detected in applications of interest (e.g., in vitro and / or in vivo research and / or clinical applications). Interested detectable markers include radioisotopes (e.g., γ or positron emitters), enzymes that produce detectable products (e.g., horseradish peroxidase, alkaline phosphatase, luciferase, etc.), fluorescent proteins, paramagnetic atoms, etc. In some aspects, antibodies or fusion proteins are conjugated to the specific binding partner of the detectable marker, such as conjugated to biotin, so that the detectable markers including avidin / streptavidin can be detected.

[0133] In certain embodiments, the agent is a labeled agent that finds use in in vivo imaging, such as near infrared (NIR) optical imaging, single photon emission computed tomography (SPECT) ± CT imaging, positron emission tomography (PET) ± CT imaging, nuclear magnetic resonance (NMR) spectroscopy, etc. Labeled agents that find use in such applications include, but are not limited to, fluorescent markers, radioisotopes, etc. In certain aspects, the labeled agent is a multimodal in vivo imaging agent that allows in vivo imaging using two or more imaging methods (e.g., see Thorp-Greenwood and Coogan (2011) Dalton Trans. 40: 6129-6143).

[0134] In certain embodiments, the labeling agent is an in vivo imaging agent that finds use in near infrared (NIR) imaging applications. Such agents include, but are not limited to, Kodak X-SIGHT dyes, Pz 247, DyLight 750 and 800 Fluors, Cy5.5 and 7 Fluors, Alexa Fluor 680 and 750 dyes, IRDye 680 and 800CW Fluors. According to some embodiments, the labeling agent is an in vivo imaging agent that finds use in SPECT imaging applications, non-limiting examples of which include 99m Tc, 111 In, 123 I. 201 Tl and 133 In certain embodiments, the labeling agent is an in vivo imaging agent that finds use in PET imaging applications, such as 11 C. 13 N. 15 O. 18 F. 64 Cu,62 Cu, 124 I. 76 Br, 82 Rb, 68 Ga et al.

[0135] For half-life extension, the antibodies and fusion proteins of the present disclosure can be conjugated to agents that provide improved pharmacokinetic profiles (e.g., by pegylation, hyperglycosylation, etc.). Modifications that can increase serum half-life are of interest. The subject antibody or fusion protein can be "pegylated" in that it contains one or more polyethylene glycol (PEG) moieties. Methods and reagents suitable for the pegylation of proteins are well known in the art and can be found, for example, in U.S. Pat. No. 5,849,860. PEGs suitable for conjugation to proteins are typically soluble in water at room temperature and have the general formula R(O-CH2-CH2) n In some embodiments, the present invention relates to a PEG-containing peptide or a PEG-containing peptide. The PEG-containing peptide is preferably a PEG-containing peptide or a PEG-containing peptide. The PEG-containing peptide is preferably a PEG-containing peptide or a PEG-containing peptide. The PEG-containing peptide is preferably a PEG-containing peptide or a PEG-containing peptide. The PEG-containing peptide is preferably a PEG-containing peptide or a PEG-containing peptide. The PEG-containing peptide is preferably a PEG-containing peptide or a PEG-containing peptide. The PEG-containing peptide is preferably a PEG-containing peptide or a PEG-containing peptide.

[0136] Where a subject antibody or fusion protein is to be isolated from a source, the antibody or fusion protein can be conjugated to one or more moieties that facilitate purification, such as members of a specific binding pair, e.g., biotin (a member of a biotin-avidin specific binding pair), lectin, etc. The antibody can also be bound to (e.g., immobilized to) a solid support including, but not limited to, polystyrene plates or beads, magnetic beads, test strips, membranes, and the like.

[0137] Where the antibody or fusion protein is to be detected in an assay, the antibody or fusion protein may comprise a detectable label, such as a radioisotope (e.g., 89 Zr; 111 In, etc.), enzymes that produce detectable products (e.g., luciferase, β-galactosidase, horseradish peroxidase, alkaline phosphatase, etc.), fluorescent proteins, chromogenic proteins, dyes (e.g., fluorescein isothiocyanate, rhodamine, phycoerythrin, etc.); fluorescent emitting metals, such as 152Eu, or other lanthanide metals, attached to the protein via a metal chelating group such as EDTA; chemiluminescent compounds, such as luminol, isoluminol, acridinium salts, etc.; bioluminescent compounds, such as fluorescein; fluorescent proteins; etc. Indirect labels include antibodies specific for the subject protein, where the antibody can be detected via a secondary antibody; and members of a specific binding pair, such as biotin-avidin, etc.

[0138] Any of the above-mentioned agents can be conjugated to an antibody or fusion protein via a joint. If present, the joint molecule can have enough length to allow the antibody or fusion protein and the medicament to be connected to move flexibly between the antibody or fusion protein and the medicament to be connected. The joint molecule can be, for example, about 6-50 atoms long. The joint molecule can also be, for example, an aryl acetylene, an ethylene glycol oligomer containing 2-10 monomeric units, a diamine, a diacid, an amino acid or a combination thereof.

[0139] Where the linker is a peptide, the linker can be of any suitable length, such as 1 amino acid (e.g., Gly) to 20 or more amino acids, 2 amino acids to 15 amino acids, 3 amino acids to 12 amino acids, including 4 amino acids to 10 amino acids, 5 amino acids to 9 amino acids, 6 amino acids to 8 amino acids, or 7 amino acids to 8 amino acids, and can be 1, 2, 3, 4, 5, 6, or 7 amino acids in length.

[0140] Flexible linkers include glycine polymers (G) n , glycine-serine polymers, glycine-alanine polymers, alanine-serine polymers and other flexible linkers known in the art. Glycine and glycine-serine polymers can be used for relatively unstructured amino acids of interest and can be used as neutral tethers between components. One of ordinary skill in the art will recognize that the design of an antibody or fusion protein conjugated to any of the above-mentioned agents can include all or part of a flexible linker, such that the linker can include a flexible linker and one or more portions that confer a less flexible structure.

[0141] According to some embodiments, the antibody or fusion protein is conjugated to the agent via a non-cleavable linker. Non-cleavable linkers of interest include, but are not limited to, thioether linkers. Examples of thioether linkers that can be employed include succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC) linkers.

[0142] In certain embodiments, antibody is conjugated with agent via cleavable joint.According to some embodiments, joint is chemically unstable joint, such as acid cleavable joint, it is stable under neutral pH (blood flow pH 7.3-7.5), but is internalized into weakly acidic endosome (pH 5.0-6.5) and lysosome (pH 4.5-5.0) of target cell (for example, cancer cell) and experiences hydrolysis afterwards.Chemically unstable joint includes but is not limited to joint based on hydrazone, joint based on oxime, joint based on carbonate, joint based on ester etc.In certain embodiments, joint is enzyme unstable joint, such as being stable in blood flow but after being internalized into target cell, experience for example by the enzyme unstable joint of the enzymatic cleavage of lysosomal protease (such as cathepsin or plasmin) in lysosome of target cell (for example, cancer cell). Enzyme-labile linkers include, but are not limited to, linkers comprising peptide bonds, e.g., dipeptide-based linkers, such as valine-citrulline (VC) linkers, such as maleimidopropionyl-valine-citrulline-p-aminobenzyl (MC-vc-PAB) linkers, valine-alanyl-p-aminobenzyloxy (Val-Ala-PAB) linkers, etc. Chemically unstable linkers, enzyme-labile linkers, and non-cleavable linkers are known and described in detail in, e.g., Ducry & Stump (2010) Bioconjugate Chem. 21: 5-13; Nolting, B. (2013) Methods Mol Biol. 1045: 71-100; Tsuchikama and An (2018) Protein & Cell 9 (1): 33-46; and elsewhere.

[0143] Many strategies can be used to connect the agent directly or indirectly to the antibody or fusion protein via a linker. For example, the agent can be derivatized by covalently attaching a linker to the agent, wherein the linker has a functional group that can react with a "chemical handle" on the antibody or fusion protein. The functional group on the linker can vary and can be selected based on compatibility with the chemical handle on the antibody or fusion protein. According to one embodiment, the chemical handle on the antibody or fusion protein is provided by incorporating a non-natural amino acid with a chemical handle into the antibody or fusion protein. Non-natural amino acids that have been found to be useful for preparing the conjugates of the present disclosure include those with azides, alkynes, olefins, amino-oxy groups, hydrazines, aldehydes (e.g., formylglycine, such as SMARTag from Catalent Pharma Solutions). TMThe present invention relates to amino acids with functional groups such as nitrone, nitrile oxide, cyclopropene, norbornene, isocyanide, aryl halide and boronic acid functional groups. Non-natural amino acids that can be incorporated into the antibodies of the conjugates of the present invention (which can be selected to provide the functional group of interest) are known and described, for example, in Maza et al., (2015) Bioconjug. Chem. 26 (9): 1884-9; Patterson et al., (2014) ACS Chem. Biol. 9: 592-605; Adumeau et al., (2016) Mol. Imaging Biol. (2): 153-65; and elsewhere. Non-natural amino acids can be incorporated into antibodies or fusion proteins via chemical synthesis or recombinant methods, for example, during translation of the antibody or fusion protein in a host cell, using a suitable orthogonal aminoacyl tRNA synthetase-tRNA pair for incorporating non-natural amino acids.

[0144] The functional group of the non-natural amino acid present in the antibody or fusion protein can be an azide, an alkyne, an alkene, an amino-oxy group, a hydrazine, an aldehyde, an asaldehyde, a nitrone, a nitrile oxide, a cyclopropene, a norbornene, an isocyanide, an aryl halide, a boronic acid, a diazo, a tetrazine, a tetrazole, a quadrocyclane, an iodobenzene or other suitable functional group, and the functional group on the linker is selected to react with the functional group of the non-natural amino acid (or vice versa). As just one example, a non-natural amino acid with an azide (e.g., 5-azido-L-norvaline, etc.) can be incorporated into an antibody or fusion protein, and the linker portion of the linker-agent portion can include an alkyne functional group, so that the antibody or fusion protein and the linker-agent portion are covalently conjugated via an azide-alkyne cycloaddition. Conjugation can be performed using, for example, a copper-catalyzed azide-alkyne cycloaddition reaction.

[0145] In certain embodiments, the chemical handle on the antibody or fusion protein does not involve non-natural amino acids. Antibodies that do not contain non-natural amino acids can be conjugated to the agent by utilizing, for example, a nucleophilic functional group of the antibody or fusion protein (such as the N-terminal amine or the primary amine of lysine, or any other nucleophilic amino acid residue) as a nucleophile in a substitution reaction with a portion with a reactive leaving group or other electrophilic group. An example would be to prepare an agent-linker portion with an N-hydroxysuccinimide (NHS) ester and allow it to react with the antibody or fusion protein under aqueous conditions at an elevated pH (about 10), or in a polar organic solvent (such as DMSO) with an added non-nucleophilic base (such as N,N-diisopropylethylamine).

[0146] It will be appreciated that the specific methods used to connect linkers, agents and / or antibodies or fusion proteins to one another may vary depending on the specific linkers, agents and / or antibodies or fusion proteins and functional groups selected and used to conjugate the various components to one another.

[0147] Methods for producing antibodies

[0148] Using the information provided herein, standard techniques well known to those skilled in the art can be used to prepare anti-uPAR antibodies and fusion proteins of the present disclosure. For example, nucleic acid sequences encoding the amino acid sequences of antibodies or fusion proteins of the present disclosure can be used to express antibodies or fusion proteins. The polypeptide sequences provided herein (see, e.g., Table 1) can be used to determine suitable nucleic acid sequences encoding antibodies or fusion proteins, and then the nucleic acid sequences are used to express one or more antibodies or fusion proteins that are specific to human uPAR. Nucleic acid sequences can be optimized to reflect the specific codon "preference" of various expression systems according to standard methods well known to those skilled in the art. Using the sequence information provided, nucleic acids can be synthesized according to many standard methods known to those skilled in the art.

[0149] Once nucleic acids encoding subject antibodies have been synthesized, they can be amplified and / or cloned according to standard methods. Molecular cloning techniques for achieving these purposes are known in the art. Various cloning and in vitro amplification methods suitable for constructing recombinant nucleic acids are known to those skilled in the art and are the subject of many textbooks and laboratory manuals.

[0150] The expression of natural nucleic acid or synthetic nucleic acid encoding the antibody and fusion protein of the present disclosure can be achieved by operably connecting the nucleic acid encoding the antibody or fusion protein to a promoter (which is constitutive or inducible), and incorporating the construct into an expression vector to produce a recombinant expression vector. The vector may be suitable for replication and integration in prokaryotes, eukaryotes, or both. Typical cloning vectors include functionally appropriate transcription and translation terminators, initiation sequences, and promoters that can be used to regulate the expression of nucleic acids encoding antibodies. The vector optionally includes a universal expression cassette containing at least one independent terminator sequence, a sequence (e.g., as found in shuttle vectors) that allows the expression cassette to replicate in both eukaryotes and prokaryotes, and a selection marker for both prokaryotic and eukaryotic systems.

[0151] In order to obtain high levels of expression of the cloned nucleic acid, expression plasmids are usually constructed, which usually contain a strong promoter to direct transcription, a ribosome binding site for translation initiation, and a transcription / translation terminator, each in a functional orientation with respect to each other and to the protein coding sequence. Examples of regulatory regions suitable for this purpose in E. coli are the promoter and operator regions of the E. coli tryptophan biosynthetic pathway, the phage lambda (P L) left promoter and L-arabinose (araBAD) operon. It is also useful to include a selection marker in the DNA vector transformed in E. coli. Examples of such markers include genes that specify resistance to ampicillin, tetracycline or chloramphenicol. Expression systems for expressing antibodies can be obtained using, for example, E. coli, Bacillus sp. and Salmonella. E. coli systems can also be used.

[0152] The antibody gene can also be subcloned into an expression vector that allows the addition of a tag (e.g., FLAG, hexa-histidine, etc.) at the C-terminus or N-terminus of the antibody (e.g., IgG, Fab, scFv, etc.) for purification. Methods for transfecting and expressing genes in mammalian cells are known in the art. Transducing cells with nucleic acids can involve, for example, incubating lipid particles containing nucleic acids with cells, or incubating viral vectors containing nucleic acids with cells within the host range of the vector. Cultures of cells used in the present disclosure, including cell lines and cultured cells from tissues (e.g., tumors) or blood samples, are well known in the art.

[0153] Once the nucleic acid encoding the subject antibody is isolated and cloned, one can express the nucleic acid in a variety of recombinant engineered cells known to those skilled in the art. Examples of such cells include bacteria, yeast, filamentous fungi, insects (e.g., those employing baculovirus vectors), and mammalian cells.

[0154] The separation and purification of subject antibodies can be accomplished according to methods known in the art. For example, by immunoaffinity purification (or using protein L or A precipitation), washing to remove non-specifically bound material, and eluting specifically bound antibodies, it is possible to separate proteins from cells genetically modified to express proteins constitutively and / or after induction, or from synthetic reaction mixtures. The separated antibodies can be further purified by other methods often used in dialysis and protein purification methods. In one embodiment, metal chelate chromatography can be used to separate antibodies. As discussed above, antibodies of the present disclosure can include modifications that are convenient to separation.

[0155] The antibody can be prepared in a substantially pure or isolated form (e.g., free of other polypeptides). The protein can be present in a composition that is enriched for the polypeptide relative to other components that may be present (e.g., other polypeptides or other host cell components). A purified antibody can be provided such that the antibody is present in a composition that is substantially free of other expressed proteins, e.g., less than 90%, typically less than 60%, and more typically less than 50% of the composition is comprised of other expressed proteins.

[0156] Antibodies produced by prokaryotic cells may need to be exposed to a chaotropic agent for proper folding. For example, during purification from E. coli, the expressed protein may be optionally denatured and then renatured. This can be achieved, for example, by dissolving the bacterially produced antibody in a chaotropic agent such as guanidine hydrochloride. The antibody is then renatured by slow dialysis or by gel filtration. Alternatively, the nucleic acid encoding the antibody may be operably linked to a secretion signal sequence such as pelB so that the antibody is secreted into the periplasm in a properly folded form.

[0157] The present disclosure also provides cells for producing antibodies of the present disclosure, wherein suitable cells include eukaryotic cells, such as mammalian cells. The cell may be a hybrid cell or "hybridoma" that is capable of replicating antibodies (e.g., monoclonal antibodies, such as IgG) in vitro. For example, the present disclosure provides a recombinant host cell (also referred to herein as a "genetically modified host cell") that is genetically modified with one or more nucleic acids comprising a nucleotide sequence encoding a heavy chain and / or light chain of an antibody of the present disclosure.

[0158] It is also contemplated herein that the technology of the recombinant DNA form of the antigen binding region for producing antibody molecules bypasses the generation of hybridomas. For example, DNA is cloned into bacteria (e.g., phage), yeast (e.g., Saccharomyces or Pichia), insect or mammalian expression systems. An example of suitable technology uses a phage lambda vector system with a leader sequence that causes the expressed antibody (e.g., Fab or scFv) to migrate to the periplasmic space (between the bacterial cell membrane and the cell wall) or to be secreted. People can quickly produce a large number of functional fragments (e.g., Fab or scFv) that bind to an antigen of interest.

[0159] Antibodies specifically binding to human uPAR can be prepared using a variety of techniques known in the art, including the use of hybridomas, recombinant, phage display technology, selected lymphocyte antibody methods (SLAM) or combinations thereof. For example, phage display methods can be used to prepare and separate antibodies. Phage display is used for high-throughput screening of protein interactions. Phage can be used to display antigen-binding domains expressed from a full set or combinatorial antibody library (e.g., human or mouse). Phage expressing antigen-binding domains binding to human uPAR can be selected or identified with human uPAR, for example, using human uPAR labeled with binding or capture on a solid surface or bead to select or identify. The phage used in these methods is typically a filamentous phage, including fd and M13 binding domains expressed from phage, wherein Fab, Fv (a separate Fv region from a light chain or heavy chain) or a disulfide-stabilized Fv antibody domain is recombinantly fused to phage gene III or gene VIII protein. The production of high affinity human antibodies by chain shuffling is known, as is combinatorial infection and in vivo recombination as a strategy for constructing large phage libraries. In another embodiment, ribosome display can be used to replace phage as a display platform. Antibodies in cell surface libraries can be screened. Such procedures provide an alternative to traditional hybridoma technology for the isolation and subsequent cloning of monoclonal antibodies.

[0160] After phage selection, the antibody coding region from the phage can be isolated and used to produce full antibodies, including human antibodies, or any desired antigen-binding fragments, and expressed in any desired host (including mammalian cells, insect cells, plant cells, yeast and bacteria). For example, techniques for recombinantly producing Fv, scFv, Fab, F(ab')2 and Fab' fragments can be used using methods known in the art.

[0161] Nucleic acids, expression vectors and cells

[0162] In view of the above section on methods of producing the antibodies and fusion proteins of the present disclosure, it should be understood that the present disclosure also provides nucleic acids, expression vectors and cells.

[0163] In certain embodiments, a nucleic acid is provided that encodes a variable heavy chain (V H ) polypeptide, variable light chain (V L ) polypeptide, or both, including any of the anti-human uPAR antibodies of the present disclosure, such as any of such antibodies described above. According to some embodiments, the antibody is a single-chain antibody (eg, scFv), and the nucleic acid encodes the single-chain antibody.

[0164] According to some embodiments, a nucleic acid is provided, the nucleic acid encoding the CAR of the present disclosure, for example, a CAR comprising: a V comprising the anti-human uPAR antibody of the present disclosure; H Peptide and V L A single chain antibody of a polypeptide; a transmembrane domain; and an intracellular signaling domain. Examples of such single chain antibodies, transmembrane domains, and intracellular signaling domains are described in detail above.

[0165] Also provide expression vector, it comprises any one in nucleic acid of the present disclosure.The expression of natural nucleic acid or synthetic nucleic acid encoding antibody and fusion protein of the present disclosure can be realized by operably connecting the nucleic acid encoding antibody or fusion protein to a promoter (which is constitutive or inducible), and incorporating the construct into the expression vector to produce a recombinant expression vector.The vector can be suitable for replication and integration in prokaryotes, eukaryotes or both.Typical cloning vectors include transcription and translation terminators, initiation sequences and promoters that are appropriately oriented in function, which can be used to regulate the expression of nucleic acid encoding antibodies.The vector optionally includes a universal expression cassette containing at least one independent terminator sequence, a sequence (e.g., as found in shuttle vectors) that allows the expression cassette to replicate in both eukaryotes and prokaryotes, and a selection marker for both prokaryotic and eukaryotic systems.

[0166] Also provided are cells comprising any of the nucleic acids and / or expression vectors disclosed herein. According to some embodiments, the cells disclosed herein include a V encoding an antibody H V of peptides and antibodies L In some such embodiments, the antibody is a single-chain antibody (e.g., scFv), and the nucleic acid encodes the single-chain antibody. According to some embodiments, a cell is provided that comprises a variable heavy chain (V H ) polypeptide and a first nucleic acid encoding an antibody variable light chain (V L ) polypeptide. In certain embodiments, such as cells, comprise a first expression vector comprising the first nucleic acid and a second expression vector comprising the second nucleic acid.

[0167] Also provided is a method for preparing an antibody or fusion protein of the present disclosure, the method being included in culturing the cell of the present disclosure under conditions suitable for cell expression of the antibody or fusion protein, wherein the antibody or fusion protein is produced. The conditions for culturing cells so that the antibody or fusion protein is expressed can vary. Such conditions can be included at a suitable temperature (e.g., 32°C-42°C, such as 37°C) and pH (e.g., pH 7.0-7.7, such as pH 7.4), in an environment with a suitable percentage of CO2 (e.g., 3% to 10%, such as 5%), in a suitable container (e.g., cell culture plate or its wells), in a suitable culture medium (e.g., cell culture medium, such as DMEM, RPMI, MEM, IMDM, DMEM / F-12, etc.) to culture cells.

[0168] Composition

[0169] As described above, the present disclosure also provides compositions. According to some embodiments, the compositions of the present disclosure include antibodies, fusion proteins or conjugates of the present disclosure. For example, the antibody, fusion protein or conjugate can be any antibody, fusion protein or conjugate described in the antibody section above, which description is incorporated herein for brevity, but is not repeated herein.

[0170] In certain aspects, the compositions of the present disclosure include antibodies, fusion proteins or conjugates present in a liquid medium. The liquid medium can be an aqueous liquid medium, such as water, a buffer solution, etc. One or more additives, such as salts (e.g., NaCl, MgCl2, KCl, MgSO4), buffers (Tris buffer, N-(2-hydroxyethyl) piperazine-N'-(2-ethanesulfonic acid) (HEPES), 2-(N-morpholino) ethanesulfonic acid (MES), 2-(N-morpholino) ethanesulfonic acid sodium salt (MES), 3-(N-morpholino) propanesulfonic acid (MOPS), N-tris[hydroxymethyl]methyl-3-aminopropanesulfonic acid (TAPS) etc.), solubilizers, detergents (e.g., nonionic detergents such as Tween-20 etc.), nuclease inhibitors, protease inhibitors, glycerol, chelating agents etc. can be present in such compositions.

[0171] Aspects of the present disclosure further include pharmaceutical compositions. In some embodiments, the pharmaceutical compositions of the present disclosure comprise the anti-human uPAR antibody of the present disclosure (or a conjugate or fusion protein comprising the same) and a pharmaceutically acceptable carrier.

[0172] Antibodies, fusion proteins or conjugates can be incorporated into various preparations for therapeutic administration. More particularly, antibodies, fusion proteins or conjugates can be formulated into pharmaceutical compositions by combining with appropriate pharmaceutically acceptable excipients or diluents, and can be formulated into preparations in solid, semisolid, liquid or gaseous form, such as tablets, capsules, powders, granules, ointments, solutions, injections, inhalants and sprays.

[0173] Formulations of antibodies, fusion proteins or conjugates for administration to an individual (eg, suitable for human administration) are generally sterile, and may further be free of detectable pyrogens or other contaminants that would contraindicate administration to a patient depending on the chosen route of administration.

[0174] In pharmaceutical dosage forms, antibodies, fusion proteins or conjugates can be administered in the form of their pharmaceutically acceptable salts, or they can also be used alone or in appropriate combination, as well as in combination with other pharmaceutically active compounds. The following methods and carriers / excipients are merely exemplary and are by no means limiting.

[0175] For oral preparations, the antibody, fusion protein or conjugate can be used alone or in combination with suitable additives to make tablets, powders, granules or capsules, for example, with conventional additives such as lactose, mannitol, corn starch or potato starch; with binders such as crystalline cellulose, cellulose derivatives, gum arabic, corn starch or gelatin; with disintegrants such as corn starch, potato starch or sodium carboxymethyl cellulose; with lubricants such as talc or magnesium stearate; and, if necessary, with diluents, buffers, wetting agents, preservatives and flavoring agents.

[0176] The antibody, fusion protein or conjugate can be formulated for parenteral (e.g., intravenous, intraarterial, intraosseous, intramuscular, intracerebral, intracerebroventricular, intrathecal, subcutaneous, etc.) administration. In certain aspects, the antibody, fusion protein or conjugate is formulated for injection by dissolving, suspending or emulsifying the antibody, fusion protein or conjugate in an aqueous solvent or a non-aqueous solvent, such as vegetable oil or other similar oils, synthetic fatty acid glycerides, esters of higher fatty acids or propylene glycol; and, if necessary, conventional additives such as solubilizers, isotonic agents, suspending agents, emulsifiers, stabilizers and preservatives are used.

[0177] Pharmaceutical compositions comprising antibodies, fusion proteins or conjugates can be prepared by mixing antibodies, fusion proteins or conjugates having a desired degree of purity with optional physiologically acceptable carriers, excipients, stabilizers, surfactants, buffers and / or tonicity agents. Acceptable carriers, excipients and / or stabilizers are nontoxic to recipients at the doses and concentrations employed, and include buffers such as phosphates, citrates and other organic acids; antioxidants including ascorbic acid, glutathione, cysteine, methionine and citric acid; preservatives (such as ethanol, benzyl alcohol, phenol, m-cresol, p-chloro-m-cresol, methyl or propyl paraben, benzalkonium chloride or a combination thereof); amino acids such as arginine, glycine, ornithine, lysine, histidine, glutamic acid, aspartic acid, isoleucine, d-glutamic acid ... acid, leucine, alanine, phenylalanine, tyrosine, tryptophan, methionine, serine, proline, and combinations thereof; monosaccharides, disaccharides, and other carbohydrates; low molecular weight (less than about 10 residues) polypeptides; proteins, such as gelatin or serum albumin; chelating agents, such as EDTA; sugars, such as trehalose, sucrose, lactose, glucose, mannose, maltose, galactose, fructose, sorbitol, raffinose, glucosamine, N-methylglucosamine, galactosamine, and neuraminic acid; and / or nonionic surfactants, such as Tween, Brij Pluronics, Triton-X, or polyethylene glycol (PEG).

[0178] The pharmaceutical composition can be in liquid form, lyophilized form, or liquid form reconstituted from a lyophilized form, wherein the lyophilized preparation is reconstituted with a sterile solution before administration. The standard procedure for reconstitution of a lyophilized composition is to add back a volume of purified water (usually equivalent to the volume removed during lyophilization); however, a solution containing an antibacterial agent can be used to produce a pharmaceutical composition for parenteral administration.

[0179] Aqueous formulations of antibodies, fusion proteins or conjugates can be prepared in a pH buffer solution, e.g., in the range of about 4.0 to about 7.0, or about 5.0 to about 6.0, or alternatively about 5.5 pH. Examples of buffers suitable for pH within this range include phosphate buffers, histidine buffers, citrate buffers, succinate buffers, acetate buffers, and other organic acid buffers. The buffer concentration can be about 1 mM to about 100 mM, or about 5 mM to about 50 mM, depending on, for example, the desired tension of the buffer and the formulation.

[0180] A tonicity agent may be included to adjust the tension of the formulation. Example tonicity agents include sodium chloride, potassium chloride, glycerol, and any component from the group of amino acids, sugars, and combinations thereof. In some embodiments, the aqueous formulation is isotonic, although hypertonic or hypotonic solutions may be suitable. The term "isotonic" refers to a solution having the same tension as some other solution to which it is compared, such as a physiological saline solution or serum. The tonicity agent may be used in an amount of about 5 mM to about 350 mM, for example, in an amount of 100 mM to 350 mM.

[0181] Surfactants may also be added to the formulation to reduce aggregation and / or minimize the formation of particles in the formulation and / or reduce adsorption. Example surfactants include polyoxyethylene sorbitan fatty acid esters (Tween), polyoxyethylene alkyl ethers (Brij), alkylphenyl polyoxyethylene ethers (Triton-X), polyoxyethylene-polyoxypropylene copolymers (Poloxamers, Pluronic) and sodium dodecyl sulfate (SDS). An example of a suitable polyoxyethylene sorbitan-fatty acid ester is polysorbate 20 (available under the trademark Tween 20 TM Polysorbate 80 (sold under the trademark Tween 80 TM Examples of suitable polyethylene-polypropylene copolymers are sold under the names F68 or Poloxamer 188 TM Examples of suitable polyoxyethylene alkyl ethers are those sold under the trademark Brij TM Exemplary concentrations of surfactants may range from about 0.001% to about 1% w / v.

[0182] Lyoprotectants may also be added to protect the antibody, fusion protein or conjugate from unstable conditions during the lyophilization process. For example, known lyoprotectants include sugars (including glucose and sucrose); polyols (including mannitol, sorbitol and glycerol); and amino acids (including alanine, glycine and glutamic acid). Lyoprotectants may be included, for example, in an amount of about 10 mM to 500 nM.

[0183] In some embodiments, the pharmaceutical composition comprises an antibody, fusion protein or conjugate, and one or more of the above identified components (e.g., surfactants, buffers, stabilizers, tonicity agents), and is substantially free of one or more preservatives, such as ethanol, benzyl alcohol, phenol, m-cresol, p-chloro-m-cresol, methyl or propyl paraben, benzalkonium chloride, and combinations thereof. In other embodiments, the preservative is included in the formulation, e.g., at a concentration in the range of about 0.001% to about 2% (w / v).

[0184] Reagent test kit

[0185] Aspects of the present disclosure further include kits.In certain embodiments, kits find use in practicing the methods of the present disclosure, including but not limited to methods of treating a condition associated with uPAR expression and / or activity in a subject in need thereof.

[0186] Therefore, in certain embodiments, the kit of the present disclosure comprises any one of the pharmaceutical compositions of the present disclosure, and instructions for administering the pharmaceutical composition to an individual in need thereof. The pharmaceutical composition contained in the kit may comprise any antibody, fusion protein and / or conjugate of the present disclosure, such as any antibody, fusion protein and / or conjugate described above. It should be understood that the kit of the present disclosure may comprise any medicament and feature described above in the section related to the subject antibody, fusion protein, conjugate and composition, which are not repeated herein for the purpose of brevity.

[0187] The kit of the present disclosure may include a certain amount of compositions, which exist in unit dose (e.g., ampoule) or multiple dose form. Therefore, in certain embodiments, the kit may include one or more (e.g., two or more) unit doses (e.g., ampoule) of compositions, which include antibodies, fusion proteins and / or conjugates of the present disclosure. As used herein, the term "unit dose" refers to physically discrete units suitable as unit doses for human and animal subjects, each unit containing a predetermined amount of compositions calculated in an amount sufficient to produce a desired effect. The amount of a unit dose depends on a variety of factors, such as the specific antibodies, fusion proteins and / or conjugates used, the effects to be achieved, and the pharmacodynamics associated with antibodies, fusion proteins and / or conjugates in individuals. In yet other embodiments, the kit may include a single multiple dose of the amount of the composition.

[0188] The instructions (e.g., instructions for use (IFU)) included in the test kit can be recorded on a suitable recording medium. For example, the instructions can be printed on a substrate (such as paper or plastic, etc.). Therefore, the instructions can be present in the test kit as a package insert, in the label of the container of the test kit or its components (i.e., related to packaging or sub-packaging). In other embodiments, the instructions exist as an electronic storage data file present in a suitable computer-readable storage medium, and the computer-readable storage medium is, for example, a portable flash drive, a DVD, a CD-ROM, a floppy disk, etc. In other embodiments, the actual instructions are not present in the test kit, but provide a means for obtaining the instructions from a remote source, for example, via the Internet. The example of this embodiment is a test kit comprising a website, in which the instructions can be viewed and / or can be downloaded from the website. Like the instructions, the means for obtaining the instructions are recorded on a suitable substrate.

[0189] How to use

[0190] Aspects of the present disclosure further include methods of using the antibodies, fusion proteins (e.g., CARs) and conjugates of the present disclosure. The methods can be used in a variety of situations, including in vitro and / or in vivo research and / or clinical applications.

[0191] In certain embodiments, methods are provided for treating a condition associated with uPAR expression and / or activity in a subject in need thereof, the method comprising administering to the subject an effective amount of a composition comprising an antibody, fusion protein (e.g., CAR) or conjugate of the present disclosure.

[0192] According to some embodiments, the condition associated with uPAR expression and / or activity is cancer. The subject method can be used to treat a variety of cancers. As used herein, "tumor" refers to the growth and proliferation of all tumor cells (whether malignant or benign), as well as all precancerous cells and cancer cells and tissues. The terms "cancer" and "cancerous" refer to or describe a physiological condition characterized by unregulated cell growth / proliferation in a mammal. According to some embodiments, cancer is characterized by cancer cells expressing uPAR on its surface. In certain embodiments, cancer includes solid tumors. According to some embodiments, solid tumors are carcinomas, lymphomas, blastomas, or sarcomas. In some embodiments, when cancer includes solid tumors, cancer is characterized by stromal cells in the tumor microenvironment expressing uPAR on its surface.

[0193] Examples of cancers that can be treated with the subject methods include, but are not limited to, carcinomas, lymphomas, blastomas, and sarcomas. More specific examples of such cancers include squamous cell carcinoma, small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma, lung squamous cell carcinoma, peritoneal cancer, hepatocellular carcinoma, gastrointestinal cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bile duct cancer, bladder cancer, hepatoma, breast cancer, colon cancer, colorectal cancer, endometrial cancer or uterine cancer, salivary gland cancer, kidney cancer, prostate cancer, vulvar cancer, thyroid cancer, hepatic carcinoma, various types of head and neck cancer, etc. In certain embodiments, the subject has a cancer selected from the group consisting of solid tumors, recurrent glioblastoma multiforme (GBM), non-small cell lung cancer, metastatic melanoma, melanoma, peritoneal cancer, epithelial ovarian cancer, glioblastoma multiforme (GBM), metastatic colorectal cancer, colorectal cancer, pancreatic ductal adenocarcinoma, squamous cell carcinoma, esophageal cancer, gastric cancer, neuroblastoma, fallopian tube cancer, bladder cancer, metastatic breast cancer, pancreatic cancer, soft tissue sarcoma, recurrent head and neck cancer squamous cell carcinoma, head and neck cancer, anaplastic astrocytoma, malignant pleural mesothelioma, breast cancer, squamous non-small cell lung cancer, rhabdomyosarcoma, metastatic renal cell carcinoma, basal cell carcinoma (basal cell epithelioma) and gliosarcoma. According to some embodiments, the subject has breast cancer, lung cancer, bladder cancer, ovarian cancer, prostate cancer, liver cancer, colon cancer, pancreatic cancer, gastric cancer, glioma or any combination thereof.

[0194] In certain embodiments, the cancer comprises a hematological malignancy. Non-limiting examples of hematological malignancies include leukemias, lymphomas, and multiple myeloma.

[0195] According to some embodiments, a method of inhibiting tumor invasion, tumor metastasis, extracellular matrix (ECM) degradation, tumor angiogenesis, tumor cell proliferation, or any combination thereof in a subject having cancer is provided, the method comprising administering to the subject an effective amount of a composition comprising an antibody, fusion protein (e.g., CAR) or conjugate of the present disclosure.

[0196] The antibodies, fusion proteins and conjugates of the present disclosure can be administered via any suitable route of administration, such as oral (e.g., in tablet form, capsule form, liquid form, etc.), parenteral (e.g., by intravenous, intraarterial, subcutaneous, intramuscular or epidural injection), topical, intranasal, intratumoral administration, etc.

[0197] The antibodies, fusion proteins and conjugates disclosed herein can be administered in a therapeutically effective amount as a composition. "Therapeutically effective amount" means a dosage sufficient to produce a desired result, e.g., an amount sufficient to produce a beneficial or desired therapeutic (including preventive) result (such as alleviation of symptoms of cancer) compared to a control. With respect to cancer, in some embodiments, a therapeutically effective amount is sufficient to slow the growth of a tumor, reduce the size of a tumor, and / or the like. An effective amount can be administered in one or more administrations.

[0198] As described above, aspects of the present disclosure include methods for treating cancer in an individual. Treatment means at least improvement of one or more symptoms associated with an individual's cancer, wherein improvement is broadly used to refer to at least a reduction in the magnitude of a parameter (e.g., symptom) associated with the treated cancer. Therefore, treatment also includes situations in which cancer or at least one or more symptoms associated therewith are completely suppressed (e.g., prevented from occurring) or stopped (e.g., terminated), such that the individual no longer suffers from cancer, or at least no longer suffers from symptoms characterizing cancer.

[0199] The antibody, fusion protein or conjugate disclosed herein can be administered to an individual alone or in combination with a second agent. The second agent of interest includes but is not limited to agents approved by the U.S. Food and Drug Administration and / or the European Medicines Agency (EMA) for use in treating cancer. In some embodiments, the second agent is an immune checkpoint inhibitor. The immune checkpoint inhibitor of interest includes but is not limited to cytotoxic T lymphocyte-associated antigen 4 (CTLA-4) inhibitors, programmed cell death-1 (PD-1) inhibitors, programmed cell death ligand-1 (PD-L1) inhibitors, lymphocyte activation gene-3 (LAG-3) inhibitors, T cell immunoglobulin domains and mucin domain 3 (TIM-3) inhibitors, indoleamine (2,3)-dioxygenase (IDO) inhibitors, T cell immune receptors with Ig and ITIM domains (TIGIT) inhibitors, V domain Ig inhibitors of T cell activation (VISTA) inhibitors, B7-H3 inhibitors and any combination thereof.

[0200] When the antibody, fusion protein or conjugate of the present disclosure is used together with the second agent, the antibody, fusion protein or conjugate and the second agent can be applied to the individual according to any suitable administration scheme. According to certain embodiments, the antibody, fusion protein or conjugate and the second agent are administered according to the dosage regimen approved for individual use. In some embodiments, the administration of the antibody, fusion protein or conjugate allows the second agent to be administered according to a dosage regimen, which relates to one or more lower and / or less frequent dosages and / or the number of cycles reduced compared to the use of the second agent when the antibody, fusion protein or conjugate is not administered. In some aspects, the administration of the second agent allows the antibody, fusion protein or conjugate to be administered according to a dosage regimen, which relates to one or more lower and / or less frequent dosages and / or the number of cycles reduced compared to the use of the antibody, fusion protein or conjugate when the second agent is not administered.

[0201] In some embodiments, one or more doses of the antibody, fusion protein or conjugate and the second agent are administered to the individual simultaneously. "Simultaneously" means that the antibody, fusion protein or conjugate and the second agent are present in the same pharmaceutical composition, or that the antibody, fusion protein or conjugate and the second agent are administered as separate pharmaceutical compositions within 1 hour or less, 30 minutes or less, or 15 minutes or less.

[0202] In some embodiments, one or more doses of the antibody, fusion protein or conjugate and the second agent are administered sequentially to the individual.

[0203] In some embodiments, the antibody, fusion protein or conjugate and the second agent are applied to individuals with different compositions and / or at different times. For example, the antibody, fusion protein or conjugate can be applied before the second agent is applied, for example, in a specific cycle. Alternatively, the second agent can be applied before the antibody, fusion protein or conjugate is applied, for example, in a specific cycle. The second agent to be applied can be applied at least 1 hour, 3 hours, 6 hours, 12 hours, 24 hours, 48 ​​hours, 72 hours or up to 5 days or longer after the first agent to be applied is applied.

[0204] In one example, before administering the antibody, fusion protein or conjugate, the second agent is administered to the individual for a desired time period. In some aspects, such a scheme "primes" cancer cells to enhance the anti-cancer effect of the antibody, fusion protein or conjugate. Such a time period separating the step of administering the second agent from the step of administering the antibody, fusion protein or conjugate has a sufficient length to allow sensitization of the cancer cells, ideally increasing the anti-cancer effect of the antibody, fusion protein or conjugate.

[0205] In some embodiments, the administration of one agent is specifically timed relative to the administration of another agent. For example, in some embodiments, an antibody, fusion protein, or conjugate is administered such that a specific effect is observed (or expected to be observed, e.g., based on a population study showing a correlation between a given dosing regimen and a specific effect of interest).

[0206] In certain aspects, the desired relative dosing regimen of the agents administered in combination can be evaluated or determined empirically, e.g., using ex vivo, in vivo, and / or in vitro models; in some embodiments, such evaluation or empirical determination is performed in vivo, in a patient population (e.g., to allow for the establishment of correlations), or alternatively, in a specific individual of interest.

[0207] In some embodiments, the antibody, fusion protein or conjugate and the second agent are administered according to an intermittent dosing regimen including at least two cycles. In the case where two or more agents are administered in combination and each agent is administered by such an intermittent cyclic regimen, the individual doses of different agents can intersect with each other. In some aspects, one or more doses of the second agent are administered within a period of time after a dose of the first agent. In some embodiments, each dose of the second agent is administered within a period of time after a dose of the first agent. In some aspects, after a period of time, each dose of the first agent is followed by a dose of the second agent. In some embodiments, two or more doses of the first agent are administered between at least one pair of doses of the second agent; in some aspects, two or more doses of the second agent are administered between at least one pair of doses of the first agent. In some embodiments, different doses of the same agent are separated by a common time interval; in some embodiments, the time intervals between different doses of the same agent vary. In some aspects, different doses of the antibody, fusion protein or conjugate and the second agent are separated by a common time interval; in some embodiments, different doses of different agents are separated by different time intervals.

[0208] An exemplary regimen for a cyclic dosing regimen that crosses two intervals may include: (a) a first dosing period during which a therapeutically effective amount of an antibody, fusion protein, or conjugate is administered to an individual; (b) a first rest period; (c) a second dosing period during which a therapeutically effective amount of a second agent is administered to an individual; and (d) a second rest period. A second exemplary regimen for a cyclic dosing regimen that crosses two intervals may include: (a) a first dosing period during which a therapeutically effective amount of a second agent is administered to an individual; (b) a first rest period; (c) a second dosing period during which a therapeutically effective amount of an antibody, fusion protein, or conjugate is administered to an individual; and (d) a second rest period.

[0209] In some embodiments, the first resting period and the second resting period can correspond to the same number of hours or days. Alternatively, in some embodiments, the first resting period and the second resting period are different, wherein the first resting period is longer than the second resting period, or vice versa. In some embodiments, each of the resting periods corresponds to 120 hours, 96 hours, 72 hours, 48 ​​hours, 24 hours, 12 hours, 6 hours, 30 hours, 1 hour or less. In some embodiments, if the second resting period is longer than the first resting period, it can be defined as days or weeks instead of hours (e.g., 1 day, 3 days, 5 days, 1 week, 2 weeks, 4 weeks or more).

[0210] If the length of the first resting period is determined by the presence or development of a particular biological event or therapeutic event, the length of the second resting period can be determined based on different factors, either individually or in combination. Exemplary such factors may include the type and / or stage of the cancer for which the therapy is administered; the properties of the antibody, fusion protein, or conjugate (e.g., pharmacokinetic properties), and / or one or more characteristics of the patient's response to therapy with the antibody, fusion protein, or conjugate. In some embodiments, the length of one or both resting periods can be adjusted based on the pharmacokinetic properties of one or another of the administered agents (e.g., as assessed via plasma concentration levels). For example, when the plasma concentration of the relevant agent is below a predetermined level, optionally after an assessment or other consideration of one or more characteristics of the individual's response, the relevant resting period may be considered to have ended.

[0211] In certain aspects, the number of cycles for which a particular agent is administered can be determined empirically. In addition, in some embodiments, the exact regimen followed (e.g., the number of doses, the spacing of doses (e.g., relative to each other or relative to another event, such as the administration of another therapy), the amount of dose, etc.) may be different for one or more cycles compared to one or more other cycles.

[0212] The antibody, fusion protein or conjugate and the second agent can be administered together or independently via any suitable route of administration. The antibody, fusion protein or conjugate and the second agent can be administered via an administration route independently selected from oral, parenteral (e.g., by intravenous, intraarterial, subcutaneous, intramuscular or epidural injection), local, intranasal, intratumoral administration, etc. According to certain embodiments, the antibody, fusion protein or conjugate and the second agent are both administered simultaneously (in the same pharmaceutical composition or a separate pharmaceutical composition) or sequentially orally or parenterally (e.g., in tablet form, capsule form, liquid form, etc.).

[0213] The following examples are offered by way of illustration and not by way of limitation.

[0214] experiment

[0215] Based on the important role of uPAR as an anticancer target, therapeutic antibodies targeting uPAR for anticancer therapy have been developed. Fully human rAb, 2G10 and 3C6 (27) were identified from the initial Fab library of people using phage display technology, and it was shown that it was effective for human TNBC cells in a xenograft model (17). Together with therapeutic radionuclides or as antibody-drug conjugates (ADC), the antitumor efficacy of antibodies is increased (17, 24, 27). However, they lack cross-reactivity, which limits their progress as clinical candidates. In the development process of therapeutic antibodies, before the first in vivo study, accurately predicting the human pharmacokinetics, toxicity and efficacy of antibodies is the basis for developing them into effective biotherapeutic entities (28). Compared with other species, cynomolgus monkeys (cyno) are genetically similar to humans, and are the most relevant non-human primate models for preclinical studies in the development of antibody drugs (29).

[0216] This paper describes the use of microfluidic platform and photoelectric tweezers to screen the mouse B lymphocytes sensitized by uPAR, and establishes an accelerated discovery method for developing new humans and cyno cross-reactive rAbs. Unique cross-reactive rAbs show antibody-dependent cellular toxicity (ADCC), ADC cytotoxicity and the inhibitory effect on cell adhesion for human breast cancer cells. In addition, the lead antibody shows their therapeutic efficacy in reducing tumor growth in the in situ animal model of human breast cancer, providing promising rAb candidates. Finally, a binding model of the lead antibody is provided, which shows the binding epitope of the lead antibody causing the unique activity for uPAR.

[0217] Example 1 - High-throughput B cell screening of human uPAR and cyno uPAR cross-reactive antibodies

[0218] Swiss Jim Lambert (SJL / J) mice (n=8) were subjected to a 60-day immunization campaign using a recombinant soluble form of human uPAR (suPAR) lacking the GPI anchor as the immunogen, with a seven-day buffer period between bleed and boost ( Figure 1A suPAR was prepared by removing endotoxin to reduce nonspecific pyrogenic responses to immunized animals and further characterized using SDS-PAGE, immunoblotting, and LC-MS / MS ( FIG. 8A to FIG. 8B The immunized mice were monitored by bleeding every two weeks and their antibody titers were subsequently determined. Antiserum binding curves showed that the production of anti-uPAR antibodies increased within the first week of immunization. Sustained antibody production was maintained throughout the campaign, with antibody titers ranging from 1 x 10 7 dilution of mouse antiserum to reach saturation ( Fig. 9Following maturation of confirmed uPAR-sensitized plasma B cells, spleen and bone marrow were harvested from each animal to allow isolation of CD45R(B220) cells using magnetic beads and flow-assisted cell sorting. - / CD138 高 Antibody-secreting cells (ASC).

[0219] To screen and select cross-reactive antibodies against human uPAR and cyno uPAR, Beacon TM The platform performs high-throughput optofluidic screening of single B cells (30). In vivo development of antibodies relies on maturation and selection of ASCs, providing antibodies with high specificity for their targets and low off-target binding to other host proteins. The Beacon platform is able to screen and select tens of thousands of B cells from immunized animals, thus accelerating the antibody discovery process (31). A total of 49,127 mouse ASCs were input into the OptoSelect TM The cells were screened against human uPAR, mouse uPAR, and cyno uPAR in a nanopen on a 3500 chip. In total, 217 binders against human uPAR were identified, 80 of which were cross-reactive with cyno uPAR, and no cells were able to produce binders reactive against mouse uPAR ( Figures 1B to 1D Interestingly, 8 ASCs produced specific binders to cyno uPAR and 137 ASCs were specific for human uPAR.

[0220] Example 2 - VH / VL sequencing, cloning and recombinant IgG expression

[0221] A total of 217 individual mouse B cells were exported from Beacon and then used the rapid amplification of cDNA ends (RACE) protocol to cover 80 pairs of VH and VL sequences from cross-reactive binders to human uPAR and cyno uPAR (32). A total of 64 clones showed amplicons within 500-700 bp with 78% recovery, and these amplicons were sequenced using next generation sequencing (NGS), resulting in 60 pairs of unique VH and VL sequences with 94% sequence recovery and 100% diversity.

[0222] Previous studies have shown that Herceptin, a humanized IgG1 monoclonal antibody targeting the HER2 protein, can promote tumor cell death by inducing ADCC through the interaction of its IgG1 Fc and Fcγ receptors on human immune cells (33-35). In order to convey this effector function to anti-uPAR antibodies, 60 unique mouse VH / VL sequences were linked to the Herceptin IgG1 constant region for the generation of rAbs in the form of chimeric antibodies. From the recovered pool, 44 initial antibodies were successfully recombinantly expressed.

[0223] Example 3 - Cell surface uPAR recognition and binding affinity of antibody candidates

[0224] Antibodies generated by immunizing animals with suPAR lacking the cell surface anchoring motif can target regions of the protein that are inaccessible to membrane-bound uPAR. However, effective targeting of cell surface receptors benefits from both recognition of epitopes that are exposed to the solvent and in a native conformational state displayed on the cell surface (36). Therefore, FACS was applied to evaluate recognition of cell surface uPAR by each antibody candidate at different concentrations of MDA-MB-231 cells, a triple-negative breast cancer cell line with high uPAR expression ( Figure 2 From 44 initial antibodies, 12 lead candidates recognized uPAR displayed on breast cancer cells in a dose-dependent manner, with the half-maximal effective binding concentration (EC 50 ) values ​​ranged from 0.39 nM to 7.6 nM ( Figure 2 All candidates were benchmarked against 2G10 and 3C6 and showed greater potency in recognizing cell surface uPAR as demonstrated by their lower EC50 values, and these values ​​were comparable to Herceptin binding to HER2 (EC 50 =3.6 nM) (Table 2) (37, 38).

[0225] Table 2 - In vitro characterization of novel anti-uPAR antibody candidates. NA = no applicable activity, dash (-) = not determined

[0226]

[0227] The binding affinity of the lead candidates to human uPAR was further characterized using biolayer interferometry (BLI). All lead candidates had equilibrium dissociation constants (K) in the pM range. D ) values ​​for human uPAR, showing stronger binding affinities in the double-digit nanomolar range than 2G10 and 3C6 (17). These results demonstrate the ability to select in vivo affinity-matured antibodies to develop tight binders with slow off-rates against uPAR.

[0228] Example 4 - Cross-reactivity profile confirmed by ELISA

[0229] To ensure that cross-reactivity of the 12 lead candidates with the Herceptin constant region was maintained, their binding to human uPAR and cyno uPAR was assessed by ELISA ( Figure 3A The results showed that all lead candidates showed cross-reactivity and strong binding to both human uPAR and cyno uPAR, with EC 50 The values ​​were in the range of 0.05-0.8 nM and 0.1-1.1 nM, respectively. Two of them showed approximately 2-fold higher binding affinity to human uPAR, seven antibodies showed 2-7-fold stronger binding to cyno uPAR, and three antibodies bound to human uPAR and cyno uPAR with comparable binding affinity. On the other hand, 2G10 and 3C6 were able to bind to human uPAR, but no reactivity was observed with cyno uPAR ( Figure 3A and Table 2 ).

[0230] Example 5 - Antibody-dependent cellular cytotoxicity (ADCC)

[0231] With the confirmation of cell surface uPAR recognition and cross-reactivity of the 12 lead antibody candidates, we next evaluated whether they mediated ADCC to promote tumor cell death as a uPAR-targeted immunotherapy approach. ADCC assays for all lead antibody candidates were first performed using MDA-MB-231 cells as target cells in the presence of NK-92MI CD16a effector cells. The resulting dose-response curves showed that all lead candidates, except 4718, were able to induce ADCC, and no cytotoxicity was observed against the isogenic huIgG1 control ( Figure 3B ). The ADCC activity of the selected eight antibody candidates (3159, 3595, 3639, 5016, 8163, 9538, 11857, 13706) that exerted effective ADCC responses in NK-92 cells was further tested in the presence of human PBMCs from three different healthy donors. Due to the inherent properties of the effector cells from each PBMC donor, this usually leads to considerable donor-donor variability in their ability to induce ADCC (39). As expected, inherent donor variability was observed between the eight lead candidates, and the results showed that in the presence of healthy PBMCs, they all promoted effector cell function against MDA-MB-231 cells in a dose-dependent manner ( Figure 4 The mean maximum percentages of ADCC responses of the eight antibodies ranged from 46% to 66%, and seven of them were more potent than 2G10, with EC 50The values ​​ranged from 0.1-13 nM and were 10 to 96 times lower (Table 2).

[0232] Example 6 - Cytotoxicity as an Antibody Drug Conjugate (ADC)

[0233] In addition to Fc-mediated ADCC as a strategy for providing anti-tumor cytotoxicity, antibody-drug conjugates (ADCs) have rapidly developed in recent decades to selectively deliver cytotoxic payloads directly to target cancer cells (40). To determine whether the eight selected antibody candidates can be internalized by targeting uPAR for ADC approaches, ADC efficacy was evaluated in vitro against MDA-MB-231 cells using Fab-αHFc-CL-MMAE, which recognizes the Fc portion of Herceptin and has a cathepsin-cleavable linker connected to monomethyl auristatin E (MMAE). Controls were performed in the absence of αHFc-CL-MMAE treatment, and no cytotoxicity was observed. Although all antibodies recognize cell surface uPAR, only four of them (3159, 8163, 11857, and 3595) showed a concentration-dependent increase in ADC cytotoxicity in the presence of αHFc-CL-MMAE. The low EC values ​​of the four antibodies were 1.17, 1.2, and 1.8, indicating that the ADCs have a high affinity for uPAR and low affinity for MMAE. 50 The values ​​ranged from 0.57 nM to 0.76 nM, indicating that they target uPAR in distinct complexes and induce efficient internalization ( Figure 5 A).

[0234] Example 7 - Inhibition of cell adhesion to vitronectin

[0235] It is known that the binding of vitronectin (VN) to uPAR induces intracellular signaling events that activate integrins to promote cancer cell adhesion and communication with the extracellular matrix (41). To investigate whether the eight lead antibody candidates have any functional inhibition of tumor cells by targeting cell surface uPAR, their ability to block uPAR-mediated cell adhesion to VN was evaluated. The results showed that the eight lead candidates were able to inhibit the adhesion of MDA-MB-231 cells to VN-coated wells in a dose-dependent manner, and five of them (3159, 6312, 8163, 9538, and 11857) had EC values ​​ranging from 0.9 μM to 5.4 μM. 50 Values ​​(Table 2 and Fig.10). Candidate 3159 showed the strongest inhibitory effect and was comparable to 3C6, which was previously identified as an inhibitor that abolished uPAR-mediated cell adhesion (42). Overall, the in vitro characterization of the lead antibodies highlighted candidates 3159, 8163, and 11857 ​​as the most promising rAbs, which possessed ADCC activity, ADC cytotoxicity by inducing efficient uPAR-rAb internalization, and functional inhibition of cell adhesion (Table 2), facilitating the investigation of their therapeutic efficacy in an orthotopic animal model of breast cancer.

[0236] Example 8 - Therapeutic efficacy in an orthotopic animal model of human breast cancer

[0237] To determine the in vivo therapeutic efficacy of the three lead antibodies, MDA-MB-231 cells were orthotopically implanted into the Foxn1 nu The mammary fat pads of nude mice were injected with each antibody (30 mg / kg) intravenously weekly to treat mice bearing tumors (75-100 mm in size). 3 ) animals. Close monitoring of tumor growth between treatment groups revealed that all antibodies were able to reduce tumor burden relative to untreated controls ( Figure 5 B). The data show that candidate 11857 ​​exhibited the strongest efficacy, with a tumor burden 3.1±0.4-fold smaller than untreated controls at day 21 (p=0.0039). Such activity was maintained at days 25 and 28, where tumor burdens were 3.3±0.3-fold (p=0.0058) and 3.6±0.6-fold (p=0.0125) smaller than untreated controls, respectively ( Figure 5 B) Although candidates 3159 and 8163 had little effect in reducing tumor volume compared to candidate 11857, they were still effective in reducing tumor growth rate relative to untreated controls ( Figure 5 C). The excellent antitumor activity of candidate 11857 ​​is also reflected in its ability to impair tumor growth rate compared with untreated controls (p = 0.0002), which is much better than that of candidates 3159 (p = 0.0141) and 8163 (p = 0.0267) ( Figure 5 C).

[0238] Example 9 - Epitope Binning Using Biolayer Interferometry

[0239] The reduction in tumor growth rate and the ability of the three lead antibodies (3159, 8163, and 11857) to impair cell adhesion led us to investigate their binding epitopes on uPAR. Epitope binning was performed by BLI, and 2G10 was included as a control. Human uPAR and cyno uPAR share 96% sequence identity ( Figure 6A), where most of the sequence variation between homologs is located in the uPA binding site, resulting in species-specific interactions between uPA and uPAR (43, 44). On the other hand, the VN binding site is located on the opposite side and is more conserved between uPAR homologs ( Figure 6 A). Previous studies have shown that 2G10 was identified as a competitor that disrupts the uPA / uPAR interaction, suggesting that it binds to a region that blocks uPA binding (45). The BLI curves showed that the three lead antibodies could bind to uPAR after the formation of the 2G10-uPAR complex. This result suggests that they target uPAR in a region that is different from the uPA binding domain where 2G10 is recognized ( Figure 6 B). To further test whether the three lead antibodies could inhibit the binding of VN to uPAR, the binding response of VN after each of them bound to uPAR was measured ( Figure 6 BLI measurements showed that binding of 3159 to uPAR completely abolished subsequent VN binding, 11857 ​​showed a partial effect in blocking VN binding, and 8163 did not affect VN binding to uPAR ( Figure 6 B). This is consistent with the results from the adhesion assay, showing that 3159 exhibited the strongest inhibitory effect on VN-mediated cell adhesion, indicating that 3159 recognizes the VN binding site and blocks the interaction between VN and uPAR. This result was also confirmed by changing the order of adding 3159 and VN to show that 3159 was able to compete with VN for binding to uPAR ( Fig.11 In addition, epitope competition assays were performed among different antibody pairs to identify whether they have different binding epitopes ( Figure 6 C). Interestingly, both 3159 and 8163 can bind to uPAR simultaneously, but once the rAb-uPAR complex is formed with 11857, each of them cannot interact with uPAR, suggesting that 11857 ​​has partially overlapping epitopes with 3159 and 8163 ( Figure 6 C). In addition, it was found that the binding epitopes of these three antibodies were different from 3C6 ( Fig.12 ).

[0240] discuss

[0241] The increasing understanding of uPAR and its molecular partners in tumorigenesis, cancer progression and metastasis provides a basis for the development of new diagnostic, prognostic and therapeutic approaches to treat a variety of tumors (46, 47). Cynomolgus monkeys have been used as valuable models to provide the most relevant information for the safety, efficacy and pharmacokinetic profiles of translational therapies for human use (48, 49). Therefore, this study proposes a rapid antibody discovery pipeline that will allow the identification of cross-reactive antibodies against human uPAR and cyno uPAR. A 60-day immunization campaign using recombinant human suPAR was able to rapidly generate uPAR-sensitized B cells in SJL mice, and the Beacon platform allowed the cultivation, manipulation and screening of single B cells within one day with a 99% guarantee of clonal origin. Similar approaches have recently been used to successfully develop neutralizing antibodies against SARS-CoV and SARS-CoV-2 (50). The methods of the invention provide an example of using immunization to bias an immune response, which is combined with screening of antigen-sensitized B cells to identify human / cyno cross-reactive antibodies with strong binding affinity and anti-tumor activity against human breast cancer, demonstrating the ability of in vivo development and affinity maturation in B cells for antibody selection.

[0242] Effective tumor-targeting antibodies induce direct and indirect effects on tumor cells mediated by their Fab variable regions and Fc constant regions, respectively (51). Targeted therapies for HER2-positive breast cancer in clinical use (i.e., Herceptin and Perjeta) target the HER2 protein and induce ADCC as part of their tumor killing mechanism by recruiting immune effector cells via the Fc domain (52). In order to confer ADCC activity on mouse antibodies exerted by human immune cells, their VH / VL domains were engineered to include the Herceptin constant region. Interestingly, ADCC of varying magnitude was observed in the presence of NK-92 cells, indicating that epitope recognition of the antibody is critical for regulating ADCC activity. These findings confirm previous studies showing how antigen binding can alter IgG conformation and affect recognition of the Fc region by FcγRIIIa and FcγRIIIb receptors on the surface of NK cells and PBMCs (53-55). Furthermore, the binding epitope of an antibody influences the angle of its Fc domain relative to the target cell surface and can control the accessibility of the Fc region to interact with effector cells to induce ADCC (56, 57).

[0243] Although ADCC is one of the main mechanisms of action for most anti-tumor mAbs currently in the clinic, recent findings suggest that antibodies that functionally inhibit their targets while inducing ADCC offer further benefits for achieving effective anti-tumor responses (58, 59). Previous studies have shown how vitronectin deficiency severely impairs tumor growth in orthotopic xenograft models of breast cancer (60). Furthermore, uPAR binding to vitronectin has been shown to modulate cell adhesion and further trigger changes in cell morphology, migration, and signaling (61–63). The reported mAb 8B12 was found to inhibit the binding of vitronectin to uPAR and effectively reduce uPAR-mediated cell migration on vitronectin-coated surfaces (18). These studies reveal how inhibition of cancer cell interactions with the ECM can affect their pro-proliferative communication and overall tumorigenesis in the tumor microenvironment. This supports the finding that inhibition of cell adhesion found in lead candidates provides an advantage in impairing tumor growth and their ability to induce ADCC (41, 64).

[0244] In addition to ADCC, several therapeutic antibodies have been redeveloped as ADCs to deliver cytotoxic drugs to antigen-positive tumor cells (65). Here, lead antibodies were evaluated in ADC cytotoxicity and showed the potential to achieve cytotoxicity in MDA-MB-231 cells. According to previous studies, uPAR can be internalized by tumor cells via clathrin-mediated endocytosis or via a clathrin-independent mechanism mediated by LRP-1, both of which are responsible for transporting uPAR to lysosomes for degradation and recycling (66, 67). This can provide additional advantages because all known internalization mechanisms of uPAR separate it from its co-receptors, which include integrins and other true ligands bound to the matrix, and thus eliminate downstream signaling (68).

[0245] Finally, based on biolayer interferometry data and inhibition assays, binding models were proposed for the three lead antibodies against uPAR ( Figure 7). Candidates 3159, 8163 and 11857 ​​are combined with uPAR by the epi-position different from the binding substances 2G10 and 3C6 reported previously, and their binding epi-position is located at the opposite side of central uPA binding cavity. The binding site of 3159 and 8163 on uPAR is independent, and 11857 ​​binding epi-position overlaps with its binding site to a considerable extent, but is not identical. They all show the inhibitory influence on cell adhesion, and 3159 is combined with the epi-position on uPAR for vitronectin combination, and therefore shows the strongest inhibitory effect. 11857 ​​is combined with a spot, causes the synergistic effect to ADCC, uPAR internalization and blocking cell adhesion, demonstrates the advantage of the antibody with ADCC and the extra functional effect of damaging tumor growth.

[0246] Materials and methods

[0247] Antigen production

[0248] The HEK293 cell line stably expressing suPAR was generously provided by the Chapman laboratory at UCSF. The cells were grown in complete DMEM medium supplemented with 10% fetal bovine serum (FBS), 100 U / mL penicillin, and 100 μg / L streptomycin (Gibco) at 5% CO2 and 37°C. For suPAR production, 3.7×10 7 Cells were seeded on 5 layers The cells were cultured in a 4% culture medium and maintained in complete medium. Protein was harvested on day 3 and purified using a Ni-NTA column followed by gel filtration using a Hiload 16 / 600 Superdex200 preparative column. The production of suPAR was confirmed by immunoblot analysis using mouse anti-human uPAR monoclonal antibody clone R-3 (Invitrogen) and HRP-conjugated goat anti-mouse IgG (H+L) antibody (Biorad). Purified suPAR samples were also characterized by LC-MS / MS and analyzed using Pierce TM High capacity endotoxin removal spin columns were further subjected to endotoxin removal. After endotoxin removal, the total endotoxin levels of the samples were characterized and only those samples that did not exceed 0.5 U / mL were approved for the preparation of immunogen injections.

[0249] Animal Immunization Strategies

[0250] The immunization strategy includes an initial intraperitoneal injection with 50 μg of antigen prepared as an emulsion in Freund's complete adjuvant (FCA). Nine Swiss Jim Lambert (SJL) mice (6-8 weeks old) received a booster injection containing 25 μg of antigen every other week for three boosts, with bleeding inserted for each booster injection for 60 days. Before the start of the immunization campaign, each mouse was pre-bleeded to control for unexpected exposure to the antigen. All blood samples were allowed to coagulate, and 100-200 μL of serum was stored at -80°C for antibody titer determination.

[0251] Harvest and Enrichment of Mouse Antibody Secreting Cells (ASC)

[0252] Animals were euthanized according to approved IACUC protocols. Spleens and bone marrow were harvested and processed into single-cell suspensions in RPMI. Total B cells were isolated by magnetic negative selection using the EasySep Mouse Pan-B Cell Kit (StemCell) to deplete non-B cells from the single-cell suspension. Non-B cells were depleted by magnetic positive selection using the EasySep Mouse CD138+ Kit (StemCell) and by CD45R (B220) - / CD138 高 Gated FACS-based positive selection of cells to enrich antibody secreting cells (ASC) from single B cell suspensions, the CD45R (B220) - / CD138 高 Cells have traditionally been used to broadly define the population of plasma cells ( 69 ).

[0253] Nanofluidic optoelectronic screening of single B cells

[0254] The Beacon platform enables direct screening of secretory antibodies from ASCs. Enriched ASCs were injected into 0.75 nL OptoSelect TM 3500 and OptoSelect TM14K chip. The platform can effectively separate single ASC into nanopen using adjustable photoelectric localization parameters. ASC is cultured alone in the chip for 1 hour, and multiple fluorescence determinations based on beads in the channel are used to screen both IgG secretion and antigen specificity. In short, beads coated with rabbit anti-mouse IgG (H+L) are input into the chip, in which the active accumulation of secretory antibodies is identified by the binding of FITC-labeled goat anti-mouse secondary antibodies to beads. Antigen specificity is assessed by inputting fluorescently labeled uPAR from humans (conjugated with Alexa Fluor 488), cynomolgus monkeys (R&D Systems) or mice (Sino Biologicals) (conjugated with Alexa Flour 647) into the chip. The binding of ASC-derived IgG to antigen is monitored by the time-dependent increase of fluorescence derived from uPAR on the beads found at the opening of the nanopen. The selected ASC is exported to a 96-well plate containing lysis buffer. HEK293T / 17 cells expressing 2G10 were used as a positive control for both IgG secretion and production of anti-uPAR antibodies.

[0255] Sequencing, recombinant cloning, expression and purification of candidate antibodies

[0256] Single B cells are exported from Beacon into a 96-well plate containing lysis buffer and mineral oil. The cDNA generation process is performed using RNA capture beads using a proprietary protocol from ChemPartner & BLI. The selected human uPAR and cyno uPAR cross-reactive binders are then amplified using a RACE PCR protocol using proprietary heavy and light chain constant reverse primers. Amplicons within 500-700bp are cloned and sequenced by next-generation sequencing (NGS). NGS library preparation is completed by indexing universal forward and reverse constant primers. The samples are then run on MiSeq (Illumina). NGS-related software is used to analyze the raw data.

[0257] The VH and VL sequences were linked to the trastuzumab constant region and cloned into the pcDNA3.4-hCg1 or pcDNA3.4-hCk mammalian expression vector. Transfection and expression of recombinant IgG were performed based on the manufacturer's protocol. Briefly, HEK293F cells were seeded in FreeStyle TMThe cells were incubated at 130 rpm and 37 °C in 8% CO2. Polyethyleneimine (PEI) was used for transfection, maintaining a DNA / PEI ratio of 1:2. A 5% peptone solution was added to 0.1 equivalent volume of the original cell suspension to increase the synthesis of recombinant protein. On day 6-7 after transfection, the IgG-rich culture medium was collected and purified using a protein A column (GE MabSelect TM SuRe TM ) and dialyzed against PBS (pH 7.4) at 4°C overnight.

[0258] Biolayer interferometry (BLI) analysis

[0259] The binding affinity of anti-uPAR antibodies was measured at 25°C using the Octet RED384 system. Octet SA (streptavidin) biosensors were immobilized with biotinylated human uPAR or cyno uPAR at 2 μg / mL (Protein Sciences) in assay buffer (PBS containing 1% BSA). After equilibration to baseline in assay buffer, the biosensor was placed in each well containing anti-uPAR antibodies and allowed to dissociate in assay buffer. Association and dissociation curves were analyzed using Octet data analysis software.

[0260] Cross-reactivity of candidate antibodies by ELISA

[0261] Nunc MaxiSorp TM Flat-bottomed 96-well plates were coated with human uPAR or cyno uPAR (3.19 μg / mL) overnight at 4°C, and the plates were washed with wash buffer and blocked with 5% nonfat dry milk. Standard logarithmic serial dilutions of each antibody candidate were added to the uPAR-coated plates and incubated overnight at 4°C. The plates were washed three times and incubated with 50 μL of HRP-conjugated goat anti-human (H+L) antibody (Biorad). After incubation for 2 hours, the plates were washed and 100 μL of 1-Step TM Turbo TMB-ELISA substrate solution (Thermo Scientific). The reaction was quenched with 2M H2SO4 at room temperature for 5 minutes, and the optical density of each well was measured at 450nm using a SpectraMax190 microplate reader. The resulting dose-response curve was used to determine the minimum dose of the antibody required to achieve 50% of the saturation signal, and quantitative comparison of binding affinity was achieved.

[0262] Recognition of cellular uPAR by candidate antibodies

[0263] MDA-MB-231 cells were harvested with TrypleE and resuspended in FACS buffer (PBS + 1% BSA) to 2 × 10 6 cells / mL, and then aliquoted into 96-well plates (100 μL, 2×10 5 Cells were pelleted by centrifugation of the microplate at 400 RCF for 5 minutes and resuspended in PBS containing serially diluted antibodies to a maximum concentration of 600 nM, followed by incubation at 4°C for 50 minutes. The cells were then washed three times with PBS and incubated with AlexaFluor488-conjugated goat anti-human IgG at 4°C in the dark for 50 minutes. Finally, the cells were washed twice with FACS buffer and resuspended in 80 μL for FACS analysis in a Bio-rad S3e cell sorter.

[0264] Inhibition of cell adhesion to vitronectin

[0265] MDA-MB-231 cells were cultured in complete medium at 37°C in a humidified atmosphere of 5% CO2. MaxiSorp 96-well plates were coated with vitronectin (corning) overnight at 4°C. The wells were washed with PBS and blocked with 1% BSA in PBS for 1 hour. 50,000 MDA-MB-231 cells were seeded in each well, and serial dilutions of antibodies or RGDS peptides were added, and the plates were incubated overnight at 5% CO2 and 37°C. All wells were washed with PBS, and ice-cold methanol was added to fix the cells at room temperature for 10 minutes. After fixation, cells were stained with 5% crystal violet solution. The wells were washed three times with PBS, and the cells were lysed with 2% SDS lysis buffer. Each lysate was transferred to a transparent 96-well plate, and the absorption at 590nm was recorded to determine the number of adherent cells.

[0266] In vitro antibody-dependent cellular cytotoxicity (ADCC) using NK cells

[0267] pass EuTDA cytotoxicity reagent (PerkinElmer) was used to detect antibody-dependent cytotoxicity of NK cells against MDA-MB-231. Briefly, MDA-MB-231 cells were harvested and incubated with 2 μL / mL of fluorescence-enhanced ligand ( BATDA labeling reagent) was incubated at 37°C for 20 minutes for labeling. After BATDA diffused into the cells, it was hydrolyzed and converted to 2,2':6',2"-terpyridine-6,6"-dicarboxylic acid (TDA) by cytosolic acetylesterase. Since TDA is a non-cell permeable hydrophobic ligand, it can be trapped inside living target cells. The solution was centrifuged and the cells were washed three times with PBS. The labeled cells were reconstituted in RPMI 1640 medium without phenol red and then seeded into 96-well U-bottom sterile microplates (100 μL, 1×10 4 Next, 50 μL of serial dilutions of each antibody candidate were added to the assay plate and incubated at 37°C for 5-10 minutes. Separately, NK-92CD16a176V effector cells were harvested and concentrated to approximately 1.2×10 6 Cells / mL were added to the assay plate, resulting in a 6:1 ratio of effector cells to target cells in each well. The plate containing antibodies, target cells, and effector cells was then incubated at 37°C and 5% CO2 for 4 hours. After incubation, the plate was centrifuged at 400 RCF for 5 minutes, and 25 μL of the supernatant was transferred to a flat-bottomed detection plate. Finally, 200 μL of Europium solution (PerkinElmer, Eu-solution) and the plate was incubated at room temperature for 15 minutes to allow the formation of a highly fluorescent stable chelate (Eu-TDA). The resulting fluorescent signal was obtained in a time-resolved fluorimeter over 5 hours. Background death control was determined by diluting the target cells with culture medium and maximum death control was determined by incubating the cells with 10 uL of lysis buffer (1% Triton X-100) for 30 minutes before centrifuging the plate.

[0268] In vitro antibody-dependent cellular cytotoxicity (ADCC) using human PBMCs isolated from healthy donors

[0269] Frozen PBMC cells are commercially available from AllCells. Cells were isolated from human blood by the Leuko Pak density gradient method and then stored in liquid nitrogen. Cells were thawed at 37°C, suspended in RPMI1640+10% FBS, and incubated overnight at 37°C. According to the manufacturer's instructions, MDA-MB-231 target cells were labeled with DELFIA BATDA. Then, effector PBMC cells from each donor were plated to 96-well plates with a ratio of 50:1 with target cells. ADCC induction was triggered after adding each antibody candidate to the mixture, which was incubated for 4 hours at 37°C. Finally, supernatants were collected and mixed with europium solution. Time-resolved fluorescence (TRF) signal intensity was used to determine the degree of cytotoxicity. Control groups were set for data normalization, including target spontaneous groups (target cells), target maximum groups (target cells cracked using Triton) and background groups (supernatants of target cells). The ADCC effect was determined by the following formula: The calculated ADCC was defined by the following formula: ADCC% = (sample cytotoxicity - spontaneous cytotoxicity of a mixture of target cells and effector cells) / (maximum cytotoxicity of target cells (Triton X-100 treatment) - spontaneous cytotoxicity of a mixture of target cells and effector cells) * 100%. GraphPad Prism was used to analyze the dose-response effect.

[0270] Epitope binning

[0271] Octet RED384 was used to perform epitope binning assays in a classic sandwich assay format. All samples were prepared in assay buffer (PBS containing 1% BSA), and primary antibodies were biotinylated for immobilization on streptavidin (SA) biosensors. Each binding cycle consisted of the following steps. First, the SA biosensor was immersed in assay buffer for sensor inspection, and a baseline was established. Next, the biotinylated antibody was loaded onto the SA biosensor. After the washing step, uPAR was combined to reach saturation. The biosensor was washed in assay buffer, then moved to the next well for association of the secondary antibody, and finally transferred to the well containing buffer for the dissociation stage. Data analysis was performed using ForteBio data analysis software, and Matlab was used to make graphs.

[0272] Antibody-drug conjugate (ADC) cytotoxicity screening

[0273] MDA-MB-231 cells were seeded in 96-well plates (Corning) at 2,500 cells / well at 37°C and 5% CO2 overnight, and the cells were grown for 5 days in the presence of serial dilutions of antibodies ranging from 0.0032nM to 10nM, in triplicate, which were combined with a final concentration of 20nM of anti-human IgG Fc-specific antibody Fab fragment (Fab-αHFc-CL-MMAE, Moradec) conjugated to monomethyl auristatin E. The number of live cells was quantified by the CellTiter-Glo luminescent cell viability assay (Promega) based on ATP luminescence detection, which was proportional to the number of cells present in each well. After incubation, luminescence was recorded using a Synergy Neo2 multi-mode microplate reader (BioTek Instruments).

[0274] Therapeutic efficacy in an orthotopic animal model of human breast cancer

[0275] A group of 16 female Foxn1 nu Mice were orthotopically implanted with 1×10 6 MDA-MB-231 cells and monitored for several days until the tumor volume reached 75-100 mm 3 Once tumor volume was reached, animals were considered eligible for therapeutic intervention starting three days after reaching such tumor volume. Therapeutic intervention began with each experimental group receiving the antibody administered intravenously at a concentration of 30 mg / kg. A 30-day treatment regimen was performed, in which animals received antibody treatment weekly over a 30-day period (days 3, 10, 17, and 24). Throughout the study, animal welfare, body weight, and tumor volume were continuously monitored. After the therapeutic intervention regimen was completed, tumors were harvested and prepared for histological analysis.

[0276] Statistical analysis

[0277] All statistical analyses were performed in GraphPad Prism version 8.0 (GraphPad Software, San Diego, CA). Dose-response curves were drawn by nonlinear fitting of raw values ​​run in a minimum of three experimental replicates. Statistical analysis for all data collection was performed using two-way ANOVA with post hoc multiple comparison Tukey test. Differences between groups were considered significant at P values ​​≤ 0.05.

[0278] Liquid chromatography tandem mass spectrometry (LC-MS / MS)

[0279] Purified recombinant human suPAR (8 μg) was denatured with 6 M urea and disulfide bonds were reduced with 10 mM DTT at 55°C for 20 minutes, followed by acetamidomethylation with 12.5 mM iodoacetamide for 1 hour in the dark. Unreacted iodoacetamide was quenched with DTT, and the pH was balanced to pH 8 and digested with trypsin (Promega catalog number VA9000) at 37°C overnight. Samples were purified using Pierce TM C18 Spin Tips (Thermo Scientific TM , catalog number 87782), desalted, dried under vacuum, and resuspended in HPLC grade water containing 0.2% TFA. LC-MS / MS analysis was performed in an LTQ Orbitrap XL mass spectrometer (Thermo) coupled to a nanoACQUITY ultra-high performance liquid chromatography (UPLC) system (Waters). The tryptic digestion products were separated on a Thermo ES901 C18 column and eluted with a linear gradient of 2%-50% in buffer B (acetonitrile, 0.5% formic acid). Survey scans were recorded over the range of 325-1500 m / z, and up to three of the most intense precursor ions (MS1 features of charge ≥ 2) were selected for higher energy collision dissociation (HCD) at m / z 200 with a resolution of 30,000 for MS / MS [CB2]. Data from uPAR peptides were acquired using Xcalibur software and processed as previously described. (Zhao et al., (2021) ACS Cent Sci. American Chemical Society 7:1638-49).

[0280] Measurement of serum antibody titers in uPAR-immunized mice

[0281] Nunc MaxiSorp TMFlat-bottomed 96-well plates (Invitrogen catalog number 44-2404-21) were coated with human uPAR (3.19 μg / mL) overnight at 4°C, and the plates were blocked overnight at 4°C with 200 μL of blocking buffer consisting of 5% bovine serum albumin. Antibody titer determination was performed by standard logarithmic serial dilutions of serum in 5% skim milk powder. All uPAR-coated plates were washed three times with wash buffer (50 mM Tris-HCl, 150 mM NaCl, pH 7.4 + 0.02% Tween 20), and each serial dilution of serum was added. The plates were incubated overnight at 4°C, washed three times with wash buffer, and goat anti-mouse IgG (H + L) -HRP conjugate (BioRad catalog number 1706516, 1:3000 dilution) was added and incubated for 2 hours at room temperature on an orbital shaker. Finally, the plates were washed and 100 μL of 1-Step TM Turbo TMB-ELISA substrate solution (Thermo Scientific, catalog number 34022) was added to each well and incubated for 15 minutes, then the reaction was quenched with 2M H2SO4. The plate was incubated at room temperature for 5 minutes, and the optical density of each well was measured at 450 nM using a SpectraMax190 microplate reader against a standard curve (10-0.07 μg / mL) of mouse anti-human uPAR monoclonal antibody clone R-3 (Invitrogen catalog number MON R-3-02).

[0282] Example 10 - Humanized 11857 ​​and 3159 antibodies

[0283] Antibody 11857 ​​was used as a parental template in humanization design. Based on antibody sequence analysis and homology modeling of mAb 3D structure, three humanized VH ("11857HC1", "11857HC2" and "11857HC3") and three humanized VL ("11857LC1", "11857LC2" and "11857LC3") sequences were designed. The sequence of humanized 11857 ​​antibody is provided in Table 1 above. The Kabat numbering system is used to define CDR sequences.

[0284] In addition, antibody 3159 was used as a parental template in humanization design. Based on antibody sequence analysis and homology modeling of mAb 3D structure, three humanized VH ("3159HC1", "3159HC2" and "3159HC3") and three humanized VL ("3159LC1", "3159LC2" and "3159LC3") sequences were designed. The sequence of humanized 11857 ​​antibody is provided in Table 1 above. CDR sequences are defined using the Kabat numbering system.

[0285] The T20 scoring analyzer was used to determine the humanization score of the humanized 11857 ​​antibody as described in Gao et al. (2013) BMC Biotechnology, 13:55.

[0286] The results are shown in Table 3 below. For humanized variable region frameworks, the humanized T20 score ranges are 84-86 (VH) and 97-99 (VK), which are close to or exceed the threshold of "humanization" according to Gao et al. The T20 score ranges for humanized full-length variable regions are 79-84 (VH) and 81-82 (VK). The T20 score for humanized full-length variable heavy chain regions exceeds the threshold of humanization. Although the T20 score of the full-length κ light chain sequence is lower than the recommended critical score, based on structural modeling, the maximum T20 score is obtained without compromising the structural confirmation of the light chain. In addition, the T20 score analyzer is used to determine the humanization score of the humanized 3159 antibodies. The results are shown in Table 3 below. For humanized variable region frameworks, the humanized T20 analyzer score ranges are 84-86 (VH) and 97-99 (VK), which are close to or exceed the threshold of "humanization" according to Gao et al. The T20 scores of the humanized full-length variable regions ranged from 79-82 (VH) and 85-86 (VK), both of which were close to or exceeded the threshold for human origin. Although the T20 score of the full-length kappa light chain sequence "3159LC1" was just below the recommended critical score, based on structural modeling, the maximum T20 score was obtained without compromising the structural confirmation of the light chain.

[0287] Table 3 – T20 humanization evaluation of 11857 ​​and 3159 heavy and light chains

[0288]

[0289] The binding of humanized 11857 ​​and 3159 antibodies to human uPAR and cynouPAR was determined by biolayer interferometry (BLI). BLI curves are provided, and their affinity (KD) values ​​are reported in Table 4. The kinetic constant range of Octet HTX is between 1mM and 10pM. Therefore, the calculated KD below 10pM should be interpreted as KD<10pM. For two antibodies (11857HC2+LC3 and 11857HC3+LC3), the production output is not enough for kinetic analysis.

[0290] Two breast cancer cell lines MDA-MB-231 (high uPAR expression) and MCF-7 (low uPAR expression) are used to verify the cell surface uPAR binding for humanized and parental 11857 ​​or 3159 by cell assay based on flow cytometry. Cells were incubated with humanized and parental 11857 ​​or 3159 and stained with anti-human Fc antibodies conjugated with APC. The MFI comparison at 10 μg / mL showed specific binding to MDA-MB-231 and low non-specific binding to the MCF7 of the test article.

[0291] Using MDA-MB-231 cell line, six kinds of humanized 11857 ​​variants and parental chimeras were determined for EC50 by cell assay based on flow cytometry, and EC50 values ​​were between 0.2119 and 1.206 (μ g / mL). For three antibodies (11857HC2+LC3, 11857HC3+LC2 and 11857HC3+LC3), production output was not enough to measure EC50. In addition, using MDA-MB-231 cell line, nine kinds of humanized 3159 variants and parental chimeras were determined for EC50 by cell assay based on flow cytometry, and EC50 values ​​were between 0.1852 and 0.5176 (μ g / mL).

[0292] Table 4 – Kinetic analysis results of parental and humanized 11857 ​​and 3159

[0293]

[0294]

[0295] In addition, flow cytometry was used to compare the expression levels of uPAR in MDA-MB-231 and UMUC3 cell lines. Cells were incubated with parental 11857 ​​and stained with PE-conjugated anti-human Fc antibodies. The results showed that the expression level of uPAR in UMUC3 was about 3 times lower than that in MDA-MB-231 cells.

[0296] Example 11 - Imaging Studies

[0297] 5 mCi of Zr-89 was labeled with 500 μg of 3159 and 11857 ​​antibodies conjugated with deferoxamine (DFO), as described above for Example 10. In this example, 11857 ​​and 3159 refer to 11857HC2+LC2 and 3159HC2+LC2, respectively. Quality control analysis showed 100% labeling and 95% yield. Imaging was performed using the UMUC3 tumor model in nude male mice. For Zr89-3159, N=4, and for Zr89-11857, N=3. Doses were administered at 200-280 μCi per mouse. PET / CT images were taken at 30 minutes, 4 hours, 19 hours, 24 hours, 48 ​​hours, 72 hours, 96 hours, and 120 hours after administration.

[0298] exist Fig.13 Representative cross-sectional and coronal positron emission tomography–computed tomography (PET / CT) slices obtained from male nu / nu mice bearing subcutaneous UMUC3 xenografts are shown in FIG. 89 Zr-3159 IgG and imaged at the indicated time points. The location of the tumor is indicated by an arrow. Fig.14 A representative maximum intensity projection acquired from a male nu / nu mouse bearing a subcutaneous UMUC3 xenograft is shown in FIG. 89 Zr-3159 IgG and imaged at the indicated time points. The location of the tumor is indicated by an arrow. Fig.15 Representative cross-sectional and coronal PET / CT slices obtained from male nu / nu mice bearing subcutaneous UMUC3 xenografts are shown in FIG. 89 Zr-11857 ​​IgG and imaged at the indicated time points. The location of the tumor is indicated by an arrow. Fig.16 A representative maximum intensity projection obtained from a male nu / nu mouse with a subcutaneous UMUC3 xenograft is shown in FIG. 89 Zr-11857 ​​IgG and imaged at the indicated time points. The location of the tumor is indicated by an arrow.

[0299] SUV mean data were obtained by performing region of interest analysis on tumors from mice in the imaging cohort ( Fig.17 ). Data are presented as mean values ​​with standard deviation. Data were obtained from 4 tumors in the 3159 cohort and 3 tumors in the 11857 ​​cohort. In addition, SUV mean data were obtained by performing region of interest analysis on tumors and various normal tissues from mice in the imaging cohort. Data are presented as mean values ​​with standard deviation. Data were obtained from 4 tumors in the 3159 cohort ( Fig.18 ) and 3 tumors in the 11857 ​​cohort ( Fig.19 ) to obtain.

[0300] exist Fig. 20 The fold change in volume (normalized to the volume on day 0) of UMUC3 tumors treated with 225AC-labeled 3159 is depicted in . To enable radiolabeling, 3159 was coupled to NHS-Macropa via a lysine residue. The radiopharmaceutical was administered via the tail vein at 0.8 μCi per mouse on day 0. Tumor volume measurements were recorded for the vehicle group (n=10) and the drug-treated group (n=18). Fig.21 The 225 Volume changes of Ac-labeled 3159-treated UMUC3 tumors.

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[0371] Therefore, the foregoing only illustrates the principle of the present disclosure.It should be understood that those skilled in the art will be able to design various arrangements, which, although not explicitly described or shown in this article, embody the principle of the present invention and are included in its spirit and scope.In addition, all examples and conditional language narrated in this article are mainly intended to help readers understand the principles of the present invention and the concepts contributed by the inventor to promote this area, and should be interpreted as not being limited to such specific examples and conditions of narration.In addition, all statements of the principles, aspects and embodiments of the present invention and their specific examples are narrated herein and are intended to cover both their structural equivalents and functional equivalents.In addition, it is intended that such equivalents include both currently known equivalents and equivalents developed in the future, i.e. any element of the performance of the same function developed, regardless of the structure.Therefore, the scope of the present invention is not intended to be limited to the exemplary embodiments shown and described herein.

Claims

1. An antibody that specifically binds to human urokinase-type plasminogen activator receptor (uPAR) and competes with an antibody for binding to uPAR, the antibody comprising: Variable heavy chain (V H ) polypeptide, the variable heavy chain (V H ) polypeptide comprises: A V comprising the amino acid sequence TSGMGVS (SEQ ID NO: 2) H CDR1, A V comprising the amino acid sequence HIYWDDDKRYNPSLKT (SEQ ID NO: 3) H CDR2, and A V comprising the amino acid sequence RVRNYFSGTSYWYFDV (SEQ ID NO: 4) H CDR3; and Variable light chain (V L ) polypeptide, the variable light chain (V L ) polypeptide comprises: A V comprising the amino acid sequence RSSQNILHRTGNTYLE (SEQ ID NO: 6) L CDR1, A V comprising the amino acid sequence KVSNRFS (SEQ ID NO: 7) L CDR2, and A V comprising the amino acid sequence FQGSYVPFT (SEQ ID NO: 8) L CDR3; Variable heavy chain (V H ) polypeptide, the variable heavy chain (V H ) polypeptide comprises: V comprising the amino acid sequence SHDMS (SEQ ID NO: 10) H CDR1 A V comprising the amino acid sequence AIDSDGGLTYYSNSRER (SEQ ID NO: 11) H CDR2, and V comprising the amino acid sequence RRASYWYFDV (SEQ ID NO: 12) H CDR3; and Variable light chain (V L ) polypeptide, the variable light chain (V L ) polypeptide comprises: A V comprising the amino acid sequence RASQNIGTSIH (SEQ ID NO: 14) L CDR1, A V comprising the amino acid sequence YASESIS (SEQ ID NO: 15) L CDR2, and A V comprising the amino acid sequence QQSNSWPT (SEQ ID NO: 16) L CDR3; Variable heavy chain (V H ) polypeptide, the variable heavy chain (V H ) polypeptide comprises: V comprising the amino acid sequence DYYMN (SEQ ID NO: 18) H CDR1, A V comprising the amino acid sequence NINPNNNGGTDYNQKFKG (SEQ ID NO: 19) H CDR2, and V comprising the amino acid sequence SYGSRFPY (SEQ ID NO: 20) H CDR3; and Variable light chain (V L ) polypeptide, the variable light chain (V L ) polypeptide comprises: V comprising the amino acid sequence RASQDITNYLS (SEQ ID NO: 22) L CDR1, A V comprising the amino acid sequence YTAVLQS (SEQ ID NO: 23) L CDR2, and A V comprising the amino acid sequence QQGHTLPWT (SEQ ID NO: 24) L CDR3; or Variable heavy chain (V H ) polypeptide, the variable heavy chain (V H ) polypeptide comprises: V comprising the amino acid sequence DYYMN (SEQ ID NO: 18) H CDR1, A V comprising the amino acid sequence NINPNNNGGTDYNQKFKG (SEQ ID NO: 19) H CDR2, and V comprising the amino acid sequence SYGSRFPY (SEQ ID NO: 20) H CDR3; and Variable light chain (V L ) polypeptide, the variable light chain (V L ) polypeptide comprises: V comprising the amino acid sequence RASQDITNYLS (SEQ ID NO: 22) L CDR1, A V comprising the amino acid sequence YTSFLQS (SEQ ID NO: 26) L CDR2, and A V comprising the amino acid sequence QQGHTLPWT (SEQ ID NO: 24) L CDR3.

2. The antibody according to claim 1, wherein the antibody comprises: Variable heavy chain (V H ) polypeptide, the variable heavy chain (V H ) polypeptide comprises: A V comprising the amino acid sequence TSGMGVS (SEQ ID NO: 2) H CDR1, A V comprising the amino acid sequence HIYWDDDKRYNPSLKT (SEQ ID NO: 3) H CDR2, and A V comprising the amino acid sequence RVRNYFSGTSYWYFDV (SEQ ID NO: 4) H CDR3; and Variable light chain (V L ) polypeptide, the variable light chain (V L ) polypeptide comprises: A V comprising the amino acid sequence RSSQNILHRTGNTYLE (SEQ ID NO: 6) L CDR1, A V comprising the amino acid sequence KVSNRFS (SEQ ID NO: 7) L CDR2, and A V comprising the amino acid sequence FQGSYVPFT (SEQ ID NO: 8) L CDR3; Variable heavy chain (V H ) polypeptide, the variable heavy chain (V H ) polypeptide comprises: V comprising the amino acid sequence SHDMS (SEQ ID NO: 10) H CDR1 A V comprising the amino acid sequence AIDSDGGLTYYSNSRER (SEQ ID NO: 11) H CDR2, and V comprising the amino acid sequence RRASYWYFDV (SEQ ID NO: 12) H CDR3; and Variable light chain (V L ) polypeptide, the variable light chain (V L ) polypeptide comprises: A V comprising the amino acid sequence RASQNIGTSIH (SEQ ID NO: 14) L CDR1, A V comprising the amino acid sequence YASESIS (SEQ ID NO: 15) L CDR2, and A V comprising the amino acid sequence QQSNSWPT (SEQ ID NO: 16) L CDR3; Variable heavy chain (V H ) polypeptide, the variable heavy chain (V H ) polypeptide comprises: V comprising the amino acid sequence DYYMN (SEQ ID NO: 18) H CDR1, A V comprising the amino acid sequence NINPNNNGGTDYNQKFKG (SEQ ID NO: 19) H CDR2, and V comprising the amino acid sequence SYGSRFPY (SEQ ID NO: 20) H CDR3; and Variable light chain (V L ) polypeptide, the variable light chain (V L ) polypeptide comprises: V comprising the amino acid sequence RASQDITNYLS (SEQ ID NO: 22) L CDR1, A V comprising the amino acid sequence YTAVLQS (SEQ ID NO: 23) L CDR2, and A V comprising the amino acid sequence QQGHTLPWT (SEQ ID NO: 24) L CDR3; or Variable heavy chain (V H ) polypeptide, the variable heavy chain (V H ) polypeptide comprises: V comprising the amino acid sequence DYYMN (SEQ ID NO: 18) H CDR1, A V comprising the amino acid sequence NINPNNNGGTDYNQKFKG (SEQ ID NO: 19) H CDR2, and V comprising the amino acid sequence SYGSRFPY (SEQ ID NO: 20) H CDR3; and Variable light chain (V L ) polypeptide, the variable light chain (V L ) polypeptide comprises: V comprising the amino acid sequence RASQDITNYLS (SEQ ID NO: 22) L CDR1, A V comprising the amino acid sequence YTSFLQS (SEQ ID NO: 26) L CDR2, and A V comprising the amino acid sequence QQGHTLPWT (SEQ ID NO: 24) L CDR3.

3. The antibody according to claim 1 or claim 2, wherein the antibody comprises: Variable heavy chain (V H ) polypeptide, the variable heavy chain (V H ) a polypeptide comprising an amino acid sequence having 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 100% identity to the amino acid sequence shown in SEQ ID NO:1; and Variable light chain (V L ) polypeptide, the variable light chain (V L ) polypeptide comprises an amino acid sequence having 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 100% identity to the amino acid sequence shown in SEQ ID NO:

5.

4. The antibody of claim 1 or claim 2, wherein the antibody comprises: a variable heavy chain (VH) polypeptide comprising an amino acid sequence having 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 100% identity to the amino acid sequence shown in SEQ ID NO:9; and A variable light (VL) polypeptide comprising an amino acid sequence having 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 100% identity to the amino acid sequence shown in SEQ ID NO:

13.

5. The antibody of claim 1 or claim 2, wherein the antibody comprises: a variable heavy chain (VH) polypeptide comprising an amino acid sequence that is 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 100% identical to the amino acid sequence shown in SEQ ID NO: 17; and A variable light (VL) polypeptide comprising an amino acid sequence having 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 100% identity to the amino acid sequence shown in SEQ ID NO:

21.

6. The antibody of claim 1 or claim 2, wherein the antibody comprises: a variable heavy chain (VH) polypeptide comprising an amino acid sequence that is 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 100% identical to the amino acid sequence shown in SEQ ID NO: 17; and A variable light (VL) polypeptide comprising an amino acid sequence having 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 100% identity to the amino acid sequence shown in SEQ ID NO:

25.

7. An antibody that specifically binds to human urokinase-type plasminogen activator receptor (uPAR) and competes with an antibody for binding to uPAR, the antibody comprising: (a) Variable heavy chain (V H ) polypeptide, the variable heavy chain (V H ) polypeptide comprises: V comprising the amino acid sequence DYYMN (SEQ ID NO: 18) H CDR1; V comprising the amino acid sequence NINPNNNGGTDYNQKFQG (SEQ ID NO: 39); H CDR2; and V comprising the amino acid sequence SYGSRFPY (SEQ ID NO: 20) H CDR3; or V comprising the amino acid sequence DYYMN (SEQ ID NO: 18) H CDR1; V comprising the amino acid sequence NINPNNNGGTDYSQKFQG (SEQ ID NO: 37); H CDR2; and V comprising the amino acid sequence SYGSRFPY (SEQ ID NO: 20) H CDR3; and Variable light chain (V L ) polypeptide, the variable light chain (V L ) polypeptide comprises: V comprising the amino acid sequence RASQDITNYLS (SEQ ID NO: 22) L CDR1; V comprising the amino acid sequence YTAVLQS (SEQ ID NO: 23) L CDR2; and V comprising the amino acid sequence QQGHTLPWT (SEQ ID NO: 24) L CDR3; or (b) Variable heavy chain (V H ) polypeptide, the variable heavy chain (V H ) polypeptide comprises: A V comprising the amino acid sequence TSGMGVS (SEQ ID NO: 2) H CDR1; V comprising the amino acid sequence HIYWDDDKRYSTSLKT (SEQ ID NO: 44); H CDR2; and V comprising the amino acid sequence RVRNYFSGTSYWYFDV (SEQ ID NO: 4) H CDR3; or A V comprising the amino acid sequence TSGMGVS (SEQ ID NO: 2) H CDR1; V comprising the amino acid sequence HIYWDDDKRYSPSLKS (SEQ ID NO: 46); H CDR2; and V comprising the amino acid sequence RVRNYFSGTSYWYFDV (SEQ ID NO: 4) H CDR3; or A V comprising the amino acid sequence TSGMGVS (SEQ ID NO: 2) H CDR1; V comprising the amino acid sequence HIYWDDDKRYNPSLKS (SEQ ID NO: 48); H CDR2; and V comprising the amino acid sequence RVRNYFSGTSYWYFDV (SEQ ID NO: 4) H CDR3; and Variable light chain (V L ) polypeptide, the variable light chain (V L ) polypeptide comprises: A V comprising the amino acid sequence KSSQNILHRTGNTYLE (SEQ ID NO: 50) L CDR1; V comprising the amino acid sequence KVSNRFS (SEQ ID NO: 7); L CDR2; and V comprising the amino acid sequence FQGSYVPFT (SEQ ID NO: 8) L CDR3; or A V comprising the amino acid sequence RSSQNILHRTGNTYLE (SEQ ID NO: 6) L CDR1; V comprising the amino acid sequence KVSNRFS (SEQ ID NO: 7); L CDR2; and V comprising the amino acid sequence FQGSYVPFT (SEQ ID NO: 8) L CDR3; or A V comprising the amino acid sequence RSSQNILHRTGNTYLD (SEQ ID NO: 53) L CDR1; V comprising the amino acid sequence KVSNRFS (SEQ ID NO: 7); L CDR2; and V comprising the amino acid sequence FQGSYVPFT (SEQ ID NO: 8) L CDR3.

8. The antibody of claim 7, wherein the antibody comprises: (a) Variable heavy chain (V H ) polypeptide, the variable heavy chain (V H ) polypeptide comprises: V comprising the amino acid sequence DYYMN (SEQ ID NO: 18) H CDR1; V comprising the amino acid sequence NINPNNNGGTDYNQKFQG (SEQ ID NO: 39); H CDR2; and V comprising the amino acid sequence SYGSRFPY (SEQ ID NO: 20) H CDR3; or a VH CDR1 comprising the amino acid sequence DYYMN (SEQ ID NO: 18); a VH CDR2 comprising the amino acid sequence NINPNNNGGTDYSQKFQG (SEQ ID NO: 37); and a VH CDR3 comprising the amino acid sequence SYGSRFPY (SEQ ID NO: 20); and Variable light chain (V L ) polypeptide, the variable light chain (V L ) polypeptide comprises: V comprising the amino acid sequence RASQDITNYLS (SEQ ID NO: 22) L CDR1; V comprising the amino acid sequence YTAVLQS (SEQ ID NO: 23) L CDR2; and V comprising the amino acid sequence QQGHTLPWT (SEQ ID NO: 24) L CDR3; or (b) Variable heavy chain (V H ) polypeptide, the variable heavy chain (V H ) polypeptide comprises: A V comprising the amino acid sequence TSGMGVS (SEQ ID NO: 2) H CDR1; V comprising the amino acid sequence HIYWDDDKRYSTSLKT (SEQ ID NO: 44); H CDR2; and V comprising the amino acid sequence RVRNYFSGTSYWYFDV (SEQ ID NO: 4) H CDR3; or A V comprising the amino acid sequence TSGMGVS (SEQ ID NO: 2) H CDR1; V comprising the amino acid sequence HIYWDDDKRYSPSLKS (SEQ ID NO: 46); H CDR2; and V comprising the amino acid sequence RVRNYFSGTSYWYFDV (SEQ ID NO: 4) H CDR3; or A V comprising the amino acid sequence TSGMGVS (SEQ ID NO: 2) H CDR1; V comprising the amino acid sequence HIYWDDDKRYNPSLKS (SEQ ID NO: 48); H CDR2; and V comprising the amino acid sequence RVRNYFSGTSYWYFDV (SEQ ID NO: 4) H CDR3; and Variable light chain (V L ) polypeptide, the variable light chain (V L ) polypeptide comprises: A V comprising the amino acid sequence KSSQNILHRTGNTYLE (SEQ ID NO: 50) L CDR1; V comprising the amino acid sequence KVSNRFS (SEQ ID NO: 7); L CDR2; and V comprising the amino acid sequence FQGSYVPFT (SEQ ID NO: 8) L CDR3; or A V comprising the amino acid sequence RSSQNILHRTGNTYLE (SEQ ID NO: 6) L CDR1; V comprising the amino acid sequence KVSNRFS (SEQ ID NO: 7); L CDR2; and V comprising the amino acid sequence FQGSYVPFT (SEQ ID NO: 8) L CDR3; or A V comprising the amino acid sequence RSSQNILHRTGNTYLD (SEQ ID NO: 53) L CDR1; V comprising the amino acid sequence KVSNRFS (SEQ ID NO: 7); L CDR2; and V comprising the amino acid sequence FQGSYVPFT (SEQ ID NO: 8) L CDR3.

9. The antibody of claim 7 or claim 8, wherein the antibody comprises: Variable heavy chain (V H ) polypeptide, the variable heavy chain (V H ) a polypeptide comprising an amino acid sequence having 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 100% identity to the amino acid sequence shown in SEQ ID NO:35; and Variable light chain (V L ) polypeptide, the variable light chain (V L ) polypeptide comprises an amino acid sequence having 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 100% identity to an amino acid sequence selected from SEQ ID NO:40, SEQ ID NO:41, or SEQ ID NO:

42.

10. The antibody of claim 7 or claim 8, wherein the antibody comprises: Variable heavy chain (V H ) polypeptide, the variable heavy chain (V H ) a polypeptide comprising an amino acid sequence having 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 100% identity to the amino acid sequence shown in SEQ ID NO:36; and A variable light (VL) polypeptide comprising an amino acid sequence having 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 100% identity to an amino acid sequence selected from SEQ ID NO:40, SEQ ID NO:41, or SEQ ID NO:

42.

11. The antibody of claim 7 or claim 8, wherein the antibody comprises: Variable heavy chain (V H ) polypeptide, the variable heavy chain (V H ) a polypeptide comprising an amino acid sequence having 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 100% identity to the amino acid sequence shown in SEQ ID NO:38; and A variable light (VL) polypeptide comprising an amino acid sequence having 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 100% identity to an amino acid sequence selected from SEQ ID NO:40, SEQ ID NO:41, or SEQ ID NO:

42.

12. The antibody of claim 7 or claim 8, wherein the antibody comprises: Variable heavy chain (V H ) polypeptide, the variable heavy chain (V H ) a polypeptide comprising an amino acid sequence having 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 100% identity to the amino acid sequence shown in SEQ ID NO:43; and Variable light chain (V L ) polypeptide, the variable light chain (V L ) polypeptide comprises an amino acid sequence having 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 100% identity to an amino acid sequence selected from SEQ ID NO:49, SEQ ID NO:51, or SEQ ID NO:

52.

13. The antibody of claim 7 or claim 8, wherein the antibody comprises: Variable heavy chain (V H ) polypeptide, the variable heavy chain (V H ) a polypeptide comprising an amino acid sequence having 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 100% identity to the amino acid sequence shown in SEQ ID NO:45; and Variable light chain (V L ) polypeptide, the variable light chain (V L ) polypeptide comprises an amino acid sequence having 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 100% identity to an amino acid sequence selected from SEQ ID NO:49, SEQ ID NO:51, or SEQ ID NO:

52.

14. The antibody of claim 7 or claim 8, wherein the antibody comprises: Variable heavy chain (V H ) polypeptide, the variable heavy chain (V H ) a polypeptide comprising an amino acid sequence having 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 100% identity to the amino acid sequence shown in SEQ ID NO:47; and Variable light chain (V L ) polypeptide, the variable light chain (V L ) polypeptide comprises an amino acid sequence having 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 100% identity to an amino acid sequence selected from SEQ ID NO:49, SEQ ID NO:51, or SEQ ID NO:

52.

15. The antibody of any one of claims 1 to 14, wherein the antibody cross-reacts with non-human animal uPAR.

16. The antibody of claim 15, wherein the non-human animal uPAR is non-human primate uPAR.

17. The antibody of claim 16, wherein the non-human primate uPAR is cynomolgus monkey uPAR.

18. The antibody according to any one of claims 1 to 17, wherein the antibody is a humanized antibody.

19. The antibody according to any one of claims 1 to 18, wherein the antibody is IgG.

20. The antibody of claim 19, wherein the antibody comprises a human Fc domain.

21. The antibody of claim 20, wherein the antibody is human IgG1.

22. The antibody according to any one of claims 1 to 18, wherein the antibody is selected from the group consisting of Fab, F(ab')2 and F(ab').

23. The antibody of any one of claims 1 to 18, wherein the antibody is a single chain antibody.

24. The antibody of claim 23, wherein the single-chain antibody is a scFv.

25. The antibody according to any one of claims 1 to 24, wherein the antibody is a bispecific antibody comprising a first antigen binding domain comprising a V domain as defined in any one of claims 1 to 18. H Peptide-V L Peptide pairs.

26. The antibody of claim 25, wherein the bispecific antibody comprises a second antigen binding domain that specifically binds to an antigen other than uPAR.

27. A fusion protein comprising: A chain of the antibody according to any one of claims 1 to 26 fused to a heterologous sequence of amino acids.

28. The fusion protein of claim 27, wherein the heterologous sequence of amino acids is fused to the C-terminus of the chain of the antibody.

29. The fusion protein of claim 27 or claim 28, wherein the antibody is a single-chain antibody according to claim 23 or 24.

30. The fusion protein of claim 29, wherein the fusion protein is a chimeric antigen receptor (CAR), the chimeric antigen receptor comprising: The single-chain antibody; a transmembrane domain; and Intracellular signaling domain.

31. A conjugate comprising: The antibody according to any one of claims 1 to 26 or the fusion protein according to any one of claims 27 to 30; and A pharmaceutical agent conjugated to the antibody or the fusion protein.

32. The conjugate of claim 31, wherein the agent is a chemotherapeutic agent, a toxin, a radiosensitizer, a radioisotope, a detectable label, or a half-life extending moiety.

33. The conjugate of claim 32, wherein the radioisotope is a therapeutic radioisotope.

34. The conjugate of claim 32, wherein the detectable label is a radiolabel.

35. The conjugate of any one of claims 32 to 34, wherein the agent is conjugated to the antibody or the fusion protein via a non-cleavable linker.

36. The conjugate of any one of claims 32 to 34, wherein the agent is conjugated to the antibody or the fusion protein via a cleavable linker.

37. The conjugate of claim 36, wherein the cleavable linker is an enzyme cleavable linker.

38. The conjugate of claim 37, wherein the linker is cleavable by a lysosomal protease.

39. The conjugate of claim 38, wherein the linker is cleavable by cathepsin or plasmin.

40. A nucleic acid encoding the variable heavy chain (V H ) polypeptide, variable light chain (V L ) polypeptide or both.

41. A nucleic acid encoding the fusion protein according to any one of claims 27 to 30.

42. An expression vector comprising the nucleic acid according to claim 40 or claim 41.

43. A cell comprising the nucleic acid of claim 40 or claim 41.

44. The cell of claim 43, wherein the nucleic acid is present in an expression vector.

45. A cell comprising: Encoding the variable heavy chain (V H ) a first nucleic acid of a polypeptide; and Encoding the variable light chain (V L )A second nucleic acid of a polypeptide.

46. ​​The cell of claim 45, comprising: a first expression vector comprising the first nucleic acid; and A second expression vector comprising the second nucleic acid.

47. A cell comprising an expression vector encoding the CAR of claim 30, wherein the cell expresses the CAR on its surface.

48. A method for producing an antibody or fusion protein according to any one of claims 1 to 30, comprising culturing the cell under conditions suitable for expressing the antibody or fusion protein according to any one of claims 44 to 46, wherein the antibody or fusion protein is produced.

49. A composition comprising: The antibody according to any one of claims 1 to 26; The fusion protein according to any one of claims 27 to 30; The conjugate according to any one of claims 31 to 39; or A cell population of cells according to claim 47.

50. The composition of claim 49, wherein the antibody, the fusion protein, the conjugate or the cell population is present in liquid culture medium.

51. A composition according to claim 49 or claim 50 comprising a pharmaceutically acceptable carrier.

52. A kit comprising: A composition according to any one of claims 49 to 51; and Instructions for administering the composition to an individual in need thereof.

53. The kit of claim 52, wherein the composition is present in one or more unit doses.

54. The kit of claim 52, wherein the composition is present in two or more unit doses.

55. A method of treating a condition associated with uPAR expression and / or activity in a subject in need thereof, the method comprising administering to the subject an effective amount of a composition according to any one of claims 49 to 51.

56. The method of claim 55, wherein the condition associated with uPAR expression and / or activity is cancer.

57. The method of claim 56, wherein the cancer is characterized by cancer cells expressing uPAR on their surface.

58. The method of claim 56 or claim 57, wherein the cancer comprises a solid tumor.

59. The method of claim 58, wherein the cancer is characterized by stromal cells in the tumor microenvironment expressing uPAR on their surface.

60. The method of claim 58 or claim 59, wherein the solid tumor is a carcinoma, lymphoma, blastoma, or sarcoma.

61. The method of any one of claims 56 to 60, wherein the cancer is breast cancer, lung cancer, bladder cancer, ovarian cancer, prostate cancer, liver cancer, colon cancer, pancreatic cancer, gastric cancer, glioma, or any combination thereof.

62. The method of claim 56 or claim 57, wherein the cancer comprises a hematological malignancy.

63. The method of claim 62, wherein the hematological malignancy is leukemia, lymphoma, or multiple myeloma.

64. A method of inhibiting tumor invasion, tumor metastasis, extracellular matrix (ECM) degradation, tumor angiogenesis, tumor cell proliferation, or any combination thereof in a subject suffering from cancer, the method comprising administering to the subject an effective amount of a composition according to any one of claims 49 to 51.

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