Anti-urokinase plasminogen activator receptor antibodies and methods of use

Abstract

Provided are antibodies that specifically bind to human urokinase-type plasminogen activator receptor (uPAR). In some cases, the antibodies are cross-reactive 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 present disclosure are also provided. Methods of using the antibodies, fusion proteins, and conjugates of the present disclosure to treat conditions associated with uPAR expression and / or activity are also provided. In some embodiments, the condition associated with uPAR expression and / or activity is cancer. Non-limiting examples of such cancers include cancers characterized by cancer cells expressing uPAR on their surface, cancers characterized by stromal cells within the tumor microenvironment expressing uPAR on their surface, and the like.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Patent Application No. 63 / 413,530, filed October 5, 2022, which is incorporated herein by reference in its entirety.

[0002] Incorporation by reference of a sequence listing provided as a sequence listing XML file The Sequence Listing is provided herewith as 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.

[0003] Introduction A key characteristic of tumor cells is their enhanced ability to degrade the extracellular matrix (ECM), enabling tumor cell motility, invasion, and metastasis. The urokinase-type plasminogen activator receptor (uPAR) is an integral membrane protein linked to the plasma membrane via a glycosylphosphatidylinositol (GPI) anchor. This well-studied receptor is involved in the binding of various partners, such as urokinase-type plasminogen activator (uPA), vitronectin (VN), and transmembrane receptors, and regulates a wide variety of cellular processes, including extracellular proteolysis, angiogenesis, cell adhesion, migration, and downstream signaling events (1). Numerous studies have demonstrated that uPAR overexpression is tumor-specific (2, 3), making it a prominent biomarker for identifying tumor invasiveness (4-6) and an attractive target for cancer therapy (7), particularly in breast cancer (8-11).

[0004] Increasing evidence suggests that uPAR and HER2 are co-amplified in both non-invasive and metastatic breast cancer and act cooperatively to promote tumor progression toward the development of a metastatic phenotype (12, 13). Furthermore, downregulation of uPAR using RNAi with anti-HER2 antibodies induces a synergistic effect that inhibits breast cancer cell growth, highlighting the potential of combination therapy as an effective treatment for breast cancer (14). Clinical outcomes have shown that FDA-approved anti-HER2 antibodies are effective in metastatic HER2-positive breast cancer, but several mechanisms of resistance to anti-HER2 therapy have been identified (15, 16). Furthermore, due to the lack of HER2 expression, HER2 is not a valid target for patients with triple-negative breast cancer (TNBC) (9), thus necessitating the development of novel therapeutic strategies. Various groups have developed a series of antagonists, such as recombinant antibodies (rAbs), small molecules, and peptides, to block the interaction of uPAR with its partners (17-22). Some of these uPAR-targeting agents have also been designed as novel preclinical immunotherapeutic agents ( 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

[0005] Provided are antibodies that specifically bind to human urokinase-type plasminogen activator receptor (uPAR). In some cases, the antibodies are cross-reactive 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 present disclosure are also provided. Methods of using the antibodies, fusion proteins, and conjugates of the present disclosure to treat conditions associated with uPAR expression and / or activity are also provided. In some embodiments, the condition associated with uPAR expression and / or activity is cancer. Non-limiting examples of such cancers include cancers characterized by cancer cells expressing uPAR on their surface, cancers characterized by stromal cells within the tumor microenvironment expressing uPAR on their surface, and the like. [Brief explanation of the drawings]

[0006] [Figure 1A] Mouse immunization campaign pipeline. [Figure 1B] A workflow for accelerating the discovery of cross-reactive anti-uPAR antibodies using the Beacon™ platform. [Figure 1C] Each ASC was cultured in an individual nanopen to allow the accumulation of secreted antibodies. After culturing the cells for 1-2 hours, beads coated with anti-mouse IgG (H+L) were imported into the channel along with fluorescently labeled uPAR (AF488-human suPAR, green; and AF647-cynomolgus uPAR, red). Because the secreted antibodies were immobilized on the beads, antigen recognition allowed for the time-dependent accumulation of fluorescent signal directly above each nanopen. [Figure 1D] An increase in the signal in the AF488 and AF647 channels was observed between T0 and T11, suggesting that the antibody was able to recognize human / cynomolgus uPAR. [Figure 2] Initial antibody binding curves for MDA-MB-231 cells expressing human uPAR on the cell surface. MFI indicates median fluorescence intensity. [Figure 3A] Cross-reactivity profile of 12 antibody candidates assessed by ELISA, showing that they are cross-reactive to human and cynomolgus uPAR, with 2G10 and 3C6 being specific binders to human uPAR. [Figure 3B] The antibody candidate can prime NK-92® MI CD16a effector cells to generate ADCC against MDA-MB-231 cells in a dose-dependent manner. Two-way ANOVA followed by post-hoc Tukey's test reveals significantly greater activity than comparable human IgG1 (huIgG1). *=p≦0.001. [Figure 4]The eight selected antibody candidates were able to induce dose-dependent cell death in MBA-MB-231 cells in the presence of human PBMCs from three healthy donors. Two-way ANOVA followed by post-hoc Tukey's test reveals a more significant activity than the relative human IgG1 (huIgG1). *=p≦0.05, **=p≦0.001. [Figure 5] A) Dose-dependent cytotoxicity was observed for candidates 3159, 8163, 11857, and 3595 in the presence of an anti-human Fc Fab conjugated to the cytotoxic MMAE via a cathepsin-cleavable linker. B) The therapeutic efficacy of novel antibody candidates was determined in an orthotopic mouse model of human breast cancer using MDA-MB-231 cells. Animals showed reduced tumor size compared to untreated controls, and significant tumor growth inhibition was observed in animals treated with 11857 ​​after 21 days of treatment. C) A significant impairment of tumor growth rate was observed in antibody-treated animals throughout 30 days of treatment, with 11857 ​​being the most active agent. Data are presented as mean ± standard deviation. Statistical analysis was performed as a two-way analysis of variance with post-hoc multiple comparisons using Dunnett's test. *p<0.05, **p<0.01, ***p<0.001. [Figure 6] A) Molecular surface representation of the human uPAR-ATF-SMB complex. The uPA N-terminal fragment (ATF) is shown as a ribbon diagram in gray, and the vitronectin (VN) SMB domain is shown as a ribbon diagram in blue (PDB ID: 3BT1). Mutational variants between human uPAR and cynomolgus monkey uPAR are highlighted in yellow. B) BLI traces identify non-overlapping epitopes between each lead antibody and 2G10 that bind 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 distinct binding sites, and candidate 11857 ​​has an epitope that partially overlaps with candidates 8163 and 3159. [Figure 7]Proposed binding model for novel antibody candidates against uPAR, highlighting their inhibitory effects on vitronectin binding and different binding epitopes compared to 2G10. Antibodies 3159 and 8163 recognize different epitopes on uPAR, and 11857 ​​has an epitope that partially overlaps with antibodies 3159 and 8163. [Figure 8A] Recombinant human suPAR expressed and characterized by SDS-PAGE and immunoblot. [Figure 8B] Further characterization by LC-MS / MS showed 59.7% coverage of the complete protein sequence (SEQ ID NO: 55). [Figure 9] Antibody titers from each animal monitored throughout a 60-day immunization campaign using recombinant human uPAR as immunogen, showing the production of anti-uPAR antibodies. [Figure 10] The eight selected lead antibody candidates were able to block the adhesion of MDA-MB-231 cells to vitronectin in a dose-response manner. [Figure 11] A BLI competition assay between candidate 3159 and vitronectin in the opposite order shows the ability of 3159 to block the binding of vitronectin to uPAR. [Figure 12] BLI competition assays for the lead antibody and 3C6 discovered by phage display indicate that they have different binding epitopes. [Figure 13] PET / CT slices obtained at different time points from mice dosed with 89Zr-DFO-3159 antibody. [Figure 14] Maximum intensity projections obtained at different time points from mice dosed with 89Zr-DFO-3159 antibody. [Figure 15] PET / CT slices obtained at different time points from mice dosed with 89Zr-DFO-11857 ​​antibody. [Figure 16] Maximum intensity projections obtained at different time points from mice dosed with 89Zr-DFO-11857 ​​antibody. [Figure 17]Tumor time activity curves containing SUVmean data from four tumors in the 3159 cohort and three tumors in the 11857 ​​cohort. [Figure 18] SUVmean data obtained by region of interest analysis for tumors and various normal tissues from 3159 mice in the cohort. [Figure 19] SUVmean data obtained by region of interest analysis for tumors and various normal tissues from mice in the 11857 ​​cohort. [Figure 20] Antitumor assessment showing fold change in UMUC3 tumor volume. [Figure 21] Antitumor evaluation showing volume changes of UMUC3 tumors. DETAILED DESCRIPTION OF THE INVENTION

[0007] Before describing the antibodies and methods of the present disclosure in more detail, it is to be understood that the antibodies and methods are not limited to particular embodiments described, as such may, of course, vary. Also, the scope of the antibodies and methods will be limited only by the appended claims, and therefore it is to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting.

[0008] Where a range of values ​​is provided, unless the context clearly dictates otherwise, it is understood that each intervening value, to the tenth of the unit of the lower limit, between the upper and lower limit of that range, and any other stated or intervening value in that stated range, is encompassed within the antibodies and methods. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges and are also encompassed within the antibodies and methods, subject to any specifically excluded limit in the stated range. When the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the antibodies and methods.

[0009]

[0023] In this specification, certain ranges are presented with the term "about" before the numerical values. In this specification, the term "about" is used to provide literal support for the exact number it precedes, as well as a number that is near or approximately the number it precedes. In determining whether a number is near or approximately a specifically recited number, the near or approximately unrecited number may be a number that, in the context provided, provides substantial equivalence to the specifically recited number.

[0010] 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 described here.

[0011] All publications and patents cited herein are incorporated by reference to disclose and describe in connection with the materials and / or methods to which the publications are cited, to the same extent as if each individual publication or patent was specifically and individually indicated to be incorporated by reference. The citation of any publication is for its disclosure prior to the filing date and should not be construed as an admission that the present antibodies and methods are not entitled to antedate such publication, which may be different from the actual publication dates that may need to be independently confirmed.

[0012] It should be noted that, as used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. It should be further noted that the claims may be drafted to exclude any optional element. Accordingly, this statement is intended to serve as a predicate to the use of such exclusive terminology as "solely," "only," and the like, or the use of a "negative" limitation in connection with the recitation of claim elements.

[0013] For clarity, it is understood that certain features of the antibodies and methods that are described in the context of separate embodiments may also be provided in combination in a single embodiment. Conversely, for brevity, various features of the antibodies and methods that are described in the context of a single embodiment may also be provided separately or in any suitable subcombination. All combinations of embodiments are specifically encompassed by the present disclosure, and to the extent such combinations encompass operable processes and / or compositions, each and every combination is disclosed herein as if it were individually and explicitly disclosed. In addition, all subcombinations listed in the embodiments describing such variables are also specifically encompassed by the present antibodies and methods, and each and every such subcombination is disclosed herein as if it were individually and explicitly disclosed herein.

[0014] As will be apparent to those skilled in the art upon reading this disclosure, each of the individual embodiments described and illustrated herein has individual elements and features which may be readily separated from or combined with the features of any of the other several embodiments without departing from the scope or spirit of the method. Any recited method may be carried out in the order of events recited or in any other order which is logically possible.

[0015] Anti-UPAR antibody The present disclosure provides anti-urokinase plasminogen activator receptor (uPAR) antibodies. uPAR (UniProt Q03405-human), also known as CD87, is encoded by the PLAUR gene and belongs to the lymphoid antigen-6 superfamily. uPAR was first identified as the 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, anchored to the cell membrane by a glycosylphosphatidylinositol (GPI) linkage, contains three homologous domains, D1, D2, and D3, and has a total molecular weight of 55-60 kDa. uPAR mediates various biological processes, including plasminogen activation, proteolysis, cell signaling, and adhesion. Under normal physiological conditions, uPAR is typically expressed at low levels. In the processes of tissue remodeling, wound healing, inflammation and embryonic development, uPAR is transiently expressed at high levels and is involved in the processes of extracellular matrix (ECM) degradation, thrombolysis, cell invasion and migration.

[0016] uPAR has multiple functional roles related to tumor progression, including tumor growth and apoptosis, metastasis, angiogenesis, multidrug resistance (MDR), and prognosis. Analysis of tumor samples has shown high uPAR expression 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 some hematological malignancies. Furthermore, uPAR is expressed at high levels on stromal cells in the tumor microenvironment, such as vascular endothelial cells, tumor-associated fibroblasts, and tumor-associated macrophages, and its expression level is closely related to tumor aggressiveness and the survival of tumor-bearing patients.

[0017] In certain embodiments, an antibody of the disclosure specifically binds to human urokinase-type plasminogen activator receptor (uPAR) and competes for binding to human uPAR with an antibody having one, two, three, four, five, or all six complementarity-determining regions (CDRs) of one or more of the anti-uPAR antibodies designated herein as antibody 3159, 8163, 11857, or 3595. In some embodiments, such an antibody comprises one, two, three, four, five, or all six CDRs of the antibody designated herein as antibody 3159, 8163, 11857, or 3595. In some embodiments, such an antibody comprises one, two, three, four, five, or all six CDRs of the antibody designated herein as antibody 3159, 8163, 11857, or 3595. H and / or V L a variable heavy chain (V) 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 of H ) polypeptide and / or variable light chain (V L ) polypeptides.

[0018] Antibodies 3159, 8163, 11857, and 3595 were selected from among a number of identified anti-human uPAR antibodies based, at least in part, on their ability to cross-react with cynomolgus monkey uPAR. Cynomolgus monkeys (cynos) are genetically similar to humans compared to other species and are the most relevant non-human primate model for conducting preclinical studies in antibody drug development. Furthermore, as demonstrated in the experimental section below, these unique cross-reactive antibodies exhibit inhibitory effects on antibody-dependent cellular cytotoxicity (ADCC), ADC cytotoxicity, and cell adhesion to human cancer cells. Also demonstrated herein, these antibodies exhibit therapeutic efficacy in reducing tumor growth in orthotopic animal models of human cancer, and binding models of these antibodies demonstrate their binding epitopes that confer unique activity against uPAR.

[0019] V of 3159, 8163, 11857, and 3595 antibodies H Polypeptides and V L The amino acid sequences of the polypeptides are provided below in Table 1. The CDR sequences defined according to Kabat are underlined. [Table 1-1] [Table 1-2] [Table 1-3] [Table 1-4] [Table 1-5] [Table 1-6] [Table 1-7]

[0020] According to some embodiments, antibodies of the present disclosure specifically bind to human uPAR and include antibodies comprising one, two, three, four, five, or all six CDRs of the antibody designated herein as antibody 3159, or compete for binding to human uPAR. CDR sequences may be defined according to Kabat. In certain embodiments, such antibodies comprise one, two, three, four, five, or all six CDRs of the antibody designated herein as antibody 3159. H V 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 polypeptide. H polypeptide, V of the antibody designated herein as antibody 3159 LV 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 polypeptide. L According to some embodiments, such antibodies comprise the V of the antibody designated herein as antibody 3159. H Polypeptide, V L a V polypeptide, or both, as compared to one or more corresponding framework regions thereof; H Polypeptide, V L The polypeptide may comprise one or more amino acid substitutions (eg, one or more conservative amino acid substitutions) in one or more framework regions of the polypeptide, or both.

[0021] In certain embodiments, antibodies of the present disclosure specifically bind to human uPAR and include antibodies that contain one, two, three, four, five, or all six CDRs of the antibody designated herein as antibody 8163, or compete for binding to human uPAR. CDR sequences may be defined according to Kabat. In certain embodiments, such antibodies contain one, two, three, four, five, or all six CDRs of the antibody designated herein as antibody 8163. H V 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 polypeptide. H polypeptide, V of the antibody designated herein as antibody 8163 L V 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 polypeptide. L According to some embodiments, such antibodies comprise the V of the antibody designated herein as antibody 8163. H Polypeptide, V La V polypeptide, or both, as compared to one or more corresponding framework regions thereof; H Polypeptide, V L The polypeptide may comprise one or more amino acid substitutions (eg, one or more conservative amino acid substitutions) in one or more framework regions of the polypeptide, or both.

[0022] According to some embodiments, antibodies of the present disclosure specifically bind to human uPAR and include antibodies comprising one, two, three, four, five, or all six CDRs of the antibody designated herein as antibody 11857, or compete for binding to human uPAR. CDR sequences may be defined according to Kabat. In certain embodiments, such antibodies comprise one, two, three, four, five, or all six CDRs of the antibody designated herein as antibody 11857. H V 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 polypeptide. H polypeptide, V of the antibody designated herein as antibody 11857 L V 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 polypeptide. L According to some embodiments, such antibodies comprise the V of the antibody designated herein as antibody 11857. H Polypeptide, V L a V polypeptide, or both, as compared to one or more corresponding framework regions thereof; H Polypeptide, V L The polypeptide may comprise one or more amino acid substitutions (eg, one or more conservative amino acid substitutions) in one or more framework regions of the polypeptide, or both.

[0023] According to some embodiments, antibodies of the present disclosure specifically bind to human uPAR and include antibodies that contain one, two, three, four, five, or all six CDRs of the antibody designated herein as antibody 3595, or compete for binding to human uPAR. CDR sequences may be defined according to Kabat. In certain embodiments, such antibodies contain one, two, three, four, five, or all six CDRs of the antibody designated herein as antibody 3595. H V 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 polypeptide. H polypeptide, V of the antibody designated herein as antibody 3595 L V 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 polypeptide. L According to some embodiments, such antibodies comprise the V of the antibody designated herein as antibody 3595. H Polypeptide, V L a V polypeptide, or both, as compared to one or more corresponding framework regions thereof; H Polypeptide, V L The polypeptide may comprise one or more amino acid substitutions (eg, one or more conservative amino acid substitutions) in one or more framework regions of the polypeptide, or both.

[0024] 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.

[0025] In certain embodiments, the V H and / or V LAntibody variants having one or more amino acid substitutions relative to the amino acid sequence are provided. Sites of interest for substitutional mutagenesis include one or more CDRs and / or one or more framework regions (FRs). Conservative substitutions are shown in the table below under the heading "Preferred Substitutions." More substantial changes are provided in the table below under the heading "Exemplary Substitutions" and are further described below with reference to amino acid side chain classes. Amino acid substitutions may be introduced into a polypeptide of interest, and the products may be screened for a desired activity, e.g., retained / improved antigen binding, reduced immunogenicity, improved developability, improved manufacturability, etc. JPEG2025535040000009.jpg131170

[0026] Amino acids can be grouped according to the following common side chain properties: (1) Hydrophobic: Norleucine, Met, Ala, Val, Leu, Ile, (2) Neutral hydrophilicity: Cys, Ser, Thr, Asn, Gln, (3) Acidic: Asp, Glu, (4) Basic: His, Lys, Arg, (5) Residues that affect chain orientation: Gly, Pro, (6) Aromatic: Trp, Tyr, Phe

[0027] Non-conservative substitutions involve exchanging a member of one of these classes for another class.

[0028] 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 D ) may be measured using candidate anti-human uPAR antibodies conjugated to a fluorophore or radioisotope, or containing an N- or C-terminal epitope tag for detection by labeled antibodies. If labeling or tagging is not feasible or desirable, competitive binding assays may be used to measure half-maximal inhibitory concentrations (IC 50), the amount of unlabeled candidate anti-human uPAR antibody at which 50% of the maximum signal of the labeled competitor is detectable can be determined. D Values ​​are measured IC 50 Ligand depletion is more pronounced when measuring high affinity interactions over a lower concentration range and can be avoided or minimized by reducing the amount of human uPAR added in the experiment or by increasing the binding reaction volume.

[0029] Whether an antibody of the present disclosure "competes" with a second antibody for binding to an antigen can be easily determined using competitive binding assays known in the art. Competing antibodies can be identified, for example, through antibody competition assays. For example, a sample of a first antibody can be bound to a solid support. A sample of a second antibody suspected of competing with the first antibody is then added. One of the two antibodies is labeled. If the labeled and unlabeled antibodies bind to distinct sites on the antigen, the labeled antibody will bind to the same level regardless of the presence of a suspected competing antibody. However, if the interaction sites are identical or overlap, the unlabeled antibody will compete, reducing the amount of labeled antibody bound to the antigen. If an excess of unlabeled antibody is present, the labeled antibody will bind very little, if at all.

[0030] For purposes of this disclosure, a competing antibody is one that reduces antibody binding to an antigen 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. Detailed procedures for conducting such competitive assays are known and can be found, for example, in Harlow and Lane, *Antibodies*, *A Laboratory Manual*, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1988, pp. 567-569, 1988, ISBN 0-87969-314-2. Such assays can be performed quantitatively using purified antibodies. A standard curve can be established by titrating one antibody against itself; i.e., the same antibody is used as both the label and the competitor. The ability of an unlabeled competing antibody to inhibit the binding of a labeled antibody to the plate can be titrated. The results can be plotted, and the concentrations required to achieve the desired degree of binding inhibition can be compared.

[0031] A human uPAR polypeptide that can be used to determine whether an antibody of the present disclosure competes with a second antibody for binding to human uPAR is described in UniProt Q03405.

[0032] 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 composed of two dimers of, in turn, a heavy chain polypeptide and a light chain polypeptide); single-chain antibodies (e.g., scFv); single-chain Fv (scFv), Fab, (Fab')2, (scFv')2, and diabodies, fragments of antibodies (e.g., whole antibodies or single-chain antibody fragments) that retain specific binding to a cell surface molecule of a target cell; chimeric antibodies; monoclonal antibodies, human antibodies, humanized antibodies (e.g., humanized whole antibodies, humanized half antibodies, or humanized antibody fragments, e.g., 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 an IgG, Fv, single chain antibody, scFv, Fab, F(ab')2, F(ab') or Fab'. The antibody may be detectably labeled, for example, with an in vivo imaging agent, a radioisotope, an enzyme that generates a detectable product, a fluorescent protein, etc. The antibody may be further conjugated to another moiety, for example, a member of a specific binding pair, such as biotin (a member of the biotin-avidin specific binding pair).

[0033] An immunoglobulin light or heavy chain variable region is composed of a framework region (FR) interrupted by three hypervariable regions, also called "complementarity-determining regions" or "CDRs." The extent of the framework regions and CDRs 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 ed. USDept. Health and Human Services, Public Health Services, Bethesda, MD (1987); Lefranc et al., IMGT, the international ImMunoGeneTics information system®. Nucl. Acids Res., 2005, 33:D593-D597 (www.imgt.org / textes / IMGTScientificChart / ), and / or V Base (vbase.mrc-cpe.cam.ac.uk / ). The sequences of the framework regions of different light or heavy chains are relatively conserved within a species. The framework region of an antibody, which is the combined framework regions of the constituent light and heavy chains, serves to position and align the CDRs, which are primarily responsible for binding to an antigen epitope.

[0034] 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 method by which it is produced. 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 a non-antibody protein, and other molecules that exhibit the immunological binding properties of the parent monoclonal antibody molecule. Methods for producing monoclonal antibodies are known in the art and are described more fully below.

[0035] Any anti-human uPAR antibody of the present disclosure may be a recombinant or modified antibody, such as a chimeric antibody, a deimmunized antibody, and / or an in vitro generated antibody. As used herein, the term "recombinant" or "modified" antibody is intended to include all antibodies prepared, expressed, generated, or isolated by recombinant means, such as (i) antibodies expressed from 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) transgenic for human immunoglobulin genes, or (iv) antibodies prepared, expressed, generated, or isolated by any other means, including splicing human immunoglobulin gene sequences into other DNA sequences. Such recombinant antibodies include, for example, chimeric antibodies, deimmunized antibodies, and / or in vitro generated antibodies.

[0036] Any anti-human uPAR antibody of the present disclosure may be isolated. "Isolated" means that the antibody is separated from all or part of the components that naturally accompany it. "Isolated" also refers to the state of an antibody that is separated from all or part of the components that accompany it during production, such as chemical synthesis, recombinant expression, culture medium, etc.

[0037] Any anti-human uPAR antibody of the present disclosure may comprise a degree and / or pattern of glycosylation that differs from the degree and / or pattern of glycosylation of an antibody that is naturally produced, 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 from one or more recombinant expression vectors transfected into a host cell, and the expressed recombinant anti-human uPAR antibody comprises a different degree of glycosylation, a different glycosylation pattern, or both, compared to the degree of glycosylation and / or glycosylation pattern of the antibody when produced naturally, e.g., when produced in an animal in response to immunization with a human uPAR antigen.

[0038] In some embodiments, the anti-human uPAR antibodies of the present disclosure comprise a heavy chain comprising an Fc region, the Fc region being the V H The Fc region and V are heterologous to each other, i.e., the Fc region includes 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. H Antibodies comprising the combination of do not occur in nature and are different from, for example, anti-human uPAR antibodies produced in animals in response to immunization with human uPAR antigen.

[0039] In certain embodiments, one or more amino acid modifications can be introduced into the Fc region of an antibody provided herein, thereby generating an Fc region variant. An Fc region variant comprises 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. An Fc region variant can also comprise a human Fc region sequence (e.g., a 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 the S228P mutation).

[0040] In certain embodiments, the Fc region is mutated to increase its affinity for FcRn at pH 6.0, resulting in an increased antibody half-life. Antibodies with improved affinity for FcRn include antibodies with substitutions of one or more of Fc region residues 252, 253, 254, 256, 428, and 434, including the so-called YTE mutation with substitutions M252Y / S254T / T256E (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)).

[0041] The phrases "specifically bind," "specific for," "immunoreactive," and "immunoreactive," as well as "antigen-binding specificity," when referring to an antibody, refer to a binding reaction with an antigen that is highly preferential for the antigen or a fragment thereof, and determines the presence of the antigen in the presence of a heterogeneous antigen population (e.g., proteins and other biological preparations, e.g., in a sample). Thus, under specified immunoassay conditions, a particular antibody binds to a specific human uPAR antigen and does not bind in significant amounts to other antigens present in the sample. Specific binding to an antigen under such conditions may require an antibody selected for its specificity for a particular antigen. For example, an anti-human uPAR antibody can specifically bind to the human uPAR antigen and does not exhibit comparable binding (e.g., no detectable binding) to other proteins present in the sample.

[0042] In some embodiments, the antibodies of the present disclosure bind to the human uPAR antigen, e.g., at about 10 5 M -1 or higher affinity or K a (i.e., the equilibrium association constant of the specific binding interaction with units of 1 / M). In certain embodiments, an antibody "specifically binds" to a human uPAR antigen if it binds to or associates with the human uPAR antigen with a binding constant of 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 More than K a "High affinity" binding is defined as binding to human uPAR with a binding 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 1011 M -1 , at least 10 12 M -1 , at least 10 13 M -1 , or more K a Alternatively, affinity may be expressed in units of M (e.g., 10 -5 M~10 -13 M or less) and the equilibrium dissociation constant (K D In some embodiments, specific binding can be defined as the binding of an antibody to a cell surface area greater than about 10 -5 M or less, about 10 -6 M or less, about 10 -7 M or less, about 10 -8 M or less, or about 10 -9 M or less, 10 -10 M, 10 -11 M or 10 -12 K below M D The binding affinity of an antibody to human uPAR can be readily determined using conventional techniques, such as 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.

[0043] In certain embodiments, the antibody of the present disclosure cross-reacts with non-human animal uPAR.For example, the anti-human uPAR antibody of the present disclosure can cross-react with non-human primate uPAR.In one non-limiting example, the non-human primate uPAR is cynomolgus monkey uPAR.Also, for example, the anti-human uPAR antibody of the present disclosure can cross-react with rodent uPAR.In some cases, the rodent uPAR is mouse uPAR.

[0044] An antibody of the present disclosure is said to be "cross-reactive" with respect to two different antigens or antigenic determinants (e.g., uPAR from two different mammalian species, such as human and cynomolgus monkey) if it is specific for both of these different antigens or antigenic determinants (as defined herein). In certain embodiments, EC 50 and / or K D An antibody that binds to antigen 1 (Ag1) is "cross-reactive" to antigen 2 (Ag2) if the affinity is within a similar range for both antigens. According to some embodiments, a monoclonal antibody that binds to Ag1 is cross-reactive to Ag2 if the ratio of affinity for Ag1 to affinity for Ag2 is less than or equal to 10 (<10) and greater than or equal to 0.1 (>0.1), which means that the affinity for Ag1 does not differ by more than 10-fold (affinity is monovalent K D Thus, an antibody of the present disclosure may have a ratio of affinity for human uPAR to affinity for cynomolgus monkey uPAR that is less than or equal to 10 (<10) and greater than or equal to 0.1 (>0.1), meaning that the difference between the affinity for human uPAR and the affinity for cynomolgus monkey uPAR is no more than 10-fold (affinity is measured using a monovalent K D Such antibodies may be used, for example, in toxicology studies conducted in cynomolgus monkeys, as the toxicity profile observed in cynomolgus monkeys will be relevant for predicting potential adverse effects in humans.

[0045] An "epitope" is a site on an antigen to which an antibody binds. Epitopes can be formed from both contiguous or noncontiguous amino acids juxtaposed by protein folding (e.g., tertiary folding). Epitopes formed from contiguous amino acids are typically retained upon exposure to denaturing solvents, whereas epitopes formed by folding are typically lost upon treatment with denaturing solvents. An epitope typically comprises at least three, more typically at least five, or 8-10 amino acids, in a linear or spatial conformation. Methods for determining the spatial conformation of epitopes 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, Glenn E. Morris, Ed. (1996). Several commercial laboratories offer epitope mapping services. An epitope bound by an antibody immunoreactive with human uPAR may, for example, be present on the surface of human uPAR, and therefore such an epitope is considered to be accessible to the surface of human uPAR, accessible to the solvent, and / or exposed on the surface of human uPAR.

[0046] 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 modified to contain at least a portion of a framework and / or constant region of a human antibody. Humanized antibodies also encompass chimeric antibodies and CDR-grafted antibodies, in which various regions may be derived from different species. A chimeric antibody may be an antibody comprising a variable region from any source linked to a human constant region (e.g., a human Fc domain). Thus, in a chimeric antibody, the variable region may be non-human, and the constant region is human. A CDR-grafted antibody is an antibody comprising CDRs from a non-human "donor" antibody linked to a framework region from a human "recipient" antibody. For example, an antibody of the present disclosure in the form of an scFv may be linked to a human constant region (e.g., an Fc domain) generated into a human immunoglobulin.

[0047] Generally, humanized antibodies generate a reduced immune response in a human host when compared to a non-humanized version of the same antibody. Antibodies can be humanized using a variety of techniques, including, for example, CDR grafting, veneering or resurfacing, chain shuffling, etc. In certain embodiments, framework substitutions are identified by modeling the interactions of CDR and framework residues to identify framework residues important for antigen binding and sequence comparison, and to identify unusual framework residues at specific positions.

[0048] Thus, any of the antibodies described herein can be humanized using available methods. Substitution of rabbit or mouse CDRs into a human variable domain framework can result in their correct spatial orientation being maintained; for example, the human variable domain framework adopts the same or a similar conformation as the rabbit or mouse variable framework from which the CDRs are derived. This can be achieved by obtaining the human variable domain from a human antibody having framework sequences that show high sequence identity with the rabbit or mouse variable framework domain from which the CDRs are derived. The heavy and light chain variable framework regions can be derived from the same or different human antibody sequences. The human antibody sequences can be those of naturally occurring human antibodies or can be consensus sequences of several human antibodies.

[0049] After identifying the complementarity-determining regions of a rabbit or mouse donor immunoglobulin and a suitable human acceptor immunoglobulin, the next step is to determine which residues from these components, if any, should be substituted to optimize the properties of the resulting humanized antibody. Generally, substitution of human amino acid residues with rabbit or mouse residues should be minimized because the introduction of rabbit or mouse residues increases the risk of the antibody eliciting a human anti-rabbit antibody (HARA) or human anti-mouse antibody (HAMA) response in humans. Art-recognized methods for determining immune response can be performed to monitor HARA or HAMA responses in specific patients or during clinical trials. Patients receiving a humanized antibody can undergo immunogenicity assessments at the initiation of the aforementioned therapy and throughout administration. HARA or HAMA responses are measured by detecting antibodies against the humanized therapeutic reagent in serum samples from patients using methods known in the art, including, for example, surface plasmon resonance technology (BIACORE) and / or solid-phase ELISA analysis. In many embodiments, the subject humanized antibody does not elicit a substantial HARA response in human subjects.

[0050] Specific amino acids from human variable region framework residues are selected for substitution based on their possible influence on CDR conformation and / or antigen binding. The unnatural juxtaposition of rabbit or mouse CDR regions with human variable framework regions can result in unnatural conformational constraints that, unless corrected by substitution of specific amino acid residues, result in 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 created starting from the solved structure of an immunoglobulin chain or its domain. The chain to be modeled is compared for amino acid sequence similarity with the chain or domain of the solved three-dimensional structure, and the chain or domain showing the greatest sequence similarity is selected as the starting point for construction of the molecular model. Chains or domains sharing at least 50% sequence identity, preferably at least 60%, 70%, 80%, 90%, or more, are selected for modeling. The solved starting structure is modified to allow for differences between the actual amino acids in the immunoglobulin chain or domain being modeled and the amino acids in the starting structure. The modified structure is then assembled into a composite immunoglobulin. Finally, the model is refined by energy minimization and by ensuring that all atoms are within appropriate distances from each other and that bond lengths and angles are within chemically acceptable limits.

[0051] For example, if framework residues as defined by Kabat constitute structural loop residues as defined by Chothia, amino acids present in rabbit or mouse antibodies can be selected for substitution into the humanized antibody. Residues "adjacent to the CDR region" include amino acid residues located immediately adjacent to one or more CDRs in the primary sequence of the humanized immunoglobulin chain, e.g., a CDR defined by Kabat or a CDR defined by Chothia (see, e.g., Chothia and Lesk JMB 196:901 (1987)). These amino acids are particularly likely to interact with amino acids within the CDRs, which, if selected from the acceptor, would distort the donor CDR and reduce affinity. Furthermore, adjacent amino acids can directly interact with the antigen (Amit et al., Science, 233:747 (1986)). Selecting these amino acids from the donor may be desirable to preserve all antigen contacts that provide affinity to the original antibody.Approaches that can be used to humanize any of the antibodies described herein include those described in Williams, D., Matthews, D. & Jones, T. Humanizing 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 antibodies. 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, BK Antibody Humanization by CDR grafting. Methods Mol. Biol. 248, 135-59 (2004), and Lefranc, M.-PP, Ehrenmann, F., Ginestoux, C., Giudicelli, V. & Duroux, P. Use of IMGT® databases and tools for antibody engineering and humanization. Methods Mol. Biol. 907, 3-37 (2012), the disclosures of which are incorporated herein by reference in their entirety for all purposes.

[0052] bispecific antibody Bispecific antibodies are also provided. In certain embodiments, the bispecific antibodies of the present disclosure are selected from the V and VIII of any of the anti-human uPAR antibodies of the present disclosure, including any of the antibodies as described herein above. H Polypeptide-V LThe bispecific antibody comprises a first antigen-binding domain comprising a polypeptide pair. The bispecific antibody may comprise a second antigen-binding domain that specifically binds to the 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.

[0053] Bispecific antibodies of the present disclosure include antibodies having a full-length antibody structure and bispecific antibody fragments. As used herein, "full-length" refers to an antibody having two full-length antibody heavy chains and two full-length antibody light chains. A full-length antibody heavy chain (HC) consists of the well-known heavy chain variable and constant domains VH, CH1, CH2, and CH3. A full-length antibody light chain (LC) consists of the well-known light chain variable and constant domains VL and CL. A full-length antibody may lack the C-terminal lysine of one or both heavy chains. The term "Fab arm" refers to one heavy chain:light chain pair that specifically binds to an antigen.

[0054] Full-length bispecific antibodies can be generated, for example, by using Fab arm exchange (or half molecule exchange) between two monospecific bivalent antibodies to introduce substitutions into the heavy chain CH3 interface of each half molecule and promote heterodimerization of two antibody half molecules with different specificities in vitro in a cell-free environment or using coexpression. The Fab arm exchange reaction results in disulfide bond isomerization and dissociation of the CH3 domains. The heavy chain disulfide bond in the hinge region of the parent monospecific antibody is reduced. The resulting free cysteine ​​in one of the parent monospecific antibodies forms a heavy chain inter-disulfide bond with a cysteine ​​residue in the second parent monospecific antibody molecule, while the parent antibody CH3 domain is released and modified by dissociation. The CH3 domain of the Fab arm can be engineered to favor heterodimerization over homodimerization. The resulting product is a bispecific antibody with two Fab arms or half molecules, each binding a different epitope.

[0055] The "knob-in-hole" strategy (see, e.g., WO2006 / 028936) can be used to generate full-length bispecific antibodies. Briefly, selected amino acids that form the interface of the CHS domain in human IgG can be mutated at positions that affect CH3 domain interactions to promote heterodimer formation. An amino acid with a small side chain (hole) is introduced into the heavy chain of an antibody that specifically binds to one antigen, and an amino acid with a large side chain (knob) is introduced into the heavy chain of an antibody that specifically binds to a second antigen. After coexpression of the two antibodies, heterodimers form as a result of the preferential interaction of the heavy chain with the "knob" and the "hole." Exemplary CH3 substitution pairs that form knobs and holes 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.

[0056] Other strategies may also be used, such as using electrostatic interactions to promote heavy chain heterodimerization by substituting positively charged residues on one CH3 surface and negatively charged residues on the second CH3 surface, as described in US2010 / 0015133, US2009 / 0182127, US2010 / 028637, or US2011 / 0123532. In other strategies, heterodimerization can be promoted by the following substitutions (expressed 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): L351 Y_F405A_Y407V T394W, T366I_K392M_T394W / F405A_Y407V, T366L_K392M_T394W / F405A_Y407V, L351 Y_ ... Y_Y407A'T366A_K409F, L351Y_Y407A / T366V_K409F, Y407A / T366A_K409F, or T350V_L351Y_F405A_Y407V / T350V_T366L_K392L_T394W.

[0057] Also provided are single-chain bispecific antibodies. In some embodiments, the single-chain bispecific antibodies of the present disclosure are bispecific scFvs. Details regarding bispecific scFvs can be found, for example, in Zhou et al. (2017) J Cancer 8(18):3689-3696.

[0058] Approaches 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 antibody 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).

[0059] Fusion proteins Fusion proteins are also provided. In certain embodiments, the fusion proteins of the present disclosure comprise any chain of an anti-uPAR antibody of the present disclosure fused to a heterologous sequence of amino acids. The heterologous sequence 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 comprise a heterologous sequence at the C-terminus of the antibody chain and a heterologous sequence at the N-terminus of the antibody chain, where the heterologous sequences may be the same or different sequences. "Heterologous" as used in the context of a nucleic acid or polypeptide generally means that the nucleic acid or polypeptide is derived from a different source (e.g., a molecule of a different sequence, a different species origin, and the like) than that to which it is associated or conjugated, such that the nucleic acid or polypeptide is not found in nature. For example, in a fusion protein, the light chain polypeptide and the reporter polypeptide (e.g., GFP, red fluorescent protein (e.g., mCherry), luciferase, etc.) are said to be "heterologous" to each other. Similarly, CDRs from a mouse antibody and a constant region from a human antibody are "heterologous" to each other.

[0060] The chains of the anti-human uPAR antibody may be fused to any heterologous sequence of interest, including, but not limited to, albumin, transferrin, XTEN, homoamino acid polymers, proline-alanine-serine polymers, elastin-like peptides, or any combination thereof. In certain embodiments, the heterologous polypeptide increases the stability and / or serum half-life of the antibody when administered to an individual in need thereof, compared to the same antibody not fused to the heterologous sequence.

[0061] In certain embodiments, the fusion proteins of the present disclosure comprise a single chain antibody, e.g., the V of any of the anti-human uPAR antibodies of the present disclosure. H Polypeptide-V L Single chain antibodies (eg, scFv) comprising a pair of polypeptides include any of the antibodies described herein above.

[0062] 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 scFvs described herein), the fusion protein is a chimeric antigen receptor (CAR) comprising the single chain antibody, a transmembrane domain, and an intracellular signaling domain.

[0063] CARs of the present disclosure may contain one or more linker sequences between various domains. A "variable region linking sequence" is an amino acid sequence that connects a heavy chain variable region to a light chain variable region and provides a spacer function to accommodate the interaction of the two sub-binding domains so that the resulting polypeptide retains specific binding affinity for the same target molecule as an antibody comprising the same light and heavy chain variable regions. A non-limiting example of a variable region linking sequence is a serine-glycine linker, such as a serine-glycine linker comprising the amino acid sequence GGGGSGGGSGGGGS(G4S)3 (SEQ ID NO: 54). In certain aspects, a linker separates one or more heavy or light chain variable domains, hinge domains, transmembrane domains, costimulatory domains, and / or primary signaling domains. In specific embodiments, a CAR comprises one, two, three, four, five, or more linkers. In specific embodiments, the length of the linker 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 intervening length. 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.

[0064] In some embodiments, the antigen-binding domain of a CAR is followed by one or more spacer domains that distance the antigen-binding domain from the effector cell surface (e.g., the surface of a T cell expressing the CAR) to allow for proper cell-cell contact, antigen binding, and / or activation. The spacer domain (and any other spacer domains, linkers, etc. described herein) can be derived from either natural, synthetic, semi-synthetic, or recombinant sources. In certain embodiments, the spacer domain is a portion of an immunoglobulin, including, but not limited to, one or more heavy chain constant regions, e.g., CH2 and CH3. The spacer domain can comprise the amino acid sequence of a naturally occurring immunoglobulin hinge region or a modified immunoglobulin hinge region. In one embodiment, the spacer domain comprises the CH2 and / or CH3 of IgG1, IgG4, or IgD. Exemplary spacer domains suitable for use in the CARs described herein include hinge regions derived from the extracellular regions of type 1 membrane proteins such as CD8α and CD4, which can be wild-type hinge regions from these molecules or variants thereof. In certain aspects, the hinge domain comprises a CD8α hinge region. In some embodiments, the hinge is a PD-1 hinge or a CD152 hinge.

[0065] The "transmembrane domain" (Tm domain) is the portion of the CAR that fuses the extracellular binding moiety and the intracellular signaling domain and anchors the CAR to the plasma membrane of a cell (e.g., an immune effector cell). The Tm domain can be derived from either natural, synthetic, semi-synthetic, or recombinant sources. In some embodiments, the Tm domain is derived from the alpha or beta chain of the 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 (e.g., comprising at least the transmembrane region or a functional portion thereof).

[0066] In one embodiment, the CAR comprises a Tm domain derived from CD8α. In a particular aspect, the CAR comprises a Tm domain derived from CD8α and a short oligopeptide or polypeptide linker, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids in length, that connects the Tm domain and the intracellular signaling domain of the CAR. For example, a glycine-serine linker can be used as such a linker.

[0067] The "intracellular signaling" domain of a CAR refers to the portion of the CAR that is involved in transducing a signal from CAR binding to a target molecule / antigen inside an immune effector cell to induce effector cell function, e.g., activation, cytokine production, proliferation, and / or cytotoxic activity, including the release of cytotoxic factors into the CAR-bound target cell, or other cellular responses elicited by target molecule / antigen binding to the extracellular CAR domain. Thus, the term "intracellular signaling domain" refers to the portion of a protein that transduces an effector function signal and directs the cell to perform a specialized function. To the extent that a truncated portion of the intracellular signaling domain is used, such a truncated portion can be used in place of the full-length intracellular signaling domain, so long as it transmits the effector function signal. The term intracellular signaling domain is meant to include any truncated portion of the intracellular signaling domain sufficient to transduce an effector function signal.

[0068] Signals generated solely through the T cell receptor (TCR) are insufficient for full activation of T cells; secondary or costimulatory signals are also required. Thus, T cell activation is mediated by two distinct classes of intracellular signaling domains: primary signaling domains that initiate antigen-dependent primary activation via the TCR (e.g., the TCR / CD3 complex), and costimulatory signaling domains that act in an antigen-independent manner to provide secondary or costimulatory signals. Thus, the CARs of the present disclosure can comprise intracellular signaling domains that include one or more "costimulatory signaling domains" and "primary signaling domains."

[0069] The primary signaling domain regulates primary activation of the TCR complex in either a stimulatory or inhibitory manner. Primary signaling domains that act in a stimulatory manner may contain a signaling motif known as an immunoreceptor tyrosine-based activation motif (or "ITAM"). Non-limiting examples of ITAM-containing primary signaling domains suitable for use in the CARs of the present disclosure include those derived from FcRγ, FcRβ, CD3γ, CD3δ, CD3ε, CD3ζ, CD22, CD79α, CD79β, and CD66δ. In certain embodiments, the CAR comprises a CD3ζ primary signaling domain and one or more costimulatory signaling domains. The intracellular primary signaling and costimulatory signaling domains are operably linked to the carboxyl terminus of the transmembrane domain.

[0070] In some embodiments, a CAR comprises one or more costimulatory signaling domains to enhance the efficacy and expansion of immune effector cells (e.g., T cells) expressing the CAR. As used herein, the term "costimulatory signaling domain" or "costimulatory domain" refers to the intracellular signaling domain of a costimulatory molecule or an active fragment thereof. Exemplary costimulatory molecules suitable for use in CARs contemplated in specific embodiments 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 costimulatory signaling domains selected from the group consisting of 4-1BB (CD137), CD28, and CD134, and a CD3ζ primary signaling domain.

[0071] CARs can comprise any of a variety of suitable domains, including, but not limited to, a leader sequence; a hinge, spacer, and / or linker domain; a transmembrane domain; a costimulatory domain; a signaling domain (e.g., a CD3ζ domain); a ribosomal skipping element; a restriction enzyme sequence; a reporter protein domain, etc. Non-limiting examples of such domains that can be included in CARs of the present disclosure include those provided in Table 6 below. As will be understood by one of 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; a restriction enzyme sequence; a reporter protein, etc.) can be modified as desired, such as to improve the functionality of the CAR.

[0072] In certain aspects, a CAR of the present disclosure comprises a single-chain antibody that binds to human uPAR (e.g., any of the scFvs of the present disclosure); a transmembrane domain from a polypeptide selected from the group consisting of: CD4, CD8α, CD154, and PD-1; one or more intracellular costimulatory signaling domains from polypeptides selected from the group consisting of: 4-1BB (CD137), CD28, and CD134; and an intracellular signaling domain from a polypeptide selected from the group consisting of: FcRγ, FcRβ, CD3γ, CD3δ, CD3ε, CD3ζ, CD22, CD79α, CD79β, and CD66δ. Such a CAR may further comprise a spacer domain between the antigen-binding portion and the transmembrane domain, such as a CD8 alpha hinge.

[0073] According to some embodiments, the variable heavy chain (VH) of an antibody described herein comprises, from N-terminus to C-terminus, H ) polypeptide, linker, antibody variable light chain (V L ), a CD8 hinge region (which in some embodiments is an extended CD8 hinge region), a CD8 transmembrane domain, a 4-1BB costimulatory domain, and a CD3ζ signaling domain. According to certain embodiments, a CAR is provided that comprises, from N- to C-terminus, a variable light chain (V) of an antibody described herein. L ) polypeptide, linker, antibody variable heavy chain (V H ), a CD8 hinge region (which in some embodiments is an extended CD8 hinge region), a CD8 transmembrane domain, a 4-1BB costimulatory domain, and a CD3ζ signaling domain. In certain embodiments, a CAR is provided that comprises, from N- to C-terminus, a variable heavy chain (V) of an antibody described herein. H ) polypeptide, linker, antibody variable light chain (V L ), a CD28 hinge region, a CD28 transmembrane domain, a 4-1BB costimulatory domain, and a CD3ζ signaling domain. According to some embodiments, provided is a CAR comprising a variable light chain (V) from the N-terminus to the C-terminus of an antibody described herein. L ) polypeptide, linker, antibody variable heavy chain (V H), a CD28 hinge region, a CD28 transmembrane domain, a 4-1BB costimulatory domain, and a CD3ζ signaling domain. H The N-terminal domain of the polypeptide may be included. For example, a leader sequence (e.g., a GM-CSFR leader sequence) may be present at the N-terminus of a CAR of the present disclosure.

[0074] CARs of the present disclosure can comprise one or more additional domains as desired. Non-limiting examples of such additional domains include a ribosomal skipping element, an enzyme domain (e.g., a domain having nuclease activity, e.g., restriction endonuclease activity), a domain that allows for detection of the CAR (e.g., a reporter protein domain (e.g., a fluorescent protein (e.g., eGFP, mCherry, etc.), a luminescent protein, and / or the like), etc. For example, in certain embodiments, provided are CARs that comprise a ribosomal skipping element, a restriction enzyme domain, and / or a reporter protein domain.

[0075] 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 a CAR is provided by two or more polypeptides, the CAR can be in the form of a biotin-binding immune receptor (BBIR) (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), a switchable CAR format with a peptide neoepitope (PNE) (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 formats with leucine zippers (see, e.g., Cho et al. (2108) Cell 173(6):1426-1438.e11), CAR-T adapter molecule (CAM)-based formats with FITC formate (see, e.g., Lee et al. (2019) Cancer Res. 79(2):387-396, and Lu et al. al.(2019)Front Oncol.9:151), anti-FITC-formate 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 other universal CAR formats.

[0076] Conjugates The present disclosure also provides conjugates. According to some embodiments, the conjugates of the present disclosure comprise any of the antibodies or fusion proteins of the present disclosure and a drug conjugated to the antibody or fusion protein. The term "conjugated" generally refers to a chemical bond, either covalent or non-covalent, typically a covalent bond, that brings one molecule of interest into close proximity with a second molecule of interest. In certain embodiments, the drug conjugated to the antibody or fusion protein is a chemotherapeutic agent, a toxin, a radiosensitizing agent, a radioisotope (e.g., a therapeutic radioisotope), a detectable label, or a half-life extending moiety.

[0077] According to some embodiments, the agent is a therapeutic agent, e.g., a chemotherapeutic agent. As used herein, a "therapeutic agent" is a physiologically or pharmacologically active substance capable of producing a desired biological effect at a target site in an animal, such as a mammal or human. Therapeutic agents can be any inorganic or organic compound. Examples include, but are not limited to, peptides, proteins, nucleic acids (including siRNA, miRNA, and DNA), polymers, and small molecules. Therapeutic agents can reduce, suppress, attenuate, decrease, stop, or stabilize the onset or progression of a disease, disorder, or cell proliferation in an animal, such as a mammal or human. Therapeutic agents of interest include agents that can affect the function of a cell / tissue to which the conjugate is bound via specific binding of the antibody portion of the conjugate to an antigen. If the cell / tissue function is pathological, an agent that reduces the function of the cell / tissue can be used. In certain aspects, the conjugates of the present disclosure include agents that reduce the function of a target cell / tissue by inhibiting cell proliferation and / or killing the cell / tissue. Such agents may vary and may include cytostatic and cytotoxic agents, for example, agents capable of killing target cell tissues whether or not they are internalized into the target cells.

[0078] In certain embodiments, the therapeutic agent is a cytotoxic agent selected from an enediyne, a lexitropsin, a duocarmycin, a taxane, a puromycin, a dolastatin, a maytansinoid, and a vinca alkaloid. 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, combretastatin, calicheamicin, maytansine, maytansine DM1, maytansine DM4, DM-1, an 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 (monomethylauristatin E), MMAF (monomethylauristatin F), pyrrolobenzodiazepine (PBD), eleutherobin, netropsin, or any combination thereof.

[0079] According to some embodiments, the agent is a toxin, e.g., hemiasterlin and hemiasterlin analogs such as HTI-286 (see, e.g., USPN 7,579,323, WO 2004 / 026293, and USPN 8,129,407, the complete disclosures of which are incorporated herein by reference), abrin, brucine, cicutoxin, diphtheria toxin, batrachotoxin, botulinum toxin, shiga toxin, endotoxin, pseudomonas exotoxin, pseudomonas endotoxin, tetanus toxin, pertussis toxin, The protein toxin is selected from anthrax toxin, cholera toxin, falcarinol, fumonisin B1, fumonisin B2, aflatoxin, maurotoxin, agitoxin, charybdotoxin, margatoxin, slotoxin, scyllatoxin, hefutoxin, calciseptin, taicatoxin, calcicludin, geldanamycin, gelonin, lotaustralin, ochratoxin A, patulin, ricin, strychnine, trichothecene, zearlenone, and tetradotoxin. Enzymatically active toxins and fragments thereof that can be used include diphtheria A chain, nonbinding active fragments of diphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain, abrin A chain, modeccin A chain, alpha-sarcin, Aleurites fordii proteins, dianthin proteins, Phytolaca americana proteins (PAPI, PAPII, and PAP-S), Momordica charantia inhibitor, curcin, crotin, Sapaonaria officinalis inhibitor, gelonin, mitogenin, restrictocin, phenomycin, enomycin, and the trichothecenes.

[0080] In certain embodiments, the agent is a radiosensitizing agent. As used herein, a "radiosensitizing agent" is an agent that enhances the ability of radiation to kill tumor cells. Non-limiting examples of radiosensitizing agents that can be conjugated to an antibody or fusion protein include cisplatin, 5-fluorouracil (5-FU), AZD7762, selumetinib, and the like.

[0081] In certain embodiments, the agent is a radioisotope useful, for example, for therapy and / or detection (e.g., imaging). Non-limiting examples of radioisotopes that can be conjugated to an antibody or fusion protein include: 225 Ac, 111 Ag, 114 Ag, 71 As, 72 As, 77 As, 211 At, 198 Au, 199 Au, 212 Bi, 213 Bi, 75 Br, 76 Br, 11 C. 13 C. 55 Co, 62 Cu, 64 Cu, 67 Cu, 165 Dy, 166 Dy, 169 Er, 18 F, 19 F, 52 Fe, 59 Fe, 66 Ga, 67 Ga, 68 Ga, 72 Ga, 154-158 Gd, 157 Gd, 159 Gd, 166 Ho, 120 I, 121 I, 123 I, 124 I, 125 I, 131 I, 110 In, 111 In, 113m In, 194 Ir,81m Kr, 177 Lu, 51 Mn, 52 Mn, 99 Mo, 13 N, 15 N, 15 O. 17 O. 32 P, 33 P, 211 Pb, 212 Pb, 109 Pd, 149 Pm, 151 Pm, 142 Pr, 143 Pr, 191 PT, 193m PT, 195m Pt, 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 Examples include, but are not limited to, Zr.

[0082] In certain embodiments, a radioisotope is conjugated to an antibody or fusion protein via a chelator, e.g., a bifunctional chelator. The bifunctional chelator may contain a metal chelating moiety that binds the radioisotope in a stable coordination complex and a reactive functional group that is covalently attached to a targeting moiety, such as any of the antibodies or fusion proteins 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 an antibody or fusion protein of the present disclosure to a radioisotope include those described in Price & Orvig (2014) Chem. Soc. Rev. 43:260 and Brechbiel (2008) QJ Nucl Med Mol Imaging 52(2):166-173.

[0083] According to some embodiments, the radioisotope is a therapeutic radioisotope. In certain embodiments, the radioisotope is an alpha-emitting radioisotope, e.g., 225 Ac, 211 At, 212 Bi / 212 Pb, 213 Bi, 223 Ra, or 227 In other embodiments, the radioisotope is a beta-emitting radioisotope, e.g., 32 P, 33 P, 67 Cu, 90 Y, 131 I or 177 This is Lu.

[0084] According to some embodiments, the agent is a labeling agent. "Labeling agent" (or "detectable label") means that the agent detectably labels the antibody or fusion protein so that the antibody or fusion protein can be detected in the intended application (e.g., in vitro and / or in vivo research and / or clinical application). Detectable labels of interest include radioisotopes (e.g., gamma- or positron-emitters), enzymes that generate a detectable product (e.g., horseradish peroxidase, alkaline phosphatase, luciferase, etc.), fluorescent proteins, paramagnetic atoms, and the like. In certain aspects, the antibody or fusion protein is conjugated to a specific binding partner of the detectable label, e.g., conjugated to biotin, so that detection can occur via the detectable label, including avidin / streptavidin.

[0085] In certain embodiments, the agent is a labeling agent that finds use in in vivo imaging, such as, for example, 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. Labeling agents that find use in such applications include, but are not limited to, fluorescent labels, radioisotopes, etc. In certain aspects, the labeling agent is a multimodal in vivo imaging agent that enables in vivo imaging using two or more imaging techniques (see, for example, Thorp-Greenwood and Coogan (2011) Dalton Trans. 40: 6129-6143).

[0086] 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, Pz247, DyLight 750 and 800 Fluor, Cy5.5 and 7 Fluor, Alexa Fluor 680 and 750 dyes, IRDye 680 and 800CW Fluor. According to some embodiments, the labeling agent is an in vivo imaging agent that finds use in SPECT imaging applications, and non-limiting examples of in vivo imaging agents 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, e.g., 11 C. 13 N, 15 O. 18 F, 64 Cu, 62 Cu, 124 I, 76 Br, 82 Rb, 68 Ga, etc.

[0087] For half-life extension, the antibodies and fusion proteins of the present disclosure can be conjugated (e.g., by PEGylation, hyperglycosylation, etc.) to agents that result in improved pharmacokinetic profiles. Modifications that can improve serum half-life are of interest. The subject antibodies or fusion proteins can be "PEGylated" to contain one or more poly(ethylene glycol) (PEG) moieties. Methods and reagents suitable for 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 generally soluble in water at room temperature and have the general formula R(O-CH2-CH2). nThe PEG has the formula OR, where R is hydrogen or a protecting group such as an alkyl or alkanol group, and n is an integer between 1 and 1000. When R is a protecting group, it generally has 1 to 8 carbons. The PEG conjugated to the antibody or fusion protein of interest can be linear. The PEG conjugated to the antibody or fusion protein of interest can also be branched. Branched PEG derivatives include those described in U.S. Pat. No. 5,643,575, "star PEGs," and multi-armed PEGs. Star PEGs have been described in the art, including, for example, U.S. Pat. No. 6,046,305.

[0088] If the antibody or fusion protein of interest is isolated from a source, the antibody or fusion protein may be conjugated to one or more moieties that facilitate purification, such as a member of a specific binding pair, e.g., biotin (a member of the biotin-avidin specific binding pair), a lectin, etc. The antibody may also be bound (e.g., immobilized) to a solid support, including, but not limited to, a polystyrene plate or bead, a magnetic bead, a test strip, a membrane, etc.

[0089] If the antibody or fusion protein is to be detected in an assay, the antibody or fusion protein may be tagged with 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 metals, e.g., 152 These may include Eu or other fluorescent metals of the lanthanide series 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 luciferin; fluorescent proteins, etc. Indirect labels include antibodies specific for the subject protein, which can be detected via a secondary antibody; and a member of a specific binding pair, such as biotin-avidin.

[0090] Any of the above agents may be conjugated to the antibody or fusion protein via a linker. If present, the linker molecule may be long enough to allow some flexible movement between the antibody or fusion protein and the linking agent. The linker molecule may be, for example, about 6 to 50 atoms in length. The linker molecule may be, for example, an aryl acetylene, an ethylene glycol oligomer containing 2 to 10 monomer units, a diamine, a diacid, an amino acid, or a combination thereof.

[0091] When the linker is a peptide, the linker can be of any suitable length, such as from 1 amino acid (e.g., Gly) to 20 or more amino acids, from 2 amino acids to 15 amino acids, from 3 amino acids to 12 amino acids, from 4 amino acids to 10 amino acids, from 5 amino acids to 9 amino acids, from 6 amino acids to 8 amino acids, or from 7 amino acids to 8 amino acids, and can be 1, 2, 3, 4, 5, 6, or 7 amino acids in length.

[0092] The flexible linker is a glycine polymer (G) n Flexible linkers include 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 when relatively unstructured amino acids are desired and can function as neutral tethers between components. Those skilled in the art will recognize that the design of antibodies or fusion proteins conjugated to any of the above agents can include linkers that are all or partially flexible, such that the linker can include a flexible linker and one or more moieties that impart less flexible structure.

[0093] In some embodiments, the antibody or fusion protein is conjugated to the drug via a non-cleavable linker. Non-cleavable linkers of interest include, but are not limited to, thioether linkers. An example of a thioether linker that can be used is succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC) linker.

[0094] In certain embodiments, the antibody is conjugated to the drug via a cleavable linker. According to some embodiments, the linker is a chemically labile linker, such as an acid-cleavable linker, that is stable at neutral pH (bloodstream pH 7.3-7.5) but undergoes hydrolysis upon internalization into the mildly acidic endosomes (pH 5.0-6.5) and lysosomes (pH 4.5-5.0) of target cells (e.g., cancer cells). Chemically labile linkers include, but are not limited to, hydrazone-based linkers, oxime-based linkers, carbonate-based linkers, ester-based linkers, and the like. In certain embodiments, the linker is an enzyme-labile linker, e.g., a linker that is stable in the bloodstream but is enzyme-labile upon internalization into target cells, e.g., by lysosomal proteases (e.g., cathepsin or plasmin) in the lysosomes of target cells (e.g., cancer cells). Enzyme-labile linkers include, but are not limited to, dipeptide-based linkers such as valine-citrulline (VC) linkers, including peptide bond-containing linkers, such as maleimidocaproyl-valine-citrulline-p-aminobenzyl (MC-vc-PAB) linkers and valyl-alanyl-para-aminobenzyloxy (Val-Ala-PAB) linkers. Chemically labile linkers, enzyme-labile linkers, and non-cleavable linkers are known and are described in detail in, for example, Ducrry & 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.

[0095] Numerous strategies are available for linking a drug directly to an antibody or fusion protein or indirectly via a linker. For example, a drug may be derivatized by covalently attaching a linker to the drug, where 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 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 an unnatural amino acid bearing a chemical handle into the antibody or fusion protein. Unnatural amino acids that can be used to prepare the conjugates of the present disclosure include those bearing a functional group selected from azide, alkyne, alkene, aminooxy, hydrazine, aldehyde (e.g., formylglycine, e.g., SMARTag™ technology from Catalent Pharma Solutions), nitrone, nitrile oxide, cyclopropene, norbornene, isocyanide, aryl halide, and boronic acid functional groups. The unnatural amino acid can be selected to provide a desired functional group. Unnatural amino acids that can be incorporated into the antibodies of the conjugates of the present disclosure are known and are described, for example, in Maza et al. (2015) Bioconjug. Chem. 26(9):1884-9, Patterson et al. (2014) ACSChem. Biol. 9:592-605, Adumeau et al. (2016) Mol. Imaging Biol. (2):153-65, and elsewhere. Unnatural amino acids can be incorporated into antibodies or fusion proteins via chemical synthesis or recombinant approaches, for example, using a suitable orthogonal amino acid acyl-tRNA synthetase-tRNA pair to incorporate the unnatural amino acid during translation of the antibody or fusion protein in a host cell.

[0096] The functional group of the unnatural amino acid present in the antibody or fusion protein can be azide, alkyne, alkene, aminooxy, hydrazine, aldehyde, asaldehyde, nitrone, nitrile oxide, cyclopropene, norbornene, isocyanide, aryl halide, boronic acid, diazo, tetrazine, tetrazole, quadrocyclane, iodobenzene, or other suitable functional group, and the functional group on the linker is selected to react with the functional group of the unnatural amino acid (or vice versa). By way of example only, an azide-bearing unnatural amino acid (e.g., 5-azido-L-norvaline, etc.) can be incorporated into an antibody or fusion protein, and the linker moiety of the linker agent moiety can include an alkyne functional group such that the antibody or fusion protein and the linker agent moiety are covalently linked via azide-alkyne cycloaddition. Conjugation can be carried out, for example, using a copper-catalyzed azide-alkyne cycloaddition reaction.

[0097] In certain embodiments, the chemical handle of the antibody or fusion protein does not involve an unnatural amino acid. Antibodies that do not contain an unnatural amino acid can be conjugated to a drug by utilizing, for example, a nucleophilic functional group of the antibody or fusion protein (e.g., the N-terminal amine of lysine or a primary amine, or any other nucleophilic amino acid residue) as the nucleophile in a substitution reaction with a moiety bearing a reactive leaving group or other electrophilic group. An example includes preparing a drug-linker moiety with an N-hydroxysuccinimidyl (NHS) ester and reacting it with an added non-nucleophilic base, such as N,N-diisopropylethylamine, under aqueous conditions at high pH (about 10) or in a polar organic solvent such as DMSO.

[0098] It will be understood that the particular approach for linking the linker, drug, and / or antibody or fusion protein to one another may vary depending on the particular linker, drug, and / or antibody or fusion protein and functional groups selected and used to conjugate the various components to one another.

[0099] Methods for producing antibodies Using the information provided herein, the anti-uPAR antibodies and fusion proteins of the present disclosure can be prepared using standard techniques well known to those skilled in the art. For example, a nucleic acid sequence encoding the amino acid sequence of an antibody or fusion protein of the present disclosure can be used to express the antibody or fusion protein. The polypeptide sequences provided herein (see, for example, Table 1) can be used to determine appropriate nucleic acid sequences encoding antibodies or fusion proteins, and the nucleic acid sequences can then be used to express one or more antibodies or fusion proteins specific to human uPAR. Nucleic acid sequences can be optimized to reflect particular codon "preferences" for 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 several standard methods known to those skilled in the art.

[0100] Once nucleic acid encoding a subject antibody is synthesized, it may be amplified and / or cloned according to standard methods. Molecular cloning techniques to achieve these ends are known in the art. A wide variety of cloning and in vitro amplification methods suitable for the construction of recombinant nucleic acids are known to those of skill in the art and are the subject of numerous textbooks and laboratory manuals.

[0101] Expression of natural or synthetic nucleic acids encoding the antibodies and fusion proteins of the present disclosure can be achieved by operably linking the nucleic acid encoding the antibody or fusion protein to a promoter (either constitutive or inducible) and incorporating the construct into an expression vector to generate a recombinant expression vector. The vector may be suitable for replication and integration in prokaryotes, eukaryotes, or both. A typical cloning vector contains functionally appropriately oriented transcription and translation terminators, initiation sequences, and a promoter useful for regulating expression of the antibody-encoding nucleic acid. The vector optionally contains a generic expression cassette containing at least one independent terminator sequence, e.g., a sequence that allows replication of the cassette in both eukaryotes and prokaryotes, as found in shuttle vectors, and a selectable marker for both prokaryotic and eukaryotic systems.

[0102] To obtain high levels of expression of cloned nucleic acids, it is common to construct expression plasmids that typically contain a strong promoter to direct transcription, a ribosome binding site for translation initiation, and a transcription / translation terminator, each in functional orientation relative 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 tryptophan biosynthetic pathway of E. coli, the promoter and operator regions of the phage lambda (P L ), and the L-arabinose (araBAD) operon. It is also useful to include a selectable marker in a DNA vector transformed in E. coli. Examples of such markers include genes specifying resistance to ampicillin, tetracycline, or chloramphenicol. Expression systems for expressing antibodies are available using, for example, E. coli, Bacillus sp., and Salmonella. E. coli systems can also be used.

[0103] Antibody genes can also be subcloned into expression vectors that allow for the addition of tags (e.g., FLAG, hexahistidine, etc.) to the C- or N-terminus of the antibody (e.g., IgG, Fab, scFv, etc.) to facilitate 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 microparticles containing the nucleic acid with the cells, or incubating a viral vector containing the nucleic acid with cells within the vector's host range. Culturing cells for use in the present disclosure, including cell lines and cultured cells from tissue (e.g., tumor) or blood samples, is well known in the art.

[0104] Once nucleic acid encoding a subject antibody has been isolated and cloned, it can be expressed in a variety of recombinantly engineered cells known to those of skill in the art, including bacterial, yeast, filamentous fungi, insect (e.g., using baculovirus vectors), and mammalian cells.

[0105] Isolation and purification of the subject antibodies can be achieved according to methods known in the art. For example, the protein can be isolated from lysates of cells genetically modified to express the protein constitutively and / or upon induction, or from synthesis reaction mixtures, by immunoaffinity purification (or precipitation using protein L or A), which involves washing to remove nonspecifically bound material and eluting the specifically bound protein. The isolated protein can be further purified by dialysis and other methods commonly used in protein purification methods. In one embodiment, the antibody can be isolated using metal chelate chromatography. The antibodies of the present disclosure may contain modifications to facilitate isolation, as discussed above.

[0106] The antibody may be prepared in a substantially pure or isolated form (e.g., free from other polypeptides). The protein may 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 may be provided such that the antibody is present in a composition that is substantially free of other expressed proteins, e.g., a composition where less than 90%, usually less than 60%, and more usually less than 50% of the composition is made up of other expressed proteins.

[0107] Antibodies produced by prokaryotic cells may require exposure to a lyotropic agent for proper folding. For example, during purification from E. coli, the expressed protein can optionally be denatured and then renatured. This can be accomplished, for example, by solubilizing bacterially produced antibodies in a lyotropic agent such as guanidine HCl. The antibody is then renatured by either slow dialysis or gel filtration. Alternatively, the nucleic acid encoding the antibody can be operably linked to a secretory signal sequence, such as pelB, so that the antibody is secreted into the periplasm in a correctly folded form.

[0108] The present disclosure also provides cells that produce the antibodies of the present disclosure, where suitable cells include eukaryotic cells, e.g., mammalian cells. The cells may be hybrid cells or "hybridomas" that are capable of reproducing antibodies (e.g., monoclonal antibodies such as IgG) in vitro. For example, the present disclosure provides recombinant host cells (also referred to herein as "genetically modified host cells") that have been genetically modified with one or more nucleic acids comprising nucleotide sequences encoding the heavy and / or light chains of an antibody of the present disclosure.

[0109] Techniques for making recombinant DNA versions of the antigen-binding region of an antibody molecule that bypass hybridoma generation are also contemplated herein. The DNA is cloned into, for example, bacterial (e.g., bacteriophage), yeast (e.g., Saccharomyces or Pichia), insect, or mammalian expression systems. One suitable technique uses a bacteriophage lambda vector system with a leader sequence that directs the expressed antibody (e.g., Fab or scFv) to the periplasmic space (between the bacterial cell membrane and cell wall) or to be secreted. Large numbers of functional fragments (e.g., Fab or scFv) that bind to the antigen of interest can be rapidly produced.

[0110] Antibodies can be prepared using a variety of techniques known in the art, including the use of hybridoma, recombinant, phage display technology, selected lymphocyte antibody method (SLAM), or a combination thereof. For example, antibodies can be generated and isolated using phage display methods. Phage display is used for high-throughput screening of protein interactions. Phage can be utilized to display antigen-binding domains expressed from repertoire or combinatorial antibody libraries (e.g., human or mouse). Phage expressing antigen-binding domains that bind to human uPAR can be selected or identified, for example, using labeled human uPAR bound or captured to a solid surface or bead. Phage used in these methods are typically filamentous phage containing fd and M13 binding domains expressed from phages bearing Fab, Fv (individual Fv regions from the light or heavy chain), or disulfide-stabilized Fv antibody domains recombinantly fused to either the phage gene III or gene VIII protein. The production of high-affinity human antibodies by chain shuffling is known as a strategy for constructing large phage libraries, as is combinatorial infection and in vivo recombination. In another embodiment, ribosome display can be used to replace bacteriophage as the display platform. Cell surface libraries may also be screened for antibodies. Such procedures provide an alternative to traditional hybridoma technology for the isolation and subsequent cloning of monoclonal antibodies.

[0111] After phage selection, the antibody coding region from the phage can be isolated and used to generate whole antibodies, including human antibodies, or any desired antigen-binding fragment, which can be 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.

[0112] Nucleic acids, expression vectors and cells In view of the section above regarding methods for producing the antibodies and fusion proteins of the present disclosure, it will be understood that the present disclosure also provides nucleic acids, expression vectors and cells.

[0113] In certain embodiments, provided are variable heavy chains (V H ) polypeptide, variable light chain (V L ) polypeptide, or both, including any of the anti-human uPAR antibodies of the present disclosure, e.g., any of such antibodies described above. According to some embodiments, the antibody is a single-chain antibody (e.g., scFv), and the nucleic acid encodes the single-chain antibody.

[0114] According to some embodiments, provided is a nucleic acid encoding a CAR of the present disclosure, e.g., a V of an anti-human uPAR antibody of the present disclosure. H Polypeptides and V L The CAR comprises a single-chain antibody comprising 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.

[0115] Also provided are expression vectors containing any of the nucleic acids of the present disclosure. Expression of natural or synthetic nucleic acids encoding the antibodies and fusion proteins of the present disclosure can be achieved by operably linking the nucleic acid encoding the antibody or fusion protein to a promoter (either constitutive or inducible) and incorporating the construct into an expression vector to generate a recombinant expression vector. The vector may be suitable for replication and integration in prokaryotes, eukaryotes, or both. A typical cloning vector contains appropriately oriented transcription and translation terminators, initiation sequences, and promoters useful for regulating the expression of the antibody-encoding nucleic acid. The vector optionally contains a generic expression cassette containing at least one independent terminator sequence, e.g., a sequence that allows replication of the cassette in both eukaryotes and prokaryotes, as found in shuttle vectors, and a selectable marker for both prokaryotic and eukaryotic systems.

[0116] Cells comprising any of the nucleic acids and / or expression vectors of the present disclosure are also provided. According to some embodiments, the cells of the present disclosure comprise the V H V for polypeptides and antibodies L In certain 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, provided is a variable heavy chain (VH) of the antibody of the present disclosure. H a first nucleic acid encoding a variable light chain (V) polypeptide of the antibody; L and a second nucleic acid encoding the polypeptide. In certain embodiments, such cells comprise a first expression vector comprising the first nucleic acid and a second expression vector comprising the second nucleic acid.

[0117] Also provided is a method for producing an antibody or fusion protein of the present disclosure, comprising culturing a cell of the present disclosure under conditions suitable for the cell to express the antibody or fusion protein, thereby producing the antibody or fusion protein. The conditions for culturing the cells to express the antibody or fusion protein can vary. Such conditions can include culturing the cells in a suitable container (e.g., a cell culture plate or well thereof) in a suitable medium (e.g., a cell culture medium such as DMEM, RPMI, MEM, IMDM, or DMEM / F-12) at a suitable temperature (e.g., 32°C to 42°C, e.g., 37°C) and pH (e.g., pH 7.0 to 7.7, e.g., pH 7.4) in an environment with a suitable percentage of CO2 (e.g., 3% to 10%, e.g., 5%).

[0118] composition As summarized above, aspects of the present disclosure also provide compositions. According to some embodiments, the compositions of the present disclosure comprise an antibody, fusion protein, or conjugate of the present disclosure. For example, the antibody, fusion protein, or conjugate may be any of the antibodies, fusion proteins, or conjugates described in the antibody section above, the descriptions of which are incorporated herein for brevity but will not be repeated.

[0119] In certain aspects, compositions of the present disclosure comprise an antibody, fusion protein, or conjugate present in a liquid medium, which may be an aqueous liquid medium such as water, a buffer solution, or the like. One or more additives, such as salts (e.g., NaCl, MgCl, KCl, MgSO), 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), and the like), solubilizers, detergents (e.g., non-ionic detergents, such as Tween-20), nuclease inhibitors, protease inhibitors, glycerol, chelating agents, and the like, may be present in such compositions.

[0120] Aspects of the present disclosure further include pharmaceutical compositions. In some embodiments, the pharmaceutical compositions of the present disclosure comprise an anti-human uPAR antibody (or a conjugate or fusion protein comprising same) of the present disclosure and a pharmaceutically acceptable carrier.

[0121] The antibody, fusion protein, or conjugate can be incorporated into various formulations for therapeutic administration. More specifically, the antibody, fusion protein, or conjugate can be formulated into a pharmaceutical composition by combining with a suitable pharmaceutically acceptable excipient or diluent, and can be formulated into preparations in solid, semi-solid, liquid, or gaseous form, such as tablets, capsules, powders, granules, ointments, solutions, injections, inhalants, and aerosols.

[0122] Formulations of antibodies, fusion proteins, or conjugates for administration to an individual (e.g., those suitable for human administration) will generally be sterile and may further be free of detectable pyrogens or other contaminants that would contraindicate their administration to a patient according to the selected route of administration.

[0123] In pharmaceutical dosage forms, the antibodies, fusion proteins, or conjugates can be administered in the form of their pharmaceutically acceptable salts, or the antibodies, fusion proteins, or conjugates can be used alone or in suitable associations, as well as in combination with other pharmaceutically active compounds. The following methods and carriers / excipients are merely examples and are in no way limiting.

[0124] For oral preparations, the antibody, fusion protein, or conjugate may be used alone or in combination with suitable additives to form 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, acacia, corn starch, or gelatin; with disintegrating agents such as corn starch, potato starch, or sodium carboxymethylcellulose; with lubricants such as talc or magnesium stearate; and, as needed, with diluents, buffers, wetting agents, preservatives, and flavoring agents.

[0125] The antibody, fusion protein, or conjugate can be formulated for parenteral (e.g., intravenous, intraarterial, intraosseous, intramuscular, intracerebral, intraventricular, 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 or non-aqueous solvent such as vegetable or other similar oils, synthetic fatty acid glycerides, esters of higher fatty acids, or propylene glycol, along with conventional additives such as solubilizers, isotonic agents, suspending agents, emulsifiers, stabilizers, and preservatives, as needed.

[0126] Pharmaceutical compositions containing antibodies, fusion proteins, or conjugates can be prepared by mixing the antibody, fusion protein, or conjugate having the desired degree of purity with optional physiologically acceptable carriers, excipients, stabilizers, surfactants, buffers, and / or tonicity agents. Acceptable carriers, excipients, and / or stabilizers are non-toxic to recipients at the dosages and concentrations employed, and include buffers such as phosphate, citric acid, and other organic acids; antioxidants including ascorbic acid, glutathione, cysteine, methionine, and citric acid; preservatives (e.g., ethanol, benzyl alcohol, phenol, m-cresol, p-chloro-m-cresol, methyl or propyl paraben, benzalkonium chloride, or combinations thereof); arginine, glycine, ornithine, lysine, histidine, glutamic acid, aspartic acid, isoleucine, le ... amino acids such as sucrose, 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, sorbose, raffinose, glucosamine, N-methylglucosamine, galactosamine, and neuraminic acid; and / or non-ionic surfactants such as Tween, Brij Pluronics, Triton-X, or polyethylene glycol (PEG).

[0127] The pharmaceutical composition may be in liquid form, lyophilized form, or liquid form reconstituted from lyophilized form, and the lyophilized preparation should be reconstituted with a sterile solution before administration. The standard procedure for reconstituting a lyophilized composition is to add back a volume of pure water (typically equal to the volume removed during lyophilization), but solutions containing antimicrobial agents can be used to produce pharmaceutical compositions for parenteral administration.

[0128] Aqueous formulations of antibodies, fusion proteins, or conjugates may be prepared in a pH buffer solution, for example, at a pH ranging from about 4.0 to about 7.0, or from about 5.0 to about 6.0, or alternatively at about 5.5. Examples of buffers suitable for a pH within this range include phosphate buffer, histidine buffer, citrate buffer, succinate buffer, acetate buffer, and other organic acid buffers. The buffer concentration may be, for example, from about 1 mM to about 100 mM, or from about 5 mM to about 50 mM, depending on the buffer and the desired tonicity of the formulation.

[0129] A tonicity agent may be included to adjust the tonicity of the formulation. Exemplary tonicity agents include sodium chloride, potassium chloride, glycerin, 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 that has the same tonicity as some other solution to which it is compared, such as physiological salt 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.

[0130] Surfactants may also be added to the formulation to reduce aggregation in the formulation, minimize the formation of fine particles, and / or reduce adsorption. Exemplary surfactants include polyoxyethylene sorbitan fatty acid esters (Tween), polyoxyethylene alkyl ethers (Brij), alkylphenyl polyoxyethylene ethers (Triton-X), polyoxyethylene-polyoxypropylene copolymers (Poloxamer, Pluronic), and sodium dodecyl sulfate (SDS). Examples of suitable polyoxyethylene sorbitan fatty acid esters are polysorbate 20 (sold under the trademark Tween 20™) and polysorbate 80 (sold under the trademark Tween 80™). Examples of suitable polyethylene-polypropylene copolymers are those sold under the names Pluronic® F68 or Poloxamer 188™. Examples of suitable polyoxyethylene alkyl ethers are those sold under the trademark Brij™. Exemplary concentrations of surfactants can range from about 0.001% to about 1% w / v.

[0131] Cryoprotectants may also be added to protect the antibody, fusion protein, or conjugate against destabilizing conditions during the lyophilization process. For example, known cryoprotectants include sugars (including glucose and sucrose), polyols (including mannitol, sorbitol, and glycerol), and amino acids (including alanine, glycine, and glutamic acid). Cryoprotectants may be included in an amount of, for example, about 10 mM to 500 nM.

[0132] In some embodiments, the pharmaceutical composition comprises an antibody, fusion protein, or conjugate and one or more of the above-identified compositions (e.g., surfactant, buffer, stabilizer, tonicity agent), and is essentially 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, a preservative is included in the formulation, e.g., at a concentration ranging from about 0.001 to about 2% (w / v).

[0133] kit Aspects of the present disclosure further include kits, hi certain embodiments, the kits are used to practice 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.

[0134] Thus, in certain embodiments, kits of the present disclosure include any of the pharmaceutical compositions of the present disclosure and instructions for administering the pharmaceutical composition to a subject in need thereof. The pharmaceutical composition included in the kit may include any of the antibodies, fusion proteins, and / or conjugates of the present disclosure, such as any of the antibodies, fusion proteins, and / or conjugates described above. As will be appreciated, kits of the present disclosure may include any of the agents and features described above in the sections relating to the subject antibodies, fusion proteins, conjugates, and compositions, which will not be repeated here for brevity.

[0135] Kits of the present disclosure may include a quantity of a composition present in a unit dose, e.g., ampoule, or multi-dose format. Thus, in certain embodiments, a kit may include one or more (e.g., two or more) unit doses (e.g., ampoules) of a composition comprising an antibody, fusion protein, and / or conjugate of the present disclosure. The term "unit dose," as used herein, refers to a physically discrete unit suitable as a unit dose for human and animal subjects, each unit containing a predetermined amount of a composition calculated to be sufficient to produce a desired effect. The amount of a unit dose depends on various factors, such as the specific antibody, fusion protein, and / or conjugate used, the effect to be achieved, and the pharmacodynamics associated with the antibody, fusion protein, and / or conjugate, in an individual. In yet other embodiments, a kit may include a single multi-dose amount of a composition.

[0136] The instructions included in the kit (e.g., instructions for use (IFU)) may be recorded on a suitable recording medium. For example, the instructions may be printed on a substrate such as paper or plastic. Thus, the instructions may be present in the kit as a package insert, on labeling of a container of the kit or a component thereof (i.e., associated with the packaging or subpackaging), etc. In other embodiments, the instructions are present as an electronic storage data file present on a suitable computer-readable storage medium, e.g., a portable flash drive, DVD, CD-ROM, diskette, etc. In still other embodiments, the actual instructions are not present in the kit, but means for obtaining the instructions from a remote source, e.g., via the Internet, are provided. An example of this embodiment is a kit that includes a web address where the instructions can be viewed and / or from which the instructions can be downloaded. Like the instructions, the means for obtaining the instructions is recorded on a suitable substrate.

[0137] How to use Aspects of the present disclosure further include methods of using the antibodies, fusion proteins (e.g., CARs), and conjugates of the present disclosure, which are useful in a variety of contexts, including in vitro and / or in vivo research and / or clinical applications.

[0138] In certain embodiments, provided is a method 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., a CAR), or conjugate of the present disclosure.

[0139] According to some embodiments, the condition associated with uPAR expression and / or activity is cancer. The subject methods can be used to treat a wide variety of cancers. As used herein, "tumor" refers to all neoplastic cell growth and proliferation, whether malignant or benign, and all pre-cancerous and cancerous cells and tissues. The terms "cancer" and "cancerous" refer to or describe a physiological condition in a mammal that is typically characterized by uncontrolled cell growth / proliferation. According to some embodiments, cancer is characterized by cancer cells that express uPAR on their surface. In particular embodiments, cancer comprises a solid tumor. According to some embodiments, the solid tumor is a carcinoma, lymphoma, blastoma, or sarcoma. In some embodiments, when the cancer comprises a solid tumor, the cancer is characterized by stromal cells within the tumor microenvironment that express uPAR on their surface.

[0140] Examples of cancers that can be treated using the subject methods include, but are not limited to, carcinoma, lymphoma, blastoma, and sarcoma. 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 or uterine cancer, salivary gland cancer, kidney cancer, prostate cancer, vulvar cancer, thyroid cancer, liver cancer, various types of head and neck cancer, and the like. In certain embodiments, the individual has a cancer selected from a solid tumor, 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 squamous cell carcinoma of the head and neck, 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.

[0141] In certain embodiments, the cancer comprises a hematological malignancy. Non-limiting examples of hematological malignancies include leukemia, lymphoma, and multiple myeloma.

[0142] According to some embodiments, provided is 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, the method comprising administering to the subject an effective amount of a composition comprising an antibody, fusion protein (e.g., a CAR), or conjugate of the present disclosure.

[0143] The antibodies, fusion proteins, and conjugates of the disclosure may be administered via any suitable route of administration, for example, orally (e.g., in tablet form, capsule form, liquid form, etc.), parenterally (e.g., by intravenous, intraarterial, subcutaneous, intramuscular, or epidural injection), topically, intranasally, intratumorally, etc.

[0144] The antibodies, fusion proteins, and conjugates of the present disclosure can be administered in compositions in therapeutically effective amounts. By "therapeutically effective amount" is meant a dosage sufficient to produce a desired result, e.g., an amount sufficient to produce a beneficial or desired therapeutic (including prophylactic) result, such as a reduction in cancer symptoms compared to a control. With respect to cancer, in some embodiments, a therapeutically effective amount is sufficient to slow tumor growth, reduce tumor size, and / or the like. An effective amount can be administered in one or more administrations.

[0145] As described above, the present disclosure includes a method for treating cancer in an individual. Treatment refers to at least an improvement in one or more symptoms associated with cancer in an individual, and improvement is used broadly to refer to at least a reduction in the parameters associated with the cancer being treated, such as the severity of the symptoms. Thus, treatment also includes a situation in which cancer or at least one or more symptoms associated therewith are completely inhibited, e.g., prevented from occurring, or stopped, e.g., terminated, so that the individual no longer suffers from cancer, or at least the symptoms that characterize cancer.

[0146] The antibody, fusion protein, or conjugate of the present disclosure can be administered to an individual alone or in combination with a second agent. Second agents of interest include, but are not limited to, agents approved by the U.S. Food and Drug Administration and / or the European Medicines Agency (EMA) for use in the treatment of cancer. In some embodiments, the second agent is an immune checkpoint inhibitor. Immune checkpoint inhibitors of interest include, but are 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 domain and mucin domain 3 (TIM-3) inhibitors, indoleamine (2,3)-dioxygenase (IDO) inhibitors, T-cell immunoreceptor with Ig and ITIM domains (TIGIT) inhibitors, V-domain Ig suppressor of T-cell activation (VISTA) inhibitors, B7-H3 inhibitors, and any combination thereof.

[0147] When an antibody, fusion protein, or conjugate of the present disclosure is administered together with a second agent, the antibody, fusion protein, or conjugate and the second agent may be administered to an individual according to any suitable dosing regimen. According to certain embodiments, the antibody, fusion protein, or conjugate and the second agent are administered according to a dosing regimen approved for their respective uses. In some embodiments, administration of the antibody, fusion protein, or conjugate allows the second agent to be administered according to a dosing regimen involving one or more lower and / or less frequent doses and / or fewer cycles than would be utilized when the second agent were administered without the antibody, fusion protein, or conjugate. In certain aspects, administration of the second agent allows the antibody, fusion protein, or conjugate to be administered according to a dosing regimen involving one or more lower and / or less frequent doses and / or fewer cycles than would be utilized when the antibody, fusion protein, or conjugate were administered without the second agent.

[0148] In some embodiments, one or more doses of the antibody, fusion protein, or conjugate and the second agent are administered to an individual simultaneously. By "simultaneously," we mean either 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 within 1 hour, 30 minutes, or 15 minutes of each other in separate pharmaceutical compositions.

[0149] In some embodiments, one or more doses of the antibody, fusion protein, or conjugate and the second agent are administered sequentially to the individual.

[0150] In some embodiments, the antibody, fusion protein, or conjugate and the second agent are administered to an individual in different compositions and / or at different times. For example, the antibody, fusion protein, or conjugate may be administered before the administration of the second agent, e.g., in a particular cycle. Alternatively, the second agent may be administered before the administration of the antibody, fusion protein, or conjugate, e.g., in a particular cycle. The second agent may be administered for a period starting at least 1 hour, 3 hours, 6 hours, 12 hours, 24 hours, 48 ​​hours, 72 hours, or up to 5 days or more after the administration of the first agent.

[0151] In one example, the second agent is administered to an individual for a desired period of time prior to administration of the antibody, fusion protein, or conjugate. In certain embodiments, such a regimen "primes" the cancer cells to enhance the anti-cancer effect of the antibody, fusion protein, or conjugate. Such a period separating the step of administering the second agent from the step of administering the antibody, fusion protein, or conjugate is long enough to allow priming of the cancer cells such that the anti-cancer effect of the antibody, fusion protein, or conjugate is increased.

[0152] In some embodiments, the administration of one agent is specifically timed relative to the administration of another agent, e.g., in some embodiments, an antibody, fusion protein, or conjugate is administered such that a particular effect is observed (or expected to be observed, e.g., based on population studies showing a correlation between a given dosing regimen and a particular desired effect).

[0153] In certain aspects, the desired relative dosing regimen of co-administered agents may be empirically evaluated or determined, 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., so that a correlation is established), or alternatively, in an individual of particular interest.

[0154] In some embodiments, the antibody, fusion protein, or conjugate and the second agent are administered according to an intermittent dosing regimen comprising at least two cycles. When two or more agents are administered in combination and each is administered according to such an intermittent cycling regimen, the individual doses of the different agents may be interspersed with one another. In certain aspects, one or more doses of the second agent are administered a fixed period after a dose of the first agent. In some embodiments, each dose of the second agent is administered a fixed period after a dose of the first agent. In certain aspects, each dose of the first agent is followed by a dose of the second agent a fixed period after the first 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, and in certain 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, and in some embodiments, the time intervals between different doses of the same agent vary. In certain aspects, the different doses of the antibody, fusion protein, or conjugate and the second agent are separated from each other by a common time interval, and in some embodiments, the different doses of the different agents are separated from each other by different time intervals.

[0155] One exemplary protocol for crossing two intermittent cyclic dosing regimens may include: (a) a first dosing period during which a therapeutically effective amount of an antibody, fusion protein, or conjugate is administered to the individual, (b) a first rest period, (c) a second dosing period during which a therapeutically effective amount of a second agent is administered to the individual, and (d) a second rest period. A second exemplary protocol for crossing two intermittent cyclic dosing regimens may include: (a) a first dosing period during which a therapeutically effective amount of a second agent is administered to the 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 the individual, and (d) a second rest period.

[0156] In some embodiments, the first rest period and the second rest period may correspond to the same number of hours or days. Alternatively, in some embodiments, the first rest period and the second rest period are different, with the first rest period being longer than the second rest period, or vice versa. In some embodiments, each of the rest 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 rest period is longer than the first rest period, it may be defined in terms of days or weeks (e.g., 1 day, 3 days, 5 days, 1 week, 2 weeks, 4 weeks, or more) rather than hours.

[0157] If the length of the first resting period is determined by the presence or development of a specific biological or therapeutic event, the length of the second resting period may be determined based on different factors, either separately or in combination. Exemplary such factors may include the type and / or stage of the cancer to which the therapy is administered, the characteristics (e.g., pharmacokinetic characteristics) of the antibody, fusion protein, or conjugate, and / or one or more characteristics of the patient's response to antibody, fusion protein, or conjugate therapy. In some embodiments, the length of one or both resting periods may be adjusted in light of the pharmacokinetic characteristics (e.g., as assessed via plasma concentration levels) of one or other of the administered agents. For example, the relevant resting period may optionally be considered complete when the plasma concentration of the relevant agent is below a predetermined level, based on an evaluation of one or more characteristics of the individual's response or other considerations.

[0158] In certain aspects, the number of cycles over which a particular agent is administered may be empirically determined, and in some embodiments, the exact regimen followed (e.g., number of doses, spacing of doses (e.g., relative to each other or to another event, such as the administration of another therapy), amount of dose, etc.) may be different for one or more cycles compared to one or more other cycles.

[0159] The antibody, fusion protein, or conjugate and the second agent may be administered together or independently via any suitable route of administration. The antibody, fusion protein, or conjugate and the second agent may be administered via a route of administration independently selected from oral, parenteral (e.g., by intravenous, intraarterial, subcutaneous, intramuscular, or epidural injection), topical, intranasal, intratumoral administration, etc. According to certain embodiments, both the antibody, fusion protein, or conjugate and the second agent are administered simultaneously (in the same pharmaceutical composition or separate pharmaceutical compositions) or sequentially orally or parenterally (e.g., in tablet, capsule, liquid form, etc.). [Example]

[0160] The following examples are offered by way of illustration and not by way of limitation.

[0161] experiment Based on the important role of uPAR as an anti-cancer target, therapeutic antibodies targeting uPAR for anti-cancer therapy are being developed. Fully human rAbs, 2G10 and 3C6 (27), were identified from a human naive Fab library using phage display technology and shown to be effective against human TNBC cells in a xenograft model (17). The anti-tumor efficacy of antibodies was increased with either therapeutic radionuclides or antibody-drug conjugates (ADCs) (17, 24, 27). However, their lack of cross-reactivity limited their progress as clinical candidates. In the therapeutic antibody development process, accurate prediction of the antibody's human pharmacokinetics, toxicity, and efficacy before initial in-human studies is fundamental for its development as an effective biological therapeutic (28). Cynomolgus monkeys (cynomolgus monkeys), genetically similar to humans compared to other species, are the most relevant non-human primate model for conducting preclinical studies in antibody drug development (29).

[0162] Described herein is the establishment of an accelerated discovery approach for the development of novel human and cynomolgus monkey cross-reactive rAbs by screening uPAR-primed mouse B lymphocytes using a microfluidic platform and photoelectric tweezers. Unique cross-reactive rAbs were shown to exhibit antibody-dependent cellular cytotoxicity (ADCC), ADC cytotoxicity, and inhibitory effects on cell adhesion to human breast cancer cells. Furthermore, lead antibodies demonstrated therapeutic efficacy in reducing tumor growth in an orthotopic animal model of human breast cancer, providing promising rAb candidates. Finally, binding models of lead antibodies are provided, demonstrating their binding epitopes that confer unique activity against uPAR.

[0163] Example 1 - High-throughput B cell screening for human and cynomolgus uPAR cross-reactive antibodies Swiss Jimenez-Lambert (SJL / J) mice (n = 8) were subjected to a 60-day immunization campaign using a recombinant soluble form of human uPAR (suPAR) lacking a GPI anchor as the immunogen, with a 7-day buffer between bleeds and boosts (Figure 1A). To reduce nonspecific febrile responses in immunized animals, suPAR was prepared by endotoxin removal and further characterized using SDS-PAGE, immunoblotting, and LC-MS / MS (Figures 8A-8B). Immunized mice were monitored by biweekly blood sampling, and their antibody titers were subsequently determined. Antiserum binding curves showed an increase in the production of anti-uPAR antibodies within the first week of immunization. Sustained antibody production was observed with 1 × 10 of mouse antisera. 7 Dilution-saturating antibody titers were maintained throughout the campaign (Figure 9). Once maturation of uPAR-primed plasma B cells was confirmed, spleens and bone marrow were harvested from each animal and purified using magnetic beads and flow-assisted cell sorting to identify CD45R(B220) B cells. - / CD138 高 This enabled the isolation of antibody-secreting cells (ASCs).

[0164] To screen and select cross-reactive antibodies against human and cynomolgus monkey uPAR, we performed high-throughput optical fluid screening of single B cells using the Beacon™ platform (30). In vivo antibody development relies on the maturation and selection of ASCs, providing high specificity for antibodies against their targets with low off-target binding to other host proteins. The Beacon platform enabled the screening and selection of thousands of B cells from immunized animals, thus accelerating the antibody discovery process (31). A total of 49,127 mouse ASCs were imported into a NanoPen on an OptoSelect™ 3500 chip and screened against human, mouse, and cynomolgus monkey uPAR. Overall, 217 binders against human uPAR were identified, 80 of which were cross-reactive to cynomolgus monkey uPAR, and none of the cells were able to produce reactive binders against mouse uPAR (Figure 1B–D). Interestingly, 8 ASCs produced binders specific for cynomolgus uPAR, and 137 ASCs were specific for human uPAR.

[0165] Example 2 - VH / VL sequencing, cloning, and recombinant IgG expression A total of 217 individual mouse B cells were exported from Beacon, and 80 VH and VL sequence pairs from cross-reactive binders to human and cynomolgus uPAR were covered using a rapid amplification of cDNA ends (RACE) protocol (32). A total of 64 clones showed amplicons within 500–700 bp with a 78% recovery rate. These amplicons were sequenced using next-generation sequencing (NGS), resulting in 60 unique VH and VL sequence pairs with 94% sequence recovery and 100% diversity.

[0166] 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). To transfer this effector function to anti-uPAR antibodies, 60 unique mouse VH / VL sequences were linked to the Herceptin IgG1 constant region to generate rAbs in a chimeric antibody format. From the recovered pool, 44 initial antibodies were successfully recombinantly expressed.

[0167] Example 3 - Cell surface uPAR recognition and binding affinity of antibody candidates Antibodies generated by immunizing animals with suPAR lacking the cell surface anchor motif can target protein regions inaccessible to membrane-bound uPAR. Nevertheless, effective targeting of cell surface receptors benefits from recognizing both solvent-exposed epitopes and the native conformational state displayed on the cell surface (36). Therefore, FACS was applied to evaluate cell surface uPAR recognition by each antibody candidate under different concentrations of MDA-MB-231 cells, a triple-negative breast cancer cell line with high uPAR expression (Figure 2). From the 44 initial antibodies, 12 lead candidates recognized uPAR displayed in breast cancer cells in a dose-dependent manner, reaching half-maximal effective binding concentrations (EC 50 ) values ​​ranged from 0.39 to 7.6 nM (Figure 2). All candidates were benchmarked against 2G10 and 3C6 and shown to be more potent in recognizing cell surface uPAR as demonstrated by their lower EC50 values, which are consistent with Herceptin binding to HER2 (EC 50 = 3.6 nM) (Table 2) (37, 38). [Table 2]

[0168] The binding affinities of the lead candidates to human uPAR were further characterized using biolayer interferometry (BLI). All lead candidates exhibited equilibrium dissociation constants (K) in the pM range. D ) values ​​to human uPAR, demonstrating stronger binding affinity than 2G10 and 3C6, in the double-digit nanomolar range. (17) These results demonstrate the power of in vivo affinity-matured antibody selection to develop potent binders with slow-off kinetics to uPAR.

[0169] Example 4 - Cross-reactivity profile confirmed by ELISA To confirm that the cross-reactivity of the 12 lead candidates was maintained with the Herceptin constant region, their binding to human and cynomolgus monkey uPAR was assessed by ELISA (Figure 3A). The results showed that all lead candidates exhibited cross-reactivity, with EC values ​​ranging from 0.05 to 0.8 nM and 0.1 to 1.1 nM, respectively. 50 We demonstrated that the antibodies exhibited strong binding to both human and cynomolgus monkey uPAR with similar binding affinities. Two of them exhibited approximately two-fold higher binding affinity to human uPAR, seven exhibited two- to seven-fold stronger binding to cynomolgus monkey uPAR, and three antibodies bound to human and cynomolgus monkey uPAR with comparable binding affinities. Meanwhile, 2G10 and 3C6 were able to bind to human uPAR, but no reactivity with cynomolgus monkey uPAR was observed (Figure 3A and Table 2).

[0170] Example 5 - Antibody-dependent cytotoxicity (ADCC) Following confirmation of cell surface uPAR recognition and cross-reactivity of the 12 lead antibody candidates, we next evaluated whether they could mediate ADCC to promote tumor cell death as a uPAR-targeted immunotherapy approach. ADCC assays of all lead antibody candidates were initially performed using MDA-MB-231 cells as target cells in the presence of NK-92 MI CD16a effector cells. The resulting dose-response curves demonstrated that all lead candidates except 4718 were able to induce ADCC, and no cytotoxicity was observed with the isogenic huIgG1 control (Figure 3B). Eight selected antibody candidates (3159, 3595, 3639, 5016, 8163, 9538, 11857, and 13706) exerted effective ADCC responses in NK-92 cells and were further tested for ADCC activity in the presence of human PBMCs from three different healthy donors. Due to the unique characteristics of effector cells from each PBMC donor, this often results in considerable donor-to-donor variability in their ability to induce ADCC (39). Inherent donor variability was observed as expected among the eight lead candidates, and results showed that they all promoted effector cell function against MDA-MB-231 cells in a dose-dependent manner in the presence of healthy PBMCs (Figure 4). The average maximum percentage of ADCC response for the eight antibodies ranged from 46 to 66%, with seven of them exhibiting 10- to 96-fold lower EC values, ranging from 0.1 to 13 nM. 50 It is more potent than 2G10, which has a value (Table 2).

[0171] Example 6 - Cytotoxicity as an Antibody Drug Conjugate (ADC) In addition to Fc-mediated ADCC as a strategy for providing antitumor cytotoxicity, antibody-drug conjugates (ADCs) have been rapidly developed in recent decades to selectively deliver cytotoxic payloads directly to target cancer cells (40). To determine whether the eight selected antibody candidates could be internalized by targeting uPAR in an ADC approach, ADC efficacy was evaluated in vitro against MDA-MB-231 cells harboring Fab-αHFc-CL-MMAE, which recognizes the Herceptin Fc moiety and has a cathepsin-cleavable linker connecting it to monomethyl auristatin E (MMAE). Controls were performed without αHFc-CL-MMAE treatment, and no cytotoxicity was observed. Although all antibodies recognized 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 of the four antibodies was significantly higher than that of the control. 50 The values ​​ranged from 0.57 to 0.76 nM, indicating that they target uPAR in a distinct complex and induce efficient internalization (Figure 5A).

[0172] Example 7 - Inhibition of cell adhesion to vitronectin Vitronectin (VN) binding to uPAR is known to activate integrins, inducing intracellular signaling events that promote cancer cell adhesion and communication to the extracellular matrix (41). To determine whether eight lead antibody candidates possess functional inhibitory effects on tumor cells by targeting cell surface uPAR, we evaluated their ability to block uPAR-mediated cell adhesion to VN. Results showed that the eight lead candidates were able to inhibit MDA-MB-231 cell adhesion to VN-coated wells in a dose-dependent manner, with five of them (3159, 6312, 8163, 9538, and 11857) exhibiting EC values ​​ranging from 0.9 to 5.4 μM. 50The results demonstrated that the rAbs possessed significant therapeutic value (Table 2 and Figure 10). Candidate 3159 exhibited the strongest inhibitory effect, comparable to that of 3C6, previously identified as an inhibitor of uPAR-mediated cell adhesion (42). Overall, in vitro characterization of the lead antibodies highlighted candidates 3159, 8163, and 11857 ​​as the most promising rAbs with ADCC activity, ADC cytotoxicity by inducing efficient uPAR-rAb internalization, and functional inhibition of cell adhesion (Table 2), prompting investigation of their therapeutic efficacy in orthotopic animal models of breast cancer.

[0173] Example 8 - Therapeutic efficacy in an orthotopic animal model of human breast cancer To determine the in vivo therapeutic efficacy of the three lead antibodies, MDA-MB-231 cells were cultured in a 24-well plate using a 500-well platelet-expressing IgG antibody expressing Foxn1. nu The tumors (75–100 mm in volume) were orthotopically implanted into the mammary fat pad of nude mice. 3 Animals bearing the antibody (30 mg / kg) were treated weekly via intravenous injection with each antibody. Close monitoring of tumor growth across treatment groups revealed that all antibodies were able to reduce tumor burden compared to untreated controls (Figure 5B). Data demonstrated that candidate 11857 ​​showed the most potent efficacy, with tumor burdens 3.1 ± 0.4-fold smaller at day 21 compared to untreated controls (p = 0.0039). Such activity was maintained at tumor burdens 3.3 ± 0.3 (p = 0.0058) and 3.6 ± 0.6 (p = 0.0125)-fold smaller than untreated controls at days 25 and 28, respectively (Figure 5B). Candidates 3159 and 8163 were less effective at reducing tumor volume compared to candidate 11857, but were nevertheless effective at reducing tumor growth rate compared to untreated controls (Figure 5C). The superior antitumor activity of candidate 11857 ​​was also reflected in its ability to impair tumor growth rate compared to untreated controls (p=0.0002), which was significantly superior to that of candidates 3159 (p=0.0141) and 8163 (p=0.0267) (Figure 5C).

[0174] Example 9 - Epitope binning using biolayer interferometry The ability of the three lead antibodies (3159, 8163, and 11857) to impair cell adhesion, coupled with the reduction in tumor growth rate, led us to investigate their binding epitopes on uPAR. Epitope binning was performed by BLI, and 2G10 was included as a control. Human and cynomolgus monkey uPAR share 96% sequence identity (Figure 6A), and the majority of sequence variation between homologs resides in the uPA-binding site, which results 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 among uPAR homologs (Figure 6A). Previous studies have identified 2G10 as a competitor that inhibits uPA / uPAR interaction, suggesting that it binds to a region that inhibits uPA binding (45). BLI curves indicated that the three lead antibodies could bind to uPAR after the formation of the 2G10-uPAR complex. These results demonstrated that they target uPAR at a region distinct from the uPA-binding domain recognized by 2G10 (Figure 6B). To further test whether the three lead antibodies could inhibit VN binding to uPAR, we measured the VN binding response after each of them bound to uPAR (Figure 6B). BLI measurements showed that 3159 binding to uPAR completely abolished subsequent VN binding, while 11857 ​​showed a partial effect in blocking VN binding, and 8163 had no effect on VN binding to uPAR (Figure 6B). This is consistent with the results of adhesion assays, which showed that 3159 exhibited the strongest inhibition of VN-mediated cell adhesion, suggesting that 3159 recognized the VN-binding site and blocked the interaction between VN and uPAR. The results were also confirmed by changing the order in which 3159 and VN were added, demonstrating that 3159 was able to compete with VN for binding to uPAR (Figure 11). Furthermore, epitope competition assays were performed on different pairs of antibodies to identify whether they had distinct binding epitopes (Fig. 6C).Interestingly, both 3159 and 8163 could simultaneously bind to uPAR, but when rAb-uPAR complexes were formed with 11857, neither of them could interact with uPAR, suggesting that 11857 ​​has an epitope that partially overlaps with those of 3159 and 8163 (Fig. 6C). Furthermore, the binding epitopes of these three antibodies were found to be different from those of 3C6, which is known to block integrin αVβ1 (Fig. 12).

[0175] Consideration Our growing understanding of uPAR and its molecular partners in tumorigenesis, cancer progression, and metastasis provides the basis for developing new diagnostic, prognostic, and therapeutic approaches to treat a wide variety of tumors (46, 47). Cynomolgus monkeys have served as a valuable model for providing the most relevant information on the safety, efficacy, and pharmacokinetic profiles of translational therapeutics for human use (48, 49). Thus, this study presented a rapid antibody discovery pipeline that enabled the identification of cross-reactive antibodies against human and cynomolgus monkey uPAR. A 60-day immunization campaign with recombinant human suPAR enabled the rapid generation of uPAR-primed B cells in SJL mice, and the Beacon platform enabled the culture, manipulation, and screening of single B cells in a single day with 99% assurance of clonal origin. A similar approach has recently been used for the successful development of neutralizing antibodies against SARS-CoV and SARS-CoV-2 (50). This approach provides an example of using immunization to bias an immune response coupled to screening of antigen-primed B cells to identify human / cynomolgus cross-reactive antibodies with strong binding affinity and anti-tumor activity against human breast cancer, demonstrating the power of in vivo development and affinity maturation of B cells for antibody selection.

[0176] Effective tumor-targeting antibodies induce direct and indirect effects on tumor cells, mediated by the Fab variable region and Fc constant region, respectively (51). Clinically used targeted therapies for HER2-positive breast cancer (i.e., Herceptin and Perjeta) target the HER2 protein and induce ADCC by recruiting immune effector cells via the Fc domain as part of their tumor-killing mechanism (52). To confer mouse antibodies with ADCC activity exerted by human immune cells, their VH / VL domains were engineered to contain the Herceptin constant region. Interestingly, different amplitudes of ADCC were observed in the presence of NK-92 cells, suggesting that antibody epitope recognition is important for regulating ADCC activity. These findings support previous studies demonstrating how antigen binding can alter IgG conformation and affect Fc region recognition 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, which may govern the accessibility of the Fc region for interaction with effector cells to induce ADCC ( 56 , 57 ).

[0177] Although ADCC is one of the primary mechanisms for most antitumor mAbs currently in clinical use, recent findings suggest that antibodies that functionally inhibit targets while inducing ADCC offer additional benefits for achieving effective antitumor responses (58, 59). Previous studies have shown that vitronectin depletion strongly impairs tumor growth in an orthotopic xenograft model of breast cancer (60). In addition, uPAR binding to vitronectin has been shown to regulate cell adhesion and further induce changes in cell morphology, migration, and signaling (61-63). The reported mAb 8B12 was found to inhibit vitronectin binding to uPAR and effectively reduce uPAR-mediated cell migration on vitronectin-coated surfaces (18). These studies shed light on how inhibiting cancer cell interactions with the ECM impacts their growth-promoting signaling in the tumor microenvironment and overall tumorigenesis. This supports the finding that the inhibition of cell adhesion found in lead candidates, along with the ability to induce ADCC, provides an advantage in impairing tumor growth ( 41 , 64 ).

[0178] In addition to ADCC, several therapeutic antibodies have been redeveloped as ADCs to deliver cytotoxic drugs to antigen-positive tumor cells (65). Here, we evaluated the lead antibody for ADC cytotoxicity and demonstrated its potential to achieve cytotoxicity in MDA-MB-231 cells. Previous studies have shown that uPAR can be internalized by tumor cells via clathrin-mediated endocytosis or via a clathrin-independent mechanism mediated by LRP-1, both of which involve transporting uPAR to lysosomes for degradation and recycling (66, 67). This may offer an additional advantage, as all known internalization mechanisms of uPAR dissociate from its coreceptors, including matrix-engaging integrins and other bonafide ligands, thus suppressing downstream signaling (68).

[0179] Finally, based on the biolayer interferometry data and inhibition assays, a binding model for the three lead antibodies against uPAR (Figure 7) is proposed. Candidates 3159, 8163, and 11857 ​​bind to uPAR at different epitopes from the previously reported binders 2G10 and 3C6, and their binding epitopes are located on opposite sides of the central uPA-binding cavity. The binding sites of 3159 and 8163 on uPAR are independent, and the binding epitope of 11857 ​​overlaps significantly with their binding site, but is not identical. All of them exhibit inhibitory effects on cell adhesion, with 3159 binding to an epitope on uPAR due to vitronectin binding and therefore exhibiting the strongest inhibitory effect. 11857 ​​binds to a spot that results in synergistic effects for blocking ADCC, uPAR internalization, and cell adhesion, demonstrating the advantage of having an antibody with ADCC and additional functional effects for impairing tumor growth.

[0180] Materials and Methods Antigen production The HEK293 cell line stably expressing suPAR was generously provided by the Chapman Lab at UCSF. Cells were grown in DMEM complete medium supplemented with 10% fetal bovine serum (FBS), 100 U / mL penicillin, and 100 μg / L streptomycin (Gibco) at 5% CO and 37°C. For suPAR production, 3.7 × 10 cells were cultured. 7Cells were seeded into five stacks of Corning® CellSTACK® culture chambers 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 prep-grade column. suPAR production 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 further underwent endotoxin removal using Pierce™ High-Capacity Endotoxin Removal Spin Columns. After endotoxin removal, samples were characterized for their total endotoxin levels, and only those not exceeding 0.5 U / mL were approved for preparation of immunogen injections.

[0181] Animal immunization strategies The immunization strategy consisted of a primary intraperitoneal injection containing 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 booster injections containing 25 μg of antigen every other week for three boosts, with blood collections interspersed between each boost injection for 60 days. To control for unexpected antigen exposure, pre-bleeding was performed on each mouse before the start of the immunization campaign. All blood samples were allowed to clot, and 100–200 μL of serum was stored at -80°C for antibody titer determination.

[0182] Collection and enrichment of mouse antibody-secreting cells (ASCs) 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) and non-B cells were depleted from the single-cell suspension. Antibody-secreting cells (ASCs) were isolated by magnetic positive selection using the EasySep Mouse CD138+ Kit (StemCell) and CD45R (B220) IgG. - / CD138 高 They were enriched from single B cell suspensions by FACS-based positive selection via cell gating, which have traditionally been used to broadly define plasma cell populations ( 69 ).

[0183] Nanofluidic optoelectronic screening of single B cells Direct screening of secreted antibodies from ASCs was achieved using the Beacon platform. Enriched ASCs were injected into 0.75 nL OptoSelect™ 3500 and OptoSelect™ 14K chips. This platform allows for efficient isolation of single ASCs into nanopens using adjustable optoelectronic positioning parameters. ASCs were individually cultured within the chip for 1 hour and screened for both IgG secretion and antigen specificity using an in-channel multiplexed bead-based fluorescent assay. Briefly, beads coated with rabbit anti-mouse IgG (H+L) were imported into the chip, and active accumulation of secreted antibodies was identified by binding of an FITC-labeled goat anti-mouse secondary antibody to the beads. Antigen specificity was assessed by importing fluorescently labeled uPAR from either human (conjugated to Alexa Fluor 488), cynomolgus monkey (R&D Systems), or mouse (Sino Biologicals) (conjugated to Alexa Fluor 647) into the chip. Binding of ASC-derived IgG to the antigen was monitored by the time-dependent increase in fluorescence from uPAR on the beads found in the mouth of the NanoPen. Selected ASCs were 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 anti-uPAR antibody production.

[0184] Sequencing, recombinant cloning, expression, and purification of candidate antibodies Single B cells were exported from Beacon into a 96-well plate containing lysis buffer and mineral oil. The cDNA generation process was performed using ChemPartner & BLI's proprietary protocol with RNA capture beads. Selected human and cynomolgus uPAR cross-reactive binders were then amplified using a RACE PCR protocol with proprietary heavy and light chain constant reverse primers. Clones exhibiting amplicons within 500-700 bp were sequenced by next-generation sequencing (NGS). NGS libraries were prepared using indexing universal forward and reverse constant primers. Samples were then run on a MiSeq (Illumina). Raw data was analyzed using NGS-related software.

[0185] 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 according to the manufacturer's protocol. Briefly, HEK293F cells were seeded in FreeStyle™ medium and incubated at 130 rpm and 37°C with 8% CO2. Transfection was performed using polyethyleneimine (PEI) to maintain a 1:2 DNA / PEI ratio. To improve recombinant protein synthesis, 5% peptone solution was added at 0.1 equivalent of the original cell suspension. On days 6–7 posttransfection, the IgG-enriched medium was collected, and the IgG was purified using a Protein A column (GE MabSelect™ SuRe™) and dialyzed overnight against PBS (pH 7.4) at 4°C.

[0186] Biolayer Interferometry (BLI) analysis The binding affinity of anti-uPAR antibodies was measured using the Octet RED384 system at 25°C. Octet SA (streptavidin) biosensors were immobilized with 2 μg / mL biotinylated human or cynomolgus monkey uPAR (Protein Sciences) in assay buffer (PBS containing 1% BSA). After equilibration to baseline in assay buffer, the biosensors were placed into each well containing anti-uPAR antibody and allowed to dissociate in assay buffer. Association and dissociation curves were analyzed using Octet Data Analysis software.

[0187] Cross-reactivity of candidate antibodies by ELISA Nunc MaxiSorp™ flat-bottom 96-well plates were coated with human or cynomolgus monkey uPAR (3.19 μg / mL) overnight at 4°C. 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 a 2-hour incubation, the plates were washed and 100 μL of 1-Step™ Turbo TMB-ELISA substrate solution (Thermo Scientific) was added to each well. The reaction was quenched with 2 M H2SO4 for 5 minutes at room temperature, and the optical density of each well was measured at 450 nm using a SpectraMax190 microplate reader. The resulting dose-response curves were used to determine the minimal dose of antibody required to achieve 50% of a saturating signal, allowing for quantitative comparison of binding affinities.

[0188] Recognition of cellular uPAR by candidate antibodies MDA-MB-231 cells were harvested by TrypleE and cultured at 2 × 10 in FACS buffer (PBS + 1% BSA). 6 After resuspending at 2 × 10 cells / mL, the cells were aliquoted into 96-well plates (100 μL, 2 × 10 5Cells 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. Cells were then washed three times with PBS and incubated with AlexaFluor488-conjugated goat anti-human IgG for 50 minutes at 4°C in the dark. Finally, cells were washed twice with FACS buffer and resuspended in 80 μL for FACS analysis on a Bio-Rad S3e cell sorter.

[0189] Inhibition of cell adhesion to vitronectin 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 into each well, serial dilutions of antibody or RGDS peptide were added, and the plates were incubated overnight at 5% CO2 and 37°C. All wells were washed with PBS, and the cells were fixed by adding ice-cold methanol for 10 minutes at room temperature. After fixation, the cells were stained using a 5% crystal violet solution. The wells were washed three times with PBS, and the cells were lysed in 2% SDS lysis buffer. Each lysate was transferred to a clear 96-well plate, and the absorbance at 590 nm was recorded to determine the number of adherent cells.

[0190] In vitro antibody-dependent cellular cytotoxicity (ADCC) using NK cells Antibody-dependent cellular cytotoxicity against MDA-MB-231 cells by NK cells was detected using the DELFIA® EuTDA cytotoxicity reagent (PerkinElmer). Briefly, MDA-MB-231 cells were harvested and labeled by incubating with 2 μL / mL of a fluorescence-enhancing ligand (PerkinElmer DELFIA® BATDA labeling reagent) at 37°C for 20 minutes. After BATDA diffused into the cells, it was hydrolyzed and converted to 2,2':6',2''-terpyridine-6,6''-dicarboxylic acid (TDA) by cytoplasmic acetylesterase. TDA is a non-cell-permeable hydrophobic ligand, so it can be trapped within biological target cells. The solution was centrifuged, and the cells were washed three times with PBS. The labeled cells were reconstituted in phenol red-free RPMI 1640 medium and then plated in a 96-well U-bottom sterile microplate (100 μL, 1 × 10 cells). 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-92 CD16a 176V effector cells were harvested and incubated at approximately 1.2 × 10 6 After concentrating the cells / mL, 50 μL was added to an assay plate, resulting in a 6:1 effector-to-target cell ratio in each well. The plate containing antibody, target, and effector cells was then incubated for 4 hours at 37°C and 5% CO2. After incubation, the plate was centrifuged at 400 RCF for 5 minutes, and 25 μL of the supernatant was transferred to a flat-bottom detection plate. Finally, 200 μL of europium solution (PerkinElmer, DELFIA® Eu-Solution) was added to each well, and the plate was incubated at room temperature for 15 minutes to allow for the formation of the highly fluorescent stabilized chelate (Eu-TDA). The resulting fluorescent signal was acquired within 5 hours using a time-resolved fluorometer. A background death control was determined by diluting target cells with medium, and a maximum death control was determined by incubating the cells in 10 μL of lysis buffer (1% Triton X-100) for 30 minutes before centrifuging the plate.

[0191] In vitro antibody-dependent cellular cytotoxicity (ADCC) using human PBMCs isolated from healthy donors Frozen PBMC cells were commercially obtained from AllCells. Cells were isolated from human blood by LeukoPak density gradient method and then stored in liquid nitrogen. Cells were thawed at 37°C, suspended in RPMI 1640 + 10% FBS, and incubated overnight at 37°C. MDA-MB-231 target cells were labeled with DELFIA BATDA according to the manufacturer's instructions. Effector PBMC cells from each donor were then plated with target cells at a 50:1 ratio in a 96-well plate. ADCC induction was triggered by adding each antibody candidate to the mixture, which was then incubated at 37°C for 4 hours. Finally, the supernatant was collected and mixed with europium solution. The degree of cytotoxicity was determined using time-resolved fluorescence (TRF) signal intensity. Control groups, including a target spontaneous group (target cells), a target maximum group (target cells lysed using Triton), and a background group (supernatant from target cells), were set up for data normalization. The ADCC effect is determined by the following formula: The calculated ADCC was defined by the following formula: % ADCC = (sample cytotoxicity - spontaneous cytotoxicity of target and effector cell mixture) / (maximum target cell cytotoxicity (Triton X-100 treatment) - spontaneous cytotoxicity of target and effector cell mixture) * 100%. The dose-response effect was analyzed using GraphPad Prism.

[0192] Epitope binning The epitope binning assay was performed using Octet RED384 in a classic sandwich assay format. All samples were prepared in assay buffer (PBS with 1% BSA), and the primary antibody was biotinylated for immobilization on a streptavidin (SA) biosensor. Each binding cycle consisted of the following steps: First, the SA biosensor was soaked in assay buffer for a sensor check to establish a baseline. Next, the biotinylated antibody was loaded onto the SA biosensor. After a washing step, uPAR bound and reached saturation. After washing the biosensor with assay buffer, it was moved to the next well for secondary antibody association and finally to a buffer-containing well for the dissociation phase. Data analysis was performed using ForteBio data analysis software, and figures were generated using Matlab.

[0193] Antibody-drug conjugate (ADC) cytotoxicity screening MDA-MB-231 cells were seeded at 2,500 cells / well in 96-well plates (Corning) overnight at 37°C and 5% CO2. Cells were grown for 5 days in the presence of serial dilutions of the antibody ranging from 0.0032 nM to 10 nM, in combination with the Fab fragment of an anti-human IgG Fc-specific antibody conjugated to monomethyl auristatin E (Fab-αHFc-CL-MMAE, Moradec) at a final concentration of 20 nM. The number of viable cells was quantified by the CellTiter-Glo Luminescent Cell Viability Assay (Promega), based on luminescent detection of ATP, which is directly proportional to the number of cells present in each well. After incubation, luminescence was recorded using a Synergy Neo2 multimode microplate reader (BioTek Instruments, Inc.).

[0194] Therapeutic efficacy in orthotopic animal models of human breast cancer 16 female Foxn1 nu 1 × 10 6 MDA-MB-231 cells were orthotopically implanted, and tumor volumes were 75–100 mm 3The animals were monitored for several days until a tumor volume of 1000 mg / kg was reached. Once a tumor volume was reached, the animals were considered eligible for therapeutic intervention, which began three days after such tumor volume was reached. Therapeutic intervention began with each experimental group receiving antibody administered intravenously at a concentration of 30 mg / kg. A 30-day treatment regimen was implemented, with animals receiving antibody treatment weekly for 30 days (days 3, 10, 17, and 24). Animal welfare, body weight, and tumor volume were continuously monitored throughout the study. At the completion of the therapeutic intervention regimen, tumors were harvested and prepared for histological analysis.

[0195] statistical analysis All statistical analyses were performed using GraphPad Prism version 8.0 (GraphPad Software, Inc., San Diego, CA). Dose-response curves were derived from nonlinear fits to raw values ​​with a minimum of three experimental replicates. Statistical analysis of all data acquisition was performed as a two-way analysis of variance with post-hoc multiple comparison Tukey's test. Differences between groups were considered significant at a P value of ≤0.05.

[0196] Liquid chromatography tandem mass spectrometry (LC-MS / MS) Purified recombinant human suPAR (8 μg) was denatured with 6 M urea, disulfide bonds were reduced with 10 mM DTT at 55°C for 20 min, and then carbamidomethylated with 12.5 mM iodoacetamide for 1 h in the dark. Unreacted iodoacetamide was quenched with DTT, the pH was equilibrated to pH 8, and trypsin digestion (Promega catalog no. VA9000) was performed overnight at 37°C. Samples were desalted using Pierce™ C18 Spin Tips (Thermo Scientific™, catalog no. 87782), vacuum dried, and resuspended in HPLC-grade water with 0.2% TFA. LC-MS / MS analysis was performed on an LTQ Orbitrap XL mass spectrometer (Thermo) coupled to a nanoACQUITY ultra-performance liquid chromatography (UPLC) system (Waters). Trypsin 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 from 325–1500 m / z, and up to three most intense precursor ions (MS1 features with a charge ≥2) were selected for higher energy collisional dissociation (HCD) at a resolution of 30,000 at m / z 200 for MS / MS [CB2]. Data from the uPAR peptide were acquired using Xcalibur software and processed as previously described (Zhao et al. (2021) ACS Cent Sci. American Chemical Society 7:1638–49).

[0197] Measurement of serum antibody titers in uPAR-immunized mice Nunc MaxiSorp™ flat-bottom 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 titers were determined by performing standard logarithmic serial dilutions of serum in 5% nonfat dry milk. 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, diluted 1:3000) was added and incubated for 2 hours at room temperature in an orbital shaker. Finally, the plate was washed, and 100 μL of 1-Step™ Turbo TMB-ELISA substrate solution (Thermo Scientific, catalog no. 34022) was added to each well and incubated for 15 minutes before quenching the reaction with 2M H2SO4. The plate was incubated for 5 minutes at room temperature, and the optical density of each well was measured at 450 nM against a standard curve (10-0.07 μg / mL) of mouse anti-human uPAR monoclonal antibody clone R-3 (Invitrogen catalog no. MON R-3-02) using a SpectraMax190 microplate reader.

[0198] Example 10 - Humanized 11857 ​​and 3159 antibodies Antibody 11857 ​​was used as the parent template in the humanization design. Based on antibody sequence analysis and homology modeling of the mAb 3D structure, three humanized VH ("11857 ​​HC1," "11857 ​​HC2," and "11857 ​​HC3") and three humanized VL ("11857 ​​LC1," "11857 ​​LC2," and "11857 ​​LC3") sequences were designed. The sequence of the humanized 11857 ​​antibody is provided in Table 1 above. The CDR sequences were defined using the Kabat numbering system.

[0199] Additionally, antibody 3159 was used as the parent template in humanization design. Based on antibody sequence analysis and homology modeling of the mAb 3D structure, three humanized VH ("3159 HC1," "3159 HC2," and "3159 HC3") and three humanized VL ("3159 LC1," "3159 LC2," and "3159 LC3") sequences were designed. The sequences of the humanized 11857 ​​antibody are provided in Table 1 above. CDR sequences were defined using the Kabat numbering system.

[0200] The humanization scores of the humanized 11857 ​​antibody were determined using a T20 score analyzer, as described in Gao et al. (2013) BMC Biotechnology, 13:55. The results are shown in Table 3 below. The humanization T20 scores of the humanized variable region frameworks ranged from 84 to 86 (VH) and 97 to 99 (VK), which were close to or above the threshold for "humanization" according to Gao et al. The T20 scores of the humanized full-length variable regions ranged from 79 to 84 (VH) and 81 to 82 (VK). The T20 scores of the humanized full-length variable heavy chain regions exceeded the threshold for humanization. Although the T20 scores of the full-length kappa light chain sequence were below the recommended cutoff score, they achieved the maximum T20 score based on structural modeling without compromising the structural validation of the light chain.

[0201] Additionally, the humanization scores of the humanized 3159 antibody were determined using the T20 score analyzer. The results are shown in Table 3 below. The humanization T20 analyzer scores ranged from 84 to 86 (VH) and 97 to 99 (VK) for the humanized variable region frameworks, which were close to or exceeded the threshold for "humanization" according to Gao et al. The T20 scores for the humanized full-length variable regions ranged from 79 to 82 (VH) and 85 to 86 (VK), all of which were close to or exceeded the threshold for humanization. The T20 score for the full-length kappa light chain sequence "3159 LC1" was just below the recommended cutoff score, but based on structural modeling, it achieved the maximum T20 score without compromising the structural validation of the light chain. [Table 3]

[0202] The humanized 11857 ​​and 3159 antibodies were assayed for binding to human and cynomolgus monkey uPAR by biolayer interferometry (BLI). BLI curves are provided, and their affinity (KD) values ​​are reported in Table 4. The kinetic constant range for Octet HTX was 1 mM to 10 pM. Therefore, a calculated KD of less than 10 pM is interpreted as a KD < 10 pM. For two antibodies (11857 ​​HC2 + LC3 and 11857 ​​HC3 + LC3), the production yield was insufficient to perform kinetic analysis.

[0203] Two breast cancer cell lines, MDA-MB-231 (high uPAR expression) and MCF-7 (low uPAR expression), were used to verify cell surface uPAR binding by a flow cytometry-based cell assay for humanized and parental 11857 ​​or 3159. Cells were incubated with humanized and parental 11857 ​​or 3159 and stained with an APC-conjugated anti-human Fc antibody. MFI comparison at 10 μg / mL demonstrated specific binding of the test article to MDA-MB-231 and low nonspecific binding to MCF7.

[0204] Six humanized 11857 ​​variants and the parent chimera were assayed by flow cytometry-based cell assay for EC50 determination using the MDA-MB-231 cell line, with EC50 values ​​ranging from 0.2119 to 1.206 (μg / mL). For three antibodies (11857 ​​HC2+LC3, 11857 ​​HC3+LC2, and 11857 ​​HC3+LC3), the production yield was insufficient to perform EC50 assays. In addition, nine humanized 3159 variants and the parent chimera were assayed by flow cytometry-based cell assay for EC50 determination using the MDA-MB-231 cell line, with EC50 values ​​ranging from 0.1852 to 0.5176 (μg / mL). [Table 4]

[0205] We also compared uPAR expression levels in MDA-MB-231 and UMUC3 cell lines using a flow cytometer. Cells were incubated with parental 11857 ​​and stained with a PE-conjugated anti-human Fc antibody. The resulting uPAR expression levels were approximately three-fold lower in UMUC3 cells compared to MDA-MB-231 cells.

[0206] Example 11 - Imaging studies 5 mCi of Zr-89 was labeled with 500 μg of desferrioxamine (DFO)-conjugated 3159 and 11857 ​​antibodies, as described above for Example 10. In this example, 11857 ​​and 3159 refer to 11857 ​​HC2+LC2 and 3159 HC2+LC2, respectively. Quality control analysis demonstrated 100% labeling and 95% yield. Imaging was performed using the UMUC3 tumor model in nude male mice. N=4 for Zr89-3159 and N=3 for Zr89-11857. Doses ranged from 200 to 280 μCi per mouse. PET / CT images were acquired at 30 min, 4 h, 19 h, 24 h, 48 h, 72 h, 96 h, and 120 h post-injection.

[0207] Representative axial and coronal positron emission tomography-computed tomography (PET / CT) slices from a male nu / nu mouse bearing a subcutaneous UMUC3 xenograft are shown in Figure 13. Mice received approximately 250 μCi of 89 Zr-3159 IgG and were imaged at the indicated time points. The location of the tumor is indicated by the arrow. A representative maximum intensity projection obtained from a male nu / nu mouse bearing a subcutaneous UMUC3 xenograft is shown in Figure 14. Mice received approximately 250 μCi of 89 Zr-3159 IgG and were imaged at the indicated time points. The location of the tumor is indicated by the arrow. Representative axial and coronal PET / CT slices from a male nu / nu mouse bearing a subcutaneous UMUC3 xenograft are shown in Figure 15. Mice received approximately 250 uCi of 89Zr-11857 ​​IgG was administered and imaged at the indicated time points. The location of the tumor is indicated by the arrow. A representative maximum intensity projection obtained from a male nu / nu mouse bearing a subcutaneous UMUC3 xenograft is shown in Figure 16. The mouse received approximately 250 uCi of 89 Zr-11857 ​​IgG was received and imaged at the indicated time points. The location of the tumor is indicated by an arrow.

[0208] SUVmean data (Figure 17) were obtained by region of interest analysis for tumors from mice in the imaging cohort. Data are presented as means with standard deviations. Data were obtained from four tumors in the 3159 cohort and three tumors in the 11857 ​​cohort. Additionally, SUVmean data were obtained by region of interest analysis for tumors and various normal tissues from mice in the imaging cohort. Data are presented as means with standard deviations. Data were obtained from four tumors in the 3159 cohort (Figure 18) and three tumors in the 11857 ​​cohort (Figure 19).

[0209] The fold change in volume (normalized to the volume on day 0) of UMUC3 tumors treated with 225Ac-labeled 3159 is shown in Figure 20. To enable radiolabeling, 3159 was coupled to NHS-Macropa via a lysine residue. The radiopharmaceutical was administered at 0.8 μCi per mouse via the tail vein on day 0. Tumor volume measurements were recorded for vehicle (n=10) and drug-treated groups (n=18). 225 The volume changes of UMUC3 tumors treated with Ac-labeled 3159 are shown in FIG.

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[0211] Accordingly, the foregoing description merely illustrates the principles of the present disclosure. Those skilled in the art will recognize that, although not explicitly described or shown herein, they can devise various configurations that embody the principles of the present invention and are within its spirit and scope. Furthermore, all examples and conditional language recited herein are intended primarily to aid the reader in understanding the principles of the present invention and concepts contributed by the inventors to further the art, and should be construed without limitation to such specifically recited examples and conditions. Furthermore, all statements herein reciting principles, aspects, and embodiments of the present invention, as well as specific examples thereof, are intended to encompass both structural and functional equivalents thereof. Additionally, such equivalents are intended to include both currently known equivalents and future-developed equivalents, i.e., any elements developed that perform the same function, regardless of 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 for binding to uPAR, Variable heavy chain (V Ｈ ) a polypeptide comprising: V containing the amino acid sequence TSGMGVS (SEQ ID NO: 2) Ｈ CDR1, V containing the amino acid sequence HIYWDDDKRYNPSLKT (SEQ ID NO: 3) Ｈ CDR2, and V containing the amino acid sequence RVRNYFSGTSYWYFDV (SEQ ID NO: 4) Ｈ V, including CDR3 Ｈ A polypeptide, Variable light chain (V Ｌ ) a polypeptide comprising: V containing the amino acid sequence RSSQNILHRTGNTYLE (SEQ ID NO: 6) Ｌ CDR1, V containing the amino acid sequence KVSNRFS (SEQ ID NO: 7) Ｌ CDR2, and V containing the amino acid sequence FQGSYVPFT (SEQ ID NO: 8) Ｌ V, including CDR3 Ｌ polypeptides, Variable heavy chain (V Ｈ ) a polypeptide comprising: V containing the amino acid sequence SHDMS (SEQ ID NO: 10) Ｈ CDR1, V containing the amino acid sequence AIDSDGGLTYYSNSRER (SEQ ID NO: 11) Ｈ CDR2, and V containing the amino acid sequence RRASYWYFDV (SEQ ID NO: 12) Ｈ V, including CDR3 Ｈ A polypeptide, Variable light chain (V Ｌ ) a polypeptide comprising: V containing the amino acid sequence RASQNIGTSIH (SEQ ID NO: 14) Ｌ CDR1, V containing the amino acid sequence YASESIS (SEQ ID NO: 15) Ｌ CDR2, and V containing the amino acid sequence QQSNSWPT (SEQ ID NO: 16) Ｌ V, including CDR3 Ｌ polypeptides, Variable heavy chain (V Ｈ ) a polypeptide comprising: V containing the amino acid sequence DYYMN (SEQ ID NO: 18) Ｈ CDR1, V containing the amino acid sequence NINPNNGGTDYNQKFKG (SEQ ID NO: 19) Ｈ CDR2, and V containing the amino acid sequence SYGSRFPY (SEQ ID NO: 20) Ｈ V, including CDR3 Ｈ A polypeptide, Variable light chain (V Ｌ ) a polypeptide comprising: V containing the amino acid sequence RASQDITNYLS (SEQ ID NO: 22) Ｌ CDR1, V containing the amino acid sequence YTAVLQS (SEQ ID NO: 23) Ｌ CDR2, and V containing the amino acid sequence QQGHTLPWT (SEQ ID NO: 24) Ｌ V, including CDR3 Ｌ a polypeptide, or Variable heavy chain (V Ｈ ) a polypeptide comprising: V containing the amino acid sequence DYYMN (SEQ ID NO: 18) Ｈ CDR1, V containing the amino acid sequence NINPNNGGTDYNQKFKG (SEQ ID NO: 19) Ｈ CDR2, and V containing the amino acid sequence SYGSRFPY (SEQ ID NO: 20) Ｈ V, including CDR3 Ｈ A polypeptide, Variable light chain (V Ｌ ) a polypeptide comprising: V containing the amino acid sequence RASQDITNYLS (SEQ ID NO: 22) Ｌ CDR1, V containing the amino acid sequence YTSFLQS (SEQ ID NO: 26) Ｌ CDR2, and V containing the amino acid sequence QQGHTLPWT (SEQ ID NO: 24) Ｌ V, including CDR3 Ｌ An antibody that competes with an antibody comprising a polypeptide.

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

3. The antibody a variable heavy chain (V) 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 set forth in SEQ ID NO: 1; Ｈ ) a polypeptide; a variable light chain (V) 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 set forth in SEQ ID NO:5; Ｌ 3. The antibody of claim 1 or 2, comprising:

4. The antibody a variable heavy chain (V) 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 set forth in SEQ ID NO:9; Ｈ ) a polypeptide; a variable light chain (V) 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 set forth in SEQ ID NO: 13; Ｌ 3. The antibody of claim 1 or 2, comprising:

5. The antibody a variable heavy chain (V) 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 set forth in SEQ ID NO: 17; Ｈ ) a polypeptide; a variable light chain (V) 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 set forth in SEQ ID NO: 21; Ｌ 3. The antibody of claim 1 or 2, comprising:

6. The antibody a variable heavy chain (V) 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 set forth in SEQ ID NO: 17; Ｈ ) a polypeptide; a variable light chain (V) 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 set forth in SEQ ID NO: 25; Ｌ 3. The antibody of claim 1 or 2, comprising:

7. An antibody that specifically binds to human urokinase-type plasminogen activator receptor (uPAR), and for binding to uPAR, (a) variable heavy chain (V Ｈ ) a polypeptide comprising: V containing the amino acid sequence DYYMN (SEQ ID NO: 18) Ｈ CDR1, V comprising the amino acid sequence NINPNNGGTDYNQKFQG (SEQ ID NO: 39) Ｈ CDR2, and V comprising the amino acid sequence SYGSRFPY (SEQ ID NO: 20) Ｈ CDR3, or V containing the amino acid sequence DYYMN (SEQ ID NO: 18) Ｈ CDR1, V comprising the amino acid sequence NINPNNGGTDYSQKFQG (SEQ ID NO: 37) Ｈ CDR2, and V comprising the amino acid sequence SYGSRFPY (SEQ ID NO: 20) Ｈ V, including CDR3 Ｈ A polypeptide, Variable light chain (V Ｌ ) a polypeptide comprising: V containing 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 V, including CDR3 Ｌ a polypeptide, or (b) variable heavy chain (V Ｈ ) a polypeptide comprising: V containing 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 V containing 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 V containing 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 V, including CDR3 Ｈ A polypeptide, Variable light chain (V Ｌ ) a polypeptide comprising: V containing 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 V containing 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 V containing 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 V, including CDR3 Ｌ An antibody that competes with an antibody comprising a polypeptide.

8. The antibody (a) variable heavy chain (V Ｈ ) a polypeptide comprising: V containing the amino acid sequence DYYMN (SEQ ID NO: 18) Ｈ CDR1, V comprising the amino acid sequence NINPNNGGTDYNQKFQG (SEQ ID NO: 39) Ｈ CDR2, and V comprising the amino acid sequence SYGSRFPY (SEQ ID NO: 20) Ｈ CDR3, or V containing the amino acid sequence DYYMN (SEQ ID NO: 18) Ｈ CDR1, V comprising the amino acid sequence NINPNNGGTDYSQKFQG (SEQ ID NO: 37) Ｈ CDR2, and V comprising the amino acid sequence SYGSRFPY (SEQ ID NO: 20) Ｈ V, including CDR3 Ｈ A polypeptide, Variable light chain (V Ｌ ) a polypeptide comprising: V containing 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 V, including CDR3 Ｌ a polypeptide, or (b) variable heavy chain (V Ｈ ) a polypeptide comprising: V containing 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 V containing 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 V containing 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 V, including CDR3 Ｈ A polypeptide, Variable light chain (V Ｌ ) a polypeptide comprising: V containing 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 V containing 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 V containing 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 V, including CDR3 Ｌ The antibody of claim 7, comprising a polypeptide.

9. The antibody a variable heavy chain (V) 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 set forth in SEQ ID NO: 35; Ｈ ) a polypeptide; a variable light chain (V) 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; Ｌ 9. The antibody of claim 7 or 8, comprising:

10. The antibody a variable heavy chain (V) 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 set forth in SEQ ID NO: 36; Ｈ ) a polypeptide; a variable light chain (V) 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; Ｌ 9. The antibody of claim 7 or 8, comprising:

11. The antibody a variable heavy chain (V) 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 set forth in SEQ ID NO: 38; Ｈ ) a polypeptide; a variable light chain (V) 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; Ｌ 9. The antibody of claim 7 or 8, comprising:

12. The antibody a variable heavy chain (V) 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 set forth in SEQ ID NO: 43; Ｈ ) a polypeptide; a variable light chain (V) 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:49, SEQ ID NO:51, or SEQ ID NO:52; Ｌ 9. The antibody of claim 7 or 8, comprising:

13. The antibody a variable heavy chain (V) 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 set forth in SEQ ID NO: 45; Ｈ ) a polypeptide; a variable light chain (V) 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:49, SEQ ID NO:51, or SEQ ID NO:52; Ｌ 9. The antibody of claim 7 or 8, comprising:

14. The antibody a variable heavy chain (V) 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 set forth in SEQ ID NO: 47; Ｈ ) a polypeptide; a variable light chain (V) 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:49, SEQ ID NO:51, or SEQ ID NO:52; Ｌ 9. The antibody of claim 7 or 8, comprising:

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 a non-human primate uPAR.

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

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

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

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

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

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

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

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

25. The antibody is a V antibody according to any one of claims 1 to 18. Ｈ Polypeptide-V Ｌ The antibody of any one of claims 1 to 24, which is a bispecific antibody comprising a first antigen-binding domain comprising a polypeptide pair.

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

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

30. the fusion protein the single chain antibody; a transmembrane domain; and an intracellular signaling domain.

31. A conjugate comprising: An antibody according to any one of claims 1 to 26 or a fusion protein according to any one of claims 27 to 30; and a drug conjugated to said antibody or fusion protein.

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

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

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

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

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

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

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

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

40. The variable heavy chain (V) of the antibody according to any one of claims 1 to 26 Ｈ ) polypeptide, variable light chain (V Ｌ ) a nucleic acid encoding a polypeptide, or both.

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

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

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

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

45. A cell, The variable heavy chain (V) of the antibody of any one of claims 1 to 6 Ｈ a first nucleic acid encoding a polypeptide; The variable light chain (V Ｌ ) a second nucleic acid encoding the polypeptide.

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

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

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

51. 51. The composition of claim 49 or 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. 53. The kit of claim 52, wherein the composition is present in one or more unit doses.

54. 53. 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, comprising administering to the subject an effective amount of a composition described in any one of claims 49 to 51.

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

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

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

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

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

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

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

64. 52. 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, comprising administering to the subject an effective amount of the composition of any one of claims 49-51.

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