DPEP-1 BINDING AGENTS AND METHODS OF USE

Abstract

DPEP-1 binding agents, including antibodies, and pharmaceutical compositions containing same are described. Also provided are methods for using and producing such binding agents, antibodies, and pharmaceutical compositions, and methods of their use to treat or prevent disorders in human subjects in need thereof. TIFF2024513473000015.tif65140

Description

[Technical field]

[0001] Related Applications This disclosure claims the benefit of U.S. Provisional Patent Application No. 63 / 172,530, filed April 8, 2021, which is incorporated by reference in its entirety.

[0002] Inclusion of sequence listing The sequence listing contained in the file entitled "27929-P63463PC00_SequenceListing_2022-04-07", which is 55,531 bytes and was created on April 7, 2022, has been submitted herewith by electronic submission and is incorporated herein by reference.

[0003] Field Disclosed herein are DPEP-1 binding agents, including antibodies, and pharmaceutical compositions containing the same. Also disclosed are methods for using and producing such agents, antibodies, and pharmaceutical compositions, as well as methods and uses for treating or preventing disorders in human subjects in need thereof. [Background technology]

[0004] background Inflammation is a host defense response to harmful stimuli that can be acute or chronic. Acute inflammation is characterized by redness, heat, swelling, and pain. The primary purpose of inflammation is to localize and eradicate irritants and promote repair of surrounding tissues. In most cases, inflammation is a necessary and beneficial process. The inflammatory response involves three main stages: first, dilation of arterioles to increase blood flow; second, structural changes in microvessels and leakage of plasma proteins from the bloodstream; and third, transmigration of leukocytes through the endothelium and accumulation at the site of injury. Transendothelial migration (TEM) of leukocytes is a key step in their recruitment to sites of inflammation, injury, and immune response. Transmigration of neutrophils to sites of inflammation is thought to require cell-cell adhesion.

[0005] Failure to dissipate harmful stimuli that promote acute inflammation can lead to chronic inflammation, and some stimuli can promote immediate chronic inflammation. In some cases, inflammation results in secondary or chronic damage. Inflammation in the tumor microenvironment is also implicated in cancer acceleration and tumor metastasis. The presence of proinflammatory molecules allows malignant cancer cells to adhere to the endothelial wall, leading to metastasis. Proinflammatory cytokines induce cancer cell proliferation and aggregation, triggering other cancer cells to secrete more cytokines, resulting in a positive feedback loop. The role of adhesion molecules in acute and chronic inflammation is an area of ​​research that is needed for the development of methods to control inflammation by modulating or blocking the adhesion of leukocytes to the endothelium.

[0006] Anti-inflammatory agents function as blockers, suppressors, or regulators of the inflammatory response. Tissue-specific control of inflammation is sometimes desirable to regulate inflammation in some tissues while maintaining responses in other tissues. Anti-inflammatory agents are used to treat a variety of acute and chronic conditions. Most people have no problems taking these agents, but some people experience side effects that can be severe. In some groups, these drugs are prescribed cautiously and only when there are no alternatives, and at the lowest dose and duration necessary.

[0007] Thus, there remains a need for additional therapeutic compounds to reduce or block inflammation as current therapeutics, especially since many of the current approaches fail to adequately treat some of the more extreme cases of inflammation. Thus, what is needed are new compositions that function as blockers, inhibitors, or regulators of the inflammatory response. Summary of the Invention

[0008] overview Using a phage display library and panning strategy, we identified single domain antibodies (sdABs) that bind to human DPEP-1 (hDPEP-1). These sdABs (sdABP01-09; SEQ ID NOs: 1-9) were able to bind to hDPEP-1 in vitro and / or recognize HEK293T cells displaying hDPEP-1 on their surface. These human DPEP-1-specific V H H was shown to have good thermal stability, good SPR binding affinity, good dose-response binding to cell-presented hDPEP-1, and was characterized by epitope binding. Each of sdABP01-09 contains a biotinylated acceptor peptide (BAP) and a polyhistidine (His6) tag. The core sequences of these sdABP01-09 without the BAP and His6 tags are shown in SEQ ID NOs: 12-20.

[0009] Disclosed herein are DPEP-1 binding agents, including antibodies, and pharmaceutical compositions comprising the same. Also disclosed are methods of using and making such DPEP-1 binding agents and compositions, and methods for screening for DPEP-1 binding agents.

[0010] In one aspect, (i) SEQ ID NOs: 21, 22, and 23; (ii) SEQ ID NOs: 24, 25, and 26; (iii) SEQ ID NOs: 27, 28, and 29; (iv) SEQ ID NOs: 30, 31, and 32; (v) SEQ ID NOs: 33, 34, and 35; (vi) SEQ ID NOs: 36, 37, and 38; (vii) SEQ ID NOs: 39, 40, and 41; (viii) SEQ ID NOs: 42, 43, and 44; or (ix) SEQ ID NOs: 45, 46, and 47 Disclosed herein are binding agents that bind to DPEP-1, including:

[0011] In one embodiment, the binding agent comprises an antibody or antigen-binding fragment thereof that binds to DPEP-1, wherein the antibody or antigen-binding fragment thereof comprises an amino acid sequence at least 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% identical to any one of SEQ ID NOs: 12-20 and 48-56. In one embodiment, the binding agent comprises the amino acid sequence of any one of SEQ ID NOs: 12-20 and 48-56.

[0012] In one embodiment, the binding agent is a monoclonal antibody, a polyclonal antibody, a chimeric antibody, a humanized antibody, or an antigen-binding fragment thereof.

[0013] In one embodiment, the binding agent is an antigen-binding fragment fused to an Fc domain.

[0014] In one embodiment, the antigen-binding fragment is an Fv, scFv, Fab, Fab', F(ab')2, dsFv, ds-scFv, sdAB, dimeric, minibody, diabody, or multimeric antigen-binding fragment. In one embodiment, the antibody fragment is an sdAB.

[0015] In one embodiment, the antibody or antigen-binding fragment comprises one or more amino acids selected from the group consisting of D-amino acids, modified amino acids, amino acid analogs, or a combination thereof.

[0016] In one embodiment, the modified amino acid comprises a modification selected from the group consisting of methylation, amidation, acetylation, and / or substitution with other chemical groups.

[0017] In one embodiment, the antibody or antigen-binding fragment is modified by pegylation, acetylation, glycosylation, biotinylation, or prenylation.

[0018] In one embodiment, the antibody or antigen-binding fragment is a human, murine, llama, rabbit, sheep, or goat antibody or antigen-binding fragment thereof.

[0019] In certain embodiments, the binding agent, antibody, or antigen-binding fragment is in a pharmaceutical composition that further comprises a pharma- ceutically acceptable carrier.

[0020] In another aspect, a method for treating or preventing a disorder in a human subject in need thereof is disclosed, comprising administering to the subject an effective amount of a binding agent, an antibody or antigen-binding fragment thereof, or a pharmaceutical composition described in the present disclosure. Also provided is the use of a binding agent, an antibody or antigen-binding fragment thereof, or a pharmaceutical composition described in the present disclosure for treating or preventing a disorder in a human subject in need thereof.

[0021] In one embodiment, the disorder is selected from the group consisting of acute kidney injury, sepsis-induced conditions, and tumor metastasis. In one embodiment, the acute kidney injury includes an ischemia-reperfusion-induced condition, a pigment-induced condition, a toxin-induced condition, or a drug-induced condition. In one embodiment, the sepsis-induced condition includes a bacterial or viral sepsis-induced condition. In one embodiment, the viral sepsis-induced condition includes a COVID-19 sepsis-induced condition. In one embodiment, the sepsis-induced condition is associated with acute respiratory distress syndrome, encephalopathy, liver failure, kidney failure, or heart failure.

[0022] In one embodiment, the tumor metastasis is associated with pancreatic cancer, renal cancer, genitourinary cancer, melanoma, prostate cancer, lung cancer, breast cancer, thyroid cancer, brain cancer, ovarian cancer, cervical cancer, endometrial cancer, primary peritoneal cancer, mesothelioma, eye cancer, muscle, lymphoma, esophageal cancer, gastric cancer, liver cancer, small intestine tumor, colon cancer, testicular cancer, skin cancer, or adrenal cancer. In one embodiment, the kidney cancer is renal cell carcinoma (RCC). In one embodiment, the genitourinary cancer is urothelial carcinoma in the bladder, kidney, pelvis, or ureter. In one embodiment, the lung cancer is non-small cell carcinoma, small cell carcinoma, or neuroendocrine carcinoma. In one embodiment, the neuroendocrine carcinoma is a carcinoid tumor. In one embodiment, the breast cancer is ductal carcinoma, lobular carcinoma, or mixed ductal and lobular carcinoma. In one embodiment, the thyroid cancer is papillary, follicular, or medullary thyroid carcinoma. In one embodiment, the brain cancer is a meningioma, astrocytoma, glioblastoma, cerebellar tumor, or medulloblastoma. In one embodiment, the ovarian cancer is a serous, mucinous, or endometrioid type. In one embodiment, the cervical cancer is an intraepithelial squamous cell carcinoma, an invasive squamous cell carcinoma, or an endocervical adenocarcinoma. In one embodiment, the endometrial cancer is an endometrioid, serous, or mucinous type. In one embodiment, the mesothelioma is pleural or peritoneal. In one embodiment, the eye cancer is a retinoblastoma. In one embodiment, the muscle cancer is a rhabdomyosarcoma or a leiomyosarcoma. In one embodiment, the esophageal cancer is an adenocarcinoma or a squamous cell carcinoma. In one embodiment, the gastric cancer is a gastric adenocarcinoma or a gastrointestinal stromal tumor. In one embodiment, the liver cancer is a hepatocellular carcinoma or a cholangiocarcinoma. In one embodiment, the small intestinal tumor is a small intestinal stromal tumor or a carcinoid tumor. In one embodiment, the colon cancer is colon adenocarcinoma, colon high grade dysplasia, or colon carcinoid tumor. In one embodiment, the skin cancer is melanoma or squamous cell carcinoma.

[0023] In one embodiment, the disorder is selected from the group consisting of inflammation, ischemia-reperfusion injury, and ischemia-reperfusion injury-related disorder. In one embodiment, the disorder is inflammation. In one embodiment, the inflammation comprises organ inflammation. In one embodiment, the inflammation is associated with an inflammatory disorder selected from the group consisting of gastritis, gout, gouty arthritis, arthritis, rheumatoid arthritis, renal failure, lupus, asthma, psoriasis, pancreatitis, allergy, fibrosis, surgical complications, anemia, fibromyalgia, cancer, heart attack, congestive heart failure, stroke, aortic stenosis, arteriosclerosis, osteoporosis, multiple sclerosis, Alzheimer's disease, Parkinson's disease, ulcer, chronic bronchitis, asthma, allergy, acute lung injury, lung inflammation, airway hyperresponsiveness, vasculitis, septic shock, inflammatory skin disease, psoriasis, atopic dermatitis, eczema, and inflammatory bowel disease. In one embodiment, the inflammatory bowel disease is Crohn's disease or ulcerative colitis.

[0024] In one embodiment, the ischemia-reperfusion injury-related disorder is related to ischemic and post-ischemic events in organs and tissues, and the disorder is selected from the group consisting of thrombotic stroke, myocardial infarction, angina pectoris, embolic vascular occlusion, peripheral vascular insufficiency, visceral artery occlusion, arterial occlusion by thrombus, arterial occlusion by embolism, arterial occlusion by non-occlusive process, mesenteric artery occlusion, mesenteric vein occlusion, ischemia-reperfusion injury to mesenteric microcirculation, ischemic acute renal failure, ischemia-reperfusion injury to brain tissue, intussusception, hemodynamic shock, tissue dysfunction, organ failure, restenosis, atherosclerosis, thrombosis, platelet aggregation, shock liver, spinal cord injury, or brain injury.In one embodiment, the arterial occlusion by non-occlusive process is arterial occlusion after low mesenteric flow or after sepsis.In one embodiment, the organ failure is heart failure, liver failure, kidney failure, etc.In one embodiment, the ischemia-reperfusion injury is caused by a surgical procedure. In one aspect, the surgical procedure is a peri-operative procedure, cardiac surgery, organ surgery, organ transplantation, angiography, cardiopulmonary resuscitation, or cerebral resuscitation.

[0025] In one embodiment, ischemia-reperfusion injury is associated with harvesting of donor organs for transplantation.In one embodiment, ischemia-reperfusion injury occurs in allograft organs during donor procurement, ex vivo handling, or transplantation into transplant recipients.In one embodiment, the subject in need thereof is an organ donor or organ recipient for transplantation.

[0026] In one embodiment, a kit is disclosed herein that includes the composition disclosed herein. In one embodiment, the kit further includes a pharma- ceutically acceptable carrier.

[0027] Other features and advantages of the present disclosure will become apparent from the following detailed description. It should be understood, however, that the detailed description, while indicating preferred implementations of the present disclosure, is given by way of illustration only, since various changes and modifications within the spirit and scope of the disclosure will become apparent to those skilled in the art from the detailed description. [Brief description of the drawings]

[0028] [Figure 1-1] Figure 1 shows the thermal unfolding curves of VHHs. The thermal unfolding midpoint temperature (Tm) of VHHs was determined using circular dichroism spectroscopy by following the unfolding of VHHs at a concentration of 200 μg / mL and a wavelength of 205 nm in 100 mM phosphate buffer pH 7.4. The raw data was converted to percent folded (%) as described in Example 2, and the Tm (temperature of denaturation midpoint) was determined by Boltzmann curve fitting to the plot of % folded vs. temperature. [Figure 1-2] This is a continuation of Figure 1-1. [Figure 1-3] This is a continuation of Figure 1-2. [Figure 2A-1]Figure 1 shows surface plasma resonance (SPR) sensorgrams showing single cycle kinetic analysis of VHH binding to human DPEP-1. Recombinant human DPEP-1 was biotinylated as described in Example 3 and captured onto a CM5 sensor chip surface using Biotin CAPture reagent, followed by flowing VHHs across the surface at concentrations ranging from 0.625-10 nM (sdABP02), 2.5-40 nM (sdABP03 / 05 / 07), 6.25-100 nM (sdABP06), and 12.5-200 nM (sdABP01 / 04). Dark lines represent data points and light lines are fits to the data. Data was generated in triplicate and one replicate set of data is shown. [Figure 2A-2] This is a continuation of Figure 2A-1. [Figure 2A-3] This is a continuation of Figure 2A-2. [Figure 2B] Figure 2B shows the on / off rate map summarizing the kinetic rate constants k, k of the VHHs obtained in Figure 2A. The diagonal line represents the equilibrium dissociation constant K. Data points are shown in triplicate. [Figure 3A-1] Binding of DPEP-1 specific VHHs to human DPEP-1 displayed on cells is shown. Flow cytometry binding analysis of biotinylated VHHs at 100 nM concentration to HEK-293T overexpressing human DPEP-1 (HEK-293T-hDPEP1+; right profile of each graph). Clostridium difficile toxin A specific A20.1 VHH (Hussack et al., J Biol Chem. 286: 8961-8976, (2011)) was included as a negative VHH control (left profile of each graph). Spectral shift to the right as seen in the case of DPEP-1 specific VHHs indicates binding to human DPEP-1. No binding was seen (i.e. no spectral shift to the right) when the indicated VHHs were tested against HEK293T-PARENTAL, which is negative for hDPEP-1 expression. [Figure 3A-2] This is a continuation of Figure 3A-1. [Figure 3B]Flow cytometric analysis of biotinylated VHHs. Dose-response binding of human DPEP-1-specific VHHs to human DPEP-1 displayed on cells was determined. Flow cytometric analysis of biotinylated VHHs was performed on HEK-293-6E cells overexpressing human DPEP-1 (hDPEP-1) with increasing VHH concentrations. Clostridium difficile toxin A-specific A20.1 VHH (Hussack et al., J Biol Chem., 286: 8961-8976

[2011] ) was included as a negative VHH control. [Figure 3C-1] The graphs in Figure 3B show groupings with similar maximum fluorescence plateaus compared to the negative control A20.1 VHH. EC50 values, the VHH concentration at 50% binding, were calculated from the graphs and recorded in Table 5. [Figure 3C-2] This is a continuation of Figure 3C-1. [Figure 3C-3] This is a continuation of Figure 3C-2. [Figure 3D] Immunoprecipitation of hDPEP-1 from HEK293-6E cells overexpressing hDPEP-1 is shown. A20.1 VHH specific for Clostridium difficile toxin A was included as a negative control. MW, protein molecular weight marker. hDPEP-1 ECD, hDPEP-1 extracellular domain. [Figure 4A] A summary depiction of epitope bins identified by SPR is shown. sdABP01, sdABP06, sdABP02, and sdABP07 defined bins (i), (ii), (iii), and (iv), respectively. sdABP03 and 05 partially overlap with bin (iv), while sdABP04 partially overlaps with bin (iii). [Figure 4B]Heatmap of epitope binning by ELISA. Competitive sandwich ELISA was performed to cluster VHHs by epitope and represented as a heatmap displaying all possible pairwise combinations of VHHs (7 x 7 = 49). Binding pairs showing high binding signal (dark) recognized non-overlapping epitopes and therefore were considered to belong to different epitope bins or VHH clusters, whereas binding pairs showing no or weak binding signal (light) recognized overlapping epitopes and therefore were considered to belong to the same epitope bin. The A20.1 VHH specific for Clostridium difficile toxin A, included as a negative control, showed no signal as expected. [Figure 5A] Representative immunofluorescence images of sdABP07 reducing monocyte infiltration (i.e., renal inflammation) in the kidneys of LysMgfp / gfp mice treated with LPS. Left panel: naive; middle panel: LPS; right panel: LPS+sdABP07. [Figure 5B] Graph showing renal inflammation quantified by the number of adherent LysM+ monocytes found per field in the kidneys of LysMgfp / gfp mice treated with LPS. sdABP07 reduced LPS-induced renal inflammation. [Figure 6-1] Figure 2 shows the SEC profile of human DPEP-1 VHH-Fc. The SEC profile demonstrates that the VHH-Fc is free of aggregation and has an elution volume (Ve) consistent with its monomeric state. SEC was performed using a Superdex® 200 Increase column. mAU, milli-absorbance units. [Figure 6-2] This is a continuation of Figure 6-1. [Figure 7]Figure 1 shows the binding profile of VHH-Fc to human DPEP-1 obtained by ELISA. All nine VHH-Fc (sdABP01-09) bound to human DPEP-1 in a dose-dependent manner. Clostridium difficile toxin A-specific A20.1 VHH-Fc (Hussack et al., J Biol Chem. 286: 8961-8976,

[2011] ) was included as a negative VHH control. Data represent the average of three independent experiments. [Figure 8A] Results from assays of binding specificity and cross-reactivity of DPEP-1 VHH-Fc by flow cytometry are shown. Flow cytometry binding analysis was performed at a VHH-Fc concentration of 125 nM on HEK-293-6E cells overexpressing human DPEP-1. Clostridium difficile toxin A-specific A20.1 VHH-Fc (Hussack et al., J Biol Chem. 286: 8961-8976, 2011) was included as a negative VHH control. pAb is a rabbit anti-human DPEP-1 polyclonal antibody positive control. Binding of VHH-Fc to cells was detected using anti-human:PE, and binding of pAb was detected by using anti-rabbit:PE. Anti-human:PE and anti-rabbit:PE show negative binding assays when VHH-Fc and pAb were omitted, respectively. The dotted lines demarcate the background signal. Measurements were performed in quadruplicate. [Figure 8B] Figure 8 shows the results from the assay of binding specificity and cross-reactivity of DPEP-1 VHH-Fc by flow cytometry. Flow cytometry binding analysis was performed on HEK-293-6E cells overexpressing mouse DPEP-1 at a VHH-Fc concentration of 125 nM. The positive and negative controls and secondary antibodies used were the same as in Figure 8A. The dotted lines demarcate the background signal. Measurements were performed in quadruplicate. [Figure 8C]Figure 8 shows the results from the assay of binding specificity and cross-reactivity of DPEP-1 VHH-Fc by flow cytometry. Flow cytometry binding analysis was performed on HEK-293-6E cells overexpressing rat DPEP-1 at a VHH-Fc concentration of 125 nM. The positive and negative controls and secondary antibodies used were the same as in Figure 8A. The dotted lines demarcate the background signal. Measurements were performed in quadruplicate. [Figure 8D] Figure 8 shows the results from an assay for binding specificity and cross-reactivity of DPEP-1 VHH-Fc by flow cytometry. Flow cytometry binding analysis was performed at a VHH-Fc concentration of 125 nM on HEK-293-6E cells overexpressing human DPEP-2. The positive and negative controls and secondary antibodies used were the same as in Figure 8A. The dotted lines demarcate the background signal. Measurements were performed in quadruplicate. [Figure 8E] Figure 8 shows the results from an assay for binding specificity and cross-reactivity of DPEP-1 VHH-Fc by flow cytometry. Flow cytometry binding analysis was performed on parental HEK-293-6E cells at a VHH-Fc concentration of 125 nM. The positive and negative controls and secondary antibodies used were the same as in Figure 8A. The dotted lines demarcate the background signal. Measurements were performed in quadruplicate. [Figure 9] Dose-response binding of VHH-Fc to human DPEP-1 expressing HEK293-6E cells obtained by flow cytometry is shown. Calculated apparent EC50 (EC50app) obtained from the graph is reported in Table 7. None of the VHH-Fc bound to parental non-DPEP-1 expressing HEK293-6E cells at 125 nM (see FIG. 8E). Clostridium difficile toxin A specific A20.1 VHH (Hussack et al., J Biol Chem., 286: 8961-8976

[2011] ) was included as a negative VHH control. Measurements were in triplicate. [Figure 10]Figure 8 shows the cross-reactivity evaluation of VHH-Fc of sdABP05 and sdABP06 against mouse DPEP-1 by flow cytometry. Dose-response curves were obtained by titrating sdABP05 and sdABP06 against human DPEP-1 expressing HEK293-6E cells (left) and mouse DPEP-1 expressing HEK293-6E cells (right). sdABP05 and sdABP06 cross-reacted with mouse DPEP-1, and sdABP07 did not (see Figure 8B). The calculated apparent EC50 (EC50app) obtained from the graph is reported in Table 9. Measurements were in duplicate. [Figure 11] Figure 1 shows epitope typing of human DPEP-1 VHH-Fc by SDS-PAGE / Western blot against denatured recombinant DPEP1 from two different commercial sources. The presence of blot (binding signal) indicates that VHH-Fc of sdABP05, sdABP06, sdABP03, and sdABP08 recognize linear epitopes. The absence of binding signal for VHH-Fc of sdABP01, sdABP02, sdABP04, sdABP07, and sdABP09 indirectly indicates that these VHH-Fc recognize conformational epitopes (data not shown). Anti-human DPEP-1 polyclonal antibody (pAb; Proteintech, Cat. No. 12222-1-AP) was included as a reference. C, human DPEP1 (Creative biomart, Cat. No. DPEP1-77H); S, human DPEP1 (SinoBiologicals, Cat. No. 13543-H08H). Images were acquired with an exposure time of 5 s (left panel) or 30 s (right panel). Molecular weights (MW) are in kDa. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0029] Detailed Description I. Definition In understanding the scope of the present disclosure, the term "comprising" and its derivatives used herein are intended to be open-ended terms that specify the presence of the stated features, elements, components, groups, integers, and / or steps, but do not exclude the presence of other unstated features, elements, components, groups, integers, and / or steps. The foregoing also applies to words with similar meanings, such as the terms "including," "having," and their derivatives. Finally, terms of degree, such as "substantially," "about," and "approximately," used herein, refer to a reasonable amount of deviation of the modified term such that the end result is not significantly altered. These terms of degree should be interpreted as including at least ±5% deviation of the modified term, if this deviation does not negate the meaning of the modified word.

[0030] When a term is provided in the singular, the inventors also contemplate aspects of the present disclosure that are described by the plural of that term.As used herein and in the appended claims, the singular forms "a", "an" and "the" include plural references unless the context clearly dictates otherwise, e.g., "antibody" includes a plurality of antibodies.Thus, for example, reference to "method" includes one or more methods and / or steps of the type described herein and / or that will be apparent to a person skilled in the art upon reading this disclosure.

[0031] The terms "administer," "administering," or "administered" refer to the act of providing an agent or therapeutic treatment to a physiological system (e.g., a subject, or to cells, tissues, and organs in vivo, in vitro, or ex vivo).

[0032] The term "affinity" as used herein refers to the strength of the sum of non-covalent interactions between a single binding site of a molecule and its binding partner. Unless otherwise indicated, as used herein, "binding affinity" refers to the intrinsic binding affinity, which reflects a 1:1 interaction between members of a binding pair. The affinity of a molecule X for its partner Y is generally determined by the equilibrium dissociation constant (K D Affinity can be measured by common methods known in the art.

[0033] The term "amino acid" refers to naturally occurring amino acids, as well as non-naturally occurring or non-standard amino acids, such as amino acid analogs, synthetic amino acids, and amino acid mimetics. These amino acids may be in the L- or D- (isomer) configuration, or may include both dextrorotatory forms. The amino acids incorporated into the antibody are referred to as "residues." Amino acids may be referred to herein by either their commonly known three-letter symbols, or the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission. The following amino acid definitions are used throughout the specification: Alanine: Ala (A), Arginine: Arg (R), Asparagine: Asn (N), Aspartic acid: Asp (D), Cysteine: Cys (C), Glutamine: Gln (Q), Glutamic acid: Glu (E), Glycine: Gly (G), Histidine: His (H), Isoleucine: Ile (I), Leucine: Leu (L), Lysine: Lys (K), Methionine: Met (M), Phenylalanine: Phe (F), Proline: Pro (P), Serine: Ser (S), Threonine: Thr (T), Tryptophan: Trp (W), Tyrosine: Tyr (Y), Valine: Val (V).

[0034] The term "binding agent" as used herein refers to a ligand, including an antibody or antigen-binding fragment thereof, that forms a complex with a receptor. The ligand may be selective or non-selective. The ligand may be an agonist (partial or complete), an antagonist (i.e., blocks the action of an agonist), an inverse agonist (i.e., exerts the opposite action of an agonist), or an allosteric modulator. The antagonist may be a competitive antagonist (i.e., binds at the same site as the agonist) or a non-competitive antagonist (i.e., binds permanently at the same site as the agonist or binds at an allosteric site that is a site other than the active site). In some embodiments, the binding agent, antibody or antigen-binding fragment thereof, or composition of the present disclosure binds to and / or inhibits DPEP-1. In some embodiments, the binding agent, antibody or antigen-binding fragment thereof, or composition of the present disclosure reduces DPEP-1 regulatory function. In some embodiments, DPEP-1 regulating functions include leukocyte recruitment, inflammation, and tumor cell adhesion. In some embodiments, the binding agents, antibodies or antigen-binding fragments thereof, or compositions of the present disclosure do not affect DPEP-1 dipeptidase activity and / or its role in regulating renal tubular transport.

[0035] The terms "diagnosed", "diagnostic" or "diagnosis" mean to identify the presence or nature of a pathological condition. Diagnostic methods include observations and assays, which vary in their sensitivity and specificity. The "sensitivity" of a diagnostic observation or assay is the percentage of diseased individuals who test positive (percent of "true positives"). Diseased individuals not detected by the observation or assay are "false negatives". Subjects who are not diseased and test negative in the observation or assay are called "true negatives". The "specificity" of a diagnostic observation or assay is 1 minus the false positive rate, where the "false positive" rate is defined as the proportion of people without the disease who test positive. While a particular diagnostic method may not provide a definitive diagnosis of a condition, it is sufficient if the method provides a positive indication that aids in diagnosis.

[0036] As used herein, the term "effective amount" refers to an amount of a therapy (e.g., a prophylactic or therapeutic agent) that is sufficient to effect beneficial or desired results, including clinical results. An effective amount can be administered in one or more administrations.

[0037] As used herein, the term "inflammatory disease" refers to diseases (treatable or preventable with the compounds described herein) including, but not limited to, a) recruitment, adhesion, or activation of leukocytes, and other disorders involving neutrophils, monocytes, lymphocytes, or macrophages, b) diseases involving pathological production of inflammatory cytokines (e.g., TNF-α, interleukin (IL)-1β, IL-2, IL-6), and / or c) activation of nuclear factors that promote the transcription of genes encoding inflammatory cytokines. Examples of these nuclear transcription factors include, but are not limited to, nuclear factor-κB (NFκB), activator protein-1 (AP-1), and nuclear factor of activated T cells (NFAT).

[0038] As used herein, the term "ischemia-reperfusion injury" refers to the damage initially caused by the restriction of blood supply to a tissue (ischemia), followed by re-supply of blood (reperfusion), and the concomitant production of free radicals, inflammation, and cell death that result in organ injury and dysfunction. In a transplant scenario, ischemia-reperfusion injury negatively impacts the function of the allograft.

[0039] The term "isolated" as used herein refers to a substance being removed from its original environment (e.g., the natural environment, if it occurs in nature). For example, a naturally occurring antibody present in a living animal is not isolated, but the same antibody separated from some or all of the coexisting substances in the natural system is isolated. In some embodiments, the antibody of the present disclosure is an isolated antibody.

[0040] The term "pharmaceutically acceptable carrier" refers to any such carrier known to those skilled in the art to be suitable for a particular mode of administration.For example, the term "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, etc., that can be used as a medium for a pharmaceutically acceptable substance.In addition, the active substance can also be mixed with other active substances that do not impair the desired action, or with substances that complement the desired action or have another action.

[0041] The term "pharmaceutically acceptable salts" as used herein refers to salts that are known to be non-toxic and are commonly used in pharmaceutical literature.Typical inorganic acids used to form such salts include hydrochloric acid, hydrobromic acid, hydroiodic acid, nitric acid, sulfuric acid, phosphoric acid, hypophosphorous acid, etc.Salts derived from organic acids such as aliphatic mono- and dicarboxylic acids, phenyl-substituted alkanoic acids, hydroxyalkanoic and hydroxyalkandioic acids, aromatic acids, aliphatic and aromatic sulfonic acids, etc. may also be used. Such pharma- ceutically acceptable salts include acetate, phenylacetate, trifluoroacetate, acrylate, ascorbate, benzoate, chlorobenzoate, dinitrobenzoate, hydroxybenzoate, methoxybenzoate, methylbenzoate, o-acetoxybenzoate, naphthalene-2-benzoate, bromide, isobutyrate, phenylbutyrate, β-hydroxybutyrate, chloride, cinnamate, citrate, formate, fumarate, glycolate, heptanoate, lactate, maleate, hydroxymaleate, malonate, mesylate, nitrate, oxalate, phthalate, phosphate, monohydrogenphosphate, dihydrogenphosphate, metaphosphate, pyrophosphate, propionate, phenylpropionate, salicylate, succinate, sulfate, bisulfate, pyrosulfate, sulfite, bisulfite, sulfonate, benzenesulfonate, These include p-bromophenylsulfonate, chlorobenzenesulfonate, ethanesulfonate, 2-hydroxyethanesulfonate, methanesulfonate, naphthalene-1-sulfonate, naphthalene-2-sulfonate, p-toluenesulfonate, xylenesulfonate, tartrate, and the like.

[0042] The term "prevent" or equivalents, such as "prevention" or "preventing", refers to reducing the occurrence of a disorder or condition in a treated sample compared to a non-treated control sample, or delaying the onset or reducing the severity of one or more symptoms of a disorder or condition compared to a non-treated control sample.As used herein, preventing ischemia-reperfusion injury includes preventing oxidative damage or preventing mitochondrial permeability transition, thereby preventing or ameliorating the harmful effects of loss of blood flow to affected organs and subsequent recovery.Preventing does not mean that a subject never develops the condition later in life, but that the probability of occurrence is reduced.

[0043] The terms "reducing," "reducing," or "reduction" in the context of a disease or condition herein refer to a decrease in the cause, symptoms, or effects of the disease or condition. Thus, in the disclosed methods and uses, "reducing" can refer to a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% decrease in the amount of reperfusion injury, or any value or range therebetween.

[0044] As used herein, the term "subject" or "patient" or cognates thereof includes all members of the animal kingdom, particularly mammals, including humans. A subject or patient is preferably a human.

[0045] The term "transplantation" refers to a surgical procedure in which cells, tissues, or organs are transferred from a donor subject to a recipient subject, or from one part of the body to another part in the same subject. A "donor subject" or "donor" is a subject that gives blood, cells, tissue, or organs to another subject by blood transfusion or organ transplantation. A donor subject may be a human or another mammal. A "recipient subject" or "recipient" is a subject that receives blood, cells, tissue, or organs from another subject by blood transfusion or organ transplantation.

[0046] As used herein, the terms "treat", "treatment" and "treating" refer to the prevention, reduction, or amelioration of the progression, severity, and / or duration of at least one symptom and / or symptom of any condition or disease. The terms "treatment" or "treating" refer to any administration or use of the compounds disclosed herein, and include (i) inhibiting a disease or disease state in an individual experiencing or exhibiting a symptom or symptom of the disease or disease state (i.e., preventing further progression of the symptom and / or symptom), or (ii) ameliorating a disease in an individual experiencing or exhibiting a symptom or symptom of the disease or disease state (i.e., reversing the symptom and / or symptom). The term "controlling" includes preventing, treating, eradicating, ameliorating, or otherwise reducing the severity of a symptom of a disease or disease state.

[0047] When referring to inflammation, the terms "treat", "treatment", and "treating" refer to the reduction or amelioration of the progression, severity, and / or duration of inflammation, or one or more symptoms thereof, resulting from the administration or use of one or more therapies (e.g., one or more prophylactic and / or therapeutic agents described herein). "Treatment" can also mean extending survival compared to the expected survival in the absence of treatment. The magnitude of this effect can be measured using standard methods, e.g., in vitro assays using purified enzymes, cell-based assays, animal models, or human studies. In exemplary embodiments, the treatment of inflammation comprises one or more of the following: reducing or improving the progression, severity, and / or duration of inflammation associated with inflammatory disorders selected from the group consisting of gastritis, gout, gouty arthritis, arthritis, rheumatoid arthritis, renal failure, lupus, asthma, psoriasis, pancreatitis, allergy, fibrosis, surgery complications, anemia, fibromyalgia, cancer, heart attack, congestive heart failure, stroke, aortic valve stenosis, arteriosclerosis, osteoporosis, multiple sclerosis, Alzheimer's disease, Parkinson's disease, ulcer, chronic bronchitis, asthma, allergy, acute lung injury, pulmonary inflammation, airway hyperresponsiveness, vasculitis, septic shock, inflammatory skin disease, psoriasis, atopic dermatitis, eczema, and inflammatory bowel disease, such as Crohn's disease and ulcerative colitis.Sepsis is an inflammatory immune response induced by infection, for example, bacterial infection or viral infection. In some embodiments, treating inflammation includes reducing or ameliorating the progression, severity, and / or duration of inflammation during sepsis in a subject. In some embodiments, sepsis includes bacterial sepsis or viral sepsis. In some embodiments, viral sepsis includes COVID-19 induced sepsis. Inflammation may also be associated with ischemia-reperfusion or acute kidney injury. In some embodiments, treating inflammation includes inflammation associated with ischemia-reperfusion injury or acute kidney injury. Treating inflammation may also include reducing inflammation or modifying the inflammation profile of tissue, for example, in lung, liver, or kidney tissue. In some embodiments, treating inflammation includes inflammation in lung, liver, or kidney tissue.

[0048] In the context of cancer, the terms "treat", "treatment" and "treating" refer to the reduction or amelioration of the progression, severity and / or duration of cancer, particularly solid tumors, or one or more symptoms thereof, resulting from the administration or use of one or more therapies (e.g., one or more prophylactic and / or therapeutic agents). In exemplary embodiments, treatment of solid tumors refers to one or more of the following: (i) reducing the number of cancer cells; (ii) increasing tumor cell apoptosis; (iii) reducing tumor size; (iv) reducing tumor volume; (v) inhibiting, delaying, slowing to some extent, and / or stopping cancer cell invasion into peripheral organs; (vi) inhibiting (e.g., slowing to some extent or stopping) tumor metastasis; (vii) inhibiting tumor growth; (viii) preventing or delaying the onset and / or recurrence of tumors; (ix) reducing cancer markers associated with the presence of cancer; and / or (ix) alleviating to some extent one or more symptoms associated with cancer. "Treatment" can also mean extending survival compared to the expected survival if not treated. Standard methods can be used to measure the magnitude of this effect, for example, in vitro assay using purified enzyme, cell-based assay, animal model, or human test.For example, immunohistochemical analysis of cancer tumors of patients can show a significant increase in tumor cell apoptosis when the compositions disclosed herein are administered to patients or used in subjects.When referring to the type of cancer that usually appears as solid tumors, a "clinically detectable" tumor is one that can be detected based on tumor mass, for example, by procedures such as CAT scan, MR imaging, X-ray, ultrasound, or palpation, and / or can be detected due to the expression of one or more cancer-specific antigens in samples that can be obtained from patients.

[0049] The recitations herein of numerical ranges by endpoints include all numbers and fractions subsumed within that range (e.g., 1 to 5 includes, for example, 1, 1.5, 2, 2.75, 3, 3.90, 4, and 5). It is also to be understood that all numbers and fractions thereof are presumed to be modified by the term "about."

[0050] II. DPEP-1 DPEP-1, also known as renal dipeptidase, microsomal dipeptidase, or dehydropeptidase-1, is a cell membrane glycosylphosphatidylinositol-anchored glycoprotein currently classified as EC 3.4.13.19 (previously EC 3.4.13.11) (Keynan et al., in Hooper (Ed.) Zinc Metalloproteases in Health and Disease Taylor and Francis, London pages 285-309 (1996), incorporated herein by reference). This zinc metalloprotease is primarily expressed in the lung, liver, and kidney, including the renal endothelium and renal proximal tubule brush border (Chaudhury et al, Cell 178(5), 1205-1221 (2019), incorporated herein by reference), and is involved in the renal metabolism of glutathione and the pulmonary metabolism of peptidyl leukotrienes in vivo. In addition, DPEP-1 is the only known example of a mammalian β-lactamase and is also involved in the metabolism of glutathione and its conjugates, as well as leukotriene D4. DPEP-1 forms disulfide-linked homodimers, with the molecular weight of the monomers ranging from about 48 to 59 kDa depending on the species of origin (Keynan et al., Biochem. 35:12511-12517 (1996), incorporated herein by reference; see also Example IVB).

[0051] Dipeptidase expression has been detected in several tissues, but is primarily expressed in the lung, liver, and kidney. There have been reports of low levels of DPEP-1 activity in total extracts from spleen, small intestine, and brain, but other reports have found no detectable activity in these organs. There are four distinct DPEP-1 mRNAs in mice, which are differentially expressed in several organs (Habib et al., J. Biol. Chem. 271:16273-16280 (1996)). Organ-specific differences in the nature and extent of N-linked glycosylation of porcine DPEP-1 have also been reported (Hooper et al., Biochem. J. 324:151-157 (1997)).

[0052] The levels of DPEP-1 activity are highest in the kidney and lung (Hirota et al., Eur. J. Biochem. 160:521-525 (1986); Habib et al., Proc. Natl. Acad. Sci. USA 95:4859-4863 (1998)). In the kidney, DPEP-1 expression is restricted to epithelial cells in the brush border region of the proximal tubule and endothelial cells surrounding the capillaries. In the lung, DPEP-1 expression has been detected in many cell types, including endothelial and epithelial cells of the conducting airways, alveolar ducts, capillaries, and the basement membrane of the alveoli and terminal bronchi (Habib et al., supra, 1996; Inamura et al., Prostaglandins Leukotrienes and Essential Fatty Acids 50:85-92 (1994)). DPEP-1 expression has also been observed in endothelial cells of submucosal microvessels in the human trachea (Yamaya et al., Resp. Physiol. 111:101-109 (1998)). The expression pattern of DPEP-1 in the lung correlates with the strong lung homing of other DPEP-1-binding peptides, such as GFE-1 (Rajotte & Ruoslahti, Journal of Biological Chemistry, 274(17), 11593-11598 (1999)).

[0053] The DPEP-1 receptor functions as a leukocyte or tumor cell adhesion molecule expressed on vascular endothelium or other parenchymal cells such as renal tubular epithelium (Choudhury et al. 2019, "Dipeptidase-1 is an adhesion receptor for neutrophil recruitment in lungs and liver" Cell 178, 1205-1221; Lau, A, et al. "Dipeptidase-1 governs renal inflammation during ischemia reperfusion injury." Science advances 8.5 (2022): eabm0142). Adhesion molecules are surface-bound glycoprotein molecules that are involved in the recruitment process and are expressed on leukocytes and / or endothelial cells. A key step in leukocyte recruitment is the firm adhesion of leukocytes on the surface of the endothelium, which positions them to migrate into the vascular wall through a series of adhesion and activation events to exert their effects on the site of inflammation. DPEP-1 can also function as an innate immune receptor during organ injury or infection. One such detection system is the so-called pattern recognition receptors (PRRs) that detect key molecular signatures of invading pathogens, i.e., pathogen-associated molecular patterns (PAMPs), or endogenous damage-associated molecular patterns (DAMPs), thereby triggering the innate immune system (Janeway, C, et al., Annu. Rev. Immunol, 20 (2002), pp. 197-216). An example of a PRR is the toll-like receptor (TLR), which detects bacterial or viral products such as LPS (TLR4) or peptidoglycan (TLR2) (Bell, JK et al., Trends Immunol, 24 (2003), pp. 528-533).

[0054] TLRs are transmembrane receptors that recognize pathogen-associated molecular patterns (PAMPs) through leucine-rich repeats (LRRs) in their extracellular domains that are involved in ligand binding and autoregulation (Kawai et al, Cell Death Differ. 13, 816-825, 2006). TLRs recognize microbial structures early in the host defense response and induce the expression of many immune and inflammatory genes whose products are tailored to drive the immune mechanisms required to eliminate invading pathogens. TLRs are also involved in the recognition of damage-associated molecular patterns (DAMPs) and are increasingly recognized as regulators of tumor-promoting inflammation and promoters of tumor survival signals. Other activators of such cellular pathways may provide valid therapeutic targets for treating pathogen- and damage-associated cellular inflammation.

[0055] As used herein, the terms "dipeptidase" and "membrane dipeptidase" are synonymous with "DPEP-1" and refer to the enzyme currently classified as EC 3.4.13.19 (formerly EC 3.4.13.11), also known as renal or microsomal dipeptidase, or dehydropeptidase-1.

[0056] The term "selectively inhibit" as used herein with respect to DPEP-1 enzyme activity means that the binding agent reduces DPEP-1 activity in a manner that is selective for DPEP-1 enzyme compared to related but different enzymes, such as other proteases. Thus, DPEP-1 binding agents are distinct from non-specific inhibitors of, for example, zinc metalloproteases. Thus, DPEP-1 binding agents can selectively reduce DPEP-1 activity, but have little or no effect on, for example, the activity of dipeptidyl peptidase IV. In one embodiment, the binding agent is a competitive inhibitor that prevents binding to DPEP-1.

[0057] The term "selectively binds" as used herein with respect to DPEP-1 binding agents, including antibodies or antigen-binding fragments, means that the binding agent reduces DPEP-1-mediated leukocyte recruitment in a manner that is selective for the DPEP-1 receptor compared to a related but different receptor. DPEP-1 binding agents also refer to reducing DPEP-1-mediated leukocyte recruitment, where DPEP-1 acts as an adhesion molecule for leukocytes or tumor cells, independent of its enzymatic activity. Thus, DPEP-1 binding agents can selectively reduce DPEP-1-mediated leukocyte recruitment, but have little or no effect on, for example, the activity of dipeptidyl peptidase IV. In one embodiment, the binding agent is a competitive or non-competitive inhibitor that prevents binding to DPEP-1.

[0058] In one embodiment, the DPEP-1 binding substance disclosed herein is an antagonist of DPEP receptor, i.e., it blocks or attenuates biological response by binding to and blocking the receptor, so as to disrupt the interaction of agonist or inverse agonist and inhibit its function.In a particular embodiment, the DPEP-1 binding substance is a competitive antagonist, i.e., it competes with agonist for active site.In another particular embodiment, the DPEP-1 binding substance is a non-competitive antagonist, i.e., it binds at a site other than active site.

[0059] The term specific binding as used herein includes both low affinity specific binding and high affinity specific binding. Specific binding can be, for example, about 10 -4 M ~ about 10 -7 K for membrane dipeptidase of M D Specific binding can also be exhibited by low affinity DPEP-1 binding molecules, e.g., having a specific affinity of at least about 10 -7 M, at least about 10 -8 M, at least about 10 -9 M, at least about 10 -10 M, or at least about 10 -11M or 10 -12 K for M or higher membrane dipeptidases D The DPEP-1 binding antibody can be, for example, about 2×10 -5 M~10 -7 K for membrane dipeptidase of M D , for example, about 10 as measured by SPR -6 ~10 -7 M, or about 10 -8 M~10 -10 M, or approximately 10 as measured by flow cytometry -9 M~5×10 -7 K of M D Both low and high affinity DPEP-1 binding molecules that selectively bind to pulmonary or renal endothelium may be useful in the methods described herein.

[0060] The term "antibody" as used herein is intended to include monoclonal antibodies, polyclonal antibodies, chimeric antibodies, humanized antibodies, and antigen-binding fragments thereof. Antibodies may be from recombinant sources and / or produced in transgenic animals. The term "antigen-binding fragment" or "antibody fragment" as used herein includes, but is not limited to, Fv (a molecule comprising VL and VH), single chain Fv (scFV; a molecule comprising VL and VH connected by a peptide linker), Fab, Fab', F(ab')2, dsFv, ds-scFv, single domain antibodies (sdAB; V H H, V H , V LThe term is intended to include molecules that contain a single variable domain with three or fewer CDRs, such as IgNAR antigen-binding variable domains (VNARs), and multivalent presentations thereof. Also included are dimers, minibodies, diabodies, and multimers, bispecific and multispecific antigen-binding fragments, and domain antibodies. Antibodies can be fragmented using conventional techniques. For example, F(ab')2 fragments can be generated by treating antibodies with pepsin. The resulting F(ab')2 fragments can be treated to reduce disulfide bridges to produce Fab' fragments. Papain digestion can result in the formation of Fab fragments. Fab, Fab' and F(ab')2, scFv, dsFv, ds-scFv, sdAB, dimers, minibodies, diabodies, bispecific antigen-binding fragments, and other fragments can also be synthesized by recombinant techniques.

[0061] Antibodies against DPEP-1 can be prepared using techniques known in the art, such as those described by Kohler and Milstein, Nature 256, 495 (1975), and in U.S. Patents RE 32,011; 4,902,614; 4,543,439; and 4,411,993, which are incorporated herein by reference. (See also Monoclonal Antibodies, Hybridomas: A New Dimension in Biological Analyses, Plenum Press, Kennett, McKearn, and Bechtol (eds.), 1980, and Antibodies: A Laboratory Manual, Harlow and Lane (eds.), Cold Spring Harbor Laboratory Press, 1988, which are also incorporated herein by reference). Within the context of this disclosure, antibodies include monoclonal antibodies, polyclonal antibodies, chimeric antibodies, humanized antibodies, antigen-binding fragments (e.g., Fab and F(ab')). 2), as well as recombinantly produced binding partners.

[0062] Single-domain antibodies (sdABs) have a single monomeric variable antibody domain. Like whole antibodies, they can selectively bind to a specific antigen. With a molecular weight of only 12-15 kDa, sdABs are much smaller than conventional antibodies (150-160 kDa) that are composed of two protein heavy chains and two light chains, and even smaller than Fab fragments (about 50 kDa, one light chain and half a heavy chain) and single-chain variable fragments (about 25 kDa, two variable domains, one from the light chain and one from the heavy chain) (see Harmsen MM and De Haard HJ (2007). "Properties, production, and applications of camelid single-domain antibody fragments". Applied Microbiology and Biotechnology. 77 (1): 13-22, incorporated herein by reference). The first sdABs were engineered from heavy-chain antibodies found in camelids, and were characterized by the V H These antibodies are called H fragments. The Camelidae family includes camels and llamas. Camelid single domain antibodies have been shown to be as specific as conventional antibodies, and in some cases more robust. Moreover, they are easily isolated using the same phage panning procedures used for traditional antibodies, allowing them to be cultured in vitro at high concentrations. Due to their smaller size and single domain, these antibodies are easier to transform into bacterial cells for bulk production. sdAB has been studied for multiple pharmaceutical applications and has potential for use in the treatment of a myriad of diseases, including but not limited to acute coronary syndromes, cancer, and Alzheimer's disease. sdAB allows for a wide range of applications in therapeutic use due to its small size, easy production, and high affinity.

[0063] In this disclosure, sdAB were generated by immunizing male Lama glama with recombinant human DPEP-1 ectodomain (17-385) (hDPEP-1; Accession Number: P16444; NCBI Reference Sequence: NM_004413) using techniques known in the art (see, e.g., Baral TN, et al., (2013) Single-domain antibodies and their utility. Curr Protoc Immunol. 2013;103:2-17; Henry KA, et al, (2016) Isolation of TGF-β-neutralizing single-domain antibodies of predetermined epitope specificity using next-generation DNA sequencing. Protein Eng Des Sel. 29:439-43; Henry KA, et al (2015) Identification of cross-reactive single-domain antibodies against serum albumin using next-generation DNA sequencing. Protein Eng Des Sel. 29:439-43, all of which are incorporated by reference herein). Sel. 28:379-83). Animals were immunized subcutaneously three times (days 0, 21, and 28) with 200 μg hDPEP-1. The priming immunization was supplemented with complete Freund's adjuvant, and the boosting immunization was supplemented with incomplete Freund's adjuvant. Blood samples were collected on days 0, 28, and 35, serum was obtained after clotting, and peripheral blood mononuclear cells were purified by density gradient centrifugation. The resulting serum was specific for hDPEP-1 and was shown to have a strong positive immune response against human DPEP-1 from two different sources (CreativeBioMart and SinoBiologicals).

[0064] Other methods are known in the art for producing polyclonal antibodies in a host, such as a rabbit or goat, by immunizing the host with an immunogen or immunogen fragment, generally with an adjuvant and optionally coupled to a carrier; the antibodies against the immunogen are collected from the serum. Furthermore, polyclonal antibodies can be absorbed to be monospecific; that is, the serum can be absorbed against the relevant immunogen so that no cross-reactive antibodies remain in the serum, making it monospecific.

[0065] To produce monoclonal antibodies, antibody-producing cells (lymphocytes) can be taken from the immunized animal and fused with myeloma cells by standard somatic cell fusion procedures to immortalize these cells and give rise to hybridoma cells. Such techniques are well known in the art and include, for example, the hybridoma technique originally developed by Kohler and Milstein (Continuous cultures of fused cells secreting antibody of predefined specificity. Nature 256:495-497, 1975), as well as the human B cell hybridoma technique (Kozbor, D, and Roder, J: The production of monoclonal antibodies from human lymphocytes. Immunology Today 4:3 72-79, 1983), the EBV hybridoma technique producing human monoclonal antibodies (Cole et al. Monoclonal Antibodies in Cancer Therapy (1985) Allen R. Bliss, Inc., pages 77-96), and the screening of combinatorial antibody libraries (Huse, WD et al, "Generation of a large combinatorial library of the immunoglobulin repertoire in phage lambda" Science 246:4935 Hybridoma cells can be screened immunochemically for production of antibodies specifically reactive with the protein or a fragment thereof and monoclonal antibodies can be isolated.

[0066] Chimeric antibody derivatives, i.e., antibody molecules that combine non-human animal variable regions and human constant regions, are also contemplated within the scope of this disclosure. Chimeric antibody molecules can, for example, contain an antigen-binding domain derived from a mouse, rat, llama, or other species of antibody, along with a human constant region. Conventional methods can be used to generate chimeric antibodies containing immunoglobulin variable regions that recognize a target (see, e.g., Morrison et al. (Chimeric Human Antibody Molecules: Mouse Antigen-Binding Domains with Human Constant Region Domains. PNAS 81:21 6851-6855, 1984), and Takeda et al. (Construction of chimaeric processed immunoglobulin genes containing mouse variable and human constant region sequences. Nature 314:452-454), as well as U.S. Pat. No. 4,816,567 to Cabilly et al.; U.S. Pat. No. 4,816,397 to Boss et al.; European Patent Publication EP 171496 to Tanaguchi et al.; European Patent Publication 0173494, British Patent GB 2177096B).

[0067] Monoclonal or chimeric antibodies that specifically react with targets as described herein can be further humanized by producing human constant region chimeras, in which part of the variable region, particularly the conserved framework regions of the antigen-binding domain, is of human origin, and only the hypervariable region is of non-human origin. Such immunoglobulin molecules can be produced by techniques known in the art (e.g., Teng et al. (Construction and Testing of Mouse--Human Heteromyelomas for Human Monoclonal Antibody Production. PNAS 80:12 7308-7312, 1983), Kozbor et al., supra; Olsson et al. (Methods in Enzymol, 92:3-16 1982), and PCT Publication WO92 / 06193 or EP 0239400). Humanized antibodies can also be produced commercially (Scotgen Limited, 2 Holly Road, Twickenham, Middlesex, Great Britain).

[0068] To produce recombinant antibodies (see generally Huston et al, 1991; Johnson and Bird, 1991; Mernaugh and Mernaugh, 1995), messenger RNA from animal antibody-producing B lymphocytes, or hybridomas, is reverse transcribed to obtain complementary DNA (cDNA). The antibody cDNA, which can be full-length or partial length, is amplified and cloned into a phage or plasmid. The cDNA can be partial length heavy and light chain cDNAs, either separate or linked by a linker. The antibody or antigen-binding fragment is expressed using a suitable expression system to obtain the recombinant antibody. The antibody cDNA can also be obtained by screening an appropriate expression library.

[0069] III. DPEP-1 Binding Agents and Compositions Binding to or blocking DPEP-1 has utility in treating or preventing disorders in human subjects. For example, binding to or blocking DPEP-1 has utility for reducing inflammation-mediated disease in the lungs and kidneys, for example during sepsis or acute kidney injury. Binding to or blocking DPEP-1 also has utility for reducing tumor metastasis. Disclosed herein are compositions that bind to or block DPEP-1, including but not limited to antibodies. DPEP-1 is a target for therapeutic intervention and has a role as a physical adhesion receptor for neutrophil sequestration, independent of its enzymatic activity (see Choudhury, SR, et al., Cell 178.5 (2019): 1205-1221, incorporated herein by reference). In particular, DPEP-1 is the major adhesion receptor on the endothelium of the lung and liver, and is a therapeutic target for, for example, neutrophil-driven pulmonary inflammatory diseases. DPEP-1 also acts as an adhesion receptor for neutrophils in the hepatic and pulmonary vasculature after stimulation with the bacterial endotoxin lipopolysaccharide (LPS) and represents the major neutrophil adhesion receptor identified on the pulmonary endothelium. The peptide LSALT is a binder of DPEP-1 and has been shown to be useful through targeting DPEP-1 in inhibiting LPS-induced recruitment of neutrophils in the pulmonary vasculature, providing therapeutic benefit in endotoxemic mouse models increasing overall survival, abrogating ischemia-reperfusion-induced acute kidney injury in mice, and reducing tumor burden in animals injected with metastatic cancer cells (Choudhury, SR, et al., Cell 178.5 (2019): 1205-1221; Lau, A, et al. Science advances 8.5 (2022): eabm0142; US20200223888A1; US10493127B2, each of which is incorporated by reference in its entirety). These provide scientific justification for predicting that the presently disclosed antibodies that bind to DPEP-1 will have similar therapeutic effects and provide clinical benefit in human subjects in treating or preventing the diseases or disorders described in this disclosure.

[0070] A. DPEP-1 binding antibody Binding substances such as antibodies that bind to DPEP-1 are disclosed herein.Variants and modified embodiments of these binding antibodies that can be used in these methods are also provided.In one embodiment, the antibody regulates the activity of DPEP-1, and more particularly inhibits the activity of DPEP-1 either competitively or non-competitively.

[0071] Using a phase display library and panning strategy, we isolated a single domain antibody (sdAB; V H We have identified sdABs (also known as hDPEP-1; i.e., sdABP01-09; SEQ ID NOs: 1-9) that can recognize HEK293T cells displaying hDPEP-1 (sdABP01-07; SEQ ID NOs: 1-7), have been shown to have good thermostability, good SPR binding affinity, good dose-response binding to hDPEP-1 displayed on cells, and have been characterized by epitope binning (by SPR: sdABP01-07; by ELISA: sdABP01-04 and 07-09). Each of sdABP01-09 contains a biotinylated acceptor peptide (BAP) and a His6 tag. The core sequences of these sdABP01-09, without the BAP and His6 tags, are shown in SEQ ID NOs: 12-20.

[0072] In one embodiment, the antigen-binding fragment is an Fv, scFv, Fab, Fab', F(ab')2, dsFv, ds-scFv, sdAB, dimeric, minibody, diabody, or multimeric antigen-binding fragment. In one embodiment, the antigen-binding fragment is an sdAB.

[0073] In some embodiments, the antibody or antigen-binding fragment thereof that binds to DPEP-1 contains one or more modifications to increase protease resistance, serum stability, and / or bioavailability. In some embodiments, the modifications are selected from pegylation, acetylation, glycosylation, biotinylation, prenylation, or substitution of the antibody or antigen-binding fragment with D-amino acids and / or non-natural amino acids. The antibody or antigen-binding fragment may contain one or more non-canonical disulfide bonds, for example, at IMGT (ImMunoGeneTics) positions 54 and 78, to increase stability, protease resistance, serum stability, and / or bioavailability (see Hussack, G et al. "Engineered single-domain antibodies with high protease resistance and thermal stability." PloS ONE 6.11 (2011): e28218, incorporated herein by reference). Thus, in some embodiments, an antibody or antigen-binding fragment thereof that binds DPEP-1 contains one or more non-canonical disulfide bonds to increase stability, protease resistance, serum stability, and / or bioavailability. In some embodiments, the one or more non-canonical disulfide bonds are at IMGT positions 54 and 78.

[0074] In certain embodiments, an antibody or antigen-binding fragment thereof that binds to DPEP-1 contains one or more L-amino acids, D-amino acids, and / or non-standard amino acids.

[0075] In various embodiments, the antibody or antigen-binding fragment thereof that binds DPEP-1 further comprises one or more amino acid residues or analogs at the C-terminus, the N-terminus, or both the C-terminus and the N-terminus. In some embodiments, the active sequence of the antibody or antigen-binding fragment is not appreciably affected by the addition of these additional amino acids.

[0076] In another embodiment, the antibody or antigen-binding fragment thereof that binds DPEP-1 further comprises 1, 2, 3, 4, or 5 amino acid residues at the N-terminus.

[0077] In various embodiments, the antibody or antigen-binding fragment is selected from an X-antibody or X-antigen-binding fragment, a XX-antibody or XX-antigen-binding fragment, a XXX-antibody or XXX-antigen-binding fragment, a XXXX-antibody or XXXX-antigen-binding fragment, or a XXXXX-antibody or XXXXX-antigen-binding fragment, where X is any naturally occurring amino acid, or where X is a non-conventional amino acid or amino acid analog as described herein and known to one of skill in the art.

[0078] In another embodiment, the antibody or antigen-binding fragment that binds DPEP-1 further comprises 1, 2, 3, 4, or 5 amino acid residues at the C-terminus and is selected from antibody-X or antigen-binding fragment-X, antibody-XX or antigen-binding fragment-XX, antibody-XXX or antigen-binding fragment-XXX, antibody-XXXX or antigen-binding fragment-XXXX, or antibody-XXXXX or antigen-binding fragment-XXXXX, where X is any naturally occurring amino acid, or X is a non-conventional amino acid or amino acid analog as described herein and known to one of skill in the art.

[0079] In one embodiment, the antibody or antigen-binding fragment that binds DPEP-1 further comprises 1, 2, 3, 4, or 5 amino acid residues at the N-terminus and C-terminus of the antibody or antigen-binding fragment and is designated as X-antibody or antigen-binding fragment-X, X-antibody or antigen-binding fragment-XX, X-antibody or antigen-binding fragment-XXX, X-antibody or antigen-binding fragment-XXXX, X-antibody or antigen-binding fragment-XXXXX, XX-antibody or antigen-binding fragment-X, XX-antibody or antigen-binding fragment-XX, XX-antibody or antigen-binding fragment-XXX, XX-antibody or antigen-binding fragment-XXXX, XX-antibody or antigen-binding fragment-XXXXX, XXX-antibody or antigen-binding fragment-X, XXX-antibody or antigen-binding fragment-XX, XXX-antibody or antigen-binding fragment-XXX, XXX-antibody or antigen-binding fragment-XXX or antigen-binding fragment-XXXX, XXX-antibody or antigen-binding fragment-XXXXX, XXXX-antibody or antigen-binding fragment-X, XXXX-antibody or antigen-binding fragment-XX, XXXX-antibody or antigen-binding fragment-XXX, XXXX-antibody or antigen-binding fragment-XXXX, XXXX-antibody or antigen-binding fragment-XXXXX, XXXXX-antibody or antigen-binding fragment-X, XXXXX-antibody or antigen-binding fragment-XX, XXXXX-antibody or antigen-binding fragment-XXX, XXXXX-antibody or antigen-binding fragment-X, XXXXX-antibody or antigen-binding fragment-XX, XXXXX-antibody or antigen-binding fragment-XXX, XXXXX-antibody or antigen-binding fragment-XXXX, or XXXXX-antibody or antigen-binding fragment-XXXXX, where X is any naturally occurring amino acid, or X is a non-conventional amino acid or amino acid analogue, as described herein and known to one of skill in the art.

[0080] Disclosed herein are compositions comprising an effective amount of an antibody or antigen-binding fragment thereof that binds to DPEP-1, the antibody or antigen-binding fragment thereof comprising any one of sdABP01, sdABP02, sdABP03, sdABP04, sdABP05, sdABP06, sdABP07, sdABP08, or sdABP09, or derivatives.

[0081] In one embodiment, the antibody or antigen-binding fragment thereof that binds DPEP-1 comprises an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% identical to any one of SEQ ID NOs: 12-20 and 48-56. In one embodiment, the antibody or antigen-binding fragment thereof that binds DPEP-1 comprises an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% identical to any one of SEQ ID NOs: 13, 15, or 18. In some embodiments, any differences in the sequence are limited to the framework regions.

[0082] As understood in the art, the amino acid position or boundary that defines the CDR region of an antibody may vary depending on the context and various definitions known in the art. Some positions in a variable region may be considered as hybrid CDRs, which can be considered to be within the CDR region under one set of criteria, but outside the CDR region under another set of criteria. In some embodiments, the CDRs in the aforementioned variable light chain and variable heavy chain can be described using IMGT, Kabat, Chothia, AbM, Contact, or Paratome schemes, or other schemes known in the art. The "Kabat" approach to define CDRs uses sequence variability and is the most commonly used (Kabat et al., 1991, "Sequences of Proteins of Immunological Interest, 5th Ed." NIH 1:688-961, incorporated herein by reference). "Chothia" uses the location of the structural loops (Chothia and Lesk, 1987, J. Mol. Biol. 196:901-17; Chothia et al., 1992, J. Mol. Biol. 227: 799-817, incorporated herein by reference). The IMGT numbering scheme is an adaptation of the Chothia numbering scheme (Lefranc et al., 1999, Nucleic Acids Research, 27:209-212; http: / / imgt.cines.fr, incorporated herein by reference). CDRs defined by "AbM" are a compromise between Kabat and Chothia and are delineated using the Oxford Molecular AbM antibody modeling software (see Martin et al., 1989, PNAS, 86:9268, incorporated herein by reference; see also www.bioinf-org.uk / abs)."Contact" CDR delineation is based on the analysis of known antibody-antigen crystal structures (see, e.g., MacCallum et al., 1996, J. Mol. Biol. 262, 732-45). The "Paratome" approach involves computational programs based on a set of consensus regions derived from the structural alignment of a non-redundant set of known antibody-antigen complexes (Kunik et al., 2012, Nucl Acids Res. W521-4, incorporated herein by reference; see also www.ofranlab.org / paratome / ). The CDRs described herein are based on the IMGT definition, but it should be understood that CDRs based on other methods are to be encompassed herein. In some embodiments, the CDRs described herein are based on the IMGT numbering.

[0083] Thus, the present disclosure: (i) SEQ ID NOs: 21, 22, and 23; (ii) SEQ ID NOs: 24, 25, and 26; (iii) SEQ ID NOs: 27, 28, and 29; (iv) SEQ ID NOs: 30, 31, and 32; (v) SEQ ID NOs: 33, 34, and 35; (vi) SEQ ID NOs: 36, 37, and 38; (vii) SEQ ID NOs: 39, 40, and 41; (viii) SEQ ID NOs: 42, 43, and 44; or (ix) SEQ ID NOs: 45, 46, and 47 The present invention provides a binding agent that binds to DPEP-1, comprising:

[0084] In some embodiments, the binding agent is an antibody or antigen-binding fragment thereof. In some embodiments, the binding agent comprises three sets of CDRs having the amino acid sequences of SEQ ID NOs: 21, 22, and 23. In some embodiments, the binding agent comprises three sets of CDRs having the amino acid sequences of SEQ ID NOs: 24, 25, and 26. In some embodiments, the binding agent comprises three sets of CDRs having the amino acid sequences of SEQ ID NOs: 27, 28, and 29. In some embodiments, the binding agent comprises three sets of CDRs having the amino acid sequences of SEQ ID NOs: 30, 31, and 32. In some embodiments, the binding agent comprises three sets of CDRs having the amino acid sequences of SEQ ID NOs: 33, 34, and 35. In some embodiments, the binding agent comprises three sets of CDRs having the amino acid sequences of SEQ ID NOs: 36, 37, and 38. In some embodiments, the binding agent comprises three sets of CDRs having the amino acid sequences of SEQ ID NOs: 39, 40, and 41. In some embodiments, the binding agent comprises three sets of CDRs having the amino acid sequences of SEQ ID NOs: 42, 43, and 44. In some embodiments, the binding agent comprises three sets of CDRs having the amino acid sequences of SEQ ID NOs: 45, 46, and 47.

[0085] In one embodiment, the antibody or antigen-binding fragment thereof that binds DPEP-1 comprises an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% identical to SEQ ID NO: 12. In one embodiment, the antibody or antigen-binding fragment thereof comprises the amino acid sequence of SEQ ID NO: 12. In one embodiment, the antibody or antigen-binding fragment thereof that binds DPEP-1 is an sdAB comprising at least one CDR having an amino acid sequence as set forth in any one of SEQ ID NOs: 21, 22, and 23. In one embodiment, an antibody or antigen-binding fragment thereof that binds to DPEP-1 comprises an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% identical to SEQ ID NO: 12, and the antibody or antigen-binding fragment thereof comprises a set of three CDRs comprising or consisting of the amino acid sequences of SEQ ID NOs: 21, 22, and 23. In some embodiments, any differences in the sequence are limited to the framework regions.

[0086] In one embodiment, the antibody or antigen-binding fragment thereof that binds DPEP-1 comprises an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% identical to SEQ ID NO: 13. In one embodiment, the antibody or antigen-binding fragment thereof comprises the amino acid sequence of SEQ ID NO: 13. In one embodiment, the antibody or antigen-binding fragment thereof that binds DPEP-1 is an sdAB comprising at least one CDR having an amino acid sequence as set forth in any one of SEQ ID NOs: 24, 25, and 26. In one embodiment, an antibody or antigen-binding fragment thereof that binds to DPEP-1 comprises an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% identical to SEQ ID NO: 13, and the antibody or antigen-binding fragment thereof comprises a set of three CDRs comprising or consisting of the amino acid sequences of SEQ ID NOs: 24, 25, and 26. In some embodiments, any differences in the sequence are limited to the framework regions.

[0087] In one embodiment, the antibody or antigen-binding fragment thereof that binds DPEP-1 comprises an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% identical to SEQ ID NO: 14. In one embodiment, the antibody or antigen-binding fragment thereof comprises the amino acid sequence of SEQ ID NO: 14. In one embodiment, the antibody or antigen-binding fragment thereof that binds DPEP-1 is an sdAB comprising at least one CDR having an amino acid sequence as set forth in any one of SEQ ID NOs: 27, 28, and 29. In one embodiment, an antibody or antigen-binding fragment thereof that binds to DPEP-1 comprises an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% identical to SEQ ID NO: 14, and the antibody or antigen-binding fragment thereof comprises a set of three CDRs comprising or consisting of the amino acid sequences of SEQ ID NOs: 27, 28, and 29. In some embodiments, any differences in the sequence are limited to the framework regions.

[0088] In one embodiment, the antibody or antigen-binding fragment thereof that binds DPEP-1 comprises an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% identical to SEQ ID NO: 15. In one embodiment, the antibody or antigen-binding fragment thereof that binds DPEP-1 is an sdAB comprising at least one CDR having an amino acid sequence as set forth in any one of SEQ ID NOs: 30, 31, and 32. In one embodiment, an antibody or antigen-binding fragment thereof that binds to DPEP-1 comprises an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% identical to SEQ ID NO: 15, and the antibody or antigen-binding fragment thereof comprises a set of three CDRs comprising or consisting of the amino acid sequences of SEQ ID NOs: 30, 31, and 32. In some embodiments, any differences in the sequence are limited to the framework regions.

[0089] In one embodiment, the antibody or antigen-binding fragment thereof that binds DPEP-1 comprises an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% identical to SEQ ID NO: 16. In one embodiment, the antibody or antigen-binding fragment thereof comprises the amino acid sequence of SEQ ID NO: 16. In one embodiment, the antibody or antigen-binding fragment thereof that binds DPEP-1 is an sdAB comprising at least one CDR having an amino acid sequence as set forth in any one of SEQ ID NOs: 33, 34, and 35. In one embodiment, an antibody or antigen-binding fragment thereof that binds to DPEP-1 comprises an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% identical to SEQ ID NO: 16, and the antibody or antigen-binding fragment thereof comprises a set of three CDRs comprising or consisting of the amino acid sequences of SEQ ID NOs: 33, 34, and 35. In some embodiments, any differences in the sequence are limited to the framework regions.

[0090] In one embodiment, the antibody or antigen-binding fragment thereof that binds DPEP-1 comprises an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% identical to SEQ ID NO: 17. In one embodiment, the antibody or antigen-binding fragment thereof that binds DPEP-1 is an sdAB comprising at least one CDR having an amino acid sequence as set forth in any one of SEQ ID NOs: 36, 37, and 38. In one embodiment, an antibody or antigen-binding fragment thereof that binds to DPEP-1 comprises an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% identical to SEQ ID NO: 17, and the antibody or antigen-binding fragment thereof comprises a set of three CDRs comprising or consisting of the amino acid sequences of SEQ ID NOs: 36, 37, and 38. In some embodiments, any differences in the sequence are limited to the framework regions.

[0091] In one embodiment, the antibody or antigen-binding fragment thereof that binds DPEP-1 comprises an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% identical to SEQ ID NO: 18. In one embodiment, the antibody or antigen-binding fragment thereof comprises the amino acid sequence of SEQ ID NO: 18. In one embodiment, the antibody or antigen-binding fragment thereof that binds DPEP-1 is an sdAB comprising at least one CDR having an amino acid sequence as set forth in any one of SEQ ID NOs: 39, 40, and 41. In one embodiment, an antibody or antigen-binding fragment thereof that binds to DPEP-1 comprises an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% identical to SEQ ID NO: 18, and the antibody or antigen-binding fragment thereof comprises a set of three CDRs comprising or consisting of the amino acid sequences of SEQ ID NOs: 39, 40, and 41. In some embodiments, any differences in the sequence are limited to the framework regions.

[0092] In one embodiment, the antibody or antigen-binding fragment thereof that binds DPEP-1 comprises an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% identical to SEQ ID NO: 19. In one embodiment, the antibody or antigen-binding fragment thereof comprises the amino acid sequence of SEQ ID NO: 19. In one embodiment, the antibody or antigen-binding fragment thereof that binds DPEP-1 is an sdAB comprising at least one CDR having an amino acid sequence as set forth in any one of SEQ ID NOs: 42, 43, and 44. In one embodiment, an antibody or antigen-binding fragment thereof that binds to DPEP-1 comprises an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% identical to SEQ ID NO: 19, and the antibody or antigen-binding fragment thereof comprises a set of three CDRs comprising or consisting of the amino acid sequences of SEQ ID NOs: 42, 43, and 44. In some embodiments, any differences in the sequence are limited to the framework regions.

[0093] In one embodiment, the antibody or antigen-binding fragment thereof that binds DPEP-1 comprises an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% identical to SEQ ID NO: 20. In one embodiment, the antibody or antigen-binding fragment thereof comprises the amino acid sequence of SEQ ID NO: 20. In one embodiment, the antibody or antigen-binding fragment thereof that binds DPEP-1 is an sdAB comprising at least one CDR having an amino acid sequence as set forth in any one of SEQ ID NOs: 45, 46, and 47. In one embodiment, an antibody or antigen-binding fragment thereof that binds to DPEP-1 comprises an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% identical to SEQ ID NO: 20, and the antibody or antigen-binding fragment thereof comprises a set of three CDRs comprising or consisting of the amino acid sequences of SEQ ID NOs: 45, 46, and 47. In some embodiments, any differences in the sequence are limited to the framework regions.

[0094] In some embodiments, the binding agent is a monoclonal antibody, a polyclonal antibody, a chimeric antibody, a humanized antibody, or an antigen-binding fragment thereof. In one embodiment, the binding agent is an antigen-binding fragment fused to an Fc domain. In some embodiments, the binding agent is an antigen-binding fragment, and the antigen-binding fragment is an Fv, scFv, Fab, Fab', F(ab')2, dsFv, ds-scFv, sdAB, dimeric, minibody, diabody, or multimeric antigen-binding fragment. In some embodiments, the antigen-binding fragment is an sdAB. In some embodiments, the antibody or antigen-binding fragment comprises one or more amino acids selected from the group consisting of D-amino acids, modified amino acids, amino acid analogs, or combinations thereof. In some embodiments, the modified amino acids comprise a modification selected from the group consisting of methylation, amidation, acetylation, and / or substitution with other chemical groups. In some embodiments, the antibody or antigen-binding fragment is modified by pegylation, acetylation, glycosylation, biotinylation, or prenylation. In some embodiments, the antibody or antigen-binding fragment is a human, murine, llama, rabbit, sheep, or goat antibody or antigen-binding fragment thereof.

[0095] In some embodiments, an antibody or antigen-binding fragment thereof that binds DPEP-1 comprises an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% identical to any one of SEQ ID NOs: 12-20 and 48-56, and the antibody or antigen-binding fragment is substituted with one or more D- or L-amino acids. In other embodiments, an antibody or antigen-binding fragment thereof that binds DPEP-1 provided herein can have 1, 2, 3, 4, or 5 amino acid residues removed from the N-terminus and / or C-terminus.

[0096] In one embodiment, the antibody or antigen-binding fragment thereof that binds to DPEP-1 comprises an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% identical to any one of SEQ ID NOs: 1-9. In one embodiment, the antibody or antigen-binding fragment thereof comprises the amino acid sequence of any one of SEQ ID NOs: 1-9.

[0097] In some embodiments, the antibody or antigen-binding fragment thereof that binds to DPEP-1 comprises a biotinylated acceptor peptide (BAP) and / or a His6 tag. TIFF2024513473000002.tif4128. In some embodiments, the His6 tag comprises the amino acid sequence of HHHHHH (SEQ ID NO: 11). In some embodiments, the antibody or antigen-binding fragment thereof that binds DPEP-1 comprises a BAP and a His6 tag. In some embodiments, the antibody or antigen-binding fragment thereof that binds DPEP-1 comprises the amino sequence of SEQ ID NOs: 10 and 11. In some embodiments, the BAP and the His6 tag are linked by a spacer having the amino acid sequence of LE. In some embodiments, the BAP and the His6 tag are at the C-terminus of the antibody or antigen-binding fragment thereof.

[0098] B. Modified Antibodies and Antibody Analogs In various embodiments, the antibody or antigen-binding fragment thereof may contain amino acids, including the carboxy-terminal and / or amino-terminal amino acids in the antibody, or may be modified by PEGylation, methylation, amidation, acetylation, prenylation, and / or substitution with other chemical groups that may alter the circulating half-life of the antibody or antigen-binding fragment without adversely affecting its activity. Examples of non-conventional or non-natural amino acids include, but are not limited to, citrulline, ornithine, norleucine, norvaline, 4-(E)-butenyl-4(R)-methyl-N-methylthreonine (MeBmt), N-methyl-leucine (MeLeu), aminoisobutyric acid, statin, and N-methyl-alanine (MeAla). The amino acid may participate in a disulfide bond. In certain embodiments, the amino acid has the general structure H 2 It has N--C(H)(R)--COOH. In certain embodiments, the amino acid is a naturally occurring amino acid. In certain embodiments, the amino acid is a synthetic or non-natural amino acid (e.g., α,α-disubstituted amino acid, N-alkyl amino acid); in some embodiments, the amino acid is a D-amino acid; in certain embodiments, the amino acid is an L-amino acid.

[0099] The fragment crystallizable region (Fc region or Fc domain) is the tail region of an antibody that interacts with cell surface receptors called Fc receptors. This property allows the antibody to activate the immune system. In IgG, IgA, and IgD antibody isotypes, the Fc domain is composed of two identical protein fragments derived from the second and third constant domains of the antibody's two heavy chains. In IgM and IgE, their Fc domains contain three heavy chain constant domains (CH domains 2-4) in each polypeptide chain. The antibodies or antigen-binding fragments thereof described herein can be fused to an Fc domain and can also be multimeric, e.g., to generate bispecific / biparatopic molecules. In some embodiments, the antibodies or antigen-binding fragments thereof that bind DPEP-1 are fused to an Fc domain. In some embodiments, the antibodies or antigen-binding fragments thereof that bind DPEP-1 are multimeric. In some embodiments, the antibodies or antigen-binding fragments thereof that bind DPEP-1 are fused to an Fc domain and are multimeric. In some embodiments, the Fc domain is from IgG, IgA, IgD, IgM, or IgE. In some embodiments, the Fc domain is a human Fc domain. In some embodiments, the Fc domain is from human IgG1, IgG2, IgG3, or IgG4.

[0100] Also provided is a fusion protein comprising an antibody or antigen-binding fragment thereof that binds DPEP-1 and an Fc domain. In some embodiments, the fusion protein is a multimer. In some embodiments, the binding agent is a fusion protein of an antigen-binding fragment that binds DPEP-1 and an Fc domain. In some embodiments, the fusion protein comprises an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% identical to any one of SEQ ID NOs: 12-20 and an Fc domain. In some embodiments, the fusion protein comprises an amino acid sequence of any one of SEQ ID NOs: 12-20 and an Fc domain. In some embodiments, the fusion protein comprises an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% identical to any one of SEQ ID NOs: 48-56. In some embodiments, the fusion protein comprises the amino acid sequence of any one of SEQ ID NOs: 48-56.

[0101] In one embodiment, the fusion protein comprises an amino acid sequence at least 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% identical to SEQ ID NO: 12 and an Fc domain. In one embodiment, the fusion protein comprises an amino acid sequence at least 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% identical to SEQ ID NO: 48. In one embodiment, the fusion protein comprises the amino acid sequence of SEQ ID NO: 48. In one embodiment, the fusion protein comprises at least one CDR having an amino acid sequence as set forth in any one of SEQ ID NOs: 21, 22, and 23, and an Fc domain. In one embodiment, the fusion protein comprises an amino acid sequence at least 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, 99.5%, 99.9% or 100% identical to SEQ ID NO: 12 and an Fc domain, and the fusion protein comprises three sets of CDRs comprising or consisting of the amino acid sequences of SEQ ID NOs: 21, 22, and 23. In one embodiment, the fusion protein comprises the amino acid sequence of SEQ ID NO: 12 and an Fc domain. In one embodiment, the fusion protein comprises an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% identical to SEQ ID NO: 48, and the fusion protein comprises a set of three CDRs comprising or consisting of the amino acid sequences of SEQ ID NOs: 21, 22, and 23. In some embodiments, the fusion protein comprises the amino acid sequence of SEQ ID NO: 48. In some embodiments, any differences in the sequence are limited to the framework regions or the Fc domain.

[0102] In one embodiment, the fusion protein comprises an amino acid sequence at least 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% identical to SEQ ID NO: 13 and an Fc domain. In one embodiment, the fusion protein comprises an amino acid sequence at least 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% identical to SEQ ID NO: 49. In one embodiment, the fusion protein comprises the amino acid sequence of SEQ ID NO: 49. In one embodiment, the fusion protein comprises at least one CDR having an amino acid sequence as set forth in any one of SEQ ID NOs: 24, 25, and 26, and an Fc domain. In one embodiment, the fusion protein comprises an amino acid sequence at least 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, 99.5%, 99.9% or 100% identical to SEQ ID NO: 13 and an Fc domain, and the fusion protein comprises three sets of CDRs comprising or consisting of the amino acid sequences of SEQ ID NOs: 24, 25, and 26. In one embodiment, the fusion protein comprises the amino acid sequence of SEQ ID NO: 13 and an Fc domain. In one embodiment, the fusion protein comprises an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% identical to SEQ ID NO: 49, and the fusion protein comprises a set of three CDRs comprising or consisting of the amino acid sequences of SEQ ID NOs: 24, 25, and 26. In some embodiments, the fusion protein comprises the amino acid sequence of SEQ ID NO: 49. In some embodiments, any differences in the sequence are limited to the framework regions or the Fc domain.

[0103] In one embodiment, the fusion protein comprises an amino acid sequence at least 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% identical to SEQ ID NO: 14 and an Fc domain. In one embodiment, the fusion protein comprises an amino acid sequence at least 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% identical to SEQ ID NO: 50. In one embodiment, the fusion protein comprises the amino acid sequence of SEQ ID NO: 50. In one embodiment, the fusion protein comprises at least one CDR having an amino acid sequence as set forth in any one of SEQ ID NOs: 27, 28, and 29, and an Fc domain. In one embodiment, the fusion protein comprises an amino acid sequence at least 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, 99.5%, 99.9% or 100% identical to SEQ ID NO: 14 and an Fc domain, and the fusion protein comprises three sets of CDRs comprising or consisting of the amino acid sequences of SEQ ID NOs: 27, 28, and 29. In one embodiment, the fusion protein comprises the amino acid sequence of SEQ ID NO: 14 and an Fc domain. In one embodiment, the fusion protein comprises an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% identical to SEQ ID NO: 50, and the fusion protein comprises a set of three CDRs comprising or consisting of the amino acid sequences of SEQ ID NOs: 27, 28, and 29. In some embodiments, the fusion protein comprises the amino acid sequence of SEQ ID NO: 50. In some embodiments, any differences in the sequence are limited to the framework regions or the Fc domain.

[0104] In one embodiment, the fusion protein comprises an amino acid sequence at least 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% identical to SEQ ID NO: 15 and an Fc domain. In one embodiment, the fusion protein comprises an amino acid sequence at least 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% identical to SEQ ID NO: 51. In one embodiment, the fusion protein comprises the amino acid sequence of SEQ ID NO: 51. In one embodiment, the fusion protein comprises at least one CDR having an amino acid sequence as set forth in any one of SEQ ID NOs: 30, 31, and 32, and an Fc domain. In one embodiment, the fusion protein comprises an amino acid sequence at least 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, 99.5%, 99.9% or 100% identical to SEQ ID NO: 15 and an Fc domain, and the fusion protein comprises three sets of CDRs comprising or consisting of the amino acid sequences of SEQ ID NOs: 30, 31, and 32. In one embodiment, the fusion protein comprises the amino acid sequence of SEQ ID NO: 15 and an Fc domain. In one embodiment, the fusion protein comprises an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% identical to SEQ ID NO: 51, and the fusion protein comprises a set of three CDRs comprising or consisting of the amino acid sequences of SEQ ID NOs: 30, 31, and 32. In some embodiments, the fusion protein comprises the amino acid sequence of SEQ ID NO: 51. In some embodiments, any differences in the sequence are limited to the framework regions or the Fc domain.

[0105] In one embodiment, the fusion protein comprises an amino acid sequence at least 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% identical to SEQ ID NO: 16 and an Fc domain. In one embodiment, the fusion protein comprises an amino acid sequence at least 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% identical to SEQ ID NO: 52. In one embodiment, the fusion protein comprises the amino acid sequence of SEQ ID NO: 52. In one embodiment, the fusion protein comprises at least one CDR having an amino acid sequence as set forth in any one of SEQ ID NOs: 33, 34, and 35, and an Fc domain. In one embodiment, the fusion protein comprises an amino acid sequence at least 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, 99.5%, 99.9% or 100% identical to SEQ ID NO: 16 and an Fc domain, and the fusion protein comprises three sets of CDRs comprising or consisting of the amino acid sequences of SEQ ID NOs: 33, 34, and 35. In one embodiment, the fusion protein comprises the amino acid sequence of SEQ ID NO: 16 and an Fc domain. In one embodiment, the fusion protein comprises an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% identical to SEQ ID NO: 52, and the fusion protein comprises a set of three CDRs comprising or consisting of the amino acid sequences of SEQ ID NOs: 33, 34, and 35. In some embodiments, the fusion protein comprises the amino acid sequence of SEQ ID NO: 52. In some embodiments, any differences in the sequence are limited to the framework regions or the Fc domain.

[0106] In one embodiment, the fusion protein comprises an amino acid sequence at least 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% identical to SEQ ID NO: 17 and an Fc domain. In one embodiment, the fusion protein comprises an amino acid sequence at least 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% identical to SEQ ID NO: 53. In one embodiment, the fusion protein comprises the amino acid sequence of SEQ ID NO: 53. In one embodiment, the fusion protein comprises at least one CDR having an amino acid sequence as set forth in any one of SEQ ID NOs: 36, 37, and 38, and an Fc domain. In one embodiment, the fusion protein comprises an amino acid sequence at least 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, 99.5%, 99.9% or 100% identical to SEQ ID NO: 17 and an Fc domain, and the fusion protein comprises three sets of CDRs comprising or consisting of the amino acid sequences of SEQ ID NOs: 36, 37, and 38. In one embodiment, the fusion protein comprises the amino acid sequence of SEQ ID NO: 17 and an Fc domain. In one embodiment, the fusion protein comprises an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% identical to SEQ ID NO: 53, and the fusion protein comprises a set of three CDRs comprising or consisting of the amino acid sequences of SEQ ID NOs: 36, 37, and 38. In some embodiments, the fusion protein comprises the amino acid sequence of SEQ ID NO: 53. In some embodiments, any differences in the sequence are limited to the framework regions or the Fc domain.

[0107] In one embodiment, the fusion protein comprises an amino acid sequence at least 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% identical to SEQ ID NO: 18 and an Fc domain. In one embodiment, the fusion protein comprises an amino acid sequence at least 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% identical to SEQ ID NO: 54. In one embodiment, the fusion protein comprises the amino acid sequence of SEQ ID NO: 54. In one embodiment, the fusion protein comprises at least one CDR having an amino acid sequence as set forth in any one of SEQ ID NOs: 39, 40, and 41, and an Fc domain. In one embodiment, the fusion protein comprises an amino acid sequence at least 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, 99.5%, 99.9% or 100% identical to SEQ ID NO: 18 and an Fc domain, and the fusion protein comprises three sets of CDRs comprising or consisting of the amino acid sequences of SEQ ID NOs: 39, 40, and 41. In one embodiment, the fusion protein comprises the amino acid sequence of SEQ ID NO: 18 and an Fc domain. In one embodiment, the fusion protein comprises an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% identical to SEQ ID NO: 54, and the fusion protein comprises a set of three CDRs comprising or consisting of the amino acid sequences of SEQ ID NOs: 39, 40, and 41. In some embodiments, the fusion protein comprises the amino acid sequence of SEQ ID NO: 54. In some embodiments, any differences in the sequence are limited to the framework regions or the Fc domain.

[0108] In one embodiment, the fusion protein comprises an amino acid sequence at least 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% identical to SEQ ID NO: 19 and an Fc domain. In one embodiment, the fusion protein comprises an amino acid sequence at least 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% identical to SEQ ID NO: 55. In one embodiment, the fusion protein comprises the amino acid sequence of SEQ ID NO: 55. In one embodiment, the fusion protein comprises at least one CDR having an amino acid sequence as set forth in any one of SEQ ID NOs: 42, 43, and 44, and an Fc domain. In one embodiment, the fusion protein comprises an amino acid sequence at least 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, 99.5%, 99.9% or 100% identical to SEQ ID NO: 19 and an Fc domain, wherein the fusion protein comprises three sets of CDRs comprising or consisting of the amino acid sequences of SEQ ID NOs: 42, 43, and 44. In one embodiment, the fusion protein comprises the amino acid sequence of SEQ ID NO: 19 and an Fc domain. In one embodiment, the fusion protein comprises an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% identical to SEQ ID NO: 55, and the fusion protein comprises a set of three CDRs comprising or consisting of the amino acid sequences of SEQ ID NOs: 42, 43, and 44. In some embodiments, the fusion protein comprises the amino acid sequence of SEQ ID NO: 55. In some embodiments, any differences in the sequence are limited to the framework regions or the Fc domain.

[0109] In one embodiment, the fusion protein comprises an amino acid sequence at least 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% identical to SEQ ID NO: 20 and an Fc domain. In one embodiment, the fusion protein comprises an amino acid sequence at least 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% identical to SEQ ID NO: 56. In one embodiment, the fusion protein comprises the amino acid sequence of SEQ ID NO: 56. In one embodiment, the fusion protein comprises at least one CDR having an amino acid sequence as set forth in any one of SEQ ID NOs: 45, 46, and 47, and an Fc domain. In one embodiment, the fusion protein comprises an amino acid sequence at least 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, 99.5%, 99.9% or 100% identical to SEQ ID NO: 20 and an Fc domain, wherein the fusion protein comprises three sets of CDRs comprising or consisting of the amino acid sequences of SEQ ID NOs: 45, 56, and 47. In one embodiment, the fusion protein comprises the amino acid sequence of SEQ ID NO: 20 and an Fc domain. In one embodiment, the fusion protein comprises an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% identical to SEQ ID NO: 56, and the fusion protein comprises a set of three CDRs comprising or consisting of the amino acid sequences of SEQ ID NOs: 45, 46, and 47. In some embodiments, the fusion protein comprises the amino acid sequence of SEQ ID NO: 56. In some embodiments, any differences in the sequence are limited to the framework regions or the Fc domain.

[0110] B. Rational Design and Structure-Function Analysis of DPEP-1 Binding Antibodies Different techniques provide different and complementary information about protein structure. Primary structure can be obtained either by biochemical methods, by direct determination of amino acid sequence from protein, or from the nucleotide sequence of corresponding gene or cDNA. Quaternary structure of large proteins or aggregates can also be determined by electron microscopy. X-ray crystallography is commonly used to obtain secondary and tertiary structure, which requires detailed information about the arrangement of atoms within a protein. Other structural techniques for evaluating the structure and function of antibodies or fragments include hydrogen-deuterium exchange mass spectrometry, bio-NMR, and cryo-EM.

[0111] The first prerequisite for elucidating the three-dimensional structure of a protein by X-ray crystallography is an ordered crystal that strongly diffracts X-rays. The crystallographic method directs a beam of X-rays on a regular repeating array of many identical molecules so that the X-rays are diffracted in a pattern from which the structure of the individual molecules can be extracted. Ordered crystals of globular protein molecules are large spherical or elliptical objects with irregular surfaces that contain large holes or channels formed between the individual molecules. These channels usually occupy more than half the volume of the crystal and are filled with disordered solvent molecules. The protein molecules are in contact with each other only in a few small areas. This is one reason why the structure of a protein determined by X-ray crystallography is generally the same as the structure of the protein in solution.

[0112] X-ray crystallography can be used to screen compounds that are not known ligands of a target biomolecule for their ability to bind to the target biomolecule. The method includes obtaining a crystal of the target biomolecule; exposing the target biomolecule crystal to one or more test samples; and obtaining an X-ray crystal diffraction pattern to determine whether a ligand / receptor complex is formed.

[0113] DPEP-1 receptor can be exposed to test antibody by either co-crystallizing biomolecules in the presence of one or more test samples, or by soaking biomolecular crystals in a solution of one or more test samples.In another embodiment, the structural information from the ligand / receptor complex is used to design ligands that bind tighter, bind more specifically, have better biological activity, or have a better safety profile.These can include small molecules, or other biotherapeutic agents such as antibodies.

[0114] The antibodies described herein can be fully characterized using mass spectrometry, high performance liquid chromatography (HPLC), and amino acid analysis (AAA).

[0115] Mass spectrometry is used to obtain distance constraints between amino acid residues of proteins, which are used in determining the protein's structure.

[0116] Without being bound to any particular mechanism, it is believed that the structure of the antibody or fragment, and any modifications that stabilize the tertiary structure, enhance binding to DPEP-1.

[0117] In one embodiment, determining the key DPEP-1 epitopes that bind to DPEP-1 antibodies or antigen-binding fragments can also be used to develop therapeutic antibodies that target DPEP-1, leukocyte adhesion, and cancer metastasis. Examples of such methods include, but are not limited to, crystallization of DPEP-1 bound to DPEP-1 antibodies or antigen-binding fragments, and analysis by X-ray diffraction. In another embodiment, photochemical cross-linking of DPEP-1 antibodies or antigen-binding fragments bound to DPEP-1, followed by protease digestion and mass spectrometry (Ngai et al, J Biol Chem, 269(3), 2165-2172 (1994)) is used to identify the key amino acids of DPEP-1 antibodies or antigen-binding fragments and DPEP-1 domains involved in the interaction. In various embodiments, in silico modeling is performed to develop novel pharmaceutical compositions.

[0118] Unless otherwise defined, scientific and technical terms and nomenclature used herein have the same meaning as those generally understood by those skilled in the art to which this disclosure pertains.Generally, procedures such as cell culture, infection, molecular biology methods, etc. are common methods used in the art.Such standard techniques can be found in reference manuals such as Ausubel et al., Current Protocols in Molecular Biology, Wiley Interscience, New York, 2001; and Sambrook et al., Molecular Cloning: A Laboratory Manual, 3rd edition, Cold Spring Harbor Laboratory Press, NY, 2001, which are incorporated herein by reference.

[0119] IV. Pharmaceutical Preparations and Medication In another aspect, the compounds or agents described herein, as well as variants and modifications thereof, are provided as pharmaceutical compositions for therapeutic use.In one embodiment, the pharmaceutical formulation comprises an isolated antibody or antigen-binding fragment, and comprises the sequence listed in Table 1.In another embodiment, the pharmaceutical formulation comprises an isolated antibody or antigen-binding fragment contained as an insert in a phage virus, and / or may further comprise 1, 2, 3, 4, 5 additional amino acid residues at the N-terminus and / or C-terminus of the antibody or antigen-binding fragment sequence.

[0120] Representative delivery regimes include oral, parenteral (including subcutaneous, intramuscular, and intravenous injection), rectal, buccal (including sublingual), transdermal, inhalation, ocular, and intranasal.In one embodiment, the delivery of the compound requires subcutaneous injection of a controlled release injectable formulation.In some embodiments, the compounds described herein are useful for subcutaneous, intranasal, and inhalation administration.

[0121] The exact dosage and composition, and the selection of the most suitable delivery regimen are influenced by, among other things, the pharmacological properties of the selected antibody, the nature and severity of the condition being treated, and the physical condition and mental acuity of the recipient.In addition, the route of administration results in different amounts of material being absorbed.The bioavailability of the administration of a compound through different routes is particularly variable, with amounts ranging from less than 1% to nearly 100% being observed.Typically, the bioavailability from routes other than intravenous, intraperitoneal, or subcutaneous injection is 50% or less.

[0122] The pharmaceutical composition or formulation of the present disclosure can be formulated with physiologically acceptable carrier or excipient to prepare pharmaceutical composition.The carrier and composition can be sterile.The formulation should be suitable for the mode of administration, for example, intravenous or subcutaneous administration.The method of formulating the composition is known in the art (for example, see Remington's Pharmaceuticals Sciences, 17th Edition, Mack Publishing Co., (Alfonso R. Gennaro, editor) (1989), which is incorporated herein by reference).

[0123] Suitable pharma- ceutically acceptable carriers include, but are not limited to, water, salt solutions (e.g., NaCl), saline, buffered saline, alcohol, glycerol, ethanol, gum arabic, vegetable oils, benzyl alcohol, polyethylene glycol, gelatin, carbohydrates such as lactose, amylose, or starch, sugars such as mannitol, sucrose, dextrose, magnesium stearate, talc, silicic acid, viscous paraffin, flavor oils, fatty acid esters, hydroxymethylcellulose, polyvinylpyrrolidone, and the like, and combinations thereof. Pharmaceutical preparations can be mixed, if desired, with auxiliary substances (e.g., lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure, buffers, coloring substances, and / or aromatic substances, and the like) that do not adversely react with the active compounds or interfere with their activity. In one embodiment, water-soluble carriers suitable for intravenous administration are used. Pharmaceutically acceptable salts retain the desired biological activity of the parent antibody or antigen-binding fragment thereof without toxic side effects.

[0124] Composition or drug can also contain a small amount of wetting agent or emulsifier, or pH buffer, if desired.Composition can be liquid solution, suspension, emulsion, sustained release formulation, or powder.Composition can also be formulated as suppository, with traditional binder and carrier such as triglyceride.

[0125] The composition or agent can be formulated according to routine procedures as a pharmaceutical composition adapted for administration to humans.For example, in one embodiment, a composition for intravenous administration is typically a solution in a sterile isotonic aqueous buffer.If necessary, the composition can also include a solubilizer and a local anesthetic to ease pain at the site of injection.Generally, the components are supplied in unit dosage form, either separately or mixed together, for example as a dry lyophilized powder or a water-free concentrate in a sealed container such as an ampoule or sachet indicating the amount of active agent.If the composition is to be administered by infusion, it can be prepared in an infusion bottle containing sterile pharmaceutical grade water, saline, or dextrose / water.If the composition is administered by injection, an ampoule of sterile water for injection or saline can be provided so that the components can be mixed before administration.

[0126] In some embodiments, the pharmaceutical composition comprises a liquid carrier, such as, but not limited to, water, saline, phosphate buffered saline, Ringer's solution, dextrose solution, serum-containing solutions, Hank's solution, other aqueous physiologically balanced solutions, oils, esters, and glycols.

[0127] Compounds, including antibodies or antigen-binding fragments, as described herein can be formulated as neutral or salt forms. As mentioned above, pharma- ceutically acceptable salts include those formed with free amino groups, such as those derived from hydrochloric acid, phosphoric acid, acetic acid, oxalic acid, tartaric acid, etc., and those formed with free carboxyl groups, such as those derived from sodium, potassium, ammonium, calcium, ferric hydroxide, isopropylamine, triethylamine, 2-ethylaminoethanol, histidine, procaine, etc.

[0128] The pharmaceutical formulations of the present disclosure contain, as a binding agent, an antibody or antigen-binding fragment, which may be mixed with an excipient, diluted by an excipient, or encapsulated in a carrier, and may be in the form of a capsule, sachet, paper, or other container, according to well-known methods and pharmaceutical compositions. The compositions may be administered by any route suitable for administration of the binding agent, antibody, or antigen-binding fragment, including parenteral, intravenous, subcutaneous, or intramuscular administration. Typically, the binding agent, antibody, or antigen-binding fragment is dissolved or suspended in a sterile injectable solution at a concentration sufficient to provide the required dose in 0.5-2 ml or less. Pharmaceutical compositions of the disclosure suitable for parenteral administration comprise one or more compounds of the disclosure in combination with one or more pharma- ceutically acceptable sterile isotonic aqueous or nonaqueous solutions, dispersions, suspensions or emulsions, or sterile powders that can be reconstituted into sterile injectable solutions or dispersions immediately prior to use, which may contain antioxidants, buffers, solutes that render the formulation isotonic with the blood of the intended recipient, or suspending or thickening agents.

[0129] Injectable depot forms are made by forming microencapsulated matrices of drugs in biodegradable polymers such as polylactide-polyglycolide. The rate of drug release can be controlled depending on the ratio of drug to polymer and the nature of the particular polymer used. Examples of other biodegradable polymers include poly(orthoesters) and poly(anhydrides). Depot injectable formulations are also prepared by entrapping the drug in liposomes or microemulsions that are compatible with body tissues. The injectable material can be sterilized, for example, by filtration through a bacteria-retaining filter.

[0130] The pharmaceutical compositions may be presented in unit-dose or multi-dose sealed containers, for example, ampoules and vials, and may be stored in a freeze-dried condition requiring only the addition of the sterile liquid carrier, for example, water for injections, immediately prior to use. Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules, and tablets of the type described above.

[0131] V. Methods of Treatment and Use The methods of treatment and use are particularly intended for inflammation-related diseases and conditions, including diseases and conditions in which inflammation is caused by ischemia / reperfusion injury to tissue or organ.Ischemia in an organ and subsequent reperfusion cause structural and functional abnormalities in the tissue of that organ and other organs.Neutrophil infiltration, hemorrhage, edema, and necrosis are all observed in tissue after ischemia / reperfusion injury.DPEP-1 target represents a previously undescribed pathway for inflammation, which opens the opportunity for dipeptidase inhibitors, such as those described herein, to be used to treat or prevent inflammation-mediated diseases and conditions.

[0132] Thus, in one embodiment, a method for treating or preventing an inflammatory disorder in a human subject in need thereof is provided, comprising administering to the subject an effective amount of a binding agent or pharmaceutical composition of the present disclosure.In another embodiment, a use of a binding agent or pharmaceutical composition of the present disclosure for treating or preventing an inflammatory disorder in a human subject in need thereof is provided.In a further embodiment, a use of a binding agent or pharmaceutical composition of the present disclosure for the manufacture of a medicament for treating or preventing an inflammatory disorder in a human subject in need thereof is provided.In a further embodiment, a binding agent or pharmaceutical composition of the present disclosure for use in treating or preventing an inflammatory disorder in a human subject in need thereof is provided.

[0133] A non-exclusive list of common diseases and medical problems directly related to inflammation includes arthritis, kidney failure, lupus, asthma, psoriasis, pancreatitis, allergies, fibrosis, surgical complications, anemia, and fibromyalgia. Other diseases associated with chronic inflammation include cancer caused by chronic inflammation; heart attacks, where chronic inflammation contributes to coronary atherosclerosis; Alzheimer's disease, where chronic inflammation destroys brain cells; congestive heart failure, where chronic inflammation causes myocardial wear and tear; stroke, where chronic inflammation promotes thromboembolic events; and aortic stenosis, where chronic inflammation damages heart valves. Arteriosclerosis, osteoporosis, Parkinson's disease, infections (sepsis), inflammatory bowel diseases, including Crohn's disease and ulcerative colitis, and multiple sclerosis.

[0134] In certain embodiments, the methods or uses described herein are useful for protecting tissues and organs from damage associated with conditions such as, but not limited to, sepsis-induced injury, e.g., bacterial or viral sepsis-induced injury, acute organ injury (e.g., acute kidney injury in the setting of hypotension).

[0135] In other embodiments, the methods or uses described herein are useful for protecting tissues and organs from damage associated with sepsis-induced conditions, such as bacterial or viral sepsis-induced conditions, acute respiratory distress syndrome, encephalopathy, sepsis-induced liver failure, sepsis-induced kidney failure, or sepsis-induced heart failure. Coronavirus disease 2019 (COVID-19) is an infectious disease caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which may lead to complications such as sepsis resulting in COVID-19 sepsis-induced conditions. Thus, in some embodiments, viral sepsis-induced conditions include COVID-19 sepsis-induced conditions.

[0136] In other embodiments, the methods or uses described herein are useful for protecting tissues and organs from damage associated with ischemia-reperfusion injury, including but not limited to perioperative procedures, heart failure, liver failure, stroke, myocardial infarction, liver shock, spinal cord injury, brain injury, etc. These binding agents, antibodies or antigen-binding fragments thereof, or compositions can also be used to prevent or treat ischemia-reperfusion injury in high-risk patients.

[0137] In other embodiments the method or use is also useful prior to angioplasty or thrombolytic therapy, or following reperfusion of an ischemic organ after transplantation, or surgery, angioplasty or thrombolytic therapy.

[0138] Other examples of surgical procedures and organs at risk for ischemia-reperfusion injury during these procedures include, but are not limited to, brain injury during carotid artery surgery, cerebrovascular surgery, and cardiac and aortic surgery; acute kidney injury during cardiac surgery or major abdominal or thoracic surgery; lung injury after thromboembolectomy or use of cardiopulmonary bypass during pulmonary and cardiac surgery; cardiac injury after revascularization (coronary artery bypass graft surgery); intestinal injury after surgery on the mesenteric artery; and skin injury after harvesting of skin grafts.

[0139] Additional surgical procedures in which the method or use of the binding agent, antibody, antigen-binding fragment, or composition of the present disclosure is useful include harvesting donor organs for transplantation.In other embodiments, the method or use is also useful for protecting allograft organs during donor procurement, ex vivo handling, and transplantation into transplant recipients.The binding agent, antibody, antigen-binding fragment, or composition of the present disclosure can be administered or used before, during, or after harvesting the donor organ to be transplanted, before or during surgical procedures in which ischemia is expected.

[0140] Thus, the present disclosure relates to a method for preventing, limiting, or treating ischemia-reperfusion injury in a subject, comprising identifying a subject experiencing an ischemic event, or an impending ischemic event, or at risk of having an ischemic event, and administering a therapeutically or prophylactically effective amount of a binding agent, antibody, antigen-binding fragment, or composition described herein. The present disclosure also relates to the use of a binding agent, antibody or antigen-binding fragment thereof, or composition described herein for preventing, limiting, or treating ischemia-reperfusion injury in a subject. In some embodiments, the subject has experienced an ischemic event, an ischemic event is impending in the subject, or the subject is at risk of having an ischemic event. The present disclosure also relates to the use of a binding agent, antibody or antigen-binding fragment thereof, or composition described herein for the manufacture of a medicament for preventing, limiting, or treating ischemia-reperfusion injury in a subject. In some embodiments, the subject has experienced an ischemic event, an ischemic event is impending in the subject, or the subject is at risk of having an ischemic event. The present disclosure further relates to a binding agent, antibody or antigen-binding fragment thereof, or composition described herein for use in preventing, limiting, or treating ischemia-reperfusion injury in a subject. In some embodiments, the subject has experienced an ischemic event, an ischemic event is imminent in the subject, or the subject is at risk of having an ischemic event.

[0141] In one embodiment, disclosed is a method or use for removing an organ from a donor, comprising administering or using a binding agent, antibody, antigen-binding fragment thereof, or composition that binds to DPEP-1 disclosed herein to the donor before removing the organ.Optionally, disclosed is a method or use for a recipient of a transplanted organ, comprising administering or using a binding agent, antibody, antigen-binding fragment thereof, or composition that binds to DPEP-1 before transplanting the organ.Method or use may further comprise monitoring the level of one or more inflammatory markers to determine whether one or more markers are below a specified level before removal, i.e., whether the organ meets a predetermined marker profile.

[0142] In certain embodiments, the organ is selected from the group consisting of heart, liver, kidney, brain, intestine (large or small intestine), pancreas, lung, stomach, bladder, spleen, ovaries, testes, skeletal muscle, and combinations thereof.

[0143] In certain embodiments, the donor is a marginal donor. In one embodiment, the marginal donor is selected from the group consisting of a complex living donor, a non-heart beating donor (NHBD), or a deceased cardiac donor.

[0144] In certain embodiments, the complex living donor is elderly, for example, greater than about 60 years of age, greater than about 65 years of age, or greater than about 70 years of age.

[0145] In another particular embodiment, the complex living donor has one or more risk factors selected from the group consisting of obesity, hypertension, diabetes, nephrolithiasis (kidney stones), a communicable infection (e.g., a viral infection), or a combination thereof.

[0146] In one embodiment, the method or use further comprises preserving the organ. Optionally, the level of one or more inflammatory markers may be measured one or more times during preservation of the organ.

[0147] In another embodiment, the method or use further comprises providing the organ to the recipient, for example by transplantation. Optionally, the level of one or more inflammatory markers may be measured after the organ is provided, for example during the period immediately following reperfusion.

[0148] The inflammatory marker can be, but is not limited to, IL-12, IP-10, IL-1β, IL-5, GM-CSF, IFNγ, or IL-1α. In one embodiment, the one or more inflammatory markers are selected from the group consisting of IL-12, IP-10, IL-1β, IL-5, GM-CSF, IFNγ, or IL-1α.

[0149] Optionally, in some embodiments, one or more additional agents (e.g., antioxidants) may be administered to or used in the donor prior to organ removal. In some embodiments, one or more additional agents (e.g., antioxidants) may be administered to or used in the recipient prior to organ transplantation. In some embodiments, one or more additional agents may be administered or used prior to, simultaneously with, or after administration of a binding agent, antibody, antigen-binding fragment, or composition that binds DPEP-1. In some embodiments, the additional agent may be, for example, a small molecule, a biological agent, or a therapeutic gas.

[0150] In certain embodiments, the methods or uses disclosed herein may result in one of the more beneficial effects, including, but not limited to, improved graft function, reduced graft dysfunction, improved graft survival (including long-term survival), reduced graft deterioration, reduced incidence of delayed graft function (DGF), and the like.

[0151] In one embodiment, the method or use results in increased graft survival compared to the survival of a graft in which a binding agent, antibody, antigen-binding fragment, or composition that binds DPEP-1 is not administered or used prior to harvest or transplantation. Graft survival may be measured, for example, at 6 months, 1 year, or 3 years after transplantation. In certain embodiments, graft survival is increased by about 5%, about 10%, about 15%, about 20%, or about 25% or more.

[0152] In one embodiment, a method or use for preserving an organ is disclosed, comprising exposing the organ to be preserved (i.e., a harvested organ awaiting transplantation) to a binding agent, antibody, antigen-binding fragment, or composition that binds DPEP-1 as disclosed herein. Optionally, the method or use further comprises monitoring the levels of inflammatory markers one or more times to determine whether they are below a specified level. In certain embodiments, the organ is preserved in an organ transplant solution. In certain embodiments, the organ is preserved at a temperature between about 0° C. and 4° C. In another embodiment, the organ is preserved at a temperature of about 37° C., i.e., under non-thermic conditions. In certain embodiments, the preserved organ is connected to or associated with an organ perfusion system.

[0153] In certain embodiments, the organ preservation methods or uses disclosed herein allow for an increase in maximum cold ischemic time for a particular organ without impairment, e.g., without an increase in delayed graft function (DGF), in certain embodiments, between about 5 and about 50%, more particularly, between about 5 and about 25%, or an increase of about 5%, about 10%, about 15%, about 20%, about 25%, or about 30% or more.

[0154] In another embodiment, a method for preventing ischemia-reperfusion-related injury in an organ transplant patient is provided, comprising administering a binding agent, antibody, antigen-binding fragment, or composition that binds to DPEP-1 disclosed herein before, simultaneously, or after providing an organ to a patient. In another embodiment, a use of a binding agent, antibody or antigen-binding fragment thereof, or composition described herein for preventing ischemia-reperfusion-related injury in an organ transplant patient is provided, comprising administering a binding agent, antibody or antigen-binding fragment thereof, or composition disclosed herein before, simultaneously, or after providing an organ to a patient. In another embodiment, a use of a binding agent, antibody or antigen-binding fragment thereof, or composition described herein for the manufacture of a medicament for preventing ischemia-reperfusion-related injury in an organ transplant patient is provided, comprising a binding agent, antibody or antigen-binding fragment thereof, or composition disclosed herein for use before, simultaneously, or after providing an organ to a patient. In another aspect, there is provided a binding agent, antibody or antigen-binding fragment thereof, or composition described herein for use in preventing ischemia-reperfusion associated injury in an organ transplant patient, comprising using the binding agent, antibody or antigen-binding fragment thereof, or composition disclosed herein prior to, concomitantly with, or after donating the organ to the patient.

[0155] In another embodiment, an organ harvest kit is disclosed that includes a binding agent, antibody, antigen-binding fragment thereof, or composition that binds to DPEP-1 as disclosed herein. Optionally, the organ harvest kit may contain more additional agents.

[0156] Without being bound to any particular mechanism, the protective effect of the binding agent, antibody, antigen-binding fragment, or composition provided herein is mediated through binding to DPEP-1 target and directly reducing DPEP-1-regulated leukocyte recruitment, inflammation, and tumor cell adhesion.The effects described herein on inflammation-mediated disease and tumor metastasis occur independently of DPEP-1 dipeptidase activity or its role in regulating tubular transport.Previous studies have required the combination of DPEP-1 antagonists to extend the half-life of antibiotic compounds to treat bacterial infections. Other studies have used the DPEP-1 antagonist cilastatin to prevent or treat organ damage by preventing renal tubular uptake of chemotherapeutic agents or other nephrotoxic agents (Humanes et al., Kidney Intl, 82:652-553 (2012);Koller et al., Biochem Biophys Res Comm 131(2):974-979 (1985)). Thus, in certain embodiments, the binding agents, antibodies, antigen-binding fragments, or compositions described herein are not used to treat or reduce tissue damage directly induced by toxic compounds, such as nephrotoxic compounds or chemotherapeutic agents. In other embodiments, the binding agents, antibodies, antigen-binding fragments, or compositions described herein are not administered or used in combination with β-lactam antibiotic compounds. In other embodiments, the binding agents, antibodies, antigen-binding fragments, or compositions described herein are not administered or used in combination with carbapenem antibiotic compounds. Nevertheless, the binding agents, antibodies, antigen-binding fragments, or compositions described herein can be used to treat or reduce renal inflammation caused by toxic compounds, such as nephrotoxic compounds or chemotherapeutic agents. Thus, in some embodiments, the binding agents, antibodies, antigen-binding fragments, or compositions described herein are used to treat or reduce inflammation induced by toxic compounds, such as nephrotoxic compounds or chemotherapeutic agents.

[0157] The present disclosure provides a method of reducing or modulating inflammation, comprising administering an effective amount of a binding agent, antibody, or antigen-binding fragment thereof, or composition that binds to DPEP-1 to reduce or modulate inflammation in a subject.The present disclosure also provides the use of a binding agent, antibody or antigen-binding fragment thereof, or composition described herein for reducing or modulating inflammation in a subject.The present disclosure also provides the use of a binding agent, antibody or antigen-binding fragment thereof, or composition described herein for the manufacture of a medicament for reducing or modulating inflammation.The present disclosure further provides a binding agent, antibody or antigen-binding fragment thereof, or composition described herein for use in reducing or modulating inflammation.

[0158] In one embodiment, the composition comprises a binding agent, an antibody or antigen-binding fragment, and / or a small molecule compound.

[0159] In one embodiment, inflammation is associated with inflammatory disorders selected from the group consisting of gastritis, gout, gouty arthritis, arthritis, rheumatoid arthritis, renal failure, lupus, asthma, psoriasis, pancreatitis, allergy, fibrosis, surgery complications, anemia, fibromyalgia, cancer, heart attack, congestive heart failure, stroke, aortic valve, arteriosclerosis, osteoporosis, multiple sclerosis, Alzheimer's disease, Parkinson's disease, ulcer, chronic bronchitis, asthma, allergy, acute lung injury, lung inflammation, airway hyperresponsiveness, vasculitis, septic shock, inflammatory skin disease, psoriasis, atopic dermatitis, eczema and inflammatory bowel disease.In some embodiments, inflammatory bowel disease is Crohn's disease or ulcerative colitis.

[0160] The present disclosure provides a method for blocking leukocyte recruitment in a subject, comprising administering an effective amount of a binding agent, an antibody or an antigen-binding fragment thereof, or a composition that binds to DPEP-1 to block leukocyte recruitment.The present disclosure also provides the use of a binding agent, an antibody or an antigen-binding fragment thereof, or a composition described herein for blocking leukocyte recruitment in a subject.The present disclosure also provides the use of a binding agent, an antibody or an antigen-binding fragment thereof, or a composition described herein for the manufacture of a medicament for blocking leukocyte recruitment in a subject.The present disclosure further provides a binding agent, an antibody or an antigen-binding fragment thereof, or a composition described herein for use in blocking leukocyte recruitment in a subject.

[0161] In one embodiment, the method or use further comprises identifying the subject that needs to be treated by diagnostic test for needing to reduce inflammation.In some embodiments, the indications for treatment include but are not limited to the clinical signs and symptoms in any patient at risk of acute kidney injury (before surgery or before administration or use of intravenous contrast agent), or in any patient with reduced urine output or increased serum creatinine, for example, in patients with systemic infection or hypotension.

[0162] The present disclosure provides a method for reducing or preventing tumor metastasis in a subject, comprising administering an effective amount of a binding agent, an antibody or an antigen-binding fragment thereof, or a composition that binds to DPEP-1, thereby reducing or preventing tumor metastasis. The present disclosure also provides the use of a binding agent, an antibody or an antigen-binding fragment thereof, or a composition described herein for reducing or preventing tumor metastasis in a subject. The present disclosure also provides the use of a binding agent, an antibody or an antigen-binding fragment thereof, or a composition described herein for the manufacture of a medicament for reducing or preventing tumor metastasis in a subject. The present disclosure further provides a binding agent, an antibody or an antigen-binding fragment thereof, or a composition described herein for use in reducing or preventing tumor metastasis in a subject. In one embodiment, DPEP-1 acts as an adhesion molecule for leukocytes on tumor cells, independent of its enzymatic activity, and binding of DPEP-1 by a selective DPEP-1 binding agent described herein reduces or prevents tumor metastasis. In another embodiment, DPEP-1 contributes to inflammation that promotes tumor metastasis, and binding of DPEP-1 by a selective DPEP-1 binding agent reduces or prevents tumor metastasis.

[0163] In certain embodiments, the tumor is selected from tumors known to cause cancer that have the potential to metastasize or are currently capable of metastasis.For example, the cancer can be pancreatic cancer, renal cancer, genitourinary cancer, melanoma, prostate cancer, lung cancer, breast cancer, thyroid cancer, brain cancer, ovarian cancer, cervical cancer, endometrial cancer, primary peritoneal cancer, mesothelioma, eye cancer, muscle, lymphoma, esophageal cancer, gastric cancer, liver cancer, small intestine tumor, colon cancer, testicular cancer, skin cancer, or adrenal cancer.In one embodiment, the renal cancer is renal cell carcinoma (RCC).In one embodiment, the genitourinary cancer is urothelial carcinoma in the bladder, kidney, pelvis, or ureter.In one embodiment, the lung cancer is non-small cell carcinoma, small cell carcinoma, or neuroendocrine carcinoma.In one embodiment, the neuroendocrine carcinoma is a carcinoid tumor.In one embodiment, the breast cancer is ductal carcinoma, lobular carcinoma, or mixed ductal and lobular carcinoma. In one embodiment, the thyroid cancer is papillary, follicular, or medullary thyroid cancer. In one embodiment, the brain cancer is meningioma, astrocytoma, glioblastoma, cerebellar tumor, or medulloblastoma. In one embodiment, the ovarian cancer is serous, mucinous, or endometrioid. In one embodiment, the cervical cancer is squamous cell carcinoma in situ, invasive squamous cell carcinoma, or endocervical adenocarcinoma. In one embodiment, the endometrial cancer is endometrioid, serous, or mucinous. In one embodiment, the mesothelioma is pleural or peritoneal. In one embodiment, the eye cancer is retinoblastoma. In one embodiment, the muscle cancer is rhabdomyosarcoma or leiomyosarcoma. In one embodiment, the esophageal cancer is adenocarcinoma or squamous cell carcinoma. In one embodiment, the gastric cancer is gastric adenocarcinoma or gastrointestinal stromal tumor. In one embodiment, the liver cancer is hepatocellular carcinoma or cholangiocarcinoma. In one embodiment, the small intestinal tumor is a small intestinal stromal tumor or a carcinoid tumor. In one embodiment, colon cancer is colon adenocarcinoma, colon high grade dysplasia or colon carcinoid tumor.In one embodiment, skin cancer is melanoma or squamous cell carcinoma.In one embodiment, the method or use further comprises identifying the subject who needs to be treated through diagnostic testing to determine the presence of DPEP-1 binding molecule on the tumor of the patient, thereby determining the need for reducing or preventing tumor metastasis.

[0164] The present disclosure provides a method for reducing or preventing leukocyte recruitment and inflammation during sepsis in a subject, comprising administering an effective amount of a binding agent, antibody, antigen-binding fragment, or composition that binds to DPEP-1, thereby reducing or preventing organ complications of sepsis. The present disclosure also provides the use of a binding agent, antibody or antigen-binding fragment thereof, or composition described herein for reducing or preventing leukocyte recruitment and inflammation during sepsis in a subject. In some embodiments, the use reduces or prevents organ complications of sepsis. The present disclosure also provides the use of a binding agent, antibody or antigen-binding fragment thereof, or composition described herein for the manufacture of a medicament for reducing or preventing leukocyte recruitment and inflammation during sepsis in a subject. In some embodiments, the medicament reduces or prevents organ complications of sepsis. The present disclosure further provides a binding agent, antibody or antigen-binding fragment thereof, or composition described herein for use in reducing or preventing leukocyte recruitment and inflammation during sepsis in a subject. In some embodiments, the binding agent, antibody or antigen-binding fragment thereof, or composition that binds DPEP-1 is for use in reducing or preventing organ complications of sepsis.

[0165] In one embodiment, the method or use further comprises identifying the subject who needs to be treated through diagnostic examination to determine the need for reducing or preventing ischemia-reperfusion injury.The indications for treatment include but are not limited to the clinical signs and symptoms of ischemia-reperfusion injury, or undergoing surgical procedures that are at high risk of ischemia-reperfusion injury.

[0166] The present disclosure includes a method of treating a symptom of ischemia-reperfusion injury in a subject, comprising administering to a patient a pharma- ceutical effective amount of a binding agent, an antibody or an antigen-binding fragment thereof, or a composition that binds to DPEP-1. The present disclosure also includes the use of a binding agent, an antibody or an antigen-binding fragment thereof, or a composition described herein for treating a symptom of ischemia-reperfusion injury in a subject. The present disclosure also includes the use of a binding agent, an antibody or an antigen-binding fragment thereof, or a composition described herein for the manufacture of a medicament for treating a symptom of ischemia-reperfusion injury in a subject. The present disclosure further includes a binding agent, an antibody or an antigen-binding fragment thereof, or a composition described herein for use in treating a symptom of ischemia-reperfusion injury in a subject.

[0167] In one embodiment, the binding agent, antibody or antigen-binding fragment, or composition is administered or used until the symptoms of ischemia-reperfusion injury are reduced or ameliorated.

[0168] In one embodiment, the isolated binding agent, antibody or antigen-binding fragment thereof, or variant thereof is administered or used at a dosage of between about 0.1 μg / kg and 100 mg / kg, hi one embodiment, the dosage is between 2 μg and 10 g.

[0169] The present disclosure provides a method for reducing or preventing ischemia-reperfusion injury-related injury in a subject, comprising administering an effective amount of a binding agent, an antibody or an antigen-binding fragment thereof, or a composition that binds to DPEP-1, thereby reducing or preventing ischemia-reperfusion injury. The present disclosure also provides the use of the binding agent, the antibody or an antigen-binding fragment thereof, or the composition described herein for reducing or preventing ischemia-reperfusion injury-related injury in a subject. The present disclosure also provides the use of the binding agent, the antibody or an antigen-binding fragment thereof, or the composition described herein for the manufacture of a medicament for reducing or preventing ischemia-reperfusion injury-related injury in a subject. The present disclosure further provides the binding agent, the antibody or an antigen-binding fragment thereof, or the composition described herein for use in reducing or preventing ischemia-reperfusion injury-related injury in a subject.

[0170] In one embodiment, the method or use reduces or prevents leukocyte recruitment and inflammation associated with ischemia-reperfusion injury.

[0171] In one embodiment, the ischemia-reperfusion injury-related disorder is related to ischemic and post-ischemic events in organs and tissues, and the disorder is selected from the group consisting of thrombotic stroke, myocardial infarction, angina pectoris, embolic vascular occlusion, peripheral vascular insufficiency, visceral artery occlusion, artery occlusion by thrombus, artery occlusion by embolism, artery occlusion by non-occlusive process, mesenteric artery occlusion, mesenteric vein occlusion; ischemia-reperfusion injury to mesenteric microcirculation; ischemic acute renal failure, ischemia-reperfusion injury to brain tissue, intussusception, hemodynamic shock, tissue dysfunction, organ failure, restenosis, atherosclerosis, thrombosis, platelet aggregation, shock liver, spinal cord injury, or brain injury.In one embodiment, the artery occlusion by non-occlusive process is artery occlusion after low mesenteric flow or after sepsis.In one embodiment, the organ failure is heart failure, liver failure, kidney failure, etc.In one embodiment, the ischemia-reperfusion injury is caused by surgical procedure. In one embodiment, the surgical procedure is a perioperative procedure, cardiac surgery, organ surgery, organ transplantation, angiography, cardiopulmonary resuscitation, or cerebral resuscitation.In one embodiment, the ischemia-reperfusion injury is associated with harvesting a donor organ for transplantation.In one embodiment, the ischemia-reperfusion injury occurs in an allograft organ during donor procurement, ex vivo handling, or transplantation into a transplant recipient.

[0172] In one embodiment, the ischemia-reperfusion injury is associated with the harvesting of a donor organ for transplantation.

[0173] In one embodiment, ischemia-reperfusion injury occurs to an allograft organ during donor procurement, ex vivo handling, or transplantation into a transplant recipient.

[0174] In various embodiments, the binding agent, antibody, antigen-binding fragment, or composition can be administered or used (i) before, during, or after harvesting a donor organ to be transplanted, or (ii) before or during a surgical procedure in which ischemia is anticipated.

[0175] The present disclosure provides a method for reducing or preventing acute kidney injury in a subject, comprising administering an effective amount of a binding agent, antibody or antigen-binding fragment thereof, or composition that binds to DPEP-1, thereby reducing or preventing acute kidney injury.The present disclosure also provides the use of a binding agent, antibody or antigen-binding fragment thereof, or composition described herein for reducing or preventing acute kidney injury in a subject.The present disclosure also provides the use of a binding agent, antibody or antigen-binding fragment thereof, or composition described herein for the manufacture of a medicament for reducing or preventing acute kidney injury in a subject.The present disclosure further provides a binding agent, antibody or antigen-binding fragment thereof, or composition described herein for use in reducing or preventing acute kidney injury in a subject.In one embodiment, the method or use reduces or prevents leukocyte recruitment and inflammation associated with acute kidney injury.

[0176] In one embodiment, the method or use comprises identifying a subject in need of treatment through diagnostic testing to determine the need for reducing or preventing acute kidney injury. Acute kidney injury may be caused by ischemia-reperfusion, sepsis, dyes, toxins, or drugs. In one embodiment, the method or use comprises treating acute kidney injury. In one embodiment, acute kidney injury comprises ischemia-reperfusion-induced, dye-induced, toxin-induced, or drug-induced. In one embodiment, acute kidney injury is the result of ischemia-reperfusion. In one embodiment, acute kidney injury is the result of sepsis. In one embodiment, acute kidney injury is the result of dyes. In one embodiment, acute kidney injury is the result of toxins. In one embodiment, acute kidney injury is the result of drugs.

[0177] The pigment causing acute kidney injury can be myoglobin or heme, which is a component of hemoglobin. In one embodiment, the acute kidney injury is toxin-induced kidney injury. In one embodiment, the acute kidney injury is drug-induced kidney injury. In one embodiment, the acute kidney injury is pigment-induced kidney injury. In one embodiment, the pigment is myoglobin or hemoglobin. In one embodiment, the pigment is heme.

[0178] In one embodiment, the acute kidney injury is contrast-induced kidney injury.

[0179] Inflammation also plays a role in chronic kidney disease, and DPEP-1 can be a target to reduce inflammation in chronic kidney disease. Thus, in one embodiment, the method or use includes a subject who needs to be treated through diagnostic tests to determine the need for reducing or preventing chronic kidney disease. In one embodiment, the method or use includes treating chronic kidney disease. In one embodiment, the chronic kidney disease is associated with diabetes, hypertension, or glomerulonephritis. In one embodiment, the chronic kidney disease is vascular disease, glomerular disease, tubulointerstitial disease, or obstructive nephropathy. In one embodiment, the chronic kidney disease is vascular disease. In one embodiment, the vascular disease is large vessel disease or small vessel disease. In one embodiment, the large vessel disease is bilateral renal artery stenosis. In one embodiment, the small vessel disease is ischemic nephropathy, hemolytic uremic syndrome, or vasculitis. In one embodiment, the chronic kidney disease is glomerular disease. In one embodiment, the glomerular disease is a primary glomerular disease or a secondary glomerular disease. In one embodiment, the primary glomerular disease is focal segmental glomerulosclerosis or IgA nephropathy. In one embodiment, the secondary glomerular disease is diabetic nephropathy or lupus nephritis. In one embodiment, the chronic kidney disease is a tubulointerstitial disease. In one embodiment, the tubulointerstitial disease is drug-induced chronic tubulointerstitial nephritis, toxin-induced chronic tubulointerstitial nephritis, or reflux nephropathy. In one embodiment, the chronic kidney disease is obstructive nephropathy. In one embodiment, the obstructive nephropathy is bilateral kidney stones or benign prostatic hyperplasia of the prostate. In one embodiment, the chronic kidney disease is polycystic kidney disease, 17q12 microdeletion syndrome, or Mesoamerican nephropathy.

[0180] In certain embodiments, the binding agent, antibody, antigen-binding fragment, or composition may be administered or used in combination with one or more additional therapeutic agents. Co-administration or use includes simultaneous administration or use in separate compositions (also called parallel administration), administration or use at different times in separate compositions, or administration or use in a composition in which both agents are present.

[0181] In one embodiment, the at least one additional therapeutic agent is selected from the group consisting of a chemotherapeutic or anti-proliferative agent, an antiviral agent, an antibiotic, an antihistamine, an emollient, systemic phototherapy, psoralen photochemotherapy, laser therapy, hormone replacement therapy, an anti-inflammatory agent, an immunomodulatory or immunosuppressant agent, a neurotrophic factor, an agent for treating cardiovascular disease, an agent for treating diabetes, an agent for treating an immunodeficiency disorder, and an immune checkpoint inhibitor.

[0182] VI. Route of Administration A composition comprising a binding agent, antibody, or antigen-binding fragment thereof that binds to DPEP-1 as described herein may be administered or used by any suitable route. In some embodiments, the binding agent, antibody, or antigen-binding fragment thereof, or composition is administered or used parenterally. In some embodiments, parenteral administration is selected from intravenous, intradermal, inhalation, transdermal (topical), intraocular, intramuscular, subcutaneous, intramuscular, and / or transmucosal administration. In some embodiments, a composition as described herein is administered or used subcutaneously. As used herein, the term "subcutaneous tissue" is defined as a layer of loose irregular connective tissue just below the skin. For example, subcutaneous administration may be performed by injecting the composition into areas including, but not limited to, the thigh region, abdominal region, buttock region, or scapular region. In some embodiments, the binding agent, antibody, or antigen-binding fragment thereof, or composition as described herein is administered or used intravenously. In other embodiments, a binding agent, antibody or antigen-binding fragment thereof, or composition that binds DPEP-1 as described herein is administered or used by direct administration to a target tissue, such as the heart or muscle (e.g., intramuscularly), tumor (intratumorally), nervous system (e.g., direct injection into the brain; intracerebroventricularly; intrathecally). Alternatively, a binding agent, antibody or antigen-binding fragment thereof, or composition that binds DPEP-1 as described herein (or a composition or medicament containing a DPEP-1-binding antibody or antigen-binding fragment thereof as described herein) can be administered or used by inhalation, parenterally, intradermally, transdermally, or transmucosally (e.g., orally or nasally). If desired, more than one route can be used simultaneously.

[0183] In some embodiments, the binding agent, antibody or antigen-binding fragment thereof, or composition that binds to DPEP-1 as described herein is administered or used orally.In some embodiments, the present disclosure provides a solid dosage form of the binding agent, antibody or antigen-binding fragment thereof that binds to DPEP-1 as described herein for oral administration, comprising (a) a binding agent, antibody or antigen-binding fragment thereof that binds to DPEP-1, (b) at least one pharma- ceutically acceptable pH-lowering agent, (c) at least one absorption enhancer that is effective for promoting the bioavailability of the binding agent, antibody or antigen-binding fragment thereof that binds to DPEP-1, and (d) a protective vehicle.In some embodiments, the solid dosage form is a capsule or tablet.

[0184] VII. Medication An effective amount of the binding agent, antibody or its antigen-binding fragment, or composition that binds to the DPEP-1 of interest is used in treatment.The dosage of the antibody or its fragment used according to the present disclosure varies depending on the antibody or its fragment and the condition being treated.The dosage is sufficient to improve the symptoms or signs of the disease being treated without causing unacceptable toxicity to the patient.In general, an effective amount of the antibody or its fragment is one that provides either subjective relief or objectively identifiable improvement of symptoms.

[0185] Various embodiments include different dosing regimens. In some embodiments, the binding agent, antibody or antigen-binding fragment thereof, or composition that binds to DPEP-1 is administered or used via continuous infusion. In some embodiments, the continuous infusion is intravenous. In other embodiments, the continuous infusion is subcutaneous. Alternatively or additionally, in some embodiments, the binding agent, antibody or antigen-binding fragment thereof, or composition that binds to DPEP-1 is administered every other month, every month, twice monthly, every three weeks, every other week, every week, twice weekly, three times weekly, daily, twice daily, or another clinically desirable dosing schedule. The dosing regimen for a single subject does not have to be at fixed intervals and can vary over time depending on the needs of the subject.

[0186] In one embodiment, local dosage is administered or used at least once a day until therapeutic results are achieved.Dosage can be administered or used twice a day, but more or less frequent dosage can be administered.Once therapeutic results are achieved, binding agent, antibody or antigen-binding fragment thereof, or composition can be tapered or discontinued.Sometimes, side effects justify discontinuing treatment.An effective amount of the binding agent, antibody or antigen-binding fragment thereof, or composition of interest is used in treatment.

[0187] When used as pharmaceuticals, the antibodies or fragments thereof of the present disclosure are administered or used in the form of pharmaceutical compositions, which represent further aspects of the present disclosure. These antibodies or fragments thereof can be administered or used by a variety of routes, including oral, rectal, transdermal, subcutaneous, intravenous, intramuscular, and intranasal, or via intratracheal instillation or aerosol inhalation.

[0188] The antibody, fragment thereof, or composition that binds to DPEP-1 is useful for reducing inflammation or modifying the inflammatory profile of tissue, for example, in the liver. The mode of administration is defined by the application of the compound and can be determined by clinical trial methods to find the optimal dose.

[0189] In one embodiment, the dosage is between about 0.01 mg / kg and about 100 mg / kg of active binding agent, antibody, or antigen-binding fragment thereof, between about 0.01 mg / kg and about 50 mg / kg, between about 0.01 mg / kg and about 25 mg / kg, or between about 0.5 mg / kg and about 10 mg / kg.

[0190] In other embodiments, the dosage is between about 0.1 mg / kg and about 100 mg / kg, between about 0.1 mg / kg and about 50 mg / kg, between about 0.1 mg / kg and about 25 mg / kg, or between about 0.1 mg / kg and about 10 mg / kg.

[0191] In other embodiments, the dosage is between about 0.5 mg / kg to about 100 mg / kg, about 0.5 mg / kg to about 50 mg / kg, about 0.5 mg / kg to about 25 mg / kg, or about 0.5 mg / kg to about 10 mg / kg.

[0192] In other embodiments, the dosage is between about 1.0 mg / kg to about 25 mg / kg, between about 1.0 mg / kg to about 50 mg / kg, between about 1.0 mg / kg to about 70 mg / kg, between about 1.0 mg / kg to about 100 mg / kg, between about 5.0 mg / kg to about 25 mg / kg, between about 5.0 mg / kg to about 50 mg / kg, between about 5.0 mg / kg to about 70 mg / kg, between about 5.0 mg / kg to about 100 mg / kg, between about 10.0 mg / kg to about 25 mg / kg, between about 10.0 mg / kg to about 50 mg / kg, between about 10.0 mg / kg to about 70 mg / kg, or between about 10.0 mg / kg to about 100 mg / kg.

[0193] In one embodiment, the dosage is between about 0.2 mg and about 10 g of active binding agent, antibody, or antigen-binding fragment thereof, between about 0.2 mg and about 5 g, between about 0.2 mg and about 2.5 g, between about 0.2 mg and about 2 g, between about 0.2 mg and about 1 g, between about 0.2 mg and about 500 mg, or between about 0.2 mg and about 250 mg.

[0194] In one embodiment, the dosage is between about 2 mg and about 10 g of active binding agent, antibody, or antigen-binding fragment thereof, between about 2 mg and about 5 g, between about 2 mg and about 2.5 g, between about 2 mg and about 2 g, between about 2 mg and about 1 g, between about 2 mg and about 500 mg, or between about 2 mg and about 250 mg.

[0195] In one embodiment, the dosage is between about 10 mg and about 10 g of active binding agent, antibody, or antigen-binding fragment thereof, between about 10 mg and about 5 g, between about 10 mg and about 2.5 g, between about 10 mg and about 2 g, between about 10 mg and about 1 g, between about 10 mg and about 500 mg, or between about 10 mg and about 250 mg.

[0196] In other embodiments, the dosage is between about 20 mg and about 500 mg, between about 20 mg and about 1 g, between about 20 mg and about 1.4 g, between about 20 mg and about 1.5 g, between about 20 mg and about 2 g, between about 20 mg and about 2.5 g, between about 20 mg and about 5 g, between about 20 mg and about 10 g, between about 20 mg and about 500 mg, between about 100 mg and about 1 g, between about 100 mg and about 1.5 g, between about 100 mg and about 2 g, between about 100 mg and about 2.5 g, between about 100 mg and about 5 g, between about 100 mg and about 10 g, between about 200 mg and about 1 g, between about 200 mg and about 1.5 g, between about 200 mg and about 2 g, between about 200 mg and about 2.5 g, between about 200 mg and about 5 g, or about 200 Between 10 mg and about 10 g.

[0197] In other embodiments, the dosage is about 2 μg, 5 μg, 10 μg, 20 μg, 25 μg, 50 μg, 75 μg, 0.1 mg, 0.2 mg, about 0.5 mg, about 1 mg, about 2 mg, about 2.5 mg, about 5 mg, about 10 mg, about 20 mg, about 25 mg, about 50 mg, about 75 mg, about 100 mg, about 150 mg, about 200 mg, about 250 mg, about 300 mg, about 400 mg, about 500 mg, about 750 mg, about 800 mg, about 1 g, about 1.5 g, about 2 g, about 2.5 g, about 3 g, about 4 g, about 5 g, about 6 g, about 7 g, about 8 g, about 9 g, or about 10 g.

[0198] In another embodiment, the dosage is between about 50 μM and about 500 μM.

[0199] In various embodiments, the compositions or salts thereof described herein are administered or used in an amount of between about 0.001 and about 20 mg / kg body weight per day, between about 0.01 and about 10 mg / kg body weight per day, between about 0.1 and about 1000 μg / kg body weight per day, or between about 0.1 and about 100 μg / kg body weight per day. The route of administration or method of use varies. For example, the antibodies or salts thereof described herein are administered or used in an amount of between about 0.1 and about 1000 μg / kg body weight per day, or between about 0.1 and about 100 μg / kg body weight per day, by subcutaneous injection. By way of example, for a 50 kg human female subject, the daily dose of active ingredient is about 5 to about 5000 μg, or about 5 to about 5000 μg by subcutaneous injection. Different doses will be required depending on the route of administration or use, the potency of the antibody, the observed pharmacokinetic profile and applicable bioavailability, as well as the active agent and the disease being treated. In an alternative embodiment where administration or use is by inhalation, the daily dose is 1000 to about 20,000 μg, twice daily. In other embodiments, the dosage is between about 2 μg / day and about 10 g / day, about 2 μg / day, 5 μg / day, 10 μg / day, 20 μg / day, 25 μg / day, 50 μg / day, 75 μg / day, 0.1 mg / day, 0.2 mg / day, about 0.5 mg / day, about 1 mg / day, about 2 mg / day, about 2.5 mg / day, about 5 mg / day, about 10 mg / day, about 20 mg / day, about 25 mg / day, about 50 mg / day, about 75 mg / day, about 100 mg / day, about 150 mg / day, about 200 mg / day, about 250 mg / day, about 300 mg / day, about 400 mg / day, about 500 mg / day, about 750 mg / day, about 800 mg / day, about 1 g / day, about 1.5 g / day, about 2 g / day, about 2.5 g / day, about 3 g / day, about 4 g / day, about 5 g / day, about 6 g / day, about 7 g / day, about 8 g / day, about 9 g / day, or about 10 g / day.

[0200] In other mammals, such as horses, dogs, and cattle, higher doses may be required, which may be delivered in a conventional pharmaceutical composition by single administration, by multiple applications, or via controlled release, as needed to achieve the most effective results.

[0201] VIII. Kits In some embodiments, the present disclosure further provides a kit or other article of manufacture that contains the binding agent, antibody or antigen-binding fragment thereof, or pharmaceutical composition that binds to DPEP-1 described herein, and instructions for its reconstitution (if lyophilized) and / or use. The kit or other article of manufacture may include a container, a syringe, a vial, and any other item, device, or apparatus that is useful for administration (e.g., subcutaneous, by inhalation). Suitable containers include, for example, bottles, vials, syringes (e.g., pre-filled syringes), ampoules, cartridges, reservoirs, or lyo-jects. The containers can be formed from a variety of materials, such as glass or plastic. In some embodiments, the container is a pre-filled syringe. Suitable pre-filled syringes include, but are not limited to, borosilicate glass syringes with baked silicone coating, borosilicate glass syringes sprayed with silicone, or plastic resin syringes without silicone.

[0202] Typically, the container may hold the formulation, as well as a label on or associated with the container that may indicate instructions for reconstitution and / or use. For example, the label may indicate that the formulation is to be reconstituted to a concentration as described above. The label may further indicate that the formulation is useful or intended for, for example, subcutaneous administration. In some embodiments, the container may contain a single dose of a stable formulation containing an antibody, antigen-binding fragment thereof, or composition that binds DPEP-1. In various embodiments, a single dose of a stable formulation is present in a volume of less than about 15 ml, about 10 ml, about 5.0 ml, about 4.0 ml, about 3.5 ml, about 3.0 ml, about 2.5 ml, about 2.0 ml, about 1.5 ml, about 1.0 ml, or about 0.5 ml. Alternatively, the container holding the formulation may be a multi-use vial that allows for repeated administration (e.g., 2-6 administrations) of the formulation. The kit or other article of manufacture may further comprise a second container containing a suitable diluent (eg, BWFI, saline, buffered saline). Upon mixing of the diluent and formulation, the final protein concentration in the reconstituted formulation can be at least about 0.2 μg / ml (e.g., at least about 0.5 μg / ml, at least about 1 μg / ml, at least about 2 μg / ml, at least about 5 μg / ml, at least about 10 μg / ml, at least about 20 μg / ml, at least about 25 μg / ml, at least about 50 μg / ml, at least about 75 μg / ml, at least about 0.1 mg / ml, at least about 0.2 mg / ml, at least about 0.5 mg / ml, at least about 1 mg / ml, at least about 2 mg / ml, at least about 2.5 mg / ml, at least about 5 mg / ml, at least about 10 mg / ml, at least about 20 mg / ml, at least about 30 mg / ml, at least about 40 mg / ml, at least about 50 mg / ml, at least about 75 mg / ml, at least about 100 mg / ml). The kit or other article of manufacture may further include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, syringes, and package inserts with instructions for use. In some embodiments, the kit or other article of manufacture may include instructions for self-administration.

[0203] IX. Screening Methods Disclosed herein are methods for screening compositions that include antibodies that bind to DPEP-1, eg, human DPEP-1.

[0204] In one embodiment, the method comprises: (a) screening a library of test antibodies for their ability to bind to DPEP-1 in a tissue; and (b) identifying antibodies that exhibit selective binding affinity. In certain embodiments, the identified antibodies are subjected to one or more additional testing methods.

[0205] In one embodiment, the method comprises: (a) screening a library of test antibodies for their ability to bind to DPEP-1 in a tissue; and (b) identifying antibodies that exhibit selective binding affinity. In certain embodiments, the identified antibodies are subjected to one or more additional testing methods.

[0206] In one embodiment, the screening method comprises identifying an antibody effective in reducing inflammation in a patient's tissue, comprising the steps of: (a) screening a library of test antibodies for their ability to bind to DPEP-1 in the tissue; (b) selecting candidate test antibodies that exhibit selective binding affinity; (c) testing the candidate antibodies for inflammation-reducing activity, and (d) selecting the candidate antibody if it reduces inflammation, thereby providing an antibody effective in reducing inflammation.

[0207] For library test antibodies that exhibit selective binding affinity over other test antibodies in the library, e.g., at least a 10-100 fold increase in binding affinity over other antibodies, the antibody with selective binding affinity is further tested for its ability to reduce inflammation in a tissue according to the methods detailed below. Test antibodies that are shown to reduce inflammation in a tissue are then identified as lead antibodies for further antibody testing and development.

[0208] In one embodiment, the tissue is lung tissue, liver tissue, or kidney tissue.

[0209] In one embodiment, a method is provided for identifying antibodies effective in blocking leukocyte recruitment in a patient's vasculature.

[0210] In one embodiment, the disclosure provides a method for identifying an antibody effective in reducing inflammation in a patient's tissue, comprising the steps of: (a) screening a library of test antibodies for their ability to bind to DPEP-1; (b) selecting antibodies that exhibit selective binding affinity; (c) testing the antibodies for leukocyte recruitment inhibitory activity, and (d) selecting the antibody if it reduces inflammation in the tissue.

[0211] In one embodiment, the tissue is lung tissue, liver tissue, or kidney tissue.

[0212] In one embodiment, the method further comprises the steps of: (e) further testing the antibody for its ability to block leukocyte recruitment in an animal bearing a solid tumor; and (f) selecting the antibody if it blocks leukocyte recruitment in step (e).

[0213] In one embodiment, the method further comprises the steps of: (e) further testing the antibody for its ability to inhibit tumor metastasis in an animal having a solid tumor; and (f) selecting the antibody if it inhibits tumor metastasis in step (e).

[0214] In one embodiment, the method further comprises the steps of: (e) further testing the antibody for its ability to inhibit tumor metastasis to the lung and liver in an animal bearing a solid tumor known to metastasize to the lung or liver; and (f) selecting the antibody if it inhibits tumor metastasis in step (e).

[0215] In one embodiment, the method further comprises: (e) further testing the antibody for its ability to treat sepsis in the subject; and (f) selecting the antibody if it treats sepsis in step (e).

[0216] In one embodiment, the method further comprises the steps of: (e) further testing the antibody for its ability to treat bacterial sepsis in the subject; and (f) selecting the antibody if it treats sepsis in step (e).

[0217] In one embodiment, the method further comprises: (e) further testing the antibody for its ability to treat acute kidney injury in a subject; and (f) selecting the antibody if it treats acute kidney injury in step (e).

[0218] In one embodiment, step (a) in the method comprises screening a library of test antibodies for their ability to bind to DPEP-1.

[0219] In one embodiment, the method further includes identifying other secondary targets involved in inflammation, including cofactors, coreceptors, circulating factors, and accessory proteins, by using a binding agent, antibody, or antigen-binding fragment thereof that binds to DPEP-1 in a protein microarray.

[0220] The following non-limiting examples are illustrative of the present disclosure. EXAMPLES

[0221] Example 1. Antibodies that bind to human DPEP-1 Male llamas (Lama glama) were immunized with recombinant human DPEP-1 (hDPEP-1; Accession Number: P16444; NCBI Reference Sequence: NM_004413) ectodomain (amino acids 17-385). Briefly, animals were subcutaneously immunized three times (days 0, 21, and 28) with 200 μg of hDPEP-1 ectodomain. The priming immunization was supplemented with complete Freund's adjuvant, and the boosting immunization was supplemented with incomplete Freund's adjuvant. Blood samples were collected on days 0, 28, and 35, serum was obtained after clotting, and peripheral blood mononuclear cells (PBMCs) were purified by density gradient centrifugation. The resulting serum from the immunized bleeds was specific for hDPEP-1 and was shown to have a strong positive immune response against human DPEP-1 from two different sources (CreativeBioMart and SinoBiologicals). PBMCs were isolated from llamas and transfected with immunization vector pMED1. H A VH phage display library was constructed (see Hussack et al., "Neutralization of Clostridium difficile toxin A with single-domain antibodies targeting the cell receptor binding domain." J Biol Chem. 286:8961-8976 (2011), incorporated herein by reference). 94 clones were amplified for generation of monoclonal phages, and specificity for hDPEP1 was assessed by ELISA. To identify unique sequences, clones were amplified using V H Nine distinct sequences were identified by sequencing with H-specific primers. These sequences were transferred from the phagemid vector to monomeric V fused to a biotinylated acceptor peptide (BAP) and a His6 tag. HThe His6 was for purification by NiNTA chromatography and the BAP allows for site-specific biotinylation for detection by streptavidin. The nine different sequences identified are shown in Table 1 (sdABP01-09 with BAP and His6 tag; SEQ ID NO: 1-9) and Table 2A (sdABP01-09 without BAP and His6 tag; SEQ ID NO: 12-20). The CDRs of these sdABs are shown in Table 2B.

[0222] Table 1. sdABP01 with BAP and His6 tags TIFF2024513473000003.tif170146The underlined sequence is V H H, which are shown separately in Table 2A. H H is linked to the BAP sequence through a short spacer GQAGQGG (encoded by a sequence containing a SFII restriction site used during the cloning process). TIFF2024513473000004.tif4128 and is linked to a His6 tag (HHHHHH; SEQ ID NO: 11) by a spacer (LE). The amino acid sequences in bold are the CDRs, which are shown separately in Table 2B.

[0223] (Table 2A) sdABP01 without short spacer, BAP, and His6 tag TIFF2024513473000005.tif135146 The bold amino acid sequences are the CDRs, which are shown separately in Table 2B.

[0224] Table 2B: CDR sequences TIFF2024513473000006.tif141146

[0225] Example 2. Human DPEP-1 specific V H Thermal stability of H (T m ) V H Thermal unfolding midpoint temperature (T m ) in 100 mM phosphate buffer, pH 7.4, at a concentration of 200 μg / mL and measured at a wavelength of 205 nm. H H unfolding was determined using circular dichroism spectroscopy (Henry et al., 2017, Front Immunol 8:1759, incorporated herein by reference). Ellipticity measurements were normalized to a percentage scale and expressed as T m was determined from a plot of % folded versus temperature and fitting the data to a Boltzmann distribution. T m The denaturation midpoint temperature was determined by Boltzmann curve fitting (Figure 1). H HT m A summary of the C. difficile toxin A specific A20.1 V H H (Hussack et al., J Biol Chem., 286: 8961-8976

[2011] ) was included as a reference. These results show that all sdABs tested have good thermostability, with the thermostability of sdABP03 being particularly good.

[0226] (Table 3)V H HT m Summary TIFF2024513473000007.tif63128

[0227] Example 3. Human DPEP-1 specific V H SPR binding affinity of H DPEP-1 specific V HThe binding affinity of H was analyzed by surface plasmon resonance (SPR). Recombinant human DPEP-1 (hDPEP-1) ectodomain was chemically biotinylated using EZ-Link™ NHS-LC-LC-Biotin (Thermo Fisher, Cat. No. 21343) according to the manufacturer's instructions. The biotinylated recombinant hDPEP-1 ectodomain was then captured onto a CM5 sensor chip surface using Biotin CAPture reagent, followed by incubation with V at concentrations ranging from 0.625–10 nM (sdABP02), 2.5–40 nM (sdABP03 / 05 / 07), 6.25–100 nM (sdABP06), and 12.5–200 nM (sdABP01 / 04). H H was flowed across the surface. The dark lines represent the data points and the light lines are fits to the data. Data were generated in triplicate. V binding to human DPEP-1 H The SPR sensorgram showing the single cycle kinetic analysis of H is shown in FIG. H The kinetic rate constant k of H a , k d The on / off rate map is shown in Figure 2B. The diagonal line indicates the equilibrium dissociation constant K D The K obtained in FIG. D Values ​​(mean ± SD) are shown in Table 4. The binding data for sdABP01 showed a poor fit to a 1:1 binding model. As a result, its K D could not be determined with confidence. Better fits were observed using a heterologous ligand binding model. The affinity (K D ) is approximately 30 nM, which is in general agreement with the affinities obtained using fits to a more traditional 1:1 binding model.

[0228] (Table 4) K from Figure 2A D Summary of values TIFF2024513473000008.tif56128

[0229] Example 4: DPEP-1-specific V against human DPEP-1 presented on cells H H bond DPEP-1-specific V for human DPEP-1 H H binding was assessed by flow cytometry. Transfected HEK293T cells overexpressing full-length human DPEP-1 were used as targets, and parental HEK293T cells lacking expression of DPEP-1 were used as controls. Each cell line was detached and washed with Accutase® solution, then 2×10 5 Cells were incubated with 100 μL of biotinylated sdAb01-07 (100 nM final concentration) for 1 h at 4 °C. Binding was detected using streptavidin-phycoerythrin (SPE, Thermo Fisher, Cat. No. S866). Positive binding of HEK-293T-hDPEP1+ is the right profile of each graph, as shown in Figure 3A. Clostridium difficile toxin A-specific A20.1 V H H (Hussack et al., J. Biol. Chem., 286: 8961-8976

[2011] ) was used as a negative V H H was included as a control (left profile of each graph). DPEP-1-specific V H A spectral shift to the right, as seen in H, indicates binding to human DPEP-1 and is quantified as an increase in geometric mean fluorescence intensity (recorded above the histograms). H When H was tested against HEK293T-PARENTAL, which is negative for hDPEP-1 expression, no binding was observed (i.e., no spectral shift to the right). HH-binding was detected using phycoerythrin-labeled streptavidin. Data was collected on a FACScalibur (BD Bioscience) and then analyzed with FlowJo v10.6.2 (TreeStar). These results indicate that all seven sdABs tested were able to recognize DPEP-1 displayed on cells. No binding was observed even at 1000 nM to parental HEK293T cells that do not express DPEP-1. Taken together, these results indicate that the sdABs identified in Example 1 are specific for human DPEP-1.

[0230] Example 5. Human DPEP-1-specific V against human DPEP-1 presented on cells H Dose-response binding of H Human DPEP-1 specific V H Dose-response binding of H was analyzed to human DPEP-1 (hDPEP-1) displayed on cells. H Flow cytometry analysis of H was performed on HEK-293-6E cells overexpressing (hDPEP-1) using V H H concentrations (see FIG. 3B). H H (Hussack et al., J. Biol. Chem., 286: 8961-8976, (2011)) as a negative V H Biotinylated V for hDPEP-1 was included as a control. H Binding of H was detected using streptavidin-phycoerythrin (SPE). The binding profile of the anti-DPEP-1 rabbit pAb (Proteintech, Cat. No. 12222-1-AP) positive control is shown as an inset. Binding of the rabbit pAb was detected using a goat anti-rabbit IgG antibody conjugated to phycoerythrin (Thermo Fisher, Cat. No. P-2771MP). Graphs in Figure 3B with similar maximum fluorescence plateaus are grouped together and shown in Figure 3C (A20.1 V HH is shown for comparison. Three independent experiments were performed and data were collected on a Beckman CytoFLEX Analyzer and subsequently analyzed with FlowJo v10.6.2 (TreeStar). EC 50 Values ​​were calculated from FIG. 3C and recorded in Table 5. Based on the binding measured by flow cytometry, four affinity groups can be identified: intermediate (sdABP01 / 03), high (sdABP02 / 04 / 07), high-to-very high (sdABP05), and very high (sdABP06). These results confirm the binding detected by SPR and also indicate that these human DPEP-1 specific V H H binds to cells overexpressing hDPEP-1.

[0231] Next, hDPEP-1 from HEK293-6E cells overexpressing full-length hDPEP-1 was analyzed using the V H H. Individual biotinylated V H H was captured on neutravidin-sepharose beads (Thermo Fisher, Cat. No. 29202) and then incubated with Triton X-100 solubilized HEK293T-DPEP-1+ cells. Pulled-down proteins were then resolved on SDS-PAGE gels, transferred to PVDF, probed with anti-DPEP-1 rabbit pAb, and detected with goat anti-rabbit:HRP (Jackson Immunoresearch, Cat. No. 111-035-144) using SuperSignal™ West Pico PLUS Chemiluminescent Substrate (Thermo Fisher, Cat. No. 34578). Pure recombinant hDPEP-1 ectodomain (30 and 6 ng / well) was included in the assay as a positive control. Immunoblotting results are shown in Figure 3D. These results confirmed the binding and V H H specifically binds to human DPEP-1 on the surface of cells.

[0232] (Table 5) ECs from Figure 3C 50 Summary of values TIFF2024513473000009.tif95128

[0233] Example 6. Epitope binning of sdABPs by SPR and ELISA The epitope bins identified by SPR are summarized in Figure 4A. sdABP01, sdABP06, sdABP02, and sdABP07 defined bins (i), (ii), (iii), and (iv), respectively. sdABP03 and sdABP05 overlap with bin (iv), while sdABP04 overlaps with bin (iii). Epitope binning of sdABPs by ELISA was as described in Rossotti 2015 et al. ("Streamlined method for parallel identification of single domain antibodies to membrane receptors on whole cells", Biochim Biophys Acta, 1850(7):1397-404, incorporated herein by reference). Figure 4B shows the epitope bins determined by epitopes. H To cluster H, we use V H Schematic diagram of the competitive sandwich ELISA, represented as a heatmap displaying all possible pairwise combinations of H (7 × 7 = 49). Binding pairs showing high binding signals (dark) recognize non-overlapping epitopes and therefore belong to different epitope bins or V H The binding pairs showing no or weak binding signals (colorless / faint) were considered to belong to the H cluster, whereas the binding pairs showing no or weak binding signals (colorless / faint) were considered to recognize overlapping epitopes and therefore to belong to the same epitope bin. A20.1 V specific for Clostridium difficile toxin A was included as a negative control. HH (Hussack et al., 2011) did not show any binding signal. Thus, sdABP02, sdABP03, sdABP04, sdABP05, and sdABP07 all appear to be clustered around one primary epitope. The ELISA epitope binning results confirmed the SPR results and further showed that sdABP08 and sdABP09 belong to the same bin (i) as sdABP01.

[0234] Example 7. Human DPEP-1-specific sdABP reduced LPS-induced renal inflammation Because sdABP07 showed promising and selective binding to human DPEP-1 under immunofluorescence staining and high affinity binding to human DPEP-1 by flow cytometry (FIG. 3C and Table 5), this antibody was selected for further testing in its ability to affect LPS-induced endotoxemia. gfp / gfr Mice were treated with LPS (5 mg / kg, IV) in the presence or absence of sdABP07 (50 μg) to induce endotoxemia. The kidneys of the animals were imaged live by intravital microscopy 4 hours after treatment. Immunofluorescence images in Figure 5A show that sdABP07 inhibits the proliferation and secretion of LysM in LPS-treated mice. gfp / gfr The kidneys of mice (right panel) showed reduced monocyte infiltration compared to mice treated with LPS alone (middle panel), indicating that sdABP07 reduced LPS-induced renal inflammation, which was quantified by the number of adherent LysM+ monocytes found per field in the kidneys of these mice (n=4-5 / group, *: p<0.05) (see FIG. 5B).

[0235] Example 8. Anti-DPEP-1 V fused to human IgG1 hinge-Fc in mammalian cells H H(V H H-Fc) production Divalent V H A codon-optimized gene for H-Fc was synthesized (GeneArt, Thermo Fisher) and the human V HIt was cloned between the leader sequence and the gene for the human IgG1 hinge / Fc sequence. H H-Fc (SEQ ID NO: 48-56) was produced by transient expression in HEK293-6E cells as described in Durocher, Y. et al., "High-level and high-throughput recombinant protein production by transient transfection of suspension-growing human 293-EBNA1 cells" Nucleic Acids Res 30, E9 (2002) and Rossotti, MA et al. "Camelid single-domain antibodies raised by DNA immunization are potent inhibitors of EGFR signaling" Biochem J 476, 39-50 (2019), each of which is incorporated herein by reference in its entirety, and purified from the culture supernatant by protein A affinity chromatography. H H-Fc was buffer exchanged into phosphate buffered saline (PBS), pH 7.4, using an Amicon® Ultra-15 Centrifugal Filter Unit (Millipore, Cat. No. UFC905024). H The purity of H-Fc was confirmed by SDS-PAGE using 4-20% Mini-PROTEAN® TGX Stain-Free™ Gel (Biorad, Cat. No. 17000435). Table 6 shows the 9 expressed DPEP-1 V H The sequence of H-Fc is listed below.

[0236] (Table 6)V H Amino acid sequence of H-Fc TIFF2024513473000010.tif185156TIFF2024513473000011.tif167156

[0237] Example 9. DPEP-1 V H Size-exclusion chromatography analysis of H-Fc Refined V H H-Fc were subjected to size exclusion chromatography (SEC) to assess their aggregation resistance. H H-Fc was injected onto a Superdex™ 200 Increase 10 / 300 GL column (Cytiva) connected to an AKTA FPLC protein purification system (Cytiva) as previously described in Hussack, G. et al., "Neutralization of Clostridium difficile toxin A with single-domain antibodies targeting the cell receptor binding domain". J Biol Chem 286, 8961-8976 (2011), which is incorporated herein by reference. PBS was used as the running buffer at 0.8 mL / min. Their V H Similar to their H counterparts, all V H H-Fc was free of any aggregates (Figure 6).

[0238] Example 10. DPEP-1 V by ELISA H H-Fc binding verification V for DPEP-1 H H-Fc binding was assessed by ELISA. Microtiter well plates were coated with 50 ng / well of recombinant human DPEP-1 ectodomain (SinoBiologicals, Cat. No. 13543-H08H) in 100 μL of PBS overnight at 4° C. Plates were blocked with PBSC (1% casein [w / v] in PBS; Thermo Fisher, Cat. No. 37528) for 1 h at room temperature, then washed five times with PBST (PBS supplemented with 0.1% (v / v) Tween 20; Thermo Fisher, Cat. No. 28352) and incubated with various concentrations of V HAfter 1 hour of incubation, the plate was washed 10 times with PBST and incubated with V H H-Fc binding was probed with 1 μg / mL HRP-conjugated goat anti-human IgG (SIGMA, Cat. No. A0170) for 1 hour. Plates were washed 10 times and incubated with 100 μL of peroxidase substrate solution (SeraCare, Cat. No. 50-76-00) for 15 minutes at room temperature. The reaction was amplified by 50 μL of 1 M H 2 SO 4 The agonist was stopped by adding 100 mM NaCl to the wells, after which the absorbance was measured at 450 nm using a Multiskan™ FC photometer (Thermo Fisher). Data were fitted to a nonlinear regression ([agonist] vs. response, variable slope (4 parameters)) using GraphPad Prism version 9 (La Jolla, CA). Figure 7 shows the nine V H We demonstrate that all of the H-Fcs bound to their antigen, human DPEP-1.

[0239] Example 11. DPEP-1 V by flow cytometry H Assessment of H-Fc binding affinity, specificity, and cross-reactivity HEK293-6E cells, 1.5 x 10 6 Cells were grown in Gibco™ FreeStyle™ F17 Expression Medium (Thermo Fisher, Cat. No. A1383501) until a density of 100 cells / mL was reached. Transient transfection of HEK293-6E cells was performed with 100 μg of pcDNA3.1 expression plasmids encoding full-length human DPEP-1, mouse DPEP-1, rat DPEP-1, or human DPEP-2 in combination with 100 μg of DNA transfection reagent PEIpro® (VWR, Cat. No. 71002-812). Cell surface expression of DPEP-1 or DPEP-2 (DPEP-1 / 2) was performed for 96 hours. V HTo assess cell binding of H-Fc by flow cytometry, DPEP-1 / 2-expressing cells were harvested, washed once by PBS centrifugation, and diluted to 1 × 10 in PBSB (PBS containing 1% [w / v] BSA and 0.05% [w / v] sodium azide [SIGMA, catalog no. S2002]). 6 50 μL of individual DPEP-expressing cells were resuspended at 100 cells / mL. H H-Fc (250 nM) on ice for 1 h. Cells were then washed twice with PBSB by centrifugation at 1200 rpm for 5 min, and then incubated with 50 μL of 2 μg / mL R-Phycoerythrin-conjugated AffiniPure Fab Fragment Goat Anti-Human IgG, Fc Fragment Specific (Jackson Immunoresearch, Cat. No. 109-117-008) diluted in PBSB for an additional 1 h on ice. After the final wash, cells were resuspended in 100 μL PBSB and data were acquired on a CytoFLEX S flow cytometer (Beckman Coulter) and analyzed by FlowJo software (FlowJo LLC, v10.6.2, Ashland). Binding of the anti-human DPEP-1 rabbit pAb (Proteintech, catalog no. 12222-1-AP) positive control was detected using a goat anti-rabbit IgG antibody conjugated to R-phycoerythrin (Thermo Fisher, catalog no. P-2771MP).

[0240] Fixed V H The results of flow cytometry performed at H-Fc concentrations showed that 9 V H All of the H-Fcs were shown to bind to DPEP-1-expressing HEK293-6E cells and not to the parental non-DPEP-1 expressing cells, indicating that V HWe clearly demonstrated that H-Fc specifically targets DPEP-1 on the surface of cells (Figures 8A and 8E). The flow cytometry experiments were expanded to include HEK293-6E cells expressing mouse DPEP-1, rat DPEP-1, and human DPEP-2. H None of the H-Fcs bound to rat DPEP-1 or human DPEP-2, and the nine V H Only two of the H-Fcs (sdABP05 and sdABP06) cross-reacted with mouse DPEP-1, so the results indicate that V H The high specificity of H-Fc was demonstrated (Figures 8B, 8C, and 8D).

[0241] Varying concentrations of V were administered to HEK293-6E cells expressing human and mouse DPEP-1. H Flow cytometry experiments performed with H-Fc revealed that apparent EC 50 The affinity for human DPEP-1-expressing HEK293-6E cells was determined to be high (low EC 50 ; range: 0.6-1.8 nM; median: 0.9 nM) (Figure 9; Table 7). H H (Table 5) and their V H EC with H-Fc counterparts (Table 7) 50 Comparison of the V H Efficacy of H(EC 50 ) was found to be dramatically increased (see Table 8). Furthermore, sdABP05 and sdABP06 were determined to cross-react with mouse DPEP-1 with similar high affinity to human DPEP-1 (Figure 10; Table 9).

[0242] Table 7. Apparent EC from Figure 9 50 Summary of values TIFF2024513473000012.tif106128

[0243] (Table 8)V H H (Table 5) and their V H EC with H-Fc counterparts (Table 7)50 Comparison TIFF2024513473000013.tif53141

[0244] Table 9. Apparent EC from Figure 10 50 Summary of values TIFF2024513473000014.tif20141

[0245] Example 12. DPEP-1 V by SDS-PAGE / Western blot analysis H Epitope typing of H. V H To determine whether H recognizes a linear or conformational epitope, V H H-Fc was subjected to epitope typing experiment by SDS-PAGE / Western blot. 100 ng of each recombinant human DPEP-1 (Creative biomart, Cat. No. DPEP1-77H or SinoBiologicals, Cat. No. 13543-H08H) per well was separated on SDS-PAGE gel and transferred to PVDF membrane, and 100 ng of each individual DPEP-1 V H H-Fc or anti-human DPEP-1 rabbit pAb control was probed for 1 hour at room temperature. The membrane was washed 5 times and HBinding of H-Fc was detected with 1 μg / mL HRP-conjugated goat anti-human IgG (SIGMA, Cat. No. A0170) diluted in PBS / 1% BSA. Binding of pAb positive control was detected with goat anti-rabbit:HRP (Jackson Immunoresearch, Cat. No. 111-035-144). After 1 h incubation, membranes were washed 5 times with PBS-0.05% Tween 20 and peroxidase activity was detected with chemiluminescent reagent (SuperSignal West Pico PLUS Chemiluminescent Substrate, Thermo Fisher, Cat. No. 34580). Images were acquired with a Molecular Imager® Gel Doc™ XR System (BioRad, Cat. No. 1708195EDU). Epitope typing experiments revealed that V H H-Fc was shown to recognize a linear epitope, whereas the rest recognized conformational epitopes (Figure 11).

[0246] Example 13. Human DPEP-1 specific sdABP or V H H-Fc inhibits metastasis in patient-derived lung cancer xenografts in vivo Patient-derived lung cancer xenografts are maintained by passage in severe combined immunodeficient (SCID) mice. Stock tumors are aseptically harvested and minced to 1 mm 3 Six specimens are implanted into the flank of each SCID mouse. The tumor size is approximately 200 mm 3 The tumors are allowed to grow untreated until the tumors reach a concentration of 0.01%. H H-Fc(sdABP01, 02, 03, 04, 05, 06, 07, 08, or 09, or their V H H-Fc), or control Ab (A20.1) (10 mg / kg, 20 mg / kg, or 40 mg / kg, respectively) are administered twice at 7-day intervals by intravenous bolus injection. Dosage is determined based on the body weight of each individual animal weighed immediately prior to dosing. Tumor growth is monitored by caliper measurements every 3-4 days. Tumor size ranges from 0.1 to 0.2 mm.2 The width is calculated as width / length / 2, where width is the minimum size value and length is the maximum size value. The sdABPs disclosed herein significantly prevent metastasis and inhibit the growth of patient-derived lung cancer xenografts transplanted into SCID mice when compared to controls. The sdABPs disclosed herein are useful for reducing tumor burden and inhibiting metastasis.

[0247] Example 14. sdABP or V H H-Fc inhibits melanoma-lung metastasis in a syngeneic animal model in vivo C57-BL6 mice (Charles River) aged 8-10 weeks were transfected with the sdABP or V H H-Fc(sdABP05, 06, or their V H 100,000 B16-F10 mouse melanoma cells are injected intravenously 5 minutes after injection of either Ab (A20.1) (10 mg / kg, 20 mg / kg, or 40 mg / kg, respectively) via tail vein injection. Animals are sacrificed 2 weeks later and lungs are harvested. Tissues are processed for histology and hematoxylin-eosin staining is performed to assess tumor burden. The sdABPs disclosed herein, particularly sdABP05 and sdABP06, and their V-Ab, are shown to be effective in treating pulmonary edema. H The H-Fc counterpart is useful for reducing tumor burden and inhibiting metastasis.

[0248] The present disclosure is not limited in scope by the specific embodiments described herein, because such embodiments are intended as single illustrations of one aspect of the present disclosure only, and any functionally equivalent embodiments are within the scope of the present disclosure. Indeed, various modifications of the present disclosure, in addition to those shown and described herein, will become apparent to those skilled in the art from the foregoing description and the accompanying drawings. It will be appreciated how various modifications can be made without departing from the present disclosure. Such modifications are intended to fall within the scope of the appended claims.

[0249] All publications, patents, and patent applications referenced herein are incorporated by reference in their entirety to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference in its entirety. The citation of any reference herein is not an admission that such reference is available as prior art to the present disclosure.

Claims

1. (i) SEQ ID NOs: 21, 22, and 23; (ii) SEQ ID NOs: 24, 25, and 26; (iii) SEQ ID NOs: 27, 28, and 29; (iv) SEQ ID NOs: 30, 31, and 32; (v) SEQ ID NOs: 33, 34, and 35; (vi) SEQ ID NOs: 36, 37, and 38; (vii) SEQ ID NOs: 39, 40, and 41; (viii) SEQ ID NOs: 42, 43, and 44; or (ix) SEQ ID NOs: 45, 46, and 47 A binding substance that binds to DPEP-1, comprising:

2. 2. The binding agent of claim 1, comprising an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% identical to any one of SEQ ID NOs: 12-20 and 48-56.

3. The binding agent of claim 2, comprising any one of the amino acid sequences of SEQ ID NOs: 12-20 and 48-56.

4. The binding agent of claim 1, which is a monoclonal antibody, a polyclonal antibody, a chimeric antibody, a humanized antibody, or an antigen-binding fragment thereof.

5. The binding agent of claim 1, which is an antigen-binding fragment fused to an Fc domain.

6. The binding agent of claim 1, wherein the binding agent is an antigen-binding fragment, and the antigen-binding fragment is an Fv, scFv, Fab, Fab', F(ab')2, dsFv, ds-scFv, sdAB, dimeric, minibody, diabody, or multimeric antigen-binding fragment.

7. The binding agent of claim 6, wherein the antigen-binding fragment is sdAB.

8. The binding agent of claim 1 , wherein the antibody or antigen-binding fragment comprises one or more amino acids selected from the group consisting of D-amino acids, modified amino acids, amino acid analogs, or combinations thereof.

9. 9. The binding agent of claim 8, wherein the modified amino acid comprises a modification selected from the group consisting of methylation, amidation, acetylation, and / or substitution with other chemical groups.

10. The binding agent of claim 1, wherein the antibody or antigen-binding fragment is modified by pegylation, acetylation, glycosylation, biotinylation, or prenylation.

11. 2. The binding agent of claim 1, wherein the antibody or antigen-binding fragment is a human, mouse, llama, rabbit, sheep, or goat antibody or antigen-binding fragment thereof.

12. A pharmaceutical composition comprising the binding agent of any one of claims 1 to 11 and at least one pharmaceutical carrier.

13. A pharmaceutical composition for treating or preventing a disorder in a human subject in need thereof, comprising a binding agent according to any one of claims 1 to 11.

14. 14. The pharmaceutical composition of claim 13, wherein the disorder is selected from the group consisting of acute kidney injury, sepsis-induced conditions, and tumor metastasis.

15. 15. The pharmaceutical composition of claim 14, wherein the acute kidney injury comprises an ischemia-reperfusion-induced condition, a pigment-induced condition, a toxin-induced condition, or a drug-induced condition.

16. 15. The pharmaceutical composition of claim 14, wherein the sepsis-inducing condition comprises a bacterial or viral sepsis-inducing condition.

17. 17. The pharmaceutical composition of claim 16, wherein the viral sepsis-induced state comprises a COVID-19 sepsis-induced state.

18. 15. The pharmaceutical composition of claim 14, wherein the sepsis-induced condition is associated with acute respiratory distress syndrome, encephalopathy, liver failure, renal failure, or heart failure.

19. 15. The pharmaceutical composition of claim 14, wherein the tumor metastasis is associated with pancreatic cancer, renal cancer, genitourinary cancer, melanoma, prostate cancer, lung cancer, breast cancer, thyroid cancer, brain cancer, ovarian cancer, cervical cancer, endometrial cancer, primary peritoneal cancer, mesothelioma, eye cancer, muscle cancer, lymphoma, esophageal cancer, gastric cancer, liver cancer, small intestine tumors, colon cancer, testicular cancer, skin cancer, or adrenal cancer.

20. 20. The pharmaceutical composition of claim 19, wherein the kidney cancer is renal cell carcinoma (RCC).

21. 20. The pharmaceutical composition of claim 19, wherein the genitourinary cancer is urothelial carcinoma of the bladder, kidney, pelvis, or ureter.

22. 20. The pharmaceutical composition of claim 19, wherein the lung cancer is non-small cell carcinoma, small cell carcinoma, or neuroendocrine carcinoma.

23. 23. The pharmaceutical composition of claim 22, wherein the neuroendocrine cancer is a carcinoid tumor.

24. 20. The pharmaceutical composition of claim 19, wherein the breast cancer is ductal carcinoma, lobular carcinoma, or mixed ductal and lobular carcinoma.

25. 20. The pharmaceutical composition of claim 19, wherein the thyroid cancer is papillary, follicular, or medullary thyroid cancer.

26. 20. The pharmaceutical composition of claim 19, wherein the brain cancer is meningioma, astrocytoma, glioblastoma, cerebellar tumor, or medulloblastoma.

27. 20. The pharmaceutical composition of claim 19, wherein the ovarian cancer is of the serous, mucinous, or endometrioid type.

28. 20. The pharmaceutical composition of claim 19, wherein the cervical cancer is squamous cell carcinoma in situ, invasive squamous cell carcinoma, or endocervical adenocarcinoma.

29. 20. The pharmaceutical composition of claim 19, wherein the endometrial cancer is of the endometrioid, serous, or mucinous type.

30. 20. The pharmaceutical composition of claim 19, wherein the mesothelioma is pleural or peritoneal.

31. 20. The pharmaceutical composition of claim 19, wherein the eye cancer is retinoblastoma.

32. 20. The pharmaceutical composition of claim 19, wherein the muscle cancer is rhabdomyosarcoma or leiomyosarcoma.

33. 20. The pharmaceutical composition of claim 19, wherein the esophageal cancer is adenocarcinoma or squamous cell carcinoma.

34. 20. The pharmaceutical composition of claim 19, wherein the gastric cancer is gastric adenocarcinoma or gastrointestinal stromal tumor.

35. 20. The pharmaceutical composition of claim 19, wherein the liver cancer is hepatocellular carcinoma or cholangiocarcinoma.

36. 20. The pharmaceutical composition of claim 19, wherein the small intestine tumor is a small intestine stromal tumor or a carcinoid tumor.

37. 20. The pharmaceutical composition of claim 19, wherein the colon cancer is colon adenocarcinoma, colon high grade dysplasia, or colon carcinoid tumor.

38. 20. The pharmaceutical composition of claim 19, wherein the skin cancer is melanoma or squamous cell carcinoma.

39. 14. The pharmaceutical composition of claim 13, wherein the disorder is selected from the group consisting of inflammation, ischemia-reperfusion injury, and ischemia-reperfusion injury-related disorders.

40. 40. The pharmaceutical composition of claim 39, wherein the disorder is inflammation.

41. 41. The pharmaceutical composition of claim 40, wherein the inflammation is associated with an inflammatory disorder selected from the group consisting of gastritis, gout, gouty arthritis, arthritis, rheumatoid arthritis, renal failure, lupus, asthma, psoriasis, pancreatitis, allergies, fibrosis, surgical complications, anemia, fibromyalgia, cancer, heart attack, congestive heart failure, stroke, aortic stenosis, arteriosclerosis, osteoporosis, multiple sclerosis, Alzheimer's disease, Parkinson's disease, ulcers, chronic bronchitis, asthma, allergies, acute lung injury, pulmonary inflammation, airway hyperresponsiveness, vasculitis, septic shock, inflammatory skin diseases, psoriasis, atopic dermatitis, eczema, and inflammatory bowel disease.

42. 42. The pharmaceutical composition of claim 41, wherein the inflammatory bowel disease is Crohn's disease or ulcerative colitis.

43. 40. The pharmaceutical composition of claim 39, wherein the ischemia-reperfusion injury-related disorder is associated with ischemic and post-ischemic events in organs and tissues, and the disorder is selected from the group consisting of thrombotic stroke, myocardial infarction; angina pectoris, embolic vascular occlusion, peripheral vascular insufficiency, visceral arterial occlusion, arterial occlusion due to thrombus, arterial occlusion due to embolism, arterial occlusion due to non-occlusive process, mesenteric artery occlusion, mesenteric vein occlusion, ischemia-reperfusion injury to mesenteric microcirculation, ischemic acute renal failure, ischemia-reperfusion injury to brain tissue, intussusception, hemodynamic shock, tissue dysfunction, organ failure, restenosis, atherosclerosis, thrombosis, platelet aggregation, shock liver, spinal cord injury, or brain injury.

44. 44. The pharmaceutical composition of claim 43, wherein the arterial occlusion due to a non-occlusive process is arterial occlusion following low mesenteric flow or following sepsis.

45. 44. The pharmaceutical composition of claim 43, wherein the organ failure is heart failure, liver failure, renal failure, etc.

46. 40. The pharmaceutical composition of claim 39, wherein the ischemia-reperfusion injury results from a surgical procedure.

47. 47. The pharmaceutical composition of claim 46, wherein the surgical procedure is a perioperative procedure, cardiac surgery, organ surgery, organ transplantation, angiography, cardiopulmonary resuscitation, or cerebral resuscitation.

48. 40. The pharmaceutical composition of claim 39, wherein the ischemia-reperfusion injury is associated with harvesting of a donor organ for transplantation.

49. 40. The pharmaceutical composition of claim 39, wherein the ischemia-reperfusion injury occurs in an allograft organ during donor procurement, ex vivo handling, or transplantation into a transplant recipient.

50. A kit comprising the binding agent of any one of claims 1 to 11 and instructions for use.

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