Compositions and methods for guiding apolipoprotein L1 to induce mammalian cell death

Abstract

Provided herein are compositions for increasing apolipoprotein L1 (ApoL1) in a target cell. The ApoL1 may be a recombinant protein or an endogenous protein, optionally in a complex comprising ApoL1. Provided herein are antibodies and other binding molecules that specifically bind to apolipoprotein L1 (ApoL1) and haptoglobin associated protein (Hpr). In preferred embodiments, the antibodies and other molecules preferably bind to an ApoL1-containing complex, such as a trypanosomal cleavage factor (TLF), under physiological conditions. In preferred embodiments, the antibodies and antigen binding fragments are bispecific, trispecific, and multispecific molecules, which can bind to the ApoL1-containing complex and further bind to a cell specific antigen. Also provided are methods of using such molecules to increase flow of an ApoL1-containing complex into a target cell expressing the cell-specific antigen. Such an increase in ApoL-containing complexes may increase cell death. In a preferred embodiment, the target cell is a cancer cell, such as a blood cancer cell or a solid tumor cell.

Description

[0001] Cross - Reference to Related Applications

[0002] This application claims the benefit and priority of U.S. Provisional Application No. 63 / 374,356, filed on September 1, 2022, which is hereby incorporated by reference in its entirety.

[0003] Reference to a Sequence Listing

[0004] The Sequence Listing, created on September 1, 2023 and having a size of 81,564 bytes, submitted as a text file named "UGA_2022-004-02_PCT_ST26.xml", is hereby incorporated by reference in accordance with 37 C.F.R. § 1.52(e)(5).

[0005] Statement Regarding Federally Sponsored Research or Development

[0006] This invention was made with government support under Grant No. RO1-AI039033 awarded by the NIH. The government has certain rights in this invention. (37 CFR § 401.14f(4)). Field of the Invention

[0007] The present invention relates to the field of targeted, induced cell death, particularly by increasing the accumulation of apolipoprotein L1 (ApoL1) in target cells. Background of the Invention

[0009] Active multiple myeloma (MM) is an incurable plasma cell malignancy that accounts for nearly 1.8% of all newly diagnosed cancers in the United States in 2021 (Siegel et al., CA Cancer J Clin., 67:7–30 (2017)). It is characterized by the proliferation of malignant, monoclonal plasma cells (>10%) in the bone marrow, accompanied by hypercalcemia, renal failure, anemia, and osteolytic lesions (Mikhael et al., Clin Lymphoma Myeloma Leuk., 20:1–7 (2020)), and it claims the lives of more than 12,000 people in the United States each year (Siegel et al., CA Cancer J Clin., 72:7–33 (2022)). Disease progression is due to resistance to single treatment strategies, including stem cell transplantation, small molecule drugs, and biologics. Additionally, an increasing subset of patients (approximately 45,000 per year) is experiencing triple and quadruple refractory responses, where all previously used strategies have become ineffective, known as relapsed / refractory multiple myeloma (RRMM), thus highlighting the need for new treatment concepts (Sonneveld et al., Haematologica, 101:396–406 (2016)).

[0010] With population aging and better diagnostic capabilities, the number of reported MM cases is expected to increase in the coming years. The emergence of resistance to prior treatments requires multiple therapy options available for additional treatment rounds. Using each line of therapy, patient responses vary, with 74% of patients having a very good partial response at first-line treatment, while only 11% have a partial response after fifth-line treatment (Sonneveld et al., Haematologica, 101:396–406 (2016)). Time to progression (TTP) is the time from the start of treatment until disease progression. TTP between treatment lines decreases with each line of treatment, from 18 months TTP after first-line treatment, 13 months at second-line treatment, 7 months at third-line treatment, and only 5 months TTP during subsequent treatment lines. Using each line of treatment, it must be evaluated based on each patient's response during prior line treatment to determine which combination therapy can be used. It is these multi-line treatments with a wide range of therapies available that allow survival rates to be improved.

[0011] The emergence of new treatment options has improved survival rates, from a 24% five-year survival rate in the 1980s to the current 50%, with a median survival between 29 months and 62 months (Wong et al., Blood, 132 Suppl 1:4773 (2018)). However, due to the need for multiple treatment regimens after multiple relapses, this improvement in survival rates has led to a substantial increase in the costs associated with treating the disease. Therefore, there remains a need for additional therapeutic treatment lines with different mechanisms of action, especially for subsequent treatment lines when standard-of-care drugs are not an option due to reduced effectiveness or declining health of the patient.

[0012] Accordingly, it is an object of the present invention to provide alternative compositions and methods for treating multiple myeloma and other cancers. Summary of the Invention

[0014] Compositions for increasing cell death of target cells in mammalian subjects (such as humans) in need thereof and methods of using the same. The method generally comprises administering to the subject an effective amount of a composition that increases apolipoprotein L1 (ApoL1) (e.g., endogenous or exogenous ApoL1) in the target cells. Compositions are provided that bind to both ApoL1 or ApoL1-containing complexes (such as trypanosome lytic factor (TLF)), optionally TLF-1 and / or TLF-2, and a cell-specific antigen. Preferred compositions are bispecific and multispecific antibodies that comprise a first antigen-binding fragment that binds to ApoL1 or an ApoL1-containing complex, optionally TLF, and a second antigen-binding fragment that binds to a cell-specific antigen.

[0015] In other embodiments, the composition comprises ApoL1 or a functional fragment or variant thereof and a targeting moiety of a cell-specific antigen. The ApoL1 or a functional fragment or variant thereof is directly or indirectly conjugated or fused to the targeting moiety. In some embodiments, the composition comprises a delivery vehicle, optionally a liposome or a polymeric nanoparticle. The targeting moiety can be conjugated or fused to the delivery vehicle. Preferred targeting moieties are antibodies and antigen-binding fragments.

[0016] The target cell can be a mammalian or non-mammalian cell. The mammalian cell can be a diseased (e.g., cancerous) or infected cell. The non-mammalian cell can be, for example, a bacterium, a fungus, or a non-mammalian eukaryotic cell. The cell can be a human cell.

[0017] The cell-specific antigen can be specific for the diseased cell. The diseased cell can be a cancer cell, such as a blood cancer cell and a solid tumor cell. In some embodiments, the subject has a disease caused by the target cell, and the composition is administered in an effective amount to treat the disease. Preferably, the cell-specific antigen is not a trypanosome-specific surface antigen, and the subject does not have trypanosomiasis.

[0018] Also provided herein are antibodies and other binding molecules that specifically bind apolipoprotein L1 (ApoL1) and haptoglobin-related protein (Hpr). In preferred embodiments, the antibodies and other molecules bind ApoL1-containing complexes, such as trypanosome lytic factor (TLF). Preferably, the antibodies and other molecules bind ApoL1-containing complexes (such as TLF) under physiological conditions, including but not limited to endogenous complexes in vivo.

[0019] The antibody or antigen-binding fragment can be or comprise an anti-ApoL1 antibody or antigen-binding fragment that comprises the three complementarity-determining regions (CDRs) of the heavy-chain variable domain of SEQ ID NO:24 or a variant thereof having at least 70%, 80%, 90%, or 95% sequence identity thereto, and the three complementarity-determining regions (CDRs) of the light-chain variable domain of SEQ ID NO:36 or SEQ ID NO:77 or a variant thereof having at least 70%, 80%, 90%, or 95% sequence identity thereto. In some embodiments, the heavy-chain and light-chain variable domain CDRs comprise:

[0020] TYAMS (SEQ ID NO:25), EISNGGLYTYYPDTVTG (SEQ ID NO:26), ENRNWYFDL (SEQ ID NO:27), RSSQSIVNSNGNTYLE (SEQ ID NO:37), and KVSNRFS (SEQ ID NO:38), FQGSHVPLT (SEQ ID NO:39), or variants thereof having at least 70%, 80%, 90%, or 95% sequence identity thereto;

[0021] GFTFSTYA (SEQ ID NO:28), ISNGGLYT (SEQ ID NO:29), IRENRNWYFDL (SEQ ID NO:30), QSIVNSNGNTY (SEQ ID NO:40), KVS, and FQGSHVPLT (SEQ ID NO:39), or variants thereof having at least 70%, 80%, 90%, or 95% sequence identity thereto; or

[0022] GFTFSTY (SEQ ID NO:31), SNGGLY (SEQ ID NO:32), ENRNWYFDL (SEQ ID NO:27), RSSQSIVNSNGNTYLE (SEQ ID NO:37), KVSNRFS (SEQ ID NO:38), and FQGSHVPLT (SEQ ID NO:39), or variants thereof having at least 70%, 80%, 90%, or 95% sequence identity thereto.

[0023] In some embodiments, the antibody or antigen-binding fragment comprises a heavy-chain variable domain and a light-chain variable domain, the heavy-chain variable domain comprising the amino acid sequence of SEQ ID NO:24 or a variant thereof having at least 70%, 80%, 90%, or 95% sequence identity thereto, and the light-chain variable domain comprising the amino acid sequence of SEQ ID NO:36 or SEQ ID NO:77 or a variant thereof having at least 70%, 80%, 90%, or 95% sequence identity thereto.

[0024] The antibody or antigen-binding fragment can be or comprise an anti-Hpr antibody or antigen-binding fragment, the anti-Hpr antibody or antigen-binding fragment comprising three complementarity-determining regions (CDRs) of the heavy-chain variable domain of SEQ ID NO:3 or a variant thereof having at least 70%, 80%, 90%, or 95% sequence identity thereto, and three complementarity-determining regions (CDRs) of the light-chain variable domain of SEQ ID NO:14 or a variant thereof having at least 70%, 80%, 90%, or 95% sequence identity thereto. In some embodiments, the heavy-chain and light-chain variable domain CDRs comprise:

[0025] NYGMN (SEQ ID NO:4), WINSYTGEATYTDDLKG (SEQ ID NO:5), EGYGDYGYSFDY (SEQ ID NO:6), RATKNIYTYLA (SEQ ID NO:16), NAKTLAE (SEQ ID NO:17), and QHHYGTPRT (SEQ ID NO:18), or variants thereof having at least 70%, 80%, 90%, or 95% sequence identity thereto;

[0026] GYIFTNYG (SEQ ID NO:7), INSYTGEA (SEQ ID NO:8), AREGYGDYGYSFDY (SEQ ID NO:9), KNIYTY (SEQ ID NO:19), NAK, and QHHYGTPRT (SEQ ID NO:18), or variants thereof having at least 70%, 80%, 90%, or 95% sequence identity thereto; or

[0027] GYIFTNY (SEQ ID NO:10), NSYTGE (SEQ ID NO:11), EGYGDYGYSFDY (SEQ ID NO:6), RATKNIYTYLA (SEQ ID NO:16), NAKTLAE (SEQ ID NO:17), and QHHYGTPRT (SEQ ID NO:18), or variants thereof having at least 70%, 80%, 90%, or 95% sequence identity thereto.

[0028] In some embodiments, the antibody or antigen-binding fragment comprises a heavy-chain variable domain and a light-chain variable domain, the heavy-chain variable domain comprising the amino acid sequence of SEQ ID NO:3 or a variant thereof having at least 70%, 80%, 90%, or 95% sequence identity thereto, and the light-chain variable domain comprising the amino acid sequence of SEQ ID NO:14 or a variant thereof having at least 70%, 80%, 90%, or 95% sequence identity thereto.

[0029] The antibody or antigen-binding fragment can be or comprise an anti-Hpr antibody or antigen-binding fragment, the anti-Hpr antibody or antigen-binding fragment comprising three complementarity-determining regions (CDRs) of the heavy-chain variable domain of SEQ ID NO:56 or a variant thereof having at least 70%, 80%, 90%, or 95% sequence identity thereto, and three complementarity-determining regions (CDRs) of the light-chain variable domain of SEQ ID NO:65 or a variant thereof having at least 70%, 80%, 90%, or 95% sequence identity thereto. In some embodiments, the heavy-chain and light-chain variable domain CDRs comprise:

[0030] DYSIH (SEQ ID NO:57), WKHTESGESTYADDFKG (SEQ ID NO:58), GANYGSLLDY (SEQ ID NO:59), RASKSVSTSGYSYMH (SEQ ID NO:66), LASNLES (SEQ ID NO:67), QHNRELPLT (SEQ ID NO:68), or a variant thereof having at least 70%, 80%, 90%, or 95% sequence identity thereto;

[0031] GFTFTDYS (SEQ ID NO:60), KHTESGES (SEQ ID NO:61), ARGANYGSLLDY (SEQ ID NO:62), KSVSTSGYSY (SEQ ID NO:69), LAS, QHNRELPLT (SEQ ID NO:68), or a variant thereof having at least 70%, 80%, 90%, or 95% sequence identity thereto; or

[0032] GFTFTDY (SEQ ID NO:63), HTESGE (SEQ ID NO:64), GANYGSLLDY (SEQ ID NO:59), RASKSVSTSGYSYMH (SEQ ID NO:66), LASNLES (SEQ ID NO:67), QHNRELPLT (SEQ ID NO:68), or a variant thereof having at least 70%, 80%, 90%, or 95% sequence identity thereto.

[0033] In some embodiments, the antibody or antigen-binding fragment comprises a heavy-chain variable domain and a light-chain variable domain, the heavy-chain variable domain comprising the amino acid sequence of SEQ ID NO:56 or a variant thereof having at least 70%, 80%, 90%, or 95% sequence identity thereto, and the light-chain variable domain comprising the amino acid sequence of SEQ ID NO:65 or a variant thereof having at least 70%, 80%, 90%, or 95% sequence identity thereto.

[0034] The antibody or antigen-binding fragment may comprise one or more constant domains from an immunoglobulin constant region (Fc), optionally wherein the constant domain is a human constant domain. In some embodiments, the constant domain is an IgA, IgD, IgE, IgG, or IgM constant domain. In some embodiments, the antibody or antigen-binding fragment comprises one or more human IgG constant domains, optionally an IgG1, IgG2, IgG3, or IgG4 domain. In some embodiments, the antibody or antigen-binding fragment is not murine IgG1 or IgG2a.

[0035] An antibody or antigen-binding fragment can be detectably labeled or comprise a conjugated toxin, drug, receptor, enzyme, receptor ligand.

[0036] In some embodiments, the antibody or antigen-binding fragment is a monoclonal antibody, human antibody, chimeric antibody, or humanized antibody. The antibody or antigen-binding fragment can be a bispecific, trispecific, or multispecific antibody. In some embodiments, a variant of the provided sequence is a humanized form of the sequence.

[0037] In preferred embodiments, the anti-ApoL1 and / or anti-Hpr antibody or antigen-binding fragment is a bispecific, trispecific, or multispecific antibody that comprises a second (or third or more) antigen-binding fragment that binds to a cell-specific antigen. Accordingly, provided are bispecific, trispecific, or multispecific antibodies that have one or more antigen-binding fragments that bind to an ApoL1-containing complex (such as TLF) and a second (third or more) antigen-binding fragment that binds to a cell-specific antigen. In some embodiments, the cell-specific antigen is a cancer or tumor antigen. The cancer antigen can be a blood cancer antigen, optionally selected from BCMA, PD-L1 / B7-HA / CD247, CTLA4, CD38, CD319 / SLAMF-7, TNFRSF17 / BCMA, SYND1 / CD138, CD229, CD47, CD123 / IL3-RA, CD19, CD20, CD22, FcRH5, GPRC5D, and CLL-1. Accordingly, in some embodiments, the composition comprises an antibody or antigen-binding fragment that binds to: BCMA, PD-L1 / B7-HA / CD247, CTLA4, CD38, CD319 / SLAMF-7, TNFRSF17 / BCMA, SYND1 / CD138, CD229, CD47, CD123 / IL3-RA, CD19, CD20, CD22, FcRH5, GPRC5D, and CLL-1.

[0038] In other embodiments, the tumor antigen is a pancreatic cancer antigen, optionally selected from Claudin 18.2, MUC1, Mesothelin (MSLN), and Myoferlin (MYOF). Accordingly, in some embodiments, the bispecific or multispecific antibody comprises an antigen-binding fragment that specifically binds to Claudin 18.2, MUC1, Mesothelin (MSLN), and Myoferlin (MYOF).

[0039] In other embodiments, the tumor antigen is a melanoma cancer antigen, optionally PMEL17. Accordingly, in some embodiments, the bispecific or multispecific antibody comprises an antigen-binding fragment that specifically binds to PMEL17.

[0040] In some embodiments, the second antigen-binding fragment is an anti-BCMA antigen-binding fragment that comprises the three CDRs of the heavy-chain variable domain of SEQ ID NO: 41 or a variant thereof having at least 70%, 80%, 90%, or 95% sequence identity thereto, and the three CDRs of the light-chain variable domain of SEQ ID NO: 42 or a variant thereof having at least 70%, 80%, 90%, or 95% sequence identity thereto. For example, the CDRs can be CDR1H: SYAMS (SEQ ID NO: 43) or a variant thereof having at least 70%, 80%, 90%, or 95% sequence identity thereto, CDR2H: AISGSGGSTYYADSVKG (SEQ ID NO: 44) or a variant thereof having at least 70%, 80%, 90%, or 95% sequence identity thereto, CDR3H: VAPYFAPFDY (SEQ ID NO: 45) or a variant thereof having at least 70%, 80%, 90%, or 95% sequence identity thereto, CDR1L: RASQSVSSSYLA (SEQ ID NO: 46) or a variant thereof having at least 70%, 80%, 90%, or 95% sequence identity thereto, CDR2L: GASSRAT (SEQ ID NO: 47) or a variant thereof having at least 70%, 80%, 90%, or 95% sequence identity thereto, and CDR3L: QQYGNPPLYT (SEQ ID NO: 48) or a variant thereof having at least 70%, 80%, 90%, or 95% sequence identity thereto. In some embodiments, the second antigen-binding fragment comprises a heavy-chain variable domain and a light-chain variable domain, the heavy-chain variable domain comprising the amino acid sequence of SEQ ID NO: 41 or a variant thereof having at least 70%, 80%, 90%, or 95% sequence identity thereto, and the light-chain variable domain comprising the amino acid sequence of SEQ ID NO: 42 or a variant thereof having at least 70%, 80%, 90%, or 95% sequence identity thereto.

[0041] In some embodiments, the antibody or antigen-binding fragment comprises the amino acid sequence of SEQ ID NO: 71 or a variant thereof having at least 70%, 80%, 90%, or 95% sequence identity thereto, and / or the amino acid sequence of SEQ ID NO: 72 or a variant thereof having at least 70%, 80%, 90%, or 95% sequence identity thereto. In a specific embodiment, the antibody or antigen-binding fragment comprises the amino acid sequences of SEQ ID NO: 71 and SEQ ID NO: 72.

[0042] In certain exemplary constructs, the antibody is an anti-ApoL1, anti-cell specific antigen IgG1-scFv bispecific chimeric antibody, or an anti-Hpr, anti-cell specific antigen IgG1-scFv bispecific chimeric antibody, optionally having Figure 8 the structure of.

[0043] In a specific embodiment, the bispecific antibody has Figure 8 the structure formed by two copies of each of the amino acid sequences of SEQ ID NO:71 and SEQ ID NO:72, for example, when co-expressing nucleic acids encoding the amino acid sequences of SEQ ID NO:71 (e.g., SEQ ID NO:73) and SEQ ID NO:72 (e.g., SEQ ID NO:74).

[0044] Also provided are nucleic acids comprising DNA and RNA encoding the disclosed antibodies and antigen-binding fragments. The nucleic acids can be operably linked to an expression control sequence. Also provided are expression vectors, coding sequences, and cells (e.g., bacterial and mammalian cells) transformed with the nucleic acids and vectors.

[0045] Also provided is a method of forming an immune complex by contacting the disclosed antibodies and antigen-binding fragments with an ApoL1-containing complex (such as TLF), and the immune complex is optionally formed thereby and optionally further complexed with the cell surface. It is believed that such immune complexes will cause increased cell death when transported into cells. Accordingly, a method of inducing cell death by contacting target cells with the immune complex is provided. The contacting can occur in vitro or in vivo.

[0046] Also provided are pharmaceutical compositions comprising an effective amount of the disclosed antibodies and antigen-binding fragments.

[0047] Also provided is a method of treating cancer, and the method can include administering to a subject an effective amount of an antibody or antigen-binding fragment. Preferably, the antibodies and antigen-binding fragments used in such methods comprise a second (or more) antigen-binding fragment that binds to a cell-specific antigen (such as a tumor antigen) and enhances the delivery of an ApoL1-containing complex (such as TLF) to cells expressing the antigen. See, for example, FIG. 9. The cancer can be a blood cancer, such as multiple myeloma, leukemia (e.g., chronic lymphocytic leukemia, acute myeloid leukemia, acute lymphoblastic leukemia), non-Hodgkin lymphoma, Hodgkin lymphoma, myelodysplastic syndrome (MDS), myeloproliferative neoplasm (MPN) (or its subcategories, such as essential thrombocythemia (ET), myelofibrosis (MF), and polycythemia vera (PV)), amyloidosis, Waldenström macroglobulinemia, or aplastic anemia, or a solid cancer. Brief Description of the Drawings

[0049] Figures 1A - 1C It is a diagram of a selected high-density lipoprotein (HDL) complex containing apolipoprotein L1 (ApoL1). All circulating innate factors in the human body are consistent with all HDL particles, which contain: a hydrophobic core and cholesterol. Trypanolytic factor 1 (TLF-1)( Figure 1A ) is lipid-rich and has the proteins ApoL1, Hpr (with bound Hb), and ApoA1. Trypanolytic factor 2 (TLF-2)( Figure 1B ) is lipid-poor and contains unique IgM in addition to the TLF-1 protein. The HDL complex may also contain ApoL1 in the absence of TLF( Figure 1C ). Not lethal at physiological concentrations, a 10x increase in the physiological concentration of TLF-1 causes indiscriminate cell lethality.

[0050] Figure 2 It is a graph showing the binding of TLF-1 to HEK293 cells. Attempts were made to estimate the binding based on the half-maximal binding of Alexa-488 TLF-1 at different concentrations at 3 °C. Saturation was not achieved. 20,000 cells for each measurement were analyzed in triplicate by flow cytometry.

[0051] Figure 3A It is a series of images (images captured by Amnis) showing the uptake of TLF-1 (20 mg / ml) in HEK293 cells within two hours. Figure 3B It is a graph for Figure 3A quantifying the images. The pixel density was calculated by ImageStream 6.0 software and plotted as a percentage of the maximum pixel density. Figure 3C It is a series of images showing the co-localization of TLF and a lysosome tracer (lysotracker) in live HEK293 cells. To study TLF turnover in mammalian cells, HEK293 cells were incubated with Alexa Fluor 488-conjugated TLF (AF488 TLF) and imaged via ImageStream. AF488 TLF was endocytosed into vesicles within HEK293 cells. Quantification of the signal intensity indicated the maximum pixel intensity, indicating that TLF was taken up into the cells during turnover at 37 °C. When the cells were kept at 3 °C, no cell surface binding was detected( Figure 3B ). Figure 3D It is a graph showing competition with 2, 10, 50, and 100x unlabeled competitors (non-lytic HDL by mass or HP-1 by molecular weight) in a low-temperature binding assay. Figure 3EFigure showing the time course of TLF-1 uptake in HEK293 cells. Median intensity of TLF-1 was measured and quantified by Flowlo 9.6.4 software. Data points represent 20,000 cells per point.

[0052] Figure 4A Figure showing the viability of HEK293 cells over time (days) after incubation with control (no TLF), non-lytic HDL without ApoL1 (NLHDL), 10 μg / ml TLF, or 75 μg / ml TLF. Figure 4B Series of microscopic images of HEK293 untreated with TLF (left), non-lytic HDL (75 μg / ml) (middle), and TLF (75 μg / ml) (right). Figure 4C Bar graph showing the percentage decrease in growth of CCL-155 multiple myeloma cells after incubation with high concentrations of purified human TLF (1.26 floating fraction, subfractionation of human serum containing TLF). Increase in high concentration TLF / HDL fraction caused decrease in cell viability, measured by CellTiterGlo. ***25% decrease at total protein content of 4.96 mg / ml. Figure 4D Through G are figures showing that exogenously added ApoL1 reduces the growth of multiple mammalian cell lines. The selected cell lines represent various cancer models, CCL-155 (RPMI 8226): multiple myeloma ( Figure 4D ), PANC-1: pancreas ( Figure 4E ), A375: melanoma ( Figure 4F ), HT144: melanoma ( Figure 4G ). As indicated, the designated cell lines were incubated with recombinant ApoL1 for three or four days. Cell viability was measured using CellTiter-Glo on the fourth day. Data show the dose-dependent effect of recombinant ApoL1 on cell growth. Using a four-parameter model, LD50 was calculated using Quest Graph EC50 calculator. Results are as follows: CCL-155: 20.2 μg / mL, PANC-1: 43 μg / mL, A375: 27.5 μg / mL, HT144: 32.8 μg / mL. Points represent each replicate, where the dashed line represents the mean. Error bars represent standard deviation. Figure 4H Figure showing the number of viable RPMI8226 (CCL-155) cells after incubation with medium containing purified human TLF after immunoprecipitation with different concentrations of anti-ApoL1 (μg / ml) antibody. Figure 4IIt is a graph showing the number of viable RPMI 8226 (CCL-155) cells after incubation with a medium containing purified human TLF following immunoprecipitation with anti-Hpr antibodies at different concentrations (μg / ml).

[0053] Figures 5A - 5F Characterization of the recombinant anti-Hpr antibody is shown. Figure 5A and 5D is a non-stained image of total proteins (TLF and recombinant ApoL1) electrophoresed under non-reducing ( Figure 5A ) and reducing ( Figure 5D ) conditions. Figure 5B and 5E is an image of a Western blot using the recombinant anti-Hpr antibody under non-reducing ( Figure 5B ) and reducing ( Figure 5E ) conditions. Figure 5C and 5F is an image of a Western blot using the anti-Hpr antibody purified with ascites Prot-G under non-reducing ( Figure 5C ) and reducing ( Figure 5F ) conditions. Figure 5G is a dot blot (native) showing the binding of recombinant and ascites-purified anti-Hpr antibodies to recombinant ApoL1.

[0054] Figures 6A - 6G Characterization of the recombinant anti-ApoL1 antibody is shown. Figure 6A and 6D is a non-stained image of total proteins (TLF and recombinant ApoL1) electrophoresed under non-reducing ( Figure 5A ) and reducing ( Figure 6D ) conditions. Figure 6B and 6E is an image of a Western blot using the recombinant anti-ApoL1 antibody under non-reducing ( Figure 6B ) and reducing ( Figure 6E ) conditions. Figure 6C and 6F is an image of a Western blot using the anti-ApoL1 antibody purified with ascites Prot-G under non-reducing ( Figure 6C ) and reducing ( Figure 6F ) conditions. Figure 6G is a dot blot (native) showing the binding of recombinant and ascites-purified anti-ApoL1 antibodies to recombinant ApoL1.

[0055] Figures 7A - 7C Characterization of the recombinant anti-BCMA scFv is shown. Figure 7A is a non-stained image of total proteins (nr- and r-BCMA). Figure 7B and 7CIt is a Western blot image using anti-BCMA clone 17A5 scFv (SEQ ID NOL51)( Figure 7B ) and anti-BCMA intact monoclonal antibody (RnD Systems Cat.MAB1931)( Figure 7C ).

[0056] Figure 8 It is an illustration of exemplary anti-ApoL1 and anti-BCMA antibodies, which have a Fab portion and anti-BCMA as ScFv (SEQ ID NO:51) fused to the C-terminus of the heavy chain of human IgG1 provided in Example 7. The Fab portion has the heavy and light chain variable regions of recombinant clone 13.11 (anti-ApoL1) with the sequences provided in Example 6. See also Example 8.

[0057] Figures 9A - 9B It shows the binding of a bispecific antibody designed according to Figure 8 / Example 8 to BCMA and apolipoprotein L1. Figure 9A It shows the steps involved in a bridging ELISA assay to evaluate the binding ability of a bispecific antibody (bsAb) between recombinant variants of ApoL1 and BCMA. Figure 9B It shows that the bsAb successfully binds to the immobilized forms of both ligands (i.e., forms a bridge between the ligands).

[0058] Figures 10A - 10B It shows the process of ApoL1-induced cell death. Using the Promega RealTime-Glo Annexin V apoptosis and necrosis assay, RPMI 8226 multiple myeloma cells were analyzed in the presence of 3.1 μg / ml ApoL1 + 15 μg / ml bsAb, 15 μg / ml bsAb alone, 3.1 μg / ml ApoL1 alone, and cells alone ("cells only"). Within four hours, an apoptotic signal increase of 50 - 127% compared to the cells only level was observed, with the highest level measured when bsAb was added ( Figure 10A ). In the same RealTime assay, necrosis was measured under the same parameters: 3.1 μg / ml ApoL1 + 15 μg / ml bsAb, 15 μg / ml bsAb alone, 3.1 μg / ml ApoL1 alone, and cells alone. From 4 hours to 16 hours, necrosis signaling was measured to be higher in the cell lines containing bsAb compared to ApoL1 alone compared to the cells only. At 16 hours, this effect peaked at 67% for cells containing ApoL1 and bsAb. The points on the graph are shown as the average of 4 replicates, where the error bars represent the standard deviation. The data was normalized by setting the baseline to cells only, and then the data was represented as the average percentage difference compared to the baseline.Figure 10B )。

[0059] Figures 11A - 11E Demonstrate the binding of ApoL1-BCMA-bsAb and ApoL1-488 to RPMI8226 (CCL-155) multiple myeloma cells. Figure 11A Are a pair of scatter plots showing the gating strategy for selecting cells with a focus cell (gradient RMS) and correct aspect ratio (aspect ratio) to select single cells and exclude speed beads (aspect ratio). Secondary gating selects cells of the correct size to further remove speed beads, as well as cells that exclude the Zombie NIR dye (i.e., a dead cell indicator). Figure 11B Are graphs and charts showing the labeled bsAb relative to the labeled isotype control. Measure the 488 excitation / 525 emission intensity of cells passing through the gating strategy to measure the bound 488-bsAb or the bound 488-IgG1 isotype control. Figure 11C Are graphs and charts showing the results of an unlabeled bsAb competition assay to help determine if the bsAb is specifically labeled. Additional unlabeled bsAb is added to the reaction to act as a competitive inhibitor. An increase in the amount of unlabeled bsAb indicates a decrease in the bound Alexa-488 intensity. CV is defined as the coefficient of variation calculated as 100x (standard deviation / mean). Figures 11D - 11E Demonstrate the secondary binding of labeled ApoL1 after the binding of recombinant ApoL1-BCMA-bsAb to multiple myeloma cells RPMI 8226 (ATCC CCL-155). Figure 11D Are a pair of scatter plots showing the gating strategy for selecting cells with a focus cell (gradient RMS) and correct aspect ratio (aspect ratio) to select single cells and exclude speed beads (aspect ratio). Secondary gating selects cells of the correct size to further remove speed beads, as well as cells that exclude the Zombie NIR dye (i.e., a dead cell indicator). Figure 11E Are graphs and charts showing the measurement results of the intensity of ApoL1-488 bound to cell samples previously incubated with ApoL1-BCMA-bsAb or with human IgG1 isotype control. CV is defined as the coefficient of variation calculated as 100x (standard deviation / mean).

[0060] Figure 12An overview of the exemplary therapeutic method and subsequent mechanism is presented. (A) A bispecific antibody (bsAb) (e.g., for TLF and a target cell marker such as BCMA) is injected into a subject, (B) binds to endogenous TLF, and (C) attaches it to the surface of a target cell (e.g., a myeloma cell). (D) Endocytosis mediated by the target cell marker (e.g., BCMA) internalizes the immune complex, thereby increasing the intracellular concentration of TLF, where the lysosome is above the viability threshold and ultimately causes (E) target cell death. Detailed Description

[0061] I. Definitions

[0062] As used herein, the term "binding" with respect to the interaction between a binding protein and an antigen means that the interaction depends on the presence of a specific structure (e.g., an antigenic determinant or epitope) on the antigen. For example, a binding protein recognizes and binds to a specific antigenic structure and generally does not bind to the antigen. For example, if a binding protein binds to epitope "A", then in a reaction containing labeled "A" and the binding protein, the presence of a molecule containing epitope "A" (or free, unlabeled "A") will reduce the amount of labeled "A" that binds to the binding protein.

[0063] As used herein, a molecule is considered capable of "immunologically specifically binding" to a second molecule if its binding to the other molecule exhibits the specificity and affinity of an antibody for its homologous antigen. If such binding involves the antigen recognition site of an immunoglobulin molecule, the antibody is considered capable of "immunologically specifically binding" to the antigen (and in particular to the target region or conformation ("epitope") of Hpr or ApoL1). If other antigens have certain sequence or conformational similarities (e.g., determined by immunoassay, assay or other assays known in the art) recognized by the antigen recognition site, an antibody that immunologically specifically binds to a particular antigen may bind to the other antigen with lower affinity but will not bind to a completely unrelated antigen. However, preferably, the antibody (and its antigen-binding fragments) will not cross-react with other antigens. An antibody can also bind to other molecules in a non-immunologically specific manner through binding domains in other regions / domains of the molecule that do not involve the antigen recognition site, such as binding to the FcR receptor through the Fc region.

[0064] As used in the context of a combined or expressed effect, the term "substantially" is intended to mean that the observed effect is physiologically or therapeutically relevant. Similarly, a molecule is considered to have substantially the same immunospecificity and / or properties as another molecule if such immunospecificity and properties are greater than 60% identical, greater than 70% identical, greater than 75% identical, greater than 80% identical, greater than 85% identical, greater than 90% identical, greater than 95% identical or greater than 97% identical).

[0065] As used herein, the term "antibody" is intended to mean an immunoglobulin molecule having an antigen recognition site in the "variable region". The term antibody includes monoclonal antibodies, multispecific antibodies, human antibodies, humanized antibodies, synthetic antibodies, chimeric antibodies, camelized antibodies (see, e.g., Muyldermans et al., 2001, Trends Biochem. Sci. 26:230; Nuttall et al., 2000, Cur. Pharm. Biotech. 1:253; Reichmann and Muyldermans, 1999, J. Immunol. Meth. 231:25; International Publication Nos. WO 94 / 04678 and WO 94 / 25591; U.S. Patent No. 6,005,079), single-chain Fv (scFv) (see, e.g., see Pluckthun, The Pharmacology of Monoclonal Antibodies, Vol. 113, Rosenberg and Moore eds. Springer-Verlag, New York, pp. 269-315 (1994)), single-chain antibodies, disulfide-linked Fv (sdFv), intracellular antibodies and anti-idiotypic (anti-Id) antibodies (including, e.g., anti-Id and anti-anti-Id antibodies against the disclosed antibodies). Specifically, such antibodies include immunoglobulin molecules of any type (e.g., IgG, IgE, IgM, IgD, IgA and IgY), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2) or any subclass.

[0066] The term "variable region" is intended to distinguish such domains of an immunoglobulin from domains that are widely shared by antibodies (such as the antibody Fc domain). The variable region refers to the portion of the light chain and / or heavy chain of an antibody as defined herein that specifically binds to an antigen and, for example, contains the amino acid sequences of the CDRs; i.e., CDR1, CDR2 and CDR3, as well as the framework regions (FRs). For example, the variable region may contain three or four FRs (e.g., FR1, FR2, FR3 and optionally FR4) and three CDRs. VH refers to the variable region of the heavy chain. VL refers to the variable region of the light chain. The variable region contains "hypervariable regions", the residues of which are responsible for antigen binding.

[0067] The hypervariable regions contain amino acid residues from "complementary determining regions" or "CDRs" (e.g., according to Kabat, typically at about residues 24 - 34 (L1), 50 - 56 (L2), and 89 - 97 (L3) in the light chain variable domain and at about residues 27 - 35 (H1), 50 - 65 (H2), and 95 - 102 (H3) in the heavy chain variable domain; Kabat et al., Sequences of Proteins of Immunological Interest, 5th ed. Public Health Service, National Institutes of Health, Bethesda, MD. (1991)) and / or those residues from "hypervariable loops" (e.g., according to Chothia, residues 26 - 32 (L1), 50 - 52 (L2), and 91 - 96 (L3) in the light chain variable domain and residues 26 - 32 (H1), 53 - 55 (H2), and 96 - 101 (H3) in the heavy chain variable domain; Chothia and Lesk, 1987, J. Mol. Biol. 196:901 - 917). Conventions for including corrected or alternative numbering systems for variable domains include not only Kabat and Chothia but also IMGT (Lefranc et al. (2003), Dev Comp Immunol 27:55 - 77), Chothia (Chothia C, Lesk AM (1987), J Mal Biol 196:901 - 917; Chothia et al. (1989), Nature 342:877 - 883), and AHo (Honegger A, Plückthun A (2001) J Mol Biol 309:657 - 670). For convenience, examples of the binding proteins of the present disclosure may also be labeled according to Kabat, Chothia, or IMGT. These examples are specifically indicated as such.

[0068] "Framework region" or "FR" residues are those variable domain residues other than the hypervariable region residues as defined herein.

[0069] As used herein, the term "antigen-binding fragment" of an antibody refers to one or more portions of an antibody that contain the complementarity-determining regions ("CDRs") of the antibody and optionally framework residues that comprise the antigen recognition site of the "variable region" of the antibody, and that exhibit the ability to bind an antigen immunospecifically. Such fragments include Fab, Fab', F(ab')2, and Fv fragments; diabodies; linear antibodies; single-chain antibody molecules; and multispecific antibodies formed from antibody fragments, and their mutants, naturally occurring variants, and fusion proteins, including the antigen recognition site of the "variable region" of an antibody and a heterologous protein (e.g., a toxin, the antigen recognition site of a different antigen, an enzyme, a receptor, or a receptor ligand, etc.). For example, the term antigen-binding fragment can be used to refer to recombinant single-chain Fv fragments (scFv) and their divalent (di-scFv) and trivalent (tri-scFV) forms. Such fragments can be produced by a variety of methods known in the art.

[0070] As used herein, the term "constant region" refers to the portion of an antibody heavy or light chain other than the variable region. In the heavy chain, the constant region typically comprises a plurality of constant domains and a hinge region. For example, an IgG constant region comprises the following linked components: constant heavy C H 1, a linker, C H 2, and C H 3. In the heavy chain, the constant region comprises the Fc. In the light chain, the constant region typically comprises one constant domain (CL1).

[0071] The term "fragment crystallizable" or "Fc" or "Fc region" or "Fc portion" (which are used interchangeably herein) refers to the region of an antibody that contains at least one constant domain and that is typically (but not necessarily) glycosylated and capable of binding to one or more Fc receptors and / or components of the complement cascade. The heavy chain constant region can be selected from any of five isotypes: α, δ, ε, γ, or μ. Exemplary heavy chain constant regions are γ1 (IgG1), γ2 (IgG2), and γ3 (IgG3), or hybrids thereof.

[0072] A "constant domain" is a domain in an antibody that has a highly similar sequence within an antibody or within antibodies of the same type (e.g., IgG or IgM or IgE). The constant region of an antibody typically comprises a plurality of constant domains. For example, the constant region of a γ, α, or δ heavy chain comprises two constant domains.

[0073] The terms "full-length antibody", "intact antibody", or "whole antibody" are used interchangeably to refer to an antibody in its substantially intact form, as opposed to an antigen-binding fragment of an antibody. Specifically, a whole antibody includes an antibody having a heavy chain and a light chain that comprise an Fc region. The constant domain can be a wild-type sequence constant domain (e.g., a human wild-type sequence constant domain) or an amino acid sequence variant thereof.

[0074] A "chimeric antibody" is a molecule in which different parts of the antibody are derived from different immunoglobulin molecules (such as an antibody having a variable region derived from a non-human antibody and a human immunoglobulin constant region). Methods for producing chimeric antibodies are known in the art. See, for example, Morrison, 1985, Science 229:1202; Oi et al., 1986, BioTechniques 4:214; Gillies et al., 1989, J. Immunol. Methods 125:191-202; and U.S. Patent Nos. 6,311,415, 5,807,715, 4,816,567, and 4,816,397. Chimeric antibodies containing one or more CDRs from a non-human species and framework regions from a human immunoglobulin molecule can be produced using a variety of techniques known in the art, including, for example, CDR grafting (EP 239,400; International Publication No. WO 91 / 09967; and U.S. Patent Nos. 5,225,539, 5,530,101, and 5,585,089), veneering or surface resurfacing (EP 592,106; EP 519,596; Padlan, 1991, Molecular Immunology 28(4 / 5):489-498; Studnicka et al., 1994, Protein Engineering 7:805; and Roguska et al., 1994, Proc. Natl. Acad. Sci. USA 91:969), and chain shuffling (U.S. Patent No. 5,565,332).

[0075] As used herein, the term "humanized antibody" refers to an immunoglobulin that contains human framework regions and one or more CDRs from a non-human (usually murine or rat) immunoglobulin. The non-human immunoglobulin that provides the CDRs is referred to as the "donor," and the human immunoglobulin that provides the framework is referred to as the "recipient."

[0076] As used herein, the term "fragment" refers to a peptide or polypeptide having an amino acid sequence that contains at least 5 contiguous amino acid residues, at least 10 contiguous amino acid residues, at least 15 contiguous amino acid residues, at least 20 contiguous amino acid residues, at least 25 contiguous amino acid residues, at least 40 contiguous amino acid residues, at least 50 contiguous amino acid residues, at least 60 contiguous amino acid residues, at least 70 contiguous amino acid residues, at least 80 contiguous amino acid residues, at least 90 contiguous amino acid residues, at least 100 contiguous amino acid residues, at least 125 contiguous amino acid residues, at least 150 contiguous amino acid residues, at least 175 contiguous amino acid residues, at least 200 contiguous amino acid residues, or at least 250 contiguous amino acid residues.

[0077] As used herein, the term "fusion protein" refers to a polypeptide formed by joining two or more polypeptides by a peptide bond formed between the amino terminus of one polypeptide and the carboxyl terminus of another polypeptide or by joining one polypeptide to another by a reaction between amino acid side chains (e.g., a disulfide bond between cysteine residues on each polypeptide). A fusion protein can be formed by chemical coupling of the component polypeptides or it can be expressed as a single polypeptide from a nucleic acid sequence encoding a single continuous fusion protein. A fusion protein can be prepared by ligating two genes in-frame into a single nucleic acid sequence using conventional techniques in molecular biology and then expressing the nucleic acid in a suitable host cell under conditions that produce the fusion protein.

[0078] As used herein, the term "variant" refers to a polypeptide or polynucleotide that differs from a reference polypeptide or polynucleotide but retains the basic properties. A typical polypeptide variant differs in amino acid sequence from another reference polypeptide. Usually, the differences are limited such that the sequences of the reference polypeptide and the variant are overall very similar and identical in many regions. The amino acid sequences of the variant and the reference polypeptide may differ by one or more modifications (e.g., substitutions, additions, and / or deletions). The substituted or inserted amino acid residues may or may not be residues encoded by the genetic code. Variants of a polypeptide can be naturally occurring, such as allelic variants, or they can be non-naturally occurring variants.

[0079] The polypeptide structures of the present disclosure can be modified and altered, and the modifications and alterations still result in a molecule having properties similar to the polypeptide (e.g., conservative amino acid substitutions). For example, certain amino acids in the sequence can be replaced by other amino acids without significant loss of activity. Because the interaction ability and properties of a polypeptide define its biological functional activity, certain amino acid sequence substitutions can be made in the polypeptide sequence, but these amino acid sequence substitutions still result in a polypeptide having similar properties.

[0080] When making such changes, the hydrophilicity index of the amino acids can be considered. It is generally understood in the art the importance of the hydrophilic amino acid index in conferring interactive biological functions to polypeptides. It is known that certain amino acids can be replaced with other amino acids having a similar hydrophilicity index or score and still result in a polypeptide having similar biological activity. Hydrophilicity indices have been assigned to each amino acid according to their hydrophobicity and charge characteristics. These indices are: isoleucine (+4.5); valine (+4.2); leucine (+3.8); phenylalanine (+2.8); cysteine / cystine (+2.5); methionine (+1.9); alanine (+1.8); glycine (-0.4); threonine (-0.7); serine (-0.8); tryptophan (-0.9); tyrosine (-1.3); proline (-1.6); histidine (-3.2); glutamate (-3.5); glutamine (-3.5); aspartate (-3.5); asparagine (-3.5); lysine (-3.9); and arginine (-4.5).

[0081] It is believed that the relative hydrophilic character of the amino acids determines the secondary structure of the resulting polypeptide, which in turn defines the interaction of the polypeptide with other molecules such as enzymes, substrates, receptors, antibodies, antigens, and cofactors. It is known in the art that an amino acid can be replaced with another amino acid having a similar hydrophilicity index and still obtain a functionally equivalent polypeptide. In such changes, substitutions of amino acids with a hydrophilicity index within ±2 are preferred, substitutions of amino acids with a hydrophilicity index within ±1 are particularly preferred, and substitutions of amino acids with a hydrophilicity index within ±0.5 are even more particularly preferred.

[0082] Similar amino acid substitutions can also be made on the basis of hydrophilicity, especially when the resulting biologically equivalent polypeptides or peptides are intended for use in immunological embodiments. The following hydrophilicity values have been assigned to amino acid residues: arginine (+3.0); lysine (+3.0); aspartic acid (+3.0 ± 1); glutamic acid (+3.0 ± 1); serine (+0.3); asparagine (+0.2); glutamine (+0.2); glycine (0); proline (-0.5 ± 1); threonine (-0.4); alanine (-0.5); histidine (-0.5); cysteine (-1.0); methionine (-1.3); valine (-1.5); leucine (-1.8); isoleucine (-1.8); tyrosine (-2.3); phenylalanine (-2.5); tryptophan (-3.4). It should be understood that an amino acid can be substituted with another amino acid having a similar hydrophilicity value and still obtain a biologically equivalent, and especially immunologically equivalent, polypeptide. In such changes, substitutions of amino acids with hydrophilicity values within ±2 are preferred, substitutions of amino acids with hydrophilicity values within ±1 are particularly preferred, and substitutions of amino acids with hydrophilicity values within ±0.5 are even more particularly preferred.

[0083] As outlined above, amino acid substitutions are generally based on the relative similarity of the amino acid side chain substituents, such as their hydrophobicity, hydrophilicity, charge, size, etc. Exemplary substitutions taking into account the various above-mentioned properties are well known to those skilled in the art and include (original residue: exemplary substitution): (Ala: Gly, Ser), (Arg: Lys), (Asn: Gln, His), (Asp: Glu, Cys, Ser), (Gln: Asn), (Glu: Asp), (Gly: Ala), (His: Asn, Gln), (Ile: Leu, Val), (Leu: Ile, Val), (Lys: Arg), (Met: Leu, Tyr), (Ser: Thr), (Thr: Ser), (Tip: Tyr), (Tyr: Trp, Phe), and (Val: Ile, Leu). Accordingly, embodiments of the present disclosure contemplate functional or biological equivalents of the polypeptides as shown above. Specifically, embodiments of the polypeptide can include variants having about 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the polypeptide of interest.

[0084] The "percent amino acid sequence identity (%)" relative to a reference polypeptide sequence is defined as the percentage of amino acid residues in the candidate sequence that are identical to the amino acid residues in the reference polypeptide sequence after aligning the sequences and introducing gaps (if necessary) to obtain the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. The alignment for determining the percent amino acid sequence identity can be achieved in various ways well known to those skilled in the art, e.g., using publicly available computer software such as BLAST, BLAST-2, ALIGN, or Megalign (DNASTAR) software. Those skilled in the art can determine the appropriate parameters for aligning the sequences, including any algorithms required to achieve the maximum alignment over the full length of the sequences being compared.

[0085] As used herein, the terms "tumor" or "neoplasm" refer to an abnormal mass of tissue containing neoplastic cells. Neoplasms and tumors can be benign, pre-cancerous, or malignant.

[0086] As used herein, the terms "cancer" or "malignant neoplasm" refer to cells that exhibit uncontrolled growth and division, invade adjacent tissues, and often metastasize to other locations in the body.

[0087] As used herein, the term "antineoplastic agent" refers to a composition (such as a drug or a biological product) that can inhibit or prevent the growth, invasion, and / or metastasis of cancer.

[0088] As used herein, the phrase "pharmaceutically acceptable" refers to compounds, polymers, and other materials and / or dosage forms that are within the scope of reasonable medical judgment suitable for contact with the tissues of humans and animals without excessive toxicity, irritation, allergic response, or other problems or complications, commensurate with a reasonable benefit / risk ratio.

[0089] As used herein, the phrase "pharmaceutically acceptable carrier" refers to a pharmaceutically acceptable material, composition, or vehicle, such as a liquid or solid filler, diluent, solvent, or encapsulating material, which participates in carrying or transporting any subject composition from one organ or part of the body to another organ or part of the body. Each carrier must be "acceptable" in the sense of being compatible with the other ingredients of the subject composition and not injurious to the patient.

[0090] As used herein, the terms "individual", "subject", and "patient" are used interchangeably to refer to any individual who is the target of administration or treatment. The subject can be a vertebrate, such as a mammal. Thus, the subject can be a human or a veterinary patient.

[0091] As used herein, the term "treatment" refers to the medical management of a patient, with the intent to cure, ameliorate, stabilize, or prevent a disease, pathologic condition, or disorder. This term encompasses active treatment, i.e., treatment directed specifically to the improvement of a disease, pathologic condition, or disorder, and also encompasses etiologic treatment, i.e., treatment directed to the elimination of the cause of the relevant disease, pathologic condition, or disorder. Additionally, this term encompasses palliative treatment, i.e., treatment designed to relieve symptoms rather than cure the disease, pathologic condition, or disorder; prophylactic treatment, i.e., treatment directed to minimizing or partially or completely inhibiting the development of the relevant disease, pathologic condition, or disorder; and supportive treatment, i.e., treatment used to supplement another specific therapy directed to the improvement of the relevant disease, pathologic condition, or disorder.

[0092] As used herein, the term "therapeutically effective amount" means an amount of a therapeutic agent that, when incorporated into the particles and / or on the particles described herein, produces some desired effect at a reasonable benefit / risk ratio applicable to any medical treatment. The effective amount can vary depending on factors such as the disease or condition being treated, the specific targeting construct being administered, the size of the subject, or the severity of the disease or condition. A person of ordinary skill in the art can determine the effective amount of a particular compound empirically, without undue experimentation. In some embodiments, the term "effective amount" means an amount of a therapeutic or prophylactic agent that reduces or eliminates the symptoms of one or more brain diseases or disorders, such as reducing the size of a tumor (e.g., tumor volume).

[0093] Unless otherwise indicated herein, the recitation of a range of values herein is merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, and each separate value is incorporated into the specification as if it were individually recited herein.

[0094] The use of the term "about" is intended to describe values that are within approximately + / - 10% above or below the stated value; in other forms, these values can range within approximately + / - 5% above or below the stated value; in other forms, these values can range within approximately + / - 2% above or below the stated value; in other forms, these values can range within approximately + / - 1% above or below the stated value. The foregoing ranges are intended to be made clear by context and do not imply further limitation.

[0095] As used herein, "optional" or "optionally" means that the subsequent described event, circumstance, or material may or may not occur or exist, and the description includes instances where the event, circumstance, or material occurs or exists and instances where it does not occur or exist.

[0096] Ranges may be expressed herein as from “about” one particular value and / or to “about” another particular value. When such a range is expressed, unless the context clearly dictates otherwise, the range from one particular value and / or to another particular value is also specifically contemplated and considered to be disclosed. Similarly, when values are expressed as approximations by use of the antecedent “about,” it is to be understood that the particular value so qualified forms another specifically contemplated embodiment that is to be considered disclosed unless the context specifically dictates otherwise. It is further understood that each of the endpoints of each range is significant both relative to the other endpoint, and independently of the other endpoint, unless the context specifically dictates otherwise. It is understood that all individual values and sub-ranges contained within a specifically disclosed range are also specifically contemplated and considered to be disclosed unless the context specifically dictates otherwise. Finally, it is understood that all ranges refer both to the recited ranges as ranges, and to the set of individual numbers from the first endpoint to the second endpoint including the first and second endpoints. In the latter case, it is understood that any individual number may be selected as a form of the quantity, value, or feature that the range refers to. In this manner, a range describes a set of numbers or values from the first endpoint to the second endpoint including the first and second endpoints, from which a single member (i.e., a single number) of the set may be selected as the quantity, value, or feature that the range refers to. The foregoing applies regardless of whether some or all of these embodiments are specifically disclosed in a particular instance.

[0097] Each compound disclosed herein is intended and should be considered to be specifically disclosed herein. Additionally, each subgroup that can be identified within the present disclosure is intended and should be considered to be specifically disclosed herein. Accordingly, it is specifically contemplated that any compound or subgroup of compounds can be specifically included in use or excluded from use, or included in or excluded from a list of compounds.

[0098] Disclosed are the components for preparing the disclosed compositions, as well as the compositions themselves for use in the methods disclosed herein. These and other materials are disclosed herein, and it should be understood that while specific references to each different individual and collective combination and permutation of these compounds are not explicitly disclosed, each is specifically contemplated and described herein. For example, if a particular polypeptide is disclosed and discussed, and a variety of modifications that can be made to the polypeptide are discussed, then each and every combination and permutation of the polypeptide and the possible modifications are specifically contemplated unless specifically stated to the contrary. Thus, if a class of molecules A, B, and C and a class of molecules D, E, and F are disclosed, and an example of the combined molecule A-D is disclosed, then each combination is considered individually and collectively, even if each combination is not separately recited, such that combinations A-E, A-F, B-D, B-E, B-F, C-D, C-E, and C-F are considered disclosed. Similarly, any subset or combination of these is also disclosed. Thus, for example, the sub-groups A-E, B-F, and C-E are considered disclosed. This concept applies to all aspects of the present application, including but not limited to the steps in the methods of preparing and using the disclosed compositions. Thus, if there are a variety of additional steps that can be performed, it should be understood that each of these additional steps can be performed using any particular embodiment or combination of embodiments of the disclosed methods.

[0099] II. Compositions

[0100] ApoL1 or ApoL1-containing complexes (such as trypanosome lytic factor (TLF: TLF-1 or TLF-2) ( Figures 1A - 1C )) are a small subclass of human high-density lipoprotein (HDL). As used herein, TLF encompasses both TLF-1 and TLF-2, but in each case can be more specifically replaced by TLF-1, TLF-2, or TLF-1 and TLF-2.

[0101] TLF is present in plasma at a level of approximately 10 μg / mL ((Samanovic et al., PLoS Pathog., 5: e1000276 (2009), Bullard et al., Virulence. 3: 72–6 (2012)). In addition to ApoL1, ApoL1-containing complexes can include one or more of haptoglobin-related protein (Hpr), apolipoprotein A1 (ApoA1), and IgM.

[0102] Hpr is important for the binding of TLF to the trypanosome-specific receptor present in approximately 350 copies in the flagellar attachment pocket of the parasite, but humans do not have a known receptor for this protein (Drain et al., J Biol Chem., 276:30254–60 (2001)). The consensus amino acid sequence of Hpr is available at UniProt accession number P00739·HPTR_HUMAN, the entire content of which is incorporated herein by reference in its entirety and provided as SEQ ID NO:49:

[0103]

[0104] ApoL1 is a member of the Bcl-2 family, the members of which play crucial roles in the regulation of the programmed cell death (PCD) pathway. ApoL1 contains a BH3 domain pro-death region, which has been identified as important for ACD because deletion of the domain in wild-type ApoL1 abolishes its cytotoxicity (Wan et al., J Biol Chem., 283:21540–9 (2008)). Four additional regions have been mapped on ApoL1: a signal peptide (SP, aa1-27), a pore-forming domain (PFD, aa60-237), a membrane-associated domain (MAD, aa238-303), and an SRA-binding domain (aa339-398). The latter three domains have been shown to be important for ApoL1 function and toxicity, even though overexpression of any one of these three domains does not cause an increase in lethality (Lan et al., Exp Mol Pathol. 99:139–44 (2015)). Among the six members within the ApoL family, ApoL1 is the only member secreted into the serum, while the other members function intracellularly (Vanhollebeke et al., Cell Mol Life Sci CMLS., 63:1937–44 (2006)). Although the exact role of ApoL1 has not been determined, two recently evolved variants, G1 and G2, have been associated with an increased risk of chronic kidney disease (Pant et al., J Biol Chem., 297 (2021), Pays et al., J Am Soc Nephrol., 31:2502–5 (2020)).

[0105] Although associated with many distinct pathologies (Pays et al., Febs J., 288:360–81 (2021)), ApoL1 has been identified as the major component responsible for killing trypanosomes (Vanhollebeke et al., Mol Microbiol., 76:806–14 (2010)). After uptake into acidic parasite endosomes and lysosomal trafficking, ApoL1 undergoes pH-mediated activation and can insert into lipid membranes, forming a closed-state pH-gated cation channel that induces irreversible osmotic damage to the parasite (Schaub et al., J Biol Chem., 297 (2021), Harrington et al., J Biol Chem., 284:13505–12 (2009)). The consensus amino acid sequence of ApoL1 is available at Uniprot accession number O14791 APOL1_HUMAN, the entire content of which is incorporated herein by reference in its entirety and provided as SEQ ID NO:50:

[0106]

[0107] Studies have shown that at concentrations above physiological levels, TLF is able to induce a similar cascade of lethal events in mammalian cells, even though the direct mechanism has not been determined (Wan et al., J Biol Chem., 283:21540–9 (2008)).

[0108] The following results show that ApoL1 and ApoL1-containing complexes, such as TLF, can be used to increase cell death in targeted cells, including but not limited to cancer cells. Accordingly, compositions for increasing ApoL1 in target cells, and methods of using the same for inducing targeted cell death, are disclosed.

[0109] A. Compositions for targeting endogenous ApoL1

[0110] Compositions for increasing the cellular internalization of endogenous ApoL1 are provided. Although not necessarily specifically for this purpose, the compositions can be used to recruit endogenous ApoL1 into cells. The compositions are generally bound to ApoL1 and / or ApoL1-containing complexes, such as Figures 1A - 1CThe complexes shown in

[0111] and include, but are not limited to, TLF. The composition also typically binds to cell-specific markers present on the cell, such as cancer antigens, thereby facilitating targeting of the captured ApoL1 and / or ApoL1-containing complexes to the target cell. Cell-specific markers and antigens are molecules that can enhance the delivery of the composition to the target cell when targeted by a targeting moiety (e.g., an antibody or antigen-binding fragment). In some embodiments, the cell-specific marker on the target cell is elevated relative to some or all other (non-target) cells, different or unique relative to some or all other (non-target cells), or a combination thereof.

[0112] As discussed in more detail below, in some embodiments, the targeting moiety targets non-mammalian cells, such as bacteria or fungi. Targets and targeting moieties for targeting such foreign cells are discussed, for example, in Mambro et al., Sci Rep 11, 19500 (2021) doi.org / 10.1038 / s41598-021-98659-5 and Soniya et al., 35(24):6636-6645 (2014), each of which is incorporated herein by reference in its entirety and describes humanized monoclonal antibodies specific for β-1,3 glucan (i.e., several pathogenic fungal components and glycerol dilaurate lipid moieties for targeting Plasmodium-infected red blood cells (iRBCs), respectively).

[0113] Typically, the targeting moiety does not target trypanosome-specific surface antigens. In a preferred embodiment, the composition is an antibody, preferably a bispecific or multispecific antibody, which comprises an antigen-binding fragment of ApoL1 or an ApoL1-containing complex, and a cell-specific marker or antigen, respectively.

[0114] 1. Sequences of antibodies that bind ApoL1 or ApoL1-containing complexes

[0115] Provided herein are CDR and heavy and / or light chain sequences that bind ApoL1-containing complexes such as TLF. In some embodiments, the antibody binds Hpr (e.g., Hpr having the sequence of SEQ ID NO:49). In some embodiments, the antibody binds ApoL1 (e.g., ApoL1 having the sequence of SEQ ID NO:50). As discussed herein, antibodies that bind, preferably immunospecifically bind, Hpr or ApoL1 are explicitly provided, which have one or more associated CDRs or variants thereof, and / or one or two associated VH and VL sequences or variants thereof, in any and all antibody forms, including but not limited to intact antibodies and antigen-binding fragments in monospecific, bispecific, and higher-order multispecific forms, optionally in their humanized or chimeric forms. Thus, in some embodiments, the antibody is or comprises a fragment having antigen-binding ability (e.g., Fab', F(ab') 2 , Fab, Fv and rIgG, recombinant single-chain Fv fragments (scFv) and their divalent (di-scFv) and trivalent (tri-scFV) forms.

[0116] The present disclosure further encompasses nucleic acid molecules (DNA or RNA) encoding any such antibody, fusion protein or fragment, and vector molecules (such as plasmids) capable of transferring or replicating such nucleic acid molecules in a cell line and expressing such antibody, fusion protein or fragment, and host cells transformed with such nucleic acids. The nucleic acid can be single-stranded, double-stranded, and can contain both single-stranded and double-stranded portions.

[0117] a. Hpr-binding sequence - clone SFII 134.3

[0118] Cloned SFII 134.3 (i.e., a murine IgG2a that binds Hpr) was sequenced, showing the following variable domains and CDRs.

[0119] i. Heavy chain

[0120] Variable domain (VH)

[0121]

[0122] Table 1: SFII 134.3VH Complementary Determining Regions

[0123]

[0124] ii. Light Chain

[0125] Variable Domain (VL)

[0126]

[0127] Table 2: SFII 134.3VL Complementary Determining Regions

[0128]

[0129] In preferred embodiments, antibodies and other molecules contain six CDRs. The CDRs can include at least one, two, three, four, five, or six consensus CDRs of the CDRs of the anti-Hpr antibody SFII134.3, such as those provided herein.

[0130] For example, in some embodiments, the antibody or other molecule contains at least one, two, three, four, five, or six CDRs of the heavy and / or light chain variable domains of SEQ ID NO:3 and / or SEQ ID NO:14, respectively.

[0131] In some embodiments, the antibody or other molecule contains at least one, two, three, four, five, or six CDRs of SFII 134.3, optionally at least one CDR-H1, one CDR-H2, one CDR-H3, one CDR-L1, one CDR-L2, and one CDR-L3, selected from:

[0132] SFII 134.3 CDR-H1: NYGMN (SEQ ID NO:4), GYIFTNYG (SEQ ID NO:7), or GYIFTNY (SEQ ID NO:10) or variants thereof having at least 70%, 80%, 90%, or 95% sequence identity thereto;

[0133] SFII 134.3 CDR-H2: WINSYTGEATYTDDLKG (SEQ ID NO:5), INSYTGEA (SEQ ID NO:8), NSYTGE (SEQ ID NO:11) or variants thereof having at least 70%, 80%, 90%, or 95% sequence identity thereto;

[0134] SFII 134.3 CDR-H3: EGYGDYGYSFDY (SEQ ID NO:6), AREGYGDYGYSFDY (SEQ ID NO:9), or a variant or humanized variant thereof having at least 70%, 80%, 90%, or 95% sequence identity thereto;

[0135] SFII 134.3 CDR-L1: RATKNIYTYLA (SEQ ID NO:16), KNIYTY (SEQ ID NO:19), or a variant thereof having at least 70%, 80%, 90%, or 95% sequence identity thereto;

[0136] SFII 134.3 CDR-L2: NAKTLAE (SEQ ID NO:17), NAK, or a variant thereof having at least 70%, 80%, 90%, or 95% sequence identity thereto; and

[0137] SFII 134.3 CDR-L3: QHHYGTPRT (SEQ ID NO:18), or a variant or humanized variant thereof having at least 70%, 80%, 90%, or 95% sequence identity thereto.

[0138] In some embodiments, the antibody or other molecule comprises the H1-H3 CDRs and L1-L3 CDRs of SFII 134.3, respectively, selected from:

[0139] Kabat: NYGMN (SEQ ID NO:4), WINSYTGEATYTDDLKG (SEQ ID NO:5), EGYGDYGYSFDY (SEQ ID NO:6), RATKNIYTYLA (SEQ ID NO:16), NAKTLAE (SEQ ID NO:17), QHHYGTPRT (SEQ ID NO:18), or a variant thereof having at least 70%, 80%, 90%, or 95% sequence identity thereto;

[0140] IMGT: GYIFTNYG (SEQ ID NO:7), INSYTGEA (SEQ ID NO:8), AREGYGDYGYSFDY (SEQ ID NO:9), KNIYTY (SEQ ID NO:19), NAK, QHHYGTPRT (SEQ ID NO:18), or a variant thereof having at least 70%, 80%, 90%, or 95% sequence identity thereto; or

[0141] Chothia: GYIFTNY (SEQ ID NO:10), NSYTGE (SEQ ID NO:11), EGYGDYGYSFDY (SEQ ID NO:6), RATKNIYTYLA (SEQ ID NO:16), NAKTLAE (SEQ ID NO:17), QHHYGTPRT (SEQ ID NO:18), or variants thereof having at least 70%, 80%, 90%, or 95% sequence identity thereto.

[0142] In some embodiments, the antibody or other molecule comprises the heavy and / or light chain variable domains of SEQ ID NO:3 and / or SEQ ID NO:14, respectively, or variants thereof having at least 70%, 80%, 90%, or 95% sequence identity thereto.

[0143] b. Hpr binding sequence - Clone SFII 14.11

[0144] Clone SFII 14.11 (i.e., a murine IgG that binds to Hpr) was sequenced and the following variable domains and CDRs were shown.

[0145] i. Heavy chain

[0146] Variable domain (VH)

[0147]

[0148] Table 3: SFII 14.11 VH Complementary Determining Regions

[0149]

[0150] ii. Light chain

[0151] Variable domain (VL)

[0152]

[0153] Table 4: SFII 14.11 VL Complementary Determining Regions

[0154]

[0155] In preferred embodiments, the antibody and other molecule contain six CDRs. The CDRs can include at least one, two, three, four, five, or six consensus CDRs of the CDRs of anti - Hpr antibody 14.11, such as those provided herein.

[0156] For example, in some embodiments, an antibody or other molecule comprises at least one, two, three, four, five, or six CDRs of the heavy and / or light chain variable domains of SEQ ID NO:56 and / or SEQ ID NO:65.

[0157] In some embodiments, an antibody or other molecule comprises at least one, two, three, four, five, or six CDRs of SFII 14.11, optionally at least one CDR-H1, one CDR-H2, one CDR-H3, one CDR-L1, one CDR-L2, and one CDR-L3, selected from:

[0158] SFII 14.11 CDR-H1: DYSIH (SEQ ID NO:57), WKHTESGESTYADDFKG (SEQ ID NO:58), or GANYGSLLDY (SEQ ID NO:59), or a variant thereof having at least 70%, 80%, 90%, or 95% sequence identity thereto;

[0159] SFII 14.11 CDR-H2: GFTFTDYS (SEQ ID NO:60), KHTESGES (SEQ ID NO:61), ARGANYGSLLDY (SEQ ID NO:62), or a variant thereof having at least 70%, 80%, 90%, or 95% sequence identity thereto;

[0160] SFII 14.11 CDR-H3: GFTFTDY (SEQ ID NO:63), HTESGE (SEQ ID NO:64), or GANYGSLLDY (SEQ ID NO:59), or a variant thereof having at least 70%, 80%, 90%, or 95% sequence identity thereto;

[0161] SFII 14.11 CDR-L1: RASKSVSTSGYSYMH (SEQ ID NO:66), KSVSTSGYSY (SEQ ID NO:69), or a variant thereof having at least 70%, 80%, 90%, or 95% sequence identity thereto;

[0162] SFII 14.11 CDR-L2: LASNLES (SEQ ID NO:67), LAS, or a variant thereof having at least 70%, 80%, 90%, or 95% sequence identity thereto; and

[0163] SFII 14.11 CDR-L3: QHNRELPLT (SEQ ID NO:68), or a variant thereof having at least 70%, 80%, 90%, or 95% sequence identity thereto or a humanized variant.

[0164] In some embodiments, the antibody or other molecule comprises the H1-H3 CDRs and L1-L3 CDRs of SFII 14.11, respectively, selected from:

[0165] Kabat: DYSIH (SEQ ID NO:57), WKHTESGESTYADDFKG (SEQ ID NO:58), GANYGSLLDY (SEQ ID NO:59), RASKSVSTSGYSYMH (SEQ ID NO:66), LASNLES (SEQ ID NO:67), QHNRELPLT (SEQ ID NO:68), or variants thereof having at least 70%, 80%, 90%, or 95% sequence identity thereto;

[0166] IMGT: GFTFTDYS (SEQ ID NO:60), KHTESGES (SEQ ID NO:61), ARGANYGSLLDY (SEQ ID NO:62), KSVSTSGYSY (SEQ ID NO:69), LAS, QHNRELPLT (SEQ ID NO:68), or variants thereof having at least 70%, 80%, 90%, or 95% sequence identity thereto; or

[0167] Chothia: GFTFTDY (SEQ ID NO:63), HTESGE (SEQ ID NO:64), GANYGSLLDY (SEQ ID NO:59), RASKSVSTSGYSYMH (SEQ ID NO:66), LASNLES (SEQ ID NO:67), QHNRELPLT (SEQ ID NO:68), or variants thereof having at least 70%, 80%, 90%, or 95% sequence identity thereto.

[0168] In some embodiments, the antibody or other molecule comprises the heavy and / or light chain variable domains of SEQ ID NO:56 and / or SEQ ID NO:65, respectively, or variants thereof having at least 70%, 80%, 90%, or 95% sequence identity thereto.

[0169] c. ApoL1 binding sequence

[0170] Sequencing of the cloned SFIII 13.11 (i.e., mouse IgG1 that binds ApoL1) revealed the following variable domains and CDRs.

[0171] i. Heavy chain

[0172] Variable domain (VH)

[0173]

[0174] Table 5: SFIII 13.11 VH Complementary Determining Regions

[0175]

[0176] ii. Light chain

[0177] Variable domain (VL)

[0178]

[0179] Or

[0180]

[0181] Table 6: SFIII 13.11 VL Complementary Determining Regions

[0182]

[0183] In preferred embodiments, the antibody and other molecules contain six CDRs. The CDRs can include at least one, two, three, four, five, or six consensus CDRs from the CDRs of the anti-ApoL1 antibody SFIII 13.11.

[0184] For example, in some embodiments, the antibody or other molecule contains at least one, two, three, four, five, or six CDRs of the heavy and / or light chain variable domains of SEQ ID NO:24 and / or SEQ ID NO:36 or SEQ ID NO:77, respectively.

[0185] In some embodiments, the antibody or other molecule contains at least one, two, three, four, five, or six CDRs of SFIII 13.11, optionally at least one CDR-H1, one CDR-H2, one CDR-H3, one CDR-L1, one CDR-L2, and one CDR-L3, selected from:

[0186] SFIII 13.11 CDR-H1: TYAMS (SEQ ID NO:25), GFTFSTYA (SEQ ID NO:28), GFTFSTY (SEQ ID NO:31), or variants thereof having at least 70%, 80%, 90%, or 95% sequence identity thereto;

[0187] SFIII 13.11 CDR-H2: EISNGGLYTYYPDTVTG (SEQ ID NO:26), ISNGGLYT (SEQ ID NO:29), SNGGLY (SEQ ID NO:32), or variants thereof having at least 70%, 80%, 90%, or 95% sequence identity thereto;

[0188] SFIII 13.11 CDR-H3: ENRNWYFDL (SEQ ID NO:27), IRENRNWYFDL (SEQ ID NO:30), or variants thereof having at least 70%, 80%, 90%, or 95% sequence identity thereto;

[0189] SFIII 13.11 CDR-L1: RSSQSIVNSNGNTYLE (SEQ ID NO:37), QSIVNSNGNTY (SEQ ID NO:40), or variants thereof having at least 70%, 80%, 90%, or 95% sequence identity thereto;

[0190] SFIII 13.11 CDR-L2: KVSNRFS (SEQ ID NO:38), KVS, or variants thereof having at least 70%, 80%, 90%, or 95% sequence identity thereto; and

[0191] SFIII 13.11 CDR-L3: FQGSHVPLT (SEQ ID NO:39), or variants thereof having at least 70%, 80%, 90%, or 95% sequence identity thereto or humanized variants.

[0192] In some embodiments, the antibody or other molecule comprises the H1-H3 CDRs and L1-L3 CDRs of SFIII 13.11, respectively, selected from:

[0193] Kabat: TYAMS (SEQ ID NO:25), EISNGGLYTYYPDTVTG (SEQ ID NO:26), ENRNWYFDL (SEQ ID NO:27), RSSQSIVNSNGNTYLE (SEQ ID NO:37), KVSNRFS (SEQ ID NO:38), FQGSHVPLT (SEQ ID NO:39), or variants thereof having at least 70%, 80%, 90%, or 95% sequence identity thereto;

[0194] IMGT: GFTFSTYA (SEQ ID NO:28), ISNGGLYT (SEQ ID NO:29), IRENRNWYFDL (SEQ ID NO:30), QSIVNSNGNTY (SEQ ID NO:40), KVS, FQGSHVPLT (SEQ ID NO:39) or variants thereof having at least 70%, 80%, 90% or 95% sequence identity thereto; or

[0195] Chothia: GFTFSTY (SEQ ID NO:31), SNGGLY (SEQ ID NO:32), ENRNWYFDL (SEQ ID NO:27), RSSQSIVNSNGNTYLE (SEQ ID NO:37), KVSNRFS (SEQ ID NO:38), FQGSHVPLT (SEQ ID NO:39) or variants thereof having at least 70%, 80%, 90% or 95% sequence identity thereto.

[0196] In some embodiments, the antibody or other molecule comprises the heavy and / or light chain variable domains of SEQ ID No:24 and / or SEQ ID NO:36 or SEQ ID NO:77, respectively, or variants thereof having at least 70%, 80%, 90% or 95% sequence identity thereto.

[0197] 2. Chimeric and Humanized Antibodies

[0198] The present disclosure specifically relates to chimeric and humanized antibodies. The constant region need not be present, but if present, it is generally substantially identical to a human immunoglobulin constant region, i.e., at least about 85 - 90% and preferably about 95% or more identical. Thus, all parts of the humanized immunoglobulin, except for the possible CDRs, are substantially identical to the corresponding parts of the native human immunoglobulin sequence. A humanized antibody is an antibody having humanized light and heavy chain immunoglobulins. For example, a humanized antibody does not cover a typical chimeric antibody because, for example, the entire variable region of a chimeric antibody is non - human. It is said that through the "humanization" process, the donor antibody has been "humanized" because the resulting humanized antibody is expected to bind the same antigen as the donor antibody that provided the CDRs.

[0199] In most cases, a humanized antibody is a human immunoglobulin (recipient antibody) in which the hypervariable region residues of the recipient have been replaced by hypervariable region residues from a non-human species (donor antibody) having the desired specificity, affinity, and capacity (such as a mouse, rat, rabbit, or non-human primate). In some cases, framework region (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues. Additionally, a humanized antibody can contain residues not found in the recipient antibody or donor antibody. These modifications are made to further improve antibody efficacy. Generally, a humanized antibody will contain substantially all (at least one, and usually two) of the variable domains, wherein all or substantially all of the hypervariable regions correspond to hypervariable loops of a non-human immunoglobulin, and all or substantially all of the FRs are FRs of a human immunoglobulin sequence. Optionally, a humanized antibody will also contain at least a portion of the immunoglobulin constant region (Fc), typically the constant region of a human immunoglobulin that immunospecifically binds to an FcγRIIB polypeptide, which has been altered by the introduction of amino acid residue substitutions, deletions, or additions (i.e., mutations).See also, e.g., European Patent Nos. EP 239,400, EP 592,106, and EP 519,596; International Publication Nos. WO 91 / 09967 and WO 93 / 17105; U.S. Patent Nos. 5,225,539, 5,530,101, 5,565,332, 5,585,089, 5,766,886, and 6,407,213; and Padlan, 1991, Molecular Immunology 28(4 / 5):489-498; Studnicka et al., 1994, Protein Engineering 7(6):805-814; Roguska et al., 1994, PNAS 91:969-973; Tan et al., 2002, J. Immunol. 169:1119-1125; Caldas et al., 2000, Protein Eng. 13:353-360; Morea et al., 2000, Methods 20:267-79; Baca et al., 1997, J. Biol. Chem. 272:10678-10684; Roguska et al., 1996, Protein Eng. 9:895-904; Couto et al., 1995, Cancer Res. 55(23 Suppl.):5973s-5977s; Couto et al., 1995, Cancer Res. 55:1717-22; Sandhu, 1994, Gene 150:409-10; Pedersen et al., 1994, J. Mol. Biol. 235:959-973; Jones et al., 1986, Nature 321:522-525; Reichmann et al., 1988, Nature 332:323-329; and Presta, 1992, Curr. Op. Struct. Biol. 2:593-596).

[0200] DNA sequences encoding preferred human receptor framework sequences include, but are not limited to, FR segments from the human germline VH segments VH1-18 and JH6, and the human germline VL segments VK-A26 and JK4. In a specific embodiment, one or more CDRs are inserted into the framework region using conventional recombinant DNA techniques. The framework region can be a naturally occurring or consensus framework region, and is preferably a human framework region (see, e.g., Chothia et al., 1998, “Structural Determinants In The Sequences Of Immunoglobulin Variable Domain,” J. Mol. Biol. 278:457-479, for a list of human framework regions).

[0201] Humanized or chimeric antibodies can include substantially all of at least one, and usually two, variable domains, wherein all or substantially all of the CDR regions correspond to the CDR regions of a non-human immunoglobulin (i.e., a donor antibody), and all or substantially all of the framework regions are framework regions of human immunoglobulin consensus sequences. Preferably, the antibody also comprises at least a portion of the immunoglobulin constant region (Fc), usually at least a portion of a human immunoglobulin constant region. The constant domain of the antibody can be selected relative to the proposed function of the antibody, particularly effector functions that may be desired. In some embodiments, the constant domain of the antibody is (or includes) a human IgA, IgD, IgE, IgG, or IgM domain. In a specific embodiment, when a humanized antibody is intended for therapeutic use and antibody effector functions (such as antibody-dependent cell-mediated cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) activities) are required, human IgG constant domains, particularly IgG1 and IgG3 isotypes, are used. In alternative embodiments, when the antibody is intended for therapeutic purposes and antibody effector functions are not required, IgG2 and IgG4 isotypes are used. The present disclosure encompasses Fc constant domains containing one or more amino acid modifications that alter antibody effector functions, such as those disclosed in U.S. Patent Application Publication Nos. 2005 / 0037000 and 2005 / 0064514.

[0202] In some embodiments, the antibody contains both a light chain and variable domains of at least the heavy chain. In other embodiments, the antibody can further comprise one or more of the CH1, hinge, CH2, CH3, and CH4 regions of the heavy chain. The antibody can be selected from any class of immunoglobulins, including IgM, IgG, IgD, IgA, and IgE, and any isotype, including IgG1, IgG2, IgG3, and IgG4. In some embodiments, when it is desired for the antibody to exhibit cytotoxic activity, the constant domain is a complement-fixing constant domain, and the class is usually IgG1. In other embodiments, when such cytotoxic activity is not required, the constant domain can be of the IgG2 class. The antibody can include sequences from more than one class or isotype, and selection of a particular constant domain to optimize desired effector functions is within the ordinary skill in the art. In some embodiments, the antibody is not murine IgG1 or murine IgG2a.

[0203] The framework and CDR regions of a humanized antibody need not precisely correspond to the parental sequences. For example, the donor CDR or consensus framework can be mutagenized by substitution, insertion, or deletion of at least one residue such that the CDR or framework residue at that site does not correspond to the consensus or donor antibody. However, such mutations are preferably not extensive. Typically, at least 75% of the residues of a humanized antibody will correspond to the residues of the parental framework region (FR) and CDR sequences, more commonly 90%, and most preferably greater than 95%. Humanized antibodies can be produced using a variety of techniques known in the art, including but not limited to CDR-grafting (European Patent No. EP 239,400; International Publication No. WO 91 / 09967; and U.S. Patent Nos. 5,225,539, 5,530,101, and 5,585,089), veneering or surface remodeling (European Patent Nos. EP 592,106 and EP 519,596; Padlan, 1991, Molecular Immunology 28(4 / 5):489-498; Studnicka et al., 1994, Protein Engineering 7(6):805-814; and Roguska et al., 1994, Proc. Natl. Acad. Sci. 91:969-973), chain shuffling (U.S. Patent No. 5,565,332), and techniques disclosed, for example, in U.S. Patent Nos. 6,407,213, 5,766,886, 5,585,089, International Publication No. WO 9317105, Tan et al., 2002, J. Immunol. 169:1119-25, Caldas et al., 2000, Protein Eng. 13:353-60, Morea et al., 2000, Methods 20:267-79, Baca et al., 1997, J. Biol. Chem. 272:10678-84, Roguska et al., 1996, Protein Eng. 9:895-904, Couto et al., 1995, Cancer Res. 55(23 Suppl):5973s-5977s, Couto et al., 1995, Cancer Res. 55:1717-22, Sandhu, 1994, Gene 150:409-10, Pedersen et al., 1994, J. Mol. Biol. 235:959-73, Jones et al., 1986, Nature 321:522-525, Riechmann et al., 1988, Nature 332:323, and Presta, 1992, Curr. Op. Struct. Biol. 2:593-596.Typically, framework residues in the framework regions will be replaced by the corresponding residues from the CDR donor antibody to alter, preferably improve, antigen binding. These framework replacements are identified by methods well known in the art, e.g., by modeling the interactions of CDR and framework residues to identify framework residues important for antigen binding and by sequence comparison to identify unusual framework residues at specific positions. (See, e.g., Queen et al., U.S. Patent No. 5,585,089; U.S. Publication Nos. 2004 / 0049014 and 2003 / 0229208; U.S. Patent Nos. 6,350,861; 6,180,370; 5,693,762; 5,693,761; 5,585,089; and 5,530,101 and Riechmann et al., 1988, Nature 332:323).

[0204] 3. Bispecific and multispecific antibodies

[0205] The antibodies used in the methods of the present disclosure can be monospecific. An antibody monospecific for ApoL1 or an ApoL1-containing complex can have a targeting moiety conjugated or otherwise linked thereto. In some embodiments, the targeting moiety is an antibody or an antigen-binding fragment thereof. Thus, also of interest are bispecific antibodies, trispecific antibodies, or antibodies with greater multispecificity that exhibit specificity for different targets in addition to ApoL1 or Hpr. For example, such antibodies can bind to both ApoL1 or Hpr and also bind to an antigen important for targeting the antibody to a specific cell type or tissue (e.g., an antigen associated with a cancer antigen of the tumor being treated).

[0206] a. Exemplary structures of bispecific and multispecific molecules

[0207] In some embodiments, the antibody is a heterodimeric bis- and tris- (or more) specific Ig antibody and Fc fusion protein. Exemplary structures include, but are not limited to, IgG, IgM, single, di-, tri- or more scFv-Fc. For example, bispecific, trispecific and multispecific forms include, but are not limited to, bispecific and trispecific IgG, IgG-scFv, IgG-dAb, scFv-Fc-scFv, knobs-into-holes (KIH)-IgG, Kappa bodies, KIH0Fc-Fab / scFv, tandem scFv, KIH trispecific, bispecific Fc fusions (N-terminal or C-terminal, with or without KIH).

[0208] In embodiments, a multispecific antibody molecule can include more than one antigen-binding site, where different sites are specific for different antigens. In embodiments, a multispecific antibody molecule can bind to more than one (e.g., two or more) epitopes on the same antigen. In embodiments, a multispecific antibody molecule includes an antigen-binding site specific for a target cell (e.g., a cancer cell) and a different antigen-binding site specific for an ApoL1-containing complex such as TLF (e.g., Hpr or ApoL1). In some embodiments, the multispecific antibody molecule is a bispecific antibody molecule. Bispecific antibody molecules can be divided into five different structural groups: (i) bispecific immunoglobulin G (BsIgG); (ii) IgG appended with an additional antigen-binding moiety; (iii) bispecific antibody fragments; (iv) bispecific fusion proteins; and (v) bispecific antibody conjugates.

[0209] BsIgG is in a form that is monovalent for each antigen. Exemplary BsIgG forms include, but are not limited to, crossMab, DAF (two-in-one), DAF (four-in-one), DutaMab, DT-IgG, knobs-into-holes common light chain, knobs-into-holes assembly, charge pair, Fab-arm exchange, SEEDbody, triomab, LUZ-Y, Fcab, kappa-lambda body, orthogonal Fab. See Spiess et al., Mol. Immunol. 67 (2015): 95-106. Exemplary BsIgGs include catumaxomab (Fresenius Biotech, Trion Pharma, Neopharm), which contains an anti-CD3 arm and an anti-EpCAM arm; and ertumaxomab (NeoviiBiotech, Fresenius Biotech), which targets CD3 and HER2.

[0210] In some embodiments, BsIgG includes heavy chains engineered to heterodimerize. For example, the heavy chains can be engineered to heterodimerize using the "knobs-into-holes" strategy, the SEED platform, a common heavy chain (e.g., in the Kk body), and using a heterodimeric Fc region. See Spiess et al., Mol. Immunol. 67 (2015): 95-106. Strategies that have been used to avoid homodimeric heavy chain pairing in BsIgG include knobs-into-holes, diabody, symmetric, charge pair, HA-TF, SEEDbody, and differential protein A affinity. See ibid. BsIgG can be produced by separately expressing the component antibodies in different host cells and subsequently purifying / assembling them into BsIgG. BsIgG can also be produced by expressing the component antibodies in a single host cell. BsIgG can be purified using affinity chromatography (e.g., using protein A and sequential pH elution).

[0211] IgG with an additional antigen-binding portion is another form of bispecific antibody molecule. For example, by attaching an additional antigen-binding unit to a monospecific IgG (e.g., at the N-terminus or C-terminus of the heavy or light chain), the monospecific IgG can be engineered to be bispecific. Exemplary additional antigen-binding units include single-domain antibodies (e.g., variable heavy or variable light chains), engineered protein scaffolds, and paired antibody variable regions (e.g., single-chain variable fragments or variable fragments). See ibid. Examples of additional IgG forms include bispecific variable domain (DVD) IgG (DVD-Ig), IgG(H)-scFv, scFv-(H)IgG, IgG(L)-scFv, scFv-(L)IgG, IgG(L,H)-Fv, IgG(H)-V, V(H)-IgG, IgG(L)-V, V(L)-IgG, KIHIgG-scFab, 2scFv-IgG, IgG-2scFv, scFv4-Ig, zybody, and DVI-IgG (quadroma). See Spiess et al. Mol. Immunol. 67 (2015): 95-106. An example of IgG-scFv is MM-141 (Merrimack Pharmaceuticals), which binds IGF-1R and HER3. Examples of DVD-Ig include ABT-981 (AbbVie), which binds IL-1α and IL-1β; and ABT-122 (AbbVie), which binds TNF and IL-17A.

[0212] Bispecific antibody fragments (BsAb) are forms of bispecific antibody molecules that lack some or all of the antibody constant domains. For example, some BsAb lack the Fc region. In some embodiments, bispecific antibody fragments include a heavy chain region and a light chain region linked by a peptide linker that allows for efficient expression of the BsAb in a single host cell. Exemplary bispecific antibody fragments include, but are not limited to, nanobodies, nanobody-HAS, BiTE, diabodies, DART, TandAb, sc diabodies, sc diabodies-CH3, diabody-CH3, triabodies, miniantibodies, minibodies, TriBi minibodies, scFv-CH3 KIH, Fab-scFv, scFv-CH-CL-scFv, F(ab')2, F(ab')2-scFv2, scFv-KIH, Fab-scFv-Fc, tetravalent HCAb, sc diabody-Fc, diabody-Fc, tandem scFv-Fc, and intracellular antibodies. See ibid. For example, the BiTE form includes tandem scFv, where the component scFv binds to CD3 on T cells and a surface antigen on cancer cells.

[0213] Bispecific fusion proteins include antibody fragments linked to other proteins, for example to increase additional specificity and / or functionality. An example of a bispecific fusion protein is an immTAC, which includes an anti-CD3 scFv linked to an affinity matured T cell receptor that recognizes HLA-presented peptides. In embodiments, the docking and locking (DNL) method can be used to generate bispecific antibody molecules with higher valency. Additionally, fusion with an albumin binding protein or human serum albumin can extend the serum half-life of antibody fragments. See ibid.

[0214] In embodiments, chemical conjugation (e.g., chemical conjugation of antibodies and / or antibody fragments) can be used to generate BsAb molecules. See above. Exemplary bispecific antibody conjugates include CovX-body forms, in which a low molecular weight drug is conjugated site-specifically to a single reactive lysine in each Fab arm or to an antibody or fragment thereof. In embodiments, conjugation improves the serum half-life of the low molecular weight drug. An exemplary CovX-body is CVX-241 (NCT01004822), which includes an antibody conjugated to two short peptides that inhibit VEGF or Ang2. See ibid.

[0215] In some embodiments, the multispecific molecule further includes a heavy chain constant region (e.g., an Fc region) selected from heavy chain constant regions of IgG1, IgG2, and IgG4 (more specifically, heavy chain constant regions of human IgG1, IgG2, or IgG4). In some embodiments, the heavy chain constant region (e.g., the Fc region) is linked to (e.g., covalently linked to) one or both of the ApoL1-containing complex-binding antibody molecule and the second antibody molecule.

[0216] In some embodiments, the heavy chain constant region (e.g., Fc region) is altered (e.g., mutated) to increase or decrease one or more of the following: Fc receptor binding, antibody glycosylation, the number of cysteine residues, effector cell function, or complement function. In some embodiments, the interface between the first heavy chain constant region and the second heavy chain constant region (e.g., Fc region) is altered (e.g., mutated) to increase or decrease dimerization, for example, relative to a non-engineered interface. In some embodiments, dimerization of the heavy chain constant region (e.g., Fc region) is enhanced by providing one or more of the following to the Fc interface of the first Fc region and the second Fc region: paired cavity-protrusion ("knobs-into-holes"), electrostatic interactions, or strand exchange, such that a greater ratio of heteropolymers:homopolymers is formed, for example, relative to a non-engineered interface. In some embodiments, the heavy chain constant region (e.g., Fc region) comprises an amino acid substitution at one or more positions selected from the following: 347, 349, 350, 351, 366, 368, 370, 392, 394, 395, 397, 398, 399, 405, 407, or 409, numbered according to the Eu numbering system (e.g., of the Fc region of human IgG1). Unless otherwise specified herein, the numbering of amino acid residues in the Fc region or constant region is according to the EU numbering system, also known as the EU index, as described in: Kabat et al., Sequences of Proteins of Immunological Interest, 5th ed. Public Health Service, National Institutes of Health, Bethesda, Md., 1991.

[0217] In some embodiments, the heavy chain constant region (e.g., Fc region) comprises an amino acid substitution selected from the following: T366S, L368A, or Y407V (e.g., corresponding to the cavity or hole), or T366W (e.g., corresponding to the protrusion or knob), numbered according to the Eu numbering system, or a combination thereof.

[0218] In some embodiments, the heavy chain constant region (e.g., Fc region) comprises one or more mutations that increase or decrease one or more of the following, relative to a naturally occurring heavy chain constant region: Fc receptor binding, antibody glycosylation, the number of cysteine residues, effector cell function, or complement function.

[0219] In some embodiments, the ApoL1-containing complex-binding molecule includes a first heavy chain constant region (e.g., a first Fc region), and the second antibody molecule includes a second heavy chain constant region (e.g., a second Fc region), wherein the first heavy chain constant region includes one or more mutations that increase the heterodimerization of the first heavy chain constant region and the second heavy chain constant region relative to the naturally occurring heavy chain constant region, and / or wherein the second heavy chain constant region includes one or more mutations that increase the heterodimerization of the second heavy chain constant region and the first heavy chain constant region relative to the naturally occurring heavy chain constant region. In some embodiments, the first heavy chain constant region and the second heavy chain constant region (e.g., the first Fc region and the second Fc region) include one or more of the following: paired cavity-protrusion ("knob-into-hole"), electrostatic interactions, or chain exchange, such that, for example, a greater ratio of heteropolymers:homopolymers is formed relative to the naturally occurring heavy chain constant region.

[0220] In some embodiments, the first heavy chain constant region and / or the second heavy chain constant region (e.g., the first and / or second Fc regions, e.g., the first and / or second IgG1 Fc regions) include amino acid substitutions at one or more positions selected from: 347, 349, 350, 351, 366, 368, 370, 392, 394, 395, 397, 398, 399, 405, 407, or 409, numbered according to the Eu numbering system. In some embodiments, the first heavy chain constant region and / or the second heavy chain constant region (e.g., the first and / or second Fc regions, e.g., the first and / or second IgG1 Fc regions) include amino acid substitutions selected from: T366S, L368A, Y407V, or Y349C (e.g., corresponding to a cavity or pore), or T366W or S354C (e.g., corresponding to a protrusion or knob), numbered according to the Eu numbering system, or combinations thereof.

[0221] In some embodiments, the multispecific molecule further includes a linker, e.g., a linker located between one or more of the following: the ApoL1-containing complex-binding molecule and the second antibody molecule, the ApoL1-containing complex-binding antibody molecule and the heavy chain constant region (e.g., the Fc region), or the second antibody molecule and the heavy chain constant region. In some embodiments, the linker is selected from: a cleavable linker, a non-cleavable linker, a peptide linker, a flexible linker, a rigid linker, a helical linker, or a non-helical linker. In some embodiments, the linker is a peptide linker. In some embodiments, the peptide linker includes Gly and Ser.

[0222] b. Cancer antigen

[0223] In some embodiments, the disclosed antibodies that bind to an ApoL1-containing complex (such as TLF) are bispecific or other multispecific molecules that also bind a cancer antigen.

[0224] In some embodiments, the cancer antigen is an antigen of a blood cancer, such as multiple myeloma, leukemia (e.g., chronic lymphocytic leukemia, acute myeloid leukemia, acute lymphoblastic leukemia), non-Hodgkin lymphoma, Hodgkin lymphoma, myelodysplastic syndrome (MDS), myeloproliferative neoplasm (MPN) (or its subcategories, e.g., essential thrombocythemia (ET), myelofibrosis (MF), and polycythemia vera (PV)), amyloidosis, Waldenström macroglobulinemia, or aplastic anemia. In other embodiments, the cancer antigen is an antigen of a solid tumor.

[0225] Common blood cancer antigens include, but are not limited to, BCMA, PD-L1, CTLA-4, CD38, CD319 / SLAMF-7, TNFRSF17 / BCMA, SYND1 / CD138, CD229, CD47, CD123 / IL3-RA, CD19, CD20, CD22, FcRH5, GPRC5D, and CLL-1. Thus, in some embodiments, the bispecific or multispecific antibody comprises an antigen-binding fragment that specifically binds to BCMA, PD-L1, CTLA-4, CD38, CD319 / SLAMF-7, TNFRSF17 / BCMA, SYND1 / CD138, CD229, CD47, CD123 / IL3-RA, CD19, CD20, CD22, FcRH5, GPRC5D, or CLL-1.

[0226] Cancer and tumor antigens with known structures and known or described functions include the following cell surface receptors: HER1 (GenBank accession number: U48722), HER2 (Yoshino et al., J. Immunol., 152:2393 (1994); Disis et al., Canc. Res., 54:16 (1994); GenBank accession numbers X03363 and M17730), HER3 (GenBank accession numbers U29339 and M34309), HER4 (Plowman et al., Nature, 366:473 (1993); GenBank accession numbers: L07868 and T64105), epidermal growth factor receptor (EGFR) (GenBank accession numbers U48722 and KO3193), vascular endothelial growth factor receptor (GenBank accession number M32977), vascular endothelial growth factor receptor (GenBank accession numbers AF022375, 1680143, U48801 and X62568), insulin-like growth factor-I (GenBank accession numbers X00173, X56774, X56773, X06043, European Patent No. GB 2241703), insulin-like growth factor-II (GenBank accession numbers X03562, X00910, M17863 and M17862), transferrin receptor (Trowbridge and Omary, Proc. Nat. Acad. USA, 78:3039 (1981); GenBank accession numbers X01060 and M11507), estrogen receptor (GenBank accession numbers M38651, X03635, X99101, U47678 and M12674), progesterone receptor (GenBank accession numbers X51730, X69068 and M15716), follicle-stimulating hormone receptor (FSH-R) (GenBank accession numbers Z34260 and M65085), retinoic acid receptor (GenBank accession numbers L12060, M60909, X77664, X57280, X07282 and X06538), MUC-1 (Barnes et al., Proc. Nat. Acad. Sci. USA, 86:7159 (1989); GenBank accession numbers M65132 and M64928), NY-ESO-1 (GenBank accession numbers AJ003149 and U87459), NA17-A (PCT Publication No.: WO 96 / 40039), Melan-A / MART-1 (Kawakami et al., Proc. Nat. Acad. Sci.USA, 91:3515 (1994); GenBank accession numbers U06654 and U06452), tyrosinase (Topalian et al., Proc. Nat. Acad. Sci. USA, 91:9461 (1994); GenBank accession number: M26729; Weber et al., J. Clin. Invest, 102:1258 (1998)), Gp-100 (Kawakami et al., Proc. Nat. Acad. Sci. USA, 91:3515 (1994); GenBank accession number: S73003, Adema et al., J. Biol. Chem., 269:20126 (1994)), MAGE (van den Bruggen et al., Science, 254:1643 (1991)); GenBank accession numbers U93163, AF064589, U66083, D32077, D32076, D32075, U10694, U10693, U10691, U10690, U10689, U10688, U10687, U10686, U10685, L18877, U10340, U10339, L18920, U03735 and M77481), BAGE (GenBank accession number: U19180; US Patent Nos. 5,683,886 and 5,571,711), GAGE (GenBank accession numbers AF055475, AF055474, AF055473, U19147, U19146, U19145, U19144, U19143 and U19142), any CTA class receptor, including the specific HOM-MEL-40 antigen encoded by the SSX2 gene (GenBank accession numbers X86175, U90842, U90841 and X86174), carcinoembryonic antigen (CEA, Gold and Freedman, J. Exp. Med., 121:439 (1985); GenBank accession numbers M59710, M59255 and M29540), PyLT (GenBank accession numbers J02289 and J02038); p97 (melanotransferrin) (Brown et al., J. Immunol., 127:539-46 (1981); Rose et al., Proc. Natl. Acad. Sci. USA, 83:1261-61 (1986), neuroblastoma antigen PTK7 and B7-DC (PD-L2).

[0227] Additional tumor-associated antigens include prostate-specific antigen (PSA) (U.S. Patent Nos. 6,677,157; 6,673,545); β-human chorionic gonadotropin (β-HCG) (McManus et al., Cancer Res., 36:3476-81 (1976); Yoshimura et al., Cancer, 73:2745-52 (1994); Yamaguchi et al., Br. J. Cancer, 60:382-84 (1989): Alfthan et al., Cancer Res., 52:4628-33 (1992)); glycosyltransferase β-1,4-N-acetylgalactosaminyltransferase (GalNAc) (Hoon et al., Int. J. Cancer, 43:857-62 (1989); Ando et al., Int. J. Cancer, 40:12-17 (1987); Tsuchida et al., J. Natl. Cancer, 78:45-54 (1987); Tsuchida et al., J. Natl. Cancer, 78:55-60 (1987)); NUC18 (Lehmann et al., Proc. Natl. Acad. Sci. USA, 86:9891-95 (1989); Lehmann et al., Cancer Res., 47:841-45 (1987)); melanoma antigen gp75 (Vijayasardahi et al., J. Exp. Med., 171:1375-80 (1990); GenBank accession number: X51455); human cytokeratin 8; high molecular weight melanoma antigen (Natali et al., Cancer, 59:55-63 (1987); keratin 19 (Datta et al., J. Clin. Oncol., 12:475-82 (1994)).

[0228] Tumor antigens of interest include antigens regarded in the art as “cancer / testis” (CT) antigens, which are immunogenic in subjects with a malignancy (Scanlan et al., Cancer Immun., 4:1 (2004)). CT antigens include at least 19 different antigen families, which contain one or more members and are capable of inducing an immune response, including but not limited to MAGEA (CT1); BAGE (CT2); MAGEB (CT3); GAGE (CT4); SSX (CT5); NY-ESO-1 (CT6); MAGEC (CT7); SYCP1 (C8); SPANXB1 (CT11.2); NA88 (CT18); CTAGE (CT21); SPA17 (CT22); OY-TES-1 (CT23); CAGE (CT26); HOM-TES-85 (CT28); HCA661 (CT30); NY-SAR-35 (CT38); FATE (CT43); and TPTE (CT44).

[0229] Additional tumor antigens that can be targeted (including tumor-associated or tumor-specific antigens) include, but are not limited to, alpha-actinin-4, Bcr-Abl fusion protein, Casp-8, beta-catenin, cdc27, cdk4, cdkn2a, coa-1, dek-can fusion protein, EF2, ETV6-AML1 fusion protein, LDLR-fucosyltransferase AS fusion protein, HLA-A2, HLA-A11, hsp70-2, KIAAO205, Mart2, Mum-1, 2, and 3, neo-PAP, class I myosin, OS-9, pml-RARα fusion protein, PTPRK, K-ras, N-ras, triosephosphate isomerase, Bage-1, Gage 3,4,5,6,7, GnTV, Herv-K-mel, Lage-1, Mage-A1,2,3,4,6,10,12, Mage-C2, NA-88, NY-Eso-1 / Lage-2, SP17, SSX-2, and TRP2-Int2, MelanA (MART-I), gp100 (Pmel 17), tyrosinase, TRP-1, TRP-2, MAGE-1, MAGE-3, BAGE, GAGE-1, GAGE-2, p15(58), CEA, RAGE, NY-ESO (LAGE), SCP-1, Hom / Mel-40, PRAME, p53, H-Ras, HER-2 / neu, BCR-ABL, E2A-PRL, H4-RET, IGH-IGK, MYL-RAR, Epstein-Barr virus antigen, EBNA, human papillomavirus (HPV) antigens E6 and E7, TSP-180, MAGE-4, MAGE-5, MAGE-6, p185erbB2, p180erbB-3, c-met, nm-23H1, PSA, TAG-72-4, CA19-9, CA72-4, CAM 17.1, NuMa, K-ras, beta-catenin, CDK4, Mum-1, p16, TAGE, PSMA, PSCA, CT7, telomerase, 43-9F, 5T4, 791Tgp72, alpha-fetoprotein, 13HCG, BCA225, BTAA, CA 125, CA 15-3 (CA 27.29\BCAA), CA 195, CA 242, CA-50, CAM43, CD68\KP1, CO-029, FGF-5, G250, Ga733 (EpCAM), HTgp-175, M344, MA-50, MG7-Ag, MOV18, NB\70K, NY-CO-1, RCAS1, SDCCAG16, TA-90 (Mac-2 binding protein\cyclophilin C-associated protein), TAAL6, TAG72, TLP, and TPS.Other tumor-associated antigens and tumor-specific antigens are known to those of skill in the art and are suitable for targeting by the disclosed fusion proteins.

[0230] In certain embodiments, the tumor antigen is a pancreatic cancer antigen, optionally selected from Claudin 18.2, MUC1, Mesothelin (MSLN), and Myoferlin (MYOF).

[0231] MYOF can be used to target pancreatic ductal adenocarcinoma (PDAC) (Gupta et al., Nat Cell Biol 23, 232–242 (2021)), non-small cell lung cancer (Song et al., Oncol Lett. 11(2):998–1006 (2016)), doi:10.3892 / ol.2015.3988, and breast cancer (Zhang et al., Nat Commun. 9(1):3726 (2018) doi:10.1038 / s41467-018-06179-0.).

[0232] Mesothelin (MSLN) has also been found in ovarian cancer, lung adenocarcinoma, malignant mesothelioma, cholangiocarcinoma, gastric cancer, and pediatric acute myeloid leukemia (Hassan and Ho et al., Eur J Cancer., 44(1):46–53 (2008), Hassan et al., J Clin Oncol. 34(34):4171-4179.doi:10.1200 / JCO.2016.68.3672 (2016)).

[0233] Thus, in some embodiments, the bispecific or multispecific antibody comprises antigen-binding fragments that specifically bind to Claudin 18.2, MUC1, Mesothelin (MSLN), and Myoferlin (MYOF).

[0234] In other embodiments, the tumor antigen is a melanoma cancer antigen, optionally PMEL17. Thus, in some embodiments, the bispecific or multispecific antibody comprises antigen-binding fragments that specifically bind to PMEL17.

[0235] Generally, the cell marker or antigen is not a trypanosome-specific surface antigen.

[0236] In preferred embodiments, the foregoing antigens are targeted by antibodies that bind to them. Accordingly, for all of the provided tumor antigens, antibodies and antigen-binding fragments that specifically bind to them are also provided. In other embodiments, the targeting moiety is not an antibody and can be, for example, another polypeptide, carbohydrate, lipid, etc., as discussed elsewhere herein.

[0237] 4. Derivatives and Conjugates

[0238] The present disclosure specifically contemplates the generation and use of derivatives of any of the above antibodies and their antigen-binding fragments. The term derivative encompasses antibodies or their antigen-binding fragments that bind immunospecifically to an antigen, but that include one, two, three, four, five or more amino acid substitutions, additions, deletions or modifications relative to the "parent" (or wild-type) molecule (also referred to as variants). Such amino acid substitutions or additions may introduce naturally occurring (i.e., DNA-encoded) or non-naturally occurring amino acid residues.

[0239] The term derivative also encompasses, for example, chimeric or humanized variants of any of the disclosed antibodies, and variants thereof having altered CH1, hinge, CH2, CH3 or CH4 regions, so as to form, for example, antibodies having a variant Fc region that exhibits enhanced or impaired effector or binding properties.

[0240] The term derivative also encompasses non-amino acid modifications (e.g., can be glycosylated (e.g., having altered mannose, 2-N-acetylglucosamine, galactose, fucose, glucose, sialic acid, 5-N-acetylneuraminic acid, 5-glycolylneuraminic acid, etc. content), acetylated, polyethylene glycolated, phosphorylated, amidated, amino acids derivatized by known protecting / blocking groups), proteolysis linked to cell ligands or other proteins, etc. In some embodiments, the altered carbohydrate modification regulates one or more of the following: antibody solubility, promotion of subcellular transport and secretion of the antibody, promotion of antibody assembly, conformational integrity, and antibody-mediated effector function. In a specific embodiment, relative to an antibody lacking carbohydrate modification, the altered carbohydrate modification enhances antibody-mediated effector function. Carbohydrate modifications that result in altered antibody-mediated effector function are well known in the art (e.g., see Shields, R.L. et al. (2002) “Lack Of Fucose On Human IgG N-Linked Oligosaccharide Improves Binding ToHuman Fcgamma RIIIAnd Antibody-Dependent Cellular Toxicity.,” J. Biol. Chem. 277(30):26733-26740; Davies J. et al. (2001) “Expression Of GnTIII In ARecombinantAnti-CD20 CHO Production Cell Line:Expression Of Antibodies With AlteredGlycoforms Leads To An Increase In ADCC Through Higher Affinity For FC GammaRIII,” Biotechnology&Bioengineering 74(4):288-294).Methods for altering carbohydrate content are known to those of skill in the art, see for example Wallick, S.C. et al. (1988) “Glycosylation Of A VHResidue Of AMonoclonal Antibody Against Alpha(1----6)Dextran Increases ItsAffinity For Antigen,” J.Exp.Med.168(3):1099-1109; Tao, M.H. et al. (1989) “StudiesOf Aglycosylated Chimeric Mouse-Human IgG.Role Of Carbohydrate In TheStructure And Effector Functions Mediated By The Human IgG Constant Region,” J.Immunol.143(8):2595-2601; Routledge, E.G. et al. (1995) “The Effect OfAglycosylation On The Immunogenicity Of A Humanized Therapeutic CD3Monoclonal Antibody,” Transplantation 60(8):847-53; Elliott, S. et al. (2003) “Enhancement Of Therapeutic Protein In Vivo Activities ThroughGlycoengineering,” Nature Biotechnol.21:414-21; Shields, R.L. et al. (2002) “Lack OfFucose On Human IgG N-Linked Oligosaccharide Improves Binding To HumanFcgamma RIII And Antibody-Dependent Cellular Toxicity.,” J.Biol.Chem.277(30):26733-26740).

[0241] In some embodiments, the humanized antibody is a derivative. Such humanized antibodies include amino acid residue substitutions, deletions, or additions in one or more non-human CDRs. Compared to non-derivative humanized antibodies, humanized antibody derivatives can have substantially the same binding, better binding, or worse binding. In specific embodiments, one, two, three, four, or five amino acid residues of the CDR have been substituted, deleted, or added (i.e., mutated).

[0242] The derivative antibody or antibody fragment can be modified by chemical modification using techniques known to those skilled in the art, which include but are not limited to specific chemical cleavage, acetylation, formulation, metabolic synthesis of tunicamycin, etc. In one embodiment, the antibody derivative will have a function similar or identical to the parental antibody. In another embodiment, the antibody derivative will exhibit altered activity relative to the parental antibody. For example, compared to the parental antibody, the derivative antibody (or its fragment) can bind more tightly to its epitope or be more resistant to proteolysis.

[0243] The derivatized antibody can be used to alter the half-life (e.g., serum half-life) of the parental antibody in mammals (preferably humans). Preferably, such alteration will result in a half-life greater than 15 days, preferably greater than 20 days, greater than 25 days, greater than 30 days, greater than 35 days, greater than 40 days, greater than 45 days, greater than 2 months, greater than 3 months, greater than 4 months, or greater than 5 months. The increased half-life of the humanized antibody or its fragment in mammals (preferably humans) results in a higher serum titer of the antibody or antibody fragment in the mammal, and thus reduces the frequency of administration of the antibody or antibody fragment and / or reduces the concentration of the antibody or antibody fragment to be administered. Antibodies or their fragments with increased in vivo half-life can be produced by techniques known to those skilled in the art. For example, antibodies or their fragments with increased in vivo half-life can be produced by modifying (e.g., substituting, deleting, or adding) amino acid residues identified as participating in the interaction between the Fc domain and the FcRn receptor. Humanized antibodies can be engineered to increase the biological half-life (see, for example, U.S. Patent No. 6,277,375). For example, humanized antibodies can be engineered in the Fc hinge domain to increase the in vivo or serum half-life.

[0244] Antibodies or fragments thereof with increased in vivo half-life can be generated by attaching polymer molecules, such as high molecular weight polyethylene glycol (PEG), to the antibody or antibody fragment. PEG can be attached to the antibody or antibody fragment with or without a multifunctional linker through site-specific conjugation of PEG to the N- or C-terminus of the antibody or antibody fragment or via the ε-amino group present on lysine residues. Linear or branched polymers that result in minimal loss of biological activity will be used for derivatization. The degree of conjugation will be closely monitored by SDS-PAGE and mass spectrometry to ensure correct conjugation of PEG molecules to the antibody. Unreacted PEG can be separated from the antibody-PEG conjugate by, for example, size exclusion or ion exchange chromatography.

[0245] Antibodies can also be modified by the methods and coupling agents described by Davis et al. (see U.S. Patent No. 4,179,337) to provide compositions that can be injected into the mammalian circulatory system with substantially no immunogenic response.

[0246] One embodiment encompasses modification of framework residues of humanized ApoL1 or Hpr antibodies. Framework residues in the framework region can be replaced with corresponding residues from a CDR donor antibody to alter, preferably improve, antigen binding. These framework substitutions are identified by methods well known in the art, for example, by modeling the interactions of CDR and framework residues to identify framework residues important for antigen binding and by sequence comparison to identify unusual framework residues at specific positions. (See, for example, U.S. Patent No. 5,585,089; and Riechmann, L. et al. (1988) “Reshaping Human Antibodies For Therapy,” Nature 332:323-327).

[0247] Yet another embodiment encompasses anti-ApoL1 and anti-Hpr antibodies (and more preferably, humanized antibodies) and antigen-binding fragments thereof that are recombinantly fused or chemically conjugated (including both covalent and non-covalent conjugation) to a heterologous molecule (i.e., an unrelated molecule). The fusion need not be direct, but can occur through a linker sequence.

[0248] In one embodiment, such heterologous molecules are polypeptides having at least 10, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90 or at least 100 amino acids. Alternatively, such heterologous molecules can be enzymes, hormones, cell surface receptors, drug moieties such as: toxins (such as abrin, ricin A, Pseudomonas exotoxin (i.e., PE-40), diphtheria toxin, ricin, gelonin or pokeweed antiviral protein), proteins (such as tumor necrosis factor, interferon (e.g., alpha-interferon, beta-interferon), nerve growth factor, platelet-derived growth factor, tissue plasminogen activator or apoptosis agents (e.g., tumor necrosis factor-alpha, tumor necrosis factor-beta)), biological response modifiers (such as for example lymphokines (e.g., interleukin-1 ("IL-1"), interleukin-2 ("IL-2"), interleukin-6 ("IL-6")), granulocyte macrophage colony-stimulating factor ("GM-CSF"), granulocyte colony-stimulating factor ("G-CSF"), or macrophage colony-stimulating factor ("M-CSF")), or growth factors (e.g., growth hormone ("GH")), cytotoxins (e.g., cytostatic or cytocidal agents such as paclitaxel, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, teniposide, vincristine, vinblastine, colchicine, doxorubicin, daunorubicin, mitoxantrone, mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, and puromycin and its analogs or homologs), antimetabolites (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil dacarbazine), alkylating agents (e.g., nitrogen mustard, thiotepa melphalan, (carmustine; BCNU) and lomustine (CCNU), cyclophosphamide, busulfan, dibromomannitol, streptozocin, mitomycin C and cis-dichlorodiamine platinum (II) (DDP) cisplatin), anthracyclines (e.g., daunorubicin (formerly called daunomycin) and doxorubicin), antibiotics (e.g., dactinomycin (formerly called actinomycin)), bleomycin, mithramycin and anthramycin (AMC)) or antimitotic agents (e.g., vincristine and vinblastine).

[0249] Techniques for conjugating such therapeutic moieties to antibodies are well known; see, e.g., Arnon et al., “Monoclonal Antibodies For Immunotargeting Of Drugs In Cancer Therapy”, Monoclonal Antibodies And Cancer Therapy, Reisfeld et al. (eds.), 1985, pp. 243-56, Alan R. Liss, Inc.); Hellstrom et al., “Antibodies For Drug Delivery”, Controlled Drug Delivery (2nd ed.), Robinson et al. (eds.), 1987, pp. 623-53, Marcel Dekker, Inc.); Thorpe, “Antibody Carriers Of Cytotoxic Agents In Cancer Therapy: A Review”, Monoclonal Antibodies ‘84: Biological And Clinical Applications, Pinchera et al. (eds.), 1985, pp. 475-506); “Analysis, Results, And Future Prospective Of The Therapeutic Use Of Radiolabeled Antibody In Cancer Therapy”, Monoclonal Antibodies For Cancer Detection And Therapy, Baldwin et al. (eds.), 1985, pp. 303-16, Academic Press; and Thorpe et al. (1982) “The Preparation And Cytotoxic Properties Of Antibody-Toxin Conjugates,” Immunol. Rev. 62:119-158.

[0250] In one embodiment, the ApoL1 and Hpr antibodies or fusion molecules include an Fc portion. The Fc portion of such molecules can vary according to isotype or subclass, can be chimeric or hybrid, and / or can be modified, for example, to improve effector function, half-life control, tissue accessibility, enhance biophysical properties (such as stability) and increase production efficiency (and at lower cost). Many modifications that can be used to construct the disclosed fusion proteins and methods for preparing them are known in the art, see, for example, Mueller, J.P. et al. (1997) “Humanized Porcine VCAM-Specific Monoclonal Antibodies With Chimeric IgG2 / G4 Constant Regions Block Human Leukocyte Binding To Porcine Endothelial Cells,” Mol. Immunol. 34(6):441-452, Swann, P.G. (2008) “Considerations For The Development Of Therapeutic Monoclonal Antibodies,” Curr. Opin. Immunol. 20:493-499 (2008), and Presta, L.G. (2008) “Molecular Engineering And Design Of Therapeutic Antibodies,” Curr. Opin. Immunol. 20:460-470. In some embodiments, the Fc region is a native IgG1, IgG2 or IgG4 Fc region. In some embodiments, the Fc region is a hybrid, such as a chimera having an IgG2 / IgG4 Fc constant region. Modifications to the Fc region include, but are not limited to, IgG4 modified to prevent binding to Fcγ receptors and complement, IgG1 modified to improve binding to one or more Fcγ receptors, IgG1 modified to minimize effector function (amino acid changes), IgG1 with altered / without glycans (usually by altering the expression host), IgG1 with altered pH-dependent binding to FcRn, and IgG4 in which the serine at amino acid residence #228 in the hinge region is changed to proline (S228P) to enhance stability. The Fc region can include the entire hinge region, or less than the entire hinge region.

[0251] The treatment outcomes of patients with non-Hodgkin lymphoma or Waldenström macroglobulinemia treated with rituximab, a chimeric mouse / human IgG1 monoclonal antibody directed against CD20, are associated with the expression of allelic variants of Fcγ receptors with different intrinsic affinities for the Fc domain of human IgG1. Specifically, patients with the high-affinity allele of the low-affinity activating Fc receptor CD16A (FcγRIIIA) exhibit a higher response rate, and in non-Hodgkin lymphoma cases, progression-free survival is improved. In another embodiment, the Fc domain can contain one or more amino acid insertions, deletions, or substitutions that reduce binding to the low-affinity inhibitory Fc receptor CD32B (FcγRIIB) and retain the wild-type level of binding to the low-affinity activating Fc receptor CD16A (FcγRIIIA) or enhance binding thereto.

[0252] Another embodiment includes IgG 2-4 heterozygotes and IgG 4 mutants that have reduced binding to FcRs, which increases their half-life. Representative IG 2-4 heterozygotes and IgG 4 mutants are described in: Angal, S. et al. (1993) “A Single Amino Acid Substitution Abolishes The Heterogeneity Of Chimeric Mouse / Human (Igg4) Antibody,” Molec. Immunol. 30(1):105-108; Mueller, J.P. et al. (1997) “Humanized Porcine VCAM-Specific Monoclonal Antibodies With Chimeric Igg2 / G4 Constant Regions Block Human Leukocyte Binding To Porcine Endothelial Cells,” Mol. Immunol. 34(6):441-452; and U.S. Patent No. 6,982,323. In some embodiments, the IgG 1 and / or IgG 2 domain is deleted, for example, Angal, s. et al. described an IgG in which serine 241 was replaced with proline 1 and IgG 2 .

[0253] Substitutions, additions or deletions in the derivatized antibody can be in the Fc region of the antibody and are thus used to modify the binding affinity of the antibody for one or more FcγRs. Methods for modifying antibodies for which binding to one or more FcγRs is modified are known in the art, see for example PCT publication numbers WO 04 / 029207, WO 04 / 029092, WO 04 / 028564, WO99 / 58572, WO 99 / 51642, WO 98 / 23289, WO 89 / 07142, WO 88 / 07089 and US Patent Nos. 5,843,597 and 5,642,821. In one specific embodiment, modification of the Fc region results in an antibody having altered antibody-mediated effector functions, altered binding to other Fc receptors (e.g., Fc activating receptors), altered antibody-dependent cell-mediated cytotoxicity (ADCC) activity, altered C1q binding activity, altered complement-dependent cytotoxicity activity (CDC), phagocytic activity or any combination thereof.

[0254] In some embodiments, the present disclosure encompasses antibodies whose Fc regions have been modified such that the molecule will exhibit altered Fc receptor (FcR) binding activity, e.g., exhibit reduced activity against activating receptors (such as FcγRIIA or FcγRIIIA) or increased activity against inhibitory receptors (such as FcγRIIB). Preferably, such antibodies will exhibit reduced antibody-dependent cell-mediated cytotoxicity (ADCC) or complement-dependent cytotoxicity (CDC) activity (relative to wild-type Fc receptors).

[0255] Modifications that affect Fc-mediated effector functions are well known in the art (see U.S. Patent No. 6,194,551 and WO 00 / 42072; Stavenhagen, J.B. et al. (2007) “Fc Optimization Of Therapeutic Antibodies Enhances Their Ability To Kill Tumor Cells In Vitro And Controls Tumor Expansion In Vivo Via Low-Affinity Activating Fcgamma Receptors,” Cancer Res. 57(18):8882-8890; Shields, R.L. et al. (2001) “High Resolution Mapping of the Binding Site on Human IgG1 for FcγRI, FcγRII, FcγRIII, and FcRn and Design of IgG1 Variants with Improved Binding to the FcγR,” J. Biol. Chem. 276(9):6591-6604). Exemplary variants of the human IgG1 Fc domain with reduced binding to FcγRIIA or FcγRIIIA but unchanged or enhanced binding to FcγRIIB include S239A, H268A, S267G, E269A, E293A, E293D, Y296F, R301A, V303A, A327G, K322A, E333A, K334A, K338A, A339A, D376A.

[0256] In some embodiments, the disclosure encompasses antibodies in which the Fc region has been deleted (e.g., Fab or F(ab) 2 etc.).

[0257] Any molecule of the present disclosure can be fused to a marker sequence, such as a peptide, to facilitate purification. In a preferred embodiment, the marker amino acid sequence is a hexahistidine peptide, i.e., the hemagglutinin "HA" tag, which corresponds to an epitope derived from the influenza hemagglutinin protein (Wilson, I.A. et al. (1984) "The Structure Of An Antigenic Determinant In A Protein," Cell, 37:767-778), and the "flag" tag (Knappik, A. et al. (1994) "An Improved Affinity Tag Based On The FLAG Peptide For The Detection And Purification Of Recombinant Antibody Fragments," Biotechniques 17(4):754-761).

[0258] The present disclosure also encompasses antibodies or antigen-binding fragments thereof conjugated to a diagnostic agent, a therapeutic agent, or any other molecule for which an increased serum half-life is desired. Antibodies can be used for diagnosis (in vivo, in situ, or in vitro), for example, to monitor the development or progression of a disease, disorder, or infection, as part of a clinical test procedure, for example, to determine the efficacy of a given treatment regimen. Detection can be facilitated by conjugating the antibody to a detectable substance. Examples of detectable substances include various enzymes, cofactors, fluorescent materials, luminescent materials, bioluminescent materials, radioactive materials, positron-emitting metals, and non-radioactive paramagnetic metal ions. The detectable substance can be directly or indirectly conjugated or linked to the antibody using techniques known in the art, either directly or through an intermediate, such as a linker known in the art, for example. See, for example, U.S. Patent No. 4,741,900 regarding metal ions that can be conjugated to an antibody for use as a diagnostic agent in accordance with the present disclosure. Such diagnosis and detection can be accomplished by conjugating the antibody to a detectable substance, which includes but is not limited to various enzymes, including but not limited to horseradish peroxidase, alkaline phosphatase, β-galactosidase, or acetylcholinesterase; cofactor complexes, such as but not limited to streptavidin / biotin and avidin / biotin; fluorescent materials, such as but not limited to umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride, or phycoerythrin; luminescent materials, such as but not limited to luminol; bioluminescent materials, such as but not limited to luciferase, luciferin, and aequorin; radioactive materials, such as but not limited to bismuth ( 213 Bi), carbon ( 14 C), chromium ( 51 Cr), cobalt ( 57 Co), fluorine ( 18 F), gadolinium ( 153 Gd, 159Gd), gallium ( 68 Ga, 67 Ga), germanium ( 68 Ge), holmium ( 166 Ho), indium ( 115 In, 113 In, 112 In, 111 In), iodine ( 131 I, 125 I, 123 I, 121 I), lanthanum ( 140 La), lutetium ( 177 Lu), manganese ( 54 Mn), molybdenum ( 99 Mo), palladium ( 103 Pd), phosphorus ( 32 P), praseodymium ( 142 Pr), promethium ( 149 Pm), rhenium ( 186 Re, 188 Re), rhodium ( 105 Rh), ruthenium ( 97 Ru), samarium ( 153 Sm), scandium ( 47 Sc), selenium ( 75 Se), strontium ( 85 Sr), sulfur ( 35 S), technetium ( 99 Tc), thallium ( 201 Ti), tin ( 113 Sn, 117 Sn), tritium ( 3 H), xenon ( 133 Xe), ytterbium ( 169 Yb, 175 Yb), yttrium ( 90 Y), zinc ( 65 Zn); using positron-emitting metals and non-radioactive paramagnetic metal ions for various positron emission tomography.

[0259] The molecules of the present disclosure can be conjugated to a second antibody to form an antibody heteroconjugate, as described by Segal in U.S. Patent No. 4,676,980. Such heteroconjugate antibodies can additionally bind to a hapten (such as fluorescein, etc.), or a cell marker, or a cytokine, or a chemokine (e.g., CCL21), etc.

[0260] The molecules of the present disclosure can be attached to a solid support, which can be specifically used for immunoassays or purification of a target antigen or other molecules capable of binding to a target antigen that has been immobilized on the support by binding to an antibody or antigen-binding fragment of the present disclosure. Such solid supports include, but are not limited to, glass, cellulose, polyacrylamide, nylon, polystyrene, polyvinyl chloride, or polypropylene.

[0261] 5. Methods for Preparing Antibodies and Antigen-Binding Fragments

[0262] The disclosed antibodies can be produced by any method known in the art for generating polypeptides (e.g., in vitro synthesis, recombinant DNA production, etc.). Preferably, the antibodies are produced by recombinant DNA technology. Recombinant immunoglobulin expression techniques can be used to produce antibodies. The recombinant production of immunoglobulin molecules (including humanized antibodies) is described in U.S. Patent No. 4,816,397 (Boss et al.), U.S. Patent Nos. 6,331,415 and 4,816,567 (both granted to Cabilly et al.), British Patent GB 2,188,638 (Winter et al.), and British Patent GB 2,209,757. Techniques for the recombinant expression of immunoglobulins (including humanized immunoglobulins) can also be found in Goeddel et al., Gene Expression Technology Methods in Enzymology Vol. 185 Academic Press (1991), and Borreback, Antibody Engineering, W.H. Freeman (1992). More information on the production, design, and expression of recombinant antibodies can be found in Mayforth, Designing Antibodies, Academic Press, San Diego (1993).

[0263] An exemplary process for producing a recombinant chimeric antibody can include the following: a) constructing an expression vector encoding and expressing an antibody heavy chain by conventional molecular biology methods, wherein the CDRs and variable regions of a murine anti-ApoL1 or anti-Hpr monoclonal antibody are fused to an Fc region derived from a human immunoglobulin, thereby producing a vector for expressing a chimeric antibody heavy chain; b) constructing an expression vector encoding and expressing an antibody light chain of a murine anti-ApoL1 or anti-Hpr monoclonal antibody by conventional molecular biology methods, thereby producing a vector for expressing a chimeric antibody light chain; c) transferring the expression vectors into a host cell by conventional molecular biology methods to produce a transfected host cell for expressing the chimeric antibody; and d) culturing the transfected cells by conventional cell culture techniques to produce the chimeric antibody.

[0264] Exemplary processes for generating recombinant humanized antibodies can include the following: a) constructing an expression vector encoding and expressing an anti-ApoL1 or anti-Hpr heavy chain by conventional molecular biology methods, wherein the minimum portions of the CDRs and variable region frameworks required to retain the binding specificity of the donor antibody are derived from a non-human immunoglobulin, such as a murine anti-ApoL1 or anti-Hpr monoclonal antibody, and the remainder of the antibody is derived from a human immunoglobulin, thereby generating a vector for expressing a humanized antibody heavy chain; b) constructing an expression vector encoding and expressing an antibody light chain by conventional molecular biology methods, wherein the minimum portions of the CDRs and variable region frameworks required to retain the binding specificity of the donor antibody are derived from a non-human immunoglobulin, such as a murine anti-ApoL1 or anti-Hpr monoclonal antibody, and the remainder of the antibody is derived from a human immunoglobulin, thereby generating a vector for expressing a humanized antibody light chain; c) transferring the expression vectors into a host cell by conventional molecular biology methods to generate a transfected host cell for expressing a humanized antibody; and d) culturing the transfected cells by conventional cell culture techniques to generate a humanized antibody.

[0265] For any of the exemplary methods, a host cell can be co-transfected with such expression vectors, which can contain different selectable markers but are preferably identical except for the heavy and light chain coding sequences. This procedure provides for equivalent expression of the heavy and light chain polypeptides. Alternatively, a single vector can be used that encodes both the heavy and light chain polypeptides. The coding sequences for the heavy and light chains can include cDNA or genomic DNA or both. Host cells for expressing recombinant antibodies can be bacterial cells (such as Escherichia coli), or more preferably eukaryotic cells (e.g., Chinese hamster ovary (CHO) cells or HEK-293 cells). The choice of expression vector depends on the choice of host cell and can be selected to have the desired expression and regulatory properties in the selected host cell. Other cell lines that can be used include, but are not limited to, CHO-K1, NSO, and PER.C6 (Crucell, Leiden, Netherlands).

[0266] Any of the above antibodies can be used to generate anti-idiotypic antibodies using techniques well known to those skilled in the art (see, for example, Greenspan, N.S. et al. (1989) “Idiotypes: Structure And Immunogenicity,” FASEB J. 7:437-444; and Nisinoff, A. (1991) “Idiotypes: Concepts And Applications,” J. Immunol. 147(8):2429-2438).

[0267] If desired, the binding properties of any of the above antibodies can be further improved by screening for variants that exhibit such desired properties. For example, such antibodies can be generated using various phage display methods known in the art. In phage display methods, functional antibody domains are displayed on the surface of phage particles carrying the polynucleotide sequences encoding them. In a specific embodiment, such phages can be used to display antigen-binding domains (such as Fab and Fv or disulfide-stabilized Fv) expressed from a repertoire or combinatorial antibody library (e.g., human or murine). Phages expressing antigen-binding domains that bind to an antigen of interest can be selected or identified using the antigen, e.g., using a labeled antigen or an antigen that is bound or captured to a solid surface or bead. The phages used in these methods are typically filamentous phages, including fd and M13. The antigen-binding domains are expressed as proteins recombinantly fused to the phage gene III or gene VIII protein. Examples of phage display methods that can be used to prepare the immunoglobulins or fragments thereof of the present disclosure include the methods disclosed in the following: Brinkman, U. et al. (1995) “Phage Display Of Disulfide-Stabilized Fv Fragments,” J. Immunol. Methods, 182:41-50, 1995; Ames, R. S. et al. (1995) “Conversion Of Murine Fabs Isolated From A Combinatorial Phage Display Library To Full Length Immunoglobulins,” J. Immunol. Methods, 184:177-186; Kettleborough, C. A. et al. (1994) “Isolation Of Tumor Cell-Specific Single-Chain Fv From Immunized Mice Using Phage-Antibody Libraries And The Re-Construction Of Whole Antibodies From These Antibody Fragments,” Eur. J. Immunol., 24:952-958, 1994; Persic, L.et al. (1997) “An Integrated Vector System For The Eukaryotic Expression Of Antibodies Or Their Fragments After Selection From Phage Display Libraries,” Gene, 187:9-18; Burton, D.R. et al. (1994) “Human Antibodies From Combinatorial Libraries,” Adv. Immunol. 57:191-280; PCT Publication WO 92 / 001047; WO 90 / 02809; WO 91 / 10737; WO 92 / 01047; WO 92 / 18619; WO 93 / 11236; WO 95 / 15982; WO 95 / 20401; and U.S. Patent Nos. 5,698,426; 5,223,409; 5,403,484; 5,580,717; 5,427,908; 5,750,753; 5,821,047; 5,571,698; 5,427,908; 5,516,637; 5,780,225; 5,658,727; 5,733,743 and 5,969,108.

[0268] As described in the above references, after phage selection, the antibody-encoding regions from the phage can be isolated and used to generate full antibodies (including humanized antibodies or any other desired fragments) and expressed in any desired host (including mammalian cells, insect cells, plant cells, yeast, and bacteria), for example, as detailed below. For example, for the recombinant production of Fab, Fab', and F(ab') 2The techniques of the fragment also employ methods known in the art (such as those disclosed in: PCT Publication WO 92 / 22324; Mullinax, R.L. et al. (1992) “Expression Of A Heterodimeric Fab Antibody Protein In One Cloning Step,” BioTechniques, 12(6):864 - 869; and Sawai et al. (1995) “Direct Production Of The Fab Fragment Derived From The Sperm Immobilizing Antibody Using Polymerase Chain Reaction And cDNA Expression Vectors,” Am.J.Reprod.Immunol. 34:26 - 34; and Better, M. et al. (1988) “Escherichia coli Secretion Of An Active Chimeric Antibody Fragment,” Science 240:1041 - 1043). Examples of techniques that can be used to generate single-chain Fv and antibodies include those disclosed in: U.S. Patent Nos. 4,946,778 and 5,258,498; Huston, J.S. et al. (1991) “Protein Engineering Of Single-Chain Fv Analogs And Fusion Proteins,” Methods in Enzymology 203:46 - 88; Shu, L. et al., “Secretion Of A Single-Gene-Encoded Immunoglobulin From Myeloma Cells,” Proc.Natl.Acad.Sci.(USA) 90:7995 - 7999; and Skerra.A. et al. (1988) “Assembly Of A Functional Immunoglobulin Fv Fragment In Escherichia coli,” Science 240:1038 - 1040.

[0269] Phage display technology can be used to increase the affinity of an antibody for ApoL1 or Hpr. This technology will be useful for obtaining high-affinity antibodies that can be used in the disclosed methods. This technology (termed affinity maturation) utilizes mutagenesis or CDR walking and reselection of such receptors or ligands (or their extracellular domains) or antigenic fragments thereof to identify antibodies that bind antigen with higher affinity compared to an initial or parental antibody (see, e.g., Glaser, S.M. et al. (1992) “Antibody Engineering By Codon-Based Mutagenesis In A Filamentous Phage Vector System,” J. Immunol. 149:3903-3913). Mutagenizing entire codons rather than single nucleotides generates a semi-randomized library of amino acid mutations. A library can be constructed that includes a pool of variant clones, each variant clone differing by a single amino acid change in a single CDR and containing variants that represent each possible amino acid substitution of each CDR residue. Mutants with increased binding affinity for antigen can be screened by contacting immobilized mutants with labeled antigen. Any screening method known in the art can be used to identify mutant antibodies with increased avidity for antigen (e.g., ELISA) (see, e.g., Wu, H. et al. (1998) “Stepwise In Vitro Affinity Maturation Of Vitaxin, An AlphavBeta3-Specific Humanized Mab,” Proc. Natl. Acad. Sci. (USA) 95(11):6037-6042; Yelton, D.E. et al. (1995) “Affinity Maturation Of The BR96 Anti-Carcinoma Antibody By Codon-Based Mutagenesis,” J. Immunol. 155:1994-2004). CDR walking that randomizes the light chain can be used (see Schier et al. (1996) “Isolation Of Picomolar Affinity Anti-C-Erbb-2 Single-Chain Fv By Molecular Evolution Of The Complementarity Determining Regions In The Center Of The Antibody Binding Site,” J. Mol. Biol. 263:551-567).

[0270] Accordingly, the present disclosure contemplates the use of random mutagenesis to identify improved CDRs. Alternatively, phage display technology can be used to increase (or decrease) CDR affinity. This technique (referred to as affinity maturation) employs mutagenesis or "CDR walking" and re-selection, using the target antigen or an antigenic fragment thereof to identify antibodies with CDRs that bind the antigen with higher (or lower) affinity compared to the initial or parental antibody (see, e.g., Glaser, S.M. et al. (1992) "Antibody Engineering By Codon-Based Mutagenesis In A Filamentous Phage Vector System," J. Immunol. 149:3903-3913). Mutagenizing entire codons rather than single nucleotides generates a semi-randomized library of amino acid mutations. A library can be constructed that includes a pool of variant clones, each variant clone differing by a single amino acid change in a single CDR and containing variants that represent each possible amino acid substitution for each CDR residue. Mutants with increased (or decreased) binding affinity for the antigen can be screened by contacting the immobilized mutants with the labeled antigen. Any screening method known in the art can be used to identify mutant antibodies with increased (or decreased) affinity for the antigen (e.g., ELISA) (see, e.g., Wu, H. et al. (1998) "Stepwise In Vitro Affinity Maturation Of Vitaxin, An Alphav Beta3-Specific Humanized Mab," Proc. Natl. Acad. Sci. (USA) 95(11):6037-6042; Yelton, D.E. et al. (1995) "Affinity Maturation Of The BR96 Anti-Carcinoma Antibody By Codon-Based Mutagenesis," J. Immunol. 155:1994-2004). CDR walking with randomized light chains can be used (see Schier et al. (1996) "Isolation Of Picomolar Affinity Anti-C-Erbb-2 Single-Chain Fv By Molecular Evolution Of The Complementarity Determining Regions In The Center Of The Antibody Binding Site," J. Mol. Biol. 263:551-567).

[0271] Methods for achieving such affinity maturation are described, for example, in: Krause, J.C. et al. (2011) “An Insertion Mutation That Distorts Antibody Binding Site Architecture Enhances Function Of A Human Antibody,” MBio. 2(1) pii: e00345-10. doi:10.1128 / mBio.00345-10; Kuan, C.T. et al. (2010) “Affinity-Matured Anti-Glycoprotein NMB Recombinant Immunotoxins Targeting Malignant Gliomas And Melanomas,” Int. J. Cancer 10.1002 / ijc.25645; Hackel, B.J. et al. (2010) “Stability And CDR Composition Biases Enrich Binder Functionality Landscapes,” J. Mol. Biol. 401(1):84-96; Montgomery, D.L. et al. (2009) “Affinity Maturation And Characterization Of A Human Monoclonal Antibody Against HIV-1 gp41,” MAbs 1(5):462-474; Gustchina, E. et al. (2009) “Affinity Maturation By Targeted Diversification Of The CDR-H2 Loop Of A Monoclonal Fab Derived From A Synthetic "Human Antibody Library And Directed Against The Internal Trimeric Coiled-Coil Of Gp41 Yields A Set Of Fabs With Improved HIV-1 Neutralization Potency And Breadth,” Virology 393(1):112-119; Finlay, W.J. et al. (2009) “Affinity Maturation Of A Humanized Rat Antibody For Anti-RAGE Therapy: Comprehensive Mutagenesis Reveals A High Level Of Mutational Plasticity Both Inside And Outside The Complementarity-Determining Regions,” J. Mol. Biol. 388(3):541-558; Bostrom, J. et al. (2009) “Improving Antibody Binding Affinity And Specificity For Therapeutic Development,” Methods Mol. Biol. 525:353-376; Steidl, S. et al. (2008) “In Vitro Affinity Maturation Of Human GM-CSF Antibodies By Targeted CDR-Diversification,” Mol. Immunol. 46(1):135-144; and Barderas, R. et al. (2008) “Affinity maturation of antibodies assisted by in silico modeling,” Proc. Natl. Acad. Sci. (USA) 105(26):9029-9034。

[0272] 6. Exemplary bispecific antibodies

[0273] An anti-ApoL1, BCMA IgG1-scFv (C-terminal of the heavy chain) chimeric antibody has been designed and has the Figure 8 structure.

[0274] The Fab portion is the variable regions of the heavy and light chains of the recombinant clone SFIII 13.11 (anti-ApoL1) having the sequences SEQ ID NO:24 (VH) and SEQ ID NO:36 or SEQ ID NO:77 (VL).

[0275] Anti-BCMA is [Clone 17A5], a human IgG1, a ScFv of κ fused to the C-terminus of the heavy chain of human IgG1. The heavy and light chain variable sequences of anti-BCMA Clone 17A5 are:

[0276] VH

[0277]

[0278] VL

[0279]

[0280] The CDR sequences of anti-BCMA Clone 17A5 (shown in bold in the above sequences) are:

[0281] CDR1H: SYAMS (SEQ ID NO:43),

[0282] CDR2H: AISGSGGSTYYADSVKG (SEQ ID NO:44),

[0283] CDR3H: VAPYFAPFDY (SEQ ID NO:45),

[0284] CDR1L: RASQSVSSSYLA (SEQ ID NO:46),

[0285] CDR2L: GASSRAT (SEQ ID NO:47),

[0286] CDR3L: QQYGNPPLYT (SEQ ID NO:48).

[0287] An exemplary sequence of the anti-BCMA scFv is

[0288]

[0289] where the CDRs are in bold and gggggsggggsggggs (lowercase) (SEQ ID NO:52) is the flexible linker.

[0290] In some embodiments, the bispecific antibody comprises the amino acid sequence

[0291] EVQLVESGGGLVKPGGSLKLSCAASGFTFSTYAMSWVRQSPEKRLEWVAEISNGGLYTYYPDTVTGRFTISRDNVKNILYLEMSSLRSEDTAIYYCIRENRNWYFDLWGAGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGSGGGGSGGGGSEIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGNPPLYTFGQGTKVEIKGGGGSGGGGSGGGGSEVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKVAPYFAPFDYWGQGTLVTVSS(SEQ ID NO:71) or a variant thereof having at least 70%, 80%, 90% or 95% sequence identity thereto;

[0292] and / or

[0293] DVLMTQTPLSLPVSLGDQASISCRSSQSIVNSNGNTYLEWYLQKPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYYCFQGSHVPLTFGAGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO:72) or a variant thereof having at least 70%, 80%, 90% or 95% sequence identity thereto.

[0294] With respect to SEQ ID NO:71,

[0295] EVQLVESGGGLVKPGGSLKLSCAASGFTFSTYAMSWVRQSPEKRLEWVAEISNGGLYTYYPDTVTGRFTISRDNVKNILYLEMSSLRSEDTAIYYCIRENRNWYFDLWGAGTTVTVSS (SEQ ID NO:24) is the heavy chain variable domain of the STII 13.11 anti-ApoL1 antibody;

[0296] ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP (SEQ ID NO:75) is the heavy chain constant region sequence;

[0297] GGGGSGGGGSGGGGS (SEQ ID NO:52) is the linker sequence; and

[0298] EIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGNPPLYTFGQGTKVEIKGGGGSGGGGSGGGGSEVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKVAPYFAPFDYWGQGTLVTVSS (SEQ ID NO:51) is the variable domain of BCMA 17A5 scFv.

[0299] For SEQ ID NO:72:

[0300] DVLMTQTPLSLPVSLGDQASISCRSSQSIVNSNGNTYLEWYLQKPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYYCFQGSHVPLTFGAGTKLEIK (SEQ ID NO:77) is the light chain variable domain of STII 13.11 anti-ApoL1 antibody; and

[0301] RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO:76) is the light chain constant region sequence.

[0302] A bispecific antibody having Figure 8 the structure can be formed from two copies of each of the amino acid sequences SEQ ID NO:71 and SEQ ID NO:72, for example, upon co-expression of nucleic acids encoding the amino acid sequences SEQ ID NO:71 (e.g., SEQ ID NO:73) and SEQ ID NO:72 (e.g., SEQ ID NO:74).

[0303] For the BMCA clone 17A5 sequence and alternative anti-BCMA sequences, see WO 2014 / 122144, which is incorporated herein by reference in its entirety, and the sequences can be used to derive alternative anti-BMCA arms of antibodies.

[0304] In another embodiment, in addition to or as an alternative to anti-ApoL1, the Fab portion comprises the heavy and light chain CDRs, or the entire heavy and light chain variable regions respectively belonging to the recombinant clones SFII 134.3 (anti-Hpr) or SFII14.11 (anti-Hpr) having the sequences SEQ ID NO:3 (VH) and SEQ ID NO:14 (VL) or the sequences SEQ ID NO:56 (VH) and SEQ ID NO:65 (VL), and their associated CDRs, as discussed in more detail elsewhere herein.

[0305] 7. Other exemplary antibodies for targeting

[0306] As introduced above, cell-specific markers and cancer antigens are targeting moieties, preferably the preferred targets of antibodies. Antibodies for targeting cell-specific markers and cancer antigens are known in the art and can be used in the disclosed compositions.

[0307] A specific example of an anti-B7-H1 (PD-L1) antibody is MDX-1105 (WO / 2007 / 005874, published January 1, 2007), a human anti-B7-H1 antibody.

[0308] For anti-B7-DC (PD-L2) antibodies, see 7,411,051, 7,052,694, 7,390,888 and U.S. Published Application No. 2006 / 0099203.

[0309] Examples of anti-CTLA4 antibodies contemplated for use in the disclosed compositions and methods include the antibodies described in PCT / US2006 / 043690 (Fischkoff et al., WO / 2007 / 056539).

[0310] Other specific exemplary antibodies (whose sequences can be used in the disclosed bispecific and multispecific antibodies) include but are not limited to Talquetamab (JNJ64407564), Indatuximab ravtansine, Daratumumab, Elotuzumab, DFRF4539A and BFCR4350A.

[0311] Daratumumab is a human IgG(κ) antibody that targets a unique epitope of CD38 (approved by the FDA in 2016).

[0312] Indatuximab ravtansine targets CD138.

[0313] Elotuzumab is an mAb targeting the extracellular domain of SLAMF7 and has shown moderate success in the phase 3 ELOQUENT-2 study.

[0314] DFRF4539A and BFCR4350A target FcRH5.

[0315] Taqtuomab is a bispecific antibody targeting GPRC5D on multiple myeloma and CD3 on T cells.

[0316] See, for example, Leow et al., J Pers Med. 11(5):334(2021). doi:10.3390 / jpm11050334 and U.S. Patent No. 10,562,968.

[0317] Antibodies targeting Claudin 18.2 are provided in WO 2022 / 136642A1.

[0318] Antibodies targeting MUC1 (Gatipotuzumab) are provided, for example, in WO 2019 / 219891 and KR20210010565A.

[0319] Examples of antibodies targeting MSLN (mesothelin) include, but are not limited to,

[0320] Anetumab ravtansine, see, for example, W02009 / 068204A1 and WO2020234114A1;

[0321] SS1P, see, for example, U.S. Patent No. 8,460,660B2;

[0322] Amatuximab or MORAb-009, see, for example, U.S. Patent No. 9,803,022; and

[0323] SD1 / SD2, see, for example, WO 2014 / 052064.

[0324] Examples of antibodies targeting PMEL17 are discussed, for example, in U.S. Patent No. 9,056,910, WO 2013 / 165940, EP 2844300.

[0325] All of the foregoing patents and applications are hereby specifically incorporated by reference in their entirety, including but not limited to their antibody sequences, and most specifically the CDRs, which may be incorporated into the disclosed compositions to target ApoL1 to cells expressing its target antigen.

[0326] B. Compositions for targeting exogenous ApoL1

[0327] Compositions for increasing the cellular internalization of exogenous ApoL1 are also provided. Although not necessarily for this purpose specifically, the compositions can be used to deliver exogenous ApoL1 (e.g., recombinant ApoL1) into cells.

[0328] The compositions generally comprise ApoL1, such as ApoL1 of SEQ ID NO:50, or a functional fragment or variant thereof having, for example, 70%, 75%, 80%, 85%, 90%, 95% sequence identity therewith. The compositions generally further comprise a targeting moiety that binds to a cell-specific marker (such as a cancer antigen) present on the cell, thereby facilitating the targeting of ApoL1 or a fragment or variant thereof to the target cell. The targeted ApoL1 can be internalized by the cell in an effective amount that increases the cell death of the target cell. ApoL1 or a fragment or variant thereof can be fused to or conjugated directly or indirectly to the targeting moiety. For example, in some embodiments, ApoL1 is a fusion protein comprising ApoL1 or a fragment or variant thereof and a targeting moiety. In some embodiments, ApoL1 or a fragment or variant thereof is conjugated to the targeting moiety. In some embodiments, ApoL1 or a fragment or variant thereof is packaged in a delivery agent (such as a nanoparticle or liposome), and the delivery agent also has a targeting moiety conjugated thereto.

[0329] Any composition can further comprise a cell-penetrating peptide.

[0330] 1. Targeting moiety

[0331] Representative targeting moieties include, but are not limited to, antibodies and antigen-binding fragments thereof, aptamers, peptides, and small molecules. The binding moiety can be conjugated to a polymer forming a nanocarrier. Generally, the binding moiety is displayed on the outer shell of the nanocarrier. The outer shell can act as a barrier to prevent the nanocarrier from being recognized by the subject's immune system, thereby increasing the half-life of the nanocarrier in the subject. The nanoparticle can contain a hydrophobic core. In the case of liposomal nanoparticles, the core can also be hydrophilic. In some embodiments, the hydrophobic core is made of a biodegradable polymeric material. The inner core carries a therapeutically effective payload and releases the therapeutically effective payload at a sustained rate after systemic, intraperitoneal, oral, pulmonary, or local administration. The nanocarrier also optionally includes a detectable label, such as a fluorophore or an NMR contrast agent that allows visualization of the nanocarrier.

[0332] In other embodiments, the targeting moiety is conjugated, linked, or directly fused to ApoL1 or a fragment or variant thereof.

[0333] The targeting moiety of the nanocarrier can be an antibody or an antigen-binding fragment thereof. The targeting moiety should have an affinity for a cell surface receptor or cell surface antigen on the target cell. The targeting moiety can cause the nanocarrier to be internalized within the target cell.

[0334] The targeting moiety can specifically recognize and bind to a target molecule that is specific for a cell type, tissue type, or organ. The target molecule can be a cell surface polypeptide, lipid, or glycolipid. The target molecule can be a receptor that is selectively expressed on a specific cell surface, tissue, or organ. The cell-specific marker can be directed against a specific type of cell, including but not limited to stem cells, skin cells, blood cells, immune cells, muscle cells, nerve cells, cancer cells, virus-infected cells, bacterial cells, fungal cells, organ-specific cells, and other eukaryotic cells. The cell marker can be specific for endothelial cells, ectodermal cells, or mesenchymal cells. Representative cell-specific markers include but are not limited to cancer-specific markers. The cell marker can be any cell-specific marker, including cancer and tumor antigens, as well as other mammalian and non-mammalian cell targets (e.g., bacterial and fungal cells), including but not limited to those provided elsewhere herein (see, e.g., above). Generally, the targeting moiety does not target trypanosome-specific surface antigens.

[0335] The targeting moiety can be a peptide. The targeting peptide can be covalently associated with a polymer, and the covalent association can be mediated by a linker. The targeting moiety can be an antigen-binding fragment of an antibody or a fusion protein thereof. The antibody can be in any form, including but not limited to those provided elsewhere herein (see, e.g., above).

[0336] 2. Exemplary Nanocarriers

[0337] There are provided nanocarrier compositions comprising ApoL1 or a fragment or variant thereof loaded into, attached to the surface of, and / or encapsulated within a delivery vehicle, and a targeting moiety.

[0338] The nanocarrier delivery vehicle can be, for example, a polymeric particle, an inorganic particle, a silica particle, a liposome, a micelle, a multilamellar vesicle, or a microbubble.

[0339] In some embodiments, the delivery vehicle is a nanoscale composition, such as from 10 nm to about 1 micrometer, excluding 1 micrometer. However, it should be understood that in some embodiments and for some uses, the particles can be smaller or larger (e.g., microparticles, etc.). Although many of the compositions disclosed herein are referred to as nanoparticle or nanocarrier compositions, it should be understood that in some embodiments and for some uses, the carrier can be slightly larger than a nanoparticle. For example, the carrier composition can be between about 1 micrometer and about 1000 micrometers. Such compositions can be referred to as microparticle compositions. For example, the nanocarrier according to the present disclosure can be a microparticle. The diameter size of the microparticle can be between, for example, 0.1 μm and 100 μm. In another example, the nanocarrier can be a superparticle. A superparticle is a particle having a diameter size greater than about 100 μm. For example, the diameter size of the superparticle can be from about 100 μm to about 1,000 μm.

[0340] Microbubbles are bubbles with diameters less than one millimeter but greater than one micrometer, which are widely used in industry, life sciences, and medicine. The composition of the bubble shell and the filling material determines imparted properties such as buoyancy, crush strength, thermal conductivity, and acoustic properties. In medicine, they can be applied to diagnosis (such as imaging) and treatment (such as drug delivery).

[0341] In some embodiments for treating cancer, it is desirable for the particles to have a size suitable for entering the tumor microenvironment. In a specific embodiment, the particles have a size suitable for entering the tumor microenvironment and / or tumor cells through the enhanced permeability and retention (EPR) effect. EPR refers to the property that molecules of certain sizes accumulate more easily in tumor tissue than in normal tissue. Thus, in an exemplary treatment of cancer, the delivery agent can be in the range of about 25 nm to about 500 nm (including the end values), or in the range of about 50 nm to about 300 nm (including the end values). In another example, the delivery agent can be in the range of about 80 nm to about 120 nm (including the end values). In another example, the delivery agent can be in the range of about 85 nm to about 110 nm (including the end values).

[0342] Polymer nanoparticles are typically formed using aqueous and non-aqueous solvents by single emulsion or double emulsion processes. Generally, after removal of the solvent, the nanoparticles contain a minimal amount of non-aqueous solvent.

[0343] In one embodiment, nanoparticles are prepared using the emulsion solvent evaporation method. The polymer material is dissolved in a water-immiscible organic solvent and mixed with a drug solution or a combination of drug solutions. The water-immiscible organic solvent can be a GRAS component such as chloroform, dichloromethane, and acetate. The drug can be dissolved in one or more of the following, but is not limited to: acetone, ethanol, methanol, isopropanol, acetonitrile, and dimethyl sulfoxide (DMSO). Then an aqueous solution is added to the resulting mixture solution to obtain an emulsion solution by emulsification. The emulsification technique can be, but is not limited to, probe sonication or homogenization by a homogenizer.

[0344] In another embodiment, nanoparticles are prepared using the nanoprecipitation method or a microfluidic device. The polymer material is mixed with a drug or a drug combination in a water-immiscible organic solvent. The water-immiscible organic solvent can be one or more of the following: acetone, ethanol, methanol, isopropanol, acetonitrile, and dimethyl sulfoxide (DMSO). Then the resulting mixture solution is added to an aqueous solution to produce a nanoparticle solution. The agent can associate with the surface of the polymer matrix of the particle, be encapsulated in the polymer matrix of the particle, be surrounded by the polymer matrix of the particle, and / or be distributed throughout the polymer matrix of the particle.

[0345] In another embodiment, nanoparticles are prepared by self-assembly of amphiphilic polymers (optionally including hydrophilic polymers and / or hydrophobic polymers) using emulsion solvent evaporation, single-step nanoprecipitation, or a microfluidic device.

[0346] Other exemplary methods for generating nanoparticles covered by the present disclosure are described in Zhou et al., Biomaterials, 33(2):583-591 (2012) and Han et al., Nanomedicine (2016).

[0347] Two methods for incorporating targeting moieties into nanoparticles include: i) conjugating a targeting ligand to the hydrophilic region of the polymer (e.g., PEG) prior to nanoparticle preparation; and ii) incorporating a targeting molecule onto the nanoparticle, where the PEG layer on the nanoparticle surface can be cleaved in the presence of a chemical or enzyme at the tissue of interest to expose the targeting molecule.

[0348] The particles can be microparticles or nanoparticles. Nanoparticles are commonly used for interstitial applications, cell penetration, and certain administration routes. Nanoparticles can have any desired size for the intended use. Nanoparticles can have any diameter from 10 nm to about 1,000 nm. Nanoparticles can have a diameter of 10 nm to 900 nm, 10 nm to 800 nm, 10 nm to 700 nm, 10 nm to 600 nm, 10 nm to 500 nm, 20 nm to 500 nm, 30 nm to 500 nm, 40 nm to 500 nm, 50 nm to 500 nm, 50 nm to 400 nm, 50 nm to 350 nm, 50 nm to 300 nm, or 50 nm to 200 nm. In some embodiments, the nanoparticles can have a diameter less than 400 nm, less than 300 nm, or less than 200 nm. The range can be between 50 nm and 300 nm.

[0349] The average diameter of the nanoparticles generally ranges between about 50 nm and about 500 nm, or between about 50 nm and about 350 nm. In some embodiments, the average diameter of the nanoparticles is about 100 nm. The ζ potential of the nanoparticles generally ranges between about -50 mV and about +50 mV, or between about -25 mV and +25 mV, or between about -10 mV and about +10 mV.

[0350] In some embodiments, the particles are brain-penetrating polymer nanoparticles that can be loaded with a drug and optimized for intracerebral convection-enhanced delivery (CED), such as those discussed in WO 2013 / 166487 and U.S. Published Application No. 2015 / 0118311. For example, the particles can be formed by emulsifying a polymer-drug solution, then removing the solvent and centrifuging at a first force to remove larger particles, and then collecting smaller particles using a second, higher force to precipitate smaller particles having a diameter less than 100 nm or an average diameter in the range of 25 - 75 nanometers that are capable of penetrating the brain interstitial space.

[0351] Partially water-miscible organic solvents such as benzyl alcohol, butyl lactate, and ethyl acetate (EA) allow for the formulation of nanoparticles by an emulsion diffusion mechanism and are capable of producing smaller nanoparticles than water-immiscible solvents such as dichloromethane (DCM). The use of partially water-miscible organic solvents increases the yield of brain-penetrating nanoparticles. Representative solvents that can be used include DCM, benzyl alcohol, butyl lactate, ethyl acetate (EA), and acetone. EA is particularly attractive due to its low toxicity.

[0352] To reduce aggregation, a sugar such as the FDA-approved disaccharide trehalose can be added to the composition. Other sugars include glucose, sucrose, and lactose. Typically, the weight ratio of the sugar to the nanoparticles ranges between 10 - 50%.

[0353] a. Polymer

[0354] The nanocarrier can be a particle containing one or more hydrophilic polymers. Hydrophilic polymers include cellulose polymers such as starch and polysaccharides; hydrophilic polypeptides; poly(amino acids) such as poly-L-glutamic acid (PGS), γ-polyglutamic acid, poly-L-aspartic acid, poly-L-serine, or poly-L-lysine; polyalkylene glycols and polyalkylene oxides such as polyethylene glycol (PEG), polypropylene glycol (PPG), and poly(ethylene oxide) (PEO); poly(oxyethylated polyols); poly(vinyl alcohols); polyvinylpyrrolidone); poly(hydroxyalkyl methacrylamide); poly(hydroxyalkyl methacrylate); poly(sugars); poly(hydroxy acids); poly(vinyl alcohol), and copolymers thereof.

[0355] The nanoparticles can contain one or more hydrophobic polymers. Examples of suitable hydrophobic polymers include polyhydroxy acids such as poly(lactic acid), poly(glycolic acid), and poly(lactic-co-glycolic acid); polyhydroxyalkanoates such as poly-3-hydroxybutyrate or poly-4-hydroxybutyrate; polycaprolactone; poly(orthoesters); polyanhydrides; poly(phosphazenes); poly(lactide-co-caprolactone); polycarbonates such as tyrosine polycarbonate; polyamides (including synthetic and natural polyamides), polypeptides, and poly(amino acids); polyesteramides; polyesters; poly(dioxanone); poly(alkylene alkylates); hydrophobic polyethers; polyurethanes; polyether esters; polyacetals; polycyanoacrylates; polyacrylates; polymethyl methacrylate; polysiloxanes; poly(ethylene oxide) / poly(propylene oxide) copolymers; polyketals; polyphosphates; polyhydroxyvalerates; polyalkylene oxalates; polyalkylene succinates; poly(maleic acid) and its copolymers.

[0356] In certain embodiments, the hydrophobic polymer is an aliphatic polyester. In some embodiments, the hydrophobic polymer is poly(lactic acid), poly(glycolic acid), or poly(lactic-co-glycolic acid).

[0357] The nanoparticles can contain one or more biodegradable polymers. The biodegradable polymers can include polymers that are insoluble or slightly soluble in water and that are chemically or enzymatically converted to water-soluble materials in the body. The biodegradable polymers can include soluble polymers crosslinked by hydrolyzable crosslinking groups such that the crosslinked polymers are insoluble or slightly soluble in water.

[0358] The biodegradable polymers in the nanoparticles can include polyamides, polycarbonates, polyalkylenes, polyalkylene glycols, poly(alkylene oxides), polyalkylene terephthalates, polyvinyl alcohols, polyvinyl ethers, polyvinyl esters, polyvinyl halides, polyvinyl pyrrolidones, polyglycolides, polysiloxanes, polyurethanes and their copolymers, alkyl celluloses, hydroxyalkyl celluloses, cellulose ethers, cellulose esters, cellulose nitrates, acrylate and methacrylate polymers, methyl cellulose, ethyl cellulose, hydroxypropyl cellulose, hydroxypropyl methyl cellulose, hydroxybutyl methyl cellulose, cellulose acetate, cellulose propionate, cellulose acetate butyrate, cellulose acetate phthalate, carboxyethyl cellulose, cellulose triacetate, sodium cellulose sulfate, poly(methyl methacrylate), poly(ethyl methacrylate), poly(butyl methacrylate), poly(isobutyl methacrylate), poly(hexyl methacrylate), poly(isodecyl methacrylate), poly(lauryl methacrylate), poly(phenyl methacrylate), poly(methyl acrylate), poly(isopropyl acrylate), poly(isobutyl acrylate), poly(octadecyl acrylate), polyethylene, polypropylene, poly(ethylene glycol), poly(ethylene oxide), polyethylene terephthalate, poly(vinyl alcohol), poly(vinyl acetate), polyvinyl chloride, polystyrene and polyvinyl pyrrolidone, their derivatives, linear and branched copolymers and their block copolymers, and their blends. Exemplary biodegradable polymers include polyesters, poly(orthoesters), poly(vinyl amines), poly(caprolactones), poly(hydroxybutyric acids), poly(hydroxyvaleric acids), polyanhydrides, poly(acrylic acids), polyglycolides, poly(carbamates), polycarbonates, polyphosphates, polyphosphazenes, their derivatives, linear and branched copolymers and their block copolymers, and their mixtures.

[0359] The nanoparticles can contain one or more amphiphilic polymers. An amphiphilic polymer can be a polymer containing a hydrophobic polymer block and a hydrophilic polymer block. The hydrophobic polymer block can contain one or more of the above hydrophobic polymers or their derivatives or copolymers. The hydrophilic polymer block can contain one or more of the above hydrophilic polymers or their derivatives or copolymers. In some embodiments, the amphiphilic polymer is a diblock polymer containing a hydrophobic end formed by a hydrophobic polymer and a hydrophilic end formed by a hydrophilic polymer. In some embodiments, a moiety can be attached to the hydrophobic end, the hydrophilic end, or both.

[0360] In some embodiments, the nanoparticles contain a first amphiphilic polymer having a hydrophobic polymer block, a hydrophilic polymer block, and a targeting moiety conjugated to the hydrophilic polymer block; and a second amphiphilic polymer having a hydrophobic polymer block and a hydrophilic polymer block but no targeting moiety. The hydrophobic polymer block of the first amphiphilic polymer and the hydrophobic polymer block of the second amphiphilic polymer may be the same or different. Similarly, the hydrophilic polymer block of the first amphiphilic polymer and the hydrophilic polymer block of the second amphiphilic polymer may be the same or different.

[0361] In some embodiments, the nanoparticles comprise a biodegradable polyester or polyanhydride, such as poly(lactic acid), poly(glycolic acid), and poly(lactic-co-glycolic acid). The nanoparticles may contain one or more of the following polyesters: homopolymers that comprise: glycolic acid units, herein referred to as “PGA”; and lactic acid units, such as poly-L-lactic acid, poly-D-lactic acid, poly-D,L-lactic acid, poly-L-lactide, poly-D-lactide, and poly-D,L-lactide, collectively referred to herein as “PLA”; and caprolactone units, such as poly(ε-caprolactone), collectively referred to herein as “PCL”; and copolymers that comprise lactic acid and glycolic acid units, such as the various forms of poly(lactic-co-glycolic acid) and poly(lactide-co-glycolide) characterized by the ratio of lactic acid:glycolic acid, collectively referred to herein as “PLGA”; and polyacrylates and their derivatives. Exemplary polymers also include copolymers of polyethylene glycol (PEG) and the above polyesters, such as the various forms of PLGA-PEG or PLA-PEG copolymers, collectively referred to herein as “PEGylated polymers”. In certain embodiments, the PEG moiety may be covalently associated with the polymer to produce a “PEGylated polymer” via a cleavable linker. Other polymers include PLGA-poly(ε-benzyloxycarbonyl-L-lysine) (PLL) (i.e., PLGA-PLL).

[0362] The nanoparticles may also contain one or more polymer conjugates having an end-to-end bond between the polymer and a targeting moiety or a detectable label. For example, the modified polymer may be a PLGA-PEG-peptide block polymer.

[0363] The nanoparticles may contain one polymer or a mixture of two or more polymers. The nanoparticles may contain other entities, such as stabilizers, surfactants, or lipids. The nanoparticles may contain a first polymer having a targeting moiety and a second polymer having no targeting moiety. By adjusting the ratio of the targeting polymer to the non-targeting polymer, the density of the targeting moieties on the exterior of the particles can be adjusted.

[0364] The nanoparticles can contain an amphiphilic polymer having a hydrophobic end, a hydrophilic end, and a targeting moiety attached to the hydrophilic end. In some embodiments, the amphiphilic macromolecule is a block copolymer having a hydrophobic polymer block, a hydrophilic polymer block covalently coupled to the hydrophobic polymer block, and a targeting moiety covalently coupled to the hydrophilic polymer block. For example, the amphiphilic polymer can have a conjugate having the structure A-B-X, where A is a hydrophobic molecule or hydrophobic polymer, B is a hydrophilic molecule or hydrophilic polymer, and X is a targeting moiety. Exemplary amphiphilic polymers include amphiphilic polymers where A is a hydrophobic biodegradable polymer, B is PEG, and X is a targeting, binding targeting moiety.

[0365] In some embodiments, the nanoparticles contain a first amphiphilic polymer having the structure A-B-X as described above and a second amphiphilic polymer having the structure A-B, where A and B in the second amphiphilic macromolecule are selected independently of A and B in the first amphiphilic macromolecule, although they can be the same.

[0366] b. Liposomes and Micelles

[0367] In some embodiments, the nanocarrier is a liposome or a micelle. A liposome is a spherical vesicle composed of concentric phospholipid bilayers separated by an aqueous compartment. Liposomes can adhere to the cell surface and form a molecular film thereon. Structurally, a liposome is a lipid vesicle composed of concentric phospholipid bilayers enclosing an aqueous substance (Gregoriadis et al., Int. J. Pharm., 300, 125 - 30 (2005); Gregoriadis and Ryman, Biochem. J., 124, 58P (1971)). Hydrophobic compounds associate with the lipid phase, while hydrophilic compounds associate with the aqueous phase.

[0368] Liposomes have the ability to form a molecular film on the surfaces of cells and tissues. Clinical studies have demonstrated the efficacy of liposomes as topical healing agents (Dausch et al., Klin Monatsbl Augenheilkd 223, 974 - 83 (2006); Lee et al., Klin Monatsbl Augenheilkd 221, 825 - 36 (2004)). Liposomes are also used in ophthalmology to improve keratitis, corneal transplant rejection, uveitis, endophthalmitis, and proliferative vitreoretinopathy (Ebrahim et al., 2005; Li et al., 2007).

[0369] Liposomes have been widely investigated as drug carriers for various chemotherapeutic agents (about 25,000 scientific papers have been published on the subject) (Gregoriadis, N Engl J Med 295, 765 - 70 (1976); Gregoriadis et al., Int. J. Pharm. 300, 125 - 30 (2005)). Water-soluble anticancer substances such as doxorubicin can be protected in the aqueous compartment of liposomes bounded by a phospholipid bilayer, while lipophilic substances such as amphotericin and capsaicin can be incorporated into the phospholipid bilayer (Aboul-Fadl, Curr Med Chem 12, 2193 - 214 (2005); Tyagi et al., J Urol 171, 483 - 9 (2004)). The topical and intravitreal delivery of cyclosporine with liposomes has been significantly improved (Lallemand et al., Eur J Pharm Biopharm 56, 307 - 18 2003). The delivery of chemotherapeutic agents has led to improved pharmacokinetics and a reduced toxicity profile (Gregoriadis, Trends Biotechnol 13, 527 - 37 (1995); Gregoriadis and Allison, FEBS Lett 45, 71 - 4 1974; Sapra et al., Curr Drug Deliv 2, 369 - 81 (2005)). More than ten liposomes and lipid-based formulations have received regulatory approval, and many liposomal drugs are in preclinical development or clinical trials (Barnes, Expert Opin Pharmacother 7, 607 - 15 (2006); Minko et al., Anticancer Agents Med Chem 6, 537 - 52 (2006)). Safety data on the acute, subchronic, and chronic toxicity of liposomes have been gleaned from the extensive clinical experience of thousands of patients using liposomes clinically.

[0370] Nanocarriers (such as liposomes and micelles) can be formed from one or more lipids that can be neutral, anionic, or cationic at physiological pH. Suitable neutral and anionic lipids include, but are not limited to, sterols and lipids such as cholesterol, phospholipids, lysolipids, lysophospholipids, sphingolipids, or polyethylene glycolated lipids. Neutral and anionic lipids include, but are not limited to, phosphatidylcholine (PC) (such as egg PC, soy PC), including, but not limited to, 1,2-diacyl-glycerol-3-phosphocholine; phosphatidylserine (PS), phosphatidylglycerol, phosphatidylinositol (PI); glycolipids; phosphosphingolipids (such as sphingomyelin) and glycosphingolipids (also known as 1-ceramidoglucoside), such as ceramide galactopyranoside, gangliosides, and cerebrosides; fatty acids, sterols containing a carboxylic acid group, such as cholesterol; 1,2-diacyl-sn-glycerol-3-phosphoethanolamine, including, but not limited to, 1,2-dioleoylphosphoethanolamine (DOPE), 1,2-docosanoylphosphoethanolamine (DHPE), 1,2-distearoylphosphatidylcholine (DSPC), 1,2-dipalmitoylphosphatidylcholine (DPPC), and 1,2-dimyristoylphosphatidylcholine (DMPC). Lipids can also include various natural (e.g., tissue-derived L-α-phosphatidyl: egg yolk, heart, brain, liver, soy) and / or synthetic (e.g., saturated and unsaturated 1,2-diacyl-sn-glycerol-3-phosphocholine, 1-acyl-2-acyl-sn-glycerol-3-phosphocholine, 1,2-didecanoyl-SN-glycerol-3-phosphocholine) lipid derivatives. In some embodiments, the liposome contains a phosphatidylcholine (PC) head group and optionally sphingomyelin. In another embodiment, the liposome contains DPPC. In additional embodiments, the liposome contains neutral lipids such as 1,2-dioleoylphosphatidylcholine (DOPC).

[0371] In certain embodiments, the liposome is produced from a single type of phospholipid. In some embodiments, the phospholipid has a phosphatidylcholine head group and can be, for example, sphingomyelin. The liposome can include sphingomyelin metabolites. Sphingomyelin metabolites for formulating liposomes include, but are not limited to, ceramides, sphingosine, or sphingosine-1-phosphate. The concentration range of sphingomyelin metabolites contained in the lipids used to formulate liposomes can be from about 0.1 mol% to about 10 mol%, or from about 2.0 mol% to about 5.0 mol%, or can be a concentration of about 1.0 mol%.

[0372] Suitable cationic lipids in liposomes include, but are not limited to, N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium salts, also known as TAP lipids, such as methyl sulfate. Suitable TAP lipids include, but are not limited to, DOTAP (dioleoyl-), DMTAP (dimyristoyl-), DPTAP (dipalmitoyl-), and DSTAP (distearoyl-). Suitable cationic lipids in liposomes include, but are not limited to, dimethyldioctadecylammonium bromide (DDAB), 1,2-diacyl-oxy-3-trimethylammonium propane, N-[1-(2,3-dioleyloxy)propyl]-N,N-dimethylamine (DODAP), 1,2-diacyl-oxy-3-dimethylammonium propane, N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA), 1,2-dialkyloxy-3-dimethylammonium propane, dioctadecylamidoglycyl spermine (DOGS), 3-[N-(N',N'-dimethylamino-ethane)carbamoyl]cholesterol (DC-Chol); 2,3-dioleyloxy-N-(2-(sperminecarboxamido)-ethyl)-N,N-dimethyl-1-propanaminium trifluoroacetate (DOSPA), β-alanyl cholesterol, cetyltrimethylammonium bromide (CTAB), diC 14-amidine, N-tert-butyl-N'-tetradecyl-3-tetradecylaminopropamidine, N-(α-trimethylammonioacetyl) docosanoyl-D-glutamate chloride (TMAG), tetracosanoyl-N-(trimethylammonioacetyl) diethanolamine chloride, 1,3-dioleoyloxy-2-(6-carboxy-spermidinyl)-propanamide (DOSPER), and N,N,N',N'-tetramethyl-N'-bis(2-hydroxyethyl)-2,3-dioleoyloxy-1,4-butanediammonium iodide. In one embodiment, the cationic lipid can be a 1-[2-(acyloxy)ethyl]-2-alkyl(alkenyl)-3-(2-hydroxyethyl)imidazoline chloride derivative, such as 1-[2-(9(Z)-octadecenoyloxy))ethyl]-2-(8(Z)-heptadecenyl-3-(2-hydroxyethyl)imidazoline chloride (DOTIM) and 1-[2-(hexadecanoyloxy)ethyl]-2-pentadecyl-3-(2-hydroxyethyl)imidazoline chloride (DPTIM). In one embodiment, the cationic lipid can be a 2,3-dialkoxypropyl quaternary ammonium derivative having a hydroxyalkyl moiety on the quaternary amine, such as 1,2-dioleoyl-3-dimethyl-hydroxyethyl ammonium bromide (DORI), 1,2-dioleoyloxypropyl-3-dimethyl-hydroxyethyl ammonium bromide (DORIE), 1,2-dioleoyloxypropyl-3-dimethyl-hydroxypropyl ammonium bromide (DORIE-HP), 1,2-dioleoyloxypropyl-3-dimethyl-hydroxybutyl ammonium bromide (DORIE-HB), 1,2-dioleoyloxypropyl-3-dimethyl-hydroxypentyl ammonium bromide (DORIE-Hpe), 1,2-dimyristoyloxypropyl-3-dimethyl-hydroxyethyl ammonium bromide (DMRIE), 1,2-dipalmitoyloxypropyl-3-dimethyl-hydroxyethyl ammonium bromide (DPRIE), and 1,2-distearoyloxypropyl-3-dimethyl-hydroxyethyl ammonium bromide (DSRIE).

[0373] The lipid can be formed from a combination of more than one lipid. For example, a charged lipid can be combined with a lipid that is nonionic or uncharged at physiological pH. Nonionic lipids include, but are not limited to, cholesterol and DOPE (1,2-dioleoyl phosphatidylethanolamine). The molar ratio of the first phospholipid (such as sphingomyelin) to the second lipid can range from about 5:1 to about 1:1, or 3:1 to about 1:1, or about 1.5:1 to about 1:1, or the molar ratio is about 1:1.

[0374] In some embodiments, the liposomes or micelles include phospholipids, cholesterol, and nitrogen-containing lipids. Examples include phospholipids, including natural phospholipids such as phosphatidylcholine, phosphatidylserine, phosphatidylglycerol, phosphatidylinositol, phosphatidylethanolamine, phosphatidic acid, cardiolipin, sphingomyelin, egg yolk lecithin, soy lecithin, and lysophosphatidylcholine, and hydrogenated products thereof obtained in a standard manner. Synthetic phospholipids can also be used, such as distearyl phosphatidylcholine, dipalmitoyl phosphatidylcholine, dipalmitoyl phosphatidylethanolamine, dipalmitoyl phosphatidylserine, stearoyl phosphatidylcholine, stearoyl phosphatidylethanolamine, and homopolymer {N'-[N-(2-aminoethyl)-2-aminoethyl] asparagine} P[Asp(DET)] and block cationic isomer poly(ethylene glycol) (PEG)-b-P[Asp(DET)].

[0375] In some embodiments, the liposomes are long-circulating liposomes or stealth liposomes, such as those reviewed in Immordino et al., Int J Nanomedicine, 1(3):297–315(2006), which is incorporated herein by reference in its entirety. For example, liposomes have been developed that are modified on their surface with a variety of molecules, including glycolipids and sialic acid. Long-circulating liposomes can include, for example, the synthetic polymer poly(ethylene glycol) (PEG) in the liposome composition. PEG on the surface of the liposome carrier can extend the blood circulation time while reducing uptake by the mononuclear phagocyte system (stealth liposomes) and serve as an anchor for targeting moieties.

[0376] Antibodies and antibody fragments are widely used as targeting moieties for liposomes due to their high specificity for the target antigen. In the case of immunoliposomes, methods for generating targeted liposomes by conjugating an antibody to the liposome surface are known in the art. Such techniques include, but are not limited to, conventional conjugation and maleimide-based techniques. See, for example, (Paszko and Senge, Curr Med Chem., 19(31):5239-77(2012), Kelly et al., Journal of Drug Delivery, Volume 2011(2011), Article ID 727241, 11 pages).

[0377] The micelles can be polymeric micelles, such as micelles composed of amphiphilic diblock or triblock copolymers made of a hydrophilic and a hydrophobic block (see, for example, Croy and Kwon, Curr Pharm Des., 12(36):4669-84(2006)).

[0378] 3. Other functional elements

[0379] Other functional elements that can be associated, linked, conjugated or otherwise directly or indirectly linked to ApoL1, an antibody or its particles or other delivery vehicles include protein transduction domains and fusion peptides.

[0380] For example, the efficiency of a particulate delivery system can also be enhanced by attaching functional ligands to the particle surface. Potential ligands include, but are not limited to, small molecules, cell-penetrating peptides (CPPs), targeting peptides, antibodies or aptamers (Yu et al., PLoS One., 6: e24077 (2011), Cu et al., J Control Release, 156: 258–264 (2011), Nie et al., J Control Release, 138: 64–70 (2009), Cruz et al., J Control Release, 144: 118–126 (2010)). Attachment of these moieties has a variety of different functions; such as inducing intracellular uptake, endosomal disruption and delivery of plasmid payloads to the nucleus. A variety of methods have been employed to tether ligands to the particle surface. One method is direct covalent attachment to functional groups on PLGA NPs (Bertram, Acta Biomater. 5: 2860–2871 (2009)). Another method utilizes amphiphilic conjugates (such as avidin palmitate) to immobilize biotinylated ligands to the NP surface (Fahmy et al., Biomaterials, 26: 5727–5736 (2005), Cu et al., Nanomedicine, 6: 334–343 (2010)). Particles generated by this method enhance uptake in cells, but reduce pDNA release and gene transfection, which may be due to surface modification hindering pDNA release. In a similar method, lipid-conjugated polyethylene glycol (PEG) is used as a multivalent linker for cell-penetrating peptides, CPPs or folic acid (Cheng et al., Biomaterials, 32: 6194–6203 (2011)).

[0381] These methods, as well as other methods discussed herein and other methods known in the art, can be combined to fine-tune particle function and efficacy. In some preferred embodiments, PEG is used as a linker to attach functional molecules to the particle. For example, DSPE-PEG(2000)-maleimide is commercially available and can be used for covalent attachment of functional molecules (such as CPPs).

[0382] "Protein transduction domain" or PTD refers to a polypeptide, polynucleotide, or organic or inorganic compound that facilitates crossing of a lipid bilayer, micelle, cell membrane, organelle membrane, or vesicle membrane. A PTD attached to another molecule facilitates the molecule's crossing of the membrane, e.g., from the extracellular space into the intracellular space, or from the cytoplasm into an organelle. A PTA can be a short basic peptide sequence, such as those present in many cellular and viral proteins. Exemplary protein transduction domains well known in the art include, but are not limited to, antennapedia PTD and TAT (transcriptional transactivator) PTD, polyarginine, polylysine, or a mixture of arginine and lysine, HIV TAT (YGRKKRRQRRR (SEQ ID NO:53) or RKKRRQRRR (SEQ ID NO:54), 11 arginine residues, VP22 peptide, and ANTp peptide (RQIKIWFQNRRMKWKK) (SEQ ID NO:55) or a positively charged polypeptide or polynucleotide having 8-15 residues, preferably 9-11 residues. Short non-peptide polymers rich in amine or guanidine groups are also capable of carrying molecules across biological membranes. Transmembrane peptides and other peptide derivatives derived from antennapedia can also be used (Cheng et al., Biomaterials, 32(26):6194-203 (2011)). The results showed that the transmembrane peptides with additional Args further enhanced uptake and endosomal escape, while IKK NBD has an antennapedia domain for penetration and a domain that blocks NFkB activation and has been safely used in the lung for other purposes (von Bismarck et al., Pulmonary Pharmacology & Therapeutics, 25(3):228-35 (2012), Kamei et al., Journal Of Pharmaceutical Sciences, 102(11):3998-4008 (2013)).

[0383] A "fusion peptide" is any peptide that has the ability to destabilize a membrane. Generally, fusion peptides have a tendency to form an amphipathic α-helical structure when a hydrophobic surface, such as a membrane, is present. The presence of a fusion peptide induces the formation of pores in the cell membrane by disrupting the ordered packing of membrane phospholipids. Some fusion peptides act to promote lipid disorder and in this way enhance the chance of membrane merger or fusion of the close proximity of two membrane-enveloped particles (e.g., cells, enveloped viruses, liposomes) having different properties. Other fusion peptides may attach to two membranes simultaneously, causing the membranes to merge and facilitating their fusion into one membrane. Examples of fusion peptides include fusion peptides from the extracellular domain of viral envelope proteins, membrane-destabilizing peptides from the juxtamembrane domain of viral envelope proteins in the cytoplasmic tail.

[0384] Other fusion peptides also typically contain amphiphilic regions. Examples of peptides containing amphiphilic regions include: melittin, bombesin, the cytoplasmic tail of HIV1 gp41, microbial and reptilian cytotoxic peptides such as bomolitin 1, pardaxin, wasp venom toxin, crabrolin, cecropin, entamoeba and staphylococcal α-toxin; viral fusion peptides from: (1) regions at the N-terminus of the transmembrane (TM) domain of viral envelope proteins, e.g., HIV-1, SIV, influenza, polio, rhinovirus and coxsackievirus; (2) regions within the TM extracellular domain, e.g., Semliki Forest virus, Sindbis virus, rotavirus, rubella virus and the fusion peptide from sperm protein PH-30; (3) regions near the membrane on the cytoplasmic side of viral envelope proteins, e.g., avian leukosis virus (ALV), feline immunodeficiency virus (FIV), Rous sarcoma virus (RSV), Moloney murine leukemia virus (MoMuLV) and spleen necrosis virus (SNV).

[0385] 4. Preparation methods

[0386] a. Conjugates

[0387] Polymer synthesis methods are described, for example, in Braun et al. (2005) Polymer Synthesis: Theory and Practice. New York, NY: Springer. Polymers can be synthesized via step-growth polymerization, chain-growth polymerization or plasma polymerization.

[0388] In some embodiments, the synthesis of amphiphilic polymers begins with a hydrophobic polymer capped with a first reactive coupling group and a hydrophilic polymer capped with a second reactive coupling group capable of reacting with the first reactive coupling group to form a covalent bond. One of the first reactive coupling group or the second reactive coupling group can be a primary amine, where the other reactive coupling group can be an amine-reactive linking group such as isothiocyanate, isocyanate, acyl azide, NHS ester, sulfonyl chloride, aldehyde, glyoxal, epoxide, ethylene oxide, carbonate, aryl halide, imidate, carbodiimide, acid anhydride and fluorophenyl ester. One of the first reactive coupling group or the second reactive coupling group can be an aldehyde, where the other reactive coupling group can be an aldehyde-reactive linking group such as hydrazide, alkoxyamine and primary amine. One of the first reactive coupling group or the second reactive coupling group can be a thiol, where the other reactive coupling group can be a thiol-reactive group such as maleimide, haloacetyl and pyridyl disulfide.

[0389] In some embodiments, a hydrophobic polymer capped with an amine or amine-reactive linking group is conjugated to a hydrophilic polymer capped with a complementary reactive linking group. For example, NHS-ester-activated PLGA can be formed by reacting PLGA-CO(OH) with NHS and a coupling reagent such as dicyclohexylcarbodiimide (DCC) or 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC). The NHS-ester-activated PLGA can react with a hydrophilic polymer capped with a primary amine such as PEG-NH 2 to form an amphiphilic PLGA-b-PEG block copolymer.

[0390] In some embodiments, the same or a similar coupling reaction is used to form a conjugate of an amphiphilic polymer and a targeting moiety. In some embodiments, the conjugate is prepared starting with a hydrophilic polymer capped with a first reactive coupling group at one end and a protecting group at the second end. The hydrophilic polymer is reacted with a targeting moiety having a reactive group complementary to the first reactive group to form a covalent bond between the hydrophilic polymer and the targeting moiety. The protecting group can then be removed to provide a second reactive coupling group, for example to allow a hydrophobic polymer block to conjugate to the conjugate of the hydrophilic polymer and the targeting moiety. Then, a hydrophobic polymer capped with a reactive coupling group complementary to the second reactive coupling group can be covalently conjugated to form the conjugate. Of course, these steps can also be carried out in the reverse order, i.e., the conjugate of the hydrophobic polymer and the hydrophilic polymer can be formed first, followed by deprotecting the targeting moiety and conjugating it to the hydrophilic polymer block.

[0391] In some embodiments, a conjugate is formed having moieties conjugated to both ends of an amphiphilic polymer. For example, an amphiphilic polymer having a hydrophobic polymer block and a hydrophilic polymer block can have a targeting moiety conjugated to the hydrophilic polymer block and an additional moiety conjugated to the hydrophobic polymer block. In some embodiments, the additional moiety can be a detectable label. In some embodiments, the additional moiety is a therapeutic, prophylactic, or diagnostic agent. For example, the additional moiety can be a moiety for radiotherapy. The conjugate can be prepared starting with a hydrophobic polymer having a first reactive coupling group at one end and a first protecting group at the other end and a hydrophilic polymer having a second reactive coupling group at one end and a second protecting group at the other end. The hydrophobic polymer can be reacted with an additional moiety having a reactive group complementary to the first reactive coupling group, thereby forming a conjugate of the hydrophobic polymer and the additional moiety. The hydrophilic polymer can be reacted with a targeting moiety having a reactive group complementary to the second reactive coupling group, thereby forming a conjugate of the hydrophilic polymer and the targeting moiety. The first protecting group and the second protecting group can be removed to generate a pair of complementary reactive coupling groups that can react to covalently link the hydrophobic polymer block to the hydrophilic polymer block.

[0392] b. Formation of nano-carriers

[0393] i. Emulsion method

[0394] In some embodiments, nanoparticles are prepared using the emulsion solvent evaporation method. For example, a polymeric material is dissolved in a water-immiscible organic solvent and mixed with a drug solution or a combination of drug solutions. In some embodiments, a solution of a therapeutic, prophylactic, or diagnostic agent to be encapsulated is mixed with the polymer solution. The polymer can be one or more of, but not limited to: PLA, PGA, PCL, their copolymers, polyacrylates, the aforementioned polyethylene glycolated polymers, the aforementioned polymer-drug conjugates, the aforementioned polymer-peptide conjugates, or various forms of the aforementioned fluorescently labeled polymers, or combinations thereof. The drug molecule can be one or more of, but not limited to: PPARγ activators (e.g., rosiglitazone, (RS)-5-[4-(2-[methyl(pyridin-2-yl)amino]ethoxy)benzyl]thiazolidine-2,4-dione, pioglitazone, (RS)-5-(4-[2-(5-ethylpyridin-2-yl)ethoxy]benzyl)thiazolidine-2,4-dione, troglitazone, (RS)-5-(4-[(6-hydroxy-2,5,7,8-tetramethylchroman-2-yl)methoxy]benzyl)thiazolidine-2,4-dione, etc.), prostaglandin E2 analogs (PGE2, (5Z,11α,13E,15S)-7-[3-hydroxy-2-(3-hydroxyoct-1-enyl)-5-oxocyclopentyl]hept-5-enoic acid, etc.), β3 adrenergic receptor agonists (CL 316243, 5-[(2R)-2-[[(2R)-2-(3-chlorophenyl)-2-hydroxyethyl]amino]propyl]-1,3-benzodioxole-2,2-dicarboxylic acid disodium hydrate, etc.), fibroblast growth factor 21 (FGF-21), irisin, RNA, DNA, chemotherapeutic compounds, nuclear magnetic resonance (NMR) contrast agents, or combinations thereof. The water-immiscible organic solvent can be one or more of, but not limited to: chloroform, dichloromethane, and acyl acetates. The drug can be dissolved in one or more of, but not limited to: acetone, ethanol, methanol, isopropanol, acetonitrile, and dimethyl sulfoxide (DMSO).

[0395] In some embodiments, the polymer solution contains one or more polymer conjugates as described above. The polymer solution can contain a first amphiphilic polymer conjugate having a hydrophobic polymer block, a hydrophilic polymer block, and a targeting moiety conjugated to the hydrophilic end. In some embodiments, the polymer solution contains one or more additional polymers or amphiphilic polymer conjugates. For example, in addition to the first amphiphilic polymer conjugate, the polymer solution can also contain one or more hydrophobic polymers, hydrophilic polymers, lipids, amphiphilic polymers, polymer-drug conjugates, or conjugates containing other targeting moieties. By controlling the ratio of the first amphiphilic polymer to the additional polymers or amphiphilic polymer conjugates, the density of the targeting moieties can be controlled. The first amphiphilic polymer can be present at 1% to 100% by weight of the polymers in the polymer solution. For example, the first amphiphilic polymer can be present at 10%, 20%, 30%, 40%, 50%, or 60% by weight of the polymers in the polymer solution.

[0396] An aqueous solution is then added to the resulting mixture solution to obtain an emulsion solution by emulsification. The emulsification technique can be, but is not limited to, probe sonication or homogenization by a homogenizer. The plaque-targeting peptide or fluorophore or drug can be associated with the surface of the polymer matrix of the particles of the present invention, encapsulated in the polymer matrix of the particles of the present invention, surrounded by the polymer matrix of the particles of the present invention, and / or distributed throughout the polymer matrix of the particles of the present invention.

[0397] ii. Nanoprecipitation method

[0398] In another embodiment, multimodal nanoparticles are prepared using a nanoprecipitation method or a microfluidic device. A polymeric material is mixed with a drug or drug combination in a water-immiscible organic solvent. The polymer can be, but is not limited to, one or more of the following: PLA, PGA, PCL, their copolymers, polyacrylates, the aforementioned polyethylene glycolated polymers, the aforementioned polymer-drug conjugates, the aforementioned polymer-peptide conjugates, or the aforementioned fluorescently labeled polymers, or various forms of combinations thereof. The drug molecule can be, but is not limited to, one or more of the following: PPARγ activators (e.g., rosiglitazone, (RS)-5-[4-(2-[methyl(pyridin-2-yl)amino]ethoxy)benzyl]thiazolidine-2,4-dione, pioglitazone, (RS)-5-(4-[2-(5-ethylpyridin-2-yl)ethoxy]benzyl)thiazolidine-2,4-dione, troglitazone, (RS)-5-(4-[(6-hydroxy-2,5,7,8-tetramethylchroman-2-yl)methoxy]benzyl)thiazolidine-2,4-dione, etc.), prostaglandin E2 analogs (PGE2, (5Z,11α,13E,15S)-7-[3-hydroxy-2-(3-hydroxyoct-1-enyl)-5-oxocyclopentyl]hept-5-enoic acid, etc.), β3 adrenergic receptor agonists (CL 316243, 5-[(2R)-2-[[(2R)-2-(3-chlorophenyl)-2-hydroxyethyl]amino]propyl]-1,3-benzodioxole-2,2-dicarboxylic acid disodium hydrate, etc.), RNA, DNA, chemotherapeutic compounds, nuclear magnetic resonance (NMR) contrast agents, or combinations thereof. The water-immiscible organic solvent can be, but is not limited to, one or more of the following: acetone, ethanol, methanol, isopropanol, acetonitrile, and dimethyl sulfoxide (DMSO). The resulting mixture solution is then added to a polymer nonsolvent (such as an aqueous solution) to produce a nanoparticle solution. A plaque-targeting peptide or fluorophore or drug can associate with the surface of the polymeric matrix of the particles of the present invention, be encapsulated within the polymeric matrix of the particles of the present invention, be surrounded by the polymeric matrix of the particles of the present invention, and / or be distributed throughout the polymeric matrix of the particles of the present invention.

[0399] iii. Microfluidics

[0400] Methods for preparing nanoparticles using microfluidics are known in the art. Suitable methods include those described by Karnik et al. in U.S. Patent Application Publication No. 2010 / 0022680A1. Generally, a microfluidic device includes at least two channels that converge into a mixing device. The channels are typically formed by lithography, etching, embossing, or molding a polymer surface. Fluid sources are attached to each channel, and pressure is applied to the sources such that the fluids flow through the channels. The pressure can be applied by syringes, pumps, and / or gravity. The inlet streams of solutions having polymers, targeting moieties, lipids, drugs, payloads, etc. converge and mix, and the resulting mixture is combined with a polymer non-solvent solution to form nanoparticles having a desired size and density on the surface portion. By varying the pressure and flow rate in the inlet channels and the nature and composition of the fluid sources, nanoparticles having reproducible size and structure can be produced.

[0401] iv. Other methods

[0402] Solvent Evaporation . In this method, the polymer is dissolved in a volatile organic solvent (such as dichloromethane). The drug (either soluble or dispersed as fine particles) is added to the solution, and the mixture is suspended in an aqueous solution containing a surfactant (such as poly(vinyl alcohol)). The resulting emulsion is stirred until most of the organic solvent has evaporated, leaving behind solid microparticles. The resulting microparticles are washed with water and dried overnight in a freeze dryer. By this method, microparticles having different sizes (0.5 - 1000 microns) and morphologies can be obtained. This method is applicable to relatively stable polymers such as polyesters and polystyrenes.

[0403] However, unstable polymers (such as polyanhydrides) may degrade during the manufacturing process due to the presence of water. For these polymers, the following two methods carried out in a completely anhydrous organic solvent are more useful.

[0404] Hot - Melt Microencapsulation . In this method, the polymer is first melted and then mixed with solid particles. The mixture is suspended in an immiscible solvent (such as silicone oil) and heated to 5°C above the polymer melting point with continuous stirring. Once the emulsion is stable, it is cooled until the polymer particles solidify. The resulting microparticles are washed by decantation with petroleum ether to obtain a free-flowing powder. Microparticles having sizes between 0.5 and 1000 microns can be obtained by this method. The outer surface of the spheres prepared by this technique is usually smooth and dense. This procedure is used to prepare microparticles made of polyesters and polyanhydrides. However, this method is limited to polymers having molecular weights between 1,000 - 50,000 daltons.

[0405] Solvent Removal。This technique is mainly designed for polyanhydrides. In this method, the drug is dispersed or dissolved in a solution of the selected polymer in a volatile organic solvent (such as dichloromethane). This mixture is suspended in an organic oil (such as silicone oil) by stirring to form an emulsion. Different from solvent evaporation, this method can be used to prepare microparticles from polymers with high melting points and different molecular weights. Through this procedure, microparticles in the range of 1 - 300 microns can be obtained. The external morphology of the spheres produced by this technique highly depends on the type of polymer used.

[0406] Spray Drying In this method, the polymer is dissolved in an organic solvent. A known amount of the active drug is suspended (for insoluble drugs) or co - dissolved (for soluble drugs) in the polymer solution. Then the solution or dispersion is spray - dried. The typical processing parameters of a small spray dryer (Buchi) are as follows: polymer concentration = 0.04 g / mL, inlet temperature = - 24 °C, outlet temperature = 13 - 15 °C, aspirator setting = 15, pump setting = 10 mL / minute, spray flow = 600 Nl / hr, and nozzle diameter = 0.5 mm. Microparticles in the range of 1 - 10 microns are obtained, and their morphology depends on the type of polymer used.

[0407] Hydrogel Particles 。Microparticles prepared from gel - type polymers (such as alginic acid) are produced by traditional ion - gelation techniques. The polymer is first dissolved in an aqueous solution, mixed with barium sulfate or some bioactive agents, and then extruded through a droplet - forming device which, in some cases, uses a nitrogen stream to interrupt the droplets. An ion - hardening bath with slow stirring (about 100 - 170 RPM) is located below the extrusion device to capture the formed micro - droplets. The microparticles are incubated in the bath for twenty to thirty minutes to allow sufficient time for gelation to occur. The particle size is controlled by using extruders of various sizes or by changing the flow rate of nitrogen or the polymer solution. Chitosan microparticles can be prepared by dissolving the polymer in an acid solution and cross - linking it with tripolyphosphate. Carboxymethyl cellulose (CMC) microparticles can be prepared by dissolving the polymer in an acid solution and precipitating the microparticles with lead ions. In the case of negatively charged polymers (e.g., alginic acid, CMC), positively charged ligands with different molecular weights (e.g., polylysine, polyethyleneimine) can be ionically linked.

[0408] v. Liposome and micelle formation

[0409] Liposomes generally have an aqueous core. The aqueous core can contain water or a mixture of water and alcohol. Suitable alcohols include, but are not limited to, methanol, ethanol, propanol (such as isopropanol), butanol (such as n-butanol, isobutene, sec-butanol, tert-butanol), pentane (such as pentanol, isobutyl methanol), hexanol (such as 1-hexanol, 2-hexanol, 3-hexanol), heptanol (such as 1-heptanol, 2-heptanol, 3-heptanol, and 4-heptanol), or octanol (such as 1-octanol) or combinations thereof.

[0410] Liposomes include, for example, small unilamellar vesicles (SUVs), large unilamellar vesicles (LANs) formed by a single lipid bilayer, and multilamellar vesicles (MLVs) formed by multiple membrane layers. The LAN is a vesicle with relatively large particles formed by a single lipid bilayer. Thus, liposomes can have one or several aqueous compartments defined by one (unilamellar) or several (multilamellar) phospholipid bilayers (Sapra et al., Curr. Drug Deliv., 2, 369-81 (2005)). Multilamellar liposomes have more lipid bilayers for hydrophobic therapeutic agents to associate with. Thus, a larger amount of therapeutic agent may be present within the liposome to reach the target cells.

[0411] Liposomes can have any particle size. For example, the average particle size can be from about 10 to about 2000 nm. In one embodiment of the present invention, the average particle size is about 10, 20, 25, 30, 40, 50, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1,000, 1,250, 1,500, 1,750, 2,000 nm (or any range between about 10 and about 2,000 nm) or greater. In one embodiment of the present invention, the average particle size is about 2,000, 1,750, 1,500, 1,250, 1,000, 900, 800, 700, 600, 500, 400, 300, 200, 100, 50, 40, 30, 25, 20, 10 nm (or any range between about 2,000 and 10 nm) or smaller. The average particle size can be about 20 to about 1,000 nm, about 100 to about 1,500 nm, about 100 to about 1,000 nm, about 100 to about 700 nm, about 200 to about 2,000 nm, about 1,000 to about 2,000 nm, or about 750 to about 1,500 nm. The particle size refers to the diameter of the particles measured by dynamic light scattering.

[0412] Liposome formulations can contain large liposomes that account for 1% to 100% of the liposome population in the formulation. In some embodiments, the large liposomes account for greater than about 50% of the liposome population in the formulation.

[0413] Methods for preparing liposomes are known in the art and can include, for example, drying lipids from organic solvents, dispersing lipids in an aqueous medium, purifying the resulting liposomes, and analyzing the final product. Some liposome preparation methods include, for example, extrusion methods, Mozafari methods, polyol dilution methods, bubble methods, and heating methods.

[0414] Micelles can be prepared in a conventional manner, such as by reverse evaporation, ether injection, surfactant-based techniques, etc. Polymer micelle formulations utilizing block copolymers having hydrophilic and hydrophobic segments have been disclosed, for example, in U.S. Application No. 2016 / 0114058, WO 2009 / 142326 A1, and WO 2010 / 013836 A1.

[0415] c. Methods for encapsulating or attaching molecules to the surface of particles

[0416] There are two main classes of molecules that can be encapsulated or attached to polymers directly or via coupling molecules: targeting molecules, attachment molecules, and therapeutic, nutritional, diagnostic, or prophylactic agents. These can be coupled using standard techniques. The targeting molecule or therapeutic molecule to be delivered can be directly coupled to the polymer or coupled to a material (such as a fatty acid incorporated into the polymer).

[0417] Functional groups refer to the conjugation of ligands to the particle surface via functional chemical groups (carboxylic acid, aldehyde, amine, thiol, and hydroxyl) present on the particle surface and on the ligand to be attached. Functional groups can be introduced into the particles in two ways.

[0418] The first way is during the preparation of the particles, for example, during the emulsion preparation of the particles by incorporating a stabilizer having a chemical functional group.

[0419] The second way is post-particle preparation, by directly crosslinking the particles and the ligand using homobifunctional or heterobifunctional crosslinkers. This second procedure can use suitable chemicals and a class of crosslinkers (such as CDI, EDAC, glutaraldehyde, etc., discussed in more detail below) or any other crosslinker that couples the ligand to the particle surface via chemical modification of the post-preparation particle surface. This second class also includes a method by which amphiphilic molecules (such as fatty acids, lipids, or functional stabilizers) can passively adsorb and adhere to the particle surface, thereby introducing functional end groups for tethering to ligands.

[0420] d. Methods for linking ApoL1 to a targeting moiety

[0421] As discussed above, in some embodiments, ApoL1 or a fragment, variant, or fusion protein thereof is linked to a targeting moiety, such as an antibody, and used to deliver ApoL1 without a nano-carrier delivery system. In addition to conjugating a targeting moiety to a bioactive molecule, the bioactive molecule can also be linked or associated with ApoL1 or a fragment, variant, or fusion protein thereof by any method known in the art. For example, ApoL1 and the targeting moiety can be co-expressed as a fusion protein in a host cell.

[0422] An antibody or an active fragment thereof can be chemically linked to a polypeptide by a peptide bond or by a chemical or peptide linker molecule of a type well known in the art. Methods for linking a drug or other small molecule drug to an antibody fragment are well known and can include the use of bifunctional chemical linkers such as N-succinimidyl (4-iodoacetyl)-aminobenzoate; sulfo-succinimidyl (4-iodoacetyl)-aminobenzoate; 4-succinimidyloxycarbonyl-.alpha.-methyl-.alpha.-(2-pyridyldithio)toluene; sulfo-succinimidyl-6-[.alpha.-methyl-.alpha.-(pyridyldithio)-toluamide]hexanoate; N-succinimidyl-3-(-2-pyridyldithio)-propionate; succinimidyl-6-[3(-(-2-pyridyldithio)-propionamido]hexanoate; sulfo-succinimidyl-6-[3(-(-2-pyridyldithio)-propionamido]hexanoate; 3-(2-pyridyldithio)-propionylhydrazide, Ellman's reagent, dichlorotriazine acid, S-(2-thiopyridyl)-L-cysteine, etc. Additional bifunctional linking molecules are discussed, for example, in U.S. Patent Nos. 5,349,066, 5,618,528, 4,569,789, 4,952,394, and 5,137,877.

[0423] The linker can be cleavable or non-cleavable. Highly stable linkers can reduce the amount of payload shed in circulation, thereby improving the safety profile and ensuring that more payload reaches the target cell. The linker can be based on chemical motifs, including disulfide, hydrazone, or peptide (cleavable) or thioether (non-cleavable), and control the distribution and delivery of the active agent to the target cell. Cleavable and non-cleavable types of linkers have been shown to be safe in preclinical and clinical trials (see, for example, Brentuximab vedotin, which includes an enzyme-sensitive linker cleavable by cathepsin; and Trastuzumab emtansine, which includes a stable, non-cleavable linker). In a specific embodiment, the linker is a peptide linker cleavable by Edman degradation ( et al., Molecular diversity, 17(3):605–11(2013)).

[0424] An uncleavable linker can retain the active agent within the cell or target microenvironment. As a result, the entire antibody, linker, and active agent enter the targeted cell, where the antibody is degraded to the amino acid level. The resulting complex between the amino acids of the antibody, the linker, and the active agent becomes the active drug. In contrast, a cleavable linker is catalyzed by an enzyme in the target cell or microenvironment in which the active agent is released. Once cleaved, the payload can escape the targeted cell and attack neighboring cells (also known as "bystander killing").

[0425] In some embodiments, there is one or more additional molecules between the active agent and the cleavage site. Other considerations include site-specific conjugation (TDC) (Axup, Proceedings of the National Academy of Sciences, 109(40):16101–6 (2012)) and conjugation techniques that can improve stability and the therapeutic index (such as those described in Lyon et al., Bioconjugate Chem., 32(10):1059–1062 (2014), and Kolodych et al., Bioconjugate Chem., 26(2):197–200 (2015)), as well as α-emitting immunoconjugates (Wulbrand et al., Multhoff, Gabriele editor, PLoS ONE. 8(5):e64730 (2013)).

[0426] III. Pharmaceutical Compositions

[0427] The composition can be formulated as a pharmaceutical composition with a suitable pharmaceutically acceptable carrier for administration to an individual in need thereof. The formulation can be administered enterally (e.g., orally) or parenterally (e.g., by injection or infusion).

[0428] The composition can be formulated for parenteral administration. As used herein, "parenteral administration" means administration by any method other than through the digestive tract or non-invasive topical or regional routes. For example, parenteral administration can include intravenous, intradermal, intraarterial, intraperitoneal, intralesional, intracranial, intraarticular, intraprostatic, intrathoracic, intratracheal, intravitreal, intratumoral, intramuscular, subcutaneous, subconjunctival, intracapsular, intrapericardial, intraumbilical, or transmucosal (nasal, vaginal, pulmonary, or rectal), e.g., by injection and infusion to a patient.

[0429] In some embodiments, the composition is administered systemically, e.g., by injection or infusion. In some embodiments, the composition is administered locally, e.g., by injection or infusion.

[0430] Parenteral formulations can be prepared as aqueous compositions using techniques known in the art. Generally, such compositions can be prepared as injectable formulations, such as solutions or suspensions; solid forms suitable for adding a reconstitution medium prior to injection to prepare a solution or suspension; emulsions, such as water-in-oil (w / o) emulsions, oil-in-water (o / w) emulsions and their microemulsions, liposomes or milk liposomes.

[0431] The carrier can be a solvent or a dispersion medium, which contains, for example, water, ethanol, one or more polyols (for example, glycerol, propylene glycol and liquid polyethylene glycol); oils, such as vegetable oils (for example, peanut oil, corn oil, sesame oil, etc.) and their combinations. Appropriate fluidity can be maintained, for example, by using coatings (such as lecithin), by maintaining the required nano-carrier size in the case of dispersions and / or by using surfactants. In many cases, it includes isotonic agents, such as sugars or sodium chloride.

[0432] Solutions and dispersions of the active compound in the form of the free acid or base or its pharmaceutically acceptable salts can be prepared in water or another solvent or dispersion medium appropriately mixed with one or more pharmaceutically acceptable excipients, which include but are not limited to surfactants, dispersants, emulsifiers, pH regulators, viscosity regulators and their combinations.

[0433] Suitable surfactants can be anionic, cationic, amphoteric or non-ionic surfactants. Suitable anionic surfactants include but are not limited to those anionic surfactants containing carboxylate, sulfonate and sulfate ions. Examples of anionic surfactants include long-chain alkyl sulfonates of sodium, potassium, ammonium and alkyl aryl sulfonates, such as sodium dodecylbenzenesulfonate; sodium dialkyl sulfosuccinate, such as sodium dodecylbenzenesulfonate; sodium dialkyl sulfosuccinate, such as bis-(2-ethylsulfonyl)-sulfosuccinate; and alkyl sulfates, such as sodium lauryl sulfate. Cationic surfactants include but are not limited to quaternary ammonium compounds, such as benzalkonium chloride, benzethonium chloride, cetrimide, stearyldimethylbenzylammonium chloride, polyoxyethylene and coconut amine. Examples of non-ionic surfactants include ethylene glycol monostearate, propylene glycol myristate, glycerol monostearate, glyceryl stearate, polyglyceryl-4 oleate, sorbitan acylates, sucrose acylates, PEG-150 laurate, PEG-400 monolaurate, polyoxyethylene monolaurate, polysorbate, polyoxyethylene octylphenyl ether, PEG-1000 cetyl ether, polyoxyethylene tridecyl ether, polypropylene glycol butyl ether, 401, stearoyl monoisopropanolamide and polyoxyethylene hydrogenated tallowamide. Examples of amphoteric surfactants include sodium N-dodecyl-β-alaninate, sodium N-lauryl-β-imino dipropionate, myristoyl amphoacetate, lauryl betaine and lauryl thio betaine.

[0434] The formulation may contain preservatives to prevent microbial growth. Suitable preservatives include, but are not limited to, parabens, chlorobutanol, phenol, sorbic acid, and thimerosal. The formulation may also contain antioxidants to prevent degradation of the active agent.

[0435] The formulation is typically buffered to a pH of 3 - 8 for parenteral administration after reconstitution. Suitable buffers include, but are not limited to, phosphate buffers, acetate buffers, and citrate buffers.

[0436] Water-soluble polymers are often used in formulations for parenteral administration. Suitable water-soluble polymers include, but are not limited to, polyvinylpyrrolidone, dextran, carboxymethylcellulose, and polyethylene glycol.

[0437] Sterile injectable solutions can be prepared by incorporating the required amount of the active compound with one or more of the excipients listed above (as needed) in a suitable solvent or dispersion medium and then filtering to sterilize. Generally, dispersions are prepared by incorporating the various sterile active ingredients into a sterile vehicle that contains a basic dispersion medium and the required other ingredients from those listed above. In the case of sterile powders for the preparation of sterile injectable solutions, exemplary preparation methods include vacuum drying and freeze-drying techniques that yield a powder of the active ingredient plus any additional desired ingredients from its previously sterile-filtered solution.

[0438] Enteral formulations are prepared using pharmaceutically acceptable carriers. As used herein, "carriers" include, but are not limited to, diluents, preservatives, binders, lubricants, disintegrants, swelling agents, fillers, stabilizers, and combinations thereof. Polymers used in dosage forms include hydrophobic or hydrophilic polymers and pH-dependent or pH-independent polymers. Hydrophobic and hydrophilic polymers include, but are not limited to, hydroxypropylmethylcellulose, hydroxypropylcellulose, hydroxyethylcellulose, carboxymethylcellulose, polyethylene glycol, ethylcellulose, microcrystalline cellulose, polyvinylpyrrolidone, polyvinyl alcohol, polyvinyl acetate, and ion exchange resins. Carriers also include all components of coating compositions, which may include plasticizers, pigments, colorants, stabilizers, and glidants. Formulations can be prepared using one or more pharmaceutically acceptable excipients, which include diluents, preservatives, binders, lubricants, disintegrants, swelling agents, fillers, stabilizers, and combinations thereof.

[0439] Controlled release formulations can be prepared as described in standard references such as “Pharmaceutical dosage form tablets”, edited by Liberman et al. (New York, Marcel Dekker, Inc., 1989), “Remington – The science and practice of pharmacy”, 20th edition, Lippincott Williams & Wilkins, Baltimore, MD, 2000, and “Pharmaceutical dosage forms and drug delivery systems”, 6th edition, Ansel et al. (Media, PA: Williams and Wilkins, 1995). These references provide information on excipients, materials, equipment, and processes for preparing sustained release dosage forms for tablets and capsules, and tablets, capsules, and granules. These references provide information on carriers, materials, equipment, and processes for preparing sustained release dosage forms for tablets and capsules, and tablets, capsules, and granules.

[0440] Stabilizers are used to inhibit or slow down drug decomposition reactions, such reactions including, for example, oxidation reactions. Suitable stabilizers include, but are not limited to: antioxidants, butylated hydroxytoluene (BHT); ascorbic acid, its salts and esters; vitamin E, tocopherol and its salts; sulfites such as sodium metabisulfite; cysteine and its derivatives; citric acid; propyl gallate and butylated hydroxyanisole (BHA).

[0441] In some embodiments, the composition is formulated for mucosal administration, such as by nasal, pulmonary, or oral delivery.

[0442] Mucosal formulations may include one or more agents for enhancing delivery across the nasal mucosa. Agents for enhancing mucosal delivery are known in the art, see for example U.S. Patent Application No. 20090252672 to Eddington and U.S. Patent Application No. 20090047234 to Touitou. Acceptable agents include, but are not limited to, calcium chelators (EDTA), nasal enzyme inhibitors (boron leucine, aprotinin), mucociliary clearance inhibitors (preservatives), nasal solubilizers (cyclodextrins, fatty acids, surfactants), and micelle formers (surfactants such as bile acids, laureth-9, and tauroursodeoxycholate (STDHF)). The composition may include one or more absorption enhancers, including surfactants, fatty acids, and chitosan derivatives, which can enhance delivery by modulating tight junctions (TJ) (B.J. Aungst et al., J. Pharm. Sci. 89(4):429 - 442 (2000)). Generally, the optimal absorption enhancer should possess the following qualities: its effect should be reversible, it should provide a rapid permeability enhancing effect on the cell membrane of the mucosa, and it should be non-cytotoxic at effective concentration levels and have no harmful and / or irreversible effects on the cell membrane or cytoskeleton of the TJ.

[0443] Provided are pharmaceutical packages and kits that include one or more containers filled with an antibody or fusion protein. Additionally, one or more other prophylactic or therapeutic agents useful for treating a disease may also be included in the pharmaceutical package or kit. One embodiment provides a pharmaceutical package or kit that includes one or more containers filled with one or more of the ingredients of a pharmaceutical composition. Optionally, associated with such containers may be a notice in a form required by a government agency regulating the manufacture, use, or sale of a pharmaceutical or biological product, which notice reflects the approval obtained by the manufacturing, using, or selling entity for human administration.

[0444] Also provided are kits useful for the following methods. In one embodiment, the kit includes one or more antibodies or fusion proteins. In another embodiment, the kit further includes one or more other prophylactic or therapeutic agents useful for treating cancer in one or more containers. In certain embodiments, the other prophylactic or therapeutic agents are chemotherapeutic agents. In other embodiments, the prophylactic or therapeutic agent is a biological or hormonal therapeutic agent.

[0445] IV. Methods of Use

[0446] A. Methods of Treatment

[0447] The disclosed compositions can be used to increase the delivery and internalization of ApoL1 to target cells. As shown in the examples below, increasing the internalization of ApoL1 in mammalian cells (e.g., by increasing the internalization of recombinant exogenous ApoL1 or endogenous ApoL1-containing complexes such as TLF) can lead to cell death, including cancer cell death. Thus, the disclosed compositions can be used to increase the level of ApoL1 in target cells and induce their death.

[0448] In a preferred embodiment, the composition is an ApoL1-containing complex-binding compound, a bispecific or multispecific antibody that specifically binds both an ApoL1-containing complex and a target cell marker. See, for example Figure 12 .

[0449] All cell types are contemplated for targeting, either alone or in combination, including but not limited to stem cells, skin cells, blood cells, immune cells, muscle cells, nerve cells, cancer cells, virus-infected cells, bacterial cells, fungal cells, organ-specific cells, and other eukaryotic cells. The cell marker can be specific for endothelial cells, ectodermal cells, or mesenchymal cells. Thus, the cells can be mammalian or non-mammalian cells. Most preferably, the cells are located in a mammal. The mammalian cells can be human cells. Thus, targeting of mammalian (e.g., human) and non-mammalian cells in a subject (e.g., a human) is contemplated. Thus, the target cells can be bacterial or fungal cells in a mammalian subject such as a human. Additionally or alternatively, the target cells can be non-mammalian cells infected by another organism such as a virus or bacteria. For example, in some embodiments, intracellular organisms, where the infected cells can be targeted by the presence of extracellular markers. The organisms can be bacteria or eukaryotes such as Plasmodium falciparum, Toxoplasma gondii, Leishmania spp., Trypanosoma cruzi, Listeria monocytogenes, Chlamydia trachomatis, Rickettsia burnetii, Mycobacterium tuberculosis, and other intracellular bacteria and eukaryotes. The extracellular eukaryotes or the extracellular stages of intracellular organisms that can be specifically targeted to cells include, for example, Toxoplasma gondii, Trichomonas vaginalis, Plasmodium falciparum.

[0450] In a preferred embodiment, cancer cells (including both blood cancer cells and solid tumor cells) are preferred target cells. The binding activity of the compound can be selected based on the target cells.

[0451] Generally, the target cells are not Trypanosoma.

[0452] 1. Method of treatment

[0453] Thus, in some embodiments, an effective amount of the disclosed composition (such as recombinant ApoL1 and a targeting moiety) or a bispecific or multispecific antibody that specifically binds Hpr or ApoL1 and a target cell antigen is administered to a subject.

[0454] As used herein, the term "effective amount" or "therapeutically effective amount" refers to a dose sufficient to treat, inhibit, or alleviate one or more symptoms of the disorder being treated or otherwise provide the desired pharmacological and / or physiological effect. The precise dose will vary depending on the selected active agent and various factors such as subject-dependent variables (e.g., age, immune system health, etc.), the disease, and the ongoing treatment. Generally, such amount is effective to induce or increase the cell death of ApoL1-mediated target cells. In some embodiments, the subject has a disease or disorder caused by the target cells, and the cell death is induced in an effective amount to treat the disease or disorder. For example, as discussed in more detail below, in some embodiments, the subject has cancer, the target cells are cancer cells, and the treatment increases the cell death of the cancer cells.

[0455] Any diseased tissue having a unique biomarker or a biomarker that is overexpressed compared to normal cells can serve as a target cell, and any such disease that would benefit from an increase in the cell death of the diseased tissue can be treated.

[0456] For example, when the target cells are blood cancer cells, bispecific antibodies that specifically bind to Hpr or ApoL1 and blood cancer antigens such as BCMA, CD38, CD319 / SLAMF-7, TNFRSF17 / BCMA, SYND1 / CD138, CD229, CD47, CD123 / IL3-RA, CD19, CD20, CD22, FcRH5, GPRC5D, CLL-1, PD-L1, or CTLA4 can be used.

[0457] Similarly, in some embodiments, the targeting moiety of the recombinant ApoL1 composition targets BCMA, CD38, CD319 / SLAMF-7, TNFRSF17 / BCMA, SYND1 / CD138, CD229, CD47, CD123 / IL3-RA, CD19, CD20, CD22, FcRH5, GPRC5D, CLL-1, PD-L1, or CTLA4.

[0458] Similarly, bispecific molecules and other multispecific molecules can target solid tumors or other target cells. Exemplary other antigens (including solid tumor antigens) for bispecific or other multispecific molecules are provided elsewhere herein.

[0459] For example, if the solid tumor is pancreatic cancer, bispecific antibodies that specifically bind Hpr or ApoL1 and pancreatic cancer antigens such as Claudin 18.2, MUC1, Mesothelin (MSLN), and Myoferrin (MYOF) can be used. Similarly, in some embodiments, the targeting moiety of the recombinant ApoL1 composition targets Claudin 18.2, MUC1, Mesothelin (MSLN), and Myoferrin (MYOF).

[0460] For example, if the solid tumor is melanoma, bispecific antibodies that specifically bind Hpr or ApoL1 and melanoma cancer antigens such as PMEL17 can be used. Similarly, in some embodiments, the targeting moiety of the recombinant ApoL1 composition targets PMEL17.

[0461] These are non-limiting examples because many other targets are provided herein and in the art, and as provided herein, the disclosed compositions and methods can be readily modified to target these antigens.

[0462] In some embodiments, the composition is administered to a subject in need thereof once every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or 31 days. In some embodiments, the composition is administered to the subject once, twice, or three times per week. In some embodiments, the composition is administered to the subject every other day. In some embodiments, the composition is administered to the subject once, twice, or three times per month. In some embodiments, the composition is administered for 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or more weeks or months.

[0463] In a specific embodiment, for antibodies and other proteins, the dose administered to a patient is typically from 0.0001 mg / kg to 100 mg / kg of patient body weight. Preferably, the dose administered to the patient is between 0.0001 mg / kg and 20 mg / kg, between 0.0001 mg / kg and 10 mg / kg, between 0.0001 mg / kg and 5 mg / kg, between 0.0001 mg / kg and 2 mg / kg, between 0.0001 mg / kg and 1 mg / kg, between 0.0001 mg / kg and 0.75 mg / kg, between 0.0001 mg / kg and 0.5 mg / kg, from 0.0001 mg / kg to 0.25 mg / kg, from 0.0001 mg / kg to 0.15 mg / kg, from 0.0001 mg / kg to 0.10 mg / kg, from 0.001 mg / kg to 0.5 mg / kg, from 0.01 mg / kg to 0.25 mg / kg, or from 0.01 mg / kg to 0.10 mg / kg of patient body weight. Generally, due to the immune response to foreign polypeptides, the half-life of human antibodies in the human body is longer than that of antibodies from other species. Therefore, lower doses of human antibodies and lower frequencies of administration are usually possible. In addition, the dose and frequency of administration of an antibody or its fragment or fusion protein can be reduced by enhancing the uptake and tissue penetration of the antibody or fusion protein through modification (such as, for example, lipidation).

[0464] 2. Disease to be treated

[0465] a. Cancer

[0466] The disclosed compositions and methods can be used to treat cancer in a subject in need thereof. In mature animals, a balance is generally maintained between cell renewal and cell death in most organs and tissues. Cells of various types in the body have a given lifespan; when these cells die, new cells are generated through the proliferation and differentiation of various types of stem cells. Under normal circumstances, the production of new cells is so regulated that the number of any particular type of cell remains constant. However, occasionally, cells that no longer respond to normal growth control mechanisms appear. The cell clones produced by these cells can expand to a considerable size, thereby producing a tumor or neoplasm. A tumor that cannot grow indefinitely and does not extensively invade the surrounding healthy tissue is benign. A tumor that continues to grow and gradually becomes invasive is malignant. The term cancer specifically refers to a malignant tumor. In addition to uncontrolled growth, malignant tumors also exhibit metastasis. In this process, small clusters of cancer cells break off from the tumor, invade blood vessels or lymphatic vessels, and are carried to other tissues, where they continue to proliferate. In this way, a primary tumor at one site may give rise to secondary tumors at another site.

[0467] The compositions and methods described herein can be used to treat a subject having a benign or malignant tumor by delaying or inhibiting the growth of the tumor in the subject, reducing the growth or size of the tumor, inhibiting or reducing the metastasis of the tumor, and / or inhibiting or reducing symptoms associated with tumor development or growth.

[0468] Malignant tumors that can be treated are classified herein according to the embryonic origin of the tissue from which the tumor derives. Leukemias and lymphomas are malignant tumors of hematopoietic cells in the bone marrow. Leukemias proliferate in single cell form, while lymphomas tend to grow as tumor masses. Malignant tumors can occur in many organs or tissues of the body, giving rise to cancers. Carcinomas are tumors that derive from endodermal or ectodermal tissues, such as the epithelial layers of the skin or internal organs and glands. Sarcomas, which occur less frequently, derive from mesodermal connective tissues, such as bone, fat, and cartilage. Malignant tumors can occur in many organs or tissues of the body, giving rise to cancers.

[0469] In a preferred embodiment, the composition is used to treat liquid tumors or blood cancers or tumors of the vasculature, such as multiple myeloma, leukemias (e.g., chronic lymphocytic leukemia, acute myeloid leukemia, acute lymphoblastic leukemia), non-Hodgkin lymphoma, Hodgkin lymphoma, myelodysplastic syndromes (MDS), myeloproliferative neoplasms (MPN) (or its subcategories, e.g., essential thrombocythemia (ET), myelofibrosis (MF), and polycythemia vera (PV)), amyloidosis, Waldenström macroglobulinemia, or aplastic anemia.

[0470] Additional types of cancers that can be treated with the provided compositions and methods include, but are not limited to, adenocarcinomas and sarcomas of bone, bladder, brain, breast, cervix, colon, esophagus, kidney, liver, lung, nasopharynx, pancreas, prostate, skin, stomach, and uterus. In some embodiments, the disclosed compositions are used to treat multiple cancer types simultaneously. The compositions can also be used to treat metastases or tumors at multiple locations.

[0471] The disclosed compositions can be used to treat cells that undergo unregulated growth, invasion, or metastasis.

[0472] A representative but non-limiting list of cancers that can be treated using the disclosed compositions includes cancers of the blood and lymphatic systems (including leukemia, Hodgkin lymphoma, non-Hodgkin lymphoma, solitary plasmacytoma, multiple myeloma), cancers of the urogenital system (including prostate cancer, bladder cancer, kidney cancer, urethral cancer, penile cancer, testicular cancer), cancers of the nervous system (including meningioma, glioma, glioblastoma, ependymoma), head and neck cancers (including squamous cell carcinomas of the oral cavity, nasal cavity, nasopharynx, oropharynx, larynx, and paranasal sinuses), lung cancers (including small cell and non-small cell lung cancer), gynecological cancers (including cervical cancer, endometrial cancer, vaginal cancer, vulvar cancer, ovarian cancer, and fallopian tube cancer), gastrointestinal cancers (including gastric cancer, small intestine cancer, colorectal cancer, liver cancer, hepatobiliary cancer, and pancreatic cancer), skin cancers (including melanoma, squamous cell carcinoma, and basal cell carcinoma), breast cancers (including ductal and lobular carcinomas and triple-negative breast cancer), and pediatric cancers (including neuroblastoma, Ewing sarcoma, Wilms tumor, medulloblastoma).

[0473] In some embodiments, the tumor is a solid tumor characterized by increased vascular permeability, and optionally, the enhanced permeability and retention (EPR) effect, relative to less vascularized tumors, increases the localization of the ApoL1-containing complex-antibody complex to the tumor site.

[0474] b. Other diseases

[0475] Disruptions in the cell death pathway at the molecular level are associated not only with the pathogenesis of cancer but also with other diseases of great social importance, such as infections, such as viral infections (e.g., HIV), bacterial infections, fungal infections, non-mammalian eukaryotic cell infections, etc., atherosclerosis, ischemia, reperfusion injury, infections, inflammation, autoimmunity, and neurological disorders (Kaminskyy and Zhivotovsky, Cell Death & Disease, Volume 9, Article number: 110 (2018)). Thus, the disclosed compositions and methods can be used to treat such diseases.

[0476] For example, neutrophils are involved in various types of tissue inflammation and diseases, and targeting them for cell death according to the disclosed compositions and methods can be used to treat autoimmune and inflammatory diseases.

[0477] In some embodiments, the compositions and methods can be used to treat infections. For example, in some embodiments, foreign cells, such as bacteria or fungi, are specifically targeted directly for cell death. In other embodiments, the infection is treated by targeting infected mammalian (e.g., host) cells (e.g., by targeting extracellular markers on the infected mammalian cells). Exemplary foreign and infected target cells were discussed above.

[0478] For example, one strategy for eliminating latent HIV-1 viral reservoirs is the shock and kill approach, which involves using latency reversing agents (LRAs) to reactivate viral gene expression (shock), followed by elimination of cells carrying the reactivated provirus (kill) (Rao et al., Nat Commun. 12(1):2475 (2021) doi:10.1038 / s41467-021-22608-z). Thus, in some embodiments, according to the disclosed compositions and methods, HIV-infected cells (such as CD4+ T cells) are targeted for cell death and thus treat HIV.

[0479] 3. Combination Therapy

[0480] The disclosed compositions can be used in combination with one or more additional active agents, which can be administered in the same or different mixtures. Exemplary additional active agents include standard chemotherapy, radiotherapy, and other anti-cancer treatments.

[0481] In some embodiments, the additional active agent is a therapeutic drug. Most chemotherapy drugs can be classified into alkylating agents, antimetabolites, anthracyclines, plant alkaloids, topoisomerase inhibitors, monoclonal antibodies, and other anti-tumor agents.

[0482] Non-limiting examples of anti-tumor drugs that damage DNA or inhibit DNA repair include carboplatin, carmustine, chlorambucil, cisplatin, cyclophosphamide, dacarbazine, daunorubicin, doxorubicin, epirubicin, idarubicin, ifosfamide, lomustine, mechlorethamine, mitoxantrone, oxaliplatin, procarbazine, temozolomide, and valrubicin.

[0483] In some embodiments, the anti-tumor drug is a histone deacetylase inhibitor, which inhibits DNA repair at the transcriptional level and disrupts chromatin structure. In some embodiments, the anti-tumor drug is a proteasome inhibitor, which inhibits DNA repair by disrupting ubiquitin metabolism in cells. Ubiquitin is a signaling molecule that regulates DNA repair. In some embodiments, the anti-tumor drug is a kinase inhibitor, which inhibits DNA repair by altering the DNA damage response signaling pathway.

[0484] Additional anti-tumor agents include, but are not limited to, alkylating agents (such as cisplatin, carboplatin, oxaliplatin, mechlorethamine, cyclophosphamide, chlorambucil, dacarbazine, lomustine, carmustine, procarbazine, chlorambucil and ifosfamide), antimetabolites (such as fluorouracil, gemcitabine, methotrexate, cytarabine, fludarabine and floxuridine), some antimitotic agents and vinca alkaloids (such as vincristine, vinblastine, vinorelbine and vindesine), anthracycline antibiotics (including doxorubicin, daunorubicin, valrubicin, idarubicin and epirubicin, and actinomycins, such as actinomycin D), cytotoxic antibiotics (including mitomycin, plicamycin and bleomycin) and topoisomerase inhibitors (including camptothecin, such as irinotecan and topotecan and epipodophyllotoxin derivatives, such as amsacrine, etoposide, etoposide phosphate and teniposide) and cytoskeleton targeting drugs, such as paclitaxel.

[0485] In some embodiments, the active agent is a radiosensitizer. Examples of known radiosensitizers include cisplatin, gemcitabine, 5-fluorouracil, pentoxifylline, vinorelbine, PARP inhibitors, histone deacetylase inhibitors and proteasome inhibitors.

[0486] In some embodiments, the additional active agent is radiation. Radiation therapy (also known as radiotherapy) is the medical use of ionizing radiation as part of cancer treatment to control malignant cells.

[0487] B. Detection methods

[0488] The disclosed ApoL1-containing complex-binding antibodies (e.g., anti-Hpr and anti-ApoL1 antibodies) and antigen-binding fragments thereof can be used, for example, to detect ApoL1-containing complexes (such as TLF) and their components (such as Hpr and ApoL1). Accordingly, the present disclosure provides for the use of one or more antibodies (or fragments thereof) that immunospecifically bind such antigens to determine the presence of ApoL1-containing complexes (such as TLF) or their components (such as Hpr and ApoL1) in a cell or tissue or other biological sample of a subject. Such antibodies and fragments are preferably used in immunoassays, such as Western blotting, enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), fluorescence-activated cell sorting (FACS), immunohistochemistry (IHC), etc.

[0489] In some embodiments, the detection method comprises: a) administering (e.g., parenterally, subcutaneously, or intraperitoneally) to a subject an effective amount of a labeled antibody or antigen-binding fragment that immunospecifically binds to a complex containing ApoL1, such as TLF; b) waiting a time interval after administration to allow the labeled molecule to preferentially localize at the site in the subject where the complex containing ApoL1, such as TLF, is located (and to clear unbound labeled molecules to background levels); c) determining the background level; and d) detecting the labeled antibody in the subject, such that detection of the labeled antibody above the background level indicates the level and / or location of the complex containing ApoL1, such as TLF, in the subject. According to this embodiment, the antibody is labeled with an imaging moiety that can be detected using imaging systems known to those of skill in the art. The background level can be determined by a variety of methods, including comparing the amount of labeled molecules detected to a standard value previously determined for a particular system.

[0490] It will be appreciated in the art that the size of the subject and the imaging system used will determine the number of imaging moieties required to produce a diagnostic image. In vivo tumor imaging is described in S.W. Burchiel et al., “Immunopharmacokinetics of Radiolabeled Antibodies and Their Fragments,” (Chapter 13, Tumor Imaging: The Radiochemical Detection of Cancer, S.W. Burchiel and B.A. Rhodes, eds., Masson Publishing Inc. (1982).

[0491] Depending on several variables, including the type of label used and the mode of administration, the time interval after administration to allow the labeled molecule to preferentially localize at the site in the subject and to clear unbound labeled molecules to background levels is 6 to 48 hours, or 6 to 24 hours, or 6 to 12 hours. In another embodiment, the time interval after administration is 5 to 20 days or 5 to 10 days.

[0492] In vivo scanning methods known in the art can be used to detect the presence of labeled molecules in a subject. These methods depend on the type of label used. A person skilled in the art will be able to determine the appropriate method for detecting a specific label. Methods and devices that can be used in the disclosed diagnostic methods include, but are not limited to, computed tomography (CT), whole body scanning (such as positron emission tomography (PET)), magnetic resonance imaging (MRI), and ultrasound examination. In a specific embodiment, the molecule is labeled with a radioisotope and detected in a patient using a radiation-responsive surgical instrument (Thurston et al., U.S. Application No. 5,441,050). In another embodiment, the molecule is labeled with a fluorescent compound and detected in a patient using a fluorescence-responsive scanning instrument. In another embodiment, the molecule is labeled with a positron-emitting metal and detected in a patient using positron emission tomography. In yet another embodiment, the molecule is labeled with a paramagnetic label and detected in a patient using magnetic resonance imaging (MRI).

[0493] The disclosed invention can be further understood by the following numbered paragraphs:

[0494] 1. A method of increasing cell death of target cells in a mammalian subject in need thereof, the method comprising administering to the subject an effective amount of a composition that increases apolipoprotein L1 (ApoL1) in the target cells.

[0495] 2. The method according to paragraph 1, wherein the composition increases the accumulation of endogenous ApoL1 in the target cells.

[0496] 3. The method according to paragraph 2, wherein the endogenous ApoL1 is a component of an ApoL1-containing complex.

[0497] 4. The method according to paragraph 3, wherein the ApoL1-containing complex is trypanosome lytic factor (TLF), optionally TLF-1 and / or TLF-2.

[0498] 5. The method according to any one of paragraphs 1 to 4, wherein the composition comprises a first antigen-binding fragment that binds to ApoL1 or an ApoL1-containing complex and a targeting moiety that targets the composition to the target cells, optionally wherein the composition is a bispecific or multispecific antibody that comprises a first antigen-binding fragment that binds to ApoL1 or an ApoL1-containing complex, optionally TLF, and a second antigen-binding fragment that binds to a cell-specific antigen.

[0499] 6. The method according to paragraph 1, wherein the composition comprises ApoL1 or a functional fragment or variant thereof and a targeting moiety that targets a cell-specific antigen.

[0500] 7. The method according to paragraph 6, wherein the composition comprises the ApoL1 or a functional fragment or variant thereof directly or indirectly conjugated or fused to the targeting moiety.

[0501] 8. The method according to paragraph 6 or 7, wherein the composition comprises a delivery vehicle, optionally a liposome or a polymeric nanoparticle.

[0502] 9. The method according to paragraph 8, wherein the targeting moiety is conjugated or fused to the delivery vehicle.

[0503] 10. The method according to any one of paragraphs 6 to 9, wherein the targeting moiety is an antibody or an antigen-binding fragment.

[0504] 11. The method according to any one of paragraphs 1 to 10, wherein the cell-specific antigen is specific for diseased cells.

[0505] 12. The method according to paragraph 11, wherein the diseased cells are cancer cells.

[0506] 13. The method according to paragraph 12, wherein the cancer cells are blood cancer cells.

[0507] 14. The method according to any one of paragraphs 1 to 13, wherein the subject has a disease caused by the target cells.

[0508] 15. The method according to paragraph 14, wherein the composition is administered in an effective amount to treat the disease.

[0509] 16. The method according to any one of paragraphs 1 to 15, wherein the cell-specific antigen is not a trypanosome-specific surface antigen.

[0510] 17. The method according to any one of paragraphs 1 to 16, wherein the subject does not have trypanosomiasis.

[0511] 18. A composition comprising ApoL1 or a functional fragment or variant thereof and a targeting moiety, wherein the targeting moiety does not target a trypanosome-specific surface antigen.

[0512] 19. The composition according to paragraph 18, wherein the ApoL1 or a functional fragment or variant thereof is directly or indirectly conjugated or fused to the targeting moiety.

[0513] 20. The composition according to paragraph 19, wherein the composition comprises a delivery vehicle, optionally a liposome or a polymeric nanoparticle, and optionally, wherein the targeting moiety is conjugated or fused to the delivery vehicle.

[0514] 21. An antibody or an antigen-binding fragment comprising:

[0515] The three complementarity-determining regions (CDRs) of the heavy-chain variable domain of SEQ ID NO:24, or variants or humanized forms thereof having at least 70%, 80%, 90% or 95% sequence identity thereto, and the three complementarity-determining regions (CDRs) of the light-chain variable domain of SEQ ID NO:36 or SEQ ID NO:77, or variants or humanized forms thereof having at least 70%, 80%, 90% or 95% sequence identity thereto,

[0516] wherein the antibody or antigen-binding fragment binds to apolipoprotein L1 (ApoL1).

[0517] 22. The antibody or antigen-binding fragment according to paragraph 21, wherein the heavy-chain and light-chain variable domain CDRs comprise:

[0518] TYAMS (SEQ ID NO:25), EISNGGLYTYYPDTVTG (SEQ ID NO:26), ENRNWYFDL (SEQ ID NO:27), RSSQSIVNSNGNTYLE (SEQ ID NO:37) and KVSNRFS (SEQ ID NO:38), FQGSHVPLT (SEQ ID NO:39) or variants or humanized forms thereof having at least 70%, 80%, 90% or 95% sequence identity thereto;

[0519] GFTFSTYA (SEQ ID NO:28), ISNGGLYT (SEQ ID NO:29), IRENRNWYFDL (SEQ ID NO:30), QSIVNSNGNTY (SEQ ID NO:40), KVS and FQGSHVPLT (SEQ ID NO:39) or variants or humanized forms thereof having at least 70%, 80%, 90% or 95% sequence identity thereto; or

[0520] GFTFSTY (SEQ ID NO:31), SNGGLY (SEQ ID NO:32), ENRNWYFDL (SEQ ID NO:27), RSSQSIVNSNGNTYLE (SEQ ID NO:37), KVSNRFS (SEQ ID NO:38) and FQGSHVPLT (SEQ ID NO:39) or variants or humanized forms thereof having at least 70%, 80%, 90% or 95% sequence identity thereto.

[0521] 23. The antibody or antigen-binding fragment according to paragraph 21 or 22, wherein the heavy-chain and light-chain variable domain CDRs comprise:

[0522] TYAMS (SEQ ID NO:25), EISNGGLYTYYPDTVTG (SEQ ID NO:26), ENRNWYFDL (SEQ ID NO:27), RSSQSIVNSNGNTYLE (SEQ ID NO:37) and KVSNRFS (SEQ ID NO:38), FQGSHVPLT (SEQ ID NO:39);

[0523] GFTFSTYA (SEQ ID NO:28), ISNGGLYT (SEQ ID NO:29), IRENRNWYFDL (SEQ ID NO:30), QSIVNSNGNTY (SEQ ID NO:40), KVS and FQGSHVPLT (SEQ ID NO:39); or

[0524] GFTFSTY (SEQ ID NO:31), SNGGLY (SEQ ID NO:32), ENRNWYFDL (SEQ ID NO:27), RSSQSIVNSNGNTYLE (SEQ ID NO:37), KVSNRFS (SEQ ID NO:38) and FQGSHVPLT (SEQ ID NO:39).

[0525] 24. An antibody or antigen-binding fragment according to any one of paragraphs 21 to 23, which comprises a heavy chain variable domain and a light chain variable domain, wherein the heavy chain variable domain comprises the amino acid sequence of SEQ ID NO:24 or a variant or humanized form thereof having at least 70%, 80%, 90% or 95% sequence identity thereto, and the light chain variable domain comprises the amino acid sequence of SEQ ID NO:36 or SEQ ID NO:77 or a variant or humanized form thereof having at least 70%, 80%, 90% or 95% sequence identity thereto.

[0526] 25. An antibody or its antigen-binding fragment according to any one of paragraphs 21 to 24, which comprises a heavy chain variable domain and a light chain variable domain, wherein the heavy chain variable domain comprises the amino acid sequence of SEQ ID NO:24, and the light chain variable domain comprises the amino acid sequence of SEQ ID NO:36 or SEQ ID NO:77.

[0527] 26. An antibody or antigen-binding fragment, which comprises:

[0528] The three complementarity-determining regions (CDRs) of the heavy chain variable domain of SEQ ID NO:3, or variants or humanized forms thereof having at least 70%, 80%, 90% or 95% sequence identity thereto, and the three complementarity-determining regions (CDRs) of the light chain variable domain of SEQ ID NO:14, or variants or humanized forms thereof having at least 70%, 80%, 90% or 95% sequence identity thereto,

[0529] wherein the antibody or antigen-binding fragment binds to haptoglobin-related protein (Hpr).

[0530] 27. The antibody or antigen-binding fragment according to paragraph 26, wherein the heavy and light chain variable domain CDRs comprise:

[0531] NYGMN (SEQ ID NO:4), WINSYTGEATYTDDLKG (SEQ ID NO:5), EGYGDYGYSFDY (SEQ ID NO:6), RATKNIYTYLA (SEQ ID NO:16), NAKTLAE (SEQ ID NO:17) and QHHYGTPRT (SEQ ID NO:18) or variants or humanized forms thereof having at least 70%, 80%, 90% or 95% sequence identity thereto;

[0532] GYIFTNYG (SEQ ID NO:7), INSYTGEA (SEQ ID NO:8), AREGYGDYGYSFDY (SEQ ID NO:9), KNIYTY (SEQ ID NO:19), NAK and QHHYGTPRT (SEQ ID NO:18) or variants or humanized forms thereof having at least 70%, 80%, 90% or 95% sequence identity thereto; or

[0533] GYIFTNY (SEQ ID NO:10), NSYTGE (SEQ ID NO:11), EGYGDYGYSFDY (SEQ ID NO:6), RATKNIYTYLA (SEQ ID NO:16), NAKTLAE (SEQ ID NO:17) and QHHYGTPRT (SEQ ID NO:18) or variants or humanized forms thereof having at least 70%, 80%, 90% or 95% sequence identity thereto.

[0534] 28. The antibody or antigen-binding fragment according to paragraph 26 or 27, wherein the heavy and light chain variable domain CDRs comprise:

[0535] NYGMN (SEQ ID NO:4), WINSYTGEATYTDDLKG (SEQ ID NO:5), EGYGDYGYSFDY (SEQ ID NO:6), RATKNIYTYLA (SEQ ID NO:16), NAKTLAE (SEQ ID NO:17), and QHHYGTPRT (SEQ ID NO:18);

[0536] GYIFTNYG (SEQ ID NO:7), INSYTGEA (SEQ ID NO:8), AREGYGDYGYSFDY (SEQ ID NO:9), KNIYTY (SEQ ID NO:19), NAK, and QHHYGTPRT (SEQ ID NO:18); or

[0537] GYIFTNY (SEQ ID NO:10), NSYTGE (SEQ ID NO:11), EGYGDYGYSFDY (SEQ ID NO:6), RATKNIYTYLA (SEQ ID NO:16), NAKTLAE (SEQ ID NO:17), and QHHYGTPRT (SEQ ID NO:18).

[0538] 29. The antibody or antigen-binding fragment according to any one of paragraphs 26 to 28, which comprises a heavy chain variable domain and a light chain variable domain, wherein the heavy chain variable domain comprises the amino acid sequence of SEQ ID NO:3 or a variant or humanized form thereof having at least 70%, 80%, 90% or 95% sequence identity thereto, and the light chain variable domain comprises the amino acid sequence of SEQ ID NO:14 or a variant or humanized form thereof having at least 70%, 80%, 90% or 95% sequence identity thereto.

[0539] 30. The antibody or its antigen-binding fragment according to any one of paragraphs 26 to 29, which comprises a heavy chain variable domain and a light chain variable domain, wherein the heavy chain variable domain comprises the amino acid sequence of SEQ ID NO:3 and the light chain variable domain comprises the amino acid sequence of SEQ ID NO:14.

[0540] 31. An antibody or antigen-binding fragment, which comprises:

[0541] The three complementarity-determining regions (CDRs) of the heavy chain variable domain of SEQ ID NO:56, or a variant or humanized form thereof having at least 70%, 80%, 90% or 95% sequence identity thereto, and the three complementarity-determining regions (CDRs) of the light chain variable domain of SEQ ID NO:65, or a variant or humanized form thereof having at least 70%, 80%, 90% or 95% sequence identity thereto,

[0542] wherein the antibody or antigen-binding fragment binds to haptoglobin-related protein (Hpr).

[0543] 32. The antibody or antigen-binding fragment according to paragraph 31, wherein the heavy and light chain variable domain CDRs comprise:

[0544] DYSIH (SEQ ID NO:57), WKHTESGESTYADDFKG (SEQ ID NO:58), GANYGSLLDY (SEQ ID NO:59), RASKSVSTSGYSYMH (SEQ ID NO:66), LASNLES (SEQ ID NO:67), QHNRELPLT (SEQ ID NO:68) or a variant or humanized form thereof having at least 70%, 80%, 90% or 95% sequence identity thereto;

[0545] GFTFTDYS (SEQ ID NO:60), KHTESGES (SEQ ID NO:61), ARGANYGSLLDY (SEQ ID NO:62), KSVSTSGYSY (SEQ ID NO:69), LAS, QHNRELPLT (SEQ ID NO:68) or a variant or humanized form thereof having at least 70%, 80%, 90% or 95% sequence identity thereto; or

[0546] GFTFTDY (SEQ ID NO:63), HTESGE (SEQ ID NO:64), GANYGSLLDY (SEQ ID NO:59), RASKSVSTSGYSYMH (SEQ ID NO:66), LASNLES (SEQ ID NO:67), QHNRELPLT (SEQ ID NO:68) or a variant or humanized form thereof having at least 70%, 80%, 90% or 95% sequence identity thereto.

[0547] 33. The antibody or antigen-binding fragment according to paragraph 31 or 32, wherein the heavy and light chain variable domain CDRs comprise:

[0548] DYSIH (SEQ ID NO:57), WKHTESGESTYADDFKG (SEQ ID NO:58), GANYGSLLDY (SEQ ID NO:59), RASKSVSTSGYSYMH (SEQ ID NO:66), LASNLES (SEQ ID NO:67), QHNRELPLT (SEQ ID NO:68);

[0549] GFTFTDYS (SEQ ID NO:60), KHTESGES (SEQ ID NO:61), ARGANYGSLLDY (SEQ ID NO:62), KSVSTSGYSY (SEQ ID NO:69), LAS, QHNRELPLT (SEQ ID NO:68); or

[0550] GFTFTDY (SEQ ID NO:63), HTESGE (SEQ ID NO:64), GANYGSLLDY (SEQ ID NO:59), RASKSVSTSGYSYMH (SEQ ID NO:66), LASNLES (SEQ ID NO:67), QHNRELPLT (SEQ ID NO:68).

[0551] 34. The antibody or antigen-binding fragment according to any one of paragraphs 31 to 33, which comprises a heavy chain variable domain and a light chain variable domain, wherein the heavy chain variable domain comprises the amino acid sequence of SEQ ID NO:56 or a variant or humanized form thereof having at least 70%, 80%, 90% or 95% sequence identity thereto, and the light chain variable domain comprises the amino acid sequence of SEQ ID NO:65 or a variant or humanized form thereof having at least 70%, 80%, 90% or 95% sequence identity thereto.

[0552] 35. The antibody or its antigen-binding fragment according to any one of paragraphs 31 to 34, which comprises a heavy chain variable domain and a light chain variable domain, wherein the heavy chain variable domain comprises the amino acid sequence of SEQ ID NO:56 and the light chain variable domain comprises the amino acid sequence of SEQ ID NO:65.

[0553] 36. The antibody or antigen-binding fragment according to any one of paragraphs 21 to 35, wherein the antibody or antigen-binding fragment binds to a complex containing ApoL1, optionally, wherein the complex containing ApoL1 is trypanosome lytic factor (TLF).

[0554] 37. The antibody or antigen-binding fragment according to paragraph 36, wherein the TLF is endogenous human TLF.

[0555] 38. An antibody or antigen-binding fragment according to paragraph 36 or 37, wherein the antibody or antigen-binding fragment can bind to the ApoL1-containing complex under physiological conditions.

[0556] 39. An antibody or antigen-binding fragment according to any one of paragraphs 36 to 38, wherein the antibody or antigen-binding fragment is capable of binding to the ApoL1-containing complex of a subject, optionally wherein the subject is a human.

[0557] 40. An antibody or antigen-binding fragment according to any one of paragraphs 21 to 39, wherein the antibody is not murine IgG1 or IgG2a.

[0558] 41. An antibody or antigen-binding fragment according to any one of paragraphs 21 to 40, which comprises one or more constant domains from an immunoglobulin constant region (Fc).

[0559] 42. An antibody or antigen-binding fragment according to paragraph 41, wherein the constant domain is a human constant domain.

[0560] 43. An antibody or antigen-binding fragment according to paragraph 42, wherein the human constant domain is an IgA, IgD, IgE, IgG or IgM domain.

[0561] 44. An antibody or antigen-binding fragment according to paragraph 43, wherein the human IgG constant domain is an IgG1, IgG2, IgG3 or IgG4 domain.

[0562] 45. An antibody or antigen-binding fragment according to any one of paragraphs 21 to 44, wherein the antibody or antigen-binding fragment is detectably labeled or comprises a conjugated toxin, drug, receptor, enzyme, receptor ligand.

[0563] 46. An antibody or antigen-binding fragment according to any one of paragraphs 21 to 45, wherein the antibody is a monoclonal antibody, human antibody, chimeric antibody or humanized antibody.

[0564] 47. An antibody or antigen-binding fragment according to any one of paragraphs 21 to 46, wherein the antibody is a bispecific, trispecific or multispecific antibody.

[0565] 48. An antibody or antigen-binding fragment according to paragraph 47, wherein the bispecific, trispecific or multispecific antibody comprises a second antigen-binding fragment that binds to a cell-specific antigen.

[0566] 49. A bispecific, trispecific or multispecific antibody that comprises a first antigen-binding fragment that binds to ApoL1 or an ApoL1-containing complex, optionally TLF, and a second antigen-binding fragment that binds to a cell-specific antigen.

[0567] 50. The antibody or antigen-binding fragment according to paragraph 48 or 49, wherein the cell-specific antigen is a cancer or tumor antigen.

[0568] 51. The antibody or antigen-binding fragment according to paragraph 50, wherein the cancer or tumor antigen is a blood cancer antigen or solid tumor antigen optionally selected from claudin 18.2, MUC1, mesothelin (MSLN), myoferritin (MYOF), and PMEL17.

[0569] 52. The antibody or antigen-binding fragment according to paragraph 51, wherein the blood cancer antigen is selected from the group consisting of BCMA, CD38, CD319 / SLAMF-7, TNFRSF17 / BCMA, SYND1 / CD138, CD229, CD47, CD123 / IL3-RA, CD19, CD20, CD22, FcRH5, GPRC5D, CLL-1, PD-L1, and CTLA4.

[0570] 53. The antibody or antigen-binding fragment according to any one of paragraphs 48 to 52, wherein the cell-specific antigen is BCMA.

[0571] 54. The antibody or antigen-binding fragment according to paragraph 28, wherein the second antigen-binding fragment comprises the heavy chain variable domain of SEQ ID NO: 41 or three CDRs of a variant or humanized form thereof having at least 70%, 80%, 90% or 95% sequence identity thereto, and the light chain variable domain of SEQ ID NO: 42 or three CDRs of a variant or humanized form thereof having at least 70%, 80%, 90% or 95% sequence identity thereto.

[0572] 55. The antibody or antigen-binding fragment according to paragraph 54, wherein the second antigen-binding fragment comprises six CDRs, and the six CDRs comprise the following amino acid sequences:

[0573] CDR1H: SYAMS (SEQ ID NO: 43) or a variant or humanized form thereof having at least 70%, 80%, 90% or 95% sequence identity thereto,

[0574] CDR2H: AISGSGGSTYYADSVKG (SEQ ID NO: 44) or a variant or humanized form thereof having at least 70%, 80%, 90% or 95% sequence identity thereto,

[0575] CDR3H: VAPYFAPFDY (SEQ ID NO:45) or a variant or humanized form thereof having at least 70%, 80%, 90% or 95% sequence identity thereto,

[0576] CDR1L: RASQSVSSSYLA (SEQ ID NO:46) or a variant or humanized form thereof having at least 70%, 80%, 90% or 95% sequence identity thereto,

[0577] CDR2L: GASSRAT (SEQ ID NO:47) or a variant or humanized form thereof having at least 70%, 80%, 90% or 95% sequence identity thereto, and

[0578] CDR3L: QQYGNPPLYT (SEQ ID NO:48) or a variant or humanized form thereof having at least 70%, 80%, 90% or 95% sequence identity thereto.

[0579] 56. The antibody or antigen-binding fragment according to any one of paragraphs 53 to 55, wherein the second antigen-binding fragment comprises a heavy chain variable domain and a light chain variable domain, the heavy chain variable domain comprises the amino acid sequence of SEQ ID NO:41 or a variant or humanized form thereof having at least 70%, 80%, 90% or 95% sequence identity thereto, and the light chain variable domain comprises the amino acid sequence of SEQ ID NO:42 or a variant or humanized form thereof having at least 70%, 80%, 90% or 95% sequence identity thereto.

[0580] 57. The antibody or antigen-binding fragment according to paragraph 56, wherein the second antigen-binding fragment comprises a heavy chain variable domain and a light chain variable domain, the heavy chain variable domain comprises the amino acid sequence of SEQ ID NO:41, and the light chain variable domain comprises the amino acid sequence of SEQ ID NO:42,

[0581] optionally wherein the second antigen-binding fragment comprises the amino acid sequence of SEQ ID NO:51.

[0582] 58. The antibody or antigen-binding fragment according to any one of paragraphs 49 to 56, which comprises the amino acid sequence of SEQ ID NO:71 or a variant or humanized form thereof having at least 70%, 80%, 90% or 95% sequence identity thereto, and / or the amino acid sequence of SEQ ID NO:72 or a variant or humanized form thereof having at least 70%, 80%, 90% or 95% sequence identity thereto.

[0583] 59. An antibody or antigen-binding fragment according to any one of paragraphs 49 to 56, which comprises the amino acid sequences of SEQ ID NO:71 and SEQ ID NO:72.

[0584] 60. An antibody or antigen-binding fragment according to any one of paragraphs 49 to 56, which comprises two copies of each of the amino acid sequences of SEQ ID NO:70 and SEQ ID NO:72

[0585] 61. An anti-ApoL1, anti-cell specific antigen IgG1-scFv bispecific chimeric antibody.

[0586] 62. An anti-Hpr, anti-cell specific antigen IgG1-scFv bispecific chimeric antibody.

[0587] 63. A nucleic acid which encodes an antibody or antigen-binding fragment according to any one of paragraphs 21 to 63.

[0588] 64. The nucleic acid according to paragraph 63, which is operably linked to an expression control sequence.

[0589] 65. An expression vector which comprises the nucleic acid according to paragraph 63 or 64.

[0590] 66. A cell which comprises the nucleic acid according to paragraph 63 or 64 or the expression vector according to paragraph 65, optionally wherein the cell is a mammalian cell.

[0591] 67. An immune complex which comprises an antibody or antigen-binding fragment according to any one of paragraphs 21 to 62 which binds to ApoL1 or an ApoL1-containing complex, optionally wherein the complex is TLF.

[0592] 68. An immune complex which comprises an antibody or antigen-binding fragment according to any one of paragraphs 47 to 62.

[0593] 69. A method of inducing cell death, the method comprising contacting a target cell with the immune complex according to paragraph 67 or 68.

[0594] 70. The method according to paragraph 69, wherein the contacting occurs in vitro.

[0595] 71. The method according to paragraph 69, wherein the contraction occurs in a subject.

[0596] 72. The method according to paragraph 71, wherein the subject has cancer.

[0597] 73. A pharmaceutical composition comprising an antibody or antigen-binding fragment according to any one of paragraphs 21 to 62.

[0598] 74. A method of treating cancer, the method comprising administering to a subject an effective amount of an antibody or antigen-binding fragment according to any one of paragraphs 21 to 62.

[0599] 75. The method according to paragraph 74, which comprises administering to the subject an effective amount of an antibody or antigen-binding fragment according to any one of paragraphs 22 to 62.

[0600] 76. The method according to paragraph 74 or 75, wherein the cancer is a blood cancer.

[0601] 77. The method according to paragraph 76, wherein the cancer is multiple myeloma, leukemia (e.g., chronic lymphocytic leukemia, acute myeloid leukemia, acute lymphoblastic leukemia), non-Hodgkin lymphoma, Hodgkin lymphoma, myelodysplastic syndrome (MDS), myeloproliferative neoplasm (MPN) (or its subcategories, e.g., essential thrombocythemia (ET), myelofibrosis (MF), and polycythemia vera (PV)), amyloidosis, Waldenström macroglobulinemia, or aplastic anemia.

[0602] 78. The method according to paragraph 74 or 75, wherein the cancer is a solid cancer.

[0603] 79. A method of treating a subject in need thereof, the method comprising administering to the subject a composition according to any one of paragraphs 1 to 62.

[0604] 80. The method according to paragraph 79, wherein the subject has cancer.

[0605] 81. The method according to any one of paragraphs 1 to 5 or 11 to 17, wherein the composition comprises an antibody according to any one of paragraphs 21 to 62.

[0606] 82. The composition or method according to any one of paragraphs 1 to 81, wherein the target cell is a mammalian cell.

[0607] 83. The composition or method according to paragraph 82, wherein the mammalian cell is an infected cell.

[0608] 84. The composition or method according to paragraph 83, wherein the infected cell is infected with a virus, bacterium or eukaryotic intracellular organism, optionally selected from HIV, Plasmodium falciparum, Toxoplasma gondii, Leishmania spp., Trypanosoma cruzi, Listeria monocytogenes, Chlamydia trachomatis, Coxiella burnetii, Mycobacterium tuberculosis and Trichomonas vaginalis.

[0609] 85. The composition or method according to any one of paragraphs 1 to 81, wherein the target cell is a non-mammalian cell.

[0610] 86. The composition or method according to paragraph 85, wherein the non-mammalian cell is a bacterium, fungus or non-mammalian eukaryotic cell.

[0611] 87. The composition or method according to paragraph 86, wherein the non-mammalian cell is not Trypanosoma.

[0612] 88. The composition or method according to any one of the preceding paragraphs, wherein the subject is a mammal, optionally a human.

[0613] Examples

[0614] Example 1: TLF does not bind to mammalian cells with high affinity.

[0615] Materials and Methods

[0616] To measure TLF-1 binding by flow cytometry, cells were grown to mid-log phase, harvested, washed and resuspended (1x10 7 / ml) in DMEM supplemented with 10% fetal bovine serum. Alexa-488 TLF-1 labeled according to the manufacturer's instructions (Invitrogen) was incubated with excess Hb on ice for 10 minutes and then added to the cells in ice-cold complete DMEM and further incubated at 3 °C for three hours. The cells were washed twice with ice-cold phosphate-buffered saline buffer (PBS) (10 mM NaPi, 137 mM NaCl, pH 7.4), kept on ice and analyzed by flow cytometry. All binding experiments were performed in triplicate, with 50,000 cells measured per experiment / data point.

[0617] Results

[0618] The mechanism by which mammalian cells tolerate TLF has never been studied. HDL endocytosis has been investigated in multiple systems, and the exact purpose and efficiency of this process are still controversial, but cholesterol transport is thought to be one of the main goals (Rohrl and Stangl, “HDL endocytosis and resecretion” Biochim Biophys Acta, 2013.1831(11): pp. 1626 - 1633). The issue of TLF endocytosis efficiency has also not been thoroughly studied, except for a single observation that TLF is internalized in macrophages infected with Leishmania parasites (Samanovic et al., “Trypanosomelytic factor, an antimicrobial high---density lipoprotein, ameliorates Leishmania infection,” PLoS Pathog, 2009.5(1): p.e1000276.). In this case, macrophage death was not reported, indicating that TLF is somehow unable to induce its toxic effects when at near - physiological levels in culture. Trypanosoma brucei binds TLF with high specificity and affinity and can be easily observed at 3C by a previously developed method (DeJesus et al., “A Single Amino Acid Substitution in the Group1 Trypanosoma brucei gambiense Haptoglobin - Hemoglobin Receptor Abolishes TLF - 1 Binding,” PLoS Pathog., 9(2013)). To identify whether there is a receptor for TLF in mammalian cells, this same cryo - binding assay was performed. Although no high - affinity binding was observed, high concentrations of AF488 TLF - 1 were detected by flow cytometry but were not saturated( Figure 2 ). See also Dejesus et al., “Evasion of Africantrypanosomes to human innate immunity,” Dissertation in fulfillment of Doctor of Philosophy, University of Georgia, submitted in 2014.

[0619] Example 2: TLF is internalized and localized to lysosomes in mammalian cells.

[0620] Materials and Methods

[0621] TLF-1 Binding and Uptake Studies

[0622] To measure TLF-1 uptake by flow cytometry, cells were grown to mid-log phase, collected, washed and resuspended (1x10 7 / ml) in DMEM supplemented with 10% fetal bovine serum. Alexa-488 TLF-1 with and without hemoglobin was added to the cells, which were then incubated at 37 °C for three hours. Uptake was stopped by placing the tubes on ice and then washing twice with ice-cold PBS. Analysis to determine the amount of TLF-1 uptake was performed using both a Cyan cytometer and an Amnis ImageStream cytometer by FlowJo software. For uptake studies, 20,000 cells were imaged per experiment by Amnis Imagestream and each experiment was performed in triplicate. Uptake was also measured by fluorescence microscopy. After incubation, the cells were washed twice with ice-cold PBS. After washing, the cells were spread onto slides, fixed with methanol at -20 °C for 5 min and analyzed by fluorescence microscopy. Images were captured by a Zeiss Image Capture Inverted Microscope and Axiovision v4.6 software. These images were subjected to the same exposure and had the same degree of contrast.

[0623] Competitive Binding Studies

[0624] Competitive binding studies using unlabeled non-lytic HDL and Hp 1-1 were performed to analyze the specificity of TLF-1 binding to HEK293 cells. Cells were collected, washed and resuspended (1x10 7 / ml) in ice-cold DMEM supplemented with 10% fetal bovine serum and then transferred to 3 °C for at least 10 minutes. Alexa-488 conjugated TLF-1 (20 nM constant) was complexed with hemoglobin (50 nM) at 4 °C for 10 minutes. Increasing concentrations of unlabeled competitor were incubated with Hb (50 nM) at 4 °C for 10 minutes. The competing ligand was then mixed with Alexa-488 conjugated TLF-1 / Hb and added to the cells at 3 °C and incubated for three hours. The cells were then transferred to ice, washed with ice-cold 1X PBS and analyzed by Cyan cytometer and FlowJo software. All competitive studies were performed in triplicate.

[0625] Results

[0626] Next, experiments were designed to study TLF uptake in HEK293 mammalian cells. First, cells were incubated with AF488-TLF at 37 °C and imaged via Amnis ImageStream. Then Amnis internalization plot analysis was used to identify granule location. AF488-TLF was observed to be endocytosed into vesicles ( Figure 3A and 3B ), where the maximum pixel intensity indicated that the TLF was inside the cell rather than associated with the cell surface.

[0627] After determining that TLF was indeed taken up by HEK293 cells, experiments were designed to determine TLF cellular localization. Co-localization of AF488-TLF with a lysosome tracer indicated that TLF was indeed localized to low pH compartments similar to lysosomes ( Figure 3C ). This was further confirmed by fluorescence microscopy. To test whether TLF uptake was due to specific binding to the haptoglobin (Hp) receptor, as in the case of Trypanosoma brucei, a competitive binding assay was performed using unlabeled ligand. As Figure 3D shown, no competition was observed with increasing amounts (both molar and mass equivalents) of unlabeled Hp. These findings are consistent with previously published literature on HDL binding in mammalian cells, including HEK293 cells, namely that unlike in human-infective trypanosomes, there is no TLF-specific receptor in the mammalian system (Xiao et al., Circ Res., 103:159–66 (2008)).

[0628] Previous studies using HEK293 cells transfected with scavenger receptor class B type I (SR-BI, a haptoglobin-hemoglobin receptor) measured that SR-BI-mediated HDL uptake reached saturation within three hours (Pagler et al., J Biol Chem., 281:11193–204 (2006)). To test whether the endocytic mechanism in wild-type HEK293 cells could reach equilibrium, a TLF uptake time course was performed. After three hours, the signal of AF488-TLF tended to stabilize, indicating equilibrium ( Figure 3E ).

[0629] Hemoglobin has been shown to be an important cofactor for the binding and uptake of TLF by African trypanosomes (Widener et al., PLoS Pathog. 3: e129 (2007)). As discussed and shown herein, no receptor capable of binding Hpr present in TLF has been identified in mammalian systems. With this in mind, experiments were designed to examine the differences in uptake rates when hemoglobin was added. The signal intensity of AF488 TLF analyzed by flow cytometry did not indicate any substantial differences in uptake rates. See also Dejesus et al., “Evasion of African trypanosomes to human innate immunity,” Dissertation in fulfillment of Doctor of Philosophy, University of Georgia, submitted in 2014. Example 3: Mammalian cells are sensitive to TLF and recombinant ApoL1.

[0630] Materials and Methods

[0631] Cell viability assay

[0632] HEK293

[0633] HEK293 cells were harvested from mid-logarithmic phase cultures, washed, and resuspended in complete DMEM medium at a final concentration of 1 x 106 / ml. After incubation at 37 °C for 72 hours, susceptibility to TLF bound to hemoglobin (Hb) was determined over a range of TLF concentrations. The number of viable cells was determined by hemocytometer under phase contrast microscopy. In addition, cell viability was quantified by flow cytometry (Cyan) using a Live / Dead Cell Viability Kit (Invitrogen). All viability assays were performed in triplicate.

[0634] Cell culture and maintenance

[0635] Cells were cultured in the indicated growth medium. Growth medium: For RPMI 8226 (CCL-155), K-562 (CCL-243), RPMI-1640 with 10% FBS; for HT144, McCoy's 5A with 10% FBS, 1% L-glutamine, 1% antibiotic / antifungal; for Panc1 and A375, DMEM with 10% FBS, 1% L-glutamine, 1% antibiotic / antifungal. Cells were maintained at 37 °C in a humidified atmosphere with 5% CO 2 2.

[0636] RPMI 8226 / ATCC CCL-155 cells

[0637] The cells were cultured in RPMI-1640 with 10% FBS and 1% antibiotic / antifungal. A total of 40 μl of cells were seeded in a white 384-well plate at a final cell density of 1e5 / mL and allowed to grow for four days in the presence of the specified additives. After the experiment was completed, the cells were processed using the CellTiter cell viability assay.

[0638] PANC-1 / ATCC CRL-1469

[0639] The cells were cultured in DMEM with 10% FBS, 1% L-glutamine, and 1% antibiotic / antifungal. A total of 80 μl of cells were seeded into a white 384-well plate at a final cell density of 5e4 / mL and allowed to grow and adhere overnight. The next day, the medium was removed and replaced with 50 μl of complete medium containing the specified additives and allowed to grow for four days. After the experiment was completed, the cells were processed using the CellTiter cell viability assay.

[0640] A375 / ATCC CRL-1619

[0641] The cells were cultured in DMEM with 10% FBS, 1% L-glutamine, and 1% antibiotic / antifungal. A total of 80 μl of cells were seeded into a white 384-well plate at a final cell density of 5e4 / mL and allowed to grow and adhere overnight. The next day, the medium was removed and replaced with complete medium containing the specified additives and allowed to grow for four days. After the experiment was completed, the cells were processed using the CellTiter cell viability assay.

[0642] HT144 / ATCC HTB-63

[0643] The cells were cultured in McCoy's with 10% FBS, 1% L-glutamine, and 1% antibiotic / antifungal. A total of 80 μl of cells were seeded into a white 384-well plate at a final cell density of 5 x 10^4 cells / mL and allowed to grow and adhere overnight. The next day, the medium was removed and replaced with complete medium containing the specified additives and allowed to grow for three days. After the experiment was completed, the cells were processed using the CellTiter cell viability assay.

[0644] CellTiter cell viability assay

[0645] An equal volume of CellTiter-Glo 2.0 (Promega Cat.G9241) was added to each well via a robotic syringe. The plate was shaken for 1 minute and the luminescence was measured via a SpectraMax iD3 plate reader (Molecular Diagnostics) after 10 minutes. The data was imported into GraphPad and standard error and t-tests were performed to measure the indicated p-values. Floating viability assay

[0646] As described previously (1), human HDL was purified by differential flotation on a sodium bromide gradient (1.26 floats / float). The floats were diluted with PBS to the indicated total protein content and mixed 1:1 with cells in a standard cell survival assay.

[0647] Recombinant ApoL1 cell viability assay

[0648] Recombinant ApoL1 (Sino Biological 13910-H08B) was resuspended in complete medium at 100 μg / mL. And added to each well at the indicated final concentration. Five replicates were performed for each concentration.

[0649] ApoL1 depletion assay

[0650] As described previously (Shiflett et al., J Biol Chem 280:32578–32585 (2005)), human HDL was purified by differential flotation on a sodium bromide gradient (1.26 floats / float). 1 mL of floats was incubated overnight at 4°C on a rotator with the indicated antibodies (anti-ApoL1 13.11, anti-Hpr 14.11 (antibody sequences provided below)) at 150 μg / mL, 75 μg / mL, 37 μg / mL, 0 μg / mL. 100 μl of Protein-G MagBeads (Genscript L00274) was added to each sample and incubated for 1 hour at RT on a rotator. The beads were removed via a magnet and the depleted floats were used in the cell survival assay, six replicates for each condition.

[0651] Results

[0652] To test whether mammalian cells are resistant to TLF in culture, a 72-hour survival assay was performed. TLF concentrations up to 20 μg / ml did not show inhibition of cell growth ( Figure 4A)。However, incubation with 75 μg / ml TLF for 72 hours caused growth attenuation. The physiological level of TLF is approximately 10 μg / ml (Samanovic et al., PLoS Pathog., 5: e1000276 (2009)). The difference in cell density between 24-hour labeling and 72 hours was also recorded by light microscopy ( Figure 4B )。See also Dejesus et al., “Evasion of African trypanosomes to human innate immunity,” Dissertation in fulfillment of Doctor of Philosophy, University of Georgia, submitted in 2014.

[0653] Next, RPMI 8226 (CCL-155) multiple myeloma (MM) cells were incubated with high-concentration TLF fractions. As measured by CellTiter-Glo, an increase in the high-concentration TLF / HDL fraction caused a decrease in cell viability (25% decrease at 4.96 mg / mL) ( Figure 4C )。

[0654] RPMI 8226 (CCL-155, multiple myeloma), PANC-1 (CRL-1469, human pancreatic cancer), A375 (CRL-1619, human malignant melanoma), and HT144 (HTB-63, human malignant melanoma) cells were incubated with recombinant ApoL1 for 3 - 4 days ( Figures 4D - 4G )。The cell viability of HT-144 was measured on the third day using CellTiter-Glo, and the cell viability of CCL-155, PANC-1, and A375 was measured on the fourth day. The LD50 values were determined by using a four-parameter model with a Quest Graph EC50 calculator. The resulting LD50 values for different cell lines were as follows: CCL-155 20.2 μg / mL, PANC-1 43 μg / mL, A375 27.5 μg / mL, and HT144 32.8 μg / mL. The data show a dose-dependent effect of recombinant ApoL1 on cell growth. For comparison, the circulating levels of ApoL1 range from ∼400 ng - 15 μg, with a median of 3 μg (Bruggeman et al., J Am Soc Nephrol., 25: 634–44 (2014)).

[0655] Depletion of TLF using ApoL1 mAb increased the survival rate of the RPMI 8226 (CCL-155) multiple myeloma cell line ( Figure 4H)。This data indicates that by increasing the amount of anti-ApoL1 mAb added during immunoprecipitation, the cell growth effect of ApoL1 containing HDL particles can be eliminated. Similar results were obtained when using anti-Hpr antibodies( Figure 4I )。

[0656] Example 4: Anti-Hpr Antibody SFII 134.3 Sequence and Binding Assay

[0657] Materials and Methods

[0658] Antibody Sequencing

[0659] SFII 134.3, a mouse anti-Hpr IgG2a, was sequenced by whole transcriptome shotgun sequencing (RNA-Seq). Total RNA was extracted from hybridoma cells and a barcoded cDNA library was generated by RT-PCR using random hexamers. Next-generation sequencing was performed on an Illumina HiSeq sequencer. Contigs were assembled and data for antibody sequences were mined to identify all viable antibody sequences (i.e., those that did not contain stop codons). The variable heavy domain and variable light domain were identified separately.

[0660] The genes were separated into heavy and light chains. For each chain, the variable domain was reported together with the signal peptide and constant domain regions.

[0661] Complementary determining regions (CDRs) were identified. CDR sequences according to the Kabat, IMGT, and Chothia formulas were provided.

[0662] Western Blot

[0663] Materials: TLF – TLF purified by column (2 ug), rApoL1 – ApoL1 protein, human recombinant (Sinobiological 13910-H08B) (1 ug), L – Chameleon Duo Ladder (Licor)-8 uL, L2 – Benchmark Protein Ladder (Thermo 10747012)-2 uL

[0664] Assay: SDS-Page 4%-15% Mini-PROTEAN TGX (200V, 30 min), transfer to nitrocellulose (75V, 45 min), block in blocking buffer (PBS) for 1 hour, dilute antibody at 1:10,000 in 0.5x blocking buffer (PBS) + 0.2% Tween - overnight, wash 4x with PBS-T, dilute secondary antibody at 1:10,000 in 0.5x blocking buffer (PBS) + 0.2% Tween – 1 hour (Licor goat anti-mouse 800, 926-32210), wash 4x with PBS-T, rinse 1x with PBS, image in 800 channel on Odyssey DLx infrared imager.

[0665] Dot blot

[0666] Materials: rApoL1 – ApoL1 protein, human recombinant (Sino biological 13910-H08B) (1 μg)

[0667] Assay: 500 ng ApoL1 in 10 μl 50% FBS, serially diluted 1:2 in 50% FBS, block in blocking buffer (PBS) for 1 hour, wash 4x with PBS-T, dilute antibody at 1:10,000 in 0.5x blocking buffer (PBS) + 0.2%, Tween - overnight, wash 4x with PBS-T, dilute secondary antibody at 1:10,000 in 0.5x blocking buffer (PBS) + 0.2% Tween – 1 hour (Licor goat anti-mouse 800, 926-32210), wash 4x with PBS-T, rinse 1x with PBS, image in 800 channel on Odyssey DLx infrared imager.

[0668] Results

[0669] Signal peptide amino acid and nucleic acid sequences

[0670]

[0671] Heavy chain variable domain (VH) and CDR amino acid and nucleic acid sequences

[0672]

[0673] Complementary determining region sequences

[0674]

[0675]

[0676]

[0677] Heavy chain constant amino acid and nucleic acid sequences

[0678]

[0679] Light chain variable domain (VL) and CDR amino acid and nucleic acid sequences

[0680]

[0681] Complementary determining region sequences

[0682]

[0683]

[0684] Light chain constant amino acid and nucleic acid sequences

[0685]

[0686] Western blot assays comparing recombinant antibody and ascites Pro-G purified antibody showed that both the recombinant and purified antibodies bound in the TLF column, rather than rApoL1, under non-reducing but not reducing conditions, and the size was related to Hpr in previous assays. See Figures 5A - 5F . In dot blot (native) assays, neither antibody bound recombinant ApoL1. See Figure 5G .

[0687] Example 5: Anti-Hpr antibody SFII 14.11 sequence

[0688] Materials and methods

[0689] Antibody sequencing

[0690] Hybridoma SFII 14.11 antibody sequencing of CDR (only variable region)

[0691] * Reverse transcription (RACE) using VL and VH PCR primers

[0692] * Subclone the PCR products into plasmids and express as single colonies

[0693] * DNA gel verification; with 5

[0694] Colony verification sequencing

[0695] Complementary determining regions (CDRs) have been identified. CDR sequences according to the Kabat, IMGT, and Chothia formulas are provided.

[0696] Results

[0697] Heavy chain variable domain (VH) and CDR amino acid and nucleic acid sequences

[0698]

[0699] Complementary determining region sequences

[0700]

[0701]

[0702] Light chain variable domain (VL) and CDR amino acid and nucleic acid sequences

[0703]

[0704] Complementary determining region sequences

[0705]

[0706]

[0707]

[0708] Example 6: Anti-ApoL1 antibody sequences and binding assays

[0709] Materials and methods

[0710] Antibody sequencing

[0711] SFIII 13.11, a murine anti-ApoL1 IgG1, was sequenced by whole transcriptome shotgun sequencing (RNA-Seq). Total RNA was extracted from hybridoma cells and a barcoded cDNA library was generated by RT-PCR using random hexamers. Next-generation sequencing was performed on an Illumina HiSeq sequencer. Contigs were assembled and data for antibody sequences were mined to identify all viable antibody sequences (i.e., those that did not contain stop codons). The variable heavy domain and variable light domain were identified separately.

[0712] The genes were separated into heavy and light chains. For each chain, the variable domain was reported together with the signal peptide and constant domain regions.

[0713] Complementary determining regions (CDRs) were identified. CDR sequences according to the Kabat, IMGT, and Chothia formulas were provided.

[0714] Western blot

[0715] Materials: TLF - purified TLF by column (2 ug), rApoL1 - ApoL1 protein, human recombinant (Sinobiological 13910 - H08B) (1 ug), L - Chameleon Duo Ladder (Licor) - 8 uL, L2 - Benchmark Protein Ladder (Thermo 10747012) - 2 uL

[0716] Assay: SDS - Page 4% - 15% Mini - PROTEAN TGX (200V, 30 min), transfer to nitrocellulose (75V, 45 min), block in blocking buffer (PBS) for 1 hour, dilute antibody 1:10,000 in 0.5x blocking buffer (PBS) + 0.2% Tween - overnight, wash 4x with PBS - T, dilute secondary antibody 1:10,000 in 0.5x blocking buffer (PBS) + 0.2% Tween - 1 hour (Licor goat anti - mouse 800, 926 - 32210), wash 4x with PBS - T, rinse 1x with PBS, image in 800 channel on Odyssey DLx infrared imager.

[0717] Dot blot

[0718] Materials: rApoL1 - ApoL1 protein, human recombinant (Sino biological 13910 - H08B) (1 ug)

[0719] Assay: 500 ng ApoL1 in 10 ul 50% FBS, serially diluted 1:2 in 50% FBS, block in blocking buffer (PBS) for 1 hour, wash 4x with PBS - T, dilute antibody 1:10,000 in 0.5x blocking buffer (PBS) + 0.2%, Tween - overnight, wash 4x with PBS - T, dilute secondary antibody 1:10,000 in 0.5x blocking buffer (PBS) + 0.2% Tween - 1 hour (Licor goat anti - mouse 800, 926 - 32210), wash 4x with PBS - T, rinse 1x with PBS, image in 800 channel on Odyssey DLx infrared imager.

[0720] Results

[0721] Signal peptide amino acid and nucleic acid sequences

[0722]

[0723] Heavy chain variable domain (VH) and CDR amino acid and nucleic acid sequences

[0724]

[0725] Complementary determining region sequences

[0726]

[0727]

[0728] Heavy chain constant amino acid and nucleic acid sequences

[0729]

[0730]

[0731] Light chain variable domain (VL) and CDR amino acid and nucleic acid sequences

[0732] Complementary determining region sequences

[0733]

[0734]

[0735] Light chain constant amino acid and nucleic acid sequences

[0736]

[0737] Western blot assays comparing recombinant antibody and ascites Pro-G purified antibody showed that both recombinant and purified antibodies bound in the TLF column and rApoL1 under both non-reducing and reducing conditions. See Figures 6A - 6F In dot blot (native) assays, both antibodies bound recombinant ApoL1. See Figure 6G .

[0738] Example 7: Anti-BCMA scFv binds BCMA

[0739] Materials and Methods

[0740] 17A5 scFv: Anti-BCMA clone 17A5 has heavy and light chain variable sequences:

[0741] VH

[0742]

[0743] VL

[0744]

[0745] The CDR sequences of anti-BCMA clone 17A5 (shown in bold in the above sequences) are:

[0746] CDR1H: SYAMS (SEQ ID NO:43),

[0747] CDR2H: AISGSGGSTYYADSVKG (SEQ ID NO:44),

[0748] CDR3H: VAPYFAPFDY (SEQ ID NO:45),

[0749] CDR1L: RASQSVSSSYLA (SEQ ID NO:46),

[0750] CDR2L: GASSRAT (SEQ ID NO:47),

[0751] CDR3L: QQYGNPPLYT (SEQ ID NO:48).

[0752] The sequence of the anti-BCMA scFv of clone 17A5 (“17A5 scFv”) is

[0753]

[0754]

[0755] wherein the CDRs are in bold and gggggsggggsggggs (lowercase) (SEQ ID NO:52) is a flexible linker.

[0756] For the sequence of BMCA clone 17A5 and alternative anti-BCMA sequences, see WO 2014 / 122144, which is incorporated herein by reference in its entirety.

[0757] Materials: BCMA–BCMA protein, human, recombinant (ECD, rFc tag) (Sino biological 10620-H15H) (0.5 ug); L–Chameleon Duo Ladder (Licor)-8 uL; L2–Benchmark Protein Ladder (Thermo 10747012)-2 uL; Control BCMA monoclonal–anti-hBCMA, mouse monoclonal, clone 1004023 (RnD Systems Cat.MAB1931)

[0758] Method: SDS-Page 4%-15% Mini-PROTEAN TGX (200V, 30 min); transfer to nitrocellulose (75V, 45 min); block in blocking buffer (PBS) for 1 hour; dilute antibody 1:10,000 in 0.5x blocking buffer (PBS) + 0.2%; Tween–overnight; wash 4x PBS-T; dilute secondary antibody 1:10,000 in 0.5x blocking buffer (PBS) + 0.2% Tween–1 hour (Licor goat anti-mouse 800, 926-32210); wash 4x PBS-T; rinse 1x PBS; image in 800 channel on Odyssey DLx infrared imager

[0759] Results

[0760] The ability of the anti-BCMA scFv of SEQ ID NO:51 to bind recombinant BCMA was tested by Western blot compared to a commercially available control monoclonal antibody (RnD Systems Cat. MAB1931). The scFv BCMA antibody binds to a similar moving target - recombinant BCMA - verifying that it specifically binds the correct target. See Figures 7A - 7C 。

[0761] Example 8: Design of Bispecific ApoL1-BCMA-bsAb Antibody

[0762] An anti-ApoL1, BCMA IgG1-scFv (heavy chain C-terminus) antibody has been designed and the chimeric antibody has Figure 8 the structure of

[0763] The Fab portion is the heavy and light chain variable regions of recombinant clone SFIII 13.11 (anti-ApoL1) with a heavy chain variable domain of SEQ ID NO:24 and a light chain variable domain of SEQ ID NO:36 or SEQ ID NO:77.

[0764] The anti-BCMA is [Clone 17A5] provided in Example 7 (SEQ ID NO:51), human IgG1, ScFv of κ fused to the C-terminus of the heavy chain of human IgG1.

[0765] The full clone 13.11x BCMAscFv: bsAbBCMA / ApoL1 sequence used in the following example is:

[0766] Heavy chain amino acid sequence

[0767]

[0768]

[0769] Heavy chain nucleic acid sequence

[0770]

[0771]

[0772] Light chain amino acid sequence

[0773]

[0774] Light chain nucleic acid sequence

[0775]

[0776]

[0777] Example 9: Bispecific ApoL1-BCMA-bsAb antibody binds to BCMA and apolipoprotein L1

[0778] Materials and methods

[0779] ELISA

[0780] Fixation and blocking

[0781] Enzyme-linked immunosorbent assay (ELISA) plates were prepared by incubating them with their respective fixation buffer (bicarbonate / carbonate coating buffer 100 mM) or blocking buffer (PBS, 0.1% Tween-20, 1% BSA) overnight at 4°C.

[0782] Bridging ELISA

[0783] For the bridging ELISA assay, ApoL1 (Sino Cat. 13910-H08B) or BCMA (Sino Cat. 10620-H15H) was resuspended at a 1:10,000 dilution in ELISA binding buffer (1X PBST + 1% BSA) and then incubated overnight at 4°C. ApoL1 was immobilized in all conditions except when BCMA was used as the primary protein (ApoL1-*). After immobilization, 100 μl of bispecific antibody (bsAb, 1 μg / ml in ELISA blocking buffer) was added and incubated for 1 hour at 37°C. Subsequently, each well was washed four times with 150 μl of 1X PBST. Next, 1 μg / mL of rabbit Fc-labeled BCMA was incubated in ELISA blocking buffer for 1 hr at 37°C, followed by four additional washes of 150 μl of 1X PBST. The secondary binding component, anti-rabbit HRP, was added at a 1:10,000 dilution and the plate was incubated for an additional hour at 37°C, followed by four additional washes of 150 μl with 1X PBST. To visualize the response, TMB one-step substrate reagent (Sigma) was applied in a 50 μl volume and incubated for no more than 15 minutes. The response was terminated by adding 50 μl of stop solution and the absorbance at 450 nm was immediately measured on a SpectraMax iD3 plate reader (Molecular Diagnostics).

[0784] ELISA using normal human serum

[0785] For ELISA involving normal human serum (NHS), the conditioned media was combined with ELISA binding buffer at a 1:1 ratio. The mixture was incubated overnight at 4°C and then incubated overnight at 4°C with blocking buffer (PBS, 1% BSA, 0.1% Tween-20). Subsequently, the prepared samples were exposed to HRP-labeled bsAb, introduced at a 1:1,000 dilution (final 1 μg / mL), and incubated for 1 hour at 37°C. After that, each well was washed four additional times with 150 μl of 1X PBST. Finally, TMB one-step substrate reagent (Sigma) was added in a 50 μl volume and incubated for a 15-minute period. The response was terminated by adding 50 μl of stop solution and the absorbance at 450 nm was immediately read on a SpectraMax iD3 plate reader (Molecular Diagnostics).

[0786] Bispecific antibody competition ELISA

[0787] Competitive ELISA was performed to study the competitive binding interaction involving B cell maturation antigen (BCMA) and bsAb. The BCMA protein was immobilized onto ELISA plate wells at a dilution of 1:10,000, and each well received 0.1 μg of the immobilized protein for 1 hour at 37 °C. During incubation, competition was set up by binding HRP-labeled bsAb at a dilution of 1:10,000 (concentration of 0.1 μg per well) with different concentrations of unlabeled bsAb (ranging from 0 to 100-fold of the original concentration, up to 10 μg per well). The two forms of bsAb were combined, added to the ELISA plate, and incubated for 1 hour at 37 °C. After that, each well was washed an additional four rounds with 150 μl of 1XPBST. Finally, TMB one-step substrate reagent (Sigma) was added in a 50 μl volume and incubated for a 5-minute period. The response was terminated by adding 50 μl of stop solution, and the absorbance at 450 nm was immediately read on a SpectraMax iD3 plate reader (Molecular Diagnostics).

[0788] Results

[0789] The experiment was designed to test the binding of the bispecific ApoL1-BCMA-bsAb antibody in Example 8. The results showed that the bispecific antibody bound effectively to its designated targets ApoL1 and BCMA, covering both native and recombinant iterations in a dose-dependent manner. The specificity of the bsAb for these targets was confirmed by its ability to precisely outcompete the HRP-labeled bsAb in a competitive bridging ELISA. As discussed in more detail below, these data demonstrate the precision and selectivity with which the bsAb engages its designated targets ( Figures 9A - 9B ).

[0790] Bridging ELISA was used to evaluate the binding ability of the bispecific antibody (bsAb) between recombinant variant ApoL1 and BCMA ( Figure 9A ). The bsAb showed successful binding to the immobilized forms (Bridge) of both ligands. In contrast, no detectable signal was recorded when the bsAb, ApoL1, or ...

Claims

1. A method of increasing cell death of target cells in a mammalian subject in need thereof, the method comprising administering to the subject an effective amount of a composition that increases apolipoprotein L1 (ApoL1) in the target cells.

2. The method according to claim 1, wherein the composition increases the accumulation of endogenous ApoL1 in the target cells.

3. The method according to claim 2, wherein the endogenous ApoL1 is a component of an ApoL1-containing complex.

4. The method according to claim 3, wherein the ApoL1-containing complex is trypanosome lytic factor (TLF), optionally TLF-1 and / or TLF-2.

5. The method according to any one of claims 1 to 4, wherein the composition comprises a first antigen-binding fragment that binds to ApoL1 or an ApoL1-containing complex and a targeting moiety that targets the composition to the target cells, optionally wherein the composition is a bispecific or multispecific antibody that comprises a first antigen-binding fragment that binds to ApoL1 or an ApoL1-containing complex, optionally TLF, and a second antigen-binding fragment that binds to a cell-specific antigen.

6. The method according to claim 1, wherein the composition comprises ApoL1 or a functional fragment or variant thereof and a targeting moiety that targets a cell-specific antigen.

7. The method according to claim 6, wherein the composition comprises the ApoL1 or a functional fragment or variant thereof directly or indirectly conjugated or fused to the targeting moiety.

8. The method according to claim 6 or 7, wherein the composition comprises a delivery agent, optionally a liposome or a polymeric nanoparticle.

9. The method according to claim 8, wherein the targeting moiety is conjugated or fused to the delivery agent.

10. The method according to any one of claims 6 to 9, wherein the targeting moiety is an antibody or an antigen-binding fragment.

11. The method according to any one of claims 1 to 10, wherein the cell-specific antigen is specific for diseased cells.

12. The method according to claim 11, wherein the diseased cells are cancer cells.

13. The method according to claim 12, wherein the cancer cells are blood cancer cells.

14. The method according to any one of claims 1 to 13, wherein the subject has a disease caused by the target cells.

15. The method according to claim 14, wherein the composition is administered in an effective amount to treat the disease.

16. The method according to any one of claims 1 to 15, wherein the cell-specific antigen is not a trypanosome-specific surface antigen.

17. The method according to any one of claims 1 to 16, wherein the subject does not have trypanosomiasis.

18. A composition comprising ApoLl or a functional fragment or variant thereof and a targeting moiety, wherein the targeting moiety does not target a trypanosome-specific surface antigen.

19. The composition according to claim 18, wherein the ApoL1 or a functional fragment or variant thereof is directly or indirectly conjugated or fused to the targeting moiety.

20. The composition according to claim 19, wherein the composition comprises a delivery vehicle, optionally a liposome or polymeric nanoparticle, optionally, wherein the targeting moiety is conjugated or fused to the delivery vehicle.

21. An antibody or antigen-binding fragment comprising: The three complementarity-determining regions (CDRs) of the heavy-chain variable domain of SEQ ID NO: 24 or a variant or humanized form thereof having at least 70%, 80%, 90% or 95% sequence identity thereto, and the three complementarity-determining regions (CDRs) of the light-chain variable domain of SEQ ID NO: 36 or SEQ ID NO: 77 or a variant or humanized form thereof having at least 70%, 80%, 90% or 95% sequence identity thereto, wherein the antibody or antigen-binding fragment binds to apolipoprotein L1 (ApoL1).

22. The antibody or antigen-binding fragment according to claim 21, wherein the heavy-chain and light-chain variable domain CDRs comprise: TYAMS (SEQ ID NO: 25), EISNGGLYTYYPDTVTG (SEQ ID NO: 26), ENRNWYFDL (SEQ ID NO: 27), RSSQSIVNSNGNTYLE (SEQ ID NO: 37) and KVSNRFS (SEQ ID NO: 38), FQGSHVPLT (SEQ ID NO: 39) or a variant or humanized form thereof having at least 70%, 80%, 90% or 95% sequence identity thereto; GFTFSTYA (SEQ ID NO: 28), ISNGGLYT (SEQ ID NO: 29), IRENRNWYFDL (SEQ ID NO: 30), QSIVNSNGNTY (SEQ ID NO: 40), KVS and FQGSHVPLT (SEQ ID NO: 39) or a variant or humanized form thereof having at least 70%, 80%, 90% or 95% sequence identity thereto; or GFTFSTY (SEQ ID NO: 31), SNGGLY (SEQ ID NO: 32), ENRNWYFDL (SEQ ID NO: 27), RSSQSIVNSNGNTYLE (SEQ ID NO: 37), KVSNRFS (SEQ ID NO: 38) and FQGSHVPLT (SEQ ID NO: 39) or a variant or humanized form thereof having at least 70%, 80%, 90% or 95% sequence identity thereto.

23. The antibody or antigen-binding fragment according to claim 21 or 22, wherein the heavy-chain and light-chain variable domain CDRs comprise: TYAMS (SEQ ID NO: 25), EISNGGLYTYYPDTVTG (SEQ ID NO: 26), ENRNWYFDL (SEQ ID NO: 27), RSSQSIVNSNGNTYLE (SEQ ID NO: 37), and KVSNRFS (SEQ ID NO: 38), FQGSHVPLT (SEQ ID NO: 39); GFTFSTYA (SEQ ID NO: 28), ISNGGLYT (SEQ ID NO: 29), IRENRNWYFDL (SEQ ID NO: 30), QSIVNSNGNTY (SEQ ID NO: 40), KVS, and FQGSHVPLT (SEQ ID NO: 39); or GFTFSTY (SEQ ID NO: 31), SNGGLY (SEQ ID NO: 32), ENRNWYFDL (SEQ ID NO: 27), RSSQSIVNSNGNTYLE (SEQ ID NO: 37), KVSNRFS (SEQ ID NO: 38), and FQGSHVPLT (SEQ ID NO: 39).

24. An antibody or antigen-binding fragment according to any one of claims 21 to 23, which comprises a heavy chain variable domain and a light chain variable domain, wherein the heavy chain variable domain comprises the amino acid sequence of SEQ ID NO: 24 or a variant or humanized form thereof having at least 70%, 80%, 90% or 95% sequence identity thereto, and the light chain variable domain comprises the amino acid sequence of SEQ ID NO: 36 or SEQ ID NO: 77 or a variant or humanized form thereof having at least 70%, 80%, 90% or 95% sequence identity thereto.

25. An antibody or antigen-binding fragment thereof according to any one of claims 21 to 24, which comprises a heavy chain variable domain and a light chain variable domain, wherein the heavy chain variable domain comprises the amino acid sequence of SEQ ID NO: 24 and the light chain variable domain comprises the amino acid sequence of SEQ ID NO: 36 or SEQ ID NO:

77.

26. An antibody or antigen-binding fragment, which comprises: Three complementarity-determining regions (CDRs) of the heavy chain variable domain of SEQ ID NO: 3 or a variant or humanized form thereof having at least 70%, 80%, 90% or 95% sequence identity thereto, and three complementarity-determining regions (CDRs) of the light chain variable domain of SEQ ID NO: 14 or a variant or humanized form thereof having at least 70%, 80%, 90% or 95% sequence identity thereto, wherein the antibody or antigen-binding fragment binds to haptoglobin-related protein (Hpr).

27. The antibody or antigen-binding fragment according to claim 26, wherein the heavy and light chain variable domain CDRs comprise: NYGMN (SEQ ID NO: 4), WINSYTGEATYTDDLKG (SEQ ID NO: 5), EGYGDYGYSFDY (SEQ ID NO: 6), RATKNIYTYLA (SEQ ID NO: 16), NAKTLAE (SEQ ID NO: 17), and QHHYGTPRT (SEQ ID NO: 18), or variants or humanized forms thereof having at least 70%, 80%, 90%, or 95% sequence identity thereto; GYIFTNYG (SEQ ID NO: 7), INSYTGEA (SEQ ID NO: 8), AREGYGDYGYSFDY (SEQ ID NO: 9), KNIYTY (SEQ ID NO: 19), NAK, and QHHYGTPRT (SEQ ID NO: 18), or variants or humanized forms thereof having at least 70%, 80%, 90%, or 95% sequence identity thereto; or GYIFTNY (SEQ ID NO: 10), NSYTGE (SEQ ID NO: 11), EGYGDYGYSFDY (SEQ ID NO: 6), RATKNIYTYLA (SEQ ID NO: 16), NAKTLAE (SEQ ID NO: 17), and QHHYGTPRT (SEQ ID NO: 18), or variants or humanized forms thereof having at least 70%, 80%, 90%, or 95% sequence identity thereto.

28. The antibody or antigen-binding fragment according to claim 26 or 27, wherein the CDRs of the heavy and light chain variable domains comprise: NYGMN (SEQ ID NO: 4), WINSYTGEATYTDDLKG (SEQ ID NO: 5), EGYGDYGYSFDY (SEQ ID NO: 6), RATKNIYTYLA (SEQ ID NO: 16), NAKTLAE (SEQ ID NO: 17), and QHHYGTPRT (SEQ ID NO: 18); GYIFTNYG (SEQ ID NO: 7), INSYTGEA (SEQ ID NO: 8), AREGYGDYGYSFDY (SEQ ID NO: 9), KNIYTY (SEQ ID NO: 19), NAK, and QHHYGTPRT (SEQ ID NO: 18); or GYIFTNY (SEQ ID NO: 10), NSYTGE (SEQ ID NO: 11), EGYGDYGYSFDY (SEQ ID NO: 6), RATKNIYTYLA (SEQ ID NO: 16), NAKTLAE (SEQ ID NO: 17), and QHHYGTPRT (SEQ ID NO: 18).

29. An antibody or antigen-binding fragment according to any one of claims 26 to 28, which comprises a heavy chain variable domain and a light chain variable domain, wherein the heavy chain variable domain comprises the amino acid sequence of SEQ ID NO: 3 or a variant or humanized form thereof having at least 70%, 80%, 90% or 95% sequence identity thereto, and the light chain variable domain comprises the amino acid sequence of SEQ ID NO: 14 or a variant or humanized form thereof having at least 70%, 80%, 90% or 95% sequence identity thereto.

30. An antibody or antigen-binding fragment thereof according to any one of claims 26 to 29, which comprises a heavy chain variable domain and a light chain variable domain, wherein the heavy chain variable domain comprises the amino acid sequence of SEQ ID NO: 3 and the light chain variable domain comprises the amino acid sequence of SEQ ID NO:

14.

31. An antibody or antigen-binding fragment, comprising: three complementarity-determining regions (CDRs) of the heavy chain variable domain of SEQ ID NO: 56 or a variant or humanized form thereof having at least 70%, 80%, 90% or 95% sequence identity thereto, and three complementarity-determining regions (CDRs) of the light chain variable domain of SEQ ID NO: 65 or a variant or humanized form thereof having at least 70%, 80%, 90% or 95% sequence identity thereto, wherein the antibody or antigen-binding fragment binds to haptoglobin-related protein (Hpr).

32. The antibody or antigen-binding fragment according to claim 31, wherein the heavy and light chain variable domain CDRs comprise: DYSIH (SEQ ID NO: 57), WKHTESGESTYADDFKG (SEQ ID NO: 58), GANYGSLLDY (SEQ ID NO: 59), RASKSVSTSGYSYMH (SEQ ID NO: 66), LASNLES (SEQ ID NO: 67), QHNRELPLT (SEQ ID NO: 68) or a variant or humanized form thereof having at least 70%, 80%, 90% or 95% sequence identity thereto; GFTFTDYS (SEQ ID NO: 60), KHTESGES (SEQ ID NO: 61), ARGANYGSLLDY (SEQ ID NO: 62), KSVSTSGYSY (SEQ ID NO: 69), LAS, QHNRELPLT (SEQ ID NO: 68) or a variant or humanized form thereof having at least 70%, 80%, 90% or 95% sequence identity thereto; or GFTFTDY (SEQ ID NO: 63), HTESGE (SEQ ID NO: 64), GANYGSLLDY (SEQ ID NO: 59), RASKSVSTSGYSYMH (SEQ ID NO: 66), LASNLES (SEQ ID NO: 67), QHNRELPLT (SEQ ID NO: 68) or a variant or humanized form thereof having at least 70%, 80%, 90% or 95% sequence identity thereto.

33. The antibody or antigen-binding fragment according to claim 31 or 32, wherein the CDRs of the heavy and light chain variable domains comprise: DYSIH (SEQ ID NO: 57), WKHTESGESTYADDFKG (SEQ ID NO: 58), GANYGSLLDY (SEQ ID NO: 59), RASKSVSTSGYSYMH (SEQ ID NO: 66), LASNLES (SEQ ID NO: 67), QHNRELPLT (SEQ ID NO: 68); GFTFTDYS (SEQ ID NO: 60), KHTESGES (SEQ ID NO: 61), ARGANYGSLLDY (SEQ ID NO: 62), KSVSTSGYSY (SEQ ID NO: 69), LAS, QHNRELPLT (SEQ ID NO: 68); or GFTFTDY (SEQ ID NO: 63), HTESGE (SEQ ID NO: 64), GANYGSLLDY (SEQ ID NO: 59), RASKSVSTSGYSYMH (SEQ ID NO: 66), LASNLES (SEQ ID NO: 67), QHNRELPLT (SEQ ID NO: 68).

34. The antibody or antigen-binding fragment according to any one of claims 31 to 33, which comprises a heavy chain variable domain and a light chain variable domain, wherein the heavy chain variable domain comprises the amino acid sequence of SEQ ID NO: 56 or a variant or humanized form thereof having at least 70%, 80%, 90% or 95% sequence identity thereto, and the light chain variable domain comprises the amino acid sequence of SEQ ID NO: 65 or a variant or humanized form thereof having at least 70%, 80%, 90% or 95% sequence identity thereto.

35. The antibody or its antigen-binding fragment according to any one of claims 31 to 34, which comprises a heavy chain variable domain and a light chain variable domain, wherein the heavy chain variable domain comprises the amino acid sequence of SEQ ID NO: 56 and the light chain variable domain comprises the amino acid sequence of SEQ ID NO:

65.

36. An antibody or antigen-binding fragment according to any one of claims 21 to 35, wherein the antibody or antigen-binding fragment binds to an ApoL1-containing complex, optionally wherein the ApoL1-containing complex is trypanosome lytic factor (TLF).

37. An antibody or antigen-binding fragment according to claim 36, wherein the TLF is endogenous human TLF.

38. An antibody or antigen-binding fragment according to claim 36 or 37, wherein the antibody or antigen-binding fragment is capable of binding to the ApoL1-containing complex under physiological conditions.

39. An antibody or antigen-binding fragment according to any one of claims 36 to 38, wherein the antibody or antigen-binding fragment is capable of binding to an ApoL1-containing complex of a subject, optionally wherein the subject is a human.

40. An antibody or antigen-binding fragment according to any one of claims 21 to 39, wherein the antibody is not murine IgG1 or IgG2a.

41. An antibody or antigen-binding fragment according to any one of claims 21 to 40, which comprises one or more constant domains from an immunoglobulin constant region (Fc).

42. An antibody or antigen-binding fragment according to claim 41, wherein the constant domain is a human constant domain.

43. An antibody or antigen-binding fragment according to claim 42, wherein the human constant domain is an IgA, IgD, IgE, IgG or IgM domain.

44. An antibody or antigen-binding fragment according to claim 43, wherein the human IgG constant domain is an IgG1, IgG2, IgG3 or IgG4 domain.

45. An antibody or antigen-binding fragment according to any one of claims 21 to 44, wherein the antibody or antigen-binding fragment is detectably labeled or comprises a conjugated toxin, drug, receptor, enzyme, receptor ligand.

46. An antibody or antigen-binding fragment according to any one of claims 21 to 45, wherein the antibody is a monoclonal antibody, human antibody, chimeric antibody or humanized antibody.

47. An antibody or antigen-binding fragment according to any one of claims 21 to 46, wherein the antibody is a bispecific, trispecific or multispecific antibody.

48. An antibody or antigen-binding fragment according to claim 47, wherein the bispecific, trispecific or multispecific antibody comprises a second antigen-binding fragment that binds to a cell-specific antigen.

49. A bispecific, trispecific or multispecific antibody that comprises a first antigen-binding fragment that binds to ApoL1 or an ApoL1-containing complex, optionally TLF, and a second antigen-binding fragment that binds to a cell-specific antigen.

50. An antibody or antigen-binding fragment according to claim 48 or 49, wherein the cell-specific antigen is a cancer or tumor antigen.

51. The antibody or antigen-binding fragment according to claim 50, wherein the cancer or tumor antigen is a blood cancer antigen or a solid tumor antigen optionally selected from Claudin 18.2, MUC1, Mesothelin (MSLN), Myoferlin (MYOF), and PMEL17.

52. The antibody or antigen-binding fragment according to claim 51, wherein the blood cancer antigen is selected from the group consisting of: BCMA, CD38, CD319 / SLAMF-7, TNFRSF17 / BCMA, SYND1 / CD138, CD229, CD47, CD123 / IL3-RA, CD19, CD20, CD22, FcRH5, GPRC5D, CLL-1, PD-L1, and CTLA4.

53. The antibody or antigen-binding fragment according to any one of claims 48 to 52, wherein the cell-specific antigen is BCMA.

54. The antibody or antigen-binding fragment according to claim 28, wherein the second antigen-binding fragment comprises the heavy chain variable domain of SEQ ID NO: 41 or three CDRs of a variant or humanized form thereof having at least 70%, 80%, 90%, or 95% sequence identity thereto, and the light chain variable domain of SEQ ID NO: 42 or three CDRs of a variant or humanized form thereof having at least 70%, 80%, 90%, or 95% sequence identity thereto.

55. The antibody or antigen-binding fragment according to claim 54, wherein the second antigen-binding fragment comprises six CDRs, and the six CDRs comprise the following amino acid sequences: CDR1H: SYAMS (SEQ ID NO: 43) or a variant or humanized form thereof having at least 70%, 80%, 90%, or 95% sequence identity thereto, CDR2H: AISGSGGSTYYADSVKG (SEQ ID NO: 44) or a variant or humanized form thereof having at least 70%, 80%, 90%, or 95% sequence identity thereto, CDR3H: VAPYFAPFDY (SEQ ID NO: 45) or a variant or humanized form thereof having at least 70%, 80%, 90%, or 95% sequence identity thereto, CDR1L: RASQSVSSSYLA (SEQ ID NO: 46) or a variant or humanized form thereof having at least 70%, 80%, 90%, or 95% sequence identity thereto, CDR2L: GASSRAT (SEQ ID NO: 47) or a variant or humanized form thereof having at least 70%, 80%, 90%, or 95% sequence identity thereto, and CDR3L: QQYGNPPLYT (SEQ ID NO: 48) or a variant or humanized form thereof having at least 70%, 80%, 90%, or 95% sequence identity thereto.

56. An antibody or antigen-binding fragment according to any one of claims 53 to 55, wherein the second antigen-binding fragment comprises a heavy-chain variable domain and a light-chain variable domain, the heavy-chain variable domain comprising the amino acid sequence of SEQ ID NO: 41 or a variant or humanized form thereof having at least 70%, 80%, 90% or 95% sequence identity thereto, and the light-chain variable domain comprising the amino acid sequence of SEQ ID NO: 42 or a variant or humanized form thereof having at least 70%, 80%, 90% or 95% sequence identity thereto.

57. An antibody or antigen-binding fragment according to claim 56, wherein the second antigen-binding fragment comprises a heavy-chain variable domain and a light-chain variable domain, the heavy-chain variable domain comprising the amino acid sequence of SEQ ID NO: 41 and the light-chain variable domain comprising the amino acid sequence of SEQ ID NO: 42, optionally wherein the second antigen-binding fragment comprises the amino acid sequence of SEQ ID NO:

51.

58. An antibody or antigen-binding fragment according to any one of claims 49 to 56, which comprises the amino acid sequence of SEQ ID NO: 71 or a variant or humanized form thereof having at least 70%, 80%, 90% or 95% sequence identity thereto, and / or the amino acid sequence of SEQ ID NO: 72 or a variant or humanized form thereof having at least 70%, 80%, 90% or 95% sequence identity thereto.

59. An antibody or antigen-binding fragment according to any one of claims 49 to 56, which comprises the amino acid sequences of SEQ ID NO: 71 and SEQ ID NO:

72.

60. An antibody or antigen-binding fragment according to any one of claims 49 to 56, which comprises two copies of each of the amino acid sequences of SEQ ID NO: 70 and SEQ ID NO:

72.

61. An anti-ApoL1, anti-cell-specific antigen IgG1-scFv bispecific chimeric antibody.

62. An anti-Hpr, anti-cell-specific antigen IgG1-scFv bispecific chimeric antibody.

63. A nucleic acid encoding an antibody or antigen-binding fragment according to any one of claims 21 to 63.

64. The nucleic acid according to claim 63, which is operably linked to an expression control sequence.

65. An expression vector comprising the nucleic acid according to claim 63 or 64.

66. A cell comprising the nucleic acid according to claim 63 or 64 or the expression vector according to claim 65, optionally wherein the cell is a mammalian cell.

67. An immune complex comprising an antibody or antigen-binding fragment according to any one of claims 21 to 62 that binds to ApoL1 or an ApoL1-containing complex, optionally wherein the complex is TLF.

68. An immune complex comprising an antibody or antigen-binding fragment according to any one of claims 47 to 62.

69. A method of inducing cell death, the method comprising contacting a target cell with an immune complex according to claim 67 or 68.

70. The method according to claim 69, wherein the contacting occurs in vitro.

71. The method according to claim 69, wherein the contraction occurs in a subject.

72. The method according to claim 71, wherein the subject has cancer.

73. A pharmaceutical composition comprising an antibody or antigen-binding fragment according to any one of claims 21 to 62.

74. A method of treating cancer, the method comprising administering to a subject an effective amount of an antibody or antigen-binding fragment according to any one of claims 21 to 62.

75. The method according to claim 74, which comprises administering to the subject an effective amount of an antibody or antigen-binding fragment according to any one of claims 22 to 62.

76. The method according to claim 74 or 75, wherein the cancer is a blood cancer.

77. The method according to claim 76, wherein the cancer is multiple myeloma, leukemia (e.g., chronic lymphocytic leukemia, acute myeloid leukemia, acute lymphoblastic leukemia), non-Hodgkin lymphoma, Hodgkin lymphoma, myelodysplastic syndrome (MDS), myeloproliferative neoplasm (MPN) (or its subcategories, e.g., essential thrombocythemia (ET), myelofibrosis (MF), and polycythemia vera (PV)), amyloidosis, Waldenström macroglobulinemia, or aplastic anemia.

78. The method according to claim 74 or 75, wherein the cancer is a solid cancer.

79. A method of treating a subject in need thereof, the method comprising administering to the subject a composition according to any one of claims 1 to 62.

80. The method according to claim 79, wherein the subject has cancer.

81. The method according to any one of claims 1 to 5 or 11 to 17, wherein the composition comprises an antibody according to any one of claims 21 to 62.

82. The composition or method according to any one of claims 1 to 81, wherein the target cell is a mammalian cell.

83. The composition or method according to claim 82, wherein the mammalian cell is an infected cell.

84. The composition or method according to claim 83, wherein the infected cell is infected with a virus, bacterium, or eukaryotic intracellular organism, which is optionally selected from HIV, Plasmodium falciparum, Toxoplasma gondii, Leishmania spp., Trypanosoma cruzi, Listeria monocytogenes, Chlamydia trachomatis, Coxiella burnetii, Mycobacterium tuberculosis, and Trichomonas vaginalis.

85. The composition or method according to any one of claims 1 to 81, wherein the target cell is a non-mammalian cell.

86. The composition or method according to claim 85, wherein the non-mammalian cell is a bacterium, fungus, or non-mammalian eukaryotic cell.

87. The composition or method according to claim 86, wherein the non-mammalian cell is not a Trypanosoma.

88. The composition or method according to any one of the preceding claims, wherein the subject is a mammal, optionally a human.

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