Compositions and methods for channeling apolipoprotein L1 to induce mammalian cell death - Patents.com

Abstract

Provided herein are compositions for increasing apolipoprotein L1 (ApoL1) in target cells. ApoL1 can be a recombinant protein or, optionally, an endogenous protein in an ApoL1-containing complex. Antibodies and other binding molecules specifically bind to apolipoprotein L1 (ApoL1) and haptoglobin-related protein (Hpr). In preferred embodiments, the antibodies and other molecules bind to ApoL1-containing complexes, such as trypanosoma lytic factor (TLF), preferably under physiological conditions. In preferred embodiments, the antibodies and antigen-binding fragments are bispecific, trispecific, and multispecific molecules that bind to ApoL1-containing complexes and can further bind to cell-specific antigens. Also provided are methods for using such molecules to increase the flux of ApoL1-containing complexes to target cells expressing cell-specific antigens.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of and priority to U.S. Provisional Application No. 63 / 374,356, filed September 1, 2022, which is incorporated herein by reference in its entirety.

[0002] Sequence Listing Reference The Sequence Listing, submitted as a text file named "UGA_2022-004-02_PCT_ST26.xml," created on September 1, 2023, and having a size of 81,564 bytes, is incorporated herein by reference in accordance with 37 CFR § 1.52(e)(5).

[0003] STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT 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)).

[0004] 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 technology]

[0005] Active multiple myeloma (MM) is an incurable malignant tumor of plasma cells, accounting for approximately 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, along with hypercalcemia, renal failure, anemia, and osteolytic lesions (see Mikhael, et al., Clin Lymphoma Myeloma Leuk., 20:1-7 (2020)). It claims the lives of more than 12,000 people in the United States annually (Siegel, et al., CA Cancer J Clin., 72:7-33 (2022)). Disease progression is due to resistance to single treatment strategies, including stem cell transplants, small molecule drugs, and biologics. Furthermore, a growing subset of patients, approximately 45,000 per year, experience triple and quadruple refractory responses, termed relapsed and refractory multiple myeloma (RRMM), in which all previously used tactics fail, thereby highlighting the need for new therapeutic concepts (Sonneveld, et al., Haematologica, 101:396-406 (2016)).

[0006] Due to an aging population and improved diagnostic capabilities, the number of reported MM cases is expected to increase over the next few years. The emergence of resistance to previous treatments necessitates the need for multiple therapeutic options to be available for further rounds of treatment. Patient responses vary with each line of therapy, with 74% of patients experiencing a very good partial response after the first line and only 11% experiencing a good partial response after the fifth line (Sonneveld, et al., Haematologica, 101:396-406 (2016)). Time to progression (TTP) is the time from the start of treatment to disease progression. TTP between treatment lines decreases with each line, from 18 months after the first line to 13 months after the second line, 7 months after the third line, and only 5 months during subsequent lines. Each line of treatment must be evaluated to determine what combination therapy can be used based on each patient's response during the previous line. It is these multiple lines of treatment with a wide range of therapies to choose from that has made the increase in survival rates possible.

[0007] The emergence of new treatment options has led to increased survival, with 5-year survival rates increasing from 24% in the 1980s to 50% today, with median survival ranging from 29 to 62 months (Wong, et al., Blood, 132 Supplement 1:4773 (2018)). However, this increase in survival has led to a significant increase in the costs associated with treating this disease due to the multiple treatment regimens required after many rounds of recurrence. Therefore, there remains a need for additional lines of treatment with different mechanisms of action, especially for subsequent lines of treatment when standard treatments are not an option due to reduced efficacy or deterioration of the patient's health. It is therefore an object of the present invention to provide alternative compositions and methods for the treatment of multiple myeloma and other cancers. [Prior art documents] [Non-patent literature]

[0008] [Non-Patent Document 1] Siegel,et al.,CA Cancer J Clin.,67:7-30(2017) [Non-patent document 2] Mikhael,et al.,Clin Lymphoma Myeloma Leuk.,20:1-7(2020) [Non-patent document 3] Siegel,et al.,CA Cancer J Clin.,72:7-33(2022) [Non-patent document 4] Sonneveld,et al.,Haematologica,101:396-406(2016) [Non-Patent Document 5] Wong,et al.,Blood,132 Supplement 1:4773(2018) Summary of the Invention [Means for solving the problem]

[0009] Compositions and methods of use thereof for increasing cell death of target cells in a mammalian subject, such as a human, in need thereof. The methods typically involve administering to a subject an effective amount of a composition that increases apolipoprotein L1 (ApoL1) (e.g., endogenous or exogenous ApoL1) in target cells. Compositions that bind to both ApoL1 or trypanosoma lytic factor (TLF), optionally an ApoL1-containing complex such as TLF-1 and / or TLF-2, and a cell-specific antigen are provided. Preferred compositions are bispecific and multispecific antibodies having 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.

[0010] In other embodiments, the composition comprises ApoL1 or a functional fragment or variant thereof and a targeting moiety against a cell-specific antigen. The ApoL1 or 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 polymeric nanoparticle. The targeting moiety can be conjugated or fused to the delivery vehicle. Preferred targeting moieties are antibodies and antigen-binding fragments.

[0011] The target cell can be a mammalian cell or a non-mammalian cell. The mammalian cell can be a diseased cell (e.g., cancerous) or infected cell. The non-mammalian cell can be, for example, a bacterial, fungal, or non-mammalian eukaryotic cell. The cell can be a human cell.

[0012] The cell-specific antigen can be specific to diseased cells. The diseased cells can be cancer cells, such as blood cancer cells and solid tumor cells. In some embodiments, the subject is suffering from a disease caused by the target cells, 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.

[0013] Also provided herein are antibodies and other binding molecules that specifically bind to apolipoprotein L1 (ApoL1) and haptoglobin-related protein (Hpr). In preferred embodiments, the antibodies and other molecules bind to ApoL1-containing complexes, such as trypanosoma lytic factor (TLF). Preferably, the antibodies and other molecules bind to ApoL1-containing complexes, such as TLF, in vivo under physiological conditions, including, but not limited to, endogenous complexes.

[0014] The antibody or antigen-binding fragment may be or comprise an anti-ApoL1 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 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 CDRs of the heavy chain variable domain and the light chain variable domain are 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; 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 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 thereof having at least 70, 80, 90, or 95% sequence identity thereto.

[0015] In some embodiments, the antibody or antigen-binding fragment comprises a 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 a 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.

[0016] The antibody or antigen-binding fragment may be or comprise an 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 CDRs of the heavy and light chain variable domains are 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 a variant 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 a variant 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 a variant thereof having at least 70, 80, 90, or 95% sequence identity thereto.

[0017] In some embodiments, the antibody or antigen-binding fragment comprises a 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 a 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.

[0018] The antibody or antigen-binding fragment may be or comprise an anti-Hpr antibody or antigen-binding fragment comprising the 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 the 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 CDRs of the heavy and light chain variable domains are: 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; 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 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.

[0019] In some embodiments, the antibody or antigen-binding fragment comprises a 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 a 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.

[0020] The antibody or antigen-binding fragment can comprise one or more constant domains from an immunoglobulin constant region (Fc), optionally where the constant domains are human. In some embodiments, the constant domain is an IgA constant domain, an IgD constant domain, an IgE constant domain, an IgG constant domain, or an IgM constant domain. In some embodiments, the antibody or antigen-binding fragment comprises one or more human IgG constant domains, optionally an IgG1 domain, an IgG2 domain, an IgG3 domain, or an IgG4 domain. In some embodiments, the antibody or antigen-binding fragment is not a murine IgG1 or IgG2a.

[0021] The antibody or antigen-binding fragment may be detectably labeled or may comprise a conjugated toxin, drug, receptor, enzyme, or receptor ligand.

[0022] In some embodiments, the antibody or antigen-binding fragment is a monoclonal antibody, a human antibody, a chimeric antibody, or a humanized antibody. The antibody or antigen-binding fragment may be a bispecific antibody, a trispecific antibody, or a multispecific antibody. In some embodiments, variants are of the provided sequences and are humanized forms.

[0023] In preferred embodiments, the anti-ApoL1 and / or anti-Hpr antibody or antigen-binding fragment is in a bispecific, trispecific, or multispecific antibody and includes a second (or third) antigen-binding fragment that binds to a cell-specific antigen. Thus, bispecific, trispecific, or multispecific antibodies are provided that have one or more antigen-binding fragments that bind to an ApoL1-containing complex, such as TLF, and a second (or 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 may optionally be a hematological cancer antigen 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. Thus, 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.

[0024] In other embodiments, the tumor antigen is a pancreatic cancer antigen, optionally selected from claudin 18.2, MUC1, mesothelin (MSLN), and myoferlin (MYOF). Thus, 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).

[0025] In other embodiments, the tumor antigen is a melanoma cancer antigen, optionally PMEL17. Thus, in some embodiments, the bispecific or multispecific antibody comprises an antigen-binding fragment that specifically binds to PMEL17.

[0026] In some embodiments, the second antigen-binding fragment is an anti-BCMA antigen-binding fragment comprising 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 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 are 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 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 a 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.

[0027] 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 particular embodiments, the antibody or antigen-binding fragment comprises the amino acid sequences of SEQ ID NO: 71 and SEQ ID NO: 72.

[0028] Particularly exemplary structures are anti-ApoL1, anti-cell-specific antigen IgG1-scFv bispecific chimeric antibody or anti-Hpr, anti-cell-specific antigen IgG1-scFv bispecific chimeric antibody, optionally having the structure of Figure 8.

[0029] In certain embodiments, the bispecific antibody has the structure of Figure 8 and is formed by two copies of the amino acid sequences of SEQ ID NO:71 and SEQ ID NO:72, respectively, for example, formed upon co-expression of a nucleic acid encoding the amino acid sequence of SEQ ID NO:71 (e.g., SEQ ID NO:73) and a nucleic acid encoding the amino acid sequence of SEQ ID NO:72 (e.g., SEQ ID NO:74).

[0030] Nucleic acids, including DNA and RNA, encoding the disclosed antibodies and antigen-binding fragments are also provided. The nucleic acids can be operably linked to expression control sequences. Expression vectors, coding sequences, and cells, e.g., bacterial and mammalian cells, transfected with the nucleic acids and vectors are also provided.

[0031] Also provided are methods of forming immune complexes by contacting the disclosed antibodies and antigen-binding fragments with an ApoL1-containing complex, such as TLF, and optionally immune complexes formed therefrom, optionally further complexed to the surface of a cell. It is believed that such immune complexes, when delivered intracellularly, result in increased cell death. Accordingly, provided are methods of inducing cell death by contacting target cells with immune complexes. Contacting can occur in vitro or in vivo.

[0032] Pharmaceutical compositions comprising effective amounts of the disclosed antibodies and antigen-binding fragments are also provided.

[0033] Methods of treating cancer are also provided and can include administering an effective amount of an antibody or antigen-binding fragment to a subject. Preferably, the antibodies and antigen-binding fragments used in such methods include a second (or more) antigen-binding fragments that bind to a cell-specific antigen, such as a tumor antigen, and enhance delivery of an ApoL1-containing complex, such as TLF, to cells expressing the antigen. See, e.g., Figure 9. The cancer can be a hematological cancer, such as multiple myeloma, leukemia (e.g., chronic lymphocytic leukemia, acute myeloid leukemia, acute lymphoblastic leukemia), non-Hodgkin's lymphoma, Hodgkin's lymphoma, myelodysplastic syndrome (MDS), myeloproliferative neoplasm (MPN) (or subcategories thereof, e.g., essential thrombocythemia (ET), myelofibrosis (MF), and polycythemia vera (PV)), amyloidosis, Waldenström's macroglobulinemia, or aplastic anemia, or a solid tumor. [Brief explanation of the drawings]

[0034] [Figure 1A] Figure 1 shows selected high-density lipoprotein (HDL) complexes containing apolipoprotein L1 (ApoL1). All human circulating innate factors are consistent with all HDL particles, which contain a hydrophobic core and cholesterol. Trypanosome lytic factor 1 (TLF-1) (Figure 1A) is lipid-rich and contains the proteins ApoL1, Hpr (with bound Hb), and ApoA1. Trypanosome lytic factor 2 (TLF-2) (Figure 1B) is lipid-poor and contains unique IGM in addition to the TLF-1 protein. HDL complexes can also contain ApoL1 in the absence of TLF (Figure 1C). While nonlethal at physiological concentrations, a 10-fold increase in TLF-1 results in indiscriminate cell killing. [Figure 1B]Figure 1 shows selected high-density lipoprotein (HDL) complexes containing apolipoprotein L1 (ApoL1). All human circulating innate factors are consistent with all HDL particles, which contain a hydrophobic core and cholesterol. Trypanosome lytic factor 1 (TLF-1) (Figure 1A) is lipid-rich and contains the proteins ApoL1, Hpr (with bound Hb), and ApoA1. Trypanosome lytic factor 2 (TLF-2) (Figure 1B) is lipid-poor and contains unique IGM in addition to the TLF-1 protein. HDL complexes can also contain ApoL1 in the absence of TLF (Figure 1C). While nonlethal at physiological concentrations, a 10-fold increase in TLF-1 results in indiscriminate cell killing. [Figure 1C] Figure 1 shows selected high-density lipoprotein (HDL) complexes containing apolipoprotein L1 (ApoL1). All human circulating innate factors are consistent with all HDL particles, which contain a hydrophobic core and cholesterol. Trypanosome lytic factor 1 (TLF-1) (Figure 1A) is lipid-rich and contains the proteins ApoL1, Hpr (with bound Hb), and ApoA1. Trypanosome lytic factor 2 (TLF-2) (Figure 1B) is lipid-poor and contains unique IGM in addition to the TLF-1 protein. HDL complexes can also contain ApoL1 in the absence of TLF (Figure 1C). While nonlethal at physiological concentrations, a 10-fold increase in TLF-1 results in indiscriminate cell killing. [Figure 2] Figure 1 is a plot showing the binding of TLF-1 to HEK293 cells. Estimated binding values ​​were determined based on half-maximal binding at 3°C ​​using various concentrations of Alexa-488 TLF-1. Saturation was not achieved. 20,000 cells per assay were analyzed in triplicate by flow cytometry. [Figure 3A] 1 is a series of images showing TLF-1 uptake (20 mg / ml) in HEK293 cells over 2 hours (images captured by Amnis). [Figure 3B]3B is a plot quantifying the images in FIG. 3A. Pixel density was calculated by ImageStream 6.0 software and plotted as a percentage of maximum pixel density. [Figure 3C] This is a series of images showing the colocalization of TLF and LysoTracker in live HEK293 cells. To examine TLF uptake in mammalian cells, HEK293 cells were incubated with Alexa Fluor 488-conjugated TLF (AF488 TLF) and imaged via ImageStream. AF488 TLF is endocytosed into vesicles within HEK293 cells. Quantification of signal intensity shows maximum pixel intensity, indicating that TLF is taken up by cells upon uptake at 37°C. When cells were maintained at 3°C, no binding to the cell surface was detected (Figure 3B). [Figure 3D] 1 is a plot showing competition with 2, 10, 50, and 100-fold unlabeled competitor (insoluble HDL by mass or HP-1 by molecular weight) in a low temperature binding assay. [Figure 3E] Figure 1 shows a plot of the time course of TLF-1 uptake in HEK293 cells. Median TLF-1 intensity was measured and quantified using Flowlo 9.6.4 software. Data points represent 20,000 cells per point. [Figure 4A] 1 is a plot showing the viability of HEK293 cells over time (days) after incubation with control (no TLF), non-soluble HDL (ApoL1 (NLHDL), 10 μg / ml TLF, or 75 μg / ml no TLF). [Figure 4B] 1 is a series of microscopy images showing HEK293 cells treated with no TLF (left), non-lytic HDL (75 μg / ml) (center), and TLF (75 μg / ml) (right). [Figure 4C]1 is a bar graph showing the percent reduction in proliferation of CCL-155 multiple myeloma cells after incubation with concentrated purified human TLF (1.26 float fraction, a subfraction of human serum that contains TLF). Increased concentration of the TLF / HDL fraction results in a reduction in cell viability, as measured by CellTiterGlo. ***25% reduction at a total protein content of 4.96 mg / ml. [Figure 4D] Figure 4 shows plots demonstrating that exogenously added ApoL1 reduces proliferation of multiple mammalian cell lines. The selected cell lines represent various cancer models: CCL-155 (RPMI8226): multiple myeloma (Figure 4D), PANC-1: pancreatic (Figure 4E), A375: melanoma (Figure 4F), and HT144: melanoma (Figure 4G). Specific cell lines were incubated with recombinant ApoL1 for 3 or 4 days, as indicated. Cell viability was assayed on day 4 using CellTiter-Glo. The data show the dose-dependent effect of recombinant ApoL1 on cell proliferation. The LD50 was calculated using the Quest Graph EC50 Calculator using a four-parameter model. The results are as follows: CCL-155: 20.2 μg / mL, PANC-1: 43 μg / mL, A375: 27.5 μg / mL, and HT144: 32.8 μg / mL. Points represent each replicate, and dashed lines represent the mean. Error bars represent standard deviation. [Figure 4H] Plot showing the number of viable RPMI8226 (CCL-155) cells after incubation with medium containing purified human TLF following immunoprecipitation using various concentrations of anti-ApoL1 (μg / ml) antibody. [Figure 4E]Figure 4 shows plots demonstrating that exogenously added ApoL1 reduces proliferation of multiple mammalian cell lines. The selected cell lines represent various cancer models: CCL-155 (RPMI8226): multiple myeloma (Figure 4D), PANC-1: pancreatic (Figure 4E), A375: melanoma (Figure 4F), and HT144: melanoma (Figure 4G). Specific cell lines were incubated with recombinant ApoL1 for 3 or 4 days, as indicated. Cell viability was assayed on day 4 using CellTiter-Glo. The data show the dose-dependent effect of recombinant ApoL1 on cell proliferation. The LD50 was calculated using the Quest Graph EC50 Calculator using a four-parameter model. The results are as follows: CCL-155: 20.2 μg / mL, PANC-1: 43 μg / mL, A375: 27.5 μg / mL, and HT144: 32.8 μg / mL. Points represent each replicate, and dashed lines represent the mean. Error bars represent standard deviation. [Figure 4H] Plot showing the number of viable RPMI8226 (CCL-155) cells after incubation with medium containing purified human TLF following immunoprecipitation using various concentrations of anti-ApoL1 (μg / ml) antibody. [Figure 4F]Figure 4 shows plots demonstrating that exogenously added ApoL1 reduces proliferation of multiple mammalian cell lines. The selected cell lines represent various cancer models: CCL-155 (RPMI8226): multiple myeloma (Figure 4D), PANC-1: pancreatic (Figure 4E), A375: melanoma (Figure 4F), and HT144: melanoma (Figure 4G). Specific cell lines were incubated with recombinant ApoL1 for 3 or 4 days, as indicated. Cell viability was assayed on day 4 using CellTiter-Glo. The data show the dose-dependent effect of recombinant ApoL1 on cell proliferation. The LD50 was calculated using the Quest Graph EC50 Calculator using a four-parameter model. The results are as follows: CCL-155: 20.2 μg / mL, PANC-1: 43 μg / mL, A375: 27.5 μg / mL, and HT144: 32.8 μg / mL. Points represent each replicate, and dashed lines represent the mean. Error bars represent standard deviation. [Figure 4H] Plot showing the number of viable RPMI8226 (CCL-155) cells after incubation with medium containing purified human TLF following immunoprecipitation using various concentrations of anti-ApoL1 (μg / ml) antibody. [Figure 4G] Figure 4 shows plots demonstrating that exogenously added ApoL1 reduces proliferation of multiple mammalian cell lines. The selected cell lines represent various cancer models: CCL-155 (RPMI8226): multiple myeloma (Figure 4D), PANC-1: pancreatic (Figure 4E), A375: melanoma (Figure 4F), and HT144: melanoma (Figure 4G). Specific cell lines were incubated with recombinant ApoL1 for 3 or 4 days, as indicated. Cell viability was assayed on day 4 using CellTiter-Glo. The data show the dose-dependent effect of recombinant ApoL1 on cell proliferation. The LD50 was calculated using the Quest Graph EC50 Calculator using a four-parameter model. The results are as follows: CCL-155: 20.2 μg / mL, PANC-1: 43 μg / mL, A375: 27.5 μg / mL, and HT144: 32.8 μg / mL. Points represent individual replicates, dashed lines represent the mean, and error bars represent the standard deviation. [Figure 4H] 1 is a plot showing the number of viable RPMI8226 (CCL-155) cells following incubation with medium containing purified human TLF after immunoprecipitation using various concentrations of anti-ApoL1 antibody (μg / ml). [Figure 4I] 1 is a plot showing the number of viable RPMI8226 (CCL-155) cells following incubation with medium containing purified human TLF after immunoprecipitation using various concentrations of anti-Hpr (μg / ml) antibody. [Figure 5A] Characterization of recombinant anti-Hpr antibodies. Figures 5A and 5D are unstained images of total proteins (TLF and recombinant ApoL1) electrophoresed under non-reducing (Figure 5A) and reducing (Figure 5D) conditions. Figures 5B and 5E are images of Western blots using recombinant anti-Hpr antibodies under non-reducing (Figure 5B) and reducing (Figure 5E) conditions. Figures 5C and 5F are images of Western blots using ascites Prot-G purified anti-Hpr antibodies under non-reducing (Figure 5C) and reducing (Figure 5F) conditions. Figure 5G is a dot blot (native) showing recombinant and ascites purified anti-Hpr antibodies binding to recombinant ApoL1. [Figure 5BC] Characterization of recombinant anti-Hpr antibodies. Figures 5A and 5D are unstained images of total proteins (TLF and recombinant ApoL1) electrophoresed under non-reducing (Figure 5A) and reducing (Figure 5D) conditions. Figures 5B and 5E are images of Western blots using recombinant anti-Hpr antibodies under non-reducing (Figure 5B) and reducing (Figure 5E) conditions. Figures 5C and 5F are images of Western blots using ascites Prot-G purified anti-Hpr antibodies under non-reducing (Figure 5C) and reducing (Figure 5F) conditions. Figure 5G is a dot blot (native) showing recombinant and ascites purified anti-Hpr antibodies binding to recombinant ApoL1. [Figure 5D]Characterization of recombinant anti-Hpr antibodies. Figures 5A and 5D are unstained images of total proteins (TLF and recombinant ApoL1) electrophoresed under non-reducing (Figure 5A) and reducing (Figure 5D) conditions. Figures 5B and 5E are images of Western blots using recombinant anti-Hpr antibodies under non-reducing (Figure 5B) and reducing (Figure 5E) conditions. Figures 5C and 5F are images of Western blots using ascites Prot-G purified anti-Hpr antibodies under non-reducing (Figure 5C) and reducing (Figure 5F) conditions. Figure 5G is a dot blot (native) showing recombinant and ascites purified anti-Hpr antibodies binding to recombinant ApoL1. [Figure 5EF] Characterization of recombinant anti-Hpr antibodies. Figures 5A and 5D are unstained images of total proteins (TLF and recombinant ApoL1) electrophoresed under non-reducing (Figure 5A) and reducing (Figure 5D) conditions. Figures 5B and 5E are images of Western blots using recombinant anti-Hpr antibodies under non-reducing (Figure 5B) and reducing (Figure 5E) conditions. Figures 5C and 5F are images of Western blots using ascites Prot-G purified anti-Hpr antibodies under non-reducing (Figure 5C) and reducing (Figure 5F) conditions. Figure 5G is a dot blot (native) showing recombinant and ascites purified anti-Hpr antibodies binding to recombinant ApoL1. [Figure 5G] Characterization of recombinant anti-Hpr antibodies. Figures 5A and 5D are unstained images of total proteins (TLF and recombinant ApoL1) electrophoresed under non-reducing (Figure 5A) and reducing (Figure 5D) conditions. Figures 5B and 5E are images of Western blots using recombinant anti-Hpr antibodies under non-reducing (Figure 5B) and reducing (Figure 5E) conditions. Figures 5C and 5F are images of Western blots using ascites Prot-G purified anti-Hpr antibodies under non-reducing (Figure 5C) and reducing (Figure 5F) conditions. Figure 5G is a dot blot (native) showing recombinant and ascites purified anti-Hpr antibodies binding to recombinant ApoL1. [Figure 6A]Characterization of recombinant anti-ApoL1 antibodies. Figures 6A and 6D are unstained images of total proteins (TLF and recombinant ApoL1) electrophoresed under non-reducing (Figure 5A) and reducing (Figure 6D) conditions. Figures 6B and 6E are images of Western blots using recombinant anti-ApoL1 antibodies under non-reducing (Figure 6B) and reducing (Figure 6E) conditions. Figures 6C and 6F are images of Western blots using ascites Prot-G purified anti-ApoL1 antibodies under non-reducing (Figure 6C) and reducing (Figure 6F) conditions. Figure 6G is a dot blot (native) showing recombinant and ascites purified anti-ApoL1 antibodies binding to recombinant ApoL1. [Figure 6BC] Characterization of recombinant anti-ApoL1 antibodies. Figures 6A and 6D are unstained images of total proteins (TLF and recombinant ApoL1) electrophoresed under non-reducing (Figure 5A) and reducing (Figure 6D) conditions. Figures 6B and 6E are images of Western blots using recombinant anti-ApoL1 antibodies under non-reducing (Figure 6B) and reducing (Figure 6E) conditions. Figures 6C and 6F are images of Western blots using ascites Prot-G purified anti-ApoL1 antibodies under non-reducing (Figure 6C) and reducing (Figure 6F) conditions. Figure 6G is a dot blot (native) showing recombinant and ascites purified anti-ApoL1 antibodies binding to recombinant ApoL1. [Figure 6D] Characterization of recombinant anti-ApoL1 antibodies. Figures 6A and 6D are unstained images of total proteins (TLF and recombinant ApoL1) electrophoresed under non-reducing (Figure 5A) and reducing (Figure 6D) conditions. Figures 6B and 6E are images of Western blots using recombinant anti-ApoL1 antibodies under non-reducing (Figure 6B) and reducing (Figure 6E) conditions. Figures 6C and 6F are images of Western blots using ascites Prot-G purified anti-ApoL1 antibodies under non-reducing (Figure 6C) and reducing (Figure 6F) conditions. Figure 6G is a dot blot (native) showing recombinant and ascites purified anti-ApoL1 antibodies binding to recombinant ApoL1. [Figure 6EF]Characterization of recombinant anti-ApoL1 antibodies. Figures 6A and 6D are unstained images of total proteins (TLF and recombinant ApoL1) electrophoresed under non-reducing (Figure 5A) and reducing (Figure 6D) conditions. Figures 6B and 6E are images of Western blots using recombinant anti-ApoL1 antibodies under non-reducing (Figure 6B) and reducing (Figure 6E) conditions. Figures 6C and 6F are images of Western blots using ascites Prot-G purified anti-ApoL1 antibodies under non-reducing (Figure 6C) and reducing (Figure 6F) conditions. Figure 6G is a dot blot (native) showing recombinant and ascites purified anti-ApoL1 antibodies binding to recombinant ApoL1. [Figure 6G] Characterization of recombinant anti-ApoL1 antibodies. Figures 6A and 6D are unstained images of total proteins (TLF and recombinant ApoL1) electrophoresed under non-reducing (Figure 5A) and reducing (Figure 6D) conditions. Figures 6B and 6E are images of Western blots using recombinant anti-ApoL1 antibodies under non-reducing (Figure 6B) and reducing (Figure 6E) conditions. Figures 6C and 6F are images of Western blots using ascites Prot-G purified anti-ApoL1 antibodies under non-reducing (Figure 6C) and reducing (Figure 6F) conditions. Figure 6G is a dot blot (native) showing recombinant and ascites purified anti-ApoL1 antibodies binding to recombinant ApoL1. [Figure 7A] Characterization of recombinant anti-BCMA scFvs. Figure 7A is an unstained image of total protein (nr- and r-BCMA). Figures 7B and 7C are images of Western blots utilizing anti-BCMA clone 17A5 scFv (SEQ ID NO: L51) (Figure 7B) and anti-BCMA intact monoclonal antibody (RnD Systems catalog no. MAB1931) (Figure 7C). [Figure 7BC] Characterization of recombinant anti-BCMA scFvs. Figure 7A is an unstained image of total protein (nr- and r-BCMA). Figures 7B and 7C are images of Western blots utilizing anti-BCMA clone 17A5 scFv (SEQ ID NO: L51) (Figure 7B) and anti-BCMA intact monoclonal antibody (RnD Systems catalog no. MAB1931) (Figure 7C). [Figure 8]FIG. 1 is a diagram of an exemplary anti-ApoL1, anti-BCMA antibody having a Fab portion having the heavy and light chain variable regions of recombinant clone 13.11 (anti-ApoL1) having the sequence provided in Example 6, and anti-BCMA as an ScFv (SEQ ID NO: 51) fused to the heavy chain C-terminus of human IgG1, as provided in Example 7. See also Example 8. [Figure 9A] Figure 8 / Example 8 shows the binding of a bispecific antibody designed according to BCMA and apolipoprotein L1. Figure 9A shows the steps involved in a bridging ELISA assay to assess the binding ability of a bispecific antibody (bsAb) between recombinant variations of ApoL1 and BCMA. Figure 9B shows that the bsAb successfully binds to immobilized forms of both ligands (i.e., forms a bridge between the ligands). [Figure 9B] Figure 8 / Example 8 shows the binding of a bispecific antibody designed according to BCMA and apolipoprotein L1. Figure 9A shows the steps involved in a bridging ELISA assay to assess the binding ability of a bispecific antibody (bsAb) between recombinant variations of ApoL1 and BCMA. Figure 9B shows that the bsAb successfully binds to immobilized forms of both ligands (i.e., forms a bridge between the ligands). [Figure 10A]ApoL1-induced cell death process was demonstrated. Using the Promega RealTime-Glo Annexin V apoptosis and necrosis assay, RPMI8226 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 4 h, the cell apoptosis signal was observed to increase by 50-127% over the level of cells alone, with the highest level measured when bsAb was added (Figure 10A). Within 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. Increased necroptotic signaling over ApoL1 alone was measured in cell lines containing bsAb from 4 to 16 hours compared to cells alone. At 16 hours, this effect peaked at 67% for cells containing ApoL1 and bsAb. Points on the graph are shown as the average of four replicates, with error bars representing standard deviation. Data were normalized by setting the baseline to cells alone and then expressed as the mean (average percentage) difference compared to baseline (Figure 10B). [Figure 10B]ApoL1-induced cell death process was demonstrated. Using the Promega RealTime-Glo Annexin V apoptosis and necrosis assay, RPMI8226 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 4 h, the cell apoptosis signal was observed to increase by 50-127% over the level of cells alone, with the highest level measured when bsAb was added (Figure 10A). Within 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. Increased necroptotic signaling over ApoL1 alone was measured in cell lines containing bsAb from 4 to 16 hours compared to cells alone. At 16 hours, this effect peaked at 67% for cells containing ApoL1 and bsAb. Points on the graph are shown as the average of four replicates, with error bars representing standard deviation. Data were normalized by setting the baseline to cells alone and then expressed as the mean (average percentage) difference compared to baseline (Figure 10B). [Figure 11A]Figure 11A demonstrates the binding of ApoL1-BCMA-bsAb and ApoL1-488 to RPMI8226 (CCL-155) multiple myeloma cells. Figure 11A is a pair of scatter plots showing the gating strategy for selecting in-focus cells (gradient RMS) and cells with the correct aspect ratio (aspect ratio) to select single cells and exclude SpeedBeads (aspect ratio). Secondary gating selects cells of the correct size, further excluding SpeedBeads and excluding cells that express the dead cell indicator Zombie NIR dye. Figure 11B is a plot and chart showing labeled bsAb versus labeled isotype control. Cells that passed the gating strategy were measured for 488 excitation / 525 emission intensity to measure bound 488-bsAb or bound 488-IgG1 isotype control. Figure 11C is a plot and chart showing the results of an unlabeled bsAb competition assay, which helps determine whether a bsAb is specifically labeled. Additional unlabeled bsAb was added to the reaction to act as a competitive inhibitor. Increasing amounts of unlabeled bsAb indicate a decrease in bound Alexa-488 intensity. CV is defined as the coefficient of variation, calculated as 100 × (standard deviation / mean). Figures 11D-11E show secondary binding of labeled ApoL1 after recombinant ApoL1-BCMA-bsAb binds to multiple myeloma cells RPMI8226 (ATCC CCL-155). Figure 11D is a pair of scatter plots illustrating the gating strategy for selecting in-focus cells (gradient RMS) and cells with the correct aspect ratio (aspect ratio) to select single cells and exclude SpeedBeads (aspect ratio). Secondary gating further removes SpeedBeads by selecting cells of the correct size and excludes cells that express the dead cell indicator, Zombie NIR dye. Figure 11E is a plot and chart showing measurements of the intensity of ApoL1-488 binding to cell samples previously incubated with ApoL1-BCMA-bsAb or a human IgG1 isotype control. CV is defined as the coefficient of variation, calculated as 100 × (standard deviation / mean). [Figure 11B]Figure 11A demonstrates the binding of ApoL1-BCMA-bsAb and ApoL1-488 to RPMI8226 (CCL-155) multiple myeloma cells. Figure 11A is a pair of scatter plots showing the gating strategy for selecting in-focus cells (gradient RMS) and cells with the correct aspect ratio (aspect ratio) to select single cells and exclude SpeedBeads (aspect ratio). Secondary gating selects cells of the correct size, further excluding SpeedBeads and excluding cells that express the dead cell indicator Zombie NIR dye. Figure 11B is a plot and chart showing labeled bsAb versus labeled isotype control. Cells that passed the gating strategy were measured for 488 excitation / 525 emission intensity to measure bound 488-bsAb or bound 488-IgG1 isotype control. Figure 11C is a plot and chart showing the results of an unlabeled bsAb competition assay, which helps determine whether a bsAb is specifically labeled. Additional unlabeled bsAb was added to the reaction to act as a competitive inhibitor. Increasing amounts of unlabeled bsAb indicate a decrease in bound Alexa-488 intensity. CV is defined as the coefficient of variation, calculated as 100 × (standard deviation / mean). Figures 11D-11E show secondary binding of labeled ApoL1 after recombinant ApoL1-BCMA-bsAb binds to multiple myeloma cells RPMI8226 (ATCC CCL-155). Figure 11D is a pair of scatter plots illustrating the gating strategy for selecting in-focus cells (gradient RMS) and cells with the correct aspect ratio (aspect ratio) to select single cells and exclude SpeedBeads (aspect ratio). Secondary gating further removes SpeedBeads by selecting cells of the correct size and excludes cells that express the dead cell indicator, Zombie NIR dye. Figure 11E is a plot and chart showing measurements of the intensity of ApoL1-488 binding to cell samples previously incubated with ApoL1-BCMA-bsAb or a human IgG1 isotype control. CV is defined as the coefficient of variation, calculated as 100 × (standard deviation / mean). [Figure 11C]Figure 11A demonstrates the binding of ApoL1-BCMA-bsAb and ApoL1-488 to RPMI8226 (CCL-155) multiple myeloma cells. Figure 11A is a pair of scatter plots showing the gating strategy for selecting in-focus cells (gradient RMS) and cells with the correct aspect ratio (aspect ratio) to select single cells and exclude SpeedBeads (aspect ratio). Secondary gating selects cells of the correct size, further excluding SpeedBeads and excluding cells that express the dead cell indicator Zombie NIR dye. Figure 11B is a plot and chart showing labeled bsAb versus labeled isotype control. Cells that passed the gating strategy were measured for 488 excitation / 525 emission intensity to measure bound 488-bsAb or bound 488-IgG1 isotype control. Figure 11C is a plot and chart showing the results of an unlabeled bsAb competition assay, which helps determine whether a bsAb is specifically labeled. Additional unlabeled bsAb was added to the reaction to act as a competitive inhibitor. Increasing amounts of unlabeled bsAb indicate a decrease in bound Alexa-488 intensity. CV is defined as the coefficient of variation, calculated as 100 × (standard deviation / mean). Figures 11D-11E show secondary binding of labeled ApoL1 after recombinant ApoL1-BCMA-bsAb binds to multiple myeloma cells RPMI8226 (ATCC CCL-155). Figure 11D is a pair of scatter plots illustrating the gating strategy for selecting in-focus cells (gradient RMS) and cells with the correct aspect ratio (aspect ratio) to select single cells and exclude SpeedBeads (aspect ratio). Secondary gating further removes SpeedBeads by selecting cells of the correct size and excludes cells that express the dead cell indicator, Zombie NIR dye. Figure 11E is a plot and chart showing measurements of the intensity of ApoL1-488 binding to cell samples previously incubated with ApoL1-BCMA-bsAb or a human IgG1 isotype control. CV is defined as the coefficient of variation, calculated as 100 × (standard deviation / mean). [Figure 11D]Figure 11A demonstrates the binding of ApoL1-BCMA-bsAb and ApoL1-488 to RPMI8226 (CCL-155) multiple myeloma cells. Figure 11A is a pair of scatter plots showing the gating strategy for selecting in-focus cells (gradient RMS) and cells with the correct aspect ratio (aspect ratio) to select single cells and exclude SpeedBeads (aspect ratio). Secondary gating selects cells of the correct size, further excluding SpeedBeads and excluding cells that express the dead cell indicator Zombie NIR dye. Figure 11B is a plot and chart showing labeled bsAb versus labeled isotype control. Cells that passed the gating strategy were measured for 488 excitation / 525 emission intensity to measure bound 488-bsAb or bound 488-IgG1 isotype control. Figure 11C is a plot and chart showing the results of an unlabeled bsAb competition assay, which helps determine whether a bsAb is specifically labeled. Additional unlabeled bsAb was added to the reaction to act as a competitive inhibitor. Increasing amounts of unlabeled bsAb indicate a decrease in bound Alexa-488 intensity. CV is defined as the coefficient of variation, calculated as 100 × (standard deviation / mean). Figures 11D-11E show secondary binding of labeled ApoL1 after recombinant ApoL1-BCMA-bsAb binds to multiple myeloma cells RPMI8226 (ATCC CCL-155). Figure 11D is a pair of scatter plots illustrating the gating strategy for selecting in-focus cells (gradient RMS) and cells with the correct aspect ratio (aspect ratio) to select single cells and exclude SpeedBeads (aspect ratio). Secondary gating further removes SpeedBeads by selecting cells of the correct size and excludes cells that express the dead cell indicator, Zombie NIR dye. Figure 11E is a plot and chart showing measurements of the intensity of ApoL1-488 binding to cell samples previously incubated with ApoL1-BCMA-bsAb or a human IgG1 isotype control. CV is defined as the coefficient of variation, calculated as 100 × (standard deviation / mean). [Figure 11E]Figure 11A demonstrates the binding of ApoL1-BCMA-bsAb and ApoL1-488 to RPMI8226 (CCL-155) multiple myeloma cells. Figure 11A is a pair of scatter plots showing the gating strategy for selecting in-focus cells (gradient RMS) and cells with the correct aspect ratio (aspect ratio) to select single cells and exclude SpeedBeads (aspect ratio). Secondary gating selects cells of the correct size, further excluding SpeedBeads and excluding cells that express the dead cell indicator Zombie NIR dye. Figure 11B is a plot and chart showing labeled bsAb versus labeled isotype control. Cells that passed the gating strategy were measured for 488 excitation / 525 emission intensity to measure bound 488-bsAb or bound 488-IgG1 isotype control. Figure 11C is a plot and chart showing the results of an unlabeled bsAb competition assay, which helps determine whether a bsAb is specifically labeled. Additional unlabeled bsAb was added to the reaction to act as a competitive inhibitor. Increasing amounts of unlabeled bsAb indicate a decrease in bound Alexa-488 intensity. CV is defined as the coefficient of variation, calculated as 100 × (standard deviation / mean). Figures 11D-11E show secondary binding of labeled ApoL1 after recombinant ApoL1-BCMA-bsAb binds to multiple myeloma cells RPMI8226 (ATCC CCL-155). Figure 11D is a pair of scatter plots illustrating the gating strategy for selecting in-focus cells (gradient RMS) and cells with the correct aspect ratio (aspect ratio) to select single cells and exclude SpeedBeads (aspect ratio). Secondary gating further removes SpeedBeads by selecting cells of the correct size and excludes cells that express the dead cell indicator, Zombie NIR dye. Figure 11E is a plot and chart showing measurements of the intensity of ApoL1-488 binding to cell samples previously incubated with ApoL1-BCMA-bsAb or a human IgG1 isotype control. CV is defined as the coefficient of variation, calculated as 100 × (standard deviation / mean). [Figure 12]

[0023] Figure 1 is an overview of an exemplary proposed therapeutic method and the mechanism that follows: (A) A bispecific antibody (bsAb) (e.g., TLF and a target cell marker such as BCMA) is injected into a subject, (B) binds to endogenous TLF, and (C) tethers it to the surface of target cells (e.g., myeloma cells). (D) Target cell marker (e.g., BCMA)-mediated endocytosis internalizes the immune complex, increasing the intracellular concentration of TLF with lysosomes above the viability threshold, ultimately resulting in (E) target cell death. DETAILED DESCRIPTION OF THE INVENTION

[0035] I. Definition As used herein, the term "bind," in reference to the interaction of a binding protein with an antigen, means that the interaction is dependent on the presence of a particular structure (e.g., an antigenic determinant or epitope) on the antigen. For example, a binding protein recognizes and binds to a particular antigenic structure rather than the antigen in general. For example, if a binding protein binds to epitope "A," the presence of a molecule containing epitope "A" (or free, unlabeled "A") in a reaction involving labeled "A" and the binding protein will reduce the amount of labeled "A" bound to the binding protein.

[0036] As used herein, a molecule is said to be capable of "immunospecifically binding" to a second molecule if such binding exhibits the specificity and affinity of the antibody for its cognate antigen. An antibody is said to be capable of "immunospecifically binding" to a target region or conformation ("epitope") of an antigen (particularly an Hpr or ApoL1 antigen) if such binding involves the antigen recognition site of an immunoglobulin molecule. An antibody that immunospecifically binds to a particular antigen may bind other antigens with lower affinity if the other antigens share some sequence or conformational similarity recognized by the antigen recognition site, as determined, for example, by immunoassay, BIACORE® assay, or other assay known in the art, but do not bind to completely unrelated antigens. Preferably, however, antibodies (and their antigen-binding fragments) do not cross-react with other antigens. Antibodies may also bind other molecules in a non-immunospecific manner, such as FcR receptors, by binding domains in other regions / domains of the molecule that do not involve an antigen recognition site, such as the Fc region.

[0037] The term "substantially" as used in the context of binding or an effect exhibited is intended to indicate that the effect observed is physiologically or therapeutically relevant. Similarly, a molecule is said to have substantially the same immunospecificity and / or properties as another molecule if such immunospecificity and properties are greater than 60% identity, greater than 70% identity, greater than 75% identity, greater than 80% identity, greater than 85% identity, greater than 90% identity, greater than 95% identity, or greater than 97% identity).

[0038] As used herein, the term "antibody" is intended to refer to an immunoglobulin molecule having a "variable region" antigen recognition site. The term antibody includes monoclonal antibodies, polyspecific 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. Pat. No. 6,005,079), single-chain Fvs (scFv) (see, e.g., Pluckthunin, The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds. Springer-Verlag, New York, 1999; and U.S. Pat. No. 6,005,079). New York, pp. 269-315 (1994)), single chain antibodies, disulfide-linked Fvs (sdFvs), intrabodies, and anti-idiotypic (anti-Id) antibodies (including, for example, anti-Ids and anti-Id antibodies to the disclosed antibodies). In particular, 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 subclass.

[0039] The term "variable region" is intended to distinguish such domains of immunoglobulins from domains broadly shared by antibodies (such as antibody Fc domains). The variable region refers to the portion of the light and / or heavy chain of an antibody as defined herein that specifically binds to an antigen and includes, for example, the amino acid sequences of the CDRs, i.e., CDR1, CDR2, and CDR3, and framework regions (FRs). For example, a variable region can include three CDRs as well as three or four FRs (e.g., FR1, FR2, FR3, and optionally FR4). VH refers to the variable region of the heavy chain. VL refers to the variable region of the light chain. The variable region includes a "hypervariable region" whose residues are involved in antigen binding.

[0040] The hypervariable region comprises amino acid residues from a "complementarity determining region" or "CDR" (e.g., typically residues about 24-34 (L1), 50-56 (L2), and 89-97 (L3) in the light chain variable domain and about 27-35 (H1), 50-65 (H2), and 95-102 (H3) in the heavy chain variable domain, according to Kabat; Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD. (1991)), and / or amino acid residues from a "hypervariable loop" (e.g., Chothia; Chothia and According to Lesk, 1987, J. Mol. Biol. 196:901-917, these include residues 26-32 (L1), 50-52 (L2), and 91-96 (L3) in the light chain variable domain, and 26-32 (H1), 53-55 (H2), and 96-101 (H3) in the heavy chain variable domain. Designations involving modified or alternative numbering systems for variable domains include Kabat and Chothia, as well as IMGT (Lefranc, et al. (2003), Dev Comp Immunol 27:55-77), Chothia (Chothia C, Lesk AM (1987), J Mol Biol 196:901-917; Chothia, et al. (1989), Nature 342:877-883), and AHo (Honegger A, Plueckthun A (2001) J Mol Biol 309:657-670). For convenience, examples of binding proteins of the present disclosure may be labeled according to Kabat, Chothia, or IMGT. These examples will be explicitly designated as such.

[0041] "Framework" or "FR" residues are those variable domain residues other than the hypervariable region residues as herein defined.

[0042] As used herein, the term "antigen-binding fragment" of an antibody refers to one or more portions of an antibody that contain the antibody's complementarity-determining regions ("CDRs"), optionally framework residues comprising the antibody's "variable region" antigen recognition sites, and that exhibit the ability to immunospecifically bind to an antigen. Such fragments include Fab, Fab', F(ab')2, and Fv fragments, diabodies, linear antibodies, single-chain antibody molecules formed from antibody fragments, and multispecific antibodies, as well as mutants, naturally occurring variants thereof, and fusion proteins comprising the antibody's "variable region" antigen recognition sites and a heterologous protein (e.g., a toxin, an antigen recognition site for 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 (scFvs), as well as bivalent (di-scFv) and trivalent (tri-scFv) forms thereof. Such fragments can be produced by various methods known in the art.

[0043] As used herein, the term "constant region" refers to a portion of an antibody heavy or light chain other than the variable region. In a heavy chain, the constant region generally includes multiple constant domains and a hinge region; for example, an IgG constant region includes the following linked components: constant heavy C H 1. Linker, C H 2, and C H In the heavy chain, the constant region comprises Fc. In the light chain, the constant region generally comprises one constant domain (CL1).

[0044] The terms "crystallizable fragment" or "Fc" or "Fc region" or "Fc portion" (which can be used interchangeably herein) refer to the region of an antibody that comprises at least one constant domain, is generally (but not necessarily) glycosylated, and is 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 the five isotypes: α, δ, ε, γ, or μ. Exemplary heavy chain constant regions are gamma 1 (IgG1), gamma 2 (IgG2), and gamma 3 (IgG3), or hybrids thereof.

[0045] A "constant domain" is a domain in antibodies that is very similar in sequence in the same type of antibody / antibodies, e.g., IgG or IgM or IgE. The constant region of an antibody generally comprises multiple constant domains, e.g., the constant region of a gamma, alpha, or delta heavy chain comprises two constant domains.

[0046] The terms "full-length antibody," "intact antibody," or "whole antibody" are used interchangeably to refer to an antibody in substantially intact form, as opposed to an antigen-binding fragment thereof. Specifically, a whole antibody includes an antibody having heavy and light chains, including an Fc region. The constant domains may be wild-type sequence constant domains (e.g., human wild-type sequence constant domains) or amino acid sequence variants thereof.

[0047] A "chimeric antibody" is a molecule in which different portions of the antibody are derived from different immunoglobulin molecules, such as antibodies 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, e.g., 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 comprising one or more CDRs from a non-human species and a framework region 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, WO 91 / 09967, and U.S. Pat. Nos. 5,225,539, 5,530,101, and 5,585,089), veneering or 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. Pat. No. 5,565,332).

[0048] As used herein, the term "humanized antibody" refers to an immunoglobulin comprising a human framework region and one or more CDRs from a non-human (usually mouse or rat) immunoglobulin. The non-human immunoglobulin providing the CDRs is called the "donor" and the human immunoglobulin providing the framework is called the "acceptor."

[0049] As used herein, the term "fragment" refers to a peptide or polypeptide comprising an amino acid sequence of 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.

[0050] As used herein, the term "fusion protein" refers to a polypeptide formed by joining two or more polypeptides via a peptide bond formed between the amino terminus of one polypeptide and the carboxyl terminus of another polypeptide, or by linking one polypeptide to another via a reaction between amino acid side chains (e.g., a disulfide bond between cysteine ​​residues on each polypeptide). Fusion proteins can be formed by chemical coupling of the constituent polypeptides, or can be expressed as a single polypeptide from a nucleic acid sequence encoding a single, contiguous fusion protein. Fusion proteins can be prepared using conventional techniques in molecular biology to join two genes in frame into a single nucleic acid, followed by expression of the nucleic acid in a suitable host cell under conditions in which the fusion protein is produced.

[0051] As used herein, the term "variant" refers to a polypeptide or polynucleotide that differs from a reference polypeptide or polynucleotide but retains essential properties. A typical variant of a polypeptide differs in amino acid sequence from another, reference polypeptide. Generally, the differences are limited, so that the sequences of the reference polypeptide and the variant are closely similar overall and, in many regions, identical. A variant and reference polypeptide may differ in amino acid sequence by one or more modifications (e.g., substitutions, additions, and / or deletions). A substituted or inserted amino acid residue may or may not be one encoded by the genetic code. A variant of a polypeptide may be a naturally occurring variant, such as an allelic variant, or a variant that is not known to occur naturally.

[0052] Modifications and changes can be made in the structure of the polypeptides of the present disclosure and still obtain molecules with similar properties (e.g., conservative amino acid substitutions). For example, certain amino acids can be substituted for other amino acids in the sequence without significant loss of activity. Because it is the interaction ability and properties of a polypeptide that define its biological functional activity, substitutions of certain amino acid sequences within the polypeptide sequence may still result in polypeptides with similar properties.

[0053] When making such changes, the hydropathic index of the amino acid can be taken into consideration. The importance of the hydropathic amino acid index in conferring interactive biological function to a polypeptide is generally understood in the art. It is known that a particular amino acid can be substituted with another amino acid having a similar hydropathic index or score and still obtain a polypeptide having similar biological activity. Each amino acid is assigned a hydropathic index based on its 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), glutamic acid (-3.5), glutamine (-3.5), aspartic acid (-3.5), asparagine (-3.5), lysine (-3.9), and arginine (-4.5).

[0054] The relative hydropathic properties of amino acids are believed to determine the secondary structure of a resulting polypeptide, which in turn defines the polypeptide's interactions with other molecules, such as enzymes, substrates, receptors, antibodies, antigens, and cofactors. It is known in the art that an amino acid can be substituted with another amino acid having a similar hydropathic index to obtain a functionally equivalent polypeptide. Such changes are preferably substituted with amino acids having a hydropathic index within ±2, particularly preferably within ±1, and even more particularly within ±0.5.

[0055] Substitutions of similar amino acids can also be made on the basis of hydrophilicity, particularly when the resulting biologically, functionally 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 is understood that an amino acid can be substituted with another amino acid having a similar hydrophilicity value and still obtain a biologically equivalent, and particularly an immunologically equivalent, polypeptide. Such changes are preferably made with amino acids having hydrophilicity values ​​within ±2, particularly preferably within ±1, and even more particularly within ±0.5.

[0056] As outlined above, amino acid substitutions are generally based on the relative similarity of the amino acid side chains of the replacement residues, e.g., their hydrophobicity, hydrophilicity, charge, size, etc. Exemplary substitutions that take into account various of the foregoing characteristics are well known to those of skill in the art and include the following (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 above polypeptides. In particular, embodiments of the polypeptides can include variants having about 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the polypeptide of interest.

[0057] With respect to a reference polypeptide sequence, "percent (%) amino acid sequence identity" is defined as the percentage of amino acid residues in a candidate sequence that are identical to the amino acid residues in the reference polypeptide sequence after aligning the sequences and, if necessary, introducing gaps to achieve the maximum percent sequence identity (without considering any conservative substitutions as part of sequence identity). Alignment for the purpose of determining percent amino acid sequence identity can be achieved in a variety of ways within the skill of the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN, Megalign (DNASTAR) software, etc. Those skilled in the art can determine the appropriate parameters for aligning sequences, including any algorithms required to achieve maximum alignment across the full length of the sequences being compared.

[0058] As used herein, the term "tumor" or "neoplasm" refers to an abnormal mass of tissue containing neoplasms. Neoplasms and tumors can be benign, pre-malignant, or malignant.

[0059] As used herein, the term "cancer" or "malignant tumor" refers to cells that exhibit uncontrolled growth and division, invade neighboring tissues, and often metastasize to other locations in the body.

[0060] As used herein, the term "anti-tumor agent" refers to a composition, such as a drug or biologic, that can inhibit or prevent cancer growth, invasion, and / or metastasis.

[0061] As used herein, the phrase "pharmaceutically acceptable" refers to compositions, polymers, other materials and / or dosage forms that are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit / risk ratio.

[0062] 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, that is involved in carrying or transporting a subject composition from one organ or part of the body to another. 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.

[0063] 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. A subject may be a vertebrate, e.g., a mammal. Thus, a subject may be a human or veterinary patient.

[0064] 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, condition, or disorder. This term includes active treatment, i.e., treatment specifically directed at ameliorating a disease, condition, or disorder, and also includes causal treatment, i.e., treatment directed at eliminating the cause of the associated disease, condition, or disorder. In addition, this term includes palliative treatment, i.e., treatment designed to relieve symptoms rather than cure the disease, condition, or disorder; preventative treatment, i.e., treatment directed at minimizing or partially or completely inhibiting the onset of the associated disease, condition, or disorder; and supportive treatment, i.e., treatment used to complement another specific therapy directed at ameliorating the associated disease, condition, or disorder.

[0065] As used herein, the term "therapeutically effective amount" refers to an amount of a therapeutic agent that, when incorporated into and / or onto a particle described herein, produces some desired effect at a reasonable benefit / risk ratio applicable to any medical treatment. The effective amount may vary depending on factors such as the disease or condition being treated, the particular targeting construct being administered, the size of the subject, or the severity of the disease or condition. One of ordinary skill in the art can empirically determine the effective amount of a particular compound without necessitating undue experimentation. In some embodiments, the term "effective amount" refers to the amount of a therapeutic or prophylactic agent to reduce or alleviate symptoms of one or more diseases or disorders of the brain, such as reducing tumor size (e.g., tumor volume).

[0066] Recitation of ranges of values ​​herein, unless otherwise indicated 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 herein as if it were individually referenced herein.

[0067] Use of the term "about" is intended to describe values ​​that are either above or below within about + / - 10% of the stated value, in other embodiments, values ​​may range from about + / - 5% above or below the stated value, in other embodiments, values ​​may range from about + / - 2% above or below the stated value, and in other forms, values ​​may range from about + / - 1% above or below the stated value. The foregoing ranges are intended to be made clear by context, and no further limitation is implied.

[0068] As used herein, "optionally" or "optionally" means that the subsequently described event, circumstance, or substance may occur or exist, or may not occur or exist, and the description includes cases where the event, circumstance, or substance occurs or exists, as well as cases where it does not occur or exist.

[0069] Ranges may be expressed herein as from "about" one particular value and / or to "about" another particular value. When such ranges are expressed, ranges from the one particular value and / or from the other particular value are considered to be specifically contemplated and disclosed unless the context specifically dictates otherwise. Similarly, when values ​​are expressed as approximations, it will be understood that by using the antecedent "about," the particular value forms another specifically contemplated embodiment that should be considered disclosed unless the context specifically dictates otherwise. Furthermore, unless the context specifically dictates otherwise, it will be understood that each endpoint of a range is significant both in relation to the other endpoint and independently of the other endpoint. It should be understood that all individual values ​​and subranges of values ​​included within explicitly disclosed ranges are also specifically contemplated and should be considered disclosed unless the context specifically dictates otherwise. Finally, all ranges should be understood to refer both to the recited range as a range and as a collection of individual numbers from the first endpoint (inclusive) to the second endpoint (inclusive). In the latter case, it should be understood that any of the individual numbers may be selected as one form of the quantity, value, or characteristic to which the range refers. In this manner, a range describes a set of consecutive numbers or values ​​from a first endpoint to (and including) a second endpoint, and a single member of the set (i.e., a single number) may be selected as the quantity, value, or characteristic to which the range refers. The above applies regardless of whether some or all of these embodiments are explicitly disclosed in a particular instance.

[0070] All compounds disclosed herein are intended to be specifically disclosed herein and should be considered to be specifically disclosed herein. Furthermore, all subgroups that can be identified within this disclosure are intended to be specifically disclosed herein and should be considered to be specifically disclosed herein. Consequently, it is specifically contemplated that any compound or subgroup of compounds can be specifically included or excluded for use, or can be included or excluded from a list of compounds.

[0071] Disclosed are the components used to prepare the disclosed compositions, as well as the compositions themselves used within the methods disclosed herein. When these and other materials are disclosed herein, and combinations, subsets, interactions, groups, etc. of these materials are disclosed, it is understood that each is specifically contemplated and described herein, even though specific reference to each of the various individual and collective combinations and permutations of these compounds may not be explicitly disclosed. For example, when particular polypeptides are disclosed and discussed, and several modifications that can be made to some polypeptides are discussed, any and all combinations and permutations of the polypeptides and possible modifications are specifically contemplated, unless specifically indicated to the contrary. Thus, if classes of molecules A, B, and C, as well as classes D, E, and F, are disclosed, and one example of a combined molecule, A-D, is disclosed, each individually and collectively, and combinations A-E, A-F, B-D, B-E, B-F, C-D, C-E, and C-F are contemplated as being disclosed, even though each is not individually described. Likewise, any subset or combination of these is also disclosed. Thus, for example, subgroups A-E, B-F, and C-E would be considered disclosed. This concept applies to all aspects of this application including, but not limited to, steps in methods of making and using the disclosed compositions. Thus, where there are various additional steps that may be performed, it is understood that each of these additional steps may be performed in any specific embodiment or combination of embodiments of the disclosed methods.

[0072] II. Composition ApoL1 or ApoL1-containing complexes such as trypanosome lytic factor (TLF: TLF-1 or TLF-2) (Figures 1A-1C) are a minor subclass of human high-density lipoproteins (HDL). As used herein, TLF encompasses both TLF-1 and TLF-2, but in each case can be substituted more specifically with TLF-1, TLF-2, or TLF-1 and TLF-2.

[0073] TLF is present in plasma at levels 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 may include one or more of haptoglobin-related protein (Hpr), apolipoprotein A1 (ApoA1), and IgM.

[0074] Hpr is important for TLF binding to a trypanosome-specific receptor present in approximately 350 copies in the flagellar binding pocket of the parasite, but humans have no known receptor for this protein (Drain, et al., J Biol Chem., 276:30254-60 (2001)). The consensus amino acid sequence of Hpr is available under Unitprot accession number P00739·HPTR_HUMAN (the entire contents of which are specifically incorporated herein by reference in their entirety) and is provided as SEQ ID NO: 49 below: MSDLGAVISLLLWGRQLFALYSGNDVTDISDDRFPKPPEIANGYVEHLFRYQCKNYYRLRTEGDGVYTLNDKKQWINKAVGDKLPECEAVCGKPKNPANPVQRILGGHLDAKGSFPWQAKMVSHHNLTTGATLINEQWLLTTAKNLFLNHSENATAKDIAPTLTLYVGKKQLVEIEKVVLHPNYHQVDIGLIKLKQKVLVNERVMPICLPSKNYAEVGRVGYVSGWGQSDNFKLTDHLKYVMLPVADQYDCITHYEGSTCPKWKAPKSPVGVQPILNEHTFCVGMSKYQEDTCYGDAGSAFAVHDLEEDTWYAAGILSFDKSCAVAEYGVYVKVTSIQHWVQKTIAEN (SEQ ID NO: 49).

[0075] ApoL1 is a member of the Bcl-2 family, whose members play a key role in regulating the programmed cell death (PCD) pathway. ApoL1 contains a BH3 domain death domain that has been identified as important for PCD, as deletion of this domain in wild-type ApoL1 eliminates its cytotoxicity (Wan, et al., J Biol Chem., 283:21540-9 (2008)). Four additional regions have been mapped to ApoL1: a signal peptide (SP, aa 1-27), a pore-forming domain (PFD, aa 60-237), a membrane-associated domain (MAD, aa 238-303), and an SRA-binding domain (aa 339-398). The last three domains have been shown to be important for ApoL1 function and toxicity, although overexpression of any of the three domains did not result in increased lethality (Lan, et al., Exp Mol Pathol. 99:139-44 (2015)). Of the six members in the ApoL family, ApoL1 is the only one secreted into serum; the other members function intracellularly (Vanhollebeke, et al., Cell Mol Life Sci CMLS., 63:1937-44 (2006)). While the exact role of ApoL1 remains to be determined, two recently evolved variants, G1 and G2, have been correlated with 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)).

[0076] Despite the many inherent obstacles arising from the involvement of ApoL1 (Pays, et al. (Febs J., 288:360-81 (2021)), it has been identified as a key component involved in trypanosome killing (Vanhollebeke, et al., Mol Microbiol., 76:806-14 (2010)). Following uptake into acidic parasitoid endosomes and lysosomal transport, ApoL1 undergoes pH-mediated activation and inserts into lipid membranes to form a closed-state pH-gated cation channel, capable of inducing 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 under Uniprot accession number O14791. APOL1_HUMAN (the entire contents of which are specifically incorporated herein by reference in their entirety) and is provided as SEQ ID NO: 50 below: MEGAALLRVSVLCIWMSALFLGVGVRAEEAGARVQQNVPSGTDTGDPQSKPLGDWAAGTMDPESSIFIEDAIKYFKEKVSTQNLLLLLTDNEAWNGFVAAAELPRNEADELRKALDNLARQMIMKDKNWHDKGQQYRNWFLKEFPRLKSELEDNIRRLRALADGVQKVHKGTTIANVVSGSLSISSGILTLVGMGLAPFTEGGSLVLLEPGMELGITAALTGITSSTMDYGKKWWTQAQAHDLVIKSLDKLKEVREFLGENISNFLSLAGNTYQLTRGIGKDIRALRRARANLQSVPHASASRPRVTEPISAESGEQVERVNEPSILEMSRGVKLTDVAPVSFFLVLDVVYLVYESKHLHEGAKSETAEELKKVAQELEEKLNILNNNYKILQADQEL (SEQ ID NO: 50).

[0077] Studies have shown that at supraphysiological concentrations, TLF can induce a similar cascade of lethal events in mammalian cells, although the direct mechanism has yet to be determined (Wan, et al., J Biol Chem., 283:21540-9 (2008)).

[0078] The following results demonstrate 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. Thus, compositions for increasing ApoL1 in target cells and methods of using the same to induce targeted cell death are disclosed.

[0079] A. Compositions for Targeting Endogenous ApoL1 Compositions for increasing cellular internalization of endogenous ApoL1 are provided. Although not necessarily used exclusively for this purpose, the compositions can be used to mobilize endogenous ApoL1 into cells. The compositions typically bind to ApoL1 and / or ApoL1-containing complexes (including, but not limited to, TLFs), such as those depicted in Figures 1A-1C. The compositions also typically bind to cell-specific markers, such as cancer antigens present on cells, thereby facilitating targeting of captured ApoL1 and / or ApoL1-containing complexes to target cells. Cell-specific markers and antigens are molecules that, when targeted by a targeting moiety (e.g., an antibody or antigen-binding fragment), can enhance delivery of the composition to target cells. In some embodiments, the cell-specific marker is elevated on target cells compared to some or all other (non-target) cells, unique or intrinsic to target cells compared to some or all other (non-target) cells, or a combination thereof.

[0080] The targeted ApoL1-containing complex can be internalized by the target cell in an amount effective to increase cell death of the target cell. Thus, the composition typically includes a binding moiety capable of specifically binding to ApoL1 or another component of the ApoL1-containing complex, and the targeting moiety can specifically recognize and bind to a target molecule (e.g., a cell-specific marker or antigen) specific to a cell type, tissue type, or organ. The binding molecule and / or targeting molecule can be a polypeptide, lipid, or glycolipid. The target molecule of the targeting moiety can be a receptor selectively expressed on a particular 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. Exemplary cell-specific markers include, but are not limited to, cancer-specific markers. The cell marker can be any cell-specific marker, including cancer antigens and tumor antigens, including but not limited to those provided elsewhere herein (see, e.g., below).

[0081] 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 specifically incorporated herein by reference in its entirety), which describe humanized monoclonal antibodies specific for β-1,3 glucan, a component of several pathogenic fungi, and a glyceryl dilaurate lipid moiety that targets malaria parasite-infected red blood cells (iRBCs), respectively.

[0082] Typically, the targeting moiety does not target a trypanosome-specific surface antigen. In a preferred embodiment, the composition is an antibody, preferably a bispecific or multispecific antibody, comprising an antigen-binding fragment against ApoL1 or an ApoL1-containing complex and a cell-specific marker or antigen, respectively.

[0083] 1. Sequence of an antibody that binds to ApoL1 or an ApoL1-containing complex Provided herein are CDRs and heavy and / or light chain sequences that bind to ApoL1-containing complexes, such as TLF. In some embodiments, the antibody binds to Hpr, e.g., Hpr having the sequence of SEQ ID NO: 49. In some embodiments, the antibody binds to ApoL1, e.g., ApoL1 having the sequence of SEQ ID NO: 50. As discussed herein, antibodies that bind, preferably immunospecifically bind, to Hpr or ApoL1 have one or more relevant CDRs or variants thereof, and / or one or both relevant VH and VL sequences or variants thereof, and are expressly provided in all antibody forms, including, but not limited to, intact antibodies and antigen-binding fragments, in monospecific, bispecific, and higher multispecific formats, and optionally as humanized or chimeric forms thereof. Thus, in some embodiments, antibodies are or comprise fragments with antigen-binding ability (e.g., Fab', F(ab')2, Fab, Fv, and rIgG), recombinant single-chain Fv fragments (scFv), and bivalent (di-scFv) and trivalent (tri-scFv) forms thereof.

[0084] The present disclosure further includes nucleic acid molecules (DNA or RNA) encoding any such antibodies, fusion proteins, or fragments, as well as vector molecules (such as plasmids) that are capable of transmitting or replicating such nucleic acid molecules and expressing such antibodies, fusion proteins, or fragments in cell lines, and host cells transformed with such nucleic acids. The nucleic acid may be single-stranded, double-stranded, or may contain both single- and double-stranded portions.

[0085] a. Hpr binding sequence - clone SFII 134.3 Clone SFII 134.3, a murine IgG2a that binds to Hpr, was sequenced to reveal the following variable domains and CDRs:

[0086] i. Heavy chain Variable domain (VH) QIQLVQSGPELKKPGETVKISCKASGYIFTNYGMNWVRQAPGKGLKWMGWINSYTGEATYTDDLKGRFAFSLESSASTAYLQINNLKNEDTATYFCAREGYGDYGYSFDYWGQGTTLTVSS (SEQ ID NO: 3) [Table 1]

[0087] ii. Light chain Variable domain (VL) DIQMTQSPASLSASVGETVTITCRATKNIYTYLAWYQQKQGKSPQFLVYNAKTLAEGVPSRFSGSGSGTQFSLKINSLQPEDFGNYYCQHHYGTPRTFGGGTKLEIK (SEQ ID NO: 14) [Table 2]

[0088] In preferred embodiments, antibodies and other molecules comprise six CDRs, which may include at least one, two, three, four, five, or six of the consensus CDRs of the anti-Hpr antibody SFII 134.3, such as those provided herein.

[0089] For example, in some embodiments, the antibody or other molecule comprises at least one, two, three, four, five, or six CDRs of the heavy chain variable domain and / or light chain variable domain of SEQ ID NO:3 and / or SEQ ID NO:14, respectively.

[0090] In some embodiments, the antibody or other molecule comprises at least one, two, three, four, five, or six CDRs of SFII 134.3 selected from the following, optionally at least one CDR-H1, one CDR-H2, one CDR-H3, one CDR-L1, one CDR-L2, and one CDR-L3: SFII 134.3 CDR-H1: NYGMN (SEQ ID NO: 4), GYIFTNYG (SEQ ID NO: 7), or GYIFTNY (SEQ ID NO: 10), or a variant thereof having at least 70, 80, 90, or 95% sequence identity thereto; SFII 134.3 CDR-H2: WINSYTGEATYTDDLKG (SEQ ID NO: 5), INSYTGEA (SEQ ID NO: 8), NSYTGE (SEQ ID NO: 11), or a variant thereof having at least 70, 80, 90, or 95% sequence identity thereto; SFII 134.3 CDR-H3: EGYGDYGYSFDY (SEQ ID NO: 6), AREGYGDYGYSFDY (SEQ ID NO: 9), or a variant thereof having at least 70, 80, 90, or 95% sequence identity thereto; 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; 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 SFII 134.3 CDR-L3:QHHYGTPRT (SEQ ID NO: 18), or a variant thereof having at least 70, 80, 90, or 95% sequence identity thereto.

[0091] In some embodiments, the antibody or other molecule comprises the H1-H3 and L1-L3 CDRs, respectively, of SFII 134.3 selected from: 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; 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 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 a variant thereof having at least 70, 80, 90, or 95% sequence identity thereto.

[0092] In some embodiments, the antibody or other molecule comprises a heavy chain variable domain and / or a light chain variable domain of SEQ ID NO: 3 and / or SEQ ID NO: 14, respectively, or a variant thereof having at least 70, 80, 90, or 95% sequence identity thereto.

[0093] b. Hpr binding sequence - clone SFII 14.11 Clone SFII 14.11, a mouse IgG that binds to Hpr, was sequenced to reveal the following variable domains and CDRs:

[0094] i. Heavy chain Variable domain (VH) QIQLVQSGPELKKPGETVKISCKASGFTFTDYSIHWVKQAPGKGLKWMGWKHTESGESTYADDFKGRFVFSLETSASTAYLQINNLKNEDTSTYFCARGANYGSLLDYWGQGTTLTVSS (SEQ ID NO: 56) [Table 3]

[0095] ii. Light chain Variable domain (VL) DIVLTQSPASLAVSLGQRATISCRASKSVSTSGYSYMHWYQQKPGQSPKLLIYLASNLESGVPARFSGSGSGTDFTLNIHPVEEEDAATYYCQHNRELPLTFGAGTKLELKR (SEQ ID NO: 65) [Table 4]

[0096] In preferred embodiments, antibodies and other molecules comprise six CDRs, which may include at least one, two, three, four, five, or six of the consensus CDRs of the anti-Hpr antibody 14.11, such as those provided herein.

[0097] For example, in some embodiments, the antibody or other molecule comprises at least one, two, three, four, five, or six CDRs of the heavy chain variable domain and / or light chain variable domain of SEQ ID NO: 56 and / or SEQ ID NO: 65, respectively.

[0098] In some embodiments, the antibody or other molecule comprises at least one, two, three, four, five, or six CDRs of SFII 14.11 selected from the following, optionally at least one CDR-H1, one CDR-H2, one CDR-H3, one CDR-L1, one CDR-L2, and one CDR-L3: 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; 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; 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; 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; 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 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.

[0099] In some embodiments, the antibody or other molecule comprises the H1-H3 and L1-L3 CDRs, respectively, of SFII 14.11 selected from: 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 a variant thereof having at least 70, 80, 90, or 95% sequence identity thereto; 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 a variant thereof having at least 70, 80, 90, or 95% sequence identity thereto; or 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 a variant thereof having at least 70, 80, 90, or 95% sequence identity thereto.

[0100] In some embodiments, the antibody or other molecule comprises a heavy chain variable domain and / or a light chain variable domain of SEQ ID NO: 56 and / or SEQ ID NO: 65, respectively, or a variant thereof having at least 70, 80, 90, or 95% sequence identity thereto.

[0101] c. ApoL1 binding sequence Clone SFIII 13.11, a murine IgG1 that binds to ApoL1, was sequenced to reveal the following variable domains and CDRs:

[0102] i. Heavy chain Variable domain (VH) EVQLVESGGGLVKPGGSLKLSCAASGFTFSTYAMSWVRQSPEKRLEWVAEISNGGLYTYYPDTVTGRFTISRDNVKNILYLEMSSLRSEDTAIYYCIRENRNWYFDLWGAGTTVTVSS (SEQ ID NO: 24) [Table 5]

[0103] ii. Light chain Variable domain (VL) DVLMTQTPLSLPVSLGDQASISCRSSQSIVNSNGNTYLEWYLQKPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYYCFQGSHVPLTFGAGTKLELK (SEQ ID NO: 36), or DVLMTQTPLSLPVSLGDQASISCRSSQSIVNSNGNTYLEWYLQKPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYYCFQGSHVPLTFGAGTKLEIK (SEQ ID NO: 77) [Table 6]

[0104] In preferred embodiments, antibodies and other molecules comprise six CDRs, which may include at least one, two, three, four, five, or six of the consensus CDRs of the anti-ApoL1 antibody SFIII 13.11.

[0105] For example, in some embodiments, the antibody or other molecule comprises at least one, two, three, four, five, or six CDRs of the heavy chain variable domain and / or light chain variable domain of SEQ ID NO:24 and / or SEQ ID NO:36 or SEQ ID NO:77, respectively.

[0106] In some embodiments, the antibody or other molecule comprises at least one, two, three, four, five, or six CDRs of SFIII 13.11 selected from the following, optionally at least one CDR-H1, one CDR-H2, one CDR-H3, one CDR-L1, one CDR-L2, and one CDR-L3: SFIII 13.11 CDR-H1: TYAMS (SEQ ID NO: 25), GFTFSTYA (SEQ ID NO: 28), GFTFSTY (SEQ ID NO: 31), or a variant thereof having at least 70, 80, 90, or 95% sequence identity thereto; SFIII 13.11 CDR-H2: EISNGGLYTYYPDTVTG (SEQ ID NO: 26), ISNGGLYT (SEQ ID NO: 29), SNGGLY (SEQ ID NO: 32), or a variant thereof having at least 70, 80, 90, or 95% sequence identity thereto; SFIII 13.11 CDR-H3: ENRNWYFDL (SEQ ID NO: 27), IRENRNWYFDL (SEQ ID NO: 30), or a variant thereof having at least 70, 80, 90, or 95% sequence identity thereto; SFIII 13.11 CDR-L1: RSSQSIVNSNGNTYLE (SEQ ID NO: 37), QSIVNSNGNTY (SEQ ID NO: 40), or a variant thereof having at least 70, 80, 90, or 95% sequence identity thereto; SFIII 13.11 CDR-L2: KVSNRFS (SEQ ID NO: 38), KVS, or a variant thereof having at least 70, 80, 90, or 95% sequence identity thereto; and SFIII 13.11 CDR-L3:FQGSHVPLT (SEQ ID NO: 39), or a variant thereof having at least 70, 80, 90, or 95% sequence identity thereto.

[0107] In some embodiments, the antibody or other molecule comprises the H1-H3 and L1-L3 CDRs, respectively, of SFIII 13.11 selected from: 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 a variant thereof having at least 70, 80, 90, or 95% sequence identity thereto; 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 a variant thereof having at least 70, 80, 90, or 95% sequence identity thereto; or 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 a variant thereof having at least 70, 80, 90, or 95% sequence identity thereto.

[0108] In some embodiments, the antibody or other molecule comprises a heavy chain variable domain and / or a light chain variable domain of SEQ ID NO:24 and / or SEQ ID NO:36 or SEQ ID NO:77, respectively, or a variant thereof having at least 70, 80, 90, or 95% sequence identity thereto.

[0109] 2. Chimeric and humanized antibodies The present disclosure particularly relates to chimeric and humanized antibodies. Constant regions need not be present, but if present, typically are substantially identical to human immunoglobulin constant regions, i.e., at least about 85-90%, preferably about 95% or more identical. Thus, all portions of a humanized immunoglobulin, except possibly for the CDRs, are substantially identical to corresponding portions of a natural human immunoglobulin sequence. A humanized antibody is an antibody having a humanized light chain and a humanized heavy chain immunoglobulin. For example, a humanized antibody does not encompass a typical chimeric antibody, since the entire variable region of a chimeric antibody is non-human. The resulting humanized antibody is expected to bind to the same antigen as the donor antibody that provided the CDRs, and therefore the donor antibody is said to be "humanized" by the process of "humanization."

[0110] In most cases, humanized antibodies are human immunoglobulins (recipient antibody) in which hypervariable region residues of the recipient are replaced by hypervariable region residues from a non-human species (donor antibody) such as mouse, rat, rabbit, or non-human primate having the desired antibody specificity, affinity, and capacity. In some instances, framework region (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues. Furthermore, humanized antibodies may comprise residues that are not found in the recipient antibody or the donor antibody. These modifications are made to further refine antibody performance. In general, humanized antibodies will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the hypervariable regions correspond to those of a non-human immunoglobulin and all or substantially all of the FRs are those of a human immunoglobulin sequence. A humanized antibody also optionally comprises at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin that immunospecifically binds to an FcγRIIB polypeptide, and has been modified by introducing amino acid residue substitutions, deletions, or additions (i.e., mutations).For example, see 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, as well as 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. 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. 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 Supp):5973s-5977s, Couto et al.,1995,Cancer Res. 55:1717-22, Sandhu, 1994, Gene 150:409-10, Pedersen et. See also 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.

[0111] DNA sequences encoding preferred human acceptor framework sequences include, but are not limited to, FR segments from human germline VH segments VH1-18 and JH6 and human germline VL segments VK-A26 and JK4. In certain embodiments, one or more of the CDRs are inserted within the framework regions using routine recombinant DNA techniques. The framework regions may be naturally occurring or consensus framework regions, and preferably, may be human framework regions (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).

[0112] A humanized or chimeric antibody may comprise substantially all, at least one, and typically two, variable domains, with all or substantially all CDRs corresponding to those of a non-human immunoglobulin (i.e., donor antibody) and all or substantially all framework regions of a human immunoglobulin consensus sequence. Preferably, the antibody also comprises at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin. The antibody constant domain may be selected with regard to the proposed function of the antibody, particularly any effector functions that may be required. In some embodiments, the antibody constant domain is (or comprises) a human IgA, IgD, IgE, IgG, or IgM domain. In certain embodiments, when a humanized antibody is intended for therapeutic use, human IgG constant domains, particularly those of the IgG1 and IgG3 isotypes, are used, and antibody effector functions such as antibody-dependent cell-mediated cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) activity are required. In alternative embodiments, IgG2 and IgG4 isotypes are used when the antibody is intended for therapeutic purposes and antibody effector function is not required. The present disclosure encompasses Fc constant domains comprising one or more amino acid modifications that alter antibody effector function, such as those disclosed in U.S. Patent Application Publication Nos. 2005 / 0037000 and 2005 / 0064514.

[0113] In some embodiments, the antibody comprises both a light chain and at least the variable domain of a heavy chain. In other embodiments, the antibody may 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 immunoglobulin, including IgM, IgG, IgD, IgA, and IgE, and any isotype, including IgG1, IgG2, IgG3, and IgG4. In some embodiments, the constant domain is a complement-fixing constant domain, typically of the IgG1 class, if it is desired that the antibody exhibit cytotoxic activity. In other embodiments, if such cytotoxic activity is not desired, the constant domain can be of the IgG2 class. The antibody can comprise sequences from multiple classes or isotypes, and selecting a particular constant domain to optimize desired effector functions is within the ordinary skill in the art. In some embodiments, the antibody is not a murine IgG1 or a murine IgG2a.

[0114] The framework and CDR regions of a humanized antibody need not correspond exactly to the parental sequences; for example, the donor CDR or consensus framework may be mutagenized by substitution, insertion, or deletion of at least one residue so that the CDR or framework residue at that site corresponds to neither the consensus nor the donor antibody. However, it is preferred that such mutations not be extensive. Usually, at least 75% of the humanized antibody residues will correspond to the parental framework region (FR) and CDR sequences, more often 90%, and most preferably greater than 95%.Humanized antibodies can be produced using various techniques known in the art, including CDR-grafting (EP 239,400, WO 91 / 09967, and U.S. Pat. Nos. 5,225,539, 5,530,101, and 5,585,089), veneering or resurfacing (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., 1995, Protein Engineering 7(6):805-814). al., 1994, Proc. Natl. Acad. Sci. 91:969-973), chain shuffling (US Pat. No. 5,565,332), and US Pat. No. 6,407,213, US Pat. No. 5,766,886, US Pat. 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. al.,1996,Protein Eng. 9:895-904; Couto et al., 1995, Cancer Res. 55(23 Supp):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. Often, framework residues in the framework regions are substituted with the corresponding residue from the CDR donor antibody to alter, and preferably improve, antigen binding.These framework substitutions are identified by methods well known in the art, such as by modeling the interactions of CDR and framework residues to identify framework residues important for antigen binding and sequence comparison to identify unusual framework residues at particular 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).

[0115] 3. Bispecific and multispecific antibodies The antibodies used in the methods of the present disclosure may be monospecific. Monospecific antibodies against ApoL1 or ApoL1-containing complexes 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, bispecific, trispecific, or more highly multispecific antibodies that exhibit specificity for different targets in addition to ApoL1 or Hpr are also of interest. For example, such antibodies can bind to both ApoL1 or Hpr and an antigen that is important for targeting the antibody to a particular cell type or tissue (e.g., an antigen associated with the cancer antigen of the tumor being treated).

[0116] a. Exemplary Structures of Bispecific and Multispecific Molecules In some embodiments, the antibodies are heterodimeric bi- and tri- (or higher) specific Ig antibodies and Fc fusion proteins. Exemplary structures include, but are not limited to, IgG, IgM, mono-, di-, tri-, or higher scFv-Fc. For example, bispecific, trispecific, and multispecific formats include, but are not limited to, bispecific and trispecific IgG, IgG-scFv, IgG-dAb, scFv-Fc-scFv, knob-in-hole (KIH)-IgG, κλ-body, KIH0Fc-Fab / scFv, tandem scFv, KIH trispecific, and bispecific Fc fusions (N- or C-terminal, with or without KIH).

[0117] In embodiments, a multispecific antibody molecule can comprise two or more antigen-binding sites, where different sites are specific for different antigens. In embodiments, a multispecific antibody molecule can bind to two or more (e.g., two or more) epitopes on the same antigen. In embodiments, a multispecific antibody molecule comprises 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 a TLF (e.g., Hpr or ApoL1). In some embodiments, a multispecific antibody molecule is a bispecific antibody molecule. Bispecific antibody molecules can be classified into five different structural groups: (i) bispecific immunoglobulin G (BsIgG), (ii) IgG with additional antigen-binding moieties appended, (iii) bispecific antibody fragments, (iv) bispecific fusion proteins, and (v) bispecific antibody conjugates.

[0118] BsIgG is a monovalent format for each antigen. Exemplary BsIgG formats include, but are not limited to, crossMab, DAF (2-in-1), DAF (4-in-1), DutaMab, DT-IgG, knob-in-hole common LC, knob-in-hole assembly, charge pair, Fab-arm exchange, SEEDbody, triomab, LUZ-Y, Fcab, kappa-lambda body, and 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 (Neovii Biotech, Fresenius Biotech), which targets CD3 and HER2.

[0119] In some embodiments, the BsIgG comprises a heavy chain engineered for heterodimerization. For example, the heavy chain can be engineered for heterodimerization using a "knobs-in-holes" strategy, the SEED platform, a common heavy chain (e.g., Kk-body), and the use of 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-in-holes, duobodies, azymetric, charge pairing, HA-TF, SEEDbodies, and differential Protein A affinity. See ibid. BsIgG can be produced by separate expression of component antibodies in different host cells and subsequent purification / assembly into BsIgG. BsIgG can also be produced by expression of component antibodies in a single host cell. BsIgG can be purified using affinity chromatography, for example, using Protein A and sequential pH elution.

[0120] IgGs with additional antigen-binding moieties added are another form of bispecific antibody molecule. For example, monospecific IgGs can be engineered to have bispecificity by adding additional antigen-binding units to the monospecific IgG (e.g., to the N- or C-terminus of either the heavy or light chain). Exemplary additional antigen-binding units include single domain antibodies (e.g., variable heavy chains 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 IgG formats that can be added include dual 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, KIH IgG-scFab, 2scFv-IgG, IgG-2scFv, scFv4-Ig, zybody, and DVI-IgG. See Spiess et al. Mol. Immunol. 67(2015):95-106. An example of an IgG-scFv is MM-141 (Merrimack Pharmaceuticals), which binds to IGF-1R and HER3. Examples of DVD-Igs include ABT-981 (AbbVie), which binds to IL-1 alpha and IL-1 beta, and ABT-122 (AbbVie), which binds to TNF and IL-17A.

[0121] Bispecific antibody fragments (BsAbs) are a form of bispecific antibody molecule that lacks some or all of the antibody constant domains. For example, some BsAbs lack the Fc region. In some embodiments, bispecific antibody fragments contain heavy and light chain regions connected by a peptide linker, allowing efficient expression of the BsAb in a single host cell. Exemplary bispecific antibody fragments include, but are not limited to, nanobodies, nanobody-HAS, BiTEs, diabodies, DARTs, TandAbs, scdiabodies, scdiabody-CH3, diabody-CH3, triplebodies, miniantibodies, minibodies, TriBiminibodies, scFv-CH3 KIH, Fab-scFv, scFv-CH-CL-scFv, F(ab')2, F(ab')2-scFv2, scFv-KIH, Fab-scFv-Fc, tetravalent HCAb, scdiabody-Fc, diabody-Fc, tandem scFv-Fc, and intrabodies. See ibid. For example, a BiTE format comprises tandem scFvs, where the component scFvs bind to CD3 on T cells and a surface antigen on cancer cells.

[0122] Bispecific fusion proteins include, for example, antibody fragments linked to other proteins to add additional specificity and / or functionality. An example of a bispecific fusion protein is immTAC, which contains an anti-CD3 scFv linked to an affinity-matured T cell receptor that recognizes an HLA-presented peptide. In embodiments, the dock-and-lock (DNL) method can be used to generate bispecific antibody molecules with higher valency. Additionally, fusion to albumin-binding proteins or human serum albumin can extend the serum half-life of antibody fragments. See Id.

[0123] In embodiments, chemical conjugation, e.g., chemical conjugation of antibodies and / or antibody fragments, can be used to generate BsAb molecules. See ibid. Exemplary bispecific antibody conjugates include the CovX-body format, in which a low-molecular-weight drug is site-specifically conjugated to a single reactive lysine on each Fab arm or antibody or fragment thereof. In embodiments, the 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 either VEGF or Ang2. See ibid.

[0124] In some embodiments, the multispecific molecule further comprises a heavy chain constant region (e.g., Fc region), which is selected from the heavy chain constant regions of IgG1, IgG2, and IgG4, more particularly, the heavy chain constant region of human IgG1, IgG2, or IgG4. In some embodiments, the heavy chain constant region (e.g., Fc region) is covalently linked to one or both of the antibody molecule that binds to the ApoL1-containing complex and the second antibody molecule, for example.

[0125] In some embodiments, the heavy chain constant region (e.g., Fc region) is modified (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 of the first and second heavy chain constant regions (e.g., Fc region) is modified (e.g., mutated) to increase or decrease dimerization, e.g., compared to an unengineered interface. In some embodiments, dimerization of the heavy chain constant regions (e.g., Fc region) is enhanced by providing one or more of paired cavity-protrusions ("knobs-in-holes"), electrostatic interactions, or strand exchange at the Fc interface of the first and second Fc regions, e.g., so that a greater ratio of heteromultimer:homomultimer forms is formed, e.g., compared to an unengineered interface. In some embodiments, the heavy chain constant region (e.g., Fc region) comprises an amino acid substitution at a position selected from one or more of, e.g., 347, 349, 350, 351, 366, 368, 370, 392, 394, 395, 397, 398, 399, 405, 407, or 409 of the Fc region of human IgG1, numbered according to the EU numbering system. Unless otherwise specified herein, numbering of amino acid residues in the Fc region or constant region is according to the EU numbering system (also referred to 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.

[0126] 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 a cavity or hole), or T366W (e.g., corresponding to a protrusion or knob), or a combination thereof, numbered according to the Eu numbering system.

[0127] 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 compared to a naturally occurring heavy chain constant region: Fc receptor binding, glycosylation of the antibody, the number of cysteine ​​residues, effector cell function, or complement function.

[0128] In some embodiments, the ApoL1-containing complex binding molecule comprises a first heavy chain constant region (e.g., a first Fc region), and the second antibody molecule comprises a second heavy chain constant region (e.g., a second Fc region), wherein the first heavy chain constant region comprises one or more mutations that increase heterodimerization of the first heavy chain constant region and the second heavy chain constant region compared to naturally occurring heavy chain constant regions, and / or the second heavy chain constant region comprises one or more mutations that increase heterodimerization of the second heavy chain constant region and the first heavy chain constant region compared to naturally occurring heavy chain constant regions. In some embodiments, the first and second heavy chain constant regions (e.g., the first and second Fc regions) comprise one or more of paired cavity-protrusion ("knob-in-hole"), electrostatic interactions, or strand exchange, e.g., to form a greater ratio of heteromultimer:homomultimer forms compared to naturally occurring heavy chain constant regions.

[0129] In some embodiments, the first and / or second heavy chain constant region (e.g., the first and / or second Fc region, e.g., the first and / or second IgG1 Fc region) comprises an amino acid substitution at a position selected from one or more of 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 and / or second heavy chain constant region (e.g., the first and / or second Fc region, e.g., the first and / or second IgG1 Fc region) comprises an amino acid substitution selected from the following: T366S, L368A, Y407V, or Y349C (e.g., corresponding to a cavity or hole), or T366W or S354C (e.g., corresponding to a protrusion or knob), numbered according to the Eu numbering system, or a combination thereof.

[0130] In some embodiments, the multispecific molecule further comprises a linker, e.g., a linker between the ApoL1-containing complex-binding molecule and the second antibody molecule, a linker between the ApoL1-containing complex-binding antibody molecule and a heavy chain constant region (e.g., Fc region), or a linker between the second antibody molecule and a 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 comprises Gly and Ser.

[0131] B. Cancer antigen In some embodiments, antibodies of the present disclosure that bind to ApoL1-containing complexes, such as TLF, are bispecific or other multispecific molecules that also bind to cancer antigens.

[0132] In some embodiments, the cancer antigen is an antigen of a hematological cancer, e.g., multiple myeloma, leukemia (e.g., chronic lymphocytic leukemia, acute myeloid leukemia, acute lymphoblastic leukemia), non-Hodgkin's lymphoma, Hodgkin's lymphoma, myelodysplastic syndrome (MDS), myeloproliferative neoplasms (MPN) (or subcategories thereof, e.g., essential thrombocythemia (ET), myelofibrosis (MF), and polycythemia vera (PV), amyloidosis, Waldenstrom's macroglobulinemia, or aplastic anemia. In other embodiments, the cancer antigen is an antigen of a solid tumor.

[0133] Common hematological 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. Accordingly, in some embodiments, a 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.

[0134] 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 ...5 (GenBank accession numbers U29339 and M34309), and HER6 (Plowman, et al., J. Immunol., 152:2393 (1994); Disis, et al., Canc. Res., 54:16 (1994); GenBank accession numbers X03363 and M17730). 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 (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, and X06043; European Patent No. GB2241703), 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 Acc. 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. WO96 / 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. 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. al., J. Biol. Chem., 269:20126 (1994)), MAGE (van den Bruggen, et al. 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, U5555, U5555, U5555, U5555, U5555, U5555, U5555, U5555, U5555, U5555, U5555, U5555, U5555, U5555, U5555, U5555, U5555, U5555, U5556, U5557, U5558, U5559 ... ,683,886 and 5,571,711), GAGE ​​(GenBank accession numbers AF055475, AF055474, AF055473, U19147, U19146, U19145, U19144, U19143, and U19142), any of the receptors of the CTA class, including the 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, M29540), PyLT (GenBank accession numbers J02289, 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).

[0135] Additional tumor-associated antigens include prostate surface antigen (PSA) (U.S. Pat. Nos. 6,677,157 and 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)), and the glycosyltransferase β-1,4-N-acetylgalactosaminyltransferase (GalNAc) (Hoon, et al., Int. J. Cancer, 43:857-62 (1989); Ando, ​​et al. 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. 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. al., J. Exp. Med., 171:1375-80 (1990), GenBank accession number: X51455), human cytokeratin 8, high molecular weight melanoma antigen (Natali, et al. al., Cancer, 59:55-63 (1987), and keratin 19 (Datta, et al., J. Clin. Oncol., 12:475-82 (1994)).

[0136] Tumor antigens of interest include those antigens considered in the art to be "cancer / testis" (CT) antigens that are immunogenic in subjects with malignant conditions (Scanlan, et al., Cancer Immun., 4:1 (2004)). CT antigens include a family of at least 19 different antigens that 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).

[0137] Additional tumor antigens that can be targeted, including tumor-associated or tumor-specific antigens, include 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, myosin class I, OS-9, pml-RARα fusion protein, PTPRK, K-ras, N-ras, triosephosphate isomerase, Bage-1, Gage3,4,5,6,7, 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-18 0, MAGE-4, MAGE-5, MAGE-6, p185erbB2, p180erbB-3, c-met, nm-23H1, PSA, TAG-72-4, CA19-9, CA72-4, CAM17.1, NuMa, K-ras, beta-catenin, CDK4, Mum-1, p16, TAGE, PSMA, PSCA, CT7, telomerase, 43-9F, 5T4, 791Tgp72, alpha-fetoprotein, 13HCG, BCA225, BTAA, CA125, CA15-3 (CA27).29 / BCAA), CA195, CA242, 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-related protein), TAAL6, TAG72, TLP, and TPS. Other tumor-associated and tumor-specific antigens are known to those of skill in the art and are suitable for targeting with the disclosed fusion proteins.

[0138] In certain embodiments, the tumor antigen is a pancreatic cancer antigen, optionally selected from claudin 18.2, MUC1, mesothelin (MSLN), and myoferlin (MYOF).

[0139] 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).

[0140] Mesothelin (MSLN) has also been found in ovarian cancer, lung adenocarcinoma, malignant mesothelioma, biliary tract cancer, gastric cancer, and childhood 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)).

[0141] 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).

[0142] In other embodiments, the tumor antigen is a melanoma cancer antigen, optionally PMEL17. Thus, in some embodiments, the bispecific or multispecific antibody comprises an antigen-binding fragment that specifically binds to PMEL17.

[0143] Typically, the cell marker or antigen is not a trypanosome-specific surface antigen.

[0144] In preferred embodiments, the aforementioned antigens are targeted with antibodies that bind to them. Thus, for all tumor antigens provided, antibodies and antigen-binding fragments that specifically bind to them are also provided. In other embodiments, the targeting moiety is not an antibody, but can be, for example, another polypeptide, carbohydrate, lipid, etc., as discussed elsewhere herein.

[0145] 4. Derivatives and conjugates The present disclosure particularly contemplates the production and use of derivatives of any of the above-described antibodies and antigen-binding fragments thereof. The term derivative encompasses antibodies or antigen-binding fragments thereof that immunospecifically bind to an antigen but that contain one, two, three, four, five, or more amino acid substitutions, additions, deletions, or modifications compared to the "parent" (or wild-type) molecule (also referred to as a variant). Such amino acid substitutions or additions can introduce naturally occurring (i.e., DNA-encoded) or non-naturally occurring amino acid residues.

[0146] The term derivative also encompasses, for example, chimeric or humanized variants of any of the disclosed antibodies, as well as variants with altered CH1, hinge, CH2, CH3, or CH4 regions to form, for example, antibodies with variant Fc regions with enhanced or impaired effector or binding properties.

[0147] The term derivative further encompasses non-amino acid modifications, such as glycosylation (e.g., containing modified mannose, 2-N-acetylglucosamine, galactose, fucose, glucose, sialic acid, 5-N-acetylneuraminic acid, 5-glycolneuraminic acid, etc.), acetylation, pegylation, phosphorylation, amidation, derivatization with known protecting / blocking groups, proteolytic cleavage, amino acids that can be linked to cellular ligands or other proteins, etc. In some embodiments, the engineered carbohydrate modification modulates one or more of the following: antibody solubilization, promoting intracellular trafficking and secretion of the antibody, promoting antibody assembly, conformational integrity, and antibody-mediated effector function. In certain embodiments, the engineered carbohydrate modification enhances antibody-mediated effector function compared to an antibody lacking the carbohydrate modification. Carbohydrate modifications that result in altered antibody-mediated effector functions are well known in the art (see, e.g., Shields, R. Let al. (2002) "Lack of Fucose on Human IgG N-Linked Oligosaccharide Improves Binding to Human Fc gamma RIII and Antibody-Dependent Cellular Toxicity," J. Biol. Chem. 277(30):26733-26740; Davies J. et al. (2001) "Expression of GnTIII in a Recombinant Anti-CD20 CHO Production Cell Line: Expression of Antibodies with Altered Glycoforms Leads to an Increase in ADCC Through Higher Affinity for Fc gamma RIII," Biotechnology & Bioengineering 74(4):288-294).Methods for modifying carbohydrate content are known to those skilled in the art (e.g., Wallick, SC et al. (1988) "Glycosylation of a VH residue of a monoclonal antibody against alpha(1-6) dextran increases its affinity for antigen," J. Exp. Med. 168(3):1099-1109; Tao, MH et al. (1989) "Studies of aglycosylated chimeric mouse-human IgG. Role of carbohydrate in the structure and effector functions mediated by the human IgG constant region," J. Immunol. 143(8):2595-2601; Routledge, E.G. et al. (1995) "The effect of aglycosylation on the immunogenicity of a humanized therapeutic CD3 monoclonal antibody," Transplantation 60(8):847-53; Elliott, S. et al. al. (2003) “Enhancement Of Therapeutic Protein In Vivo Activities Through Glycoengineering,” Nature Biotechnol. 21:414-21, Shields, RLet al. (2002) “Lack Of Fucose On Human IgG N-Linked Oligosaccharide Improves Binding To Human Fcgamma RIII And Antibody-Dependent Cellular Toxicity.,” J. Biol. Chem. 277(30):26733-26740).

[0148] In some embodiments, a humanized antibody is a derivative. Such a humanized antibody comprises substitution, deletion, or addition of amino acid residues in one or more non-human CDRs. A derivative of a humanized antibody may have substantially the same binding, better binding, or worse binding compared to a non-derivative humanized antibody. In certain embodiments, one, two, three, four, or five amino acid residues in the CDRs are substituted, deleted, or added (i.e., mutated).

[0149] Derivative antibodies or antibody fragments can be modified by chemical modification using techniques known to those skilled in the art, including, but not limited to, specific chemical cleavage, acetylation, formulation, metabolic synthesis using tunicamycin, etc. In one embodiment, an antibody derivative has similar or identical function as the parent antibody. In another embodiment, an antibody derivative exhibits altered activity compared to the parent antibody. For example, a derivative antibody (or fragment thereof) may bind to its epitope more strongly or be more resistant to proteolysis than the parent antibody.

[0150] Derivatized antibodies can be used to alter the half-life (e.g., serum half-life) of a parent antibody in a mammal, preferably a human. Preferably, such an alteration will result in a half-life of 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. Increasing the half-life of a humanized antibody or fragment thereof of the present disclosure in a mammal, preferably a human, will result in a higher serum titer of the antibody or antibody fragment in the mammal, thus reducing the frequency of administration of the antibody or antibody fragment and / or reducing the concentration of the antibody or antibody fragment administered. Antibodies or fragments thereof with increased in vivo half-lives can be generated by techniques known to those skilled in the art. For example, antibodies or fragments thereof with increased in vivo half-lives can be generated by modifying (e.g., substituting, deleting, or adding) amino acid residues identified to be involved in the interaction between the Fc domain and the FcRn receptor. Humanized antibodies can be engineered to increase biological half-life (see, e.g., U.S. Patent No. 6,277,375). For example, humanized antibodies can be engineered in the Fc hinge domain to increase in vivo or serum half-life.

[0151] Antibodies or fragments thereof with increased in vivo half-lives can be generated by conjugating 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 through site-specific conjugation to the N- or C-terminus of the antibody or antibody fragment, or via epsilon-amino groups present on lysine residues, with or without a multifunctional linker. Linear or branched polymer derivatization that minimizes loss of biological activity will be used. The degree of conjugation will be closely monitored by SDS-PAGE and mass spectrometry to ensure proper 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.

[0152] Antibodies can also be modified by the methods and coupling agents described by Davis et al. (see U.S. Pat. No. 4,179,337) to provide compositions that can be injected into the circulatory system of a mammal without substantially immunogenic response.

[0153] One embodiment encompasses modifications of framework residues of a humanized ApoL1 or Hpr antibody. Framework residues within the framework regions can be substituted with corresponding residues from the CDR donor antibody to alter, preferably improve, antigen binding. These framework substitutions are identified by methods well known in the art, such as modeling the interactions of CDR and framework residues to identify framework residues important for antigen binding and sequence comparison to identify unusual framework residues at specific positions. (See, e.g., U.S. Patent No. 5,585,089, and Riechmann, L. et al. (1988) "Reshaping Human Antibodies For Therapy," Nature 332:323-327.)

[0154] 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 heterologous (i.e., unrelated) molecules. Fusion is not necessarily direct, but may occur through linker sequences.

[0155] In one embodiment, such a heterologous molecule is a polypeptide 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. Such a heterologous molecule may alternatively be an enzyme, hormone, cell surface receptor, or drug moiety.For example: toxins (e.g., abrin, ricin A, Pseudomonas exotoxin (i.e., PE-40), diphtheria toxin, ricin, gelonin, or pokeweed antiviral protein), proteins (e.g., tumor necrosis factor, interferons (e.g., α-interferon, β-interferon), nerve growth factor, platelet-derived growth factor, tissue plasminogen activator, or apoptotic agents (e.g., tumor necrosis factor-α, tumor necrosis factor-β), biological response modifiers (e.g., lymphokines (e.g., interleukin-1 (“IL-1”), interleukin-2), interleukin-3), interleukin-4, interleukin-5, interleukin-6), interleukin-7, interleukin-8, interleukin-9, interleukin-10, interleukin-11), interleukin-12, interleukin-13, interleukin-14, interleukin-15, interleukin-16), interleukin-17, interleukin-18, interleukin-19, interleukin-20, interleukin-21), interleukin-22, interleukin-23, interleukin-24, interleukin-16, interleukin-17, interleukin-18, interleukin-19 ... 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., cytostatics or cytocidal agents, e.g., paclitaxel, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicine, anti-inflammatory drugs (e.g., benzodiazepines ... NU® (carmustine; BSNU) and lomustine (CCNU), cyclophosphamide, busulfan, dibromomannitol, streptozotocin, mitomycin C, and cis-dichlorodiamineplatinum(II) (DDP) cisplatin), anthracyclines (e.g., daunorubicin (formerly daunomycin) and doxorubicin), antibiotics (e.g., dactinomycin (formerly actinomycin), bleomycin, mithramycin, and anthramycin (AMC)), or antimitotics (e.g., vincristine and vinblastine).

[0156] Techniques for conjugating such therapeutic moieties to antibodies are well known, see, for example, Arnon et al., "Monoclonal Antibodies For Immunotargeting Of Drugs In Cancer Therapy," in MONOCLONAL ANTIBODIES AND CANCER THERAPY, Reisfeld et al. (eds.), 1985, pp. 243-56, Alan R. Liss, Inc.), Hellstrom et al., "Antibodies For Drug Delivery," in 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," in MONOCLONAL ANTIBODIES '84: BIOLOGICAL AND CLINICAL APPLICATIONS, Pinchera et al. al. (eds.), 1985, pp. 475-506), "Analysis, Results, And Future Prospective Of The Therapeutic Use Of Radiolabeled Antibodies In Cancer Therapy," in 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.

[0157] In one embodiment, the ApoL1 and Hpr antibodies or fusion molecules comprise an Fc portion. The Fc portion of such molecules can be varied by 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 improve production efficiency (and lower cost). Many modifications useful in constructing the disclosed fusion proteins and methods for making them are known in the art. See, for example, Mueller, JP et al. (1997) "Humanized Porcine VCAM-Specific Monoclonal Antibodies With Chimeric IgG2 / G4 Constant Regions Block Human Leukocyte Binding to Porcine Endothelial Cells," Mol. Immun. 34(6):441-452; Swann, PG (2008) "Considerations For The Development Of Therapeutic Monoclonal Antibodies," Curr. Opin. Immun. 20:493-499 (2008); and Presta, LG (2008) "Molecular Engineering And Design Of Therapeutic Antibodies," Curr. Opin. Immun. 20:460-470. In some embodiments, the Fc region is the Fc region of a native IgG1, IgG2, or IgG4. In some embodiments, the Fc region is hybrid, eg, chimeric, having an IgG2 / IgG4 Fc constant region.Modifications to the Fc region include, but are not limited to, an IgG4 modified to prevent binding to Fc gamma receptors and complement, an IgG1 modified to improve binding to one or more Fc gamma receptors, an IgG1 modified (amino acid changes) to minimize effector function, a modified / glycan-free IgG1 (typically by altering the expression host), and an IgG1 with modified pH-dependent binding to FcRn, as well as an IgG4 with a serine at amino acid position 228 in the hinge region altered to enhance stability (S228P). The Fc region can include the entire hinge region or less than the entire hinge region.

[0158] Treatment outcomes in patients treated with rituximab (a chimeric mouse / human IgG1 monoclonal antibody against CD20) for non-Hodgkin's lymphoma or Waldenström's macroglobulinemia correlated with the individual's expression of allelic variants of Fcγ receptors with different inherent affinities for the Fc domain of human IgG1. In particular, patients with high-affinity alleles of the low-affinity activating Fc receptor CD16A (FcγRIIIA) exhibited higher response rates and, in the case of non-Hodgkin's lymphoma, improved progression-free survival. In another embodiment, the Fc domain can contain one or more amino acid insertions, deletions, or substitutions to reduce binding to the low-affinity inhibitory Fc receptor CD32B (FcγRIIB) and retain or enhance wild-type levels of binding to the low-affinity activating Fc receptor CD16A (FcγRIIIA).

[0159] Another embodiment is an IgG that reduces binding to FcR and increases its half-life. 2-4 Representative IgGs include hybrids and IgG4 variants. 2-4Hybrid and IgG4 variants 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. Immun. 34(6):441-452, and U.S. Pat. No. 6,982,323. In some embodiments, the IgG1 and / or IgG2 domains are deleted. For example, Angal, S. et al. describe IgG1 and IgG2 variants in which serine 241 is replaced with proline.

[0160] The substitutions, additions, or deletions in the derivatized antibodies may be in the Fc region of the antibody, thereby serving to modify the binding affinity of the antibody to one or more FcγRs. Methods for modifying antibodies with modified binding to one or more FcγRs are known in the art. See, for example, PCT Publication Nos. WO04 / 029207, WO04 / 029092, WO04 / 028564, WO99 / 58572, WO99 / 51642, WO98 / 23289, WO89 / 07142, WO88 / 07089, and U.S. Patent Nos. 5,843,597 and 5,642,821. In a specific embodiment, modifications of the Fc region result in antibodies with altered antibody-mediated effector function, 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 (CDC), phagocytosis, or any combination thereof.

[0161] In some embodiments, the disclosure encompasses antibodies in which the Fc region has been modified, such that the molecule exhibits decreased activity towards activating receptors, e.g., FcγRIIA or FcγRIIIA, or increased activity towards inhibitory receptors, such as FcγRIIB. Preferably, such antibodies will exhibit decreased antibody-dependent cellular cytotoxicity (ADCC) or complement-dependent cytotoxicity (CDC) activity (compared to wild-type Fc receptors).

[0162] Modifications that affect Fc-mediated effector function are well known in the art (see U.S. Pat. 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 that have 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, and D376A.

[0163] In some embodiments, the disclosure encompasses antibodies in which the Fc region may be deleted (e.g., Fab or F(ab)2, etc.).

[0164] Any of the molecules 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, 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).

[0165] The present disclosure also encompasses antibodies or antigen-binding fragments thereof conjugated to diagnostic or therapeutic agents, or any other molecule for which increased serum half-life is desired. Antibodies can be used diagnostically (in vivo, in situ, or in vitro), for example, to determine the effectiveness of a given treatment regimen, as part of a clinical testing procedure, or to monitor the onset or progression of a disease, disorder, or infection. Detection can be facilitated by coupling the antibody to a detectable substance. Examples of detectable substances include various enzymes, artificial groups, fluorescent materials, luminescent materials, bioluminescent materials, radioactive materials, positron-emitting metals, and non-radioactive paramagnetic metal ions. Detectable substances can be coupled or conjugated to the antibody directly or indirectly via an intermediate (e.g., a linker known in the art) using techniques known in the art. See, for example, U.S. Pat. No. 4,741,900 for metal ions that can be conjugated to antibodies for use as diagnostics according to the present disclosure. Such diagnosis and detection can be achieved by coupling the antibody to a detectable substance, which can include various enzymes (enzymes including, but not limited to, horseradish peroxidase, alkaline phosphatase, beta-galactosidase, or acetylcholinesterase), artificial group conjugates (such as streptavidin / biotin and avidin / biotin), luminescent materials (such as 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 (including, but not limited to, bismuth ( 213 Bi), carbon ( 14 C), chromium ( 51 Cr), Cobalt ( 57 Co), fluorine ( 18 F), gadolinium ( 153 Gd, 159 Gd), 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), various positron emitting metals using positron emission tomography, and non-radioactive paramagnetic metal ions.

[0166] The molecules of the present disclosure can be conjugated to a second antibody to form an antibody heteroconjugate as described in U.S. Patent No. 4,676,980 to Segal. Such heteroconjugate antibodies can be further conjugated to a hapten (such as fluorescein), a cell marker, a cytokine, or a chemokine (e.g., CCL21), or the like.

[0167] The molecules of the present disclosure can be bound to solid supports, which are particularly useful for immunoassays or purification of target antigens or other molecules that can bind to target antigens immobilized on a support via 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.

[0168] 5. Methods for Producing Antibodies and Antigen-Binding Fragments The disclosed antibodies can be produced by any method known in the art useful for producing polypeptides, e.g., in vitro synthesis, recombinant DNA production, etc. Preferably, the antibodies are produced by recombinant DNA technology. The antibodies can be produced using recombinant immunoglobulin expression techniques. Recombinant production of immunoglobulin molecules, including humanized antibodies, is described in U.S. Pat. No. 4,816,397 (Boss et al.), U.S. Pat. Nos. 6,331,415 and 4,816,567 (both Cabilly et al.), British Patent No. GB ​​2,188,638 (Winter et al.), and British Patent No. 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). Further information regarding the generation, design, and expression of recombinant antibodies can be found in Mayforth, Designing Antibodies, Academic Press, San Diego (1993).

[0169] An exemplary process for producing a recombinant chimeric antibody can include: a) constructing, by conventional molecular biology methods, an expression vector that encodes and expresses an antibody heavy chain in which the CDRs and variable region of a mouse anti-ApoL1 or anti-Hpr monoclonal antibody are fused to an Fc region derived from a human immunoglobulin, thereby producing a vector for expressing the chimeric antibody heavy chain; b) constructing, by conventional molecular biology methods, an expression vector that encodes and expresses the antibody light chain of a mouse anti-ApoL1 or anti-Hpr monoclonal antibody, thereby producing a vector for expressing the chimeric antibody light chain; c) transferring the expression vector into a host cell by conventional molecular biology methods to produce a transfected host cell for expression of the chimeric antibody; and d) culturing the transfected cell by conventional cell culture techniques to produce the chimeric antibody.

[0170] An exemplary process for producing a recombinant humanized antibody includes: a) constructing, by conventional molecular biology methods, an expression vector that encodes and expresses an anti-ApoL1 or anti-Hpr heavy chain in which the minimum portion of the CDRs and variable region frameworks required to retain donor antibody binding specificity 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 producing a vector for expression of the humanized antibody heavy chain; and b) constructing, by conventional molecular biology methods, an expression vector that encodes and expresses an anti-ApoL1 or anti-Hpr heavy chain in which the minimum portion of the CDRs and variable region frameworks required to retain donor antibody binding specificity 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 producing a vector for expression of the humanized antibody heavy chain. This method can include constructing an expression vector that encodes and expresses an antibody light chain in which a minimal portion of the variable region framework is derived from a non-human immunoglobulin, such as a mouse anti-ApoL1 or anti-Hpr monoclonal antibody, and the remainder of the antibody is derived from a human immunoglobulin, thereby producing a vector for expression of a humanized antibody light chain; c) transferring the expression vector into a host cell by conventional molecular biology methods to produce a transfected host cell for expression of the humanized antibody; and d) culturing the transfected cell by conventional cell culture techniques to produce the humanized antibody.

[0171] For either exemplary method, host cells can be co-transfected with expression vectors that can contain different selectable markers, but that are preferably identical except for the heavy and light chain coding sequences. This procedure provides for equal expression of heavy and light chain polypeptides. Alternatively, a single vector encoding both heavy and light chain polypeptides can be used. The heavy and light chain coding sequences can comprise cDNA or genomic DNA, or both. Host cells used to express recombinant antibodies can be either 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 characteristics 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).

[0172] Any of the above antibodies can be used to generate anti-idiotype antibodies using techniques well known to those skilled in the art (see, e.g., 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).

[0173] The binding properties of any of the above antibodies can be further improved, if necessary, by screening for variants exhibiting such desired characteristics. 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 certain embodiments, such phage can be utilized to display antigen-binding domains, such as Fab, Fv, or disulfide-stabilized Fv, expressed from repertoire or combinatorial antibody libraries (e.g., human or murine). Phage expressing antigen-binding domains that bind to the antigen of interest can be selected or identified using, for example, labeled antigen or antigen bound or captured to a solid surface or bead. Phage used in these methods are typically filamentous phage, including fd and M13. The antigen-binding domain is expressed as a protein recombinantly fused to either the phage gene III or gene VIII protein. Examples of phage display methods that can be used to generate the immunoglobulins or fragments thereof of the present disclosure include Brinkman, U. et al. (1995) "Phage Display of Disulfide-Stabilized Fv Fragments," J. Immunol. Methods, 182:41-50, 1995; Ames, R. 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, CA 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, DRet al. (1994) “Human Antibodies From Combinatorial Libraries,” Adv. Immunol. 57:191-280, PCT Publications WO92 / 001047, WO90 / 02809, WO91 / 10737, WO92 / 01047, WO92 / 18619, WO93 / 11236, WO95 / 15982, WO95 / 20401, and U.S. Patent Nos. 5,698,426, 5,223,409, Examples of such compounds include Nos. 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.

[0174] After phage selection, as described in the above references, the antibody coding region from the phage can be isolated and used to generate whole antibodies, including humanized antibodies, or any other desired fragments, as described in detail below, and expressed in any desired host, including mammalian cells, insect cells, plant cells, yeast, and bacteria. For example, techniques for recombinantly producing Fab, Fab', and F(ab')2 fragments can be employed using methods known in the art (e.g., as disclosed in PCT Publication No. WO 92 / 22324; Mullinax, R., et al. (1992) "Expression of a Heterodimeric Fab Antibody Protein in One Cloning Step," BioTechniques, 12(6):864-869; 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 produce single-chain Fvs and antibodies include those described in U.S. Pat. Nos. 4,946,778 and 5,258,498, Huston, J. 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.

[0175] Phage display technology can be used to increase the affinity of antibodies for ApoL1 or Hpr. This technology may be useful for obtaining high-affinity antibodies that can be used in the disclosed methods. This technique, called affinity maturation, uses mutagenesis or CDR walking and reselection using such receptors or ligands (or their extracellular domains) or antigenic fragments thereof to identify antibodies that bind with higher affinity to an antigen compared to the initial or parent antibody (see, e.g., Glaser, SM et al. (1992) "Antibody Engineering By Codon-Based Mutagenesis In A Filamentous Phage Vector System," J. Immunol. 149:3903-3913). By mutagenizing entire codons, rather than single nucleotides, a repertoire of semi-randomized amino acid mutations is obtained. Libraries can be constructed to contain a pool of variant clones, each of which differs by a single amino acid modification in a single CDR, and includes variants representing each possible amino acid substitution for each CDR residue. Mutants with increased binding affinity to the antigen can be screened by contacting the immobilized mutants with labeled antigen. Any screening method known in the art (e.g., ELISA) can be used to identify mutant antibodies with increased affinity to the antigen (see, for example, 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, DE et al. (1995) "Affinity Maturation of the BR96 Anti-Carcinoma Antibody by Codon-Based Mutagenesis," J. Immunol. 155:1994-2004).CDR walking can be used to randomize the light chain (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).

[0176] Thus, 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, called affinity maturation, uses mutagenesis or "CDR walking" using a target antigen or its antigenic fragment and reselection to identify antibodies with CDRs that bind with higher (or lower) affinity to the antigen compared to the initial or parent antibody (see, e.g., Glaser, SM et al. (1992) "Antibody Engineering By Codon-Based Mutagenesis In A Filamentous Phage Vector System," J. Immunol. 149:3903-3913). By mutagenizing entire codons, rather than single nucleotides, a repertoire of semi-randomized amino acid mutations is obtained. Libraries can be constructed to contain a pool of variant clones, each of which differs by a single amino acid modification in a single CDR, including variants representing 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 labeled antigen. Any screening method known in the art (e.g., ELISA) can be used to identify mutant antibodies with increased (or decreased) affinity for the antigen (see 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, DE et al. (1995) "Affinity Maturation of the BR96 Anti-Carcinoma Antibody by Codon-Based Mutagenesis," J. Immunol. 155:1994-2004).CDR walking can be used to randomize the light chain (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).

[0177] Methods for achieving such affinity maturation are described, for example, in: Krause, JC 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, DL et al. 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 Naieve 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, WJet 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.

[0178] 6. Exemplary Bispecific Antibodies An anti-ApoL1, BCMA IgG1-scFv (heavy chain C-terminus) chimeric antibody was designed and has the structure shown in Figure 8.

[0179] The Fab portion is the heavy and light chain variable regions of recombinant clone SFIII 13.11 (anti-ApoL1) having SEQ ID NO: 24 (VH) and SEQ ID NO: 36 or SEQ ID NO: 77 (VL).

[0180] Anti-BCMA is an ScFv of human IgG1 kappa [clone 17A5] 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 as follows: VH [ka] VL [ka]

[0181] The CDR sequences of anti-BCMA clone 17A5, which are in bold in the sequence above, are as follows: CDR1H: SYAMS (SEQ ID NO: 43), CDR2H: AISGSGGSTYYADSVKG (SEQ ID NO: 44), CDR3H: VAPYFAPFDY (SEQ ID NO: 45), CDR1L: RASQSVSSSYLA (SEQ ID NO: 46), CDR2L: GASSRAT (SEQ ID NO: 47), CDR3L: QQYGNPPLYT (SEQ ID NO: 48).

[0182] An exemplary sequence of an anti-BCMA scFv is: [ka] where the CDRs are in bold and ggggsggggsggggs (lowercase) (SEQ ID NO: 52) is the flexible linker.

[0183] In some embodiments, the bispecific antibody comprises the amino acid sequence (SEQ ID NO: 71) or a variant thereof having at least 70, 80, 90, or 95% sequence identity thereto; and / or DVLMTQTPLSLPVSLGDQASISCRSSQSIVNSNGNTYLEWYLQKPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYYCFQGSHVPLTFGAGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 72) or a variant thereof having at least 70, 80, 90, or 95% sequence identity thereto.

[0184] With respect to SEQ ID NO: 71: EVQLVESGGGLVKPGGSLKLSCAASGFTFSTYAMSWVRQSPEKRLEWVAEISNGGLYTYYPDTVTGRFTISRDNVKNILYLEMSSLRSEDTAIYYCIRENRNWYFDLWGAGTTVTVSS (SEQ ID NO: 24) is the heavy chain variable domain of the STII 13.11 anti-ApoL1 antibody; ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP (SEQ ID NO: 75) is the heavy chain constant region sequence; GGGGSGGGGSGGGGS (SEQ ID NO: 52) is a linker sequence, EIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGNPPLYTFGQGTKVEIKGGGGSGGGGSGGGGSEVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKVAPYFAPFDYWGQGTLVTVSS (SEQ ID NO: 51) is (the variable domain of) the BCMA17A5 scFv.

[0185] With respect to SEQ ID NO: 72: DVLMTQTPLSLPVSLGDQASISCRSSQSIVNSNGNTYLEWYLQKPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYYCFQGSHVPLTFGAGTKLEIK (SEQ ID NO: 77) is the light chain variable domain of the STII 13.11 anti-ApoL1 antibody; RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 76) is the light chain constant region sequence.

[0186] Bispecific antibodies having the structure of Figure 8 can be formed by two copies of the amino acid sequences SEQ ID NO:71 and SEQ ID NO:72, respectively, for example, upon co-expression of a nucleic acid encoding the amino acid sequence of SEQ ID NO:71 (e.g., SEQ ID NO:73) and a nucleic acid encoding the amino acid sequence of SEQ ID NO:72 (e.g., SEQ ID NO:74).

[0187] See WO2014 / 122144 (specifically incorporated herein by reference in its entirety) for the BMCA clone 17A5 sequence, and alternative anti-BCMA sequences that can be used to derive alternative anti-BMCA arms of antibodies.

[0188] In another embodiment, in addition to or instead of anti-ApoL1, the Fab portion comprises the heavy and light chain CDRs, or the entire heavy and light chain variable regions, of recombinant clone SFII 134.3 (anti-Hpr) or SFII 14.11 (anti-Hpr) having the sequences of SEQ ID NO: 3 (VH) and SEQ ID NO: 14 (VL), or SEQ ID NO: 56 (VH) and SEQ ID NO: 65 (VL), respectively, and their associated CDRs, as discussed in more detail elsewhere herein.

[0189] 7. Other Exemplary Antibodies for Targeting As introduced above, cell-specific markers and cancer antigens are preferred targets for targeting moieties, preferably antibodies. Antibodies for targeting cell-specific markers and cancer antigens are known in the art and can be used in the disclosed compositions.

[0190] A specific example of an anti-B7-H1 (PD-L1) antibody is the human anti-B7-H1 antibody MDX-1105 (WO / 2007 / 005874, published January 11, 2007).

[0191] 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.

[0192] Examples of anti-CTLA4 antibodies contemplated for use in the disclosed compositions and methods include those described in PCT / US2006 / 043690 (WO / 2007 / 056539 to Fischkoff et al.).

[0193] Other specific exemplary antibodies that can be utilized in the bispecific and multispecific antibodies whose sequences are disclosed include, but are not limited to, talquetamab (JNJ64407564), indatuximab ravtansine, daratumumab, elotuzumab, DFRF4539A, and BFCR4350A.

[0194] Daratumumab is a human IgG (kappa) antibody that targets a unique epitope on CD38 (FDA approved in 2016).

[0195] Indatuximab ravtansine targets CD138.

[0196] Elotuzumab is a mAb directed against the extracellular domain of SLAMF7 that has shown moderate success in the phase 3 ELOQUENT-2 trial.

[0197] DFRF4539A and BFCR4350A target FcRH5.

[0198] Talquetamab is a bispecific antibody that targets GPRC5D on multiple myeloma and CD3 on T cells.

[0199] 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.

[0200] Antibodies targeting claudin 18.2 antibodies are provided in WO2022 / 136642A1.

[0201] Antibodies targeting MUC1 (gatipotuzumab) are provided, for example, in WO2019 / 219891 and KR2021 / 0010565A.

[0202] Examples of antibodies targeting MSLN (mesothelin) antibodies include: anetumab ravtansine (see, e.g., WO2009 / 068204A1 and WO2020 / 234114A1); SS1P (see, e.g., U.S. Patent No. 8,460,660 B2), amatuximab or MORAb-009 (see, e.g., U.S. Patent No. 9,803,022), and SD1 / SD2 (see, for example, WO2014 / 052064), but are not limited to these.

[0203] Examples of antibodies that target PMEL17 are discussed, for example, in U.S. Pat. No. 9,056,910, WO 2013 / 165940, and EP 2844300.

[0204] All of the aforementioned patents and applications are specifically incorporated by reference in their entirety, including, but not limited to, their antibody sequences, particularly CDRs, which may be incorporated into the disclosed compositions for targeting ApoL1 to cells expressing their target antigens.

[0205] B. Compositions for Targeting Exogenous ApoL1 Compositions for increasing cellular internalization of exogenous ApoL1 are also provided. Although not necessarily used exclusively for this purpose, the compositions can be used to deliver exogenous ApoL1, such as recombinant ApoL1, to cells.

[0206] The composition typically comprises ApoL1, such as ApoL1 of SEQ ID NO: 50, or a functional fragment or variant thereof having, for example, 70%, 75%, 80%, 85%, 90%, or 95% sequence identity thereto. The composition typically further comprises a targeting moiety that binds to a cell-specific marker, such as a cancer antigen, present on the cell, thereby facilitating targeting of ApoL1 or its fragment or variant to the target cell. The targeted ApoL1 can be internalized by the cell in an amount effective to increase cell death of the target cell. ApoL1 or its fragment or variant can be fused to, or directly or indirectly conjugated 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 its fragment or variant is conjugated to the targeting moiety. In some embodiments, ApoL1 or a fragment or variant thereof is packaged into a delivery vehicle, such as a nanoparticle or liposome, which delivery vehicle further has a targeting moiety conjugated thereto.

[0207] Any of the compositions can further comprise a cell membrane penetrating peptide.

[0208] 1. Targeting part Representative targeting moieties include, but are not limited to, antibodies and their antigen-binding fragments, aptamers, peptides, and small molecules. The binding moiety can be conjugated to the polymer that forms the nanocarrier. Typically, the binding moiety is displayed on the outer shell of the nanocarrier. The outer shell can act as a shield 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 include 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 polymer material. The inner core carries a therapeutic payload and releases the therapeutic 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 NMR imaging agent, that allows visualization of the nanocarrier.

[0209] In other embodiments, the targeting moiety is conjugated, linked, or directly fused to ApoL1, or a fragment or variant thereof.

[0210] The targeting moiety of the nanocarrier can be an antibody or an antigen-binding fragment thereof. The targeting moiety must have affinity for a cell surface receptor or antigen on the target cell. The targeting moiety can result in internalization of the nanocarrier within the target cell.

[0211] The targeting moiety can specifically recognize and bind to a target molecule specific to 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 selectively expressed on a particular cell surface, tissue, or organ. The cell-specific marker can be directed to 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 to endothelial cells, ectodermal cells, or mesenchymal cells. Exemplary cell-specific markers include, but are not limited to, cancer-specific markers. The cell marker can be any cell-specific marker, including cancer antigens 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). Typically, the targeting moiety does not target trypanosome-specific surface antigens.

[0212] The targeting moiety may be a peptide. The targeting peptide may be covalently associated with the polymer, and the covalent association may be mediated by a linker. The targeting moiety may be an antibody or antigen-binding fragment of the fusion protein. The antibody may be in any format, including, but not limited to, those provided elsewhere herein (see, e.g., above).

[0213] 2. Exemplary Nanocarriers Nanocarrier compositions are provided that include ApoL1 or a fragment or variant thereof and a targeting moiety loaded onto, bound to the surface of, and / or encapsulated within a delivery vehicle.

[0214] 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.

[0215] In some embodiments, the delivery vehicle is a nanoscale composition, e.g., from 10 nm to about 1 micron (but not including 1 micron). However, it should be understood that in some embodiments and depending on the application, the particles may be smaller or larger (e.g., microparticles, etc.). While many of the compositions disclosed herein are referred to as nanoparticle or nanocarrier compositions, it should be understood that in some embodiments and depending on the application, the carrier may be somewhat larger than a nanoparticle. For example, the carrier composition may be from about 1 micron to about 1000 microns. Such compositions may be referred to as microparticle compositions. For example, nanocarriers according to the present disclosure may be microparticles. Microparticles may have a size, e.g., of 0.1 to 100 μm in diameter. In another example, nanocarriers may be superparticles. Superparticles are particles having a size greater than about 100 μm in diameter. For example, superparticles may have a size of about 100 μm to about 1,000 μm in diameter.

[0216] Microbubbles are air bubbles with diameters less than 1 millimeter but larger than 1 micrometer, and they have a wide range of applications in industry, life sciences, and medicine. The composition of the bubble shell and filler material determines their properties, such as buoyancy, compressive strength, thermal conductivity, and acoustic properties. In medicine, they are used for diagnostics, such as imaging, and therapeutics, such as drug delivery.

[0217] In some embodiments for treating cancer, it is desirable for the particles to be sized appropriately to access the tumor microenvironment. In certain embodiments, the particles are sized appropriately to access the tumor microenvironment and / or tumor cells via the enhanced permeability and retention (EPR) effect. EPR refers to the tendency for molecules of a certain size to accumulate in tumor tissue to a greater extent than in normal tissue. Thus, in an exemplary treatment for cancer, the delivery vehicle may be in the range of about 25 nm to about 500 nm, inclusive, or about 50 nm to about 300 nm, inclusive. In another example, the delivery vehicle may be in the range of about 80 nm to about 120 nm, inclusive. In another example, the delivery vehicle may be in the range of about 85 nm to about 110 nm, inclusive.

[0218] Polymeric nanoparticles are typically formed using a single or double emulsion process using aqueous and non-aqueous solvents. Typically, the nanoparticles contain a minimal amount of non-aqueous solvent after solvent removal.

[0219] In one embodiment, nanoparticles are prepared using an emulsion solvent evaporation method. The polymeric 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, or acyl acetate. The drug can be dissolved in one or more of the following, but not limited to, acetone, ethanol, methanol, isopropyl alcohol, acetonitrile, and dimethyl sulfoxide (DMSO). An aqueous solution is then added to the resulting mixed solution to obtain an emulsion solution by emulsification. The emulsification technique can be, but is not limited to, probe sonication or homogenization using a homogenizer.

[0220] In another embodiment, the nanoparticles are prepared using nanoprecipitation or a microfluidic device. A polymeric material is mixed with a drug or drug combination in a water-miscible organic solvent. The water-miscible organic solvent can be one or more of acetone, ethanol, methanol, isopropyl alcohol, acetonitrile, and dimethyl sulfoxide (DMSO). The resulting mixed solution is then added to an aqueous solution to obtain a nanoparticle solution. The drug may be associated with the surface of the polymer matrix of the particles, encapsulated within the polymer matrix of the particles, surrounded by the polymer matrix of the particles, and / or dispersed throughout the polymer matrix of the particles.

[0221] In another embodiment, the nanoparticles are prepared by the self-assembly of amphiphilic polymers, optionally including hydrophilic and / or hydrophobic polymers, using emulsion solvent evaporation, single-step nanoprecipitation, or microfluidic devices.

[0222] Other exemplary methods for producing nanoparticles encompassed by the present disclosure are described in Zhou, et al., Biomaterials, 33(2):583-591 (2012) and Han, et al., Nanomedicine (2016).

[0223] Two methods for incorporating targeting moieties into nanoparticles include i) conjugation of targeting ligands to hydrophilic regions of polymers (e.g., PEG) prior to nanoparticle preparation, and ii) incorporation of targeting molecules onto nanoparticles, where the PEG layer on the nanoparticle surface can be cleaved in the presence of chemicals or enzymes in the tissue of interest to expose the targeting molecules.

[0224] Particles can be microparticles or nanoparticles. Nanoparticles are often utilized for tissue application, cell penetration, and specific 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 diameters 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, nanoparticles can have diameters less than 400 nm, less than 300 nm, or less than 200 nm. The range can be 50 nm to 300 nm.

[0225] The average diameter of the nanoparticles is typically about 50 nm to about 500 nm, or about 50 nm to about 350 nm. In some embodiments, the average diameter of the nanoparticles is about 100 nm. The zeta potential of the nanoparticles is typically about -50 mV to about +50 mV, or about -25 mV to about +25 mV, or about -10 mV to about +10 mV.

[0226] In some embodiments, the particles are brain-penetrating polymeric nanoparticles that can be loaded with drugs and optimized for intracranial convection-enhanced delivery (CED), such as those discussed in WO 2013 / 166487 and U.S. Published Application No. 2015 / 011831. For example, the particles can be formed by emulsifying a polymer-drug solution, then removing the solvent, centrifuging at a first force to remove larger particles, and then using a second, higher force to collect the smaller particles, precipitating smaller particles with diameters less than 100 nm or average diameters in the range of 25 to 75 nanometers that can penetrate the brain interstitial space.

[0227] Partially water-miscible organic solvents, such as benzyl alcohol, butyl lactate, and ethyl acetate (EA), enable the formulation of nanoparticles via an emulsion-diffusion mechanism and can produce smaller nanoparticles than water-immiscible solvents, such as dichloromethane (DCM). The use of partially water-miscible organic solvents improves 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.

[0228] To reduce aggregation, sugars 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 sugar to nanoparticles is 10-50%.

[0229] a. polymer Nanocarriers may also be particles comprising one or more hydrophilic polymers, including cellulosic polymers (e.g., starch and polysaccharides), hydrophilic polypeptides, poly(amino acids) (e.g., poly-L-glutamic acid (PGS), gamma-polyglutamic acid, poly-L-aspartic acid, poly-L-serine, or poly-L-lysine), polyalkylene glycols and polyalkylene oxides (e.g., polyethylene glycol (PEG), polypropylene glycol (PPG), and poly(ethylene oxide) (PEO)), poly(oxyethylated polyols), poly(olefinyl alcohols), polyvinylpyrrolidone), poly(hydroxyalkylmethacrylamides), poly(hydroxyalkylmethacrylates), poly(saccharides), poly(hydroxy acids), poly(vinyl alcohols), and copolymers thereof.

[0230] The nanoparticles can comprise one or more hydrophobic polymers. Examples of suitable hydrophobic polymers include polyhydroxy acids (e.g., poly(lactic acid), poly(glycolic acid), and poly(lactic-co-glycolic acid)), polyhydroxyalkanoates (e.g., poly3-hydroxybutyrate or poly4-hydroxybutyrate), polycaprolactone, poly(orthoesters), polyanhydrides, poly(phosphazenes), poly(lactide-co-caprolactone), polycarbonates (e.g., tyrosine polycarbonate), polyamides (including synthetic and natural polyamides), polypeptides, and poly(amino acids). Poly(ethylene glycol) acrylates, propylene glycol acrylates, propylene glycol diacrylates, propylene glycol ...

[0231] In certain embodiments, the hydrophobic polymer is an aliphatic polyester, hi some embodiments, the hydrophobic polymer is poly(lactic acid), poly(glycolic acid), or poly(lactic-co-glycolic acid).

[0232] The nanoparticles can include one or more biodegradable polymers. Biodegradable polymers can include water-insoluble or sparingly soluble polymers that are chemically or enzymatically converted into water-soluble materials in the body. Biodegradable polymers can include soluble polymers crosslinked with hydrolyzable crosslinking groups, rendering the crosslinked polymers water-insoluble or sparingly soluble.

[0233] Biodegradable polymers in the nanoparticles include polyamides, polycarbonates, polyalkylenes, polyalkylene glycols, polyalkylene oxides, polyalkylene terephthalates, polyvinyl alcohols, polyvinyl ethers, polyvinyl esters, polyvinyl halides, polyvinylpyrrolidone, polyglycolides, polysiloxanes, polyurethanes and their copolymers, alkyl celluloses, hydroxyalkyl celluloses, cellulose ethers, cellulose esters, nitrocellulose, polymers of acrylic and methacrylic esters, methyl cellulose, ethyl cellulose, hydroxypropyl cellulose, hydroxypropylmethyl cellulose, hydroxybutylmethyl cellulose, cellulose acetate, cellulose propionate, cellulose acetate butyrate, cellulose acetate phthalate, carboxyethyl cellulose, cellulose triacetate, cellulose sulfate sodium salt, poly(methyl methacrylate), poly(ethyl methacrylate), poly(butyl methacrylate), poly(isobutyl methacrylate), poly(hexyl methacrylate), acrylate), 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), poly(ethylene terephthalate), poly(vinyl alcohol), poly(vinyl acetate), polyvinyl chloride, polystyrene, and polyvinylpyrrolidone, derivatives thereof, linear and branched copolymers and block copolymers thereof, and mixtures thereof. Exemplary biodegradable polymers include polyesters, poly(orthoesters), poly(ethyleneimine), poly(caprolactone), poly(hydroxybutyrate), poly(hydroxyvalerate), polyanhydrides, poly(acrylic acid), polyglycolide, poly(urethane), polycarbonates, polyphosphate esters, polyphosphazenes, derivatives thereof, linear and branched copolymers and block copolymers thereof, and mixtures thereof.

[0234] The nanoparticles can include one or more amphiphilic polymers. The amphiphilic polymer can be a polymer including a hydrophobic polymer block and a hydrophilic polymer block. The hydrophobic polymer block can include one or more of the hydrophobic polymers described above or their derivatives or copolymers. The hydrophilic polymer block can include one or more of the hydrophilic polymers described above or their derivatives or copolymers. In some embodiments, the amphiphilic polymer is a diblock polymer including a hydrophobic end formed from a hydrophobic polymer and a hydrophilic end formed from a hydrophilic polymer. In some embodiments, a moiety can be attached to the hydrophobic end, the hydrophilic end, or both.

[0235] In some embodiments, the nanoparticles comprise 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 that do not include a targeting moiety. The hydrophobic polymer block of the first amphiphilic polymer and the hydrophobic polymer block of the second amphiphilic polymer can 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 can be the same or different.

[0236] In some embodiments, the nanoparticles comprise biodegradable polyesters or polyanhydrides, such as poly(lactic acid), poly(glycolic acid), and poly(lactic-co-glycolic acid). The nanoparticles may comprise one or more of the following polyesters: homopolymers containing glycolic acid units, referred to herein as "PGA," homopolymers containing 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 homopolymers containing caprolactone units, such as poly(ε-caprolactone), collectively referred to herein as "PCL," and copolymers containing lactic and glycolic acid units, such as various forms of poly(lactic-co-glycolic acid) and poly(lactide-co-glycolide), characterized by the ratio of lactic acid to glycolic acid, and polyacrylates and derivatives thereof, collectively referred to herein as "PLGA." Exemplary polymers also include copolymers of polyethylene glycol (PEG) and the aforementioned polyesters, such as various forms of PLGA-PEG or PLA-PEG copolymers, collectively referred to herein as "PEGylated polymers." In certain embodiments, the PEG region is covalently associated with the polymer to yield a "PEGylated polymer" via a cleavable linker. Other polymers include PLGA-poly(ε-carbobenzoxyl-L-lysine) (PLL) (i.e., PLGA-PLL).

[0237] The nanoparticles can also include one or more polymer conjugates, including an end-to-end linkage between the polymer and the targeting moiety or detectable label. For example, the modified polymer can be a PLGA-PEG-peptide block polymer.

[0238] The nanoparticles can comprise a mixture of one or more polymers. The nanoparticles can contain other substances such as stabilizers, surfactants, or lipids. The nanoparticles can comprise a first polymer having a targeting moiety and a second polymer without a targeting moiety. By adjusting the ratio of the targeting polymer to the non-targeting polymer, the density of the targeting moiety on the outside of the particle can be adjusted.

[0239] The nanoparticles can comprise 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 attached to the hydrophobic polymer block, and a targeting moiety covalently attached to the hydrophilic polymer block. For example, the amphiphilic polymer can have a conjugate with the structure ABX, where A is a hydrophobic molecule or polymer, B is a hydrophilic molecule or polymer, and X is a targeting moiety. Exemplary amphiphilic polymers include those in which A is a hydrophobic biodegradable polymer, B is PEG, and X is a targeting moiety that binds to the targeting moiety.

[0240] In some embodiments, the nanoparticles comprise a first amphiphilic polymer having the structure ABX described above and a second amphiphilic polymer having the structure AB, where A and B in the second amphiphilic macromolecule are independently selected from A and B in the first amphiphilic macromolecule, although they may be the same.

[0241] b. Liposomes and micelles In some embodiments, the nanocarrier is a liposome or micelle. Liposomes are spherical vesicles composed of concentric phospholipid bilayers separated by aqueous compartments. Liposomes can attach to cell surfaces and form molecular membranes. Structurally, liposomes are lipid vesicles composed of concentric phospholipid bilayers surrounding an aqueous interior (Gregoriadis, et al. Int. J. Pharm., 300, 125-30 2005; Gregoriadis and Ryman, Biochem. J., 124, 58P (1971)). Hydrophobic compounds are associated with the lipid phase, while hydrophilic compounds are associated with the aqueous phase.

[0242] Liposomes have the ability to form molecular membranes on the surfaces of cells and tissues. Clinical studies have demonstrated the effectiveness 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 have also been used in ophthalmology to improve keratitis, corneal transplant rejection, uveitis, endophthalmitis, and proliferative vitreoretinopathy (Ebrahim, et al., 2005; Li, et al., 2007).

[0243] Liposomes have been widely studied as drug carriers for various chemotherapeutic agents (approximately 25,000 scientific papers have been published on this topic) (Gregoriadis, N Engl J Med 295, 765-70 (1976); Gregoriadis, et al., Int. J. Pharm. 300, 125-30 (2005)). Water-soluble anticancer agents such as doxorubicin can be protected within the aqueous compartment(s) of the liposome separated by the phospholipid bilayer(s), while lipid-soluble agents 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)). Topical and intravitreal delivery of cyclosporine has been dramatically improved by liposomes (Lallemand, et al., Eur J Pharm Biopharm 56, 307-18 2003). Delivery of chemotherapy drugs results in 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 10 liposome and lipid-based formulations have been approved by regulatory agencies, 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 are obtained from extensive clinical experience using liposomes in clinics on thousands of patients.

[0244] 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, sphingolipids, or PEGylated lipids.Neutral and anionic lipids include, but are not limited to, 1,2-diacyl-glycero-3-phosphocholine, phosphatidylcholine (PC) (e.g., egg PC, soybean PC); phosphatidylserine (PS), phosphatidylglycerol, phosphatidylinositol (PI); glycolipids; sphingophospholipids such as sphingomyelin, and glycosphingolipids such as ceramide galactopyranoside, ganglioside, and cerebroside (also known as 1-ceramidylglucoside); fatty acids containing a carboxylic acid group; Included are, but are not limited to, sterols (e.g., cholesterol); 1,2-dioleylphosphoethanolamine (DOPE), 1,2-dihexadecylphosphoethanolamine (DHPE), 1,2-distearoylphosphatidylcholine (DSPC), 1,2-dipalmitoylphosphatidylcholine (DPPC), and 1,2-dimyristoylphosphatidylcholine (DMPC). Lipids can also include various natural (e.g., L-α-phosphatidylcholine derived from tissues such as egg yolk, heart, brain, liver, and soybean) and / or synthetic (e.g., saturated and unsaturated 1,2-diacyl-sn-glycero-3-phosphocholine, 1-acyl-2-acyl-sn-glycero-3-phosphocholine, and 1,2-diheptanoyl-SN-glycero-3-phosphocholine) derivatives of lipids. In some embodiments, the liposomes comprise a phosphatidylcholine (PC) head group and, optionally, sphingomyelin. In other embodiments, the liposomes comprise DPPC. In further embodiments, the liposomes comprise a neutral lipid such as 1,2-dioleoylphosphatidylcholine (DOPC).

[0245] In certain embodiments, liposomes are formed from a single type of phospholipid. In some embodiments, the phospholipid has a phosphatidylcholine head group, such as sphingomyelin. The liposomes may contain a sphingomyelin metabolite. Sphingomyelin metabolites used to formulate the liposomes include, but are not limited to, ceramide, sphingosine, or sphingosine 1-phosphate. The concentration of the sphingomyelin metabolite contained in the lipids used to formulate the liposomes may range from about 0.1 mol% to about 10 mol%, or from about 2.0 mol% to about 5.0 mol%, or may be about 1.0 mol%.

[0246] Suitable cationic lipids for liposomes include, but are not limited to, N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium salts (also referred to as TAP lipids), such as methyl sulfate salts. Suitable TAP lipids include, but are not limited to, DOTAP (dioleoyl-), DMTAP (dimyristoyl-), DPTAP (dipalmitoyl-), and DSTAP (distearoyl-). Suitable cationic lipids for liposomes include, but are not limited to, dimethyldioctadecylammonium bromide (DDAB), 1,2-diacyloxy-3-trimethylammonium propane, N-[1-(2,3-dioleoyloxy)propyl]-N,N-dimethylamine (DODAP), 1,2-diacyloxy-3-dimethylammonium propane, N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA ... Dioctadecylamidoglycylspermine (DOGS), 3-[N-(N',N'-dimethylaminoethane)carbamoyl]cholesterol (DC-Chol); 2,3-dioleoyloxy-N-(2-(sperminecarboxamido)ethyl)-N,N-dimethyl-1-propanaminium trifluoroacetate (DOSPA), β-alanylcholesterol, cetyltrimethylammonium bromide (CTAB), diC 14-amidine, N-ferf-butyl-N'-tetradecyl-3-tetradecylamino-propionamidine, N-(alpha-trimethylammonioacetyl)didodecyl-D-glutamic acid chloride (TMAG), ditetradecanoyl-N-(trimethylammonioacetyl)diethanolamine chloride, 1,3-dioleoyloxy-2-(6-carboxy-spermyl)propylamide (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)-imidazolinium chloride derivative, such as 1-[2-(9(Z)-octadecenoyloxy)ethyl]-2-(8(Z)-heptadecenyl-3-(2-hydroxyethyl)imidazolinium chloride (DOTIM), and 1-[2-(hexadecanoyloxy)ethyl]-2-pentadecyl-3-(2-hydroxyethyl)imidazolinium chloride (DPTIM). In one embodiment, the cationic lipid can be a 2,3-dialkyloxypropyl quaternary ammonium compound derivative containing a hydroxyalkyl moiety on the quaternary amine, such as 1,2-dioleoyl-3-dimethyl-hydroxyethylammonium bromide (DORI), 1,2-dioleoyl The hydroxypropyl 1,2-dioleyloxypropyl-3-dimethyl-hydroxyethyl ammonium bromide (DORIE), 1,2-dioleyloxypropyl-3-dimethyl-hydroxypropyl ammonium bromide (DORIE-HP), 1,2-dioleyloxypropyl-3-dimethyl-hydroxybutyl ammonium bromide (DORIE-HB), 1,2-dioleyloxypropyl-3-dimethylhydroxypentyl ammonium bromide (DORIE-Hpe), 1,2-dimyristyloxypropyl-3-dimethylhydroxylethyl ammonium bromide (DMRIE), 1,2-dipalmityloxypropyl-3-dimethylhydroxyethyl ammonium bromide (DPRIE), and 1,2-disteryloxypropyl-3-dimethylhydroxyethyl ammonium bromide (DSRIE).

[0247] The lipid may be formed from a combination of multiple lipids; for example, a charged lipid may be combined with a lipid that is non-ionic or uncharged at physiological pH. Non-ionic lipids include, but are not limited to, cholesterol and DOPE (1,2-dioleoylglycerylphosphatidylethanolamine). The molar ratio of the first phospholipid, such as sphingomyelin, to the second lipid may range from about 5:1 to about 1:1, or from 3:1 to about 1:1, or from about 1.5:1 to about 1:1, or the molar ratio is about 1:1.

[0248] In some embodiments, the liposome or micelle comprises a phospholipid, cholesterol, and a nitrogen-containing lipid, examples of which include phospholipids, including natural phospholipids such as phosphatidylcholine, phosphatidylserine, phosphatidylglycerol, phosphatidylinositol, phosphatidylethanolamine, phosphatidic acid, cardiolipin, sphingomyelin, egg yolk lecithin, soybean lecithin, and lysolecithin, as well as their hydrogenated products obtained in standard manners. It is also possible to use synthetic phospholipids such as dicetyl phosphate, distearoylphosphatidylcholine, dipalmitoylphosphatidylcholine, dipalmitoylphosphatidylethanolamine, dipalmitoylphosphatidylserine, eleostearoylphosphatidylcholine, and eleostearoylphosphatidylethanolamine, as well as homopoly{N'--[N-(2-aminoethyl)-2-aminoethyl]aspartamide}P[Asp(DET)] and block cationic poly(ethylene glycol)(PEG)-bP[Asp(DET)].

[0249] In some embodiments, the liposomes are long-circulating or stealth liposomes, such as those reviewed in Immordino, et al., Int J Nanomedicine, 1(3):297-315 (2006), which is specifically incorporated herein by reference in its entirety. Liposomes have been developed with surfaces modified with various molecules, including glycolipids and sialic acid. Long-circulating liposomes can include, for example, synthetic polymers such as poly(ethylene glycol) (PEG) in the liposome composition. PEG on the surface of the liposome carrier can extend blood circulation time while reducing uptake by the mononuclear phagocyte system (stealth liposomes) and can function as an anchor for targeting moieties.

[0250] Antibodies and antibody fragments are widely used as targeting moieties for liposomes due to their high specificity for target antigens. Methods for producing targeted liposomes, called immunoliposomes, by coupling antibodies to the liposome surface are known in the art. Such techniques include, but are not limited to, conventional coupling and maleimide-based techniques. For example, see Paszko and Senge, Curr Med Chem., 19(31):5239-77(2012); Kelly, et al., Journal of Drug Delivery, Volume 2011(2011), article ID 727241, page 11.

[0251] The micelles can be polymeric micelles, such as those composed of amphiphilic diblock or triblock copolymers consisting of a solvent-philic block and a solvent-phobic block (see, e.g., Croy and Kwon, Curr Pharm Des., 12(36):4669-84 (2006)).

[0252] 3. Other Functional Elements Other functional elements that can be directly or indirectly associated, linked, conjugated, or otherwise attached to ApoL1, antibodies, or particles or other delivery vehicles thereof include protein transduction domains and membrane fusogenic peptides.

[0253] For example, the efficiency of particle delivery systems can also be improved 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 serves a variety of different functions, including inducing cellular uptake, endosomal disruption, and delivery of plasmid payloads to the nucleus. Many methods exist for tethering ligands to particle surfaces. One approach is direct covalent attachment to functional groups on PLGA NPs (Bertram, Acta Biomater. 5:2860-2871 (2009)). Another approach utilizes amphiphilic conjugates such as avidin palmitate to immobilize biotinylated ligands on the NP surface (Fahmy, et al., Biomaterials, 26:5727-5736 (2005); Cu, et al., Nanomedicine, 6:334-343 (2010)). This approach produces particles that enhance cellular uptake but reduce pDNA release and gene transfection. This is likely due to the surface modification blocking pDNA release. In a similar approach, lipid-conjugated polyethylene glycol (PEG) is used as a polyvalent linker for penetratin, CPP, or folic acid (Cheng, et al., Biomaterials, 32:6194-6203 (2011)).

[0254] These methods, as well as other methods discussed herein and known in the art, can be combined to tailor the function and effectiveness of particles. In some preferred embodiments, PEG is used as a linker to link functional molecules to particles. For example, DSPE-PEG(2000)-maleimide is commercially available and can be used to covalently attach functional molecules such as CPPs.

[0255] A "protein transduction domain" or PTD refers to a polypeptide, polynucleotide, or organic or inorganic compound that facilitates passage across a lipid bilayer, a micelle, a cell membrane, an organelle membrane, or a vesicle membrane. A PTD attached to another molecule facilitates passage of the molecule across a membrane (e.g., movement from the extracellular space to the intracellular space, or from the cytoplasm into an organelle). A PTD can be a short basic peptide sequence, such as those present in many cellular and viral proteins. Exemplary protein transduction domains known in the art include, but are not limited to, the Antennapedia PTD and TAT (transactivator of transcription) PTD, polyarginine, polylysine, or a mixture of arginine and lysine, the HIV TAT (YGRKKRRQRRR (SEQ ID NO: 53) or RKKRRQRRR (SEQ ID NO: 54), 11 arginine residues, the VP22 peptide, and the ANTp peptide (RQIKIWFQNRRMKWKK) (SEQ ID NO: 55), or positively charged polypeptides or polynucleotides having 8-15 residues, preferably 9-11 residues. Short non-peptide polymers rich in amine or guanidinium groups can also carry molecules across biological membranes. Penetratin and other derivatives of peptides derived from Antennapedia (Cheng, et al. al., Biomaterials, 32(26):6194-203 (2011) can also be used. Results show that penetratin with an additional Arg residue further enhances uptake and endosomal escape, and that it contains an antennapedia domain for penetration and an IKK NBD 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)).

[0256] A "fusogenic peptide" is any peptide capable of destabilizing membranes. Generally, fusogenic peptides tend to form amphipathic alpha-helical structures in the presence of hydrophobic surfaces, such as membranes. The presence of a fusogenic peptide induces pore formation in cell membranes by disrupting the ordered packing of membrane phospholipids. Some fusogenic peptides act to enhance lipid disorder, thus increasing the likelihood of membrane fusion or coalescence between two closely located membrane-enveloped particles of various natures (e.g., cells, enveloped viruses, liposomes). Other fusogenic peptides can simultaneously bind to two membranes, causing their fusion and promoting their fusion into one. Examples of fusogenic peptides include fusion peptides from viral envelope protein ectodomains and membrane-destabilizing peptides from the membrane-proximal domains of viral envelope proteins from their cytoplasmic tails.

[0257] Other membrane fusion peptides often contain amphipathic regions. Examples of amphipathic region-containing peptides include melittin, magainin, and HIV1. These include the cytoplasmic tail of gp41, microbial and reptilian cytotoxic peptides (e.g., bomolitin 1, paradaxin, mastoparan, clavulolin, cecropin, entamoeba, and staphylococcal alpha toxin); (1) viral fusion peptides from the N-terminal region of the transmembrane (TM) domain of viral envelope proteins (e.g., HIV-1, SIV, influenza, polio, rhinovirus, and coxsackievirus); (2) viral fusion peptides from the internal region of the TM ectodomain (e.g., Semliki Forest virus, Sindbis virus, rotavirus, and rubella virus), and the fusion peptide from sperm protein PH-30; and (3) viral fusion peptides from the membrane-proximal region of 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)).

[0258] 4. Preparation method a. conjugate Methods for polymer synthesis 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 polymerization, or plasma polymerization.

[0259] In some embodiments, amphiphilic polymers are synthesized from a hydrophobic polymer terminated with a first reactive coupling group and a hydrophilic polymer terminated with a second reactive coupling group capable of reacting with the first reactive coupling group to form a covalent bond. Either the first or second reactive coupling group can be a primary amine, and 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, oxirane, carbonate, aryl halide, imide ester, carbodiimide, anhydride, and fluorophenyl ester. Either the first or second reactive coupling group can be an aldehyde, and the other reactive coupling group can be an aldehyde-reactive linking group such as hydrazide, alkoxyamine, and primary amine. Either the first reactive coupling group or the second reactive coupling group can be a thiol, and the other reactive coupling group can be a sulfhydryl reactive group such as maleimide, haloacetyl, and pyridyl disulfide.

[0260] In some embodiments, a hydrophobic polymer terminated with an amine or amine-reactive linking group is coupled to a hydrophilic polymer terminated 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 ethyl(dimethylaminopropyl)carbodiimide (EDC). NHS-ester-activated PLGA can be reacted with a hydrophilic polymer terminated with a primary amine, such as PEG-NH2, to form an amphiphilic PLGA-b-PEG block copolymer.

[0261] In some embodiments, conjugates of amphiphilic polymers with targeting moieties are formed using the same or similar coupling reaction. In some embodiments, the conjugates are made from hydrophilic polymers terminated 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 is then removed to provide a second reactive coupling group, for example, allowing a hydrophobic polymer block to be coupled to the conjugate of the hydrophilic polymer and the targeting moiety. A hydrophobic polymer terminated with a reactive coupling group complementary to the second reactive coupling group can then be covalently attached to form the conjugate. Of course, this step can also be performed in reverse order, i.e., a conjugate of the hydrophobic polymer and the hydrophilic polymer can be formed first, followed by deprotection and coupling of the targeting moiety to the hydrophilic polymer block.

[0262] 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 used in radiotherapy. Conjugates can be prepared from 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 coupling group complementary to the first reactive coupling group, thereby forming a conjugate of the hydrophobic polymer to the additional moiety. The hydrophilic polymer can be reacted with a targeting moiety having a reactive coupling group complementary to the second reactive coupling group, thereby forming a conjugate of the hydrophilic polymer to the targeting moiety. The first protecting group and the second protecting group can be removed to provide a pair of complementary reactive coupling groups that can react to covalently bond the hydrophobic polymer block to the hydrophilic polymer block.

[0263] b. Nanocarrier formation i. Emulsion method In some embodiments, the nanoparticles are prepared using an 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 the following, but is not limited to: various forms of PLA, PGA, PCL, copolymers thereof, polyacrylate, the aforementioned PEGylated polymers, the aforementioned polymer-drug conjugates, the aforementioned polymer-peptide conjugates, or the aforementioned fluorescently labeled polymers, or combinations thereof. The drug molecule can be one or more of the following, but is not limited to: PPAR gamma 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 (such as PGE2, (5Z,11α,13E,15S)-7-[3-hydroxy-2-(3-hydroxyoct-1-enyl)-5-oxocyclopentyl]hept-5-enoic acid), beta-3 adrenergic receptor agonists (such as CL316243, 5-[(2R)-2-[[(2R)-2-(3-chlorophenyl)-2-hydroxyethyl]amino]propyl]-1,3-benzodioxole-2,2-dicarboxylic acid disodium hydrate), fibroblast growth factor 21 (FGF-21), irisin, RNA, DNA, chemotherapeutic compounds, nuclear magnetic resonance (NMR) imaging agents, or combinations thereof. The water-immiscible organic solvent can be one or more of the following, but is not limited to: chloroform, dichloromethane, and acyl acetate. The drug may be dissolved in one or more of the following, but is not limited to: acetone, ethanol, methanol, isopropyl alcohol, acetonitrile, and dimethyl sulfoxide (DMSO).

[0264] In some embodiments, the polymer solution comprises one or more polymer conjugates as described above. The polymer solution may comprise 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 comprises one or more additional polymers or amphiphilic polymer conjugates. For example, the polymer solution may comprise, in addition to the first amphiphilic polymer conjugate, one or more hydrophobic polymers, hydrophilic polymers, lipids, amphiphilic polymers, polymer-drug conjugates, or conjugates comprising other targeting moieties. The density of the targeting moiety can be controlled by controlling the ratio of the first amphiphilic polymer to the additional polymer or amphiphilic polymer conjugate. The first amphiphilic polymer may be present in the polymer solution at 1% to 100% by weight of the polymer. For example, the first amphiphilic polymer may be present in the polymer solution at 10%, 20%, 30%, 40%, 50%, or 60% by weight of the polymer.

[0265] An aqueous solution is then added to the resulting mixed solution to obtain an emulsion solution by emulsification. The emulsification technique can be, but is not limited to, probe sonication or homogenization with a homogenizer. The plaque-targeting peptide, fluorophore, or drug may be associated with the surface of the polymer matrix of the particle, encapsulated within the polymer matrix of the particle, surrounded by the polymer matrix of the particle, and / or dispersed throughout the polymer matrix of the particle of the present invention.

[0266] ii. Nanoprecipitation method In another embodiment, the multimodal nanoparticles are prepared using nanoprecipitation or microfluidic devices. A polymeric material is mixed with a drug or drug combination in a water-miscible organic solvent. The polymer can be one or more of the following, but is not limited to: PLA, PGA, PCL, copolymers thereof, polyacrylate, various forms of the aforementioned PEGylated polymers, the aforementioned polymer-drug conjugates, the aforementioned polymer-peptide conjugates, or the aforementioned fluorescently labeled polymers, or combinations thereof. The drug molecule can be one or more of the following, but is not limited to: PPAR gamma 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 (such as PGE2, (5Z,11α,13E,15S)-7-[3-hydroxy-2-(3-hydroxyoct-1-enyl)-5-oxocyclopentyl]hept-5-enoic acid), beta-3 adrenergic receptor agonists (such as CL316243, 5-[(2R)-2-[[(2R)-2-(3-chlorophenyl)-2-hydroxyethyl]amino]propyl]-1,3-benzodioxole-2,2-dicarboxylic acid disodium hydrate), RNA, DNA, chemotherapy compounds, nuclear magnetic resonance (NMR) contrast agents, or combinations thereof. The water-miscible organic solvent may be one or more of, but is not limited to, acetone, ethanol, methanol, isopropyl alcohol, acetonitrile, and dimethyl sulfoxide (DMSO). The resulting mixed solution is then added to a polymer non-solvent, such as an aqueous solution, to obtain a nanoparticle solution.The plaque targeting peptide or fluorophore or drug may be associated with the surface of the polymer matrix of the particle, encapsulated within the polymer matrix of the particle, surrounded by the polymer matrix of the particle, and / or dispersed throughout the polymer matrix of the particles of the invention.

[0267] iii. Microfluidics Methods for producing nanoparticles using microfluidics are known in the art. Suitable methods include those described in U.S. Patent Application Publication No. 2010 / 0022680 A1 by Karnik et al. Generally, microfluidic devices contain at least two channels that converge into a mixing device. The channels are typically formed by lithography, etching, embossing, or molding of a polymer surface. A fluid source is attached to each channel, and application of pressure to the source induces fluid flow in the channel. Pressure can be applied by a syringe, pump, and / or gravity. Inlet streams of solutions containing 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 with desired surface size and density moieties. By varying the pressure and flow rate in the inlet channel and the nature and composition of the fluid source, nanoparticles can be produced with reproducible sizes and structures.

[0268] iv. Other methodologies Solvent evaporation. In this method, the polymer is dissolved in a volatile organic solvent such as methylene chloride. 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 solid microparticles. The resulting microparticles are washed with water and dried overnight in a freeze dryer. This method can yield microparticles with different sizes (0.5-1000 microns) and morphologies. This method is useful for relatively stable polymers such as polyester and polystyrene.

[0269] However, unstable polymers such as polyanhydrides may be degraded during the manufacturing process due to the presence of water. For these polymers, the following two methods, which are carried out in completely anhydrous organic solvents, are more useful.

[0270] 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 with continuous stirring to 5°C above the melting point of the polymer. Once the emulsion is stabilized, it is cooled until the polymer particles solidify. The resulting microparticles are washed by decantation with petroleum ether to obtain a free-flowing powder. This method yields microparticles ranging in size from 0.5 to 1000 microns. The outer surface of spheres prepared by this technique is usually smooth and dense. This procedure is used to prepare microparticles composed of polyesters and polyanhydrides. However, this method is limited to polymers with molecular weights between 1,000 and 50,000 daltons.

[0271] Solvent Removal. This technique is primarily 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 methylene chloride. This mixture is suspended by stirring in an organic oil (such as silicone oil) to form an emulsion. Unlike solvent evaporation, this method can be used to create microparticles from polymers with high melting points and different molecular weights. This procedure can produce microparticles ranging from 1 to 300 microns. The external morphology of the spheres produced by this technique is highly dependent on the type of polymer used.

[0272] Spray drying: In this method, a polymer is dissolved in an organic solvent. A known amount of active drug is suspended (insoluble drug) or co-dissolved (soluble drug) in the polymer solution. The solution or dispersion is then spray-dried. Typical process parameters for a mini spray dryer (Buchi) are: polymer concentration = 0.04 g / mL, inlet temperature = -24°C, outlet temperature = 13-15°C, aspirator setting = 15, pump setting = 10 mL / min, spray flow rate = 600 Nl / hr, and nozzle diameter = 0.5 mm. Microparticles in the 1-10 micron range are obtained, with a morphology depending on the type of polymer used.

[0273] Hydrogel Microparticles. Microparticles composed of gel-type polymers, such as alginate, are produced by conventional ionic gelation techniques. The polymer is first dissolved in an aqueous solution and mixed with barium sulfate or some bioactive agent. Then, in some instances, it is extruded through a microdroplet-forming device, which uses a stream of nitrogen gas to separate the droplets. A slowly stirred (approximately 100-170 RPM) ionic hardening bath is placed below the extrusion device to capture the microdroplets as they form. The microparticles are left to incubate in the bath for 20-30 minutes to allow sufficient time for gelation to occur. Microparticle size is controlled by using various sizes of extruders or by varying either the flow rate of the nitrogen gas or the polymer solution. Chitosan microparticles can be prepared by dissolving the polymer in an acidic solution and crosslinking with tripolyphosphate. Carboxymethylcellulose (CMC) microparticles can be prepared by dissolving the polymer in an acidic solution and precipitating the microparticles with lead ions. In the case of negatively charged polymers (eg, alginate, CMC), positively charged ligands of different molecular weights (eg, polylysine, polyethyleneimine) can be ionically bound.

[0274] v. Liposome and micelle formation Liposomes typically have an aqueous core. The aqueous core can contain water or a mixture of water and an alcohol. Suitable alcohols include, but are not limited to, methanol, ethanol, propanol (e.g., isopropanol), butanol (e.g., n-butanol), isobutene, sec-butanol, tart-butanol, pentane (e.g., amyl alcohol, isobutylcarbinol), hexanol (e.g., 1-hexanol, 2-hexanol, 3-hexanol), heptanol (e.g., 1-heptanol, 2-heptanol, 3-heptanol, and 4-heptanol), or octanol (e.g., 1-octanol), or a combination thereof.

[0275] Liposomes include, for example, small unilamellar vesicles (SUVs) formed by a single lipid bilayer, large unilamellar vesicles (LANs), which are relatively large vesicles formed by a single lipid bilayer, and multilamellar vesicles (MLVs) formed by multiple membrane layers. Thus, liposomes can have one or more aqueous compartments delineated by either 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. Therefore, potentially more therapeutic agents are available within the liposome to reach target cells.

[0276] Liposomes can be of any particle size, for example, with an average particle size of about 10 to about 2,000 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 less. 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. Particle size refers to the diameter of the particles as measured by dynamic light scattering.

[0277] The liposome formulation may comprise large liposomes ranging from 1-100% of the liposome population in the formulation, hi some embodiments, the large liposomes represent greater than about 50% of the liposome population in the formulation.

[0278] Methods for producing liposomes are known in the art and may include, for example, drying lipids from organic solvents, dispersing lipids in aqueous media, purifying the resulting liposomes, and analyzing the final product. Some methods for liposome production include, for example, extrusion, Mozafari, polyol dilution, bubble, and heat methods.

[0279] Micelles can be prepared in conventional manner, for example, by reverse phase evaporation, ether injection, surfactant-based techniques, etc. Polymeric micelle formulations utilizing block copolymers having hydrophilic and hydrophobic segments are disclosed, for example, in U.S. Application No. 2016 / 0114058, WO2009 / 142326A1, and WO2010 / 013836A1.

[0280] c. Methods for encapsulating or binding molecules to the surface of particles There are two main groups of molecules that can be encapsulated or attached to polymers, either directly or via coupling molecules: targeting molecules, adhesion molecules, and therapeutic, nutritional, diagnostic, or preventative agents. These can be coupled using standard techniques. The targeting or therapeutic molecule to be delivered can be coupled directly to the polymer or to a material, such as a fatty acid, that is incorporated into the polymer.

[0281] Functionality refers to the conjugation of a ligand to the surface of a particle via functional chemical groups (carboxylic acids, aldehydes, amines, sulfhydryls, and hydroxyls) present on the surface of the particle and on the ligand to be bound. Functionality can be introduced to particles in two ways.

[0282] The first is during the preparation of the particles, for example during emulsion preparation of the particles by incorporation of stabilizers with functional chemical groups.

[0283] The second is post-particle preparation by directly crosslinking the particles and ligands using homo- or heterobifunctional crosslinkers. This second procedure can use suitable chemical and crosslinker classes (e.g., CDI, EDAC, glutaraldehyde, etc., discussed in more detail below) or any other crosslinker that couples the ligand to the particle surface via post-particle surface chemical modification. This second class also includes processes in which amphiphilic molecules, such as fatty acids, lipids, or functional stabilizers, can be passively adsorbed and attached to the particle surface, thereby introducing functional end groups for tethering to the ligand.

[0284] d. Methods of Linking ApoL1 to a Targeting Moiety 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 nanocarrier delivery system. In addition to conjugating the targeting moiety to a biologically active molecule, the latter can be bound 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 expressed together in a host cell as a fusion protein.

[0285] Antibodies or active fragments thereof can be chemically linked to the polypeptide by peptide bonds or by chemical or peptide linker molecules of a type well known in the art. Methods for linking drugs or other small molecule agents to antibody fragments are well known and include N-succinimidyl(4-iodoacetyl)-aminobenzoate; sulfosuccinimidyl(4-iodoacetyl)-aminobenzoate; 4-succinimidyl-oxycarbonyl-A-invert-(2-pyridyldithio)toluene; sulfosuccinimidyl-6-[.alpha.-methyl-.A-invert-(pyridyldithiol)-toluamido]hexanoate; N-succinimidyl-3-(-2-pyridyldithio)-proprionate; succinimidyl-6-[3(-(-2-pyridyldithio) Bifunctional linking molecules may include the use of bifunctional chemical linkers such as sulfosuccinimidyl-6-[3-(-(-2-pyridyldithio)-propionamido]hexanoate; sulfosuccinimidyl-6-[3-(-(-2-pyridyldithio)-propionamido]hexanoate; 3-(2-pyridyldithio)-propionyl hydrazide, Ellman's reagent, dichlorotriazine acid, S-(2-thiopyridyl)-L-cysteine, and the like. Additional bifunctional linking molecules are discussed, for example, in U.S. Pat. Nos. 5,349,066, 5,618,528, 4,569,789, 4,952,394, and 5,137,877.

[0286] The linker may be cleavable or non-cleavable. A highly stable linker can reduce the amount of payload lost during circulation, thus improving the safety profile and ensuring that more payload reaches target cells. The linker can control the distribution and delivery of the active agent to target cells based on chemical motifs, including disulfides, hydrazones, or peptides (cleavable), or thioethers (non-cleavable). Cleavable and non-cleavable linkers have been proven safe in preclinical and clinical trials (see, for example, brentuximab vedotin, which contains an enzyme-sensitive linker cleavable by cathepsins, and trastuzumab emtansine, which contains a stable non-cleavable linker). In certain embodiments, the linker is a peptide linker cleavable by Edman degradation (Bachor, et al., Molecular diversity, 17(3):605-11 (2013)).

[0287] A non-cleavable linker can retain the active agent within the cell or target microenvironment. As a result, the entire antibody, linker, and active agent enter the target cell, and 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 an active drug. In contrast, a cleavable linker is catalyzed by an enzyme in the target cell or microenvironment to release the active agent. Once cleaved, the payload can escape from the target cell and attack neighboring cells (also known as "bystander killing").

[0288] In some embodiments, one or more additional molecules are present between the active agent and the cleavage site. Other considerations include site-specific conjugation (TDC) (conjugation techniques such as those described in Axup, Proceedings of the National Academy of Sciences, 109(40):16101-6 (2012), and Lyon, et al., Bioconjugate Chem., 32(10):1059-1062 (2014), and Kolodych, et al., Bioconjugate Chem., 26(2):197-200 (2015) (which can improve stability and therapeutic index), and alpha-emitting immunoconjugates (Wulbrand, et al., Multhoff, Gabriele, ed., PLoS ONE. 8(5):e64730 (2013)).

[0289] III. Pharmaceutical Compositions The composition can be formulated into a pharmaceutical composition together with a 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).

[0290] The composition can be formulated for parenteral administration. "Parenteral administration," as used herein, refers to administration by any method other than through the digestive tract or a non-invasive local or regional route. For example, parenteral administration can include administration to a patient intravenously, intradermally, intraarterially, intraperitoneally, intralesionally, intracranially, intraarticularly, intraprostatically, intrapleurally, intratracheally, intravitreally, intratumorally, intramuscularly, subcutaneously, subconjunctivally, intravesically, intrapericardially, intraumbilically, or transmucosally (nasal, vaginal, pulmonary, or rectal), for example, by injection or infusion.

[0291] In some embodiments, the composition is administered systemically, for example, by injection or infusion. In some embodiments, the composition is administered locally, for example, by injection or infusion.

[0292] Parenteral formulations can be prepared as aqueous compositions using techniques known in the art.Typically, such compositions can be prepared as injectable formulations, for example, as solutions or suspensions, as solid forms suitable for use in preparing solutions or suspensions when adding a reconstitution vehicle before injection, as emulsions such as water-in-oil (w / o) emulsions, oil-in-water (o / w) emulsions, and microemulsions thereof, liposomes, or emulsomes.

[0293] The carrier may be a solvent or dispersion medium containing, for example, water, ethanol, one or more polyols (e.g., glycerol, propylene glycol, and liquid polyethylene glycol), oils, such as vegetable oils (e.g., peanut oil, corn oil, sesame oil, etc.), and combinations thereof. Proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by maintaining the required nanocarrier size in the case of dispersions, and / or by the use of surfactants. Isotonic agents, for example, sugars or sodium chloride, are often included.

[0294] Solutions and dispersions of the active compounds as free acids or bases or pharmacologically acceptable salts thereof can be prepared in water or another solvent or dispersion medium suitably mixed with one or more pharmaceutically acceptable excipients, including, but not limited to, surfactants, dispersing agents, emulsifying agents, pH adjusters, viscosity adjusters, and combinations thereof.

[0295] Suitable surfactants may be anionic, cationic, amphoteric, or nonionic surfactants. Suitable anionic surfactants include, but are not limited to, those containing carboxylate, sulfonate, and sulfate ions. Examples of anionic surfactants include sodium, potassium, and ammonium long-chain alkyl sulfonates and alkylaryl sulfonates, such as sodium dodecylbenzenesulfonate; sodium dialkyl sulfosuccinates, such as sodium dodecylbenzenesulfonate; sodium dialkyl sulfosuccinates, such as sodium bis-(2-ethylthiol)-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, cetrimonium bromide, stearyl dimethylbenzyl ammonium chloride, polyoxyethylene, and coconut amine. Examples of nonionic surfactants include ethylene glycol monostearate, propylene glycol myristate, glyceryl monostearate, glyceryl stearate, polyglyceryl-4-oleate, sorbitan acylate, sucrose acylate, PEG-150 laurate, PEG-400 monolaurate, polyoxyethylene monolaurate, polysorbate, polyoxyethylene octylphenyl ether, PEG-1000 cetyl ether, polyoxyethylene tridecyl ether, polypropylene glycol butyl ether, Poloxamer® 401, stearoyl monoisopropanolamide, and polyoxyethylene hydrogenated tallow amide. Examples of amphoteric surfactants include sodium N-dodecyl beta-alanine, sodium N-lauryl beta-iminodipropionate, myristoamphoacetate, lauryl betaine, and lauryl sulfobetaine.

[0296] The formulation may contain a preservative to prevent the growth of microorganisms. Suitable preservatives include, but are not limited to, parabens, chlorobutanol, phenol, sorbic acid, and thimerosal. The formulation may also contain an antioxidant to prevent the decomposition of the active agent(s).

[0297] Formulations, upon reconstitution, are typically buffered for parenteral administration to a pH of 3 to 8. Suitable buffers include, but are not limited to, phosphate buffers, acetate buffers, and citrate buffers.

[0298] 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.

[0299] Sterile injectable solution can be prepared by incorporating the required amount of active compound into a suitable solvent or dispersion medium, which contains one or more of the excipients listed above as needed, and then sterilizing by filtration.Generally, dispersion is prepared by incorporating various sterilized active ingredients into a sterile medium containing a basic dispersion medium and the other necessary ingredients listed above.For the preparation of sterile powder for preparing sterile injectable solution, exemplary preparation methods include vacuum drying and freeze-drying technology, which can obtain a powder of active ingredient and any additional desired ingredients from its previously sterile-filtered solution.

[0300] Enteral preparations are prepared using pharmaceutically acceptable carriers.As generally 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.

[0301] Carriers also include all components of the coating composition, which may include plasticizers, pigments, colorants, stabilizers, and glidants. The formulations can be prepared using one or more pharmaceutically acceptable excipients, including diluents, preservatives, binders, lubricants, disintegrants, swelling agents, fillers, stabilizers, and combinations thereof.

[0302] Controlled-release dosage formulations can be prepared as described in standard references such as Liberman et al. (New York, Marcel Dekker, Inc., 1989), "Remington—The science and practice of pharmacy," 20th ed., 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 tablets and capsules, as well as delayed-release dosage forms of tablets, capsules, and granules. These references provide information on excipients, materials, equipment, and processes for preparing tablets and capsules, as well as delayed-release dosage forms of tablets, capsules, and granules.

[0303] Stabilizers are used to inhibit or retard drug decomposition reactions, including, by way of example, oxidative 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).

[0304] In some embodiments, the compositions are formulated for mucosal administration, such as nasal, pulmonary, or buccal delivery.

[0305] Mucosal formulations may contain one or more agents to enhance delivery through the nasal mucosa. Agents for enhancing mucosal delivery are known in the art, see, for example, U.S. Patent Application No. 2009 / 0252672 to Eddington and U.S. Patent Application No. 2009 / 47234 to Touitou. Acceptable agents include, but are not limited to, calcium chelators (EDTA), nasal enzyme inhibitors (boroleucine, aprotinin), mucociliary clearance inhibitors (preservatives), nasal membrane solubilizers (cyclodextrins, fatty acids, surfactants), and micelle formation (bile acids, Laureth 9, and surfactants such as tallow dehydrofusidate (STDHF)). The composition may contain one or more absorption enhancers, including surfactants, fatty acids, and chitosan derivatives, which can enhance delivery by modulating tight junctions (TJs) (BJ Aungst, et al., J. Pharm. Sci. 89(4):429-442(2000)). In general, an optimal absorption enhancer should be reversible in its effects, provide a rapid penetration-enhancing effect on mucosal cell membranes, and be non-cytotoxic at effective concentration levels without harmful and / or irreversible effects on TJ cell membranes or the cytoskeleton.

[0306] Pharmaceutical packs and kits are provided that comprise one or more containers filled with the antibody or fusion protein. Additionally, one or more other prophylactic or therapeutic agents useful for treating disease can also be included in the pharmaceutical pack or kit. One embodiment provides a pharmaceutical pack or kit that comprises one or more containers filled with one or more of the ingredients of the pharmaceutical composition. Optionally, a notice in a form prescribed by a government agency regulating the manufacture, use, or sale of pharmaceuticals or biological products may be associated with such container(s), reflecting approval by the agency of manufacture, use, or sale for human administration.

[0307] Also provided are kits that can be used in the following methods. In one embodiment, the kit comprises one or more antibodies or fusion proteins. In another embodiment, the kit further comprises, in one or more containers, one or more other prophylactic or therapeutic agents useful in the treatment of cancer. In certain embodiments, the other prophylactic or therapeutic agent is a chemotherapeutic agent. In other embodiments, the prophylactic or therapeutic agent is a biological or hormonal therapeutic agent.

[0308] IV.How to use A. Treatment method The disclosed compositions can be used to increase the delivery and internalization of ApoL1 into target cells. As shown in the Examples below, increasing the internalization of ApoL1, for example, by increasing the internalization of recombinant exogenous ApoL1 or endogenous ApoL1-containing complexes, such as TLF, in mammalian cells can result in 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.

[0309] In a preferred embodiment, the composition is an ApoL1-containing complex binding compound, which is a bispecific or multispecific antibody that specifically binds to both an ApoL1-containing complex and a target cell marker (see, e.g., Figure 12).

[0310] Targeting of all cell types, alone or in combination, is contemplated, 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 may be specific for endothelial cells, ectodermal cells, or mesenchymal cells. Thus, the cell may be a mammalian cell or a non-mammalian cell. Most preferably, the cell is within a mammal. The mammalian cell may be a human cell. Thus, targeting of mammalian cells (e.g., human) and non-mammalian cells in a subject (e.g., human) is contemplated. Thus, the target cell may be a bacterial cell or a fungal cell in a mammalian subject, such as a human. Additionally, or alternatively, the target cell may be a non-mammalian cell infected with another organism, such as a virus or bacteria. For example, in some embodiments, the target cell is an intracellular organism, where the infected cell may be targeted by the presence of an extracellular marker. The organism can be a bacterium or a eukaryote, such as Plasmodium falciparum, Toxoplasma gondii, Leishmania sp., Trypanosoma cruzi, Listeria monocytogenes, Chlamydia trachomatis, Coxiella burnetti, Mycobacterium tuberculosis, and other intracellular bacteria and eukaryotes. Extracellular eukaryotes or extracellular stages of intracellular organisms whose cells can be specifically targeted include, for example, Toxoplasma gondii, Trichomonas vaginalis, and Plasmodium falciparum.

[0311] In a preferred embodiment, cancer cells, including both hematological cancer cells and solid tumor cells, are preferred target cells. The binding activity of the compounds can be selected based on the target cells.

[0312] Typically, the target cell is not a trypanosome.

[0313] 1.Treatment method Thus, in some embodiments, a subject is administered an effective amount of a disclosed composition, e.g., recombinant ApoL1 and a targeting moiety, or a bispecific or multispecific antibody that specifically binds to Hpr or ApoL1 and a target cell antigen.

[0314] As used herein, the term "effective amount" or "therapeutically effective amount" refers to a dosage 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 exact dosage will vary depending on various factors, such as the active agent selected, as well as subject-dependent variables (e.g., age, immune system health, etc.), the disease, and the treatment being affected. Typically, this amount is effective to induce or increase ApoL1-mediated cell death of the target cells. In some embodiments, the subject has a disease or disorder caused by the target cells, and cell death is induced in an amount effective 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 cell death of the cancer cells.

[0315] Any diseased tissue with a unique biomarker, or a biomarker that is overexpressed compared to normal cells, can serve as a target cell to treat any such disease that would benefit from increasing cell death in the diseased tissue.

[0316] For example, when the target cells are blood cancer cells, a bispecific antibody that specifically binds to Hpr or ApoL1 and a blood cancer antigen (e.g., 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.

[0317] 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.

[0318] Similarly, bispecific and other multispecific molecules can target solid tumors or other target cells. Exemplary other antigens include solid tumor antigens, for which bispecific or other multispecific molecules are provided elsewhere herein.

[0319] For example, if the solid tumor is pancreatic cancer, a bispecific antibody that specifically binds to Hpr or ApoL1 and a pancreatic cancer antigen (e.g., claudin 18.2, MUC1, mesothelin (MSLN), and myoferlin (MYOF)) can be used. Similarly, in some embodiments, the targeting moiety of the recombinant ApoL1 composition targets claudin 18.2, MUC1, mesothelin (MSLN), and myoferlin (MYOF).

[0320] For example, if the solid tumor is melanoma, a bispecific antibody that specifically binds Hpr or ApoL1 and a melanoma cancer antigen (e.g., PMEL17) can be used. Similarly, in some embodiments, the targeting moiety of the recombinant ApoL1 composition targets PMEL17.

[0321] These are non-limiting examples, as many other targets are provided herein and in the art, and as provided herein, the disclosed compositions can be readily modified to target these antigens.

[0322] 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 a subject once, twice, or three times weekly. In some embodiments, the composition is administered to a subject every other day. In some embodiments, the composition is administered to a subject once, twice, or three times monthly. In some embodiments, the composition is administered for 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 weeks or months, or more.

[0323] In certain embodiments, for antibodies and other proteins, the dosage administered to a patient is typically between 0.0001 mg / kg and 100 mg / kg of the patient's body weight. Preferably, the dosage administered to a patient is 0.0001 mg / kg to 20 mg / kg, 0.0001 mg / kg to 10 mg / kg, 0.0001 mg / kg to 5 mg / kg, 0.0001 to 2 mg / kg, 0.0001 to 1 mg / kg, 0.0001 mg / kg to 0.75 mg / kg, 0.0001 mg / kg to 0.5 mg / kg, 0.0001 mg / kg to 0.25 mg / kg, 0.0001 to 0.15 mg / kg, 0.0001 to 0.10 mg / kg, 0.001 to 0.5 mg / kg, 0.01 to 0.25 mg / kg, or 0.01 to 0.10 mg / kg of the patient's body weight. Generally, human antibodies have a longer half-life in the human body than antibodies derived from other species due to the immune response to foreign polypeptides. Thus, lower doses of human antibodies and less frequent administration are often possible. Furthermore, the dosage and frequency of administration of antibodies or fragments thereof, or fusion proteins can be reduced by enhancing uptake and tissue penetration of the antibodies or fusion proteins by modifications such as, for example, lipidation.

[0324] 2. Disease to be treated Cancer The disclosed compositions and methods can be used to treat cancer in subjects in need of such treatment. In mature animals, a balance between cell renewal and cell death is typically maintained in most organs and tissues. Various types of mature cells in the body have a fixed lifespan, and 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 regulated so that the number of any particular type of cell remains constant. However, occasionally, cells arise that no longer respond to normal growth control mechanisms. These cells give rise to clones of cells that can grow to significant sizes, producing tumors or neoplasms. Tumors that are unable to grow indefinitely and do not extensively invade healthy surrounding tissues are benign. Tumors that continue to grow and invade slowly are malignant. The term cancer specifically refers to malignant tumors. In addition to uncontrolled growth, malignant tumors exhibit metastasis. During this process, small clusters of cancerous cells shed from the tumor, invade blood or lymphatic vessels, and travel to other tissues, where they continue to grow. In this way, a primary tumor at one site can give rise to a secondary tumor at another site.

[0325] The compositions and methods described herein are useful for treating a subject with a benign or malignant tumor by slowing or inhibiting tumor growth in the subject, reducing tumor growth or size, inhibiting or reducing tumor metastasis, and / or inhibiting or reducing symptoms associated with tumor development or growth.

[0326] Malignant tumors that can be treated are classified herein according to the embryonic origin of the tissue from which the tumor originates. Leukemia and lymphoma are malignant tumors of hematopoietic cells in the bone marrow. Leukemias grow as single cells, while lymphomas tend to grow as tumor masses. Malignant tumors can appear in many organs or tissues of the body to establish cancer. Carcinomas are tumors that arise from endodermal or ectodermal tissues, such as the skin or the epithelial lining of internal organs and glands. Sarcomas, which occur less frequently, originate from mesodermal connective tissues, such as bone, fat, and cartilage. Malignant tumors can appear in many organs or tissues of the body to establish cancer.

[0327] In a preferred embodiment, the composition is used to treat liquid tumors or blood cancers or tumors of the vasculature, such as multiple myeloma, leukemia (e.g., chronic lymphocytic leukemia, acute myeloid leukemia, acute lymphoblastic leukemia), non-Hodgkin's lymphoma, Hodgkin's lymphoma, myelodysplastic syndromes (MDS), myeloproliferative neoplasms (MPN) (or subcategories thereof, e.g., essential thrombocythemia (ET), myelofibrosis (MF), and polycythemia vera (PV), amyloidosis, Waldenstrom's macroglobulinemia, or aplastic anemia.

[0328] Additional types of cancer that can be treated with the provided compositions and methods include, but are not limited to, adenocarcinomas and sarcomas of the 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.

[0329] The disclosed compositions can be used to treat cells undergoing uncontrolled proliferation, invasion, or metastasis.

[0330] A representative, but non-limiting, list of cancers that can be treated using the disclosed compositions includes cancers of the blood and lymphatic system (including leukemia, Hodgkin's lymphoma, non-Hodgkin's lymphoma, solitary plasmacytoma, and multiple myeloma), cancers of the genitourinary system (including prostate cancer, bladder cancer, kidney cancer, urethral cancer, penile cancer, and testicular cancer), cancers of the nervous system (including meningiomas, gliomas, glioblastomas, and ependymomas), cancers of the head and neck (including squamous cell carcinomas of the oral cavity, nasal cavity, nasopharyngeal cavity, oropharynx, larynx, and paranasal sinuses), and cancers of the urinary system (including squamous cell carcinomas of the oral cavity, nasal cavity, nasopharyngeal cavity, oropharynx, larynx, and paranasal sinuses). cancer), lung cancer (including small cell and non-small cell lung cancer), gynecological cancer (including cervical, endometrial, vaginal, vulvar, ovarian, and fallopian tube cancer), gastrointestinal cancer (including gastric, small intestine, colorectal, hepatic ductal, hepatobiliary, and pancreatic cancer), skin cancer (including melanoma, squamous cell, and basal cell carcinoma), breast cancer (including ductal, lobular, and triple-negative breast cancer), and childhood cancer (including neuroblastoma, Ewing's sarcoma, Wilms' tumor, and medulloblastoma).

[0331] In some embodiments, the tumor is a solid tumor characterized by increased vascular permeability, and optionally, the enhanced permeability and retention (EPR) effect increases localization of the ApoL1-containing complex-antibody conjugate to the tumor site compared to tumors with fewer blood vessels.

[0332] b. Other diseases Impairment of cell death pathways at the molecular level may be associated with the development of not only cancer but also other diseases of great social importance, such as infectious diseases such as viral infections (e.g., HIV), bacterial infections, fungal infections, and non-mammalian eukaryotic cell infections, arteriosclerosis, ischemia, reperfusion injury, infectious diseases, inflammation, autoimmunity, and neurological disorders (Kaminskyy and Zhivotovsky, Cell Death & Disease, volume 9, Article number: 110 (2018)). Therefore, the disclosed compositions and methods can be used to treat such diseases.

[0333] For example, neutrophils are involved in various types of tissue inflammation and disease, and targeting them for cell death in accordance with the disclosed compositions and methods can be used to treat autoimmune and inflammatory diseases.

[0334] In some embodiments, the compositions and methods can be used to treat infectious diseases. For example, in some embodiments, foreign cells, such as bacteria or fungi, are specifically and directly targeted for cell death. In other embodiments, the infection is treated by targeting infected mammalian (e.g., host) cells, for example, by targeting extracellular markers on infected mammalian cells. Exemplary foreign and infected target cells are discussed above.

[0335] For example, one strategy for eliminating the latent HIV-1 reservoir is the shock-and-kill approach, which uses latency-reactivating agents (LRAs) to reactivate viral gene expression (shock) and then eliminate cells harboring the reactivated provirus (kill) (Rao, et al., Nat Commun. 12(1):2475 (2021) doi:10.1038 / s41467-021-22608-z). Thus, in some embodiments, HIV-infected cells, such as CD4+ T cells, are targeted for cell death according to the disclosed compositions and methods, thus treating HIV.

[0336] 3. Combination therapy 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, radiation therapy, and other anti-cancer treatments.

[0337] In some embodiments, the additional active agent is a therapeutic agent. Most chemotherapeutic agents can be classified as alkylating agents, antimetabolites, anthracyclines, plant alkaloids, topoisomerase inhibitors, monoclonal antibodies, and other anti-tumor agents.

[0338] 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.

[0339] In some embodiments, the antitumor drug is a histone deacetylase inhibitor that suppresses DNA repair at the transcription level and disrupts chromatin structure.In some embodiments, the antitumor drug is a proteasome inhibitor that suppresses DNA repair by disrupting ubiquitin metabolism in cells.Ubiquitin is a signaling molecule that regulates DNA repair.In some embodiments, the antitumor drug is a kinase inhibitor that suppresses DNA repair by changing DNA damage response signaling pathway.

[0340] Additional antineoplastic agents include alkylating agents (e.g., cisplatin, carboplatin, oxaliplatin, mechlorethamine, cyclophosphamide, chlorambucil, dacarbazine, lomustine, carmustine, procarbazine, chlorambucil, and ifosfamide), antimetabolites (fluorouracil, gemcitabine, methotrexate, cytosine arabinoside, fludarabine, and floxuridine), some antimitotic agents, and vinca alkaloids (e.g., vincristine, vinblastine, vinorelbine, and vindesine), anthracyclines, and cyclosporines. These include, but are not limited to, cyclosporins (including doxorubicin, daunorubicin, valrubicin, idarubicin, and epirubicin, and actinomycins (such as actinomycin D)), cytotoxic antibiotics (including mitomycin, plicamycin, and bleomycin), and topoisomerase inhibitors (including camptothecins such as irinotecan and topotecan, epipodophyllotoxin derivatives such as amsacrine, etoposide, etoposide phosphate, and teniposide), and cytoskeleton-targeting agents (e.g., paclitaxel).

[0341] 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.

[0342] 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.

[0343] B. Detection Methods The disclosed ApoL1-containing complex-binding antibodies, e.g., anti-Hpr and anti-ApoL1 antibodies, and antigen-binding fragments thereof, can be used to detect ApoL1-containing complexes, such as TLF, and their components, such as Hpr and ApoL1. Thus, the present disclosure provides for assaying the presence of ApoL1-containing complexes, such as TLF, or their components, such as Hpr and ApoL1, in cells or tissues or other biological samples of a subject using one or more antibodies (or fragments thereof) that immunospecifically bind to such antigens. 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), and the like.

[0344] In some embodiments, the detection method includes: 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 an ApoL1-containing complex, such as TLF; b) waiting a period of time after administration to allow the labeled molecule to preferentially concentrate at sites in the subject where ApoL1-containing complexes, such as TLF, are located (and to allow unbound labeled molecule to be removed 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 an ApoL1-containing complex, such as TLF, in the subject. According to this embodiment, the antibody is labeled with an imaging moiety that is detectable using imaging systems known to those skilled in the art. The background level can be determined by various methods, including comparing the amount of detected labeled molecule to a standard value previously determined for a particular system.

[0345] It will be understood in the art that the size of the subject and the imaging system used will determine the amount of imaging moiety 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 in TUMOR IMAGING: THE RADIOCHEMICAL DETECTION OF CANCER, S.W. Burchiel and B.A. Rhodes, eds., Masson Publishing Inc. (1982)).

[0346] Depending on several variables, including the type of label used and the mode of administration, the time interval after administration is 6 to 48 hours, or 6 to 24 hours, or 6 to 12 hours, to allow the labeled molecule to be preferentially concentrated at the site of interest and for unbound labeled molecule to be cleared to background levels. In another embodiment, the time interval after administration is 5 to 20 days or 5 to 10 days.

[0347] The presence of the labeled molecule can be detected in a subject using methods known in the art for in vivo scanning. These methods depend on the type of label used. One of ordinary skill in the art will be able to determine the appropriate method for detecting a particular label. Methods and devices that can be used in the disclosed diagnostic methods include, but are not limited to, whole-body scans such as computed tomography (CT), position emission tomography (PET), magnetic resonance imaging (MRI), and ultrasound. In certain embodiments, the molecule is labeled with a radioisotope and detected in a patient using a radiation-responsive surgical instrument (Thurston et al., U.S. Pat. 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).

[0348] The disclosed invention can be further understood by the following numbered paragraphs: 1. A method for increasing cell death of target cells in a mammalian subject in need thereof, comprising administering to the subject an effective amount of a composition that increases apolipoprotein L1 (ApoL1) in the target cells.

[0349] 2. The method of paragraph 1, wherein the composition increases the accumulation of endogenous ApoL1 in the target cells.

[0350] 3. The method of paragraph 2, wherein the endogenous ApoL1 is a component of an ApoL1-containing complex.

[0351] 4. The method of paragraph 3, wherein the ApoL1-containing complex is trypanosoma lytic factor (TLF), optionally TLF-1 and / or TLF-2.

[0352] 5. The method of 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 a target cell, and optionally the composition is a bispecific or multispecific antibody, wherein the first antigen-binding fragment binds to ApoL1 or an ApoL1-containing complex, optionally TLF, and the second antigen-binding fragment binds to a cell-specific antigen.

[0353] 6. The method of paragraph 1, wherein the composition comprises ApoL1 or a functional fragment or variant thereof and a targeting moiety against a cell-specific antigen.

[0354] 7. The method of paragraph 6, wherein said composition comprises said ApoL1 or a functional fragment or variant thereof directly or indirectly conjugated or fused to said targeting moiety.

[0355] 8. The method of paragraph 6 or 7, wherein the composition comprises a delivery vehicle, optionally a liposome or a polymeric nanoparticle.

[0356] 9. The method of paragraph 8, wherein the targeting moiety is conjugated or fused to the delivery vehicle.

[0357] 10. The method of any one of paragraphs 6 to 9, wherein the targeting moiety is an antibody or antigen-binding fragment.

[0358] 11. The method of any one of paragraphs 1 to 10, wherein the cell-specific antigen is specific to a diseased cell.

[0359] 12. The method of paragraph 11, wherein the diseased cells are cancer cells.

[0360] 13. The method of paragraph 12, wherein the cancer cells are blood cancer cells.

[0361] 14. The method of any one of paragraphs 1 to 13, wherein the subject is suffering from a disease caused by the target cells.

[0362] 15. The method of paragraph 14, wherein the composition is administered in an amount effective to treat the disease.

[0363] 16. The method of any one of paragraphs 1 to 15, wherein the cell-specific antigen is not a trypanosome-specific surface antigen.

[0364] 17. The method of any one of paragraphs 1-16, wherein the subject does not have trypanosomiasis.

[0365] 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.

[0366] 19. The composition of paragraph 18, wherein said ApoL1 or functional fragment or variant thereof is directly or indirectly conjugated or fused to said targeting moiety.

[0367] 20. The composition of paragraph 19, wherein the composition comprises a delivery vehicle, optionally a liposome or a polymeric nanoparticle, and optionally the targeting moiety is conjugated or fused to the delivery vehicle.

[0368] 21. An antibody or antigen-binding fragment, three complementarity determining regions (CDRs) of a 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 three complementarity determining regions (CDRs) of a 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; The antibody or antigen-binding fragment binds to apolipoprotein L1 (ApoL1).

[0369] 22. The CDRs of the heavy chain variable domain and the light chain variable domain are: 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 22. The antibody or antigen-binding fragment of paragraph 21, comprising 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.

[0370] 23. The CDRs of the heavy chain variable domain and the light chain variable domain are: 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 23. The antibody or antigen-binding fragment of paragraph 21 or 22, comprising 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).

[0371] 24. The antibody or antigen-binding fragment of any one of paragraphs 21 to 23, comprising a heavy chain variable domain comprising 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 a light chain variable domain comprising 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.

[0372] 25. The antibody or antigen-binding fragment thereof according to any one of paragraphs 21 to 24, comprising a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 24 and a light chain variable domain comprising the amino acid sequence of SEQ ID NO: 36 or SEQ ID NO: 77.

[0373] 26. An antibody or antigen-binding fragment, three complementarity determining regions (CDRs) of a 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 a 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; An antibody or antigen-binding fragment, wherein the antibody or antigen-binding fragment binds to haptoglobin-related protein (Hpr).

[0374] 27. The CDRs of the heavy chain variable domain and the light chain variable domain are: 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 a variant or humanized form 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 a variant or humanized form thereof having at least 70, 80, 90, or 95% sequence identity thereto; or 27. The antibody or antigen-binding fragment of paragraph 26, comprising 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 a variant or humanized form thereof having at least 70, 80, 90, or 95% sequence identity thereto.

[0375] 28. The CDRs of the heavy chain variable domain and the light chain variable domain are: 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 28. The antibody or antigen-binding fragment of paragraph 26 or 27, comprising 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).

[0376] 29. The antibody or antigen-binding fragment of any one of paragraphs 26 to 28, comprising a heavy chain variable domain comprising 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 a light chain variable domain comprising 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.

[0377] 30. The antibody or antigen-binding fragment thereof according to any one of paragraphs 26 to 29, comprising a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 3 and a light chain variable domain comprising the amino acid sequence of SEQ ID NO: 14.

[0378] 31. An antibody or antigen-binding fragment, three complementarity determining regions (CDRs) of a 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 a 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; An antibody or antigen-binding fragment, wherein the antibody or antigen-binding fragment binds to haptoglobin-related protein (Hpr).

[0379] 32. The CDRs of the heavy chain variable domain and the light chain variable domain are: 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 32. The antibody or antigen-binding fragment of paragraph 31, comprising 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.

[0380] 33. The CDRs of the heavy chain variable domain and the light chain variable domain are: 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 33. The antibody or antigen-binding fragment of paragraph 31 or 32, comprising 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).

[0381] 34. The antibody or antigen-binding fragment of any one of paragraphs 31 to 33, comprising a heavy chain variable domain comprising 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 a light chain variable domain comprising 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.

[0382] 35. The antibody or antigen-binding fragment thereof according to any one of paragraphs 31 to 34, comprising a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 56 and a light chain variable domain comprising the amino acid sequence of SEQ ID NO: 65.

[0383] 36. The antibody or antigen-binding fragment of any one of paragraphs 21 to 35, wherein the antibody or antigen-binding fragment binds to an ApoL1-containing complex, and optionally the ApoL1-containing complex is trypanosoma lytic factor (TLF).

[0384] 37. The antibody or antigen-binding fragment of paragraph 36, wherein said TLF is endogenous human TLF.

[0385] 38. The antibody or antigen-binding fragment of paragraph 36 or 37, wherein the antibody or antigen-binding fragment is capable of binding to the ApoL1-containing complex under physiological conditions.

[0386] 39. The antibody or antigen-binding fragment of any one of paragraphs 36 to 38, wherein the antibody or antigen-binding fragment is capable of binding to an ApoL1-containing complex in a subject, and optionally the subject is a human.

[0387] 40. The antibody or antigen-binding fragment of any one of paragraphs 21 to 39, wherein the antibody is not a murine IgG1 or IgG2a.

[0388] 41. The antibody or antigen-binding fragment of any one of paragraphs 21 to 40, comprising one or more constant domains from an immunoglobulin constant region (Fc).

[0389] 42. The antibody or antigen-binding fragment of paragraph 41, wherein the constant domains are human constant domains.

[0390] 43. The antibody or antigen-binding fragment of paragraph 42, wherein the human constant domain is an IgA domain, an IgD domain, an IgE domain, an IgG domain, or an IgM domain.

[0391] 44. The antibody or antigen-binding fragment of paragraph 43, wherein the human IgG constant domain is an IgG1 domain, an IgG2 domain, an IgG3 domain, or an IgG4 domain.

[0392] 45. The antibody or antigen-binding fragment of 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, or receptor ligand.

[0393] 46. ​​The antibody or antigen-binding fragment of any one of paragraphs 21 to 45, wherein the antibody is a monoclonal antibody, a human antibody, a chimeric antibody, or a humanized antibody.

[0394] 47. The antibody or antigen-binding fragment of any one of paragraphs 21 to 46, wherein the antibody is a bispecific, trispecific, or multispecific antibody.

[0395] 48. The antibody or antigen-binding fragment of paragraph 47, wherein the bispecific, trispecific, or multispecific antibody comprises a second antigen-binding fragment that binds to a cell-specific antigen.

[0396] 49. A bispecific, trispecific, or multispecific antibody comprising 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.

[0397] 50. The antibody or antigen-binding fragment of paragraph 48 or 49, wherein the cell-specific antigen is a cancer or tumor antigen.

[0398] 51. The antibody or antigen-binding fragment of paragraph 50, wherein the cancer or tumor antigen is optionally a hematological cancer antigen or solid tumor antigen selected from claudin 18.2, MUC1, mesothelin (MSLN), myoferlin (MYOF), and PMEL17.

[0399] 52. The antibody or antigen-binding fragment of paragraph 51, wherein the hematological 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.

[0400] 53. The antibody or antigen-binding fragment of any one of paragraphs 48 to 52, wherein the cell-specific antigen is BCMA.

[0401] 54. The antibody or antigen-binding fragment of paragraph 28, wherein the second antigen-binding fragment comprises the three CDRs of the heavy chain variable domain 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 three CDRs of the light chain variable domain of SEQ ID NO: 42, or a variant or humanized form thereof having at least 70, 80, 90, or 95% sequence identity thereto.

[0402] 55. The second antigen-binding fragment has the following amino acid sequence: 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 55. The antibody or antigen-binding fragment of paragraph 54, comprising six CDRs, including CDR3L: QQYGNPPLYT (SEQ ID NO: 48) or a variant or humanized form thereof having at least 70, 80, 90, or 95% sequence identity thereto.

[0403] 56. The antibody or antigen-binding fragment of any one of paragraphs 53 to 55, wherein the second antigen-binding fragment comprises a 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 a 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.

[0404] 57. The second antigen-binding fragment comprises a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 41 and a light chain variable domain comprising the amino acid sequence of SEQ ID NO: 42; 57. The antibody or antigen-binding fragment of Paragraph 56, optionally wherein the second antigen-binding fragment comprises the amino acid sequence of SEQ ID NO:51.

[0405] 58. The antibody or antigen-binding fragment of any one of paragraphs 49 to 56, comprising 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.

[0406] 59. The antibody or antigen-binding fragment of any one of paragraphs 49 to 56, comprising the amino acid sequences of SEQ ID NO: 71 and SEQ ID NO: 72.

[0407] 60. The antibody or antigen-binding fragment of any one of paragraphs 49 to 56, comprising two copies of the amino acid sequences of SEQ ID NO: 70 and SEQ ID NO: 72, respectively.

[0408] 61. Anti-ApoL1, anti-cell-specific antigen IgG1-scFv bispecific chimeric antibody.

[0409] 62. Anti-Hpr, anti-cell-specific antigen IgG1-scFv bispecific chimeric antibody.

[0410] 63. A nucleic acid encoding the antibody or antigen-binding fragment of any one of paragraphs 21 to 63.

[0411] 64. The nucleic acid of paragraph 63, operably linked to an expression control sequence.

[0412] 65. An expression vector comprising a nucleic acid according to paragraph 63 or 64.

[0413] 66. A cell comprising a nucleic acid according to paragraph 63 or 64, or an expression vector according to paragraph 65, optionally wherein the cell is a mammalian cell.

[0414] 67. An immune complex comprising the antibody or antigen-binding fragment of any one of paragraphs 21 to 62 bound to ApoL1 or an ApoL1-containing complex, optionally wherein the complex is TLF.

[0415] 68. An immune complex comprising the antibody or antigen-binding fragment of any one of paragraphs 47 to 62.

[0416] 69. A method of inducing cell death, comprising contacting a target cell with an immunoconjugate according to paragraph 67 or 68.

[0417] 70. The method of paragraph 69, wherein said contacting occurs in vitro.

[0418] 71. The method of paragraph 69, wherein the contraction occurs in vivo in a subject.

[0419] 72. The method of paragraph 71, wherein the subject has cancer.

[0420] 73. A pharmaceutical composition comprising the antibody or antigen-binding fragment of any one of paragraphs 21 to 62.

[0421] 74. A method of treating cancer, comprising administering to said subject an effective amount of the antibody or antigen-binding fragment of any one of paragraphs 21 to 62.

[0422] 75. The method of Paragraph 74, comprising administering to the subject an effective amount of the antibody or antigen-binding fragment of any one of Paragraphs 22 to 62.

[0423] 76. The method of paragraph 74 or 75, wherein the cancer is a hematological cancer.

[0424] 77. The method of paragraph 76, wherein the cancer is multiple myeloma, leukemia (e.g., chronic lymphocytic leukemia, acute myeloid leukemia, acute lymphoblastic leukemia), non-Hodgkin's lymphoma, Hodgkin's lymphoma, myelodysplastic syndrome (MDS), myeloproliferative neoplasm (MPN) (or subcategories thereof, e.g., essential thrombocythemia (ET), myelofibrosis (MF), and polycythemia vera (PV), amyloidosis, Waldenström's macroglobulinemia, or aplastic anemia.

[0425] 78. The method of paragraph 74 or 75, wherein the cancer is a solid tumor.

[0426] 79. A method of treating a subject in need thereof, comprising administering to said subject a composition described in any one of paragraphs 1-62.

[0427] 80. The method of paragraph 79, wherein the subject has cancer.

[0428] 81. The method of any one of paragraphs 1 to 5 or 11 to 17, wherein the composition comprises an antibody of any one of paragraphs 21 to 62.

[0429] 82. The composition or method of any one of paragraphs 1 to 81, wherein the target cell is a mammalian cell.

[0430] 83. The composition or method of paragraph 82, wherein the mammalian cell is an infected cell.

[0431] 84. The composition or method of paragraph 83, wherein the infected cells are optionally infected with a virus, bacterium, or eukaryotic intracellular organism selected from HIV, Plasmodium falciparum, Toxoplasma gondii, Leishmania sp., Trypanosoma cruzi, Listeria monocytogenes, Chlamydia trachomatis, Coxiella burnetti, Mycobacterium tuberculosis, and Trichomonas vaginalis.

[0432] 85. The composition or method of any one of paragraphs 1 to 81, wherein the target cell is a non-mammalian cell.

[0433] 86. The composition or method of paragraph 85, wherein the non-mammalian cell is a bacterial, fungal, or non-mammalian eukaryotic cell.

[0434] 87. The composition or method of paragraph 86, wherein the non-mammalian cell is not a trypanosome.

[0435] 88. A composition or method according to any one of the preceding paragraphs, wherein the subject is a mammal, optionally a human. [Example]

[0436] Example 1: TLF does not bind to mammalian cells with high affinity. Materials and Methods To measure TLF-1 binding by flow cytometry, cells were grown to mid-log phase, harvested, washed, and resuspended in DMEM supplemented with 10% fetal bovine serum (1 × 10 7 Alexa-488 TLF-1, labeled according to the manufacturer's instructions (Invitrogen), was incubated with excess Hb for 10 minutes on ice, then added to cells in ice-cold complete DMEM and further incubated for 3 hours at 3°C. Cells were washed twice with ice-cold phosphate-buffered saline (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.

[0437] result The mechanisms that enable mammalian cells to tolerate TLF have not previously been explored. HDL endocytosis has been studied in multiple systems, and although the exact purpose and efficiency of this process remain controversial, cholesterol transport is thought to be one of its primary goals (Rohrl and Stangl, “HDL endocytosis and resecretion,” Biochim Biophys Acta, 2013, 1831(11):1626-1633). The issue of TLF endocytosis efficiency has not been fully explored, except for a single observation that TLF was internalized in Leishmania-infected macrophages (Samanovic, et al., “Trypanosome lytic factor, an antimicrobial high-density lipoprotein, ameliorates Leishmania infection,” PLoS Pathog, 2009, 5(1):e1000276). In this case, no macrophage cell death was reported, thereby indicating that TLF is somehow unable to elicit its toxic effects when present at near physiological levels in culture.

[0438] Trypanosoma brucei brucei binds to TLF with high specificity and affinity, which can be easily observed at 3C using a previously developed method (DeJesus, et al., "A Single Amino Acid Substitution in the Group 1 Trypanosoma brucei gambiense Haptoglobin-Hemoglobin Receptor Abolishes TLF-1 Binding," PLoS Pathog., 9 (2013)). To determine whether a receptor for TLF exists in mammalian cells, we performed this same low-temperature binding assay. Although high-affinity binding was not observed, high concentrations of AF488 TLF-1 were detectable but not saturating by flow cytometry (Figure 2). See also DeJesus, et al., "Evasion of African trypanosomes to human innate immunity," Dissertation in fulfillment of Doctor of Philosophy, University of Georgia, submitted 2014.

[0439] Example 2: In mammalian cells, TLF is internalized and localized to lysosomes. Materials and Methods TLF-1 binding and uptake studies To measure TLF-1 uptake by flow cytometry, cells were grown to mid-log phase, harvested, washed, and resuspended in DMEM supplemented with 10% fetal bovine serum (1 × 10 7Alexa-488 TLF-1, performed with or without hemoglobin, was added to the cells and subsequently incubated at 37°C for 3 hours. Uptake was stopped by placing the tubes on ice and then washing twice with ice-cold PBS. The amount of TLF-1 uptake was determined using both a Cyan cytometer and an Amnis ImageStream cytometer, analyzed with FlowJo software. For uptake studies, 20,000 cells per experiment were imaged with 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 glass slides, fixed with methanol at -20°C for 5 minutes, and analyzed by fluorescence microscopy. Images were captured using a Zeiss Image Capture inverted microscope and Axiovision v4.6 software. Images were subjected to the same exposure and contrast to the same extent.

[0440] Competitive binding study The specificity of TLF-1 binding to HEK293 cells was analyzed using competitive binding studies with unlabeled, non-soluble HDL and Hp1-1. Cells were harvested, washed, and resuspended (1 x 10) in ice-cold DMEM supplemented with 10% fetal bovine serum. 7 1 / ml) and then transferred for at least 10 minutes at 3°C. Alexa-488-conjugated TLF-1 (constant 20 nM) was complexed with hemoglobin (50 nM) for 10 minutes at 4°C. Increasing concentrations of unlabeled competitor were incubated with Hb (50 nM) for 10 minutes at 4°C. Competitive ligands were then mixed with Alexa-488-conjugated TLF-1 / Hb, added to the cells at 3°C, and incubated for 3 hours. Cells were then transferred to ice, washed with ice-cold 1x PBS, and analyzed using a Cyan cytometer and FlowJo software. All competition experiments were performed in triplicate.

[0441] result Next, we designed an experiment to examine TLF uptake in HEK293 mammalian cells. First, cells were incubated with AF488 TLF at 37°C and imaged via Amnis ImageStream. Particle location was then determined using Amnis internalization plot analysis. AF488 TLF was observed to be endocytosed into vesicles with maximum pixel intensity (Figures 3A and 3B), indicating intracellular rather than cell surface association.

[0442] After confirming that TLF was indeed internalized in HEK293 cells, we designed experiments to determine TLF intracellular localization. Colocalization of AF488 TLF with Lysotracker indicates that TLF localizes to a low-pH compartment similar to lysosomes (Figure 3C). This was further confirmed by fluorescence microscopy. To test whether TLF internalization is due to a specific haptoglobin (Hp) receptor (as in Trypanosoma brucei), we performed a competitive binding assay using unlabeled ligand. As shown in Figure 3D, no competition was observed with increasing amounts of unlabeled Hp (both molar and mass equivalents). These findings are consistent with previously published literature on HDL binding in mammalian cells, including HEK293 cells, which, unlike human-infectious trypanosomes, lacks a specific TLF receptor in mammalian systems (Xiao, et al., Circ Res., 103:159-66 (2008)).

[0443] Previous studies using HEK293 cells transfected with scavenger receptor class B type 1 (SR-BI, haptoglobin-hemoglobin receptor) measured SR-BI-mediated HDL uptake, which reached saturation within 3 hours (Pagler, et al., J Biol Chem., 281:11193-204 (2006)). To test whether the wild-type HEK293 cell endocytic machinery could reach equilibrium, a time course of TLF uptake was performed. By 3 hours, the signal for AF488-TLF plateaued, indicating equilibrium (Figure 3E).

[0444] Hemoglobin has been shown to be an important cofactor for TLF binding and uptake in African trypanosomes (Widener, et al., PLoS Pathog. 3:e129 (2007)). As discussed and shown herein, a receptor capable of binding Hpr present in TLF has not been identified in mammalian systems. With this in mind, experiments were designed to examine differences in uptake rates upon the addition of hemoglobin. The signal intensity of AF488 TLF analyzed using flow cytometry did not show 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 2014.

[0445] Example 3: Mammalian cells are sensitive to TLF and recombinant ApoL1. Materials and Methods Cell viability assay HEK293 HEK293 cells were harvested from mid-logarithmic cultures, washed, and resuspended in complete DMEM medium at a final concentration of 1 × 106 / ml. Sensitivity to hemoglobin (Hb)-bound TLF was measured over a range of TLF concentrations after 72 hours of incubation at 37°C. The number of viable cells was determined by counting with a hemacytometer using a phase-contrast microscope. Cell viability was further quantified by flow cytometry (Cyan) using a Live / Dead Cell Viability Kit (Invitrogen). All viability assays were performed in triplicate.

[0446] Cell culture and maintenance Cells were cultured in the indicated growth media: RPMI-1640 containing 10% FBS for RPMI8226 (CCL-155) and K-562 (CCL-243); McCoy's 5A containing 10% FBS, 1% L-glutamine, and 1% antibiotic / antimycotic for HT144; and DMEM containing 10% FBS, 1% L-glutamine, and 1% antibiotic / antimycotic for Panc1 and A375. Cells were maintained at 37°C in a humidified atmosphere with 5% CO2.

[0447] RPMI8226 / ATCC CCL-155 cells Cells were cultured in RPMI-1640 containing 10% FBS, 1% antibiotic / antimycotic. Cells were seeded into white 384-well plates in a total of 40 μl at a final cell density of 1e5 / mL and grown for 4 days with the indicated supplements. Upon completion of the experiment, cells were processed using a cell titer cell viability assay.

[0448] PANC-1 / ATCC CRL-1469 Cells were cultured in DMEM containing 10% FBS, 1% L-glutamine, and 1% antibiotic / antimycotic. Cells were seeded into white 384-well plates in a total volume of 80 μl at a final cell density of 5e4 / mL and allowed to grow and attach overnight. The following day, the medium was removed and replaced with 50 μl of complete medium containing the indicated additives and allowed to grow for 4 days. Upon completion of the experiment, cells were processed using a cell titer cell viability assay.

[0449] A375 / ATCC CRL-1619 Cells were cultured in DMEM containing 10% FBS, 1% L-glutamine, and 1% antibiotic / antimycotic. Cells were seeded into white 384-well plates in a total volume of 80 μl at a final cell density of 5e4 / mL and allowed to grow and attach overnight. The following day, the medium was removed and replaced with complete medium containing the indicated supplements and allowed to grow for 4 days. Upon completion of the experiment, cells were processed using a cell titer cell viability assay.

[0450] HT144 / ATCC HTB-63 Cells were cultured in McCoy's containing 10% FBS, 1% L-glutamine, and 1% antibiotic / antimycotic. Cells were seeded into white 384-well plates in a total volume of 80 μl at a final cell density of 5 x 10^4 cells / mL and allowed to grow and attach overnight. The next day, the medium was removed and replaced with complete medium containing the indicated supplements and allowed to grow for 3 days. Upon completion of the experiment, cells were processed using a cell titer cell viability assay.

[0451] Cell titer cell viability assay An equal volume of CellTiter-Glo 2.0 (Promega catalog G9241) was added to each well via a robotic injector. The plate was shaken for 1 minute, and luminescence was measured 10 minutes later via a SpectraMax iD3 pl...

Claims

1. A composition for using in mammalian subjects where it is necessary to increase cell death of target cells, characterized in that the composition is administered in an effective amount that increases apolipoprotein L1 (ApoL1) in the target cells.

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

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

4. The composition according to claim 3, wherein the ApoL1-containing complex is a trypanosoma lysis factor (TLF), optionally TLF-1 and / or TLF-2.

5. The composition according to claim 1, comprising a first antigen-binding fragment that binds to ApoL1 or an ApoL1-containing complex, and a targeting portion that targets the composition to target cells, wherein the composition is optionally a bispecific antibody or a multispecific antibody, the first antigen-binding fragment binds to ApoL1 or an ApoL1-containing complex, optionally to TLF, and the second antigen-binding fragment binds to a cell-specific antigen.

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

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

8. The composition according to claim 6, wherein the composition comprises a delivery vehicle, optionally liposomes, or polymer nanoparticles.

9. The composition according to claim 8, wherein the targeted portion is conjugated or fused to the delivery vehicle.

10. The composition according to claim 6, wherein the targeted portion is an antibody or an antigen-binding fragment.

11. The composition according to claim 1, wherein the cell-specific antigen is specific to diseased cells.

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

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

14. The composition according to claim 1, wherein the subject is suffering from a disease caused by the target cells.

15. The composition according to claim 14, characterized in that the composition is administered in an effective amount for treating the disease.

16. The composition according to claim 1, wherein the cell-specific antigen is not a trypanosome-specific surface antigen.

17. The composition according to claim 1, wherein the subject does not have trypanosomiasis.

18. A composition comprising ApoL1 or a functional fragment or variant thereof, and a targeting portion, wherein the targeting portion 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 targeted portion.

20. The composition according to claim 19, wherein the composition comprises a delivery vehicle, optionally liposomes or polymer nanoparticles, and optionally the targeting portion is conjugated or fused to the delivery vehicle.

21. An antibody or antigen-binding fragment, It includes 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 with it, and 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 with it, The antibody or antigen-binding fragment is an antibody or antigen-binding fragment that binds to apolipoprotein L1 (ApoL1).

22. The CDRs of the heavy chain variable domain and the light chain variable domain are 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 their variants or humanized forms having at least 70, 80, 90, or 95% sequence identity. 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 their variants or humanized forms having at least 70, 80, 90, or 95% sequence identity, or The antibody or antigen-binding fragment according to claim 21, comprising 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.

23. The CDRs of the heavy chain variable domain and the light chain variable domain are 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 The antibody or antigen-binding fragment according to claim 21, comprising 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. The antibody or antigen-binding fragment according to claim 21, comprising: a heavy chain variable domain containing 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 therewith; and a light chain variable domain containing 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 therewith.

25. The antibody or antigen-binding fragment according to claim 21, comprising a heavy chain variable domain containing the amino acid sequence of SEQ ID NO: 24 and a light chain variable domain containing the amino acid sequence of SEQ ID NO: 36 or SEQ ID NO:

77.

26. An antibody or antigen-binding fragment, It includes 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 with it, 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 with it, The antibody or antigen-binding fragment is an antibody or antigen-binding fragment that binds to haptoglobin-related protein (Hpr).

27. The CDRs of the heavy chain variable domain and the light chain variable domain are 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 their variants or humanized forms having at least 70, 80, 90, or 95% sequence identity. 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 their variants or humanized forms having at least 70, 80, 90, or 95% sequence identity, or The antibody or antigen-binding fragment according to claim 26, comprising 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 a variant or humanized form thereof having at least 70, 80, 90, or 95% sequence identity.

28. The CDRs of the heavy chain variable domain and the light chain variable domain are 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 The antibody or antigen-binding fragment according to claim 26, comprising 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. The antibody or antigen-binding fragment according to claim 26, comprising: a heavy chain variable domain containing 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 therewith; and a light chain variable domain containing 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 therewith.

30. The antibody or antigen-binding fragment according to claim 26, comprising a heavy chain variable domain containing the amino acid sequence of SEQ ID NO: 3 and a light chain variable domain containing the amino acid sequence of SEQ ID NO:

14.

31. An antibody or antigen-binding fragment, It includes 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 with it, 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 with it, The antibody or antigen-binding fragment is an antibody or antigen-binding fragment that binds to haptoglobin-related protein (Hpr).

32. The CDRs of the heavy chain variable domain and the light chain variable domain are 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 their variants or humanized forms having at least 70, 80, 90, or 95% sequence identity. 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 their variants or humanized forms having at least 70, 80, 90, or 95% sequence identity, or The antibody or antigen-binding fragment according to claim 31, comprising 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.

33. The CDRs of the heavy chain variable domain and the light chain variable domain are 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 The antibody or antigen-binding fragment according to claim 31, comprising 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), and QHNRELPLT (SEQ ID NO: 68).

34. The antibody or antigen-binding fragment according to claim 31, comprising: a heavy chain variable domain containing 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 therewith; and a light chain variable domain containing 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 therewith.

35. The antibody or antigen-binding fragment according to claim 31, comprising a heavy chain variable domain containing the amino acid sequence of SEQ ID NO: 56 and a light chain variable domain containing the amino acid sequence of SEQ ID NO:

65.

36. The 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, and optionally the ApoL1-containing complex is a trypanosoma lysis factor (TLF).

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

38. The antibody or antigen-binding fragment according to claim 36, wherein the antibody or antigen-binding fragment can bind to the ApoL1-containing complex under physiological conditions.

39. The antibody or antigen-binding fragment according to claim 36, wherein the antibody or antigen-binding fragment can bind to an ApoL1-containing complex in a target, and optionally the target is a human.

40. The antibody or antigen-binding fragment according to any one of claims 21 to 35, wherein the antibody is not mouse IgG1 or IgG2a.

41. An antibody or antigen-binding fragment according to any one of claims 21 to 35, comprising one or more constant domains from an immunoglobulin constant region (Fc).

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

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

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

45. The antibody or antigen-binding fragment according to any one of claims 21 to 35, wherein the antibody or antigen-binding fragment is detectably labeled or conjugated to a toxin, drug, receptor, enzyme, or receptor ligand.

46. The antibody or antigen-binding fragment according to any one of claims 21 to 35, wherein the antibody is a monoclonal antibody, a human antibody, a chimeric antibody, or a humanized antibody.

47. The antibody or antigen-binding fragment according to any one of claims 21 to 35, wherein the antibody is a bispecific antibody, a tripspecific antibody, or a multispecific antibody.

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

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

50. The antibody or antigen-binding fragment according to claim 48, 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 hematological cancer antigen or solid tumor antigen optionally selected from claudin 18.2, MUC1, mesothelin (MSLN), myoferin (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 claim 48, wherein the cell-specific antigen is BCMA.

54. The antibody or antigen-binding fragment according to claim 48, wherein the second antigen-binding fragment comprises 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, or a humanized form thereof, and 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, or a humanized form thereof.

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

56. The antibody or antigen-binding fragment according to claim 54, wherein the second antigen-binding fragment comprises a heavy chain variable domain including 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 therewith, and a light chain variable domain including 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 therewith.

57. The second antigen-binding fragment comprises a heavy chain variable domain containing the amino acid sequence of SEQ ID NO: 41 and a light chain variable domain containing the amino acid sequence of SEQ ID NO:

42. The antibody or antigen-binding fragment of claim 56, wherein the second antigen-binding fragment optionally comprises the amino acid sequence of SEQ ID NO:

51.

58. The antibody or antigen-binding fragment according to claim 49, comprising 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 therewith, 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 therewith.

59. The antibody or antigen-binding fragment according to claim 49, comprising the amino acid sequences of SEQ ID NO: 71 and SEQ ID NO:

72.

60. The antibody or antigen-binding fragment according to claim 49, each comprising two copies of the amino acid sequences of SEQ ID NO: 70 and SEQ ID NO: 72, respectively.

61. A bispecific chimeric antibody against ApoL1 and the anti-cell-specific antigen IgG1-scFv.

62. A bispecific chimeric antibody against HPR and the cell-specific antigen IgG1-scFv.

63. A nucleic acid encoding an antibody or antigen-binding fragment according to any one of claims 21 to 35 and 49.

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

65. An expression vector comprising the nucleic acid described in claim 63.

66. A cell comprising the nucleic acid described in claim 63, or an expression vector comprising the nucleic acid described in claim 63, wherein the cell is optionally a mammalian cell.

67. An immune complex comprising an antibody or antigen-binding fragment according to any one of claims 21 to 35 and 49, conjugated to ApoL1 or an ApoL1-containing complex, wherein the complex is optionally a TLF.

68. An immune complex comprising an antibody or antigen-binding fragment according to any one of claims 21 to 35 and 49.

69. A composition comprising an immune complex comprising an antibody or antigen-binding fragment according to any one of claims 21 to 35 and 49, conjugated to ApoL1 or an ApoL1-containing complex, wherein the complex is optionally a TLF, or a composition comprising an immune complex comprising an antibody or antigen-binding fragment according to any one of claims 21 to 35 and 49, for use in a method of inducing cell death, wherein the method comprises contacting target cells with the immune complex.

70. The composition according to claim 69, wherein the contact occurs in vitro.

71. The composition according to claim 69, wherein the shrinkage occurs in vivo in the subject.

72. The composition 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 35 and 49.

74. A composition for treating cancer in a subject, comprising an antibody or antigen-binding fragment according to any one of claims 21 to 35 and 49.

75. The composition according to claim 74, wherein the cancer is a blood cancer.

76. The composition according to claim 75, 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).

77. The composition according to claim 74, wherein the cancer is a solid tumor.

78. A composition comprising the composition according to any one of claims 1 to 20, or the antibody or antigen-binding fragment according to any one of claims 21 to 35 and 49, for treating a subject in need of treatment.

79. The composition according to claim 78, wherein the subject has cancer.

80. The composition according to any one of claims 1 to 17, wherein the target cell is a mammalian cell.

81. The composition according to claim 80, wherein the mammalian cells are infected cells.

82. The composition according to claim 81, wherein the infected cells are infected with a virus, bacterium, or eukaryotic intracellular organism selected arbitrarily from HIV, Plasmodium falciparum, Toxoplasma gondii, Leishmania sp., Trypanosoma cruzi, Listeria monocytogenes, Chlamydia trachomatis, Coxiella burnetti, Mycobacterium tuberculosis, and Trichomonas virginalis.

83. The composition according to any one of claims 1 to 17, wherein the target cell is a non-mammalian cell.

84. The composition according to claim 83, wherein the non-mammalian cell is a bacterium, fungus, or non-mammalian eukaryotic cell.

85. The composition according to claim 84, wherein the non-mammalian cells are not trypanosomes.

86. A composition comprising the composition according to any one of claims 1 to 17, or the antibody or antigen-binding fragment according to any one of claims 21 to 35 and 49, for the treatment of a subject in need of treatment, wherein the subject is a mammal, optionally human.

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