Cellular uptake peptide-therapeutic payload compositions and methods of use

Abstract

The present disclosure relates, inter alia, to compositions that include an internalization peptide, also known as an intracellular uptake peptide, connected to a pharmaceutical agent, also known as a therapeutic payload, by a linker.

Description

[Technical field]

[0001] The present disclosure relates to peptides that are utilized, for example, in drug delivery. CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to and the benefit of U.S. Provisional Patent Application No. 63 / 295,978, filed January 3, 2022, U.S. Provisional Patent Application No. 63 / 394,135, filed August 1, 2022, and U.S. Provisional Patent Application No. 63 / 417,106, filed October 18, 2022, the entire contents of which are incorporated herein by reference in their entireties.

[0002] Sequence Listing This application contains a Sequence Listing that has been submitted in XML format via EFS-Web. The contents of the XML copy entitled "SKX-004_Sequence Listing", created on Jan. 2, 2023, and 111,437 bytes in size, are hereby incorporated by reference in their entirety. [Background technology]

[0003] Peptides have various advantages as delivery agents, including their small size, e.g., increasing tumor adhesion, their more consistent 1:1 peptide:drug stoichiometry (e.g., compared to antibody-drug conjugates (ADCs)), and their ease of manufacture (e.g., compared to ADCs).

[0004] There is a need for more and improved peptides for therapeutic drug delivery. Summary of the Invention

[0005] Thus, the present disclosure relates in some aspects to a composition comprising an internalization peptide, also known as an intracellular uptake peptide, connected to a pharmaceutical, also known as a therapeutic payload by a linker. In some embodiments, the composition can deliver the pharmaceutical / therapeutic payload to the cell and internalize it (e.g., by macropinocytosis). In some embodiments, the composition further comprises an organelle uptake peptide, e.g., a nuclear localization signal (NLS). Thus, in some embodiments, the composition can be internalized in the cell and delivered to the organelle of interest inside the cell, for example, to allow the pharmaceutical / therapeutic payload to contact the site of action.

[0006] In some embodiments, the composition further comprises one or more cleavage sites (e.g., matrix metalloproteinase or furin cleavage sites). In some embodiments, the composition further comprises one or more cleavage sites that allow for site-specific removal of more components of the composition when the composition is inside a cell. In some embodiments, the cleavage site is transferred between the internalization / cellular uptake peptide and the organelle uptake peptide (which is itself linked to the pharmaceutical / therapeutic payload), thereby allowing for cleavage of the internalization / cellular uptake peptide from the organelle uptake peptide and the pharmaceutical / therapeutic payload. In some embodiments, cleavage occurs via an intracellular enzyme.

[0007] In embodiments, the composition is as shown in FIG. 8, FIG. 11, FIG. 14, FIG. 15, or FIG.

[0008] In several aspects, methods are provided for delivering a pharmaceutical / therapeutic payload to a cell by internalization, the methods comprising contacting a cell in vitro, ex vivo, or in vivo with a composition comprising a pharmaceutical / therapeutic payload linked to an internalization / cellular uptake peptide, the composition optionally comprising one or more cleavage sites that allow for site-specific removal of more components of the composition when they are inside the cell.

[0009] In several aspects, methods for organelle-specific delivery of a pharmaceutical / therapeutic payload are provided, comprising contacting a cell in vitro, ex vivo, or in vivo with a composition comprising a pharmaceutical / therapeutic payload linked to an internalization / cellular uptake peptide and an organelle uptake peptide, the composition optionally comprising one or more cleavage sites that allow for site-specific removal of more components of the composition when they are inside the cell. [Brief description of the drawings]

[0010] [Figure 1] Shown are the sequences of 429 (IGF-1r peptide, aka SKwx-429) (SEQ ID NO: 48) and OV-8 (isolated from Ovcar-3 cells, aka Skwx-OV-8) (SEQ ID NO: 49), as well as two peptide-drug conjugates. DAR = drug-antibody ratio. [Diagram 2] OV-8 internalization in various cell lines Skwx-OV-8 has been shown to be internalized in several cancer cell lines of different lineages. [Diagram 3] 1 shows dose response curves for various cell lines with peptide drug conjugates ("PDCs"): (1) A549: human lung cancer, (2) SW480: human colorectal cancer, (3) HCT116: human colorectal cancer, and (4) G292: human osteosarcoma. [Figure 4] 1 shows that peptide-drug conjugates kill tumor cells. [Diagram 5] The effect of PDC on G292 cells is shown. [Figure 6] 1 shows that MAE and PDC disrupt microtubules. [Figure 7] The 429 peptide-gemcitabine construct is shown. [Figure 8]1 shows a non-limiting schematic diagram of various conjugated peptides of the present disclosure. The internalization peptides can be, among others, Skwx-429 and Skwx-Ov-8 (FIG. 1). An optional FLAG sequence can be added to determine whether enzymatic cleavage has occurred. The MMP-9 cleavage peptide is shown, but Furin can be used as well. The nuclear localization signal (NLS) is the sequence shown. FITC (optional) can be added to the C-terminus to visualize the reaction. [Figure 9] Nuclear localization of the 429 construct in three cell lines (G292 [osteosarcoma], U87MG [glioblastoma], and HCT116 [colorectal cancer]) is shown. [Figure 10] Nuclear staining detected by anti-FLAG antibody is shown. As can be seen, the construct still accumulates in the nucleus of U87 cells, although it does not appear to be cleaved, i.e., the entire 429 construct may be localized to the nucleus. [Figure 11] Immunofluorescence data are shown demonstrating that the entire 429 construct was localized to the nucleus. [Figure 12] 1 shows a construct with two HDAC inhibitors as exemplary drug payloads. [Figure 13] Shown is the amino acid sequence of SmarcB1 (SEQ ID NO: 122), which shows the potential region (SEQ ID NO: 123) that binds to myc. SmarcB1 is a tumor suppressor gene that binds to and represses the proto-oncogene myc. SmarcB1 mutations are associated with ATRT (atypical teratoid rhabdoid tumor), a pediatric tumor with poor prognosis. [Figure 14] A proof-of-concept prototype conjugate for delivering the SmarcB1-myc binding sequence to the nucleus is shown (SEQ ID NO: 111). [Figure 15] FIG. 1 shows non-limiting schematic diagrams of various conjugated peptides of the present disclosure. [Figure 16]AB show the effect of MMAE drug conjugates on G292 cells. A shows images of G292 cells treated with 50 nM MMAE, 500 nM 429-MMAE, and 50 nM 429-MMAE. B shows a bar graph of cell viability with various dosages of 429-MMAE and ovalbumin (OV)-MMAE treatment. [Figure 17] 1 shows confocal fluorescence microscopy images of the effect of 429-MMAE on microtubules in G292 cells. [Figure 18] Figures A-C show that 429-MMAE affects cell viability and apoptosis in A431 cells. A shows images of A431 cells treated with 50 nM MMAE, 500 nM 429-MMAE, and 50 nM 429-MMAE. B shows a bar graph illustrating the viability of A431 cells after various 429-MMAE and OV-MMAE treatments. C shows a bar graph illustrating the apoptosis of A431 cells after various 429-MMAE and OV-MMAE treatments. [Figure 19] 1 shows confocal fluorescence microscopy images of 429-MMAE disrupting microtubules in A431 cells. [Figure 20] A to B show dose response curves of A413, HCT-116, and G292 cells treated with 429-MMAE. A shows dose response curves of A431 and HCT-116 cells treated with 429-MMAE. B shows dose response curves of A431 and G292 cells treated with 429-MMAE. [Figure 21] Figures A-C show that 429-MMAE induces apoptosis in CHLA06 cells. A is a bar graph showing the results of a caspase assay 24 hours after treating CHLA06 cells with various doses of MMAE and 429-MMAE. B is a bar graph showing the results of a caspase assay 48 hours after treating CHLA06 cells with various doses of MMAE and 429-MMAE. C shows confocal fluorescence microscopy images of 29-MMAE inducing apoptosis in CHLA06 cells. [Figure 22]AB show that 429-MMAE-1 inhibits CHLA06 cells. A is a bar graph comparing the results of CHLA06 cell viability after various 429-MMAE-1 treatments. B shows confocal fluorescence microscopy images of CHLA06 cells treated with various dosages of 429-MMAE-1. [Diagram 23] AB shows that 429-P (429-NLS-panobinostat) inhibits CHLA06 cells. A shows confocal fluorescence microscopy images of CHLA06 cells treated with various doses of 429-P. B is a bar graph showing the viability of CHLA06 cells treated with various doses of panobinostat and 429-P. [Figure 24] A-B show dose response curves of A413 and CHLA06 cells treated with 429-P. A shows the dose response curve of A431 cells treated with 429-P and panobinostat. B shows the dose response curve of CHLA06 cells treated with 429-P and panobinostat. [Diagram 25] Confocal microscopy images of 429-SMARCB1 internalized into CHLA-06 cells are shown. [Figure 26] Confocal microscopy images of 429-SMARCB1 co-localized with histones in CHLA-06 cells are shown. [Figure 27] A-C show that 429-SMARCB1 inhibits Myc transcription. A shows a bar graph comparing the viability of HCT116 Myc-reporter cells after various doses of panobinostat. B shows a bar graph comparing the viability of HCT116 Myc-reporter cells after various doses of 429-SMARCB1. C shows a line graph showing the viability of HCT116 Myc-reporter cells after various doses of 429-SMARCB1. [Figure 28] AB show that 429-SMARCB1 affects Myc transcription and viability. A shows a bar graph comparing transcription of HCT116 Myc-reporter cells with various treatments. B shows a bar graph comparing viability of HCT116 Myc-reporter cells after control and 429-SMARCB1 treatment. [Figure 29] 1 shows a PAGE gel comparing the expression of SMARCB1 in atypical teratoid / rhabdoid tumor cell lines. [Diagram 30] AB show that SMARCB1 and tazemetostat reduce cell viability and increase caspase activity in CHLA06 cells. A is a bar graph showing the results of a cell viability assay after treating CHLA06 cells with various treatments. B is a bar graph showing the results of a caspase assay after treating CHLA06 cells with various treatments. [Diagram 31] 1 shows a 429-SMARCB1 mimetic conjugate (SEQ ID NO: 111) for delivering SmarcB1-myc binding sequences to the nucleus. [Diagram 32] AB show that 429-SMARCB1 mimetic conjugates and tazemetostat significantly increase caspase activity in CHLA06 cells. A shows a graph comparing various 429-SMARCB1 mimetic conjugates and tazemetostat treatment in CHLA06 cells. B shows a line graph showing various 429-SMARCB1 mimetic conjugates and tazemetostat treatment in CHLA06 cells. [Diagram 33] AB show that 429-SMARCB1 mimetic conjugates and tazemetostat significantly increase caspase activity in CHLA02 cells. A shows a graph comparing various 429-SMARCB1 mimetic conjugates and tazemetostat treatment in CHLA02 cells. B shows a line graph showing various 429-SMARCB1 mimetic conjugates and tazemetostat treatment in CHLA02 cells. [Diagram 34] AB shows that 429-SMARCB1 mimetic conjugates affect viability in CHLA06 cells. A is a bar graph comparing the viability of CHLA06 cells treated with various dosages of 429-SMARCB1 mimetic conjugates. B is a confocal microscopy image of CHLA06 cells treated with 429-SMARCB1 mimetic conjugates. [Diagram 35] AB show that 429-SMARCB1 mimetic conjugates inhibit Myc transcription. A shows a bar graph comparing the viability of HCT116 Myc-reporter cells after treatment with various dosages of 429-SMARCB1 mimetic conjugates. B shows a bar graph comparing the levels of Myc transcription after treatment with various dosages of 429-SMARCB1 mimetic conjugates. [Diagram 36] AB show the effect of 429 ScHv-SMARCB1 antibody and OV SMARCB1 antibody. A shows confocal microscopy images of cells treated with 429 antibody. B shows a bar graph comparing cell viability after treatment with various 429 constructs. [Figure 37A] 1 shows the effect of 429-MMAE in nude mice. A line graph comparing the effect of various dosages of 429-MMAE on tumor volume is shown. [Figure 37B] 1 shows the effect of 429-MMAE in nude mice. 2 shows a bar graph comparing the effect of various dosages of 429-MMAE on tumor volume. [Figure 37C] 1 shows the effect of 429-MMAE in nude mice. 2 shows a bar graph comparing the effect of various dosages of 429-MMAE on tumor weight. [Figure 38A] 1 shows a comparison of various small molecule inhibitors of IGF-1R and 429-MMAE on various cell lines. 2 shows a line graph comparing the dose response of IGF-1R inhibitors and 429-MMAE on CHLA06 cells. [Figure 38B] 1 shows a comparison of various small molecule inhibitors of IGF-1R and 429-MMAE on various cell lines. 2 shows a line graph comparing the IC50 of IGF-1R inhibitors and 429-MMAE on G292 cells. [Figure 38C] 1 shows a comparison of various small molecule inhibitors of IGF-1R and 429-MMAE on various cell lines. 2 shows a line graph comparing the IC50 of IGF-1R inhibitors and 429-MMAE on HCT116 cells. [Figure 38D]1 shows a comparison of various small molecule inhibitors of IGF-1R and 429-MMAE on various cell lines. A line graph comparing the IC50 of MMAE and 429-MMAE on WS1 cells is shown. [Figure 38E] 1 shows a comparison of various small molecule inhibitors of IGF-1R and 429-MMAE on various cell lines. 2 shows a line graph comparing the IC50 of MMAE and 429-MMAE on CCD841 cells. [Figure 39] AB show apoptosis of CHLA06 at 48 and 72 hours by IGFR inhibitors. A shows a line graph comparing the IC50 of 429-MMAE and small molecule inhibitors at 48 hours for inducing apoptosis of CHLA06 cells. B shows a line graph comparing the IC50 of 429-MMAE and small molecule inhibitors at 72 hours for inducing apoptosis of CHLA06 cells. [Diagram 40] 1 shows a line graph comparing the dose response curves of 429-MMAE with and without IGF. [Diagram 41] A-C show a comparison of 429-MMAE (drug peptide ratio = 1) and 429-MMAE (drug peptide ratio = 2) in various cell lines. A shows a line graph comparing apoptosis of CHLA06 at 48 hours after treatment with 429-MMAE (drug peptide ratio = 1) and 429-MMAE (drug peptide ratio = 2). B shows a line graph comparing dose response of CHLA06 at 5 days after treatment with 429-MMAE (drug peptide ratio = 1) and 429-MMAE (drug peptide ratio = 2). C shows a line graph comparing dose response of CHLA04 at 5 days after treatment with 429-MMAE (drug peptide ratio = 1) and 429-MMAE (drug peptide ratio = 2). [Figure 42A] 1 shows a comparison of 429-MMAE and 429-SM in various cell lines. 2 shows a line graph comparing apoptosis of CHLA06 after 48 hours of treatment with 429-MMAE and 429-SM. [Figure 42B]1 shows a comparison of 429-MMAE and 429-SM in various cell lines. 2 shows a line graph comparing HCT myc at 48 hours after treatment with 429-MMAE and 429-SM. [Figure 42C] 1 shows a comparison of 429-MMAE and 429-SM in various cell lines. 2 shows a line graph comparing the dose response of A431 after treatment with 429-MMAE and 429-SM. [Fig.42D] 1 shows a comparison of 429-MMAE and 429-SM in various cell lines. 2 shows a line graph comparing the dose response of HCT116 after treatment with 429-MMAE and 429-SM. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0011] In some aspects, a composition is provided that includes an internalization peptide, also known as an intracellular uptake peptide, connected to a pharmaceutical, also known as a therapeutic payload by a linker. Thus, in some embodiments, the composition can deliver the pharmaceutical / therapeutic payload to a cell and internalize it (e.g., by macropinocytosis). In some embodiments, the composition further includes an organelle uptake peptide, such as a nuclear localization signal (NLS). Thus, in some embodiments, the composition can be internalized in the cell and delivered to the organelle of interest inside the cell, for example, to allow the pharmaceutical / therapeutic payload to contact the location of action.

[0012] In some embodiments, the composition further comprises one or more cleavage sites (e.g., matrix metalloproteinase or furin cleavage sites). In some embodiments, the composition further comprises one or more cleavage sites that allow for site-specific removal of more components of the composition when they are inside a cell. In some embodiments, the cleavage site is transferred between the internalization / cellular uptake peptide and the organelle uptake peptide (which is itself linked to the pharmaceutical / therapeutic payload), thereby allowing for cleavage of the internalization / cellular uptake peptide from the organelle uptake peptide and the pharmaceutical / therapeutic payload. In some embodiments, cleavage occurs via an intracellular enzyme.

[0013] This composition is alternatively referred to herein as a "peptide-drug conjugate" or "PDC."

[0014] In some embodiments,

number

[0015] In some embodiments,

number

[0016] In embodiments, m is 1 and V has the amino acid sequence of DFYGCLLDLSLGVPSGVRRRCITA (SEQ ID NO: 104), or a variant thereof.

[0017] In embodiments, m is 1 and V has the amino acid sequence of RPWILHLGRERSTDDQTPVHRMSLMHPLYHTRGKSYKVEG (SEQ ID NO: 105), or a variant thereof.

[0018] In embodiments, Z is or comprises 1, or 2, or 3, or 4, or 5, or 6, or 7, or 8, or 9, or 10, or more therapeutic payloads.

[0019] In embodiments, the composition is of the structure shown in FIG.

[0020] In embodiments, the composition is of the structure shown in FIG.

[0021] In embodiments, the composition is of the structure shown in FIG.

[0022] In embodiments, the composition is of the structure shown in FIG.

[0023] In some embodiments, the composition is of the structure shown in FIG.

[0024] In embodiments, the composition comprises: DFYGCLLDLSLGVPSLGWRRRCITADYKDDDDKWLPSSIPKKKRKV (SEQ ID NO: 113) (1 429-M), FSFGNSSLRKLPGVVRRRRCARGVWRDYKDDDDKWLPSSIPKKKRKV (SEQ ID NO: 114) (No. 3 OvB8-M), DFYGCLLDLSLGVPSLGWRRRCITADYKDDDDKPRRARSPKKKRKV (SEQ ID NO: 115) (429-F), or FSFGNSSLRKLPGVVRRRRCARGVWRDYKDDDDKPRRARSPKKKRKV (SEQ ID NO: 116) (No. 4 OVB8-F), or variants thereof, where DYKDDDDK (SEQ ID NO: 109) = FLAG sequence, WLPSSI (SEQ ID NO: 107) = MMP9 cleavage site, PRRARS (SEQ ID NO: 108) = Furin cleavage site, and PKKKRKV (SEQ ID NO: 106) = nuclear localization signal. In some embodiments, any of the FLAG sequence, MMP9 cleavage site, Furin cleavage site, and nuclear localization signal may be removed from the composition.

[0025] In some embodiments, the composition further comprises a targeting moiety. In some embodiments, the targeting moiety is directed to IGFR or EGFR. In some embodiments, the targeting moiety is directed to type 1 receptor (IGF-1R), epidermal growth factor receptor (EGFR), insulin receptor, GRP78 / BiP, or transferrin receptor.

[0026] Internalization peptides / cell-uptake peptides The internalization peptide / cell uptake peptide may be referred to as an "internalization peptide," "cell penetrating peptide," or "CP3."

[0027] In some embodiments, the internalization / internalization peptide can bind to a marker, e.g., a receptor, to cause internalization by the cell. In some embodiments, the internalization / internalization peptide can be internalized by the cell by pinocytosis, e.g., macropinocytosis.

[0028] In some embodiments, the internalization / cellular uptake peptide is or includes the amino acid sequence DFYGCLLDLSLGVPSLGWRRRCITA (SEQ ID NO: 48), or a variant thereof. In some embodiments, the internalization / cellular uptake peptide is or includes "429", or a variant thereof. See FIG. 1.

[0029] In some embodiments, the internalization / cellular uptake peptide is or comprises the amino acid sequence of FSFGNSSLRKLPGVVRRRRCARGVWR (SEQ ID NO: 49), or a variant thereof. In some embodiments, the internalization / cellular uptake peptide is or comprises "OV-8", or a variant thereof. See FIG. 1.

[0030] In some embodiments, the internalization / cellular uptake peptide is DFYGCLLDLSLGVPSLGWRRRCITA (SEQ ID NO: 48) ("1 429-M"); FSFGNSSLRKLPGVVRRRRCARGVWR (SEQ ID NO: 49) ("No. 3 OvB8-M"); DFYGCLLDLSLGVPSLGWRRRCITA (SEQ ID NO: 48) ("429-F"); FSFGNSSLRKLPGVVRRRRCARGVWR (SEQ ID NO: 49) ("No. 4 OVB8-F"); or a variant thereof.

[0031] In some embodiments, the internalization / cellular uptake peptide is YQVVSVVWHRGLLRPVLRGSLKPWYIFVRIHISWGQVHVG (SEQ ID NO: 43) ("CPP 1"), TGWWFFWSYVCSVRLLAVTCSFWCTEQGEESLPRAGTHTH (SEQ ID NO:44) ("CPP 4"), YTLGLGKSQRWNLFRNFFCYMPRVTVFCKSCRCVSFQTSG (SEQ ID NO: 45) ("CPP 5"), HYLLIRCARGFCEVRLVDAMCSITIVIDCVLFTSQRERAE (SEQ ID NO:46) ("CPP 6"), or a variant thereof.

[0032] In some embodiments, the internalization / internal uptake peptides can bind to and / or be internalized by cancer cells. In some embodiments, the cancer cells are selected from colorectal cancer cells (e.g., CaCo2), neuroblastoma cells (e.g., SK-N-BE(2)), and / or epidermal cancer cells (e.g., A431, derived from an epidermoid carcinoma of the vulva from an 85-year-old woman). In some embodiments, the cancer cells are or are derived from OVCAR, MIA, A549, A431, G292, HCT116, CHLA02, CHLA04, CHLA05, or CHLA06 cell lines.

[0033] In embodiments, the internalization / cellular uptake peptide is or comprises an amino acid sequence in Table 1, or a variant thereof. [Table 1] TIFF2025503584000005.tif99159

[0034] In some embodiments, the internalization / cellular uptake peptide is capable of binding to Ovcar-3 cells. In some embodiments, the internalization / cellular uptake peptide is PRGWRRCLGWSVGPASGSGP (SEQ ID NO:50), FSFGNSSLRKLPGVVRRRRCARGVWR (SEQ ID NO:49), CLGVCISGDPEQADMWGCRP (SEQ ID NO:51), ILSRSQYLVFGYAVVARGSR (SEQ ID NO:52), GSGLCSVCQCGGMYSRVVLGQRRWM (SEQ ID NO:53), GVSPIVHGDAGLLQPRNTRL (SEQ ID NO:54), CRQRGDGGAMRRGSVCLRGW (SEQ ID NO:55), GDGTGVQRAAVGFDLTGHSP (SEQ ID NO:56), AVSACGRCRHCRVPGVPCWLYCVCT (SEQ ID NO:57), PSTMSIAGGGFCGHPRRGTI (SEQ ID NO:58), DQCAGHDSVQRHALHGLAVM (SEQ ID NO:59), GASPCVFRVDSTSGGPLVFPDLQST (SEQ ID NO: 60), RREDSLTRVRQALREALGAG (SEQ ID NO:61), and LDVGVAQHRGGTVIRVAMRA (SEQ ID NO: 62), or a variant thereof.

[0035] In embodiments, the internalization / cellular uptake peptide is or comprises the amino acid sequence FSFGNSSLRKLPGVVRRRRCARGVWR (SEQ ID NO: 49), or a variant thereof.

[0036] In some embodiments, the internalization / internalization peptide is capable of binding to MIA cells. In some embodiments, the internalization / internalization peptide is GIRRGGLGRQRGWGRRRKPI (SEQ ID NO: 63), RAPGGFTRTNSVRRATGSM (SEQ ID NO: 64), YGKVGGWRLATVGRRTESRI (SEQ ID NO: 65), KQVGLINGSACSCDMYQRVV (SEQ ID NO: 66), VNSVKGVHRRAFSEQPLGSG (SEQ ID NO:67), DAGRAPALGWFFFGRRRFGE (SEQ ID NO: 68), GVPGCGSHGADWGREIGATSTGMAL (SEQ ID NO: 69), GVCRWMRPHFRMPRRWRLGR (SEQ ID NO: 70), RSSGMCIIARRHVLSCHSLS (SEQ ID NO:71), SGVGTCPTRPWVPGVCRQVS (SEQ ID NO:72), RVIVSGLSGRVRMGVKRRRI (SEQ ID NO: 73), FMRHARPTRGVVRRIGHGV (SEQ ID NO: 74), QLYACLRLVWGRQASAGWRVTMTG (SEQ ID NO: 75), LERIVGQWYSEGLSGDWANL (SEQ ID NO: 76), GASRCFSLCSGSEGAESRRQATTV (SEQ ID NO:77), HLGCSMPINVFGVCIWKCA (SEQ ID NO: 78), RRLAVRLRVMLRRGPVCIRV (SEQ ID NO: 79), LVDACIGSCQPEGSPPARPPFQLA (SEQ ID NO: 80), RCVGCCKYLIAQAYRFTVAGGAAQYA (SEQ ID NO: 81), AGVHCGLTNVHGVGVRSYRRLYA (SEQ ID NO: 82), VNGWGLGRRGMGIDYRWAVH (SEQ ID NO: 83), and QSARCAVTMGLDALNLRRGISPVLD (SEQ ID NO: 84), or a variant thereof.

[0037] In some embodiments, the internalization / internal uptake peptide is capable of binding to A549 cells. In some embodiments, the internalization / internal uptake peptide is YPWQQVLVPPPGNVHAFATI (SEQ ID NO: 85), TAVAGVGVSAGQLQGGTAAG (SEQ ID NO: 86), FFGLSFPASVAVSDSGVQMR (SEQ ID NO: 87), SSRAVSWMVQLRVQRGSGLV (SEQ ID NO: 88), SRLSLVQSCSVSWGHQTVGT (SEQ ID NO: 89), VARWCQVNWAYGRRLAPPVG (SEQ ID NO: 90), FLLCSLPLTQPPYRVIGAYA (SEQ ID NO:91), GGPSGIGWQHAWWVFGRQGL (SEQ ID NO: 92), LQRQIAGPRSRQRGRGSRYR (SEQ ID NO: 93), FEDMRLGWRNGQPPGGVAIW (SEQ ID NO: 94), DSWQGARVQPGVGAHTAQQR (SEQ ID NO: 95), GIKTVRGYGVWRWHGGSPRM (SEQ ID NO: 96), IRVWCQWLRTKLAAGQVNCA (SEQ ID NO:97), SYGVGGRHSGGQGEALVYSV (SEQ ID NO: 98), RAQNCGNWAAVQRINVSSMA (SEQ ID NO: 99), GRAPEQTPCIGWRGGLIKDL (SEQ ID NO: 100), SGSRNFAAARWSHRGFCVPW (SEQ ID NO: 101), IAFSRRGRGWWCQMPGGSRV (SEQ ID NO: 102), and GCCSAGLPSSLLSTPQSRSR (SEQ ID NO: 103), or a variant thereof.

[0038] In some embodiments, the internalization / cellular uptake peptide lacks any tags (eg, FLAG tag, His6 tag, etc.).

[0039] Drug / Therapeutic Payload In some embodiments, the therapeutic payload is a small molecule drug, a biologic, or a radionuclide.

[0040] In several embodiments, the therapeutic payload is a biologic selected from a toxin, an antibody, or an oligonucleotide (such as, but not limited to, an antisense inhibitor).

[0041] In some embodiments, the therapeutic payload is a SmarcB1 mimetic. In some embodiments, the therapeutic payload has the amino acid sequence of Figure 13 (SEQ ID NO: 122 or 123) or Figure 14 (SEQ ID NO: 111). In some embodiments, the composition comprising a SmarcB1 mimetic is the composition of Figure 14 (SEQ ID NO: 111).

[0042] In embodiments, the SmarcB1 mimetic is or includes the amino acid sequence of MMMMALSKTFGQKPVKFQLEDDGEFYMIGSEVGNYLRMFRGSLYKRYPSLWRRLATVEERKKIVASSHGKKTKPNTKDHGYTTLATSVTLLKASEVEEILDGNDEKYKAVSISTEPPTYLREQKAKRNSQWVPTLPNSSHHLDAVPCSTTINRNRMGRDKKRTFPLCFDDHDPAVIHENASQPEVLVPIRLDMEIDGQKLRDAFTWNMNEKLMTPEMFSEILCDDLDLNPLTFVPAIASAIRQQIESYPTDSILEDQSDQRVIIKLNIHVGNISLVDQFEWDMSEKENSPEKFALKLCSELGLGGEFVTTIAYSIRGQLSWHQKTYAFSENPLPTVEIAIRNTGDADQWCPLLETLTDAEMEKKIRDQDRNTRRMRRLANTAPAW (SEQ ID NO: 122).

[0043] In embodiments, the SmarcB1 mimetic is or includes the amino acid sequence of LVPIRLDMEIDGQKLRDAFTWNMNEKLMTPEMFSEILCDDLDLNPLTFVPAIASAIRQQI (SEQ ID NO: 123).

[0044] In some embodiments, the therapeutic payload is monomethylauristatin E (MMAE), for example, as shown in FIG.

[0045] In some embodiments, the therapeutic payload is an anti-cancer drug, hi some embodiments, the anti-cancer drug is a chemotherapeutic agent.

[0046] In some embodiments, the chemotherapeutic agent is an alkylating agent such as thiotepa and CYTOXAN cyclophosphamide; an alkyl sulfonate such as busulfan, improsulfan, and piposulfan; an aziridine such as benzodopa, carboquone, meturedopa, and uredopa; an ethylenimine and a methylamelamine, including altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide, and trimethylolomelamine; an acetogenin (e.g., bullatacin and bullatacinone); a camptothecin (including the synthetic analog topotecan); a bryostatin; a caIIy statin; a CC-1065 (including its adozelesin, carzelesin, and bizelesin synthetic analogs); a cryptophycin (e.g., cryptophycin 1 and cryptophycin 8); a dolastatin; a duocarmycin (including the synthetic analogs KW-2189 and CB 1-TM1); erytherobin; pancratistatin; sarcodictyin; spongiostatin; chlorambucil, chlornaphazine, cholophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembitine, phenesterine, prednimustine, trofosfamide, nitrogen mustards such as uracil mustard; carmustine, chlorozoto nitrosoureas such as cin, fotemustine, lomustine, nimustine, and ranimnustine; enediyne antibiotics (e.g., the calicheamicins, particularly calicheamicin gamma II and calicheamicin omega II (see, e.g., Agnew, Chem. Intl. Ed. Engl., 33:183-186 (1994)); the dynemicins, including dynemicin A; bisphosphonates such as clodronic acid; esperamicin;and neocarzinostatin chromophores and related chromoprotein enediyne antibiotic chromophores), aclacinomycin, actinomycin, autramycin, azaserine, bleomycin, cactinomycin, carabicin, caminomycin, carzinophilin, chromomycinis, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, ADRIAMYCIN doxorubicin (morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2 -pyrrolino-doxorubicin, and deoxydoxorubicin), mitomycins such as epirubicin, esorubicin, idarubicin, marcelomycin, and mitomycin C, antibiotics such as mycophenolic acid, nogalamycin, olivomycin, peplomycin, potfilomycin, puromycin, queramycin, rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, and zorubicin; metabolic antagonists such as methotrexate and 5-fluorouracil (5-FU); denopterin, methotrexate, folic acid analogues such as pteropterin and trimetrexate; purine analogues such as fludarabine, 6-mercaptopurine, thiamiprine, and thioguanine; pyrimidine analogues such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, and floxuridine; androgens such as calsterone, dromostanolone propionate, epithiostanol, mepitiostane, and testolactone; synthetic adrenal cortex folates such as minoglutethimide, mitotane, and trilostane. Anti-adrenal;folic acid supplements such as folinic acid;aceglatone;aldophosphamide glycosides;aminolevulinic acid;eniluracil;amsacrine;bestrabulil;bisantrene;edatraxate;demecolcine;diaziquone;elformitin;elliptinium acetate;epothilone;etoglucide;gallium nitrate;hydroxyurea;lentinan;lonidynin;maytansinoids such as maytansine and ansamitocin;mitoguazone;mitoxantrone;mopidamol;nitraelin;pentostatin;phenamet;Pirarubicin; losoxantrone; podophyllic acid; 2-ethylhydrazide; procarbazine; PSK polysaccharide complex (JHS Natural Products, Eugene, Oreg.); razoxane; rhizoxin; sizofuran; spirogermanium; tenuazonic acid; triaziquone; 2,2',2''-trichlorotriethylamine; trichothecenes (e.g., T-2 toxin, veracrine A, roridin A, and anguidine); urethane; vindesine; dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine; arabinoside ("Ara-C"); cyclophosphamide; thiotepa; taxoids, e.g., TAXOL paclitaxel (Bristol-Myers Squibb Oncology, Princeton, NJ), ABRAXANE Cremophor-free, albumin-bound nanoparticle paclitaxel formulation (American Pharmaceutical Partners, Schaumberg, 111.), and TAXOTERE docetaxel (Rhone-Poulenc Rorer,Antony,France);Chlorambucil;GEMZAR gemcitabine;6-thioguanine;Mercaptopurine;Methotrexate;Platinum analogues such as cisplatin, oxaliplatin, and carboplatin;Vinblastine;Platinum;Etoposide (VP-16);Ifosfamide;Mitoxantrone;Vincristine;NAVELBINE.Vinorelbine;Novantrone;Teniposide;Edatrexate;Daunomycin;Aminopterin;Xeloda;Ibandronate;Irinotecan (Camptosar, CPT-11) (including treatment regimens of irinotecan with 5-FU and leucovorin);Topoisomerase inhibitors RFS 2000;difluoromethylornithine (DMFO);retinoids such as retinoic acid;capecitabine;combretastatin;leucovorin (LV);oxaliplatin, including oxaliplatin treatment regimens (FOLFOX);lapatinib (Tykerb);Selected from inhibitors of PKC-α, Raf, H-Ras, EGFR (e.g., erlotinib (Tarceva) which reduces cell proliferation) and VEGF-A, and pharma- ceutically acceptable salts, acids, or derivatives of any of the above;

[0047] In some embodiments, the chemotherapeutic agent is a histone deacetylase (HDAC) inhibitor (HDI). In some embodiments, the HDI is selected from vorinostat, panobinostat, tazemetostat, mocetinostat, romidepsin, trichostatin A, and trapoxin A. In some embodiments, the therapeutic payload is as shown in FIG.

[0048] In embodiments, the anticancer agent is monomethylauristatin E (MMAE).

[0049] In embodiments, the anticancer drug is an antisense oligonucleotide that binds to kras, gemcitabine, or cisplatin.

[0050] In some embodiments, the therapeutic payload is gemcitabine, for example, as shown in FIG.

[0051] In some embodiments, the therapeutic payload is an HDAC inhibitor.

[0052] In some embodiments, the therapeutic payload is panobinostat.

[0053] In some embodiments, the therapeutic payload is tazemetostat.

[0054] In some embodiments, the drug peptide ratio is the ratio between the therapeutic payload and the internalization peptide / cellular uptake peptide. In some embodiments, the drug peptide ratio is 1, or 2, or 3, or 4, or 5, or 6, or 7, or 8, or 9, or 10, or more. In some embodiments, the 429-MMAE drug peptide ratio is 1, or 2, or 3, or 4, or 5, or 6, or 7, or 8, or 9, or 10, or more.

[0055] Organelle Uptake Peptides In embodiments, the compositions include an organelle uptake peptide that enables uptake or internalization of the composition by an organelle.

[0056] In embodiments, the organelle uptake peptide is a targeting peptide or a signal peptide.

[0057] In some embodiments, the organelle uptake peptide is a nuclear localization signal, optionally a classical nuclear localization signal. In some embodiments, the nuclear localization signal is a monopartite classical nuclear localization signal. In some embodiments, the nuclear localization signal has the amino acid sequence of PKKKRKV (SEQ ID NO: 106), or a variant thereof. In some embodiments, the nuclear localization signal is a bipartite classical nuclear localization signal. In some embodiments, the nuclear localization signal is a non-classical nuclear localization signal.

[0058] In some embodiments, the organelle uptake peptide is an endoplasmic reticulum localization signal.

[0059] Cutting site In some embodiments, the composition further comprises one or more cleavage sites (e.g., a matrix metalloproteinase or a furin cleavage site). In some embodiments, V or W or X further comprises a cleavage site.

[0060] In some embodiments, the composition further comprises one or more cleavage sites that allow for site-specific removal of more components of the composition when they are inside a cell. In some embodiments, the cleavage site is transferred between the internalization / cellular uptake peptide and the organelle uptake peptide (which is itself linked to the pharmaceutical / therapeutic payload), thereby allowing for cleavage of the internalization / cellular uptake peptide from the organelle uptake peptide and the pharmaceutical / therapeutic payload. In some embodiments, cleavage occurs via an intracellular enzyme.

[0061] In embodiments, the cleavage site is a matrix metalloproteinase cleavage site, for example, one of the following: [Table 2]

[0062] In some embodiments, the cleavage site is a matrix metalloproteinase 9 cleavage site. In some embodiments, the cleavage site has the amino acid sequence of WLPSSI (SEQ ID NO: 107).

[0063] In some embodiments, the cleavage site is a furin cleavage site, hi some embodiments, the cleavage site has the amino acid sequence of PRRARS (SEQ ID NO: 108).

[0064] Targeting Moieties / Cell Surface Receptor Targeting Peptides In embodiments, the composition comprises a targeting moiety / cell surface receptor targeting peptide.

[0065] In some embodiments, the targeting moiety / cell surface receptor targeting peptide binds to a cancer cell surface receptor or a microvascular endothelial cell surface receptor. In some embodiments, the cancer cell surface receptor is insulin-like growth factor type 1 receptor (IGF-1R), epidermal growth factor receptor (EGFR), insulin receptor, GRP78 / BiP, or transferrin receptor.

[0066] In several embodiments, the targeting moiety / cell surface receptor targeting peptide is IGFR or EGFR specific, e.g., RPWILHLGRERSTDDQTPVHRMSLMHPLYHTRGKSYKVEG (SEQ ID NO: 105) or a variant thereof), optionally linked to an MMP9 cleavage site, which is further linked to an internalization peptide, e.g., Ov8 or Skwx-429, and optionally further linked to a pharmaceutical / therapeutic payload.

[0067] In embodiments, the composition comprises: [Table 3] Includes.

[0068] In some embodiments, the composition lacks any tags (eg, FLAG tags, His6 tags, etc.).

[0069] GPNMB and drug peptide conjugates In some aspects, the present disclosure provides a composition comprising GPNMB recognition domain linked to a therapeutic payload.In some embodiments, the composition comprises 1, or 2, or 3, or 4, or 5, or 6, or 7, or 8, or 9, or 10, or more GPNMB recognition domains linked to a therapeutic payload.In some embodiments, the composition comprises a peptide, a low molecular weight protein, or an antibody (e.g., a single chain antibody) that binds to GPNMB.In some embodiments, the composition is internalized by GPNMB expressed on the surface of a cell.

[0070] In some embodiments, the single chain antibody is a single chain variable fragment (scFv). In some embodiments, the composition comprises a single chain antibody, a low molecular weight protein, or a single polypeptide that contains at least one variable binding domain (e.g., a variable domain of a mammalian heavy or light chain immunoglobulin, a camelid VHH, or a cartilaginous fish (e.g., a shark) Ig-NAR domain) that can specifically bind to an antigen, such as GPNMB. Non-limiting examples of single chain antibodies include single domain antibodies.

[0071] In some embodiments, the term "single domain antibody" refers to a polypeptide that contains one camelid VHH or at least one cartilaginous fish Ig-NAR domain capable of specifically binding to an antigen. Non-limiting examples of single domain antibodies are described, for example, in U.S. Publication No. 2010 / 0092470.

[0072] In some embodiments, the low molecular weight protein ranges in size from about 2 kDa to about 4 kDa, in some embodiments, the low molecular weight protein ranges in size from about 2 kDa to about 3.5 kDa, from about 2 kDa to about 3 kDa, or from about 2 kDa to about 2.5 kDa.

[0073] In some embodiments, the peptide or small molecular weight protein is an agonist. In some embodiments, the peptide or small molecular weight protein is an antagonist.

[0074] In some embodiments, the antibody ranges in size from about 5 kDa to about 30 kDa, from about 5 kDa to about 25 kDa, from about 5 kDa to about 20 kDa, or from about 5 kDa to about 15 kDa, in some embodiments, the antibody (e.g., a single chain antibody) is a 12 kDa fragment.

[0075] In some embodiments, the peptide or small molecular weight protein retains the biological function of an antibody. In some embodiments, the small molecular weight protein and the antibody composition are interchangeable. In some embodiments, the peptide or small molecular weight protein is derived from the binding region of an antibody. In some embodiments, the target-specific small molecular weight protein is incorporated into the binding region of an antibody (e.g., a single chain antibody).

[0076] In some embodiments, the single chain antibody is a single chain variable fragment (scFv). In some embodiments, the composition comprises a single chain antibody, a low molecular weight protein, or a single polypeptide that contains at least one variable binding domain that can specifically bind to an antigen (e.g., a variable domain of a mammalian heavy or light chain immunoglobulin, a camelid VHH, or a cartilaginous fish (e.g., a shark) Ig-NAR domain). Non-limiting examples of single chain antibodies include single domain antibodies.

[0077] In some embodiments, the composition comprises a single chain antibody having a heavy chain variable domain, or a fragment thereof. The variable domain determines the specificity of the antibody. Each variable region contains three hypervariable regions, also known as complementarity determining regions (CDRs), flanked by four relatively conserved framework regions (FRs). The three CDRs, designated CDR1, CDR2, and CDR3, contribute to the binding specificity of the antibody. In some embodiments, the single chain antibody is a chimeric antibody. In some embodiments, the single chain antibody is a humanized antibody.

[0078] In embodiments, the disclosure provides compositions that specifically bind to glycoprotein non-metastatic melanoma protein b (GPNMB), the compositions being a single chain antibody, or fragment thereof, comprising CDR1, CDR2, and CDR3, wherein CDR1 is or comprises MIHYLGWSKV (SEQ ID NO: 1), or YMGSRQNDMG (SEQ ID NO: 2), or LNRAPYFPNA (SEQ ID NO: 3), or WLSNNFWAW (SEQ ID NO: 4), or a variant thereof, and the variant is about 1, or about 2, or and CDR2 is or comprises QIKMWWAMMH (SEQ ID NO:5), or TSFWIKARKW (SEQ ID NO:6), or WASWRWFRDH (SEQ ID NO:7), or QTLKTSWWWT (SEQ ID NO:8), or a variant thereof, the variant comprising about 1, or about 2, or about 3, or about 4, or about 5 mutations, the mutations being selected from substitutions or deletions; and CDR3 is LPGGNNFNLYR SFIMTLW (SEQ ID NO: 9), or WQLGSGGLHVWVSCHMYGGVYGKTSELALTWPGMWTRTHLW (SEQ ID NO: 10), or DSPRLDSIWSMSHAMGHL (SEQ ID NO: 11), or YLVRFRLDGVVWHEVVGNVNEYRTAWASIFDWLAKFQLGS (SEQ ID NO: 12), or ILNSCNPTWTRLQFKFCPALLMNGPIFAWFLWAAWVSSAM (SEQ ID NO: 13), or SDEYRFDRSVESVWQDLVIYQSHVLWWT the single chain antibody, or fragment thereof, being or comprising TVHRYLAAWIQW (SEQ ID NO: 14), or VNWINMHSAKWMWWTHGV (SEQ ID NO: 15), or TPTLNYGNFNERDMDTVG (SEQ ID NO: 16), or a variant thereof, wherein the variant comprises about 1, or about 2, or about 3, or about 4, or about 5 mutations, wherein the mutations are selected from substitutions or deletions; or a peptide, LPGGNNFNLYRSFIMTLW (SEQ ID NO: 9), or a variant thereof;WQLGSGGLHVWVSCHMYGGVYGKTSELALTWPGMWTRTHLW (SEQ ID NO: 10), or variants thereof; DSPRLDSIWSMSHAMGHL (SEQ ID NO: 11), or variants thereof; YLVRFRLDGVVWHEVVGNVNEYRTAWASIFDWLAKFQLGS (SEQ ID NO: 12), or variants thereof; ILNSCNPTWTRLQFKFCPALLMNGPIFAWFLWAAWVSSAM (SEQ ID NO: 13), or variants thereof; CVLGDYHYLYTGSADHTLRE SLSLLTRCRFRRVIYQQQLC (SEQ ID NO: 120), or variants thereof; FTHISRFGEQHPTLVNLFYPRWQYIFIFTRNALCGQYVWD (SEQ ID NO: 17), or variants thereof; LSDFEINKRCVLGVFYYLGEGSRGHILSFFHPNVQQFRVP (SEQ ID NO: 18), or variants thereof; GDFGNRYIQLLVIVLSNGQIFVQKLLSMRPFTINGDRLRQ (SEQ ID NO: 19), or variants thereof; ASVTLFRLVPMQNAWVTV LPSFAEFVMARPIEERATWGGS (SEQ ID NO: 20), or variants thereof; WQLGSGGLHVWVSCHMYGGVYGKTSELALTWPGMWTRTHL (SEQ ID NO: 21), or variants thereof; DSTMTLLARANSHASLLNVYLNINQKVEDVFFMLFLGSPP (SEQ ID NO: 22), or variants thereof; LATWIQGSKIWSNETYSISIQVSWLSLFVMGQNLYYLLSV (SEQ ID NO: 23), or variants thereof; SDEYRFDRSVESVWQDL VIYQSHVLWWTTVHRYLAAWIQW (SEQ ID NO: 14), or variants thereof; VNWINMHSAKWMWWTHGV (SEQ ID NO: 15), or variants thereof; TPTLNYGNFNERDMDTVG (SEQ ID NO: 16), or variants thereof; QKVLYTAVPFKHGSYIFCDRIGFIFNYICEFSRSWSTWSM (SEQ ID NO: 24), or variants thereof; VGNSIQYVKAVQTQYWAAITSWIPITRFTTRVMFLLPQVM (SEQ ID NO: 25), or variants thereof;QVTARCLQIIVLYKLGNNGVTSLFHVYAMSSVPKFSVKWS (SEQ ID NO: 26), or variants thereof; GQVCSPFVTSALKLFGADFGWWSLVLMKAVSWVLYITFNA (SEQ ID NO: 27), or variants thereof; TLDHVRQAYAALSYSTVRCTFVSQMTIFYFGRVMFKALCN (SEQ ID NO: 28), or variants thereof; AYHLQLEQLRCVSQNVVTSFLDFALTKALALRSVLLGVWM (SEQ ID NO: 29), or variants thereof; QCFQDDLCVLPVASLLDHTRSMSHLFEGWLPSRSFGFRNG (SEQ ID NO: 30), or variants thereof; STAVVIWQDW FRTFRMAVVVSSARDEAVDRVCLAWPARYM (SEQ ID NO: 31), or a variant thereof; LSAADMYKHKIVGEAGEPIFYMVRNWLQKQWPMNFILMSL (SEQ ID NO: 32), or a variant thereof; FLWQWSADSMGAVDACSHKILGLFYHSQNIFARPFLRSYG (SEQ ID NO: 33), or a variant thereof; NIFIPPVGYYLTKQCGTNNFWFYQVLCRRWGADSILFWER (SEQ ID NO: 34), or a variant thereof, wherein the variant comprises about 1, or about 2, or about 3, or about 4, or about 5 mutations, wherein the mutations are selected from substitutions or deletions;

[0079] In several embodiments, the GPNMB single chain antibody comprises CDRs, wherein CDR1 is or comprises MIHYLGWSKV (SEQ ID NO: 1) or a variant thereof, CDR2 is or comprises QIKMWWAMMH (SEQ ID NO: 5) or a variant thereof, and CDR3 is or comprises LPGGNNFNLYRSFIMTLW (SEQ ID NO: 9) or a variant thereof, wherein the variant comprises about 1, or about 2, or about 3, or about 4, or about 5 mutations, wherein the mutations are selected from substitutions or deletions.

[0080] In several embodiments, the GPNMB single chain antibody comprises CDRs, wherein CDR1 is or comprises MIHYLGWSKV (SEQ ID NO: 1) or a variant thereof, CDR2 is or comprises QIKMWWAMMH (SEQ ID NO: 5) or a variant thereof, and CDR3 is or comprises WQLGSGGLHVWVSCHMYGGVYGKTSELALTWPGMWTRTHLW (SEQ ID NO: 10) or a variant thereof, wherein the variant comprises about 1, or about 2, or about 3, or about 4, or about 5 mutations, wherein the mutations are selected from substitutions or deletions.

[0081] In several embodiments, the GPNMB single chain antibody comprises CDRs, wherein CDR1 is or comprises YMGSRQNDMG (SEQ ID NO: 2) or a variant thereof, CDR2 is or comprises TSFWIKARKW (SEQ ID NO: 6) or a variant thereof, and CDR3 is or comprises DSPRLDSIWSMSHAMGHL (SEQ ID NO: 11) or a variant thereof, wherein the variant comprises about 1, or about 2, or about 3, or about 4, or about 5 mutations, wherein the mutations are selected from substitutions or deletions.

[0082] In several embodiments, the GPNMB single chain antibody comprises CDRs, wherein CDR1 is or comprises YMGSRQNDMG (SEQ ID NO: 2) or a variant thereof, CDR2 is or comprises TSFWIKARKW (SEQ ID NO: 6) or a variant thereof, and CDR3 is or comprises YLVRFRLDGVVWHEVVGNVNEYRTAWASIFDWLAKFQLGS (SEQ ID NO: 12) or a variant thereof, wherein the variant comprises about 1, or about 2, or about 3, or about 4, or about 5 mutations, wherein the mutations are selected from substitutions or deletions.

[0083] In several embodiments, the GPNMB single chain antibody comprises CDRs, wherein CDR1 is or comprises LNRAPYFPNA (SEQ ID NO: 3) or a variant thereof, CDR2 is or comprises WASWRWFRDH (SEQ ID NO: 7) or a variant thereof, and CDR3 is or comprises ILNSCNPTWTRLQFKFCPALLMNGPIFAWFLWAAWVSSAM (SEQ ID NO: 13) or a variant thereof, wherein the variant comprises about 1, or about 2, or about 3, or about 4, or about 5 mutations, wherein the mutations are selected from substitutions or deletions.

[0084] In several embodiments, the GPNMB single chain antibody comprises CDRs, wherein CDR1 is or comprises WLSNNFWAW (SEQ ID NO: 4) or a variant thereof, CDR2 is or comprises QTLKTSWWWT (SEQ ID NO: 8) or a variant thereof, and CDR3 is or comprises SDEYRFDRSVESVWQDLVIYQSHVLWWTTVHRYLAAWIQW (SEQ ID NO: 14) or a variant thereof, wherein the variant comprises about 1, or about 2, or about 3, or about 4, or about 5 mutations, wherein the mutations are selected from substitutions or deletions.

[0085] In several embodiments, the GPNMB single chain antibody comprises CDRs, wherein CDR1 is or comprises LNRAPYFPNA (SEQ ID NO: 3) or a variant thereof, CDR2 is or comprises WASWRWFRDH (SEQ ID NO: 7) or a variant thereof, and CDR3 is or comprises VNWINMHSAKWMWWTHGV (SEQ ID NO: 15) or a variant thereof, wherein the variant comprises about 1, or about 2, or about 3, or about 4, or about 5 mutations, wherein the mutations are selected from substitutions or deletions.

[0086] In several embodiments, the GPNMB single chain antibody comprises CDRs, wherein CDR1 is or comprises WLSNNFWAW (SEQ ID NO: 4) or a variant thereof, CDR2 is or comprises QTLKTSWWWT (SEQ ID NO: 8) or a variant thereof, and CDR3 is or comprises TPTLNYGNFNERDMDTVG (SEQ ID NO: 16) or a variant thereof, wherein the variant comprises about 1, or about 2, or about 3, or about 4, or about 5 mutations, wherein the mutations are selected from substitutions or deletions.

[0087] In some embodiments, the GPNMB single chain antibody, or fragment thereof, further comprises variable region framework (FW) sequences juxtaposed between the CDRs by the formula (FW1)-(CDR1)-(FW2)-(CDR2)-(FW3)-(CDR3)-(FW4), wherein the variable region FW sequence in the heavy chain variable region is a heavy chain variable region FW sequence and the variable region FW sequence in the light chain variable region is a light chain variable region FW sequence.

[0088] In some embodiments, the variable region FW sequences are human variable region FW sequences.

[0089] In several embodiments, the GPNMB single chain antibody comprises the amino acid sequence MAQVQLVESGGGVVQPGRSLRLSCAASMIHYLGWSKVWFRQAPGKEREFVAQIKMWWAMMHYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARLPGGNNFNLYRSFIMTLWWGQGTLVTVSSGPGGQ (SEQ ID NO: 35), or an amino acid sequence having at least about 90%, or at least about 95%, or at least about 97%, or at least about 98%, or at least about 99% identity thereto.

[0090] In several embodiments, the GPNMB single chain antibody comprises an amino acid sequence of MAQVQLVESGGGVVQPGRSLRLSCAASMIHYLGWSKVWFRQAPGKEREFVAQIKMWWAMMHYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARWQLGSGGLHVWVSCHMYGGVYGKTSELALTWPGMWTRTHLWWGQGTLVTVSSGPGGQ (SEQ ID NO: 36), or an amino acid sequence having at least about 90%, or at least about 95%, or at least about 97%, or at least about 98%, or at least about 99% identity thereto.

[0091] In several embodiments, the GPNMB single chain antibody comprises an amino acid sequence of MAQVQLVESGGGVVQPGRSLRLSCAASYMGSRQNDMGWFRQAPGKEREFVATSFWIKARKWYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDSPRLDSIWSMSHAMGHLWGQGTLVTVSSGPGGQ (SEQ ID NO: 37), or an amino acid sequence having at least about 90%, or at least about 95%, or at least about 97%, or at least about 98%, or at least about 99% identity thereto.

[0092] In several embodiments, the GPNMB single chain antibody comprises an amino acid sequence of MAQVQLVESGGGVVQPGRSLRLSCAASYMGSRQNDMGWFRQAPGKEREFVATSFWIKARKWYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARYLVRFRLDGVVWHEVVGNVNEYRTAWASIFDWLAKFQLGSWGQGTLVTVSSGPGGQ (SEQ ID NO: 38), or an amino acid sequence having at least about 90%, or at least about 95%, or at least about 97%, or at least about 98%, or at least about 99% identity thereto.

[0093] In several embodiments, the GPNMB single chain antibody comprises an amino acid sequence of MQVQLVESGGGVVQPGRSLRLSCAASLNRAPYFPNAWFRQAPGKEREFVAWASWRWFRDHYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARILNSCNPTWTRLQFKFCPALLMNGPIFAWFLWAAWVSSAMWGQGTLVTVSSGPGGQ (SEQ ID NO: 39), or an amino acid sequence having at least about 90%, or at least about 95%, or at least about 97%, or at least about 98%, or at least about 99% identity thereto.

[0094] In several embodiments, the GPNMB single chain antibody comprises an amino acid sequence of MQVQLVESGGGVVQPGRSLRLSCAASSWLSNNFWAWWFRQAPGKEREFVAQTLKTSWWWTYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARSDEYRFDRSVESVWQDLVIYQSHVLWWTTVHRYLAAWIQWWGQGTLVTVSSGPGGQ (SEQ ID NO: 40), or an amino acid sequence having at least about 90%, or at least about 95%, or at least about 97%, or at least about 98%, or at least about 99% identity thereto.

[0095] In several embodiments, the GPNMB single chain antibody comprises an amino acid sequence of MQVQLVESGGGVVQPGRSLRLSCAASLNRAPYFPNAWFRQAPGKEREFVAWASWRWFRDHYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARVNWINMHSAKWMWWTHGVWGQGTLVTVSSGPGGQ (SEQ ID NO: 41), or an amino acid sequence having at least about 90%, or at least about 95%, or at least about 97%, or at least about 98%, or at least about 99% identity thereto.

[0096] In several embodiments, the GPNMB single chain antibody comprises the amino acid sequence MQVQLVESGGGVVQPGRSLRLSCAASSWLSNNFWAWWFRQAPGKEREFVAQTLKTSWWWTYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARTPTLNYGNFNERDMDTVGWGQGTLVTVSSGPGGQ (SEQ ID NO: 42), or an amino acid sequence having at least about 90%, or at least about 95%, or at least about 97%, or at least about 98%, or at least about 99% identity thereto.

[0097] In some embodiments, the therapeutic payload is an anti-cancer drug.

[0098] In some embodiments, the therapeutic payload is a small molecule drug, a biologic, or a radionuclide.

[0099] In several embodiments, the therapeutic payload is a biologic selected from a toxin, an antibody, or an oligonucleotide (such as, but not limited to, an antisense inhibitor).

[0100] In some embodiments, the therapeutic payload is monomethylauristatin E (MMAE).

[0101] In some embodiments, the therapeutic payload is a histone deacetylase (HDAC) inhibitor, hi some embodiments, the HDAC inhibitor is selected from vorinostat, panobinostat, tazemetostat, mocetinostat, romidepsin, trichostatin A, and trapoxin A.

[0102] In embodiments, the therapeutic payload is gemcitabine, cisplatin, or an antisense oligonucleotide that binds to kras.

[0103] In some embodiments, the therapeutic payload is a SmarcB1 mimetic.

[0104] In some embodiments, the therapeutic payload is an anti-cancer drug, hi some embodiments, the anti-cancer drug is a chemotherapeutic agent.

[0105] In some embodiments, the chemotherapeutic agent is an alkylating agent such as thiotepa and CYTOXAN cyclophosphamide; an alkyl sulfonate such as busulfan, improsulfan, and piposulfan; an aziridine such as benzodopa, carboquone, meturedopa, and uredopa; an ethylenimine and a methylamelamine, including altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide, and trimethylolomelamine; an acetogenin (e.g., bullatacin and bullatacinone); a camptothecin (including the synthetic analog topotecan); a bryostatin; a caIIy statin; a CC-1065 (including its adozelesin, carzelesin, and bizelesin synthetic analogs); a cryptophycin (e.g., cryptophycin 1 and cryptophycin 8); a dolastatin; a duocarmycin (including the synthetic analogs KW-2189 and CB 1-TM1); erytherobin; pancratistatin; sarcodictyin; spongiostatin; chlorambucil, chlornaphazine, cholophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembitine, phenesterine, prednimustine, trofosfamide, nitrogen mustards such as uracil mustard; carmustine, chlorozoto nitrosoureas such as cin, fotemustine, lomustine, nimustine, and ranimnustine; enediyne antibiotics (e.g., the calicheamicins, particularly calicheamicin gamma II and calicheamicin omega II (see, e.g., Agnew, Chem. Intl. Ed. Engl., 33:183-186 (1994)); the dynemicins, including dynemicin A; bisphosphonates such as clodronic acid; esperamicin;and neocarzinostatin chromophores and related chromoprotein enediyne antibiotic chromophores), aclacinomycin, actinomycin, autramycin, azaserine, bleomycin, cactinomycin, carabicin, caminomycin, carzinophilin, chromomycinis, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, ADRIAMYCIN doxorubicin (morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2 -pyrrolino-doxorubicin, and deoxydoxorubicin), mitomycins such as epirubicin, esorubicin, idarubicin, marcelomycin, and mitomycin C, antibiotics such as mycophenolic acid, nogalamycin, olivomycin, peplomycin, potofilomycin, puromycin, queramycin, rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, and zorubicin; antimetabolites such as methotrexate and 5-fluorouracil (5-FU); denopterin, methotrexate, puromycin, and the like; folic acid analogues such as teropterin and trimetrexate; purine analogues such as fludarabine, 6-mercaptopurine, thiamiprine and thioguanine; pyrimidine analogues such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine and floxuridine; androgens such as calsterone, dromostanolone propionate, epithiostanol, mepitiostane and testolactone; adrenal cortex synthetic hormones such as minoglutethimide, mitotane and trilostane. Anti-inflammatory drugs;Folic acid supplements such as folinic acid;Aceglatone;Aldophosphamide glycosides;Aminolevulinic acid;Eniluracil;Amsacrine;Vestravcil;Bisantrene;Edatraxate;Demecolcine;Diaziquone;Elformitin;Elliptinium acetate;Epothilone;Etoglucide;Gallium nitrate;Hydroxyurea;Lentinan;Lonidynin;Maytansinoids such as maytansine and ansamitocin;Mitoguazone;Mitoxantrone;Mopidammol;Nitraerin;Pentostatin;Fenamet;Pirarubicin;Rosoxantrone;Podophyllic acid; 2-ethylhydrazide; procarbazine; PSK polysaccharide complex (JHS Natural Products, Eugene, Oreg.); razoxane; rhizoxin; sizofuran; spirogermanium; tenuazonic acid; triaziquone; 2,2',2''-trichlorotriethylamine; trichothecenes (e.g., T-2 toxin, veracrine A, roridin A, and anguidine); urethane; vindesine; dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine; arabinoside ("Ara-C"); cyclophosphamide; thiotepa; taxoids, such as TAXOL paclitaxel (Bristol-Myers Squibb Oncology, Princeton, NJ), ABRAXANE Cremophor-free, albumin-bound paclitaxel nanoparticle formulation (American Pharmaceutical Partners, Schaumberg, 111.), and TAXOTERE docetaxel (Rhone-Poulenc Rorer, Antony, France); chlorambucil; GEMZAR gemcitabine; 6-thioguanine; mercaptopurine; methotrexate; platinum analogs such as cisplatin, oxaliplatin, and carboplatin; vinblastine; platinum; etoposide (VP-16); ifosfamide; mitoxantrone; vincristine; NAVELBINE vinorelbine; novantrone; teniposide; edatrexate; daunomycin; aminopterin; xeloda; ibandronate; irinotecan (Camptosar, CPT-11) (including treatment regimens of irinotecan in conjunction with 5-FU and leucovorin); topoisomerase inhibitors RFS 2000;difluoromethylornithine (DMFO);retinoids such as retinoic acid;capecitabine;combretastatin;leucovorin (LV);oxaliplatin, including oxaliplatin treatment regimens (FOLFOX);lapatinib (Tykerb);Selected from inhibitors of PKC-α, Raf, H-Ras, EGFR (e.g., erlotinib (Tarceva) which reduces cell proliferation) and VEGF-A, and pharma- ceutically acceptable salts, acids, or derivatives of any of the above;

[0106] Linker In some embodiments, the composition comprises one or more linkers.

[0107] In some embodiments, the linker is of the type used in ADCs, see, for example, Su, et al. Acta Pharmaceutica Sinica B, Volume 11, Issue 12, December 2021, Pages 3889-390, the entire contents of which are incorporated herein by reference. In some embodiments, the linker is a cleavable linker. In some embodiments, the linker has a chemical trigger in its structure that can be cleaved to release the components of the composition. In some embodiments, the linker is non-cleavable.

[0108] In some embodiments, the linker is a valine-citrulline (Val-Cit) linker. In some embodiments, the linker is mc-Val-Cit. In some embodiments, the linker is MC-Val-Cit-PAB. In some embodiments, the linker is MC-Val-Cit-PAB-PNP. In some embodiments, the linker is as shown in FIG. 1. In some embodiments, the linker is as shown in FIG. 12. In some embodiments, the linker is a triglycyl peptide linker (CX).

[0109] In some embodiments, the linker is a flexible linker. In some embodiments, the linker consists essentially of glycine and serine residues. In some embodiments, the linker is a (GGS) n where: nis 1, or 2, or 3, or 4, or 5. In embodiments, the linker is GGSGGSGGSG (SEQ ID NO: 118), or a variant thereof, wherein the variant comprises about 1, or about 2, or about 3, or about 4, or about 5 mutations, wherein the mutations are selected from substitutions or deletions.

[0110] In some embodiments, the linker further comprises a cleavage site, in some embodiments, the cleavage site is a matrix metalloproteinase cleavage site, optionally a matrix metalloproteinase 9 cleavage site, optionally having the amino acid sequence of WLPSSI (SEQ ID NO: 107), or a furin cleavage site, optionally having the amino acid sequence of PRRARS (SEQ ID NO: 108).

[0111] Exemplary Constructs In several embodiments, the composition comprises the amino acid sequence DFYGCLLDLSLGVPSLGWRRRCITADYKDDDDKWLPSSIPKKKRKVDGQKLRDAFTWNMNEKLMTPEMFSEILCDDLD (SEQ ID NO: 110), or an amino acid sequence having at least about 90%, or at least about 95%, or at least about 97%, or at least about 98%, or at least about 99% identity thereto.

[0112] In several embodiments, the composition comprises the amino acid sequence DFYGCLLDLSLGVPSLGWRRRCITAWLPSSIPKKKRKVDGQKLRDAFTWNMNEKLMTPEMFSEILCDDLD (SEQ ID NO: 111), or an amino acid sequence having at least about 90%, or at least about 95%, or at least about 97%, or at least about 98%, or at least about 99% identity thereto.

[0113] In several embodiments, the composition comprises the amino acid sequence DFYGCLLDLSLGVPSLGWRRRCITAWLPSSIPKKKRKVDGQKLRDAFTWNMNEKLMTPEMFSEILADDLD (SEQ ID NO: 121), or an amino acid sequence having at least about 90%, or at least about 95%, or at least about 97%, or at least about 98%, or at least about 99% identity thereto.

[0114] In several embodiments, the composition comprises the amino acid sequence of QVQLVESGGGVVQPGRSLRLSCAASGGGGSGGGGSWFRQAPGKEREFVAGGGGSGGGGSYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDFYGCLLDLSLGVPSLGWRRRCITAWGQGLVTVSSGPGGQWLPSSIPKKKRKVDGQKLRDAFTWNMNEKLMTPEMFSEILCDDLD (SEQ ID NO: 112), or an amino acid sequence having at least about 90%, or at least about 95%, or at least about 97%, or at least about 98%, or at least about 99% identity thereto.

[0115] In some embodiments, the compositions are suitable for administration with one or more additional therapeutic agents, hi some embodiments, the additional agent is tazemetostat.

[0116] In some embodiments, the compositions are suitable for administration with one or more additional therapies. In some embodiments, other therapies that may be used in combination with the compositions provided herein include, for example, radiation therapy, chemotherapy, hormone therapy, and the use of angiogenesis inhibitors. Additional combination partners that may be useful include checkpoint inhibitors (e.g., anti-PD1 / L1, anti-CTLA-4, anti-LAG3, anti-B7-H3, anti-B7-H4, anti-TIM3, anti-TIGIT, anti-CD47, anti-TMIGD2, anti-BTLA, anti-CEACAM, or anti-GARP), other costimulatory antibodies (e.g., anti-OX40, anti-ICOS, anti-CD137, anti-GITR, or anti-CD40), cancer vaccines (e.g., viral-based vaccines, peptide vaccines, whole cell vaccines, or RNA-based vaccines), and targeted agents (e.g., HERCEPTIN® (trastuzumab), TARCEVA® (erlotinib), AVASTIN® (bevacizumab), or IMBRUVICA® (ibrutinib)).

[0117] variant In embodiments, a variant as used herein has or comprises about 1, or about 2, or about 3, or about 4, or about 5, or about 6, or about 7, or about 8, or about 9, or about 10 mutations. In embodiments, the mutations are selected from substitutions or deletions.

[0118] In embodiments, a variant as used herein has at least about 90%, or at least about 93%, or at least about 95%, or at least about 97%, or at least about 98% identity to the sequences described herein.

[0119] In embodiments, the sequences described herein or at least 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 99.5, 99.8, 99.9% identity to the sequences described herein (or at least about 70% identity to the sequences described herein, or at least Disclosed herein are one or more (e.g., about 1, or about 2, or about 3, or about 4, or about 5, or about 6, or about 7, or about 8, or about 9, or about 10, or about 15, or about 20, or about 30) substitutions to a sequence having about 75%, or at least about 80%, or at least about 85%, or at least about 90%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99% identity).In embodiments, one or more amino acids of the sequences described herein are naturally occurring amino acids, e.g., hydrophilic amino acids (e.g., positively charged polar hydrophilic amino acids, e.g., arginine (R) or lysine (K); polar and charge neutral hydrophilic amino acids, e.g., asparagine (N), glutamine (Q), serine (S), threonine (T), proline (P), and cysteine ​​(C); negatively charged polar hydrophilic amino acids, e.g., aspartic acid (D) or glutamic acid (E); or aromatic positively charged polar hydrophilic amino acids, e.g., histidine (H)), or hydrophobic amino acids (e.g., hydrophobic aliphatic amino acids, e.g., glycine (G), alanine (A), leucine (L), isoleucine (I), methionine (M), or valine (V); hydrophobic aromatic amino acids, e.g., phenylalanine (V), (F), tryptophan (W), or tyrosine (Y), or non-classical amino acids (e.g., selenocysteine, pyrrolidine, N-formylmethionine, β-alanine, GABA and δ-aminolevulinic acid, 4-aminobenzoic acid (PABA), D-isomers of common amino acids, 2,4-diaminobutyric acid, α-aminoisobutyric acid, 4-aminobutyric acid, Abu, 2-aminobutyric acid, γ-Abu, ε-Ahx, 6-aminohexanoic acid, Aib, 2-aminoisobutyric acid, 3-aminopropionic acid, ornithine, norleucine, norvaline, hydroxyproline, sarcosme, citrulline, homocitrulline, cysteic acid, t-butylglycine, t-butylalanine, phenylglycine, cyclohexylalanine, β-alanine, fluoro-amino acids, designer amino acids, e.g., β-methylamino acids, C α-methyl amino acids, N α-methyl amino acids, and amino acid analogs in general).

[0120] In some embodiments, the amino acid mutation is an amino acid substitution, which may include conservative and / or non-conservative substitutions. "Conservative substitutions" may be made, for example, based on similarity in polarity, charge, size, solubility, hydrophobicity, hydrophilicity, and / or amphipathic nature of the amino acid residues involved. The 20 naturally occurring amino acids may be grouped into the following six standard amino acid groups: (1) hydrophobic: Met, Ala, Val, Leu, Ile; (2) neutral hydrophilic: Cys, Ser, Thr; Asn, Gln; (3) acidic: Asp, Glu; (4) basic: His, Lys, Arg; (5) residues that affect chain orientation: Gly, Pro; and (6) aromatic: Trp, Tyr, Phe. As used herein, a "conservative substitution" is the exchange of one amino acid with another amino acid listed in the same group of the six standard amino acid groups shown above. For example, the exchange of Asp with Glu retains one negative charge in the so modified polypeptide. Additionally, glycine and proline may be substituted for each other based on their ability to disrupt an α-helix. As used herein, a "non-conservative substitution" is the exchange of one amino acid with another amino acid listed in a different group of the six standard amino acid groups (1) to (6) shown above.

[0121] Polynucleotides, Vectors, and Host Cells In some embodiments, the disclosure provides a composition comprising a polynucleotide comprising a nucleic acid sequence encoding any one of the amino acids, peptides, antibodies, bispecific molecules, and / or compositions described herein. In some embodiments, the polynucleotide is RNA or DNA. In some embodiments, the RNA is messenger RNA (mRNA) or modified mRNA.

[0122] In embodiments, the disclosure provides compositions, including vector compositions, comprising any one of the polynucleotides described herein.

[0123] In some embodiments, the present disclosure provides compositions comprising host cells comprising the polynucleotides described herein or the vectors described herein. The cells may be, for example, cultured in vitro or genetically engineered. The host cells may be obtained from normal or diseased subjects, including healthy humans, from private research deposits, from public culture collections such as the American Type Culture Collection, or from commercial suppliers.

[0124] In embodiments, the present disclosure provides a pharmaceutical composition comprising a composition described herein and a pharma- ceutically acceptable excipient or carrier.

[0125] Furthermore, the present disclosure also provides pharmaceutical compositions comprising the compositions described herein in combination with a pharma- ceutically acceptable carrier. A "pharma-ceutically acceptable carrier" (also called an "excipient" or "carrier") is a pharma-ceutically acceptable solvent, suspending agent, stabilizer, or any other pharmacologically inert vehicle for delivering one or more therapeutic compounds to a subject (e.g., a mammal such as a human, non-human primate, dog, cat, sheep, pig, horse, cow, mouse, rat, or rabbit) that is non-toxic to cells or subjects exposed thereto at the dosage and concentration used. A pharma-ceutically acceptable carrier may be liquid or solid, and may be selected with the proposed mode of administration in mind to provide the desired bulk, consistency, and other relevant transport and chemical properties when combined with one or more of the therapeutic compounds and any other components of a given pharmaceutical composition. Typical pharma- ceutically acceptable carriers that do not adversely react with amino acids include, by way of example and not limitation, water, saline, binders (e.g., polyvinylpyrrolidone or hydroxypropylmethylcellulose), fillers (e.g., lactose and other sugars, gelatin, or calcium sulfate), lubricants (e.g., starch, polyethylene glycol, or sodium acetate), disintegrants (e.g., starch or sodium starch glycolate), and wetting agents (e.g., sodium lauryl sulfate). Pharmaceutically acceptable carriers also include pH buffered aqueous solutions or liposomes (small vesicles composed of various types of lipids, phospholipids, and / or surfactants that are useful for delivery of drugs to mammals).Further examples of pharma- ceutically acceptable carriers include buffers such as phosphate, citrate, and other organic acids, antioxidants such as ascorbic acid, proteins such as low molecular weight (less than about 10 residues) polypeptides, serum albumin, gelatin, or immunoglobulins, hydrophilic polymers such as polyvinylpyrrolidone, amino acids such as glycine, glutamine, asparagine, arginine, or lysine, monosaccharides, disaccharides, and other carbohydrates, including glucose, mannose, or dextrins, chelating agents such as EDTA, sugar alcohols such as mannitol or sorbitol, salt-forming counterions such as sodium, and / or non-ionic surfactants such as TWEEN™, polyethylene glycol (PEG), and PLURONICS™.

[0126] Pharmaceutical compositions can be formulated by mixing one or more active agents with one or more physiologically acceptable carriers, diluents, and / or auxiliaries, and optionally other agents that are normally incorporated into formulations to provide improved transfer, delivery, tolerance, etc. Pharmaceutical compositions can be formulated, for example, into lyophilized formulations, aqueous solutions, dispersions, or solid preparations such as tablets, dragees, or capsules. Numerous suitable formulations can be found in the formulary known to all medicinal chemists: Remington's Pharmaceutical Sciences (18th ed., Mack Publishing Company, Easton, PA (1990)), in particular Chapter 87 therein by Block, Lawrence. These formulations include, for example, powders, pastes, ointments, jellies, waxes, oils, lipids, lipid (cationic or anionic)-containing vesicles (such as LIPOFECTIN™), DNA conjugates, anhydrous absorbent pastes, oil-in-water and water-in-oil emulsions, emulsions carbowax (polyethylene glycols of various molecular weights), semi-solid gels, and semi-solid mixtures containing carbowax. Any of the above mixtures may be suitable for the treatments and therapies described herein, so long as the active agent in the formulation is not inactivated by the formulation and the formulation is physiologically compatible and tolerable with the route of administration. For additional information relating to formulations, excipients, and carriers well known to medicinal chemists, see also Baldrick, Regul Toxicol Pharmacol 32:210-218, 2000; Wang, Int J Pharm 203:1-60, 2000; Charman J Pharm Sci 89:967-978, 2000, and Powell et al. PDA J Pharm Sci Technol 52:238-311, 1998), and citations therein.

[0127] Pharmaceutical compositions include, but are not limited to, solutions, emulsions, aqueous suspensions, and liposome-containing formulations. These compositions can be produced from a variety of components, including, for example, preformed liquids, self-emulsifying solids, and self-emulsifying semisolids. Emulsions are often biphasic systems that are composed of two immiscible liquid phases that are intimately mixed and dispersed with each other. In general, emulsions are either water-in-oil (w / o) or oil-in-water (o / w) types. Emulsion formulations are widely used for oral delivery of therapeutic agents due to their ease of formulation and the effectiveness of solubilization, absorption, and bioavailability.

[0128] The compositions and formulations may contain sterile aqueous solutions, which may also contain buffers, diluents, and other suitable additives (e.g., penetration enhancers, carrier compounds, and other pharma- ceutically acceptable carriers). The compositions may additionally contain other auxiliary components conventionally found in pharmaceutical compositions. Thus, the compositions may also contain compatible pharma- ceutical active substances, such as, for example, antipruritic agents, astringents, local anesthetics, or anti-inflammatory agents, or additional substances useful in physically formulating various dosage forms of the compositions provided herein, such as dyes, flavoring agents, preservatives, antioxidants, opacifiers, thickeners, and stabilizers. In addition, the compositions may be mixed with auxiliary substances, such as, for example, lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure, buffers, coloring agents, flavoring agents, and aromatic substances. However, when added, such substances should not unduly interfere with the biological activity of the polypeptide components in the compositions provided herein. The formulations may be sterilized if desired.

[0129] In some embodiments, the compositions provided herein may be in the form of a solution or powder, with or without a diluent to create an injectable suspension. The compositions may contain additional components, including, but not limited to, a medicament acceptable vehicle, such as saline, water, lactic acid, mannitol, or a combination thereof.

[0130] Any suitable method can be used to administer the compositions described herein to a mammal. Administration can be, for example, parenteral (e.g., by subcutaneous, intrathecal, intracerebroventricular, intramuscular, or intraperitoneal injection, or by intravenous drip). Administration can be rapid (e.g., by injection) or occur over a period of time (e.g., by slow infusion or administration of a sustained release formulation). In some embodiments, administration can be topical (e.g., transdermal, sublingual, ophthalmic, or intranasal), pulmonary (e.g., by inhalation or insufflation of a powder or aerosol), or oral.

[0131] How to use In several aspects, methods are provided for delivering a pharmaceutical / therapeutic payload to a cell by internalization, the methods comprising contacting a cell in vitro, ex vivo, or in vivo with a composition comprising a pharmaceutical / therapeutic payload linked to an internalization / cellular uptake peptide, the composition optionally comprising one or more cleavage sites that allow for site-specific removal of more components of the composition when they are inside the cell.

[0132] In several aspects, methods for organelle-specific delivery of a pharmaceutical / therapeutic payload are provided, comprising contacting a cell in vitro, ex vivo, or in vivo with a composition comprising a pharmaceutical / therapeutic payload linked to an internalization / cellular uptake peptide and an organelle uptake peptide, the composition optionally comprising one or more cleavage sites that allow for site-specific removal of more components of the composition when they are inside the cell.

[0133] In several embodiments, a method for delivering a pharmaceutical / therapeutic payload to a cell by internalization is provided, the method comprising contacting a cell in vitro, ex vivo, or in vivo with a composition comprising a pharmaceutical / therapeutic payload linked to a GPNMB recognition domain, the composition optionally comprising one or more cleavage sites that allow site-specific removal of more components of the composition when they are inside the cell.

[0134] In some aspects and embodiments, the compositions of the present disclosure bind to GPNMB expressed on cells. In some embodiments, the compositions of the present disclosure reduce, inhibit, or prevent the expression of GPNMB on cells, optionally cancer stem cells. In some embodiments, the cells are cancer stem cells or metastatic tumor cells.

[0135] In embodiments, the compositions of the present disclosure inhibit the expression of GPNMB on cancer cells, thereby preventing tumors from metastasizing and / or growing.

[0136] In embodiments, the compositions of the present disclosure reduce, inhibit, or prevent tumor invasion, metastasis, or angiogenesis in triple-negative breast cancer.

[0137] In embodiments, the present disclosure provides a method for inhibiting or reducing tumor growth and / or inducing or increasing immune tolerance, the method comprising administering an effective amount of a composition of any one of the embodiments to a patient in need thereof, or contacting a cell with a composition of any one of the embodiments (e.g., ex vivo, using allogeneic or autologous cells).

[0138] In some aspects, a method for treating or preventing disease is provided, comprising administering an effective amount of the composition or its pharmaceutical composition to a patient in need thereof, wherein the disease is treatable by targeting cancer cell surface receptor or microvascular endothelial cell surface receptor.In some embodiments, the disease is treatable by targeting tumor, optionally atypical teratoid rhabdoid tumor.

[0139] In aspects, methods are provided for treating or preventing cancer. In embodiments, the cancer is a solid tumor or a hematological cancer.

[0140] Tumor cells, or cancer cells, refer to the uncontrolled proliferation of cells or tissues and / or abnormal increase in cell survival and / or inhibition of apoptosis that prevents the body's organs and systems from functioning normally. For example, tumor cells include benign and malignant cancers, polyps, hyperplasias, and dormant tumors or micrometastases.

[0141] In some embodiments, the cancers include basal cell carcinoma, biliary tract cancer; bladder cancer; bone cancer; brain and central nervous system cancer; breast cancer; cancer of the peritoneum; cervical cancer; choriocarcinoma; colon and rectal cancer; connective tissue cancer; digestive system cancer; endometrial cancer; esophageal cancer; eye cancer; head and neck cancer; gastric cancer (including gastrointestinal cancer); glioblastoma; liver cancer; hepatocellular carcinoma; intraepithelial neoplasia; kidney cancer or renal cancer. cancer);larynx cancer;leukemia;liver cancer;lung cancer (e.g., small cell lung cancer, non-small cell lung cancer, adenocarcinoma of the lung, and squamous cell carcinoma of the lung);melanoma;myeloma;neuroblastoma;oral cancer (lips, tongue, mouth, and pharynx);ovarian cancer;pancreatic cancer;prostate cancer;retinoblastoma;rhabdomyosarcoma;rectal cancer;respiratory system cancer;salivary gland cancer;sarcoma;skin cancer;squamous cell carcinoma;gastric cancer;testicular cancer;thyroid cancer;uterine or endometrial cancer;urinary system cancer;vulvar cancer;Hodgkin's and non-Hodgkin's lymphomas, and B-cell lymphomas (low-grade / follicular non-Hodgkin's lymphoma (NHL);small lymphocytic (SL) NHL;intermediate-grade / follicular NHL;intermediate-grade and lymphomas, including diffuse NHL; high-grade immunoblastic NHL; high-grade lymphoblastic NHL; high-grade small noncleaved cell NHL; bulky disease NHL; mantle cell lymphoma; AIDS-related lymphoma; and Waldenstrom's macroglobulinemia; chronic lymphocytic leukemia (CLL); acute lymphoblastic leukemia (ALL); hairy cell leukemia; chronic myeloblastic leukemia; and other carcinomas and sarcomas; and post-transplant lymphoproliferative disorder (PTLD), as well as abnormal blood vessel proliferation associated with nematoses, edema (e.g., associated with brain tumors), and Meigs' syndrome.

[0142] In embodiments, the disease is treatable by targeting the pediatric tumor.

[0143] In embodiments, the disease is treatable by targeting nervous system tumors.

[0144] In embodiments, the disease is treatable by targeting pediatric nervous system tumors.

[0145] In embodiments, the compositions are administered using convection-enhanced delivery.

[0146] kit The present disclosure also provides kits comprising the subject composition, or the subject composition and one or more other additives, in one or more containers.

[0147] Kits containing the pharmaceutical compositions of the invention are also provided in several embodiments.

[0148] As used herein, the word "comprising" and variations thereof are intended to be open-ended, such that the recitation of items in a list does not exclude other similar items that may also be useful in the materials, compositions, devices, and methods of the present technology. Similarly, the terms "can" and "may" and variations thereof are intended to be open-ended, such that a recitation that an embodiment can or may include certain elements or features does not exclude other embodiments of the present technology that do not contain those elements or features. Although the open-ended term "comprising" is used herein to describe and claim the present disclosure as a synonym for terms such as including, containing, or having, the present technology, or embodiments thereof, may alternatively be described using more restrictive terms such as "consisting of" or "consisting essentially of" the listed components.

[0149] Unless otherwise defined, all technical and scientific terms in this specification have the same meaning as commonly understood by those skilled in the art to which this disclosure belongs. Although any method and material similar or equivalent to the methods and materials described herein can be used in the practice or testing of this disclosure, the preferred methods and materials are described herein. All publications, patents, and patent publications cited herein are incorporated by reference in their entirety for all purposes.

[0150] The present disclosure is further illustrated by the following non-limiting examples. EXAMPLES

[0151] Example 1: Peptide discovery and biological assays Peptide Discovery Cell-penetrating peptides have been discovered based on determining their ability to be internalized into cells using a variety of assays depending on whether the target is known or unknown.

[0152] Biological assays The biological activity of the peptides was determined using various assays. To determine proliferation activity, cells were plated at 5 × 10 per well in 96-well microtiter plates. 3 Cells were plated onto the plate. Samples and controls were added and plates were incubated at 37°C for 24, 48, and 72 hours. Viability was determined by adding WST-1 and reading at 450 nM for 1-4 hours. Alternatively, viability was determined using kits such as the EZViable Calcein AM Cell Viability Assay (BioVision) or the Live / Dead Cell Imaging Kit (ThermoFisher). Induction of cellular events such as apoptosis was determined using reagents such as the Cell Event Caspase 3 / 7 Reagent (ThermoFisher).

[0153] FIG. 1 shows the sequences of 429 (an IGF-1r peptide) and OV-8 (isolated from Ovcar-3 cells) used herein.

[0154] Figure 2 shows the internalization of OV-8 in various cell lines. Skwx-OV-8 has been shown to be internalized in several cancer cell lines of different lineages.

[0155] FIG. 3 shows dose response curves for various cell lines with peptide drug conjugates ("PDC"): (1) A549: human lung cancer, (2) SW480: human colorectal cancer, (3) HCT116: human colorectal cancer, and (4) G292: human osteosarcoma. On day 0, cells were plated at 5000 cells / well in 100 ul of D10. 100 ul of PDC was added and titrated 1:1 in D10 (starting concentration=500 nM). At 72 hours, WST-1 was added and plates were read in a Promega GloMax at 450 nM for 5 hours.

[0156] Figure 4 shows phase contrast images of HCT116 with 500 nM vehicle control, peptide 429 without MMAE, and test samples, demonstrating that the peptide-drug conjugates kill tumor cells.

[0157] Figure 5 shows the effect of PDC on G292 cells. Cells were plated as in Figure 3 and treated with calcein AM for 1-2 hours at 37°C.

[0158] Figure 6 shows that MAE and PDC disrupt microtubules. Cells were stained with ViaFluor 647 live cell microtubule dye (Biotium). Cells were treated as in Figure 3 and stained with ViaFluor for 60 minutes at 37°C.

[0159] FIG. 9 shows the nuclear localization of the 429 construct in three cell lines (G292 [osteosarcoma], U87MG [glioblastoma], and HCT116 [colorectal cancer]).

[0160] Figure 10 shows nuclear staining detected by anti-FLAG antibody. As can be seen, the construct does not appear to be cleaved but still accumulates in the nucleus of U87 cells, i.e., the entire 429 construct may localize to the nucleus.

[0161] Figure 11 shows immunofluorescence data demonstrating that the entire 429 construct was localized to the nucleus. Cells were stained and visualized with 429-MMP9-NLS-FITC for 3-4 hours at 37°C.

[0162] Example 2: Identification of drug conjugates for proof-of-concept animal studies To deliver drugs or biological therapeutics into cells, pocket biologics (peptides or antibodies) with internalization moieties, linkers, and localization signals were developed. Drugs or biological therapeutics are conjugated to the localization signal and delivered to the desired site by the nuclear localization signal. 429-MMAE-1 and 429-NLS-panobinostat constructs were developed for in vivo studies. G292 and A431 cell lines were used in the in vivo studies. G292 cells were derived from a primary osteosarcoma of a 9-year-old Caucasian female. The cells exhibit a B-type phenotype for G6PD and the karyotype is subtetraploid with chromosomal markers. A431 cells were established from an epidermoid carcinoma of the skin (epidermis) of an 85-year-old female patient. Epidermal growth factor (EGF) stimulation of A431 cells induces rapid tyrosine phosphorylation of intracellular signaling proteins that control cellular processes such as growth, proliferation, and apoptosis. 429-MMAE had effects in various cell lines (Figures 16A-22B). 429-MMAE affected cell viability in G292 cell lines (Figures 16A-17). 50 nm, 125 nm, 250 nm, 500 nm concentrations of 429-MMAE and 50 nm, 125 nm, 250 nm, 500 nm concentrations of OV-MMAE conjugates were compared with DMSO and 50 nm MMAE in G292 cells. G292 cells treated with various dosages of 429-MMAE had significantly lower cell viability compared to treatment with DMSO (Figures 16A-16B). The stained G292 cells treated with 429-MMAE were observed under a confocal fluorescence microscope, and images showed that 429-MMAE affected microtubules in G292 cells (Figure 17).

[0163] 429-MMAE also affected viability and apoptosis in A431 cells (Figure 18A-19). 429-MMAE at 50 nm, 125 nm, 250 nm, 500 nm concentrations and OV-MMAE at 50 nm, 125 nm, 250 nm, 500 nm concentrations were compared with DMSO and 50 nm MMAE in A431 cells. A431 cells treated with various doses of 429-MMAE had significantly lower cell viability compared to treatment with DMSO (Figure 18B). A431 cells treated with various doses of 429-MMAE had significantly higher cell apoptosis compared to treatment with DMSO (Figure 18C). 429-MMAE also disrupted microtubules in A431 cells (Figure 19). The dose response curves of 1:3 dilution of MMAE were compared between A431 cells and HCT116 cells (Figure 20A). A431 cells have an IC of 71.2 pM 50 HCT-116 cells had an IC value of 500.4. 50 The dose response curves of 429-MMAE in A431 and G292 were compared (FIG. 20B). A431 cells had an IC 50 G292 cells had an IC value of 24.1 nM. 50 The results showed that the 429-MMAE induced apoptosis in CHLA06 cells as shown by caspase assay (Figures 21A-22B). Treatment with 500 nM 429-1 MMAE induced more apoptosis in CHLA06 cells compared to DMSO at 24 hours (Figure 21A). Treatment with 100 nM and 500 nM 429-1 MMAE induced more apoptosis in CHLA06 cells compared to DMSO at 48 hours (Figure 21B). 421-1 MMAE corresponds to 429-MMAE with a drug-antibody ratio (DAR) of 1. Figure 22A compares the viability of CHLA06 cells after treatment with various dosages of MMAE and 429-1 MMAE.

[0164] Without being bound to a particular theory, 429-MMAE had a higher IC compared to MMAE alone. 50This indicates that the linker is stable and the drug is not released. The smaller size of the 429-MMAE conjugate leads to rapid tumor attachment, and the expected shorter half-life is favorable for many indications, as the goal is to process and clear the drug as quickly as possible. The 429-MMAE conjugate has lower manufacturing costs compared to conventional antibody-drug conjugates. The 429-MMAE conjugate is suitable for treating pediatric nervous system tumors, as the drug administration is intratumoral and not systemic.

[0165] The 429-panobinostat construct can localize to the nucleus of cells as an uncleaved construct and target atypical teratoid rhabdoid tumors (ATRT) (Figures 23A-24B). 429-NLS-panobinostat (429-P) inhibits CHLA06 cells. 100 nm, 50 nm, 25 nm, 10 nm, and 5 nm concentrations of the 429-panobinostat construct significantly affected cell viability compared to DMSO (Figure 23B). Dose response curves show that the 429-panobinostat construct is more effective than panobinostat alone (Figures 24A-24B). IC of A431 cells treated with 429-panobinostat 50 IC of 32.6 nM and A431 treated with panobinostat is 108 nM. 429-IC of CHLA06 cells treated with panobinostat 50 is 12.3 nM and A431 treated with panobinostat is >500 nM. 429-MMAE and 429-NLS-panobinostat inhibit the AT / RT cell line CHLA06.

[0166] 429-SMARCB1 is internalized in CHLA06 cells as shown in confocal fluorescence microscopy images (FIG. 25). 429-SMARCB1 also colocalizes with histones as shown in confocal fluorescence microscopy images (FIG. 26). 429-SMARCB1 inhibits Myc transcription. FIG. 27A shows the results of HCT116 Myc-reporter cells treated with various doses of panobinostat. FIG. 27B-C shows that HCT116 Myc-reporter cells treated with increasing doses of 429-SMARCB1 cause greater inhibition of Myc transcription. 429-SMARCB1 affects both Myc transcription and viability as demonstrated by FIG. 28A-B. 50 nM 429-SMARCB1, 5 nM 429-SMARCB1, and 500 nM 429-MMAE-1 all significantly affected Myc transcription compared to the control (Figure 28A). 429-SMARCB1 also affected HCT116 cell viability compared to the control (Figure 28B). 429-SMARCB1 and tazemetostat reduced cell viability and increased caspase activity in CHLA06 cells (Figures 30A-B).

[0167] The structure of the 429-SMARCB1(F) mimetic conjugate is shown in Figure 31. SMARCB1 mimetic(F) and tazemetostat significantly increased caspase activity in CHLA06 cells (Figure 32A-B). SMARCB1 mimetic(F) and tazemetostat significantly increased caspase activity in CHL02 cells (Figure 33A-B). 50ug of 429-SMARCB1(F) also significantly affected the viability of CHLA06 cells compared to the 429 control (429 is a delivery peptide that does not contain a mimetic (Figure 34A-B)). 429-SMARCB1(F) also inhibited the transcription of Myc and the viability of HCT116 cells (Figure 35A-B).

[0168] We also developed 429 ScHv-SMARCB1 antibody with the structure "Antibody-429 CDR3-MMP9-NLS-SmarcB1". 429 ScHv-SMARCB1 antibody significantly affected cell viability compared to DMSO control (Figure 37A-B). 429-Antibody and OV-Antibody are scHvs containing peptides in CDR3, 429-Antibody-S and OV-Antibody-S are scHVs containing SMARCB1 mimics, and 429-MMAE is used as a positive control (Figure 38B).

[0169] Without being bound by any particular theory, 429-SMARCB1 mimetic conjugates induced apoptosis in HCT116, CHLA02, and CHLA06 cells. 429-SMARCB1 mimetic conjugates inhibited myc activation in HCT116 cells. There was a synergistic apoptotic effect when 429-SMARCB1 was used in combination with tazemetostat in ATRT cell lines. 429-antibody-SMARCB1 constructs inhibited the proliferation of cancer cells.

[0170] Example 3: Testing of drug conjugates 429-MMAE Animal Studies The 429-MMAE drug conjugate was shown to inhibit A431 xenografts in nude mice (Figures 37A-37C). A431 cells were used as xenografts. Animals received vehicle (PBS), one injection of 429 peptide (50ug / injection) or 429-137 (25ug / injection), or four weekly injections of 429-136 (25ug / injection) by intratumoral route. Tumors were measured twice weekly and were randomly assigned to each tumor type when tumors reached 1000mm. 3Animals were sacrificed when tumor volume reached 100 mg / kg. Figure 37A shows a comparison of tumor volume after treatment with 429-MMAE at two different dosages (SKWX803 corresponds to 429-MMAE). Figure 37B shows a comparison of tumor volume 23 days after treatment with 429-MMAE (429-136 corresponds to 429-MMAE). Animals given one injection of 429-MMAE (25ug / injection; 1mg / kg) and four weekly injections of 429-MMAE (25ug / injection; 1mg / kg) showed significantly lower tumor growth compared to control vehicle or control 429 peptide. The weight of excised tumors in mice treated with one injection of 429-MMAE (25ug / injection; 1mg / kg) and four weekly injections of 429-MMAE (25ug / injection; 1mg / kg) was significantly lighter compared to control vehicle or 429 peptide (Figure 37C). Animal weights remained constant throughout the study. There was no apparent toxicity with the 429-MMAE conjugate.

[0171] Effects of 429-MMAE on normal and cancer cell lines Three small molecule inhibitors of IGF-1R were compared with 429-MMAE on various cell lines (Figures 38A-38E). Figure 38A shows a comparison of dose response of CHLA06 after treatment with 429-MMAE, PPP (picropodophyllin), linsitinib, and NVP-ADW742. Both linsitinib and NVP-ADW742 had no effect on CHLA06 cells. PPP was active in CHLA06 cells, but less effective in terms of IC50 compared to 429-MMAE. Linsitinib, NVP-ADW742, PPP, and 429-MMAE were also tested in G292 and HCT 116 cell lines (Figures 38B-38C). 429-MMAE was more effective in inhibiting cell proliferation in both G292 and HCT 116 cell lines compared to the three IGF-1R inhibitors in terms of IC50. 429-MMAE was also better at inducing apoptosis in CHLA06 cells compared to the IGF-1R inhibitors at 48 and 72 hours post-treatment (Figures 39A-39B).

[0172] 429-MMAE and MMAE were also tested in two normal cell lines, WS1 and CCD841 (Figures 38D-38E). 429-MMAE had no apparent effect on CCD841 and was 5-10 times less effective on WS1 cells. WS1 cells were derived from normal human skin. WS1 cells have a doubling potential of 67 population doublings. CCD841 cells were isolated from normal human colon tissue. Without being bound by a particular theory, it appears that a therapeutic index exists between the effects of 429-MMAE on tumor and normal cell lines, which may increase the maximum therapeutic dose in the clinical setting.

[0173] 429-MMAE was also tested in the presence or absence of IGF-1 (Figure 40). Figure 40 shows the shift in IC50 in A431 cells in the presence of IGF-1. Without being bound to a particular theory, the shift between cells in the presence or absence of IGF suggests that IGF-1 blocks binding of the conjugate.

[0174] 429-MMAE was also tested at different drug peptide ratios (DRP) (i.e., number of drug molecules per peptide) on CHLA04 and CHLA06 cells (Figures 41A-41C). 429-136 corresponds to 429-MMAE with a DPR of 1 (average 1.36). 429-193 corresponds to 429-MMAE with a DPR of 2 (average 1.93). The drug linker is conjugated to a cysteine ​​resident. Since the 429 peptide has two cysteines, the 429 peptide can have a DPR of 1 or 2. The apoptosis of CHLA-06, the dose response of CHLA-06, and the dose response of CHLA04 were compared between these two 429-MMAE constructs.

[0175] 429-MMAE was also compared to 429-SM in various cell lines. 429-SM corresponds to 429-Smarc-MMAE and consists of a 429-linker-NLS-SMARCB1 mimic with one or two MMAE molecules conjugated via cysteines. CHLA06 cells were plated overnight at 10,000 cells / well in B27-5. Samples were added and incubated at 37°C for 48 hours. Caspase 3 / 7 reagent was added and the plate was read after 30 minutes at room temperature. 429-SM induced apoptosis of CHLA06 cells across the entire dose response (Figure 42A). Induction of apoptosis correlates directly with drug concentration (i.e., the higher the drug concentration, the greater the apoptotic effect). 429-SM also inhibited HCT myc transcription (Figure 42B). 429-SM was also effective in A431 and HCT116 cells (FIGS. 42C-42D).

[0176] Other embodiments While the present invention has been described in connection with the detailed description, it should be understood that the above description is intended to be illustrative and not limiting of the scope of the invention, which is defined by the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.

[0177] The content of any individual section may be equally applicable to all sections.

[0178] Incorporation by Reference All patents and publications referenced herein are hereby incorporated by reference in their entirety.

[0179] The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application, and nothing herein should be construed as an admission that the present invention is not entitled to antedate such publications by virtue of prior invention.

[0180] As used herein, all headings are for organizational purposes only and are not intended to limit the disclosure in any way.

Claims

【Request 1】 【Number 1】 A composition according to the formula: J comprises an intracellular uptake peptide, and optionally a cell surface receptor targeting peptide and / or an organelle uptake peptide and / or a protein detection peptide; if a cell surface receptor targeting peptide and / or an organelle uptake peptide and / or a protein detection peptide are present, optionally there are cleavage sites between each of these peptides and also between these peptides and the intracellular uptake peptide; the intracellular uptake peptide is capable of binding to a marker that is internalized by the cell, optionally expressed on the cancer cell, optionally selected from OVCAR, MIA, A549, A431, G292, HCT116, CHLA02, CHLA04, CHLA05, CHLA06, or DFYGCLLDLSLGVPSLGWRRRCITA (SEQ ID NO: 48) ("429"); YQVVSVVWHRGLLRPVLRGSLKPWYIFVRIHISWGQVHVG (SEQ ID NO: 43), TGWWFFWSYVCSVRLLAVTCSFWCTEQGEESLPRAGTHTH (SEQ ID NO: 44), YTLGLGKSQRWNLFRNFFCYMPRVTVFCKSCRCVSFQTSG (SEQ ID NO: 45), HYLLIRCARGFCEVRLVDAMCSITIVIDCVLFTSQRERAE (SEQ ID NO: 46), QGSLISCWVTFVQESWDAYSLCSDLFKCQGGQVFSICEPE (SEQ ID NO: 47), FSFGNSSLRKLPGVVRRRRCARGVWR (SEQ ID NO:49) ("OV-8"); DFYGCLLDLSLGVPSLGWRRRCITA (SEQ ID NO: 48) ("1 429-M"); FSFGNSSLRKLPGVVRRRRCARGVWR (SEQ ID NO: 49) ("No. 3 OvB8-M"); DFYGCLLDLSLGVPSLGWRRRCITA (SEQ ID NO: 48) ("429-F"), and FSFGNSSLRKLPGVVRRRRCARGVWR (SEQ ID NO: 49) ("No. 4 OVB8-F"); or a variant thereof, or having an amino acid sequence selected from Table 1 or Figure 1, or a variant thereof; Z comprises a therapeutic payload; The composition wherein Y is a linker covalently linking J and Z. 【Request 2】 【Number 2】 wherein V comprises a cell surface receptor targeting peptide; m is 0 or 1; W comprises an intracellular uptake peptide; X comprises an organelle uptake peptide; The composition of claim 1 , wherein n is 0 or 1.

3. 3. The composition of claim 1 or 2, wherein Z is or comprises 1, or 2, or 3, or 4, or 5, or 6, or 7, or 8, or 9, or 10, or more therapeutic payloads.

4. The composition of claims 1 to 3, wherein the therapeutic payload is an anti-cancer drug.

5. The composition of any one of claims 1 to 4, wherein the therapeutic payload is a small molecule drug, a biologic, or a radionuclide.

6. 6. The composition of claim 5, wherein the therapeutic payload is a biologic selected from a toxin, an antibody, or an oligonucleotide.

7. The composition of claim 4 , wherein the anticancer drug is monomethyl auristatin E (MMAE).

8. The composition of claim 4 , wherein the anti-cancer agent is a histone deacetylase (HDAC) inhibitor.

9. 9. The composition of claim 8, wherein the HDAC inhibitor is selected from vorinostat, panobinostat, tazemetostat, mocetinostat, romidepsin, trichostatin A, and trapoxin A.

10. The composition of claim 5 , wherein the payload is an antisense oligonucleotide that binds to kras, gemcitabine, or cisplatin.

11. The composition of claim 5 , wherein the therapeutic payload is a SmarcB1 mimetic.

12. the cellular uptake peptide is capable of binding to Ovcar-3 cells; and / or PRGWRRCLGWSVGPASGSG (SEQ ID NO:50), FSFGNSSLRKLPGVVRRRRCARGVWR (SEQ ID NO:49), CLGVCISGDPEQADMWGCRP (SEQ ID NO:51), ILSRSQYLVFGYAVVARGSR (SEQ ID NO:52), GSGLCSVCQCGGMYSRVVLGQRRWM (SEQ ID NO:53), GVSPIVHGDAGLLQPRNTRL (SEQ ID NO:54), CRQRGDGGAMRRGSVCLRGW (SEQ ID NO:55), GDGTGVQRAAVGFDLTGHSP (SEQ ID NO:56), AVSACGRCRHCRVPGVPCWLYCVCT (SEQ ID NO:57), PSTMSIAGGGCFGHPRRGTI (SEQ ID NO:58), DQCAGHDSVQRHALHGLAVM (SEQ ID NO:59), GASPCVFRVDSTSGGPLVFPDLQST (SEQ ID NO: 60), RREDSLTRVRQALREALGAG (SEQ ID NO:61), and 10. The composition of any one of the preceding claims, having an amino acid sequence selected from LDVGVAQHRGGTVIRVAMRA (SEQ ID NO: 62), or a variant thereof.

13. 2. The composition of any one of the preceding claims, wherein the intracellular uptake peptide has an amino acid sequence that is FSFGNSSLRKLPGVVRRRRCARGVWR (SEQ ID NO: 49), or a variant thereof.

14. the intracellular uptake peptide is capable of binding to MIA cells; and / or GIRRGGLGGRQRGWGRRRKPI (SEQ ID NO:63), RAPGGFTRTNSVRRATGSM (SEQ ID NO: 64), YGKVGGWRLATVGRRTESRI (SEQ ID NO:65), KQVGLINGSACSCDMYQRVV (SEQ ID NO:66), VNSVKGVHRRAFSEQPLGSG (SEQ ID NO:67), DAGRAPALGWFFFGRRRFGE (SEQ ID NO:68), GVPGCGSHGADWGREIGATSTGMAL (SEQ ID NO: 69), GVCRWMRPHFRMPRRWRLGR (SEQ ID NO: 70), RSSGMCIIARRHVLSCHSLS (SEQ ID NO:71), SGVGTCPTRPWVPGVCRQVS (SEQ ID NO:72), RVIVSGLSGRVRMGVKRRRI (SEQ ID NO:73), FMRHARPTRGVVRRIGHGV (SEQ ID NO:74), QLYACLRLVWGRQASAGWRVTMTG (SEQ ID NO:75), LERIVGQWYSEGLSGDWANL (SEQ ID NO: 76), GASRCFSLCSGSEGAESRQATTV (SEQ ID NO:77), HLGCSMPINVFGVCIWKCA (SEQ ID NO:78), RRLAVRLRVMLRRGPVCIRV (SEQ ID NO:79), LVDACIGSCQPEGSPPARPPFQLA (SEQ ID NO: 80), RCVGCCKYLIAQAYRFTVAGGAAQYA (SEQ ID NO: 81), AGVHCGLTNVGVGVRSYRRLYA (SEQ ID NO: 82), VNGWGLGRRGMGIDYRWAVH (SEQ ID NO: 83), and The composition of any one of claims 1 to 11, having an amino acid sequence selected from QSARCAVTMGLDALNLRRGISPVLD (SEQ ID NO: 84), or a variant thereof.

15. the intracellular uptake peptide is capable of binding to A549 cells; and / or YPWQQVLVPPPGNVHAFATI (SEQ ID NO: 85), TAVAGVGVSAGQLQGGTAAG (SEQ ID NO: 86), FFGLSFPASVAVSDSGVQMR (SEQ ID NO: 87), SSRAVSWMVQLRVQRGSGLV (SEQ ID NO: 88), SRLSLVQSCSVSWGHQTVGT (SEQ ID NO: 89), VARWCQVNWAYGRRLAPPVG (SEQ ID NO:90), FLLCSLPLTQPPYRVIGAYA (SEQ ID NO:91), GGPSGIGWQHAWWVFGRQGL (SEQ ID NO: 92), LQRQIAGPRSRQRGRGSRYR (SEQ ID NO:93), FEDMRLGWRNGQPPGGVAIW (SEQ ID NO:94), DSWQGARVQPGVGAHTAQQR (SEQ ID NO:95), GIKTVRGYGVWRWHGGSPRM (SEQ ID NO: 96), IRVWCQWLRTKLAAGQVNCA (SEQ ID NO: 97), SYGVGGRHSGGQGEALVYSV (SEQ ID NO: 98), RAQNCGNWAAVQRINVSSMA (SEQ ID NO:99), GRAPEQTPCIGWRGGLIKDL (SEQ ID NO: 100), SGSRNFAAARWSHRGFCVPW (SEQ ID NO: 101), IAFSRRGRGWWCQMPGGSRV (SEQ ID NO: 102), and The composition of any of claims 1 to 11, having an amino acid sequence selected from GCCSAGLPSSLLSTPQSRSR (SEQ ID NO: 103), or a variant thereof.

16. The composition of claim 2, wherein m is 1 and the cell surface receptor targeting peptide binds to a cancer cell surface receptor or a microvascular endothelial cell surface receptor.

17. 17. The composition of claim 16, wherein the cancer cell surface receptor is insulin-like growth factor type 1 receptor (IGF-1R), epidermal growth factor receptor (EGFR), insulin receptor, GRP78 / BiP, or transferrin receptor.

18. 20. The composition of claim 2 or 17, wherein m is 1 and V has the amino acid sequence of DFYGCLLDLSLGVPSGVRRRCITA (SEQ ID NO: 104), or a variant thereof.

19. 20. The composition of claim 2 or 17, wherein m is 1 and V has the amino acid sequence of RPWILHLGRERSTDDQTPVHRMSLMHPLYHTRGKSYKVEG (SEQ ID NO: 105), or a variant thereof.

20. The composition of claim 2 , wherein the organelle uptake peptide is a targeting peptide or a signal peptide.

21. 21. The composition of claim 20, wherein the organelle uptake peptide is a nuclear localization signal, optionally a classical nuclear localization signal.

22. 22. The composition of claim 21, wherein the nuclear localization signal is a monopartite classical nuclear localization signal.

23. The composition of claim 22, wherein the nuclear localization signal has the amino acid sequence of PKKKRKV (SEQ ID NO: 106).

24. 22. The composition of claim 21, wherein the nuclear localization signal is a bipartite classical nuclear localization signal.

25. 22. The composition of claim 21, wherein the nuclear localization signal is a non-classical nuclear localization signal.

26. The composition of claim 2 , wherein the organelle uptake peptide is an endoplasmic reticulum localization signal.

27. The composition of claim 2 , wherein V, W or X further comprises a cleavage site.

28. 28. The composition of claim 27, wherein the cleavage site is a matrix metalloproteinase cleavage site.

29. 28. The composition of claim 27, wherein the cleavage site is a matrix metalloproteinase 9 cleavage site.

30. 28. The composition of claim 27, wherein the cleavage site has the amino acid sequence of WLPSSI (SEQ ID NO: 107).

31. 28. The composition of claim 27, wherein the cleavage site is a furin cleavage site.

32. 32. The composition of claim 31 , wherein the cleavage site has the amino acid sequence of PRRARS (SEQ ID NO: 108).

33. The composition of claim 1 , wherein the protein detection peptide is a FLAG tag having the amino acid sequence DYKDDDDK (SEQ ID NO: 109).

34. 2. The composition of any one of the preceding claims, wherein the variant comprises about 1, or about 2, or about 3, or about 4, or about 5 mutations, wherein the mutations are selected from substitutions or deletions.

35. 2. The composition of any one of the preceding claims, wherein the linker is a flexible linker.

36. 36. The composition of claim 35, wherein the linker consists essentially of glycine and serine residues.

37. The linker is (GGS) ｎ where: ｎ The composition of claim 35 or 36, wherein is 1, or 2, or 3, or 4, or 5.

38. 38. The composition of any one of claims 35-37, wherein the linker is GGSGGSGGSG (SEQ ID NO: 118), or a variant thereof, wherein the variant comprises about 1, or about 2, or about 3, or about 4, or about 5 mutations, wherein the mutations are selected from substitutions or deletions.

39. 2. The composition of any one of the preceding claims, wherein the linker comprises a cleavage site.

40. 40. The composition of claim 39, wherein the cleavage site is a matrix metalloproteinase cleavage site, optionally a matrix metalloproteinase 9 cleavage site, optionally having the amino acid sequence of WLPSSI (SEQ ID NO: 107), or a furin cleavage site, optionally having the amino acid sequence of PRRARS (SEQ ID NO: 108).

41. A composition comprising the amino acid sequence of DFYGCLLDLSLGVPSLGWRRRCITADYKDDDDKWLPSSIPKKKRKVDGQKLRDAFTWNMNEKLMTPEMFSEILCDDLD (SEQ ID NO: 110), or an amino acid sequence having at least about 90%, or at least about 95%, or at least about 97%, or at least about 98%, or at least about 99% identity thereto.

42. A composition comprising the amino acid sequence of DFYGCLLDLSLGVPSLGWRRRCITAWLPSSIPKKKRKVDGQKLRDAFTWNMNEKLMTPEMFSEILCDDLD (SEQ ID NO: 111), or an amino acid sequence having at least about 90%, or at least about 95%, or at least about 97%, or at least about 98%, or at least about 99% identity thereto.

43. A composition comprising the amino acid sequence of QVQLVESGGGVVQPGRSLRLSCAASGGGGSGGGGSWFRQAPGKEREFVAGGGGSGGGGGSYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDFYGCLLDLSLGVPSLGWRRRCITAWGQGLVTVSSGPGGQWLPSSIPKKKRKVDGQKLRDAFTWNMNEKLMTPEMFSEILCDDLD (SEQ ID NO: 112), or an amino acid sequence having at least about 90%, or at least about 95%, or at least about 97%, or at least about 98%, or at least about 99% identity thereto.

44. The composition of any of claims 1 to 43, wherein the composition is suitable for administration together with one or more additional therapeutic agents.

45. 45. The composition of claim 44, wherein the additional therapeutic agent is tazemetostat.

46. 13. A polynucleotide comprising a nucleic acid sequence encoding an amino acid sequence according to any of the preceding claims, or a variant thereof.

47. A vector comprising the polynucleotide of claim 46.

48. A host cell comprising the vector of claim 47.

49. A pharmaceutical composition comprising the composition of any one of claims 1 to 43 and a pharma- ceutical acceptable excipient or carrier.

50. A method for treating or preventing a disease, comprising administering an effective amount of a composition according to any one of claims 1 to 43 or a pharmaceutical composition according to claim 49 to a patient in need thereof.

51. 51. The method of claim 50, wherein the disease is treatable by targeting a cancer cell surface receptor or a microvascular endothelial cell surface receptor.

52. 51. The method of claim 50, wherein the disease is treatable by targeting a tumor, optionally an atypical teratoid rhabdoid tumor.

53. The disease is basal cell carcinoma, biliary tract cancer; bladder cancer; Bone cancer; cancer of the brain and central nervous system; breast cancer; cancer of the peritoneum; cervical cancer; choriocarcinoma; colon and rectal cancer; cancer of the connective tissue; cancer of the digestive system; endometrial cancer; esophageal cancer; eye cancer; head and neck cancer; gastric cancer (including gastrointestinal cancer); glioblastoma; liver cancer; hepatocellular carcinoma; intraepithelial neoplasia; kidney or renal cancer; laryngeal cancer; leukemia; liver cancer; Lung cancer (e.g., small cell lung cancer, non-small cell lung cancer, adenocarcinoma of the lung, and squamous cell carcinoma of the lung); melanoma; myeloma; neuroblastoma; oral cancer (lips, tongue, mouth, and throat); ovarian cancer; pancreatic cancer; prostate cancer; retinoblastoma; rhabdomyosarcoma; rectal cancer; Cancer of the respiratory system; cancer of the salivary glands; sarcoma; skin cancer; squamous cell carcinoma; stomach cancer; testicular cancer; thyroid cancer; uterine or endometrial cancer; cancer of the urinary system; vulva cancer; Hodgkin's and non-Hodgkin's lymphomas, as well as B-cell lymphomas (low-grade / follicular non-Hodgkin's lymphoma (NHL); small lymphocytic (SL) NHL; intermediate-grade / follicular NHL; intermediate-grade diffuse NHL; high-grade immunoblastic NHL; high-grade lymphoblastic NHL; high-grade small noncleaved cell NHL; bulky disease NHL; mantle cell lymphoma) 53. The method of any one of claims 50 to 52, wherein the cancer is optionally selected from lymphomas, including lymphomas including leukemia, AIDS-related lymphomas, and Waldenstrom's macroglobulinemia; chronic lymphocytic leukemia (CLL); acute lymphoblastic leukemia (ALL); hairy cell leukemia; chronic myeloblastic leukemia; and other carcinomas and sarcomas; and post-transplant lymphoproliferative disorders (PTLDs), as well as abnormal blood vessel proliferation associated with phacomatosis, edema (e.g., associated with brain tumors), and Meigs' syndrome.

54. 54. The method of any one of claims 50 to 53, wherein the administering is accomplished using convection-enhanced delivery.

55. A composition comprising a glycoprotein non-metastatic melanoma protein b (GPNMB) recognition domain linked to a therapeutic payload.

56. 56. The composition of claim 55, wherein the GPNMB recognition domain is linked to one, or two, or three, or four, or five, or six, or seven, or eight, or nine, or ten, or more therapeutic payloads.

57. The GPNMB recognition domain is (a) a single chain antibody, or a fragment thereof, comprising CDR1, CDR2, and CDR3, CDR1 is or comprises MIHYLGWSKV (SEQ ID NO:1), or YMGSRQNDMG (SEQ ID NO:2), or LNRAPYFPNA (SEQ ID NO:3), or WLSNNFWAW (SEQ ID NO:4), or a variant thereof, said variant comprising about 1, or about 2, or about 3, or about 4, or about 5 mutations, said mutations being selected from substitutions or deletions; CDR2 is or comprises QIKMWWAMMH (SEQ ID NO:5), or TSFWIKARKW (SEQ ID NO:6), or WASWRWFRDH (SEQ ID NO:7), or QTLKTSWWWT (SEQ ID NO:8), or a variant thereof, said variant comprising about 1, or about 2, or about 3, or about 4, or about 5 mutations, said mutations being selected from substitutions or deletions; If CDR3 is LPGGNNFNLYRSFIMTLW (SEQ ID NO: 9), or WQLGSGGLHVWVSCHMYGGVYGKTSELALTWPGMWTRTHLW (SEQ ID NO: 10), or DSPRLDSIWSMSHAMGHL (SEQ ID NO: 11), or YLVRFRLDGVVWHEVVGNVNEYRTAWASIFDWLAKFQLGS (SEQ ID NO: 12), or ILNSCNPTWTRLQFKFCPALLMNGPIFAWFLWAAWVSSAM (SEQ ID NO: 13), or SDEYRFDRSVESVWQDLVIYQSHVLWWTTVHRYLAAWIQW (SEQ ID NO: 14), or VNWINMHSAKWMWWTHGV (SEQ ID NO: 15), or TPTLNYGNFNERDMDTVG (SEQ ID NO: 16), or a variant thereof, wherein said variant comprises about 1, or about 2, or about 3, or about 4, or about 5 mutations, wherein said mutations are selected from substitutions or deletions; or (b) a peptide, LPGGNNFNLYRSFIMTLW (SEQ ID NO: 9), or variants thereof; WQLGSGGLHVWVSCHMYGGVYGKTSELALTWPGMWTRTHLW (SEQ ID NO: 10), or variants thereof; DSPRLDSIWSMSHAMGHL (SEQ ID NO: 11), or variants thereof; YLVRFRLDGVVWHEVVGNVNEYRTAWASIFDWLAKFQLGS (SEQ ID NO: 12), or variants thereof; ILNSCNPTWTRLQFKFCPALLMNGPIFAWFLWAAWVSSAM (SEQ ID NO: 13), or CVLGDYHYLYTGSADHTLRESLSLLTRCRFRRVIYQQQLC (SEQ ID NO: 120), or variants thereof; FTHISRGFEQHPTLVNLFYPRWQYIFIFTRNALCGQYVWD (SEQ ID NO: 17), or variants thereof; LSDFEINKRCVLGVFYYLGEGSRGHILSFFHPNVQQFRVP (SEQ ID NO: 18), or variants thereof; GDFGNRYIQLLVIVLSNGQIFVQKLLSMRPFTINGDRLRQ (SEQ ID NO: 19), or variants thereof ;ASVTLFRLVPMQNAWVTVLPSFAEFVMARPIEERATWGGS (SEQ ID NO: 20), or variants thereof;WQLGSGGLHVWVSCHMYGGVYGKTSELALTWPGMWTRTHL (SEQ ID NO: 21), or variants thereof;DSTMTLLARANSHASLLNVYLNINQKVEDVFFMLFLGSPP (SEQ ID NO: 22), or variants thereof;LATWIQGSKIWSNETYSISIQVSWLSLFVMGQNLYYLLSV (SEQ ID NO: 23), or variants thereof;SDEYRFD RSVESVWQDLVIYQSHVLWWTTVHRYLAAWIQW (SEQ ID NO: 14), or variants thereof; VNWINMHSAKWMWWTHGV (SEQ ID NO: 15), or variants thereof; TPTLNYGNFNERDMDTVG (SEQ ID NO: 16), or variants thereof; QKVLYTAVPFKHGSYIFCDRIGFIFNYICEFSRSWSTWSM (SEQ ID NO: 24), or variants thereof; VGNSIQYVKAVQTQYWAAITSWIPITRFTTRVMFLLPQVM (SEQ ID NO: 25), or variants thereof;QVTARCLQIIVLYKLGNNGVTSLFHVYAMSSVPKFSVKWS (SEQ ID NO: 26), or variants thereof; GQVCSPFVTSALKLFGADFGWWSLVLMKAVSWVLYITFNA (SEQ ID NO: 27), or variants thereof; TLDHVRQAYAALSYSTVRCTFVSQMTIFYFGRVMF KALCN (SEQ ID NO: 28), or variants thereof; AYHLQLEQLRCVSQNVVTSFLDFALTKALALRSVLLGVWM (SEQ ID NO: 29), or variants thereof; QCFQDDLCVLPVASLLDHTRSMSHLFEGWLPSRSFGFRNG (SEQ ID NO: 30), or variants thereof; STAVVIWQDW the peptide is selected from: FRTFRMAVVVSSARDEAVDRVCLAWPARYM (SEQ ID NO: 31), or a variant thereof; LSAADMYKHKIVGEAGEPIFYMVRNWLQKQWPMNFILMSL (SEQ ID NO: 32), or a variant thereof; FLWQWSADSMGAVDACSHKILGLFYHSQNIFARPFLRSYG (SEQ ID NO: 33), or a variant thereof; NIFIPPVGYYLTKQCGTNNFWFYQVLCRRWGADSILFWER (SEQ ID NO: 34), or a variant thereof, wherein said variant comprises about 1, or about 2, or about 3, or about 4, or about 5 mutations, wherein said mutations are selected from substitutions or deletions; 57. The composition of claim 55 or 56, which is or comprises:

58. CDR1 is or comprises MIHYLGWSKV (SEQ ID NO: 1) or a variant thereof; CDR2 is or comprises QIKMWAMMH (SEQ ID NO:5) or a variant thereof; CDR3 is or comprises LPGGNNFNLYRSFIMTLW (SEQ ID NO: 9) or a variant thereof; 58. The composition of claim 57, wherein the variant comprises about 1, or about 2, or about 3, or about 4, or about 5 mutations, wherein the mutations are selected from substitutions or deletions.

59. CDR1 is or comprises MIHYLGWSKV (SEQ ID NO: 1) or a variant thereof; CDR2 is or comprises QIKMWAMMH (SEQ ID NO:5) or a variant thereof; CDR3 is or comprises WQLGSGGLHVWVSCHMYGGVYGKTSELALTWPGMWTRTHLW (SEQ ID NO: 10) or a variant thereof; 58. The composition of claim 57, wherein the variant comprises about 1, or about 2, or about 3, or about 4, or about 5 mutations, wherein the mutations are selected from substitutions or deletions.

60. CDR1 is or comprises YMGSRQNDMG (SEQ ID NO:2) or a variant thereof; CDR2 is or comprises TSFWIKARKW (SEQ ID NO: 6) or a variant thereof; CDR3 is or comprises DSPRLDSIWSMSHAMGHL (SEQ ID NO: 11) or a variant thereof; 58. The composition of claim 57, wherein the variant comprises about 1, or about 2, or about 3, or about 4, or about 5 mutations, wherein the mutations are selected from substitutions or deletions.

61. CDR1 is or comprises YMGSRQNDMG (SEQ ID NO:2) or a variant thereof; CDR2 is or comprises TSFWIKARKW (SEQ ID NO: 6) or a variant thereof; CDR3 is or comprises YLVRFLDGVVWHEVVGNVNEYRTAWASIFDWLAKFQLGS (SEQ ID NO: 12) or a variant thereof; 58. The composition of claim 57, wherein the variant comprises about 1, or about 2, or about 3, or about 4, or about 5 mutations, wherein the mutations are selected from substitutions or deletions.

62. CDR1 is or comprises LNRAPYFPNA (SEQ ID NO:3) or a variant thereof; CDR2 is or comprises WASWRWFRDH (SEQ ID NO:7) or a variant thereof; CDR3 is or comprises ILNSCNPTWTRLQFKFCPALLMNNGPIFAWFLWAAWVSSAM (SEQ ID NO: 13) or a variant thereof; 58. The composition of claim 57, wherein the variant comprises about 1, or about 2, or about 3, or about 4, or about 5 mutations, wherein the mutations are selected from substitutions or deletions.

63. CDR1 is or comprises WLSNNFWAW (SEQ ID NO:4) or a variant thereof; CDR2 is or comprises QTLKTSWWWT (SEQ ID NO: 8) or a variant thereof; CDR3 is or comprises SDEYRFDRSVESVWQDLVIYQSHVLWWTTVHRYLAAWIQW (SEQ ID NO: 14) or a variant thereof; 58. The composition of claim 57, wherein the variant comprises about 1, or about 2, or about 3, or about 4, or about 5 mutations, wherein the mutations are selected from substitutions or deletions.

64. CDR1 is or comprises LNRAPYFPNA (SEQ ID NO:3) or a variant thereof; CDR2 is or comprises WASWRWFRDH (SEQ ID NO:7) or a variant thereof; CDR3 is or comprises VNWINMHSAKWMWWTHGV (SEQ ID NO: 15) or a variant thereof; 58. The composition of claim 57, wherein the variant comprises about 1, or about 2, or about 3, or about 4, or about 5 mutations, wherein the mutations are selected from substitutions or deletions.

65. CDR1 is or comprises WLSNNFWAW (SEQ ID NO:4) or a variant thereof; CDR2 is or comprises QTLKTSWWWT (SEQ ID NO: 8) or a variant thereof; CDR3 is or comprises TPTLNYGNFNERDMDTVG (SEQ ID NO: 16) or a variant thereof; 58. The composition of claim 57, wherein the variant comprises about 1, or about 2, or about 3, or about 4, or about 5 mutations, wherein the mutations are selected from substitutions or deletions.

66. 66. The composition of any one of claims 57-65, wherein the single chain antibody, or fragment thereof, further comprises variable region framework (FW) sequences juxtaposed between the CDRs by the formula (FW1)-(CDR1)-(FW2)-(CDR2)-(FW3)-(CDR3)-(FW4), wherein the variable region FW sequence in the heavy chain variable region is a heavy chain variable region FW sequence and the variable region FW sequence in the light chain variable region is a light chain variable region FW sequence.

67. The composition of any one of claims 57-66, wherein the variable region FW sequences are human.

68. The single chain antibody is 59. The composition of claim 57 or 58, comprising an amino acid sequence of MAQVQLVESGGGVVQPGRSLRLSCAASMIHYLGWSKVWFRQAPGKEREFVAQIKMWWAMMHYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARLPGGNNNFNLYRSFIMTLWWGQGTLVTVSSGPGGQ (SEQ ID NO: 35), or an amino acid sequence having at least about 90%, or at least about 95%, or at least about 97%, or at least about 98%, or at least about 99% identity thereto.

69. The single chain antibody is 60. The composition of claim 57 or 59, comprising an amino acid sequence of MAQVQLVESGGGVVQPGRSLRLSCAASMIHYLGWSKVWFRQAPGKEREFVAQIKMWWAMMHYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARWQLGSGGLHVWVSCHMYGGVYGKTSELALTWPGMWTRTHLWWGQGTLVTVSSGPGGQ (SEQ ID NO: 36), or an amino acid sequence having at least about 90%, or at least about 95%, or at least about 97%, or at least about 98%, or at least about 99% identity thereto.

70. The single chain antibody is 61. The composition of claim 57 or 60, comprising an amino acid sequence of MAQVQLVESGGGVVQPGRSLRLSCAASYMGSRQNDMGWFRQAPGKEREFVATSFWIKARKWYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDSPRLDSIWSMSHAMGHLWGQGTLVTVSSGPGGQ (SEQ ID NO: 37), or an amino acid sequence having at least about 90%, or at least about 95%, or at least about 97%, or at least about 98%, or at least about 99% identity thereto.

71. The single chain antibody is 62. The composition of claim 55 or 61, comprising an amino acid sequence of MAQVQLVESGGGVVQPGRSLRLSCAASYMGSRQNDMGWFRQAPGKEREFVATSFWIKARKWYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARYLVRFLDGVVWHEVVGNVNEYRTAWASIFDWLAKFQLGSWGQGTLVTVSSGPGGQ (SEQ ID NO: 38), or an amino acid sequence having at least about 90%, or at least about 95%, or at least about 97%, or at least about 98%, or at least about 99% identity thereto.

72. The single chain antibody is 63. The composition of claim 55 or 62, comprising an amino acid sequence of MQVQLVESGGGVVQPGRSLRLSCAASLNRAPYFPNAWFRQAPGKEREFVAWASWRWFRDHYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARILNSCNPTWTRLQFKFCPALLMNNGPIFAWFLWAAWVSSAMWGQGTLVTVSSGPGGQ (SEQ ID NO: 39), or an amino acid sequence having at least about 90%, or at least about 95%, or at least about 97%, or at least about 98%, or at least about 99% identity thereto.

73. The single chain antibody is 64. The composition of claim 55 or 63, comprising an amino acid sequence of MQVQLVESGGGVVQPGRSLRLSCAASSWLSNNFWAWFRQAPGKEREFVAQTLKTSWWTYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARSDEYRFDRSVESVWQDLVIYQSHVLWWTTVHRYLAAWIQWWGQGTLVTVSSGPGGQ (SEQ ID NO: 40), or an amino acid sequence having at least about 90%, or at least about 95%, or at least about 97%, or at least about 98%, or at least about 99% identity thereto.

74. The single chain antibody is 65. The composition of claim 55 or 64, comprising an amino acid sequence of MQVQLVESGGGVVQPGRSLRLSCAASLNRAPYFPNAWFRQAPGKEREFVAWASWRWFRDHYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARVNWINMHSAKWMWWTHGVWGQGTLVTVSSGPGGQ (SEQ ID NO: 41), or an amino acid sequence having at least about 90%, or at least about 95%, or at least about 97%, or at least about 98%, or at least about 99% identity thereto.

75. The single chain antibody is 66. The composition of claim 55 or 65, comprising an amino acid sequence of MQVQLVESGGGVVQPGRSLRLSCAASSWLSNNFWAWFRQAPGKEREFVAQTLKTSWWTYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARTPTLNYGNFNERDMMDTVGWGQGTLVTVSSGPGGQ (SEQ ID NO: 42), or an amino acid sequence having at least about 90%, or at least about 95%, or at least about 97%, or at least about 98%, or at least about 99% identity thereto.

76. 57. The composition of claim 56, wherein the therapeutic payload is an anti-cancer drug.

77. 80. The composition of claim 56 or 76, wherein the therapeutic payload is a small molecule drug, a biologic, or a radionuclide.

78. 78. The composition of claim 77, wherein the therapeutic payload is a biologic selected from a toxin, an antibody, or an oligonucleotide.

79. 77. The composition of claim 76, wherein the anticancer agent is monomethyl auristatin E, vetodin monomethyl auristatin F, or mafodotin.

80. 77. The composition of claim 76, wherein the anti-cancer agent is a histone deacetylase (HDAC) inhibitor.

81. 81. The composition of claim 80, wherein the HDAC inhibitor is selected from vorinostat, panobinostat, tazemetostat, mocetinostat, romidepsin, trichostatin A, and trapoxin A.

82. 78. The composition of claim 77, wherein the therapeutic payload is gemcitabine, cisplatin, or an antisense oligonucleotide that binds to kras.

83. 78. The composition of claim 77, wherein the therapeutic payload is a SmarcB1 mimetic.

84. The composition of any one of claims 56 to 83, wherein the GPNMB recognition domain and the therapeutic payload are linked by a linker, optionally consisting essentially of glycine and serine residues.

85. 85. The composition of claim 84, wherein the linker is (GGS)n, where n is 1, or 2, or 3, or 4, or 5.

86. 86. The composition of claim 85, wherein the linker is GGSGGSGGSG (SEQ ID NO: 118), or a variant thereof, wherein the variant comprises about 1, or about 2, or about 3, or about 4, or about 5 mutations, and the mutations are selected from substitutions or deletions.

87. The composition of any one of claims 56 to 86, wherein the linker comprises a cleavage site.

88. 88. The composition of claim 87, wherein the cleavage site is a matrix metalloproteinase cleavage site, optionally a matrix metalloproteinase 9 cleavage site, optionally having the amino acid sequence of WLPSSI (SEQ ID NO: 107), or a furin cleavage site, optionally having the amino acid sequence of PRRARS (SEQ ID NO: 108).

89. The composition of any one of claims 56 to 88, wherein the composition binds to GPNMB expressed on a cell.

90. The composition of any one of claims 56 to 89, wherein the composition reduces, inhibits or prevents expression of GPNMB on cells, optionally cancer stem cells.

91. A polynucleotide comprising a nucleic acid sequence encoding an amino acid sequence according to any one of claims 56 to 90, or a variant thereof.

92. A composition comprising a vector composition comprising the polynucleotide of claim 91.

93. 93. A composition comprising a host cell comprising the polynucleotide of claim 91 or the vector of claim 92.

94. A pharmaceutical composition comprising the composition of any one of claims 56 to 93 and a pharma- ceutical acceptable excipient or carrier.

95. A method for treating or preventing cancer, comprising administering an effective amount of a composition according to any one of claims 56 to 93 or a pharmaceutical composition according to claim 94 to a subject in need thereof.

96. 96. The method of claim 95, wherein the cancer is triple-negative breast cancer.

97. 96. The method of claim 95, wherein the cancer is a solid tumor.

98. 96. The method of claim 95, wherein the cancer is a hematological cancer.

99. The cancer may be selected from the group consisting of vascular cancer, eye tumors, basal cell carcinoma, biliary tract cancer; bladder cancer; bone cancer; brain and central nervous system cancer; breast cancer; peritoneal cancer; cervical cancer; choriocarcinoma; colon and rectal cancer; connective tissue cancer; digestive system cancer; endometrial cancer; esophageal cancer; eye cancer; head and neck cancer; gastric cancer (including gastrointestinal cancer); glioblastoma; liver cancer; hepatocellular carcinoma; intraepithelial neoplasia; kidney or renal cancer; laryngeal cancer; leukemia; liver cancer; Lung cancer (e.g., small cell lung cancer, non-small cell lung cancer, adenocarcinoma of the lung, and squamous cell carcinoma of the lung); melanoma; myeloma; neuroblastoma; oral cancer (lips, tongue, mouth, and throat); ovarian cancer; pancreatic cancer; prostate cancer; retinoblastoma; rhabdomyosarcoma; rectal cancer; Cancer of the respiratory system; cancer of the salivary glands; sarcoma (e.g., Kaposi's sarcoma); skin cancer; squamous cell carcinoma; stomach cancer; testicular cancer; thyroid cancer; uterine or endometrial cancer; cancer of the urinary system; vulva cancer; Hodgkin's and non-Hodgkin's lymphomas, and B-cell lymphomas (low-grade / follicular non-Hodgkin's lymphoma (NHL); small lymphocytic (SL) NHL; intermediate-grade / follicular NHL; intermediate-grade diffuse NHL; high-grade immunoblastic NHL; high-grade lymphoblastic NHL; high-grade small noncleaved cell NHL; bulky disease NHL; mantle 99. The method of any one of claims 95-98, wherein the cancer is selected from one or more of lymphomas, including lymphomas, including acute lymphoblastic leukemia (including idiopathic cell lymphoma; AIDS-related lymphoma; and Waldenstrom's macroglobulinemia); chronic lymphocytic leukemia (CLL); acute lymphoblastic leukemia (ALL); hairy cell leukemia; chronic myeloblastic leukemia; and other carcinomas and sarcomas; and post-transplant lymphoproliferative disorders (PTLDs), as well as abnormal blood vessel proliferation associated with phacomatosis, edema (e.g., associated with brain tumors), and Meigs' syndrome.

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