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WO2025199531A2 - Nanoparticules ciblées et leurs utilisations contre le cancer - Google Patents

Nanoparticules ciblées et leurs utilisations contre le cancer

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Publication number
WO2025199531A2
WO2025199531A2 PCT/US2025/021146 US2025021146W WO2025199531A2 WO 2025199531 A2 WO2025199531 A2 WO 2025199531A2 US 2025021146 W US2025021146 W US 2025021146W WO 2025199531 A2 WO2025199531 A2 WO 2025199531A2
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WO
WIPO (PCT)
Prior art keywords
seq
cancer
nanoparticle
fragment
targeting
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
PCT/US2025/021146
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English (en)
Other versions
WO2025199531A3 (fr
Inventor
Richard B. Meagher
Eileen Jeanne Kennedy
Suresh AMBATI
Zachary A. LEWIS
Xiaorong LIN
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
University of Georgia
University of Georgia Research Foundation Inc UGARF
Original Assignee
University of Georgia
University of Georgia Research Foundation Inc UGARF
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Application filed by University of Georgia, University of Georgia Research Foundation Inc UGARF filed Critical University of Georgia
Publication of WO2025199531A2 publication Critical patent/WO2025199531A2/fr
Publication of WO2025199531A3 publication Critical patent/WO2025199531A3/fr
Pending legal-status Critical Current
Anticipated expiration legal-status Critical

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Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/69—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit
    • A61K47/6905—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a colloid or an emulsion
    • A61K47/6911—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a colloid or an emulsion the form being a liposome
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/10—Dispersions; Emulsions
    • A61K9/127—Synthetic bilayered vehicles, e.g. liposomes or liposomes with cholesterol as the only non-phosphatidyl surfactant
    • A61K9/1271—Non-conventional liposomes, e.g. PEGylated liposomes or liposomes coated or grafted with polymers

Definitions

  • the BACKGROUND Cancer is the leading cause of death around the globe accounting for about 15% of all deaths in 2020. In the U.S. ⁇ 600,000 people die of cancer every year (CDC). In 2024, the United States is facing an alarming rise in cancer incidence, set to surpass 2 million new cases. Prostate, breast, endometrial, pancreatic, kidney, and melanoma cancers contribute to this surge.
  • the nanoparticles comprise a C-type lectin receptor or a fragment thereof that is coupled to the surface of the nanoparticle, wherein the C-type lectin receptor or a fragment thereof binds an antigen expressed by a cancer cell.
  • the anticancer agent is encapsulated in the nanoparticle.
  • the C-type lectin receptor or fragment thereof is coupled to the surface of the nanoparticle by inserting the C-type lectin receptor or fragment thereof into the surface of the nanoparticle.
  • the C-type lectin receptor or fragment thereof is coupled to the surface of the nanoparticle by attaching the C-type lectin receptor or fragment thereof to the surface of the nanoparticle.
  • the C-type lectin receptor or fragment thereof is selected from the group consisting of Dectin-1 or a fragment PATENT ATTORNEY DOCKET NO.0U6500-1491008 (010WO1) thereof, Dectin-2 or a fragment thereof, Dectin-3 or a fragment thereof, and DC-SIGN or a fragment thereof.
  • the C-type lectin receptor or a fragment thereof is a soluble C- type lectin receptor or a fragment thereof.
  • the soluble C-type lectin receptor or a fragment thereof is a soluble human Dectin-1 comprising an amino acid sequence having at least 85%, 90%, 95%, or 99% identity to SEQ ID NO: 1, or a fragment thereof and is not a full-length Dectin-1 protein.
  • the soluble human Dectin-1 comprises SEQ ID NO: 1 or a fragment thereof and is not a full-length Dectin-1 protein.
  • the soluble C-type lectin receptor or a fragment thereof is a soluble human Dectin-2 comprising an amino acid sequence having at least 85%, 90%, 95%, or 99% identity to SEQ ID NO: 2, or a fragment thereof and is not a full-length Dectin-2 protein.
  • the soluble human Dectin-2 comprises SEQ ID NO: 2 or a fragment thereof and is not a full-length Dectin-2 protein.
  • the soluble C-type lectin receptor or a fragment thereof is a soluble human Dectin-3 comprising an amino acid sequence having at least 85%, 90%, 95%, or 99% identity to SEQ ID NO: 3, or a fragment thereof and is not a full-length Dectin-3 protein.
  • the soluble human Dectin-3 comprises SEQ ID NO: 3 or a fragment thereof and is not a full-length Dectin-3 protein.
  • the C-type lectin receptor comprises a Dendritic Cell-Specific Intercellular adhesion molecule-3-Grabbing Non-integrin (DC-SIGN) polypeptide or a fragment thereof.
  • DC-SIGN polypeptide is not a full-length DC- SIGN polypeptide.
  • the C-type lectin receptor fragment comprises a carbohydrate recognition domain (CRD).
  • the DC-SIGN polypeptide comprises a DC-SIGN CRD (SEQ ID NO: 21) and one or more neck regions of DC-SIGN selected from the group consisting of (NR1) SEQ ID NO: 22, (NR2) SEQ ID NO: 23, (NR3) SEQ ID NO: 24, (NR4) SEQ ID NO: 25, (NR5) SEQ ID NO: 26, (NR6) SEQ ID NO: 27, PATENT ATTORNEY DOCKET NO.0U6500-1491008 (010WO1) (NR7) SEQ ID NO: 28 and (NR8) SEQ ID NO: 29.
  • the DC-SIGN CRD (SEQ ID NO: 21) and the one or more neck regions are joined with one or more linkers.
  • the concentration of the anticancer agent is reduced as compared to the concentration of the anticancer agent in a nanoparticle that does not comprise a C-type lectin receptor or a fragment thereof coupled to the surface of the nanoparticle.
  • the antigen is a tumor-specific antigen.
  • the tumor-specific antigen is solid tumor antigen.
  • the solid tumor antigen is a breast tumor antigen, a prostrate tumor antigen or a lung tumor antigen.
  • the tumor-specific antigen is a glycan.
  • the cancer-targeting nanoparticle is a liposome.
  • a nanoparticle comprising: (a) a C-type lectin receptor or fragment thereof that binds an antigen on a cancer cell; and (b) a signal-generating molecule, wherein the C-type lectin receptor or fragment thereof is coupled to the surface of the nanoparticle and the signal-generating molecule generates a signal when the C-type lectin receptor or fragment thereof binds the antigen on the cancer cell.
  • the C-type lectin receptor or fragment thereof is selected from the group consisting of Dectin-1 or a fragment thereof, Dectin-2 or a fragment thereof, Dectin- 3, and DC-SIGN or a fragment thereof.
  • the signal-generating molecule is linked to the C-type lectin receptor or fragment thereof.
  • the C-type lectin receptor or a fragment thereof is a soluble C-type lectin receptor or a fragment thereof.
  • the soluble C-type lectin receptor or a fragment thereof is a human Dectin-1 comprising an amino acid sequence having at least 85%, 90%, 95%, or 99% identity to SEQ ID NO: 1, or a fragment thereof.
  • the soluble human Dectin-1 comprises SEQ ID NO: 1 or a fragment thereof.
  • the soluble C-type lectin receptor or a fragment thereof is a soluble human Dectin-2 comprising an amino acid sequence having at least 85%, 90%, 95%, or 99% identity to SEQ ID NO: 2, or a fragment thereof.
  • the soluble human Dectin-2 comprises SEQ ID NO: 2 or a PATENT ATTORNEY DOCKET NO.0U6500-1491008 (010WO1) fragment thereof.
  • the soluble C-type lectin receptor or a fragment thereof is a soluble human Dectin-3 comprising an amino acid sequence having at least 85%, 90%, 95%, or 99% identity to SEQ ID NO: 3, or a fragment thereof.
  • the soluble human Dectin-3 comprises SEQ ID NO: 3 or a fragment thereof.
  • the C-type lectin receptor comprises a Dendritic Cell-Specific Intercellular adhesion molecule-3-Grabbing Non-integrin (DC-SIGN) polypeptide or a fragment thereof.
  • DC-SIGN Dendritic Cell-Specific Intercellular adhesion molecule-3-Grabbing Non-integrin
  • the DC-SIGN polypeptide comprises a DC-SIGN CRD (SEQ ID NO: 21) and one or more neck regions of DC-SIGN selected from the group consisting of (NR1) SEQ ID NO: 22, (NR2) SEQ ID NO: 23, (NR3) SEQ ID NO: 24, (NR4) SEQ ID NO: 25, (NR5) SEQ ID NO: 26, (NR6) SEQ ID NO: 27, (NR7) SEQ ID NO: 28 and (NR8) SEQ ID NO: 29.
  • the DC-SIGN polypeptide comprises a DC- SIGN CRD (SEQ ID NO: 21), NR1 (SEQ ID NO: 22) and NR2 (SEQ ID NO: 23).
  • the DC-SIGN polypeptide has at least 90% identity to SEQ ID NO: 32.
  • the DC-SIGN polypeptide comprises a DC-SIGN CRD (SEQ ID NO: 21), NR7 (SEQ ID NO: 28) and NR8 (SEQ ID NO: 29).
  • the DC-SIGN polypeptide has at least 90% identity to SEQ ID NO: 30.
  • the DC-SIGN CRD (SEQ ID NO: 21) and the one or more neck regions are joined with one or more linkers.
  • the signal-generating molecule is incorporated into or attached to the surface of the nanoparticle.
  • the signal-generating molecule is a fluorescent dye or fluorescent polypeptide.
  • the C-type lectin receptor or fragment thereof is linked to the C-terminal and/or an N-terminal fragment of a fluorescent protein, an antibody or a fragment thereof or an enzyme.
  • the cancer-targeting nanoparticles described herein are cancer- targeting liposomes.
  • the liposome comprises from about 40 to about 70 mole percent phosphatidylcholine relative to total lipid content.
  • the phosphatidylcholine is fully hydrogenated soy phosphatidylcholine (18:0-18:1 PC, 1-stearoyl- 2-oleoyl-sn-glycero-3-phosphocholine).
  • the liposome comprises about 20% to 50% mole percent cholesterol relative to total lipid content. In some embodiments, the liposome comprises about 1 to about 6 mole percent polyethylene glycol (PEG) relative to total lipid content.
  • PEG polyethylene glycol
  • the PEG is mPEG-2000-DSPE (18:0 PEG2000 PE) (sodium;[(2R)-2,3-di(octadecanoyloxy)propyl] 2-(2-methoxyethoxycarbonylamino)ethyl phosphate).
  • the liposome comprises about 5 to 25 mole percent anti- cancer agent relative to total lipid content.
  • the liposome comprises about PATENT ATTORNEY DOCKET NO.0U6500-1491008 (010WO1) 0.3 to about 1.2 mole percent C-type lectin receptor or a fragment thereof relative to total lipid content. Also provided are pluralities of any of the cancer-targeting nanoparticles described herein.
  • a pharmaceutical composition comprising any of the pluralities of nanoparticles described herein. Uses of the pharmaceutical compositions for the treatment of cancer are also provided. Also provided is a method of treating or preventing cancer in a subject comprising administering to the subject having cancer or at risk of developing cancer an effective amount of a plurality of cancer-targeting nanoparticles described herein. Further provided is a method of treating or preventing cancer in a subject comprising administering to the subject having cancer or at risk of developing cancer, any of the pharmaceutical compositions described herein. In some embodiments, the subject has breast cancer, prostate cancer or lung cancer. In some methods, a second therapeutic agent or therapy is administered to the subject. In some methods, the second therapy is surgery, a second anticancer agent, and/or radiation.
  • the second therapeutic agent is a second anticancer agent.
  • the anticancer agent is a drug, a peptide or an antibody.
  • the drug is a chemotherapeutic drug.
  • the chemotherapeutic drug is doxorubicin. Also provided is a method for detecting cancer in a subject or a sample from a subject comprising: (a) contacting the subject or a sample from the subject with the plurality of any of the nanoparticles comprising a C-type lectin receptor or fragment thereof that binds an antigen on a cancer cell; and a signal-generating molecule, and (b) detecting a signal, wherein a signal indicates the presence of cancer.
  • the signal is a fluorescent signal. In some embodiments, the signal is directly or indirectly detected. DESCRIPTION OF THE FIGURES
  • the present application includes the following figures. The figures are intended to illustrate certain embodiments and/or features of the compositions and methods, and to supplement any description(s) of the compositions and methods. The figures do not limit the scope of the compositions and methods, unless the written description expressly indicates that such is the case.
  • FIG.1 is a schematic of a Dectin-2 (DEC2)-targeted doxorubicin (DOX) loaded lipid nanoparticle, DEC2-DOX-LL, shown binding to a cognate glycoprotein ligand on the surface of a HR-negative breast cancer cell.
  • DEC2 Dectin-2
  • DOX doxorubicin
  • a DOX-loaded liposome is PATENT ATTORNEY DOCKET NO.0U6500-1491008 (010WO1) coated with the glycan recognition domain and stalk region of Dectin-2.
  • the Dectin-2 monomers (purple) are tethered to the liposome via the DSPE moiety of a DEC2-PEG-DSPE conjugate. They float freely on the liposomal membrane and form dimers as they bind cognate ligands.
  • the liposomes are also coated with red fluorescent rhodamine B (red star) via a DHPE moiety to monitor their binding to cells.
  • the crystal structure of DOX (also red) is shown inside of the liposome.
  • the mole percent ratios of liposomal lipid:Dectin- 2:rhodamine B:DOX are 100:1:2:16, respectively.
  • FIGS. 2A-H shows that exemplary Dectin-1, -2 and -3 coated liposomes (DEC1-Ls, DEC2-Ls and DEC3-Ls, respectively) bound specifically and quantitatively to triple negative breast cancer cell line MDA-MB-231, while untargeted control liposomes did not.
  • DCSIGN DCS12-Ls did not bind significantly. However, this was likely due to a defective preparation because, in subsequent replicate experiments with a different preparation, the DCS12-Ls did bind to these cells.
  • FIGS. 3A-G show that exemplary Dectin-2, Dectin-2 and Dectin-3 coated liposomes DEC1-Ls, DEC2-Ls and DEC3-Ls bind efficiently to the prostate cancer cell line PC3, which was derived from a prostate bone marrow adenocarcinoma. Representative images reveal CTL targeted liposome bound to in vitro grown cells.
  • a scatter bar plot quantifies the area of red fluorescent liposome binding in pixels and p values relative to the BSA-L controls are indicated.
  • N 10 randomly photographed images quantified for each bar. Photographs were taken at 10X magnification, combining phase & red fluorescence. Zero red pixel values were set to 5 pixels to allow log 10 plotting of data.
  • FIGS.4A-G shows that exemplary DEC1-Ls, DEC2-Ls and DEC3-Ls bound efficiently to the lung cancer cell line A549 cell line, which was derived from a lung carcinoma.
  • A,B,C,D Representative images of CTL targeted liposome binding in vitro.
  • E & F No significant binding was detected for two untargeted liposome controls, BSA-Ls or uncoated Ls.
  • the liposomal protein concentration during staining for the five protein coated liposomes was 1 ug/200 uL.
  • G A scatter bar plot quantifies the area of red fluorescent liposome binding in pixels and p values relative to the BSA-L controls are indicated.
  • N 10 randomly photographed images quantified for each bar. Cell nuclei were stained with Hoechst. Photographs were taken at 10X magnification, combining blue & red fluorescence. Zero red pixel values were set to 5 to allow p-value estimates and log 10 plotting of data.
  • FIGS. 5A-G show that binding to artificially immortalized human kidney embryonic HEK293T control cells was relatively weak and of low statistical significance. A,B,C,D.
  • FIGS.6A-G show that exemplary DEC1-Ls, DEC2-Ls and DEC3-Ls bound efficiently and strongly to the triple positive breast cancer cell line MCF7.
  • A,B,C,D Representative images of CTL targeted liposome binding in vitro.
  • DEC1-Ls, DEC2-Ls and DEC3-Ls bound efficiently and significantly when compared to BSA-Ls.
  • E & F Little binding was detected for two untargeted liposome controls, BSA-Ls or uncoated Ls. The liposomal protein concentration during staining for the five protein coated liposomes was 1 ug/100 uL.
  • G A scatter bar plot quantifies the area of red fluorescent liposome binding in pixels and p values relative to the BSA-L PATENT ATTORNEY DOCKET NO.0U6500-1491008 (010WO1) controls are indicated.
  • N 10 randomly photographed images quantified for each bar using data from the red channel. Photographs were taken at 10X magnification, combining blue & red fluorescence.
  • FIGS. 7A-C show cell killing of triple negative breast cancer cells by C-type lectin receptor targeted liposomes and DOX in the nucleus.
  • MDA-MB-231 cells were treated with DOXIL® and DOXIL targeted by four different C-type lectin receptor polypeptides (CTLs) and assayed for metabolic activity after three days treatment.
  • CTLs C-type lectin receptor polypeptides
  • FIGS. 8A-E show human DCIS-H samples in a BrCaProg3 tissue array stained with DEC2-DOX-LLs, DOX-LLs, and H&E. (A) DEC2-DOX-LL staining of DCIS-H tissue.
  • C DEC2-DOX-LL staining of non-cancerous breast tissue with ductal morphology (NB-NC). The top row contains the merged red fluorescent liposome and blue, fluorescent DAPI stained images. The bottom row shows the red fluorescent liposome channel alone. Exposures in the red channel were at 100% light intensity for 220 msec and in the blue channel were at 22% light intensity for 5 msec.
  • E Quantification of liposome binding.
  • FIGS.9A-D show HuCAT292 patient TNBC tumor sections stained with DEC2-DOX- LLs, DOX-LLs, and H&E.
  • A DEC2-DOX-LL staining of FFPE section of HuCAT292 tumor.
  • B DOX-LL staining of HuCAT292 same tumor.
  • C H&E staining of an adjacent section from the same tumor. The top row of A & B contains the merged red fluorescent liposome and blue, fluorescent DAPI stained images. The bottom row shows the red fluorescent liposome channel alone.
  • FIGS.10A-B show that DEC2-DOX-LLs labeled the metastatic triple negative breast cancer (TNBC) cell line MDA-MB-468 far more efficiently than untargeted DOX-LLs.
  • TNBC metastatic triple negative breast cancer
  • LBD4 liposome dilution buffer 4
  • FIGS.11A-B show that DEC2-DOX-LLs labeled the metastatic triple negative breast cancer (TNBC) cell line MDA-MB-231 far more efficiently than untargeted DOX-LLs. Staining and photographic conditions are provided in the legend for FIGS 10A-B.
  • FIGS.12A-B show that DEC2-DOX-LLs labeled the metastatic HER2 overexpressing breast cancer cell line SKBR3 far more efficiently than untargeted DOX-LLs. Staining and photographic conditions are provided in the legend for FIGS 10A-B.
  • FIGS. 13A-B show that DEC2-DOX-LLs labeled the metastatic non-small cell lung carcinoma (NSCLC) cell line A549 far more efficiently than untargeted DOX-LLs. Staining and photographic conditions are provided in the legend for FIGS 10A-B.
  • LLB2 liposome dilution buffer 2
  • BME beta-mercaptoethanol
  • Nanoparticle Delivery of Anti-Cancer Agents Although nanoparticles have emerged as a technology for delivering therapeutic agents to cells, and some have been approved for clinical use, the translation of nanotechnology for targeting and treating cancer in human patients has been limited.
  • the present disclosure provides cancer-targeted nanoparticles that overcome longstanding difficulties in cancer drug delivery.
  • the cancer- targeted nanoparticles described herein can be used to specifically deliver an anti-cancer agent to cancer cells in a subject and/or enhance accumulation of the anti-cancer agent in the targeted cancer cells (e.g., in or on a tumor), while reducing side effects in the subject.
  • Nanoparticles for the diagnosis, treatment or prevention of cancer.
  • nanoparticles can be, but are not limited to, lipid nanoparticles, for example, liposomes or non-liposomal lipid nanoparticles (for example, lipid nanoparticles with a non-aqueous core (LNPs)), dendrimers, polymeric micelles, nanocapsules or nanospheres, to name a few.
  • LNPs non-aqueous core
  • the nanoparticles described herein comprise a cancer- targeting molecule, for example, a polypeptide or a fragment thereof, that targets the nanoparticle to an antigen expressed on cancer cells.
  • the nanoparticle is a cancer-targeting nanoparticle comprising a C-type lectin receptor or a fragment thereof that is coupled to the surface of the nanoparticle, wherein the C-type lectin receptor or a fragment thereof targets an antigen expressed by a cancer cell.
  • the cancer-targeting molecule targets the antigen on a cancer cell by specifically or selectively binding to the antigen.
  • the terms specifically bind or selectively binds mean binding that is measurably different from a non-specific or non- selective interaction.
  • Specific binding can be measured, for example, by determining binding of a molecule to a target antigen compared to binding of a control molecule.
  • Specific binding can be determined by competition with a control molecule that is similar to the target antigen, such as an excess of non-labeled target antigen. In that case, specific binding is indicated if the binding of the labeled target to a probe is competitively inhibited by the excess unlabeled target antigen.
  • the target antigen is a cell surface antigen, for example, a tumor-specific antigen (e.g., a tumor-specific protein or a tumor-specific glycan).
  • the tumor-specific protein or tumor-specific glycan is a solid tumor antigen, for example, and not to be limiting, a breast tumor antigen, a prostrate tumor antigen or a lung tumor antigen.
  • a tumor specific antigen is a protein or other molecule (for example, a glycan) that is found only on cancer cells and not found to any appreciable extent on normal cells.
  • the tumor specific antigen is involved in neoblastic transformation of cells.
  • a cancer-targeting molecule for example, a C-type lectin receptor or a fragment thereof
  • a C-type lectin receptor or fragment thereof can be incorporated into the surface (e.g., the outer bilayer) of a liposome or attached to a liposome.
  • Incorporation can occur by insertion or intercalation of the cancer- PATENT ATTORNEY DOCKET NO.0U6500-1491008 (010WO1) targeting molecule, e.g., a C-type lectin receptor or a fragment thereof, into the lipid bilayer.
  • Attachment to a liposome can occur, for example, by affinity to a molecule incorporated into the outer lipid bilayer of the liposome.
  • the liposome can be coated with biotin (for example, DSPE-PEG-biotin inserted into the lipid bilayer) and the C-type lectin receptor or a fragment thereof linked to streptavidin.
  • the C-type lectin receptor or a fragment thereof can be linked or conjugated to a lipid carrier (e.g., DSPE-PEG) prior to insertion of the C-type lectin receptor or a fragment thereof, via the DSPE lipid moiety, into the outer surface of the liposome.
  • a C-type lectin receptor or a fragment thereof can also be conjugated to a nanoparticle by a number of methods known in the art (e.g., Arruebo et al. “Antibody-Conjugated Nanoparticles for Biomedical Applications,” Journal of Nanomaterials vol. 2009, Article ID 439389 (2009)).
  • Liposomes can also be conjugated to cancer-targeting molecules via a streptavidin/biotin bond, thiol/maleimide chemistry, azide/alkyne chemistry, tetrazine/cyclooctyne chemistry, and other click chemistries. These chemical handles are prepared either during phosphoramidite synthesis or post-synthesis.
  • click chemistry refers to biocompatible reactions intended primarily to join substrates of choice with specific biomolecules. Click chemistry reactions are not disturbed by water, generate minimal and non-toxic byproducts, and are characterized by a high thermodynamic driving force that drives it quickly and irreversibly to high yield of a single reaction product, with high reaction specificity.
  • Exemplary human C-Type Lectin receptor polypeptides that can target nanoparticles to cancer cells, for example, by binding an antigen(s) on cancer cells include, but are not limited to, Dectin-1, Dectin-2, Dectin-3, and DC-SIGN.
  • the C-Type Lectin polypeptide or a fragment thereof comprises a carbohydrate recognition domain (CRD) and is not a full-length C-type lectin receptor (e.g., not a full-length Dectin-1, Dectin-2, Dectin-3 or DC-SIGN polypeptide). Fragments of Dectin-1, Dectin-2, Dectin-3, or DC-SIGN, for example fragments comprising a CRD, can also be coupled to the surface of any of the nanoparticles described herein. Table 1 sets forth the corresponding UniProt Nos. for the amino acid sequences of these exemplary C-type Lectin polypeptides.
  • amino acid sequences set forth in the Uniprot Nos of Table 1 are incorporated herein by reference.
  • Amino acid sequences having at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% sequence identity with the amino acids sequences set forth in the Uniprot Nos of Table 1 are PATENT ATTORNEY DOCKET NO.0U6500-1491008 (010WO1) also provided.
  • the amino acid sequence is a cancer cell binding protein sequence that has at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% sequence identity with the amino acids sequences set forth in the Uniprot Nos of Table 1.
  • C-type Lectins Exemplary C-type lectin polypeptides that bind to cancer cells include but are not limited to soluble human Dectin-1, Dectin-2 and Dectin-3 polypeptides comprising SEQ ID NO: 1, 2, and 3, respectively.
  • Other exemplary targeting molecules include but are not limited to soluble mouse Dectin-1, Dectin-2 and Dectin-3 polypeptides comprising SEQ ID NO: 4, 5, 6, respectively.
  • Fragments of SEQ ID NOs: 1, 2, 3, 4, 5, or 6, for example, fragments comprising a deletion of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acids from the C-terminal and/or N-terminal end of a polypeptide comprising or consisting of SEQ ID NOs: 1, 2, 3, 4, 5, PATENT ATTORNEY DOCKET NO.0U6500-1491008 (010WO1) or 6, are also provided and can be used in any of the nanoparticles (e.g. liposomes), polypeptides or compositions described herein.
  • Other exemplary constructs that can be used in any of the nanoparticles described herein are set forth as follows.
  • SEQ ID NO: 7 is a nucleic acid sequence encoding an exemplary codon optimized soluble mouse Dectin-1 (sDectin-1).
  • a vector pET-45b+ sequence of 9 codons is boxed with the start codon underlined.
  • the mouse sDectin-1 sequence (CLEC7A, GenBank No. AAS37670.1) is shown in plain text; an Ala codon GCT and stop codons TAA and TTA are underlined, with stop codons in bold.
  • Alternative gene name MmsDectin1lyshis Alternative gene name MmsDectin1lyshis.
  • the length of the nucleotide sequence is 604 base pairs, with 597 base pairs encoding a protein of 199 amino acids in length.
  • the N-terminal amino acid sequence and (His) 6 (HHHHHH) (SEQ ID NO: 37) affinity tag is boxed.
  • the Gly Ser (GS) flexible linker residues and reactive lys (K) residues appear in bold with lysines in italic.
  • Mouse sDectin-1 amino acid residues appear in plain text, ending in a C-terminal Ala residue (A) in bold, the PATENT ATTORNEY DOCKET NO.0U6500-1491008 (010WO1) codon for which was used to put stop codons and a PacI site in frame.
  • a stop codon in any of the polypeptide sequences disclosed herein can be removed, to produce a polypeptide that does not include one or more stop codons.
  • the protein comprising the mouse sDectin-1 polypeptide is 199 amino acids in length, with a molecular weight (MW) of 22,389.66 g/mole. The theoretical pI is 7.74. It is understood that any protein described herein comprising an affinity tag, for example, a (His)6 affinity tag, can be modified to remove the His tag. Therefore, polypeptides (i.e.
  • any C-type lectin receptor or fragment thereof or DC- SIGN polypeptide or fragment thereof) that do not comprise a histidine tag are provided herein.
  • any nucleotide sequence described herein can further comprise a protease cleavage site for post-translational and/or post-purification removal of the affinity tag.
  • the soluble mouse Dectin-1 polypeptide comprises amino acids 23-198 of SEQ ID NO: 8 or amino acids 23-199 of SEQ ID NO: 8.
  • the soluble mouse Dectin-1 polypeptide comprises SEQ ID NO: 46 MAHHHHHHYGTGSGKGKGSGSGFWRHNSGRNPEEKDSFLSRNKENHKPTESSLDE KVAPSKASQTTGGFSQSCLPNWIMHGKSCYLFSFSGNSWYGSKRHCSQLGAHLLKID NSKEFEFIESQTSSHRINAFWIGLSRNQSEGPWFWEDGSAFFPNSFQVRNAVPQESLL HNCVWIHGSEVYN
  • the vector pET-45b+ sequence of 9 codons is boxed with the start codon underlined.
  • Sites for cloning into pET-45B+ KpnI (GGTACC)(SEQ ID NO: 38) and PacI (TTAATTAA) (SEQ ID NO: 36), respectively, are underlined. Codons for Gly Ser (G,S) flexible linker residues appear in bold and the codons for reactive lys (K) residues (AAG) appear in bold, with lysine codons in italic.
  • Codon optimized sDectin-2 from the CLEC6A mouse Dectin 2 gene appears in plain text, with an Ala codon (GCT) and stop codons, TAA and TTA, underlined and stop codons in bold.
  • the alternative gene name is MmsDectin2lyshis.
  • the length of the nucleic acid sequence is 574 base pairs, PATENT ATTORNEY DOCKET NO.0U6500-1491008 (010WO1) with 567 base pairs encoding a protein that is 190 amino acids in length.
  • the nucleic acid encoding the codon-optimized mouse sDectin-2 exemplary was cloned into pET-45B+.
  • This polypeptide comprises a mouse sDectin-2 protein.
  • the N terminal amino acid and (His) 6 (HHHHHH)(SEQ ID NO: 37) affinity tag from pET-45B+ is boxed.
  • the Gly Ser (GS) flexible linker residues and reactive lys (K) residues appear in bold, with lysines in italic.
  • Mouse sDectin-2 amino acid residues appear in plain text ending in a C-terminal Ala residue (A) (bold), the codon for which was used to put stop codons and PacI site in frame.
  • the polypeptide comprising the mouse sDectin-2 that is produced has 189 amino acids, with a MW of 21,699.25 g/mole and a theoretical pI of 6.33.
  • the soluble mouse Dectin-2 polypeptide comprises amino acids 23-188 of SEQ ID NO: 10, or amino acids 23-189 of SEQ ID NO: 10.
  • the soluble mouse Dectin-2 polypeptide comprises SEQ ID NO: 47 MAHHHHHHYGTGSGKGKGSGSGIMDQPSRRLYELHTYHSSLTCFSEGTMVSEKMW GCCPNHWKSFGSSCYLISTKENFWSTSEQNCVQMGAHLVVINTEAEQNFITQQLNES LSYFLGLSDPQGNGKWQWIDDTPFSQNVRFWHPHEPNLPEERCVSIVYWNPSKWG WNDVFCDSKHNSICEMKKIYLA (SEQ ID NO: 10)
  • the soluble mouse Dectin-2 polypeptide comprises SEQ ID NO: 47 MAHHHHHHYGTGSGKGKGSGSGIMDQPSRRLYELHTYHSSLTCFSEGTMVSEKMW GCCPNHWKSFGSSCYLISTKENFWSTSEQNCVQMGAHLVVINTEAEQNFITQQLNES LSYFLGLSDPQGNGKWQWIDDTPFSQNVRFWHPHEPNLPEERCVSIVYWNPSKWG WNDVF
  • NP_034949.3 is shown in plain text, with an Ala codon (GCT) and stop codons TAA and TTA underlined. Stop codons are shown in bold.
  • the alternative gene name is MmsDectin3lyshis.
  • the length of the nucleotide sequence is 604 base pairs, with 597 base pairs encoding a protein that is 199 amino acids in length.
  • the nucleic acid encoding the exemplary codon-optimized mouse sDectin-3 was cloned into pET-45B+.
  • This polypeptide comprises a mouse sDectin-3 protein.
  • the N terminal amino acid and (His) 6 (HHHHHH)(SEQ ID NO: 37) affinity tag from pET-45B+ is boxed.
  • Gly Ser (GS)flexible linker residues and reactive lys (K) residues are shown in bold, with lysines in italic.
  • Mouse sDectin-3 amino acid residues are shown in plain text (amino acids 23-199 of SEQ ID NO: 12), ending in a C-terminal Ala residue (A) in bold, the codon for which was used to put stop codons and PacI site in frame.
  • the polypeptide is 199 amino acids in length with a MW of 23,023.72 g/mole and a theoretical pI or 6.52.
  • the soluble mouse Dectin-3 polypeptide comprises amino acids 23-198 of SEQ ID NO: 12 or amino acids 23-199 of SEQ ID NO: 12.
  • the soluble mouse Dectin-3 polypeptide comprises SEQ ID NO: 48 MAHHHHHHYGTGSGKGKGKGSGSGHYFLRWTRGSVVKLSDYHTRVTCIREEPQPGAT GGTWTCCPVSWRAFQSNCYFPLNDNQTWHESERNCSGMSSHLVTINTEAEQNFVTQ LLDKRFSYFLGLADENVEGQWQWVDKTPFNPHTVFWEKGESNDFMEEDCVVLVHV HEKWVWNDFPCHFEVRRICKLPGITFNWKPSKA (SEQ ID NO: 48) SEQ ID NO: 13
  • the human sDectin-1 DNA sequence is expressed from vector pET-45B+.
  • the vector pET-45b+ sequence and His tag of 9 codons is boxed with the start codon underlined.
  • Cloning sites BamHI (GGATCC)(SEQ ID NO: 38) and PacI (TTAATTAA)(SEQ ID NO: 36), respectively, are underlined. Codons for enterokinase processing site in lower case font, Codons for Gly Ser (G,S) flexible linker residues and reactive lys (K) residues (AAA and AAG) are shown in bold with lysine codons in italic.
  • the human sDectin-1 sequence (CLEC7A, GenBank Accession No.
  • NM_197947 is shown in plain text, codon optimized for expression.
  • An alternate name for this sequence is HssDectin1lyshis.
  • the nucleotide sequence encoding human sDectin-1 has a length of 649 base pairs, encoding a polypeptide that is 214 amino acids in length.
  • the nucleic acid encoding the exemplary codon-optimized human sDectin-1 was cloned into pET-45B+.
  • SEQ ID NO: 14 is an amino acid sequence encoded by SEQ ID NO: 13. This is a polypeptide comprising a human sDectin-1 protein. The N-terminal amino acid and (His)6 (HHHHHH)(SEQ ID NO: 37) affinity tag from pET-45B+ is boxed. The enterokinase processing site in lower case font. The Gly Ser (GS)flexible linker residues and reactive lys (K) residues are shown in bold, with lysines in italic.
  • Human sDectin-1 amino acid residues are shown in plain text, ending in a C-terminal Ala residue (A) in bold, the codon for which was used to put stop codons and PacI site in frame.
  • the polypeptide is 214 amino acids in length, with a MW of 23,703.20 g/mole and a theoretical pI of 6.22.
  • the soluble human Dectin-1 polypeptide fragment comprises amino acids 35-213 of SEQ ID NO: 14 or amino acids 35-214 of SEQ ID NO: 14.
  • a polypeptide comprising a human sDectin-1 protein comprises SEQ ID NO: 40 or a fragment thereof (for example, amino acids 23-201 of SEQ ID NO: 40, or amino acids 23-202 of SEQ ID NO: 40).
  • a polypeptide comprising a human sDectin-1 protein comprises SEQ ID NO: 43 or a fragment thereof. The membrane spanning and/or stalk region of human Dectin-1 is underlined.
  • SEQ ID NO: 15 is a nucleic acid sequence encoding an exemplary codon optimized soluble human Dectin-2 (sDectin-2) (SEQ ID NO: 16).
  • the human sDectin-2 nucleotide sequence is expressed from vector pET-45B+.
  • the length of the nucleotide sequence is about 616 base pairs with 580 base pairs encoding a protein of 203 amino acids in length.
  • the vector pET-45b+ sequence of 9 codons including the His tag is boxed with the start codon underlined.
  • Codons for Gly Ser (G,S) flexible linker residues are shown in bold, and reactive lys (K) residues (AAG) are shown in bold, with lysines in italic.
  • Codon optimized sDectin-2 from the CLEC6A human Dectin 2 gene (cDNA GenBank Accession No. NM_001317999) is shown in plain text.
  • An Ala codon (GCT) and stop codons, TAA and TTA, are underlined, with stop codons shown in bold.
  • the alternative gene name is HssDectin2lyshis.
  • the nucleic acid encoding the codon-optimized human sDectin-2 exemplary was cloned into pET-45B+.
  • This polypeptide comprises a human sDectin-2 protein.
  • the N-terminal amino acid and (His) 6 (HHHHHH)(SEQ ID NO: 37) affinity tag from pET-45B+ are boxed.
  • the enterokinase processing site is in lower case font.
  • Gly Ser (GS) flexible linker residues and reactive lys (K) PATENT ATTORNEY DOCKET NO.0U6500-1491008 (010WO1) residues are shown in bold, with lysines in italic.
  • Human sDectin-2 amino acid residues are shown in plain text (GenBank Accession No.
  • NP_001007034.1 amino acids 36-203 of SEQ ID NO: 10
  • A C-terminal Ala residue
  • the polypeptide is 203 amino acids in length, with a MW of 22,969 g/mole and a theoretical pI of 5.91.
  • the soluble human Dectin-2 polypeptide fragment comprises amino acids 35-202 of SEQ ID NO: 16 or amino acids 35-203 of SEQ ID NO: 16.
  • a polypeptide comprising a human sDectin-2 protein comprises SEQ ID NO: 41 or a fragment thereof (for example, amino acids 23-186 of SEQ ID NO: 41, or amino acids 23-187 of SEQ ID NO: 41).
  • a polypeptide comprising a human sDectin-2 protein comprises SEQ ID NO: 44 or a fragment thereof.
  • the membrane spanning and/or stalk region of human Dectin-2 is underlined.
  • SEQ ID NO: 17 is a nucleic acid sequence encoding an exemplary codon optimized soluble human Dectin-3 (sDectin-3) (SEQ ID NO: 18).
  • the human sDectin-3 DNA sequence is expressed from vector pET-45B+ in E. coli.
  • the vector pET-45b+ sequence of 9 codons with hist tag is boxed, with the start codon underlined.
  • Sites for cloning into pET-45b+ BamHI (GGATCC)(SEQ ID NO: 39) and PacI (TTAATTAA)(SEQ ID NO: 36), respectively, are underlined.
  • Codons for enterokinase processing site are in lower case font.
  • PATENT ATTORNEY DOCKET NO.0U6500-1491008 (010WO1) Codons for Gly Ser (G,S) flexible linker residues are shown in bold and reactive lys (K) residues (AAG) are shown in bold, with lysines in italic.
  • Codon optimized sDectin-3 from the CLEC4D human Dectin-3 gene (GenBank Accession NM_080387) is shown in plain text.
  • An Ala codon (GCT) and stop codons, TAA and TTA, are underlined, with stop codons in bold.
  • the alternative gene name is HssDectin3lyshis.
  • the nucleotide sequence has a length of 628 base pairs, encoding a polypeptide of 207 amino acids in length.
  • the nucleic acid encoding the exemplary codon-optimized human sDectin-3 was cloned into pET-45B+.
  • This polypeptide comprises the human Dectin-3 protein.
  • the N-terminal amino acid and (His)6 (HHHHHH)(SEQ ID NO: 37) affinity tag from pET-45B+ is boxed.
  • the enterokinase processing site is in lower case font.
  • the Gly Ser (GS)flexible linker residues and reactive lys (K) residues are shown in bold, with lysines in italic.
  • the human sDectin-3 amino acid residues (GenBank Accession No.
  • NP_525126 are shown in plain text (amino acids 35-207 of SEQ ID NO: 12), ending in a C-terminal Ala residue (A) in bold, the codon for which was used to put stop codons and a PacI site in frame.
  • the protein is 207 amino acids in length with a MW of 23,662 g/mole and a theoretical pI of 7.64.
  • the soluble human Dectin-3 polypeptide fragment comprises amino acids 35-206 of SEQ ID NO: 18.
  • a polypeptide comprising a human sDectin-3 protein comprises SEQ ID NO: 42 or a fragment thereof (for example, amino acids 23-194 of SEQ ID NO: 42, or amino acids 23-195 of SEQ ID NO: 42).
  • a polypeptide comprising a human sDectin-3 protein comprises SEQ ID NO: 45 or a fragment thereof. The membrane spanning and/or stalk region of human Dectin-3 is underlined.
  • the targeting molecule is a Dendritic Cell-Specific Intercellular adhesion molecule-3-Grabbing Non-integrin (DC-SIGN) polypeptide or a fragment thereof comprising a carbohydrate recognition domain (CRD), and wherein the targeting molecule is incorporated into the outer surface of the nanoparticle.
  • DC-SIGN Dendritic Cell-Specific Intercellular adhesion molecule-3-Grabbing Non-integrin
  • DC- SIGN Dendritic Cell-Specific Intercellular adhesion molecule-3-Grabbing Non-integrin
  • DC- SIGN Dendritic Cell-Specific Intercellular adhesion molecule-3-Grabbing Non-integrin
  • DC- SIGN is a type II membrane protein, with a single CRD, expressed on the surface of immature dendritic cells (DCs), involved in initiation of the primary immune response.
  • Human DC-SIGN is a 404 amino acid residue polypeptide that comprises an N-terminal cytoplasmic tail, a transmembrane domain, and an extracellular domain comprising eight neck regions (NR1-NR8) and a carbohydrate recognition domain (CRD).
  • the full-length sequence of DC-SIGN is set forth under UniProtKB No. Q9NNX6, and set forth herein as SEQ ID NO: 19.
  • the CDR and one or more neck regions of DC-SIGN can be joined with or without a linker having about 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acids.
  • linkers include but are not limited to GSG n wherein n is an integer, and GSGSG (SEQ ID NO: 49).
  • Any of the DC-SIGN polypeptides provided herein can also be linked to a tag, for example, a histidine tag, as described below.
  • the DC-SIGN polypeptide fragment comprises, consists essentially of, or consists of, a DC-SIGN CRD (SEQ ID NO: 21), NR7 (SEQ ID NO: 28) and NR8 (SEQ ID NO: 29).
  • SEQ ID NO: 30 is an exemplary sequence comprising a DC-SIGN CRD (SEQ ID NO: 21), NR7 (SEQ ID NO: 28) and NR8 (SEQ ID NO: 29). Fragments of SEQ ID NO: 30 are also provided.
  • SEQ ID NO: 31 encodes the amino acid sequence of SEQ ID NO: 30.
  • the fragment comprises, consists essentially of, or consists of, a DC-SIGN (CRD) SEQ ID NO: 21, NR1 (SEQ ID NO: 22) and NR2 (SEQ ID NO: 23).
  • the fragment comprises, consists essentially of, or consists of, SEQ ID NO: 32 (DSC12 construct) as set PATENT ATTORNEY DOCKET NO.0U6500-1491008 (010WO1) forth below.
  • SEQ ID NO: 32 SEQ ID NO: 32
  • An N terminal amino acid and (His)6 (HHHHHH) (SEQ ID NO: 37) affinity tag from a pET-45B+ vector is boxed.
  • Gly Ser (GS)flexible linker residues and reactive lys (K) residues for linking to lipid carrier are in bold with lysines in italic.
  • Human DC-SIGN amino acid residues appear in plain text and includes 2x23 a.a.
  • SEQ ID NO: 32 is another exemplary sequence comprising a DC-SIGN (CRD) SEQ ID NO: 1, NR1 (SEQ ID NO: 2) and NR2 (SEQ ID NO: 3). Fragments of SEQ ID NO: 32 are also provided.
  • SEQ ID NO: 33 encodes SEQ ID NO: 32.
  • DC-SIGN polypeptides include, but are not limited to: • a polypeptide comprising, consisting essentially of, or consisting of a DC-SIGN CRD (SEQ ID NO: 21), (NR1) SEQ ID NO: 22, (NR2) SEQ ID NO: 23, (NR3) SEQ ID NO: 24, (NR4) SEQ ID NO: 25, (NR5) SEQ ID NO: 26, (NR6) SEQ ID NO: 27, (NR7) SEQ ID NO:
  • nucleic acid refers to deoxyribonucleic acids (DNA) or ribonucleic acids (RNA) and polymers thereof in either single- or double-stranded form. Unless specifically limited, the term encompasses nucleic acids containing known analogues of natural nucleotides that have similar binding properties as the reference nucleic acid and are metabolized in a manner similar to naturally occurring nucleotides.
  • nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (e.g., degenerate codon substitutions), alleles, orthologs, SNPs, and complementary sequences as well as the sequence explicitly indicated.
  • degenerate codon substitutions may be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed-base and/or deoxyinosine residues (Batzer et al., Nucleic Acid Res. 19:5081 (1991); Ohtsuka et al., J. Biol. Chem. 260:2605-2608 (1985); and Rossolini et al., Mol. Cell.
  • Probes 8:91-98 (1994) In each case, where specific nucleic acid or polypeptide sequences are recited, embodiments comprising a sequence having at least 70% (e.g., 70%, 75%,80%, 85%. 90%, 95%, 99%) identity to the recited sequence are also provided. Identity or similarity with respect PATENT ATTORNEY DOCKET NO.0U6500-1491008 (010WO1) to a sequence is defined as the percentage of amino acid residues in the candidate sequence that are identical (i.e., same residue) with the starting amino acid residues, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity.
  • Identity or similarity with respect PATENT ATTORNEY DOCKET NO.0U6500-1491008 (010WO1) to a sequence is defined as the percentage of amino acid residues in the candidate sequence that are identical (i.e., same residue) with the starting amino acid residues, after aligning the sequences and introducing gaps, if necessary, to achieve the
  • polypeptide and nucleic acid sequences having at least 70% e.g., 70%, 75%, 80%, 85%. 90%, 95%, 99%
  • polypeptides and nucleic acid sequences having at least 60% e.g. 60%, 65%, 70%, 75%, 80%, 85%. 90%, 95%, 99%
  • sequences having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity to any one of SEQ ID NOs: 1-48 can be used in any of the nanoparticles (for example, liposomes) described herein.
  • Nucleic acids encoding the polypeptides described herein are also provided.
  • Methods of alignment of sequences for comparison are well known in the art. Optimal alignment of sequences for comparison can be conducted, for example, by the local homology algorithm of Smith and Waterman (Adv. Appl. Math.2:482, 1970), by the homology alignment algorithm of Needleman and Wunsch (J. Mol. Biol.48:443, 1970), by the search for similarity method of Pearson and Lipman (Proc. Natl.
  • the hydropathic index of amino acids may also be considered.
  • the importance of the hydropathic amino acid index in conferring interactive biologic function on a protein is generally understood in the art (Kyte and Doolittle; (1982) J Mol Biol. 157(1):105-32). It is accepted that the relative hydropathic character of the amino acid contributes to the secondary structure of the resultant protein, which in turn defines the interaction of the protein with other molecules, for example, enzymes, substrates, receptors, DNA, antibodies, antigens and the like.
  • a liposome can have a size of about 50 nm to about 1000 nm, about 50 nm to about 900 nm, about 50 nm to about 800 nm, about 50 nm to about 700 nm, about 50 nm to about 600 nm, about 50 nm to about 500 nm, about 50 nm to about 400 nm, about 50 nm to about 300 nm, about 50 nm to about 200 nm, or about 50 nm to about 100 nm. Also provided are pluralities or populations of liposomes, wherein the liposomes in the plurality have an average size of about 1000 nanometers (nm) or less.
  • the liposomes in the plurality can have an average size of about 75 nm to 120 nm, about 75 nm to about 110 nm, or about 75 nm to about 100 nm. In some embodiments, the liposomes in the plurality can have am average size of about 50 nm to about 100 nm, about 50 nm to 95 nm, about 50 nm to about 90 nm, about 50 nm to about 85 nm, 50 nm to about 80 nm, about 50 nm to 75 nm, about 70 nm to about 90 nm, or about 50 nm to about 75 nm.
  • the anticancer or antineoplastic agent can be, but is not limited to a drug, an antisense oligonucleotide, a shRNA, a siRNA, a mRNA, a peptide, an aptamer, a drug, or a small molecule, to name a few.
  • drug loaded liposomes for example, DOX- loaded liposomes
  • DOX- loaded liposomes can be made by using a transmembrane ammonium sulfate pH gradient.
  • newly formed dehydrated liposomes can be hydrated in a highly pH basic 250 mM solution of (NH4) 2 SO 4 , such that this basic ammonium salt is both in the liposomal lumen and in the media surrounding the liposomes.
  • doxorubicin is added to this medium. Because doxorubicin is a weak base and positively charged at pH 6.5, it moves into the liposomes and is exchanged for ammonium ion as it moves out into the media (Haran et al., Biochim Biophys Acta, 1151, 201-15 (1993); Mayer et al., Cancer Lett, 53, 183-90 (1990)). Doxorubicin crystalizes as it is concentrated in the lumen of the liposomes, removing it from the equilibrium formula, which further enhances uptake.
  • liposomes with high concentrations of luminal PATENT ATTORNEY DOCKET NO.0U6500-1491008 (010WO1) doxorubicin can be made using this method (e.g., about 16 mol % relative to moles of liposomal lipid).
  • lipid components can be used to make liposomes. These include neutral lipids that exist either in an uncharged or neutral zwitterionic form at physiological pH. Such lipids include, for example, diacylphosphatidylcholine, diacylphosphatidylethanolamine, ceramide, sphingomyelin, dihydrosphingomyelin, cephalin, and cerebrosides.
  • the liposomes comprise phosphatidylcholine or a derivative thereof (e.g., a phosphatidyl choline having an acyl group having 6 to 22 carbon atoms, a diacylphosphatidylcholine, and/or diacylphosphatidylethanolamine).
  • Synthetic derivatives of any of the lipids described herein can also be used to make lipid nanoparticles.
  • Lipid nanoparticles can also comprise a sterol, for example, cholesterol.
  • Lipid nanoparticles can also comprise a cationic lipid which carries a net positive charge at about physiological pH.
  • Such cationic lipids include, but are not limited to, N,N-dioleyl-N,N-dimethylammonium chloride (DODAC); N-(2,3-dioleyloxy)propyl-N,N-N-triethylammonium chloride (DOTMA); N,N- distearyl-N,N-dimethylammonium bromide (DDAB); N-(2,3-dioleoyloxy)propyl)-N,N,N- trimethylammonium chloride (DOTAP); 1,2-Dioleyloxy-3-trimethylaminopropane chloride salt (DOTAP.Cl); 3.beta.-(N--(N',N'-dimethylaminoethane)-carbamoyl)cholesterol ("DC- Chol”), N-(1-(2,3-dioleyloxy)propyl)-N-2-(sperminecarboxamido)ethyl)-N
  • Anionic lipids are also suitable for use in lipid nanoparticles described herein. These include, but are not limited to, phosphatidylglycerol, cardiolipin, diacylphosphatidylserine, diacylphosphatidic acid, N-dodecanoyl phosphatidylethanoloamine, N-succinyl phosphatidylethanolamine, N-glutaryl phosphatidylethanolamine, lysyl phosphatidylglycerol, and other anionic modifying groups joined to neutral lipids.
  • the liposome comprises phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, phosphatidic acid, phosphatidylglycerol, palmitoyloleoyl phosphatidylcholine, lysophosphatidylcholine, lysophosphatidylethanolamine, dimyristoylphosphatidylcholine (DMPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylcholine, distearoylphosphatidylcholine (DSPC), dilinoleoylphosphatidylcholine, a 1,2-distearoyl-sn- PATENT ATTORNEY DOCKET NO.0U6500-1491008 (010WO1) glycero-3-phosphoethanolamine (DSPE) conjugated polyethylene glycol (DSPE-PEG), a
  • PEG can be PEG-molecular weight (MW500) to PEG-MW20000.
  • any of the lipids described herein can be attached or conjugated to a C-type lectin receptor polypeptide or a fragment thereof that binds a target antigen.
  • a lipid can be functionalized with a reactive group such as, for example, N-hydroxysuccinimide (NHS).
  • NHS N-hydroxysuccinimide
  • pegylated versions of any of the lipids described herein can be conjugated to a C-type lectin receptor polypeptide or a fragment thereof that binds a target antigen on a cancer cell, or an antigen expressed on a cancer cell.
  • the targeted antigen is a ligand of any of the C-type lectin receptor polypeptides or a fragments thereof described herein.
  • the C-type lectin receptor polypeptide or a fragment thereof binds to a glycan on the surface of the cancer cell.
  • the C-type lectin receptor polypeptide or a fragment thereof specifically binds to a tumor-associated glycan.
  • the nanoparticle for example, a liposome, comprises about 40 to 70, 45 to 70, 50 to 70, 55 to 70, 60 to 70, or 65 to 70, 40 to 60, 45 to 60, 50 to 60, or 55 to 60 moles percent phosphatidylcholine lipid (e.g., fully hydrogenated soy phosphatidylcholine (18:0-18:1 PC, 1-stearoyl-2-oleoyl-sn-glycero-3-phosphocholine) (HSPC) or 18:10 (n10)- 16:00 PC, 15:0-18:1 PC).
  • phosphatidylcholine lipid e.g., fully hydrogenated soy phosphatidylcholine (18:0-18:1 PC, 1-stearoyl-2-oleoyl-sn-glycero-3-phosphocholine) (HSPC) or 18:10 (n10)- 16:00 PC, 15:0-18:1 PC).
  • the nanoparticles for example, a liposome, comprise about 40, 45, 50, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, or 70 mole percent phosphatidylcholine lipid (e.g., fully hydrogenated soy phosphatidylcholine (18:0-18:1 PC, 1-stearoyl-2-oleoyl-sn-glycero-3-phosphocholine) (HSPC) or 18:10 (n10)-16:00 PC, 15:0- 18:1 PC) relative to total lipid content.
  • HSPC fully hydrogenated soy phosphatidylcholine
  • HSPC 1-stearoyl-2-oleoyl-sn-glycero-3-phosphocholine
  • the nanoparticle for example, a liposome, comprises about 20 to about 50, about 25 to about 50, about 30 to about 50, about 35 to about 50, about 40 to about 50, about 45 to about 50, about 20 to about 40, about 25 to about 40, about 30 to about 40, or about 35 to about 40 mole percent cholesterol (e.g., CAS 57-88-0).
  • the nanoparticle, for example, a liposome comprises about 20, 25, 30, 35, 40, 45, or 50 mole percent cholesterol relative to total lipid content.
  • the nanoparticle for example, a liposome, comprises about 1 to about 6 , about 2 to about 6, about 3 to about 6, about 4 to about 6 or about 5 to about 6 mole percent polyethylene glycol (e.g., methyl PEG-2000-DSPE (18:0 PEG2000 PE) PATENT ATTORNEY DOCKET NO.0U6500-1491008 (010WO1) (sodium;[(2R)-2,3-di(octadecanoyloxy)propyl] 2-(2-methoxyethoxycarbonylamino)ethyl phosphate) (CAS 147867-65-0)) relative to total lipid.
  • polyethylene glycol e.g., methyl PEG-2000-DSPE (18:0 PEG2000 PE) PATENT ATTORNEY DOCKET NO.0U6500-1491008 (010WO1) (sodium;[(2R)-2,3-di(octadecanoyloxy)propyl] 2-(2-meth
  • the liposome comprises 18:1 PEG2000 PE, 16:0 PEG2000 PE, or 14:0 PEG2000 PE instead of 18:0 PEG2000 PE relative to total lipid content.
  • any of the nanoparticles described herein, for example, liposomes can contain about 0.2 to about 25 mole percent anticancer agents.
  • the nanoparticle can contain about 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 moles percent or greater of an anticancer agent or imaging agent relative to total lipid content of the liposome.
  • the nanoparticles can comprise about 0.2:100, 0.3:100, 0.4:100, 0.5:100, 0.6:100, 0.7:100, 0.8:100, 0.9:100, 1:100, 2:100, 3:100, 4:100, 5:100, 6:100, 7:100, 8:100, 9:100, 10:100, 11:100, 12:100, 13:100, 14:100, 15:100, 16:100, 17:100, 18:100, 19:100, 20:100 mole ratio or greater of anticancer agent or imaging agent relative to total lipid content.
  • the nanoparticle for example, a liposome can contain about 5 to about 10 mole percent, about 5 to about 15 mole percent, about 5 to about 20 mole percent, or about 5 to about 25 mole percent of an anticancer agent or imaging agent relative to lipid.
  • the nanoparticles can comprise about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 mole percent of an anticancer agent or imaging agent relative to lipid.
  • the nanoparticles comprise about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 mole percent of an anticancer agent relative to lipid, wherein the anticancer agent is selected from the group consisting of doxorubicin, paclitaxel, taxol, cytarabine, mifamurtide, irinotecan, verteporfin, daunorubicin, and vincristine.
  • the nanoparticle for example, a liposome, comprises about 0.2 to about 2 mole percent C-type lectin receptor or a fragment thereof relative to total lipid content.
  • the liposome can comprise about 0.2 to about 2, about 0.3 to about 2, about 0.4 to about 2, 0.5 to about 2, 0.6 to about 2, 0.7 to about 2, 0.8 to about 2, 0.9 to about 2, 1.0 to about 2, 1.1 to about 2, 1.2 to about 2, 1.3 to about 2, 1.4 to about 2, 1.5 to about 2, 1.6 to about 2, 1.7 to about 2, 1.8 to about 2 or 1.9 to about 2 mole percent C-type lectin receptor or a fragment thereof relative to total lipid content.
  • the nanoparticle comprises about 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9 or 2.0 mole percent C-type lectin receptor or a fragment thereof relative to total lipid content.
  • PATENT ATTORNEY DOCKET NO.0U6500-1491008 (010WO1) in some embodiments, the nanoparticle, for example, a liposome, comprises about 0.1 to about 5 mole percent imaging agent relative to total lipid content.
  • the nanoparticle comprises about 0.1, 0.20.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5 or 5 mole percent imaging agent relative to lipid.
  • the imaging agent is a fluorescent imaging agent, such as, for example, rhodamine or fluorescein isothiocyanate. Table 2 provides components and corresponding mole percent ratios for exemplary cancer-targeted nanoparticles comprising doxorubicin and other anticancer agents.
  • mole ratio is the ratio between the amounts in moles of two components, for example, the ratio between the number of moles of a targeting molecule (for example, a C- type lectin receptor or a fragment thereof, and the number of moles of lipid (targeting molecules: moles of lipid) or the number of moles of an anticancer agent and the number of moles of lipid (moles of anticancer agent: moles of lipid).
  • the nanoparticles can comprise a 0.002:100, 0.05:100, 0.1:100, 0.5:100, 1:100, 2:100, 3:100, 4:100, 5:100, 10: 100, 15:100, 20:100, 25:100 mole ratio of targeting molecule to liposomal lipid or greater.
  • mole percent ratio or mole percentage expresses the concentration of a substance in a mixture as the number of moles of that substance divided by the total number of moles, multiplied by 100.
  • Pluralities of two or more of any of the nanoparticles described herein are also provided.
  • a plurality of liposomes can comprise from about two to about 1 x 10 14 (100 trillion) liposomes.
  • a plurality can have at least 100, 250, 500, 750, 1000, 5000, 10,000, 25,000, 50,000,100,000, 500,000, 1 million or more liposomes.
  • at least 80%, 85%, 90%, 95%, or 99% of the liposomes in the plurality comprise and anticancer agent or an imaging agent.
  • cancer-targeting molecules from two cancer-targeting molecules to about 1,000, 1,500, 2,000, 2,500, 3,000, 3,500, 4,000, 4,500, 5,000, 5,500, 6,000, 6,500, 7,000, 7,500, 8,000, 8,500, 9,000, 9,500 or 10,000 cancer-targeting molecules (e.g., a C-type lectin receptor or fragment thereof) can be coupled to the liposomes provided herein.
  • cancer-targeting molecules e.g., a C-type lectin receptor or fragment thereof
  • about two molecules to about 3,000 cancer-targeting molecules are incorporated into nanoparticles that are between about 50 nm and 100 nm in diameter.
  • Those of skill in the art would know how to calculate the number of C-type lectin receptor polypeptides or fragments thereof that can be incorporated into a nanoparticle, for example between about two and 10,000 C-type lectin receptor polypeptides or fragments thereof or greater depending on the size of the nanoparticle.
  • a nanoparticle comprising: (a) a C-type lectin receptor or fragment thereof that binds an antigen on a cancer cell; and (b) a signal-generating molecule, wherein the C-type lectin receptor or fragment thereof is coupled to the surface of the nanoparticle and the signal-generating molecule generates a signal when the C-type lectin receptor or fragment thereof binds the antigen on the cancer cell.
  • the signal-generating molecule is linked to the C-type lectin receptor or fragment thereof.
  • the signal-generating molecule is incorporated into or attached to the outer surface of the nanoparticle, for example, a liposome.
  • the signal-generating molecule is a fluorescent dye or fluorescent polypeptide.
  • the C-type lectin receptor or fragment thereof is linked to the C-terminal and/or an N-terminal fragment of a fluorescent protein.
  • a plurality of any of the nanoparticles described herein that comprise a signal-generating molecule comprising: (a) contacting the subject or a sample from the subject with a plurality of any of the cancer-targeted nanoparticles comprising a signaling molecule described herein; (b) detecting a signal, wherein a signal indicates the presence of cancer.
  • the C- type lectin receptor or fragment thereof is linked to a fluorescent protein, an antibody or a fragment thereof, or an enzyme.
  • the signal is directly or indirectly detected.
  • a biological sample is a sample derived from a subject and includes, but is not limited to, any cell, tissue or biological fluid.
  • the sample can be, but is not limited PATENT ATTORNEY DOCKET NO.0U6500-1491008 (010WO1) to, blood, plasma, serum, sputum, urine, saliva, bronchoalveolar lavage fluids, biopsy (e.g., tissue or cells isolated from organ tissue, for example, from lung, liver, kidney, skin etc.), vaginal secretion, nasal secretion, skin, gastric secretion, or bone marrow specimens.
  • Methods of Treatment Also provided are methods for treating or preventing cancer in a subject. The methods comprise administering to the subject having cancer or at risk of developing cancer an effective amount of a plurality of any of the nanoparticles described herein.
  • each nanoparticle in the plurality comprises an anticancer or antineoplastic agent and a C-type lectin receptor or fragment thereof that binds a target antigen on a cancer cell, wherein the C-type lectin receptor or fragment thereof is coupled to the outer surface of the liposome and the anticancer or antineoplastic agent is encapsulated in the liposome.
  • At least 80%, 85%, 90%, 95%, or 99% of the nanoparticles in the plurality comprise an anticancer or antineoplastic agent and a C-type lectin receptor or fragment thereof that binds a target antigen on a cancer cell, wherein the C-type lectin receptor or fragment thereof is coupled to the outer surface of the liposome and the anticancer or antineoplastic agent is encapsulated in the liposome.
  • treat, treating, and treatment refer to a method of reducing or delaying one or more effects or symptoms of cancer.
  • the subject can be diagnosed with cancer.
  • Treatment can also refer to a method of reducing the underlying pathology rather than just the symptoms.
  • the effect of the administration to the subject can have the effect of, but is not limited to, reducing one or more symptoms of the disease, a reduction in the severity of the disease, the complete ablation of the disease, or a delay in the onset or worsening of one or more symptoms.
  • a disclosed method is considered to be a treatment if there is about a 10% reduction in one or more symptoms of the disease in a subject when compared to the subject prior to treatment or when compared to a control subject or control value.
  • the reduction can be about a 10, 20, 30, 40, 50, 60, 70, 80, 90, 100%, or any amount of reduction in between.
  • prevent, preventing, or prevention is meant a method of precluding, delaying, averting, obviating, forestalling, stopping, or hindering the onset, incidence, severity, or recurrence of a disease or disorder.
  • the disclosed method is considered to be a prevention if there is a reduction or delay in onset, incidence, severity, or recurrence of cancer in a subject susceptible to cancer or recurrence of cancer compared to control subjects susceptible to cancer or recurrence of cancer that did not receive treatment.
  • the reduction or PATENT ATTORNEY DOCKET NO.0U6500-1491008 (010WO1) delay in onset, incidence, severity, or recurrence of cancer can be about a 10, 20, 30, 40, 50, 60, 70, 80, 90, 100%, or any amount of reduction in between.
  • the methods provided herein optionally further include administering an effective amount of a second therapeutic agent or therapy to the subject.
  • the second therapeutic agent or therapy can be administered to the subject prior to, simultaneously with, or subsequent to administration of the plurality of nanoparticles.
  • the second therapeutic therapy is surgery.
  • the second therapeutic agent is a second anticancer agent (e.g., a chemotherapeutic, immunotherapeutic, or cellular therapy (e.g., CAR T cell therapy).
  • cancer is a disease characterized by the rapid and uncontrolled growth of aberrant cells. Cancer cells can spread locally or through the bloodstream and lymphatic system to other parts of the body. The cancer can be a solid tumor.
  • the cancer is a blood or hematological cancer, such as a leukemia
  • Solid tumors include, by way of example, bone and connective tissue sarcomas (e.g., bone sarcoma, osteosarcoma, chondrosarcoma, Ewing's sarcoma, malignant giant cell tumor, fibrosarcoma of bone, chordoma, periosteal sarcoma, soft-tissue sarcomas, angiosarcoma (hemangiosarcoma), fibrosarcoma, Kaposi's sarcoma, leiomyosarcoma, liposarcoma, lymphangiosarcoma, neurilemmoma, rhabdomyosarcoma, synovial sarcoma), brain tumors (e.g., glioma, astrocytoma, brain stem glioma, ependymoma, oligodendro
  • cancers include myxosarcoma, osteogenic sarcoma, endotheliosarcoma, lymphangio endothelio sarcoma, mesothelioma, synovioma, hemangioblastoma, epithelial carcinoma, cystadenocarcinoma, bronchogenic carcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma and papillary adenocarcinomas.
  • anticancer agents include, but are not limited to amsacrine, bleomycin, busulfan, capecitabine, carboplatin, carmustine, chlorambucil, cisplatin, cladribine, clofarabine, crisantaspase, cyclophosphamide, cytarabine, dacarbazine, dactinomycin, daunorubicin, docetaxel, doxorubicin, epirubicin, etoposide, everolimus, fludarabine, fluorouracil, gemcitabine, hydroxycarbamide, idarubicin, ifosfamide, irinotecan, leucovorin, doxorubicin, daunorubicin, lomustine, melphalan, mercaptopurine, mesna, methotrexate, mitomycin, mitoxantrone, oxaliplatin, paclitaxel, pemetrexed,
  • the terms “anticancer agent”, “anticancer drug” and “antineoplastic agent” are used interchangeably throughout.
  • the anticancer agent is selected from the group consisting of temozolomide, procarbazine, carmustine, lomustine, and vincristine.
  • subject is meant an individual.
  • the subject can be an adult subject or a pediatric subject.
  • Pediatric subjects include subjects ranging in age from birth to eighteen years of age. Thus, pediatric subjects of less than about 10 years of age, five years of age, two years of age, one year of age, six months of age, three months of age, one month of age, one week of age or one day of age are also included as subjects.
  • the subject is an animal, for example, a mammal such as a primate, and, more preferably, a human.
  • a mammal such as a primate
  • Non- human primates are subjects as well.
  • the term subject includes domesticated animals, such as cats, dogs, etc., livestock (for example, cattle, horses, pigs, sheep, goats, etc.) and laboratory animals (for example, ferret, chinchilla, mouse, rabbit, rat, gerbil, guinea pig, etc.).
  • livestock for example, cattle, horses, pigs, sheep, goats, etc.
  • laboratory animals for example, ferret, chinchilla, mouse, rabbit, rat, gerbil, guinea pig, etc.
  • Pharmaceutical Compositions Any of the nanoparticles or pluralities of nanoparticles provided herein can be in a pharmaceutical composition.
  • any of the pharmaceutical compositions are provided herein, including use of an effective amount of the pharmaceutical composition for the treatment or prevention of cancer.
  • effective amount as used throughout, is defined as any amount necessary to produce a desired physiologic response, for example, treating or preventing cancer.
  • the dosage ranges for administration are those large enough to produce the desired effect in which one or more symptoms of the disease or disorder are affected (e.g., reduced or delayed). The dosage should not be so large as to cause substantial adverse side effects, such as unwanted cross-reactions, unwanted cell death, and the like.
  • the dosage will vary with the type of inhibitor, the species, age, body weight, general health, sex and diet of the subject, the mode and time of administration, rate of excretion, drug combination, and severity of the particular condition and can be determined by one of skill in the art.
  • the dosage can be adjusted by the individual physician in the event of any contraindications. Dosages can vary and can be administered in one dose or multiple doses administered daily or at extended intervals. PATENT ATTORNEY DOCKET NO.0U6500-1491008 (010WO1) Any of the liposomes described herein can be provided in a composition, for example, a pharmaceutical composition.
  • the composition can include one or more liposomes disclosed herein.
  • compositions include, for example, a pharmaceutical composition comprising a therapeutically effective amount of any of the liposomes described herein and a pharmaceutical carrier.
  • carrier means a compound, composition, substance, or structure that, when in combination with a compound or composition, aids or facilitates preparation, storage, administration, delivery, effectiveness, selectivity, or any other feature of the compound or composition for its intended use or purpose.
  • a carrier can be selected to minimize any degradation of the active ingredient and to minimize any adverse side effects in the subject.
  • pharmaceutically acceptable carriers include sterile biocompatible pharmaceutical carriers, including, but not limited to, saline, buffered saline, artificial cerebral spinal fluid, dextrose, and water.
  • compositions comprising any of the liposomes described herein can be prepared according to standard techniques and further comprise a pharmaceutically acceptable carrier.
  • a pharmaceutically acceptable carrier e.g., normal saline will be employed as the pharmaceutically acceptable carrier.
  • suitable carriers include, e.g., water, buffered water or saline, 0.4% saline, 0.3% glycine, dextrose, and the like, including glycoproteins for enhanced stability, such as albumin, lipoprotein, and globulin. These compositions are usually sterile.
  • the pharmaceutical compositions can also contain a pharmaceutically acceptable excipient. Such excipients include any pharmaceutical agent that does not itself induce an immune response harmful to the individual receiving the composition, and which may be administered without undue toxicity.
  • Pharmaceutically acceptable excipients include, but are not limited to, liquids such as water, saline, glycerol, sugars and ethanol.
  • Pharmaceutically acceptable salts can be included therein, for example, mineral acid salts such as hydrochlorides, hydrobromides, phosphates, sulfates, and the like; and the salts of organic acids such as acetates, propionates, malonates, benzoates, and the like.
  • auxiliary substances such as wetting or emulsifying agents, pH buffering substances, and the like, may be present in such vehicles.
  • compositions can contain pharmaceutically acceptable auxiliary substances as required to approximate physiological conditions, such as pH adjusting and buffering agents, tonicity adjusting agents and the like, for example, sodium acetate, sodium lactate, sodium chloride, potassium chloride, and calcium chloride.
  • the liposome suspension may include lipid-protective agents which protect lipids against free-radical and lipid-peroxidative damages on storage.
  • Lipophilic free-radical quenchers such as alphatocopherol and water-soluble iron-specific chelators, such as ferrioxamine, are suitable.
  • concentration of the nanoparticles (e.g., liposomes) in the pharmaceutical formulations can vary widely, i.e., from less than about 0.05%, usually at or at least about 2- 5% to as much as 10 to 30% by weight and will be selected primarily by fluid volumes, viscosities, in accordance with the particular mode of administration selected.
  • the liposomes may be dried or lyophilized and resuspended to a desired concentration in water or buffers at time of use.
  • the amount of active agent in the liposome or plurality of liposomes administered depends upon the particular label used, the disease state being diagnosed and the judgment of the clinician but is generally between about 0.01 and about 150 mg per kilogram of body weight, preferably between, about 0.1 and about 30 mg/kg of body weight, about 0.1 and about 20 mg/kg (e.g., 1 mg/kg, 2mg/kg, 3 mg/kg, 4 mg/kg, 5 mg/kg , 6 mg/kg, 7 mg/kg, 8 mg/kg, 9 mg/kg , 10 mg/kg, 11 mg/kg, 12mg/kg, 13 mg/kg, 14 mg/kg, 15 mg/kg , 16 mg/kg, 17 mg/kg, 18 mg/kg, 19 mg/kg, 20 mg/kg) of body weight, about 0.1 to about 10 mg/kg (e.g., 1 mg/kg, 2
  • compositions disclosed herein are administered in a number of ways depending on whether local or systemic treatment is desired, and on the area to be treated.
  • the compositions are administered via any of several routes of administration, including orally, intranasally, via inhalation, via nebulizer, parenterally, intravenously, intraperitoneally, intracranially, intraspinally, intrathecally, intraventricularly, intramuscularly, subcutaneously, intracavity, transdermally, or via convection-enhanced delivery.
  • compositions can also be delivered locally to the area in need of treatment, for example by topical application or local PATENT ATTORNEY DOCKET NO.0U6500-1491008 (010WO1) injection.
  • the pharmaceutical compositions can also be delivered via pump or at a surgical site.
  • Effective doses for any of the administration methods described herein can be extrapolated from dose-response curves derived from in vitro or animal model test systems.
  • a cancer-targeting nanoparticle comprising: a. an anticancer agent; and b. a C-type lectin receptor or a fragment thereof that is coupled to the surface of the nanoparticle, wherein the C-type lectin receptor or a fragment thereof binds an antigen expressed by a cancer cell.
  • PATENT ATTORNEY DOCKET NO.0U6500-1491008 (010WO1) The cancer-targeting nanoparticle of any of embodiments 1 or 2 , wherein the C-type lectin receptor or fragment thereof is coupled to the surface of the nanoparticle by inserting the C-type lectin receptor or fragment thereof into the surface of the nanoparticle.
  • the cancer-targeting nanoparticle of any of embodiments 1or 2 wherein the C-type lectin receptor or fragment thereof is coupled to the surface of the nanoparticle by attaching the C-type lectin receptor or fragment thereof to the surface of the nanoparticle.
  • DC-SIGN Dendritic Cell-Specific Intercellular adhesion molecule-3- Grabbing Non-integrin
  • CCD carbohydrate recognition domain
  • the cancer-targeting nanoparticle of embodiment 15, wherein the DC-SIGN polypeptide comprises a DC-SIGN CRD (SEQ ID NO: 21), NR7 (SEQ ID NO: 28) and NR8 (SEQ ID NO: 29).
  • the cancer-targeting nanoparticle of any one of embodiments 1-20, wherein the C- type lectin receptor or fragment thereof is conjugated to a lipid carrier.
  • the cancer-targeting nanoparticle of any one of embodiments 1-20 wherein the concentration of the anticancer agent is reduced as compared to the concentration of PATENT ATTORNEY DOCKET NO.0U6500-1491008 (010WO1) the anticancer agent in a nanoparticle that does not comprise a C-type lectin receptor or a fragment thereof coupled to the surface of the nanoparticle.
  • PATENT ATTORNEY DOCKET NO.0U6500-1491008 (010WO1)
  • PATENT ATTORNEY DOCKET NO.0U6500-1491008 (010WO1)
  • a plurality of cancer-targeting nanoparticles according to any one of embodiments 1- 43 Use of the cancer-targeting nanoparticle of any one of embodiments 30-43 or the plurality of embodiment 44 for the treatment of cancer.
  • a pharmaceutical composition comprising a plurality of cancer-targeting nanoparticles according to any one of embodiments 1-43.
  • a nanoparticle comprising: g.
  • a C-type lectin receptor or fragment thereof that binds an antigen on a cancer cell; and h. a signal-generating molecule, wherein the C-type lectin receptor or fragment thereof is coupled to the surface of the nanoparticle and the signal-generating molecule generates a signal when the C-type lectin receptor or fragment thereof binds the antigen on the cancer cell.
  • the nanoparticle of embodiment 48 wherein the signal-generating molecule is linked to the C-type lectin receptor or fragment thereof.
  • the nanoparticle of embodiment 50 wherein the the soluble C-type lectin receptor or a fragment thereof is a soluble human Dectin-3 comprising an amino acid sequence having at least 85%, 90%, 95%, or 99% identity to SEQ ID NO: 3, SEQ ID NO: 18, SEQ. ID NO: 42 or a fragment thereof.
  • DC-SIGN Dendritic Cell-Specific Intercellular adhesion molecule-3- Grabbing Non-integrin
  • the nanoparticle of embodiment 57 wherein the DC-SIGN polypeptide comprises a DC-SIGN CRD (SEQ ID NO: 21) and one or more neck regions of DC-SIGN selected from the group consisting of (NR1) SEQ ID NO: 22, (NR2) SEQ ID NO: 23, (NR3) SEQ ID NO: 24, (NR4) SEQ ID NO: 25, (NR5) SEQ ID NO: 26, (NR6) SEQ ID NO: 27, (NR7) SEQ ID NO: 28 and (NR8) SEQ ID NO: 29.
  • a method of treating or preventing cancer in a subject comprising administering to the subject having cancer or at risk of developing cancer an effective amount of the pharmaceutical composition of embodiment 46.
  • the method of embodiment 68, wherein the subject has breast cancer, prostate cancer or lung cancer.
  • PATENT ATTORNEY DOCKET NO.0U6500-1491008 (010WO1) .
  • the method of embodiment 68 or 69, wherein a second therapeutic agent or therapy is administered to the subject.
  • the method of embodiment 70, wherein the second therapeutic agent or therapy is surgery, radiation or immunotherapy. .
  • any one of embodiments 68-72, wherein the anticancer agent is a drug, a peptide or an antibody. .
  • the use of embodiment 76 or 77, wherein the anticancer agent is a drug, a peptide or an antibody. .
  • the use of embodiment 78, wherein the drug is a chemotherapeutic drug. .
  • chemotherapeutic drug is doxorubicin.
  • a method for detecting cancer in a subject or a sample from a subject comprising: a) contacting the subject or a sample from the subject with the plurality of nanoparticles of any one of embodiments 47-67; and b) detecting a signal, wherein a signal indicates the presence of cancer.
  • the method of embodiment 81, wherein the signal is a fluorescent signal.
  • Liposome preparations used to stain cells were prepared as described previously (Ambati et al. mSPhere 4:1-16 (2019)) starting with commercial 100 nm diameter pegylated liposomes from FormuMax Sci. Inc. (Sunnyvale, CA) (F10203A, DSPC:CHOL:mPEG-DSPE (50:45:5 mol/mol). All liposomes were remotely loaded with 2 mol percent DHPE-rhodamine B relative to 100 moles percent liposomal lipid.
  • the cells were washed thrice with media and photographed at 10X magnification taking bright field and red epifluorescent images on ECHO RSVF1000/REVOLVE R4 microscope (VWR International, Radnor PA) in the inverted PATENT ATTORNEY DOCKET NO.0U6500-1491008 (010WO1) position.
  • the cells were costained with DAPI (4′,6-diamidino-2-phenylindole) in which case the DAPI channel was recorded in place of the visible light channel.
  • the rhodamine red channel was merged with the bright field channel to generate the images presented.
  • This liposome contains the CRD and stalk region of a CTL such as Dectin-2 conjugated to the lipid carrier DSPE-PEG, which was then inserted into the liposomal membrane via the DSPE lipid moiety.
  • DHPE-rhodamine B is inserted via its DHPE lipid moiety so that binding to cells can be monitored and quantified by fluorescence.
  • CRD and Rhodamine were loaded at 1 and 2 mol percent, respectively, relative to moles of liposomal lipid in a 100 nm liposome.
  • an anti-cancer cell nanoparticle (FIG.1) liposomes pre- loaded with an anti-cancer drug, for example, Doxil® (a.k.a., Doxosome), which is loaded with 16 mol % doxorubicin (DOX) relative to moles of liposomal lipid, were used.
  • Doxil® a.k.a., Doxosome
  • DOX 16 mol % doxorubicin
  • BSA coated BSA-Ls an untargeted protein coated control, and plain liposomes, lacking any protein as an additional control, were prepared. All six of the preparations of liposomes do not contain the DOX shown in FIG.1.
  • Representative breast, prostate, and lung cancer cell lines (Table 1) and control cell lines were grown on the surface of 24 well plastic microtiter plates to between 50% and 90% confluence and washed once with growth media. The cancer cell lines employed did not lend themselves to cytometric assays of reagent binding, because they tend to grow best attached to a surface. Cells were stained for 1 hr with various targeted and untargeted liposomes and washed extensively.
  • CTL liposome binding appears to be specific for only one or a few glycoprotein membrane rafts on the surface of any one cell.
  • Biological and experimental replicates of this experiment showed nearly identical results.
  • DEC2-Ls bound up to 80% of the cells in some microscopic fields.
  • a similar result was obtained for DEC1-Ls, DEC2-Ls and DEC3-Ls staining of a triple positive breast cancer cell line MCF7. This demonstration of such strong specific binding encouraged examination of cancer cell lines representative of other cancers.
  • Prostate cancer The experiment illustrated in FIG.3 examined DCS-12-Ls, DEC1-Ls, DEC2-Ls and DEC3-Ls binding to the prostate epithelial adenocarcinoma cancer cell line PC3.
  • Untargeted fluorescent liposomes did not bind significantly.
  • Other prostate cell lines such as, but not limited to, DU-145 and LNCaP-LN3, can also be examined using methodology as described above..
  • FFPE prostate patient tumor samples are available from TissueArray.com and other sources.
  • FIG.4 examines DCS12-Ls, DEC1-Ls, DEC2-Ls and DEC3-Ls binding to the non-small cell lung carcinoma (NSCLC) cancer line A594.
  • NSCLC non-small cell lung carcinoma
  • H-1975 and HCC-827 Other metastatic lung cancer cell lines that can be examined using methodology described above include H-1975 and HCC-827.
  • FFPE NSCLC patient tumor samples are available from TissueArray and other sources.
  • PATENT ATTORNEY DOCKET NO.0U6500-1491008 (010WO1) Control cells As a negative control, binding to HEK293T cells was examined.
  • These human embryonic kidney cells were artificially immortalized first by transformation with sheared fragments of adenovirus type 5 DNA that delivered oncogene analogs E1A and E1B and then with SV40 T antigen gene (Kovesdi and Hedley, Viruses 2: 1681-1703 (2010)).
  • DCS12-DOXIL, DEC1-DOXIL, DEC3- DOXIL, and DEC3-DOXIL delivering 10 uM DOX all showed order of magnitude improvements in cell killing of the triple negative breast cancer cells relative to DOXIL (FIG. 7A, second bar vs bar 3, 4, 5, and 6).
  • C-type lectin targeted liposomes delivering 5 uM DOX were less effective relative to DOXIL® (FIG.3B). Clearly, C-type lectin receptor targeting dramatically improved the performance of DOXIL.
  • Dectin-1, Dectin-2, Dectin-3 (MCL) and DSC-12 coated liposomes bind and kill cancer cells, for example, triple negative breast cancer cells. It was surprising that, although Dectin-1, Dectin-2, and Dectin-3 coated liposomes bound to relatively small patches on the surface of cancer cell lines, these C-type receptor targeted liposomes were highly efficient at killing cancer cells, as shown in FIG.7.
  • PATENT ATTORNEY DOCKET NO.0U6500-1491008 (010WO1) EXAMPLE II DEC2-DOX-LLs specifically stain cancer cells in human Ductal Carcinoma In Situ (DCIS) tissue sections.
  • FFPE Formalin Fixed Paraffin Embedded human breast cancer tissue arrays
  • CHTN_BrCaProg3 Cooperative Human Tissue Network
  • Reagents LDB2 tissue blocking, labeling and washing buffer (20 mM HEPES, 10 mM triethanolamine, 150 mM NaCl, 10 mM CaCl 2 , 1 mM BME, 5% BSA w/v, pH 8.0).
  • DAPI at 5 ug/1000 uL in LDB2.
  • DAPI diluted to 1 ug/100 uL in LDB2.
  • Liposomes DEC2-DOX-LLs and DOX-LLs tagged with lissamine rhodamine B were prepared as described above.
  • DEC2-DOX-LLs we diluted so that DEC2 protein concentration was at 1.0 ug/150 uL (1:150 w/v) in LDB2 liposome dilution buffer. Control DOX-LL were diluted equivalently.
  • Staining the FFPE tissue arrays Microscope slides with the FFPE tissue BrCaProg3 arrays or FFPE patient TNBC tumor sections were deparaffinized at the University of Georgia’s (UGA) Veterinary Diagnostic Laboratory. The area with tissue sections was encircled with a Liquid repellent pencil-Liquid blocker (Agar Scientific Inc., Rotherham, UK). All procedures were carried out at room temperature.
  • FFPE tissue samples was blocked for 30 min to 1 hr with ⁇ 600 uL of LDB2 that covered the area within the repellent border. This solution was replaced with ⁇ 600 uL of liposomes diluted into LDB2 and incubated for one hr. The samples were washed PATENT ATTORNEY DOCKET NO.0U6500-1491008 (010WO1) briefly twice with 600 uL of LDB2 and then stained for 30 min with DAPI. The sample was washed twice more. All but 20 to 50 uL of the LDB2 was removed and a coverslip was placed over the array. The slides were sealed with clear finger-nail polish, which was let dry for 20 min.
  • the purple area shows the concentration of red fluorescent DEC2-DOX-LL staining in the areas with tightly packed blue, fluorescent nuclei.
  • the red liposome channel alone is shown below (FIG.8A bottom).
  • An H&E-stained section from a duplicate BrCaProg3 array shows that the typical morphology of DCIS-H tumors (FIG.8D) matches the morphology of the DEC2-DOX-LL- stained tissue. DOX-LLs did not stain tissue with the DCIS-H, morphology (FIG.8B), although some background punctate red fluorescent staining was observed in most images.
  • DEC2-DOX-LL did not significantly stain control non-neoplastic breast from patients without breast carcinoma (a.k.a., NB-NC) with ductal morphology (FIG.8C), although again some background red fluorescent staining was observed in most images.
  • FFPE tissue tumor sections from patients with TNBC breast cancer were stained with DEC2-DOX- LLs and control DOX-LLs.
  • Example images with the red liposomes and blue, fluorescent nuclei are shown in FIG.9 (A & B, top row).
  • TNBC tumor cells have been characterized as small and disorganized with very little cytoplasm (Schmadeka et al., 2014. Am J Clin Path, 141:462-477).
  • H&E-stained images of an adjacent tissue section confirmed that regions within adjacent tissue sections had cells with this expected morphology (FIG.8C). Using DAPI staining as a guide in the microscope, regions of the tissue having cells with this morphology were easily identified and photographed.
  • FIG 9A Close examination of FIG 9A shows DEC2-DOX-LLs staining in a tight ring around the nuclei, matching this phenotype and as expected for cells with scant cytoplasm.
  • Untargeted DOX-LLs staining showed some red fluorescent background, but background staining was not concentrated in TNBC cells (FIG. 9B).
  • FIGS. 14A-13B also show that DEC2-DOX-LLs labeled TNBC tissue within a TNBC patient tumor FFPE breast tissue section far more efficiently than untargeted DOX-LLs.
  • TNBC metastatic triple negative breast cancer
  • TNBC triple negative breast cancer
  • NSCLC metastatic non-small cell lung carcinoma
  • FIGS.14A-B showed that DEC2-DOX-LLs label TNBC tissue within a TNBC patient tumor FFPE breast tissue section HuCAT292 far more efficiently than untargeted DOX-LLs. Similar results are expected with other metastatic breast cancer cells lines such as, but not limited to, other TNBC cell lines (for example, BT-20, HCC-79, and BT-549). Non- metastatic breast cancer cell lines such as, but not limited to MCF-7, can also be examined using methodology as described above. Additional tumor samples that can be examined, for example, other FFPE TNBC patient tumor samples, are available from TissueArray and other sources.
  • Dectin-2 targeted rhodamine tagged DEC2-DOX- LLs specifically stained two types of breast cancer cells in FFPE tissue sections, with little background staining by DOX-LLs.
  • DEC2-DOX-LLs specifically stained cancerous mammary ductal DCIS-H tissue in patient tissue arrays, but not breast tissue from healthy individuals.
  • DOX-LLs did not bind cancerous mammary ductal DCIS-H tissue in these same patient tissue samples.
  • DEC2-DOX-LLs specifically stained TNBC cells in patient tissue samples, and DOX-LLs did not.
  • mice xenografted human tumors e.g., MDA-MB-231 and BT474
  • Charles River Labs can also be used in staining studies (as described above) for any of the C-type lectin receptors or fragments thereof described herein.
  • TNBC breast tumor sections such as HUCAT292 are commercially available from TissueArray.
  • Mouse Xenograft Model The efficacy of DEC2-DOX-LL DectiSomes in mouse xenograft models of three deadly metastatic cancers, non-small cell lung carcinoma (NSCLC), prostate cancer, and triple negative breast cancer (TNBC) can be determined.
  • NSCLC non-small cell lung carcinoma
  • TNBC triple negative breast cancer
  • Dectin-2 coated DOXIL DectiSomes are delivered intravenously (i.v.) to mice with various metastatic tumors, the drug can reach tumor cells and kill or inhibit the tumor growth more effectively than commercial DOXIL (DOX-LLs).
  • DOX-LLs commercial DOXIL
  • a significant reduction in the dose of DOX required when it is delivered by DEC2-DOX-LLs is expected.
  • Establishing improved efficacy at a reduced dose in mice, using multiple xenograft models can be extrapolated into efficacy with reduced toxicity to patients relative to untargeted DOXIL or other cancer drugs.
  • mice PATENT ATTORNEY DOCKET NO.0U6500-1491008 (010WO1)
  • Successful xenografts of human cancer cells into mice require that the mice are immunodeficient (Sargent et al. “Genetically diverse mouse platform to xenograft cancer cells,” Dis Model Mech 15, 10.1242/dmm 049457 (2022)).
  • NOD/ShiLtJ-Rag1em5Lutzy/J inbred RAG KO mice or NOD SCID NOD.Cg-Prkdcscid/J mice (JAX:001303) can be used for these experiments. They are both deficient in both B and T cells, and readily xenograft (Hudson et al.
  • mice Leukemia 12, 2029-33.10.1038/sj.leu.2401236 (1998).
  • Well-characterized metastatic cell lines to establish tumors in mice including NSCLC cell lines (A549, H-1975, and HCC-827), prostate cancer cell lines (PC-3, DU-145, and LNCaP-LN3), and the TNBC cell lines (MDA-MB-231, MDA-MB-468, BT-20, HCC-70 and BT-549), can be used.
  • Mice will be given a subcutaneous injection (s.c.) in both flanks with 5x10 6 cells on Day 0 (D0) (Jones et al., Leukemia 12, 2029-33.10.1038/sj.leu.2401236 2005)).
  • V t (length x width 2 ) x 2) at D21 (3 weeks).
  • three-week tumor volumes can vary. Although variation can occur, one of skill in the art would know how to obtain adequate tumor volumes for these studies.
  • the ability of DEC2-DOX-LLs to suppress tumor growth relative to DOXIL (DOX- LLs) will be studied. A mock treatment control will be included. On D7, D14 and D21 after initiating s.c. tumors, mice will be given a retro-orbital (intravenous) injection of the liposome reagents.
  • Treatment will be started with both type of liposomes delivering 4 mg/kg DOX, but doses can range from 1 to 10 mg/kg.
  • a set of mock treated control mice with tumors will be given liposomal buffer. Reductions in tumor size greater than 50% at D28 and 50% survival at D42 can be expected. It is possible that higher leves of tumor size reduct and survival will be observed during these time frames. Survival studies will be carried out for 5 to 8 weeks, but the time necessary to reach a humane endpoint will likely vary widely among NSCLC, prostate, and TNBC cancers and cell lines. Power analysis. An exemplary power analysis for TNBC tumors is provided below. Following various therapeutic treatments (Georgievski et al., 2024 Cell Death Dis 15, 328.
  • a subject that has cancer can be treated using any of the pharmaceutical compositions or methods described herein.
  • an effective amount of any of the plurality of C-type receptor targeted nanoparticles comprising an anticancer agent described herein can be administered intravenously to a subject.
  • Intravenous delivery can be perfomed by infusion of the plurality of nanoparticles over the course of 15 minutes to an hour or more.
  • the subject can be dosed in this manner, for example, once every few weeks (for example, every three or four weeks), for as long as the cancer in the subject PATENT ATTORNEY DOCKET NO.0U6500-1491008 (010WO1) does not progress, shows no evidence of cardiotoxicity and continues to tolerate treatment.
  • the plurality of nanoparticles is delivered via intracarotid, intranasal, intracranial, intraperitoneal, or convection-enhanced delivery.
  • the subject will undergo monthly treatments for two, three, four or months.
  • a clinician can assess whether the subject is responding to treatment or not. If the subject is not responding or the subject is experiencing adverse effects, the dosage of the anticancer agent can be altered or a second therapeutic agent can be added to the subject treatment regimen.

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Abstract

L'invention concerne des nanoparticules ciblant le cancer pour le traitement ou la prévention du cancer.
PCT/US2025/021146 2024-03-22 2025-03-24 Nanoparticules ciblées et leurs utilisations contre le cancer Pending WO2025199531A2 (fr)

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