NANOPARTICLE IMMUNOCONJUGATES

Abstract

Disclosed herein are nanoparticle immunoconjugates useful for therapeutics and/or diagnostics. The immunoconjugates have diameter (e.g., average diameter) no greater than 20 nanometers (e.g., as measured by dynamic light scattering (DLS) in aqueous solution, e.g., saline solution). In certain embodiments, the conjugates are silica-based nanoparticles with single chain antibody fragments attached thereto.

Claims

1-44. (canceled)

45. An immunoconjugate comprising: a nanoparticle coated with an organic polymer; an antibody fragment conjugated to the organic polymer-coated nanoparticle, a therapeutic agent conjugated to the organic polymer-coated nanoparticle through a linker, wherein the nanoparticle has a diameter no greater than 20 nanometers, wherein the nanoparticle comprises a silica-based core and a silica shell surrounding at least a portion of the core, wherein the antibody fragment is a single chain variable fragment (scFv).

46. The immunoconjugate of claim 45, wherein the linker is a cleavable linker.

47. The immunoconjugate of claim 46, wherein the cleavable linker is selected from a group consisting of peptide, hydrazine and disulfide linkers.

48. The immunoconjugate of claim 45, wherein the linker is a peptide linker.

49. The immunoconjugate of claim 45, wherein the linker is a peptide linker that is cleaved by lysosomal proteases.

50. The immunoconjugate of claim 49, wherein the lysosomal protease is cathepsin-B.

51. The immunoconjugate of claim 45, wherein the linker is a dipeptide linker.

52. The immunoconjugate of claim 51, wherein the dipeptide linker is a valine-citrulline linker.

53. The immunoconjugate of claim 45, wherein the antibody fragment is from about 25 kDa to about 30 kDa.

54. The immunoconjugate of claim 45, wherein the nanoparticle comprises a fluorescent compound within the core.

55. The immunoconjugate of claim 45, wherein the nanoparticle has from one to ten antibody fragments attached thereto.

56. The immunoconjugate of claim 45, wherein the nanoparticle has a diameter no greater than 15 nanometers.

57. The immunoconjugate of claim 45, wherein the nanoparticle has a diameter in a range from 1 nm to 20 nm.

58. The immunoconjugate of claim 45, wherein the antibody fragment comprises anti-VEGF-A.

59. The immunoconjugate of claim 45, wherein the immunoconjugate comprises one or more imaging agents.

60. The immunoconjugate of claim 59, wherein the one or more imaging agents comprise a PET tracer.

61. The immunoconjugate of claim 59, wherein the one or more imaging agents comprise a fluorophore.

62. The immunoconjugate of claim 4, wherein the therapeutic agent comprises a chemotherapy drug.

63. The immunoconjugate of claim 45, wherein the therapeutic agent comprises a radioisotope.

64. The immunoconjugate of claim 63, wherein the radioisotope is a member selected from the group consisting of .sup.99mTc, .sup.111In, .sup.64Cu, .sup.67Ga, .sup.186Re, .sup.188Re, .sup.153Sm, .sup.177Lu, .sup.67Cu, .sup.123I, .sup.124I, .sup.125I, .sup.11C, .sup.13N, .sup.15O, .sup.18F, .sup.186Re, .sup.188Re, .sup.153Sm, .sup.166Ho, .sup.177Lu, .sup.149Pm, .sup.90Y, .sup.213Bi, .sup.103Pd, .sup.109Pd, .sup.159Gd, .sup.140La, .sup.198Au, .sup.199Au, .sup.169Yb, .sup.175Yb, .sup.165Dy, .sup.166Dy, .sup.67Cu, .sup.105Rh, .sup.111Ag, .sup.89Zr, .sup.225Ac, and .sup.192Ir.

Description

BRIEF DESCRIPTION OF DRAWINGS

[0075] The foregoing and other objects, aspects, features, and advantages of the present disclosure will become more apparent and better understood by referring to the following description taken in conduction with the accompanying drawings, in which:

[0076] FIG. 1 shows a schematic illustration showing the synthesis of .sup.89Zr-labeled C′dot radioimmunoconjugate using a chelator-based radiolabeling technique. PEGylated and maleimide-functionalized C′ dot (C′ dot-PEG-Mal, 1) was first reacted with reduced glutathione (GSH) to introduce the —NH.sub.2 groups for the following-up bioconjugates, forming C′ dot-PEG-GSH (2). Then the nanoparticle was conjugated with DBCO-PEG4-NHS ester and DFO-NCS, forming C′ dot-PEG-DBCO (3) and DFO-C′ dot-PEG-DBCO (4), respectively. Azide-functionalized small targeting ligands, such as single-chain variable fragment (scFv-azide) (or single-domain antibody, sdAb-azide), was conjugated to the nanoparticle based on strain-promoted azide-alkyne cycloaddition, forming DFO-C′ dot-PEG-scFv (5). The final C′dot radioimmunoconjugate (.sup.89Zr-DFO-C′ dot-PEG-scFv, 6) was by labeling it with .sup.89Zr-oxalate. The embodiments illustrated in FIG. 1 are not limited to scFv and can include various types of antibody fragments, e.g., sdAbs.

[0077] FIGS. 2A and 2B show in vivo (FIG. 2A) coronal and (FIG. 2B) sagittal PET images of .sup.89Zr-DFO-C′ dot-PEG at different post-injection time points (10 min, 1 h, Day 1, Day 3 and Day 6) in a healthy nude mouse. The reaction ratio between C′ dot-PEG-Mal and GSH was kept at 1:20. The PET images were acquired by using a Focus 120 MicroPET scanner.

[0078] FIG. 3 shows biodistribution data of .sup.89Zr-DFO-C′ dot-PEG in a healthy nude mouse on Day 6. Less than 2% ID/g of bone (and joint) uptake was observed.

[0079] FIGS. 4A and 4B show a chelator-free .sup.89Zr radiolabeling experimental example.

[0080] FIG. 4A shows .sup.89Zr labeling yields of C′ dot-PEG-Mal under varied pH conditions at 75° C.

[0081] FIG. 4B shows .sup.89Zr labeling yields of C′ dot-PEG-Mal using varied combinations of C′ dot to .sup.89Zr-oxalate ratio.

[0082] FIGS. 5A and 5B show in vivo coronal PET images of [89Zr]C′ dot-PEG at different post-injection time points (10 min, Day 1, Day 3 and Day 6) in a healthy nude mouse. [.sup.89Zr]C′ dot-PEG was synthesized by using a chelator-free radiolabeling technique. The PET images were acquired by using a Focus 120 MicroPET scanner.

[0083] FIG. 5A shows PET images acquired without EDTA (ethylenediaminetetraacetic acid).

[0084] FIG. 5B shows PET images acquired with EDTA

[0085] FIG. 6 shows biodistribution data of [.sup.89Zr]C′ dot-PEG in healthy nude mice (n=3) on Day 7. Over 10% ID/g of bone (and joint) uptake was observed in this case, indicating a less stable radiolabeling using a chelator-free method (when compared with that of chelator-based method).

[0086] FIG. 7 shows biodistribution data of .sup.89Zr-DFO-C′ dot, .sup.89Zr-DFO-C′ dot-DBCO and .sup.89Zr-DFO-C′ dot-PEG-sdAb in healthy nude mice at 48 h post-injection. An improved pharmacokinetic profile (with prolonged blood circulation half-life and lower liver uptake) can be achieved by optimizing the number of DFO, DBCO and sdAb from each C′ dot.

[0087] FIG. 8 shows an exemplary schematic of thiol-maleimide chemistry.

[0088] FIG. 9 shows an exemplary schematic of alkene-tetrazine chemistry.

DETAILED DESCRIPTION

[0089] Throughout the description, where compositions are described as having, including, or comprising specific components, or where methods are described as having, including, or comprising specific steps, it is contemplated that, additionally, there are compositions of the present invention that consist essentially of, or consist of, the recited components, and that there are methods according to the present invention that consist essentially of, or consist of, the recited processing steps.

[0090] It should be understood that the order of steps or order for performing certain action is immaterial so long as the invention remains operable. Moreover, two or more steps or actions may be conducted simultaneously.

[0091] The mention herein of any publication, for example, in the Background section, is not an admission that the publication serves as prior art with respect to any of the claims presented herein. The Background section is presented for purposes of clarity and is not meant as a description of prior art with respect to any claim.

[0092] Molecular therapeutics (e.g., antibodies) can modulate the immune system toward antitumor activity by manipulating immune checkpoints (e.g., the monoclonal antibody ipilimumab inhibits CTLA4, a negative regulatory molecule that inhibits function of the immune system). The rationale is to trigger preexisting, but dormant, antitumor immune responses. Other molecules and pathways have acted as immune switches. PD-1, another negative regulatory receptor expressed on T cells, has also been targeted. Switching a single immune checkpoint may not be sufficient to induce an antitumor response, explaining some of the failures of targeting single immune regulatory checkpoints like PD-1 or CTLA4. However, without wishing to be bound to any theory, treatment can be bolstered by the addition of RT, which is thought, in some cases, to have immunomodulatory properties. In these cases, tumors outside of RT treatment fields have been found to shrink as a result of a putative systemic inflammatory or immune response provoked by RT, highlighting the potential for radiation to spark a systemic antitumor immune response. Augmenting immune activity may also potentiate the local effects of RT.

[0093] By raising the concentration alone of these immunoconjugates, disease can be treated. A therapeutic radiolabel can also be added to further treat disease. In certain embodiments, the immunoconjugate act as a therapeutic at high concentrations, and without a therapeutic radiolabel. In certain embodiments, the radiolabel is attached to the same nanoparticle in an all-in-one multi-therapeutic platform. Alternatively, therapeutic radioisotopes can be administered independently.

[0094] Described herein are target-specific nanoparticle immunoconjugates (e.g., single chain antibody fragments bound to the particle surface) for targeted diagnostic and/or therapeutic platforms. In certain embodiments, the nanoparticle immunoconjugates are less than 20 nm (e.g., 6 to 10 nm) in diameter. This small size is found to offer advantages in therapeutic and/or imaging applications. For example, the disclosed immunoconjugates may offer improved targeting of diseased tissue and reduced non-specific uptake by organs (e.g., by the liver). The smaller immunoconjugates may also demonstrate reduced immune reactivity, thereby further improving efficacy.

[0095] In certain embodiments, the nanoparticle comprises silica, polymer (e.g., poly(lactic-co-glycolic acid) (PLGA)), and/or metal (e.g., gold, iron).

[0096] In certain embodiments, the silica-based nanoparticle platform comprises ultrasmall nanoparticles or “C dots,” which are fluorescent, organo-silica core shell particles that have diameters controllable down to the sub-10 nm range with a range of modular functionalities. C dots are described by U.S. Pat. No. 8,298,677 B2 “Fluorescent silica-based nanoparticles”, U.S. Publication No. 2013/0039848 A1 “Fluorescent silica-based nanoparticles”, and U.S. Publication No. US 2014/0248210 A1 “Multimodal silica-based nanoparticles”, the contents of which are incorporated herein by reference in their entireties. Incorporated into the silica matrix of the core are near-infrared dye molecules, such as Cy5.5, which provides its distinct optical properties. Surrounding the core is a layer or shell of silica. The silica surface is covalently modified with silyl-polyethylene glycol (PEG) groups to enhance stability in aqueous and biologically relevant conditions. These particles have been evaluated in vivo and exhibit excellent clearance properties owing largely to their size and inert surface. Among the additional functionalities incorporated into C dots are chemical sensing, non-optical (PET) image contrast and in vitro/in vivo targeting capabilities, which enable their use in visualizing lymph nodes for surgical applications, and melanoma detection in cancer.

[0097] C dots are synthesized via an alcohol-based modified Stöber process. C′dots are synthesized in water.

[0098] C dots or C′dots provide a unique platform for drug delivery due to their physical properties as well as demonstrated human in vivo characteristics. These particles are ultrasmall and benefit from EPR effects in tumor microenvironments, while retaining desired clearance and pharmacokinetic properties. To this end, described herein is a nanoparticle drug delivery system in which, in certain embodiments, drug constructs are covalently attached to C dots or C′dots (or other nanoparticles).

[0099] C dots or C′dots can serve as highly specific and potent multi-therapeutic targeted particle probes to combine antibody fragments with therapeutic radiolabels (e.g., .sup.177Lu, .sup.225Ac, .sup.90Y, .sup.89Zr) on a single platform. Alternatively, C dot or C′dot coupling of targeting peptides, such as alphaMSH, known to be immunomodulatory and anti-inflammatory in nature, can also be combined with C dot or C′dot radiotherapeutic (and/or other particle-based) platforms to achieve enhanced efficacy. In certain embodiments, the concentration of the radioisotope and/or antibody fragment is higher in therapeutic applications compared to diagnostic applications.

[0100] In contrast to other multimodal platforms, immunoconjugates can comprise different moieties that are attached to the nanoparticle itself. For example, in certain embodiments, a radioisotope is attached to the nanoparticle and an antibody fragment is attached to the nanoparticle—that is, in these embodiments, the radiolabel is not attached to the antibody fragment itself. As another example, immunoconjugates can comprise a targeting ligand attached to the nanoparticle, a radioisotope attached to the nanoparticle, and an antibody fragment attached to the nanoparticle. The stoichiometric ratios of different moieties attached to the C dot will affect the biodistribution of the nanoparticle immunoconjugate.

[0101] The immunoconjugates, e.g., C dot-antibody (mAb) and -antibody-fragment (vFab) conjugates, can be prepared using either of two approaches. Scheme 1 comprises thiol-maleimide chemistry, as shown in FIG. 8. Scheme 1 is designed around proteins modified to contain maleimide groups. Scheme 2 comprises alkene-tetrazine chemistry as shown in FIG. 9.

[0102] In Scheme 1 as shown in FIG. 8, C dots containing Cy5 dye, surface functionalized with PEG and maleimide groups (C dots-(Cy5)-PEG-mal) were prepared as previously described in Bradbury et al., 2014. Silanes modified with the Cy5 fluorophore were prepared and titrated with tetramethylorthosilane (TMOS) into a dilute solution of NH.sub.4OH (molar ratio TMOS:Cy5:NH3:H20 is 1:0.001:0.44:1215) and allowed to mix for 24 hours (Urata C, Aoyama Y, Tonegawa A, Yamauchi Y, Kuroda K. Dialysis process for the removal of surfactants to form colloidal mesoporous silica nanoparticles. Chem Commun (Camb). 2009; (34):5094-6) (Yamada H, Urata C, Aoyama Y, Osada S, Yamauchi Y, Kuroda K. Preparation of Colloidal Mesoporous Silica Nanoparticles with Different Diameters and Their Unique Degradation Behavior in Static Aqueous Systems, Chem. Mater. 2012; 24(8):1462-71.) (Wang J, Sugawara-Narutaki A, Fukao M, Yokoi T, Shimojima A, Okubo T. Two-phase synthesis of monodisperse silica nanospheres with amines or ammonia catalyst and their controlled self-assembly. ACS Appl Mater Interfaces. 2011; 3(5):1538-44.) This resulted in a Cy5 encapsulated silica particle, the surface of which was further PEGylated and functionalized with maleimide groups by treatment with PEG-silane (500 g/mole) (Suzuki K, Ikari K, Imai H. Synthesis of silica nanoparticles having a well-ordered mesostructured using a double surfactant system. J Am Chem Soc. 2004; 126(2):462-3) and maleimide-PEG-silane (molar ratio PEG-silane:TMOS:mal-PEG-silane of 1:2.3:0.006). The maleimide groups can then be effectively transformed into amine groups by reacting the particles with compounds that contain a thiol and amine (e.g., cysteine methyl ester or cysteamine-HCl). The resulting C dot-(Cy5)-PEG-amine can then be subsequently modified with a succinimidyl 3-(2-pyridyldithio)propionate (SPDP). The pyridyldithiol serves at least two purposes: one, it can be used to quantitate conjugation efficiencies; two, it may serves as a ‘protecting group’ to minimize oxidation of thiol groups; etc. TCEP can then be used to remove the group releasing a pyridine 2-thione, which can be measured by HPLC or UV-absorption for quantitation. The resulting C dot-(Cy5)-PEG-thiol can then be reacted with protein-maleimide leading to the desired C dot-(Cy5)-PEG-mAb or C dot-(Cy5)-PEG-vFab.

[0103] In Scheme 2 as shown in FIG. 9, alkene-tetrazine chemistry is utilized for protein attachment. Here, the mAb or vFab is modified with a click reactive groups, such as methyltetrazine-PEG.sub.4-NHS ester. The C dot-(Cy5)-PEG-amine, as described in FIG. 8 (Scheme 1), is then modified with the appropriate click partner, (e.g., TCO-PEG4-NHS ester). In the final step, the methyltetrazine-mAb or -vFab can then be reacted with the C dot-(Cy5)-PEG-TCO leading to the C dot-(Cy5)-PEG-mAb or C dot-(Cy5)-PEG-vFab product.

[0104] Antibody fragments (fAbs) provide advantages (e.g., size, no Fc region for reduced immunogenicity, scalability, and adaptability) compared to standard monoclonal antibodies (mAbs). fAbs are the stripped-down binding region of an antibody which is usually expressed as a single continuous sequence in an expression host (e.g., E. Coli). In certain embodiments, a fAb or mAb can be as small as 15 kDa (+/−5 kDa) (e.g., about 3 nm). In other embodiments, a fAb or mAb can be up to 150 kDa (e.g., up to 20 nm). In one embodiment, a fAb is approximately 60 kDa (e.g., +/−15 kDa). A fAb comprises an immunoglobin heavy-chain variable and constant domain linked to the corresponding domains of an immunoglobin light chain. In another embodiment, the antibody format can be a single chain variable fragment (scFv) fragment that is approximately 30 kDa (e.g., +/−10 kDa). A scFv fragment comprises a heavy-chain variable domain linked to a light-chain variable domain. In other embodiments, the antibody format can be a single domain antibody (sdAb) fragment that is approximately 15 kDa (e.g., +/−5 kDa). A sdAb fragment comprises a single heavy-chain variable domain. In certain embodiments, the antibody fragment is an anti-CEA scFv for targeting different tumors.

[0105] In certain embodiments, various linkers are used. In certain embodiments, a cleavable linker (e.g., peptide, hydrazine, or disulfide) is used. In certain embodiments, a noncleavable linker (e.g., thioether) is used. In certain embodiments, a peptide linker is selectively cleaved by lysosomal proteases (e.g., cathepsin-B). In certain embodiments, a valine-citrulline dipeptide linker is used.

[0106] In certain embodiments, different linkers as described in U.S. Pat. Nos. 4,680,338, 5,122,368, 5,141,648, 5,208,020, 5,416,064, 5,475,092, 5,543,390, 5,563,250 5,585,499, 5,880,270, 6,214,345, 6,436,931, 6,372,738, 6,340,701, 6,989,452, 7,129,261, 7,375,078, 7,498,302, 7,507,420, 7,691,962, 7,910,594, 7,968,586, 7,989,434, 7,994,135, 7,999,083, 8,153,768, 8,236,319, Zhao, R.; et al, (2011) J. Med. Chem. 36, 5404; Doronina, S.; et al, (2006) Bioconjug Chem, 17, 114; Hamann, P.; et al. (2005) Bioconjug Chem. 16, 346, the contents of which are hereby incorporated by reference herein, are used.

[0107] In certain embodiments, the mAbs and/or fAbs are U.S. approved for certain uses. Non-limiting examples of mAbs and fAbs include anti-GPIIb/IIIa, anti-VEGF-A, and anti-TNF-α. ReoPro (abciximab) is an anti-GPIIb/IIIa, chimeric fAb, IgG1-κ developed by Centocor/Eli Lilly as described by Nelson and Reichert, “Development trends for therapeutic antibody fragments,” Nature Biotechnology, 27(4), 2009. Lucentis (ranibizumab) is an anti-VEGF-A, humanized Fab IgG1-κ developed by Genentech (Nelson and Reichert, 2009) that is used to prevent wet age-related macular degeneration. Cimzia (certolizumab pegol), is an Anti-TNF-α, PEGylated humanized fAb developed by UCB (Nelson and Reichert, 2009) that is used to prevent moderate to severe Crohn's disease.

[0108] In certain embodiments, PET (Positron Emission Tomography) tracers are used as imaging agents. In certain embodiments, PET tracers comprise .sup.89Zr, .sup.64Cu, [.sup.18F] fluorodeoxyglucose.

[0109] In certain embodiments, fluorophores comprise fluorochromes, fluorochrome quencher molecules, any organic or inorganic dyes, metal chelates, or any fluorescent enzyme substrates, including protease activatable enzyme substrates. In certain embodiments, fluorophores comprise long chain carbophilic cyanines. In other embodiments, fluorophores comprise DiI, DiR, DiD, and the like. Fluorochromes comprise far red, and near infrared fluorochromes (NIRF). Fluorochromes include but are not limited to a carbocyanine and indocyanine fluorochromes. In certain embodiments, imaging agents comprise commercially available fluorochromes including, but not limited to Cy5.5, Cy5 and Cy7 (GE Healthcare); AlexaFlour660, AlexaFlour680, AlexaFluor750, and AlexaFluor790 (Invitrogen); VivoTag680, VivoTag-S680, and VivoTag-5750 (VisEn Medical); Dy677, Dy682, Dy752 and Dy780 (Dyomics); DyLight547, DyLight647 (Pierce); HiLyte Fluor 647, HiLyte Fluor 680, and HiLyte Fluor 750 (AnaSpec); IRDye 800CW, IRDye 800RS, and IRDye 700DX (Li-Cor); and ADS780WS, ADS830WS, and ADS832WS (American Dye Source) and Kodak X-SIGHT 650, Kodak X-SIGHT 691, Kodak X-SIGHT 751 (Carestream Health).

[0110] In certain embodiments, click reactive groups are used (for ‘click chemistry’). Examples of click reactive groups include the following: alkyne, azide, thiol (sulfydryl), alkene, acrylate, oxime, maliemide, NHS (N-hydroxysuccinimide), amine (primary amine, secondary amine, tertiary amine, and/or quarternary ammonium), phenyl, benzyl, hydroxyl, carbonyl, aldehyde, carbonate, carboxylate, carboxyl, ester, methoxy, hydroperoxy, peroxy, ether, hemiacetal, hemiketal, acetal, ketal, orthoester, orthocarbonate ester, amide, carboxyamide, imine (primary ketimine, secondary ketamine, primary aldimine, secondary aldimine), imide, azo (diimide), cyanate (cyanate or isocyanate), nitrate, nitrile, isonitrile, nitrite (nitrosooxy group), nitro, nitroso, pyridyl, sulfide, disulfide, sulfinyl, sulfonyl, sulfino, sulfo, thiocyanate, isothiocyanate, caronothioyl, thione, thial, phosphine, phosphono, phosphate, phosphodiester, borono, boronate, bomino, borinate, halo, fluoro, chloro, bromo, and/or iodo moieties.

[0111] Cancers that may be treated include, for example, prostate cancer, breast cancer, testicular cancer, cervical cancer, lung cancer, colon cancer, bone cancer, glioma, glioblastoma, multiple myeloma, sarcoma, small cell carcinoma, melanoma, renal cancer, liver cancer, head and neck cancer, esophageal cancer, thyroid cancer, lymphoma, and/or leukemia.

[0112] In certain embodiments, targeting peptide ligands, such as alpha-MSH, attached to C dots, can serve as immunomodulators alongside other therapies to enhance treatment response.

[0113] In certain embodiments, in addition to administration of an immunoconjugate described herein, a method of treatment may include administration of antibodies, small molecule drugs, radiation, pharmacotherapy, chemotherapy, cryotherapy, thermotherapy, electrotherapy, phototherapy, ultrasonic therapy and/or surgery.

[0114] In certain embodiments, the immunoconjugate comprises a therapeutic agent, e.g., a drug (e.g., a chemotherapy drug) and/or a therapeutic radioisotope. As used herein, “therapeutic agent” refers to any agent that has a therapeutic effect and/or elicits a desired biological and/or pharmacological effect, when administered to a subject.

[0115] In certain embodiments, the radioisotope is a radiolabel that can be monitored/imaged (e.g., via PET or single-photon emission computed tomography (SPECT)). Example radioisotopes that can be used include beta emitters (e,g. .sup.177Lutetium) and alpha emitters (e.g., .sup.225Ac). In certain embodiments, one or more of the following radioisotopes are used: .sup.99mTc, .sup.111In, .sup.64Cu, .sup.67Ga, .sup.186Re, .sup.188Re, .sup.153Sm, .sup.177Lu, .sup.67Cu, .sup.123I, .sup.124I, .sup.125I, .sup.11C, .sup.13N, .sup.15O, .sup.18F, .sup.186Re, .sup.188Re, .sup.153Sm, .sup.166Ho, .sup.177Lu, .sup.149Pm, .sup.90Y, .sup.213Bi, .sup.103Pd, .sup.109Pd, .sup.159Gd, .sup.140La, .sup.198Au, .sup.199Au, .sup.169Yb, .sup.175Yb, .sup.165Dy, .sup.166Dy, .sup.67Cu, .sup.105Rh, .sup.111Ag, .sup.89Zr, .sup.225Ac, and .sup.192Ir.

[0116] In certain embodiments, the immunoconjugate comprises one or more drugs, e.g., one or more chemotherapy drugs, such as sorafenib, paclitaxel, docetaxel, MEK162, etoposide, lapatinib, nilotinib, crizotinib, fulvestrant, vemurafenib, bexorotene, and/or camptotecin.

[0117] In certain embodiments, the immunoconjugate comprises a chelator, for example, 1,4,8,11-tetraazabicyclo[6.6.2]hexadecane-4,11-diyl)diacetic acid (CB-TE2A); desferoxamine (DFO); diethylenetriaminepentaacetic acid (DTPA); 1,4,7,10-tetraazacyclotetradecane-1,4,7,10-tetraacetic acid (DOTA); thylenediaminetetraacetic acid (EDTA); ethylene glycolbis(2-aminoethyl)-N,N,N′,N′-tetraacetic acid (EGTA); 1,4,8,11-tetraazacyclotetradecane-1,4,8,11-tetraacetic acid (TETA); ethylenebis-(2-4 hydroxy-phenylglycine) (EHPG); 5-Cl-EHPG; 5Br-EHPG; 5-Me-EHPG; 5t-Bu-EHPG; 5-sec-Bu-EHPG; benzodiethylenetriamine pentaacetic acid (benzo-DTPA); dibenzo-DTPA; phenyl-DTPA, diphenyl-DTPA; benzyl-DTPA; dibenzyl DTPA; bis-2 (hydroxybenzyl)-ethylene-diaminediacetic acid (HBED) and derivatives thereof; Ac-DOTA; benzo-DOTA; dibenzo-DOTA; 1,4,7-triazacyclononane N,N′,N″-triacetic acid (NOTA); benzo-NOTA; benzo-TETA, benzo-DOTMA, where DOTMA is 1,4,7,10-tetraazacyclotetradecane-1,4,7,10-tetra(methyl tetraacetic acid), benzo-TETMA, where TETMA is 1,4,8,11-tetraazacyclotetradecane-1,4,8,11-(methyl tetraacetic acid); derivatives of 1,3-propylenediaminetetraacetic acid (PDTA); triethylenetetraaminehexaacetic acid (TTHA); derivatives of 1,5,10-N,N′,N″-tris(2,3-dihydroxybenzoyl)-tricatecholate (LICAM); and 1,3,5-N,N′,N″-tris(2,3- dihydroxybenzoyl)aminomethylbenzene (MECAM), or other metal chelators.

[0118] In certain embodiments, the immunoconjugate comprises more than one chelator.

[0119] In certain embodiments the radioisotope-chelator pair is .sup.89Zr-DFO. In certain embodiments the radioisotope-chelator pair .sup.177Lu-DOTA. In certain embodiments, the is radioisotope-chelator pair is .sup.225Ac-DOTA.

[0120] In certain embodiments, the therapeutic agent (e.g., drug and/or radioisotope) is attached to the nanoparticle or the antibody fragment (protein), or both, using a bioorthogonal conjugation approach (e.g., amine/NHS-ester, thiol/maleimide, azide/alkyne click, or tetrazine/TCO click). For radiolabeling using radiometals, the radiometal chelator can be first attached to either particle or protein or both, followed by the radiometal. Alternatively, the radiometal/chelator complex can be performed, followed by attachment onto the particle or protein or both. Radioiodination can also be achieved using standard approaches where a tyrosine or phenolic group on the particle or protein or both is modified by electrophilic addition chemistry.

[0121] In certain embodiments, the immunoconjugate is administered to a subject suffering from a particular disease or condition (e.g., cancer) for treatment of the disease or condition.

EXPERIMENTAL EXAMPLES

Preparation of the C Dot-(Cy5)-PEG-Maleimide:

[0122] A maleimide and NHS ester functionalized polyethylene glycol (mal-dPEG.sub.12-NHS) was conjugated with aminosilane (APTES) in DMSO (molar ratio mal-PEG-NHS:APTES:DMSO 1:0.9:60). The reaction mixture was left under nitrogen at room temperature for 48 hours to generate silane functionalized mal-dPEG (mal-dPEG-APTES). A maleimide functionalized Cy5 (mal-Cy5) was reacted with a thiol-silane (MPTMS) in DMSO (molar ratio Cy5:MPTMS:DMOS 1:25:1150). The reaction was left under nitrogen at room temperature for 24 hours to generate a silane functionalized Cy5 (Cy5-MPTMS). TMOS and Cy5-MPTMS were then titrated into an ammonia hydroxide solution (˜pH 8) (molar ratio TMOS:Cy5:NH3:H2O 1:0.001:0.44:1215). The solution was stirred at 600 rpm at room temperature for 24 hours to form homogeneous Cy5 encapsulated silica nanoparticles. The mal-dPEG-APTES and silane functionalized polyethylene glycol (PEG-silane, MW around 500, Gelest) were then added into the synthesis solution to PEGylate and surface-functionalize the particles (PEG-silane:TMOS:mal-PEG-APTES 1:2.3:0.006). The solution was stirred at 600 rpm at room temperature for 24 hours followed by incubation at 80° C. for another 24 hours without stirring. The solution was dialyzed in 2000 mL with deionized water for two days (10 k MWCO), filtered with 200 nm syringe filters, and finally chromatographically purified (Superdex 200) resulting in the desired mal-C dots.

Preparation of C Dot Immunoconjugates

[0123] Studies were performed to conjugate single chain antibody fragments (scFv)s to the C dot core silica nanoparticles. An scFv that bound matrix metalloproteinase 12 (MMP-12) was expressed in E. coli. The construct contained C-terminal His and FLAG tags for nickel affinity chromatography and immune-detection. A mutant scFv was constructed in which the last amino acid of the polypeptide chain was converted to a cysteine (Cys). The change was confirmed by sequencing the mutant gene. Expression and nickel affinity purification of the wild type scFv and the C-terminal Cys containing mutant was confirmed by sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis (PAGE), visualized with Coomassie blue stain at a molecular weight consistent with the scFv. Western blot analysis of the scFv SDS PAGE gel was performed with an anti-FLAG tag HRP conjugate. The Western blot analysis confirmed that the identity of the gel band was the scFv.

[0124] The scFv were clones modified with azide containing bifunctional linkers. The wild type scFv was modified with N-hydroxy-succinimide (NHS) ester-polyethylene glycol (PEG).sub.4-azide. Without wishing to be bound to any theory, modification of wild type scFv with NHS ester-PEG.sub.4-azide results in the random incorporation of PEG.sub.4-azide on to free amines on surface lysine residues. The C-terminal scFv Cys construct was conjugated with maleimide-PEG.sub.3-azide for site specific PEG.sub.3-azide introduction on to the Cys sulfhydryl. The scFv constructs were analyzed for azide incorporation by reaction with a Dibenzocyclooctyne (DBCO)-PEG-Cy5 fluorescent probe. Azides react with DBCOs via a metal free click chemistry reaction to form a covalent linkage. Unreacted DBCO-Cy5 dye was removed from the reaction mixtures by 40 kDa cutoff size exclusion spin columns. The successful introduction of an azide group on the surface of the scFvs was confirmed by visualizing the wild type and C-terminal Cys scFv-PEG-Cy5 fluorescent dye constructs using a BioRad Versa-Doc imager.

[0125] The azide conjugated scFv were then reacted with C dots containing 1-3 DBCOs on their surfaces. The reaction was allowed to continue for 12 h at room temperature. Unconjugated scFv was purified from conjugated scFv-C dots using multiple techniques including phosphate buffered saline washes in 50,000 molecular weight cut off spin columns, G-200 size exclusion column chromatography or size exclusion spin columns and velocity sedimentation thought a sucrose cushion. Velocity sedimentation and size exclusion chromatography appear to be the most scalable methods of purification. The purified scFv C-dot conjugates were analyzed by dot blot scFv immune-detection/particle fluorescence assays, gel electrophoresis and fluorescent ELISAs with immobilized MMP-12.

[0126] These methods can be applied to other types of antibody fragments, e.g., sdAbs.

[0127] FIG. 1 shows a schematic illustration showing the synthesis of .sup.89Zr-labeled C′dot radioimmunoconjugate using a chelator-based radiolabeling technique. PEGylated and maleimide-functionalized C′ dot (C′ dot-PEG-Mal, 1) was first reacted with reduced glutathione (GSH) to introduce the —NH.sub.2 groups for the following-up bioconjugates, forming C′ dot-PEG-GSH (2). Then the nanoparticle was conjugated with DBCO-PEG4-NHS ester and DFO-NCS, forming C′ dot-PEG-DBCO (3) and DFO-C′ dot-PEG-DBCO (4), respectively. Azide-functionalized small targeting ligands, such as single-chain variable fragment (scFv-azide) (or single-domain antibody, sdAb-azide), was conjugated to the nanoparticle based on strain-promoted azide-alkyne cycloaddition, forming DFO-C′ dot-PEG-scFv (5). The final C′dot radioimmunoconjugate (.sup.89Zr-DFO-C′ dot-PEG-scFv, 6) was by labeling it with .sup.89Zr-oxalate. The schematic illustrated in FIG. 1 is not limited to scFv and can include various types of antibody fragments, e.g., sdAbs.

[0128] FIGS. 2A and 2B show in vivo (FIG. 2A) coronal and (FIG. 2B) sagittal PET images of .sup.89Zr-DFO-C′ dot-PEG at different post-injection time points (10 min, 1 h, Day 1, Day 3 and Day 6) in a healthy nude mouse. The reaction ratio between C′ dot-PEG-Mal and GSH was kept at 1:20. The PET images were acquired by using a Focus 120 MicroPET scanner.

[0129] FIG. 3 shows biodistribution data of .sup.89Zr-DFO-C′ dot-PEG in a healthy nude mouse on Day 6. Less than 2% ID/g of bone (and joint) uptake was observed.

[0130] FIGS. 4A and 4B show a chelator-free .sup.89Zr radiolabeling experimental example.

[0131] FIG. 4A shows .sup.89Zr labeling yields of C′ dot-PEG-Mal under varied pH conditions at 75° C.

[0132] FIG. 4B shows .sup.89Zr labeling yields of C′ dot-PEG-Mal using varied combinations of C′ dot to .sup.89Zr-oxalate ratio.

[0133] FIGS. 5A and 5B show in vivo coronal PET images of [89Zr]C′ dot-PEG at different post-injection time points (10 min, Day 1, Day 3 and Day 6) in a healthy nude mouse. [.sup.89Zr]C′ dot-PEG was synthesized by using a chelator-free radiolabeling technique.

[0134] The PET images were acquired by using a Focus 120 MicroPET scanner.

[0135] FIG. 5A shows PET images acquired without EDTA (ethylenediaminetetraacetic acid).

[0136] FIG. 5B shows PET images acquired with EDTA

[0137] FIG. 6 shows biodistribution data of [.sup.89Zr]C′ dot-PEG in healthy nude mice (n=3) on Day 7. Over 10% ID/g of bone (and joint) uptake (highlighted with a red box) was observed in this case, indicating a less stable radiolabeling using a chelator-free method (when compared with that of chelator-based method).

[0138] FIG. 7 shows biodistribution data of .sup.89Zr-DFO-C′ dot, .sup.89Zr-DFO-C′ dot-DBCO and .sup.89Zr-DFO-C′ dot-PEG-sdAb in healthy nude mice at 48 h post-injection. An improved pharmacokinetic profile (with prolonged blood circulation half-life and lower liver uptake) can be achieved by optimizing the number of DFO, DBCO and sdAb from each C′ dot.