Bispecific Nanoparticle Bioconjugates

Abstract

The present invention relates to nanoparticle bioconjugates comprising binding proteins for binding to antigens on cancer cells and immune cells, and methods of treatment involving their use and administration.

Description

[Technical field]

[0001] The present invention relates to nanoparticle bioconjugates, compositions comprising same, and therapeutic methods and uses thereof.

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority from Australian provisional application AU 2022901088, the contents of which are incorporated herein by reference in their entirety. [Background technology]

[0003] Targeted immunotherapy using monoclonal antibodies (mAbs) has been highly successful due to its unparalleled specificity and efficacy. Their growth in the past two decades has been unprecedented, with a wide range of applications from oncology to inflammatory diseases. Recently, different types of antibody-based products have emerged, including bispecific antibodies (biAbs), which mainly target different types of cancer. BiAbs can simultaneously bind to both tumor cells and immune cells (e.g., killer T cells), and tumor cells are rapidly lysed. BiAbs have shown great therapeutic potential as an emerging immunotherapy. Currently, two biAbs have been approved as cancer therapies, and hundreds of new candidates are in clinical trials.

[0004] Currently available biAbs suffer from poor conformational and formulation stability and have short serum half-lives leading to their rapid elimination from the body, for example, blinatumomab has a half-life of 2.1 hours. Hundreds of different biAb constructs have been reported so far, and most of these constructs suffer from stability and half-life problems. This represents a significant limitation for the use of these molecules for the treatment of cancer, where a longer serum half-life is essential for better therapeutic outcomes.

[0005] There is a need for improved bispecific binding molecules that have increased conformation and / or formulation stability.

[0006] The reference to any prior art in this specification is not an admission or suggestion that this prior art forms part of common general knowledge in any jurisdiction, or that this prior art would be understood by, considered relevant, and / or reasonably expected to be combined with other prior art by a person of ordinary skill in the art. Summary of the Invention

[0007] The present invention provides a nanoparticle bioconjugate comprising: a first nanoparticle conjugated with a first binding protein for specifically binding to a target moiety on a cancer cell; a second nanoparticle conjugated with a second binding protein for specifically binding to a target moiety on an immune cell; The nanoparticles are arranged in a dumbbell or flower-like configuration; A nanoparticle bioconjugate is provided, wherein the bioconjugate is capable of binding to a cancer cell via a first binding protein and to an immune cell via a second binding protein.

[0008] In certain embodiments, the first nanoparticles are metal nanoparticles and the second nanoparticles are magnetic nanoparticles. In alternative embodiments, the first nanoparticles are magnetic nanoparticles and the second nanoparticles are metal nanoparticles. The metal nanoparticles can be magnetic or non-magnetic.

[0009] Preferably, the architecture of the nanoparticle bioconjugates is in the form of a central metal nanoparticle surrounded by three to four or more magnetic nanoparticles in a flower-like configuration.

[0010] Alternatively, the nanoparticle bioconjugates can be in the form of a central (core) metal nanoparticle surrounded by three to four or more magnetic nanoparticles in a flower-like configuration.

[0011] In an alternative embodiment, the nanoparticle bioconjugate may comprise a first and a second nanoparticle in a "dumbbell-like" architecture, whereby the nanoparticle conjugate comprises a metal nanoparticle adjacent to or in intimate contact with a magnetic nanoparticle.

[0012] It will therefore be understood that the nanoparticle bioconjugates of the present invention are not in the form of spherical nanoparticles.

[0013] In any embodiment of the present invention, the magnetic nanoparticles may be selected from Fe3O4 (iron oxide) nanoparticles, Fe(CO)5 (iron pentacarbonyl) nanoparticles, or FeCl2 (iron chloride) nanoparticles.

[0014] In any embodiment, the metal nanoparticles may be selected from gold (Au), silver (Ag), platinum (Pt) or palladium (Pd), copper (Cu), nickel (Ni), cobalt (Co), or alloys or nanoparticles of two or more thereof.

[0015] Preferably, the metal nanoparticles are gold or silver nanoparticles. In a particularly preferred embodiment, the metal nanoparticles are gold nanoparticles.

[0016] In any embodiment, the nanoparticle bioconjugate is composed of iron and gold nanoparticles, preferably the bioconjugate comprises a central gold nanoparticle surrounded by three to four or more iron nanoparticles to form a flower-shaped nanoparticle.

[0017] In any embodiment, the nanoparticle bioconjugate is composed of iron and silver nanoparticles, preferably the bioconjugate comprises a central silver nanoparticle surrounded by three to four or more iron nanoparticles to form a flower-shaped nanoparticle.

[0018] In any embodiment, the nanoparticle bioconjugates of the invention can be from about 5 nm to about 100 nm. In a preferred embodiment, the nanoparticle bioconjugates can be from about 35 nm to about 80 nm, preferably about 40 nm.

[0019] In any embodiment, the first and second binding proteins are covalently bound to the first and second nanoparticles. It will be understood that any conjugation method can be used to conjugate the first and second binding proteins to the nanoparticles. Such methods are well known to those skilled in the art and in the art.

[0020] In any embodiment, the first and second antigen binding proteins are covalently attached to the first and second nanoparticles via different chemistries. It will be understood that the conjugation method will depend on the available functional groups and surface modification of the nanoparticles.

[0021] In certain embodiments, the binding protein may be directly conjugated to the nanoparticle. In alternative embodiments, the binding protein may be conjugated via a linker moiety. The linker moiety may be any of the commercially available linkers further described herein, or other linkers for conjugating peptide or protein moieties to nanoparticles.

[0022] In certain instances, the binding protein may be covalently conjugated to a nanoparticle, preferably a functionalized gold nanoparticle, via a polyethylene glycol (PEG) moiety, an alkyne, an amine, an azide, a biotin, a carboxyl, a hydroxyl, a methyl, a maleimide, a neutravidin, an NHS, a hydrazone, or a thiol linker group.

[0023] In certain instances, the binding protein may be covalently conjugated to a nanoparticle, preferably a magnetic nanoparticle such as iron, to an amine, azide, carboxyl, maleimide, or hydrazone linker group.

[0024] In any embodiment, the thiol linker can be at least one of thioctic acid, monothioctic acid, dithioctic acid, and trithioctic acid.

[0025] The first binding protein can be any binding protein that can specifically bind to a target moiety of a cancer cell. The cancer cell can be any selected from breast tumor, colorectal tumor, adenocarcinoma, mesothelioma, bladder tumor, prostate tumor, germ cell tumor, liver cancer / bile duct, carcinoma, neuroendocrine tumor, pituitary tumor, small round cell tumor, squamous cell carcinoma, melanoma, atypical fibroxanthoma, seminoma, nonseminoma, stromal Leydig cell tumor, Sertoli cell tumor, skin tumor, kidney tumor, testicular tumor, brain tumor, ovarian tumor, stomach tumor, oral tumor, bladder tumor, bone tumor, cervical tumor, esophageal tumor, laryngeal tumor, liver tumor, lung tumor, vaginal tumor, and Wilm's tumor.

[0026] In any embodiment, the targeting moiety on the cancer cell can be an antigen selected from a sugar, lipid, nucleic acid, peptide, or protein. Preferably, the targeting moiety is endogenous to the cancer cell.

[0027] The binding protein for binding to the target moiety may comprise, essentially consist of, or consist of an antigen binding domain. Alternatively, the binding protein may comprise, essentially consist of, or consist of a receptor or ligand for binding to cancer cells, or a portion thereof. In any embodiment, the target or target molecule may be any one of those described herein.

[0028] Examples of cancer cell antigens that may be specifically bound by the first antigen binding protein include HER2, EGFR, mesothelin, PSMA, GPC3, MUC1, GD2, CEA, EpCAM, LeY, PCSA, CD19, CAIX, CD20, Clec9a, CD276, PD-L1, and PD-L2. Other examples of cancer cell antigens are further described herein.

[0029] In a particularly preferred embodiment, the antigen is HER2 and the cancer cell is any cancer cell that overexpresses HER2 or is HER2 positive. Thus, in a preferred embodiment, the first binding protein is for binding to HER2 on a HER2 positive cancer cell, such as a breast cancer or gastric cancer.

[0030] Examples of HER2 antibodies and binding proteins derived therefrom are known in the art and include pertuzumab and trastuzumab.

[0031] In an alternative embodiment, the antigen is EGFR and the cancer cell is any cancer cell that overexpresses the epidermal growth factor receptor (EGFR). Thus, in an alternative preferred embodiment, the first binding protein is for binding to EGFR on an EGFR-positive cancer cell, such as a squamous cell carcinoma of the head and neck (SCCHN), glioma, nasopharyngeal carcinoma, or pancreatic cancer cell.

[0032] Examples of EGFR antibodies and binding proteins derived therefrom are known in the art and include panitumumab and nimotuzumab.

[0033] In any embodiment of the invention, the first antigen binding protein is selected from trastuzumab, pertuzumab, panitumumab, nimotuzumab, cetuximab, or an antigen-binding fragment thereof.

[0034] The second binding protein can be any binding protein that can specifically bind to a target portion of an immune cell. The immune cell can be any immune cell, optionally selected from a T cell, an antigen-presenting cell (APC), or a natural killer (NK) cell. Preferably, the immune cell is a T cell.

[0035] In any embodiment, the target moiety on the immune cell can be an antigen selected from a sugar, lipid, nucleic acid, peptide, or protein. In any embodiment, the target moiety on the immune cell can be an antigen selected from a sugar, lipid, nucleic acid, peptide, or protein. Preferably, the target moiety is endogenous to the immune cell. The binding protein for binding to the target moiety can comprise, consist essentially of, or consist of an antigen binding domain. Alternatively, the binding protein can comprise, consist essentially of, or consist of a receptor or ligand, or a portion thereof, for binding to the immune cell. In any aspect, the target or target molecule can be any one described herein.

[0036] In a preferred embodiment of the invention, the immune cell is a T cell and the target moiety on the T cell bound by the second binding protein is any one selected from CD3, CD2, CD4, CD7, CD8, PD1, CTLA4, KIR, CD16, CD94, CD161, NTBA, CD19, CD20, CD22, CD30, CD33, CD38, CD40L, CD44, CD56, CD79b, CD80, CD86, CD135, CD137, CD138, CD154, EphA2, EGFR, and any combination thereof.

[0037] Thus, in a preferred embodiment, the immune cell is a T cell and the second binding protein is selected from an anti-CD3 antibody, an anti-CD2 antibody, an anti-CD4 antibody, an anti-CD7 antibody, an anti-CD8 antibody, an anti-PD1 antibody, an anti-CTLA4 antibody, an anti-KIR antibody, an anti-CD16 antibody, an anti-CD94 antibody, an anti-CD161 antibody, an anti-NTBA antibody, a recombinant human NTBA, an anti-CD19 antibody, an anti-CD20 antibody, an anti-CD22 antibody, an anti-CD30 antibody, an anti-CD33 antibody, an anti-CD38 antibody, an anti-CD40L antibody, an anti-CD44 antibody, an anti-CD56 antibody, an anti-CD79b antibody, an anti-CD80 antibody, an anti-CD86 antibody, an anti-CD135 antibody, an anti-CD137 antibody, an anti-CD138 antibody, an anti-CD154 antibody, an anti-EphA2 antibody, an anti-EGFR antibody, or a fragment thereof.

[0038] In some embodiments of the invention, when the immune cell is a T cell, the binding protein can be an anti-CD3 antibody (e.g., OKT3, UCHT1, IP26, SK7, HIT3a, or other clones) and / or an anti-CD4 antibody and / or an anti-CD8 antibody and / or an anti-PD1 antibody and / or an anti-CTLA4 antibody, etc., or a combination thereof.

[0039] When the immune cell is a NK cell, the binding protein is preferably an anti-KIR antibody and / or an anti-CD16 antibody and / or an anti-CD94 antibody and / or an anti-CD161 antibody and / or an anti-CD56 antibody, etc., or a combination thereof.

[0040] It will be understood that the first and / or second binding protein can be any suitable polypeptide capable of binding to an antigen. For example, the antigen binding protein can be an antibody, an antibody fragment, an engineered antibody, a chimeric antibody, a heteroconjugate antibody, or a combination thereof.

[0041] In certain embodiments, the binding protein is an antibody. Optionally, the antibody is selected from IgA, IgD, IgE, IgG, and IgM. Preferably, the antibody is an IgG. The IgG can be IgG1, IgG2, IgG3, or IgG4.

[0042] In any embodiment, the binding protein may be an antibody fragment, optionally selected from a recombinant antibody fragment, a diabody, a triabody, a chimeric antibody, a F(ab')2 fragment, a Fab' fragment, a Fab'-SH fragment, a Fab fragment, a sFv fragment, a dsFv fragment, a bispecific sFv fragment, a bispecific dsFv fragment, a single chain Fv protein (scFv), a disulfide stabilized Fv protein, or a combination thereof.

[0043] In certain embodiments, the first and second binding proteins may be the same antibody format. For example, both binding proteins may be in the form of IgG or any other antibody format described herein, including any antigen-binding fragment of IgG (such as scFv, Fab', or others). In alternative embodiments, the first and second antigen-binding proteins are different antibody formats. For example, in certain embodiments, the first binding protein may be in the form of a monoclonal immunoglobulin (mAb, IgG) and the second binding protein may be in the form of an antibody fragment, such as scFv. The first and / or second binding proteins may be monospecific or multispecific (such as bispecific or trispecific).

[0044] In further embodiments, the bioconjugate may further comprise a moiety to enable cell penetration, such as a cell penetrating peptide (CPP). Examples of suitable cell penetrating peptides are known to those of skill in the art, and non-limiting examples include the human immunodeficiency virus 1 (HIV1) Tat protein (e.g., Tat 48-60 Or Tat 47-57 ,Transmission (e.g., Antp 43-48 ), transportan, oligoarginines (e.g., R8), MAP, MPG, and peptides from SAP (see, e.g., Veldhoen et al. 2008 Int. J. Mol. Sci. 9(7), 1276-1320, https: / / doi.org / 10.3390 / ijms9071276, which is incorporated by reference in its entirety). The CPP may be conjugated to the nanoparticle via a suitable linker moiety, such as those described herein for conjugation of binding proteins.

[0045] In some embodiments, the bioconjugate comprises an HIV-TAT cell penetrating peptide, preferably Tat 47-57(SEQ ID NO: 12). The HIV-TAT CPP can be attached to the nanoparticle via a thiol linker, such as thiol-PEG-NHS (see, e.g., Cruz and Kayser 2019, Cancers 116(6), page 870, https: / / doi.org / 10.3390 / cancers11060870, incorporated by reference in its entirety).

[0046] The invention also provides pharmaceutical compositions comprising any of the nanoparticle bioconjugates described herein, optionally in combination with a pharma- ceutically acceptable excipient or carrier.

[0047] The invention also provides a method of treating or preventing cancer in a subject comprising administering to a subject in need thereof a nanoparticle bioconjugate or pharmaceutical composition described herein. As used herein, a method of treating cancer includes a method of inhibiting, preventing, or minimizing the spread or progression of cancer, including inhibiting or preventing metastasis of cancer.

[0048] Still further, the present invention provides the use of a nanoparticle bioconjugate of the invention in the manufacture of a medicament for the treatment or prevention of cancer.

[0049] The present invention further provides a nanoparticle bioconjugate or a pharmaceutical composition of the invention for use in the treatment or prevention of cancer.

[0050] The present invention further provides kits for the production of the nanoparticle bioconjugates of the invention. Preferably, the kits include nanoparticles, binding proteins, and instructions for combining them to obtain bioconjugates capable of binding to cancer cells and immune cells.

[0051] As used herein, unless the context otherwise requires, the term "comprise", as well as variations of terms such as "comprising", "comprises", "comprised", etc., are not intended to exclude additional additives, components, integers or steps.

[0052] Further aspects of the invention and further embodiments of the aspects described in the preceding paragraphs will become apparent from the following description, given by way of example and with reference to the accompanying drawings, in which: [Brief description of the drawings]

[0053] [Figure 1] A. Schematic diagram showing the conjugation of a first and a second antigen binding protein, preferably in a flower-shaped configuration, to nanoparticles and their use for targeting T cells and cancer cells simultaneously. B. Pyrolytic synthesis of magnetic gold nanoparticles. Iron and gold precursors are dissolved in an organic solvent and chemical precipitation is achieved at 310° C. under vigorous stirring in the presence of N2 gas. After cooling, the nanoparticles were purified and stored. [Diagram 2] Characterization of Fe3O4@Au nanoparticles. A. Transmission electron microscopy (TEM) images of representative flower-like nanoparticles. (i) TEM of representative hydrophobic tail nanoparticles (Fe3O4@Au / OA). The diameters of the gold core and iron are about 11 and 14 nm, respectively. (ii) TEM of representative hydrophilic tail nanoparticles with dopamine layer (Fe3O4@Au / Dop). The average diameter of the shell is 4 nm. B. FTIR spectra of hydrophobic Fe3O4@Au nanoparticles (upper line) and hydrophilic Fe3O4@Au nanoparticles (lower line). The wavenumbers were recorded from 600 to 4000 cm-1. [Diagram 3] MALDI-TOF mass spectrometry of Nmab-Fe3O4@Au-αCD3scFv NPs. Mass spectra of Fe3O4@Au NPs before and after coupling with anti-EGFR mAB (nimotuzumab, 149 KDa) and anti-CD3 scFv (24 KDa). [Figure 4]Normalized UV-Vis absorbance spectroscopy of modified nanoparticles: A. Hydrophobic Fe3O4@Au NPs showing their strong absorbance at 573 nm while shifted to 390 nm after modification. B. NmabNPs (light green) and BisNPs (red) showing the total amount of antibody loaded, NmabNPs (blue), BisNPs (green) showing very strong absorbance of conjugated antibody after centrifugation at about 260 nm. [Diagram 5] Nanodrop protein measurements of conjugated Fe3O4@Au NPs: A. PBS baseline, B. Bare NPs negative control, C. Bis-Fe3O4@Au NPs (conjugated bispecific NPs), D. Fe3O4@Au-αCD3scFv NPs, E. Nmab-Fe3O4@Au NPs. [Figure 6] Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis of conjugated Fe3O4@Au NPs. Lane 1, protein size marker; lane 2, free nimotuzumab; lane 3, blank control; lane 4, anti-CD3 scFv; lane 5, NPs; lane 6, Nmab-Fe3O4@Au NPs; lane 7, Bis-Fe3O4@Au NPs (bispecific nanoparticles conjugated to nimotuzumab and anti-CD3 scFv; otherwise referred to as Nmab-Fe3O4@Au-αCD3scFv). [Figure 7] Cytotoxicity assay of co-cultured A431 and Jurkat cells at a ratio of 1:5 (target:effector) with Fe3O4@Au nanoparticles (0-5000 μg / ml iron). The viability of the co-cultured cells was not significantly affected until treated with Fe3O4@Au NPs at Fe concentrations >2500 μg / ml. Data are reported as mean ± SD. [Figure 8]Cytotoxicity assay of co-cultured A431 and Jurkat cells at a ratio of 1:5 (target:effector) after incubation with different concentrations of: A. Nimotuzumab. Cell viability was not significantly affected until treatment with 1000 μg / ml of Nimotuzumab; B. Nmab-Fe3O4@Au nanoparticles; and C. Bis-Fe3O4@Au nanoparticles (nanoparticles conjugated to Nimotuzumab and anti-CD3 scFv). Viability of co-cultured cells was significantly decreased after incubation with Bis-Fe3O4@Au NPs compared to Nmab-Fe3O4@Au nanoparticles with ≧300 μg / ml of iron. Data are reported as mean ± SD. Nimotuzumab 2000μg / ml vs. control***p 0.0001, Nmab-Fe3O4@Au NPs 300μg / ml vs. control***p 0.0003, and Nmab-Fe3O4@Au NPs 700μg / ml vs. control****p<0.0001, Bis-Fe3O4@Au NPs 300 and 700μg / ml vs. control****p<0.0001 (one-way ANOVA post hoc Fisher LSD). [Figure 9] Cytotoxicity assay of co-cultured A431 and Jurkat cells in a ratio of 1:5 (target:effector) after incubation with nimotuzumab (1, left column), Nmab-Fe3O4@Au nanoparticles (2, center column), and Bis-Fe3O4@Au nanoparticles (3, right column, bispecific NPs conjugated with anti-EGFR Nmab and αCD3scFv) at the same nimotuzumab concentration. The viability of the co-cultured cells was not significantly affected until treatment with 1000 μg / ml nimotuzumab for cells treated with antibody alone, but the viability was significantly decreased after incubation with Bis-Fe3O4@Au nanoparticles compared to Nmab-Fe3O4@Au NPs with ≧43 μg / ml nimotuzumab. Data are reported as mean ± SD. [Figure 10]Cellular uptake of functionalized Fe3O4@Au NPs into cancer cell lines. A. Intracellular iron and gold content was quantitatively measured by ICP-MS after 12 h incubation with 50 μg / ml iron. B. Intracellular iron was detected by Prussian blue staining after 12 h incubation with 50 μg / ml Fe3O4@Au NPs (a: Fe3O4@Au NPs, b: Nmab-Fe3O4@Au NPs, c: Bis-Fe3O4@Au NPs, bispecific NPs conjugated with anti-EGFR Nmab and αCD3scFv). Uptake data are reported as mean ± SD (x3). [Figure 11] Cytotoxicity assay of co-cultured SkBr3 and Jurkat cells at a ratio of 1:5 (target:effector) after incubation with: A. free Tmab, B. Tmab-Fe3O4@Au NPs, and C. Bis-Tmab-Fe3O4@Au-CD3 scFv NPs (bispecific NPs conjugated with anti-HER2 Tmab and αCD3 scFv). The viability of co-cultured cells was significantly decreased after incubation with Bis-Tmab-Fe3O4@Au-CD3 scFv NPs compared to Tmab-Fe3O4@Au NPs with ≧300 μg / ml iron. Data are reported as mean ± SD. D: Cytotoxicity assay of co-cultured SkBr3 and Jurkat cells at a ratio of 1:5 (target:effector) after incubation with different concentrations of free Tmab, Tmab-Fe3O4@Au NPs (Tmab NPs), or Bis-Tmab-Fe3O4@Au-αCD3scFv NPs (Bis NPs, bispecific NPs conjugated with anti-HER2 Tmab and αCD3scFv). The viability of the co-cultured cells was significantly decreased after incubation with Bis-Tmab-Fe3O4@Au αCD3scFv NPs compared to Tmab-Fe3O4@Au NPs and compared to free Tmab at ≧43 μg / ml. Free Tmab had no significant effect on cell viability below 200 μg / ml. Data are reported as mean ± SD. [Figure 12]Characterization of Tmab-Fe3O4@Au NPs. A. Representative TEM image of flower-shaped NPs. B. Hydrodynamic diameter of the NPs after conjugation with an average size of 149.3 nm. [Figure 13] Cytokine release profile of EGFR-A431 targeting T cells. In 96-well plates, 4x103 cells / well cells were seeded, then Jurkat E6-1 cells were added at an effector:target (E:T) ratio of 5:1 and incubated with anti-CD3 scFv, Bis-Fe3O4@Au NPs (NPs conjugated with anti-EGFR Nmab and αCD3scFv), and Nmab-Fe3O4@Au NPs for 24 hours. Cytokine IL-2 secreted into the supernatant was determined by ELISA. Data are reported as mean ± SD. Bis-NPs vs. anti-CD3 scFv conjugated NPs, Nmab-NPs, and control unconjugated NPs ***p<0.0001 (one-way ANOVA post-hoc Fisher LSD). [Figure 14] Representative TEM images of dumbbell-like Fe3O4@Au nanoparticles. The diameters of the gold and iron nanoparticles are about 5 and 10 nm, respectively. [Figure 15] Cytotoxicity assays performed on co-cultures of A431 and Jurkat cells in a ratio of 1:5 (target:effector) with the following treatments of different concentrations of dumbbell-like NPs: A. bare NPs, B. Bis-Fe3O4@Au NPs (bispecific NPs conjugated with anti-EGFR Nmab and αCD3scFv), and C. Nmab-Fe3O4@Au NPs. The viability of the co-cultured cells was significantly decreased after incubation with Bis-Fe3O4@Au NPs compared to Nmab-Fe3O4@Au NPs with ≧300 μg / ml iron. [Figure 16] Characterization of spherical gold nanoparticles. A. TEM image of a representative Au nanoparticle. B. UV-Vis absorption spectroscopy of gold nanoparticles at 521 nm. [Figure 17]Cytotoxicity assays performed on co-cultures of A431 and Jurkat cells in a ratio of 1:5 (target:effector) with the following treatments of spherical AuNPs at different concentrations: A. Nmab-AuNPs, B. Bis AuNPs (bispecific NPs conjugated with anti-EGFR Nmab and αCD3scFv). The viability of the co-cultured cells was not significantly affected after incubation with Bis-AuNPs compared to Nmab-AuNPs. [Figure 18] Cytotoxicity assays performed on co-cultures of MDA-MB 468 and Jurkat cells in a ratio of 1:5 (target:effector) after treatment with different concentrations of: A. free Nmab, B. Nmab-Fe3O4@Au NPs, and C. Bis-Nmab-Fe3O4@Au-αCD3scFv NPs (bispecific NPs conjugated with anti-EGFR Nmab and αCD3scFv). The viability of co-cultured cells was significantly decreased after incubation with Bis-Nmab-Fe3O4@Au-αCD3scFv NPs compared to Nmab-Fe3O4@Au NPs with ≧300 μg / ml iron. Data are reported as mean ± SD. ****p<0.0001 (one-way ANOVA post-hoc Fisher LSD). [Figure 19] Cytotoxicity assay of co-cultured MDA-MB 468 and Jurkat cells at a ratio of 1:5 (target:effector) after incubation with different concentrations of free Nmab, Nmab-Fe3O4@Au NPs (Nmab NPs), or Bis-Nmab-Fe3O4@Au-αCD3scFv (bispecific NPs conjugated with anti-EGFR Nmab and αCD3scFv). The viability of the co-cultured cells was significantly decreased after incubation with Bis-Nmab-Fe3O4@Au-αCD3scFv NPs compared to Nmab-Fe3O4@Au NPs and compared to free Nmab at ≧43 μg / ml. Free Nmab had no significant effect on cell viability below 100 μg / ml. Data are reported as mean ± SD. [Figure 20]Cytotoxicity assays performed on co-cultures of A431 and Jurkat cells at a ratio of 1:5 (target:effector) with different concentrations of αCD3scFv-Fe3O4@Au-Nmab NPs. A. The viability of the co-cultured cells was significantly decreased after incubation with αCD3scFv-Fe3O4@Au-Nmab NPs with 43 μg / ml of Nmab. B. The viability of the co-cultured cells was significantly decreased after incubation with αCD3scFv-Fe3O4@Au-Nmab NPs with ≧300 μg / ml of iron. [Figure 21] Cytotoxicity assay of co-cultured MDA-MB 468 and Jurkat cells in a ratio of 1:5 (target:effector) after incubation with: A. free Tmab, B. Tmab-Fe3O4@Au NPs, and C. Bis-Tmab-Fe3O4@Au-αCD3scFv NPs αCD3scFv (bispecific NPs conjugated with anti-HER2 Tmab and αCD3scFv). The viability of co-cultured cells was significantly decreased after incubation with Bis-Tmab-Fe3O4@Au-αCD3scFv NPs compared to Tmab-Fe3O4@Au NPs with ≧300 μg / ml iron. Data are reported as mean ± SD. *p<0.0001, **p<0.0001, ****p<0.0001 (one-way ANOVA post-hoc Fisher LSD). [Figure 22]Cytotoxicity assay of co-cultured MDA-MB 468 and Jurkat cells at a ratio of 1:5 (target:effector) after incubation with different concentrations of free Tmab, Tmab-Fe3O4@Au NPs (Tmab NPs), or Bis-Tmab-Fe3O4@Au-αCD3scFv NPs (Bis NPs, bispecific NPs conjugated with anti-HER2 Tmab and αCD3scFv). The viability of the co-cultured cells was significantly decreased after incubation with Bis-Tmab-Fe3O4@Au-αCD3scFv NPs compared to Tmab-Fe3O4@Au NPs and compared to free Tmab at ≧43 μg / ml. Free Tmab had no significant effect on cell viability below 200 μg / ml. Data are reported as mean ± SD. [Figure 23] Cytotoxicity assays performed on co-cultures of A431 and Jurkat cells with different concentrations of bispecific anti-EGFR and anti-CD3 NPs, panitumumab (Pmab)-Fe3O4@Au-αCD3scFv, in a ratio of 1:5 (target:effector). A. The viability of the co-cultured cells was significantly decreased after incubation with Pmab-Fe3O4@Au-αCD3scFv NPs with 10 μg / ml of Pmab. B. The viability of the co-cultured cells was significantly decreased after incubation with Pmab-Fe3O4@Au-αCD3scFv NPs with ≧70 μg / ml of iron. [Figure 24] Characterization of silver-iron nanoparticles (Fe3O4@Ag NPs). A. TEM image of representative silver-iron nanoparticles. B. UV-Vis absorption spectroscopy of silver-iron nanoparticles at 409 nm. [Diagram 25]Cytotoxicity assays performed on co-cultures of A431 and Jurkat cells with different concentrations of bispecific anti-EGFR and anti-CD3 NPs conjugated with CPP, Nmab-Fe3O4@Au-αCD3scFv / CPP, in a ratio of 1:5 (target:effector). A. The viability of co-cultured A431 and Jurkat cells was significantly decreased after incubation with Nmab-Fe3O4@Au-αCD3scFv / CPP NPs at iron concentrations of ≧70 μg / ml. B. The viability of co-cultured A431 and Jurkat cells was significantly decreased after incubation with Nmab-Fe3O4@Au-αCD3scFv / CPP NPs at Nmab concentrations of ≧10 μg / ml. [Figure 26] Cytotoxicity assays performed on co-cultures of A431 and Jurkat cells with different concentrations of Nmab-Fe3O4@Au-αCD3scFv NPs in the presence and absence of CPPs at a ratio of 1:5 (target:effector). The viability of the co-cultured cells was significantly decreased after incubation with Nmab-Fe3O4@Au-αCD3scFv / CPP NPs with iron concentrations of ≥ 35 μg / ml (CPPBisNp, right column) compared to Nmab-Fe3O4@Au-αCD3scFv NPs with conjugated CPPs (Bis NPs, left column). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0054] It will be understood that the invention disclosed and defined herein extends to all alternative combinations of two or more of the individual features mentioned or apparent from the text or drawings, all of which different combinations constitute various alternative aspects of the invention.

[0055] Currently available bispecific binding agents, especially those for engaging T cells and cancer cells (so-called bispecific T cell engagers, or "BiTes"), suffer from poor conformational and formulation stability and have short serum half-lives leading to their rapid clearance from the body. Hundreds of different BiTe constructs have been reported so far, and most of these constructs suffer from such stability and half-life problems. This represents a significant limitation for the use of these antibodies for the treatment of cancer, where a longer serum half-life is essential for better therapeutic outcomes.

[0056] The present invention seeks to overcome one or more limitations of prior art bispecific binding agents. The present invention relates to nanoparticle-based bispecific binding agents (nanobioconjugates) that include both immune cell engagers and cancer-specific engagers, where the nanobioconjugates are capable of interacting with both immune cells (e.g., T cells, NK cells, etc.) and cancer cells.

[0057] The present invention is believed to have a variety of advantages over existing bispecifics, including greater conformation and formulation stability. Furthermore, the size of the nanoparticles means that they are more likely to bypass renal filtration, and therefore the nanobioconjugates of the present invention are more likely to have longer serum half-lives.

[0058] Advantageously, the nanoparticles of the present invention provide a first and a second binding protein (for binding to cancer cells and immune cells, respectively) conjugated to different nanoparticle types. The presence of two different nanoparticle types (such as iron and gold) in the composite nanostructure facilitates easy attachment of different functional groups and allows the conjugation of two different antibodies, or fragments thereof, thereby providing design flexibility. The use of different nanoparticle types also allows the use of different chemistries to conjugate the antigen binding proteins. This provides several advantages, including the ability to control the ratio of each binding protein and efficiency (e.g., fewer processing steps) in preparing the nanoparticle conjugates.

[0059] The use of magnetic nanoparticles also facilitates the purification and preparation of bioconjugates for therapeutic use.

[0060] definition Unless otherwise defined, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0061] For purposes of interpreting this specification, the following definitions are of general application and, whenever appropriate, terms used in the singular will also include the plural and vice versa.

[0062] The term "bispecific" means that the nanoparticle bioconjugate of the present invention can specifically bind to at least two different antigenic determinants. Typically, a bispecific binding molecule comprises two antigen binding sites, each of which is specific for a different antigenic determinant. In certain embodiments, a bispecific antigen binding molecule can simultaneously bind two antigenic determinants, particularly two antigenic determinants expressed on two different cells.

[0063] As used herein, "antigen" refers to a molecule or molecular structure that can be bound by a specific antibody or T-cell receptor. Antigens can be proteins, peptides (chains of amino acids), polysaccharides (monosaccharides / simple sugar chains), lipids, or nucleic acids.

[0064] In any embodiment, the binding protein is any protein that can associate with, recognize, or bind to a target moiety on a cancer or immune cell. Preferably, the target moiety is a protein endogenous to the target cell. The binding protein may comprise, essentially consist of, or consist of an antigen-binding domain. Alternatively, the binding protein may comprise, essentially consist of, or consist of a receptor or a portion thereof for binding to the target moiety.

[0065] As used herein, a "targeting moiety" can be any moiety present on a target cell (e.g., a cancer or immune cell) that can be bound by a binding protein. In any aspect, the targeting moiety (i.e., the target of the binding protein) is a protein that is presented, displayed, or otherwise accessible on the cell surface, or secreted by the cancer or immune cell. The targeting moiety can be constitutively presented, displayed, or otherwise accessible on the cell surface, or only transiently presented, displayed, or otherwise accessible on the cell surface. In one embodiment, the target is a protein that is transported and secreted from the cell, that is not tethered to or part of the cell. The target can be constitutively secreted, or only transiently secreted.

[0066] The binding protein may include a receptor for a ligand or a binding partner for binding to a target molecule. It will be understood that the binding partner may be any form of molecule that allows binding to a cancer or immune cell. For example, the binding partner may include or consist of a ligand, receptor, target, or antigen binding domain. The binding partner may be a naturally occurring molecule that binds to a target. The binding partner may be non-naturally occurring or may be modified from a naturally occurring molecule (e.g., by directed evolution). The binding partner may be naturally occurring but heterologous to the intracellular retention moiety or linker.

[0067] In any embodiment, the binding protein comprises, consists essentially of, or consists of an antigen-binding domain of an antibody or an antigen-binding fragment thereof. The antibody or antigen-binding fragment may be modified or engineered, or is a human antibody. Examples of modified or engineered antibodies are chimeric antibodies, humanized antibodies, multiparatopic antibodies (e.g., biparatopic antibodies), and multispecific antibodies (e.g., bispecific antibodies). Examples of antigen-binding fragments include Fab, Fab', F(ab')2, Fv, single chain antibodies (e.g., scFv), minibodies, and diabodies.

[0068] In any embodiment, the binding protein may bind to two different target moieties or two different epitopes on the same target moiety.

[0069] An "antibody" or "immunoglobulin" or "Ig" is a gamma globulin protein found in the blood, or other body fluids, of vertebrates that functions in the immune system to bind antigens, thus identifying and / or neutralizing foreign substances. Antibodies are generally heterotetrameric glycoproteins composed of two identical light (L) chains and two identical heavy (H) chains. Each L chain is linked to an H chain by one covalent disulfide bond. The two H chains are linked to each other by one or more disulfide bonds, depending on the H chain isotype. Each H and L chain also has regularly spaced intrachain disulfide bridges.

[0070] The H and L chains define specific Ig domains. More specifically, each H chain contains at the N-terminus a variable domain (V H ), followed by three constant domains (CH) for each of the α and γ chains, and four CH domains for the μ and ε isotypes. Each L chain has at its N-terminus a variable domain (V L ), followed at the other end by a constant domain (CL). L V H and C L is aligned with the first constant domain of the heavy chain (CHL).

[0071] Antibodies can be assigned to different classes or isotypes. There are five classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, with heavy chains designated α, δ, ε, γ, and μ, respectively. The γ and α classes are further divided into subclasses based on relatively minor differences in 3 / 4 sequence and function, for example, humans express the following subclasses: IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. Light chains from any vertebrate species can be assigned to one of two clearly distinct types, called kappa and lambda, based on the amino acid sequence of their constant domains.

[0072] The constant domain comprises the Fc portion, which includes the carboxy-terminal portions of both H chains held together by disulfides. The effector functions of antibodies, such as ADCC, are determined by sequences in the Fc region, which is also the region recognized by Fc receptors (FcRs) found on certain types of cells.

[0073] V H and V L The pairing of these together forms a "variable region" or "variable domain" which comprises the amino terminal domain of either the heavy or light chain of an antibody. The variable domain of the heavy chain is referred to as the "V H The variable domain of the light chain may be referred to as "V L ". The V domain contains the "antigen binding site" which influences antigen binding and defines the specificity of a particular antibody for that particular antigen. V regions span approximately 110 amino acid residues and consist of relatively invariant stretches called framework regions (FRs) (generally about 4) of 15-30 amino acids separated by shorter regions of extreme variability called "hypervariable regions" (generally about 3), each generally 9-12 amino acids long. The FRs adopt a predominantly β-sheet configuration, and the hypervariable regions form loops that connect, and in some cases form part of, the β-sheet structure.

[0074] The terms "full length antibody," "intact antibody," or "whole antibody" are used interchangeably to refer to an antibody in its substantially intact form, as opposed to an antigen-binding fragment of an antibody. Specifically, whole antibodies include those having heavy and light chains, including the Fc region. The constant domains may be wild-type sequence constant domains (e.g., human wild-type sequence constant domains) or amino acid sequence variants thereof.

[0075] As used herein, "variable region" refers to a portion of the light and / or heavy chain of an antibody defined herein that is capable of specifically binding to an antigen and includes the amino acid sequences of the complementarity determining regions (CDRs), i.e., CDR1, CDR2, and CDR3, and framework regions (FRs). For example, a variable region includes three or four FRs (e.g., FR1, FR2, FR3, and optionally FR4) together with three CDRs. VH refers to the variable region of the heavy chain. VL refers to the variable region of the light chain.

[0076] As used herein, the term "complementarity determining region" (syn.CDR, i.e., CDR1, CDR2, and CDR3) refers to the amino acid residues of an antibody variable region whose presence is a major contributor to specific antigen binding. Each variable region domain (VH or VL) typically has three CDRs identified as CDR1, CDR2, and CDR3. In one example, the amino acid positions assigned to the CDRs and FRs are defined according to the Kabat Sequences of Proteins of Immunological Interest, National Institutes of Health, Bethesda, Md., 1987 and 1991 (also referred to herein as the "Kabat numbering system"). In another example, the amino acid positions assigned to the CDRs and FRs are defined according to the Enhanced Chothia Numbering Scheme (http: / / www.bioinfo.org.uk / mdex.html). The present invention is not limited to FRs and CDRs defined by the Kabat numbering system, but may be any number defined according to the canonical numbering system or the methods of Chothia and Lesk J. Mol. Biol. 196:901-917, 1987; Chothia et al., Nature 342:877-883, 1989; and / or Al-Lazikani et al. al., J. Mol. Biol. 273:927-948, 1997, Honnegher and Plukthun J. Mol. Biol. 309:657-670, 2001, or the IMGT system discussed in Giudicelli et al., Nucleic Acids Res. 25:206-211 1997. In one example, the CDRs are defined according to the Kabat numbering system. Optionally, the heavy chain CDR2 according to the Kabat numbering system does not include the five C-terminal amino acids listed herein, or any one or more of these amino acids are substituted with another naturally occurring amino acid.In this regard, Padlan et al., FASEB J., 9:133-139, 1995, established that the five C-terminal amino acids of heavy chain CDR2 are generally not involved in antigen binding.

[0077] "Hypervariable region" refers to the regions of an antibody variable domain that are hypervariable in sequence and / or form structurally defined loops. Generally, antibodies contain six hypervariable regions, V H Three in (H1, H2, H3), and V L Includes three in (L1, L2, L3).

[0078] "Framework" or "FR" residues are those variable domain residues other than the hypervariable region residues as herein defined.

[0079] An "antigen-binding site" generally refers to a molecule that includes at least the hypervariable and framework regions required to confer antigen-binding function to a V domain. The antigen-binding site may be in the form of an antibody or antibody fragment, such as mAb, single domain (SD)-mAb, dAb, Fab, SD-Fab, Fd, SD-Fv, Fv, F(ab')2, or scFv, in the manner described herein. As used herein, terminal antigen-binding site, antigen-binding portion, and antigen-binding domain may be used interchangeably. It will be understood that an antigen-binding protein may comprise or consist of an antigen-binding domain.

[0080] As used herein, the term "Fv" is intended to mean any protein, whether composed of multiple polypeptides or a single polypeptide, in which the VL and VH associate to form a complex having an antigen-binding domain, i.e., capable of specifically binding to an antigen. The VH and VL forming the antigen-binding domain may be in a single polypeptide chain or in different polypeptide chains. Furthermore, the Fv of the invention (and any protein of the invention) may have multiple antigen-binding domains that may or may not bind to the same antigen. This term is to be understood to encompass fragments derived directly from antibodies, and proteins corresponding to such fragments produced using recombinant means. In some instances, the VH is not linked to a heavy chain constant domain (CH)1 and / or the VL is not linked to a light chain constant domain (CL). Exemplary Fv-containing polypeptides or proteins include Fab fragments, Fab' fragments, F(ab') fragments, scFvs, diabodies, triabodies, tetrabodies, or higher order complexes, or any of the foregoing linked to a constant region or domain thereof, e.g., a CH2 or CH3 domain, e.g., a minibody. A "Fab fragment" consists of a monovalent antigen-binding fragment of an immunoglobulin and can be produced by digestion of a whole antibody with the enzyme papain, resulting in a fragment consisting of an intact light chain and a portion of the heavy chain, or can be produced using recombinant means. An "Fab' fragment" of an antibody can be obtained by treating a whole antibody with pepsin, followed by reduction, resulting in a molecule consisting of an intact light chain and a portion of the heavy chain containing the VH and a single constant domain. Two Fab' fragments are obtained per antibody treated in this manner. Fab' fragments can also be produced by recombinant means. An "F(ab')2 fragment" of an antibody consists of a dimer of two Fab' fragments held together by two disulfide bonds, and can be obtained by treating a whole antibody molecule with the enzyme pepsin without subsequent reduction. A "Fab2" fragment is a recombinant fragment containing, for example, two Fab fragments linked using a leucine zipper or CH3 domain.A "single-chain Fv" or "scFv" is a recombinant molecule containing the variable region fragment of an antibody (Fv) in which the variable region of the light chain and the variable region of the heavy chain are covalently linked by a suitable flexible polypeptide linker.

[0081] The terms "binds to", "specifically binds to", or "specific for" with respect to a targeting moiety refer to a binding domain that recognizes and binds to a particular ligand or antigen and does not substantially recognize or bind to other molecules in a sample. In the context of an antigen-binding protein, an antigen-binding domain or targeting moiety that specifically binds to an antigen from one species may also bind to that antigen from another species. This cross-species reactivity is typical of many antibodies and therefore does not violate the definition that an antigen-binding domain is specific. An antigen-binding domain that specifically binds to an antigen may also bind to different allelic forms of the antigen (allelic variants, splice variants, isoforms, etc.) or homologous variants of this antigen from the same gene family. This cross-reactivity is typical of many antibodies and therefore does not violate the definition that an antigen-binding domain is specific.

[0082] An "intact" or "whole" antibody comprises an antigen-binding site as well as a C L and at least heavy chain constant domains, CH1, CH2, and CH3. The constant domains may be native sequence constant domains (e.g., human native sequence constant domains) or amino acid sequence variants thereof.

[0083] "Whole antibody fragments containing the variable domain" include SD-mAbs, Fab, Fab', F(ab')2, and Fv fragments; diabodies; linear antibodies, single-chain antibody molecules; and multispecific antibodies formed from antibody fragments.

[0084] The "Fab fragment" is a fragment of the variable region domain of the H chain (V H ) and the first constant domain of one heavy chain (CHI). Each Fab fragment is monovalent with respect to antigen binding, i.e., it has a single antigen-binding site.

[0085] "Fab' fragments" differ from Fab fragments by having additional few residues at the carboxy terminus of the CH1 domain including one or more cysteines from the antibody hinge region. Fab'-SH is the designation herein for Fab' in which the cysteine ​​residues of the constant domains bear a free thiol group.

[0086] The "F(ab')2 fragment" roughly corresponds to two disulfide-linked Fab fragments which have divalent antigen-binding activity and are still capable of cross-linking antigen.

[0087] "Fv" is the minimum antibody fragment which contains a complete antigen-recognition and binding site. This fragment consists of a dimer of one heavy- and one light-chain variable domain in tight, non-covalent association.

[0088] In a single-chain Fv (scFv) species, one heavy and one light chain variable domain can be covalently linked by a flexible peptide linker such that the light and heavy chains can associate in a "dimeric" structure similar to a two-chain Fv species. From the folding of these two domains emerge six hypervariable loops (three loops each from the H and L chains) that contribute amino acid residues for antigen binding and confer antigen-binding specificity to the antibody.

[0089] "Single-chain Fv," also abbreviated as "sFv" or "scFv," is a Fv polypeptide chain consisting of VFs linked together to form a single polypeptide chain. H and V L Preferably, the scFv polypeptide has a V domain that enables the scFv to form the desired structure for antigen binding. H Domain and V L It further comprises a polypeptide linker between the domains.

[0090] A "single variable domain" is one half of an Fv (containing only the three CDRs specific for an antigen) that has the ability to recognize and bind antigen, although generally with lower affinity than the entire binding site.

[0091] "Diabody" refers to an antibody fragment that has two antigen-binding sites, which are bound to the same polypeptide chain (V H -V L ) in the light chain variable domain (V L ) connected to a heavy chain variable domain (V H Small antibody fragments include V H Domain and V L These are prepared by constructing sFv fragments (see preceding paragraph) with a short linker (about 5-10 residues) between the domains, such that inter-chain but not intra-chain pairing of the V domains is achieved, resulting in bivalent fragments, i.e., fragments with two antigen-binding sites.

[0092] Diabodies can be bivalent or bispecific. Bispecific diabodies are diabodies that combine the V H and V L Heterodimers of two "crossover" sFv fragments in which the domains are present on different polypeptide chains. Triabodies and tetrabodies are also commonly known in the art.

[0093] In any embodiment, the binding proteins can be derived from human antibodies or can be humanized forms of non-human antibodies.

[0094] "Human antibody" refers to an antibody having an amino acid sequence that corresponds to that of an antibody produced by a human. Human antibodies can be produced using a variety of techniques known in the art, including phage display libraries. Human antibodies can be prepared by administering an antigen to a transgenic animal that has been modified to produce such an antibody in response to antigen challenge, but whose endogenous locus has been disabled.

[0095] "Humanized" forms of non-human (e.g., rodent) antibodies are chimeric antibodies that contain minimal sequence derived from the non-human antibody. For the most part, humanized antibodies are human immunoglobulins (recipient antibody) in which residues from a hypervariable region of the recipient are replaced by residues from a hypervariable region of a non-human species (donor antibody) such as mouse, rat, rabbit, or non-human primate having the desired antibody specificity, affinity, and capacity. In some cases, framework region (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues. Furthermore, humanized antibodies may comprise residues that are not found in the recipient antibody or the donor antibody. These modifications are made to further improve antibody performance. In general, humanized antibodies will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the hypervariable loops correspond to those of a non-human immunoglobulin and all or substantially all of the FRs are those of a human immunoglobulin sequence. The humanized antibody optionally also will comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin.

[0096] "Monoclonal antibody" refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor amounts. Monoclonal antibodies are highly specific, being directed against a single antigenic site or determinant on the antigen. In addition to their specificity, monoclonal antibodies are advantageous in that they may be synthesized uncontaminated by other antibodies. Monoclonal antibodies may be prepared by the hybridoma method. "Monoclonal antibodies" may also be isolated from phage antibody libraries using molecular engineering techniques.

[0097] As used herein, the term "antigen" is intended to include a substance that binds to or induces the production of one or more antibodies, and may include, but is not limited to, proteins, peptides, polypeptides, oligopeptides, lipids, carbohydrates, and combinations thereof, such as glycosylated proteins or glycolipids. As used herein, the term "antigen" refers to a molecular entity that can be expressed on a target cell and recognized by the adaptive immune system, including, but not limited to, antibodies or TCRs, or engineered molecules, including, but not limited to, transgenic TCRs, CARs, scFvs or multimers thereof, Fab fragments or multimers thereof, antibodies or multimers thereof, single chain antibodies or multimers thereof, or any other molecule that can perform binding to a structure with high affinity.

[0098] "Epitope" generally refers to the portion of an antigen that is bound by the antigen-binding site of an antibody. An epitope may be "linear" in the sense that the hypervariable loops of the antibody CDRs that form the antigen-binding site bind to a sequence of amino acids as in the primary protein structure. In certain embodiments, the epitope is a "conformational epitope", i.e., the hypervariable loops of the CDRs bind to residues as they are presented in a tertiary or quaternary protein structure.

[0099] The term "immune cell" or "immune effector cell" refers to a cell that may be part of the immune system and that performs a specific effector function, such as an alpha-beta T cell, a NK cell, a NKT cell, a B cell, a Breg cell, a Treg cell, an innate lymphoid cell (ILC), a cytokine-induced killer (CIK) cell, a lymphokine-activated killer (LAK) cell, a gamma-delta T cell, a mesenchymal stem cell or a mesenchymal stromal cell (MSC), a monocyte or a macrophage, or any hematopoietic progenitor cell, such as an early precursor subset that can mature or differentiate into a pluripotent stem cell and a somatic cell.

[0100] The term "pharmaceutical composition" refers to a preparation that is in a form that is effective for the biological activity of the active ingredients contained therein and does not contain additional ingredients that are unacceptably toxic to the subject to which the formulation is administered.

[0101] "Pharmaceutically acceptable carrier" refers to an ingredient in a pharmaceutical composition, other than an active ingredient, that is non-toxic to a subject. Pharmaceutically acceptable carriers include, but are not limited to, buffers, excipients, stabilizers, or preservatives.

[0102] As used herein, the term "subject" refers to a mammal, such as a mouse, rat, cow, pig, goat, chicken, dog, monkey, or human. Preferably, the subject is a human. The subject may be a subject (patient) suffering from a disorder such as cancer, but the subject may also be a healthy subject. As used herein, "subject," "individual," and "patient" may be used interchangeably.

[0103] A "therapeutically effective amount" of an agent, e.g., a pharmaceutical composition, refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic or prophylactic result. A therapeutically effective amount of an agent, for example, eliminates, reduces, delays, minimizes, or prevents the deleterious effects of a disease.

[0104] As used herein, the term "treating" a disorder means reducing the frequency or severity of at least one sign or symptom of the disease or disorder experienced by a subject.

[0105] The term "prevent" as used herein is intended to refer to at least a reduction in the likelihood of the risk of (or susceptibility to) acquiring a disease or disorder (i.e., not developing at least one clinical symptom of the disease in an individual who may be exposed to or predisposed to the disease but has not yet experienced or displayed symptoms of the disease). Biological and physiological parameters for identifying such patients are provided herein and are well known by physicians. For example, prevention of abnormal immune responses may be characterized by the absence of increased release of cytokines following treatment with a cellular immunotherapeutic agent.

[0106] Binding Proteins The nanoparticle bioconjugates of the invention comprise a first nanoparticle conjugated with a first binding protein for binding to a target moiety on a cancer cell, and a second nanoparticle conjugated with a second binding protein for binding to a target moiety on an immune cell, preferably an antigen on a T cell.

[0107] The first and second binding proteins (or binding moieties) of the bioconjugates of the invention can be in the form of any antigen binding protein described herein, including antibodies (including human or humanized antibodies), variants or fragments thereof.

[0108] In any embodiment, the binding protein comprises or consists of an antigen-binding domain of an antibody or an antigen-binding fragment thereof. The antibody or antigen-binding fragment may be modified or engineered or is a human antibody. Examples of modified or engineered antibodies are chimeric antibodies, humanized antibodies, multiparatopic antibodies (e.g., biparatopic antibodies), and multispecific antibodies (e.g., bispecific antibodies). Examples of antigen-binding fragments include Fab, Fab', F(ab')2, Fv, single chain antibodies (e.g., scFv), minibodies, and diabodies.

[0109] In any embodiment, the antigen binding protein comprises: (i) single chain Fv fragment (scFv), (ii) a dimeric scFv (di-scFv), or (iii) It may be in the form of one of (i) or (ii) bound to the constant region of an antibody, Fc, or heavy chain constant domain (CH)2 and / or CH3.

[0110] Further, as described herein, the antigen binding protein may be (i) diabody, (ii) triabodies, (iii) tetrabodies, (iv) nanobodies, (v) iBody, (vi) a VH domain, (vii) Fab; (viii) F(ab')2, (ix) Fv, or (x) It may be in one of the forms (i) to (ix) bound to the constant region of an antibody, Fc, or heavy chain constant domain (CH)2 and / or CH3.

[0111] The above antigen-binding protein may also be referred to as an antigen-binding domain of an antibody.

[0112] In certain instances, the first and second antigen binding proteins are of the same architecture, e.g., both proteins can be in the form of an scFv or other similar antibody fragment, or both proteins can be in the form of an IgG, including any class of IgG.

[0113] In an alternative example, the first and second antigen binding proteins are of different architectures, for example the first antigen binding protein (i.e. for binding to a cancer cell antigen) is preferably in the form of an IgG and the second antigen binding protein (i.e. for binding to an antigen on an immune cell, preferably a T cell) may be in the form of an antibody fragment, such as an scFv.

[0114] The T cell antigen binding protein preferably comprises at least one binding molecule capable of binding to an activating T cell antigen. In certain embodiments, the T cell binding protein comprises no more than one antigen binding portion capable of specifically binding to an activating T cell antigen. Thus, in one embodiment, the T cell antigen binding protein provides monovalent binding to an activating T cell antigen.

[0115] In certain embodiments, the T cell antigen is CD3, particularly human or cynomolgus CD3, most particularly human CD3. In some embodiments, the T cell antigen is the epsilon subunit of CD3.

[0116] Antibodies for binding to CD3 are known in the art, as are antibodies for binding to CD3 epsilon. In certain embodiments, the binding protein optionally comprises part or all of the antigen-binding domain of a known CD3 binding protein selected from H2C, OKT3, UCGT1, and FN18.

[0117] In one embodiment, the T cell antigen binding scFv can compete with monoclonal antibody H2C (described in PCT Publication No. 2008 / 119567) for binding to an epitope on CD3. In another embodiment, the T cell antigen binding scFv can compete with monoclonal antibody V9 (described in Rodrigues et al, Int J Cancer Suppl 7, 45-50 (1992) and U.S. Patent No. 6,054,297) for binding to an epitope on CD3. In yet another embodiment, the T cell antigen binding scFv can compete with monoclonal antibody FN18 (described in Nooij et al., Eur J Immunol 19, 981-984 (1986)) for binding to an epitope on CD3.

[0118] In one embodiment, the T cell binding protein is specific for CD3 and comprises three CDRs of a heavy chain variable region sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO:1, and / or three CDRs of a light chain variable region sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO:3, or a variant thereof that retains functionality.

[0119] In any embodiment, the T cell binding protein comprises a heavy chain variable region sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO:1, and a light chain variable region sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO:3, or a variant thereof that retains functionality, including a humanized variant thereof. In one embodiment, the anti-CD3 binding protein comprises the sequence of an OKT3 antibody fragment having the code 1SY6, obtained from the Protein Data Bank (https: / / www.rcsb.org / structure / 1sy6).

[0120] In another particular embodiment, the T cell antigen is CD16, particularly human or cynomolgus CD16, most particularly human CD16. Antibodies for binding to CD16 are known in the art.

[0121] In one embodiment, the T cell binding protein is specific for CD16 and comprises three CDRs of a heavy chain variable region sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 10, and / or three CDRs of a light chain variable region sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 11, or a variant thereof that retains functionality.

[0122] In any embodiment, the T cell binding protein comprises a heavy chain variable region sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 10, and a light chain variable region sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 11, or a variant thereof that retains functionality, including a humanized variant thereof. In one embodiment, the anti-CD16 binding protein comprises the sequence of an anti-CD16 antibody fragment having catalog number #1151-V obtained from ViroGen).

[0123] In alternative embodiments, the T cell antigen is selected from the group consisting of CD2, CD4, CD7, CD8, PD1, CTLA4, KIR, CD16, CD94, CD161, NTBA, CD19, CD20, CD22, CD30, CD33, CD38, CD40L, CD44, CD56, CD79b, CD80, CD86, CD135, CD137, CD138, CD154, EphA2, EGFR, or any combination thereof. It will be appreciated that this is not an exhaustive list of T cell antigens and it is within the skill of the art to be able to identify suitable T cell antigens and corresponding antigen binding proteins for binding thereto.

[0124] The antibody for binding to such T cell antigens is also known in the art.For example, the antibody for binding to CD38 includes daratumumab, felzalutamab, isatuximab, mezagitamab; the antibody for binding to CD7 includes grisnilimab; the antibody for binding to PD1 includes valstilimab, budigalimab, camrelizumab, cemiplimab, cetrelimab, dostallimab, ezabenlimab, geptanolimab, rodapolimab, izularimab; the antibody for binding to CTLA-4 includes ipilimumab, nurlimab, pabunalimab, quabonlimab, tremelimumab, budaliimab, zalifrelimab; and the antibody for binding to CD30 includes iratumumab, SGN-30, 5F11. One skilled in the art can utilize the antigen-binding domain from any known antibody to produce nanobioconjugates according to the present invention.

[0125] A cancer cell antigen binding protein will be understood to be any protein that can specifically bind to an antigen on a cancer cell, for example a tumor-associated antigen or a tumor-specific antigen.

[0126] As used herein, "tumor-associated antigen" or cancer cell antigen refers to an antigen expressed by a cancer cell (the term "tumor antigen" may also be used to refer to the same). Tumor antigens are proteins produced by tumor cells that elicit an immune response, particularly a T-cell mediated immune response. Tumor antigens are well known in the art and include, for example, glioma-associated antigen, carcinoembryonic antigen (CEA), β-human chorionic gonadotropin, alpha fetoprotein (AFP), lectin-reactive AFP, thyroglobulin RAGE-1, MN-CAIX, human telomerase reverse transcriptase, RU1, RU2 (AS), intestinal carboxylesterase, mut hsp70-2, M-CSF, hK4 prostase, prostate specific antigen (PSA), PAP, NY-ESO-1, LAGE-1a, p53, P501S prostein, PSMA, Her2 / neu, survivin and telomerase, prostate cancer tumor antigen-1 (PCTA-1), MAGE, ELF2M, neutrophil elastase, ephrin B2, CD22, insulin growth factor (IGF)-I, IGF-II, IGF-I receptor, and mesothelin.

[0127] The cancer cell antigen binding protein is preferably capable of specifically binding to any one of the following cancer cell antigens: endothelial growth factor receptor (EGFR), human epidermal growth factor receptor 2 (HER2), fibroblast activation protein (FAP), melanoma-associated chondroitin sulfate proteoglycan (MCSP), carcinoembryonic antigen (CEA), carbonic anhydrase IX (CAIX), prostate-specific antigen (PSMA), CD33, CD19, CD20, mesothelin, GPC3, MUC1, GD2, CEA, EpCAM, LeY, PCSA, Clec9a, CD276, PD-L1 and PD-L2, or any other cancer antigen described herein.

[0128] In one embodiment, the tumor antigen comprises one or more antigenic cancer epitopes associated with malignant tumors. Malignant tumors express several proteins that can serve as target antigens for immune attack. These molecules include, but are not limited to, tissue-specific antigens such as MART-1, tyrosinase, and GP 100 in melanoma, and prostatic acid phosphatase (PAP) and prostate-specific antigen (PSA) in prostate cancer. Other target molecules belong to the group of transformation-associated molecules, such as the oncogene HER-2 / Neu / ErbB-2. Yet another group of target antigens are oncofetal antigens, such as carcinoembryonic antigen (CEA). In B-cell lymphomas, tumor-specific idiotypic immunoglobulins constitute true tumor-specific immunoglobulin antigens that are unique to individual tumors. B-cell differentiation antigens, such as CD19, CD20, and CD37, are other candidates for target antigens in B-cell lymphomas. Several of these antigens (CEA, HER-2, CD19, CD20, idiotype) have been used as targets for passive immunotherapy with monoclonal antibodies with limited success.

[0129] The type of tumor antigen referred to in the present invention can also be a tumor-specific antigen (TSA). A TSA is unique to tumor cells and does not occur on other cells in the body. A tumor-associated antigen (TAA) is not unique to tumor cells, but instead is also expressed on normal cells under conditions that cannot induce a state of immune tolerance to the antigen. Expression of an antigen on a tumor can occur under conditions that allow the immune system to respond to the antigen. TAA can be antigens that are expressed on normal cells during fetal development, when the immune system is immature and unable to respond, or they can be antigens that are usually present at very low levels on normal cells, but are expressed at much higher levels on tumor cells. Those tumor-associated antigens of greatest clinical interest are differentially expressed compared to the corresponding non-tumor tissue, allowing preferential recognition of tumor cells by specific T cells or immunoglobulins.

[0130] Non-limiting examples of TSA or TAA antigens include differentiation antigens such as MART-1 / MelanA (MART-1), gp 100 (Pmel 17), tyrosinase, TRP-1, TRP-2, and tumor-specific multilineage antigens such as MAGE-1, MAGE-3, BAGE, GAGE-1, GAGE-2, p15; overexpressed embryonic antigens such as CEA; overexpressed oncogenes and mutated tumor suppressor genes such as p53, Ras, HER-2 / neu; unique tumor antigens resulting from chromosomal translocations such as BCR-ABL, E2A-PRL, H4-RET, 1GH-IGK, MYL-RAR; and viral antigens such as Epstein-Barr virus antigen EBVA and human papillomavirus (HPV) antigens E6 and E7. Other large, protein-based antigens include TSP-180, MAGE-4, MAGE-5, MAGE-6, RAGE, NY-ESO, p185erbB2, p180erbB-3, c-met, nm-23H 1, PSA, TAG-72, CA 19-9, CA 72-4, CAM 17.1, NuMa, K-ras, beta-catenin, CDK4, Mum-1, p 15, p 16, 43-9F, 5T4, 791Tgp72, alpha-fetoprotein, beta-HCG, BCA225, BTAA, CA 125, CA 15-3\CA 27.29\BCAA, CA195, CA242, CA-50, CAM43, CD68\P 1, CO-029, FGF-5, G250, Ga733\EpCAM, HTgp-175, M344, MA-50, MG7-Ag, MOV 18, NB / 70K, NY-CO-1, RCAS 1, SDCCAG16, TA-90\Mac-2 binding protein\cyclophilin C-related protein, TAAL6, TAG72, TLP, and TPS.Particularly preferred examples of target cell antigens according to the present invention include CD33 (Siglec-3), CD123 (IL3RA), CD135 (FLT-3), CD44 (HCAM), CD44V6, CD47, CD184 (CXCR4), CLEC12A (CLL1), LeY, FRp, MICA / B, CD305 (LAIR-1), CD366 (TIM-3), CD96 (TACTILE), CD133, CD56, CD29 (ITGB1), CD44 (HCAM), CD47 (IAP), CD66 (CEA), CD112 (Nectin2), CD117 (c-Kit), CD133, CD146 (MCAM), CD155 (PVR), CD171 (LI CAM), CD221 (IGF1), CD227 (MUC1), CD243 (MRD1), CD246 (ALK), CD271 (LNGFR), CD19, CD20, GD2, and in particular EGFR, mesothelin, GPC3, MUC1, HER2, GD2, CEA, EpCAM, LeY, PCSA, and CD276.

[0131] Those skilled in the art will be familiar with antigen binding domains described in the literature for binding to any one or more of the cancer antigens listed above. Certain non-limiting examples follow, but it will be understood that the scope of the present invention should not be limited to the specific antigen binding proteins described herein. Also, once a person skilled in the art has determined which cancer cells require treatment, he or she can easily refer to known literature on the cancer cell type to determine suitable antigens present on the cancer cells for targeting. The person skilled in the art can then utilize any known or novel antigen binding domain for binding to a cancer antigen for conjugation to the nanoparticles described herein to generate suitable bioconjugates for the treatment of cancer. The ability of the bioconjugate to bind to the target antigen can be confirmed using common laboratory techniques, including those referenced herein in the examples.

[0132] In one embodiment, the cancer cell antigen binding protein is capable of specifically binding to epidermal growth factor receptor (EGFR). In another embodiment, the binding protein comprises an scFv molecule that can compete with monoclonal antibody GA201 for binding to an epitope of EGFR (see PCT Publication WO 2006 / 082515, which is incorporated herein by reference in its entirety).

[0133] In one embodiment, a binding protein specific for EGFR comprises three CDRs of a heavy chain variable region sequence that are at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO:6, and / or three CDRs of a light chain variable region sequence that are at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO:7, or a functional variant thereof.

[0134] In certain embodiments, the binding protein specific for EGFR comprises a heavy chain variable region sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO:6, and a light chain variable region sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO:7, or a variant thereof that retains functionality. In one embodiment, the binding protein specific for EGFR comprises nimotuzumab antibody (Nmab) having the code 3GKW obtained from the Protein Data Bank (https: / / www.rcsb.org / structure / 3gkw).

[0135] In one embodiment, a binding protein specific for EGFR comprises three CDRs of a heavy chain variable region sequence that are at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO:9, and / or three CDRs of a light chain variable region sequence that are at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO:8, or a functional variant thereof.

[0136] In certain embodiments, the binding protein specific for EGFR comprises a heavy chain variable region sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 9, and a light chain variable region sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 8, or a variant thereof that retains functionality. In one embodiment, the binding protein specific for EGFR comprises the panitumumab antibody (Pmab) having the code 5SX4 obtained from the Protein Data Bank (https: / / www.rcsb.org / structure / 5sx4).

[0137] In one embodiment, the cancer cell antigen binding protein is capable of specifically binding to HER2. In one embodiment, the binding protein specific for HER2 comprises three CDRs of a heavy chain variable region sequence that are at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 4, and / or three CDRs of a light chain variable region sequence that are at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 5, or a functionally retained variant thereof. In any embodiment, the binding protein comprises a heavy chain variable region sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO:4, and a light chain variable region sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO:5, or a variant thereof that retains functionality. In one embodiment, the binding protein specific for HER2 comprises the trastuzumab antibody (Tmab) having the code DB00072, obtained from the Online Drug Bank (https: / / go.drugbank.com / drugs / DB00072).

[0138] Magnetic and Metal Nanoparticles Those skilled in the art will appreciate that a variety of nanoparticles may be used to generate the bioconjugates of the present invention. Typically, nanoparticles that are superparamagnetic, crystalline, physically and chemically stable, environmentally safe, and biocompatible are preferred.

[0139] In any embodiment of the present invention, the magnetic nanoparticles may be selected from Fe3O4 (magnetite), FeCl2, Fe(Co)5 (iron pentacarbonyl), maghemite (y-Fe2O3), or hematite (a-Fe2O3) nanoparticles. In a preferred embodiment, the magnetic nanoparticles are Fe3O4. In some embodiments, the average diameter of the magnetic nanoparticles is about 14 nm.

[0140] In any embodiment, the metal nanoparticles may be selected from gold (Au), silver (Ag), platinum (Pt) or palladium (Pd), copper (Cu), nickel (Ni), cobalt (Co), or alloys or nanoparticles of two or more thereof.

[0141] Preferably, the metal nanoparticles are gold or silver nanoparticles. In a particularly preferred embodiment, the metal nanoparticles are gold nanoparticles.

[0142] In any embodiment, the nanoparticle bioconjugate is composed of iron and gold nanoparticles, preferably the bioconjugate comprises a central gold nanoparticle surrounded by three to four or more iron nanoparticles to form a flower-shaped nanoparticle.

[0143] In any embodiment, the nanoparticle bioconjugates of the invention can be from about 5 nm to about 200 nm. In any embodiment, the nanoparticle bioconjugates of the invention are from about 35 nm to 80 nm, preferably about 40 nm.

[0144] In any embodiment, the nanoparticle bioconjugates of the present invention may have a polymer coating to increase their dispersibility and / or stability. Examples of polymers for nanoparticle surface modification include PEG, polydopamine, dextrose, PVA, PAA, starch, or chitosan. In a preferred embodiment, the nanoparticle bioconjugates of the present invention have a PEG coating.

[0145] Conjugation of antigen-binding domains to nanoparticles Those of skill in the art will be familiar with standard techniques for conjugating antigen binding proteins to nanoparticles, including the metal or magnetic nanoparticles described herein.

[0146] Standard techniques, such as those described herein in the Examples, can be used to covalently bind antigen-binding proteins to nanoparticles. Further examples of methods for conjugating nanoparticles to antigen-binding proteins include those described in the art (see, for example, Jazayeri et al. (2016) Various methods of gold nanoparticles (GNPs) conjugation to antibodies. Sensing and Bio-Sensing Research 9:17-22, and Cruz and Kayser (2019) Synthesis and Enhanced Cellular Uptake In Vitro of Anti-HER2 Multifunctional Gold Nanoparticles. Cancer 11(870):1-22, which are incorporated herein by reference).

[0147] Those skilled in the art will understand that the conjugation method depends on the functional groups available on the nanoparticle coating, the linker used, and the ligand to be attached to the nanoparticle. Examples of suitable conjugation methods include carboxyl linkage (using carbodiimide to include carboxyl groups present on the target binding protein), maleimide linkage, azide linkage, biotin linkage, hydroxyl linkage, methyl linkage, neutravidin linkage, NHS (N-hydroxysuccinimide) moiety linkage, thiol linkage, PEG moiety linkage, amide linkage, hydrazone linkage, amine linkage (using carbodiimide to include amino groups present on the target binding protein).

[0148] In a preferred embodiment, the conjugation method is covalent conjugation involving amide, hydrazone, and / or amine bonds, achieved by carbodiimide and / or malemide chemistry.

[0149] In some embodiments, conjugation is accomplished using a commercially available nanoparticle conjugation kit, such as a carboxyl magnetic iron oxide nanoparticle kit or an amine iron oxide nanoparticle kit (e.g., supplied by Ocean NanoTech), or a modified protocol based on the methods described in such kits. Again, these techniques would be within the skill set of one of ordinary skill in the art.

[0150] In any embodiment, the antigen binding protein may be conjugated to a nanoparticle using the methods described herein in the Examples.

[0151] In a preferred embodiment, the first and second binding proteins are covalently conjugated to the nanoparticle via different chemistries. The use of different chemistries facilitates the preparation of composite nanoparticles.

[0152] In one example, one binding protein may be covalently conjugated to a magnetic nanoparticle via a polyethylene glycol (PEG) moiety and the other binding protein may be covalently conjugated to a metal nanoparticle via a thiol linker, optionally where the thiol is at least one of thioctic acid, monothioctic acid, dithioctic acid, and trithioctic acid.

[0153] Conditions Treated, Dosage, and Administration The bispecific nanoparticles of the invention have particular utility for the treatment or prevention of cancer.

[0154] A wide range of examples include breast tumors, colorectal tumors, adenocarcinoma, mesothelioma, bladder tumors, prostate tumors, germ cell tumors, liver / bile duct, carcinoma, neuroendocrine tumors, pituitary tumors, small round cell tumors, squamous cell carcinoma, melanoma, atypical fibroxanthoma, seminoma, nonseminoma, stromal Leydig cell tumors, Sertoli cell tumors, skin tumors, kidney tumors, testicular tumors, brain tumors, ovarian tumors, stomach tumors, oral cavity tumors, bladder tumors, bone tumors, cervical tumors, esophageal tumors, laryngeal tumors, liver tumors, lung tumors, vaginal tumors, and Wilms' tumor.

[0155] Examples of specific cancers include, but are not limited to, adenocarcinoma, adenoma, adenofibroma, adenolymphoma, odontoma, AIDS-related cancer, acoustic neuroma, acute lymphocytic leukemia, acute myeloid leukemia, adenocystic carcinoma, adrenal cortical carcinoma, primary myelofibrosis, alopecia, alveolar soft part sarcoma, ameloblastoma, angiokeratoma, eosinophilic angiolymphocytosis, sclerosing hemangioma, hemangiomatosis, apdoma, anal cancer, angiosarcoma, aplastic anemia, astrocytoma, ataxia telangiectasia, basal cell carcinoma (skin), bladder cancer, bone cancer, intestinal cancer, brain stem glioma, brain and CNS tumors, breast cancer, branchiomas, CNS tumors, Carcinoid tumor, cervical cancer, childhood brain tumor, childhood cancer, childhood leukemia, childhood soft tissue sarcoma, chondrosarcoma, choriocarcinoma, chronic lymphocytic leukemia, chronic myeloid leukemia, colorectal cancer, cutaneous T-cell lymphoma, carcinoma (e.g., Walker, basal cell, basosquamous, Brown-Pierce, ductal, Ehrlich, Krebs2, Merkel cell, mucinous, non-small cell lung, oat cell, papillary, scirrhous, bronchiolar, bronchogenic, squamous cell, and transitional cell), carcinosarcoma, cervical dysplasia, phyllodes cystosarcoma, cementoma, chordoma, stroma, chondrosarcoma, chondroblastoma, craniopharyngioma, cholangiomas , cholesteatoma, cystadenocarcinoma, cystadenoma, dermatofibrosarcoma protuberans, desmoplastic small round cell tumor, ductal carcinoma, dysgerminoma, endocrine carcinoma, endometrial carcinoma, ependymoma, esophageal cancer, Ewing's sarcoma, extrahepatic bile duct cancer, eye cancer, eye: melanoma, retinoblastoma, fallopian tube cancer, Fanconi anemia, fibroma, fibrosarcoma, gallbladder cancer, gastric cancer, gastrointestinal cancer, gastrointestinal-carcinoid tumor, genitourinary cancer, germ cell tumor, gestational trophoblastic disease, glioma, gynecological cancer, giant cell tumor, ganglioneuroma, glioma, glomus hemangioma, granulosa cell tumor, ginandroblastoma, hematological malignancies, hairy cell white Hematologic malignancies, head and neck cancer, hepatocellular carcinoma, hereditary breast cancer, histiocytosis, Hodgkin's disease, human papillomavirus, hydatidiform mole, hypercalcemia, hypopharyngeal cancer, hamartoma, hemangioendothelioma, hemangioma, hemangiopericytoma, angiosarcoma, angiosarcoma, histiocytic disorders, malignant histiocytosis, histiocytoma, liver cancer, hidradenoma, chondrosarcoma, immunoproliferative small, lipoma, intraocular melanoma, islet cell carcinoma, Kaposi's sarcoma, kidney cancer, Langerhans cell histiocytosis, laryngeal cancer, leiomyosarcoma, leukemia, Li-Fraumeni syndrome, lip cancer, liposarcoma, liver cancer, lung cancer, lymphedema, lymphoma, Hodgkin's lymphoma, non-Hodgkin's lymphoma,Leiomyosarcoma, leukemia, leukosarcoma, Leydig cell tumor, liposarcoma, leiomyoma, leiomyosarcoma, lymphangioma, lymphangiocytoma, lymphadioma, lymphangioleiomyoma, lymphangiosarcoma, male breast cancer, malignant rhabdoid tumor of the kidney, medulloblastoma, melanoma, Merkel cell carcinoma, mesothelioma, metastatic cancer, oral cancer, multiple endocrine neoplasia, mycosis fungoides, myelodysplastic syndrome, myeloma, myeloproliferative disorder, malignant carcinoid syndrome, carcinoid heart disease, medulloblastoma, meningioma, melanoma, mesenchymoma, mesonephroma, mesothelioma dermoma, myoblastoma, fibroid, myosarcoma, myxoma, myxosarcoma, nasal cancer, nasopharyngeal cancer, nephroblastoma, neuroblastoma, neurofibromatosis, Nijmegen destruction syndrome, non-melanoma skin cancer, non-small cell lung cancer-(NSCLC), schwannoma, neuroblastoma, neuroepithelioma, neurofibromatosis, neurofibroma, neuroma, tumors (e.g. bone, breast, digestive system, colorectal, liver), eye cancer, esophageal cancer, oral cancer, oropharyngeal cancer, osteosarcoma, ostomy ovarian cancer, pancreatic cancer, paranasal sinus cancer, parathyroid cancer, ear subgland cancer, penile cancer, peripheral neuroectodermal tumor, pituitary cancer, polycythemia vera, prostate cancer, osteoma, osteosarcoma, ovarian cancer, papilloma, paraganglioma, nonchromaffin paraganglioma, pinealoma, plasmacytoma, proto-oncogene, rare cancers and related disorders, renal cell carcinoma, retinoblastoma, rhabdomyosarcoma, Rothmund-Thompson syndrome, reticuloendotheliosis, rhabdomyoma, salivary gland cancer, sarcoma, schwannoma, Sezary syndrome, skin cancer, small cell lung cancer (SCLC), small intestine cancer, soft tissue sarcoma, spinal tumor, squamous These include epithelial carcinoma (skin), gastric carcinoma, synovial sarcoma, sarcomas (e.g., Ewing experimental, Kaposi, and mast cell sarcoma), Sertoli cell tumor, synovium, testicular cancer, thymic carcinoma, thyroid cancer, transitional cell carcinoma (bladder), transitional cell carcinoma (renal pelvis / ureter), choriocarcinoma, teratoma, theca cell tumor, thymoma, trophoblastic tumor, urethral cancer, urinary system cancer, uroplakin, uterine sarcoma, uterine cancer, vaginal cancer, vulvar cancer, Waldenstrom's macroglobulinemia, and Wilms' tumor.

[0156] In some instances, the nanoparticle bioconjugates described herein can be administered orally, parenterally, by inhalation spray, adsorption, absorption, topically, rectally, nasally, buccally, intravaginally, intraventricularly, via an implanted reservoir in a dosage formulation containing a conventional non-toxic pharma- ceutically acceptable carrier, or by any other convenient dosage form. As used herein, the term "parenteral" includes subcutaneous, intravenous, intramuscular, intraperitoneal, intrathecal, intraventricular, intrasternal, and intracranial injection or infusion techniques.

[0157] Methods for preparing nanoparticle bioconjugates into suitable forms (e.g., pharmaceutical compositions) for administration to a subject are known in the art and include, for example, those methods described in Remington's Pharmaceutical Sciences (18th ed., Mack Publishing Co., Easton, Pa., 1990) and the US Pharmacopeia: National Formulary (Mack Publishing Company, Easton, Pa., 1984).

[0158] The pharmaceutical compositions of the present invention are particularly useful for parenteral administration, such as intravenous administration or administration into a body cavity or cavity of an organ or joint. The composition for administration will generally comprise a solution of the antigen-binding site dissolved in a pharma- ceutically acceptable carrier, e.g., an aqueous carrier. A variety of aqueous carriers, e.g., buffered saline, etc., can be used. The composition may contain pharma- ceutically acceptable auxiliary substances necessary to approximate physiological conditions, such as pH adjusting and buffering agents, toxicity adjusting agents, etc., e.g., sodium acetate, sodium chloride, potassium chloride, calcium chloride, sodium lactate, etc. The concentration of the antigen-binding site of the present invention in these formulations may vary widely and will be selected primarily based on fluid volumes, viscosities, body weight, etc., according to the particular mode of administration selected and the needs of the patient. Exemplary carriers include water, saline, Ringer's solution, dextrose solution, and 5% human serum albumin. Non-aqueous vehicles, such as mixed oils and ethyl oleate, may also be used. Liposomes may also be used as carriers. The vehicle may contain minor amounts of additives that enhance isotonicity and chemical stability, e.g., buffers and preservatives.

[0159] When formulated, the nanoparticle bioconjugates of the invention will be administered in a manner compatible with the dosage formulation and in such amount as will be therapeutically / prophylactically effective. The formulations are easily administered in a variety of dosage forms, such as the types of injectable solutions described above, although other pharma- ceutically acceptable forms are also contemplated, such as tablets, pills, capsules, or other solids for oral administration, suppositories, pessaries, nasal solutions, or sprays, aerosols, inhalants, liposomal forms, and the like. Pharmaceutical "slow-release" capsules or compositions may also be used. Sustained-release formulations are generally designed to provide a constant drug level over an extended period of time and may be used to deliver the antigen-binding moieties of the invention.

[0160] Suitable dosages of the nanoparticle bioconjugates of the invention will vary depending on the particular nanoparticle bioconjugate, the condition being treated, and / or the subject being treated. It is within the ability of a skilled physician to determine suitable dosages, for example, by starting with a suboptimal dosage and incrementally modifying the dosage to determine an optimal or useful dosage. Alternatively, data from cell culture assays or animal studies can be used to determine appropriate dosages for treatment / prophylaxis, and a suitable dose can be determined based on the ED of the active compound with little or no toxicity. 50 The therapeutically / prophylactically effective dose can be estimated initially from cell culture assays. Dosages are within the range of circulating concentrations including the IC 50 The compound may be formulated in animal models to achieve a circulating plasma concentration range that includes the concentration or amount of compound that achieves a half-maximal inhibition of symptoms (i.e., the concentration or amount of compound that achieves a half-maximal inhibition of symptoms). Such information can be used to more accurately determine useful doses in humans. Levels in plasma can be measured, for example, by high performance liquid chromatography.

[0161] In some instances, the methods of the invention comprise administering a prophylactically or therapeutically effective amount of a nanoparticle bioconjugate described herein.

[0162] The term "therapeutically effective amount" is an amount that, when administered to a subject in need of treatment, improves the subject's prognosis and / or condition and / or reduces or inhibits one or more symptoms of a clinical condition described herein below the level observed and tolerated as a clinical diagnosis or clinical feature of the condition. The amount administered to a subject will depend on the specific characteristics of the condition being treated, the type and stage of the condition being treated, the mode of administration, and the characteristics of the subject, such as general health, other diseases, age, sex, genotype, and weight. Those skilled in the art will be able to determine appropriate dosages depending on these and other factors. Thus, the term should not be construed as limiting the invention to a specific amount, e.g., weight or amount of protein, but rather the invention encompasses any amount of antigen-binding site sufficient to achieve the described result in a subject.

[0163] As used herein, the term "prophylactically effective amount" is intended to mean an amount of protein sufficient to prevent or inhibit or delay the onset of one or more detectable symptoms of a clinical condition. One of skill in the art will recognize that such amounts will vary depending, for example, on the particular antigen-binding site administered and / or the particular subject and / or the type or severity or level of the condition and / or predisposition (genetic or otherwise) to the condition. Thus, this term should not be construed as limiting the invention to a particular amount, e.g., weight or amount of antigen-binding site, but rather the invention encompasses any amount of antigen-binding site sufficient to achieve the described result in a subject.

[0164] The present invention additionally includes kits comprising the nanoparticle bioconjugates of the invention. The kits may additionally include a pharma- ceutically acceptable carrier.

[0165] Optionally, the kits of the invention are packaged with instructions for use in the methods described herein according to any of the examples.

[0166] In a preferred embodiment, the kit includes components for producing a nanoparticle bioconjugate of the invention, eg, a nanoparticle component and a binding protein for conjugating thereto. EXAMPLES

[0167] Here, we report the development of a flower-like nanoparticle platform for the combined delivery of two antigen-binding proteins. These nanoparticles, called bispecific nanoflowers, contain binding domains for binding to antigens on tumor cells (e.g., for binding to EGFR using antibodies nimotuzumab or panitumumab) while simultaneously triggering T cell activation via binding to T cells (e.g., by using anti-CD3 scFv or anti-CD16 scFv), also conjugated to the nanoparticle. By chemically conjugating the antibodies in the nanoparticle platform, the nanoflowers of the present invention are expected to produce a synergistic effect resulting from the two binding proteins tested in vitro in cancer cells.

[0168] We believe this is the first proof of concept demonstrating the synergistic effect of dual antibody targeted immunotherapy in cancer via iron oxide-gold nanoparticles. In one example, the developed nanodelivery system shows that the exemplary bispecific iron oxide-gold nanoparticles are effective bispecific bioconjugates to retarget T cells towards EGFR expressing cells in vitro at a rate 69.7% higher than that of free nimotuzumab.

[0169] Example 1: Materials and Methods material Hydrogen tetrachloroaurate(III) trihydrate (HAUCL4●3H2O), iron pentacarbonyl (Fe(CO)5), oleylamine, oleic acid, 1-octadecene, α,ω-bis{2-[(3-carboxy-1-oxopropyl)amino]ethyl}PEG (Mr=3,000), N-hydroxysuccinimide (NHS), N-(3-dimethylaminopropyl)N0-ethylcarbodiimide hydrochloride (EDC), 4-aminophenyl β-D-glucopyranoside, (2-(N-morpholino)ethanesulfonic acid) (MES), and PD-10 desalting columns were purchased from Sigma-Aldrich (Sydney, NSW, AUS). 1,2 hexadecanediol was purchased from TLC chemicals. MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide), penicillin, and streptomycin solutions were purchased from Invitrogen. All chemicals used in the synthesis were used without further purification. Nimotuzumab (145 kDa) monoclonal antibody was from Biocon, (Bangalore, India), also referred to herein as Nmab. Trastuzumab (also referred to herein as Tmab) was from Genentech. Panitumumab (also referred to herein as Pmab) was from Amgen. Single chain variable fragments (28-30 KDa) for anti-CD3 scFv were synthesized by the inventors and purchased from Virogen (Brighton, MA, USA). Single chain variable fragments for anti-CD16 scFv were purchased from Virogen (Brighton, MA, USA).

[0170] Instrumentation and characterization of NPs Nanoparticles (NPs) were visualized by transmission electron microscopy (TEM) using a JEOL microscope (JEOL JEM 1400, Gatan Inc., USA) operating at an accelerating voltage of 120 kV. 8 μl of diluted (dilution ratio 1:3) NPs were applied to carbon-coated copper grids. The grids were left to dry at room temperature for 2 h and imaged using a transmission electron microscope.

[0171] A UV-Visible spectrophotometer (UV-Vis, Shimadzu, Japan) was used for the absorption spectra of Fe3O4@Au NPs, Fe3O4@AgNPs, and antibody conjugation in hexane or aqueous solutions depending on the nature of the NPs. The hydrodynamic size distribution and zeta potential of the NPs were measured in triplicate by a particle size analyzer Zeta-sizer Nano ZS90 (Malvern, Worcestershire, UK) at a scattering angle of 90 at 25 °C, adjusted for the reflectance index of the dispersant. The particle size distribution, average particle size, and polydispersity index are reported. Fourier transform infrared spectroscopy (FTIR) was recorded on a Shimadzu IRT racer-100 spectrophotometer. The final concentration of the NPs was determined using inductively coupled plasma mass spectrometry using a PerkinElmer Nexion 300X ICP-MS instrument (PerkinElmer, Waltham, MA, USA). For this, the NPs were nebulized with aqua regia. All mass spectra were acquired using a matrix-assisted laser desorption / ionization / time-of-flight (MALDI-TOF) Bruker Autoflex Speed ​​LRF with a 1000 Hz Smartbeam-II laser. Spectra were recorded in specular reflectance mode using an accelerating voltage of 9 kV, linear positive ion mode, ion source 1: 19.5 kV, ion source 2: 16.5 kV, matrix suppression deflection of 18000 Da and suppression up to and including 18000 Da, mass range: 20,000-400,000 Da. Data analysis was performed using Flex Control version 3.4, Flex Analysis version 3.4.

[0172] SDS-PAGE was used to evaluate NP-antibody conjugation, samples were loaded on SDS-PAGE and electrophoresis was performed at 100V for 30 minutes followed by incubation with Coomassie Brilliant Blue to detect protein bands. Visualization and analysis of protein bands was performed on a Bio-Rad Gel Doc™ XR system and analyzed using Bio-Rad Image software. Conjugation was confirmed using a Nano-Drop 1000 (Thermo-Scientific) spectrophotometer measurement, which was performed by measuring absorbance at 280 nm with a 0.1×PBS baseline. Enzyme-linked immunoassay (ELISA) was performed and absorbance measurements were taken at 450 nm through a microplate reader. "Naked" and conjugated NPs were measured in the presence of a positive control (Herceptin®) and a negative control (assay buffer).

[0173] The UV spectra of Bis-Fe3O4@Au-NPs (bispecific NPs conjugated to anti-EGFR Nmab and anti-CD3 scFv) and Nmab-Fe3O4@Au NPs (i.e. nanoparticles containing only anti-EGFR Nmab) are shown in Figure 4. It is possible to determine the presence of conjugated antibody by subtracting the amount of conjugated antibody obtained after centrifugation from the total amount of antibody loaded before centrifugation. A sharp peak at about 260 nm determines the presence of conjugated antibody.

[0174] cell line A431 is a human epithelial carcinoma cell line with known overexpression of EGFR. Jurkat, clonal E6-1 cells (TIB-125) are acute T-cell leukemia that are CD3 positive. Jurkat cells were purchased from the American Type Culture collection (ATCC, MEL, VIC, AUS). MDA-MB 231 is an epithelial, human breast cancer cell line that provides a cellular model of metastatic breast adenocarcinoma. MDA-MB 468 is another epithelial, human breast cancer cell line that provides a cellular model of metastatic breast adenocarcinoma.

[0175] Cells were cultured at 37 °C in a humidified incubator supplied with 5% CO2 in flasks containing Dulbecco's Modified Eagle Medium (DMEM) low glucose medium, or Jurkat's RPMI-1640, supplemented with 10% fetal bovine serum (FBS) and 1% penicillin / streptomycin. Cells were passaged once every 2-3 days depending on their growth rate. Cells were cultured until approximately 80-90% confluence before harvesting. During harvesting, cells were washed with PBS, followed by trypsinization with 2 ml of trypsin and incubation for 10 min to detach the cells from the flask. Trypsin was neutralized by adding 8 ml of fresh supplemented medium, and then 2 ml of harvested cells were transferred to a new flask and resuspended in fresh medium. Cell viability was determined before each experiment using a hemocytometer.

[0176] Preparation of anti-CD3 scFv A specific His6-tagged anti-CD3 scFv antibody construct was designed from OKT3 mAb and expressed according to the method of a previous study (Kipriyanov et al. (1997) J Immunol Methods 15(200):67-77). The anti-CD3 scFv sequence was obtained from the Protein Data Bank under the code (1SY6). The construct contained a linker peptide (Gly4Ser)3 (SEQ ID NO:2) between the light and heavy variable chains. The construct was cloned into a gWiz expression plasmid, which was transformed into competent Escherichia coli. The anti-CD3 scFv was produced using standard techniques for mammalian expression systems using HEK 293 cells.

[0177] The antibody was purified using a HisTrap column equilibrated with PBS containing 10 mM imidazole. The purified antibody was dialyzed into PBS containing 15 mM sodium phosphate, 0.15 M NaCl (pH 7.4).

[0178] [Table 2-1]

[0179] [Table 2-2]

[0180] Fe 3 O 4 Preparation of @Au NPs Fe3O4@Au NPs were synthesized following a previously reported procedure with minor modifications (Yu, Chen et al. 2005). Fe3O4@Au NPs were synthesized by a pyrolysis method under inert conditions (Fig. 1). First, 6 mmol of oleic acid, 6 mmol of oleylamine, and 10 mmol of 1,2-hexadecanediol were mixed with 20 ml of 1-octadecene (ODE) in a 50 ml round-bottom flask equipped with a thermometer, flow control adapter, reflux condenser, and rubber septum, and stirred at 160 °C for 30 min under gentle nitrogen flow. Then, under a blanket of nitrogen, 0.3 ml of Fe(CO)5 was rapidly injected into the solution. After 3 min, a degassed gold precursor solution consisting of 40 mg of HAuCl4·H2O, 1.5 ml of oleylamine, and 5 ml of ODE was added dropwise to the hot solution at 180 °C within 10 min to ensure complete mixing. The solution turned deep red immediately after injection, indicating the formation of gold nanoparticles. The solution was then slowly heated to 310 °C and refluxed for 45 min. After cooling at room temperature, 40 ml of isopropanol was added to the solution and centrifuged at 8000 rpm for 10 min to remove large particles. The precipitate was redispersed in hexane and centrifuged again at 7000 rpm to remove any undispersed material. Ethanol was then added to the solution and centrifuged again three times to obtain a brown dispersion. The Fe3O4@Au NPs were dissolved in hexane in the presence of oleylamine for further use (Figure 1B).

[0181] Fe 3 O 4 @Modification of Au NPs Transfer of hydrophobic nanoparticles to aqueous media (to improve solubility) was performed using 30 mg of α,ω-bis{2-[(3-carboxy-1-oxopropyl)amino]ethyl}PEG (Mr=3,000), 2 mg of N-hydroxysuccinimide, 3 mg of dicyclohexylcarbodiimide, and 1.5 mg of dopamine hydrochloride dissolved in a mixture of 1 ml of dimethylformamide, 2 ml of CHCl3, and 10 mg of anhydrous Na2CO3. The solution was stirred for 2 h at 37 °C, 5 mg of Fe3O4@Au NPs were added, and the resulting solution was stirred overnight at 37 °C under a nitrogen blanket. The modified NPs were precipitated by adding 5 ml of hexane and ethanol, collected by centrifugation at 16,500 × g, and surfactants and other salts were removed via dialysis (molecular weight cutoff, 10 kDa) in PBS for 24 h. Finally, they were filtered through a 0.22 μm sterile filter. The final iron concentration of the particles was determined by inductively coupled plasma mass spectrometry (ICP-MS).

[0182] statistical analysis Results are expressed as mean ± SD. Data were analyzed using GraphPad Prism (GraphPad Software, Version 9, La Jolla, CA, USA). One-way ANOVA ad hoc Fisher LSD was used to calculate significance, and *P<0.05 was considered significant.

[0183] Example 2: Synthesis and characterization of Fe3O4@Au NPs The 40 nm Fe3O4@Au NPs were synthesized via pyrolysis at high temperature in the presence of organic solvents, which produced monodispersed and highly stable NPs.

[0184] The synthesis of flower-shaped Fe3O4@Au NPs was characterized using transmission electron microscopy (TEM). Au NPs with an average core size of 11 nm were observed. The Fe3O4 NPs appeared as three to four petal shapes with an average diameter of 14 nm.

[0185] The organic ligands were then replaced with dopamine, forming a 4 nm thin layer of polydopamine, leaving catechol groups on the NP surface for further conjugation. Figure 2A shows a TEM image of 40 nm Fe3O4@Au NPs (due to the heavy atom effect, gold NPs appear as black). The NPs were well dispersed after the ligand exchange, and the hydrodynamic size of the dopamine-coated NPs in solution from DLS was 136 ± 2.49 nm, which is much larger than that of unmodified NPs 35.19 ± 0.04 nm (Table 1). The size increase was probably due to either hydrogen bond formation during the ligand exchange or the formation of a thin layer of polydopamine.

[0186] After surface modification, the zeta potential of the modified flower-shaped NPs was about -30 mV. Figure 2B shows the FTIR (Fourier transform infrared spectroscopy) spectra of Fe3O4@Au NPs before and after modification. -1 The characteristic peak at 1653 cm corresponds to the catechol of dopamine. -1 The characteristic peak C=O band at 0.05C was an indication of successful activation of dopamine molecules on the NP surface. NPs are commonly capped with dopamine to improve their dispersibility and stability.

[0187] Medical and biological applications require water solubility of Fe3O4@Au NPs. For this reason, the oleic acid coating of the NPs was modified by ligand exchange to a water-soluble ligand. In this embodiment, PEG was used to align the oil phase of the NPs with the aqueous phase, replace dopamine with oleic acid on the surface of the iron NPs, and then the amino group of dopamine was reacted with the carboxyl group of PEG under the catalysis of EDC-sulfo-NHS system.

[0188] To produce Fe3O4@Au NPs conjugated with nimotuzumab or dopamine, the EDC-sulfo-NHS system was used to activate the carboxyl groups of the PEG-modified Fe3O4@Au NPs as previously described. Excess EDC and sulfo-NHS were removed before the addition of nimotuzumab to avoid self-crosslinking of the antibody. After activation using the EDC-sulfo-NHS system, nimotuzumab was ligated to PEG (M) via a condensation reaction involving the formation of a peptide bond. r = 2,000) and conjugated to the modified Fe3O4@Au NPs through dopamine.

[0189] [Table 1]

[0190] Example 3: Fe3O4@Au NPs conjugated to anti-EGFR mAb (Nmab) and anti-CD3 scFv (mAb-Fe3O4@Au-αCD3 NPs) Nmab-Fe 3 O 4 Fe-Ag@Au-αCD3scFv NPs 3 O 4 @Conjugation of Au NPs Anti-EGFR antibody nimotuzumab was conjugated to Fe3O4NPs using the carbodiimide method, and anti-CD3 scFv antibody was conjugated to Au NPs using a thiol linker as briefly described herein. 1 mg of Fe3O4@Au NPs in sulfonic acid methyl ester buffer (pH 6) was mixed with 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC, 1.1 mmol), and sulfo-NHS (1 mmol) was added to the solution to activate the carboxyl groups on the PEG ligands for 30 min. After gentle mixing for 30 min at room temperature, the solution was subjected to a PD-10 column to remove excess EDC and sulfo-NHS. Then, 100 μg of nimotuzumab was added to the conjugate for 3 h with gentle shaking to functionalize the Fe3O4 side of the nanoflowers. After 3 hours of incubation, 5 μl of anti-CD3 scFv antibody was added to the mixture, and the mixture was placed on a shaker for overnight incubation at room temperature to functionalize the Au side. Prior to immobilization of anti-CD3 scFv, a thiol linker was added to the anti-CD3 scFv. The antibody-conjugated NPs were separated from unbound antibody and Fe3O4@Au NPs using 300K ultrafiltration.

[0191] Mass spectrometry analysis of Nmab-Fe3O4@Au-αCD3 scFv NPs revealed a specific peak of nimotuzumab at m / z ≈148 KDa (Figure 3), which was not detected in Au-Fe3O4 NPs. In addition, collected supernatants were analyzed for unbound antibody by UV-vis measurements (Figure 4). NanoDrop 1000 spectrophotometer measurements for protein confirmed the conjugation as a single peak detected in the conjugated sample (Figure 5). SDS-PAGE was also used to confirm antibody-NP conjugation (Figure 6).

[0192] Effector: Nmab-Fe assessed by cytokine production using target cells 3 O 4 Efficacy of @Au-αCD3scFv NPs On the day before the assay, A431 cells were cultured at 4 × 10 3Cells / well were seeded in triplicate in 96-well microtiter plates. Jurkat E6-1 cells were co-cultured with EGFR-positive A431 cells at an effector:target (E:T) ratio of 5:1 in the presence of unconjugated NPs, anti-CD3 scFv (5 μg / ml), Nmab-Fe3O4@Au NPs, or Bis-Fe3O4@Au NPs (bispecific NPs conjugated to Nmab and αCD3 scFv). After 24 hours, aliquots of culture supernatants were taken and the levels of IL-2 were measured using a commercially available ELISA test kit (Sydney, NSW, AUS). Samples were analyzed on a plate reader.

[0193] Figure 13 shows the specifically induced production of IL-2 (97.5±10 pg / ml) after 24 h of stimulation with Bis-Fe3O4@Au NPs (bispecific NPs conjugated to Nmab and αCD3 scFv). In contrast, NPs conjugated only to αCD3 scFv activated low levels of IL-2 production (5.5±0.7 pg / ml) from binding to T cells, and no IL-2 stimulation was observed after treatment with unconjugated NPs or Nmab-Fe3O4@Au NPs. These results indicate that Bis-Fe3O4@Au NPs are more potent activators of T cells in vitro than NPs conjugated to anti-CD3 scFv alone.

[0194] Target: Nmab-Fe assessed by cytotoxicity assay using effector cells 3 O 4 Efficacy of @Au-αCD3scFv NPs To evaluate the efficacy of bispecific nanoparticle conjugation, a cytotoxicity assay was performed. Logarithmic growth phase A431 cells containing Fe3O4@Au NP particles were incubated at 4 × 10 3Adherent cells were grown by seeding cells / well in triplicate in 96-well microtiter plates. The MTT colorimetric assay was used. This cell viability test is based on the reduction of the tetrazolium salt MTT (3-(4,5-dimethylthiazol-2-yl-2,5-diphenyltetrazolium bromide) by mitochondrial reductase in metabolically active cells. Cells were seeded on 96-well culture plates at a density of 4000 cells per well in DMEM and different concentrations of buffer were added. At 12 hours, the medium was removed and replaced with 100 μl culture medium containing 1% FBS.

[0195] The viability of the co-cultured cells was not significantly affected until treated with Fe3O4@Au NPs at Fe concentrations >2500 μg / ml (Fig. 7).

[0196] A series of MTT assays were then performed to evaluate the cytotoxic effect of Fe3O4@Au nanoparticles conjugated to nimotuzumab and / or anti-CD3 scFv on A431 and Jurkat clone E6-1 cells at different concentrations for five treatment groups: Group A, Nmab; Group B, Fe3O4@Au NPs; Group C, Nmab-Fe3O4@Au NPs; Group D, Nmab-Fe3O4@Au-αCD3 scFv NPs; Group E, Cisplatin; and Group F, treatment with fresh culture medium. Treatment and effector cells were removed after 72 hours of incubation. MTT solution (5 mg / ml in PBS) was then added to each well to assess cell viability. After 2 hours at 37°C, the solution was removed. 100 μl of DMSO was added to dissolve the cells. After 30 min of incubation at 37°C and thorough mixing, the viability was measured by a microplate reader at 570 nm. The spectrophotometer was calibrated to zero absorbance by using culture medium without cells. Cell viability was calculated and plotted against concentration, and data were evaluated using Prism 9 (GraphPad Software version 8, La Jolla, CA, USA).

[0197] The cytotoxicity of Nmab-Fe3O4@Au-αCD3scFv NPs (i.e., nanoparticles containing antigen-binding proteins for binding to EGFR and CD3) was significantly increased compared to Fe3O4@Au NPs.

[0198] Figure 8 shows that the viability of the co-cultured cells was significantly decreased after incubation with Nmab-Fe3O4@Au-αCD3scFv NPs compared to the singly conjugated Nmab-Fe3O4@Au nanoparticles.

[0199] FIG. 9 similarly shows that cell viability was significantly reduced after incubation with Nmab-Fe3O4@Au-αCD3scFv NPs compared to nanoparticles conjugated to Nmab (i.e., naked antibody at the same antibody concentration) or nimatozumab alone.

[0200] To further evaluate the cytotoxic effect of Fe3O4@Au nanoparticles conjugated to Nmab and / or anti-CD3 scFv in relation to additional target cancer cells, MTT assays were performed on MDA_MB468 breast cancer cells with Jurkat effector cells at different concentrations for three treatment groups: Group A, free Nmab (nimotuzumab); Group B, Nmab-Fe3O4@Au NPs: Group C, Nmab-Fe3O4@Au-αCD3scFv NPs. The control group was treated with fresh culture medium alone. One day before the assay, logarithmic growth phase MDA-MB468 cells containing Fe3O4@Au NP particles were inoculated with 4 × 10 3Adherent cells were grown by seeding at 1000 cells / well in triplicate in 96-well microtiter plates. Cells were then seeded on 96-well culture plates at a density of 4000 cells per well in RPMI medium. At 12 hours, medium was removed and replaced with 100 μl culture medium containing 1% FBS. Treatments were added and then removed after 72 hours of incubation, and effector cells were washed off with PBS. MTT solution (5 mg / ml in PBS) was then added to each well to assess cell viability. After 2-4 hours of incubation at 37°C, the supernatant was removed. 100 μl of DMSO was added to lyse the cells. After 30 minutes of incubation at 37°C and thorough mixing, viability was measured by a microplate reader at 570 nm. The spectrophotometer was calibrated to zero absorbance by using culture medium without cells. Cell viability was calculated and plotted versus concentration, and data were evaluated using Prism 9 (GraphPad Software version 8, La Jolla, CA, USA).

[0201] The cytotoxicity of co-cultured cells treated with Nmab-Fe3O4@Au-αCD3scFv NPs was significantly increased compared to Fe3O4@Au NPs.

[0202] FIG. 18 shows that the viability of the co-cultured cells was significantly decreased after incubation with Nimotuzumab-Fe3O4@Au-αCD3scFv NPs compared to the Nimotuzumab-Fe3O4@Au nanoparticles conjugated alone.

[0203] FIG. 19 similarly shows that cell viability was significantly reduced after incubation with nimotuzumab-Fe3O4@Au-αCD3scFv NPs compared to nanoparticles conjugated to Nmab (i.e., naked antibody at the same antibody concentration) or Nmab alone.

[0204] αCD3scFv-Fe 3 O 4 Fe-Ag@Au-Nmab NPs were generated using anti-EGFR monoclonal antibody and anti-CD3 scFv. 3 O 4 @Conjugation of Au NPs To evaluate an alternative configuration of bispecific nanoparticles, where the first binder is bound to gold and the second binder is bound to Fe3O4, anti-CD3 scFv was conjugated to Fe3O4NPs using the carbodiimide method and anti-EGFR antibody nimotuzumab was conjugated to Au NPs using a thiol linker, as briefly described herein. 1 mg of Fe3O4@Au NPs in sulfonic acid methyl ester buffer (pH 6) was mixed with 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC, 1.1 mmol) and sulfo-NHS (1 mmol) was added to the solution to activate the carboxyl groups on the PEG ligands for 30 min. After gentle mixing at room temperature for 30 min, the solution was subjected to a PD-10 column to remove excess EDC and sulfo-NHS. 5 μg of scFv CD3 was then added to the conjugate and gently shaken for 3 h to functionalize the Fe3O4 side of the nanoflowers. After 3 h incubation, 100 μl of Nmab antibody was added to the mixture and the mixture was placed on a shaker for overnight incubation at room temperature to functionalize the Au side. A thiol linker was added to Nmab prior to immobilization of Nimotuzumab. The antibody-conjugated NPs were separated from unbound antibody and Fe3O4@Au NPs using 300K ultrafiltration.

[0205] Target: αCD3scFv-Fe assessed by cytotoxicity assay using effector cells 3 O 4 @Efficacy of Au-Nmab NPs To evaluate the efficacy of bispecific nanoparticle conjugation, a cytotoxicity assay was performed. One day before the assay, logarithmic growth phase A431 cells were cultured at 4 × 10 3Adherent cells were grown by seeding at 1000 cells / well in triplicate in 96-well microtiter plates. The MTT colorimetric assay was used as described herein. Cells were seeded on 96-well culture plates at a density of 4000 cells per well in RPMI medium. At 12 h, the medium was removed and replaced with 100 μl culture medium containing 1% FBS. A series of MTT assays were then performed to evaluate the cytotoxic effect of αCD3scFv-Fe3O4@Au-Nmab nanoparticles at different concentrations of Nmab or iron compared to a control group treated with fresh culture medium. Treatments were removed after 72 h of incubation and effector cells were washed off with PBS. MTT solution (5 mg / ml in PBS) was then added to each well to assess cell viability. After 2-4 h at 37 °C, the MTT solution was removed. 100 μl of DMSO was added to dissolve the cells. After 30 min of incubation at 37°C and thorough mixing, the viability was measured by a microplate reader at 570 nm. The spectrophotometer was calibrated to zero absorbance by using culture medium without cells. Cell viability was calculated and plotted against concentration, and data were evaluated using Prism 9 (GraphPad Software version 8, La Jolla, CA, USA).

[0206] The cytotoxicity of co-cultured cells treated with αCD3scFv-Fe3O4@Au-Nmab NPs was significantly increased compared to no treatment (control).

[0207] FIG. 20 shows that the viability of the co-cultured cells was significantly decreased after incubation with αCD3scFv-Fe3O4@Au-Nmab NPs compared to the control.

[0208] Fe 3 O 4 Cellular uptake of @Au NPs We demonstrated the cellular uptake of functionalized Fe3O4@Au NPs into cancer cell lines (Figure 10). Intracellular iron and gold content was quantitatively measured by ICP-MS after 12 h of incubation with 50 μg / ml iron. Compared to untreated cells, the iron concentration of cells treated with Bis-Fe3O4@Au NPs was significantly increased to 373.92 ± 36.1 fg / cell after incubation (Figure 10A). Prussian blue staining (Figure 10B) was used to qualitatively determine the iron content inside co-cultured Jurkat and A431 cells treated with Bis-Fe3O4@Au NPs (bispecific NPs conjugated to Nmab and αCD3scFv) or Nmab-Fe3O4@Au NPs, or untreated.

[0209] Example 4: Fe3O4@Au NPs conjugated to anti-HER2 mAb (trastuzumab) and anti-CD3 scFv (mAb-Fe3O4@Au-αCD3 NPs) Fe 3 O 4 Preparation of @Au NPs Fe3O4@Au NPs were synthesized by pyrolysis method according to the procedure described above with minor modifications. Briefly, 6 mmol of oleic acid, 6 mmol of oleylamine, and 10 mmol of 1,2-hexadecanediol were mixed with 20 ml of 1-octadecene (ODE) in a 50 ml round-bottom flask equipped with a thermometer, flow control adapter, reflux condenser, and rubber septum, and stirred at 160 °C under gentle nitrogen flow for 30 min. Then, under a blanket of nitrogen, 0.3 ml of Fe(CO)5 was rapidly injected into the solution. After 3 min, a degassed gold precursor solution consisting of 40 mg of HAuCl4·3H2O, 1.5 ml of oleylamine, and 5 ml of ODE was added dropwise to the hot solution at 180 °C within 10 min to ensure complete mixing. The solution turned deep red immediately after injection, indicating the formation of gold nanoparticles. The solution was then slowly heated to 310 °C and refluxed for 45 min. After cooling at room temperature, 40 mL of isopropanol was added to the solution and centrifuged at 8000 rpm for 10 min to remove large particles. The precipitate was redispersed in hexane and centrifuged again at 7000 rpm to remove any undispersed material. Ethanol was then added to the solution and centrifuged again three times to obtain a brown dispersion. The Fe3O4@Au NPs were stored in hexane in the presence of oleylamine for further use.

[0210] Fe to improve solubility 3 O 4 @Modification of Au NPs To improve the solubility of iron-gold NPs, transfer of hydrophobic nanoparticles into aqueous media was performed using a modified protocol. Briefly, 30 mg of α,ω-bis{2-[(3-carboxy-1-oxopropyl)amino]ethyl}PEG (Mr=3,000), 2 mg of N-hydroxysuccinimide, 3 mg of dicyclohexylcarbodiimide, and 1.5 mg of dopamine hydrochloride were dissolved in a mixture of 1 ml of dimethylformamide, 2 ml of CHCl3, and 10 mg of anhydrous Na2CO3. The solution was stirred for 2 h at 37 °C, 5 mg of Fe3O4@Au NPs were added, and the resulting solution was stirred overnight at 37 °C under N2 flow. The modified NPs were precipitated by adding 5 mL of hexane and ethanol, collected by centrifugation at 16,500 × g, and surfactants and other salts were removed via dialysis (molecular weight cutoff, 10 kDa) in PBS for 24 h. Finally, they were filtered through a 0.22 μm sterile filter. The final iron concentration of the particles was determined by inductively coupled plasma mass spectrometry (ICP-MS).

[0211] Fe binding to monoclonal antibody trastuzumab (Tmab) and anti-CD3 scFv antibody 3 O 4 @Conjugation of Au NPs Trastuzumab (Tmab) was conjugated to Fe3O4NPs using the carbodiimide method. Meanwhile, anti-CD3 scFv antibody was conjugated to Au NPs using a thiol linker as briefly described herein. First, 1 mg of Fe3O4@Au NPs in sulfonic acid methyl ester buffer (pH 6) was mixed with 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC, 1.1 mmol), and sulfo-NHS (1 mmol) was added to the solution to activate the carboxyl groups on the PEG ligands for 30 min. After gentle stirring at room temperature for 30 min, the solution was subjected to a PD-10 desalting column to remove excess EDC and sulfo-NHS. Then, Tmab (200 μg) was added to the mixture and gently stirred for 3 h to functionalize the Fe3O4 side of the nanoflowers. After 3 h of incubation, 5 μl of anti-CD3 scFv antibody was added to the mixture, and the mixture was placed on a shaker for overnight incubation at room temperature to functionalize the Au side. A thiol linker was added to the anti-CD3 scFv before adding it to the NP mixture. The antibody-conjugated NPs were separated from unbound antibody and Fe3O4@Au NPs using 300K ultrafiltration centrifuged at 1000 rpm for 5 min. The collected supernatant was analyzed for unbound antibody by absorbance measurement using a Shimadzu 2600 UV-Vis spectrophotometer (Shimadzu, Japan). The binding efficiency was calculated as the difference between the initial addition and the amount of unbound antibody determined in the supernatant.

[0212] Target: Tmab-conjugated Fe assessed by cytotoxicity assay using effector cells 3 O 4 @Effectiveness of Au NPs SkBr3 cells were seeded in 96-well plates (5000 cells / well). The plates were incubated with 5% CO2 and cultured at 37°C overnight to promote adhesion. Jurkat cells were added at a ratio of 5:1 effector (Jurkat): target (SkBr3) cells. Antibody-conjugated nanoparticles were added at various concentrations. Target SkBr3 cells and RPMI-1640 complete medium group served as control. After 72 hours of culture, effector cells were removed with washing and MTT was added for further incubation for 2 hours. The supernatant was discarded and 100 μl of dimethyl sulfoxide was added. After thorough mixing, the absorbance value of each well was measured at 570 nm using a Perkin Elmer VictorX plate reader. The viability percentage was then calculated as [SkBr3 (treated cells)-background] / [SkBr3 (untreated cells)-background]×100. (where SkBr3 is the absorbance at 570 mm).

[0213] To evaluate the efficacy of bispecific NPs conjugation, cytotoxicity assays were performed with different concentrations of Bis-Tmab-Fe3O4@Au-αCD3scFv (bispecific NPs conjugated to Tmab and αCD3scFv) (Figure 11A-C) and Tmab-Fe3O4@Au (Figure 11D) NPs (NPs conjugated to Tmab alone) in co-cultures of SkBr3 and Jurkat cells at a ratio of 1:5 (target:effector). Both Bis-Tmab-Fe3O4@Au-CD3 and Tmab-Fe3O4@Au induced cytotoxicity of target cells (SkBr3 cells) by effector cells (Jurkat cells). Bis-Tmab-Fe3O4@Au-CD3 NPs showed significantly higher cytotoxicity than Tmab-Fe3O4@Au NPs.

[0214] To further evaluate the cytotoxic effect of Fe3O4@Au nanoparticles conjugated to Tmab and / or anti-CD3 scFv in relation to additional target cancer cells, MTT assay was performed on MDA-MB231 breast cancer cells with Jurkat effector cells at different concentrations for three treatment groups: Group A, free Tmab; Group B, Tmab-Fe3O4@Au NPs: Group C, Tmab-Fe3O4@Au-αCD3scFv NPs. The control group was treated with fresh culture medium alone. One day before the assay, logarithmic growth phase MDA-MB231 cells containing Fe3O4@Au NP particles were inoculated with 4 × 10 3 Adherent cells were grown by seeding at 1000 cells / well in triplicate in 96-well microtiter plates. Cells were then seeded on 96-well culture plates at a density of 4000 cells per well in RPMI medium. At 12 hours, medium was removed and replaced with 100 μl culture medium containing 1% FBS. Treatments were added and then removed after 72 hours of incubation, and effector cells were washed off with PBS. MTT solution (5 mg / ml in PBS) was then added to each well to assess cell viability. After 2-4 hours at 37°C, the solution was removed. 100 μl DMSO was added to lyse the cells. After 30 minutes of incubation at 37°C and thorough mixing, viability was measured by a microplate reader at 570 nm. The spectrophotometer was calibrated to zero absorbance by using culture medium without cells. Cell viability was calculated and plotted versus concentration, and data were evaluated using Prism 9 (GraphPad Software version 8, La Jolla, CA, USA).

[0215] The cytotoxicity of co-cultured cells treated with Tmab-Fe3O4@Au-αCD3scFv NPs was significantly increased compared to Fe3O4@Au NPs.

[0216] FIG. 21 shows that the viability of the co-cultured cells was significantly decreased after incubation with bispecific Tmab-Fe3O4@Au-αCD3scFv NPs compared to the singly conjugated Tmab-Fe3O4@Au nanoparticles.

[0217] FIG. 22 similarly shows that cell viability was significantly reduced after incubation with Tmab-Fe3O4@Au-αCD3scFv NPs compared to nanoparticles conjugated to free Tmab (i.e., naked antibody at the same antibody concentration) or Tmab alone.

[0218] Example 5: Fe3O4@Au NPs conjugated to anti-EGFR mAb (panitumumab, Pmab) and anti-CD3 scFv (mAb-Fe3O4@Au-αCD3 scFv NPs) Fe to monoclonal antibody Pmab and anti-CD3 scFv antibody 3 O 4 @Conjugation of Au NPs To evaluate the efficacy of bispecific nanoparticles using alternative anti-EGFR binders, anti-EGFR monoclonal antibody panitumumab was conjugated to Fe3O4NPs using the carbodiimide method and anti-CD3 scFv antibody was conjugated to Au NPs using a thiol linker as briefly described herein. 1 mg of Fe3O4@Au NPs in sulfonic acid methyl ester buffer (pH 6) was mixed with 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC, 1.1 mmol) and sulfo-NHS (1 mmol) was added to the solution to activate the carboxyl groups on the PEG ligands for 30 min. After gentle mixing at room temperature for 30 min, the solution was subjected to a PD-10 column to remove excess EDC and sulfo-NHS. 100 μg of panitumumab was then added to the conjugate with gentle shaking to functionalize the Fe3O4 side of the nanoflowers and shaken for 3 h. After 3 h incubation, 5 μl of anti-CD3 scFv antibody was added to the mixture, and the mixture was placed on a shaker for overnight incubation at room temperature to functionalize the Au side. Prior to immobilization of anti-CD3 scFv, a thiol linker was added to the anti-CD3 scFv. The antibody-conjugated NPs were separated from unbound antibody and Fe3O4@Au NPs using 300K ultrafiltration.

[0219] Target: Pmab-conjugated Fe assessed by cytotoxicity assay using effector cells 3 O 4 @Effectiveness of Au NPs To evaluate the efficacy of bispecific nanoparticle conjugation, a cytotoxicity assay was performed. One day before the assay, logarithmic growth phase A431 cells containing Fe3O4@Au NP particles were inoculated at 4 × 10 3Adherent cells were grown by seeding cells / well in triplicate in 96-well microtiter plates. The MTT colorimetric assay was used. This cell viability test is based on the reduction of the tetrazolium salt MTT (3-(4,5-dimethylthiazol-2-yl-2,5-diphenyltetrazolium bromide) by mitochondrial reductase in metabolically active cells. Cells were seeded on 96-well culture plates at a density of 4000 cells per well in DMEM medium. At 12 hours, the medium was removed and replaced with 100 μl culture medium containing 1% FBS.

[0220] MTT assay was then performed to evaluate the cytotoxic effect of Pmab-Fe3O4@Au-αCD3scFv nanoparticles on A431 and Jurkat cells at different concentrations of Pmab or iron. The control group was treatment with cell medium alone. The treatment was removed after 72 h of incubation, and the effector cells were washed off with PBS. Then, MTT solution (5 mg / ml in PBS) was added to each well to evaluate cell viability. After 2-4 h at 37 °C, the solution was removed. 100 μl of DMSO was added to lyse the cells. After 30 min of incubation at 37 °C and thorough mixing, the viability was measured by a microplate reader at 570 nm. The spectrophotometer was calibrated to zero absorbance by using culture medium without cells. Cell viability was calculated and plotted against concentration, and the data was evaluated using Prism 9 (GraphPad Software version 8, La Jolla, CA, USA).

[0221] The cytotoxicity of co-cultured cells treated with Pmab-Fe3O4@Au-αCD3scFv NPs was significantly increased compared to Fe3O4@Au NPs.

[0222] FIG. 23 shows that the viability of the co-cultured cells was significantly decreased after incubation with bispecific Pmab-Fe3O4@Au-αCD3scFv NPs compared to the singly conjugated Pmab-Fe3O4@Au nanoparticles.

[0223] Example 6: Bispecific Fe3O4@Au NPs conjugated to cell penetrating peptides (CPPs) Bispecific Fe3O4@Au NPs conjugated to cell-penetrating peptides (mAb-Fe3O4@Au NPs-αCD3 / CPP NPs) were synthesized by conjugating Fe3O4@Au NPs to anti-EGFR monoclonal antibody (Nmab), anti-CD3 scFv, and CPP.

[0224] HIV-TAT cell penetrating peptide PEGylated (CPP-PEG-SH) HIV-TAT (residues 47-57) cell penetrating peptide was dissolved to a concentration of 1 mg / mL (641 M) in NaHCO3 0.1 M, pH 8.0. A 10 mg / mL (2 mM) solution of NHS-PEG-SH (5 kDa) in NaHCO3 0.1 M pH 8.0 was added to the HIV-TAT peptide at a 4:1 NHS linker / CPP molar ratio and incubated overnight at 4 °C. Unreacted CPP was removed by centrifugation through a 3 kDa cutoff filter and the PEGylated CPP was buffer exchanged into 0.01 M phosphate buffered saline pH 7.4 containing 1 mM EDTA.

[0225] Nanoparticle surface functionalization Anti-EGFR antibody nimotuzumab was conjugated to Fe3O4NPs using the carbodiimide method, and anti-CD3 scFv antibody and HIV-TAT CPP were conjugated to Au NPs using thiol linkers as briefly described herein. 1 mg of Fe3O4@Au NPs in sulfonic acid methyl ester buffer (pH 6) was mixed with 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC, 1.1 mmol), and sulfo-NHS (1 mmol) was added to the solution to activate the carboxyl groups on the PEG ligands for 30 min. After gentle mixing at room temperature for 30 min, the solution was subjected to a PD-10 column to remove excess EDC and sulfo-NHS. Then, 100 μg of nimotuzumab was added to the conjugate and incubated for 3 h with gentle shaking to functionalize the Fe3O4 side of the nanoflowers. After 3 hours of incubation, 5 μl of anti-CD3 scFv antibody was added to the mixture and the mixture was placed on a shaker for overnight incubation at room temperature to functionalize the Au side. A thiol linker was added to the anti-CD3 scFv before immobilization of the anti-CD3 scFv. PEGylated CPP was added after the buffer was exchanged into PBS 0.01M pH 7.4 containing 1 mM EDTA. The antibody-conjugated NPs were separated from unbound antibody and unconjugated Fe3O4@Au NPs using 300K ultrafiltration.

[0226] Target: Nmab-Fe assessed by cytotoxicity assay using effector cells 3 O 4 Effectiveness of @Au-αCD3 scFv / CPP NPs To evaluate the efficacy of CPP bispecific nanoparticle conjugation, a cytotoxicity assay was performed. Logarithmic growth phase A431 cells containing Fe3O4@Au NP particles were incubated at 4 × 10 3Adherent cells were grown by seeding cells / well in triplicate in 96-well microtiter plates. The MTT colorimetric assay was used. This cell viability test is based on the reduction of the tetrazolium salt MTT (3-(4,5-dimethylthiazol-2-yl-2,5-diphenyltetrazolium bromide) by mitochondrial reductase in metabolically active cells. Cells were seeded on 96-well culture plates at a density of 4000 cells per well in DMEM and different concentrations of buffer were added. At 12 h, the medium was removed and replaced with 100 μl culture medium containing 1% FBS. A431 cells were designated as target cells and Jurkat E6-1 were considered as effector cells. Jurkat E6-1 cells were incubated with Nmab-Fe3O4@Au-αCD3 scFv / CPP A431 cells were added with or without NPs at an effector:target (E:T) ratio of 5:1. Untreated cells were selected as control. After 72 h of culture, effector cells were washed with PBS and MTT was added for further incubation for 2-4 h. The supernatant was discarded and 100 μl of dimethyl sulfoxide was added. After thorough mixing, the absorbance value of each well was measured at 570 nm using a Perkin Elmer VictorX plate reader (Melbourne, VIC, Australia). The percent viability was then calculated as [A431 (treated cells)-background] / [A431 (untreated cells)-background]×100, where A431 is the absorbance at 570 mm.

[0227] Figure 25 shows that the viability of co-cultured A431 and Jurkat cells was significantly decreased after incubation with Nmab-Fe3O4@Au-αCD3scFv / CPP NPs at iron concentrations of ≧70 μg / ml (Figure 25A) and with Nmab-Fe3O4@Au-αCD3scFv / CPP NPs at Nmab concentrations of ≧10 μg / ml (Figure 25B).

[0228] The cytotoxicity of co-cultured cells treated with Nmab-Fe3O4@Au-αCD3 scFv / CPP NPs was significantly increased compared to Nmab-Fe3O4@Au-αCD3 scFv NPs without CPP.

[0229] FIG. 26 shows that the viability of co-cultured cells was significantly decreased after incubation with bispecific Nmab-Fe3O4@Au-αCD3 scFv / CPP NPs compared to Nmab-Fe3O4@Au-αCD3 scFv NPs without CPP.

[0230] Example 7: Preparation of bispecific silver-iron nanoparticles (Fe3O4@Ag NPs) Fe3O4@Ag NPs were synthesized by pyrolysis method following a previously reported procedure with minor modifications. Briefly, 6 mmol of oleic acid, 6 mmol of oleylamine, and 10 mmol of 1,2-hexadecanediol were mixed with 20 ml of 1-octadecene (ODE) in a 50 ml round-bottom flask equipped with a thermometer, flow control adapter, reflux condenser, and rubber septum, and stirred at 120 °C under gentle nitrogen flow for 30 min. Then, under a blanket of nitrogen, 0.3 ml of Fe(CO)5 solution was rapidly injected into the solution. After 5 min, a degassed silver precursor solution consisting of 35 mg of AgNO3, 1 ml of oleylamine, and 5 ml of toluene solution was added dropwise to the hot solution at 180 °C within 10 min to ensure complete mixing. The solution turned dark brown immediately after injection, indicating the formation of silver nanoparticles. The solution was then slowly heated to 205 °C and refluxed for 90 min. After cooling at room temperature, 40 mL of isopropanol was added to the solution and centrifuged at 8000 rpm for 10 min to remove large particles. The precipitate was redispersed in hexane and centrifuged again at 7000 rpm to remove any undispersed material. Ethanol was then added to the solution and centrifuged again three times to obtain a brown dispersion. The Fe3O4@Ag NPs were stored in hexane in the presence of 0.5 μl of oleylamine for further use, including conjugation to antigen-binding proteins, as described elsewhere herein.

[0231] The synthesis of Fe3O4@Ag NPs was characterized using transmission electron microscopy (TEM) (Figure 24A). Ag NPs with an average core size of 9 nm were observed. The Fe3O4NPs appeared as dumbbell shapes. Figure 24B shows the UV-Vis spectrum of Fe3O4@Ag NPs with a peak at 409 nm. The hydrodynamic size of the NPs was 8.23 ​​± 0.03 nm (Table 3).

[0232] [Table 3]

[0233] Example 8: Dumbbell-like Fe3O4@Au NPs conjugated to anti-EGFR mAb (Nmab) and anti-CD3 scFv (mAb-Fe3O4@Au-αCD3 NPs) TEM was used to confirm the morphology and synthesis of dumbbell-like Fe3O4@Au NPs. Methods for making dumbbell-shaped nanoparticles are known to those skilled in the art and involve slight modifications to the methods used to produce flower-shaped nanoparticles. Briefly, reducing the molar ratio of iron pentacarbonyl in the preparation of nanoparticles promotes the production of dumbbell-shaped compared to flower-shaped nanoparticles.

[0234] As shown in Figure 14, Au NPs are observed as spheres with an average core size of about 5 nm (shown in black due to the heavy atom effect), while Fe3O4 NPs show a diameter of about 10 nm. Cytotoxicity assays confirmed that the viability of co-cultured cells was significantly decreased after incubation with dumbbell-like Bis-Nmab-Fe3O4@Au-αCD3scFv NPs (bispecific NPs conjugated with Nmab and anti-CD3 scFv) compared to spherical Nmab-Fe3O4@Au NPs with ≧300 μg / ml iron or bare unconjugated NPs (Figure 15).

[0235] Example 9: Spherical Au NPs conjugated to anti-EGFR mAb (Nmab) and anti-CD3 scFv (Nmab-Fe3O4@Au-CD3 NPs) Preparation of spherical Au NPs Briefly, 150 μL of 22 mM trisodium citrate, 50 μL of 2.5 mM tannic acid, and 50 μL of 150 mM potassium carbonate were added to a conical flask containing 30 mL of Milli-Q water and stirred vigorously at 60° C. for 5 min. Then, 1.25 mL of 12.5 mM HAuCL4 solution was slowly added and stirred at 60° C. for 2 min. As soon as the color changed from light yellow to dark, the temperature was reduced to 40° C. and allowed to stir gently for 15 min. The Au NPs were cooled at room temperature and then stored at 40° C.

[0236] Conjugation of @Au NPs to monoclonal antibodies anti-EGFR mAb (Nmab) and anti-CD3 scFv antibody For PEGylation of Au NPs, 1 ml (100 μg) of Au NPs and 20 μL of thiol-PEG-NHS linker stock solution (10 mg / ml) were mixed in a small tube and incubated overnight in a refrigerator under gentle rotation. Anti-CD3 scFv (5 μL) and Nmab (200 μg) antibodies were then added to the NP mixture at 10 μL and 20 μL, respectively. The antibody-conjugated NPs were separated from unbound antibody and Au NPs using ultrafiltration tubes.

[0237] TEM was used to confirm the morphology and synthesis of spherical Au NPs. As shown in Figure 16A, the Au NPs were observed as spheres with an average size of about 17 nm and were black in color due to the heavy atom effect.

[0238] Cytotoxicity assays were performed by co-culture of A431 and Jurkat cells in a ratio of 1:5 (target:effector) treated with different concentrations of (A) spherical Nmab-Fe3O4@Au NPs, or (B) spherical Bis-Fe3O4@Au NPs (bispecific NPs conjugated with Nmab and anti-CD3 scFv). Cell viability (measured as absorbance) was not significantly affected after incubation with Bis-Fe3O4@Au NPs (Figure 17B) compared to Nmab@Au NPs (Figure 17A). These results indicate that antigen-binding protein-conjugated spherical gold nanoparticles did not significantly induce cell killing when compared to conjugated flower-shaped nanoparticles. The results demonstrate that flower-shaped iron-gold NPs are likely to provide superior therapeutic outcomes compared to nanoparticles of alternative configurations.

[0239] Example 10: Fe3O4@Au NPs conjugated to anti-EGFR mAb (Nmab) and anti-CD16 scFv (mAb-Fe3O4@Au-αCD16 scFv NPs) Fe to nimotuzumab and anti-CD16 scFv 3 O 4 @Au NPs conjugation was performed using Nmab-Fe 3 O 4 @This is carried out to generate Au-αCD16 scFv NPs. The anti-EGFR antibody nimotuzumab is conjugated to Fe3O4NPs using the carbodiimide method previously described, and the anti-CD16 scFv antibody is conjugated to Au NPs using a thiol linker as briefly described herein. 1 mg of Fe3O4@Au NPs in sulfonic acid methyl ester buffer (pH 6) is mixed with 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC, 1.1 mmol), and sulfo-NHS (1 mmol) is added to the solution to activate the carboxyl groups on the PEG ligands for 30 min. After gentle mixing for 30 min at room temperature, the solution is subjected to a PD-10 column to remove excess EDC and sulfo-NHS. 100 μg of nimotuzumab is then added to the conjugate for 3 h with gentle shaking to functionalize the Fe3O4 side of the nanoflowers. After 3 hours of incubation, 5 μl of anti-CD16 scFv antibody is added to the mixture, and the mixture is placed on a shaker for overnight incubation at room temperature to functionalize the Au side. A thiol linker is added to the anti-CD16 scFv prior to immobilization of the anti-CD16 scFv. The antibody-conjugated NPs are separated from unbound antibody and unconjugated Fe3O4@Au NPs using 300K ultrafiltration.

[0240] Mass spectrometry analysis of Nmab-Fe3O4@Au-αC16scFv NPs is used to reveal a specific peak of nimotuzumab at m / z approx. 148KDa, which is not detectable in Au-Fe3O4NPs. In addition, the collected supernatant is analyzed for unbound antibody by UV-vis measurements as described herein. NanoDrop 1000 spectrophotometer measurements for protein confirm the conjugation as a single peak detected in the conjugated sample. SDS-PAGE can also be used to confirm antibody-NP conjugation.

[0241] Nmab-Fe 3 O 4 @The efficacy of Au-αCD3scFv NPs is evaluated by cytokine production using effector:target cells Logarithmically growing A431 cells were cultured at 4 × 10 3Adherent cells are grown by seeding cells / well in triplicate in 96-well microtiter plates. Jurkat E6-1 cells are co-cultured with EGFR-positive A431 cells at an effector:target (E:T) ratio of 5:1 in the presence of unconjugated NPs, anti-CD16 scFv (5 μg / ml), Nmab-Fe3O4@Au NPs, or Bis-Fe3O4@Au NPs (bispecific NPs conjugated to Nmab and αCD16 scFv). After 24 hours, aliquots of culture supernatants are taken and the levels of IL-2 are measured using a commercially available ELISA test kit (Sydney, NSW, AUS). Samples are analyzed on a plate reader.

[0242] The results are expected to show specifically induced production of IL-2 after 24 hours of stimulation with Bis-Fe3O4@Au NPs (bispecific NPs conjugated to Nmab and αCD16 scFv). In contrast, NPs conjugated only to αCD16 scFv are expected to activate no to low levels of IL-2 production from binding to T cells, and no IL-2 stimulation is expected to be observed after treatment with unconjugated NPs or Nmab-Fe3O4@Au NPs. The results will show that Bis-Fe3O4@Au NPs are more potent activators of T cells in vitro than NPs conjugated to anti-CD16 scFv alone.

[0243] Nmab-Fe 3 O 4 @The efficacy of Au-αCD16 scFv NPs is evaluated by a cytotoxicity assay using target:effector cells To evaluate the efficacy of bispecific nanoparticle conjugation, a cytotoxicity assay is performed. Logarithmic growth phase A431 cells containing Fe3O4@Au NP particles are cultured at 4 × 10 3Adherent cells are grown by seeding cells / well in triplicate in 96-well microtiter plates. The MTT colorimetric assay is used. This cell viability test is based on the reduction of the tetrazolium salt MTT (3-(4,5-dimethylthiazol-2-yl-2,5-diphenyltetrazolium bromide) by mitochondrial reductase in metabolically active cells. Cells are seeded on 96-well culture plates at a density of 4000 cells per well in DMEM and different concentrations of buffer are added. At 12 hours, the medium is removed and replaced with 100 μl culture medium containing 1% FBS.

[0244] The viability of the co-cultured cells would not be significantly affected until treated with Fe3O4@Au NPs with Fe concentrations >2500 μg / ml.

[0245] A series of MTT assays are then performed to evaluate the cytotoxic effect of Fe3O4@Au nanoparticles conjugated to nimotuzumab and / or anti-C16 scFv on A431 and Jurkat cells at different concentrations for five treatment groups: Group A, Nmab; Group B, Fe3O4@Au NPs; Group C, Nmab-Fe3O4@Au NPs; Group D, Nmab-Fe3O4@Au-αCD16 scFv NPs; Group E, cisplatin; and Group F, treatment with fresh culture medium. The treatments are removed after 72 hours of incubation and the effector cells are washed off with PBS. MTT solution (5 mg / ml in PBS) is then added to each well to evaluate cell viability. After 2-4 hours at 37°C, the solution is removed. 100 μl of DMSO is added to lyse the cells. After 30 min incubation at 37° C. and thorough mixing, the viability is measured by a microplate reader at 570 nm. The spectrophotometer is calibrated to zero absorbance by using culture medium without cells. Cell viability is calculated and plotted against concentration, and the data is evaluated using Prism 9 (GraphPad Software version 8, La Jolla, Calif., USA).

[0246] The cytotoxicity of Nmab-Fe3O4@Au-αCD16scFv NPs (i.e., nanoparticles containing antigen-binding proteins for binding to EGFR and CD16) would be significantly increased compared to Fe3O4@Au NPs.

[0247] The results would show that the viability of the co-cultured cells was significantly decreased after incubation with Nmab-Fe3O4@Au-αCD16scFv NPs compared to the singly conjugated Nmab-Fe3O4@Au nanoparticles.

[0248] The results are similarly expected to show that cell viability was significantly reduced after incubation with Nmab-Fe3O4@Au-αCD16scFv NPs compared to nanoparticles conjugated to Nmab (i.e., naked antibody at the same antibody concentration) or nimatozumab alone.

[0249] To further evaluate the cytotoxic effect of Fe3O4@Au nanoparticles conjugated to Nmab and / or anti-CD16 scFv in relation to additional target cancer cells, MTT assays are performed on MDA_MB468 breast cancer cells with Jurkat effector cells at different concentrations for three treatment groups: Group A, free Nmab (nimotuzumab); Group B, Nmab-Fe3O4@Au NPs: Group C, Nmab-Fe3O4@Au-αCD16scFv NPs. The control group is treatment with fresh culture medium alone. One day before the assay, logarithmic growth phase MDA-MB468 cells containing Fe3O4@Au NP particles were inoculated with 4 × 10 3Adherent cells are grown by seeding at 100 cells / well in triplicate in 96-well microtiter plates. Cells are seeded on 96-well culture plates at a density of 4000 cells per well in RPMI medium. At 12 hours, medium is removed and replaced with 100 μl culture medium containing 1% FBS. Treatments are added and then removed after 72 hours of incubation, and effector cells are washed off with PBS. MTT solution (5 mg / ml in PBS) is then added to each well to assess cell viability. After 2-4 hours at 37°C, the solution is removed. 100 μl DMSO is added to lyse the cells. After 30 minutes of incubation at 37°C and thorough mixing, viability is measured by a microplate reader at 570 nm. The spectrophotometer is calibrated to zero absorbance by using culture medium without cells. Cell viability is calculated and plotted versus concentration, and data are evaluated using Prism 9 (GraphPad Software version 8, La Jolla, Calif., USA).

[0250] The cytotoxicity of co-cultured cells treated with Nmab-Fe3O4@Au-αCD16 scFv NPs would be significantly increased compared to Fe3O4@Au NPs.

[0251] The results would show that the viability of the co-cultured cells was significantly decreased after incubation with Nimotuzumab-Fe3O4@Au-αCD16scFv NPs compared to the singly conjugated Nmab-Fe3O4@Au nanoparticles.

[0252] The results would also show that cell viability was significantly reduced after incubation with nimotuzumab-Fe3O4@Au-αCD16scFv NPs compared to nanoparticles conjugated to Nmab (i.e., naked antibody at the same antibody concentration) or Nmab alone.

Claims

1. 1. A nanoparticle bioconjugate comprising: a first nanoparticle conjugated with a first binding protein for specifically binding to a target moiety on a cancer cell; a second nanoparticle conjugated with a second binding protein for specifically binding to a target moiety on an immune cell; the nanoparticles are arranged in a dumbbell or flower-like configuration; A nanoparticle bioconjugate, wherein the bioconjugate is capable of binding to a cancer cell via the first binding protein and to an immune cell via the second binding protein.

2. 2. The nanoparticle bioconjugate of claim 1, wherein the first nanoparticle is a metallic nanoparticle and the second nanoparticle is a magnetic nanoparticle.

3. 2. The nanoparticle bioconjugate of claim 1, wherein the first nanoparticle is a magnetic nanoparticle and the second nanoparticle is a metallic nanoparticle.

4. 3. The nanoparticle bioconjugate of claim 1 or 2, wherein the metal nanoparticles are magnetic or non-magnetic.

5. 5. The nanoparticle bioconjugate of any one of claims 1 to 4, wherein the architecture of the nanoparticle bioconjugate is in the form of a central metal nanoparticle surrounded by 3-4 or more magnetic nanoparticles in a flower-like configuration.

6. 5. The nanoparticle bioconjugate of any one of claims 1 to 4, wherein the nanoparticle bioconjugate is in the form of a central magnetic nanoparticle surrounded by 3-4 or more metal nanoparticles in a flower-like configuration.

7. 5. The nanoparticle bioconjugate of any one of claims 1 to 4, wherein the nanoparticle bioconjugate comprises the first and second nanoparticles in a "dumbbell-like" architecture, whereby preferably the nanoparticle conjugate comprises a metal nanoparticle adjacent to or in close contact with a magnetic nanoparticle.

8. The nanoparticle bioconjugate of any one of claims 1 to 7, wherein the nanoparticle bioconjugate is not in the form of a spherical nanoparticle.

9. The magnetic nanoparticles are Fe ３ O ４ (Iron oxide) nanoparticles, Fe(CO) ５ (iron pentacarbonyl) nanoparticles, or FeCl ２ 9. The nanoparticle bioconjugate of any one of claims 1 to 8, selected from (iron chloride) nanoparticles.

10. 10. The nanoparticle bioconjugate of any one of claims 1 to 9, wherein the metal nanoparticles are selected from gold (Au), silver (Ag), platinum (Pt) or palladium (Pd), copper (Cu), nickel (Ni), cobalt (Co), or alloys or nanoparticles of two or more thereof.

11. The nanoparticle bioconjugate of any one of claims 1 to 10, wherein the metal nanoparticles are gold or silver nanoparticles, preferably gold nanoparticles.

12. 12. The nanoparticle bioconjugate of any one of claims 1 to 11, wherein the nanoparticle bioconjugate is composed of iron and gold nanoparticles, preferably the bioconjugate comprises a central gold nanoparticle surrounded by 3-4 or more iron nanoparticles to form a flower-shaped nanoparticle.

13. The nanoparticle bioconjugate of any one of claims 1 to 12, wherein the nanoparticle bioconjugate is from about 5 nm to about 100 nm.

14. The nanoparticle bioconjugate of any one of claims 1 to 13, wherein the nanoparticle bioconjugate is about 35 nm to 80 nm, preferably about 40 nm.

15. The nanoparticle bioconjugate of any one of claims 1 to 14, wherein the first and second binding proteins are covalently attached to the first and second nanoparticles.

16. 16. The nanoparticle bioconjugate of any one of claims 1 to 15, wherein one or both of the first and second binding proteins are directly conjugated to the nanoparticle or are conjugated via a linker moiety.

17. 17. The nanoparticle bioconjugate of any one of claims 1 to 16, wherein the binding protein is conjugated to the nanoparticle via a linker moiety selected from a polyethylene glycol (PEG) moiety, an alkyne, an amine, an azide, a biotin, a carboxyl, a hydroxyl, a methyl, a maleimide, a neutravidin, NHS, a hydrazone, or a thiol linker group.

18. The nanoparticle bioconjugate of any one of claims 1 to 17, wherein the first binding protein is any binding protein capable of specifically binding to a target moiety on a cancer cell.

19. 19. The nanoparticle bioconjugate of any one of claims 1 to 18, wherein the binding protein for binding to the target moiety on a cancer cell comprises, consists essentially of, or consists of an antigen-binding domain.

20. 19. The nanoparticle bioconjugate of any one of claims 1 to 18, wherein the binding protein for binding to the target moiety on a cancer cell comprises, consists essentially of, or consists of a receptor or ligand, or part thereof, for binding to the cancer cell.

21. The nanoparticle bioconjugate of any one of claims 1 to 20, wherein the targeting moiety on the cancer cell is selected from a sugar, a lipid, a nucleic acid, a peptide, or a protein.

22. 22. The nanoparticle bioconjugate of any one of claims 1 to 21, wherein the cancer cell is selected from breast tumor, colorectal tumor, adenocarcinoma, mesothelioma, bladder tumor, prostate tumor, germ cell tumor, liver cancer / bile duct, carcinoma, neuroendocrine tumor, pituitary tumor, small round cell tumor, squamous cell carcinoma, melanoma, atypical fibroxanthoma, seminoma, nonseminoma, stromal Leydig cell tumor, Sertoli cell tumor, skin tumor, kidney tumor, testicular tumor, brain tumor, ovarian tumor, gastric tumor, oral cavity tumor, bladder tumor, bone tumor, cervical tumor, esophageal tumor, laryngeal tumor, liver tumor, lung tumor, vaginal tumor, and Wilm's tumor.

23. 23. The nanoparticle bioconjugate of any one of claims 1 to 22, wherein the target moiety on the cancer cell is a cancer cell antigen selected from HER2, EGFR, mesothelin, GPC3, MUC1, GD2, CEA, EpCAM, LeY, PCSA, CD19, CD20, Clec9a, CD276, PD-L1, and PD-L2.

24. 24. The nanoparticle bioconjugate of any one of claims 1 to 23, wherein the antigen is HER2 and the cancer cells are any cancer cells that overexpress HER2 or are HER2 positive, such as breast cancer or gastric cancer.

25. 24. The nanoparticle bioconjugate of any one of claims 1 to 23, wherein the antigen is EGFR and the cancer cells are any cancer cells that overexpress the epidermal growth factor receptor (EGFR), such as squamous cell carcinoma of the head and neck (SCCHN), glioma, breast cancer, nasopharyngeal carcinoma, or pancreatic cancer cells.

26. 26. The nanoparticle bioconjugate of any one of claims 1 to 25, wherein the second binding protein is any binding protein capable of specifically binding to a target moiety on an immune cell.

27. 27. The nanoparticle bioconjugate of any one of claims 1 to 26, wherein the immune cell is selected from a T cell, an antigen presenting cell (APC), or a natural killer (NK) cell, preferably the immune cell is a T cell.

28. 28. The nanoparticle bioconjugate of any one of claims 1 to 27, wherein the target moiety on the immune cell is selected from a sugar, a lipid, a nucleic acid, a peptide, or a protein.

29. 29. The nanoparticle bioconjugate of any one of claims 1 to 28, wherein the binding protein for binding to the target moiety comprises, consists essentially of, or consists of an antigen-binding domain.

30. 29. The nanoparticle bioconjugate of any one of claims 1 to 28, wherein the binding protein for binding to the target moiety comprises, consists essentially of, or consists of a receptor or ligand, or part thereof, for binding to the immune cell.

31. 31. The nanoparticle bioconjugate of any one of claims 1-30, wherein the immune cell is a T cell and the target moiety on the T cell bound by the second binding protein is selected from CD3, CD2, CD4, CD7, CD8, PD1, CTLA4, KIR, CD16, CD94, CD161, NTBA, CD19, CD20, CD22, CD30, CD33, CD38, CD40L, CD44, CD56, CD79b, CD80, CD86, CD135, CD137, CD138, CD154, EphA2, EGFR, and any combination thereof.

32. 31. The nanoparticle bioconjugate of any one of claims 1 to 30, wherein the immune cell is a NK cell and the binding protein is for binding to an antigen selected from KIR, CD16, CD94, CD161, or CD56.

33. The nanoparticle bioconjugate of any one of claims 1 to 32, wherein one or both of the binding proteins is an antibody, or an antigen-binding fragment thereof.

34. 34. The nanoparticle bioconjugate of any one of claims 1 to 33, wherein one or both of the binding proteins is an antibody selected from IgA, IgD, IgE, IgG, IgM, preferably the antibody is IgG.

35. 34. The nanoparticle bioconjugate of any one of claims 1 to 33, wherein one or both of the binding proteins is an antibody fragment selected from a recombinant antibody fragment, a diabody, a triabody, a chimeric antibody, a F(ab')2 fragment, a Fab' fragment, a Fab'-SH fragment, a Fab fragment, a sFv fragment, a dsFv fragment, a bispecific sFv fragment, a bispecific dsFv fragment, a single chain Fv protein (scFv), a disulfide stabilized Fv protein, or a combination thereof.

36. The nanoparticle bioconjugate of any one of claims 1 to 35, wherein the first and second binding proteins are the same antibody format.

37. The nanoparticle bioconjugate of any one of claims 1 to 36, wherein the bioconjugate further comprises a cell penetrating peptide.

38. 38. A pharmaceutical composition comprising any of the nanoparticle bioconjugates of any one of claims 1 to 37, optionally in combination with a pharma- ceutically acceptable excipient or carrier.

39. 39. A method of treating or preventing cancer in a subject, comprising administering to a subject in need thereof a nanoparticle bioconjugate of any one of claims 1 to 37, or a pharmaceutical composition of claim 38.

40. 40. Use of the nanoparticle bioconjugate of any one of claims 1 to 37 in the manufacture of a medicament for the treatment or prevention of cancer.

41. A nanoparticle bioconjugate according to any one of claims 1 to 37, or a pharmaceutical composition according to claim 38, for use in the treatment or prevention of cancer.

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