Bispecific nanoparticle bioconjugates

Abstract

The present invention relates to nanoparticle bioconjugates comprising binding proteins for binding to antigens of cancer cells and immune cells, as well as uses and methods of treatment comprising administering the same.

Description

Technical Field

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

[0002] Cross-reference to earlier applications

[0003] This application claims priority from Australian provisional application AU 2022901088, the contents of which are hereby incorporated by reference in their entirety. Background Art

[0004] Targeted immunotherapy using monoclonal antibodies (mAbs) has been extremely successful due to their unparalleled specificity and efficacy. Its growth over 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), mainly targeting different types of cancer. biAbs can bind to tumor cells and immune cells (e.g., killer T cells) simultaneously, resulting in dramatic lysis of tumor cells. biAbs have shown great therapeutic potential as emerging immunotherapies. Currently, two biAbs are approved as cancer therapies, and hundreds of new candidates are in clinical trials.

[0005] Currently available biAbs have low conformational and formulation stability and short serum half-lives, resulting in rapid elimination from the body, e.g., blinatumomab has a half-life of 2.1 hours. Hundreds of different biAb constructs have been reported so far, and most of these have stability and half-life issues. This represents a significant limitation to the use of these molecules for the treatment of cancer, where a longer serum half-life is critical for better therapeutic outcomes.

[0006] There is a need for improved bispecific binding molecules with increased conformational and / or formulation stability.

[0007] The reference to any prior art in this specification does not constitute an acknowledgement or implication that this prior art forms part of the common general knowledge in any jurisdiction, nor does it mean that this prior art can be reasonably understood, regarded as relevant, and / or combined with other prior art by a person skilled in the art. Summary of the invention

[0008] The present invention provides a nanoparticle bioconjugate, wherein the nanoparticle bioconjugate comprises:

[0009] a first nanoparticle, a first binding protein for specifically binding to a target portion on a cancer cell conjugated to the first nanoparticle, and

[0010] a second nanoparticle, wherein a second binding protein for specifically binding to a target portion on an immune cell is conjugated to the second nanoparticle;

[0011] wherein the nanoparticles are arranged in a dumbbell-like configuration or a flower-like configuration, and

[0012] The bioconjugate is capable of binding to cancer cells via the first binding protein and binding to immune cells via the second binding protein.

[0013] In certain embodiments, the first nanoparticle is a metal nanoparticle and the second nanoparticle is a magnetic nanoparticle. In alternative embodiments, the first nanoparticle is a magnetic nanoparticle and the second nanoparticle is a metal nanoparticle. The metal nanoparticle may be magnetic or non-magnetic.

[0014] Preferably, 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.

[0015] Alternatively, the nanoparticle bioconjugate may be in the form of a central (core) magnetic nanoparticle surrounded by 3-4 or more metallic nanoparticles in a flower-like configuration.

[0016] In alternative embodiments, the nanoparticle bioconjugate may comprise the first nanoparticle and the second nanoparticle in a "dumbbell-shaped" architecture, whereby the nanoparticle conjugate comprises a metallic nanoparticle adjacent to or in close contact with a magnetic nanoparticle.

[0017] Therefore, it should be understood that the nanoparticle bioconjugates of the present invention are not in the form of spherical nanoparticles.

[0018] In any embodiment of the present invention, the magnetic nanoparticles may be selected from: Fe 3 O 4 (Iron Oxide) Nanoparticles, Fe(CO) 5 (Pentacarbonyl Iron) nanoparticles or FeCl 2 (Ferric chloride) nanoparticles.

[0019] 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.

[0020] Preferably, the metal nanoparticles are gold nanoparticles or silver nanoparticles. In a particularly preferred embodiment, the metal nanoparticles are gold nanoparticles.

[0021] In any embodiment, the nanoparticle bioconjugate comprises iron nanoparticles and gold nanoparticles, preferably wherein the bioconjugate comprises a central gold nanoparticle surrounded by 3-4 or more iron nanoparticles to form a flower-shaped nanoparticle.

[0022] In any embodiment, the nanoparticle bioconjugate comprises iron nanoparticles and silver nanoparticles, preferably wherein the bioconjugate comprises a central silver nanoparticle surrounded by 3-4 or more iron nanoparticles to form a flower-shaped nanoparticle.

[0023] In any embodiment, the nanoparticle bioconjugate of the present invention may be between about 5 nm and about 100 nm. In a preferred embodiment, the nanoparticle bioconjugate may be between about 35 nm and 80 nm, preferably about 40 nm.

[0024] In any embodiment, the first binding protein and the second binding protein are covalently conjugated to the first nanoparticle and the second nanoparticle. It should be understood that any conjugation method can be used to conjugate the first binding protein and the second binding protein to the nanoparticle. Such methods are well known to those skilled in the art.

[0025] In any embodiment, the first antigen binding protein and the second antigen binding protein are covalently conjugated to the first nanoparticle and the second nanoparticle by different chemistries. It should be understood that the conjugation method will depend on the available functional groups and the surface modification of the nanoparticles.

[0026] In certain embodiments, the associated proteins can be directly conjugated to the nanoparticle. In alternative embodiments, the associated proteins can be conjugated via a linker moiety. The linker moiety can be any commercially available linker as further described herein, or can be other linkers for conjugating a peptide or protein moiety to a nanoparticle.

[0027] In certain examples, the binding protein can be covalently conjugated to the nanoparticle, preferably a functionalized gold nanoparticle, via a polyethylene glycol (PEG) moiety, alkyne, amine, azide, biotin, carboxyl, hydroxyl, methyl, maleimide, neutravidin, NHS, hydrazone, or thiol linker group.

[0028] In certain examples, the binding protein can be covalently conjugated to the nanoparticle, preferably a magnetic nanoparticle (such as iron), via an amine, azide, carboxyl, maleimide or hydrazone linker group.

[0029] In any embodiment, the thiol linker can be at least one of lipoic acid, monolipoic acid, dilipoic acid, and trilipoic acid.

[0030] The first binding protein can be any binding protein that can specifically bind to a target portion of a cancer cell. The cancer cell can be any one selected from the following: breast tumor, colorectal tumor, adenocarcinoma, mesothelioma, bladder tumor, prostate tumor, germ cell tumor, hepatoma / cholangiocarcinoma, neuroendocrine tumor, pituitary neoplasm, small round cell tumor, squamous cell carcinoma, melanoma, atypical fibroxanthoma, seminoma, non-seminoma, 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.

[0031] In any embodiment, the target moiety on the cancer cell may be an antigen selected from the group consisting of: a sugar, a lipid, a nucleic acid, a peptide or a protein. Preferably, the target moiety is endogenous to the cancer cell.

[0032] The binding protein for binding to the target moiety may comprise, consist essentially of, or consist of an antigen binding domain. In an alternative, the binding protein may comprise, consist essentially of, or consist of a receptor or ligand or a portion thereof for binding to the cancer cell. In any aspect, the target or target molecule may be any of those described herein.

[0033] Examples of cancer cell antigens that can 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.

[0034] In a particularly preferred embodiment, the antigen is HER2, and the cancer cell is any cancer cell that overexpresses HER2 or is HER2 positive. Therefore, in a preferred embodiment, the first binding protein is used to bind to HER2 on HER2 positive cancer (such as breast cancer or gastric cancer) cells.

[0035] Examples of HER2 antibodies and binding proteins derived therefrom are known in the art and include pertuzumab and trastuzumab.

[0036] In an alternative embodiment, the antigen is EGFR, and the cancer cell is any cancer cell that overexpresses epidermal growth factor receptor (EGFR). Therefore, in an alternative preferred embodiment, the first binding protein is used to bind to EGFR on EGFR-positive cancer cells, such as head and neck squamous cell carcinoma (SCCHN), glioma, nasopharyngeal carcinoma or pancreatic cancer cells.

[0037] Examples of EGFR antibodies and binding proteins derived therefrom are known in the art and include panitumumab and nimotuzumab.

[0038] In any embodiment of the present invention, the first antigen binding protein is selected from: trastuzumab, pertuzumab, panitumumab, nimotuzumab, cetuximab or an antigen binding fragment thereof.

[0039] The second binding protein may be any binding protein that can specifically bind to a target portion of an immune cell. The immune cell may be any immune cell, optionally selected from one of the following: a T cell, an antigen presenting cell (APC) or a natural killer (NK) cell. Preferably, the immune cell is a T cell.

[0040] In any embodiment, the target moiety on the immune cell can be an antigen selected from the following: sugar, lipid, nucleic acid, peptide or protein. In any embodiment, the target moiety on the immune cell can be an antigen selected from the following: 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 include, consist essentially of or consist of an antigen binding domain. In an alternative, the binding protein can include, 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 of those described herein.

[0041] In a preferred embodiment of the present invention, the immune cell is a T cell, and the target portion on the T cell bound by the second binding protein is selected from any one of the following: 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.

[0042] Therefore, in a preferred embodiment, the immune cell is a T cell, and the second binding protein is selected from: anti-CD3 antibody, anti-CD2 antibody, anti-CD4 antibody, anti-CD7 antibody, anti-CD8 antibody, anti-PD1 antibody, anti-CTLA4 antibody, anti-KIR antibody, anti-CD16 antibody, anti-CD94 antibody, anti-CD161 antibody, anti-NTBA antibody, recombinant human NTBA, anti-CD19 antibody, anti-CD20 antibody, anti-CD22 antibody, anti-CD30 antibody, anti-CD33 antibody, anti-CD38 antibody, anti-CD40L antibody, anti-CD44 antibody, anti-CD56 antibody, anti-CD79b antibody, anti-CD80 antibody, anti-CD86 antibody, anti-CD135 antibody, anti-CD137 antibody, anti-CD138 antibody, anti-CD154 antibody, anti-EphA2 antibody, anti-EGFR antibody or a fragment thereof.

[0043] In some embodiments of the present invention, when the immune cells are T cells, the binding protein can be anti-CD3 antibodies (e.g., OKT3, UCHT1, IP26, SK7, HIT3a or other clones) and / or anti-CD4 antibodies and / or anti-CD8 antibodies and / or anti-PD1 antibodies and / or anti-CTLA4 antibodies, etc. or a combination thereof.

[0044] If 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.

[0045] It should be understood that the first binding protein and / or the 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, a genetically engineered antibody, a chimeric antibody, a heterologous conjugate antibody, or a combination thereof.

[0046] In certain embodiments, the binding protein is an antibody. Optionally, the antibody is selected from IgA, IgD, IgE, IgG, IgM. Preferably, the antibody is IgG. The IgG may be IgG1, IgG2, IgG3, IgG4.

[0047] In any embodiment, the binding protein can be an antibody fragment, which is optionally selected from: a recombinant antibody fragment, a bifunctional antibody, a trifunctional antibody, 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.

[0048] In certain embodiments, the first binding protein and the second binding protein can be the same antibody format. For example, both binding proteins can 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 other). In alternative embodiments, the first antigen binding protein and the second antigen binding protein are different antibody formats. For example, in certain embodiments, the first binding protein can be in the form of a monoclonal immunoglobulin (mAb, IgG), and the second binding protein can be in the form of an antibody fragment, such as scFv. The first binding protein and / or the second binding protein can be monospecific or multispecific (such as bispecific or trispecific).

[0049] In further embodiments, the bioconjugate may further comprise a moiety for achieving cell penetration, such as a cell penetrating peptide (CPP). Examples of suitable cell penetrating peptides are known to those skilled in the art, and non-limiting examples include peptides from the human immunodeficiency virus 1 (HIV1) Tat protein (e.g., Tat 48-60 or Tat 47-57 ; penetration (for example, Antp 43-48 ); transporter; oligoarginine (e.g., R 8 ); MAP; MPG; SAP (see, e.g., Veldhoen et al. 2008 Int. J. Mol. Sci. 9(7), 1276-1320, https: / / doi.org / 10.3390 / ijms9071276; the document is incorporated by reference in its entirety). The CPP can be conjugated to the nanoparticle via a suitable linker moiety, such as the linker moiety described herein for conjugation to a binding protein.

[0050] In some embodiments, the bioconjugate further comprises an HIV-TAT cell penetrating peptide, preferably Tat 47-57 (SEQ ID NO: 12). The HIV-TAT CPP can be linked to the nanoparticle via a thiol linker, such as thiol-PEG-NHS (see, for example, Cruz and Kayser 2019, Cancers 116(6), p. 870, https: / / doi.org / 10.3390 / cancers11060870 ; said document is incorporated by reference in its entirety).

[0051] The present invention also provides a pharmaceutical composition comprising any of the nanoparticle bioconjugates described herein, optionally in combination with a pharmaceutically acceptable excipient or carrier.

[0052] The present invention also provides a method for treating or preventing cancer in a subject, the method comprising administering to a subject in need thereof a nanoparticle bioconjugate or pharmaceutical composition as described herein. As used herein, methods for treating cancer include methods for inhibiting, preventing or minimizing the spread or progression of cancer, including inhibiting or preventing the metastasis of cancer.

[0053] Still further, the present invention provides a use of the nanoparticle bioconjugate of the present invention for preparing a medicament for treating or preventing cancer.

[0054] The present invention further provides a nanoparticle bioconjugate or pharmaceutical composition of the present invention for use in treating or preventing cancer.

[0055] The present invention further provides a kit for producing the nanoparticle bioconjugate of the present invention. Preferably, the kit comprises nanoparticles, binding proteins and instructions for combining them to obtain bioconjugates capable of binding to cancer cells and immune cells.

[0056] As used herein, the term "comprise" and variations of the term such as "comprising," "comprises," and "comprised" are not intended to exclude additional additives, components, integers or steps, unless the context requires otherwise.

[0057] Further aspects of the invention and further embodiments of the aspects described in the preceding paragraphs will become apparent from the following description, which is given by way of example and with reference to the accompanying drawings. BRIEF DESCRIPTION OF THE DRAWINGS

[0058] Figure 1: A. Schematic diagram depicting the conjugation of the first antigen binding protein and the second antigen binding protein to nanoparticles, preferably in a flower-shaped arrangement, and their use for simultaneously targeting T cells and cancer cells. B. Thermal decomposition synthesis of magnetic gold nanoparticles. Iron precursors and gold precursors are dissolved in an organic solvent and in the presence of N 2 In the case of gas, chemical precipitation was achieved under vigorous stirring at 310° C. After cooling, the nanoparticles were purified and stored.

[0059] Figure 2 :Fe 3 O 4 @Characterization of Au nanoparticles. A. Transmission electron microscopy (TEM) images of representative flower-like nanoparticles. (i) Representative hydrophobic tail nanoparticles (Fe 3 O 4@Au / OA) TEM. The diameters of the gold core and iron are approximately 11 and 14 nm, respectively. (ii) Representative hydrophilic tail nanoparticles with dopamine layer (Fe 3 O 4 @Au / Dop) TEM. The average shell diameter is 4nm. B. Hydrophobic Fe 3 O 4 @Au nanoparticles (upper line) and hydrophilic Fe 3 O 4 @ FTIR spectrum of Au nanoparticles (lower line). Recorded between 600 and 4000 cm -1 The number of waves between.

[0060] Figure 3 :Nmab-Fe 3 O 4 MALDI-TOF mass analysis of @Au-αCD3scFv NPs. Fc before and after conjugation with anti-EGFR mAB (nimotuzumab; 149 KDa) and anti-CD3 scFv (24 KDa). 3 O 4 @Mass spectrum of Au NPs.

[0061] Figure 4 : Normalized UV-visible absorption spectroscopy of modified nanoparticles: A. Hydrophobic Fe 3 O 4 @Au NPs show their strong absorption at 573nm, which shifts to 390nm after modification. B. NmabNPs (light green) and dual NPs (red); showing the total amount of loaded antibodies; NmabNPs (blue); dual NPs (green); showing very strong absorption of conjugated antibodies after centrifugation at about 260n.

[0062] Figure 5 : Conjugated Fe 3 O 4 Nanodrop protein measurement results of @Au NP. A. PBS baseline, B. Naked NP negative control, C. Dual Fe 3 O 4 @Au NP (conjugated bispecific NP), D.Fe 3 O 4 @Au-αCD3scFv NP, E.Nmab-Fe 3 O 4 @AuNP.

[0063] Figure 6 : Conjugated Fe 3 O 4Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis of @Au NP. Lane 1, protein size marker; Lane 2, free nimotuzumab; Lane 3, blank control; Lane 4, anti-CD3 scFv; Lane 5, NP; Lane 6, Nmab-Fe 3 O 4 @Au NP; Lane 7, double Fe 3 O 4 @Au NP (bispecific nanoparticles conjugated with nimotuzumab and anti-CD3 scFv; otherwise known as Nmab-F 3 O 4 @Au-αCD3scFv).

[0064] Figure 7 : In Fe 3 O 4 Cytotoxicity assay of co-cultured A431 and Jurkat cells at a 1:5 ratio (target:effector) with Au nanoparticles (0-5000 μg / ml iron). 3 O 4 @Before Au NP treatment, the viability of co-cultured cells was not significantly affected. Data are reported as mean ± SD.

[0065] Figure 8 : Cytotoxicity assay of co-cultured A431 and Jurkat cells at a 1:5 ratio (target:effector) after incubation with different concentrations of: A. Nimotuzumab. Cell viability was not significantly affected before treatment with 1000 μg / ml of Nimotuzumab. B. Nmab-Fe 3 O 4 @Au nanoparticles; and C.BiFe 3 O 4 @Au nanoparticles (nanoparticles conjugated with nimotuzumab and anti-CD3 scFv). With Nmab-Fe with ≥300 μg / ml iron 3 O 4 @Au nanoparticles compared with double Fe 3 O 4 After incubation with @Au NPs, the viability of co-cultured cells was significantly reduced. Data are reported as mean ± SD. Nimotuzumab at 2000 μg / ml increased the viability of the cells relative to the control. *** p 0.0001; 300 μg / ml Nmab-Fe 3 O 4 @Au NP relative to the control, *** p 0.0003; and 700 μg / ml Nmab-Fe3 O 4 @Au NP relative to the control, **** p<0.0001; 300 and 700 μg / ml of biFe 3 O 4 @Au NP relative to the control, **** p<0.0001 (one-way ANOVA post hoc Fisher LSD).

[0066] Fig. 9 : At the same nimotuzumab concentration, in the presence of nimotuzumab (1, left column), Nmab-Fe 3 O 4 @Au nanoparticles (2, middle column) and biFe 3 O 4 Cytotoxicity assay of co-cultured A431 and Jurkat cells at a 1:5 ratio (target:effector) after incubation with @Au nanoparticles (3, right column, bispecific NPs conjugated with anti-EGFR Nmab and αCD3 scFv). 3 O 4 Compared with cells treated with antibodies alone, the viability of co-cultured cells was not significantly affected before treatment with 1000 μg / ml nimotuzumab, whereas the viability of co-cultured cells was not significantly affected after treatment with dual Fe 3 O 4 After incubation with @Au nanoparticles, the activity decreased significantly. Data are reported as mean ± SD.

[0067] Fig.10 :The functionalized Fe 3 O 4 @Au NP cellular uptake into cancer cell lines. A. Intracellular iron and gold content was quantified by ICP-MS after incubation with 50 μg / ml Fe for 12 h. B. Intracellular iron and gold content was quantified by ICP-MS after incubation with 50 μg / ml Fe 3 O 4 After incubation with Au NPs for 12 h, intracellular iron (a:Fe 3 O 4 @AuNP; b:Nmab-Fe 3 O 4 @Au NP; c: double Fe 3 O 4 @Au NPs, bispecific NPs conjugated with anti-EGFR Nmab and αCD3 scFv). Uptake data are reported as mean ± SD (x3).

[0068] Fig.11 : Cytotoxicity assay of co-culture SkBr3 and Jurkat cells at a 1:5 ratio (target:effector) after incubation with: A. free Tmab, B. Tmab-Fe 3 O 4 @Au NP and C. Dual Tmab-Fe 3 O 4 @Au-CD3scFv NP (bispecific NP conjugated with anti-HER2 Tmab and αCD3scFv). 3 O 4 @Au NPs compared with dual Tmab-Fe 3 O 4 The viability of co-cultured cells was significantly reduced after incubation with @Au-CD3 scFv NPs. Data are reported as mean ± SD. D: After incubation with different concentrations of free Tmab, Tmab-Fe 3 O 4 @Au NP(Tmab NP) or double Tmab-Fe 3 O 4 Cytotoxicity assay of co-cultured SkBr3 and Jurkat cells at a 1:5 ratio (target:effector) after incubation with @Au-αCD3scFv NPs (double NPs; (bispecific NPs conjugated with anti-HER2 Tmab and αCD3scFv)). 3 O 4 Compared with @Au NPs, and compared with ≥43μg / ml free Tmab, compared with dual Tmab-Fe 3 O 4 After incubation with @AuαCD3scFv NP, the viability of co-cultured cells was significantly reduced. Free Tmab had no significant effect on cell viability below 200 μg / ml. Data are reported as mean ± SD.

[0069] Fig.12 :Tmab-Fe 3 O 4 @ Characterization of Au NPs. A. Representative TEM image of flower-shaped NPs. B. The average size of the hydrodynamic diameter of the NPs after conjugation was 149.3 nm.

[0070] Fig.13 : Cytokine release profile of EGFR-A431 targeting T cells. In a 96-well plate, 4x10 3cells / well, and then Jurkat E6-1 cells were added at an effector:target (E:T) ratio of 5:1 and incubated with anti-CD3 scFv, dual Fc 3 O 4 @Au NP (bispecific NP conjugated with anti-EGFR Nmab and αCD3 scFv) and Nmab-Fe 3 O 4 @Au NPs were incubated together for 24 hours. The cytokine IL-2 secreted into the supernatant was determined by ELISA. Data are reported as mean ± SD. Dual NPs relative to anti-CD3 scFv-conjugated NPs, Nmab-NPs, and control unconjugated NPs, *** p<0.0001 (one-way ANOVA post hoc Fisher's LSD).

[0071] Fig.14 .Dumbbell-shaped Fe 3 O 4 @ Representative TEM images of Au nanoparticles. The diameters of gold nanoparticles and iron nanoparticles are approximately 5 and 10 nm, respectively.

[0072] Fig.15 Figure : Cytotoxicity assays performed in co-cultures of A431 and Jurkat cells at a 1:5 ratio (target:effector) at different concentrations of dumbbell-shaped NPs treated with: A. naked NPs, B. dual Fe 3 O 4 @Au NP (bispecific NP conjugated with anti-EGFR Nmab and αCD3 scFv) and C.Nmab-Fe 3 O 4 @Au NPs. With Nmab-Fe with ≥300μg / ml iron 3 O 4 @Au NPs, compared with bi-Fe 3 O 4 After incubation with @Au NPs, the viability of the co-culture cells was significantly reduced.

[0073] Fig.16 : Characterization of spherical gold nanoparticles. A. TEM image of representative Au nanoparticles. B. UV-visible absorption spectroscopy of gold nanoparticles at 521 nm.

[0074] Fig.17Figure 2: Cytotoxicity assays in co-cultures of A431 and Jurkat cells at different concentrations of spherical AuNPs treated with the following at a 1:5 ratio (target:effector): A. Nmab-AuNPs, B. Dual AuNPs (bispecific NPs conjugated to anti-EGFR Nmab and αCD3scFv). Compared to Nmab-AuNPs, the viability of co-culture cells was not significantly affected after incubation with dual AuNPs.

[0075] Fig.18 : Cytotoxicity assays performed in co-cultures of MDA-MB 468 cells and Jurkat cells at a 1:5 ratio (target:effector) under the following treatments at different concentrations: A. free Nmab, B. Nmab-Fe 3 O 4 @Au NP and C. Double Nmab-Fe 3 O 4 @Au-αCD3scFv NP (bispecific NP conjugated with anti-EGFR Nmab and αCD3scFv). With Nmab-Fe with ≥300μg / ml iron 3 O 4 @Au NPs, compared with double Nmab-Fe 3 O 4 The viability of co-cultured cells was significantly reduced after incubation with @Au-αCD3scFv NPs. Data are reported as mean ± SD. **** p<0.0001 (one-way ANOVA post hoc Fisher's LSD).

[0076] Fig.19 :In the presence of different concentrations of free Nmab, Nmab-Fe 3 O 4 @Au NP(Nmab NP) or double Nmab-Fe 3 O 4 Cytotoxicity assay of co-cultured MDA-MB 468 and Jurkat cells at a 1:5 ratio (target:effector) after incubation with @Au-αCD3scFv (bispecific NP conjugated with anti-EGFR Nmab and αCD3scFv). 3 O 4 Compared with ≥43 μg / ml free Nmab, the 3 O 4The viability of co-cultured cells was significantly reduced after incubation with @Au-αCD3scFv NP. Free Nmab had no significant effect on cell viability at concentrations below 100 μg / ml. Data are reported as mean ± SD.

[0077] Fig. 20 :At different concentrations of αCD3scFv-Fe 3 O 4 Cytotoxicity assays performed in co-culture of A431 and Jurkat cells at a 1:5 ratio (target:effector) with Au-Nmab NPs. A. In co-culture with αCD3 scFv-Fc with 43 μg / ml Nmab 3 O 4 The viability of the co-cultured cells was significantly reduced after incubation with @Au-Nmab NPs. B. Incubation with αCD3scFv-Fe with ≥300 μg / ml iron 3 O 4 After incubation with @Au-Nmab NPs, the viability of co-culture cells was significantly reduced.

[0078] Fig.21 : Cytotoxicity assay of co-cultured MDA-MB468 and Jurkat cells at a 1:5 ratio (target:effector) after incubation with: A. free Tmab, B. Tmab-Fe 3 O 4 @Au NP and C. Dual Tmab-Fe 3 O 4 @Au-αCD3scFv NPαCD3scFv (bispecific NP conjugated with anti-HER2 Tmab and αCD3scFv). Tmab-Fe with ≥300μg / ml iron 3 O 4 @Au NPs, compared with dual Tmab-Fe 3 O 4 The viability of co-cultured cells was significantly reduced after incubation with @Au-αCD3scFv NPs. Data are reported as mean ± SD. * p<0.0001, ** p<0.0001, **** p<0.0001 (one-way ANOVA post hoc Fisher's LSD).

[0079] Fig. 22 :In the presence of different concentrations of free Tmab, Tmab-Fe 3 O 4 @Au NP(Tmab NP) or double Tmab-Fe 3 O 4Cytotoxicity assay of co-cultured MDA-MB 468 and Jurkat cells at a 1:5 ratio (target:effector) after incubation with @Au-αCD3scFv NPs (Dual NPs; bispecific NPs conjugated with anti-HER2 Tmab and αCD3scFv). 3 O 4 Compared with ≥43 μg / ml free Tmab, the 3 O 4 After incubation with @Au-αCD3scFv NPs, the viability of co-cultured cells was significantly reduced. Free Tmab had no significant effect on cell viability below 200 μg / ml. Data are reported as mean ± SD.

[0080] Fig.23 : A431 and Jurkat cells were incubated with different concentrations of bispecific anti-EGFR and anti-CD3 NPs, panitumumab (Pmab)-Fe 3 O 4 Cytotoxicity assays performed when co-cultured with @Au-αCD3scFv. A. In co-culture with Pmab-Fc with 10 μg / ml Pmab 3 O 4 The viability of the co-cultured cells was significantly reduced after incubation with @Au-αCD3scFv NPs. 3 O 4 After incubation with @Au-αCD3scFv NP, the viability of co-culture cells was significantly reduced.

[0081] Fig.24 :Silver-iron nanoparticles (Fe 3 O 4 @Ag NP) characterization. A. TEM image of representative silver iron nanoparticles. B. UV-visible absorption spectroscopy of silver iron nanoparticles at 409 nm.

[0082] Fig.25 : A431 and Jurkat cells were incubated at a 1:5 ratio (target:effector) with different concentrations of bispecific anti-EGFR and anti-CD3 NPs conjugated to CPP, Nmab-Fe 3 O 4 Cytotoxicity assays when co-cultured with @Au-αCD3scFv / CPP. A. In the presence of Nmab-Fe 3 O 4The viability of co-cultured A431 and Jurkat cells was significantly reduced after incubation with @Au-αCD3scFv / CPP NPs. 3 O 4 After incubation with @Au-αCD3scFv / CPP NP, the viability of co-cultured A431 and Jurkat cells was significantly reduced.

[0083] Fig.26 : In the presence and absence of CPP, Nmab-Fe 3 O 4 Cytotoxicity assay in co-culture of A431 and Jurkat cells at a 1:5 ratio (target:effector) with Au-αCD3scFv NPs. 3 O 4 Compared with @Au-αCD3scFv NP (double NP, left column), Nmab-Fe 3 O 4 After incubation with @Au-αCD3scFv / CPP NP (CPP double NP, right column), the viability of the co-cultured cells was significantly reduced. DETAILED DESCRIPTION

[0084] It should be understood that the invention disclosed and defined in this specification extends to all alternative combinations of two or more of the individual features mentioned or evident in the text or drawings. All these different combinations constitute various alternative aspects of the invention.

[0085] Currently available bispecific binders, in particular bispecific binders for engaging T cells and cancer cells (so-called bispecific T cell engagers, or "BiTe"), have low conformational and formulation stability and short serum half-life, leading to their rapid elimination 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 issues. This represents a significant limitation for the use of these antibodies for the treatment of cancer, where a longer serum half-life is crucial for better therapeutic outcomes.

[0086] The present invention seeks to overcome one or more limitations of prior art bispecific binders. The present invention relates to nanoparticle-based bispecific binders (nanobioconjugates) comprising both immune cell engagers and cancer-specific engagers, wherein the nanobioconjugates are capable of interacting with both immune cells (such as T cells, NK cells, etc.) and cancer cells.

[0087] The present invention is believed to have various advantages over existing bispecifics, including greater conformational and formulation stability. Further, it is believed that the nanobioconjugates of the present invention may have a longer serum half-life, as the size of the nanoparticles means that they may bypass renal filtration.

[0088] Advantageously, nanoparticles of the present invention provide the 1st conjugated protein and the 2nd conjugated protein (for combining with cancer cells and immune cells, respectively) put together with different nanoparticle types. There are two different nanoparticle types (such as iron and gold) in the composite nanostructure, which contribute to the easy connection of different functional groups, so that two different antibodies or their fragments can be put together, and thus provide design flexibility. In addition, different nanoparticle types are used to enable different chemicals to be put together with antigen-binding proteins. This provides several advantages, including the ability to control every kind of conjugated protein ratio, and the efficiency (for example, fewer processing steps) of preparing nanoparticle conjugates.

[0089] Furthermore, the use of magnetic nanoparticles facilitates the purification and preparation of bioconjugates for therapeutic use.

[0090] definition

[0091] Unless defined otherwise, 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.

[0092] For the purposes of interpreting this specification, the following definitions will generally apply and, where appropriate, terms used in the singular will also include the plural, and vice versa.

[0093] The term "bispecific" means that the nanoparticle bioconjugate of the present invention is able to specifically bind to at least two different antigenic determinants. Typically, a bispecific binding molecule comprises two antigen binding sites, each of which is specific to different antigenic determinants. In certain embodiments, a bispecific antigen binding molecule is able to simultaneously bind to two antigenic determinants, particularly two antigenic determinants expressed on two different cells.

[0094] 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 (chains of monosaccharides / simple sugars), lipids, or nucleic acids.

[0095] In any aspect, the binding protein is any protein that can associate, 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, consist essentially of or consist of an antigen binding domain. In an alternative, the binding protein may comprise, consist essentially of or consist of a receptor or portion thereof for binding to the target moiety.

[0096] As used herein, "target moiety" can be any part that can be bound by binding proteins present on target cells (e.g., cancer or immune cells). In any aspect, the target 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 cancer or immune cells. The target 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 so that it is not tethered to a cell or a part of the cell. The target can be constitutively secreted, or only transiently secreted.

[0097] The binding protein can include a receptor for a ligand or a binding partner for binding to a target molecule. It should be understood that the binding partner can be in the form of any molecule that enables binding to cancer or immune cells. For example, the binding partner can include or consist of a ligand, a receptor, a target or an antigen binding domain. The binding partner can be a naturally occurring molecule that binds to a target. The binding partner can be non-naturally occurring, or can be modified by naturally occurring molecules (e.g., by directed evolution). The binding partner can be naturally occurring, but can also be heterologous to a portion or a linker retained in a cell.

[0098] In any aspect, the binding protein comprises, consists essentially of, or consists of an antigen binding domain of an antibody or antigen binding fragment thereof. The antibody or antigen binding fragment may have been modified or engineered, or may be 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 bifunctional antibodies.

[0099] In any aspect, the binding protein can bind to two different target moieties or to two different epitopes on the same target moiety.

[0100] "Antibodies" or "immunoglobulins" or "Ig" are gamma globulins present in the blood or other body fluids of vertebrates that function in the immune system to bind to antigens, thereby identifying and / or neutralizing foreign bodies. Antibodies are typically 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. Depending on the H chain isotype, the two H chains are interconnected by one or more disulfide bonds. Each H and L chain also has regularly spaced intrachain disulfide bridges.

[0101] The H and L chains define specific Ig domains. More specifically, each H chain has a variable domain (V H ), followed by three constant domains (CH) of each of the α and γ chains, and four CH domains of the μ and ε isoforms. Each L chain has a variable domain (V) at the N-terminus. L ), followed by a constant domain (CL) at its other end. L With V H Aligned, and C L Aligned with the first constant domain of the heavy chain (CHL).

[0102] Antibodies can be assigned to different classes or isotypes. There are five classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, with heavy chains named α, δ, ε, γ, and μ, respectively. Based on relatively minor differences in 3 / 4 sequence and function, the γ and α classes are further divided into subclasses, for example, humans express the following subclasses: IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The L chain from any vertebrate species can be assigned to one of two distinctly different types, called κ and λ, based on the amino acid sequence of its constant domain.

[0103] The constant domain includes an Fc portion comprising the carboxyl terminal portions of two H chains held together by disulfide bonds. The effector functions of antibodies (such as ADCC) are determined by sequences in the Fc region, which is also the portion recognized by Fc receptors (FcRs) present on certain types of cells.

[0104] V H and V L The variable domains of the heavy and light chains of the antibodies are referred to as "V" and "V" chains. H The variable domain of the light chain may be referred to as the "V L". The V domain contains the "antigen binding site" that affects antigen binding and defines the specificity of a particular antibody for its particular antigen. The V region spans about 110 amino acid residues and consists of relatively invariant stretches called framework regions (FR) of 15-30 amino acids (usually about 4), which are separated by shorter regions of great variability called "hypervariable regions" (usually about 3), each of which is usually 9-12 amino acids in length. The FRs mainly adopt a β-sheet configuration, and the hypervariable regions form loops connecting the β-sheet structure, and in some cases form part of the β-sheet structure.

[0105] The terms "full-length antibody", "intact antibody" or "whole antibody" are used interchangeably to refer to an antibody in substantially complete form, rather than an antigen-binding fragment of an antibody. Specifically, intact antibodies include antibodies having heavy and light chains that include an Fc region. The constant domains may be wild-type sequence constant domains (e.g., human wild-type sequence constant domains) or amino acid sequence variants thereof.

[0106] As used herein, "variable region" refers to the part of the light chain and / or heavy chain of an antibody as defined herein that is capable of specifically binding to an antigen, and includes the complementary determining region (CDR); i.e., CDR1, CDR2 and CDR3, and the amino acid sequence of the framework region (FR). For example, the variable region comprises three or four FRs (e.g., FR1, FR2, FR3 and optionally FR4) and three CDRs. VH refers to the variable region of the heavy chain. VL refers to the variable region of the light chain.

[0107] As used herein, the term "complementarity determining region" (syn.CDR; i.e., CDR1, CDR2 and CDR3) refers to the amino acid residues of the variable region of an antibody, the presence of which is a major factor in specific antigen binding. Each variable region domain (VH or VL) generally has three CDRs identified as CDR1, CDR2 and CDR3. In one example, the amino acid positions assigned to CDR and FR are defined according to the Kabat sequences (Kabat Sequences of Proteins of Immunological Interest) of proteins with immunological significance, the National Institutes of Health (National Institutes of Health, Bethesda, Md.) of Bethesda, Maryland, 1987 and 1991 (also referred to herein as "Kabat numbering system") definitions. In another example, the amino acid positions assigned to CDR and FR 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 includes all numbering systems, including the canonical numbering system or the numbering system 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., J. Mol. Biol. 273:927-948, 1997; the numbering system of Honnegher and Plükthun, 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 replaced by another naturally occurring amino acid. In this regard, Padlan et al., Journal of the Federation of American Societies for Experimental Biology (FASEB J.), 9: 133-139, 1995 determined that the five C-terminal amino acids of the heavy chain CDR2 are generally not involved in antigen binding.

[0108] "Hypervariable region" refers to the region of the variable domain of an antibody that is highly variable in sequence and / or forms a loop with defined structure. Typically, an antibody comprises six hypervariable regions: V H Three of them (H1, H2, H3) and V L Three of them (L1, L2, L3).

[0109] "Framework" or "FR" residues are those variable domain residues other than the hypervariable region residues as herein defined.

[0110] "Antigen binding site" generally refers to a molecule that includes at least the hypervariable region and framework region required to confer antigen binding function to the V domain. In the methods described herein, the antigen binding site can 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). As used herein, terminal antigen binding site, antigen binding portion and antigen binding domain can be used interchangeably. It should be understood that the antigen binding protein can comprise or consist of an antigen binding domain.

[0111] As used herein, the term "Fv" shall mean any protein, whether comprising multiple polypeptides or a single polypeptide, wherein VL and VH associate and form a complex with an antigen binding domain, i.e., can specifically bind to an antigen. The VH and VL forming the antigen binding domain may be in a single polypeptide chain or in different polypeptide chains. In addition, the Fv of the present invention (and any protein of the present invention) may have multiple antigen binding domains, which may or may not bind to the same antigen. This term should be understood to encompass fragments directly derived from antibodies and proteins corresponding to such fragments produced using a recombinant approach. In some instances, VH is not connected to a heavy chain constant domain (CH) 1 and / or VL is not connected to a light chain constant domain (CL). Exemplary Fv-containing polypeptides or proteins include Fab fragments, Fab' fragments, F(ab') fragments, scFv, bifunctional antibodies, trifunctional antibodies, tetrafunctional antibodies or higher order complexes, or any of the aforementioned connected to its constant region or domain (e.g., CH2 or CH3 domain), e.g., mini antibodies. "Fab fragments" consist of monovalent antigen-binding fragments of immunoglobulins and can be produced by papain digestion of whole antibodies to produce fragments consisting of a portion of a complete light chain and a heavy chain, or can be produced using recombinant methods. "Fab' fragments" of antibodies can be obtained by treating whole antibodies with pepsin, followed by reduction to produce molecules consisting of a portion of a complete light chain and a heavy chain, the heavy chain comprising VH and a single constant domain. Two Fab' fragments of each antibody treated in this way are obtained. Fab' fragments can also be produced by recombinant methods. "F(ab')2 fragments" of antibodies consist of dimers of two Fab' fragments held together by two disulfide bonds, and are obtained by treating whole antibody molecules with pepsin without subsequent reduction. "Fab2" fragments are a recombinant fragment comprising two Fab fragments connected using, for example, a leucine zipper or a CH3 domain. "Single-chain Fv" or "scFv" is a recombinant molecule containing a variable region fragment (Fv) of an antibody, in which the variable region of the light chain and the variable region of the heavy chain are covalently connected by a suitable flexible polypeptide linker.

[0112] The terms "bind to," "specifically bind to," or "have specificity for" with respect to a targeting moiety refer to a binding domain that recognizes and binds to a specific 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 an antigen from another species. This cross-species reactivity is a typical feature of many antibodies, and is therefore not contrary to the definition of having specificity for an antigen binding domain. 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 a typical feature of many antibodies, and is therefore not contrary to the definition of having specificity for an antigen binding domain.

[0113] An "intact" or "whole" antibody is one that contains both the antigen binding site and the C L and an antibody comprising at least heavy chain constant domains CH1, CH2 and CH3. The constant domain may be a native sequence constant domain (eg, a human native sequence constant domain) or an amino acid sequence variant thereof.

[0114] "Whole antibody fragments including variable domains" include SD-mAb, Fab, Fab', F(ab')2 and Fv fragments; bifunctional antibodies; linear antibodies, single-chain antibody molecules; and multispecific antibodies formed from antibody fragments.

[0115] The "Fab fragment" consists of a complete L chain and the variable region domain of the H chain (V H ) and the first constant domain of a heavy chain (CHI). Each Fab fragment is monovalent in terms of antigen binding, i.e. it has a single antigen binding site.

[0116] "Fab' fragment" differs from the Fab fragment by having an additional few residues at the carboxyl terminus of the CH1 domain, which includes 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.

[0117] A "F(ab')2 fragment" roughly corresponds to two disulfide-linked Fab fragments having divalent antigen-binding activity and is still capable of cross-linking antigens.

[0118] "Fv" is the smallest antibody fragment that contains a complete antigen recognition and binding site. This fragment consists of a dimer of one heavy chain and one light chain variable region domain in tight, non-covalent association.

[0119] In single-chain Fv (scFv) species, one heavy chain variable domain and one light chain variable domain can be covalently linked by a flexible peptide linker so that the light chain and the heavy chain can associate in a "dimer" structure similar to that of a two-chain Fv species. Six hypervariable loops (3 loops each from the H and L chains) are generated from the folding of these two domains, which contribute amino acid residues to antigen binding and confer antigen binding specificity to the antibody.

[0120] "Single-chain Fv" is also abbreviated as "sFv" or "scFv" and is comprised of V sequences linked to form a single polypeptide chain. H and V L Preferably, the scFv polypeptide further comprises a V H With V L A polypeptide linker between the domains that enables the scFv to form the desired structure for antigen binding.

[0121] A "single variable domain" is half of an Fv (comprising only three CDRs specific for the antigen) that has the ability to recognize and bind to an antigen, although generally at a lower affinity than the entire binding site.

[0122] "Bifunctional antibodies" refer to antibody fragments with two antigen-binding sites, which are contained in the same polypeptide chain (V H -V L ) in the light chain variable domain (V L ) connected to the heavy chain variable domain (V H ). By H With V L Small antibody fragments are prepared by constructing sFv fragments (see previous paragraph) with short linkers (about 5-10 residues) between the domains, allowing interchain but not intrachain pairing of the V domains, resulting in bivalent fragments, i.e., fragments with two antigen-binding sites.

[0123] Bifunctional antibodies can be bivalent or bispecific. Bispecific bifunctional antibodies are heterodimers of two "crossover" sFv fragments, in which the V H and V L The domains are present on different polypeptide chains. Triabodies and tetrabodies are also generally known in the art.

[0124] In any embodiment, the binding protein can be derived from a human antibody, or can be a humanized form of a non-human antibody.

[0125] A "human antibody" refers to an antibody having an amino acid sequence corresponding to that of an antibody produced by a human. Human antibodies can be produced using various 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 antibodies in response to antigenic challenge, but whose endogenous loci have been disabled.

[0126] The humanized form of "non-human" (e.g., rodent) antibody is a chimeric antibody containing the minimum sequence derived from a non-human antibody. In most cases, a humanized antibody is a human immunoglobulin (recipient antibody), wherein the residues from the hypervariable region of the recipient are replaced by residues of a non-human species (donor antibody) with desired antibody specificity, affinity and ability, such as mouse, rat, rabbit or non-human primate hypervariable region. In some cases, the framework region (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues. In addition, the humanized antibody may be included in the recipient antibody or in the residues not present in the donor antibody. These modifications are made in order to further improve antibody performance. Generally, a humanized antibody will include substantially all at least one, and typically two variable domains, wherein all or substantially all hypervariable loops correspond to the hypervariable loops of non-human immunoglobulins, and all or substantially all FRs are FRs of human immunoglobulin sequences. Humanized antibodies will optionally also include at least a portion of an immunoglobulin (typically a human immunoglobulin) constant region (Fc).

[0127] "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 small amounts. Monoclonal antibodies are highly specific for a single antigenic site or determinant on an antigen. In addition to their specificity, monoclonal antibodies are advantageous in that they can be synthesized without contamination by other antibodies. Monoclonal antibodies can be prepared by the hybridoma method. "Monoclonal antibodies" can also be isolated from phage antibody libraries using molecular engineering techniques.

[0128] As used herein, the term "antigen" is intended to include substances that bind to or induce the production of one or more antibodies, and may include, but are 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 can be recognized by the adaptive immune system, including, but not limited to, an antibody or TCR, or an engineered molecule, including, but not limited to, a transgenic TCR, a CAR, a scFv or multimer thereof, a Fab fragment or multimer thereof, an antibody or multimer thereof, a single-chain antibody or multimer thereof, or any other molecule that can bind to a structure with high affinity.

[0129] An "epitope" generally refers to the portion of an antigen that binds to the antigen binding site of an antibody. An epitope can 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 in the primary protein structure. In certain embodiments, the epitope is a "conformational epitope," i.e., an epitope in which the hypervariable loops of the CDRs bind to residues as they are present in the tertiary or quaternary protein structure.

[0130] The term "immune cell" or "immune effector cell" refers to a cell that can be part of the immune system and performs a specific effector function, such as α-β T cells, NK cells, NKT cells, B cells, Breg cells, Treg cells, innate lymphoid cells (ILC), cytokine-induced killer (CIK) cells, lymphokine-activated killer (LAK) cells, γ-δ T cells, mesenchymal stem cells or mesenchymal stromal cells (MSC), monocytes or macrophages, or any hematopoietic progenitor cells, such as pluripotent stem cells and early progenitor cell subsets that may mature or differentiate into somatic cells.

[0131] The term "pharmaceutical composition" refers to a preparation that is in a form that permits the biological activity of the active ingredients contained therein to be effective and that contains no additional components that would be unacceptably toxic to a subject to which the formulation would be administered.

[0132] "Pharmaceutically acceptable carrier" refers to a component of a pharmaceutical composition other than the active ingredient that is non-toxic to a subject. Pharmaceutically acceptable carriers include, but are not limited to, buffers, excipients, stabilizers or preservatives.

[0133] As used herein, the term "subject" refers to a mammal, such as a mouse, rat, cow, pig, goat, chicken, dog, monkey or human. Preferentially, the subject is a human. The subject may be a subject (patient) suffering from a condition such as cancer, but the subject may also be a healthy subject. As used herein, the terms "subject", "individual" and "patient" may be used interchangeably.

[0134] A "therapeutically effective amount" of an agent, such as a pharmaceutical composition, refers to an amount effective, measured at dosages and for periods of time necessary, to achieve the desired therapeutic or preventive result. A therapeutically effective amount of an agent, for example, eliminates, reduces, delays, minimizes or prevents an adverse effect of a disease.

[0135] As used herein, the term "treat" (treatment of) a condition means reducing the frequency or severity of at least one sign or symptom of the disease or condition experienced by a subject.

[0136] As used herein, the term "prevention" refers to at least reducing the likelihood of acquiring the risk (or susceptibility) of a disease or condition (i.e., preventing an individual who may be exposed to or susceptible to a disease but has not yet experienced or displayed symptoms of the disease from developing at least one of the clinical symptoms of the disease). Biological and physiological parameters for identifying such patients are provided herein and are also well known to physicians. For example, prevention of an abnormal immune response may be characterized by the absence of an increase in the release of cytokines following treatment with a cellular immunotherapeutic agent.

[0137] Binding protein

[0138] The nanoparticle bioconjugate of the present invention comprises a first nanoparticle and a second nanoparticle, wherein a first binding protein for binding to a target portion of a cancer cell is conjugated to the first nanoparticle, and a second binding protein for binding to a target portion of an immune cell, preferably an antigen of a T cell, is conjugated to the second nanoparticle.

[0139] The first binding protein and the second binding protein (or binding moiety) of the bioconjugate of the invention may be in the form of any antigen binding protein described herein, including antibodies (including human or humanized antibodies), variants or fragments thereof.

[0140] In any aspect, the binding protein comprises or consists of an antigen binding domain of an antibody or antigen binding fragment thereof. The antibody or antigen binding fragment may have been modified or engineered, or be a human antibody. Examples of antibodies that have been modified or engineered 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 antibody (e.g., scFv), miniantibodies, and diabodies.

[0141] In any aspect, the antigen binding protein may be in the form of:

[0142] (i) single-chain Fv fragment (scFv);

[0143] (ii) dimeric scFv (di-scFv); or

[0144] (iii) one of (i) or (ii) linked to the constant region of an antibody, Fc or heavy chain constant domain (CH)2 and / or CH3.

[0145] Further, as described herein, the antigen binding protein may be in the form of:

[0146] (i) Bifunctional antibodies;

[0147] (ii) trifunctional antibodies;

[0148] (iii) tetrafunctional antibodies;

[0149] (iv) Nanobodies;

[0150] (v)ibody;

[0151] (vi) VH domain

[0152] (vii) Fab;

[0153] (vii) F(ab')2;

[0154] (ix) Fv; or

[0155] (x) one of (i) to (ix) linked to the constant region of an antibody, Fc or heavy chain constant domain (CH)2 and / or CH3.

[0156] The above antigen binding protein may also be referred to as an antibody antigen binding domain.

[0157] In some examples, the first antigen binding protein and the second antigen binding protein have the same structure, for example, both proteins can be in the form of scFv or other similar antibody fragments. Alternatively, both proteins can be in the form of IgG, including any class of IgG.

[0158] In alternative examples, the first antigen binding protein and the second antigen binding protein have different structures. For example, the first antigen binding protein (i.e., for binding to cancer cell antigens) is preferably in the form of IgG, while the second antigen binding protein (i.e., for binding to antigens on immune cells, preferably T cells) can be in the form of antibody fragments such as scFv.

[0159] The T cell antigen binding protein preferably comprises at least one binding molecule capable of binding to an activated T cell antigen. In a specific embodiment, the T cell binding protein comprises no more than one antigen binding moiety capable of specifically binding to an activated T cell antigen. Thus, in one embodiment, the T cell antigen binding protein provides a monovalent binding to an activated T cell antigen.

[0160] In particular 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.

[0161] Antibodies for binding to CD3 are known in the art, as are antibodies for binding to CD3ε. In certain embodiments, the binding protein comprises a portion or all of the antigen binding domain of a known CD3 binding protein, and the binding protein is optionally selected from H2C, OKT3, UCGT1 and FN18.

[0162] In one embodiment, the T cell antigen binding scFv can compete with monoclonal antibody H2C (described in PCT Publication No. WO2008 / 119567) for binding to an epitope of 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. Pat. No. 6,054,297) for binding to an epitope of 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 of CD3.

[0163] 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 variants thereof that retain function.

[0164] 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 functionally retained variant thereof (including a humanized variant thereof). In one embodiment, the anti-CD3 binding protein comprises the sequence of the OKT3 antibody fragment with the code 1SY6 obtained from the Protein Data Bank (https: / / www.rcsb.org / structure / 1sy6).

[0165] In another specific embodiment, the T cell antigen is CD 16, particularly human or cynomolgus CD 16, most particularly human CD 16. Antibodies for binding to CD 16 are known in the art.

[0166] 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 variants thereof that retain function.

[0167] 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 variants thereof (including humanized variants thereof) that retain function. In one embodiment, the anti-CD16 binding protein comprises the sequence of an anti-CD16 antibody fragment obtained from ViroGen with catalog number #1151-V.

[0168] In an alternative embodiment, 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 should be understood that this is not an exhaustive list of T cell antigens, and that it is within the scope of those skilled in the art to be able to identify suitable T cell antigens and corresponding antigen binding proteins that bind thereto.

[0169] Antibodies for binding to such T cell antigens are known in the art. For example, antibodies for binding to CD38 include Daratumumab, felzartamab, isatuximab, mezagitamab; antibodies for binding to CD7 include Grisninimab; antibodies for binding to PD1 include balstilimab, budigalimab, camrelizumab, cemiplimab, cetrelimab, dostarlimab, ezabelimab, and sirolimab. nlimab), geptanolimab, lodapolimab, izuralimab; antibodies for binding to CTLA-4 include ipilimumab, nurulimab, pavunalimab, quavonlimab, tremelimumab, vudalimab, zalifrelimab; antibodies for binding; antibodies for binding to CD30 include Iratumumab, SGN-30, 5F11. Those skilled in the art can use the antigen binding domain from any known antibody to produce the nanobioconjugate according to the present invention.

[0170] A cancer cell antigen binding protein will be understood to be any protein that is capable of specifically binding to an antigen on a cancer cell, such as a tumor-associated antigen or a tumor-specific antigen.

[0171] As used herein, "tumor-associated antigens" or cancer cell antigens refer to antigens expressed by cancer cells (the term "tumor-antigen" may also be used to refer to the same antigens). 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 prostate, prostate-specific antigen (PSA), PAP, NY-ESO-1, LAGE-1a, p53, P501S protein, PSMA, Her2 / neurin, 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.

[0172] The cancer cell antigen binding protein is preferably a protein that can specifically bind to any 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.

[0173] In one embodiment, tumor antigen comprises one or more antigenic cancer epitopes related to malignant tumor.Malignant tumor expresses many proteins that can serve as target antigens of 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 transformation-related molecule groups, such as oncogene HER-2 / Neu / ErbB-2.Another group of target antigens is carcinoembryonic antigen, such as carcinoembryonic antigen (CEA).In B cell lymphoma, tumor-specific idiotype immunoglobulin constitutes the real tumor-specific immunoglobulin antigen unique to single tumor.B cell differentiation antigens such as CD 19, CD20 and CD37 are other candidates of target antigens in B cell lymphoma.Some antigens (CEA, HER-2, CD19, CD20, idiotype) in these antigens have been used as targets of passive immunotherapy using monoclonal antibodies, but the success rate is limited.

[0174] The type of tumor antigen mentioned in the present invention can also be a tumor-specific antigen (TSA). TSA is specific to tumor cells and does not occur on other cells in the body. Tumor-associated antigens (TAA) are not specific to tumor cells, but are expressed on normal cells under conditions where an immune tolerance state to the antigen cannot be induced. The expression of antigens on tumors can occur under conditions that enable the immune system to respond to the antigen. TAAs can be antigens expressed on normal cells during fetal development when the immune system is immature and unable to respond, or can be antigens that are usually present at very low levels on normal cells but expressed at much higher levels on tumor cells. Compared with corresponding non-tumor tissues, those tumor-associated antigens with the greatest clinical significance are expressed differently and allow tumor cells to be preferentially recognized by specific T cells or immunoglobulins.

[0175] Non-limiting examples of TSA or TAA antigens include the following: differentiation antigens, such as MART-1 / MelanA (MART-1), gp 100 (Pmel 17), tyrosinase, TRP-1, TRP-2, and tumor-specific multi-lineage 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 caused by 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, CA19-9, CA72-4, CAM 17.1, NuMa, K-ras, β-catenin, CDK4, Mum-1, p15, p16, 43-9F, 5T4, 791Tgp72, alpha-fetoprotein, β-HCG, BCA225, BTAA, CA125, 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, RCAS1, SDCCAG16, TA-90\Mac-2 binding protein\cyclophilin C-associated 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 (fibronectin 2), 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 especially EGFR, mesothelin, GPC3, MUC1, HER2, GD2, CEA, EpCAM, LeY, PCSA and CD276.

[0176] Those skilled in the art will be familiar with the antigen binding domains described in the literature, which are used to bind to any one or more of the cancer antigens listed above. The following are some non-limiting examples, but it should be understood that the scope of the present invention should not be limited to the specific antigen binding proteins described herein. In addition, once those skilled in the art have determined which cancer cell needs to be treated, those skilled in the art can easily determine the suitable antigen for targeting present on the cancer cell with reference to the known literature on the cancer cell type. Then, those skilled in the art can utilize any known or new antigen binding domains that bind to cancer antigens for conjugation with nanoparticles described herein to produce suitable bioconjugates for treating cancer. The ability of bioconjugates to bind to target antigens can be confirmed using common laboratory techniques, including the techniques cited in the examples herein.

[0177] In one embodiment, the cancer cell antigen binding protein can specifically bind to epidermal growth factor receptor (EGFR). In another embodiment, the binding protein comprises a scFv molecule that can compete with monoclonal antibody GA201 for binding to the epitope of EGFR (see PCT Publication No. WO 2006 / 082515, which is incorporated herein by reference in its entirety).

[0178] In one embodiment, the binding protein specific for EGFR 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:6 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:7, or variants thereof that retain function.

[0179] In certain embodiments, the binding protein with specificity 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 function. In one embodiment, the binding protein with specificity for EGFR comprises the nimotuzumab antibody (Nmab) with the code 3GKW obtained from the protein database (https: / / www.rcsb.org / structure / 3gkw).

[0180] In one embodiment, the binding protein specific for EGFR 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:9 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:8, or variants thereof that retain function.

[0181] In certain embodiments, the binding protein with specificity 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 function. In one embodiment, the binding protein with specificity for EGFR comprises a panitumumab antibody (Pmab) with the code 5SX4 obtained from the protein database (https: / / www.rcsb.org / structure / 5sx4).

[0182] 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 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 at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 5, or variants thereof that retain function. 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 function. In one embodiment, the binding protein that is specific for HER2 comprises a trastuzumab antibody (Tmab) with the code DB00072 obtained from an online drug library (https: / / go.drugbank.com / drugs / DB00072).

[0183] Magnetic nanoparticles and metallic nanoparticles

[0184] Those skilled in the art will appreciate that a variety of nanoparticles may be used to produce the bioconjugates of the present invention. In general, nanoparticles having superparamagnetism, crystallinity, physical and chemical stability, environmental safety, and biocompatibility are preferred.

[0185] In any embodiment of the present invention, the magnetic nanoparticles may be selected from: Fe 3 O 4 (Magnetite), FeCl 2 、Fe(Co) 5 (pentacarbonyl iron), hematite (y-Fe 2 O 3 ) or hematite (a-Fe 2 O 3 In a preferred embodiment, the magnetic nanoparticles are Fe 3 O 4 In some embodiments, the average diameter of the magnetic nanoparticles is about 14 nm.

[0186] 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.

[0187] Preferably, the metal nanoparticles are gold nanoparticles or silver nanoparticles. In a particularly preferred embodiment, the metal nanoparticles are gold nanoparticles.

[0188] In any embodiment, the nanoparticle bioconjugate comprises iron nanoparticles and gold nanoparticles, preferably wherein the bioconjugate comprises a central gold nanoparticle surrounded by 3-4 or more iron nanoparticles to form a flower-shaped nanoparticle.

[0189] In any embodiment, the nanoparticle bioconjugate of the present invention may be between about 5 nm and about 200 nm. In any embodiment, the nanoparticle bioconjugate of the present invention may be between about 35 nm and 80 nm, preferably about 40 nm.

[0190] In any embodiment, the nanoparticle bioconjugate of the present invention can have a polymer coating to improve its dispersibility and / or stability. Examples of polymers for nanoparticle surface modification include PEG, polydopamine, dextran polymers, PVA, PAA, starch or chitosan. In a preferred embodiment, the nanoparticle bioconjugate of the present invention has a PEG coating.

[0191] Conjugation of antigen binding domains to nanoparticles

[0192] Those skilled in the art will be familiar with standard techniques for conjugating antigen binding proteins to nanoparticles, including metallic nanoparticles or magnetic nanoparticles as described herein.

[0193] For example, standard techniques, such as the techniques described herein in the examples, can be used to covalently connect antigen-binding proteins to nanoparticles. Other examples of methods for nanoparticles to be conjugated to antigen-binding proteins include those described in the art (see, for example, Jazayeri et al. (2016) Various methods of gold nanoparticles (GNPs) conjugated 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 for Anti-HER2 Multifunctional Gold Nanoparticles. Cancer 11 (870): 1-22; The document is incorporated herein by reference).

[0194] Those skilled in the art will appreciate that the conjugation method will depend 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 bonds (including conjugation to carboxyl groups present on target binding proteins using carbodiimides), maleimide bonds, azide bonds, biotin bonds, hydroxyl bonds, methyl bonds, neutral avidin bonds, NHS (N-hydroxysuccinimide) moiety bonds, thiol bonds, PEG moiety bonds, amide bonds, hydrazone bonds, amine bonds (including conjugation to amino groups present on target binding proteins using carbodiimides).

[0195] In a preferred embodiment, the conjugation method is covalent conjugation involving amide, hydrazone and / or amine bonds; achieved via carbodiimide and / or maleimide chemistries.

[0196] In some embodiments, conjugation is achieved using a commercially available nanoparticle conjugation kit (e.g., supplied by Ocean Nanotech), such as a carboxyl magnetic iron oxide nanoparticle kit or an amine iron oxide nanoparticle kit, or a modified protocol based on the methods described in such kits. Again, these techniques will be within the skill of those skilled in the art.

[0197] In any embodiment, the antigen binding protein can be conjugated to the nanoparticles using the methods as described herein in the Examples.

[0198] In any embodiment, the first binding protein and the second binding protein are covalently conjugated to the nanoparticles by different chemistries. The use of different chemistries facilitates the preparation of composite nanoparticles.

[0199] In one example, one binding protein can be covalently conjugated to the magnetic nanoparticles via a polyethylene glycol (PEG) moiety, and the other binding protein can be covalently conjugated to the metal nanoparticles via a thiol linker, wherein optionally the thiol is at least one of lipoic acid, monolipoic acid, dilipoic acid, and trilipoic acid.

[0200] Conditions to be treated, dosage and administration

[0201] The bispecific nanoparticles of the present invention are particularly suitable for treating or preventing cancer.

[0202] Broad examples include breast tumors, colorectal tumors, adenocarcinomas, mesotheliomas, bladder tumors, prostate tumors, germ cell tumors, hepatocellular carcinoma / cholangiocarcinoma, neuroendocrine tumors, pituitary neoplasms, 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 tumors, bladder tumors, bone tumors, cervical tumors, esophageal tumors, laryngeal tumors, liver tumors, lung tumors, vaginal tumors, and Wilm's tumor.

[0203] Examples of specific cancers include, but are not limited to, adenocarcinoma, adenoma, adenofibroma, adenolymphoma, adenoma, AIDS-related cancers, acoustic neuroma, acute lymphoblastic leukemia, acute myeloid leukemia, adenoid cystic carcinoma, adrenocortical carcinoma, agnogenic myeloid metaplasia, metaplasia), alopecia, alveolar soft tissue sarcoma, ameloblastoma, angiokeratoma, angiolymphoid hyperplasia with eosinophilia, angiomatous sclerosis, angiomatosis, apudoma, 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, branchioma, CNS tumors, carcinoid tumors, cervical cancer, childhood brain tumors, childhood cancer, childhood leukemia, childhood soft tissue sarcoma, chondrosarcoma, choriocarcinoma, chronic lymphocytic leukemia, chronic myeloid leukemia, colorectal cancer, cutaneous T-cell lymphoma, carcinomas (e.g., Walker's carcinoma, basal cell carcinoma, basosquamous cell carcinoma, Brown-Pearce carcinoma, ductal carcinoma, Ehrlich tumor tumour), Krebs 2 carcinoma, Merkel cell carcinoma, mucinous carcinoma, non-small cell lung cancer, oat cell carcinoma, papillary carcinoma, scirrhous carcinoma, bronchiolar carcinoma, bronchogenic carcinoma, squamous cell carcinoma and transitional cell carcinoma), carcinosarcoma, cervical dysplasia, cystosarcoma phyllodes, cementoma, chordoma, bud tumor, chondrosarcoma, chondroblastoma, craniopharyngioma, cholangioma, cholesteatoma, cylindroma, cystadenocarcinoma, cystadenoma, dermatofibrosarcoma-protuberans, desmoplastic small round cell tumor, ductal carcinoma, dysgerminoma, endocrine carcinoma, endometrial carcinoma, ependymoma, esophageal carcinoma, Ewing's sarcoma sarcoma), extrahepatic bile duct cancer, eye cancer, eye: melanoma, retinoblastoma, fallopian tube cancer, Fanconi anaemia, fibroma, fibrosarcoma, gallbladder cancer, stomach cancer, gastrointestinal cancer, gastrointestinal carcinoid, genitourinary cancer, germ cell tumors, gestational trophoblastic disease, glioma, gynecological cancer, giant cell tumor, ganglioneuroma, glioma, glomus tumor, granulosa cell tumor, ovarian gynandroblastoma, hematological malignancies, hairy cell leukemia, head and neck cancer, hepatocellular carcinoma, hereditary breast cancer, histiocytosis, Hodgkin's diseasedisease), human papillomavirus, hydatidiform mole, hypercalcemia, hypopharyngeal cancer, hamartoma, hemangioendothelioma, hemangioma, hemangiopericytoma, hemangiosarcoma, hemangiosarcoma, histiocytic disorder, malignant histiocytosis, histiocytoma, hepatocellular carcinoma, sweat adenoma, chondrosarcoma, immunoproliferative small cell, opoma, 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, leukocytic sarcoma, leydig cell tumor, liposarcoma, leiomyoma, leiomyosarcoma, lymphangioma, lymphangiocytoma, lymphangioma, lymphangiofibroma, lymphangiosarcoma, male breast cancer, malignant rhabdoid tumor of kidney, medulloblastoma, melanoma, Merkel cell cancer, mesothelioma, metastatic cancer, oral cancer, multiple endocrine neoplasms, mycosis fungoides, myelodysplastic syndrome, myeloma, myeloproliferative disorders, malignant carcinoid syndrome, carcinoid heart disease, medulloblastoma, meningioma, melanoma, mesenchymal tumor, mesonephroma, mesothelioma, myoblastoma, myoma, sarcoma, myxoma, myxosarcoma, nasal cancer, nasopharyngeal cancer, Wilms tumor, neuroblastoma, neurofibromatosis, Nijmegen breakage syndrome syndrome), non-melanoma skin cancer, non-small cell lung cancer (nsclc), schwannoma, neuroblastoma, neuroepithelioma, neurofibromatosis, neurofibrocarcinoma, 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 cancer, parathyroid cancer, parotid gland cancer, penile cancer, peripheral neuroectodermal tumors, pituitary cancer, polycythemia vera, prostate cancer, bone tumor, osteosarcoma, ovarian cancer, papilloma, paraganglioma, non-pheochromocytic paraganglioma, pineal tumor, plasmacytoma, proto-oncogenes, rare cancers and related conditions, renal cell carcinoma, retinoblastoma, rhabdomyosarcoma, Rothmund-Thomson syndromesyndrome), reticuloendotheliosis, rhabdoid tumor, salivary gland cancer, sarcoma, schwannoma, Sezary syndrome, skin cancer, small cell lung cancer (sclc), small intestinal cancer, soft tissue sarcoma, spinal cord tumor, squamous cell carcinoma (skin), gastric cancer, synovial sarcoma, sarcomas (e.g., Ewing's experimental sarcoma, Kaposi's sarcoma, and mast cell sarcoma), Sertoli cell tumor, synovioma, testicular cancer, thymic cancer, thyroid cancer, transitional cell carcinoma (bladder), transitional tumors-(renal-pelvic- / -ureter), trophoblastic carcinoma, teratoma, thecal cell tumor, thymoma, trophoblastic tumor, urethral cancer, urinary system cancer, uroplakins, uterine sarcoma, uterine cancer, vaginal cancer, vulvar cancer, Waldenstrom's-macroglobulinemia, and Wilms' tumor.

[0204] In some examples, the nanoparticle bioconjugates as described herein can be administered orally, parenterally, by inhalation spray, absorption, absorption, topically, rectally, nasally, buccally, vaginally, intraventricularly, by implanted reservoirs in dosage formulations containing conventional nontoxic pharmaceutically acceptable carriers, 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.

[0205] Methods for preparing nanoparticle bioconjugates into a form suitable for administration to a subject (e.g., a pharmaceutical composition) are known in the art and include, for example, methods described in Remington's Pharmaceutical Sciences (18th ed., Mack Publishing Co., Easton, Pa., 1990) and US Pharmacopeia: National Formulary (Mack Publishing Co., Easton, Pa., 1984).

[0206] The pharmaceutical composition of the present invention is particularly suitable for parenteral administration, such as intravenous administration or intraluminal administration of a body cavity or organ or joint. The composition for administration generally comprises a solution of an antigen binding site dissolved in a pharmaceutically acceptable carrier, such as an aqueous carrier. Various aqueous carriers can be used, such as buffered saline, etc. The composition can contain pharmaceutically acceptable auxiliary substances required for close physiological conditions, such as pH regulators and buffers, toxicity regulators, etc., such as 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 can vary over a wide range and will be selected based on the volume of fluid, viscosity, body weight, etc., according to the specific 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 can also be used. Liposomes can also be used as carriers. The vehicle can contain a small amount of additives that enhance isotonicity and chemical stability, such as buffers and preservatives.

[0207] After formulation, the nanoparticle bioconjugates of the present invention will be administered in a manner compatible with the dosage formulation and in a therapeutically / preventively effective amount. The formulation is easily administered in various dosage forms, such as injectable solutions of the type described above, but other pharmaceutically acceptable forms, such as tablets, pills, capsules or other oral solids, suppositories, vaginal suppositories, nasal solutions or sprays, aerosols, inhalants, liposome forms, etc., can also be envisioned. Drug "slow release" capsules or compositions can also be used. Slow release formulations are typically designed to provide constant drug levels over an extended period of time and can be used to deliver the antigen binding sites of the present invention.

[0208] The appropriate dosage of the nanoparticle bioconjugates of the invention will vary depending on the specific nanoparticle bioconjugate, the condition to be treated, and / or the subject being treated. A skilled physician is capable of determining an appropriate dosage, for example, by starting with a suboptimal dosage and incrementally modifying the dosage to determine an optimal or useful dosage. Alternatively, to determine an appropriate dosage for treatment / prevention, data from cell culture assays or animal studies are used, wherein the appropriate dosage is within a circulating concentration range that includes the ED of the active compound with little or no toxicity. 50 The dosage may vary within this range depending upon the dosage form employed and the route of administration utilized. The therapeutically / prophylactically effective dose can be estimated initially from cell culture assays. In animal models, the dose can be formulated to achieve an IC including as determined in cell culture. 50 (i.e., the concentration or amount of the 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.

[0209] In some examples, the methods of the invention comprise administering a prophylactically or therapeutically effective amount of a nanoparticle bioconjugate described herein.

[0210] The term "therapeutically effective amount" refers to an amount that improves the prognosis and / or status of a subject and / or reduces or inhibits one or more symptoms of a clinical condition described herein to a level below that observed and accepted as a clinical diagnosis or clinical characteristic of the condition when administered to a subject in need of treatment. The amount applied to the subject will depend on the specific characteristics of the condition to be treated, the type and stage of the condition to be treated, the mode of administration, and the characteristics of the subject, such as general health, other diseases, age, sex, genotype, and body weight. Those skilled in the art will be able to determine a suitable dosage based on these and other factors. Therefore, this term should not be construed as limiting the present invention to a specific amount, e.g., the weight or amount of protein, but the present invention encompasses any amount of antigen binding sites sufficient to achieve the results in a subject.

[0211] As used herein, the term "prophylactically effective amount" should be understood to refer to a sufficient amount of protein to prevent or inhibit or delay the onset of detectable symptoms of one or more clinical conditions. Those skilled in the art will appreciate that such amounts will vary depending on, for example, the specific antigen binding site administered and / or the specific subject and / or the type or severity or level of the condition and / or the cause (genetic or otherwise) of the condition. Therefore, this term should not be interpreted as limiting the invention to a specific amount, e.g., weight or amount of antigen binding sites, but rather the invention encompasses any amount of antigen binding sites sufficient to achieve the described result in a subject.

[0212] The present invention further comprises a kit comprising the nanoparticle bioconjugate of the present invention. The kit may further comprise a pharmaceutically acceptable carrier.

[0213] Optionally, according to any embodiment, the kits of the invention are packaged with instructions for use in the methods described herein.

[0214] In preferred embodiments, the kit comprises components for producing the nanoparticle bioconjugates of the invention, such as components of the nanoparticles and a binding protein for conjugation thereto.

[0215] Examples

[0216] Here, the inventors 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 the antibodies nimotuzumab or panitumumab) while causing T cell activation by binding to T cells (e.g., by using anti-CD3 scFv or anti-CD16 scFv), also conjugated to the nanoparticles. By chemically conjugating to antibodies in the nanoparticle platform, the nanoflowers of the present invention are expected to produce a synergistic effect of the two binding proteins, which was examined in vitro in cancer cells.

[0217] The inventors believe that this is the first proof of concept to demonstrate the synergistic effect of dual antibody targeted immunotherapy against cancer through iron oxide-gold nanoparticles. In one example, the developed nanodelivery system showed that the exemplary bispecific iron oxide-gold nanoparticles were an effective bispecific bioconjugate for retargeting T cells to EGFR-expressing cells in vitro at a rate 69.7% higher than that of free nimotuzumab.

[0218] Example 1: Materials and Methods

[0219] Material

[0220] Tetrachloroauric acid (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-pyranoglucopyranoside, (2-(N-morpholino)ethanesulfonic acid) (MES), PD-10 desalting column 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 is from Biocon (Bangalore, India) and is also referred to herein as Nmab. Trastuzumab (also referred to herein as Tmab) is from Genentech. Panitumumab (referred to herein as Pmab) is from Amgen. Single chain variable fragments (28-30 KDa) of anti-CD3 scFv were synthesized by the inventors and purchased from Virogen (Brighton, MA, USA). Single chain variable fragments of anti-CD16 scFv were purchased from Virogen (Brighton, MA, USA).

[0221] Instrumentation and characterization of NPs

[0222] Nanoparticles (NPs) were visualized by transmission electron microscopy (TEM) using a JEOL microscope (JEOL JEM 1400, Gatan Inc., USA) at an accelerating voltage of 120 kV. 8 μl of diluted (dilution ratio 1:3) NPs were applied to a carbon-coated copper grid. The grid was placed at room temperature for 2 hours to dry and imaged using a transmission electron microscope.

[0223] The Fe content was determined using a UV-Vis spectrophotometer (Shimadzu, Japan). 3 O 4 @Au NP, Fe 3 O 4@Absorption spectra of AgNP and antibody conjugated in hexane solution or aqueous solution, depending on the nature of NP. The hydrodynamic size distribution and zeta potential of NP were measured by a particle size analyzer Zeta-sizer Nano ZS90 (Malvern, Worcestershire, UK) at a scattering angle of 90 at 25 ° C, in triplicate, adjusted for the reflectivity index of the dispersant. This reports the particle size distribution, average particle size and polydispersity index. Fourier transform infrared spectra (FTIR) were recorded on a Shimadzu IRTracer-100 spectrophotometer. Inductively coupled plasma mass spectrometry using a PerkinElmer 300X ICP-MS instrument (PerkinElmer, Waltham, MA, USA) was used to determine the final concentration of NP. For this reason, NPs were atomized with aqua regia. All mass spectra were acquired using a matrix-assisted laser desorption ionization / time of flight (MALDI-TOF) Bruker AutoflexSpeed ​​LRF with a 1000 Hz Smartbeam-II laser. Spectra were recorded in normal 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 and suppression: up to 18000 Da, mass range: 20,000-400,000 Da. Data analysis was performed using flex control version 3.4, flex analysis version 3.4.

[0224] To evaluate NP antibody conjugation using SDS-PAGE, samples were loaded onto SDS-PAGE and electrophoresis was performed at 100 V for 30 minutes followed by incubation with Coomassie Brilliant Blue to detect protein bands. TM Protein bands were visualized and analyzed using the XR system and analyzed using Bio-Rad Image software. Nano-Drop 1000 (Thermo Scientific) spectrophotometer measurements were used to confirm conjugation by measuring absorbance at 280 nm with a 0.1X PBS baseline. Enzyme-linked immunosorbent assays (ELISAs) were performed with absorbance measurements at 450 nm using a microplate reader. "Nude" NPs and conjugated NPs were measured with positive and negative controls (assay buffer).

[0225] Double Fe 3 O 4@Au-NP (bispecific NP conjugated with anti-EGFR Nmab and anti-CD3 scFv) and Nmab-Fe 3 O 4 The UV spectrum of @Au NPs (i.e., nanoparticles containing only anti-EGFR Nmab) is as follows Figure 4 As shown. The presence of conjugated antibodies can be determined by subtracting the amount of conjugated antibodies obtained after centrifugation from the total amount of antibodies loaded before centrifugation. The presence of conjugated antibodies is determined by a sharp peak at approximately 260 nm.

[0226] Cell lines

[0227] A431 is a human epithelial cancer cell line with known overexpression of EGFR. Jurkat, clone E6-1 cells (TIB-125) are a CD3-positive acute T-cell leukemia. Jurkat cells are 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 cell model for metastatic breast cancer. MDA-MB 468 is another epithelial human breast cancer cell line that provides a cell model for metastatic breast cancer.

[0228] at 37°C in a 5% CO 2 In a humidified incubator, cells were cultured in flasks containing Dulbecco's modified Eagle's medium (DMEM) low glucose medium or Jurkat's RPMI-1640, which was supplemented with 10% fetal bovine serum (FBS) and 1% penicillin / streptomycin. Depending on the growth rate of the cells, passage was performed every 2-3 days. Before collection, the cells were cultured to a confluence of approximately 80% to 90%. During collection, the cells were washed with PBS, then trypsinized with 2 ml of trypsin, and incubated for 10 minutes to separate the cells from the flask. Trypsin was neutralized by adding 8 ml of fresh supplemented culture medium, and then 2 ml of the collected cells were transferred to a new flask and resuspended with fresh culture medium. Before each experiment, cell viability was determined using a hemocytometer.

[0229] Preparation of anti-CD3 scFv

[0230] The construct of the specific His6-tagged anti-CD3 scFv antibody was designed from the OKT3 mAb and expressed according to the method of previous studies (Kipriyanov et al. (1997) Journal of Immunology Methods 15(200):67-77). The anti-CD3 scFv sequence was obtained from the protein database with the code (1SY6). The construct included a linker peptide (Gly4Ser) 3 (SEQ ID NO:2) between the light variable chain and the heavy variable chain. The construct was cloned into the gWiz expression plasmid and then transformed into competent Escherichia coli. Standard techniques using the mammalian expression system of HEK 293 cells were used to produce the anti-CD3 scFv.

[0231] The antibody was purified using a His Trap 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.

[0232] Table 2: Antibody and peptide sequences

[0233]

[0234]

[0235] Fe 3 O 4 Preparation of @Au NPs

[0236] Fe was synthesized according to a previously reported procedure with slight modifications. 3 O 4 @Au NP (Yu, Chen et al. 2005). Synthesis of Fe by thermal decomposition under inert conditions 3 O 4 @Au NPs (Figure 1). Initially, 6 mmol oleic acid, 6 mmol oleylamine and 10 mmol 1,2-hexadecanediol were mixed with 20 ml 1-octadecene (ODE) in a 50 ml round-bottom flask equipped with a thermometer, a flow control adapter, a reflux condenser and a rubber septum and stirred at 160 °C for 30 min under a gentle nitrogen flow. Then, under nitrogen blanketing, 0.3 ml Fe(CO) 5 After 3 minutes, 40 mg HAuCl 4 ·3H 2A degassed gold precursor solution consisting of 10 ml of O, 1.5 ml of oleylamine and 5 ml of ODE was added dropwise to the hot solution at 180°C over 10 minutes to ensure adequate mixing. The solution immediately turned deep red after injection, indicating the formation of gold nanoparticles. The solution was then slowly heated to 310°C and refluxed for 45 minutes. After cooling at room temperature, 40 ml of isopropanol was added to the solution and centrifuged at 8000 rpm for 10 minutes 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. In the presence of oleylamine, Fe 3 O 4 @Au NPs were dissolved in hexane for further use ( Figure 1B ).

[0237] Fe 3 O 4 Modification of @Au NPs

[0238] Dissolve in 1 ml dimethylformamide, 2 ml CHCl 3 and 10 mg anhydrous Na 2 CO 3 30 mg α,ω-bis{2-[(3-carboxy-1-oxopropyl)amino]ethyl}PEG (Mr=3,000), 2 mg N-hydroxysuccinimide, 3 mg dicyclohexylcarbodiimide and 1.5 mg dopamine hydrochloride in a mixture of 1% α,ω-bis{2-[(3-carboxy-1-oxopropyl)amino]ethyl}PEG (Mr=3,000), 2 mg N-hydroxysuccinimide, 3 mg dicyclohexylcarbodiimide and 1.5 mg dopamine hydrochloride were added to transfer the hydrophobic nanoparticles into an aqueous medium (to improve solubility). The solution was stirred at 37°C for 2 hours, and 5 mg Fe 3 O 4 @Au NPs, and the resulting solution was stirred at 37 ° C overnight under nitrogen. The modified NPs were precipitated by adding 5 ml of hexane and ethanol and collected by centrifugation at 16,500 × g, and the surfactants and other salts were removed by dialysis in PBS (molecular weight cutoff, 10 kDa) for 24 hours. Finally, it was 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).

[0239] Statistical analysis

[0240] Results are expressed as mean ± SD. Data were analyzed using GraphPad Prism (GraphPad Software, Version 9, La Jolla, CA, USA). One-way ANOVA post hoc Fisher LSD was used to calculate significance. * P < 0.05 was considered significant.

[0241] Example 2: Fe 3 O4 Synthesis and characterization of @Au NPs

[0242] A 40 nm Fe 3 O 4 @Au NPs, which yields monodisperse highly stable NPs.

[0243] Transmission electron microscopy (TEM) was used to characterize the flower-shaped Fe 3 O 4 @Synthesis of Au NPs. Au NPs were observed to be spheres with an average core size of 11 nm. 3 O 4 The NPs showed a 3-4-petal shape with an average diameter of 14 nm.

[0244] Subsequently, the organic ligands were replaced by dopamine forming a 4 nm polydopamine thin layer, leaving the catechol groups on the NP surface for further conjugation. Figure 2 A shows a 40 nm Fe 3 O 4 TEM image of @Au NPs (gold NPs are shown in black due to heavy atom effects). After ligand exchange, the NPs were well dispersed and the hydrodynamic size of the dopamine-coated NPs in the DLS solution was 136 ± 2.49 nm, which is much larger than the unmodified NPs 35.19 ± 0.04 nm (Table 1). The increase in size may be due to the formation of hydrogen bonds or the formation of a thin layer of polydopamine during ligand exchange.

[0245] After surface modification, the zeta potential of the flower-shaped NPs after modification was approximately −30 mV. Figure 2 B shows Fe before and after modification 3 O 4 FTIR (Fourier transform infrared spectroscopy) spectrum of @Au NP. 3300cm -1 The characteristic peak at 1653cm corresponds to the catechol of dopamine. -1 The characteristic peak C=O band at indicates the successful activation of dopamine molecules on the surface of NPs. NPs are usually protected with dopamine to improve their dispersibility and stability.

[0246] Medical and biological applications require Fe 3 O 4 @Au NPs have a water solubility. For this reason, the oleic acid coating of the NPs was modified to a water-soluble ligand by ligand exchange. In the present example, PEG was used to align the oil phase of the NPs with the water phase, dopamine was replaced with oleic acid on the surface of the iron NPs, and then the amino groups of dopamine were reacted with the carboxyl groups of PEG under the catalysis of the EDC-sulfo-NHS system.

[0247] To generate Fe bound to nimotuzumab or dopamine 3 O 4 @Au NPs, EDC-sulfo-NHS system for activation of Fe modified with PEG 3 O 4 @The carboxyl groups of Au NPs were prepared as described above. Before adding nimotuzumab, excess EDC and sulfo-NHS were removed to avoid self-crosslinking of the antibody. After activation using the EDC-sulfo-NHS system, nimotuzumab was activated by PEG (M r = 2,000) and dopamine via a peptide bond-forming condensation reaction with the modified Fe 3 O 4 @Au NP conjugation.

[0248] Table 1: Fe 3 O 4 @Hydrodynamic diameter size (Z-average), PDI, zeta potential (ζ) and absorption maximum (λmax) and TEM size (nm) of Au NPs.

[0249]

[0250] NP: nanoparticle format, PDI: polydispersity index, TEM: transmission electron microscopy.

[0251] Example 3: Anti-EGFR mAb (Nmab) and anti-CD3 scFv (mAb-Fc 3 O 4 @Au-αCD3 NP) conjugated Fe 3 O 4 @Au NP

[0252] Fe 3 O 4 @Au NPs were conjugated with nimotuzumab and anti-CD3 scFv to generate Nmab-Fe 3 O 4 @Au-αCD3scFvNP

[0253] As briefly described in this article, the anti-EGFR antibody nimotuzumab was conjugated to Fe 3 O 4 NPs, while anti-CD3 scFv antibodies were linked to Au NPs using a thiol linker. 1 mg Fe 3 O 4@Au NPs were 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 minutes. After gentle mixing at room temperature for 30 minutes, the solution was subjected to a PD-10 column to remove excess EDC and sulfo-NHS. 100 μg of nimotuzumab was then added to the conjugate for 3 hours while gently shaking to allow the Fe 3 O 4 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 incubation at room temperature overnight to functionalize the Au side. Prior to immobilization of the anti-CD3 scFv, a thiol linker was added to the anti-CD3 scFv. Unbound antibody and Fe 3 O 4 @Au NPs were separated from antibody-conjugated NPs.

[0254] Nmab-Fe 3 O 4 @ Mass spectrometry analysis of Au-αCD3 scFvNP revealed that Nimotuzumab has a specific peak at m / z approximately 148KDa ( Figure 3 ), which is in Au-Fe 3 O 4 In addition, the unbound antibody in the collected supernatant was analyzed by UV-vis measurement ( Figure 4 NanoDrop 1000 spectrophotometer readings of the protein confirmed a single peak of conjugation detected in the conjugated sample ( Figure 5 ). SDS-PAGE was also used to confirm antibody NP conjugation ( Figure 6 ).

[0255] Use of effector:target cells assessed by cytokine production by Nmab-Fe 3 O 4 Efficacy of @Au-αCD3scFv NPs

[0256] One day before the assay, A431 cells were plated at 4 × 10 3 10 cells / well were plated 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 NP; anti-CD3 scFv (5 μg / ml); Nmab-F 3 O 4 @Au NP; or double Fe 3 O 4@AuNP (bispecific NP conjugated with Nmab and αCD3 scFv). After 24 hours, aliquots of the culture supernatant were collected and the levels of IL-2 were measured by using a commercially available ELISA test kit (Sydney, New South Wales, Australia). The samples were analyzed using a plate reader.

[0257] Fig.13 shows that the use of double Fe 3 O 4 The production of IL-2 (97.5±10 pg / ml) was specifically induced by Au NP (bispecific NP conjugated with Nmab and αCD3 scFv) after 24 hours of stimulation. In contrast, NP conjugated with αCD3 scFv alone activated low-level IL-2 production (5.5±0.7 pg / ml) by binding to T cells, and was not significantly increased after stimulation with unconjugated NP or Nmab-FcFv. 3 O 4 No IL-2 stimulation was observed after treatment with @Au NPs. These results suggest that in vitro 3 O 4 @Au NPs are more potent T cell activators than NPs conjugated with anti-CD3 scFv alone.

[0258] Target: Effector cells evaluated by cytotoxicity assay of Nmab-Fe 3 O 4 Efficacy of @Au-αCD3scFv NPs

[0259] To evaluate the efficacy of bispecific nanoparticle conjugation, a cytotoxicity assay was performed. 3 O 4 @Au NP particles in the logarithmic growth phase of A431 cells at 4x10 3 Cells / well were plated in triplicate in 96-well microtiter plates to grow adherent cells. 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 at a density of 4000 cells per well in 96-well culture plates and various concentrations of DMEM buffer were added. At 12 hours, the medium was removed and replaced with 100 μl of medium containing 1% FBS.

[0260] When using Fe concentration>2500μg / ml 3 O 4 Before Au NP treatment, the viability of co-culture cells was not significantly affected ( Figure 7 ).

[0261] A series of MTT assays were then performed at different concentrations to evaluate the expression of Fc-binding proteins conjugated to nimotuzumab and / or anti-CD3 scFv for the five treatment groups. 3 O 4 @Au nanoparticles cytotoxic effects on A431 and Jurkat clone E6-1 cells: Group A, Nmab; Group B, Fe 3 O 4 @Au NP; Group C, Nmab-Fe 3 O 4 @Au NP; Group D, Nmab-Fe 3 O 4 @Au-αCD3 scFv NP; Group E, cisplatin; and Group F, treated with fresh medium. After incubation for 72 hours, treatment and effector cells were removed. 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 DMSO was added to dissolve the cells. After incubation at 37°C for 30 minutes and thorough mixing, viability was measured at 570 nm by a microplate reader. The spectrophotometer was calibrated to zero absorbance using cell-free culture medium. Cell viability was calculated and plotted against concentration, and the data were evaluated using Prism9 (GraphPad software version 8, La Jolla, California, USA).

[0262] with Fe 3 O 4 Compared with @Au NP, Nmab-Fe 3 O 4 @Au-αCD3scFv NPs (i.e., nanoparticles comprising antigen binding proteins for binding to EGFR and CD3) showed a significant increase in cytotoxicity.

[0263] Figure 8 Shown, with a single conjugated Nmab-Fe 3 O 4 @Au nanoparticles compared with Nmab-Fe 3 O 4 After incubation with @Au-αCD3scFv NP, the viability of the co-cultured cells was significantly reduced.

[0264] Fig. 9 Similarly, it was shown that the nanoparticles conjugated with Nmab (i.e., naked antibody at the same antibody concentration) or nimotuzumab alone had a significantly higher affinity to Nmab-Fe 3 O 4 After incubation with @Au-αCD3scFv NP, cell viability was significantly reduced.

[0265] To further evaluate Fc conjugated to Nmab and / or anti-CD3 scFv 3 O 4 @ Cytotoxic effect of Au nanoparticles. Regarding additional cancer cells, MTT assay was 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-Fe 3 O 4 @Au NP; Group C, Nmab-Fe 3 O 4 @Au-αCD3scFv NP. The control group was treated with fresh culture medium alone. One day before the assay, 3 O 4 @Au NP particles in the logarithmic growth phase of MDA-MB468 cells at 4x10 3 Cells / wells were plated in triplicate in 96-well microtiter plates to grow adherent cells. The cells were then seeded onto 96-well culture plates in RPMI medium at a density of 4000 cells per well. At 12 hours, the medium was removed and replaced with 100 μl of medium containing 1% FBS. Treatment was added, then removed after 72 hours of incubation, and the 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 incubation at 37°C for 2-4 hours, the supernatant was removed. 100 μl of DMSO was added to dissolve the cells. After incubation at 37°C for 30 minutes and thorough mixing, viability was measured at 570 nm by a microplate reader. The spectrophotometer was calibrated to zero absorbance by using a cell-free culture medium. Cell viability was calculated and plotted for concentration, and data were evaluated using Prism 9 (GraphPad software version 8, La Jolla, California, USA).

[0266] with Fe 3 O 4 Compared with @Au NP, Nmab-Fe 3 O 4 @The cytotoxicity of co-cultured cells treated with Au-αCD3scFv NPs was significantly increased.

[0267] Fig.18 Shown, with a single conjugated Nmab-Fe 3 O 4 @Au nanoparticles compared with Nimotuzumab-Fe 3 O 4 After incubation with @Au-αCD3scFv NP, the viability of the co-cultured cells was significantly reduced.

[0268] Fig.19 Similarly, it was shown that the NPs conjugated to nimotuzumab-Fc showed a significant difference in the expression of NPs compared to Nmab (i.e., naked antibody at the same antibody concentration) or nanoparticles conjugated to Nmab alone. 3 O 4 After incubation with @Au-αCD3scFv NP, cell viability was significantly reduced.

[0269] Fe 3 O 4 @Au NPs were conjugated with anti-EGFR monoclonal antibody and anti-CD3 scFv to generate αCD3scFv-Fe 3 O 4 @Au- Nmab NP

[0270] As briefly described herein, in order to evaluate alternative configurations of bispecific nanoparticles in which the first conjugate is linked to gold and the second conjugate is linked to Fe 3 O 4 The anti-CD3 scFv was linked to the Fc using the carbodiimide method. 3 O 4 NPs, while the anti-EGFR antibody Nimotuzumab was linked to Au NPs using a thiol linker. 1 mg Fe 3 O 4 @Au NPs were 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 minutes. After gentle mixing at room temperature for 30 minutes, the solution was subjected to a PD-10 column to remove excess EDC and sulfo-NHS. Then 5 μg of scFv CD3 was added to the conjugate and gently shaken for 3 hours to allow the Fe of the nanoflowers to 3 O 4 After 3 hours of incubation, 100 μl of Nmab antibody was added and the mixture was placed on a shaker for overnight incubation at room temperature to functionalize the Au side. Prior to immobilization of Nimotuzumab, a thiol linker was added to Nmab. Unbound antibody and Fe were removed using 300K ultrafiltration. 3 O 4 @Au NPs were separated from antibody-conjugated NPs.

[0271] Target used: αCD3scFv-Fc assessed by cytotoxicity assay on effector cells 3 O 4 @Au-Nmab NP efficacy

[0272] To evaluate the efficacy of bispecific nanoparticle conjugation, a cytotoxicity assay was performed. A431 cells in logarithmic growth phase were cultured at 4 × 10 3Cells / well were plated in triplicate in 96-well microtiter plates to grow adherent cells. MTT colorimetric assay was used as described herein. Cells were seeded at a density of 4000 cells per well in 96-well culture plates in RPMI medium. At 12 hours, the medium was removed and replaced with 100 μl medium containing 1% FBS. A series of MTT assays were then performed to evaluate the effects of αCD3scFv-Fe at different concentrations of Nmab or iron compared to controls treated with fresh medium. 3 O 4 @Cytotoxic effect of Au-Nmab nanoparticles. After 72 hours of incubation, the treatment was removed and the 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 MTT solution was removed. 100 μl of DMSO was added to dissolve the cells. After incubation at 37°C for 30 minutes and thorough mixing, viability was measured at 570 nm by a microplate reader. The spectrophotometer was calibrated to zero absorbance using a cell-free culture medium. Cell viability was calculated and plotted for concentration, and data were evaluated using Prism 9 (GraphPad Software Version 8, La Jolla, California, USA).

[0273] Compared with no treatment (control), αCD3scFv-Fc 3 O 4 @The cytotoxicity of co-cultured cells treated with Au-Nmab NPs was significantly increased.

[0274] Fig. 20 It is shown that, compared with the control, in the presence of αCD3scFv-Fe 3 O 4 After incubation with @Au-Nmab NPs, the viability of the co-cultured cells was significantly reduced.

[0275] Fe 3 O 4 Cellular uptake of @Au NPs

[0276] It was demonstrated that the functionalized Fe 3 O 4 @Au NP cellular uptake into cancer cell lines ( Fig.10 After incubation with 50 μg / ml iron for 12 h, the intracellular iron and gold contents were quantified by ICP-MS. Compared with untreated cells, the iron concentration of cells treated with dual Fe3O4@Au NPs after incubation increased significantly to 373.92 ± 36.1 fg / cell ( Fig.10 A). Prussian blue staining ( Fig.10 B) For quantitative determination of the use of double Fe 3 O 4@Au NP (bispecific NP conjugated with Nmab and αCD3scFv) or Nmab-Fe 3 O 4 @Au NP-treated or untreated co-cultured Jurkat and A431 cells.

[0277] Example 4: Anti-HER2 mAb (trastuzumab) and anti-CD3 scFv (mAb-F 3 O 4 @Au-αCD3 NP) conjugated Fe 3 O 4 @Au NP

[0278] Fe 3 O 4 Preparation of @Au NPs

[0279] Fe was synthesized by thermal decomposition according to the procedure described above with slight modifications. 3 O 4 @Au NP. Briefly, 6 mmol oleic acid, 6 mmol oleylamine and 10 mmol 1,2-hexadecanediol were mixed with 20 ml 1-octadecene (ODE) in a 50 ml round-bottom flask equipped with a thermometer, flow control adapter, reflux condenser, rubber septum and stirred at 160 °C under a gentle nitrogen flow for 30 min. Then, under nitrogen blanketing, 0.3 ml Fe(CO) 5 After 3 minutes, 40 mg HAuCl 4 A degassed gold precursor solution consisting of 3H2O, 1.5ml oleylamine and 5ml ODE was added dropwise to the hot solution at 180°C over 10 minutes to ensure adequate mixing. The solution immediately turned deep red after injection, indicating the formation of gold nanoparticles. The solution was then slowly heated to 310°C and refluxed for 45 minutes. After cooling at room temperature, 40mL of isopropanol was added to the solution and centrifuged at 8000rpm for 10 minutes to remove large particles. The precipitate was redispersed in hexane and centrifuged again at 7000rpm to remove any undispersed material. Ethanol was then added to the solution and centrifuged again three times to obtain a brown dispersion. In the presence of oleylamine, Fe 3 O 4 @Au NPs were stored in hexane for further use.

[0280] Fe 3 O 4 @Au NP modification can improve solubility

[0281] To improve the solubility of FeAu NPs, a modified protocol was used to transfer the hydrophobic nanoparticles into aqueous media. 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 1 ml of dimethylformamide, 2 ml of CHCl 3 and 10 mg anhydrous Na 2 CO 3 The solution was stirred at 37 °C for 2 hours and 5 mg Fe 3 O 4 @AuNP, and the resulting solution was stirred at 37 ° C overnight under N2 flow. The modified NPs were precipitated by adding 5 mL of hexane and ethanol and collected by centrifugation at 16,500 × g, and the surfactants and other salts were removed by dialysis in PBS (molecular weight cutoff, 10 kDa) for 24 hours. Finally, it was 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).

[0282] Fe 3 O 4 @Au NP conjugation with monoclonal antibody trastuzumab (Tmab) and anti-CD3 scFv antibody

[0283] Trastuzumab (Tmab) was combined with Fe 3 O 4 NPs. The anti-CD3 scFv antibody was linked to Au NPs using a thiol linker as briefly described here. First, 1 mg Fe 3 O 4 @Au NPs were 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 minutes. After gentle stirring at room temperature for 30 minutes, the solution was subjected to a PD-10 desalting column to remove excess EDC and sulfo-NHS. Tmab (200 μg) was then added to the mixture and gently stirred for 3 hours to allow the Fe 3 O 4 After 3 hours of incubation, 5 μl of anti-CD3 scFv antibody was added 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. Unbound antibody and Fe were removed using a 300K ultrafiltration centrifugation at 1000 rpm for 5 minutes. 3 O 4@ Antibody-conjugated NPs were separated from Au NPs. Unbound antibodies in the collected supernatants were analyzed by absorbance measurement using a Shimadzu 2600 UV-Vis spectrophotometer (Shimadzu Corporation, Japan). The binding efficiency was calculated as the difference between the amount of initially added and unbound antibodies determined in the supernatant.

[0284] Tmab-conjugated Fc assessed by cytotoxicity assay using effector cells 3 O 4 @AuNP efficacy

[0285] SkBr3 cells were seeded in 96-well plates (5000 cells / well). The plates were incubated with 5% CO 2 Incubate and culture overnight at 37 ° C to promote adhesion. Jurkat cells were added at a ratio of 5: 1 effector cells (Jurkat): target cells (SkBr3). Antibody-conjugated nanoparticles were added at different concentrations. Target SkBr3 cells and RPMI-1640 complete medium groups served as controls. After 72 hours of culture, effector cells were removed by 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 percentage of viability was then calculated as [SkBr3 (treated cells) - background] / [SkBr3 (untreated cells) - background] × 100. (Where SkBr3 is the absorbance at 570 nm).

[0286] To evaluate the efficacy of bispecific NP binding, the dual Tmab-Fe 3 O 4 @Au-αCD3scFv (bispecific NP conjugated with Tmab and αCD3scFv) ( Fig.11 AC) and Tmab-Fe 3 O 4 @Au( Fig.11 D) Cytotoxicity assay was performed in co-culture of SkBr3 and Jurkat cells at a 1:5 ratio (target:effector) with NPs (NPs conjugated with Tmab alone). 3 O 4 @Au-CD3 and Tmab-Fe 3 O 4 @Au Both induce cytotoxicity of target cells (SkBr3 cells) via effector cells (Jurkat cells). Dual Tmab-Fe 3 O 4 @Au-CD3NP has significantly higher cytotoxicity than Tmab-Fe 3 O 4 @Cytotoxicity of AuNPs.

[0287] To further evaluate Fc conjugated to Tmab and / or anti-CD3 scFv 3 O 4 @ Cytotoxic effect of Au nanoparticles. Regarding 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-Fe 3 O 4 @Au NP; Group C, Tmab-Fe 3 O 4 @Au-αCD3scFv NP. The control group was treated with fresh culture medium alone. One day before the assay, 3 O 4 @Au NP particles in the logarithmic growth phase of MDA-MB231 cells at 4x10 3 Cells / wells were plated in triplicate in 96-well microtiter plates to grow adherent cells. The cells were then seeded onto 96-well culture plates in RPMI medium at a density of 4000 cells per well. At 12 hours, the medium was removed and replaced with 100 μl of medium containing 1% FBS. Treatment was added, then removed after 72 hours of incubation, and the 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 of DMSO was added to dissolve the cells. After incubation at 37°C for 30 minutes and thorough mixing, viability was measured at 570 nm by a microplate reader. The spectrophotometer was calibrated to zero absorbance by using a cell-free culture medium. Cell viability was calculated and plotted for concentration, and data were evaluated using Prism 9 (GraphPad software version 8, La Jolla, California, USA).

[0288] with Fe 3 O 4 Compared with @Au NP, Tmab-Fe 3 O 4 @The cytotoxicity of co-cultured cells treated with Au-αCD3scFv NPs was significantly increased.

[0289] Fig.21 Shown, with a single conjugated Tmab-Fe 3 O 4 @Au nanoparticles compared with bispecific Tmab-Fe 3 O 4 After incubation with @Au-αCD3scFv NP, the viability of the co-cultured cells was significantly reduced.

[0290] Fig. 22 Similarly, it was shown that the Tmab-Fe nanoparticles showed a significant increase in the expression of free Tmab (i.e., naked antibody at the same antibody concentration) or nanoparticles conjugated to Tmab alone. 3 O 4 After incubation with @Au-αCD3scFv NP, cell viability was significantly reduced.

[0291] Example 5: Anti-EGFR mAb (Panitumumab, Pmab) and anti-CD3 scFv (mAb-Fc 3 O 4 @Au-αCD3 scFvNP) conjugated Fe 3 O 4 @Au NP

[0292] Fe 3 O 4 Conjugation of @Au NPs with monoclonal antibody Pmab and anti-CD3 scFv antibody

[0293] As briefly described in this article, to evaluate the efficacy of bispecific nanoparticles using alternative anti-EGFR conjugates, the anti-EGFR monoclonal antibody panitumumab was conjugated to Fc using the carbodiimide method. 3 O 4 NPs, while anti-CD3 scFv antibodies were linked to Au NPs using a thiol linker. 1 mg Fe 3 O 4 @Au NPs were 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 minutes. After gentle mixing at room temperature for 30 minutes, 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 and shaken for 3 hours while gently shaking to allow the Fe 3 O 4 After 3 hours of incubation, 5 μl of anti-CD3 scFv antibody was added and the mixture was placed on a shaker for incubation overnight at room temperature to functionalize the Au side. Prior to immobilization of the anti-CD3 scFv, a thiol linker was added to the anti-CD3 scFv. Unbound antibody and Fe 3 O 4 @Au NPs were separated from antibody-conjugated NPs.

[0294] Pmab-conjugated Fc was evaluated by cytotoxicity assay using effector cells 3 O 4 @AuNP efficacy

[0295] To evaluate the efficacy of bispecific nanoparticle conjugation, a cytotoxicity assay was performed. 3 O 4@Au NP particles in the logarithmic growth phase of A431 cells at 4x10 3 Cells / well were plated in triplicate in 96-well microtiter plates to grow adherent cells. 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 at a density of 4000 cells per well in 96-well culture plates in DMEM medium. At 12 hours, the medium was removed and replaced with 100 μl medium containing 1% FBS.

[0296] MTT assay was then performed to evaluate the effect of Pmab-Fe 3 O 4 @Au-αCD3scFv nanoparticles cytotoxic effect on A431 and Jurkat cells. The control group was treated with cell culture medium alone. The treatment was removed after 72 hours of incubation, and the 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 dissolve the cells. After incubation at 37°C for 30 minutes and thorough mixing, viability was measured at 570 nm by a microplate reader. The spectrophotometer was calibrated to zero absorbance using a cell-free culture medium. Cell viability was calculated and plotted for concentration, and the data were evaluated using Prism 9 (GraphPad Software Version 8, La Jolla, California, USA).

[0297] with Fe 3 O 4 Compared with @Au NP, Pmab-Fe 3 O 4 @The cytotoxicity of co-cultured cells treated with Au-αCD3scFv NPs was significantly increased.

[0298] Fig.23 Shown, with a single conjugated Pmab-Fe 3 O 4 Compared with the bispecific Pmab-Fe@Au nanoparticles, 3 O 4 After incubation with @Au-αCD3scFv NP, the viability of the co-cultured cells was significantly reduced.

[0299] Example 6: Bispecific Fc conjugated to a cell penetrating peptide (CPP) 3 O 4 @Au NP

[0300] By adding Fe 3 O4 @Au NPs were conjugated with anti-EGFR monoclonal antibody (Nmab), anti-CD3 scFv, and CPP, and bispecific Fc-binding peptides conjugated to cell penetrating peptides were synthesized. 3 O 4 @Au NP(mAb-Fe 3 O 4 @Au NPs-αCD3 / CPP NP).

[0301] HIV-TAT Cell Penetrating Peptide PEGylated (CPP-PEG-SH)

[0302] HIV-TAT (residues 47-57) cell-penetrating peptide was dissolved in 0.1 M NaHCO, pH 8.0. 3 The concentration was 1 mg / mL (641 M). At a 4:1 NHS-linker / CPP molar ratio, 0.1 M NaHCO pH 8.0 was added. 3 A solution of 10 mg / mL (2 mM) NHS-PEG-SH (5 kDa) in PBS was added to the HIV-TAT peptide 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.

[0303] Nanoparticle surface functionalization

[0304] As briefly described in this article, the anti-EGFR antibody nimotuzumab was conjugated to Fe 3 O 4 NPs, while anti-CD3 scFv antibody and HIV-TAT CPP were linked to Au NPs using a thiol linker. 1 mg Fe 3 O 4 @Au NPs were 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 minutes. After gentle mixing at room temperature for 30 minutes, the solution was subjected to a PD-10 column to remove excess EDC and sulfo-NHS. 100 μg of nimotuzumab was then added to the conjugate and incubated for 3 hours while gently shaking to allow the Fe 3 O 4Side functionalization. After incubation for 3 hours, 5 μl of anti-CD3 scFv antibody was added and the mixture was placed on a shaker for incubation overnight at room temperature to functionalize the Au side. Before immobilization of the anti-CD3 scFv, a thiol linker was added to the anti-CD3 scFv. After buffer exchange with 1 mM EDTA, pH 7.4 0.01 M PBS, the PEGylated CPP was added. The unbound antibody and unconjugated Fc were removed using 300K ultrafiltration. 3 O 4 @Au NPs were separated from antibody-conjugated NPs.

[0305] Target: Effector cells evaluated by cytotoxicity assay of Nmab-Fe 3 O 4 @Au-αCD3scFv / CPPNP effect

[0306] To evaluate the efficacy of CPP bispecific nanoparticle conjugation, a cytotoxicity assay was performed. 3 O 4 @Au NP particles in the logarithmic growth phase of A431 cells at 4x10 3 Cells / well were plated in triplicate in 96-well microtiter plates to grow adherent cells. 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 at a density of 4000 cells per well in 96-well culture plates and various concentrations of DMEM buffer were added. At 12 hours, the medium was removed and replaced with 100 μl of medium containing 1% FBS. A431 cells were designated as target cells and Jurkat E6-1 were considered effector cells. In the presence or absence of Nmab-Fe 3 O 4 @Au-αCD3 scFv / CPPNP, Jurkat E6-1 cells were added to A431 cells at an effector: target (E:T) ratio of 5: 1. Untreated cells were selected as controls. After 72 hours of culture, the effector cells were washed off with PBS and MTT was added for further incubation for 2-4 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 (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 nm).

[0307] Fig.25It is shown that when Nmab-Fe3O4@Au-αCD3scFv / CPP NPs ( Fig.25 A) after incubation; and with Nmab-Fe3O4@Au-αCD3scFv / CPP NPs at a concentration of ≥10 μg / ml Nmab ( Fig.25 B) After incubation together, the viability of co-cultured A431 and Jurkat cells was significantly reduced.

[0308] Nmab-Fe without CPP 3 O 4 @Au-αCD3 scFv NP compared with Nmab-Fe 3 O 4 @The cytotoxicity of co-cultured cells treated with Au-αCD3 scFv / CPPNP was significantly increased.

[0309] Fig.26 It is shown that Nmab-Fe 3 O 4 @Au-αCD3 scFv NP compared with bispecific Nmab-Fe 3 O 4 After incubation with @Au-αCD3 scFv / CPP NP, the viability of co-cultured cells was significantly reduced.

[0310] Example 7: Bispecific Silver-Iron Nanoparticles (Fe 3 O 4 Preparation of Ag NPs

[0311] Fe was synthesized by thermal decomposition according to a previously reported procedure with slight modifications. 3 O 4 @Ag NP. Briefly, 6 mmol oleic acid, 6 mmol oleylamine and 10 mmol 1,2-hexadecanediol were mixed with 20 ml 1-octadecene (ODE) in a 50 ml round-bottom flask equipped with a thermometer, flow control adapter, reflux condenser, rubber septum and stirred at 120 °C under a gentle nitrogen flow for 30 min. Then, under nitrogen blanketing, 0.3 ml Fe(CO) 5 The solution was quickly injected into the solution. After 5 minutes, 35 mg AgNO 3A degassed silver precursor solution consisting of 1 ml of oleylamine and 5 ml of toluene solution was added dropwise to the hot solution at 180°C over 10 minutes to ensure thorough mixing. The solution immediately turned dark brown after injection, indicating the formation of silver nanoparticles. The solution was then slowly heated to 205°C and refluxed for 90 minutes. After cooling at room temperature, 40 mL of isopropanol was added to the solution and centrifuged at 8000 rpm for 10 minutes 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. As described elsewhere in this article, Fe 3 O 4 @Ag NPs were stored in hexane in the presence of 0.5 μl oleylamine for further use, including for conjugation with antigen binding proteins.

[0312] Transmission electron microscopy (TEM) was used to characterize Fe 3 O 4 Synthesis of Ag NPs( Fig.24 A). The average core size of Ag Np was observed to be 9 nm. 3 O 4 NP appears as a dumbbell shape. Fig.24 B shows Fe 3 O 4 The UV-Vis spectrum of @Ag NPs has a peak at 409 nm. The hydrodynamic size of the NPs is 8.23 ​​± 0.03 nm (Table 3).

[0313] Table 3: Fe 3 O 4 @Hydrodynamic diameter size (Z-average), PDI, zeta potential (ζ) and absorption maximum (λ max) and TEM size (nm) of Ag NPs.

[0314]

[0315] NP: nanoparticle format, PDI: polydispersity index, TEM: transmission electron microscopy.

[0316] Example 8: Anti-EGFR mAb (Nmab) and anti-CD3 scFv (mAb-Fc 3 O 4 Dumbbell-shaped Fe@Au-αCD3 NP conjugated 3 O 4 @Au NP

[0317] TEM was used to confirm the dumbbell-shaped Fe 3 O 4@Au NP Morphology and Synthesis. Methods for preparing dumbbell-shaped nanoparticles are known to the skilled person and include minor modifications to methods for producing flower-shaped nanoparticles. In short, reducing the molar ratio of iron pentacarbonyl in the preparation of the nanoparticles favors the production of dumbbell shapes compared to flower-shaped nanoparticles.

[0318] like Fig.14 As shown, Au NPs were observed to be spheres with an average core size of about 5 nm (shown in black due to heavy atom effects), while Fe 3 O 4 The diameter of the NPs was shown to be about 10 nm. Cytotoxicity assays confirmed that the spherical Nmab-Fe 3 O 4 Compared with the dumbbell-shaped dual-Nmab-Fe@Au NPs or bare unconjugated NPs with ≥300 μg / ml iron, 3 O 4 @Au-αCD3scFv NP (bispecific NP conjugated with Nmab and anti-CD3 scFv) incubated together, the viability of co-cultured cells was significantly reduced ( Fig.15 ).

[0319] Example 9: Anti-EGFR mAb (Nmab) and anti-CD3 scFv (Nmab-Fc 3 O 4 Spherical AuNPs conjugated with @Au-CD3 NP

[0320] Preparation of spherical Au NPs

[0321] 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 minutes. Then, 1.25 mL of 12.5 mM HAuCL4 solution was slowly added and stirred at 60°C for 2 minutes. Once the color changed from light yellow to black, the temperature was reduced to 40°C and gently stirred for 15 minutes. The Au NPs were allowed to cool at room temperature and then stored at 40°C.

[0322] Conjugation of @Au NPs with monoclonal anti-EGFR mAb (Nmab) and anti-CD3 scFv antibody

[0323] 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 cold room under gentle rotation. Anti-CD3 scFv (5 μL) and Nmab (200 μg) antibodies were then added to the NP mixture, 10 μL and 20 μL, respectively. Antibody-conjugated NPs were separated from unbound antibodies and Au NPs using ultrafiltration tubes.

[0324] TEM was used to confirm the morphology and synthesis of the spherical Au NPs. Fig.16 As shown in A, Au NPs were observed to be spherical with an average size of about 17 nm and appeared black due to the heavy atom effect.

[0325] In the experiment with different concentrations of (A) spherical Nmab-Fe 3 O 4 @Au NP or (B) spherical double Fe 3 O 4 Cytotoxicity assays were performed on co-cultures of A431 and Jurkat cells treated with Nmab@Au NPs (bispecific NPs conjugated with Nmab and anti-CD3 scFv) at a 1:5 ratio (target:effector). Fig.17 A) Compared with double Fe 3 O 4 @AuNP( Fig.17 B) After incubation together, cell viability (measured as absorbance) was not significantly affected. 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 suggest that flower-shaped iron gold NPs may provide superior therapeutic effects compared to nanoparticles of alternative configurations.

[0326] Example 10: Fc conjugated with anti-EGFR mAb (Nmab) and anti-CD16 scFv 3 O 4 @Au NP(mAb-Fe 3 O 4 @Au-αCD16 scFv NP)

[0327] Fe 3 O 4 Conjugation of @Au NPs with nimotuzumab and anti-CD16 scFv to generate Nmab-Fe 3 O 4 @Au-α CD16 scFv NP

[0328] As briefly described herein, the anti-EGFR antibody nimotuzumab was conjugated to Fc using the carbodiimide method described above. 3 O 4NPs, while anti-CD16 scFv antibodies were linked to Au NPs using a thiol linker. 1 mg Fe 3 O 4 @Au NPs were 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 minutes. After gentle mixing at room temperature for 30 minutes, 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 hours while gently shaking to allow the Fe 3 O 4 After 3 hours of incubation, 5 μl of anti-CD16 scFv antibody was added to the mixture and the mixture was placed on a shaker for incubation overnight at room temperature to functionalize the Au side. Prior to immobilization of the anti-CD16 scFv, a thiol linker was added to the anti-CD16 scFv. Unbound antibody and unconjugated Fc were removed using 300K ultrafiltration. 3 O 4 @Au NPs were separated from antibody-conjugated NPs.

[0329] Nmab-Fe 3 O 4 Mass spectrometry analysis of @Au-αC16scFv NPs revealed a specific peak of nimotuzumab at m / z approximately 148 KDa, which is 3 O 4 NPs were not detected. In addition, unbound antibody in the collected supernatant was analyzed by UV-vis measurement as described herein. NanoDrop 1000 spectrophotometer readings of proteins will confirm the single peak conjugation detected in the conjugated samples. SDS-PAGE can also be used to confirm antibody NP conjugation.

[0330] Use of effector:target cells assessed by cytokine production by Nmab-Fe 3 O 4 Efficacy of @Au-αCD3scFvNP

[0331] One day before the assay, A431 cells in logarithmic growth phase were cultured at 4 × 10 3 Cells / well were plated in triplicate in 96-well microtiter plates to grow adherent cells. 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-CD16 scFv (5 μg / ml); Nmab-Fe 3 O 4 @Au NP; or double Fe 3 O 4@Au NP (bispecific NP conjugated with Nmab and αCD16 scFv). After 24 hours, aliquots of the culture supernatant were collected and the levels of IL-2 were measured by using a commercially available ELISA test kit (Sydney, New South Wales, Australia). The samples were analyzed with a plate reader.

[0332] The results are expected to show that the use of double Fe 3 O 4 The production of IL-2 specifically induced by @Au NP (bispecific NP conjugated with Nmab and αCD16 scFv) after 24 hours of stimulation. In contrast, NP conjugated only with αCD16 scFv is expected to produce low levels of IL-2 by binding to T cells without activating it, and is expected to be higher in the presence of unconjugated NP or Nmab-Fc 3 O 4 No IL-2 stimulation was observed after Au NP treatment. 3 O 4 @Au NPs are more potent T cell activators than NPs conjugated with anti-CD16 scFv alone.

[0333] Target: Effector cells evaluated by cytotoxicity assay of Nmab-Fe 3 O 4 @Au-αCD16 scFv NP effect

[0334] To evaluate the efficacy of bispecific nanoparticle conjugation, a cytotoxicity assay was performed. 3 O 4 @Au NP particles in the logarithmic growth phase of A431 cells at 4x10 3 Cells / well were plated in triplicate in 96-well microtiter plates to grow adherent cells. 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 at a density of 4000 cells per well in 96-well culture plates and various concentrations of DMEM buffer were added. At 12 hours, the medium was removed and replaced with 100 μl of medium containing 1% FBS.

[0335] When using Fe concentration>2500μg / ml 3 O 4 @Before Au NP treatment, the viability of co-culture cells was not significantly affected.

[0336] A series of MTT assays were then performed at different concentrations to evaluate the expression of Fc receptors conjugated to nimotuzumab and / or anti-C16 scFv for the five treatment groups. 3 O 4@Au nanoparticles cytotoxic effects on A431 and Jurkat cells: Group A, Nmab; Group B, Fe 3 O 4 @Au NP; Group C, Nmab-Fe 3 O 4 @Au NP; Group D, Nmab-Fe 3 O 4 @Au-αCD16scFv NP; Group E, cisplatin; and Group F, treated with fresh medium. Treatment was removed after 72 hours of incubation, and the 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 dissolve the cells. After incubation at 37°C for 30 minutes and thorough mixing, viability was measured at 570 nm by a microplate reader. The spectrophotometer was calibrated to zero absorbance using cell-free culture medium. Cell viability was calculated and plotted against concentration, and the data were evaluated using Prism9 (GraphPad software version 8, La Jolla, California, USA).

[0337] with Fe 3 O 4 Compared with @Au NP, Nmab-Fe 3 O 4 @Au-αCD16scFv NPs (i.e., nanoparticles comprising antigen binding proteins for binding to EGFR and CD16) will have significantly increased cytotoxicity.

[0338] The results will show that with a single conjugated Nmab-Fe 3 O 4 @Au nanoparticles compared with Nmab-Fe 3 O 4 After incubation with @Au-αCD16scFv NP, the viability of the co-cultured cells was significantly reduced.

[0339] The results are expected to show similarly that the nanoparticles conjugated with Nmab-Fe compared to Nmab (ie, naked antibody at the same antibody concentration) or nimotuzumab alone. 3 O 4 After incubation with @Au-αCD16scFv NP, cell viability was significantly reduced.

[0340] To further evaluate Fc conjugated to Nmab and / or anti-CD16 scFv 3 O 4@ Cytotoxic effect of Au nanoparticles. Regarding additional target cancer cells, MTT assay was 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-Fe 3 O 4 @Au NP; Group C, Nmab-Fe 3 O 4 @Au-αCD16scFv NP. The control group was treated with fresh culture medium alone. One day before the assay, 3 O 4 @Au NP particles in the logarithmic growth phase of MDA-MB468 cells at 4x10 3 Cells / wells were plated in triplicate in 96-well microtiter plates to grow adherent cells. Cells were seeded on 96-well culture plates in RPMI medium at a density of 4000 cells per well. At 12 hours, the medium was removed and replaced with 100 μl of medium containing 1% FBS. Treatment was added, then removed after 72 hours of incubation, and the 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 of DMSO was added to dissolve the cells. After incubation at 37°C for 30 minutes and thorough mixing, viability was measured at 570 nm by a microplate reader. The spectrophotometer was calibrated to zero absorbance by using a cell-free culture medium. Cell viability was calculated and plotted for concentration, and data was evaluated using Prism 9 (GraphPad software version 8, La Jolla, California, USA).

[0341] with Fe 3 O 4 Compared with @Au NP, Nmab-Fe 3 O 4 @The cytotoxicity of co-cultured cells treated with Au-αCD16 scFv NPs will be significantly increased.

[0342] The results will show that with a single conjugated Nmab-Fe 3 O 4 @Au nanoparticles compared with Nimotuzumab-Fe 3 O 4 After incubation with @Au-αCD16scFv NP, the viability of the co-cultured cells was significantly reduced.

[0343] The results will similarly show that the nanoparticles conjugated to nimotuzumab-Fc have a significant difference compared to Nmab (i.e., naked antibody at the same antibody concentration) or nanoparticles conjugated to Nmab alone. 3O 4 After incubation with @Au-αCD16scFv NP, cell viability was significantly reduced.

Claims

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

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

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

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

5. The nanoparticle bioconjugate according to any one of claims 1 to 4, wherein the framework 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. The nanoparticle bioconjugate according to 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 metallic nanoparticles in a flower-like configuration.

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

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

9. The nanoparticle bioconjugate according to any one of claims 1 to 8, wherein the magnetic nanoparticles are selected from: Fe3O4 (iron oxide) nanoparticles, Fe(CO)5 (iron pentacarbonyl) nanoparticles or FeCl2 (ferric chloride) nanoparticles.

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

12. The nanoparticle bioconjugate according to any one of claims 1 to 11, wherein the nanoparticle bioconjugate comprises iron nanoparticles and gold nanoparticles, preferably wherein 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 between about 5 nm and about 100 nm.

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

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

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

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

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

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

20. 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 a portion thereof for binding to the cancer cell.

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

22. The nanoparticle bioconjugate of any one of claims 1 to 21, wherein the cancer cell is any one selected from the group consisting of breast tumor, colorectal tumor, adenocarcinoma, mesothelioma, bladder tumor, prostate tumor, germ cell tumor, hepatocellular tumor / cholangiocarcinoma, neuroendocrine tumor, pituitary neoplasm, small round cell tumor, squamous cell carcinoma, melanoma, atypical fibroxanthoma, seminoma, non-seminoma, stromalleydigcelltumour, 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.

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 the group consisting of HER2, EGFR, mesothelin, GPC3, MUC1, GD2, CEA, EpCAM, LeY, PCSA, CD19, CD20, Clec9a, CD276, PD-L1, and PD-L2.

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

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

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

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 wherein the immune cell is a T cell.

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

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. 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 a portion thereof for binding to the immune cell.

31. The nanoparticle bioconjugate of any one of claims 1 to 30, wherein the immune cell is a T cell and the target portion on the T cell bound by the second binding protein is selected from the group consisting of: 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. 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 the group consisting of KIR, CD16, CD94, CD 161 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 antigen-binding fragment thereof.

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 wherein the antibody is IgG.

35. 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 the group consisting of a recombinant antibody fragment, a bifunctional antibody, a trifunctional antibody, 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 binding protein and the second binding protein are in 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. A pharmaceutical composition comprising any of the nanoparticle bioconjugates according to any one of claims 1 to 37, optionally in combination with a pharmaceutically acceptable excipient or carrier.

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

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

41. The nanoparticle bioconjugate of any one of claims 1 to 37 or the pharmaceutical composition of claim 38 for use in treating or preventing cancer.

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