Multifunctional delivery system and uses thereof

Abstract

The present invention provides a multifunctional system for synchronous delivery of separate immunoglobulins, particularly antibodies and antibody fragments, to specific cancerous cells and tissues located outside the brain. The multifunctional system is based on an inorganic core particle conjugated to a first immunoglobulin via a first polymer linker; conjugated to a second immunoglobulin via a second polymer linker; conjugated to a permeation enhancing moiety via a third polymer linker; and conjugated to a fourth monofunctional capping linker or spacer. Also provided are processes for preparing the multifunctional system, pharmaceutical compositions comprising the multifunctional system, and its use in therapeutic and / or diagnostic methods.

Description

[Technical field]

[0001] FIELD OF THEINVENTION The present invention is in the field of immunotherapy and relates to delivery systems for therapeutic and diagnostic applications. [Background technology]

[0002] 2. Background of the Invention Recent advances in cell biology and immunology have led to a paradigm shift from a "single drug, single target" approach to combination therapy and multi-targeting drug approaches in the treatment of various intractable diseases. However, although certain drug combinations may theoretically be therapeutically effective in the treatment of various diseases, their clinical success is limited due to differences in the pharmacokinetics and tissue distribution of each drug in the combination.

[0003] To overcome these limitations, different approaches have been developed. Bispecific antibodies (bsAbs) are artificial proteins that combine the specificities of two antibodies in a single immunoglobulin molecule to simultaneously block multiple receptors or ligands. bsAbs can also bring targets into close proximity to support the formation of protein complexes on one cell or induce cell-cell contacts.

[0004] Recently, nanoparticles have emerged as a promising platform for the co-delivery of multiple drugs. Zhang, Tian et al. (Advanced healthcare materials 8.18(2019):1900543) provide multi-targeted nanoparticles for synergistic drug delivery through the blood-brain barrier against brain metastases of triple-negative breast cancer cells and tumor-associated macrophages.

[0005] Dixit et al. (Molecular pharmaceutics 12.9(2015):3250-3260) disclosed dual receptor-targeted theragnostic nanoparticles for localized delivery and activation of photodynamic therapy drugs in glioblastoma.

[0006] US Pat. No. 10,182,986 relates to a method for delivering nanoparticles across the blood-brain barrier to the brain of a subject by administering to the subject nanoparticles having a nanoparticle core and a targeting agent.

[0007] U.S. Patent No. 10,478,132 discloses gold nanoparticles conjugated to particles of about 4,000 to 20,000 molecules of 2-deoxy-D-glucose capable of binding to glucose transporter 1 (GLUT-1) for tumor imaging.

[0008] Shilo, Malka et al. (Nanoscale 6.4 (2014): 2146-2152) developed a technology for imaging and therapeutic applications that targets the transport of insulin-targeted gold nanoparticles (INS-GNPs) through the blood-brain barrier.

[0009] Trifunctional bispecific antibodies are artificially engineered immunoglobulins that can direct T cells to tumor cells and also induce the recruitment and activation of accessory cells through their Fc region. The simultaneous activation of different mechanisms at the tumor site leads to efficient destruction of tumor cells. Binding of the Fc portion of the targeting bispecific antibody to the Fc receptors of antigen-presenting cells, dendritic cells and macrophages results in the processing of cancer antigens, the presentation of cancer-associated peptides to helper T cells and the induction of memory T cells. However, the development of trifunctional bispecific antibodies is cumbersome, costly and time-consuming, and regulatory approval requires a long road.

[0010] Human epidermal growth factor receptor 2 (HER2) belongs to the receptor tyrosine kinase family consisting of four members: HER1 (also known as EGFR), HER2 (also known as Neu), HER3 and HER4. HER2 is a 185 kDa transmembrane glycoprotein that contains three components: an extracellular ligand-binding domain, a transmembrane domain, and an intracellular domain with tyrosine kinase activity. Unlike the other members, HER2 has no known natural ligand to which it binds. It exerts its function through EGFR-HER2 heterodimers, HER2-HER3 heterodimers, and HER2-HER2 homodimers. Amplification of the HER2 gene or overexpression of the HER2 receptor plays an important role in cell transformation, carcinogenesis, and prognosis of many types of cancer. HER2-positive tumors account for approximately 20-30% of breast cancers, 20% of advanced gastric or gastroesophageal junction cancers, 5-15% of bladder cancers, 5-15% of cervical cancers, 12-15% of gallbladder cancers, 8-35% of endometrial cancers, 6-7% of ovarian cancers, and 15-37% of salivary duct cancers. In particular, in patients with breast cancer, the most common malignant tumor and second leading cause of death from major cancers in women, detection of the expression level of HER2 is routine and useful for diagnosis, and HER2 is considered an applicable target for antitumor therapy (Yu et al. 2017, Experimental Hematology & Oncology Vol. 6, 31).

[0011] Up to now, several HER2-directed therapies, including trastuzumab, pertuzumab, T-DM1, lapatinib and afatinib (tyrosine kinase inhibitors that blocked EGFR and HER2), have been approved for HER2-positive breast cancer and non-small cell lung cancer. HER2-targeted immunotherapy consists of monoclonal antibodies (e.g., trastuzumab, pertuzumab), bispecific antibodies (e.g., MM-111, ertumaxomab) and activated T cells with anti-HER2 bispecific antibodies (HER2Bi-aATC). Trastuzumab is a classical drug for the treatment of HER2-positive metastatic breast cancer. The combined application of pertuzumab, trastuzumab and paclitaxel has been proposed as a standard therapy for HER2-positive advanced breast cancer. Resistance to anti-HER2 antibodies has led to disease progression. HER2-directed bispecific antibodies may be a promising therapeutic approach for these patients. Ertumaxomab enhanced the interaction between immune effector cells and tumor cells. MM-111 binds to HER2 and HER3 simultaneously and blocks downstream signaling. HER2Bi-aATC is also an alternative therapeutic approach for HER2-positive cancers.

[0012] Trastuzumab, as a classical anti-HER2 antibody, blocked HER2 homodimerization via binding to domain IV of HER2. As for pertuzumab, it can prevent the formation of heterodimerization via binding to HER2 subdomain II. Due to the separate but complementary modes of action, the combination of the two drugs could enhance the blockade of downstream signaling, including phosphoinositide 3-kinase / protein kinase B / mammalian target of rapamycin (PI3K / Akt / mTOR) and RAS / Raf / mitogen-activated protein kinase (MAPK). In addition, anti-HER2 monoclonal antibodies could increase endocytosis of HER2 receptors, inhibit angiogenesis, and induce tumor cell lysis via antibody-dependent cell-mediated cytotoxicity (ADCC). Synergistic antitumor functions of HER2 antibodies with other antitumor agents have been observed in both in vitro and in vivo studies. However, about 70% of patients were resistant to trastuzumab, and some showed primary resistance. To address this obstacle, researchers have proposed several corresponding strategies: maintenance of trastuzumab therapy after progression, combination of HER2 inhibitors, and development of novel anti-HER2 monoclonal antibodies. Bispecific antibodies, such as blinatumomab, have achieved great success in hematological malignancies. Among these, the widely studied HER2-targeted bispecific antibodies are also considered as a notable solution.

[0013] Ertumaxomab is an intact bispecific antibody that simultaneously targets HER2 on tumor cells and CD3 on T cells, and can activate accessory cells via its Fc fragment to exert ADCC functions.

[0014] Trifunctional antibodies can transiently link immune effector cells to tumor cells and have shown antitumor activity. MM-111 is another bispecific antibody that specifically targets HER2 / HER3 heterodimers, blocks the binding of heregulin (HRG) to HER3, and then inhibits the downstream signaling pathway of HER3. Activated T cells equipped with HER2-targeting bispecific antibodies (HER2Bi-aATC) showed significant inhibition in drug-resistant solid tumors.

[0015] There remains an unmet need for a multifunctional system that allows for the simultaneous and synchronized delivery of multiple immunoglobulins to tumor tissues and cells to improve the efficacy of cancer treatment. Summary of the Invention

[0016] The present invention provides a multifunctional system for co-delivery of at least two distinct immunoglobulin molecules to a target tissue or cell. The present invention further provides a method for preparing and using the multifunctional system for the treatment of cancer and other diseases. The multifunctional system of the present invention is particularly useful for the treatment of tumors outside the brain, or in some embodiments, outside the central nervous system (CNS).

[0017] The multifunctional system of the present invention is based on a core particle conjugated to a first and a second immunoglobulin molecule via a first and a second linker, respectively, and to a transporter or permeation enhancing moiety via a third linker. Although inorganic core particles are used with the added benefit of being a co-diagnostic, organic cores are expected to provide similar benefits. The transporter or permeation enhancing moiety may, for example, facilitate or enhance transport through the blood-tumor barrier, facilitate or enhance transport through cancer cell membranes, allow attachment of the delivery system to cancerous cells (e.g., via specific receptors), and / or improve glucose metabolism of cancer cells when configured to treat tumors outside the brain or CNS. Additional active groups optionally present on the particle surface are capped with a fourth monofunctional linker.

[0018] The immunoglobulin molecules conjugated to the core particles can include various kinds of antibodies and their fragments. In particular, it is shown herein that a combination of two different monoclonal antibodies against human epidermal growth factor receptor 2 (HER2) on a single core particle further conjugated with insulin as a permeation enhancer moiety can inhibit cancer cell proliferation in vitro and suppress tumor growth in vivo with higher efficiency compared to the efficiency of a mixture of similar particles conjugated with each antibody separately. Surprisingly, it is also shown that even in cells expressing low levels of a specific tumor antigen (e.g., HER2), two different antibodies against this antigen, when conjugated to a delivery system, can inhibit cancer cell proliferation with the help of insulin, which allows binding to cells via the insulin receptor.

[0019] Moreover, using two different fluorescently labeled antibodies conjugated to core particles, it is found that the antibodies co-localize in specific regions.Therefore, the multifunctional delivery system of the present invention not only facilitates tissue penetration of immunoglobulin molecules, but also realizes their synchronous distribution in tissue or tumor.Advantageously, the synchronous distribution of different antibodies can significantly improve the therapeutic effect of drug combination.

[0020] The present invention is further based in part on the discovery that GNPs carrying an optimized number of antibodies per particle have improved anti-proliferative activity compared to GNPs with higher or lower densities of antibody molecules.

[0021] The present invention allows the use of different combinations of antibodies and antibody fragments as effective tools for anti-cancer immunotherapy against a wide range of malignancies. The antibody-containing GNPs according to the present invention are capable of binding to cancer cells or immune cells.

[0022] One beneficial feature of the system of the present invention is that the activity of the therapeutic agent conjugated to the delivery system remains intact and does not need to be detached from the nanoparticle after delivery to the target tissue, for example, by using a cleavable linker. The capping moiety conjugated to the remaining potentially active group on the particle allows for the desired distance between the immunoglobulin and other molecules conjugated to the particle.

[0023] Surprisingly, it has been found that particles containing a relatively small number of immunoglobulin molecules in addition to a penetration-enhancing moiety can bind to tumor cells and immune cells and induce a desired response, such as inhibiting tumor cell proliferation or enhancing tumor cell apoptosis. In addition, cancer cells that are minimally affected by bispecific nanoparticles with two types of immunoglobulin molecules were affected by the inclusion of a penetration-enhancing moiety on the surface. Surprisingly, breast cancer cells expressing low levels of HER2 (hereinafter "HER2 low") were also affected by particles carrying two different types of anti-HER2 antibodies and insulin as a penetration enhancer.

[0024] Another advantage of the delivery system of the present invention is that specific combinations of antibodies are versatile and can be defined for a particular cancer type or a particular patient based on the tumor antigens and / or immune cell receptors and checkpoint molecules of the particular tumor to be targeted. Unique linkers of the present invention can be tailored to specifically conjugate the antibody of interest.

[0025] According to one aspect, (a) an inorganic particle bound to at least (i) a first polymeric linker; (ii) a second polymeric linker; (iii) a third polymeric linker; and (iv) a fourth polymeric monofunctional linker; (b) a first immunoglobulin molecule conjugated to a first linker; (c) a second immunoglobulin molecule separate from the first immunoglobulin molecule and conjugated to a second linker; (d) a permeation-enhancing moiety conjugated to a third linker. A multifunctional particle comprising: Here, the first and second immunoglobulin molecules are separate, and a total of about 2 to 400 immunoglobulin molecules are conjugated to each particle via the first and second linkers, thereby providing multifunctional particles.

[0026] According to some embodiments, each of the first and second immunoglobulin molecules is covalently conjugated to a first and second polymeric linker, which may be the same or different.

[0027] According to some embodiments, the first and second polymer linkers are the same. According to some embodiments, the permeation enhancing moiety is conjugated to the third polymer linker by a bond selected from a covalent bond, a semi-covalent bond, and a non-covalent bond.

[0028] According to some embodiments, the covalent bond is an amide bond or a disulfide bond.

[0029] According to some embodiments, at least one of the linkers is linear.

[0030] According to some embodiments, at least one of the linkers is a polymer.

[0031] According to some embodiments, the first and second linkers are linear polymer linkers.

[0032] According to some embodiments, the third linker is a linear polymer linker.

[0033] According to some embodiments, the length of the third linear polymer linker is substantially different from the length of the first and second linear polymer linkers.

[0034] According to some embodiments, the molecular weight of the third polymer linker differs from the molecular weight of the first and second polymer linkers by at least about 1000 Da.

[0035] According to some embodiments, the length of the third linear polymer linker is substantially longer than the length of the first and second linear polymer linkers.

[0036] According to some embodiments, the first and second linkers are not cleavable under physiological conditions.

[0037] According to some embodiments, the third linker is not cleavable under physiological conditions.

[0038] According to some embodiments, the third linker is cleavable under physiological conditions.

[0039] According to some embodiments, the molecular weight of the first, second and third polymer linkers is in the range of 1,000 to 10,000 Da. In some embodiments, the molecular weight of the third polymer linker is higher than the molecular weight of the first and second polymer linkers.

[0040] According to some embodiments, the molecular weight of each of the first and second linear polymer linkers is in the range of 3500-4000 Da, and the molecular weight of the third linear polymer linker is 4500 Da or greater.

[0041] According to some embodiments, the third linear polymer linker is composed of repeating monomer units, and at least one of the first and second linear polymer linkers is composed of the same repeating monomer units as the third linear polymer linker, where the third linear polymer linker has a different number of repeating monomer units than at least one of the first and second linear polymer linkers. In some embodiments, the first, second and third linear polymer linkers are composed of the same repeating monomer units, where the third linear polymer linker has a different number of repeating monomer units than that of the first and second linear polymer linkers.

[0042] According to some embodiments, the first linker and the second linker are the same.

[0043] According to some embodiments, the first and second linkers are attached to the inorganic particle via a sulfide bond, and the first and second immunoglobulin molecules are conjugated to the respective linkers via an amide bond.

[0044] According to some embodiments, each of the first and second immunoglobulin molecules is directed to a tumor-associated antigen (TAA), a tumor-associated receptor, an immune cell receptor, or an immune checkpoint protein.

[0045] According to some embodiments, the first and second immunoglobulin molecules are independently selected from the group consisting of antibodies, antibody fragments comprising at least an antigen binding site, antibody conjugates, and combinations thereof.

[0046] According to some embodiments, the two different antibodies are capable of binding to the same cancer-specific or cancer-associated cell surface antigen on a tumor cell.

[0047] According to some embodiments, at least one of the antibodies binds to a cancer-specific or cancer-associated cell surface antigen. According to some particular embodiments, the tumor antigen or tumor-associated antigen is selected from the group consisting of HER family receptors, EGFR, mesenchymal epithelial transition factor, PSMA, Nectin-4, CD155, CD3, EGFRvIII, Vγ9, CD16, CD133, IL-15, and CD19, CD20, CD30, CD38, CD38, and CD138. Each possibility represents a separate embodiment of the present invention.

[0048] According to some particular embodiments, the two antibodies bind to a Herceptin family receptor. According to some particular embodiments, the two antibodies bind to HER2.

[0049] According to some embodiments, each of the two different antibodies can bind to a different cancer-specific or cancer-associated cell surface antigen on a tumor cell.

[0050] According to some specific embodiments, one antibody binds to HER2 and one binds to HER3.

[0051] According to some embodiments, the GNP comprises a first antibody capable of binding to an immune cell (e.g., a T cell or a NK cell) and a second antibody capable of binding to at least one cancer-specific or cancer-associated cell surface antigen on a tumor cell.

[0052] According to some embodiments, one of the antibodies is specific for an NK receptor selected from the group consisting of natural cytotoxicity receptors (NCRs), NKp30, NKp44, NKp46, CD16, CD314, and CD94 / NKG2C, each possibility representing a separate embodiment of the present invention.

[0053] According to some particular embodiments, at least one of the antibodies is specific for a checkpoint molecule. According to still other embodiments, the antibody is specific for a checkpoint molecule selected from the group consisting of PD-1, PD-L1, CTLA-4, 4-1BB, OX40, TIM3, TIGIT, LAG-3, and CD47. Each possibility represents a separate embodiment of the present invention.

[0054] Non-limiting examples of targets of the antibodies of the multifunctional GNPs of the invention include HER family receptors, EGFR, mesenchymal epithelial transition factor, PSMA, nectin-4, CD155, CEA, CD3, EpCam, EGFRvIII, Vγ9, CD16, CD133, IL-15, CD19, CD20, CD30, CD38, CD38 and CD138, IGF-1 and IGF-2, VEGF, Ang2, cMET, DLL4, CD137, IGF-RI, PMEL, B7H3, GPA33, GPC3, PD-1, PD-L1, CTLA-4, 4-1BB, OX40, TIM3, TIGIT, LAG-3, and CD47, NCR, NKp30, NKp44, NKp46, CD16, CD314, and CD94 / NKG2C.

[0055] According to some embodiments, at least one of the antibodies binds to PD-1.

[0056] According to some embodiments, at least one of the antibodies binds to PD-L1.

[0057] According to some particular embodiments, each multifunctional GNP is conjugated to a pair of antibodies targeting antigens selected from the group consisting of HER2 and HER2, HER2 and HER3, HER2 and PD-1, HER2 and CTLA-4, PD-1 and PD-L1, PD-1 and CTLA-4, CEA and CD3, PSMA and CD3, EGFRvIII and CD3, EpCam and CD3, HER2 and Vγ9, CD16 and CD133, CD16 and IL-15, CD15 and CD19, CD16 and CD133, IGF-1 and IGF-2, VEGF and Ang2, EGFR and cMET, DLL4 and VEGF, HER2 and CD3, PD-1 and LAG3, PD-L1 and CD137, PSMA and CD3, IGF-RI and HER3, PMEL and CD3, B7H3 and CD3, GPA33 and CD3, GPC3 and CD3. Each possibility represents a separate embodiment of the present invention.

[0058] According to some embodiments, one of the antibodies binds to PD-1 and the other binds to PD-L1.

[0059] According to some embodiments, at least one of the antibodies is capable of binding to a T cell, NK cell, dendritic cell, or macrophage, or to an Fc receptor on an activated immune cell.

[0060] According to some embodiments, the immune cells are selected from the group consisting of NK cells, T cells, NKT cells, macrophages, and any combination thereof.

[0061] According to some embodiments, the antibody is a monoclonal antibody.

[0062] According to some embodiments, the GNPs comprise at least one antibody selected from the group consisting of a non-human antibody, a chimeric antibody, a humanized antibody, a human antibody, and any combination thereof.

[0063] According to some particular embodiments, the antibody is a chimeric monoclonal antibody.

[0064] According to some embodiments, the chimeric antibody comprises a constant region of human origin selected from the group consisting of IgG1, IgG2, IgG3, and IgG4.

[0065] According to some embodiments, at least one antibody is a humanized antibody.

[0066] According to some embodiments, about 2-40 antibodies are conjugated to each particle via a linker. According to some embodiments, about 2-20 antibody molecules are conjugated to each particle via a linker. According to some embodiments, about 2-10 antibody molecules are conjugated to each particle via a linker. According to still other embodiments, about 4-40 antibody molecules are conjugated to each particle via a linker. According to still other embodiments, about 5-30 antibody molecules are conjugated to each particle via a linker. According to some particular embodiments, about 10-20 or about 15-25 antibody molecules are conjugated to each particle via a linker.

[0067] According to some embodiments, about 20-400 antibody fragments are conjugated to each particle via a linker. According to still other embodiments, about 40-400 antibody fragments are conjugated to each particle via a linker. According to still other embodiments, about 50-300 antibody fragments are conjugated to each particle via a linker. According to some particular embodiments, about 100-200 or about 50-350 antibody molecules are conjugated to each particle via a linker.

[0068] According to some embodiments, the third linear polymer linker constitutes about 10 mol % to 40 mol % of the total polymer linkers attached to the inorganic particles.

[0069] According to some embodiments, each of the first and second linear polymer linkers independently constitutes between about 5 mol % and 40 mol % of the total polymer linkers attached to the inorganic particles.

[0070] According to some embodiments, the first, second, and third linear polymer linkers independently comprise a polymer selected from the group consisting of polyethers, polyacrylates, polyanhydrides, polyvinyl alcohols, polysaccharides, poly(N-vinylpyrrolidone), polyglycerin (PG), poly(N-(2-hydroxypropyl)methacrylamide), polyoxazolines, poly(amino acid)-based hybrids, recombinant polypeptides, derivatives, and combinations thereof. According to certain embodiments, at least one of the first, second, and third linear polymer linkers is a polyether. In some exemplary embodiments, the polyether is polyethylene glycol (PEG). Polyethylene glycol is a polyether selected from the group consisting of thiolated PEG acids (HS-PEG-COOH) and thiolated PEG amines (HS-PEG-NH 2 ), where the thiolated end is attached to an inorganic particle and the acid or amine end is conjugated to the respective immunoglobulin molecule or permeation enhancing moiety.

[0071] According to some embodiments, the multifunctional particle further comprises a non-functional capping moiety that is directly attached to the inorganic particle or is attached via a linker or spacer. According to some embodiments, the linker is a monofunctional polymer linker to which the capping moiety is attached. According to some embodiments, the polymer linker comprises a polymer selected from the group consisting of polyethers, polyacrylates, polyanhydrides, polyvinyl alcohols, polysaccharides, poly(N-vinylpyrrolidone), polyglycerin (PG), poly(N-(2-hydroxypropyl)methacrylamide), polyoxazolines, poly(amino acid)-based hybrids, recombinant polypeptides, derivatives, and combinations thereof. In some exemplary embodiments, the fourth polymer linker comprises a polyether, and the polyether is methoxypolyethylene glycol (mPEG).

[0072] According to some embodiments, the inorganic particles are nanoparticles selected from the group consisting of metal nanoparticles, metal oxide nanoparticles, ceramic nanoparticles, and any combination thereof. The metal can be selected from the group consisting of gold, silver, platinum, iron, and any combination thereof. The metal oxide can be selected from the group consisting of iron oxide, magnesium oxide, nickel oxide, cobalt oxide, aluminum oxide, zinc oxide, copper oxide, manganese oxide, and any combination thereof. In some particular embodiments, the inorganic particles are selected from the group consisting of gold nanoparticles, iron (III) oxide nanoparticles, and iron (II, III) oxide nanoparticles. In further particular embodiments, the inorganic particles are gold nanoparticles.

[0073] According to some embodiments, the inorganic particles are nanoparticles having a diameter of 10 to 160 nm.

[0074] In some embodiments, the moiety linked to the third linker is a molecule generally related to sugar metabolism. In some embodiments, the moiety linked to the third linker is a permeation enhancer or a transporter.

[0075] According to some embodiments, the moiety linked to the third linker comprises a molecule that facilitates or enhances glucose entry into the cell or cellular metabolism.

[0076] According to some embodiments, the penetration enhancing moiety is capable of increasing tumor cell metabolism. According to some embodiments, the penetration enhancing moiety is selected from the group consisting of insulin, an antibody specific for the insulin receptor, a polypeptide that specifically binds to the insulin receptor, insulin-like growth factor 1, an antibody specific for insulin-like growth factor receptor 1, a polypeptide that specifically binds to insulin-like growth factor receptor 1, a cell penetrating peptide (CPP), and any combination thereof. In some embodiments, the moiety is selected from insulin and glucose. According to some embodiments, the glucose is 2-deoxy-D-glucose.

[0077] According to some embodiments, the permeation enhancing moiety actively enhances permeation through the blood-tumor barrier.

[0078] According to some embodiments, the multifunctional particle further comprises an additional immunoglobulin conjugated to the inorganic particle via a linker or spacer.

[0079] According to some exemplary embodiments, the inorganic particles are gold nanoparticles, the first linear polymer linker and the second linear polymer linker are each independently a thiolated PEG3500 acid or a thiolated PEG3500 amine, the third linear polymer linker is a thiolated PEG5000 acid or a thiolated PEG5000 amine, and the permeation enhancing moiety is insulin.

[0080] According to some exemplary embodiments, the inorganic particles are gold nanoparticles, the first linear polymer linker is a thiolated PEG1000 acid or a thiolated PEG1000 amine, the second linear polymer linker is a thiolated PEG3500 acid or a thiolated PEG3500 amine, the third linear polymer linker is a thiolated PEG5000 acid or a thiolated PEG5000 amine, and the permeation enhancing moiety is insulin.

[0081] According to another aspect, the preparation process of multifunctional particles according to the above various embodiments is provided. According to some embodiments, the process comprises the following consecutive steps: (a) partially coating the surface of inorganic particles with a first linear polymer linker, and then conjugating the first linear polymer linker to a first biologically active molecule; (b) partially coating the surface of inorganic particles with a second linear polymer linker, and then conjugating the second linear polymer linker to a second biologically active molecule; and (c) partially coating the surface of inorganic particles with a third linear polymer linker, and then conjugating the third linear polymer linker to a permeation enhancing moiety, where steps (a), (b) and (c) can be carried out in any order.

[0082] According to some embodiments, the process for preparing the multifunctional particles comprises the sequential steps of: (a) partially coating the surface of the inorganic particles with a first linear polymer linker and a second linear polymer linker, followed by conjugating the first and second linear polymer linkers to a first and second bioactive molecule, where the first and second linear polymer linkers are identical and the first bioactive molecule is distinct from the second bioactive molecule; and (b) partially coating the surface of the inorganic particles with a third linear polymer linker, followed by conjugating the third linear polymer linker to a permeation enhancing moiety, where the length of the third linear polymer linker is substantially different from the length of the first and second linear polymer linkers and the molecular weight of the third polymer linker differs from the molecular weight of the first and second polymer linkers by at least about 1000 Da, where steps (a) and (b) can be performed in any order.

[0083] According to some embodiments, the particles are gold nanoparticles (GNPs), and the process for preparing multifunctional gold nanoparticles comprises: (a) reacting HAuCl 4 (b) simultaneous incubation of reduced GNPs having one type of monofunctional linker and two different types of heterofunctional linkers; (c) activation of the GNPs to obtain free COOH groups; (d) conjugation of a permeation enhancing moiety; and (d) conjugation of two different antibodies by incubating with a solution containing their mixture.

[0084] According to some embodiments, analysis of the GNPs is performed after each step using methods known in the art.

[0085] According to some embodiments, the monofunctional linker is mPEG-SH. According to certain embodiments, the monofunctional linker is mPEG5000-SH or mPEG6000-SH, and is added so as to cover about 60 to 95% of the particle surface.

[0086] According to some embodiments, the heterofunctional linker is COOH-PEG-SH. According to some embodiments, one heterofunctional linker is CGOH-PEG5000-SH and is added at a concentration that covers about 15% of the particle surface. According to some embodiments, another heterofunctional linker is COOH-PEG3500-SH and is added at a concentration that covers about 5% of the particle surface.

[0087] According to some embodiments, activation of the terminal acid PEG (linker) for conjugating immunoglobulin molecules is performed by mixing the GNPs with (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide HCl (EDC) and / or sulfo-NHS.

[0088] According to some embodiments, the permeation enhancing moiety is insulin, the conjugation of which is carried out by incubation with activated GNPs at a concentration of about 50-500 IU / ml for 1-5 hours.

[0089] According to some embodiments, the antibodies are two different antibodies against human HER2. According to some embodiments, the two different antibodies against HER2 are trastuzumab and pertuzumab. According to some embodiments, the two different antibodies against HER2 are trastuzumab and pertuzumab, and the first and second polymer linkers are greater than 3500 Da. According to some embodiments, the two antibodies are incubated overnight with activated GNPs conjugated to a permeation enhancing moiety at a concentration of 1-50 mg / ml.

[0090] According to some embodiments, analysis of the GNPs is performed using dynamic light scattering (DLS).

[0091] According to some embodiments, quantification of antibodies and permeation enhancing moieties (e.g., insulin) attached to PEG groups on the GNPs is performed by enzyme-linked immunosorbent assay (ELISA) of the supernatant containing unbound proteins remaining after precipitation by centrifugation of the GNPs.

[0092] According to some embodiments, a first polymer linker has a first functional end group configured to attach a first biologically active molecule, a second polymer linker has a second functional end group configured to attach a second biologically active molecule, and a third polymer linker has a third functional end group configured to attach a permeation enhancing moiety, wherein at least two of the first, second and third functional end groups are identical.

[0093] According to some embodiments, the process further comprises partially coating the surface of the inorganic particle with a capping moiety, optionally attached via a fourth polymer linker, wherein the fourth polymer linker is a monofunctional linker.

[0094] According to some embodiments, each of the first linear polymer linker and the second linear polymer linker is added in an amount suitable to cover 5%-40% of the surface of the inorganic particle, and the third linear polymer linker, if added, is in an amount suitable to cover 5%-40% of the surface of the inorganic particle. According to some embodiments, the capping moiety is used to cover about 20-85% of the surface of the inorganic particle. According to some embodiments, the capping moiety comprises a methyl group attached to the linker. According to some embodiments, the capping moiety comprises a methyl group attached to a PEG linker.

[0095] The multifunctional GNPs according to the invention are provided according to some embodiments for the treatment of cancer.

[0096] According to some embodiments, the multifunctional GNPs are for use in eliminating or inhibiting the progression, metastatic spread of cancer.

[0097] According to yet another aspect, there is provided a pharmaceutical composition comprising a multifunctional particle according to the various embodiments presented above and a pharma- ceutically acceptable carrier or excipient.

[0098] In some embodiments, the pharmaceutical composition is provided for use in treating and / or monitoring a cancer or tumor located outside the brain of a subject in need thereof. In some embodiments, the pharmaceutical composition is used in inhibiting or eliminating the progression of cancer or for preventing or inhibiting the formation or spread of cancer metastases.

[0099] According to some embodiments, the solid tumor or tumor metastasis is located outside the CNS.

[0100] According to some particular embodiments, the solid tumor is selected from the group consisting of breast, lung, bladder, pancreas and ovarian.

[0101] According to some embodiments of the invention, the cancer is selected from the group consisting of lung cancer, breast cancer, colorectal cancer, melanoma, ovarian cancer, pancreatic cancer, colon cancer, cervical cancer, kidney cancer, thyroid cancer, prostate cancer, renal cancer, pharyngeal cancer, laryngeal cancer, bladder cancer, liver cancer, fibrosarcoma, endometrial cell carcinoma, glioblastoma, and sarcoma, with each possibility representing a separate embodiment of the present invention.

[0102] According to certain embodiments, the cancer is selected from the group consisting of breast cancer, colorectal cancer, lung cancer, renal cancer, melanoma, and prostate cancer. Each possibility represents a separate embodiment of the present invention.

[0103] According to other embodiments, the solid tumor is a breast adenocarcinoma or the metastasis derives from a primary breast adenocarcinoma.

[0104] According to some embodiments, the breast cancer is metastatic breast cancer.

[0105] According to some embodiments, the breast cancer is HER2 positive.

[0106] According to some embodiments, the breast cancer is HER2 negative. According to some embodiments, the breast cancer is characterized as HER2 low.

[0107] According to some embodiments, the breast cancer is triple-negative breast cancer (TNBC).

[0108] The present invention also provides, according to some embodiments, a multifunctional particle for use in treating or monitoring a primary tumor or metastasis located outside the brain, comprising: (a) an inorganic particle bound to at least (i) a first linear polymeric linker; (ii) a second linear polymeric linker; (iii) a third linear polymeric linker; and (iv) a fourth polymeric monofunctional linker; (b) a first immunoglobulin molecule conjugated to a first linear polymer linker; (c) a second immunoglobulin molecule covalently conjugated to a second linear polymer linker; and (d) a permeation-enhancing moiety conjugated to a third linear polymer linker. Including, Provided herein is a pharmaceutical composition comprising a multifunctional particle, in which the first and second immunoglobulin molecules are distinct, and a pharma- ceutically acceptable carrier or excipient.

[0109] According to some embodiments there is provided a multifunctional particle for use in treating or monitoring a primary tumor or metastasis located outside the brain, comprising: (a) an inorganic particle bound to at least (i) a first linear heterofunctional polymeric linker; (ii) a second linear heterofunctional polymeric linker; (iii) a third linear heterofunctional polymeric linker; and (iv) a fourth polymeric monofunctional linker; (b) a first immunoglobulin molecule covalently conjugated to a first linear polymer linker; (c) a second immunoglobulin molecule covalently conjugated to a second linear polymer linker; and (d) a permeation-enhancing moiety conjugated to a third linear polymer linker. Including, Here, the first and second immunoglobulin molecules are separate, and a pharmaceutical composition is provided comprising multifunctional particles in which a total of about 2 to 400 immunoglobulin molecules are conjugated to each particle via a first and a second linker.

[0110] According to some embodiments, the pharmaceutical composition is formulated for at least one of intravenous (IV), intranasal (IN), and intraperitoneal (IP) administration.

[0111] According to some embodiments, the pharmaceutical compositions described herein are administered as part of a cancer treatment regimen selected from the group consisting of chemotherapy, immunotherapy, biological therapy, hormonal therapy, radiation therapy, bone marrow transplant, surgery, and any combination thereof.

[0112] According to some embodiments, the pharmaceutical composition according to the invention is for use in cancer immunotherapy or in boosting an immune response.

[0113] According to some embodiments, the pharmaceutical composition further comprises killer cells (eg, T cells, NK cells, NKT cells, and / or macrophages).

[0114] According to another aspect, the present invention provides a method for treating a subject having a cancer or tumor located outside the brain, comprising administering to the subject a pharmaceutical composition comprising the multifunctional GNPs disclosed herein.

[0115] According to some embodiments, a method of treating a subject having a cancer or tumor located outside of the brain comprises administering to the subject a multifunctional particle comprising: (a) an inorganic particle bound to at least (i) a first linear polymeric linker; (ii) a second linear polymeric linker; (iii) a third linear polymeric linker; and (iv) a fourth polymeric monofunctional linker; (b) a first immunoglobulin molecule conjugated to a first linear polymer linker; (c) a second immunoglobulin molecule covalently conjugated to a second linear polymer linker; and (d) a permeation-enhancing moiety conjugated to a third linear polymer linker. Including, wherein the first and second immunoglobulin molecules are distinct and comprise administering to the subject a pharmaceutical composition comprising a multifunctional particle.

[0116] According to some embodiments, the treatment reduces tumor size or the number of metastases in the subject.

[0117] According to some embodiments, the tumor or metastasis is located outside the CNS.

[0118] According to one embodiment, the method comprises administering multifunctional GNPs comprising at least one antibody that targets the Her2 / neu protein.

[0119] According to some embodiments, the method comprises administering a multifunctional GNP comprising two different antibodies targeting Her2 / neu, each GNP being conjugated to two different antibodies against HER2.

[0120] The methods of the present invention include both standalone treatments and combinations with any anti-cancer treatment.

[0121] According to a particular embodiment, the method comprises administering to a subject in need thereof a pharmaceutical composition comprising at least one multifunctional GNP and administering at least one anti-cancer drug. Such administration can be performed simultaneously or at different times.

[0122] Any tumor characterized by expressing a specific tumor antigen or tumor-associated antigen, and any cancer amenable to T-cell therapy, may be treatable with the multifunctional GNPs of the present invention.

[0123] According to some embodiments, the cancer is or comprises a solid tumor located outside the brain.

[0124] According to some embodiments, the solid tumor is a metastatic solid tumor.

[0125] According to some embodiments, the solid tumor is a primary resistant solid tumor or tumor metastasis located outside the brain.

[0126] According to some embodiments, the solid tumor or tumor metastasis is located outside the CNS.

[0127] According to some particular embodiments, the solid tumor is selected from the group consisting of breast, lung, bladder, pancreas and ovarian.

[0128] According to some embodiments of the invention, the cancer is selected from the group consisting of lung cancer, breast cancer, colorectal cancer, melanoma, ovarian cancer, pancreatic cancer, colon cancer, cervical cancer, kidney cancer, thyroid cancer, prostate cancer, renal cancer, pharyngeal cancer, laryngeal cancer, bladder cancer, liver cancer, fibrosarcoma, endometrial cell carcinoma, glioblastoma, and sarcoma, with each possibility representing a separate embodiment of the present invention.

[0129] According to certain embodiments, the cancer is selected from the group consisting of breast cancer, colorectal cancer, lung cancer, renal cancer, melanoma, and prostate cancer. Each possibility represents a separate embodiment of the present invention.

[0130] According to other embodiments, the solid tumor is a breast adenocarcinoma or the metastasis derives from a primary breast adenocarcinoma.

[0131] According to some embodiments, the breast cancer is metastatic breast cancer.

[0132] According to some embodiments, the breast cancer is HER2 positive.

[0133] According to some embodiments, the breast cancer is HER2 negative. According to some embodiments, the breast cancer is characterized as HER2 low.

[0134] According to some embodiments, the breast cancer is triple-negative breast cancer (TNBC).

[0135] According to a further aspect, the present invention provides a method of immunotherapy of cancer comprising administering a multifunctional GNP that blocks a negative regulator, such as a checkpoint inhibitor or a regulatory T cell inhibitor. According to some embodiments, the checkpoint inhibitor is selected from CTLA-4 and PD-1 / PD-L1.

[0136] According to some embodiments, the multifunctional GNPs are administered intravenously or intratumorally.

[0137] According to some embodiments, the method is part of a treatment regimen that includes an additional cancer treatment, according to some embodiments, the additional cancer treatment is selected from the group consisting of chemotherapy, immunotherapy, biological therapy, hormonal therapy, radiation therapy, bone marrow transplant, surgery, and any combination thereof.

[0138] According to an additional aspect, the present invention provides a method of enhancing an immune response in a subject in need thereof, comprising administering to the subject a pharmaceutical composition described herein.

[0139] According to some embodiments, the method of treating cancer includes administering or administering at least one additional anti-cancer therapy. According to certain embodiments, the additional anti-cancer therapy is surgery, chemotherapy, radiation therapy, or immunotherapy. In certain embodiments, the additional therapy is radiation therapy.

[0140] According to some embodiments, the method of treating cancer comprises administering an antibody and an additional anti-cancer agent, according to some embodiments, the additional anti-cancer agent is selected from the group consisting of an immunomodulatory agent, an agent that inhibits an immune co-inhibitory receptor, an activated lymphocyte cell, a kinase inhibitor, and a chemotherapeutic agent.

[0141] According to some embodiments, the additional immune modulator is an antibody against an immune checkpoint molecule. According to some embodiments, the additional immune modulator is an antibody against an immune checkpoint molecule selected from the group consisting of human programmed cell death protein 1 (PD-1), PD-L1 and PD-L2, carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1), lymphocyte activation gene 3 (LAG3), CD137, OX40 (also called CD134), killer cell immunoglobulin-like receptor (KIR), TIGIT, PVR, CTLA-4, NKG2A, GITR, and any other checkpoint molecule or combination thereof. Each possibility represents a separate embodiment of the present invention.

[0142] According to some embodiments, the anticancer drug is selected from the group consisting of erbitux, cytarabine, fludarabine, fluorouracil, mercaptopurine, methotrexate, thioguanine, gemcitabine, vincristine, vinblastine, vinorelbine, carmustine, lomustine, chlorambucil, cyclophosphamide, cisplatin, carboplatin, ifosfamide, mechlorethamine, melphalan, thiotepa, dacarbazine, bleomycin, dactinomycin, daunorubicin, doxorubicin, idarubicin, mitomycin, mitoxantrone, plicamycin, etoposide, teniposide, and any combination thereof. Each possibility represents a separate embodiment of the present invention.

[0143] According to some embodiments of the invention, the subject is a human subject.

[0144] According to some embodiments, the methods of treating cancer involve preventing or reducing the formation, growth or spread of metastases in a subject.

[0145] According to a further aspect, there is provided a method for simultaneous delivery of at least two antibodies to a specific tissue, body region, cancerous cell or specific spatial region of a tumor in a subject, comprising administering to said subject a pharmaceutical composition according to the various embodiments presented above. According to some embodiments, upon administration, the at least two antibodies show synchronous distribution within the target tissue or body region or cancerous cell.

[0146] According to some embodiments, the pharmaceutical composition is administered to the subject by at least one of oral administration, intravenous (IV) administration, intranasal administration (IN) administration, and intraperitoneal (IP) administration.

[0147] According to some embodiments, the method further comprises imaging the tissue or body region or cancerous cells of the subject, thereby evaluating the accumulation of the multifunctional particles in the subject. The imaging can be performed using an imaging system selected from the group consisting of computed tomography (CT), X-ray imaging, magnetic resonance imaging (MRI), positron emission tomography (PET), single photon emission computed tomography (SPECT), ultrasound (US), and any combination thereof.

[0148] According to some embodiments, the body area is a breast or multiple breasts.

[0149] According to some embodiments, the cancer located outside the brain is a primary or secondary cancer, the inorganic particles are radiosensitizers, and the method further comprises radiation therapy.

[0150] According to an aspect, the present invention provides a method for diagnosing or prognosing cancer in a subject, comprising determining the expression level of a tumor antigen or an immune cell receptor in a biological sample of the subject using the multifunctional GNPs of the present invention.

[0151] The multifunctional GNPs according to the present invention are also provided in a kit for determining the expression or presence of a tumor antigen or an immune cell receptor in a biological sample.

[0152] Further embodiments and the full scope of the present invention will become apparent from the detailed description given hereinafter. It should be understood, however, that the detailed description and specific examples, while indicating preferred embodiments of the invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description. [Brief description of the drawings]

[0153] [Figure 1] FIG. 1 depicts a schematic diagram of a gold nanoparticle (GNP; 1) bound to (i) a first polymer linker (2) conjugated to insulin (4); (ii) a second polymer linker (3) conjugated to a first antibody (5) and a second antibody (6); and (iii) a capping polymer moiety (7). [Diagram 2] Figure 2 shows the in vivo effect of bispecific GNPs on HER2 positive cancer cells. GNPs conjugated with insulin and two different anti-HER2 antibodies were tested in the HER2 positive breast cancer cell line BT474, compared to a mixture of GNPs conjugated with insulin and a single antibody. Untreated cells served as control. Cells were incubated for 5 days with the following conditions: 1. Control - untreated, 2. Mixture of GNPs with Trastuzumab & GNPs with Pertuzumab, 3. Bispecific GNPs with both Trastuzumab & Pertuzumab conjugated to the same particle. [Figure 3A] Figure 3A shows the in vitro efficacy of bispecific nanocomplexes on human breast cancer cell line BT474, which expresses high levels of HER2 receptor. Cells were incubated for 5 days with the following conditions: (i) control - untreated; (ii) bispecific GNPs with both anti-HER2 antibodies, Trastuzumab & Pertuzumab, conjugated to the same particle without permeation enhancing molecules; and (iii) bispecific GNPs with both Trastuzumab & Pertuzumab conjugated to the same particle in addition to insulin molecules as permeation enhancers. [Figure 3B]Figure 3B shows the in vitro efficacy of bispecific nanocomplexes on human breast cancer cell line MCF7, which expresses relatively low levels of HER2 receptors. Cells were incubated for 5 days with the following conditions: (i) control - untreated; (ii) bispecific GNPs with both anti-HER2 antibodies, Trastuzumab & Pertuzumab, conjugated to the same particle without permeation enhancing molecules; and (iii) bispecific GNPs with both Trastuzumab & Pertuzumab conjugated to the same particle in addition to insulin molecules as permeation enhancers. [Figure 4] Figure 4 shows the results of determining the optimal density, e.g., number of antibody molecules per particle. Different compositions of nanocomplexes with increasing numbers of attached antibodies were tested in vitro for growth inhibition of BT474 human breast cancer cells overexpressing the HER2 receptor. Cells were incubated for 5 days with the following conditions: (i) control - untreated; (ii) GNPs with 2 antibody molecules attached to each particle; (iii) GNPs with 11 antibody molecules attached to each particle; (iv) GNPs with 18 antibody molecules attached to each particle; (v) GNPs with 20 antibody molecules attached to each particle and (vi) GNPs with 30 antibody molecules attached to each particle. [Diagram 5] Figure 5 shows tumor growth inhibition in vivo using GNPs conjugated with the anti-HER2 antibodies trastuzumab and pertuzumab and insulin as a penetration enhancer. Mice were subcutaneously administered breast cancer cells BT474 and two weeks later, the mice were IP injected once a week for four consecutive weeks. The treatment groups tested were a control group, a group that received a mixture of free antibodies, a group that received a mixture of GNPs, one carrying trastuzumab and the other carrying pertuzumab, and a fourth group that received bifunctional GNPs (both antibodies conjugated to the same particle). [Figure 6]Figure 6 shows the utility of the multifunctional system in immunotherapy by simultaneous delivery on GNPs, anti-PD-1 targeting a receptor on T cells, and anti-PD-L1 binding to a receptor on tumor cells. Peripheral blood mononuclear cells (PBMCs) were activated with anti-CD3 and anti-CD28, and then incubated with a mixture of free anti-PD-1 and anti-PD-L1 antibodies for 6 days, or with GNPs conjugated with both anti-PD-1 and anti-PD-L1 antibodies for 6 days. After 6 days, the medium was removed to detect cytokines, and PBMCs were collected and added to H1299 lung cancer cells and incubated for 18 hours to determine T cell cytotoxicity on target cells. Cell proliferation was measured by ELISA. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0154] The present invention provides a platform for the synchronized delivery of a combination of separate immunoglobulins to specific tissues, cancerous cells or body regions, a process for the preparation of said system, pharmaceutical compositions comprising said system, and their use for therapeutic and diagnostic applications. In particular, the present invention provides a multifunctional system for the delivery of antibodies to malignant tumors or cancer cells or to specific spatial regions of a tumor and / or for recruiting immune cells, such as killer cells, to cancer cells outside the brain or outside the CNS.

[0155] The multifunctional delivery system of the present invention is based on a core particle conjugated to a first immunoglobulin via a first polymer linker, to a second separate immunoglobulin via a second polymer linker, and to a permeation enhancing moiety via a third polymer linker, which may facilitate or enhance transport through the blood-tumor barrier or, according to some embodiments, may be a cellular internalization transporter or enhancer, such as a cellular metabolic enhancer that promotes increased glucose consumption by cells, particularly cancer cells. Each of the first and second immunoglobulin molecules may be an intact antibody, or an antibody fragment or construct that includes at least an antigen binding site. Without being bound by any theory or mechanism, it is hypothesized that the transporter facilitates the permeation of the conjugated particle through the blood-tumor barrier and / or through other biological membranes, and the cellular metabolic enhancer facilitates the permeation of the entire conjugated system via a receptor, such as the insulin receptor. Advantageously, improved therapeutic and / or diagnostic effects can be achieved by simultaneously delivering two or more distinct immunoglobulins with different cellular targets and / or different mechanisms of action (e.g., tumor receptors and immune cell activators) on a single vehicle. Thus, the delivery system of the present invention may be useful in the treatment and / or diagnosis of a wide range of pathologies, particularly malignant tumors.

[0156] The present invention is based in part on the surprising discovery that two different anti-HER2 antibodies on a single core particle further conjugated to insulin as a permeation enhancer moiety can inhibit cancer cell proliferation in vitro and suppress tumor growth in vivo with greater efficiency compared to the efficiency of a mixture of similar particles conjugated to each of the antibodies separately. This has also been shown for the combination of an anti-PD-1 antibody and an anti-PD-L1 antibody.

[0157] According to the principles of the present invention, the first and second immunoglobulins are conjugated to the outer surface of the core particle via a polymer linker, rather than being loaded or encapsulated within the particle core. Importantly, the activity of the immunoglobulin molecules is maintained despite being conjugated to the core particle, and therefore the agents do not necessarily need to be released from the system upon penetration into malignant cells or tissues.

[0158] As a diagnostic, this approach in some embodiments allows for early and accurate detection of certain malignancies and other diseases or disorders. For example, when the multifunctional particle contains two or more immunoglobulin molecules that target the system to diseased or damaged cells, the core particle is a contrast agent that allows the particle to be tracked in vivo using appropriate imaging modalities.

[0159] As therapeutic agents, in some embodiments, this approach allows for the delivery of efficient combinations of therapeutic agents. In some embodiments, the combination of separate therapeutic immunoglobulins on a single platform results in optimized synergistic effects of the drug combination. In particular, this approach is suitable for the treatment of cancer using a combination of different binding moieties directed against tumor antigen / multiple tumor antigens and / or immune cells.

[0160] In some embodiments, a combined therapeutic and diagnostic use is possible, for example, by using a therapeutically active immunoglobulin, such as an antibody, conjugated to a core particle, e.g., a gold nanoparticle, that constitutes a contrast agent for diagnostic imaging.

[0161] Multifunctional system According to one aspect, a multifunctional system for simultaneous delivery of separate immunoglobulins to tissues and cells outside the brain is provided, comprising: (a) a core particle attached to at least: (i) a first polymer linker; (ii) a second polymer linker; and (iii) a third polymer linker; (b) a first immunoglobulin molecule conjugated to a first polymer linker; (c) a second immunoglobulin molecule separate from the first immunoglobulin, which is conjugated to a second polymer linker; and (d) a permeation-enhancing moiety conjugated to a third polymer linker. A multi-function system is provided that includes:

[0162] According to another aspect, (a) a core particle attached to at least: (i) a first polymer linker; (ii) a second polymer linker; and (iii) a third polymer linker; and (b) a permeation-enhancing moiety conjugated to a third polymer linker. Including, Here, a multifunctional system is provided, in which the first and second polymer linkers each have a free functional end group configured to conjugate a first immunoglobulin and a second separate immunoglobulin molecule.

[0163] In some embodiments, the length of the third polymer linker is substantially different from the length of at least one of the first and second polymer linkers, In some embodiments, the length of the third polymer linker is substantially longer than the length of at least one of the first and second polymer linkers.

[0164] The term "multifunctional system" may be used interchangeably herein with the terms "multifunctional particle", "multifunctional GNP" and "co-delivery system" and refers to a system that can achieve at least two purposes or perform a single advanced function by incorporating at least two functional units. The system of the present invention incorporates multifunctional units with separate purposes, including at least a first and a second immunoglobulin molecule with separate binding sites and, in some embodiments, separate targets and / or separate activities, and a permeation enhancing moiety, e.g., an internalization transporter moiety that aids in delivering the system through a biological membrane, e.g., the blood-tumor barrier.

[0165] As used herein, the term "co-delivery" can be used interchangeably with the term "simultaneous delivery" and means that two separate immunoglobulins are delivered simultaneously in a single composition to their target, e.g., a tumor region outside the brain, and in some embodiments, outside the CNS, a cancerous cell, or a specific region or tissue in the subject's body. In some embodiments, "co-delivery" means synchronous delivery, i.e., upon administration, the separate active agents exhibit synchronized pharmacokinetics and biodistribution. In some related embodiments, the two immunoglobulins exhibit synchronized distribution within a tissue or region of the body, or within a solid tumor or tumor metastasis. As used herein, the term "synchronous distribution" means that the two active agents co-localize within the same region, tissue, population of cells, or tumor. In some embodiments, the two immunoglobulins accumulate in a specific spatial region of the same malignant tumor or tumor of the subject. In still other embodiments, the two immunoglobulins bring two different cell types closer together. In some embodiments, the different cell types include cancer cells and immune cells.

[0166] In some embodiments, synchronized pharmacokinetics and biodistribution results in optimized synergy of the drug combination. In certain related embodiments, one of the immunoglobulins is a monoclonal antibody against a tumor antigen and the second immunoglobulin is a monoclonal antibody that activates immune cells. According to some embodiments, the two active agents are antibodies against the same or different tumor antigens.

[0167] The terms "delivery" and "delivered" encompass both delivering immunoglobulin(s) by releasing the active agent(s) from the delivery system (e.g., by using a cleavable linker) and delivering immunoglobulin(s) while conjugated to the delivery system (e.g., by covalent conjugation). Advantageously, the compositions of the multifunctional system of the invention do not interfere with the functionality of the immunoglobulins, and therefore do not necessarily have to be released from the system.

[0168] The term "separate" as used herein means that a first immunoglobulin molecule is distinct from a second immunoglobulin molecule. The term "separate" should be understood to encompass different molecules of the same type, such as two antibodies with different specificities. Furthermore, the term "separate" should be understood to encompass different molecules that contain similar fragments. For example, whole antibodies (e.g., IgG) and fragments of the antibodies (e.g., Fc / Fab regions or scFv) are considered separate active agents.

[0169] As used herein, the term "core particle" refers to a particle that constitutes the central portion of the co-delivery system. In some embodiments, the core particle is a nanoparticle. The term "nanoparticle" refers to a particle having a diameter of 1-1000 nm.

[0170] In some embodiments, the core particle is selected from the group consisting of metal particles, metal oxide particles, metal carbide particles, lipid particles, carbon-based particles, ceramic particles, polymer particles, and liposomes. Each possibility represents a separate embodiment of the present invention. In some embodiments, the core particle is an inorganic particle. In some embodiments, the inorganic particle is selected from the group consisting of metal particles, metal oxide particles, and ceramic particles. In some embodiments, the inorganic particle is selected from the group consisting of metal particles and metal oxide particles. In some embodiments, the inorganic particle is a metal particle. In other embodiments, the inorganic particle is a metal oxide particle. In certain embodiments, the inorganic particle is selected from gold particles and iron oxide particles.

[0171] In some embodiments, the metal particles are magnetic particles. In some embodiments, the inorganic particles are magnetic particles. In some embodiments, the magnetic particles are contrast agents for magnetic resonance imaging (MRI). Any magnetic particles suitable for use as MRI contrast agents can be used in the compositions and methods of the present invention. The magnetic particles can be formed, at least in part, from any material that is affected by a magnetic field. Examples of suitable materials include, but are not limited to, magnetite, hematite, ferrite, and materials that include one or more of iron, cobalt, manganese, nickel, chromium, gadolinium, neodymium, dysprosium, samarium, erbium, iron carbide, iron, or combinations thereof.

[0172] In some embodiments, the inorganic particles are contrast agents for computed tomography (CT) or X-ray imaging. In some embodiments, the inorganic particles are metal particles that can be used as CT or X-ray imaging contrast agents. As will be apparent to those skilled in the art, any metal and / or combination of metals suitable for use in CT or X-ray imaging can be used in the metal particles of the present invention in embodiments related to diagnostic use. In some embodiments, the metals that can be used to form the particles of the present invention are heavy metals or metals with high Z numbers. Examples of suitable metals include, but are not limited to, gold, silver, platinum, palladium, cobalt, iron, copper, tin, tantalum, vanadium, molybdenum, tungsten, osmium, iridium, rhenium, hafnium, thallium, lead, bismuth, gadolinium, dysprosium, holmium, and uranium, or combinations thereof.

[0173] In some embodiments, the multifunctional particle comprises essentially (a) (i) a first linear polymer linker; (ii) a second linear polymer linker; and (iii) an inorganic particle attached to a third linear polymer linker; (b) a first immunoglobulin molecule conjugated to a first linear polymer linker; (c) a second immunoglobulin molecule conjugated to a second linear polymer linker; and (d) a permeation-enhancing moiety conjugated to a third linear polymer linker. It consists of: Wherein the length of the third linear polymer linker is substantially different from the length of the first and second linear polymer linkers, and the first bioactive molecule is distinguished from the second bioactive molecule, and wherein the inorganic particle is a contrast agent that can be detected by imaging modalities selected from computed tomography (CT), X-ray imaging, magnetic resonance imaging (MRI), positron emission tomography (PET), single photon emission computed tomography (SPECT), ultrasound (US), and any combination thereof. Advantageously, in such an embodiment, the multifunctional particle can be used in diagnostic applications without the need to conjugate a labeling molecule.

[0174] According to some embodiments, the inorganic particles are metal particles selected from the group consisting of gold particles, silver particles, platinum particles, iron particles, copper particles, and mixtures or combinations thereof. Each possibility represents a separate embodiment. In some embodiments, the metal particles are gold (Au) particles.

[0175] In some embodiments, the inorganic particles are metal oxide particles. In some embodiments, the metal oxide particles are iron oxide (Fe 2 O 3 or Fe 3 O 4 ), magnesium oxide, nickel oxide, cobalt oxide, aluminum oxide, zinc oxide, copper oxide, and manganese oxide, or any combination thereof. Each possibility represents a separate embodiment of the present invention. In some embodiments, the metal oxide particles comprise an iron oxide selected from iron (III) oxide and iron (II,III) oxide. In some embodiments, the metal oxide particles are iron oxide particles, where the iron oxide is selected from iron (III) oxide and iron (II,III) oxide.

[0176] In some embodiments, the core particle is selected from the group consisting of a lipid particle, a carbon-based particle, a ceramic particle, a polymer particle, and a liposome.

[0177] In some embodiments, the core particle is a radiosensitizer. The term "radiosensitizer" as used herein refers to an agent that makes cells (especially cancer cells) more sensitive to radiation therapy. Typically, materials with high atomic number such as gold (Z=79) enhance radiation sensitivity. Thus, gold nanoparticles are an example of a core particle that is a radiosensitizer.

[0178] According to some embodiments, the core particle has a diameter of 1-200 nm, 1-180 nm, 1-160 nm, 1-140 nm, 1-120 nm, 1-100 nm, 1-90 nm, 1-80 nm, 1-70 nm, 1-60 nm, 1-50 nm, 1-40 nm, 2-100 nm, 2-60 nm, 2-50 nm, 2-40 nm, 2-30 nm, 2-20 nm, 2-10 nm, 3-100 nm, 3-60 nm, 3-80 nm, 3-90 nm, 3-120 nm, 3-140 nm, 3-160 nm, 3-200 nm, 3-300 nm, 3-40 nm, 3-50 nm, 3-60 nm, 3-70 nm, 3-80 nm, 3-90 nm, 3-100 nm, 3-120 nm, 3-140 nm, 3-160 nm, 3-180 nm, 3-200 nm, 3-300 nm, 3-40 nm, 3-50 nm, 3-60 nm, 3-70 nm, 3-8 ...200 nm, 3-300 nm, 3 0nm, 3~50nm, 3~40nm, 3~30nm, 3~20nm, 4~100nm, 4~60nm, 4~50nm, 4~40nm, 5~200nm, 6~190nm, 7~180nm, 8 ~170nm, 10~160nm, 20~160nm, 10~150nm, 10~140nm, 10~120nm, 10~110nm, 10~100nm, 10~90nm, 10~80nm, 12~70nm, 14~60nm, 15~50nm, 15~40nm, 15~30nm, 20~30nm, 15~30nm, 20~90nm, 20~80nm, 20~70nm, 20~60m , 20~50nm, 20~40nm, 20~30nm, 30~70nm, 30~60nm, 40~60nm, 10~200nm, 20~200nm, 30~200nm, 40~200nm, 5 Nanoparticles having a diameter of 0-200 nm, 60-200 nm, 70-200 nm, 80-200 nm, 90-200 nm, 100-200 nm, 110-190 nm, 120-170 nm, 130-160 nm, 100-160 nm, 80-160 nm, 60-160 nm, 40-160 nm, 20-160 nm, 10-160 nm, 20-150 nm or 30-150 nm. Each possibility represents a separate embodiment of the present invention.According to some embodiments, the core particle is a nanoparticle having a diameter of at least 1 nm, at least 2 nm, at least 3 nm, at least 4 nm, at least 5 nm, at least 10 nm, at least 12 nm, at least 15 nm, at least 18 nm, at least 20 nm, at least 25 nm, at least 30 nm, at least 35 nm, at least 40 nm, at least 45 nm, at least 50 nm, at least 60 nm, at least 70 nm, at least 80 nm, at least 90 nm, at least 100 nm, at least 110 nm, at least 120 nm, at least 130 nm, at least 140 nm, or at least 150 nm. Each possibility represents a separate embodiment of the present invention. According to some embodiments, the core particle is a nanoparticle having a diameter of at most 5 nm, at most 10 nm, at most 15 nm, at most 20 nm, at most 30 nm, at most 40 nm, at most 50 nm, at most 60 nm, at most 70 nm, at most 80 nm, at most 90 nm, at most 100 nm, at most 120 nm, at most 140 nm, at most 160 nm, at most 180 nm, or at most 200 nm. Each possibility represents a separate embodiment of the present invention.

[0179] According to some embodiments, the multifunctional particles, i.e., the entire co-delivery system, may be 5-500 nm, 6-400 nm, 8-300 nm, 10-300 nm, 10-200 nm, 10-180 nm, 10-160 nm, 10-150 nm, 10-100 nm, 20-90 nm, 20-80 nm, 20-70 nm, 20-60 nm, 25-100 nm, 25-90 nm, 25-80 nm, 25-70 nm, 25-60 nm, 25-50 nm, 25-60 nm, 25-70 nm, 25-80 nm, 25-9 ...90 nm, 25-90 nm, 25-90 nm, 25-60 nm, 25-50 nm, 25-50 nm, 25-60 nm, 25-70 nm, 25-80 nm, 25-90 nm, 25-80 nm, 25-90 nm, 25-90 nm, 25-90 nm, 25-90 nm, 25-90 nm, 25-90 nm, 25-90 nm, 25-90 nm, 25-90 nm, 25-90 nm, 25-90 nm, 25-90 nm, 25-90 0 nm, 30 to 60 nm, 40 to 200 nm, 40 to 150 nm, 40 to 120 nm, 40 to 100 nm, 40 to 80 nm, 40 to 60 nm, 50 to 300 nm, 50 to 250 nm, 50 to 200 nm, 50 to 180 nm, 50 to 150 nm, 60 to 200 nm, 70 to 180 nm, 80 to 180 nm, 90 to 170 nm, 100 to 160 nm, 100 to 200 nm, 150 to 200 nm or 150 to 180 nm. According to some embodiments, the multifunctional particles have a diameter of 2-200 nm, 1-100 nm, 1-150 nm, 1-200 nm, 2-50 nm, 2-100 nm, 2-150 nm, 4-50 nm, 4-100 nm, 4-150 nm, or 4-200 nm, with each possibility representing a separate embodiment of the present invention. According to some embodiments, the multifunctional particles have a diameter of at least 1 nm, at least 2 nm, at least 5 nm, at least 10 nm, at least 15 nm, at least 20 nm, at least 25 nm, at least 30 nm, at least 35 nm, at least 40 nm, at least 45 nm, at least 50 nm, at least 55 nm, at least 60 nm, at least 70 nm, at least 80 nm, at least 90 nm, at least 100 nm, at least 110 nm, at least 120 nm, at least 130 nm, at least 140 nm, at least 150 nm, at least 160 nm, at least 180 nm, or at least 200 nm. Each possibility represents a separate embodiment of the present invention.According to some embodiments, the multifunctional particles have a diameter of at most 5 nm, at most 20 nm, at most 30 nm, at most 40 nm, at most 50 nm, at most 60 nm, at most 70 nm, at most 80 nm, at most 90 nm, at most 100 nm, at most 110 nm, at most 120 nm, at most 130 nm, at most 140 nm, at most 150 nm, at most 180 nm, at most 200 nm, at most 250 nm, at most 300 nm, at most 350 nm, at most 400 nm, at most 450 nm, or at most 500 nm. Each possibility represents a separate embodiment of the present invention.

[0180] As used herein, the term "diameter" of a particle / nanoparticle can be used interchangeably with the term "size" of a particle / nanoparticle and refers to the maximum linear distance between two points on the surface of the particle / nanoparticle being described. The term "diameter" as used herein encompasses the size of spherical particles and non-spherical particles and can refer to the actual size of a particle or its hydrodynamic diameter, including the contribution from a solvated sphere. Any method known in the art can be used to determine the size of a particle, such as transmission electron microscopy (TEM), scanning electron microscopy (SEM), and dynamic light scattering (DLS). The term "diameter" can refer to the average diameter of a plurality of particles measured by any of the above techniques.

[0181] The core particle is coated with a polymer layer comprising at least three types of polymers: a first polymer linker having a functional end group capable of binding to a first immunoglobulin, a second polymer linker having a functional end group capable of binding to a second immunoglobulin, and a third polymer linker that is conjugated to a permeation enhancing moiety.

[0182] The term "coating" as used herein is intended to mean that a layer, e.g., a polymer layer comprising multiple polymer moieties, is chemically attached to the surface of a core particle, thereby at least partially covering the core particle. A "particle coated with a polymer layer" means that each polymer moiety in the polymer layer is chemically attached to the particle via a functional end group, e.g., a thiol group, of the polymer moiety. In some embodiments, the thiol group of the polymer is conjugated to the gold particle by an Au-S bond. The chemical attachment can be covalent, semi-covalent, or non-covalent.

[0183] The term "polymer moiety" may be used interchangeably with the term "polymer" and refers to a molecule containing two or more repeating subunits linked in a linear, branched, hyperbranched, dendritic or cyclic arrangement, or any combination thereof. In some embodiments, the term "polymer moiety" refers to a molecule containing at least three repeating subunits linked in a linear, branched, hyperbranched, dendritic or cyclic arrangement, or any combination thereof. Examples of subunits include alkylenes, arylenes, heteroalkylenes, amino acids, nucleic acids, sugars, and the like. Examples of polymer moieties include, but are not limited to, poly(ethylene glycol) groups, poly(ethylene amine) groups, and poly(amino acid) groups. The terms "polymer moiety" and "polymer" also encompass polymer linkers. As used herein, the term "polymer linker" refers to a polymer moiety that originally contains at least one functional / reactive group that allows for attachment to a substance, e.g., a particle. In some embodiments, the polymer linker is a bifunctional polymer having at least two functional / reactive groups that allow for attachment to at least two substances, thereby linking between the at least two substances. In some embodiments, the polymer linker is a monofunctional polymer having one functional / reactive group that allows for attachment to one substance, e.g., the core particle. It should be understood that the terms "monofunctional", "bifunctional", "functional" and the like, as used herein, refer to the polymer linker in its original form prior to attachment to the core particle and / or the transporter / permeabilization moiety or respective active agent.

[0184] In some embodiments, the core particle is attached to a first polymer linker. In some embodiments, the core particle is attached to a second polymer linker. In some embodiments, the core particle is attached to a third polymer linker. In some embodiments, the core particle is attached to a first, second and third polymer linker.

[0185] The term "bonded" can be used interchangeably with the term "conjugate". In some embodiments, the bond is a covalent conjugate. The terms "covalently attached", "covalently attached", "covalently linked" and "covalent bond" are used interchangeably herein and refer to the formation of a chemical bond characterized by the sharing of electron pairs between atoms. For example, a covalently attached drug coating refers to a drug coating that forms a chemical bond with the surface of the functionalized material, as compared to attaching to the surface via other means, such as adhesion or electrostatic interactions. It will be understood that a drug (e.g., a polymer) that is covalently attached to a surface can also be attached via other means in addition to covalent attachment.

[0186] In some embodiments, the polymer moiety and / or linker is attached to the outer surface of the core particle via a chemical attachment selected from the group consisting of covalent attachment, semi-covalent attachment, and non-covalent attachment. Each possibility represents a separate embodiment of the present invention. In some embodiments, the polymer moiety and / or linker is attached to the outer surface of the core particle via semi-covalent attachment. As used herein, the term "semi-covalent attachment" refers to a coordinate bond in which the shared electron pair forming the bond originates from the same atom. In the present disclosure, semi-covalent attachment can occur between a metal particle, e.g., a gold particle, and a thiol group.

[0187] In some embodiments, at least one of the first, second and third polymer linkers is a linear polymer linker. In some embodiments, the first polymer linker is a linear polymer linker. In some embodiments, the second polymer linker is a linear polymer linker. In some embodiments, the third polymer linker is a linear polymer linker. In some embodiments, the first and second polymer linkers are linear polymer linkers. In some embodiments, the first and third polymer linkers are linear polymer linkers. In some embodiments, the second and third polymer linkers are linear polymer linkers. In some embodiments, the first, second and third polymer linkers are linear polymer linkers. In some embodiments, the linear polymer linker is a bifunctional linear polymer having two types of functional / reactive groups at both ends of the linear polymer. In some embodiments, each of the first, second and third polymer linkers is independently a linear bifunctional polymer linker having two types of functional / reactive groups on both ends of the linear polymer.

[0188] As used herein, the term "linear" polymer / polymer linker refers to a polymer / polymer linker in which, in some embodiments, at least 80% of the monomer units are linked linearly, i.e., in the form of a single polymer chain. In further embodiments, the term "linear" polymer / polymer linker refers to a polymer / polymer linker in which at least 90% of the monomer units are linked linearly. In further embodiments, the term "linear" polymer / polymer linker refers to a polymer / polymer linker in which about 100% of the monomer units are linked linearly. As used herein, the term "single polymer chain" refers to a polymer chain comprising monomers linked together such that the monomer units are attached to each other via two atoms, one on each monomer unit.

[0189] In some embodiments, the multifunctional system further comprises an additional polymer moiety attached to the core particle. In some embodiments, the additional polymer moiety is a linear polymer. In some embodiments, the additional polymer moiety is a monofunctional polymer. In some embodiments, the additional polymer moiety is a monofunctional polymer linker. The additional polymer moiety is thus in some embodiments a fourth polymer linker attached to the core particle. In some embodiments, the fourth linker is used as a capping moiety. In some embodiments, the core particle is attached to the first, second, third and fourth polymer linkers. In some embodiments, the fourth polymer linker is monofunctional, i.e., has a single functional end group originally configured to conjugate the polymer linker to the core particle. In some embodiments, the fourth polymer linker is a linear monofunctional polymer.

[0190] In some embodiments, the first polymer linker comprises a polymer selected from the group consisting of, but not limited to, polyethers, polyacrylates, polyanhydrides, polyvinyl alcohols, polysaccharides, poly(N-vinylpyrrolidone), polyglycerin (PG), poly(N-(2-hydroxypropyl)methacrylamide), polyoxazolines, poly(amino acid)-based hybrids, recombinant polypeptides, derivatives, and combinations thereof, with each possibility representing a separate embodiment of the present invention.

[0191] The term "derivative," as used herein, refers to a compound whose core structure is the same as the parent compound, or whose core structure closely resembles the parent compound but has chemical or physical modifications, such as different or additional groups, including, but not limited to, alkoxy groups, carboxy groups, amine groups, methoxy groups, and thiol groups.

[0192] In some embodiments, the first polymer linker comprises a polyether. In some embodiments, the first polymer linker is a polyether. In some embodiments, the polyether is polyethylene glycol (PEG) or a derivative thereof.

[0193] Where appropriate, the abbreviation (PEG) is used in combination with a numerical suffix to indicate the average molecular weight of the PEG. A form of PEG or a PEG species is a PEG or PEG derivative having a particular average molecular weight.

[0194] As used herein, "PEG or derivatives thereof" refers to any compound that includes at least one polyethylene glycol moiety. PEG exists in linear and branched forms, including multi-arm and / or grafted polyethylene glycols. The term "PEG derivatives" as used herein refers to PEG modified by alkylation of the terminal hydroxyl group. In some embodiments, the terminal hydroxyl group is alkylated with a linear or branched C1-C6 alkyl. PEG may further include functional groups. PEG may be monofunctional, bifunctional, or multifunctional polyethylene glycol.

[0195] Exemplary functional groups include, but are not limited to, the following: hydroxyl, carboxyl, thiol, amine, phosphate, phosphonate, sulfate, sulfite, sulfonate, sulfoxide, sulfone, amide, ester, ketone, aldehyde, cyano, alkyne, azide, and alkene, or combinations thereof.

[0196] In some embodiments, the first polymer linker comprises a thiol (-SH) end group. In some embodiments, the first polymer linker is chemically attached to the core particle via the thiol (-SH) end group. In some embodiments, the first polymer linker is conjugated to the first immunoglobulin via an amide bond. In some embodiments, the core particle is conjugated to the first polymer linker via a sulfide bond, and the first immunoglobulin is conjugated to the first polymer linker via an amide bond. In some embodiments, the core particle is an inorganic particle and is conjugated to the first polymer linker via a sulfide bond, and the first immunoglobulin is conjugated to the first polymer linker via an amide bond. In some embodiments, the first polymer linker in the co-delivery system has the structure -SR-CONH-, where R is a polymer chain made of repeating monomer units. In other embodiments, the first polymer linker in the co-delivery system has the structure -SR-NHCO-, where R is a polymer chain made of repeating monomer units. In some embodiments, the first polymer linker is a thiolated PEG acid (HS-PEG-COOH) or a thiolated PEG amine (HS-PEG-NH 2 HS and COOH / NH 2 It should be understood that the terminal group refers to the polymer linker prior to conjugation with the core particle and the immunoglobulin. In some embodiments, the thiol group is chemically attached to the core particle and the acid or amine group is covalently conjugated to the first immunoglobulin. In some embodiments, the first polymer linker in the co-delivery system has a structure selected from -S-PEG-C(O)- and -S-PEG-NH-.

[0197] In some embodiments, the first polymer linker is a non-cleavable linker. In some embodiments, the first polymer linker is non-cleavable under physiological conditions.

[0198] The term "non-cleavable" as used herein refers to a stable bond that is not acid or base sensitive, is not sensitive to reducing or oxidizing agents, and is not sensitive to enzymes that may be found within a cell or in the circulatory system. In some embodiments, the non-cleavable polymer linker lacks a pH sensitive hydrazone. In some embodiments, the non-cleavable polymer linker lacks a disulfide bond. In some embodiments, the non-cleavable polymer linker lacks an ester bond. It should be understood that the term "polymer linker is non-cleavable" is meant to encompass the bond between the core particle and the polymer linker; the bond between each polymer linker and each active agent; or the bond between each polymer linker and the permeation enhancing moiety, as well as any bonds within the polymer linker itself.

[0199] In some embodiments, the first polymer linker has a molecular weight (MW) of 2,000 to 7,000 Daltons (Da). In some embodiments, the first polymer linker has a molecular weight (MW) of 500 to 10,000 Da, 1,000 to 10,000 Da, 600 to 9,500 Da, 700 to 9,000 Da, 800 to 8,500 Da, 800 to 6,000 Da, 800 to 5,000 Da, 800 to 4,000 Da, 800 to 3,000 Da, 800 to 2,000 Da, 900 to 8,000 Da, 1,000 to 7,000 Da, 1,500 to 6,500 Da, 2,000 to 6,0 00Da, 3,000~6,000Da, 4,000~6,000Da, 1,000~2,000Da, 1,000~3,000Da, 1,000~4,000Da, 1,000~5,000Da, 1,000~7,000 Da, 3,400~7,000Da, 2,000~3,000Da, 2,000~5,000Da, 2,000~7,000Da, 2,000~10,000Da, 3,000~3,400Da, 3,000~4,000Da The first polymer linker has a molecular weight (MW) within a range selected from the group consisting of 3,000-5,000 Da, 3,000-7,000 Da, 3,000-10,000 Da, 5,000-7,000 Da, 5,000-10,000 Da, and 7,000-10,000 Da. Each possibility represents a separate embodiment. According to some embodiments, the first polymer linker has a MW of at least 1,000 Da, at least 1,500 Da, at least 2,000 Da, at least 2,500 Da, at least 3,000 Da, at least 3,400 Da, at least 4,000 Da, at least 5,000 Da, at least 6,000 Da, at least 7,000 Da, or at least 8,000 Da. Each possibility represents a separate embodiment. According to some embodiments, the first polymer linker has a MW of up to 2,000 Da, up to 3,000 Da, up to 4,000 Da, up to 5,000 Da, up to 6,000 Da, up to 7,000 Da, or up to 10,000 Da. Each possibility represents a separate embodiment.

[0200] In some embodiments, the second polymer linker comprises a polymer selected from the group consisting of polyethers, polyacrylates, polyanhydrides, polyvinyl alcohols, polysaccharides, poly(N-vinylpyrrolidone), polyglycerin (PG), poly(N-(2-hydroxypropyl)methacrylamide), polyoxazolines, poly(amino acid)-based hybrids, recombinant polypeptides, derivatives, and combinations thereof, with each possibility representing a separate embodiment of the present invention.

[0201] In some embodiments, the second polymer linker comprises a polyether. In some embodiments, the second polymer linker is a polyether. In some embodiments, the polyether is polyethylene glycol (PEG) or a derivative thereof.

[0202] In some embodiments, the second polymer linker comprises a thiol (-SH) end group. In some embodiments, the second polymer linker is chemically attached to the core particle via a thiol (-SH) end group. In some embodiments, the second polymer linker is conjugated to the second immunoglobulin via an amide bond. In some embodiments, the core particle is conjugated to the second polymer linker via a sulfide bond, and the second immunoglobulin is conjugated to the second polymer linker via an amide bond. In some embodiments, the core particle is an inorganic particle and is conjugated to the second polymer linker via a sulfide bond, and the second immunoglobulin is conjugated to the second polymer linker via an amide bond. In some embodiments, the second polymer linker in the co-delivery system has the structure -SR-CONH-, where R is a polymer chain made of repeating monomer units. In other embodiments, the second polymer linker in the co-delivery system has the structure -SR-NHCO-, where R is a polymer chain made of repeating monomer units. In some embodiments, the second polymer linker is a thiolated PEG acid (HS-PEG-COOH) or a thiolated PEG amine (HS-PEG-NH2 ) is selected from HS and COOH / NH 2 It should be understood that the terminal group refers to the polymer linker prior to conjugation with the core particle and the immunoglobulin. In some embodiments, the thiol group is chemically attached to the core particle and the acid or amine group is covalently conjugated to the second immunoglobulin. In some embodiments, the second polymer linker in the co-delivery system has a structure selected from -S-PEG-C(O)- and -S-PEG-NH-.

[0203] In some embodiments, the second polymer linker is a non-cleavable linker. In some embodiments, the second polymer linker is non-cleavable under physiological conditions.

[0204] In some embodiments, the second polymer linker has a molecular weight (MW) of 2,000 to 7,000 Da. In some embodiments, the second polymer linker is 500 to 10,000 Da, 600 to 9,500 Da, 700 to 9,000 Da, 800 to 8,500 Da, 800 to 6,000 Da, 800 to 5,000 Da, 800 to 4,000 Da, 800 to 3,000 Da, 800 to 2,000 Da, 900 to 8,000 Da, 1,000 to 7,000 Da, 1,500 to 6,500 Da, 2,000 to 6,000 Da, 3,000 to 6,000 Da, 4,000 to 6,000 Da, 1,000 to 2,000 Da, 1,000 to 3,000 Da, 1,0 and 7,000-10,000 Da. Each possibility represents a separate embodiment. According to some embodiments, the second polymer linker has a MW of at least 1,000 Da, at least 1,500 Da, at least 2,000 Da, at least 2,500 Da, at least 3,000 Da, at least 3,400 Da, at least 4,000 Da, at least 5,000 Da, at least 6,000 Da, at least 7,000 Da, or at least 8,000 Da. Each possibility represents a separate embodiment. According to some embodiments, the second polymer linker has a MW of up to 2,000 Da, up to 3,000 Da, up to 4,000 Da, up to 5,000 Da, up to 6,000 Da, up to 7,000 Da, or up to 10,000 Da. Each possibility represents a separate embodiment.

[0205] According to some embodiments, the first polymer linker and the second polymer linker comprise different polymers. According to some embodiments, the first polymer linker and the second polymer linker are different polymers. In some embodiments, the first polymer linker and the second polymer linker comprise the same polymer. In some embodiments, the first polymer linker and the second polymer linker are the same.

[0206] In some embodiments, the first and second polymer linkers comprise the same polymer selected from the group consisting of polyethers, polyacrylates, polyanhydrides, polyvinyl alcohols, polysaccharides, poly(N-vinylpyrrolidone), polyglycerol (PG), poly(N-(2-hydroxypropyl)methacrylamide), polyoxazolines, poly(amino acid)-based hybrids, recombinant polypeptides, derivatives, and combinations thereof. In some embodiments, both the first and second polymer linkers comprise PEG. In some embodiments, both the first and second polymer linkers are PEG. In some embodiments, both the first and second polymer linkers comprise thiolated PEG. In some embodiments, the first and second polymer linkers comprise thiolated PEG acids (HS-PEG-COOH) or thiolated PEG amines (HS-PEG-NH 2 In some embodiments, the first and second polymer linkers comprise a thiolated PEG acid (HS-PEG-COOH) or a thiolated PEG amine (HS-PEG-NH 2 In some embodiments, both the first polymer linker and the second polymer linker are thiolated PEG acids (HS-PEG-COOH). In some embodiments, both the first polymer linker and the second polymer linker are thiolated PEG amines (HS-PEG-NH 2 ).

[0207] In some embodiments, the third polymer linker comprises a polymer selected from the group consisting of polyethers, polyacrylates, polyanhydrides, polyvinyl alcohols, polysaccharides, poly(N-vinylpyrrolidone), polyglycerin (PG), poly(N-(2-hydroxypropyl)methacrylamide), polyoxazolines, poly(amino acid)-based hybrids, recombinant polypeptides, derivatives, and combinations thereof, with each possibility representing a separate embodiment of the present invention.

[0208] In some embodiments, the third polymer linker comprises a polyether. In some embodiments, the third polymer linker is a polyether. In some embodiments, the polyether is polyethylene glycol (PEG) or a derivative thereof.

[0209] In some embodiments, the third polymer linker comprises a thiol (-SH) end group. In some embodiments, the third polymer linker is chemically attached to the core particle via the thiol (-SH) end group. In some embodiments, the third polymer linker is conjugated to the permeation moiety via an amide bond. In some embodiments, the core particle is bound to the third polymer linker via a sulfide bond, and the permeation moiety is conjugated to the third polymer linker via an amide bond. In some embodiments, the core particle is an inorganic particle, bound to the third polymer linker via a sulfide bond, and the permeation enhancing moiety is conjugated to the third polymer linker via an amide bond. In some embodiments, the third polymer linker in the co-delivery system has the structure -SR-CONH-, where R is a polymer chain made of repeating monomer units. In other embodiments, the third polymer linker in the co-delivery system has the structure -SR-NHCO-, where R is a polymer chain made of repeating monomer units. In some embodiments, the third polymer linker is a thiolated PEG acid (HS-PEG-COOH) or a thiolated PEG amine (HS-PEG-NH 2) is selected from HS and COOH / NH 2 It should be understood that the terminal group refers to the polymer linker prior to conjugation with the core particle and the permeation enhancing moiety. In some embodiments, the thiol group is chemically attached to the core particle and the acid or amine group is covalently conjugated to the permeation enhancing moiety. In some embodiments, the third polymer linker in the co-delivery system has a structure selected from -S-PEG-C(O)- and S-PEG-NH-.

[0210] In some embodiments, the third polymer linker has a molecular weight (MW) of 2,000 to 7,000 Da. In some embodiments, the third polymer linker is 500 to 10,000 Da, 600 to 9,500 Da, 700 to 9,000 Da, 800 to 8,500 Da, 800 to 6,000 Da, 800 to 5,000 Da, 800 to 4,000 Da, 800 to 3,000 Da, 800 to 2,000 Da, 900 to 8,000 Da, 1,000 to 7,000 Da, 1,500 to 6,500 Da, 2,000 to 6,000 Da, 3,000 to 6,000 Da, 4,000 to 6,000 Da, 1,000 to 2,000 Da, 1,000 to 3,000 Da, 1,0 and 7,000-10,000 Da. Each possibility represents a separate embodiment. According to some embodiments, the third polymer linker has a MW of at least 1,000 Da, at least 1,500 Da, at least 2,000 Da, at least 2,500 Da, at least 3,000 Da, at least 3,400 Da, at least 4,000 Da, at least 5,000 Da, at least 6,000 Da, at least 7,000 Da, or at least 8,000 Da. Each possibility represents a separate embodiment. According to some embodiments, the third polymer linker has a MW of up to 2,000 Da, up to 3,000 Da, up to 4,000 Da, up to 5,000 Da, up to 6,000 Da, up to 7,000 Da, or up to 10,000 Da. Each possibility represents a separate embodiment.

[0211] In some embodiments, the third polymer linker is a non-cleavable linker. In some embodiments, the third polymer linker is non-cleavable under physiological conditions.

[0212] In some embodiments, at least one of the first, second and third polymer linkers comprises a cleavable linker. In some embodiments, at least one of the first and second polymer linkers comprises a cleavable linker. In some embodiments, each of the first and second polymer linkers comprises an independently cleavable linker. According to some embodiments, the cleavable linker comprises a bond that is susceptible to cleavage by an endogenous molecule located or expressed in a particular malignant tumor, body region or tissue, such as the breast or other non-CNS tissue or location. In some embodiments, the cleavable linker is PEG succinimidyl succinate (PEGSS). According to some embodiments, the endogenous molecule is glutathione. According to some embodiments, the endogenous molecule is selected from the group comprising proteases, nucleases, hydronium ions and reducing agents. In some embodiments, the endogenous molecule is selected from neuroserpin and serpin B. Each possibility represents a separate embodiment.

[0213] According to some embodiments, the multifunctional particle further comprises a cleavage molecule inducer. According to some embodiments, the cleavage molecule inducer is selected from the group including N-acetyl-l-cysteine ​​(NAC), glutathione monoester, γ-glutamylcysteine, γ-glutamylcysteine ​​synthetase, and glutathione synthetase. Each possibility represents a separate embodiment.

[0214] According to some embodiments, the endogenous molecule is glutathione and the cleavage molecule inducer is selected from the group consisting of N-acetyl-l-cysteine ​​(NAC), glutathione monoester, gamma-glutamylcysteine, gamma-glutamylcysteine ​​synthetase, and glutathione synthetase. Each possibility represents a separate embodiment.

[0215] According to some embodiments, at least one of the first polymer linker and the second polymer linker is different from the third polymer linker. In some embodiments, at least one of the first polymer linker and the second polymer linker comprises the same polymer as the third polymer linker. In some embodiments, the first polymer linker, the second polymer linker and the third polymer linker comprise the same polymer. In further embodiments, the first polymer linker is composed of repeating monomer units and the third polymer linker is composed of the same repeating monomer units as the first linear polymer linker. In some related embodiments, the first linear polymer linker has a different number of repeating monomer units than the third linear polymer linker. In some embodiments, the second polymer linker is composed of repeating monomer units and the third polymer linker is composed of the same repeating monomer units as the second linear polymer linker. In some related embodiments, the second linear polymer linker has a different number of repeating monomer units than the third linear polymer linker. In some embodiments, the first and second polymer linkers are identical and composed of repeating monomer units, and the third polymer linker is composed of the same repeating monomer units as the first and second linear polymer linkers. In some related embodiments, the first and second linear polymer linkers have a different number of repeating monomer units than the third linear polymer linker.

[0216] In some embodiments, the first, second and third polymer linkers comprise the same polymer selected from the group consisting of polyethers, polyacrylates, polyanhydrides, polyvinyl alcohols, polysaccharides, poly(N-vinylpyrrolidone), polyglycerol (PG), poly(N-(2-hydroxypropyl)methacrylamide), polyoxazolines, poly(amino acid)-based hybrids, recombinant polypeptides, derivatives and combinations thereof. In some embodiments, the first, second and third polymer linkers comprise PEG. In some embodiments, the first, second and third polymer linkers are PEG. In some embodiments, the first, second and third polymer linkers comprise thiolated PEG. In some embodiments, the first, second and third polymer linkers comprise thiolated PEG acid (HS-PEG-COOH) or thiolated PEG amine (HS-PEG-NH 2 In some embodiments, the first, second and third polymer linkers comprise a thiolated PEG acid (HS-PEG-COOH) or a thiolated PEG amine (HS-PEG-NH 2 In some embodiments, the first, second and third polymer linkers are thiolated PEG acids (HS-PEG-COOH). In some embodiments, the first, second and third polymer linkers are thiolated PEG amines (HS-PEG-NH 2 ).

[0217] In some embodiments, the first immunoglobulin is covalently conjugated to the first polymer linker through the first functional end group of the linker, the second immunoglobulin is covalently conjugated to the second polymer linker through the second functional end group of the linker, and the permeation enhancing moiety is covalently conjugated to the third polymer linker through the third functional end group of the linker. Exemplary functional end groups include, but are not limited to, thiol groups, carboxyl groups, and amine groups. In some embodiments, at least two of the first functional end group, the second functional end group, and the third functional end group are the same. In some embodiments, the first functional end group and the second functional end group are the same. In some embodiments, the first functional end group and the third functional end group are the same. In some embodiments, the second functional end group and the third functional end group are the same. In some embodiments, the first functional end group, the second functional end group, and the third functional end group are the same.

[0218] In some embodiments, the first functional end group and the second functional end group are different. In some embodiments, the first functional end group and the third functional end group are different. In some embodiments, the second functional end group and the third functional end group are different.

[0219] In some embodiments, the first, second and third polymer linkers are linear. According to the principles of the present invention, the length of the third polymer linker is substantially different from the length of at least one of the first and second polymer linkers. In some embodiments, the length of the third polymer linker is substantially different from the length of the first polymer linker. In some embodiments, the length of the third polymer linker is substantially different from the length of the second polymer linker. In some embodiments, the length of the third polymer linker is substantially different from the length of both the first and second polymer linkers. In some embodiments, the length of the first polymer linker is substantially similar to the length of the second polymer linker, and the length of the third polymer linker is substantially different from the length of both the first and second polymer linkers.

[0220] In some embodiments, the term "length" of a polymer segment or linker refers to the length of the polymer, which depends on the number of monomers incorporated therein, the length of each monomer unit, the polymer chain structure (e.g., whether the polymer is linear or branched), spatial conformation, deformation of valence angles (or bond angles), and the degree of stretching or coiling.

[0221] The length of a polymer can be calculated as known in the art, for example, as described in Introduction to Physical Polymer Science, 4th Edition, LHSperling, 1st Edition: November 4, 2005, Chapter 3. In addition, various computational modeling methods can be used to evaluate the length of a polymer, as known in the art, which can be performed using, among others, Hyperchem, ACD / 3D, MOE 2010.10, or Chem 3D software. Physical characterization methods, such as, for example, light scattering, can also be used to evaluate the length of a coiled polymer. It should be understood that when evaluating differences in the length of polymer linkers, the same length definition (or length measurement method) must be used for the polymer linkers being compared.

[0222] The term "length" when referring to a linear polymer can refer to different definitions of length. According to some embodiments, the term "length" refers to the length of displacement, also referred to herein as the "end-to-end" length, which is the distance between the two ends of the polymer chain of a coiled polymer. The end-to-end length can be calculated, for example, by the Flory radius: [ka] (where F=Flory radius, a=monomer size, n=degree of polymerization) It can be expressed as:

[0223] According to some embodiments, the term "length" refers to the contour length, which is the distance between the ends of the polymer chain when the polymer is stretched. The contour length can be considered as the length of the maximum possible displacement. The contour length (also referred to herein as the "old contour length") can be calculated by dividing the MW of the polymer by the MW of the monomer unit and multiplying by the length of the monomer unit. To take into account the bond angle, the contour length (also referred to herein as the "new contour length") can be calculated by dividing the MW of the polymer by the MW of the monomer unit and multiplying by the length of the monomer unit and further multiplying by the cosine of ((bond angle theta-180) / 2).

[0224] As explained above, the length of a linear polymer can be estimated based on its molecular weight and the chemical structure of the monomeric units. To evaluate the difference between polymeric linkers that contain the same polymer (i.e., the same type but composed of different numbers of monomeric units), the molecular weight of the polymeric linker can be conveniently used. Thus, in some embodiments, the molecular weight of the third polymeric linker is substantially different from the molecular weight of at least one of the first and second linear polymeric linkers. In some embodiments, the molecular weight of the third polymeric linker is substantially different from the molecular weight of the first polymeric linker. In some embodiments, the molecular weight of the third polymeric linker is substantially different from the molecular weight of the second polymeric linker. In some embodiments, the molecular weight of the third polymeric linker is substantially different from the molecular weight of the first and second polymeric linkers.

[0225] As used herein, the term "substantially different" refers to a difference of at least 5%, at least 6%, at least 7%, at least 8%, at least 9%, at least 10%, at least 12%, at least 15%, at least 18%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, or at least 50%. Each possibility represents a separate embodiment of the invention. The term "substantially higher" means that a first value is higher than a second value, and the difference between the first value and the second value is at least 5%, at least 6%, at least 7%, at least 8%, at least 9%, at least 10%, at least 12%, at least 15%, at least 18%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, or at least 50%. Each possibility represents a separate embodiment of the invention.

[0226] In some embodiments, the molecular weight of the monomeric unit of the third polymer linker is substantially similar to the molecular weight of the monomeric unit of at least one of the first and second polymer linkers. As used herein, the term "substantially similar" refers to a similarity of at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, or at least 95%. Each possibility represents a separate embodiment of the present invention.

[0227] In some embodiments, the third polymer linker and at least one of the first and second polymer linkers comprise similar polymers. In some embodiments, the third linear polymer linker is composed of repeating monomer units, and at least one of the first and second linear polymer linkers is composed of the same repeating monomer units as the third linear polymer linker, where the third linear polymer linker has a different number of repeating monomer units than at least one of the first and second linear polymer linkers. In some embodiments, the third polymer linker and at least one of the first and second polymer linkers are similar except for the length of the third and the first and / or second polymer linkers.

[0228] In some embodiments, the third polymer linker and / or the first and second polymer linkers are at least about 100 Da, at least about 150 Da, at least about 200 Da, at least about 250 Da, at least about 300 Da, at least about 350 Da, at least about 400 Da, at least about 450 Da, at least about 500 Da, at least about 550 Da, at least about 600 Da, at least about 650 Da, at least about 700 Da, at least about 800 Da, at least about 900 Da, at least about 1000 Da, at least about 150 Da, at least about 200 Da, at least about 300 Da, at least about 400 Da, at least about 500 Da, at least about 650 Da, at least about 700 Da, at least about 800 Da, at least about 900 Da, at least about 1000 Da, at least about 150 Da, at least about 200 Da, at least about 300 Da, at least about 400 Da, at least about 450 Da, at least about 500 Da, at least about 550 Da, at least about 600 Da, at least about 650 Da, at least about 700 Da, at least about 1000 Da, at least about 15 ... Each possibility has a molecular weight difference of at least about 750 Da, at least about 800 Da, at least about 850 Da, at least about 900 Da, at least about 950 Da, at least about 1000 Da, at least about 1100 Da, at least about 1200 Da, at least about 1300 Da, at least about 1400 Da, at least about 1500 Da, at least about 1600 Da, at least about 1700 Da, at least about 1800 Da, at least about 1900 Da, or at least about 2000 Da. Each possibility represents a separate embodiment of the present invention.

[0229] In some embodiments, the difference between the length of the third polymer linker and the length of at least one of the first and second linear polymer linkers is configured to allow exposure of the permeation enhancing moiety on the outer surface of the co-delivery system facing the tumor cell membrane or the outer surface of a solid tumor. It should be understood that the immunoglobulin is not enclosed or encapsulated within the core particle, but rather is attached to its outer surface via a polymer linker, as is the internalization moiety, which is also attached to the surface of the same core particle via a polymer linker. Without being bound by theory or mechanism of action, it is contemplated that attaching the permeation enhancing / internalization moiety via a polymer chain having a similar length to the first and / or second polymer linker can prevent sufficient exposure of the internalization moiety on the outer surface of the co-delivery system, thereby limiting the system from passing through cell membranes and the blood-tumor barrier.

[0230] Furthermore, without being bound by theory or mechanism of action, it is contemplated that the active immunoglobulin that is not enclosed or encapsulated within the core particle remains accessible for effective binding to its antigen despite being bound to the multifunctional system. Advantageously, the specific composition of the multifunctional system of the present invention, which ensures the formation of conjugate particles with a specific hierarchical structure, not only allows the delivery of combinations of various types of immunoglobulins, but does not necessarily require cleavage of the link between the active agent and the core particle after crossing the biological membrane, since it does not interfere with the functionality of the immunoglobulins.

[0231] Thus, in some embodiments, the molecular weight of the third polymer linker is higher than the molecular weight of at least one of the first and second polymer linkers. In further embodiments, the molecular weight of the third polymer linker is higher than the molecular weight of both the first and second polymer linkers. In some embodiments, the molecular weight of the third polymer linker is higher than the molecular weight of the first and / or second polymer linkers, provided that the molecular weight of the first and / or second polymer linkers is less than 4950 Da. In some embodiments, the molecular weight of the third polymer linker is higher than the molecular weight of the first and / or second polymer linkers, provided that the molecular weight of the first and / or second polymer linkers is less than 4900 Da. In some embodiments, the molecular weight of the third polymer linker is higher than the molecular weight of the first and / or second polymer linkers, provided that the molecular weight of the first and / or second polymer linkers is less than 4800 Da. In some embodiments, the molecular weight of the third polymer linker is higher than the molecular weight of the first and / or second polymer linker, provided that the molecular weight of the first and / or second polymer linker is less than 4780 Da. In some embodiments, the third polymer linker is a PEG derivative having a molecular weight of about 5000 Da, and at least one of the first and second polymer linkers is a PEG derivative having a molecular weight of about 3500 Da. In some embodiments, the third polymer linker is a PEG derivative having a molecular weight of about 5000 Da, and both the first and second polymer linkers are PEG derivatives having a molecular weight of about 3500 Da.

[0232] In some embodiments, the third polymer linker has a molecular weight higher than that of at least one of the first and second polymer linkers. In some embodiments, the MW of the polymer linker is directly dependent on the relative molecular weights of the immunoglobulin and the permeation enhancing moiety. In some embodiments, the first immunoglobulin molecule has a higher MW than the transporter moiety and the first polymer linker has a lower MW than the third polymer linker. In some embodiments, the second immunoglobulin molecule has a higher MW than the transporter moiety and the second polymer linker has a lower MW than the third polymer linker.

[0233] In some embodiments, the third polymer linker is longer than the first and / or second polymer linker. In some embodiments, the third polymer linker has a longer end-to-end distance than the first and / or second polymer linker. In some embodiments, the third polymer linker has a longer contour distance than the first and / or second polymer linker.

[0234] In some embodiments, the third polymer linker has a lower MW than the MW of at least one of the first and second polymer linkers. In some related embodiments, the MW of at least one of the first and second polymer linkers is at least about 4000 Da. In further related embodiments, the difference between the MW of the first polymer linker and the MW of at least one of the first and second polymer linkers is at least about 2000 Da. Without being bound by theory or mechanism of action, it is contemplated that the significantly longer first and / or second linkers allow for folding (or a higher rate of coiling) of the polymer chains, such that the actual distance between each immunoglobulin and the core particle is smaller than between the permeation moiety and the core particle, so that the immunoglobulin is at least partially shielded by the transporter moiety exposed on the surface of the multifunctional particle during membrane permeation. In some related embodiments, the end-to-end distance of the third polymer linker is greater than the end-to-end distance of the first and / or second polymer linker despite the higher MW of the first and / or second polymer linker.

[0235] In some embodiments, the distance between the first immunoglobulin and the core particle and the distance between the second immunoglobulin and the core particle are smaller than the distance between the transporter moiety and the core particle. In some embodiments, at least one terminal group of the third polymer linker is similar to at least one terminal group of the first polymer linker. In some embodiments, at least one terminal group of the third polymer linker is similar to at least one terminal group of the second polymer linker. In some embodiments, the two terminal groups of the third polymer linker are similar to the two terminal groups of the first polymer linker. In some embodiments, the two terminal groups of the third polymer linker are similar to the two terminal groups of the second polymer linker. In some embodiments, the two terminal groups of the first polymer linker are similar to the two terminal groups of the second polymer linker.

[0236] In some embodiments, the core particle is linked to an additional fourth polymer. In some embodiments, the polymer is a monofunctional polymer linker. In some embodiments, the core particle is coated with a polymer layer comprising a first polymer linker, a second polymer linker, a third polymer linker, and an additional fourth polymer linker, where the additional polymer linker is monofunctional. The terms "fourth polymer" and "fourth polymer linker" can be used interchangeably. In some embodiments, the fourth polymer functions as a spacer moiety. In some embodiments, the fourth polymer is a linear polymer linker. In some embodiments, the fourth polymer is selected from the group consisting of polyethers, polyacrylates, polyanhydrides, polyvinyl alcohols, polysaccharides, poly(N-vinylpyrrolidone), polyglycerin (PG), poly(N-(2-hydroxypropyl)methacrylamide), polyoxazolines, poly(amino acid)-based hybrids, recombinant polypeptides, derivatives, and combinations thereof.

[0237] As used herein, the term "monofunctional" means that the polymer before being conjugated to the core particle has only one type of functional group that is configured to conjugate the polymer to the core particle.Therefore, the monofunctional polymer linker is not conjugated and cannot be conjugated to any moiety other than the core particle.

[0238] In some embodiments, the fourth polymer comprises the same monomer units as the first and / or second polymer. In some embodiments, the fourth polymer comprises the same monomer units as the third polymer linker. In some embodiments, the first, second, third and fourth polymers comprise the same monomer units. In some embodiments, the fourth polymer is attached to the core particle via a thiol end group of the polymer. In some embodiments, the fourth polymer is a polyether. In some embodiments, the polyether is methoxypolyethylene glycol (mPEG) or a derivative thereof. In some embodiments, the mPEG is thiolated (mPEG-SH) and the thiolated mPEG is attached to the core particle via a thiol end group.

[0239] In some embodiments, the fourth polymer has a MW of 1,000-7,000 Da. In some embodiments, the fourth polymer has a MW of 500-1,000 Da, 500-3,000 Da, 500-7,000 Da, 500-10,000 Da, 1,000-3,000 Da, 1,000-4,000 Da, 1,000-5,000 Da, 1,000-7,000 Da, 1,000-10,000 Da, 3,000-5,000 Da, 3,000-7,000 Da, 3,000-10,000 Da, 7. Each possibility represents a separate embodiment. According to some embodiments, the fourth polymer has a MW of at least 1,000 Da, at least 2,000 Da, at least 3,000 Da, at least 4,000 Da, at least 5,000 Da, at least 6,000 Da, at least 7,000 Da, or at least 8,000 Da. Each possibility represents a separate embodiment. According to some embodiments, the fourth polymer has a MW of up to 1,000 Da, up to 2,000 Da, up to 3,000 Da, up to 4,000 Da, up to 5,000 Da, up to 6,000 Da, up to 7,000 Da, or up to 10,000 Da. Each possibility represents a separate embodiment.

[0240] In some embodiments, the length of the fourth polymer is substantially similar to the length of at least one of the first polymer linker, the second polymer linker, and the third polymer linker. In some embodiments, the length of the fourth polymer is substantially similar to the length of the first polymer linker. In some embodiments, the length of the fourth polymer is substantially similar to the length of the second polymer linker. In some embodiments, the length of the fourth polymer is substantially similar to the length of the third polymer linker. In some embodiments, the length of the fourth polymer is substantially similar to the length of the polymer linker (first, second, or third), which is longer than the length of at least one of the other polymer linkers. In some embodiments, the molecular weight of the fourth polymer is substantially similar to the molecular weight of the polymer linker (first, second, or third) that has a higher molecular weight than at least one of the other polymer linkers. In some embodiments, the MW of the fourth polymer is substantially similar to the MW of the first polymer linker. In some embodiments, the MW of the fourth polymer is substantially similar to the MW of the second polymer linker. In some embodiments, the MW of the fourth polymer is substantially similar to the MW of the third polymer linker.

[0241] Without being bound by theory or mechanism of action, the effectiveness of the co-delivery system of the present invention also depends on the molar ratio of the different polymer linkers, which dictates the density of the permeation enhancing moieties and immunoglobulins within the co-delivery system.

[0242] In some embodiments, the first polymer linker comprises about 5-70 mol%, 5-60 mol%, 5-40 mol%, 8-60 mol%, 10-60 mol%, 10-55 mol%, 10-50 mol%, 10-40 mol%, 10-30 mol%, 10-25 mol%, 10-20 mol%, 15-60 mol%, 15-55 mol%, 15-50 mol%, 15-45 mol%, 15-40 mol%, 15-50 mol%, 15-60 mol%, 15-55 mol%, 15-50 mol%, 15-45 mol%, 15-40 mol%, 15-50 mol%, 15-60 mol%, 15-70 mol%, 15-80 mol%, 15-80 mol%, 15-90 mol%, 15-10 ... %. Each possibility represents a separate embodiment of the present invention. In some embodiments, the first polymer linker comprises at least 2 mol%, at least 4 mol%, at least 5 mol%, at least 6 mol%, at least 8 mol%, at least 10 mol%, at least 12 mol%, at least 15 mol%, at least 18 mol%, at least 20 mol%, at least 25 mol%, at least 30 mol%, at least 35 mol%, at least 40 mol%, at least 50 mol%, or at least 60 mol% of the total polymer attached to the core particle, with each possibility representing a separate embodiment.

[0243] In some embodiments, the second polymer linker comprises about 5-70 mol%, 5-60 mol%, 5-40 mol%, 8-60 mol%, 10-60 mol%, 10-55 mol%, 10-50 mol%, 10-40 mol%, 10-30 mol%, 10-25 mol%, 10-20 mol%, 15-60 mol%, 15-55 mol%, 15-50 mol%, 15-45 mol%, 15-40 mol%, 15-50 mol%, 15-60 mol%, 15-55 mol%, 15-50 mol%, 15-45 mol%, 15-40 mol%, 15-50 mol%, 15-60 mol%, 15-70 mol%, 15-80 mol%, 15-80 mol%, 15-90 mol%, 15-10 ... %. Each possibility represents a separate embodiment of the present invention. In some embodiments, the second polymer linker comprises at least 2 mol%, at least 4 mol%, at least 5 mol%, at least 6 mol%, at least 8 mol%, at least 10 mol%, at least 12 mol%, at least 15 mol%, at least 18 mol%, at least 20 mol%, at least 25 mol%, at least 30 mol%, at least 35 mol%, at least 40 mol%, at least 50 mol%, or at least 60 mol% of the total polymer attached to the core particle. Each possibility represents a separate embodiment.

[0244] In some embodiments, the third polymer linker comprises about 5-70 mol%, 5-60 mol%, 5-40 mol%, 8-60 mol%, 10-60 mol%, 10-55 mol%, 10-50 mol%, 10-40 mol%, 10-30 mol%, 10-25 mol%, 10-20 mol%, 15-60 mol%, 15-55 mol%, 15-50 mol%, 15-45 mol%, 15-40 mol%, 15-50 mol%, 15-60 mol%, 15-55 mol%, 15-50 mol%, 15-45 mol%, 15-40 mol%, 15-50 mol%, 15-60 mol%, 15-70 mol%, 15-80 mol%, 15-80 mol%, 15-90 mol%, 15-10 ... %. Each possibility represents a separate embodiment of the present invention. In some embodiments, the third polymer linker comprises at least 2 mol%, at least 4 mol%, at least 5 mol%, at least 6 mol%, at least 8 mol%, at least 10 mol%, at least 12 mol%, at least 15 mol%, at least 18 mol%, at least 20 mol%, at least 25 mol%, at least 30 mol%, at least 35 mol%, at least 40 mol%, at least 50 mol%, or at least 60 mol% of the total polymer attached to the core particle. Each possibility represents a separate embodiment.

[0245] In some embodiments, the fourth polymer comprises about 5-90 mol%, 5-85 mol%, 5-80 mol%, 10-80 mol%, 20-78 mol%, 25-75 mol%, 30-75 mol%, 40-75 mol%, 50-75 mol%, 60-75 mol%, 60-70 mol%, 60-80 mol%, 5-60 mol%, 10-60 mol%, 10-55 mol%, 10-50 mol%, 10-40 mol%, 15-60 mol%, 15-55 mol%, 15-50 mol%, 15-45 mol%, or 15-40 mol% of the total polymer attached to the core particle. Each possibility represents a separate embodiment of the present invention. In some embodiments, the fourth polymer comprises 60-80 mol% of the total polymer attached to the core particle. In some embodiments, the fourth polymer comprises 50-80 mol% of the total polymer attached to the core particle. In some embodiments, the fourth polymer comprises at least 2 mol%, at least 4 mol%, at least 5 mol%, at least 6 mol%, at least 8 mol%, at least 10 mol%, at least 12 mol%, at least 15 mol%, at least 18 mol%, at least 20 mol%, at least 25 mol%, at least 30 mol%, at least 35 mol%, at least 40 mol%, at least 45 mol%, at least 50 mol%, at least 55 mol%, at least 60 mol%, at least 65 mol%, or at least 70 mol% of the total polymer attached to the core particle. Each possibility represents a separate embodiment.

[0246] In some embodiments, the first polymer linker constitutes about 5-45 mol % of the total polymer attached to the core particle, the second polymer linker constitutes about 5-45 mol % of the total polymer attached to the core particle, the third polymer linker constitutes about 10-45 mol % of the total polymer attached to the core particle, and the fourth polymer constitutes about 40-80 mol % of the total polymer attached to the core particle.

[0247] In some embodiments, the first polymer linker comprises about 200-2000 molecules per particle, the second polymer linker comprises about 200-2000 molecules per particle, the third polymer linker comprises about 400-2000 molecules per particle, and the fourth polymer comprises about 1800-3000 molecules per particle.

[0248] It has been found that particles containing about 2-40 antibody molecules per particle are capable of binding to their cellular targets and inducing the desired effect. The delivery system of the present invention, according to some embodiments, comprises particles carrying about 2-50 antibody molecules. According to some embodiments, about 4-40 antibody molecules are conjugated to each particle via a linker. According to still other embodiments, about 5-30 antibody molecules are conjugated to each particle via a linker. According to some specific embodiments, about 2-20, 2-10, 5-15, 10-20, or about 15-25 antibody molecules are conjugated to each particle via a linker.

[0249] However, when antibody fragments are conjugated to particles, their respective numbers are higher. Thus, the delivery system of the present invention, according to some embodiments, comprises particles carrying about 20-400 antibody fragments. According to some embodiments, about 40-400 antibody fragments are conjugated to each particle via a linker. According to still other embodiments, about 50-300 antibody fragments are conjugated to each particle via a linker. According to some particular embodiments, about 20-200, 20-100, 100-200 or about 150-250 antibody fragments are conjugated to each particle via a linker.

[0250] According to some embodiments, the total amount of immunoglobulin molecules bound to the nanoparticles does not exceed about 0.2% of the total particle surface volume. According to some embodiments, the amount of transporter does not exceed about 0.5% of the particle surface volume. According to some embodiments, the amount of transporter does not exceed about 1% of the particle surface volume.

[0251] According to some embodiments, the fourth linker is conjugated to about 90%-99% of the total surface volume of the particle.

[0252] According to some embodiments, about 20-500 antibody fragments are conjugated to each particle via a linker. According to still other embodiments, about 40-400 antibody fragments are conjugated to each particle via a linker. According to still other embodiments, about 50-300 antibody fragments are conjugated to each particle via a linker. According to some specific embodiments, about 20-200, 20-100, 100-200, or about 50-350 antibody fragments are conjugated to each particle via a linker.

[0253] In some embodiments, the first polymer linker constitutes about 10-40 mol % of the total polymer attached to the core particle, the second polymer linker constitutes about 10-40 mol % of the total polymer attached to the core particle, the third polymer linker constitutes about 10-40 mol % of the total polymer attached to the core particle, and the fourth polymer constitutes about 40-70 mol % of the total polymer attached to the core particle.

[0254] In some embodiments, the first and second polymer linkers together comprise about 10%-60 mol%, 10-50 mol%, 10-45 mol%, 10-40 mol%, 10-30 mol%, or 10-20 mol% of the total polymer linkers attached to the core particle, with each possibility representing a separate embodiment of the present invention.

[0255] It is understood that the mole % of each polymer is dependent upon the other polymers attached to the core particle such that the total mole % of polymers does not exceed 100%.

[0256] In some embodiments, the (w / w / w / w) ratio of the first polymer linker, the second polymer linker, the third polymer linker, and the fourth polymer is 2.5:2.5:5:85 to 20:20:30:30 of the total polymer linkers attached to the core particle. According to some particular embodiments, the (w / w / w / w) ratio of the first polymer linker, the second polymer linker, the third polymer linker, and the fourth polymer is 2.5:2.5:15:80. According to other embodiments, the (w / w / w / w) ratio of the first polymer linker, the second polymer linker, the third polymer linker, and the fourth polymer is 5:5: 10:80 or 10:10:20:60. Each possibility represents a separate embodiment of the present invention.

[0257] According to the present invention, the co-delivery system comprises a permeation enhancing moiety conjugated to a third polymer linker. The terms "permeation enhancing moiety", "transporter", "permeation enhancer", "permeability enhancer", "permeability enhancing", "internalization transporter" and "internalization moiety enhancer" are used interchangeably herein and refer to a moiety that can facilitate or enhance the permeation or internalization of a delivery system through a biological membrane, e.g., the blood-tumor barrier, or enable the attachment of the delivery system to cancerous cells via a specific receptor, e.g., the insulin receptor. In some embodiments, the transporter also enhances glucose uptake by tumor cells and / or enhances tumor cell metabolism.

[0258] In some embodiments, the permeation enhancing moiety is selected from, but is not limited to, insulin, an antibody specific for an insulin receptor, or a portion of such an antibody, e.g., a Fab fragment, a polypeptide that specifically binds to an insulin receptor, insulin-like growth factor 1, an antibody specific for insulin-like growth factor receptor 1, or a portion of such an antibody, a polypeptide that specifically binds to insulin-like growth factor receptor 1, a cell penetrating peptide (CPP), and glucose or a glucose derivative. Each possibility represents a separate embodiment of the present invention.

[0259] A "cell penetrating peptide (CPP)" is a peptide with enhanced ability to cross the cell membrane bilayer without causing significant lethal membrane damage.

[0260] Other cellular proteins capable of promoting endocytosis known in the art can also be used as permeation enhancing moieties. In some embodiments, the permeation enhancing moiety is a moiety that promotes or enhances glucose uptake or consumption by tumor cells. In some embodiments, the permeation enhancing moiety is selected from insulin, insulin derivatives, glucose, or glucose derivatives. According to some embodiments, the permeation enhancer is 2-deoxy-D-glucose.

[0261] In some embodiments, the MW of the permeation enhancing moiety is about 150 to about 8000 Daltons. In some embodiments, the MW of the permeation enhancer is about 2 kD to about 8 kD. In some embodiments, the MW of the permeation enhancer is about 5 kD.

[0262] According to the principles of the present invention, the first polymer linker is conjugated to a first immunoglobulin and the second polymer linker is conjugated to a second separate immunoglobulin. As used herein, the term "immunoglobulin" refers to an agent intended to be delivered into a subject's cells, tissues or tumors located outside the brain or outside the CNS and can be used as a therapeutic, targeting or diagnostic agent. In some embodiments, each of the first and second immunoglobulins is independently selected from a biologically active molecule and a labeling molecule. According to some embodiments, the immunoglobulin molecule is characterized by poor tissue penetration.

[0263] In some embodiments, the immunoglobulin molecules are flanked by respective polymer linkers. The terms "immunoglobulin" and "immunoglobulin molecule" are used interchangeably herein and refer to compounds or molecules that can bind to specific cell receptors / antigens / markers, thereby targeting the system to specific cells. In some embodiments, the immunoglobulin molecule is a therapeutic antibody or antibody fragment. In some embodiments, the immunoglobulin molecule has therapeutic applications. In some embodiments, the immunoglobulin molecule has diagnostic applications. In some embodiments, the immunoglobulin molecule has both therapeutic and diagnostic applications. In some embodiments, the immunoglobulin molecule is an intact antibody, scFv, or antibody fragment. Each possibility represents a separate embodiment of the present invention.

[0264] In some embodiments, the first immunoglobulin and / or the second immunoglobulin is a monoclonal antibody. In some particular embodiments, the antibody is selected from the group consisting of anti-IgG1, anti-IbA1, anti-HER2+ (trastuzumab & pertuzumab), anti-EGFR (cetuximab), anti-GD2, and checkpoint inhibitor antibodies, such as anti-PD-1, anti-PD-L1, and anti-CTLA-4, or fragments thereof.

[0265] As used herein, the term "antibody" refers to a polypeptide or group of polypeptides that contain at least one binding domain formed from the folding of a polypeptide chain with a three-dimensional binding space with an internal surface shape and charge distribution complementary to the antigenic determinant characteristic of an antigen. Antibodies typically have a tetrameric form, containing two identical pairs of polypeptide chains, each pair having one "light" and one "heavy" chain. The variable regions of each light / heavy chain pair form the antibody binding site. Antibodies can be oligoclonal, polyclonal, monoclonal, chimeric, camelized, CDR-grafted, multispecific, bispecific, catalytic, humanized, fully human, anti-idiotypic, and antibodies that may be labeled in soluble or conjugated form, alone or in combination with other amino acid sequences, as well as fragments, variants, or derivatives thereof, including epitope-binding fragments. Antibodies can be from any species. The term antibody also includes binding fragments, including, but not limited to, Fv, Fab, Fab', F(ab')2 single chain antibodies (svFC), dimeric variable regions (diabodies), nanobodies, and disulfide-linked variable regions (dsFv). In particular, antibodies include immunoglobulin molecules and immunologically active fragments of immunoglobulin molecules, i.e., molecules that contain an antigen-binding site. Antibody fragments may or may not be fused to another immunoglobulin domain, including, but not limited to, an Fc region or a fragment thereof. Those skilled in the art will further recognize that other fusion products may be generated, including, but not limited to, scFv-Fc fusions, variable region (e.g., VL and VH) Fc fusions, and scFv-scFv-Fc fusions.

[0266] The term "antibody" is used in the broadest sense and includes monoclonal antibodies (including full-length or intact monoclonal antibodies), polyclonal antibodies, multivalent antibodies, chimeric antibodies, humanized antibodies, and antibody fragments of sufficient length to exhibit the desired biological activity, i.e., antibody fragments that bind to a target of interest.

[0267] The mAbs of the invention can be of any immunoglobulin class, including IgG, IgM, IgE, IgA, and IgD.

[0268] Chimeric, humanized, and human antibodies, according to some embodiments, comprise a human constant region selected from the group consisting of IgG1, IgG2, IgG3, and IgG4.

[0269] A "humanized" antibody is an antibody in which all or substantially all CDR amino acid residues are derived from a non-human CDR and all or substantially all FR amino acid residues are derived from a human FR. A humanized antibody may optionally contain at least a portion of an antibody constant region derived from a human antibody. A "humanized form" of a non-human antibody refers to a variant of a non-human antibody that has undergone humanization, typically to reduce immunogenicity in humans while retaining the specificity and affinity of the parent non-human antibody. According to some embodiments, some FR residues in a humanized antibody are replaced with the corresponding residues from a non-human antibody (e.g., the antibody from which the CDR residues are derived), e.g., to restore or improve the specificity or affinity of the antibody.

[0270] A "human antibody" is an antibody having an amino acid sequence that corresponds to that of an antibody produced by a human or a human cell, or to that of a non-human source that utilizes a human antibody repertoire, including a human antibody library, or other human antibody coding sequence. The term excludes humanized forms of non-human antibodies that contain non-human antigen-binding regions, such as those in which all or substantially all CDRs are non-human.

[0271] In some embodiments, the first immunoglobulin and / or the second immunoglobulin are antibodies. In some embodiments, the antibody is an antibody that specifically binds to a receptor present on the surface of a target cell in the body, e.g., a tumor cell or an immune cell. In some embodiments, the antibody is an antibody that specifically binds to a receptor present on cells of a particular tissue or body region, e.g., a cancerous region or cancerous cell. In some embodiments, the antibody is an antibody that specifically binds to a receptor present on the surface of a diseased cell or a cancerous cell located outside the brain. In some embodiments, the antibody is a bispecific antibody. In some embodiments, both the first immunoglobulin molecule and the second immunoglobulin molecule are bispecific antibodies. In some embodiments, the first immunoglobulin and / or the second immunoglobulin are antibodies that have therapeutic activity against cancer.

[0272] Exemplary antibodies include, but are not limited to, anti-HER2+ (trastuzumab & pertuzumab), anti-EGFR (cetuximab), checkpoint inhibitor antibodies (anti-PD-1, anti-PD-L1, anti-CTLA-4), and anti-GD2.

[0273] According to some embodiments, the at least one antibody in the delivery system is selected from the group consisting of 1-40-β-amyloid, 4-1BB (CD137), 5AC, activated F9, F10, activin receptor-like kinase 1, ACVR2B, adenocarcinoma antigen, AGS-22M6, alpha fetoprotein, angiopoietin 2, angiopoietin 3, anthrax toxin, AOC3 (VAP-1), B7-H3, Bacillus anthracis anthrax, BAFF, beta amyloid, C5, CA-125, CA-125 (mimetics), calcitonin, Canis lupusfamiliaris), IL31, carbonic anhydrase 9 (CA-IX), cardiac myosin, CCL11 (eotaxin-1), CCR2, CCR4, CCR5, CD11, CD18, CD125, CD140a, CD147 (basigin), CD15, CD152, CD154 (CD40L), CD19, CD2, CD20, CD200, CD22, CD23 (IgE receptor), CD25 (α chain of IL-2 receptor), CD27, CD4, CD6, CD28 , CD3, CD3 epsilon, CD30 (TNFRSF8), CD33, CD37, CD38, CD4, CD40, CD44v6, CD5, CD51, CD52, CD56, CD6, CD70, CD74, CD79B, CD80, CEA, CEA-related antigen, CFD, CGRP, ch4D5, CLDN18.2, Clostridium difficile, coagulation factor A, coagulation factor III, CSF1R, CSF2, CTGF, CTLA-4, CXCR4 (CD1 84), cytomegalovirus, cytomegalovirus glycoprotein B, dabigatran, DLL3, DLL4, DPP4, DR5, Escherichia coli Shiga toxin type 1, Escherichia coli Shiga toxin type 2, EGFL7, EGFR, endoglin, endotoxin, EpCAM, ephrin receptor A3, episialin, ERBB3 (HER3), respiratory syncytial virus F protein, FAP, FGF23, fibronectin extra domain-B, folate hydrolase, folate receptor alpha, Frizzled receptor, GCGR, GD2 ganglioside, GDF-8, glypican 3, GMCSF receptor alpha chain, GPNMB, growth differentiation factor 8, GUCY2C, hemagglutinin, hepatitis B surface antigen, HER1, HER2 / neu, HGF, HHGFR, histone complex, HIV-1, HLA-DR, HNGF, human beta amyloid, human TNF, ICAM-1 (CD54), ICOSL, IFN-α, IFN-γ, IgE, IgEFc region, IGF-1 receptor (CD221), IGF1, IGF2, IGHE, IL17A, IL17A and IL17F, IL20, IL-1, IL-12, IL-23, IL-13, IL-17, IL17A and IL17F, IL1A, IL-1β, IL2, IL-22, IL23, IL23A, IL31RA, IL-4, IL-5, IL-6, IL-6 receptor, IL9, ILGF2, influenza A virus hemagglutinin HA, integrin α4, integrin α4β7, integrin α5β1, integrin α7β7, integrin αvβ3, interferon gamma-inducible protein, interferon gamma-inducible protein, -feron receptor, interferon α / β receptor, ITGA2 (CD49b), kallikrein, KIR2D, KLRC1, Lewis Y antigen, LFA-1 (CD11a), LINGO-1, lipoteichoic acid, LOXL2, L-selectin (CD62L), LTA, MCP-1, mesothelin, MIF, MS4A1, MSLN, MUC1, mucin CanAg, myelin-associated glycoprotein, myostatin, NCA-90 (granulocyte antigen), neuronal apoptosis-regulating proteinase 1, NGF, N-glycolylneuraminic acid, NOGO-A, Notch1, Notch receptor, NRP1, rabbit (Oryctolagus cuniculus, OX-40, oxLDL, PCSK9, PD-1, PDCD1, PDGF-Rα, phosphate-sodium cotransporter, phosphatidylserine, platelet-derived growth factor receptor beta, prostate cancer cells, Pseudomonas aeruginosa, Pseudomonas aeruginosa type III secretion system, rabies virus glycoprotein, RANKL, respiratory syncytial virus, RHD, rhesus factor, RON, RTN4, scatter factorThe target is selected from the group consisting of IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-18, IL-19, IL-20, IL-21, IL-22, IL-23, IL-24, IL-25, IL-26, IL-27, IL-38, IL-39, IL-40, IL-41, IL-42, IL-43, IL-44, IL-45, IL-45, IL-46, IL-47, IL-48, IL-49, IL-59, IL-50, IL-51, IL-52, IL-53, IL-54, IL-55, IL-55, IL-56, IL-57, IL-58, IL-59 ...

[0274] According to some embodiments, the antibody is selected from the group consisting of abagovomab, avituzumab, abrilumab, actoxumab, adalimumab, adecatumumab, aducanumab, afacevicumab, afutuzumab, ALD518, alemtuzumab, alirocumab, altumomab pentetate, amatuximab, anetumab Lovetansine, Anifrolumab, Anrukinzumab (IMA-638), Apolizumab, Asclinbacumab, Acelizumab, Atezolizumab, Atinumab, Atolizumab (tocilizumab), Atolimumab, Avelumab, Bapineuzumab, Basiliximab, Bavituximab, Begelomab, Belimumab, Benralizumab, Bertilimumab, Besilesomab, Bevacizumab, Bezlotoxumab, Bimagrumab, Bimekizumab, Bivatuzumab Mertansine, Bleselumab, Brontuzumab, Brosozumab, Bococizumab, Brazikumab, Brentuximab Vedotin, Briakinumab, Brodalumab, Brolucizumab, Brontixtuzumab, Burosumab, Cabilalizumab, Canakinumab, Cantuzumab mertansine, Cantuzumab ravtansine, Caplacizumab, Capromab pendetide, Carmab, Carotuximab, cBR96-doxorubicin immunoconjugate, Cedelizumab, Sergituzumab amnaleukin, Cetuximab, Ch.14.18, Cixutumumab, Clazakizumab, Clenoliximab, Crivatuzumab tetraxetan, Codrituzumab, Coltuximab Ravtansine, Conatumumab, Concizumab, Crenezumab, Clotezumab, CR6261, Dacetuzumab, Daclizumab, Dalotuzumab, Dapirorizumab pegol, Daratumumab, Dectrecumab, Demcizumab, Denintuzumab mafodotin, Denosumab, Depatuxizumab mafodotin, Delrotuximab Biotin, detumomab, dinutuximab, diridabumab, domagrozumab, drozitumab, durigotumab, dupilumab, durvalumab, dusigitumab, ecromeximab, eculizumab, edovacomab, edrecolomab, efalizumab, eldelumab, elgemtumab, elotuzumab, ersilimomab, emactuzumab, emibetuzumab, emicizumab, enavatuzumab, enfortumab vedotin, enlimomabPegol, enoblitzumab, enokizumab, enoticumab, encituximab, epitumomab situxetan, epratuzumab, erenumab, etaracizumab, etrolizumab, evinacumab, evolocumab, exbivirumab, fanolesomab, faralimomab, farletuzumab, fasinumab, felvizumab, fezakinumab, fibatuzumab, ficlatuzumab, figitumumab, filibumab, framvotumab, fretikumab, fontolizumab, foralumab, foravirumab, fresolimumab, furanumab, futuximab, galcanezumab, galiximab, ganitumab, gantenerumab, gavilimomab, gemtuzumab Ozogamicin, Gevokizumab, Direntuximab, Glenbatumumab vedotin, Golimumab, Gomiliximab, Guselkumab, Ibalizumab, Ibritumomab tiuxetan, Icrucumab, Idarucizumab, IMAB362, Imalumab, Imusiromab, Imgatuzumab, Incracumab, Indatuximab ravtansine, Indusatumab vedotin, Inebilizumab, Infliximab, Intetumumab, Inolimomab, Inotuzumab ozogamicin, Ipilimumab, Iratumumab, Isatuximab, Itolizumab, Ixekizumab, Keliximab, Labetuzumab, Lampalizumab, Lanadelumab, Landgrozumab, Laprituximab emtansine, lebrikizumab, remaresomab, lendalizumab, lenzilumab, lerdelimumab, lexatumumab, ribivirumab, rifatuzumab vedotin, ligelizumab, rilotomab satetratetan, lintuzumab, lirilumab, roderucizumab, lokivetomab, lorvotuzumab mertansine, lucatumumab, lurizumab pegol, rumiliximab, lumuletuzumab, MABp1, mapatumumab, margetuximab, mavrilimumab, matuzumab, mepolizumab, metelimumab, milatuzumab, minletumomab, mirvetuximab Soravtansine, mitumomab, mogamulizumab, monalizumab, morolimumab, motavizumab, moxetumomab passudotox, muromonab-CD3, namilumab, naratuximabEmtansine, Narutasin, Natalizumab, Nabicixizumab, Nabivumab, Nebacumab, Necitumumab, Nemolizumab, Nerelimomab, Nesvacumab, Nimotuzumab, Nivolumab, Oviltoxiximab, Obinutuzumab, Ocaratuzumab, Ocrelizumab, Odulimomab, Ofatumumab, Olaratumab, Olokizumab, Omalizumab, Onartuzumab, Ontuxizumab, Opicinumab, Oretuzumab, Govomab, olticumab, otelixizumab, otlertuzumab, oxelumab, ozanezumab, ozoralizumab, pagibaximab, palivizumab, pamrevlumab, vectibix, pancomab, panobacumab, palsatuzumab, pascolizumab, pasotuxizumab, pateclizumab, patritumab, pembrolizumab, pemtumomab, perakizumab, pertuzumab, pidilizumab, pinatuzumab Vedotin, Pintumomab, Placumab, Prozalizumab, Pogalizumab, Polatuzumab Vedotin, Ponezumab, Prezalizumab, Priliximab, Pritoxaximab, Pritumumab, PRO 140, Kirizumab, Racotumomab, Ladletumab, Rafivirumab, Ralpancizumab, Ramucirumab, Raxibacumab, Refanezumab, Regavirumab, Reslizumab, Rilotumumab, Linukumab, Risankizumab, Rituximab, Rivabazumab Pegol, Lobatumumab, Loredumab, Romosozumab, Rontalizumab, Robalpituzumab Tesiline, Rovelizumab, Ruplizumab, Sacituzumab Govitecan, samaryzumab, sapelizumab, sarilumab, satumomab pendetide, secukinumab, seribantumab, setoxaximab, sevirumab, sibrotuzumab, SGN-CD19A, SGN-CD33A, sifalimumab, siltuximab, simtuzumab, siplizumab, sirukumab, sofituzumab vedotin, solanezumab, sonepcizumab, sontuzumab, stamulumab, subizumab, tabalumab, tacatuzumab tetraxetan, talizumab, tamtubetomab, tanezumab, taplitumomabPaptox, talextumab, tefibazumab, tenatumomab, teneliximab, teplizumab, teprotumumab, tesidolumab, tetulomab, tezepelumab, TGN1412, ticilimumab (tremelimumab), tildrakizumab, tigatuzumab, timolumab, tisotumab vedotin, TNX-650, tocilizumab (atlizumab), toralizumab, tosatoxumab, tositumomab, tobetumab, tralokinumab, trastuzumab, trastuzumab emtansine, tregalizumab, tremelimumab, trevoglumab, tucotuzumab The agent is selected from the group consisting of cermoleukin, tubilumab, ublituximab, urocupulumab, urelumab, urtoxazumab, ustekinumab, utomilumab, vadastuximab taliline, bundletuzumab vedotin, vanticutumab, vanucizumab, vapaliximab, varlilumab, batelizumab, vedolizumab, veltuzumab, beparimomab, besenkumab, visilizumab, bovalilizumab, volociximab, borsetuzumab mafodotin, votumumab, zentuzumab, zalutumumab, zanolimumab, zatuximab, diralimumab, zolimomab alitox, and any combination thereof.

[0275] In some embodiments, the antibody or antibody fragment or bispecific antibody has a molecular weight (MW) of 15-150 kD, 15-50 kD, 100-120 kD, 100-150 kD, 100-200 kD, 100-250 kD, 150-200 kD, 150-250 kD, 200-250 kD. Each possibility represents a separate embodiment. In some embodiments, the antibody has a MW of at least 100 kD, at least 110 kD, at least 120 kD, at least 130 kD, at least 140 kD, at least 150 kD, at least 160 kD, at least 180 kD, at least 200 kD, at least 250 kD. Each possibility represents a separate embodiment. In some embodiments, the antibody has a MW of 150-200 kD. In some embodiments, the antibody has a MW of 130-180 kD. In some embodiments, the antibody has a MW of 140-160 kD.

[0276] In some specific embodiments, the antibodies have a MW of 150-200 kD and each polymer linker comprises a PEG with a MW of at least 1,000 Da, at least 2,000 Da, at least 2,500 Da, or at least 3,000 Da. In some embodiments, the antibodies have a MW of 150-200 kD and each polymer linker comprises a PEG with a MW of up to 2,000 Da, up to 2,500 Da, up to 3,000 Da, up to 3,500 Da, up to 4,000 Da, up to 5,000 Da, or up to 6,000 Da. In some embodiments, the antibodies have a MW of 150-200 kD and each polymer linker comprises a PEG with a MW of 1,000 Da to 4,000 Da. In some such embodiments, the internalizing moiety is insulin having a MW of 5-6 kD and the third polymer linker comprises a PEG having a MW of at least 4,000 Da.

[0277] In some embodiments, the first and / or second immunoglobulin is a therapeutic agent that is effective in treating a tumor, cancer, or malignancy located outside the brain, hi some embodiments, the first and / or second immunoglobulin is an antibody used in the treatment or diagnosis of cancer, wherein the cancer is other than brain cancer.

[0278] In some embodiments, at least one of the first immunoglobulin and the second immunoglobulin is a labeled molecule. The term "labeled molecule" as used herein refers to a molecule that can generate a signal detectable by an appropriate detection means, such as, but not limited to, a radioactive molecule and a fluorescent molecule. In some embodiments, the labeled molecule has a diagnostic use. In some embodiments, the labeled molecule is a diagnostic agent. In some embodiments, the labeled molecule is a small molecule. In some embodiments, the labeled molecule is an antibody.

[0279] According to the principles of the present invention, the multifunctional system allows for the synchronized co-delivery of two immunoglobulins to specific regions, tissues, or cells of the body, particularly to specific spatial regions of tumor cells or tumors located outside the brain or outside the CNS. In some embodiments, at least one of the first immunoglobulin and the second immunoglobulin is poorly membrane permeable in its original free form. In some embodiments, both the first immunoglobulin and the second immunoglobulin are poorly membrane permeable in their original free form.

[0280] In some embodiments, each of the first and second immunoglobulins is a therapeutic agent having therapeutic or immunological activity against cancer or against a non-CNS-related disease or disorder. One advantage of the co-delivery system is the possibility of inducing synergistic effects. With regard to the co-delivery of different active agents, the therapeutic outcome can be either additive (i.e., the outcome expected by combining the effects of each drug separately) or synergistic (i.e., the combination produces a significant benefit over that expected by adding the separate effects). In some embodiments, the combination of the first and second immunoglobulins produces an additive therapeutic effect. In other embodiments, the combination of the first and second immunoglobulins produces a synergistic therapeutic effect.

[0281] In some embodiments, the first immunoglobulin is a therapeutic antibody and the second active agent is a targeting antibody capable of binding to a specific surface receptor or ligand, thereby targeting the system to a specific body region or to a specific cell population within the body, leading to enhanced and targeted therapy. In some related embodiments, the second antibody further has therapeutic or immunological activity against malignancies or non-CNS related diseases or disorders.

[0282] In some embodiments, at least one of the first and second immunoglobulins is a molecule with intracellular targeting capability, i.e., a molecule that targets an intracellular macromolecule. In some related embodiments, the immunoglobulin is conjugated to the core particle via a cleavable linker.

[0283] Complex diseases, including cancer, are often multifactorial and are known to involve redundant or synergistic actions of disease mediators or upregulation of different receptors involving crosstalk between their signaling networks (Kontermann, R. In: MAbs. Taylor & Francis, 2012. p. 182-197). As a result, blocking multiple different pathological factors and pathways may greatly improve therapeutic efficacy. This result can be achieved by combining different immunoglobulins or using dual targeting strategies.

[0284] In some embodiments, both the first and second immunoglobulins can bind to a specific surface receptor or ligand, e.g., a specific tumor antigen or a specific receptor or other moiety on an immune cell. Thus, in some embodiments, the multifunctional system of the invention allows for the combination of two different immunoglobulin, e.g., antibody, specificities in a single system to simultaneously interfere with different surface receptors or ligands in a tumor or cancer cell. Without being bound by any theory or mechanism of action, it is hypothesized that a dual-targeted particle (e.g., a dual-antibody particle) can bring different targets into close proximity to support protein complex formation on a cell or induce cell-cell contact. In some embodiments, the complex comprises an immune cell and a cancer cell. In some embodiments, the first and second active agents are antibodies, and at least one of the first and second immunoglobulins is a bispecific antibody. Thus, in some embodiments, the multifunctional system of the invention allows for simultaneous interference with three or more targets. In some related embodiments, at least one of the first and second immunoglobulins further has therapeutic activity against cancer located outside the brain or a non-CNS-related disease or disorder. In some embodiments, both the first and second active immunoglobulins further have therapeutic activity against cancer located outside the brain, or a non-CNS related disease or disorder.

[0285] In some embodiments, each of the first and second immunoglobulins is an antibody or an active fragment thereof that comprises at least an antigen-binding site, with the proviso that the first and second antibodies or antibody fragments are different. In some related embodiments, the first and second active agents comprise different antibodies. In other related embodiments, the first and second immunoglobulins comprise or consist of different fragments of the same antibody. For example, in some embodiments, the first immunoglobulin comprises or consists of the Fab region of an antibody and the second immunoglobulin comprises or consists of the Fc region of the same antibody. In other embodiments, the first immunoglobulin comprises or consists of a whole antibody (e.g., IgG) and the second immunoglobulin comprises or consists of a fragment of the same antibody. For example, in some embodiments, the first immunoglobulin comprises or consists of a whole antibody (e.g., IgG) and the second immunoglobulin comprises or consists of the Fc region of the same antibody.

[0286] In some embodiments, the core particle is a gold nanoparticle. In some embodiments, the first linear polymer linker is a thiolated PEG3500 acid or a thiolated PEG35000 amine. In some embodiments, the second linear polymer linker is a thiolated PEG3500 acid or a thiolated PEG3500 amine. In some embodiments, the third linear polymer linker is a thiolated PEG5000 acid or a thiolated PEG5000 amine. In some embodiments, the permeation enhancing moiety is insulin.

[0287] In some embodiments, the core particle is a gold nanoparticle. In some embodiments, the first linear polymer linker is a thiolated PEG3500 acid or a thiolated PEG35000 amine. In some embodiments, the second linear polymer linker is a thiolated PEG1000 acid or a thiolated PEG1000 amine. In some embodiments, the third linear polymer linker is a thiolated PEG5000 acid or a thiolated PEG5000 amine. In some embodiments, the permeation enhancing moiety is insulin.

[0288] In some embodiments, the multifunctional particle further comprises at least one additional immunoglobulin attached to the core particle via an additional polymer linker. The different possibilities for the at least one additional active agent and the respective polymer linkers are similar to those described above for the first and second active agents and the first and second polymer linkers.

[0289] In some embodiments, the present invention provides a plurality of multifunctional particles as described above in any of its embodiments.

[0290] Preparation process According to another aspect, there is provided a process for the preparation of the multifunctional particles of the present invention in any of its embodiments described above, comprising: a) partially coating the surface of a core particle with a first polymer linker, followed by conjugating the first polymer linker to a first immunoglobulin; b) partially coating the surface of the core particle with a second polymer linker, followed by conjugating the second polymer linker to a second immunoglobulin; and c) partially coating the surface of the core particle with a third polymer linker, followed by conjugating the third polymer linker to a permeation enhancing moiety. Including, Processes are provided in which steps (a), (b) and (c) can be performed in any order.

[0291] The term "partial coating," as used herein, refers to the conjugation of a plurality of respective polymer linkers to the surface of a particle such that the plurality of linkers partially cover the surface of the particle at a density level below the saturation level of the bare particle.

[0292] Any method known in the art can be used to determine the amount of polymer required to achieve full density (i.e., 100%) coating of the particles, and accordingly the amount required for partial coating. For example, adding different amounts of polymer to the particle solution and measuring the concentration of free polymer in the supernatant after centrifugation is a widely used method. Alternatively, any characterization method sensitive to changes in coating density can be used, such as Zeta potential and DLS. Furthermore, theoretical calculations can be performed to determine the amount of polymer required to achieve complete coating, depending on the surface area of ​​the particles. For example, thiol-PEG molecules can be applied to a 0.35 nm thick gold nanoparticle surface. 2 It has been previously shown that the thiol-PEG linker occupies a footprint area of ​​100 nm (Qian, Ximeni et al. Nature biotechnology 26.1 (2008): 83-90). Therefore, the amount of thiol-PEG linker required to cover 100% of the surface of gold nanoparticles (GNPs) can be calculated based on the average diameter of the GNPs.

[0293] In some embodiments, each of the first polymer linker, the second polymer linker, and the third polymer linker comprises 5-70%, 5-60%, 5-40%, 8-60%, 10-60%, 10-55%, 10-50%, 10-40%, 10-30%, 10-25%, 10-20%, 15-60%, 15-55%, 15-50%, 15-45% of the surface of the core particle. , 15-40%, 15-30%, 15-25%, 15-20%, 2-10%, 2-20%, 2-50%, 2-60%, 2-70%, 5-10%, 5-20%, 5-70%, 10-20%, 10-50%, 10-70%, 20-50%, 20-40%, 30-50%, 30-60%, 30-70%, 50-60% or 50-70%. Each possibility represents a separate embodiment of the present invention.

[0294] In some embodiments, step (a) comprises removing 5-70%, 5-60%, 5-40%, 8-60%, 10-60%, 10-55%, 10-50%, 10-40%, 10-30%, 10-25%, 10-20%, 15-60%, 15-55%, 15-50%, 15-45%, 15-40%, 15-30% of the surface of the core particle. , 15-25%, 15-20%, 2-10%, 2-20%, 2-50%, 2-60%, 2-70%, 5-10%, 5-20%, 5-70%, 10-20%, 10-50%, 10-70%, 20-50%, 20-40%, 30-50%, 30-60%, 30-70%, 50-60% or 50-70% coating. Each possibility represents a separate embodiment of the present invention.

[0295] In some embodiments, step (b) comprises removing 5-70%, 5-60%, 5-40%, 8-60%, 10-60%, 10-55%, 10-50%, 10-40%, 10-30%, 10-25%, 10-20%, 15-60%, 15-55%, 15-50%, 15-45%, 15-40%, 15-30% of the surface of the core particle. , 15-25%, 15-20%, 2-10%, 2-20%, 2-50%, 2-60%, 2-70%, 5-10%, 5-20%, 5-70%, 10-20%, 10-50%, 10-70%, 20-50%, 20-40%, 30-50%, 30-60%, 30-70%, 50-60% or 50-70% coating. Each possibility represents a separate embodiment of the present invention.

[0296] In some embodiments, step (c) comprises removing 5-70%, 5-60%, 5-40%, 8-60%, 10-60%, 10-55%, 10-50%, 10-40%, 10-30%, 10-25%, 10-20%, 15-60%, 15-55%, 15-50%, 15-45%, 15-40%, 15-30% of the surface of the core particle. , 15-25%, 15-20%, 2-10%, 2-20%, 2-50%, 2-60%, 2-70%, 5-10%, 5-20%, 5-70%, 10-20%, 10-50%, 10-70%, 20-50%, 20-40%, 30-50%, 30-60%, 30-70%, 50-60% or 50-70% coating. Each possibility represents a separate embodiment of the present invention.

[0297] In some embodiments, steps (a)-(c) are performed sequentially in any order. One skilled in the art will be able to determine the optimal order of steps depending on different parameters, such as the type of core particle, the specific polymer linker, the immunoglobulin used, the permeation enhancing moiety, etc. In some embodiments, the process further comprises centrifugation after each of steps (a), (b) and (c).

[0298] In some embodiments, the first polymer linker and the second polymer linker are the same. In some related embodiments, steps (a) and (b) are carried out simultaneously by partially coating the surface of the core particle with the first polymer linker and the second polymer linker together, and then conjugating the first and second active agents to the polymer linkers. In some related embodiments, the step of partially coating the surface of the core particle with the first and second polymer linkers together comprises coating 10-70%, 10-60%, 10-40%, 10-60%, 10-60%, 10-55%, 10-50%, 10-45%, 10-40%, 10-30%, 10-25%, 10-20%, 15-60%, 15-55%, 15-50%, 15-45%, 15-40%, 15-30%, 15-25%, 15-20%, 10-20%, 10-50%, 10-70%, 20-50%, 20-40%, 30-50%, 30-60%, 30-70%, 50-60%, or 50-70% of the surface of the core particle. Each possibility represents a separate embodiment of the present invention. In further related embodiments, conjugating the first and second active agents to the polymer linker comprises adding a mixture of the first and second active agents in a desired molar ratio to the particle solution.

[0299] In some embodiments, the process further comprises partially coating the surface of the core particle with a fourth polymer linker. In some embodiments, the fourth polymer linker is a monofunctional linker used to limit access to reactive groups on the particle.

[0300] According to a related embodiment, there is provided a process for the preparation of multifunctional particles, comprising the steps of: a) partially coating the surface of a core particle with a first polymer linker, followed by conjugating the first polymer linker to a first immunoglobulin; b) partially coating the surface of the core particle with a second polymer linker, followed by conjugating the second polymer linker to a second immunoglobulin; c) partially coating the surface of the core particle with a third polymer linker, followed by conjugating the third polymer linker to a permeation enhancing moiety; and d) partially coating the surface of the core particle with a fourth polymer linker, the fourth polymer linker being a monofunctional linker. Including, Here, a process is provided in which steps (a), (b), (c) and (d) can be performed in any order.

[0301] In some embodiments, the particles are gold nanoparticles (GNPs) and the process comprises: (a) dissolving HAuCl 4 (b) reduction of the GNPs; (b) simultaneous incubation of the reduced GNPs with one type of monofunctional linker and three types of heterofunctional linkers; (c) activation of the acid groups of the linkers; (d) conjugation of a permeation enhancing moiety; and (d) conjugation of two different antibodies by incubating with a solution containing a mixture thereof.

[0302] According to some embodiments, analysis of the GNPs is performed after each step using methods known in the art.

[0303] In some embodiments, the monofunctional linker is mPEG-SH. According to a particular embodiment, the monofunctional linker is mPEG5000-SH or mPEG6000-SH, and is added to cover about 80% of the particle surface.

[0304] In some embodiments, the heterofunctional linker is COOH-PEG-SH. According to some embodiments, one heterofunctional linker is COOH-PEG5000-SH and is added at a concentration that covers about 15% of the particle surface. According to some embodiments, another heterofunctional linker is COOH-PEG3500-SH and is added at a concentration that covers about 5% of the particle surface.

[0305] In some embodiments, activation of the GNPs is carried out by mixing the GNPs with (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide HCl (EDC).

[0306] The core particles, first polymer linker, second polymer linker, third polymer linker, fourth polymer linker, permeation enhancing moiety, and first and second immunoglobulins suitable for use in the preparation process are as described above in connection with the various aspects and embodiments of the codelivery system.

[0307] Pharmaceutical Compositions In yet another aspect, a pharmaceutical composition is provided that includes a multifunctional particle according to the various embodiments above and a pharma- ceutical acceptable carrier. In some embodiments, the pharmaceutical composition includes a plurality of multifunctional particles according to the various embodiments above and a pharma- ceutical acceptable carrier.

[0308] As used herein, a "pharmaceutical acceptable formulation", "pharmaceutical composition" or "pharmaceutical acceptable composition" may include any of a number of carriers, such as solvents, dispersion media, coating agents, surfactants, antioxidants, preservatives (e.g., antibacterial agents, antifungal agents), isotonicity agents, absorption retardants, salts, preservatives, drugs, drug stabilizers, gels, binders, excipients, disintegrants, lubricants, sweeteners, flavorings, dyes, such materials and combinations thereof, as known to those skilled in the art (Remington, 1990). Pharmaceutical compositions containing the particles described herein as active ingredients can be prepared according to conventional pharmaceutical compounding techniques. See, for example, Remington's Pharmaceutical Sciences, 18th Edition, Mack Publishing Co., Easton, Pa. (1990). See also Remington: The Science and Practice of Pharmacy, 21st ed., Lippincott Williams & Wilkins, Philadelphia, Pa. (2005).

[0309] The composition may contain different types of carriers depending on whether it is administered in solid, liquid or aerosol form and whether it needs to be sterile for administration route such as injection.Those skilled in the art will be familiar with the techniques of making sterile solutions for injection or any other route of application.Sterile injectable solutions are prepared by incorporating the required amount of active compound in a suitable solvent with various other ingredients familiar to those skilled in the art.

[0310] The carriers may, in total, comprise from about 0.1% to about 99.99999% by weight of the pharmaceutical compositions presented herein.

[0311] According to some embodiments, the pharmaceutical composition is formulated for systemic administration. According to some embodiments, the pharmaceutical composition is formulated for systemic administration selected from intravenous and intranasal administration. According to some embodiments, the pharmaceutical composition is formulated for intravenous administration. According to some embodiments, the pharmaceutical composition is formulated for intranasal administration.

[0312] Compositions contemplated herein may take the form of a solution, suspension, emulsion, aerosol, combinations thereof, or any other pharma- ceutically acceptable composition generally known in the art.

[0313] In some embodiments, the carrier is a solvent. In a non-limiting example, the composition may be placed in a solvent. Such solvents include any suitable solvent known in the art, such as water, saline, phosphate buffered saline, etc.

[0314] The formulation of the composition may vary depending on the route of administration. For example, for parenteral administration in an aqueous solution, the solution should be suitably buffered if necessary and the liquid diluent first rendered isotonic with sufficient saline or glucose. Sterile aqueous media that can be employed will be known to those of skill in the art in light of the present disclosure.

[0315] Supplementary active ingredients can also be incorporated into the composition. For human administration, preparations must meet the sterility and general safety and purity standards required by the FDA Office of Biological Sciences standards. Administration can be by any known route.

[0316] In some embodiments, the pharmaceutical composition comprises at least about 0.001 g to about 1 g of the particles disclosed herein per kilogram of subject. In some embodiments, the pharmaceutical composition comprises at least about 0.001 g to about 0.5 g of the particles disclosed herein per kilogram of subject.

[0317] The pharmaceutical composition may contain various antioxidants to retard oxidation of one or more components. In addition, prevention of microbial action may be provided by preservatives such as various antibacterial and antifungal agents, including, but not limited to, parabens (e.g., methylparaben, propylparaben), chlorobutanol, phenol, sorbic acid, thimerosal, or combinations thereof. The composition must be stable under the conditions of manufacture and storage and preserved against the contaminating action of microorganisms, such as bacteria and fungi. It will be understood that exotoxin contamination should be kept at a minimum safe level, e.g., less than 0.5ng / mg protein.

[0318] In embodiments in which the composition is in liquid form, the carrier can be a solvent or dispersion medium, including, but not limited to, water, ethanol, polyols (e.g., glycerin, propylene glycol, liquid polyethylene glycol, and the like), lipids (e.g., triglycerides, vegetable oils, liposomes), and combinations thereof. In many cases, it will be preferable to include isotonic agents, such as sugars, sodium chloride, or combinations thereof.

[0319] In other embodiments, nasal solutions or sprays, aerosols or inhalants may be used. Nasal solutions are usually aqueous solutions designed to be administered to the nasal passages in drops or sprays.

[0320] Solid compositions for oral administration are also contemplated. In these embodiments, the solid compositions may include, for example, solutions, suspensions, emulsions, tablets, pills, capsules, sustained release formulations, buccal compositions, troches, elixirs, suspensions, syrups, or combinations thereof.

[0321] Sterile injectable solutions are prepared by incorporating the required amount of active compound (e.g., nanoparticles) in an appropriate solvent along with various other ingredients listed above. The liquid medium should be suitably buffered if necessary, and the liquid diluent should first be rendered isotonic with sufficient saline or glucose prior to injection.

[0322] Dosage can be repeated as necessary, as determined by those skilled in the art.Therefore, in some embodiments of the methods described herein, a single dose is contemplated.In other embodiments, two or more doses are contemplated.When two or more doses are administered to a subject, the time interval between doses can be any time interval determined by those skilled in the art.

[0323] According to some embodiments, a pharmaceutical composition comprising at least one delivery system according to the present invention and a pharmaceutical composition comprising an additional immunomodulator or kinase inhibitor are used for the treatment of cancer by separate administration.

[0324] Toxicity and therapeutic efficacy of the compositions described herein can be determined, for example, in cell cultures or experimental animals, by standard pharmaceutical procedures, for example, by determining the IC50 (concentration resulting in 50% inhibition) and the maximum tolerated dose of the compound. The data obtained from these cell culture assays and animal studies can be used in formulating a range of dosages for use in humans. The dosage may vary depending on the dosage form employed, the dosing regimen selected, the composition of the agent used in treatment, and the route of administration utilized, among other relevant factors. The exact formulation, route of administration, and dosage can be selected by the individual physician in view of the patient's condition. Depending on the severity and responsiveness of the condition to be treated, dosing can also be a single dose of a sustained release composition, with the course of treatment lasting from several days to several weeks, or until a cure or abatement of the disease state occurs. The amount of the composition administered will, of course, depend on the subject being treated, the severity of the affliction, the manner of administration, the judgment of the prescribing physician, and all other relevant factors.

[0325] Therapeutic and Diagnostic Uses of the Compositions According to some aspects, pharmaceutical compositions are provided comprising the multifunctional particles of the present invention for use in co-delivery of a first and a second immunoglobulin to a specific population of cells in a subject in need thereof, or to a location within the body or tissue but outside the brain, and in some embodiments, outside the CNS.

[0326] According to some embodiments, the co-delivery is to a primary tumor, a malignant tumor, or a killer cell.

[0327] According to another aspect, the present invention provides a method for synchronous delivery of a first immunoglobulin and a second immunoglobulin to diseased or malignant tissue or cells outside the CNS of a subject in need thereof, comprising administering to the subject a pharmaceutical composition comprising the multifunctional particles described above in all embodiments thereof.

[0328] According to some embodiments, the pharmaceutical composition is for use in treating cancers, tumors or cancer cells located outside the brain, and in some embodiments, outside the CNS.

[0329] According to some embodiments, the tumor is a primary tumor.

[0330] According to other embodiments, the cancer cells are metastases located outside the brain. According to yet other embodiments, the cancer cells are metastases located outside the CNS.

[0331] The term "cancer" refers to or describes a physiological condition in a mammal that is typically characterized by uncontrolled cell proliferation. Examples of cancer include, but are not limited to, carcinoma, adenocarcinoma, blastoma, and sarcoma. More specific examples of such cancers include breast cancer, colon cancer, prostate cancer, melanoma, lung cancer, thyroid cancer, liver cancer, bladder cancer, kidney cancer, cervical cancer, pancreatic cancer, ovarian cancer, uterine cancer, sarcoma, bile duct cancer, or endometrial cancer.

[0332] According to some aspects and embodiments, there is provided a method of treating cancer in a subject in need thereof, comprising administering to the subject a pharmaceutical composition of the invention.

[0333] According to some aspects and embodiments, a method for monitoring cancer in a subject in need thereof is provided, comprising administering to the subject a pharmaceutical composition of the present invention and imaging a specific region or tissue, or a specific population of cells, of the subject. In some related embodiments, the pharmaceutical composition comprises the above multifunctional particles, in which the core particles are gold nanoparticles, and imaging is performed using CT, which allows the detection of the multifunctional particles in a specific body region other than the CNS. In other embodiments, the pharmaceutical composition comprises the above multifunctional particles, in which at least one immunoglobulin is a labeled molecule, e.g., labeled with a fluorescent or radioactive molecule, which allows detection by a suitable imaging modality. In some embodiments, the method for monitoring a disease or disorder comprises repeated administrations and / or repeated imaging sessions.

[0334] According to some embodiments, the method of treating cancer is part of a treatment regimen that includes at least one additional anti-cancer therapy, according to certain embodiments, the additional anti-cancer therapy is selected from surgery, chemotherapy, radiation therapy, or immunotherapy.

[0335] The pharmaceutical compositions according to the present invention may be used as part of a combination therapy with at least one anti-cancer agent.

[0336] As used herein, the terms "combination," "combination therapy," or "combination treatment" can refer to either the simultaneous administration of items to be combined, or the sequential administration of items to be combined. As described herein, when combination refers to the sequential administration of items, the items can be administered in any temporal order.

[0337] According to some embodiments, the additional anti-cancer agent is an immunomodulatory agent, an activated lymphocyte cell, a kinase inhibitor, or a chemotherapeutic agent.

[0338] According to some embodiments, the anti-cancer agent is selected from the group consisting of antimetabolites, antimitotic agents, taxanes, topoisomerase inhibitors, topoisomerase II inhibitors, aspartic acid agents, alkylating agents, antitumor antibiotics, and combinations thereof.

[0339] According to some embodiments, the anti-cancer agent is an immune modulator, such as an antibody against an immune checkpoint molecule, whether agonist or antagonist.

[0340] Checkpoint immunotherapy blockade has proven to be an exciting new arena for cancer treatment. Immune checkpoint pathways consist of various costimulatory and inhibitory molecules that work in concert to maintain self-tolerance and protect tissues from damage by the immune system under physiological conditions. Tumors exploit certain checkpoint pathways to evade the immune system. Thus, inhibition of such pathways has emerged as a promising anti-cancer therapeutic strategy. According to some embodiments, the immune modulator is selected from the group consisting of antibodies that inhibit CTLA-4, anti-human programmed cell death protein 1 (PD-1), PD-L1 and PD-L2 antibodies, activated cytotoxic lymphocyte cells, lymphocyte activators, antibodies against CEACAM, antibodies against TIGIT, and RAF / MEK pathway inhibitors, anti-lymphocyte activation gene 3 (LAG3) antibodies, anti-CD137 antibodies, anti-OX40 (CD134) antibodies, and antibodies against killer cell immunoglobulin-like receptors (KIR).

[0341] In some embodiments, pharmaceutical compositions according to the invention are for use in enhancing the immune response, i.e., increasing the responsiveness of the immune system and inducing or prolonging its memory.

[0342] As used herein, the term "subject" refers to any animal (e.g., mammal), including, but not limited to, humans, non-human primates, rodents, etc. (e.g., who are intended to be the recipient of a particular treatment). Typically, the terms "subject" and "patient" are used interchangeably herein, unless otherwise indicated.

[0343] In some embodiments, the subject is a human subject. In some embodiments, the subject is at risk of developing cancer. In some embodiments, the subject is diagnosed with cancer. In some embodiments, the subject is diagnosed with a primary cancer located outside the brain. In some embodiments, the subject is diagnosed with a non-CNS-related cancer.

[0344] As used herein, a subject at risk of suffering from a disease, disorder, or condition is a subject who shows one or more signs or symptoms indicative of a disease, disorder, or condition, or a subject who is being screened for a disease, disorder, or condition (e.g., during a routine physical examination). A subject at risk of suffering from a disease, disorder, or condition may also have one or more risk factors. A subject at risk of suffering from a disease, disorder, or condition includes an individual who has not been previously tested for the disease, disorder, or condition. However, a subject at risk of suffering from a disease, disorder, or condition also includes an individual who has been given a preliminary diagnosis but has not been subjected to a confirmatory test (e.g., biopsy and / or tissue diagnosis) or the stage of the disease, disorder, or condition is unknown. This term further includes people who have previously suffered from a disease, disorder, or condition (e.g., individuals who have gone into remission).

[0345] As used herein, a subject diagnosed with cancer may be diagnosed using any suitable method, including, but not limited to, biopsy, x-ray, blood test, and the diagnostic methods of the present invention.

[0346] As used herein, the terms "treatment," "treating," or "amelioration" of a disease, disorder, or condition refer to alleviating at least one symptom thereof, reducing its severity, or inhibiting its progression. Treatment does not necessarily mean that the disease, disorder, or condition is completely cured. To be an effective treatment, a composition useful herein need only reduce the severity of the disease, disorder, or condition, reduce the severity of symptoms associated therewith, or provide an improvement in the quality of life of the patient or subject.

[0347] Those in need of treatment include those already with the disorder as well as those in which the disorder is to be prevented.

[0348] The term "administering" or "administration" of a substance, compound, agent, or pharmaceutical composition to a subject can be performed using one of a variety of methods known to those skilled in the art. For example, a pharmaceutical composition can be administered enterally or parenterally. Enteral administration refers to administration via the gastrointestinal tract, including orally, sublingually, or rectally. Parenteral administration includes intravenous, intradermal, intramuscular, intraperitoneal, subcutaneous, intraocular, sublingual, intranasal, by inhalation, intraspinal, intracerebral, and transdermal (by absorption, e.g., through the skin tract) administration. The agent or delivery system can also be suitably introduced by rechargeable or biodegradable polymeric or other devices, e.g., patches and pumps, or formulations that provide sustained, sustained, or controlled release of the compound or agent. Administration can also be performed, for example, once, multiple times, and / or over one or more extended periods of time. In some embodiments, administration includes both direct administration, including self-administration, and indirect administration, including the act of formulating the pharmaceutical composition. For example, as used herein, a physician who instructs a patient to self-administer a pharmaceutical composition or to have the composition administered by another person, and / or who provides a patient with a prescription for a drug, is administering a composition to a patient.

[0349] The administration of the composition to the subject can be performed using any method known to those skilled in the art. The mode of administration can vary based on the application. For example, the mode of administration can vary depending on the particular cell, body region, or subject to be imaged. For example, the administration of the composition can be performed intravenously, intracerebrally, intracranially, intrathecally, intraventricularly, intrasubstantia nigra or substantia nigra region, intradermally, intraarterially, intraperitoneally, intralesionally, intratracheally, intranasally, intramuscularly, intraperitoneally, subcutaneously, orally, topically, locally, by inhalation (e.g., aerosol inhalation), by injection, by infusion, by intrathecal injection, by transmucosal injection, by intracarotid injection, by continuous infusion, by local perfusion directly bathing the target cells, via catheter, via lavage, or by other methods known to those skilled in the art, or any combination of the above.

[0350] In some embodiments, the pharmaceutical composition is administered to a subject by a systemic route of administration. In some embodiments, the systemic administration is selected from intravenous (IV) administration and intranasal (IN) administration. In some embodiments, the particles are administered intravenously. In some embodiments, the particles are administered intranasally.

[0351] The effective amount of the pharmaceutical composition is determined based on the intended purpose and the subject to be treated. The amount administered may also vary based on the specific route of administration used. The composition is preferably administered in a safe and effective amount. As used herein, the term "safe and effective amount" refers to an amount of the composition sufficient for the intended purpose without undue adverse side effects (such as toxicity, irritation, or allergic reaction).

[0352] In some embodiments, the core particles are radiosensitizers, and the method of treating cancer further comprises irradiating tumor cells (where the particles accumulate) with ionizing radiation, thereby receiving locally enhanced radiation therapy within the tumor cells. In some embodiments, the composition is used for thermal ablation of tumor cells where the composition accumulates using infrared waves, without causing damage to surrounding normal tissues or substantial toxicity to the subject. As used herein, "ablation" refers to the destruction of cells. Methods of irradiating tissues with metal particles to enhance the effects of radiation therapy are known in the art.

[0353] The present invention further discloses methods for diagnosing and prognosing cancer.

[0354] According to an aspect, the present invention provides a method for the diagnosis and / or prognosis of cancer in a subject, comprising determining the expression level of a protein in a biological sample of the subject using at least one diagnostic composition as described herein.

[0355] The term "biological sample" encompasses various sample types obtained from an organism that may be used in diagnostic or monitoring assays. The term encompasses blood and other liquid samples from an organism, solid tissue samples such as biopsy samples, or tissue cultures or cells derived therefrom, and their progeny. In addition, the term may encompass circulating tumor or other cells. The term specifically encompasses clinical samples, and further includes biological fluids including cell cultures, cell supernatants, cell lysates, serum, plasma, urine, amniotic fluid, aqueous humor and vitreous of eye samples, and tissue samples. The term also encompasses samples that have been manipulated in any way after procurement, such as by treatment with a reagent, solubilization, or enrichment for a particular component. The method of the present invention may further comprise the step of comparing the expression level to a control level.

[0356] In some embodiments, the method further comprises imaging the particular region of the subject. In some embodiments, the imaging is performed using an imaging system selected from the group consisting of computed tomography (CT), X-ray imaging, magnetic resonance imaging (MRI), positron emission tomography (PET), single photon emission computed tomography (SPECT), ultrasound (US), and any combination thereof.

[0357] In some embodiments, imaging is performed to assess accumulation of the co-delivery system in a tissue or tumor of the subject.

[0358] In some embodiments, the subject is afflicted with a tumor or cancer and imaging is performed to determine the stage of the disease or disorder. In some embodiments, the subject afflicted with a tumor or cancer is being treated with a drug and imaging is used to follow up on the treatment.

[0359] In some embodiments, the imaging diagnostic step is performed 0.5 to 96 hours after the administration step. In some embodiments, the imaging diagnostic step is performed 0.5 to 48 hours after the administration step. In some embodiments, the imaging diagnostic step is performed 0.5 to 24 hours after the administration step. In some embodiments, the imaging diagnostic step is performed 0.5 to 12 hours after the administration step. In some embodiments, the imaging diagnostic step is performed 1 to 12 hours after the administration step. In some embodiments, the imaging diagnostic step is performed 1 to 6 hours after the administration step. In some embodiments, the imaging diagnostic step is performed within 96 hours after the administration step. In some embodiments, the imaging diagnostic step is performed within 48 hours after the administration step. In some embodiments, the imaging diagnostic step is performed within 24 hours after the administration step. In some embodiments, the imaging diagnostic step is performed within 12 hours after the administration step. In some embodiments, the imaging diagnostic step is performed within 6 hours after the administration step.

[0360] kit In some embodiments, the present invention provides a kit comprising one or more compositions disclosed herein. In some embodiments, the present invention provides a kit useful for the methods disclosed herein. For example, the kit may comprise a container having a sterile reservoir that contains any of the compositions disclosed herein. In some embodiments, the kit further comprises instructions. For example, the kit may comprise instructions (e.g., indications, dosage, method, etc.) for administering the composition to a subject. In yet another example, the kit may comprise instructions regarding the application of the compositions and methods of the present invention to imaging diagnostic systems, such as computed tomography (CT), ultrasound (US), magnetic resonance imaging (MRI).

[0361] The description of various embodiments of the present invention is presented for illustrative purposes, but is not intended to be exhaustive or limited to the disclosed embodiments. Many modifications and variations will be apparent to those skilled in the art without departing from the scope and spirit of the described embodiments. The terms used in this specification have been selected to best explain the principles of the embodiments, practical applications or technical improvements to the technology found in the market, or to enable those skilled in the art to understand the embodiments disclosed herein.

[0362] Any concentration range, percentage range, or ratio range recited herein should be understood to include any integer within that range, and fractional concentrations, percentages or ratios thereof, such as tenths and hundredths of integers, unless otherwise indicated.

[0363] Any numerical range recited herein with respect to any physical characteristic, such as polymer subunits, size or length, should be understood to include any integer within the recited range, unless otherwise indicated.

[0364] As used herein, the term "about" when in conjunction with a value refers to plus or minus 10% of the reference value. For example, a molecular weight of about 1000 Da refers to a molecular weight of 1000 Da±100 Da.

[0365] It should be noted that, as used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, a reference to "a particle" includes a plurality of such particles, and a reference to "the particle" includes a reference to one or more particles. It should be further noted that the claims may be drafted to exclude optional elements. Thus, this statement is intended to serve as a predicate basis for using exclusive language, such as "exclusively," "only," and the like, in connection with the recitation of claim elements or the use of "negative" limitations.

[0366] The term "plurality" means "two or more" unless expressly specified otherwise.

[0367] When a convention similar to "at least one of A, B, and C, etc." is used, such a configuration is generally intended in the sense that one of ordinary skill in the art would understand the convention (e.g., "a system having at least one of A, B, and C" includes, but is not limited to, systems having only A, only B, only C, A and B, A and C, B and C, and / or A, B and C, etc.). It will be further understood by one of ordinary skill in the art that virtually any choice of language and / or phrase presenting two or more alternative terms, whether in the specification, claims, or drawings, should be understood to contemplate the possibility of including one of the terms, either of the terms, or both terms. For example, the phrase "A or B" will be understood to include the possibilities of "A" or "B" or "A and B."

[0368] It is understood that certain features of the invention that are described for clarity in the context of separate embodiments may also be provided in combination in a single embodiment. Conversely, various features of the invention that are described for brevity in the context of a single embodiment may also be provided separately or in any suitable subcombination. All combinations of the embodiments relating to the invention are specifically embraced by the invention and are disclosed herein as if each and every combination was individually and explicitly disclosed. In addition, all subcombinations of the various embodiments and elements thereof are specifically embraced by the invention and are disclosed herein as if each and every such subcombination was individually and explicitly disclosed herein.

[0369] The following examples are intended to illustrate the methods of making and using the compounds and methods of the present invention, and are not to be construed as limiting in any way. The present invention will now be described in conjunction with specific embodiments thereof, but it is apparent that many modifications and variations will be apparent to those skilled in the art. Accordingly, it is intended to embrace all such modifications and variations that fall within the spirit and broad scope of the appended claims.

[0370] Working Example Generally, the nomenclature used herein and the laboratory procedures utilized in the present invention include chemical synthesis, molecular, biochemical, microbiological and recombinant DNA techniques which are well known in the art and are provided throughout the specification and / or are fully described in the literature.

[0371] Example 1: Multifunctional gold nanoparticles (GNPs) codelivide two types of antibodies FIG. 1 illustrates a schematic of a non-limiting exemplary multifunctional particle showing: (i) a first polymer linker (2) conjugated to a permeation enhancing moiety (e.g., insulin); (ii) a second polymer linker (3) conjugated to a first antibody (5) and a second antibody (6); and (iii) a gold nanoparticle (GNP; 1) bound to a capping polymer moiety (7).

[0372] Preparation and characterization of GNPs conjugated with anti-IgG1, anti-Iba1 and insulin (IgG1&Iba1&Ins-GNPs) GNP synthesis: 20 nm spherical GNPs were synthesized using HAuCl 4 It was prepared by reduction of 50% w / v HAuCl in 200 ml of distilled water with citric acid. 4 A total of 414 μl of the solution was boiled in an oil bath on a heating plate with stirring. After boiling, 4.04 ml of 10% sodium citrate solution was added and the mixture was stirred with boiling for an additional 10 minutes. After removing the solution from the plate and cooling to room temperature, the solution was centrifuged until the nanoparticles precipitated.

[0373] Conjugation of PEG5000 to GNPs: GNPs were first partially coated (60% of the particle surface) with mPEG-SH (approximately 5 kDa; 40% of the particle surface) and heterofunctional HS-PEG-COOH (approximately 5 kDa; 20% of the particle surface). The amount of mPEG-SH and HS-PEG-COOH required for partial coating was such that the thiol-PEG molecules were 0.35 nm thick on the gold nanoparticle surface. 2 The conjugation was carried out by adding a mixture of HS-PEG-COOH (193 μl, 50 mg / ml) and mPEG-SH (387 μl, 50 mg / ml) to the GNP solution and mixing for 2 h. The solution was then ultracentrifuged at 15,000 RPM for 20 min and then again at 20,000 RPM for 15 min. The precipitate containing the PEG-coated GNPs (total 60% coating) was transferred to a vial.

[0374] Conjugation of insulin: To facilitate the transport of multifunctional GNPs through the membrane, insulin was covalently conjugated to the carboxyl groups of HS-PEG-COOH by adding an excess amount of insulin along with EDC (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide HCl) and NHS (N-hydroxysulfosuccinimide sodium salt) on ice and mixed for 2 h. The solution was then centrifuged twice at 15,000 RPM for 30 min (kept at low temperature) and the lower phase containing Ins-PEG-GNPs was transferred to a vial.

[0375] Conjugation of PEG3500 to GNPs: To further conjugate the antibody to the GNPs, 271 μl of HS-PEG-COOH (approximately 3.5 kDa) solution (50 mg / ml) was added to the partially coated GNPs to coat the remaining 40% of the particle surface. The solution was then mixed for 2 hours at 4° C., followed by repeated centrifugation at 15,000 RPM for 30 minutes.

[0376] Conjugation of anti-IgG1 and anti-Iba1: Fluorescently labeled anti-Iba1 and fluorescently labeled anti-IgG1 were covalently conjugated to the free carboxyl groups of HS-PEG-COOH (approximately 3.5 kDa) by adding a 1:1 molar mixture of fluorescently labeled anti-Iba1 (Ab195032-Rb Mono&Hu IBA-1-647) and fluorescently labeled anti-IgG1 (mouse monoclonal IgG1 Alexa Fluor 488 isotype control clone 11711) with EDC and NHS. The solution was then stirred for 2 hours at 4°C, followed by centrifugation to remove unbound antibody until a final concentration of Au reached 25 mg / ml.

[0377] To confirm the chemical conjugation of the antibodies, the multifunctional double antibody nanoparticles were imaged using a fluorescent microscope.

[0378] In control experiments, similar particles were prepared with only one conjugated antibody, i.e., anti-IgG1 or anti-Iba1. Percentage coverage of the different coating molecules in the control particles: 20% PEG5000 (conjugated to insulin), 20% PEG3500 (conjugated to the respective antibody) and 60% mPEG5000.

[0379] Example 2: Preparation of multifunctional GNPs coated with insulin and two anti-HER2 antibodies, trastuzumab and pertuzumab As a non-limiting example, gold nanoparticles carrying two different anti-HER2 antibodies and insulin were generated. The exemplary particles were coated with a polymer layer (2-3 in FIG. 1) containing two different polymer linkers (5-S-PEG-C(O)-, approx. 5 kDa and -S-PEGC(O)-, approx. 3.5 kDa), the first linker conjugated to insulin (4) and the second linker conjugated to two different anti-HER2 antibodies (5-6), trastuzumab and pertuzumab. An additional (7) polymer moiety (-S-PEG-O-CH3 approx. 6 kDa) was used to attach to the surface or gold nanoparticles to adjust the density of other moieties on the particle.

[0380] GNP synthesis HAuCl 4 Spherical GNPs of 20 nm were prepared by citrate reduction of 42.77% w / v HAuCl in 200 ml double distilled water (DDW). 4 A total of 414 μl of was boiled in an oil bath on a heating plate with stirring. After boiling, 4.04 ml of 10% w / v trisodium citrate in DDW was added. The solution was removed from the oil bath and allowed to cool at room temperature with stirring.

[0381] Conjugation of COOH-PEG5000-SH, COOH-PEG3500-SH, and mPEG6000-SH to GNPs GNPs were incubated with mPEG5000-SH (approximately 5 kDa; 80% of the particle surface), heterofunctional COOH-PEG5000-SH (approximately 5 kDa; 15% of the particle surface) and heterofunctional COOH-PEG3500-SH (approximately 3.5 kDa; 5% of the particle surface). The amount of PEG moieties required for the percentage of coating was 0.35 nm for thiol-PEG molecules on the gold nanoparticle surface. 2 The conjugation was carried out by adding a mixture of COOH-PEG5000-SH (127 μl, 50 mg / ml in DDW), mPEG5000-SH (809 μl, 50 mg / ml in DDW) and COOH-PEG3500-SH (30 μl, 50 mg / ml in DDW) to the GNP solution and mixing overnight. The solution was then centrifuged at 50,000 G for 20 min, and the precipitate was then redispersed in DDW and centrifuged at 50,000 G for 20 min. The precipitate containing the PEG-coated GNPs was transferred to a vial.

[0382] Activation of GNPs was performed by mixing them with EDC (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide HCl, 30 mg / ml in DDW, 100 μl) and sulfo-NHS (N-hydroxysulfosuccinimide sodium salt, 30 mg / ml in DDW, 100 μl), followed by centrifugation at 50,000 G for 20 min. The precipitate containing the activated COOH groups was transferred to a vial.

[0383] Conjugation of insulin to HS-PEG5000-COOH was then performed by adding insulin (195IU, 100IU / ml) to the GNP solution for 3 hours. The solution containing trastuzumab and pertuzumab (total 15mg) was then placed in 2ml of borate buffer (PH8, 0.1M) and then added to the GNP-insulin solution to conjugate the remaining COOH-PEG3500-SH with overnight mixing. The solution was then centrifuged at 10,000G for 20 minutes. The precipitate was subsequently redissolved in saline and then centrifuged at 10,000G for 20 minutes.

[0384] Antibody and insulin coated GNPs (Abs&Ins-GNPs) were characterized using dynamic light scattering (DLS) after each step of preparation. The hydrodynamic size and zeta potential of the GNPs confirmed the successful coating.

[0385] Quantification of antibodies (Ab) and insulin attached to the PEG groups on the GNPs was tested by enzyme-linked immunosorbent assay (ELISA) testing the supernatant containing unbound proteins retained by centrifugation.

[0386] Example 3: In vitro effects of double antibody GNPs on HER2-positive cancer cells GNPs conjugated with insulin and the different anti-HER2 antibodies trastuzumab and pertuzumab were prepared according to the protocol described in Example 3. For comparison, GNPs conjugated with insulin and a single antibody (either trastuzumab or pertuzumab) were also prepared.

[0387] The HER2-positive breast cancer cell line, BT474, was used to determine the efficacy of GNPs conjugated with each antibody as monotherapy and in combination. Cells were treated with different concentrations of trastuzumab-GNPs, pertuzumab-GNPs, or trastuzumab & pertuzumab-GNPs. Untreated cells served as control.

[0388] The effects of different treatments on the cells were examined using cell cycle arrest measurements, apoptosis and proliferation assays. Each treatment was performed in triplicate.

[0389] In specific experiments, cells were incubated for 5 days under the following conditions: 1. control-untreated, 2. mixture of GNPs with trastuzumab & GNPs with pertuzumab (each GNP was prepared using insulin as the permeation enhancing moiety), 3. bispecific GNPs with both trastuzumab & pertuzumab (and insulin) conjugated to the same particle. Cell proliferation assay was performed to find the efficacy of the complex in inhibiting tumor cell proliferation. As shown in Figure 2, bispecific GNP complex showed better efficacy in inhibiting cancer cell proliferation than a mixture of two types of antibodies, each conjugated to a separate particle.

[0390] Example 4: In vitro effects of double antibody GNPs on breast cancer cells The bispecific nanoconjugates were tested in human breast cancer cell lines BT474 and MCF7, which have high (BT474) and relatively low (MCF7) expression of the HER2 receptor.

[0391] Cells were incubated for 5 days with the following conditions: (i) control-untreated; (ii) bispecific GNPs with both anti-HER2 antibodies, Trastuzumab & Pertuzumab, conjugated to the same particle without transporter or permeation enhancing molecules; and (iii) bispecific GNPs with both Trastuzumab & Pertuzumab, conjugated to the same particle in addition to insulin molecules. Cell proliferation assays were performed to determine the effectiveness of the complexes in inhibiting tumor cell proliferation. As shown in Figures 3A and 3B, bispecific GNP complexes with insulin molecules on the delivery system were more effective in inhibiting cell proliferation, while GNPs without insulin but with antibodies were less effective. Without being bound by any mechanism of action, it is assumed that insulin molecules on the delivery system bind to insulin receptors on cancer cells and inhibit the proliferation of these cancer cells by enhancing the contact and binding of anti-HER2 antibodies to cells expressing HER2 receptors, which are less frequent on these cells.

[0392] Example 5: Determining the optimal density of antibody per particle Different compositions of nanocomplexes with increasing numbers of attached antibodies were tested in vitro for inhibition of proliferation of BT474 human breast cancer cells overexpressing the HER2 receptor.

[0393] Cells were incubated for 5 days with the following conditions: (i) control-untreated; (ii) GNPs with 2 antibodies attached to each particle; (iii) GNPs with 11 antibodies attached to each particle; (iv) GNPs with 18 antibodies attached to each particle; (v) GNPs with 20 antibodies attached to each particle and (vi) GNPs with 30 antibodies attached to each particle. Cell proliferation assays were performed to determine the effectiveness of the different conjugates in inhibiting the proliferation of HER2-positive tumor cells. As shown in Figure 4, conjugates carrying 10-20 antibodies were the most active in inhibiting cancer cell proliferation with 62-50% inhibition.

[0394] Example 6: Bispecific GNP complexes for cancer therapy: In vivo studies The efficacy of the platform as a multifunctional drug carrier was tested in vivo for the inhibition of tumor growth. In this experiment, the antibodies trastuzumab and pertuzumab, which are considered the first-line combination treatment for breast cancer tumors, were used. GNPs were conjugated with both antibodies and insulin, which enhance tumor targeting and penetration via binding to the insulin receptor present on tumor cells, and tested using BT474 cells in a subcutaneous metastatic breast cancer tumor mouse model. Two weeks after tumor inoculation, the mice were divided into four groups: a control group, a group that received a mixture of free antibodies, a group that received a mixture of GNPs with trastuzumab and GNPs with pertuzumab, and a fourth group that received bifunctional GNPs (both antibodies conjugated to the same particle). The therapeutic substances were injected IP once a week for four consecutive weeks. The results shown in Figure 5 show that the multifunctional particles are significantly (p<0.01) more efficient in reducing tumor size than both the free antibodies and the mixture of monofunctional particles.

[0395] Example 7: Bispecific GNP immunotherapeutic conjugates for inhibiting tumor cell proliferation In this experiment, immunotherapeutic antibodies: anti-PD-1, which targets a receptor on T cells, and anti-PD-L1, which targets a receptor on tumor cells, were tested conjugated alone or together with GNPs.

[0396] The first step involved the activation of immune cells, which were then incubated 6 days later with H1299 lung cancer cells to test the combinatorial activity in inhibiting tumor cell proliferation.

[0397] Anti-CD3 and anti-CD28 were added to peripheral blood mononuclear cells (PBMCs) to induce T cell activation. The following substances were then added to the cell wells (each in triplicate), with an incubation period of 6 days: (i) a mixture of free anti-PD-1 and anti-PD-L1 antibodies; (ii) GNPs conjugated to both anti-PD-1 and anti-PD-L1 antibodies, produced according to the method in Example 3.

[0398] After 6 days, the medium was removed to detect cytokines, and PBMCs were collected and added to H1299 lung cancer cells to determine T cell cytotoxicity on target cells. PBMCs were incubated with H1299 cells for 18 hours, and then cell proliferation was measured by ELISA.

[0399] As shown in Figure 6, in the presence of multispecific GNPs carrying anti-PD-1 and anti-PD-L1, the mixture of PBMC cells and cancer cells had better efficacy in inhibiting the proliferation of cancer cells.

[0400] While the present invention has been described in conjunction with specific embodiments thereof, it is evident that many alterations, modifications and variations will be apparent to those skilled in the art. Accordingly, it is intended to embrace all such alterations, modifications and variations that fall within the spirit and broad scope of the appended claims.

Claims

1. (a) an inorganic particle bound to at least (i) a first linear heterofunctional polymer linker; (ii) a second linear heterofunctional polymer linker; (iii) a third linear heterofunctional polymer linker; and (iv) a fourth polymeric monofunctional linker; (b) a first immunoglobulin molecule covalently conjugated to said first linear polymer linker; (c) a second immunoglobulin molecule covalently conjugated to the second linear polymer linker; and (d) a permeation-enhancing moiety conjugated to said third linear polymer linker. A multifunctional particle comprising: the first and second immunoglobulin molecules are separate, and a total of about 2-400 immunoglobulin molecules are conjugated to each particle via the first and second linkers; Multifunctional particles.

2. The multifunctional particle of claim 1, wherein the length of the third linear polymer linker is substantially different from the length of the first and second linear polymer linkers, and the molecular weight of the third polymer linker differs from the molecular weight of the first and second polymer linkers by at least about 1000 Da.

3. The multifunctional particle of claim 1 or 2, wherein the first, second, third and fourth polymer linkers are not cleavable under physiological conditions.

4. The multifunctional particle according to any one of claims 1 to 3, wherein the molecular weights of the first, second and third linear polymer linkers are in the range of 1,000 to 10,000 Da.

5. The multifunctional particle according to any one of claims 1 to 4, wherein the molecular weight of each of the first and second linear polymer linkers is 3500 Da or greater.

6. The multifunctional particle according to any one of claims 1 to 5, wherein the first polymer linker and the second polymer linker are the same.

7. The multifunctional particle according to any one of claims 1 to 6, wherein the molecular weight of each of the third and fourth polymer linkers is in the range of 4500 to 8000 Da.

8. The multifunctional particle according to any one of claims 1 to 7, wherein the first and second linear polymer linkers are attached to the inorganic particle via semi-covalent bonds, and the first and second immunoglobulin molecules are conjugated to the respective linear polymer linkers via amide bonds.

9. The multifunctional particle of any one of claims 1 to 8, wherein the fourth linker is conjugated to about 90% to 99% of the total surface volume of the particle.

10. The multifunctional particle of any one of claims 1 to 9, wherein the first and second immunoglobulin molecules are each independently an antibody molecule, and a total of about 2 to 40 antibody molecules are conjugated to each particle via the first and second linkers.

11. The multifunctional particle according to any one of claims 1 to 9, wherein the first and second immunoglobulin molecules are each independently an antibody fragment molecule, and a total of about 20 to 400 antibody fragment molecules are conjugated to each particle via the first and second linkers.

12. The multifunctional particle of any one of claims 1 to 11, wherein at least one of the first and second immunoglobulin molecules is capable of binding to a cancer-specific or cancer-associated cell surface antigen on a tumor cell.

13. The multifunctional particle of any one of claims 1 to 12, wherein the first and second immunoglobulin molecules are each independently an antibody or antibody fragment capable of binding to a cancer-specific or cancer-associated cell surface antigen on a tumor cell.

14. The multifunctional particle according to any one of claims 12 and 13, wherein the tumor antigen or tumor-associated antigen is selected from the group consisting of HER family receptors, EGFR, mesenchymal epithelial transition factor, PSMA, nectin-4, CD155, CD3, EGFRvIII, Vγ9, CD16, CD133, IL-15, and CD19, CD20, CD30, CD38, CD38 and CD138.

15. The multifunctional particle of claim 13 or 14, wherein the two types of antibodies or antibody fragments are capable of binding to the same or similar cancer-specific or cancer-associated cell surface antigens on tumor cells.

16. The multifunctional particle of claim 15 , wherein the two types of antibodies or antibody fragments are capable of binding to HER2.

17. The multifunctional particle of claim 13 or 14, wherein each of the two types of antibodies or antibody fragments is capable of binding to a different cancer-specific or cancer-associated cell surface antigen on tumor cells.

18. The multifunctional particle according to any one of claims 1 to 11, wherein at least one of the first and second immunoglobulin molecules is capable of binding to an immune cell selected from the group consisting of an NK cell, a T cell, an NKT cell, and a macrophage, or of binding to a checkpoint molecule on an immune cell or a tumor cell.

19. A multifunctional particle described in any one of claims 1 to 18, comprising a first antibody or antibody fragment capable of binding to at least one cancer-specific or cancer-associated cell surface antigen on a tumor cell, and a second antibody or antibody fragment capable of binding to an immune cell or a checkpoint molecule on an immune cell or a tumor cell.

20. The multifunctional particle according to any one of claims 18 or 19, wherein the checkpoint molecule is selected from the group consisting of PD-1, PD-L1, CTLA-4, 4-1BB, OX40, TIM3, TIGIT, LAG-3, and CD47.

21. The multifunctional particle of claim 20 , wherein one of the antibodies or antibody fragments is capable of binding to PD-1 and the other is capable of binding to PD-L1.

22. HER2 and HER2, HER2 and HER3, HER2 and PD-1, HER2 and CTLA-4, PD-1 and PD-L1, PD-1 and CTLA-4, CEA and CD3, PSMA and CD3, EGFRvIII and CD3, EpCam and CD3, HER2 and Vγ9, CD16 and CD133, CD16 and IL-15, CD15 and CD19, CD16 and CD133, IGF-1 and IGF-2, VEGF and Ang2, EGFR and cMET, DLL4 and VEGF, HER2 and CD3, PD-1 and LAG3, PD-L1 and CD137, PSMA and CD3, IGF-RI and HER3, PMEL and CD3, B7H3 and CD3, GPA33 and CD3, and GPC3 and CD3. The multifunctional particle of any one of claims 1 to 21, comprising a pair of antibodies or antibody fragments targeting antigens selected from the group consisting of HER2 and Ang2, EGFR and cMET, DLL4 and VEGF, HER2 and CD3, PD-1 and LAG3, PD-L1 and CD137, PSMA and CD3, IGF-RI and HER3, PMEL and CD3, B7H3 and CD3, GPA33 and CD3, and GPC3 and CD3.

23. The multifunctional particle according to any one of claims 10 to 22, wherein the immunoglobulin is selected from the group consisting of a non-human antibody, a chimeric antibody, a humanized antibody, a human antibody, and any combination thereof.

24. 28. The multifunctional particle of any one of claims 1 to 27, wherein the first, second, third, and fourth polymer linkers independently comprise a polymer selected from the group consisting of polyethers, polyacrylates, polyanhydrides, polyvinyl alcohols, polysaccharides, poly(N-vinylpyrrolidone), polyglycerin (PG), poly(N-(2-hydroxypropyl)methacrylamide), polyoxazolines, poly(amino acid)-based hybrids, recombinant polypeptides, derivatives, and combinations thereof.

25. 30. The multifunctional particle of claim 28, wherein at least one of the first, second, third and fourth polymer linkers is a polyether, and the polyether is polyethylene glycol (PEG).

26. 26. The multifunctional particle of claim 25, wherein the fourth polymer linker is methoxypolyethylene glycol (mPEG).

27. 27. The multifunctional particle according to any one of claims 1 to 26, wherein the inorganic particles are nanoparticles selected from the group consisting of metal nanoparticles, metal oxide nanoparticles, ceramic nanoparticles, and any combination thereof.

28. 28. The multifunctional particle of claim 27, wherein the inorganic nanoparticles are selected from the group consisting of gold nanoparticles, iron (III) oxide nanoparticles, and iron (II, III) oxide nanoparticles.

29. The multifunctional particle according to any one of claims 1 to 28, wherein the permeation enhancing moiety is selected from the group consisting of insulin, an antibody specific to an insulin receptor, a polypeptide that specifically binds to an insulin receptor, insulin-like growth factor 1, an antibody specific to insulin-like growth factor receptor 1, a polypeptide that specifically binds to insulin-like growth factor receptor 1, a cell penetrating peptide (CPP), glucose and glucose derivatives, and any combination thereof.

30. 30. The multifunctional particle of claim 29, wherein the permeation enhancing moiety is insulin.

31. The multifunctional particle according to any one of claims 1 to 30, wherein the inorganic particles are nanoparticles having a diameter of 10 to 160 nm.

32. 1. A process for preparing multifunctional particles, comprising: (a) partially coating the surface of an inorganic particle with a first linear polymer linker and a second linear polymer linker, and subsequently conjugating said first and said second linear polymer linkers to a first immunoglobulin molecule and a second immunoglobulin molecule, wherein said first linear polymer linker and said second linear polymer linker are identical and said first immunoglobulin molecule is distinct from said second immunoglobulin molecule; (b) partially coating the surface of the inorganic particles with a third linear polymer linker, and subsequently conjugating the third linear polymer linker to a permeation enhancing moiety; and (c) partially coating the surface of the inorganic particles with a fourth polymeric monofunctional linker; The successive steps of a length of said third linear polymer linker is substantially different from the length of said first and said second linear polymer linkers, a molecular weight of said third polymer linker differs from the molecular weight of said first and said second polymer linkers by at least about 1000 Da, and steps (a), (b) and (c) may be performed in any order; process.

33. The particles are gold nanoparticles (GNPs) and the process comprises: (a) dissolving HAuCl ４ (b) simultaneous incubation of the reduced GNPs with one type of monofunctional linker and three types of heterofunctional linkers; (c) activation of the free terminal acid groups of the linkers; (d) conjugation of the permeation enhancing moiety; and (e) conjugation of two different antibodies or antibody fragments by incubating with a solution containing a mixture thereof.

34. A pharmaceutical composition comprising the multifunctional particle of any one of claims 1 to 31 and a pharma- ceutical acceptable carrier or excipient.

35. 35. The pharmaceutical composition of claim 34, formulated for at least one of intravenous (IV), intranasal (IN), intraperitoneal (IP), and intratumoral administration.

36. 36. A pharmaceutical composition according to any one of claims 34 or 35 for use in the prevention, treatment and / or monitoring of cancer or tumour in a subject in need thereof.

37. 1. A pharmaceutical composition for use in treating or monitoring a primary tumor or metastasis located outside the brain, comprising a pharma- ceutically acceptable carrier or excipient and a multifunctional particle, The multifunctional particles include (a) an inorganic particle bound to at least (i) a first linear polymeric linker; (ii) a second linear polymeric linker; (iii) a third linear polymeric linker; and (iv) a fourth polymeric monofunctional linker; (b) a first immunoglobulin molecule conjugated to said first linear polymer linker; (c) a second immunoglobulin molecule covalently conjugated to the second linear polymer linker; and (d) a permeation enhancing moiety conjugated to said third linear polymer linker; Including, the first and second immunoglobulin molecules are separate, and a total of about 2-400 immunoglobulin molecules are conjugated to each particle via the first and second linkers; Pharmaceutical compositions.

38. 38. The pharmaceutical composition of claim 37, wherein the primary tumor or metastasis is a solid tumor selected from breast, brain, lung, melanoma, prostate, bladder, pancreatic and ovarian tumors.

39. 39. The pharmaceutical composition of any one of claims 37 and 38, wherein the breast tumor is selected from the group consisting of a resistant tumor, a metastatic tumor, a HER2 expressing tumor, a HER2 low tumor, and a triple negative breast tumor.

40. 40. The pharmaceutical composition according to any one of claims 37 to 39, wherein the treatment comprises administering or carrying out at least one additional anti-cancer therapy selected from chemotherapy, immunotherapy, biological therapy, hormonal therapy, radiation therapy, bone marrow transplantation, surgery, and any combination thereof.

41. A method for preventing, treating and / or monitoring cancer, including primary tumors or tumor metastases located outside the brain, in a subject in need thereof, comprising administering to the subject a pharmaceutical composition comprising a multifunctional particle described in any one of claims 1 to 31.

42. 42. The method of claim 41, wherein the primary tumor is a solid tumor.

43. 43. The method of claim 42, wherein the solid tumor is selected from breast, lung, melanoma, prostate, bladder, pancreatic and ovarian tumors.

44. 44. The method of any one of claims 42 and 43, wherein the solid tumor is selected from a resistant tumor and a metastatic tumor.

45. The method of any one of claims 41 to 44, wherein the primary tumor is a breast tumor selected from the group consisting of a HER2 expressing tumor, a HER2 low tumor, and a triple negative breast tumor.

46. 46. ​​The method of any one of claims 41 to 45, further comprising administering or performing at least one additional anti-cancer therapy selected from chemotherapy, immunotherapy, biological therapy, hormonal therapy, radiation therapy, bone marrow transplant, surgery, and any combination thereof.

47. 47. The method of any one of claims 41-46, wherein the therapeutic result results in the prevention or reduction of the formation, growth, or spread of metastases in said subject.

48. A method of enhancing an immune response in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a multifunctional particle according to any one of claims 18 to 21.

49. A method for simultaneously delivering at least two types of immunoglobulin molecules to a specific region, cell, or tissue outside the brain of a subject, comprising administering to the subject a pharmaceutical composition comprising a multifunctional particle described in any one of claims 1 to 31.

50. 1. A method for preventing, treating and / or monitoring a primary tumor or tumor metastasis located outside the brain in a subject in need thereof, comprising administering to said subject a pharmaceutical composition comprising a multifunctional particle; The multifunctional particles are (a) an inorganic particle bound to at least (i) a first linear polymeric linker; (ii) a second linear polymeric linker; (iii) a third linear polymeric linker; and (iv) a fourth polymeric monofunctional linker; (b) a first immunoglobulin molecule conjugated to said first linear polymer linker; (c) a second immunoglobulin molecule covalently conjugated to the second linear polymer linker; and (d) a permeation-enhancing moiety conjugated to said third linear polymer linker. Including, said first and said second immunoglobulin molecules are distinct; method.

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