Multispecific human albumin nanoparticles modified with antibody fragments and loaded with cytotoxic substances.

Abstract

The present invention relates to serum albumin nanoparticles, preferably human serum albumin nanoparticles, optionally loaded with a cytotoxic drug, whose surface is modified with at least one biomolecule capable of recognizing a target receptor overexpressed or selectively expressed on the surface of cells, preferably cancer cells. In particular, the serum albumin nanoparticles (Alb-NPs) are modified with at least one modification chain comprising a linker bound to the nanoparticle by an -S-thioether bond, and at least one biomolecule. The at least one biomolecule is bound to the linker via an amide bond formed by a transamidation (or transglutamination) reaction mediated by the enzyme transglutaminase between (i) the -NH2 residue of the X group of the linker and the CO-NH2 residue of a glutamine contained in a peptide consensus sequence inserted into the at least one biomolecule; or (ii) the -NH2 residue of a lysine contained in a peptide sequence inserted into the at least one biomolecule and the -CO-NH2 residue of a glutamine inserted into a consensus sequence that is part of the linker.

Description

[Technical Field]

[0001] The present invention relates to serum albumin nanoparticles, preferably human nanoparticles, which are at least one biomolecule capable of recognizing a target receptor overexpressed or selectively expressed on the surface of cells, preferably cancer cells, and which are optionally loaded with a cytotoxic drug whose surface has been modified. [Background technology]

[0002] Human albumin (HSA) is the most efficient and versatile natural carrier protein for drugs and endogenous small molecules introduced into the circulatory system. Molecular binding to HSA improves its pharmacokinetic profile and reduces its rapid elimination and toxicity via the renal system. HSA is the most abundant plasma protein (35–50 g / L human serum) and has a molecular weight of 66.5 kDa. Like most plasma proteins, it is synthesized in the liver and produced at a rate of 0.7 mg / hour per gram of liver (i.e., 10–15 g / day). HSA has an average plasma half-life of 19 days. Its long half-life largely depends on its ability to bind to FcRn receptors with high affinity [1]. HSA acts as a solubilizer for long-chain fatty acids and is therefore essential for lipid metabolism, binding to bilirubin, heme degradation products, and many therapeutic agents, to name just a few, such as penicillin, sulfonamides, indole compounds, and benzodiazepines [2]. In specific methods, copper(II) and nickel(II) can be complexed, and in relatively non-specific methods, calcium(II) and zinc(II) can be complexed, acting as transport vehicles for these metal ions in the blood.

[0003] HSA is a highly soluble and extremely stable acidic protein; stable in the pH range of 4–9, soluble in 40% ethanol, and can be heated to 60°C for up to 10 hours without structural loss. Its ability to be preferentially absorbed in tumor and inflammatory tissues, its immediate and broad availability, its biodegradability, and its inherent lack of toxicity and immunogenicity, along with these properties, make it an ideal molecule for the delivery of therapeutic agents and other molecules conjugated to it covalently or non-covalently.

[0004] HSAs can preferentially accumulate in tumor and inflammatory tissues due to the presence of damaged capillaries and the absence or defect of the lymphatic drainage system. This property, known as "passive tumor targeting," takes advantage of the increased ability of molecules with molecular weights greater than approximately 40 kDa to permeate and be retained in tumor tissue, thanks to the absence of permeable vessel walls, whereas in healthy tissue blood vessels only small molecules can pass through the endothelial barrier. The pore size of tumor microvessels ranges from 100 to 1200 nm in diameter [3,4], while HSAs have an effective diameter of 7.2 nm, which allows them to extravasate only in tumor tissue and not in normal tissue. This phenomenon leads to greater uptake of macromolecules such as HSAs in tumor tissue, which are usually excreted fairly rapidly, and is advantageous for their use as carriers for low molecular weight anticancer drugs that would exert cytotoxic activity even in healthy tissue.

[0005] Albumin accumulation has been observed in many animal models of solid tumors, including sarcomas, ovarian cancer, and Novikof hepatocarcinoma [5]. In a syngeneic breast cancer model, in pathological proliferations of mouse mammary intraepithelial neoplasia, and in tumors derived from epithelial-mesenchymal transition, permeability to albumin is about four times higher than that to liposomes or other similar synthetic nanoparticles of size 100 nm [6], and it is also very interesting that the phenomenon may be favorably influenced by the longer plasma half-life of HSA.

[0006] Thanks to all these properties, the conjugation of small molecules and therapeutic peptides or hormones, such as insulin or cytokines, to albumin has become a widely used and effective approach to improve their pharmacokinetic profile and semi-specific transport to affected tissues.

[0007] In addition to passive accumulation in tumor tissue, HSA is preferably taken up by many cancer cells, which further facilitates its use for the intracellular release of therapeutic agents (therapeutics) that bind to it. In fact, it has been shown that cancer cells absorb and metabolize extracellular proteins such as HSA through the mechanism of macropinocytosis to meet their increased metabolic needs. In cancer cells expressing the oncogene Ras, an internal plasma membrane protein, whose overexpression and overactivation are associated with virtually all phenotypic malignancies, extracellular proteins are used more frequently as a source of amino acids to support cell proliferation [7,8]. For example, pancreatic ductal adenocarcinoma cells can grow indefinitely in a medium containing physiologically appropriate concentrations of albumin, which do not contain essential amino acids but are preferentially taken up by cancer cells compared to normal cells [9]. Hypoalbuminemia has also been identified as a common feature in patients with advanced solid tumors

[10] .

[0008] Some cancer cells can internalize HSA not only through nonspecific macropinocytosis mechanisms but also through receptor-mediated mechanisms. The preferential internalization of HSA in cancer cells is known to be primarily due to binding to proteins abundant in cell membrane microdomains, or caveolae, which are characterized by a unique lipid composition that allows specific molecules such as antibodies, complement factors, and blood coagulation factors—which cannot pass through by filtration or diffusion—to cross the membrane, either via caveolin, Cav-1, or other means.

[0009] Cav-1 is overexpressed in a wide variety of cancer types, including pancreatic cancer, prostate cancer, and breast cancer, and Cav-1 overexpression is associated with cancer progression [11,12].

[0010] Several approaches based on the use of HSAs have been described for targeted cancer therapy. For example, it has been proposed to load them in a non-covalent form by covalently binding HSAs to therapeutic agents or by utilizing the affinity of protein binding sites specific to small molecules or to albumin-binding domains (ABDs). When inserted into larger macromolecules such as nanobodies (NB, 15 kDa) or other proteins such as human TRAIL (30 kDa), the ABD confers binding affinity to the HSA, forming a highly stable complex [13,14]. The primary method of covalent binding to HSAs instead utilizes the presence of the amino acid cysteine ​​34, which has a reduced form and therefore electrophilically and selectively reactive thiol.

[0011] A more commonly used strategy involves conjugating the payload onto the lysine side chain. This strategy utilizes the formation of amide or imine bonds (or simple CN bonds after reductive amination) and has advantages with respect to cysteine ​​34, allowing for the binding of more payloads. However, the lack of selectivity of the reaction impairs binding to the FcRn receptor and can reduce the plasma half-life of the payload-HSA conjugate

[15] . Through this reaction, it is difficult to control the number and site specificity of changes, and therefore it should be considered that the resulting molecules are heterogeneous from a structural standpoint and difficult to characterize and frame from a regulatory standpoint.

[0012] Another widely used method for conjugating small molecules to HSAs involves drug encapsulation in recombinant or naturally extracted protein-based nanoparticles. Human serum albumin nanoparticles (NP-HSA) are known for this purpose. The advantage of using NP-HSA lies in its ability to conjugate and / or capture a very large payload, which is released over time as a function of conditions. Thus, the payload can be maintained in the bloodstream for a considerably longer period, protected from degradation, and more selectively unloaded to the treatment site of the target (e.g., in tumors), with reduced interaction with healthy tissue.

[0013] Methods for synthesizing albumin nanoparticles can generally be classified as desolvation, emulsification, thermal gelation, drying, and self-assembly techniques [2,16]. The size of NP-HSA is an important parameter for its biological function, as particles that are too large in diameter will not be able to penetrate the blood vessels of tumor tissue or interact with caveolae. However, the specificity of HSA to tumor tissue is considerably lower compared to molecules such as antibodies that recognize surface receptors, which have high selectivity and efficacy. Therefore, to confer further selectivity to cancer cells, albumin nanoparticles can be modified with various specific molecules (targeting agents or TAs) that recognize specific target receptors that are overexpressed or selectively expressed on the surface of cancer cells. NPs of mannosylated HSA have been used for this purpose to selectively target drug-resistant colon cancer cells and tumor-associated macrophages, resulting in the expression of high levels of mannose and SPARC receptors. In a similar approach, folic acid-modified bovine serum albumin nanoparticles were developed for targeted administration of paclitaxel.

[0014] To confer high recognition specificity, NP-HSA was modified with monoclonal antibodies against antigens overexpressed in cancer cells and tissues, such as monoclonal antibodies against αν integrin, which is highly expressed in various cancer cells.

[17]

[0015] Similarly, it can be hypothesized to use antibodies or antibody fragments that recognize one or more surface antigens selectively expressed on cancer cells. Some of these antigens, such as Her2, are effective targets for anticancer therapy with monoclonal antibodies [18,19]. In the case of Her2, monoclonal antibodies such as trastuzumab or patuzumab may be used, as they recognize different epitopes of the protein but can both provide therapeutic effects in patients with antigen-expressing breast cancer. Antibodies against Her2 are particularly useful for modifying NP-HSA because the receptor is rapidly taken up into cells and can deliver antibodies, antibody fragments, and antibodies conjugated with cytotoxic drugs (ADCs) into the cells for targeted therapy and more effective therapy. Antibodies against other markers selectively expressed on cancer cells may be used for the same purpose, even if the markers are not taken up into cells with the same efficiency. In this case, the antibody simply acts as a recognition signal, guiding NP-HSA near the cancer cell, which can then take up NP-HSA into the cell via caveolae. The presence of two different recognition signals enhances specificity, allowing particles to be taken up into cells via a single marker.

[0016] Further molecules targeted for these applications include the protein Cripto-1, which is known to be overexpressed on the surface of cancer cells in breast, colon, gastric, and other types of cancer

[20] . Monoclonal antibodies that highly selectively recognize human proteins on the cell surface have been widely reported in the literature

[20] .

[0017] ADCs are a new class of highly potent biological drugs constructed by conjugating small molecule anticancer drugs or other therapeutic agents to antibodies using permanent or unstable linkers. These antibodies target specific antigens primarily found on target cells.

[0018] Thanks to the antibody, the ADC selectively binds to the cancer cell receptor. The receptor-ADC complex is typically taken into the cell by endocytosis, and after antibody degradation, the linker is cleaved, releasing the cytotoxic drug that induces cell death via various mechanisms of action, such as DNA binding or interaction with tubulin. Release from the linker can occur both near the cytotoxic substance and near the antibody, while the linker remains fixed to the cytotoxic substance. The cytotoxic substance used in these approaches, usually a small molecule, must be very potent (≤1 nM), otherwise the concentration of the antibody or fragment carrying it becomes too high and wasteful. Therefore, it is essential that the cytotoxic substance is released in its original form to prevent the presence of the linker or linker fragments from altering its activity. Drug administration using NP-HSA has already shown considerable preclinical and clinical success. NP-HSA has been used to capture various drugs, such as sorafenib, 5-FU, and paclitaxel [21-23]. In fact, using NP-HSA, these are trapped in a mesh of nanoparticles, making it possible to increase the number of drug molecules that are actually loaded, which can then be released intact over time by simple diffusion.

[0019] In this context, the use of NP-HSA modified with antibodies or antibody fragments is particularly useful because NP-HSA acts as a container capable of transporting many copies of cytotoxic substances that are guided to cancer cells by antibodies and released without structural changes.

[0020] Therefore, the need for albumin nanoparticles capable of delivering cytotoxic drugs to cancer cells, and more specifically, albumin nanoparticles compared to known nanoparticles, is felt in this field. [Overview of the project] [Means for solving the problem]

[0021] The present invention relates to serum albumin nanoparticles (Alb-NP) modified with at least one biomolecule capable of recognizing a target receptor overexpressed or selectively expressed on the surface of cells, preferably cancer cells. The at least one biomolecule is immobilized on the nanoparticle surface by a linker and an amide group transfer (or transglutamination) reaction mediated by the enzyme transglutaminase.

[0022] The biomolecule is preferably selected from the group consisting of antibodies, Fabs, scFvs, nanobodies (NB) and mixtures thereof. Preferably, the nanoparticles are modified with at least two different biomolecules selected from the group consisting of antibodies, Fabs, scFvs, nanobodies (NB), peptides.

[0023] Preferably, the serum albumin used in the production of the nanoparticles is human serum albumin (HSA).

[0024] The at least one biomolecule is bound to the albumin nanoparticle via a linker to which the nanoparticle is derivatized. Derivatization of the nanoparticle with the linker occurs by formation of a covalent bond between the Z group of the linker containing an electrophilic functional group and the side chain of albumin cysteine containing a SH nucleophilic group (thiol).

[0025] Preferably, the covalent bond formed between the Z group of the linker and the thiol group of albumin cysteine is a -S- thioether bond.

[0026] The linker's terminal portion contains an X-NH2 group, which binds to a consensus sequence of a biomolecule containing at least one glutamine (Q;GLN). Specifically, the X-NH2 residue of the linker binds to the glutamine in the consensus sequence via an amide transfer (or transglutamation) reaction mediated by the enzyme transglutaminase (MTG). Alternatively, an -NH2 residue may be introduced to one end of at least one biomolecule, for example, in the form of a lysine residue inserted into a peptide sequence, and a consensus sequence containing at least one glutamine (Q;GLN) may be inserted at the end of the linker in place of the X-NH2 group. In this case, the consensus sequence containing at least one glutamine is part of the linker. The amide transfer reaction mediated by the enzyme transglutaminase occurs between the -NH2 residue present in at least one biomolecule and the glutamine in the consensus sequence that forms part of the linker.

[0027] Preferably, serum albumin nanoparticles modified according to the present invention are loaded with at least one cytotoxic drug, such as 5-FU, capecitabine, cytarabine, fludarabine, cladribine, paclitaxel, doxorubicin, daunorubicin, epirubicin, docetaxel, vinblastine, vincristine, vinorelbine, mercaptopurine, methotrexate, larcitrexed, etoposide, teniposide, camptothecin, irinotecan, topotecan, and combinations thereof. In other words, the nanoparticles incorporate at least one cytotoxic drug therein, and the surface of the particles is modified as described above.

[0028] The present invention also relates to the use of serum albumin nanoparticles modified according to the present invention and loaded with cytotoxic drugs, or to pharmaceutical compositions comprising nanoparticles for the treatment of disease conditions, preferably cancerous conditions. In particular, such disease conditions are selected from melanoma, breast cancer, metastatic breast cancer, glioma, glioblastoma, adenocarcinoma, intestinal cancer, pancreatic cancer, bone cancer, kidney cancer, colon cancer, gastric cancer, chronic lymphocytic leukemia, non-small cell lung cancer, advanced and / or metastatic kidney cancer, head and neck cancer, advanced melanoma, non-Hodgkin lymphoma, metastatic melanoma, lung cancer, chronic lymphocytic leukemia (CLL), non-Hodgkin lymphoma, and age-related macular degeneration.

[0029] The present invention also relates to the use thereof for the production of preferably human, serum albumin nanoparticles derivatized with the linker according to the present invention, and albumin nanoparticles modified according to the present invention.

[0030] The present invention also relates to a method for synthesizing modified nanoparticles, comprising the steps of functionalizing nanoparticles with the linker of the present invention by forming a thioether bond, and attaching the linker to at least one biomolecule by an amide group transfer or transglutamination reaction mediated by the enzyme transglutaminase. [Brief explanation of the drawing]

[0031] [Figure 1] The dose-response curves for competitive binding to HER2 obtained with trastuzumab Fab and with the same Fab bound to human albumin particles are shown (Example 10). [Figure 2A-B] The dose-response curves for trastuzumab Fab and the same Fab bound to human albumin particles, obtained at 2 hours (A) and 24 hours (B), are shown (Example 11). [Figure 3A] The dose-response curves for trastuzumab Fab and the same Fab bound to human albumin particles, obtained at 2 hours, are shown for binding to Her2 receptor-expressing BT474 cells and non-receptor-expressing MDA-MB-231 cells (Example 12). [Figure 3B-C]The dose-response curves for trastuzumab Fab (B) and trastuzumab (C) obtained by incubation for 2 hours are shown for binding to Her2 receptor-expressing BT474 cells and non-receptor-expressing MDA-MB-231 cells (Example 12). [Figure 4] The dose-response curves for binding to Cripto-1-expressing NTERA cells at 2 and 24 hours are shown for recombinant Fab of anti-Cripto-1 antibody 10D1 and the same Fab conjugated to human albumin particles (Example 13). [Figure 5A-B] The binding of anti-Cripto-1 antibody 1B4 and anti-Her2 trastuzumab to Her2-negative and Cripto-1-positive MDA-MB-231 cells (A) and Her2-positive and Cripto-1-positive BT474 cells (B) is shown (Example 14). [Figure 5C] The dose-response curves for the binding of bispecific NP-HSA functionalized with 10F1Fab and trastuzumab Fab to Her2-positive and Cripto-1-positive BT474 cells, and Her2-negative and Cripto-1-positive MDA-MB-231 cells are shown (Example 14). [Figure 6A-D]Binding of bispecific P-HSA functionalized with trastuzumab Fab and 10D1Fab to Her2-positive and Cripto-1-positive BT474 cells, and Her2-negative and Cripto-1-positive MDA-MB-231 cells (A); Binding of a combination of NP-HSA functionalized with trastuzumab Fab and NP-HSA functionalized with 10D1Fab to Her2-positive and Cripto-1-positive BT474 cells, and Her2-negative and Cripto-1-positive MDA-MB-231 cells (A); Binding of bispecific P-HSA functionalized with trastuzumab Fab to Her2-positive and Cripto-1-positive BT474 cells, and Her2-negative and Cripto-1-positive MDA-MB-231 cells (A); Binding of a combination of NP-HSA functionalized with trastuzumab Fab and NP-HSA functionalized with 10D1 (B) Binding to A-MB-231 cells; binding of NP-HSA functionalized with 10D1Fab to Her2-positive and Cripto-1-positive BT474 cells, and Her2-negative and Cripto-1-positive MDA-MB-231 cells (C); binding of NP-HSA functionalized with trastuzumab Fab to Her2-positive and Cripto-1-positive BT474 cells, and Her2-negative and Cripto-1-positive MDA-MB-231 cells (D) (Example 15). [Figure 7A-H] A-C show the binding of FITC-NP-HSA to Cripto-1 expressing NTERA2 cells at concentrations of 10, 100, and 1000 ng / mL, respectively; D shows the binding of FITC-unfunctionalized NP-HSA to the same cells at 1000 ng / mL; E-G show the binding of FITC-NP-HSA-Fab to Cripto-1 expressing NTERA2 cells at concentrations of 10, 100, and 1000 ng / mL; H shows the binding of FITC-unfunctionalized NP-HSA-Fab to the same cells. [Modes for carrying out the invention]

[0032] All amino acids shown herein are preferably in the L configuration.

[0033] In the first embodiment, the present invention is A linker bonded to the nanoparticles by a -S-thioether bond, and • At least one type of biomolecule, This relates to serum albumin nanoparticles (Alb-NPs) modified with at least one modification chain, including [specific component].

[0034] At least one of those biomolecules, (i) Between the -NH2 residue of the X group of the linker and the CO-NH2 residue of glutamine contained in the peptide consensus sequence inserted into at least one biomolecule; or (ii) Between a -NH2 residue of lysine contained in a peptide sequence inserted into at least one biomolecule and a -CO-NH2 residue of glutamine inserted into a consensus sequence which is part of the linker; the linker is formed via an amide bond created by an amide transfer (or transglutamation) reaction mediated by the enzyme transglutaminase.

[0035] In case (i), the linker is given by equation (IV):

[0036] [ka] Derived from the precursor, In the formula, the -NH2 residue of the X group forms an amide bond with the CO-NH2 residue of the consensus sequence of glutamine (Q;GLN), which is contained in at least one biomolecule, and the consensus sequence is given by the following formula (VI): Formula AA1-AA2-Q-AA3-AA4 (VI) It has, In the formula, AA1 is either leucine (L; Leu) or absent; AA2 is either leucine (L; Leu) or threonine (T; Thr); Q is glutamine having the formula -CO-(CH2)2-CH-(NH)-CO-; AA3 is either serine (S; Ser) or glycine (G; Gly); AA4 is proline (P; Pro), alanine (A; Ala), or absent. Preferably, AA1 and AA4 are not absent simultaneously.

[0037] The consensus sequence is preferably selected from LQSP, TQGA, and LLQG.

[0038] In case (ii), the linker is given by the following equation (VII):

[0039] [ka] It is derived from a precursor containing the consensus sequence of formula (VI), which is linked to a spacer by amino acid AA1 or AA2.

[0040] In both cases, the Z group is bonded to the surface of the nanoparticle via an -S-thioether bond.

[0041] The serum albumin nanoparticles modified according to the present invention are of formula (I) or (II):

[0042] [ka] JPEG0007867490000004.jpg85153 It holds.

[0043] In equation (I), the following fragments can be identified:

[0044] [ka] In equation (II), the following fragments can be identified:

[0045] [ka] The amide group transfer reaction mediated by the enzyme transglutaminase is illustrated below (Diagram I):

[0046] [ka] With respect to formulas (I) and (II), the serum albumin nanoparticles represented by the symbol Alb-NP are bovine serum albumin nanoparticles (NP-BSA) or human serum albumin nanoparticles (NP-HSA). Preferably, they are NP-HSA.

[0047] Albumin nanoparticles have a -SH thiol group of a side chain of albumincysteine ​​34, which is attached to the Z group of the linker by a thioether bond (-S-) to a cysteine ​​residue.

[0048] The precursor of albumin nanoparticles (Alb-NPs) is given by the following formula (III):

[0049] [ka] It is represented by [this].

[0050] The symbol ... in formula (I) represents the presence of multiple modification chains attached to the nanoparticle by thioether bonds. In formula (I), only one modification chain of the nanoparticle is fully shown for the sake of simplicity in the depiction.

[0051] The nanoparticles have an average diameter (or Z-average size) of approximately 100-500 nm, preferably 100-400 nm or 300-400 nm, as measured by dynamic light scattering (DLS) technology.

[0052] Nanoparticles have a polydispersity index (PDI) that falls within the range of 0.02 to 0.05.

[0053] Albumin nanoparticles are preferably loaded with at least one cytotoxic drug, such as 5-FU, capecitabine, cytarabine, fludarabine, cladribine, paclitaxel, doxorubicin, daunorubicin, epirubicin, docetaxel, vinblastine, vincristine, vinorelbine, mercaptopurine, methotrexate, larcitrexed, etoposide, teniposide, camptothecin, irinotecan, topotecan, and combinations thereof.

[0054] The term "loaded" means that the cytotoxic drug is incorporated into the nanoparticle during its manufacture and before subsequent modification in the linker and biomolecules.

[0055] Albumin nanoparticles are produced by methods known in the art, for example, by desolvation and subsequent stabilization with a crosslinking agent, which may be glutaraldehyde or diazirine. Another method for producing albumin nanoparticles is high-pressure homogenization.

[0056] Other known manufacturing methods include emulsification, thermal gelation, drying, and self-assembly.

[0057] When a cytotoxic drug is loaded onto nanoparticles, the latter is dissolved or suspended in a starting albumin solution and then incorporated into the nanoparticles while they are being formed by the method described above.

[0058] The linker used to modify the nanoparticles of the present invention shown in formula (I) comprises a Z group, a spacer, and an X-NH- group. The linker is of formula (IV):

[0059] [ka] Derived from the precursor, In the formula, the Z group is derived from a functional group containing an electrophilic group that can react with the -SH group of albumin cysteine, preferably cysteine ​​34, and can form a thioether bond in the pH range of 4 to 9.

[0060] Preferably, the Z group can be introduced into the linker starting from the derivatives shown in Table 1 below:

[0061] [Table 1]

[0062] Preferably, Z is derived from a functional group selected from 1-bromoacetic acid, 1-chloroacetic acid, and 6-maleimidohexanoic acid.

[0063] The spacer is an inert molecule under physiological and extreme pH conditions (e.g., pH < 3 or pH > 9) that acts as a spacer between the X-NH2 group and the subsequent Z group. The spacer is a flexible molecule that can reach a size of at least 10 angstroms in its expanded configuration.

[0064] Spacers are preferably: ·-NH-(CH2-O) n -CH2-CO-(n is in the range of 2 to 10, preferably 2, 3, 4, and 5); ·-NH-(CH2-CH2-O) n -CH2-CO- (where n is in the range of 2 to 10, preferably 2, 3, 4, and 5) is selected. Or the spacer is Y m It is the basis, Y is: ·-NH-(CH2) n -CO-amino acids (where n is in the range of 3 to 10, preferably in the range of 3 to 5, and preferably n=3, 4, or 5), i.e., aminocaproic acid ε, aminopentanoic acid δ, and aminobutyric acid γ, respectively; Glycine, alanine; • Selected from and combinations thereof, m is a number that falls within the range of 1 to 5.

[0065] Therefore, Y m This indicates a polypeptide consisting of a single amino acid or a specified amino acid, and having dimensions that fall between a dipeptide and a pentapeptide.

[0066] The -CO- group of the spacer forms an amide bond with the -NH- group of the subsequent X unit, and the -NH- group of the spacer forms an amide bond with the acid group of the Z precursor.

[0067] Preferably, the spacer is -NH-(CH2-CH2-O) n -CH2-CO- (where n ranges from 2 to 5), or it is glycine.

[0068] The X-NH2 group of formula (I) contains a primary amine group bonded to an X group containing an alkyl or methoxyalkyl chain, or another non-reactive flexible chain, under normal physiological conditions or under extreme pH conditions (e.g., pH < 3 or pH > 9). Preferably, X is an alkyl chain containing at least 3 methylene groups, or a chain containing at least 2 methoxyethylene groups.

[0069] Preferably, the -X-NH2- group is: · -NH-(CH2) n -NH2 (where n ranges from 3 to 10, preferably 3, 4, and 5); · -NH-(O-CH2) n -NH2 (where n ranges from 2 to 10, preferably 2, 3, 4, and 5); · -NH-(O-CH2-CH2) n -NH2 (where n ranges from 2 to 10, preferably 2, 3, 4, and 5); · L-lysine or D-lysine amino acid; · L-ornithine or D-ornithine amino acid; · C-terminal amidated L-lysine or D-lysine amino acid; · C-terminal amidated L-ornithine or D-ornithine amino acid; can be selected from.

[0070] The X-NH2 group is linked to the spacer group via an amide bond formed between the amine group of X and the carboxyl group coming from the spacer.

[0071] When the X-NH2 group is an amino acid selected from L-lysine, L-ornithine, L-lysine amide, L-ornithine, D-lysine, D-ornithine, D-lysine amide, and D-ornithine amide, the amine group forming the amide bond is the α-amine group.

[0072] Preferably, the X-NH2 group is selected from L-lysine amino acids and C-terminally amidated L-lysine amino acids.

[0073] In one embodiment, the Z group is selected from 1-bromoacetic acid, 1-chloroacetic acid, and 6-maleimidohexanoic acid; the spacer is -NH-(CH2-CH2-O) n The group is -CH2-CO- (where n is in the range of 2 to 5), or it is glycine; the X-NH2 group is an L-lysine amino acid or a C-terminally amidated L-lysine amino acid.

[0074] In one embodiment, albumin nanoparticles, preferably NP-HSA, have a Z group selected from 1-bromoacetic acid, 1-chloroacetic acid, and 6-maleimidohexanoic acid; a spacer -NH-(CH2-CH2-O) n The derivatives are derivatized with a linker containing -CH2-CO- (where n is in the range of 2 to 5), or glycine; an X-NH2 group selected from L-lysine amino acids, D-lysine amino acids, C-terminally amidated L-lysine amino acids, and C-terminally amidated D-lysine amino acids; and preferably, the derivatized nanoparticles are loaded with at least one cytotoxic drug selected from 5-FU, sorafenib, paclitaxel, doxorubicin, and combinations thereof.

[0075] An example of a linker precursor according to formula (I) is formula (V):

[0076] [ka] This is Br-CH2-CO-O2Oc-K-NH2 produced according to Example 8.

[0077] Formula (V) is expressed as follows, by denoting various residues according to formula (I):

[0078] [ka] It is shown as follows.

[0079] Another example of a linker precursor is given by formula (VI):

[0080] [ka] This is Mal-Gly-Lys-CONH2 manufactured according to Example 9.

[0081] Formula (VI) is expressed as follows, by denoting various residues according to formula (I):

[0082] [ka] It is shown as follows.

[0083] In the case of equation (II), the linker is equation (VII):

[0084] [ka] Derived from the precursor, In the formula, Z and the spacer are defined as above with respect to formula (IV), and the X-NH2 group is defined in formula (VIII): AA1-AA2-Q-AA3-AA4 type (VIII) Substituted by a consensus peptide sequence containing at least one glutamine (Q;GLN), During the ceremony, AA1 is either leucine (L; Leu) or absent; AA2 is either leucine (L; Leu) or threonine (T; Thr); Q is glutamine; AA3 is either serine (S; Ser) or glycine (G; Gly); AA4 is either proline (P; Pro), alanine (A; Ala), or they do not exist simultaneously. Preferably, AA1 and AA4 are not absent at the same time.

[0085] The consensus sequence is preferably selected from LQSP, TQGA, and LLQG.

[0086] The consensus sequence is attached to the spacer by an amide bond between the -CO- terminus of the spacer and the -NH- group of amino acid AA1 (if present) or AA2.

[0087] The reaction between albumin cysteine ​​present on the nanoparticle surface and the linker occurs in a buffered aqueous solution within the range of 4-9, preferably with a pH of 8 or higher. The bonding reaction generally completes at room temperature (25°C) within a time interval not exceeding 16 hours.

[0088] The number of linker molecules that can be bound to nanoparticles varies depending on the number of thiol groups exposed on their surface. For example, approximate calculations based on NP-HSA with an average diameter of 100 nm and HSA molecules with similar diameters contained within 7-10 nm can be used to estimate the amount of reactive thiol groups contained in 2-3 nmol per mg of NP-HSA produced by the method described in the literature.

[0089] Albumin nanoparticles can be treated with Traut's reagent, 2-iminothiolane, to increase the number of free thiols on the nanoparticles and therefore increase the density of linkers that can be introduced onto their surface.

[0090] The linker according to the present invention contains a consensus sequence containing a Z group, a spacer and an X group, or glutamine linked by an amide bond. The advantages of having a linker with amide bonds distributed throughout the chain are its ability to impart solubility to the resulting modified nanoparticles, the ease of linker synthesis by both chemical synthesis methods and biological methods, such as enzymes, and its high stability even under extreme chemical-physical conditions (e.g., pH ranges from 1 to 9).

[0091] Derivatization of nanoparticles with a linker makes it possible to obtain nanoparticles functionalized with an amine group derived from the linker, which can react with at least one biomolecule containing a consensus sequence containing glutamine (linker of formula (IV)) or a peptide sequence containing lysine (linker of formula (VII)). The reaction between the amine group of the linker in the case of formula (IV), or the amine group of the peptide sequence contained in the biomolecule (in the case of linker of formula (VII)), and the glutamine in the consensus sequence is an amide transfer (or transglutamination) reaction mediated by the enzyme transglutaminase. Preferably, the transglutaminase is a bacterial transglutaminase called MTG.

[0092] This reaction makes it possible to covalently bond the polypeptide chain of a biomolecule to the surface of albumin nanoparticles that hold amine groups introduced by derivatization with the linker of the present invention, via isopeptide amide bonds, using a biomolecule orientation site-specific method.

[0093] Since the glutamine and lysine residues of the albumin molecule are not reactive to the enzyme transglutaminase, it is important to note that albumin nanoparticles, while possessing multiple lysine and glutamine, as well as amine groups derived from the N-terminus of the albumin molecule, do not undergo transglutaminase-mediated transglutamation reactions. Some biomolecules are known to contain transglutaminase-reactive glutamine residues. Such molecules can be used similarly in these applications.

[0094] In the case of albumin nanoparticles of formula (I) and linkers of formula (IV), the consensus sequence contained in the biomolecule is a peptide sequence containing at least one glutamine (Q;GLN) of formula (VIII) as defined above.

[0095] In the case of albumin nanoparticles of formula (II), the peptide sequence containing lysine (K;Lys) is as follows (IX): (AA)w -K-(AA) p Formula (IX) It has, In the formula, w and p are integers between 0 and 8, preferably between 1 and 5, and w and p are never equal to 0 at the same time; AA represents an amino acid selected from alanine (A; Ala), tyrosine (Y; Tyr), phenylalanine (F; Phe), glycine (G; Gly), tryptophan (W; Trp), and serine (S; Ser); K stands for lysine (Lys).

[0096] Preferably, w is equal to 0 and p is equal to 1 to 3; more preferably, w is equal to 0 and p is equal to 3.

[0097] The peptide sequence is preferably: Select from KAYA, KGYA, KSYA, KAFA, KGFA, KSFA, KAWA, KGWA, KSWA, KAYG, KGYG, KSYG, KAFG, KGFG, KSFG, KAWG, KGWG, KSWG, KAYS, KGYS, KSYS, KAFS, KGFS, KSFS, KAWS, KGWS, KSWS.

[0098] In one embodiment, albumin nanoparticles, preferably NP-HSA, have a Z group selected from 1-bromoacetic acid, 1-chloroacetic acid, and 6-maleimidohexanoic acid; a spacer -NH-(O-CH2-CH2) n Derivatized with a linker containing -CO- (where n is in the range of 3 to 5) or glycine; an X-NH2 group selected from L-lysine amino acids or C-terminally amidated L-lysine amino acids; the terminal X-NH2 group of the linker is linked by an amide bond to glutamine in a consensus sequence selected from LQSP, TQGA, or LLQG found in biomolecules.

[0099] Preferably, such nanoparticles are loaded with at least one cytotoxic drug selected from 5-FU, capecitabine, cytarabine, fludarabine, cladribine, paclitaxel, doxorubicin, daunorubicin, epirubicin, docetaxel, vinblastine, vincristine, vinorelbine, mercaptopurine, methotrexate, larcitrexed, etoposide, teniposide, camptothecin, irinotecan, topotecan, and combinations thereof.

[0100] The R1 and R2 groups in formula (I) represent biomolecules selected from antibodies, Fabs, scFvs, nanobodies (NBs), and mixtures thereof.

[0101] R1 and R2 may be the same or different from each other.

[0102] The antibody is preferably a monoclonal antibody, preferably selected from trastuzumab or partuzumab, a monoclonal antibody against HER2, a surface protein selectively expressed by breast cancer cells; trastuzumab and partuzumab that recognize different epitopes of the protein HER2; cetuximab, an anti-EGFR antibody; an anti-Cripto-1 monoclonal antibody, such as anti-Cripto-1 antibody 1B4 or anti-Cripto-1 antibody 10D1, which have been recently reported in the literature

[24] , or other antibodies against antigens selectively expressed on the surface of cancer cells.

[0103] The Fab is preferably a recombinant Fab produced from a monoclonal antibody that binds to a receptor for a biological target, such as an antibody against a cancer antigen selectively expressed on the surface of cancer cells, for example, a recombinant Fab of trastuzumab (prepared as described in Selis et al.

[25] ) or patuzumab, or another Fab obtained by introducing mutations into the polypeptide sequences of known monoclonal antibodies such as anti-Cripto-1 antibodies 1B4 and 10D1. Such a Fab may be prepared recombinantly as described in Selis et al.

[25] to obtain a consensus sequence at the C-terminus of the heavy chain, such as a TQGA sequence, and may be enzymatically conjugated to a linker present on the surface of albumin nanoparticles.

[0104] ScFv is a functional fragment of an antibody selected from trastuzumab, patuzumab, cetuximab, anti-Cripto-1 monoclonal antibodies, such as anti-Cripto-1 antibody 1B4 or anti-Cripto-1 antibody 10D1, or antibodies against cancer antigens selectively expressed on the surface of cancer cells.

[0105] The nanobody (NB) is selected from NBs targeting VEGFR2, such as 3VGR19 NB

[26] , Her2, such as 5F7GGC NB

[27] , or EGFR, such as EGa1

[27] .

[0106] With respect to formula (I), methods for introducing a consensus sequence into a biomolecule are known in the art, for example, from

[25] .

[0107] The consensus sequence is introduced into the biomolecule at a location far from the active region of the molecule itself.

[0108] The advantage of using a consensus sequence introduced into a biomolecule at a position far from the active region of the molecule itself lies in its ability to bind the biomolecule to the linker of formula (IV) present in the nanoparticles of the present invention, so that the active region (e.g., complementarity-determining region (CDRS)) is positioned outward from the nanoparticle. Outward orientation of the active region of the biomolecule promotes the recognition and interaction with receptors present on target cells, such as those present on cancer cells.

[0109] Similarly, in the case of formula (II), a method for introducing a lysine-containing peptide sequence onto a biomolecule is known in the art.

[0110] Peptide sequences are introduced into biomolecules at a location far from the active region of the molecule itself.

[0111] The advantage of using peptide sequences introduced into biomolecules at a position far from the active region of the molecule itself lies in their ability to bind biomolecules to the linker of formula (VII) present in the nanoparticles of the present invention, so that their active regions (e.g., complementarity-determining regions (CDRS)) are all positioned to face outward from the nanoparticle. The outward orientation of the active region of the biomolecule promotes the recognition and interaction with receptors present on target cells, such as those present on cancer cells.

[0112] Using the method for immobilizing biomolecules on a linker described in the present invention, it is possible to immobilize 2 to 80 μg, preferably 5 to 50 μg, of biomolecules per mg of albumin nanoparticles.

[0113] For example, if the biomolecule is Fab with a molecular weight of approximately 50 kDa, it is possible to fix approximately 200 pmol of biomolecule per mg of nanoparticles; if it is scFv with a molecular weight of approximately 25 kDa, the density will be 400 pmol / mg nanoparticles; and if it is NB with a molecular weight of approximately 12.5 kDa, the density will be 800 pmol / mg nanoparticles.

[0114] The biomolecular density per unit weight of nanoparticles is 6-40%, preferably 0.2-8%, for Fab, 0.4-16% for scFvs, and 1-33% for NB.

[0115] In one embodiment, albumin nanoparticles, preferably NP-HSA, have a Z group selected from 1-bromoacetic acid, 1-chloroacetic acid, and 6-maleimidohexanoic acid; a spacer -NH-(CH2-CH2-O) n Derivatized with a linker containing -CH2-CO- (where n is in the range of 3 to 5) or glycine; an X-NH2 group selected from L-lysine amino acids and C-terminally amidated L-lysine amino acids; the terminal group X-NH2 is amide-bonded to glutamine in a consensus sequence selected from LQSP, TQGA, and LLQG contained in the biomolecule, the biomolecule being selected from antibodies, Fabs, scFvs, nanobodies (NBs) and mixtures thereof.

[0116] Preferably, a cytotoxic drug selected from 5-FU, capecitabine, cytarabine, fludarabine, cladribine, paclitaxel, doxorubicin, daunorubicin, epirubicin, docetaxel, vinblastine, vincristine, vinorelbine, mercaptopurine, methotrexate, larcitrexed, etoposide, teniposide, camptothecin, irinotecan, topotecan, and combinations thereof is loaded onto the nanoparticles.

[0117] In a particularly preferred embodiment, the R1 group and the R2 group are different from each other.

[0118] For example, R1 and R2 could be two different types of Fab, called Fab1 and Fab2; two different types of scFv, called scFv1 and scFv2; two different types of NB, called NB1 and NB2; or two different types of antibodies or two different peptides, called Ab1 and Ab2. Alternatively, R1 and R2 could be a hybrid combination of biomolecules, such as Fab1 and scFv1, Fab1 and NB2, or scFv1 and NB2, or Ab1 and NB1.

[0119] The combination of biomolecules is selected from the biomolecules listed above.

[0120] Preferably, R1 is recombinant anti-Cripto-1 Fab 10D1 and R2 is anti-HER2 Fab, or R1 is trastuzumab and R2 is cetuximab; R1 is trastuzumab and R2 is rituximab; R1 is trastuzumab and R2 is ipilimumab; R1 is trastuzumab and R2 is alemtuzumab; R1 is trastuzumab and R2 is nivolumab; R1 is trastuzumab and R2 is pembrolizumab; R1 is trastuzumab and R2 is panitumumab; R1 is trastuzumab and R2 is ibritumomab tiuxetan (tiuxetan); R1 is trastuzumab and R2 is tocitumomab; R1 is trastuzumab and R2 is bevacizumab; R1 is trastuzumab and R2 is ofatumumab; R1 is recombinant anti-Cripto-1 Fab 10D1 and R2 is cetuximab; R1 is recombinant anti-Cripto-1 Fab 10D1 and R2 is rituximab; R1 is recombinant anti-Cripto-1 Fab 10D1 and R2 is ipilimumab; R1 is recombinant anti-Cripto-1 Fab 10D1 and R2 is alemtuzumab; R1 is recombinant anti-Cripto-1 Fab 10D1 and R2 is nivolumab; R1 is recombinant anti-Cripto-1 Fab R1 is 10D1 and R2 is pembrolizumab; R1 is recombinant anti-Cripto-1 Fab 10D1 and R2 is panitumumab; R1 is recombinant anti-Cripto-1 Fab 10D1 and R2 is ibritumomab tiuxetan; R1 is recombinant anti-Cripto-1 Fab 10D1 and R2 is tositumomab; R1 is recombinant anti-Cripto-1 Fab 10D1 and R2 is bevacizumab; R1 is recombinant anti-Cripto-1 Fab 10D1 and R2 is ofatumumab.

[0121] Modification of nanoparticles with at least two types of biomolecules is carried out using equimolar solutions of the molecules.

[0122] For example, nanoparticles can be produced that retain at least 10 μg of the Fab1 and Fab2 mixture on their surface.

[0123] This aspect of the present invention is particularly relevant because the nanoparticles obtained in this way can recognize at least two different receptors on the surface of cancer cells that overexpress them, thereby increasing the delivery specificity of anticancer drugs.

[0124] In one embodiment, albumin nanoparticles, preferably NP-HSA, have a Z group selected from 1-bromoacetic acid, 1-chloroacetic acid, and 6-maleimidohexanoic acid; a spacer -NH-(CH2-CH2-O) n The molecule is derivatized with a linker containing -CH2-CO- (where n is in the range of 3 to 5) or glycine; an X-NH2 group selected from L-lysine amino acids and C-terminally amidated L-lysine amino acids; the terminal group X-NH2 is amide-bonded to glutamine in a consensus sequence selected from LQSP, TQGA, and LLQG contained in the biomolecule. The biomolecule is represented by R1 and R2 in formula (I), where R1 and R2 are the same or different from each other.

[0125] Preferably, a cytotoxic drug selected from 5-FU, capecitabine, cytarabine, fludarabine, cladribine, paclitaxel, doxorubicin, daunorubicin, epirubicin, docetaxel, vinblastine, vincristine, vinorelbine, mercaptopurine, methotrexate, larcitrexed, etoposide, teniposide, camptothecin, irinotecan, topotecan, and combinations thereof is loaded onto the nanoparticles.

[0126] In a preferred embodiment, the nanoparticles are NP-HSAs derivatized with a linker of formula (IV) or formula (VII) and conjugated to recombinant anti-HER2 Fab and / or recombinant anti-Cripto Fab 10D1 by a consensus sequence preferably containing at least one glutamine of formula (VIII).

[0127] In one embodiment of the present invention, albumin nanoparticles loaded with or unloaded with a cytotoxic drug can be derivatized with a fluorescent dye that binds to the lysine side chain of the albumin molecule, subsequently treated with the linker of the present invention, and then subjected to a bioconjugation reaction with transglutaminase to immobilize a biomolecule via a consensus sequence. A suitable fluorescent dye for this purpose is, for example, fluorescein isothiocyanate (FITC), which can react with the primary amine group of the lysine of the albumin molecule in the absence of chemical reagents that are aggressive or harmful to the albumin molecule.

[0128] Preferably, the modified albumin nanoparticles according to the present invention have the following formula:

[0129] [ka] (Its manufacture is described in Example 4);

[0130] [ka] (Its manufacture is described in Example 5);

[0131] [ka] (Its manufacture is described in Example 6);

[0132] [ka] (Its manufacture is described in Example 7);

[0133] [ka] (Its synthesis is described in Example 16) Selected from.

[0134] In a further embodiment, the present invention relates to a pharmaceutical composition comprising albumin nanoparticles, preferably NP-HSA, modified according to the present invention and loaded with at least one cytotoxic drug, and a pharmaceutically acceptable excipient.

[0135] In a further aspect, the present invention relates to albumin nanoparticles modified according to the present invention and loaded with cytotoxic drugs, or to pharmaceutical compositions containing them for use in the treatment of cancerous conditions preferably selected from melanoma, breast cancer, metastatic breast cancer, glioma, glioblastoma, adenocarcinoma, intestinal cancer, pancreatic cancer, bone cancer, kidney cancer, colon cancer, gastric cancer, chronic lymphocytic leukemia, non-small cell lung cancer, advanced and / or metastatic kidney cancer, head and neck cancer, advanced melanoma, non-Hodgkin lymphoma, metastatic melanoma, lung cancer, chronic lymphocytic leukemia (CLL), non-Hodgkin lymphoma, and age-related macular degeneration.

[0136] Preferably, the albumin nanoparticles are NP-HSA and are modified with at least two different types of biomolecules. That is, R1 and R2 in formula (I) are different from each other.

[0137] The present invention also relates to a method for treating cancerous conditions preferably selected from melanoma, breast cancer, metastatic breast cancer, glioma, glioblastoma, adenocarcinoma, intestinal cancer, pancreatic cancer, bone cancer, kidney cancer, colon cancer, gastric cancer, chronic lymphocytic leukemia, non-small cell lung cancer, advanced and / or metastatic kidney cancer, head and neck cancer, advanced melanoma, non-Hodgkin lymphoma, metastatic melanoma, lung cancer, chronic lymphocytic leukemia (CLL), non-Hodgkin lymphoma, and age-related macular degeneration, comprising administering an effective amount of nanoparticles modified according to the present invention and loaded with a cytotoxic drug, or a composition containing them, to a patient in need.

[0138] Preferably, the albumin nanoparticles are NP-HSA and are modified with at least two different types of biomolecules. That is, R1 and R2 in formula (I) are different from each other. [Examples]

[0139] Example 1 Production of NP-HSA and NP-HSA-5FU by desolvation and stabilization using glutaraldehyde.

[0140] For the production of HSA nanoparticles, a desolvation method [29,30] was used as described below: 100 mg of human albumin (HSA fatty acid-free, Sigma, MO, USA) was solubilized in 2 mL of deionized water, and 0.1 M NaOH was added to adjust the pH to 8.6. Under constant stirring, 8 mL of 99.8% ethanol (Sigma, MO, USA) was added dropwise to the solution at a flow rate of 1 mL / min. For the production of 5-FU-loaded nanoparticles, 100 mg of HSA and 4 mg of 5FU (Sigma Aldrich, MO, USA) were solubilized in 2.2 mL of purified water, and the pH was adjusted to 8.6. The solution was maintained in incubation for 2 hours under constant stirring before proceeding with desolvation with ethanol. After the desolvation process, the NP-HSA was stabilized by adding 38 μL of 25% glutaraldehyde, grade II (MO, Sigma) in H2O under constant electromagnetic stirring, and left to stand with stirring for 24 hours. Next, NP-HSA was purified by three washing cycles with H2O by centrifugation to remove ethanol and glutaraldehyde. Finally, it was redispersed in PBS, stored at +4°C, and characterized by size (diameter nm), polydispersity (polydispersity index, PDI), and zeta potential (in mV) using a Malvern Zetasizer Ultra dynamic light scattering (DLS) system (Malvern Instruments, Worcestershire, UK). For size measurement and PDI determination, nanoparticles were diluted in PBS to a concentration of 0.1 mg / mL NP-HSA and analyzed by multi-angle scattering at +25°C using a micro-cuvette (UV-Cuvette, Brand®, Wertheim, Germany). Zeta potentials for determining surface loading were measured by diluting NP-HSA in ultrapure water to a concentration of 0.1 mg / mL NP and analyzing it at +25°C using a disposable capillary cell DTS1070 (Malvern Instruments, Worcestershire, UK). All size and zeta potential measurements were performed triplicately and reported as mean ± standard deviation.

[0141] NP-HSA was analyzed by DLS and found to have an average diameter of 130 nm, along with a polydispersity index (PDI) of 0.04.

[0142] Example 2 Production of NP-HSA by desolvation and stabilization using diazirine.

[0143] For the preparation of NP-HSA, a desolvation method [29,30] was used as described below: 100 mg of HSA fatty acid-free albumin (Sigma, MO, USA) was solubilized in 2 mL of purified water, and 0.1 M NaOH was added to adjust the pH to 8.6. Under constant stirring, 8 mL of 99.8% ethanol (Sigma, MO, USA) was added dropwise to the solution at a flow rate of 1 mL / min. After the desolvation process, the nanoparticles were stabilized under constant electromagnetic stirring by adding the photocrosslinking agent: NHS-diaziline (SDA) (Sigma, MO, USA). In particular, 1.68 mg of NHS-diaziline was solubilized in 0.5 mL of anhydrous DMSO and added to the suspension so that the molar ratio of HSA / NHS-diaziline reached 1:5, and the suspension was incubated for 3 hours. Next, the suspension was dialyzed using an MWCO 6000Da cellulose membrane, and finally, after removing excess NHS-diaziline, the suspension was exposed to ultraviolet light (360 nm) for 10 minutes under constant stirring to photostabilize NP-HSA. Then, NP-HSA was purified by three washing cycles with H2O by centrifugation, and finally redispersed in PBS, stored at +4°C, and characterized by size (nm), PDI, and zeta potential (mV) using a Malvern Zetasizer Ultra (Malvern Instruments, Worcestershire, UK). For size measurement and PDI determination, NP-HSA was diluted in PBS to a concentration of 0.1 mg / mL NP and analyzed by multi-angle scattering at +25°C using a microcuvette (UV-Cuvette, Brand®, Wertheim, Germany). To determine the zeta potential for surface loading, NP-HSA was diluted in ultrapure water to a concentration of 0.1 mg / mL NP and analyzed at +25°C using a disposable capillary cell DTS1070 (Malvern Instruments, Worcestershire, UK).

[0144] NP-HSA was analyzed by DLS and found to have an average diameter of 340 nm, along with a PDI of 0.04.

[0145] Example 3 Manufacturing of NP-HSA by high-pressure homogenization

[0146] For the production of NP-HSA by high-pressure homogenization, 100 mg of HSA fatty acid-free albumin (Sigma, MO, USA) was solubilized in purified water to a concentration of 30 mg / mL

[31] . 3% (v / v) chloroform was added to the solution obtained in this way, and the initial homogenization was carried out for 5 minutes using ULTRA-TURRAX® T-25 (IKA, Germany). Then, under high pressure, the initial emulsion was homogenized using Emulsiflex EF-B15 (Avestin Inc., Ottawa, Canada) with a pressure of 20,000 psi and a homogenization cycle number equal to 12. The resulting colloidal dispersion was subjected to a rotary evaporator at a vacuum pressure of 400 mmHg for 30 minutes at +40°C to remove chloroform. Next, NP-HSA was characterized by size (nm), PDI, and zeta potential (mV) using a Malvern Zetasizer Ultra (Malvern Instruments, Worcestershire, UK). For size and PDI determination, NP-HSA was diluted in PBS to a concentration of 0.1 mg / mL NP and analyzed by multi-angle scattering at +25°C using a micro-cuvette (UV-Cuvette, Brand®, Wertheim, Germany). For zeta potential to determine surface loading, NP-HSA was diluted in ultrapure water to a concentration of 0.1 mg / mL NP and analyzed at +25°C using a disposable capillary cell DTS1070 (Malvern Instruments, Worcestershire, UK).

[0147] NP-HSA was analyzed by DLS and found to have an average diameter of 340 nm, along with a PDI of 0.04.

[0148] Example 4 Preparation of NP-HSA, which is derivatized with HSA cysteine ​​34 having the linker maleimide-glycine-L-lysine-CONH2 and conjugated to recombinant anti-HER2 Fab using MTG.

[0149] For the preparation of NP-HSA modified with anti-HER2 Fab, recombinant Fab prepared as described in

[25] , having a TQGA sequence sensitive to the action of MTG, was used. The external SH group of the NP-HSA obtained as Example 1 or 2 was modified with the peptide linker maleimide-Gly-Lys-NH2, where the notation Lys-CONH2 indicates C-terminal amidated lysine. In the notation reported in formula (I), the "Z" unit is the maleimide group, the glycine amino acid represents the "spacer", and therefore Lys-CONH2 represents the "X-NH2" group. A 1:5 HSA / linker molar ratio was used for the derivatization reaction. After incubation at room temperature and under constant stirring for 16 hours, the modified NP, named NP-HSA-Cys-Gly-Lys, was washed three times in H2O to remove excess reagent and resuspended in phosphate buffer. Anti-HER2 Fab, prepared as described in

[25] , containing a TQGA tetrapeptide at its C-terminus so that the lysine introduced into NP can be conjugated by MTG, was reacted with NP-HSA-Cys-Gly-Lys. For the conjugation reaction, 40 μg / mL of anti-HER2 Fab was reacted with 1 mg / mL of NP-HSA-Cys-Gly-Lys in 1 mL of pH 7.3 phosphate buffer in the presence of 0.25 U / mL of MTG. The Fab conjugation reaction to NP-HSA-Cys-Gly-Lys was monitored at various time points (1 to 16 hours) by RP-HPLC, and the supernatant was analyzed to evaluate the decrease in the concentration of free Fab in the dissolved state. From the RP-HPLC analysis, it was found that the amount of conjugated Fab after 16 hours was 10 μg / mg of NP-HSA-Cys-Gly-Lys. Next, to remove excess MTG and Fab, the resulting NP-HSA was purified by three cycles of centrifugation with H2O. Finally, they were redispersed in PBS, stored at +4°C, and characterized by size (nm), PDI, and zeta potential (mV) using a Malvern Zetasizer Ultra (Malvern Instruments, Worcestershire, UK).An Agilent 1100 Series instrument (Agilent Technologies, Santa Clara, CA) equipped with a C4 Vydac analytical column, 4.6 × 250 mm, particle size 5 μm, was used for RP-HPLC analysis. To quantify anti-HER2 Fab, the following method was used: mobile phase A: H2O + 0.1% TFA and mobile phase B: ACN + 0.1% TFA, with a gradient of 30% to 45% B over 25 minutes, flow rate 0.7 mL / min. To determine the size and PDI, the NP-HSA thus obtained was diluted in PBS to a concentration of 0.1 mg / mL and analyzed by multi-angle scattering at +25°C using a micro-cuvette (UV-Cuvette, Brand®, Wertheim, Germany). To determine the zeta potential for surface loading, nanoparticles were diluted in ultrapure water to a concentration of 0.1 mg / mL of NP-HSA and analyzed at +25°C using a DTS1070 capillary cell (Malvern Instruments, Worcestershire, UK). All size and zeta potential determinations were performed in triplicate and are presented as mean ± standard deviation.

[0150] Analysis of NP-HSA and NP-HSA bonded to Fab using DLS revealed that the average diameter ranged from 130 nm (PDI 0.04) for unmodified NP-HSA to 145 nm (PDI 0.09) for NP-HSA bonded to Fab.

[0151] Example 5 Preparation of NP-HSA, which is derivatized with HSA cysteine ​​34 having the linker maleimide-Gly-Lys-NH2 and conjugated to recombinant anti-Cripto Fab 10D1 using MTG.

[0152] To produce NP-HSA conjugated with anti-Cripto Fab using MTG, the SH group of NP obtained in Example 1 was modified with the peptide linker Mal-Gly-Lys-NH2. A 1:5 HSA / linker molar ratio was used for the functionalization reaction. After incubation at room temperature and under constant stirring for 16 hours, the modified NP-HSA was washed three times in H2O to remove excess reagent and resuspended in phosphate buffer. Anti-Cripto-1 Fab containing a TQGA tetrapeptide at its C-terminus so that it could be conjugated by MTG to the lysine introduced into NP-HSA was reacted with NP-HSA-Cys-Gly-Lys. Anti-Cripto Fab

[24] was produced using the roprietary sequence of the anti-Cripto-1 antibody 10D1 as described in

[25] . For the conjugation reaction, 40 μg / mL of anti-Cripto-1 Fab was reacted with 1 mg / mL of HSA-NP in 1 mL of pH 7.3 phosphate buffer in the presence of 0.25 U / mL of MTG. The Fab conjugation reaction to NP-HSA-Cys-Gly-Lys was monitored at various time points (1–16 hours) by RP-HPLC, and the supernatant was analyzed to evaluate the decrease in the concentration of dissolved free Fab. From the RP-HPLC analysis, it was found that the amount of conjugated Fab after 16 hours was 10 μg / mg of NP-HSA. Next, the nanoparticles were purified by three washing cycles with H2O by centrifugation to remove excess MTG and Fab. Finally, they were redispersed in PBS, stored at +4°C, and characterized by size (nm), PDI, and zeta potential (mV) using a Malvern Zetasizer Ultra (Malvern Instruments, Worcestershire, UK). An Agilent 1100 Series instrument (Agilent Technologies, Santa Clara, CA) equipped with a C4 Vydac analytical column, 4.6 × 250 mm, particle size 5 μm, was used for RP-HPLC analysis. For the quantification of anti-HER2 Fab, the following method was used: mobile phase A: H2O + 0.1% TFA and mobile phase B: ACN + 0.1% TFA, with a gradient of 30% to 45% B over 25 minutes, flow rate 0.7 mL / min.To measure size and determine PDI, nanoparticles were diluted in PBS to a concentration of 0.1 mg / mL of NP and analyzed by multi-angle scattering at +25°C using a micro-cuvette (UV-Cuvette, Brand®, Wertheim, Germany). To measure zeta potential to determine surface load, nanoparticles were diluted in ultrapure water to a concentration of 0.1 mg / mL of NP-HSA and analyzed at +25°C using a DTS1070 capillary cell (Malvern Instruments, Worcestershire, UK). All size and zeta potential measurements were performed in triple replication and are presented as mean ± standard deviation.

[0153] Underivativeized NP-HSA and NP-HSA bonded to Fab were analyzed by DLS, revealing that the average diameter ranged from 130 nm (PDI 0.04) for unmodified NP to 145 nm (PDI 0.09) for NP-HSA bonded to Fab.

[0154] Example 6 Preparation of NP-HSA, which is derivatized with the linker Br-CH2-CO-O2Oc-K-NH2 and HSA cysteine ​​34, and then conjugated to recombinant anti-HER2 Fab using MTG.

[0155] To produce NP-HSA conjugated with anti-HER2 Fab using MTG, the SH group of the NP-HSA obtained in Example 1 was modified with the peptide linker Br-CH2-CO-O2Oc-K-NH2. In the notation reported in formula (I), the "Z" unit is a bromoacetic acid group, the notation O2Oc means the unnatural amino acid 8-amino-3,6-dioxo-octanoic acid and represents a "spacer", and therefore Lys-NH2 represents an "X-NH2" group.

[0156] A 1:5 HSA / linker molar ratio was used for the functionalization reaction. After incubation at room temperature and under constant stirring for 16 hours, the modified NP-HSA, defined as NP-HSA-Cys-O2Oc-Lys, was washed three times in H2O to remove excess reagent and resuspended in phosphate buffer. Anti-HER2 Fab, containing a TQGA tetrapeptide at its C-terminus to enable conjugation by MTG to the lysine introduced into NP-HSA, was reacted with NPs-Cys-O2Oc-Lys [32-35]. For the conjugation reaction, 40 μg / mL of anti-HER2 Fab was reacted with 1 mg of HSA NP-HSA-Cys-O2Oc-Lys in 1 mL of pH 7.3 phosphate buffer in the presence of 0.25 U of MTG. The Fab conjugation reaction to NPs-Cys-O2Oc-Lys was monitored at various time points (1–16 hours) using RP-HPLC, and the supernatant was analyzed to evaluate the decrease in the concentration of free Fab in the dissolved state. From the RP-HPLC analysis, it was found that the amount of conjugated Fab after 16 hours was 10 μg / mg of NP-HSA. Next, the nanoparticles were purified by three washing cycles with H2O by centrifugation to remove excess MTG and Fab. Finally, they were redispersed in PBS, stored at +4°C, and characterized by size (nm), polydispersity (PDI), and zeta potential (mV) using a Malvern Zetasizer Ultra (Malvern Instruments, Worcestershire, UK). An Agilent 1100 Series instrument (Agilent Technologies, Santa Clara, CA) equipped with a C4 Vydac analytical column, 4.6 × 250 mm, particle size 5 μm was used for the RP-HPLC analysis. To quantify anti-HER2 Fab, the following method was used: mobile phase A: H2O + 0.1% TFA and mobile phase B: ACN + 0.1% TFA, with a gradient of 30% to 45% B over 25 minutes, flow rate 0.7 mL / min. For size measurement and PDI determination, NP-HSA was diluted in PBS to a concentration of NP 0.1 mg / mL and analyzed by multi-angle scattering at +25°C using a micro-cuvette (UV-Cuvette, Brand®, Wertheim, Germany).To determine the surface load, nanoparticles were diluted in ultrapure water to a concentration of 0.1 mg / mL and analyzed at +25°C using a DTS1070 capillary cell (Malvern Instruments, Worcestershire, UK). All size and zeta potential measurements were performed in triple replication and are presented as mean ± standard deviation.

[0157] Underivativeized NP-HSA and NP-HSA bonded to Fab were analyzed by DLS, revealing that the average diameter ranged from 130 nm (PDI 0.04) for unmodified NP to 145 nm (PDI 0.09) for NP-HSA bonded to Fab.

[0158] Example 7 Preparation of NP-HSA, which is derivatized with the linker Br-CH2-CO-O2Oc-K-NH2 and HSA cysteine ​​34, and then conjugated to recombinant anti-Cripto-1 Fab 10D1 and anti-HER2 Fab using MTG.

[0159] To produce HSA nanoparticles conjugated to two anti-HER2 and anti-Cripto-1 Fabs using MTG, the SH group of the NP-HSA obtained in Example 1 was first modified with the peptide linker Br-CH2-CO-O2Oc-K-NH2 described in the previous example. A 1:5 HSA / linker molar ratio was used for the functionalization reaction. After incubation at room temperature and under constant stirring for 16 hours, the modified NP-HSA (NP-HSA-Cys-O2Oc-Lys) was washed three times in H2O to remove excess reagent and resuspended in phosphate buffer. NP-HSA-Cys-O2Oc-Lys was simultaneously reacted with 1:1 mixtures of two different Fabs: anti-HER2 and anti-Cripto-1, both containing a TQGA tag at the C-terminus so that the lysine introduced into the NP-HSA could be conjugated by MTG. For the conjugation reaction, 20 μg of anti-HER2 Fab and 20 μg of anti-Cripto Fab were reacted with 1 mg of HSA NPs-O2Oc-Lys in 5 mL of pH 7.3 phosphate buffer in the presence of 0.25 U of MTG. The Fab conjugation reaction to NP-HSA-Cys-O2Oc-Lys was monitored at various time points (1 to 16 hours) by RP-HPLC, and the supernatant was analyzed to evaluate the decrease in the concentrations of the two soluble Fabs. From the RP-HPLC analysis, it was found that after 16 hours, the amount of the two Fab conjugates was 5 μg for anti-Cripto-1 Fab and 2 μg for trastuzumab Fab per 1 mg of NP. Next, to remove inactive MTG and Fab, the nanoparticles were purified by three washing cycles with H2O by centrifugation. Finally, the samples were redispersed in PBS, stored at +4°C, and characterized by size (nm), polydispersity (PDI), and zeta potential (mV) using a Malvern Zetasizer Ultra (Malvern Instruments, Worcestershire, UK). Analysis was performed using RP-HPLC on an Agilent 1100 Series instrument (Agilent Technologies, Santa Clara, CA) equipped with a C4 Vydac analytical column, 4.6 × 250 mm, and 5 μm particle size.To quantify the two types of Fab, the following method was used: mobile phase A: H2O + 0.1% TFA and mobile phase B: ACN + 0.1% TFA, with a gradient of 30% to 45% B over 25 minutes, and a flow rate of 0.7 mL / min.

[0160] To measure size and determine PDI, nanoparticles were diluted in PBS to a concentration of 0.1 mg / mL NP and analyzed by multi-angle scattering at +25°C using a micro-cuvette (UV-Cuvette, Brand®, Wertheim, Germany). To measure zeta potential to determine surface load, nanoparticles were diluted in ultrapure water to a concentration of 0.1 mg / mL NP and analyzed at +25°C using a DTS1070 capillary cell (Malvern Instruments, Worcestershire, UK). All size and zeta potential measurements were performed in triplicate and are presented as mean ± standard deviation.

[0161] Underivativeized NP-HSA and NP-HSA bonded to Fab were analyzed by DLS, revealing that the average diameter ranged from 130 nm (PDI 0.04) for unmodified NP to 145 nm (PDI 0.09) for NP-HSA bonded to Fab.

[0162] Example 8 Chemical synthesis of the linker Br-CH2-CO-O2Oc-K-NH2

[0163] The linker structure is Br-CH2-CO-O2Oc-L-Lys-CONH2, where Br-CH2-CO- represents the N-terminal bromoacetic acid residue ("Z" unit), O2Oc represents the unnatural amino acid 8-amino-3,6-dioxo-octanoic acid, and L-Lys-CONH2 represents the natural C-terminal amidated L-lysine amino acid. The peptide was prepared by solid-phase synthesis using the Fmoc method with MBHA Rink amide resin (IRIS BIOTECH, 0.74 mmol / g). The synthesis was carried out on a 590 μmol scale. The resin was washed three times (5 mL x 3) in dimethylformamide (DMF, Romil, DELTEK) and then washed for 10 minutes with 20% (v / v) piperidine (ROMIL, DELTEK) in DMF to remove the Fmoc group from the resin. Next, the L-lysine amino acid was bound in the form of an Fmoc-L-Lys(Boc)-OH derivative (IRIS BIOTECH) dissolved in DMF at a concentration of 0.5 M. The amino acid used in a 5-fold excess was pre-activated with HATU / DIEA and an excess of 1:2 (mol / mol) (IRIS BIOTECH) as described in reference

[36] , and left in contact with the resin at room temperature for 1 hour. The resin was washed three times with DMF, and then treated with 5 mL of 40% (v / v) piperidine in DMF for 20 minutes. The resin was drained and washed three times with DMF. Next, the resin was treated with the conductor Fmoc-O2Oc-OH (8-(9-fluorenylmethyloxycarbonyl-amino)-3,6-dioxaoctanoic acid, IRIS BIOTECH) dissolved in DMF at a concentration of 0.5 M, and pre-activated with an excess amount of HBTU / DIEA 1:2 (mol / mol) at room temperature for 1 hour, as described in reference

[36] . Finally, the resin was washed three times with DMF and treated with 5 mL of 40% (v / v) piperidine in DMF for 20 minutes. The resin was drained and washed three times with DMF. Finally, it was treated with 25 equivalents of bromoacetic acid at a concentration of 1 M in DCM (dichloromethane, ROMIL, DELTEK) with DIEA (1:1 (mol / mol)) added, at room temperature for 2 hours. Finally, the resin was washed with DCM, DMF, and again with DCM, and then dried under vacuum.To remove the peptide, the resin was treated with 5 mL of TFA (trifluoroacetic acid) / Tis (triisopropylsilane) / H2O90 / 5 / 5 (v / v / v) solution at room temperature for 3 hours. The resin was filtered off, the peptide was precipitated with cold ethyl ether, isolated by centrifugation, and lyophilized with H2O and ACN (acetonitrile, ROMIL, DELTEK). The crude material was purified by preparative RP-HPLC and finally characterized by LC-MS using an Xbridge C18 column (50 × 2.1 mm ID, 5 μm) on an LC-MS ESI-TOF system (6230 ESI-TOF mass spectrometer) connected to an HPLC 1290 Infinity system. A gradient from 1% solvent B (ACN, 0.05% TFA) to 80% B was observed over 10 minutes at a flow rate of 0.2 mL / min. Solvent A was H2O, 0.05% TFA. Approximately 180 mg (95%, HPLC) of pure product was obtained, with a yield of 74%. The determined experimental mass was 410.13 atomic mass units (amu), which was in near perfect agreement with the experimental mass of 410.12 amu.

[0164] Example 9 Synthesis of the linker Mal-Gly-Lys-CONH2

[0165] The linker structure is Mal-Gly-Lys-CONH2, where Mal represents the N-terminal 6-maleimidohexanoic acid ("Z" unit), Gly represents the natural glycine amino acid, and L-Lys-CONH2 represents the natural C-terminal amidated L-lysine amino acid. The peptide was prepared by solid-phase synthesis using MBHA Rink amide resin (0.74 mmol / g) with the Fmoc method. Synthesis was performed on a 590 μmol scale. The resin was washed three times (5 mL x 3) in DMF and treated with 20% (v / v) piperidine in DMF for 10 minutes to remove the Fmoc group from the resin. Next, the L-lysine amino acid was bound in the form of an Fmoc-L-Lys(Boc)-OH derivative dissolved in DMF at a concentration of 0.5 M. The amino acids used in 5 times the normal amount were pre-activated with HATU / DIEA and an excess of 1:2 (mol / mol) (IRIS BIOTECH) as described in reference

[36] , and left in contact with the resin at room temperature for 1 hour. The resin was washed three times with DMF, and then treated with 5 mL of 40% (v / v) piperidine in DMF for 20 minutes. The resin was drained and washed three times with DMF. Next, the resin was treated with the derivative Fmoc-Gly-OH (9-fluorenylmethyloxycarbonylglycine, IRIS BIOTECH) dissolved in DMF at a concentration of 0.5 M, and pre-activated with an excess of 1:2 (mol / mol) HBTU / DIEA at room temperature for 1 hour as described in reference

[36] . Finally, the resin was washed three times with DMF and treated with 5 mL of 40% (v / v) piperidine in DMF for 20 minutes. The resin was drained and washed three times with DMF. Finally, the resin was treated with 25 equivalents of 1 M 6-maleimidohexanoic acid (Sigma-Aldrich code: 755842) in DMF (1:1 (mol / mol)) supplemented with DIEA at room temperature for 2 hours. Lastly, the resin was washed with DCM, DMF, and again with DCM, and then dried under vacuum. For peptide elimination, the resin was treated with 5 mL of TFA (trifluoroacetic acid) / Tis (triisopropylsilane) / H2O 90 / 5 / 5 (v / v / v) solution at room temperature for 3 hours. The resin was filtered off, the peptide was precipitated with cold ethyl ether, isolated by centrifugation, and lyophilized with H2O and ACN.Crude materials were purified by preparative RP-HPLC and finally characterized by LC-MS using an Xbridge C18 column (50 × 2.1 mm ID, 5 μm) on an LC-MS ESI-TOF system (6230 ESI-TOF mass spectrometer) connected to an HPLC 1290 Infinity system. A gradient from 1% solvent B (ACN, 0.05% TFA) to 80% solvent B was observed over 10 minutes at a flow rate of 0.2 mL / min. Solvent A was H2O, 0.05% TFA.

[0166] Approximately 163 mg of pure product (95%, HPLC) was obtained, with a yield of approximately 70%. The determined experimental mass was 394.64 amu, which was in near perfect agreement with the experimental mass of 394.61 amu.

[0167] Example 10 Competitive binding of recombinant Her2 binding between trastuzumab Fab and trastuzumab-modified NP-HSA.

[0168] The amount of receptor-binding antagonist can be indirectly detected by substitution testing using the same ligand or a surrogate marked with the same reporter. Substitution testing was performed using trastuzumab Fab-functionalized NP-HSA, recombinant receptor Her2-Fc (recombinant human ErbB2 Fc Chimera / R&D 1129-ER-050) immobilized on the surface of a multiwell plate, and antibody biotin-labeled trastuzumab, as prepared as described in Examples 4 and 6. The experiment was conducted by measuring the ability of trastuzumab Fab-functionalized NP-HSA to replace the binding of biotin-labeled trastuzumab to the receptor immobilized on the surface of a 96-well multiwell plate. In parallel experiments, experiments were performed using Fab that was not bound to NP-HSA. Her2-Fc receptors were immobilized overnight at 4°C in a 96-well polystyrene multiwell plate by incubation (100 μL / well) of a 0.5 μg / mL solution in phosphate buffer. Blocking was performed by incubation (2 hours at 37°C) with a 1% BSA solution (bovine serum albumin / Merck A7906, 300 μL / mL) in phosphate buffer. Subsequently, biotin-labeled trastuzumab solution (provided by the CNR Institute of Biostructures and Bioimaging), containing NP-HSA-Fab (NP-HSA 10 μg / mg) at increasing molar concentrations between 0.39 nM (approx. 2 μg / mL) and 100 nM (0.5 mg / mL), was added to various wells at a fixed concentration of 100 pM. The experiment was performed in quadruple (four wells per concentration). Detection of biotin-labeled trastuzumab bound to immobilized receptors was performed using a solution of streptavidin-peroxidase (Merck S5512) at a concentration of 50 ng / mL (100 μL / well), followed by incubation with the substrate TMB (3,3',5,5'-tetramethylbenzidine / Merck T0440).The reaction was blocked with 1N H2SO4, and absorbance was read at 450 nm using a multiplate reader (Model 680, BIO-RAD). The absorbance values ​​obtained at 450 nm were subtracted from the blank values ​​(absorbance of wells not immobilized with Her2-Fc), averaged, and expressed as a percentage compared to the biotin-labeled trastuzumab-only control. The data were fitted to an S-curve using GraphPad Prism 6.0 software to calculate the inhibitory IC50. 50 The result was determined. The experiment was repeated three times and the average was calculated. IC 50 The data are reported in Table 2 below. The inhibition curve is shown in Figure 1. This data demonstrates that HP-NSA functionalized with Fab has the same ability to bind to the Fab receptor alone.

[0169] [Table 2]

[0170] Example 11 Binding of NP-HSA conjugated to trastuzumab Fab and unconjugated recombinant Fab to Her2-positive BT474 cells.

[0171] Her2-positive BT474 cells, a human breast cancer cell line, were plated in 96-well plates (Falcon® 96-well Clear TC-Treated Microplates, Thermo Scientific) at a cell density of 10,000 cells / well. After incubation overnight at +37°C with 5% CO2, the cells were fixed with 4% formaldehyde at room temperature for 15 minutes, then washed twice with PBS (1×), and finally treated with 3% hydrogen peroxide at room temperature for 10 minutes. After washing twice with PBS (1×), the cells were treated with PBS-A containing 3% BSA (Sigma Aldrich, A3294) at room temperature for 1 hour. The wells were double-treated with NP-HSA conjugated to trastuzumab Fab, prepared as described in Examples 4 and 6, using NP-HSA and trastuzumab Fab in the concentration range of 0.12–1.0 μM, and the final volume was 100 μL / well using PBS-A. Cells were incubated with the sample at room temperature for 2 and 24 hours, washed three times with PBS (1×), and then treated with HRP-conjugated mouse IgG (Sigma-Aldrich) capable of recognizing human Fab. Finally, the wells were treated with GAM-HRP (Sigma-Aldrich) at a 1:1000 dilution at room temperature for 1 hour. The cells were washed three times with PBS and treated with OPD (Sigma Aldrich P9187) (100 μL / well) in the dark for 5 minutes. The reaction was blocked using 2.5 M H2SO4 (50 μL / well). The absorbance of the sample was read at 490 nm using a BioTek microplate reader (Winooski, VT, USA). The values ​​were averaged, a blank was subtracted (values ​​for wells treated with defunctionalized NP-HSA), and the binding constant was determined by plotting using GraphPad prism 6.0. The binding curve is shown in Figure 2AB. This data demonstrates that, at the same concentration of Fab, NP-HSA functionalized with trastuzumab Fab can bind to the receptor on cells with higher efficiency than Fab alone, both at 2 hours and 24 hours.

[0172] Example 12 Binding of NP-HSA conjugated to trastuzumab Fab and unconjugated recombinant Fab to Her2-positive BT474 cells and Her2-negative MDA-MB-231 cells.

[0173] Her2-positive BT474 cells were plated at a cell density of 10,000 cells / well in 96-well plates (Falcon® 96-well Clear TC-Treated Microplates, Thermo Scientific). Her2-negative MDA-MB-231 cells and human breast cancer cell lines were plated at a cell density of 5,000 cells / well in the same second plate. After incubation overnight at +37°C with 5% CO2, the cells were fixed with 4% formaldehyde at room temperature for 15 minutes, then washed twice with PBS (1×), and finally treated with 3% hydrogen peroxide at room temperature for 10 minutes. After washing twice with PBS (1×), the cells were treated with PBS containing 3% BSA, PBS-A (Sigma Aldrich, A3294) at room temperature for 1 hour. The wells were double-treated with trastuzumab Fab-junctioned NP-HSA, prepared as described in Examples 4 and 6, in a concentration range of 0.03 μM to 50 nM. To verify receptor expression on BT474 cells and its absence on MDA-MB-231 cells, experiments were conducted in parallel with defunctionalized NP-HSA at the same concentrations using trastuzumab Fab at concentrations of 500, 200, and 100 μg / mL and whole trastuzumab antibody at concentrations of 10, 1.0, and 0.1 μg / mL, dissolved in 100 μL / well of PBS-A. Cells were incubated with the sample at room temperature for 2 and 24 hours, washed three times in PBS (1×), and treated with mouse IgG conjugated with HRP (Sigma-Aldrich) capable of recognizing human Fab. Finally, the wells were treated with GAM-HRP (Sigma-Aldrich) at a 1:1000 dilution for 1 hour at room temperature. Cells were washed three times with PBS and treated with OPD (Sigma-Aldrich P9187) (100 μL / well) for 5 minutes in the dark. The reaction was blocked using 2.5M H2SO4 (50 μL / well). The absorbance of the sample was read at 490 nm using a BioTek microplate reader (Winooski, VT, USA). The values ​​were averaged, a blank was subtracted (values ​​from wells treated with defunctionalized NP-HSA), and the binding constant was determined by plotting using Graph Pad prism 6.0.The binding curves are shown in Figure 3ABC. Data from Figure 3A demonstrate that NP-HSA functionalized with trastuzumab Fab binds only minimally to non-receptor-expressing MDA-MB-231 cells, but can bind to BT474 cells expressing the Her2 receptor on their surface. Data from Figures 3B and 3C, obtained using very high concentrations of recombinant Fab and whole antibodies, demonstrate the absence of Her2 receptor expression on MDA-MB-231 control cells. Defunctionalized NP-HSA did not provide a detectable signal.

[0174] Example 13 Binding of recombinant anti-Cripto Fab 10D1-conjugated NP-HSA and unconjugated recombinant Fab to NTERA cells.

[0175] Cripto-1-positive cells, NTERA, were plated in 96-well plates (Falcon® 96-well Clear TC-Treated Microplates, Thermo Scientific) at a cell density of 5000 cells / well. After incubation overnight at +37°C with 5% CO2, the cells were fixed with 4% formaldehyde at room temperature for 15 minutes, then washed twice with PBS (1×), and finally treated with 3% hydrogen peroxide at room temperature for 10 minutes. After washing twice with PBS (1×), the cells were treated with 3% PBS-A at room temperature for 1 hour. Two sets of wells were treated with NP-HSA functionalized with anti-Cripto Fab 10D1, prepared as described in Example 5, as well as Fab alone and empty NP-HSA, in a concentration range of 0.000117 μM to 0.5 μM, and the final volume was 100 μL / well with 3% PBS-BSA solution. Cells were incubated with the sample at room temperature for 2 and 24 hours, washed three times in PBS (1×), and then treated with anti-human mouse IgG conjugated with HRP (Sigma-Aldrich) capable of recognizing Fab. Finally, GAM-HRP (Sigma-Aldrich) was added to each well at a 1:1000 dilution at room temperature for 1 hour. Cells were washed three times with PBS and treated with OPD (100 μL / well) in the dark for 5 minutes. Subsequently, the reaction was blocked with 2.5 M H2SO4. The absorbance of the sample was read at 490 nm using a BioTek microplate reader (Winooski, VT, USA). The binding curves are shown in Figure 4, demonstrating that NP-HSA functionalized with Fab 10D1 could bind to NTERA cells at both 2 and 24 hours in a dose-response and saturation manner, whereas single Fab could not bind under the same conditions. Defunctionalized NP-HSA did not provide a detectable signal.

[0176] Example 14 Binding of bispecific NP-HSA, functionalized with recombinant anti-Cripto Fab 10D1 and recombinant trastuzumab Fab, to Her2-positive BT474 cells and Her2-negative MDA-MB-231 cells.

[0177] Cripto-1 and Her2-positive cells, BT474, were plated in 96-well plates (Falcon® 96-well Clear TC-Treated Microplates, Thermo Scientific) at a cell density of 10,000 cells / well. Alternatively, Cripto-1-positive and Her2-negative MDA-MB-231 cells were plated in 96-well plates at a cell density of 5,000 cells / well. After incubation overnight at +37°C with 5% CO2, the cells were fixed with 4% formaldehyde at room temperature for 15 minutes, then washed twice with PBS (1×), and finally treated with 3% hydrogen peroxide at room temperature for 10 minutes. After washing twice with PBS (1×), the cells were treated with 3% PBS-A at room temperature for 1 hour. Two sets of wells were treated with NP-HSAs functionalized with two recombinant anti-Cripto-1 Fab 10D1 and trastuzumab Fab, prepared as described in Example 7, having Fab 10D1 density 5.0 μg / mg NP-HSA and trastuzumab Fab density 2.0 μg / mg NP-HSA. Functionalized NP-HSAs were used at increasing concentrations, expressed as total Fab equivalent concentrations from 0.097 nM (0.343 μg / mL NP-HSA) to 50 nM (175 μg / mL NP-HSA). In parallel, the same cells were treated with all 1B4 antibodies (anti-Cripto-1,

[30] and trastuzumab) at concentrations of 0.1 and 1.0 μg / mL. All samples were used in 3% PBS-BSA at a final volume of 100 μL / well. Cells were incubated with the samples at room temperature for 2 hours, washed three times in PBS (1×), and then treated with anti-human mouse IgG conjugated with HRP (Sigma-Aldrich) capable of recognizing Fab. Finally, GAM-HRP (Sigma-Aldrich) was added to each well at a 1:1000 dilution at room temperature for 1 hour. Cells were washed three times in PBS and then treated with OPD (100 μL / well) in the dark for 5 minutes. The reaction was then blocked with 2.5 M H2SO4. The absorbance of the samples was read at 490 nm using a BioTek microplate reader (Winooski, VT, USA).Figure 5AB shows binding data for the total B4 antibody anti-Cripto-1 and trastuzumab to two cell lines at concentrations of 0.1 μg / mL and 1.0 μg / mL, demonstrating that BT474 cells express the Her2 receptor and less Cripto-1, and that MDA-MB-231 cells express Cripto-1 only to a small degree. Figure 5C shows dose-response binding curves, demonstrating that the presence of anti-Cripto-1 Fab alone allows NP-HSA functionalized with two recombinant Fabs to bind to both Her2-negative BT474 and MDA-MB-231 cells. Defunctionalized NP-HSA did not provide a detectable signal.

[0178] Example 15 Binding of bispecific NP-HSA functionalized with recombinant anti-Cripto Fab 10D1 and recombinant trastuzumab Fab to Her2-positive BT474 cells and Her2-negative MDA-MB-231 cells compared to binding obtained with a mixture of two types of NP-HSA.

[0179] Cripto-1 and Her2-positive cells, BT474, were plated in 96-well plates (Falcon® 96-well Clear TC-Treated Microplates, Thermo Scientific) at a cell density of 10,000 cells / well. Cripto-1-positive and Her2-negative MDA-MB-231 cells were plated in 96-well plates at a cell density of 5,000 cells / well. After incubation overnight at +37°C with 5% CO2, the cells were fixed with 4% formaldehyde at room temperature for 15 minutes, then washed twice with PBS (1×), and finally treated with 3% hydrogen peroxide at room temperature for 10 minutes. After washing twice with PBS (1×), the cells were treated with 3% PBS-A at room temperature for 1 hour. Two sets of wells were treated with NP-HSA functionalized bispecificity NP-HSA prepared as described in Example 7 at a density of 3.0 μg / mg NP-HSA (10D1) and 2 μg / mg NP-HSA (trastuzumab), NP-HSA functionalized with Fab 10D1 alone as described in Example 5 at an increasing concentration of Fab from 24 pM to 12 nM at a density of 10.0 μg / mg NP-HSA, and NP-HSA functionalized with trastuzumab Fab alone at a density of 10.0 μg / mg NP-HSA as described in Example 4 and Example 6, and two types of anti-Cripto-1 mAb trastuzumab and 1B4

[30] used at concentrations of both 1.0 μg / mL and 0.1 μg / mL, at an increasing concentration of total Fab from 24 pM to 12 nM. All samples were prepared using 3% PBS-BSA at a final volume of 100 μL / well. Cells were incubated with the sample at room temperature for 2 hours, washed three times in PBS (1×), and then treated with anti-human mouse IgG conjugated with HRP (Sigma-Aldrich) capable of recognizing Fab. Finally, GAM-HRP (Sigma-Aldrich) was added to each well at a 1:1000 dilution at room temperature for 1 hour. Cells were washed three times with PBS and then treated with OPD (100 μL / well) in the dark for 5 minutes. The reaction was then blocked with 2.5 M H2SO4.The absorbance of the sample was read at 490 nm using a BioTek microplate reader (Winooski, VT, USA). The dose-response binding curves are shown in Figure 6ABCD, demonstrating that bispecific NP-HSA functionalized with two recombinant Fabs can bind to both BT474 and MDA-MB-231 cells with the same efficiency as a combination of two types of NP-HSA functionalized with two different Fabs. Defunctionalized NP-HSA did not provide a detectable signal.

[0180] Example 16 Production of NP-HSA functionalized with fluorescein and the antibody anti-Cripto-1 Fab10D1, and experimentation of its binding to NTERA2 cells expressing Cripto-1 by cytofluorimetry.

[0181] NP-HSA was prepared as described in Example 1. 5.0 mg of NP-HSA was suspended in 1.0 mL of 0.1 M borate buffer (pH 9.0). 0.14 mg of fluorescein isothiocyanate (FITC, Sigma Aldrich code F4274) was added to the suspension to achieve an HSA:FITC ratio of 1:5 (mol / mol), and solubilized in 50 μL of anhydrous DMSO. The suspension was stirred at 37°C for 3 hours.

[0182] The NP-HSA conjugation reaction to FITC was monitored at various time points (1–3 hours) by RP-HPLC, and the supernatant was analyzed to assess the decrease in the concentration of dissolved free FITC. From the RP-HPLC analysis, the amount of FITC conjugate 3 hours after the reaction was found to be 8 μg / mg NP. Next, the nanoparticles were purified by three washing cycles with H2O by centrifugation to remove excess FITC. Finally, they were redispersed in PBS and stored at +4°C. The nanoparticles were defined as FITC-NP-HSA. Aliquots (4.0 mg) of FITC-NP-HSA were used for MTG conjugation with anti-Cripto-1 Fab 10D1 to obtain FITC-NP-HSA conjugated to two Fabs (FITC-NP-HSA-Fab). Using an HSA / linker ratio of 1:5 (mol / mol), 4.0 mg of FITC-NP-HSA was treated with the peptide linker Br-CH2-CO-O2Oc-K-NH2 (see Examples 6 and 8). After incubation at room temperature and under constant stirring for 16 hours, the modified NP (FITC-NP-HSA-Lys) was washed three times in H2O to remove excess reagent, and then resuspended in phosphate buffer. FITC-NP-HSA-Lys was functionalized with anti-Cripto Fab 10D1 (see Example 7); this functionalization was carried out using MTG, taking advantage of the presence of the C-terminal TQGA tag. For the functionalization reaction, 40 μg of Fab was used in 1 mL of final volume phosphate buffer (pH 7.3) in the presence of 0.25 U of MTG. The Fab conjugation reaction to FITC-NP-HSA-Lys was monitored at various time points (1 to 16 hours) using RP-HPLC, and the supernatant was analyzed to evaluate the decrease in the concentration of free Fab in the dissolved state. From the RP-HPLC analysis, it was found that the amount of Fab conjugate after 16 hours was 10 μg / mg NP-HSA. The NP-HSA thus obtained, called FITC-NP-HSA-Fab, was then purified by three washing cycles with H2O by centrifugation to remove excess MTG and Fab.Finally, the samples were redispersed in PBS, stored at +4°C, and characterized by size (nm), polydispersity (PDI), and zeta potential (mV) using a Malvern Zetasizer Ultra (Malvern Instruments, Worcestershire, UK). An Agilent 1100 Series instrument (Agilent Technologies, Santa Clara, CA) equipped with a C4 Vydac analytical column, 4.6 × 250 mm, particle size 5 μm was used for RP-HPLC analysis. For quantification of Fab in soluble form, the following method was used: mobile phase A: H2O + 0.1% TFA and mobile phase B: ACN + 0.1% TFA, with a gradient of 30% to 45% B over 25 minutes, flow rate 0.7 mL / min.

[0183] For cytoflowmetry analysis, NTERA2 cells were plated at 40% density in 10% FBS DMEM / F12 with the addition of glutamine and antibiotics. The following day, the cells were washed with PBS and serum-free medium containing the following nanoparticles: FITC-NP-HSA and FITC-NP-HSA-Fab. NP-HSA marked with Fab but without FITC was used as a negative control. The amount of NP used was normalized to FITC-NP-HSA-Fab, and used to have anti-Cripto Fab concentrations of 1000 ng / mL, 100 ng / mL, and 10 ng / mL. Cells were incubated with NP at 37°C for 4 hours, then washed and resuspended in 0.5% BSA PBS. Samples were obtained using a "BD FACS ARIAIII cell sorter" and analyzed for FITC size and intensity. From the results, the percentage of FITC-positive cells (gate P3) and the mean fluorescence intensity (P1 MFI) of the total population analyzed are reported. At 100 ng / mL, a slight signal was observed favorably with FITC-NP-HSA-Fab (1.5%) compared to FITC-NP-HSA without Fab (0.5%). At 1000 ng / mL, the signal was considerably stronger for both samples (preparations). There was no substantial difference in the number of marked cells between Fab-modified NPs (53.5%) and unmodified NPs (63.6%). The fluorescence intensity observed with FITC-NP-HSA-Fab was considerably higher than that observed with FITC-NP-HSA, at MFI3313 and 752, respectively. Subsequently, a 4.4-fold higher fluorescence signal was observed in Fab-modified NP-HSA-treated cells compared to unmodified NP-HSA, suggesting that the presence of anti-Cripto-1 Fab allows for greater NP-HSA input into some cells. References

[0184] [1] Sleep,D.;Cameron,J.;Evans,L.R.,Albumin as a versatile platform for drug half-life extension.Biochim Biophys Acta,2013,1830,(12),5526-5534.

[0185] [2] Hoogenboezem,E.N.;Duvall,C.L.,Harnessing albumin as a carrier for cancer therapies.Adv Drug Deliv Rev,2018,130,73-89.

[0186] [3] Birn,H.;Christensen,E.I.,Renal albumin absorption in physiology and pathology.Kidney Int,2006,69,(3),440-449.

[0187] [4] Birn,H.;Fyfe,J.C.;Jacobsen,C.;Mounier,F.;Verroust,P.J.;Orskov,H.;Willnow,T.E.;Moestrup,S.K.;Christensen,E.I.,Cubilin is an albumin binding protein important for renal tubular albumin reabsorption.J Clin Invest,2000,105,(10),1353-1361.

[0188] [5] Merlot,A.M.;Kalinowski,D.S.;Richardson,D.R.,Unraveling the mysteries of serum albumin-more than just a serum protein.Front Physiol,2014,5,299.

[0189] [6] Miller,M.A.;Gadde,S.;Pfirschke,C.;Engblom,C.;Sprachman,M.M.;Kohler,R.H.;Yang,K.S.;Laughney,A.M.;Wojtkiewicz,G.;Kamaly,N.;Bhonagiri,S.;Pittet,M.J.;Farokhzad,O.C.;Weissleder,R.,Predicting therapeutic nanomedicine efficacy using a companion magnetic resonance imaging nanoparticle.Sci Transl Med,2015,7,(314),314ra183.

[0190] [7] Commisso,C.;Davidson,S.M.;Soydaner-Azeloglu,R.G.;Parker,S.J.;Kamphorst,J.J.;Hackett,S.;Grabocka,E.;Nofal,M.;Drebin,J.A.;Thompson,C.B.;Rabinowitz,J.D.;Metallo,C.M.;Vander Heiden,M.G.;Bar-Sagi,D.,Macropinocytosis of protein is an amino acid supply route in Ras-transformed cells.Nature,2013,497,(7451),633-637.

[0191] [8] Campbell,P.M.;Der,C.J.,Oncogenic Ras and its role in tumor cell invasion and metastasis.Semin Cancer Biol,2004,14,(2),105-114.

[0192] [9] Kamphorst,J.J.;Nofal,M.;Commisso,C.;Hackett,S.R.;Lu,W.;Grabocka,E.;Vander Heiden,M.G.;Miller,G.;Drebin,J.A.;Bar-Sagi,D.;Thompson,C.B.;Rabinowitz,J.D.,Human pancreatic cancer tumors are nutrient poor and tumor cells actively scavenge extracellular protein.Cancer Res,2015,75,(3),544-553.

[0193]

[10] Hauser,C.A.;Stockler,M.R.;Tattersall,M.H.,Prognostic factors in patients with recently diagnosed incurable cancer: a systematic review.Support Care Cancer,2006,14,(10),999-1011.

[0194]

[11] Chatterjee,M.;Ben-Josef,E.;Robb,R.;Vedaie,M.;Seum,S.;Thirumoorthy,K.;Palanichamy,K.;Harbrecht,M.;Chakravarti,A.;Williams,T.M.,Caveolae-Mediated Endocytosis Is Critical for Albumin Cellular Uptake and Response to Albumin-Bound Chemotherapy.Cancer Res,2017,77,(21),5925-5937.

[0195]

[12] Suzuoki,M.;Miyamoto,M.;Kato,K.;Hiraoka,K.;Oshikiri,T.;Nakakubo,Y.;Fukunaga,A.;Shichinohe,T.;Shinohara,T.;Itoh,T.;Kondo,S.;Katoh,H.,Impact of caveolin-1 expression on prognosis of pancreatic ductal adenocarcinoma.Br J Cancer,2002,87,(10),1140-1144.

[0196]

[13] Li,R.;Yang,H.;Jia,D.;Nie,Q.;Cai,H.;Fan,Q.;Wan,L.;Li,L.;Lu,X.,Fusion to an albumin-binding domain with a high affinity for albumin extends the circulatory half-life and enhances the in vivo antitumor effects of human TRAIL.J Control Release,2016,228,96-106.

[0197]

[14] Zhang,Y.;Sun,T.;Jiang,C.,Biomacromolecules as carriers in drug delivery and tissue engineering.Acta Pharm Sin B,2018,8,(1),34-50.

[0198]

[15] Kuhlmann,M.;Hamming,J.B.R.;Voldum,A.;Tsakiridou,G.;Larsen,M.T.;Schmokel,J.S.;Sohn,E.;Bienk,K.;Schaffert,D.;Sorensen,E.S.;Wengel,J.;Dupont,D.M.;Howard,K.A.,An Albumin-Oligonucleotide Assembly for Potential Combinatorial Drug Delivery and Half-Life Extension Applications.Mol Ther Nucleic Acids,2017,9,284-293.

[0199]

[16] Elzoghby,A.O.;Samy,W.M.;Elgindy,N.A.,Albumin-based nanoparticles as potential controlled release drug delivery systems.J Control Release,2012,157,(2),168-182.

[0200]

[17] Wagner,S.;Rothweiler,F.;Anhorn,M.G.;Sauer,D.;Riemann,I.;Weiss,E.C.;Katsen-Globa,A.;Michaelis,M.;Cinatl,J.,Jr.;Schwartz,D.;Kreuter,J.;von Briesen,H.;Langer,K.,Enhanced drug targeting by attachment of an anti alphav integrin antibody to doxorubicin loaded human serum albumin nanoparticles.Biomaterials,2010,31,(8),2388-2398.

[0201]

[18] Greally,M.;Kelly,C.M.;Cercek,A.,HER2: An emerging target in colorectal cancer.Curr Probl Cancer,2018,42,(6),560-571.

[0202]

[19] da Silva,J.L.;Cardoso Nunes,N.C.;Izetti,P.;de Mesquita,G.G.;de Melo,A.C.,Triple negative breast cancer: A thorough review of biomarkers.Crit Rev Oncol Hematol,2020,145,102855.

[0203]

[20] Sandomenico,A.;Ruvo,M.,Targeting Nodal and Cripto-1: Perspectives Inside Dual Potential Theranostic Cancer Biomarkers.Curr Med Chem,2019,26,(11),1994-2050.

[0204]

[21] Sharma,A.;Kaur,A.;Jain,U.K.;Chandra,R.;Madan,J.,Stealth recombinant human serum albumin nanoparticles conjugating 5-fluorouracil augmented drug delivery and cytotoxicity in human colon cancer,HT-29 cells.Colloids Surf B Biointerfaces,2017,155,200-208.

[0205]

[22] Zong,Y.;Wu,J.;Shen,K.,Nanoparticle albumin-bound paclitaxel as neoadjuvant chemotherapy of breast cancer: a systematic review and meta-analysis.Oncotarget,2017,8,(10),17360-17372.

[0206]

[23] Zhang,J.Y.;He,B.;Qu,W.;Cui,Z.;Wang,Y.B.;Zhang,H.;Wang,J.C.;Zhang,Q.,Preparation of the albumin nanoparticle system loaded with both paclitaxel and sorafenib and its evaluation in vitro and in vivo.J Microencapsul,2011,28,(6),528-536.

[0207]

[24] Foca,G.;Iaccarino,E.;Foca,A.;Sanguigno,L.;Untiveros,G.;Cuevas-Nunez,M.;Strizzi,L.;Leonardi,A.;Ruvo,M.;Sandomenico,A.,Development of conformational antibodies targeting Cripto-1 with neutralizing effects in vitro.Biochimie,2019,158,246-256.

[0208]

[25] Selis , F. ;Sandomenico , A. ;Cantile , M. ;Sanna , R. ;Calvanese , L. ; [ PubMed ] Falco, S.;Foca,A.;Caporale,A.;Iaccarino,E.;Truppo,E.;Scaramuzza,S.;Tonon,G.;Ruvo,M.

[0209]

[26] Behdani, M.; Zeinali, S.; Khanahmad, H.; Karimipour, M.; Asadzadeh, N.; Azadmanesh, K.; functional Nanobody against the vascular endothelial growth factor receptor-2;angiogenesis cell receptor.Mol Immunol,2012,50,(1-2),35-41.

[0210]

[27] Pruszynski,M.;Koumarianou,E.;Vaidyanathan,G.;Revets,H.;Devoogdt,N.;Lahoutte,T.;Zalutsky,MR,Targeting Breast Carcinoma with Radioiodinated Anti-HER2 Nanobody.Nucl Med Biol,2013,40,(1),52-59.

[0211]

[28] Oliveira,S.;Schiffelers,R.M.;van der Veeken,J.;van der Meel,R.;Vongpromek,R.;van Bergen En Henegouwen,P.M.;Storm,G.;Roovers,R.C.,Downregulation of EGFR by a novel multivalent nanobody-liposome platform.J Control Release,2010,145,(2),165-175.

[0212]

[29] Weber,C.;Coester,C.;Kreuter,J.;Langer,K.,Desolvation process and surface characterisation of protein nanoparticles.Int J Pharm,2000,194,(1),91-102.

[0213]

[30] Langer,K.;Balthasar,S.;Vogel,V.;Dinauer,N.;von Briesen,H.;Schubert,D.,Optimization of the preparation process for human serum albumin (HSA) nanoparticles.Int J Pharm,2003,257,(1-2),169-180.

[0214]

[31] Lomis,N.;Westfall,S.;Farahdel,L.;Malhotra,M.;Shum-Tim,D.;Prakash,S.,Human Serum Albumin Nanoparticles for Use in Cancer Drug Delivery: Process Optimization and In Vitro Characterization.Nanomaterials (Basel),2016,6,(6).

[0215]

[32] Caporale,A.;Monti,A.;Selis,F.;Sandomenico,A.;Tonon,G.;Ruvo,M.;Doti,N.,A comparative analysis of catalytic activity and stability of microbial transglutaminase in controlled denaturing conditions.J Biotechnol,2019,302,48-57.

[0216]

[33] Caporale,A.;Selis,F.;Sandomenico,A.;Jotti,G.S.;Tonon,G.;Ruvo,M.,The LQSP tetrapeptide is a new highly efficient substrate of microbial transglutaminase for the site-specific derivatization of peptides and proteins.Biotechnol J,2015,10,(1),154-161.

[0217]

[34] Doti,N.;Caporale,A.;Monti,A.;Sandomenico,A.;Selis,F.;Ruvo,M.,A recent update on the use of microbial transglutaminase for the generation of biotherapeutics.World J Microbiol Biotechnol,2020,36,(4),53.

[0218]

[35] Selis,F.;Foca,G.;Sandomenico,A.;Marra,C.;Di Mauro,C.;Saccani Jotti,G.;Scaramuzza,S.;Politano,A.;Sanna,R.;Ruvo,M.;Tonon,G.,Pegylated Trastuzumab Fragments Acquire an Increased in Vivo Stability but Show a Largely Reduced Affinity for the Target Antigen.Int J Mol Sci,2016,17,(4),491.

[0219]

[36] Caporale,A.;Doti,N.;Sandomenico,A.;Ruvo,M.,Evaluation of combined use of Oxyma and HATU in aggregating peptide sequences.J Pept Sci,2017,23,(4),272-281.

Claims

1. Serum albumin nanoparticles (Alb-NPs) modified with at least one modification chain: - A linker bonded to the nanoparticles by an -S-thioether bond, • At least one type of biomolecule, Includes, At least one of the aforementioned biomolecules is bound to the linker via an amide bond formed by an amide group transfer (or transglutamation) reaction mediated by the enzyme transglutaminase. The aforementioned nanoparticles are given by the following formula (I): 【Chemistry 1】 It has, In the formula, Alb-NP is a serum albumin nanoparticle having a thiol functional group (-SH) on its surface that reacts with the electrophilic group of Z to form the -S-thioether bond; Z-Spacer-X-NH- is the linker: Z is derived from an electrophilic functional group that can react with the -SH group of albumin, thereby forming the -S-thioether bond, and the functional group is selected from 2-bromoacetic acid, 3-bromopropanoic acid, 3-chloropropanoic acid, 4-bromobutyric acid, 5-chlorobutyric acid, 5-bromopentanoic acid, 5-chloropentanoic acid, 4-bromomethylbenzoic acid, 4-chloromethylbenzoic acid, 2-maleimidoacetic acid, 3-maleimidopropionic acid, 4-maleimidobutyric acid, 5-maleimopentanoic acid, 6-maleimidohexanoic acid, 3-maleimidobenzoic acid, 4-maleimidobenzoic acid, 4-(2-N-maleimido)methylbenzoic acid, 1-bromoacetic acid and 1-chloroacetic acid; The aforementioned spacer is: -NH-(CH ２ -O) ｎ -CH ２ -CO- (n is between 2 and 10); -NH-(CH ２ -CH ２ -O) ｎ -CH ２ -CO- (where n is between 2 and 10); Or the spacer is Y ｍ It is the basis, Y is: ・ -NH-(CH ２ ) ｎ -CO-amino acid (n is from 3 to 10); Glycine, alanine; - and any combination thereof; selected from m is a number between 1 and 5; The -NH- group of the spacer forms an amide bond with the carboxylic acid group of the precursor of Z, and the -CO- group of the spacer forms an amide bond with the -NH- group of the next unit X; The -X-NH- group is: -NH-(CH ２ ) ｎ -NH- (where n is in the range of 3 to 10); ・-NH-(O-CH ２ ) ｎ -NH- (where n is in the range of 2 to 10); ・-NH-(O-CH ２ -CH ２ ) ｎ -NH- (where n is in the range of 2 to 10); L-lysine amino acid; L-ornithine amino acid; • C-terminally amidated L-lysine amino acid; Selected from C-terminally amidated L-ornithine amino acids, The two -NH- groups of the -X-NH- group form an amide bond with the -CO- group of the spacer, and formula (VI): AA １ -AA ２ -Q-AA ３ -AA ４ Formula (VI) The consensus sequence having the following characteristics forms an amide bond with glutamine, During the ceremony: AA １ is either leucine (L; Leu) or absent; AA ２ It is either leucine (L; Leu) or threonine (T; Thr); Q is the formula -CO-(CH ２ ) ２ It is a glutamine containing -CH-(NH)-CO-; AA ３ It is either serine (S; Ser) or glycine (G; Gly); AA ４ It is either proline (P; Pro), alanine (A; Ala), or absent; R1 and R2, which are different from or identical to each other, are selected from Fab, scFv, nanobody (NB), antibody and combinations thereof, or The albumin nanoparticles are given by formula (II): 【Chemistry 2】 It has, In the formula, Z - Spacer - AA １ -AA ２ -Q-AA ３ -AA ４ is a linker; Alb-NP, Z, spacer, R1 and R2 are defined by formula (I) above; Formula -CO-(CH ２ ) ２ AA with Q equal to glutamine having -CH-(NH)-CO- １ -AA ２ -Q-AA ３ -AA ４ This is as defined by formula (I) above, and the -CO- terminus of the spacer and, if present, the amino acid AA １ of, or AA ２ The spacer is bonded by an amide bond between the -NH- group and the -NH- group; The peptide sequence containing lysine (K; Lys) is formula (IX): (AA) ｗ -K-(AA) ｐ Formula (IX) It has, In the formula, w and p are integers between 0 and 8, provided that w and p are never simultaneously equal to 0; AA represents an amino acid selected from alanine (A; Ala), tyrosine (Y; Tyr), phenylalanine (F; Phe), glycine (G; Gly), tryptophan (W; Trp), and serine (S; Ser); K stands for lysine (Lys), and these are albumin nanoparticles.

2. The albumin nanoparticles according to claim 1, wherein the nanoparticles are human albumin nanoparticles (NP-HSA) or bovine albumin nanoparticles (NP-BSA).

3. Albumin nanoparticles according to claim 1 or 2, wherein the nanoparticles have an average diameter (or Z-average size) of 100 to 500 nm as measured by dynamic light scattering (DLS) technique.

4. Albumin nanoparticles according to any one of claims 1 to 3, loaded with at least one cytotoxic drug selected from 5-FU, capecitabine, cytarabine, fludarabine, cladribine, paclitaxel, doxorubicin, daunorubicin, epirubicin, docetaxel, vinblastine, vincristine, vinorelbine, mercaptopurine, methotrexate, larcitrexed, etoposide, teniposide, camptothecin, irinotecan, topotecan, and combinations thereof.

5. Albumin nanoparticles according to any one of claims 1 to 4, wherein R1 and R2 are different from each other, and R1 is of two different types: Fab1 and Fab2; scFv is of two different types: scFv1 and scFv2; NB is of two different types: NB1 and NB2; antibody is of two different types: Ab1 and Ab2; or R1 and R2 are a hybrid combination of biomolecules.

6. Albumin nanoparticles according to any one of claims 1 to 5, wherein the antibody is a monoclonal antibody selected from antibody DI17E6, trastuzumab, partuzumab, cetuximab, and anti-Cripto-1 monoclonal antibody; the Fab is selected from recombinant Fab of trastuzumab, recombinant Fab of partuzumab, and recombinant Fab of anti-Cripto-1 monoclonal antibody; the scFv is a functional fragment of antibody DI17E6, trastuzumab, partuzumab, cetuximab, and anti-Cripto-1 monoclonal antibody; and the nanobody (NB) is selected from anti-VEGFR2 NB; anti-Her2 NB; and anti-EGFR NB.

7. Albumin nanoparticles according to any one of claims 1 to 6, wherein Z is derived from a functional group selected from 1-bromoacetic acid, 1-chloroacetic acid, and 6-maleimidohexanoic acid.

8. The aforementioned spacer is -NH-(CH ２ -CH ２ -O) ｎ -CH ２ Albumin nanoparticles according to any one of claims 1 to 7, wherein the nanoparticle is -CO- (where n is included in 2 to 5) or glycine.

9. Albumin nanoparticles according to any one of claims 1 to 8, wherein the X-NH- group is selected from L-lysine amino acids and C-terminally amidated L-lysine amino acids.

10. Albumin nanoparticles according to any one of claims 1 to 9, wherein the consensus sequence is selected from LQSP, TQGA, and LLQG.

11. The above formula (IX) (A) ｗ -K-(AA) ｐ In, Albumin nanoparticles according to any one of claims 1 to 8, wherein w is equal to 0 and p is equal to 1 to 3.

12. The serum albumin nanoparticle according to any one of claims 1 to 11, wherein the nanoparticle is covalently bound to a fluorescent dye on its surface. 【Request Item 13】 【Chemistry 3】 【change】 Serum albumin nanoparticles according to any one of claims 1 to 12, selected from the above.

14. The aforementioned at least one type of biomolecule, (i) -NH of the X group of the linker ２ The residue and the glutamine-CO-NH group included in the peptide consensus sequence inserted into the at least one biomolecule. ２ Between the residues; or (ii) Lysine-NH contained in the peptide sequence inserted into the at least one biomolecule ２ The residue and the glutamine -CO-NH that is inserted into the consensus sequence which is part of the linker. ２ A method for producing albumin nanoparticles according to any one of claims 1 to 13, wherein the nanoparticles are bound to a linker via an amide bond formed by an amide group transfer (or transglutamation) reaction mediated by the enzyme transglutaminase with respect to a residue.

15. A pharmaceutical agent for treating cancerous conditions, comprising albumin nanoparticles as described in claim 4.

16. The pharmaceutical product according to claim 15, wherein the cancerous condition is selected from melanoma, breast cancer, metastatic breast cancer, glioma, glioblastoma, adenocarcinoma, intestinal cancer, pancreatic cancer, bone cancer, kidney cancer, colon cancer, gastric cancer, chronic lymphocytic leukemia, non-small cell lung cancer, advanced and / or metastatic kidney cancer, head and neck cancer, advanced melanoma, non-Hodgkin lymphoma, metastatic melanoma, lung cancer, chronic lymphocytic leukemia (CLL), non-Hodgkin lymphoma, and age-related macular degeneration.

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